Contents and Abstracts of Latest Issue of BBB

(Vol.65 No.9 2001)

Chemotaxis of Fungal Zoospores, with Special Reference
to Aphanomyces cochlioides
Md. Tofazzal ISLAM and Satoshi TAHARA† p.1933

Super-channel in Bacteria: Function and Structure of a Macromolecule
Import System Mediated by a Pit-dependent ABC Transporter
Wataru HASHIMOTO, Keiko MOMMA, Yumiko MISHIMA, Bunzo MIKAMI,＊
and Kousaku MURATA† p.1949

Production of Catechol from Benzoate by the Wild Strain Ralstonia
Species Ba-0323 and Characterization of Its Catechol 1,2-Dioxygenase
Chuan Lu WANG,1 Shinji TAKENAKA,2 Shuichiro MURAKAMI,2 and Kenji AOKI2,†　p.1957

The Relationship Between a Leaf-rolling Moth (Dactylioglypha tonica)
and Fungi Covering the Cocoon
Nobutaka IMAMURA,† Takahiro ISHIKAWA, Keiichi TAKEDA, Hiroshi FUKAMI,
Aya KONNO,＊ and Ritsuo NISHIDA＊＊ p.1965

Amino Acid Supplementation Affects Hematological and Biochemical
Parameters in Elite Rugby Players
Masaru OHTANI,1† Kimiaki MARUYAMA,2 Masaaki SUGITA,3 and Kando KOBAYASHI1 p.1970

Enzyme-assisted Preparation of D-tert.-Leucine
Kurt LAUMEN,† Oreste GHISALBA, and Kurt AUER p.1977

Volatilization of Mercury under Acidic Conditions from Mercury-polluted
Soil by a Mercury-resistant Acidithiobacillus ferrooxidans SUG 2-2
Fumiaki TAKEUCHI,1 Kenji IWAHORI,2 Kazuo KAMIMURA,3 Atsunori NEGISHI,4
Terunobu MAEDA,4 and Tsuyoshi SUGIO2,† p.1981

Composition, Characteristic and Activity of Rare Earth Element-Bound
Polysaccharide from Tea
Dongfeng WANG,1,2,† Changhong WANG,1,3 Guiwen ZHAO,2 Zhenggui WEI,2
Ye TAO,4 and Xingguo LIANG5 p.1987

Relationship between Solubility of Grifolan, a Fungal 1,3-β-D-Glucan,
and Production of Tumor Necrosis Factor by Macrophages in Vitro
Ken-ichi ISHIBASHI, Noriko N. MIURA, Yoshiyuki ADACHI, Naohito OHNO,†
and Toshiro YADOMAE p.1993

N-Linked Oligosaccharides of Glycoproteins from Ginkgo biloba Pollen,
An Allergenic Pollen†
Yoshinobu KIMURA, Masashi SUZUKI, and Mariko KIMURA p.2001

Construction of a Bacillus subtilis (natto) with High Productivity
of Vitamin K2 (Menaquinone-7) by Analog Resistance
Yoshinori TSUKAMOTO,† Makoto KASAI, and Hiroyuki KAKUDA p.2007

Incorporation of Sulfonylurea into N-Isopropylacrylamide
as an Extracellular Matrix for an Artificial Pancreas
Kun NA,1 Hoo-Kyun CHOI,1 Dong-Woon KIM,2 Toshihiro AKAIKE,3
and Keun Hong PARK1† p.2016

Thermostabilization of Ovalbumin in a Developing Egg
by an Alkalinity-regulated, Two-step Process
Hajime HATTA,1 Masayo NOMURA,1 Nobuyuki TAKAHASHI,2,† and Masaaki HIROSE2,†
p.2021

Purification and Some Properties of a β-Glucosidase
from Trichoderma harzianum Type C-4
Soo-In YUN, Choon-Soo JEONG, Dae-Kyun CHUNG,1 and Hye-Seon CHOI† p.2028

CH-19 Sweet, a Non-Pungent Cultivar of Red Pepper, Increased Body
Temperature and Oxygen Consumption in Humans
Koichiro OHNUKI, Shoji NIWA,＊ Satoshi MAEDA,＊ Naohiko INOUE,
Susumu YAZAWA,＊ and Tohru FUSHIKI p.2033

Capability of Wild Rosa rugosa and Its Varieties and Hybrids
to Produce Sesquiterpene Components in Leaf Glandular Trichomes
Yasuyuki HASHIDOKO,1,2 Keiko ENDOH,1 Toshihiro KUDO,3 and Satoshi TAHARA1,2 p.2037

Enzymatically Enantioselective Hydrolysis of Prochiral
1,3-Diacyloxyglycerol Derivatives
Yoshihiko YASOHARA,† Noriyuku KIZAKI, Kenji MIYAMOTO,＊
Junzo HASEGAWA, and Takehisa OHASHI＊ p.2044

Production and Characterization of Recombinant Phanerochaete
chrysosporium Cellobiose Dehydrogenase
in the Methylotrophic Yeast Pichia pastoris
Makoto YOSHIDA,1 Tsuyoshi OHIRA,1 Kiyohiko IGARASHI,1,2 Hiromichi NAGASAWA,1
Katsumi AIDA,1 B. Martin HALLBERG,2 Christina DIVNE,2
Takeshi NISHINO,3 and Masahiro SAMEJIMA1† p.2050

Identification of Catalytic and Substrate-binding Site Residues
in Bacillus cereus ATCC7064 Oligo-1,6-glucosidase
Kunihiko WATANABE,† Kazue MIYAKE, and Yuzuru SUZUKI p.2058

Synthesis of Methyl (5Z,9Z,17R)-17-Methylnonadeca-5,9-dienoate,
the (R)-Enantiomer of the Structure Proposed for a Metabolite
of the Philippine Sponge Plakinastrella sp.
Miho TAKAGI, Hirosato TAKIKAWA, and Kenji MORI† p.2065

Effects of Bisphenol A and its Derivatives on the Response of GABAA
Receptors Expressed in Xenopus Oocytes
Hitoshi AOSHIMA,＊,† Sheikh Julfikar HOSSAIN,＊ Hideshige IMAMURA,＊ Ryuzou SHINGAI＊＊ p.2070

Note
Effect of Dimethylsulfoxide on Hydrolysis of Lipase
Wakako TSUZUKI,† Akemi UE, and Yoshiaki KITAMURA p.2078

Note
Effects of Chicken Essence Tablets on Resting Metabolic Rate
Takeshi IKEDA, Yasufumi NISHIJIMA,＊ Yoshinobu KISO,＊ Hiroshi SHIBATA,＊
Hiroyuki ONO,＊＊ and Toshio MORITANI p.2083

Note
Hibiscus Acid as an Inhibitor of Starch Digestion
in the Caco-2 Cell Model System
Chanida HANSAWASDI, Jun KAWABATA,† and Takanori KASAI p.2087

Note
Cloning and Nucleotide Sequence of the Pullulanase Gene
of Thermus thermophilus HB8 and Production
of the Enzyme in Escherichia coli
Kanae TOMIYASU, Kumiko YATO, Megumi YASUDA, Takashi TONOZUKA,1
Akiko IBUKA, and Hiroshi SAKAI2 p.2090

Note
Mass Production of Pure Gibberellin A1 by Phaeosphaeria sp. L487
and the Fungal Preparation of [U-13C]Gibberellin A1
Hiromichi KENMOKU,1 Tadaomi OOZONE,2 Tomoharu SUGAI,2 and Takeshi SASSA1,2,†
p.2095

Note
Primary Culture of Porcine Mammary Epithelial Cells as a Model System
for Evaluation of Milk Protein Expression
Haruto KUMURA,† Atsushi TANAKA, Youichi ABO, Saori YUI,1 Kei-ichi SHIMAZAKI,
Eiji KOBAYASHI,2 and Kazuhiko SAYAMA1 p.2098

Note
IgE-reactive 60 kDa Glycoprotein Occurring in Wheat Flour
Jun WATANABE,1,2 Soichi TANABE,3 Kei SONOYAMA,1,† Mitsuji KURODA,4
and Michiko WATANABE4 p.2102

Note
Synthesis of (-)-Methyl Shikimate via Enzymatic Resolution
of (1S＊, 4R＊, 5R＊)-4-Hydroxy-6-oxabicyclo[3.2.1]oct-2-en-7-one
Takayuki ORITANI, Rumi UEDA, and Hiromasa KIYOTA＊ p.2106

Note
Simple Synthesis of Climacostol, a Defensive Secretion
by the Ciliate Climacostomum virens
Yumi ABE1 and Kenji MORI1,2,† p.2110

Note
Cellular Localization of the Signaling Components
of Arabidopsis His-to-Asp Phosphorelay
Aya IMAMURA, Yuriko YOSHINO, and Takeshi MIZUNO† p.2113

Preliminary Communication
Synthesis of Macrotetrolide α, a Designed Polynactin Analog Composed
of (+)- and (-)-Bishomononactic Acids
Tadaatsu HANADATE, Hiromasa KIYOTA,† and Takayuki ORITANI p.2118

-1-
Chemotaxis of Fungal Zoospores, with Special Reference
to Aphanomyces cochlioides

Md. Tofazzal ISLAM and Satoshi TAHARA†

Laboratory of Ecological Chemistry, Division of Applied Bioscience, Graduate School of Agriculture,
Hokkaido University, Kita-ku, Sapporo 060-8589, Japan

Zoospores of phytopathogenic fungi accumulate at the potential infection sites of host roots by chemotaxis. The aggregated spores then adhere, encyst, germinate, and finally penetrate into the root tissues to initiate infection. Some of the host-specific attractants have already been identified. The host-specific attractants also induce cell differentiation of certain zoospores under laboratory conditions. This indicates that a signal released from the roots of the host plant guides the pest propagules for orientation and prepares them for establishing a host-pathogen relationship by necessary physiological changes. Some non-host plant secondary metabolites were found to markedly regulate behavior and viability of zoospores, suggesting that non-host compounds may also play a role in protecting the non-host plants from the attack of zoosporic fungi. We hypothesized that zoospores perceive the host signal(s) by specific G-protein-coupled receptors and translate it into responses by way of the phosphoinositide-Ca2+ signaling cascade. The details of the signal transduction mechanism in fungal zoospores are yet to be discovered. In this report, we review the signaling and communications between phytopathogenic fungal zoospores and host and non-host plants with special reference to Aphanomyces cochlioides.
Key words: Aphanomyces cochlioides; zoospore; chemotaxis; signaling substances; G-proteins

-2-
Super-channel in Bacteria: Function and Structure of a Macromolecule
Import System Mediated by a Pit-dependent ABC Transporter

Wataru HASHIMOTO, Keiko MOMMA, Yumiko MISHIMA, Bunzo MIKAMI,＊
and Kousaku MURATA†

Department of Basic and Applied Molecular Biotechnology, Graduate School of Agriculture,
Kyoto University, Uji, Kyoto 611-0011, Japan
＊Department of Quality Design and Development, Graduate School of Agriculture, Kyoto University,
Uji, Kyoto 611-0011, Japan

In a soil isolate, Sphingomonas sp. A1, the transport of a macromolecule (alginate: 27 kDa) is mediated by a pit-dependent ATP-binding cassette (ABC) transporter. The transporter is different from other ABC transporters so far analyzed in that its function is dependent on the pit, a mouth-like organ formed on the cell surface only when the cells are compelled to assimilate macromolecules, and in that it allows direct import of macromolecules into cells. The ABC transporter coupled with the pit, which functions as a funnel and/or concentrator of macromolecules to be imported, was designated as the <0192><0192>Super-channel′′, and in this review, we discuss the three-dimensional structure and specific function of the <0192><0192>Super-channel′′ for macromolecule import found for the first time in a bacterium.
Key words: super-channel; pit; ABC transporter; macromolecule import; three-dimensional structure

-3-
Production of Catechol from Benzoate by the Wild Strain Ralstonia
Species Ba-0323 and Characterization of Its Catechol 1,2-Dioxygenase

Chuan Lu WANG,1 Shinji TAKENAKA,2 Shuichiro MURAKAMI,2 and Kenji AOKI2,†

1Division of Science of Biological Resources, The Graduate School of Science and Technology
and 2Laboratory of Applied Microbiology, Department of Biofunctional Chemistry,
Faculty of Agriculture, Kobe University, Rokko, Kobe 657-8501, Japan

Received October 24, 2000; Received May 1, 2001
Ralstonia sp. Ba-0323, a wild strain isolated from soil, produced catechol from benzoate and accumulated it outside the cells. The bacterium produced a maximal amount of catechol (1.6 mg/ml) from 3 mg/ml of sodium benzoate in a 20-h growing culture. The conversion rate of benzoate to catechol was 70％ on a molar basis. The catechol production by the resting cells increased in the presence of glycerol, and the maximal amount of catechol produced from 3 mg/ml of sodium benzoate reached 1.9 mg/ml at the conversion rate of 83％ after 8 h of incubation. Catechol 1,2-dioxygenase, which catalyzed the ring cleavage of catechol, was purified to homogeneity from a cell extract of Ralstonia sp. Ba-0323 growing on benzoate and characterized. The specific activity of the purified enzyme was much lower than those of the dioxygenases from other microorganisms reported. The Km for catechol of the purified enzyme was much higher than those of other dioxygenases. In addition, the NH2-terminal amino acid sequence of the enzyme was less similar to the other catechol 1,2-dioxygenases than they are to each other.
Key words: benzoate-assimilating bacterium; catechol accumulation; catechol 1,2-dioxygenase; specific activity

-4-
The Relationship Between a Leaf-rolling Moth (Dactylioglypha tonica)
and Fungi Covering the Cocoon

Nobutaka IMAMURA,† Takahiro ISHIKAWA, Keiichi TAKEDA, Hiroshi FUKAMI,
Aya KONNO,＊ and Ritsuo NISHIDA＊＊

Department of Bioscience and Biotechnology, Faculty of Science and Engineering, Ritsumeikan University,
Noji-higashi, Kusatsu, Shiga 525-8577, Japan
＊Research Center for the Industrial Utilization of Marine Organisms, Sodeshi, Shimizu 424-0037, Japan
＊＊Division of Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku,
Kyoto 606-8502, Japan

Received December 6, 2000; Accepted April 24, 2001
To discover the relationship between a leaf-rolling moth and the fungi densely covering its cocoons, the rolled nest leaves were collected in two districts in Japan and antibacterial properties of the fungi were examined. Cocoons and fungi isolated from the nest were classified into 5 categories by the growth stages of the insects, and 7 categories based on taxonomic properties and pigment productivity, respectively. The dominant genus was Penicillium in each location. However, the composition of the fungal categories was different and seemed to depend on their circumstances. From all cocoons with larvae, the strains that belonged to the same fungal category and produced the same antibiotic (deoxyherqueinone) were isolated. From these results, the species-specific relationship between the insect and fungi or fungal products was considered to be not extremely tight, and it was suggested the period of the larval spinning of the cocoon is a key stage of this unique relationship.
Key words: antibiotic; Dactylioglypha tonica; fungi covering the cocoon; Penicillium; species-specific relationship

-5-
Amino Acid Supplementation Affects Hematological and Biochemical
Parameters in Elite Rugby Players

Masaru OHTANI,1† Kimiaki MARUYAMA,2 Masaaki SUGITA,3 and Kando KOBAYASHI1

1The Department 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
3Department of Health and Physical Education, Mie University, Tsu 514-8507, Japan

Received December 22, 2000; Accepted May 17, 2001
Individual amino acid supplementation affects various types of athletic performance. However, little information on combinations of amino acids is currently available. This study evaluated an amino acid mixture containing L-leucine, L-isoleucine, L-valine, L-arginine, and L-glutamine to 3.6 g of total amino acids per dose. Twenty-three rugby players were given 3.6 g, twice, daily of the amino acid mixture for 90 days (June-August 1994) and blood samples were collected for analyses in September 1993, March 1994, September 1994, and September 1995. After 90 days of supplementation, almost all of the athletes reported improvement in vigor and earlier recovery from fatigue. Significant increases (P＜0.05) were observed in hemoglobin, RBC count, hematocrit, and serum iron by amino acid supplementation. Significant increases (P＜0.05) were also noted in total cholesterol and low-density lipoprotein along with decreased (P＜0.05) alkaline phosphatase. All values reverted to original levels when measured after one year of continued training without supplementation.
Key words: amino acids; exercise; hematology; blood chemistry; sports medicine

-6-
Enzyme-assisted Preparation of D-tert.-Leucine

Kurt LAUMEN,† Oreste GHISALBA, and Kurt AUER

Novartis Pharma AG, Core Technology Area, CH-4002 Basel, Switzerland

Received January 9, 2001; Accepted April 25, 2001
Optically pure D-tert.-leucine was obtained by the enzymatic hydrolysis of (±)-N-acetyl-tert. leucine chloroethyl ester after about 50％ conversion, this being catalyzed by a protease from Bacillus licheniformis (Alcalase<09-30>), and subsequent acidic saponification of the recovered ester. Among the methyl, ethyl, octyl, chloroethyl and trichloroethyl esters, the chloroethyl ester exhibited the highest rate of hydrolysis.
Key words: D-tert.-leucine; Bacillus licheniformis; enzymatic hydrolysis; protease; Alcalase<09-30>

-7-
Volatilization of Mercury under Acidic Conditions from Mercury-polluted
Soil by a Mercury-resistant Acidithiobacillus ferrooxidans SUG 2-2

Fumiaki TAKEUCHI,1 Kenji IWAHORI,2 Kazuo KAMIMURA,3 Atsunori NEGISHI,4
Terunobu MAEDA,4 and Tsuyoshi SUGIO2,†

1Administration Center for Environmental Science and Technology, 2Division of Science and Technology
for Energy Conversion, Graduate School of Natural Science and Technology, 3Department
of Biological Function, Faculty of Agriculture, Okayama University, Tsushima Naka,
Okayama 700-8530, Japan
4Technical Research Institute, Hazama Corporation, 515-1 Nishimukai, Karima, Tsukuba 305-0822, Japan

Received January 9, 2001; Accepted April 27, 2001
Volatilization of mercury under acidic conditions from soil polluted with mercuric chloride (1.5 mg Hg/kg soil) was studied with resting cells of a mercury-resistant strain, Acidithiobacillus ferrooxidans SUG 2-2. When resting cells of SUG 2-2 (0.01 mg of protein) were incubated for 10 d at 30°C in 20 ml of 1.6 mM sulfuric acid (pH 2.5) with ferrous sulfate (3％) and mercury-polluted soil (1 g), which contained 7.5 nmol of Hg, approximately 4.1 nmol of mercury was volatilized, indicating that 54％ of the total mercury in the soil was volatilized. The amount of mercury volatilized from the soil was dependent on the concentration of Fe2+ added to the medium. When elemental sulfur, sodium tetrathionate, and pyrite were used as an electron donor for the mercury reduction, 16, 2.4 and 0.84％, respectively, of the total mercury added to the solution were volatilized. The optimum pH and temperature for mercury volatilization were 2.5 and 30°C. Approximately 92％ of the total mercury in a salt solution (pH 2.5) with resting cells of SUG 2-2 (0.01 mg of protein), ferrous sulfate (3％) and mercury-polluted soil (1 g) was volatilized by further addition of both resting cells and Fe2+ and by incubating for 30 d at 30°C.
Key words: Acidithiobacillus ferrooxidans; mercury; resistance; volatilization of mercury; iron-oxidizing bacterium

-8-
Composition, Characteristic and Activity of Rare Earth Element-Bound
Polysaccharide from Tea

Dongfeng WANG,1,2,† Changhong WANG,1,3 Guiwen ZHAO,2 Zhenggui WEI,2
Ye TAO,4 and Xingguo LIANG5

1Key Laboratory of Tea Biotechnology, Agricultural Ministry, the People′s Republic of China,
Anhui Agricultural University, Hefei 230036, China
2Department of Chemistry, University of Science and Technology of China, Hefei 230036, China
3Food Engineering Department, Ocean University of Qingdao, Qingdao 266003, China
4Beijing High-energy Physical Institute, Beijing 100039, China
5Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Kamaba,
Meguro-ku, Tokyo, Japan

Received January 18, 2001; Accepted May 23, 2001
The compositions and structural characteristics of rare earth elements-bound polysaccharides from tea (REE-TPS) were studied with the methods of Inductively Coupled Plasma Mass Spectrometry (ICP-MS), Gas Chromatography (GC) and Extended X-ray Absorption Fine Structure (EXAFS) spectroscopy. The results show that polysaccharide from tea (TPS) was a sort of glycoprotein and coordinated with Rare Earth Elements (REE) closely. The sugar fraction was composed of Rha, Ara, Xyl, Fuc, Glc, and Gal. There existed almost all natural amino acids with Glx, Asx, and Hyp as the major parts in the protein fraction. The REEs in REE-TPS were mainly composed of La, Ce, and Nd, especially, more than 75％ of them was La. The coordination atom of the first coordination shell of La in REE-TPS was oxygen, the coordination number of which was 6, and the average distance between the atoms was 2.52 <0197>. The second shell was formed from sulfur atoms, the coordination number and the average distance were 3 and 2.91 <0197>, respectively. The bio-experiments show that REE-TPS could decrease the content of blood glucose in mice significantly.
Key words: tea; rare earth elements; polysaccharide; composition analysis; bioactivity

-9-
Relationship between Solubility of Grifolan, a Fungal 1,3-β-D-Glucan,
and Production of Tumor Necrosis Factor by Macrophages in Vitro

Ken-ichi ISHIBASHI, Noriko N. MIURA, Yoshiyuki ADACHI, Naohito OHNO,†
and Toshiro YADOMAE

Laboratory for Immunopharmacology for Microbial Products, School of Pharmacy, Tokyo University
of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan

Received January 29, 2001; Accepted April 4, 2001
Grifolan, GRN, is a fungal antitumor β-glucan isolated from Grifola frondosa. Various studies suggested that the underlying mechanism of the antitumor activity of GRN is strongly related to immune modulation. In the previous publication (Adachi et al., 1994; Okazaki et al., 1995), we have shown that GRN activates macrophages to produce tumor necrosis factor (TNF) in vitro. In this study, the structural unit essential to produce TNF was examined by chemical modifications of GRN. GRN suspended in distilled water was treated at 150°C for up to 3 h. Addition of the resulting turbid solution to the RAW 264.7 macrophage-like cell line produced TNF, and the relative activity was diminished in relation to the heat treatment period. The fractions with a heating period longer than 15 min did not show any activity. After centrifugation of the resulting solution, significant activity was shown by precipitate fractions, suggesting that the insoluble form of GRN is important for TNF production. Interestingly, the precipitate fraction obtained from 9 min of treatment also had significant activity. In addition, admixing the soluble fraction with the particles significantly inhibited the TNF production. In contrast to these observations, the high-molecular-mass subfraction of the soluble fraction prepared by ultrafiltration produced significant amounts of TNF. Similar phenomena were shown with sodium hydroxide treatment and dimethylsulfoxide treatment. These facts strongly suggested that insoluble as well as a high molecular mass soluble form of GRN are required for TNF production by macrophages.
Key words: β-glucan; grifolan; tumor necrosis factor; macrophage; molecular mass

-10-
N-Linked Oligosaccharides of Glycoproteins from Ginkgo biloba Pollen,
An Allergenic Pollen†

Yoshinobu KIMURA, Masashi SUZUKI, and Mariko KIMURA

Department of Bioresources Chemistry, Faculty of Agriculture, Okayama University,
Okayama 700-8530, Japan

Received February 2, 2001; Accepted April 16, 2001
The pollen of Ginkgo biloba is one of the allergens that cause pollen allergy symptoms. The plant complex type N-glycans bearing β1--2 xylose and/or α1--3 fucose residue(s) linked to glycoallergens have been considered to be critical epitopes in various immune reactions. In this report, the structures of N-glycans of total glycoproteins prepared from Ginkgo biloba pollens were analyzed to confirm whether such plant complex type N-glycans occur in the pollen glycoproteins. The glycoproteins were extracted by SDS-Tris buffer. N-Glycans liberated from the pollen glycoprotein mixture by hydrazinolysis were labeled with 2-aminopyridine and the resulting pyridylaminated (PA-)N-glycans were purified by a combination of size-fractionation HPLC and reversed-phase HPLC. The structures of the PA-
sugar chains were analyzed by a combination of two-dimensional sugar chain mapping, IS-MS, and MS/MS. The plant complex type structures (GlcNAc2Man3Xyl1Fuc1GlcNAc2 (31％), GlcNAc2Man3Xyl1GlcNAc2 (5％), Man3Xyl1Fuc1GlcNAc2 (13％), GlcNAc1Man3Xyl1Fuc1GlcNAc2 (8％), and GlcNAc1Man3Xyl1GlcNAc2 (17％)) have been found among the N-glycans of the glycoproteins of Ginkgo biloba pollen, which might be candidates for the epitopes involved in Ginkgo pollen allergy. The remaining 26％ of the total pollen N-glycans have the typical high-mannose type structures: Man8GlcNAc2 (11％) and Man6GlcNAc2 (15％).
Key words: plant glycoprotein; antigenic N-glycan; sugar chains; pollen allergy; Ginkgo biloba

-11-
Construction of a Bacillus subtilis (natto) with High Productivity
of Vitamin K2 (Menaquinone-7) by Analog Resistance

Yoshinori TSUKAMOTO,† Makoto KASAI, and Hiroyuki KAKUDA

Central Research Institute, Mitsukan Group Corporation, 2-6 Nakamura-cho, Handa-shi,
Aichi 475-8585, Japan

Received February 5, 2001; Accepted April 13, 2001
To invent a functional natto promoting bone formation, the construction of a strain with high productivity of vitamin K2 (menaquinone-7: MK-7), which is important in the carboxylation of a kind of bone protein participating in bone formation, osteocalcin, was investigated. To screen for a strain appropriate to making natto (a Japanese traditional fermented soybean food) with high productivity of MK-7, a combination of analog resistance to the compounds on the biosynthetic pathway of menaquinones with mutation was done. Consequently, strain OUV23481, with 2-fold higher productivity (1,719 μg/100 g natto) of MK-7 than that of a commercial strain, was constructed as a mutant with analog resistance to 1-hydroxy-2-naphthoic acid (HNA), p-fluoro-D,L-phenylalanine (pFP), m-fluoro-D,L-phenylalanine (mFP), and β-2-thienylalanine (βTA). This strain was classified as Bacillus subtilis (natto). The natto made using this strain was evaluated to have a good quality as natto in all the viewpoints of appearance, flavor, taste, texture, and stringiness.
Key words: menaquinone-7; Bacillus subtilis (natto); mutation; analog resistance; osteocalcin

-12-
Incorporation of Sulfonylurea into N-Isopropylacrylamide
as an Extracellular Matrix for an Artificial Pancreas

Kun NA,1 Hoo-Kyun CHOI,1 Dong-Woon KIM,2 Toshihiro AKAIKE,3
and Keun Hong PARK1†

1College of Pharmacy, Chosun University, 375 Seoseok-dong, Dong-ku, Kwangju 501-759, Korea
2Department of Environmental Science, Kwangyang Colllege, Kwangyang 545--703, Korea
3Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama 226, Japan

Received February 5, 2001; Accepted April 19, 2001
High-molecular-weight N-isopropylacrylamide
copolymers with small amounts of sulfonylurea (SU, typically 2--4 mol％ in the feed) were synthesized by free radical polymerization in benzene. SU-incorporated polymer solutions (5, 6, 8, and 10％ w/v) in a culture medium (pH 7.4, 0.15 M ionic strength) with islet cells were mixed and poured into Millicells which supported gel formation. In order to increase the gelation temperature, the SU-incorporated copolymer gel, p(NiPAAm-co-SU), was blended with the p(NiPAAm-co-AAc) polymer at a ratio of 4 to 96. Interaction between the islet cells and the synthetic matrix of SU-incorporated copolymer gel resulted in effective cell viability and such cell functions as insulin secretion. To verify the specific interaction between the SU (K+ channel closer)-incorporated copolymer and islet cells, the cells were pretreated with diazoxide, an agonist of the ATP-sensitive K<Oh>+ channel (K+ channel opener), before interaction between the polymer and islet cells. This treatment suppressed the action of SU on the islet cells. The results from this study provide evidence that the SU-incorporated copolymer stimulated insulin secretion by specific interaction between SU moieties in the polymer and the islet cells.
Key words: gel; islet; ligand; receptor; sulfonylurea

-13-
Thermostabilization of Ovalbumin in a Developing Egg
by an Alkalinity-regulated, Two-step Process

Hajime HATTA,1 Masayo NOMURA,1 Nobuyuki TAKAHASHI,2,† and Masaaki HIROSE2,†

1Department of Food and Nutrition, Kyoto Women′s University, Kyoto 605-8501, Japan
2Research Institute for Food Science, Kyoto University, Uji, Kyoto 611-0011, Japan

Received February 7, 2001; Accepted May 9, 2001
Native ovalbumin has been known to convert into a heat-stable form, S-ovalbumin, either in an avian shell egg or in an isolated ovalbumin solution. Recently, similar conversion of ovalbumin in fertile eggs was also reported. We found that the conversion into S-ovalbumin was slower in fertile eggs than in unfertile eggs under the same incubation conditions on the basis of calorimetric analyses for the samples isolated from those eggs. During the incubation, there were differential pH changes of white in the fertile and unfertile eggs. When the pH of purified ovalbumin was manually adjusted so as to simulate the pH changes of egg white during the incubation, the course of the conversion into S-ovalbumin was very similar to that either in fertile or unfertile eggs. Therefore, we conclude that thermostabilization of ovalbumin in fertile eggs proceeds by a certain mechanism which depends on the alkalinity of egg white.
Key words: ovalbumin; S-ovalbumin; alkaline treatment; fertile egg; embryogenesis

-14-
Purification and Some Properties of a β-Glucosidase
from Trichoderma harzianum Type C-4

Soo-In YUN, Choon-Soo JEONG, Dae-Kyun CHUNG,1 and Hye-Seon CHOI†

Department of Biological Sciences, University of Ulsan, Ulsan 680-749, Korea
1Institute and Department of Genetic Engineering, Kyung-Hee University, Suwon, Korea

Received 5, 2001; Accepted May 7, 2001
Type C-4 strain of Trichoderma harzianum was isolated as a microorganism with high cellulolytic activity. β-Glucosidase is involved in the last step of cellulose saccharification by degrading cellobiose to glucose, and plays an important role in the cellulase enzyme system with a synergic action with endoglucanase and cellobiohydrolase for cellulose degradation. β-Glucosidase from T. harzianum type C-4 was purified to homogeneity through Sephacryl S-300, DEAE-Sephadex A-50, and Mono P column chromatographies. It was a single polypeptide with the molecular mass of 75,000 by SDS-PAGE. The enzyme was very active at pH 5.0 and 45°C. No significant inhibition was observed in the presence of metal ions, thiol reagents, or EDTA. The enzyme was stable in the presence of 5％ ox gall and digestive enzymes. p-Nitrophenyl-β-D-cellobioside worked as a substrate for the enzyme as much as p-nitrophenyl-β-glucopyranoside. Glucose and gluconolactone showed competitive inhibition with a Ki of 1 mM and 1.8 μM, respectively, while galactose, mannose, and xylose did not inhibit the enzyme significantly.
Key words: animal feed; cellulolytic activity; β-glucosidase; Trichoderma harzianum type C-4

-15-
CH-19 Sweet, a Non-Pungent Cultivar of Red Pepper, Increased Body
Temperature and Oxygen Consumption in Humans

Koichiro OHNUKI, Shoji NIWA,＊ Satoshi MAEDA,＊ Naohiko INOUE,
Susumu YAZAWA,＊ and Tohru FUSHIKI

Laboratory of Nutrition Chemistry, Department of Applied Life Sciences, Graduate School of Agriculture,
Kyoto University, Kyoto 606-8502, Japan
＊Laboratory of Vegetable and Ornamental Horticulture, Division of Agriculture, Graduate School
of Agriculture, Kyoto University, Kyoto 606-8585, Japan

Received March 6, 2001; Accepted April 16, 2001
We investigated the effect of CH-19 Sweet, a non-pungent cultivar of red pepper, on body temperature and oxygen consumption in humans. CH-19 Sweet was given to 11 healthy volunteers, and core body temperature, body surface temperature and oxygen consumption were measured. The control group ingested California-Wandar, which contained neither capsaicin nor capsiate. The core body temperature in the CH-19 Sweet group was significantly higher than that in the control group (P＜0.01). The forehead temperature measured by infrared thermography in the CH-19 Sweet group was significantly higher than that in the control group. The body surface temperature was increased for about 20 min after consumption of CH-19 Sweet intake, and the neck temperature was significantly higher (P＜0.001) than when the subjects consumed California-Wandar. We also measured respiratory gas by indirect calorimetry while subjects wore a face mask. A significant difference was detected in oxygen consumption between the two groups, and the value was significantly higher in the CH-19 Sweet group (P＜0.03). These results suggest that CH-19 Sweet increased thermogenesis and energy consumption.
Key words: CH-19 Sweet; capsiate; capsaicin; body temperature; oxygen consumption

-16-
Capability of Wild Rosa rugosa and Its Varieties and Hybrids
to Produce Sesquiterpene Components in Leaf Glandular Trichomes

Yasuyuki HASHIDOKO,1,2 Keiko ENDOH,1 Toshihiro KUDO,3 and Satoshi TAHARA1,2

1Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Kita-ku,
Sapporo 060-8589, Japan
2CREST, Japan Science and Technology Corporation, Honmachi 4-1-8, Kawaguchi 332-0012, Japan
3Yurigahara Park, Sapporo Parks Green Development Association, 210 Shinorocho-Taihei, Kita-ku,
Sapporo 002-8051, Japan

Received March 8, 2001; Accepted April 19, 2001
The sesquiterpene contents in leaves of wild Rosa rugosa and of sixty-one hybrid rugosas were quantitatively measured by a GC analysis. In this group of samples, the greater the number of glandular trichomes the hybrid rugosas possessed on their leaves, the larger the amount of sesquiterpenes they accumulated. In contrast, those having no leaf glandular hairs contained only a trace amount of sesquiterpene components. The concentrations of bisaborosaol A (1) and carota-1,4-dienaldehyde (2) as representative sesquiterpenes of R. rugosa were positively correlated with the density of the glandular trichomes. Furthermore, an approximately regular correlation was observed between the concentrations of 1 and 2 in most of the sesquiterpene-producing hybrid rugosas, regardless of their productivity. This suggests that a major part of these hybrid rugosas have inherited from R. rugosa the ability to produce two skeletally different sesquiterpenes in parallel with a phenotype to develop leaf glandular trichomes. This investigation also led to discovering 1-dominant (e.g., Amelie Gravereaux and Purple Pavement), 2-dominant (e.g., David Thompson), and other-dominant (e.g., Martin Frobisher) types of sesquiterpene-producing hybrid rugosas.
Key words: Rosa rugosa; hybrid rugosa; glandular trichome; bisabolane sesquiterpene; carotane sesquiterpene

-17-
Enzymatically Enantioselective Hydrolysis of Prochiral
1,3-Diacyloxyglycerol Derivatives

Yoshihiko YASOHARA,† Noriyuku KIZAKI, Kenji MIYAMOTO,＊
Junzo HASEGAWA, and Takehisa OHASHI＊

Fine Chemicals Research Laboratories, Kaneka Corporation, 1-8 Miyamae, Takasago, Hyogo 676-8688, Japan
＊Lifescience Research Laboratories, Kaneka Corporation, 1-8 Miyamae, Takasago, Hyogo 676-8688, Japan

Received March 9, 2001; Accepted April 25, 2001
An enzymatically enantioselective ester hydrolysis of prochiral 1,3-diacyloxy-2-substituted-2-propanol to chiral 1-acyloxy-2,3-propanediol was studied. The (R)-monoester was prepared by selection of a suitable lipase and alkyl chain length of the substrate diester. Lipase D from Rhizopus delemer gave (R)-1-isobutyryloxy-2-(2,4-difluorophenyl)-2,3-propanediol with 97％ee and 87％ yield at 15°C and pH 5.5. The (R)-monoester is a key intermediate of azole antifungal agents.
Key words: enantioselective hydrolysis; lipase; glycerol derivative

-18-
Production and Characterization of Recombinant Phanerochaete
chrysosporium Cellobiose Dehydrogenase
in the Methylotrophic Yeast Pichia pastoris

Makoto YOSHIDA,1 Tsuyoshi OHIRA,1 Kiyohiko IGARASHI,1,2 Hiromichi NAGASAWA,1
Katsumi AIDA,1 B. Martin HALLBERG,2 Christina DIVNE,2
Takeshi NISHINO,3 and Masahiro SAMEJIMA1†

1Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku,
Tokyo 113-8657, Japan
2Department of Cell and Molecular Biology, Structural Biology, Biomedical Centre, Uppsala University,
751 24 Uppsala, Sweden
3Department of Biochemistry and Molecular Biology, Nippon Medical School, Bunkyo-ku,
Tokyo 113-8602, Japan

Received March 13, 2001; Accepted April 20, 2001
The hemoflavoenzyme cellobiose dehydrogenase (CDH) from the white-rot fungus Phanerochaete chrysosporium has been heterologously expressed in the methylotrophic yeast Pichia pastoris. After 4 days of cultivation in the induction medium, the expression level reached 1800 U/L (79 mg/L) of CDH activity, which is considerably higher than that obtained previously for wild-type CDH (wtCDH) and recombinant CDH (rCDH) produced by P. chrysosporium. Analysis with SDS-PAGE and Coomassie Brilliant Blue (CBB) staining revealed a major protein band with an approximate molecular mass of 100 kDa, which was identified as rCDH by Western blotting. The absorption spectrum of rCDH shows that the protein contains one flavin and one heme cofactor per protein molecule, as does wtCDH. The kinetic parameters for rCDH using cellobiose, ubiquinone, and cytochrome c, as well as the cellulose-binding properties of rCDH were nearly identical to those of wtCDH. From these results, we conclude that the rCDH produced by Pichia pastoris retains the catalytic and cellulose-binding properties of the wild-type enzyme, and that the Pichia expression system is well suited for high-level production of rCDH.
Key words: cellobiose dehydrogenase (CDH); hemoflavoprotein; Pichia pastoris; Phanerochaete chrysosporium; heterologous expression

-19-
Identification of Catalytic and Substrate-binding Site Residues
in Bacillus cereus ATCC7064 Oligo-1,6-glucosidase

Kunihiko WATANABE,† Kazue MIYAKE, and Yuzuru SUZUKI

Department of Applied Biochemistry, Kyoto Prefectural University, Shimogamo, Sakyo,
Kyoto 606-8522, Japan

Received April 5, 2001; Accepted May 14, 2001
Three active site residues (Asp199, Glu255, Asp329) and two substrate-binding site residues (His103, His328) of oligo-1,6-glucosidase (EC 3.2.1.10) from Bacillus cereus ATCC7064 were identified by site-directed mutagenesis. These residues were deduced from the X-ray crystallographic analysis and the comparison of the primary structure of the oligo-1,6-glucosidase with those of Saccharomyces carlsbergensis α-glucosidase, Aspergillus oryzae α-amylase and pig pancreatic α-amylase which act on α-1,4-glucosidic linkages. The distances between these putative residues of B. cereus oligo-1,6-glucosidase calculated from the X-ray analysis data closely resemble those of A. oryzae α-amylase and pig pancreatic α-amylase. A single mutation of Asp199→Asn, Glu255→Gln, or Asp329→Asn resulted in drastic reduction in activity, confirming that three residues are crucial for the reaction process of α-1,6-glucosidic bond cleavage. Thus, it is identified that the basic mechanism of oligo-1,6-glucosidase for the hydrolysis of α-1,6-glucosidic linkage is essentially the same as those of other amylolytic enzymes belonging to Family 13 (α-amylase family). On the other hand, mutations of histidine residues His103 and His328 resulted in pronounced dissimilarity in catalytic function. The mutation His328→Asn caused the essential loss in activity, while the mutation His103→Asn yielded a mutant enzyme that retained 59％ of the k0/Km of that for the wild-type enzyme. Since mutants of other α-amylases acting on α-1,4-glucosidic bond linkage lost most of their activity by the site-directed mutagenesis at their equivalent residues to His103 and His328, the retaining of activity by His103→Asn mutation in B. cereus oligo-1,6-glucosidase revealed the distinguished role of His103 for the hydrolysis of α-1,6-glucosidic bond linkage.
Key words: oligo-1,6-glucosidase; active site; substrate-binding site; site-directed mutagenesis

-20-
Synthesis of Methyl (5Z,9Z,17R)-17-Methylnonadeca-5,9-dienoate,
the (R)-Enantiomer of the Structure Proposed for a Metabolite
of the Philippine Sponge Plakinastrella sp.

Miho TAKAGI, 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 April 10, 2001; Accepted May 2, 2001
The (R)-enantiomer (1) of methyl (5Z,9Z)-17-methylnonadeca-5,9-dienoate, the structure proposed for a metabolite of the Philippine sponge, Plakinastrella sp., was synthesized. The 1H- and 13C-NMR spectra of the synthetic material were different from those reported for the natural product. The proposed structure 1 is therefore incorrect.
Key words: marine natural product; methyl-branched fatty acid; optically active fatty acid; Plakinastrella sp.; sponge

-21-
Effects of Bisphenol A and its Derivatives on the Response of GABAA
Receptors Expressed in Xenopus Oocytes

Hitoshi AOSHIMA,＊,† Sheikh Julfikar HOSSAIN,＊ Hideshige IMAMURA,＊ Ryuzou SHINGAI＊＊

＊Department of Physics, Biology and Informatics, Faculty of Science, Yamaguchi University, 1677-1 Yoshida,
Yamaguchi 753-8512, Japan and ＊＊Department of Welfare Engineering, Faculty of Engineering,
Iwate University, 4 Ueda, Morioka 020-8551, Japan

Received April 25, 2001; Accepted May 8, 2001
To study the effects of bisphenol-A (BPA) known to have estrogenic actions, and its derivatives, 3,5-dimethylphenol (DMP) and p-t-butylphenol (TBP), on ionotropic γ-aminobutyric acid (GABA) receptors, GABAA receptors were expressed in Xenopus oocytes by injecting both poly(A)+RNA prepared from rat whole brain and cRNAs synthesized from cloned cDNAs of α1 and β1 subunit of the bovine receptors, and their electrical responses were measured by the voltage clamping method. BPA caused the potentiation and inhibition of the former receptor-responses, while it caused only inhibition of the latter ones. In the presence of low concentrations of GABA, DMP and TBP potentiated the responses of both receptors. DMP and TBP also increased the rate of decay of the response, possibly by desensitization of the receptors when GABA solution was continuously bath-applied. Diethyl terephthalate (DTP), which is also known to have estrogenic actions, had little effect on both the responses and the decay of both receptors.
Key words: bisphenol-A; 3,5-dimethylphenol; GABAA receptor; p-t-butylphenol; Xenopus oocyte

-22-
Note
Effect of Dimethylsulfoxide on Hydrolysis of Lipase

Wakako TSUZUKI,† Akemi UE, and Yoshiaki KITAMURA

National Food Research Institute, Kannondai 2-1-12,
Tsukuba, Ibaraki 305-8642, Japan

Received November 14, 2000; Accepted May 9, 2001
To establish an industrially feasible reaction process, the effect of dimethylsulfoxide (DMSO) added to an aqueous solution on the hydrolysis of lipase was investigated using fluorescent substrates. Several lipases from microorganisms were improved in their hydrolysis activities against 4-methylumbelliferyl oleate by DMSO. Variation was found in the effect of DMSO depending on the species of lipase. After the high stability of the lipase from Pseudomonas fluorescens in DMSO solution was confirmed, hydrolysis by this lipase of four acyl-4-methylumbelliferones was studied kinetically at different DMSO concentrations. DMSO added to an aqueous solution increased the Vmax of this lipase for a substrate with strong hydrophobicity, and decreased that value for a substrate with an opposite property. On the other hand, DMSO had a very small effect on Km for each substrate. A fluorometric study suggested that DMSO induced a change of the chemical environment that surrounded tryptophan residues of the lipase. Such conformational change would be one of the causes of the DMSO-induced alteration of its reactive property. These results suggest that the addition of DMSO may be a novel method of <0192>solvent engineering′ of this enzyme.
Key words: lipase; hydrolysis; dimethylsulfoxide

-23-
Note
Effects of Chicken Essence Tablets on Resting Metabolic Rate

Takeshi IKEDA, Yasufumi NISHIJIMA,＊ Yoshinobu KISO,＊ Hiroshi SHIBATA,＊
Hiroyuki ONO,＊＊ and Toshio MORITANI

Laboratory of Applied Physiology, Graduate School of Human and Environmental Studies,
Kyoto University, Yoshidanihonmatsu-cho, Sakyo-ku, Kyoto-city, Kyoto 606-8316, Japan
＊Institute for Health Care Science, Suntory Ltd., Osaka 618-8503, Japan
＊＊Department of Research and Development, Cerebos Pacific Ltd., Singapore 237994, Singapore

Received January 9, 2001; Accepted April 13, 2001
Resting energy expenditure (REE) values after consuming chicken essence tablets were significantly higher than those observed after consuming skim milk protein tablets (control trial). The increased thermogenic effects continued at least for a period of one hour and gradually decreased towards the baseline. The REE values during control treatment did not show such an augmented response.
Key words: chicken essence; thermogenic effects; resting energy expenditure; skim milk protein

-24-
Note
Hibiscus Acid as an Inhibitor of Starch Digestion
in the Caco-2 Cell Model System

Chanida HANSAWASDI, Jun KAWABATA,† and Takanori KASAI

Laboratory of Food Biochemistry, Division of Applied Bioscience, Graduate School of Agriculture,
Hokkaido University, Kita-ku, Sapporo 060-8589, Japan

Received January 17, 2001; Accepted May 22, 2001
Hibiscus acid, an α-amylase inhibitor isolated from roselle tea, and its derivatives were compared in an inhibition test for starch digestion. An α-amylase-added Caco-2 system was established as a useful model to evaluate the effects of α-glucosidase inhibitors on starch digestion. Hibiscus acid showed weak inhibition in this model system, and the methyl ester derivatives showed even weaker or no acitivity.
Key words: hibiscus acid; Hibiscus sabdariffa; roselle tea; starch digestion inhibitor; Caco-2

-25-
Note
Cloning and Nucleotide Sequence of the Pullulanase Gene
of Thermus thermophilus HB8 and Production
of the Enzyme in Escherichia coli

Kanae TOMIYASU, Kumiko YATO, Megumi YASUDA, Takashi TONOZUKA,1
Akiko IBUKA, and Hiroshi SAKAI2

Department of Food and Nutritional Sciences, Graduate School of Nutritional and Environmental Sciences,
University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan

Received January 23, 2001; Accepted April 18, 2001
A 3.4-kb SphI fragment carrying the pullulanase gene of Thermus thermophilus HB8 was cloned. Based on the nucleotide sequence of it and the flanking region analyzed by direct sequencing of the inverse PCR product, an expression vector was constructed. The E. coli cells harboring the plasmid produced an about 80-kDa protein having pullulanase activity, the optimum temperature of which was 70°C.
Key words: pullulanase; Thermus thermophilus; debranching enzyme; gene cloning; inverse PCR

-26-
Note
Mass Production of Pure Gibberellin A1 by Phaeosphaeria sp. L487
and the Fungal Preparation of [U-13C]Gibberellin A1

Hiromichi KENMOKU,1 Tadaomi OOZONE,2 Tomoharu SUGAI,2 and Takeshi SASSA1,2,†

1The United Graduate School of Agricultural Sciences, Iwate University (Yamagata University),
Tsuruoka 997-8555, Japan
2Department of Bioresource Engineering, Faculty of Agriculture, Yamagata University, Wakaba-cho,
Tsuruoka 997-8555, Japan

Received February 6, 2001; Accepted April 20, 2001
The mass production of pure gibberellin A1 (GA1) by shake-culturing Phaeosphaeria sp. L487 was investigated. Its GA1 production was markedly influenced by natural nitrogen sources and NH4NO3. When the fungus was cultured in an 8％ glucose--1.5％ oatmeal--0.1％ NH4NO3--0.5％ KH2PO4--0.1％ MgSO4・7H2O medium for 3 weeks, the amount of GA1 in the culture filtrate was up to ca. 200 μg/ml: the addition of safflower oil to the culture medium two weeks after inoculation prolonged the GA1-production period to produce 300 μg/ml. Further preparation of [U-13C]GA1 as a tool for the analysis of a complex of GA1 and its binding protein was attempted by using the fungus. The fungal culture in a [U-13C]glucose-oatmeal medium gave 6 mg of crystalline 13C-enriched GA1. Its 13C-enrichment of ca. 75％ and 1JCC values were determined by NMR spectrometry.
Key words: gibberellin A1; Phaeosphaeria sp. L487; 13C-labeled gibberellin A1; gibberellin A1 fermentation

-27-
Note
Primary Culture of Porcine Mammary Epithelial Cells as a Model System
for Evaluation of Milk Protein Expression

Haruto KUMURA,† Atsushi TANAKA, Youichi ABO, Saori YUI,1 Kei-ichi SHIMAZAKI,
Eiji KOBAYASHI,2 and Kazuhiko SAYAMA1

Laboratory of Dairy Science, Research Group of Animal Product Science, Division of Bioresources
and Product Science, The Graduate School of Agriculture, Hokkaido University, Kita 9 Nishi 9,
Sapporo 060-8589, Japan
1Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya,
Shizuoka 422-8529, Japan
2Animal Genome Research Team, Department of Animal Breeding and Genetics National Institute
of Animal Industry, Ikenodai 2, Kukisaki, Inashiki, Ibaraki 305-0901, Japan

Received March 8, 2001; Accepted May 21, 2001
Porcine mammary epithelial cells were isolated to culture on collagen gel followed by gel floating treatment to evaluate differentiation under the culture conditions of serum-free medium, supplemented with combinations of insulin, hydrocortisone, and prolactin. After the culture period, the mammary cells attached to the collagen gels were recovered to observe expression of β-casein, β-lactoglobulin, and lactoferrin by reverse transcriptase polymeric chain reaction method. Expression of β-casein was observed in the presence of insulin, hydrocortisone, and prolactin whereas transcription of β-lactoglobulin and lactoferrin occured irrespective of hydrocortisone and prolactin. Immunoblot analysis demonstrated synthesis and secretion of lactoferrin in the fraction of recovered cells and the culture medium.
Key words: primary culture; porcine mammary epithelial cell; milk protein expression

-28-
Note
IgE-reactive 60 kDa Glycoprotein Occurring in Wheat Flour

Jun WATANABE,1,2 Soichi TANABE,3 Kei SONOYAMA,1,† Mitsuji KURODA,4
and Michiko WATANABE4

1Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan
2Japan Society for the Promotion of Science, Chiyoda-ku, Tokyo 102-8471, Japan
3Faculty of Applied Biological Science, Hiroshima University, Hiroshima 739-8528, Japan
4Faculty of Education, Tokyo Gakugei University, Tokyo 184-8501, Japan

Received March 9, 2001; Accepted April 23, 2001
A new IgE-reactive glycoprotein with a molecular size of 60 kDa was isolated from wheat flour. The N-terminal amino acid sequence of the protein was LDPDESEXVTRYFRIR. The 8th amino acid residue would have been Asn to which the peroxidase-type glycochain was attached. The IgE-binding activity of the glycoprotein was rendered negligible by the enzymatic treatment applied for hypoallergenic flour production.
Key words: wheat allergen; allergenic glycoprotein; peroxidase type glycochain

-29-
Note
Synthesis of (-)-Methyl Shikimate via Enzymatic Resolution
of (1S＊, 4R＊, 5R＊)-4-Hydroxy-6-oxabicyclo[3.2.1]oct-2-en-7-one

Takayuki ORITANI, Rumi UEDA, and Hiromasa KIYOTA＊

Laboratory of Applied Bioorganic Chemistry, Division of Life Science, Graduate School of Agricultural
Science, Tohoku University, 1-1 Tsutsumidori-Amamiya, Aoba-ku, Sendai 981-8555, Japan

Received March 16, 2001; Accepted April 26, 2001
The synthesis of methyl (-)-shikimate [(-)-2] was achieved via lipase-catalyzed optical resolution of (1S＊, 4R＊, 5R＊)-4-hydroxy-6-oxabicyclo[3.2.1]oct-2-en-7-one (3). Transesterification of (±)-3 and vinyl acetate with lipase MY and subsequent hydrolysis gave optically pure (-)-3. This compound was converted to (-)-2 in two steps.
Key words: lipase; (1S＊, 4R＊, 5R＊)-4-hydroxy-6-oxabicyclo[3.2.1]oct-2-en-7-one; shikimic acid; hydrolysis; transesterification

-30-
Note
Simple Synthesis of Climacostol, a Defensive Secretion
by the Ciliate Climacostomum virens

Yumi ABE1 and Kenji MORI1,2,†

1Department of Mathematics and Science Education, Graduate School of Science, and
2Department of Chemistry, Faculty of Science, Science University of Tokyo, Kagurazaka 1-3,
Shinjuku-ku, Tokyo 162-8601, Japan

Received April 9, 2001; Accepted May 7, 2001
Climacostol ｛1,3-dihydroxy-5-[(Z)-2′-nonenyl]
benzene, 1｝, a defensive secretion by the protozoan ciliate Climacostomum virens against predators, was synthesized in a 43％ overall yield in three steps by starting
from methyl 1,3-bis(tert-butyldimethylsilyloxy)
phenylacetate (3).
Key words: alkenyl resorcinol; ciliate; Climacostomum virens; defensive toxin; resorcinolic lipid

-31-
Note
Cellular Localization of the Signaling Components
of Arabidopsis His-to-Asp Phosphorelay

Aya IMAMURA, Yuriko YOSHINO, and Takeshi MIZUNO†

Laboratory of Molecular Microbiology, School of Agriculture, Nagoya University, Chikusa-ku,
Nagoya, Nagoya 464-8601, Japan

Received April 13, 2001; Accepted May 12, 2001
In the higher plant, Arabidopsis thaliana, histidine-to-aspartate (His-to-Asp) phosphorelay signal transduction systems play crucial roles in propagation of environmental stimuli, including plant hormones. This plant has 11 sensor His-kinases, 5 histidine-containing phosphotransfer (HPt) factors (AHPs), and 20 response regulators (ARRs). To gain new insight into the functions of these phosphorelay components, their intracellular localization was examined with use of GFP-fusion proteins, constructed for certain representatives of HPt factors (AHP2) and type-A and type-B ARRs (ARR6/ARR7 and ARR10, respectively). The results showed that AHP2 is mainly located in the cytoplasmic space, while both the types of ARRs have an ability to enter preferentially into the nuclei, if not exclusively. Together with the results from an in vitro phosphorelay assay with AHP2 and ARRs, these results are discussed, in terms of a geneal framework of the Arabidopsis His-to-Asp phosphorelay network.
Key words: Arabidopsis thaliana; His-to-Asp phosphorelay; response regulators; HPt factors; His-kinases

-32-
Preliminary Communication
Synthesis of Macrotetrolide α, a Designed Polynactin Analog Composed
of (+)- and (-)-Bishomononactic Acids

Tadaatsu HANADATE, Hiromasa KIYOTA,† and Takayuki ORITANI

Laboratory of Applied Bioorganic Chemistry, Division of Life Science, Graduate School of Agricultural
Science, Tohoku University, 1-1 Tsutsumidori-Amamiya, Aoba-ku, Sendai 981-8555, Japan

Received April 27, 2001; Accepted May 28, 2001
Macrotetrolide α, a designed analog of polynactin composed of (+)- and (-)-bishomononactic acids, was synthesized. These monomers were prepared via optical resolution of the corresponding (S)-O-acetylmandelates. Assembly of the monomers to macrotetrolide α took seven steps without any loss of the intermediates.
Key words: total synthesis; polynactin; macrotetrolide; bishomononactate; optical resolution