Contents and Abstracts of Latest Issue of BBB

(Vol.62 No.7 1998)

γ-Glutamy1 Transfer Reactions by Glutaminase from Pseudomonas nitroreducens IFO 12694 and Their Application for the Syntheses of Theanine and γ-Glutamy1methy1amide
Takashi TACHIKI, Takeshi YAMADA, Katsushige MIZUNO, Masashi UEDA, Ju-ichi SHIODE, and Hiroshi FUKAMI 1279

Inhibitory Effects of Apple Polyphenol on Induced Histamine Release from RBL-2H3 Cells and Rat Mast Cells
Tomomasa KANDA,　Hiroshi AKIYAMA,＊ Akio YANAGIDA, Masayuki TANABE, Yukihiro GODA,＊ Masatake TOYODA,＊ Reiko TESHIMA,＊ and Yukio SAITO＊ 1284

Acetobacter xylinum Mutant with High Cellulose Productivity and an Ordered Structure
Kunihiko WATANABE,　Mari TABUCHI, Atsushi ISHIKAWA,Hiroshi TAKEMURA, Takayasu TSUCHIDA, Yasushi MORINAGA, and Fumihiro YOSHINAGA 1290

Preparation and Characterization of 8a-(Phosphatidylcholine-dioxy)-α-tocopherones and their Formation during the Peroxidation of Phosphatidylcholine in Liposomes
Ryo YAMAUCHI, Hiromi MIZUNO, and Koji KATO 1293

Oxidation Products of β-Carotene during the Peroxidation of Methyl Linoleate in the Bulk Phase
Ryo YAMAUCHI, Kakue TSUCHIHASHI, and Koji KATO 1301

Calcium Bioavailability of a Total Bone Extract (TBE) and Its Effects on Bone Metabolism in Rats
Takehito MIURA＊ and Masuo NAKANO 1307

Characterization of the Immobilized β-Galactosidase C from Bacillus circulans and the Production of β(1→3)-linked Disaccharides
Andreas NAUNDORF, Myl｀ene CAUSSETTE, and Katsumi AJISAKA＊ 1313

Asparagine-linked Glycosylation of the Rat Leukemia Inhibitory Factor Expressed by Simian COS7 Cells
Jun-ichi AIKAWA,1, Ei-ichiro SATO,2, Shigeru KYUWA,3 Eimei SATO,3,Ken SASAI,2 Kunio SHIOTA,2 and Tomoya OGAWA1,2 1318

Characterization of Truncated Human Mannan-Binding Protein (MBP) Expressed in Escherichia coli
Souji EDA,1 Yasuhiko SUZUKI,1 Takao KAWAI,2 Katsuki OHTANI,1 Tetsuo KASE,3 Takashi SAKAMOTO,4 and Nobutaka WAKAMIYA4＊ 1326

Anomer-Selective Glucosylation of l-Menthol by Yeast α-Glucosidase
Hiroyuki NAKAGAWA, Masaaki YOSHIYAMA,＊ Susumu SHIMURA,＊ Kohtaro KIRIMURA,
and Shoji USAMI 1332

Purification and IgE-Binding Epitopes of a Major Allergen in the Gastropod Turbo cornutus
Masaru ISHIKAWA, Masami ISHIDA, Kuniyoshi SHIMAKURA, Yuji NAGASHIMA,and Kazuo SHIOMI 1337

A Novel Fungal Endo-β-N-acetylglucosaminidase that Specifically Acts on Plant Glycoproteins
Yoko SAKAMOTO, Yoshiro KURIMURA, and Yasunobu TSUJI 1344

Three Different Types of α-Amylases from Aspergillus awamori KT-11:Their Purifications, Properties, and Specificities
Trisanti ANINDYAWATI, Ruth MELLIAWATI,＊ Kazuo ITO, Masaru IIZUKA,
and Noshi MINAMIURA 1351

Isolation of Genomic DNA Containing a Cytosolic Ascorbate Peroxidase Gene (ApxSC) from the Strawberry (Fragaria x ananassa)
In-Jung KIM and Won-Il CHUNG＊ 1358

New Monoterpentriols from the Fruiting Body of Flammulina velutipes
Yuichi HIRAI, Michimasa IKEDA＊, Tetsuya MURAYAMA, and Toshiharu OHATA 1364

Effects of Dietary Shrimp, Squid and Octopus on Serum and Liver Lipid Levels in Mice
Kazunari TANAKA, 　Tadashi SAKAI,＊ Ikuo IKEDA,＊＊ Katsumi IMAIZUMI,＊＊
and Michihiro SUGANO＊＊＊ 1369

Functional Interaction of Isr1, a Predicted Protein Kinase, with the Pkc1 Pathway in Saccharomyces cerevisiae
Kohji MIYAHARA1, Dai HIRATA, and Tokichi MIYAKAWA 1376

Analysis of the Dielectric Relaxation of a Gelatin Solution
Satosi IWAMOTO and Hitoshi KUMAGAI 1381

Breeding of a 5-Fluorouridine-resistant Mutant with Increased Cellulose Production from Acetobacter xylinum subsp. nonacetoxidans
Atsushi ISHIKAWA,＊, 　Naoto TONOUCHI, 　Takayasu TSUCHIDA, and Fumihiro YOSHINAGA 1388

Primary Structure of Streptomyces griseus Metalloendopeptidase II
Shuichi KOJIMA, 　Takashi KUMAZAKI,1 Shin-ichi ISHII,1 and Kin-ichiro MIURA 1392

Stimulation by Histamine of TPA-dependent Hepatocyte Growth Factor Production in Promyelocytic Leukemia HL-60 Cells
Mitsuru SUZUKI,1 Kunio MATSUMOTO,2 Toshikazu NAKAMURA,2 and Kiwao NAKANO1 1399

Molecular Cloning and Expression in Saccharomyces cerevisiae of Tobacco NADPH-Cytochrome P450 Oxidoreductase cDNA
Takashi YAMADA, Hiromasa IMAISHI, Atsuhiro OKA,＊ and Hideo OHKAWA 1403

Note
Inhibition of Icosanoid Production in MC/9 Mouse Mast Cells by n-3 Polyunsaturated Fatty Acids Isolated from Edible Marine Algae
Kenji ISHIHARA, 　Masakazu MURATA, Masaki KANENIWA, Hiroaki SAITO,
Kazuki SHINOHARA,＊ and Mari MAEDA-YAMAMOTO＊＊ 1412

Note
Purification and Characterization of Cysteine Protease from Pleurotus ostreatus
Hyun-Hee SHIN and Hye-Seon CHOI 1416

Note
Possible Formation of Dehydro-L-ascorbic Acid from 2,3-Diketo-L-gulonic Acid in an Aqueous Solution
Noriko MIYAKE and Tadao KURATA 1419

Note
Occurrence of Bound Salt in Freeze-concentrated Soy Sauce
Michiko WATANABE, Soichi TANABE, Kazuo FURIHATA,＊ Akira OKUBO,＊
and Soichi ARAI＊＊ 1422

Note
Suppression of the SOS-Inducing Activity of Trp-P-1 and Aflatoxin B1 by Meso-dihydroguaiaretic Acid from Machilus thunbergii in the Salmonella typhimurium TA1535/pSK1002 umu Test
Mitsuo MIYAZAWA,1 Yoshiharu OKUNO,1 Kayo OSHIRO,1 Hiroyuki KASAHARA,1
Hideo SHIMAMURA,1 Sei-ichi NAKAMURA,2 and Hiromu KAMEOKA1 1425

Note
β-Glucosidase Activity in the Rat Small Intestine toward Quercetin Monoglucosides
Kana IOKU, Yutana PONGPIRIYADACHA,＊ Yotaro KONISHI,＊＊ Yoko TAKEI,
Nobuji NAKATANI,＊＊ and Junji TERAO＊＊＊ 1428

Note
Ornatipolide, a Novel Phenolic Metabolite from the Basidiomycete, Boletus ornatipes
Hisao SHIBATA,＃ Takashi FUKUDA, Tomonari WADA, Yasufumi MORITA,＊
Toshihiro HASHIMOTO,＊＊ and Yoshinori ASAKAWA＊＊ 1432

Note
Lipase-catalyzed Kinetic Resolution of (±)-cis,cis-Spiro[4.4]nonane-1,6-diol
Yoshitaka MATSUSHIMA,＊ Takeshi KITAHARA, and Kenji MORI＊＊ 1435

Note
Increase of Lipid Hydroperoxides in the Rat Liver and Kidney after Administering Ferric Nitrilotriacetate
Kazumi IKEDA,＊ Fang SUN,＊ Kyoko TANAKA,＊ Sadako TOKUMARU,
and Shosuke KOJO＊,＊＊ 1438

Note
Comparison of Volatile Compounds from Chungkuk-Jang and Itohiki-Natto
Tadayoshi TANAKA,1 Kanako MURAMATSU,2 Haeng-ran KIM,2, Tazuko WATANABE,1
Michiyo TAKEYASU,2 Yukiko KANAI,2 and Kan KIUCHI2 1440

Note
Application of Two-dimensional Mapping for an Analysis of Galactosyllactoses in Yogurt
Tadao SAITO,＊ Tomoya TSUJI, Haruki KITAZAWA, Yasushi KAWAI,
and Takatoshi ITOH 1445

Note
Effect of Jasmonates and Related Compounds on Seed Germination of Orobanche minor Smith and Striga hermonthica (Del.) Benth
Koichi YONEYAMA,・ Masaru OGASAWARA, Yasutomo TAKEUCHI, Makoto KONNAI,
Yukihiro SUGIMOTO,＊ Hideharu SETO,＊＊ and Shigeo YOSHIDA＊＊ 1448

Note
Acetylation of Bacterial Cellulose: Preparation of Cellulose Acetate Having a High Degree of Polymerization
Mari TABUCHI, Kunihiko WATANABE,＊ Yasushi MORINAGA, and Fumihiro YOSHINAGA 1451

Note
ATP-Dependent Inactivation of Escherichia coli γ-Glutamylcysteine Synthetase by L-Glutamic Acid γ-Monohydroxamate
Makoto KATOH, Jun HIRATAKE, and Jun′ichi ODA 1455

Note
Suppression of Senescence in Normal Human Fibroblasts by Introduction of Dominant-Negative p53 Mutants or Human Papilloma Virus Type 16 E6 Protein
Akiko IDE, Michihiko FUJII, Kazuhiko NAKABABASHI, and Dai AYUSAWA 1458

Preliminary Communication
Safety Assessment of Genetically Engineered Food: Detection and Monitoring of Glyphosate-Tolerant Soybeans
Nobuaki SHIRAI,＊ Keiko MOMMA, Sachiko OZAWA, Wataru HASHIMOTO, Makoto KITO,
Shigeru UTSUMI, and Kousaku MURATA� 1461

Preliminary Communication
An N-terminal Region of LukF of Staphylococcal Leukocidin/γ-Hemolysin Crucial for the Biological Activity of the Toxin
Jun KANEKO, Ma. Anita L. MASCARENAS, Md. Nazmul HUDA, Toshio TOMITA,
and Yoshiyuki KAMIO＊ 1465

Preliminary Communication
Stereochemical Difference in Secoisolariciresinol Formation between Cell-free Extracts from Petioles and from Ripening Seeds of Arctium lappa L.
Shiro SUZUKI, Toshiaki UMEZAWA,＊ and Mikio SHIMADA 1468

-1-
Rapid and Frequent Somatic Embryogenesis from Single Cells of Regenerated Carrot Plantlets

Hiroshi YASUDA, Tsuyoshi SATOH, and Hiroshi MASUDA
Department of Agricultural Chemistry, Obihiro University of Agriculture and Veterinary Medicine,
Obihiro 080-8555, Hokkaido, Japan

Received September 8, 1997
Embryogenesis was induced from regenerated carrot plantlets in a shorter time and at a higher frequency than that observed previously from hypocotyl explants of carrot seedlings (Masuda et al. J. Plant Physiol. 145, 531 (1995)). Serial observations of embryogenesis from single cells to globular embryos via cell clusters were possible in the conditioned medium. The ability to produce embryos seemed to be acquired during the transition from torpedo-shaped embryos to regenerated plantlets, when differentiation of the epidermal cells of regenerated plantlets occurred.
carrot; regenerated plantlet; scanning electron microscopy; somatic embryogenesis

-2-
γ-Glutamyl Transfer Reactions by Glutaminase from Pseudomonas nitroreducens
IFO 12694 and Their Application for the Syntheses of Theanine and
γ-Glutamylmethylamide

Takashi TACHIKI, Takeshi YAMADA, Katsushige MIZUNO, Masashi UEDA,
Ju-ichi SHIODE, and Hiroshi FUKAMI
Department of Bioscience and Biotechnology, Faculty of Science and Engineering, Ritsumeikan University,
Nojihigashi 1-1-1, Kusatsu 525-8577, Japan

Received September 16, 1997
In a mixture containing γ-glutamyl donor (donor) and γ-glutamyl acceptor (acceptor), the glutaminase of Pseudomonas nitroreducens IFO 12694 simultaneously catalyzed a γ-glutamyl transfer reaction and hydrolysis of the donor. The variation of the activities responding to the concentration of glutathione and glycylglycine indicated that the enzyme might be classified in a group of glutaminases that shows hydrolysis prior to transfer reaction. On the other hand, the results with glutamine and ethylamine or methylamine indicated that the enzyme was active in the transfer reaction with suppressed hydrolysis of glutamine, and suggested the possibility of using the reaction for producing γ-glutamylethylamide (theanine) or γ-glutamylmethylamide (γ-GMA). In fact, in a mixture containing high concentrations of substrates (0.7 M glutamine, 1.5 M ethylamine or methylamine) and 0.5 unit/ml glutaminase (borate buffer pH 11), 270 mM (47 g/L) theanine or 250 mM (38 g/L) γ-GMA was formed in 7 h of incubation at 30°C.
theanine; γ-glutamylmethylamide; glutaminase; γ-glutamyl transfer reaction; Pseudomonas nitroreducens

-3-
Inhibitory Effects of Apple Polyphenol on Induced Histamine Release
from RBL-2H3 Cells and Rat Mast Cells

Tomomasa KANDA, Hiroshi AKIYAMA,＊ Akio YANAGIDA, Masayuki TANABE,
Yukihiro GODA,＊ Masatake TOYODA,＊ Reiko TESHIMA,＊ and Yukio SAITO＊
Institute for Production Research and Development, The Nikka Whisky Distilling Co., Ltd.,
967 Matsuyama, Masuo, Kashiwa-shi, Chiba 277-0033, Japan
＊National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan

Received November 12, 1997
The anti-allergic activities of polyphenol fractions extracted from immature fruits of apple (Rosaceae, Malus sp.) were evaluated by in vitro assays. A crude apple polyphenol (CAP) fraction, which had been obtained from the juice of immature apples by reverse-phase column chromatography, was further purified by LH-20 column chromatography to obtain an apple condensed tannin (ACT) fraction consisting of linear oligomeric epicatechins from the dimer to pentadecamer. ACT strongly inhibited the release of histamine from rat basophilic leukemia (RBL-2H3) cells stimulated by the antigen-stimulation and from rat peritoneal mast cells stimulated by compound 48/80. The IC50 values for histamine release were 30 μg/ml and 25 μg/ml, respectively. ACT also inhibited hyaluronidase activity and the increase in intracellular free calcium concentration in RBL-2H3 cells stimulated with the antigen. These results suggest that ACT affected early signal transduction including the calcium influx.
apple polyphenol; anti-allergic activity; histamine release; hyaluronidase; calcium influx

-4-
Acetobacter xylinum Mutant with High Cellulose Productivity and an Ordered Structure

Kunihiko WATANABE, Mari TABUCHI, Atsushi ISHIKAWA, Hiroshi TAKEMURA,
Takayasu TSUCHIDA, Yasushi MORINAGA, and Fumihiro YOSHINAGA
Bio-Polymer Research Co., Ltd., 3-2-1 Sakato, Takatsu-ku, Kawasaki 213-0012, Japan

Received November 19, 1997
Acetobacter xylinum subsp. sucrofermentans BPR2001, a cellulose-producing bacterium, that was newly isolated from a natural source, produced large amounts of the water-soluble polysaccharide, acetan. UDP-glucose is known to be the direct precursor in the synthetic pathways of both cellulose and acetan. We attempted to breed mutant strains and succeeded in obtaining one, BPR3001A, which produced 65％ more bacterial cellulose and accumulated 83％ less acetan than the parent strain, BPR2001. The cellulose formed was found to be structurally ordered, with higher degrees of polymerization and crystallinity and larger crystallite size than those produced by BPR2001 and other conventional strains. Furthermore, a processed dry sheet of this cellulose exhibited a higher Young′s modulus than that of the wild strain. The ordered structure of the cellulose obtained was probably due to the decreased amount of acetan which may reflect the ribbon assembly of cellulose fibrils without prevention of hydrogen bonding between microfibrils.
Acetobacter xylinum; mutant; bacterial cellulose; acetan; structure

-5-
Preparation and Characterization of 8a-(Phosphatidylcholine-dioxy)- α-tocopherones and their Formation during the Peroxidation
of Phosphatidylcholine in Liposomes

Ryo YAMAUCHI, Hiromi MIZUNO, and Koji KATO
Department of Food Science, Faculty of Agriculture, Gifu University, Gifu 501-1193, Japan

Received December 8, 1997
α-Tocopherol was reacted with the phosphatidylcholines (PCs), 1-palmitoyl-2-linoleoyl-3-sn-PC (PLPC), 1-palmitoyl-2-linolenoyl-3-sn-PC, 1-palmitoyl-2-arachidonoyl-3-sn-PC (PAPC) and 1-stearoyl-2-arachidonoyl-3-sn-PC, in the presence of the free radical initiator, 2,2′-azobis(2,4-dimethylvaleronitrile), at 37°C. The addition products of α-tocopherol with the PC peroxyl radicals were isolated and identified as 8a-(PC-dioxy)-α-tocopherones, in which the peroxyl radicals derived from each PC molecule attacked the 8a-position of the α-tocopheroxyl radical. The antioxidative efficiency of α-tocopherol against the peroxidation of PLPC and PAPC in liposomes was assessed by the formation of the reaction products of α-tocopherol. When α-tocopherol was oxidized in the presence of the water-soluble free radical initiator, 2,2′-azobis(2-amidinopropane) dihydrochloride, epoxy-α-tocopherylquinones were mainly produced together with 8a-(PC-dioxy)-α-tocopherones and α-tocopherylquinone. The yield of α-tocopherylquinone was increased by treating each sample with dilute acid which indicates the presence of tocopherone precursors other than the 8a-(PC-dioxy)-α-tocopherones. The same products were also detected from iron-dependent peroxidation, although the yields were very low.
α-tocopherol; antioxidant; lipid peroxidation; liposomes; phosphatidylcholines

-6-
Oxidation Products of β-Carotene during the Peroxidation of Methyl Linoleate
in the Bulk Phase

Ryo YAMAUCHI, Kakue TSUCHIHASHI, and Koji KATO
Department of Food Science, Faculty of Agriculture, Gifu University, Gifu 501-1193, Japan

Received December 15, 1997
Methyl linoleate containing β-carotene was autoxidized or photooxidized at 37°C in the bulk phase, and the oxidation products of β-carotene were analyzed by high-performance liquid chromatography. Formyl β-carotenes, β-carotene 5,6-epoxide, and cyclic ethers of β-carotene were detected as the oxidation products during the peroxidation of methyl linoleate initiated by a free radical initiator. These products, which were also detected in the methyl linoleate autoxidized without an initiator, were detectable only in much smaller amounts than the consumed β-carotene. In the chlorophyll-sensitized photooxidation process, the products were β-carotene 5,8-endoperoxide and β-carotene 5,6-epoxide. α-Tocopherol partially inhibited the formation of the 5,6-epoxide, but had no effect on the main product, the 5,8-endoperoxide. These results indicate that β-carotene reacted with singlet oxygen to form the 5,8-endoperoxide as the primary product during the photooxidation of methyl linoleate, and that β-carotene trapped lipid-peroxyl radicals to form oxygenated products which decomposed immediately during the autoxidation process.
β-carotene; autoxidation; photooxidation; singlet oxygen; antioxidant

-7-
Calcium Bioavailability of a Total Bone Extract (TBE) and Its Effects on Bone Metabolism in Rats

Takehito MIURA＊ and Masuo NAKANO
Department of Bioresource Science, Obihiro University of Agriculture and Veterinary Medicine,
Obihiro, Hokkaido 080-8555, Japan
＊United Graduate School of Agriculture Sciences, Iwate University, Morioka, Iwate 020-8550, Japan

Received December 22, 1997
The effects of a total bone extract (TBE), a new mineral preparation made from bovine bone rendered soluble by lactic acid and citric acid under decompression, on the bone metabolism and apparent calcium absorption were examined for an ovariectomized (OVX) rat model of osteoporosis. The apparent calcium absorption from TBE was significantly higher than that from calcium carbonate. There were no significant differences in serum biochemical indices. Although there were no significant differences in the dry weight, ash and calcium content in the tibia, or in the bone mineral density of the femur, TBE feeding increased the cortical thickness index of the femur. A positive effect of TBE on bone formation is thus suggested.
calcium; ovariectomy; osteoporosis; cortical thickness index; bone mineral density

-8-
Characterization of the Immobilized β-Galactosidase C from Bacillus circulans and the Production of β(1→3)-linked Disaccharides

Andreas NAUNDORF, Myl｀ene CAUSSETTE, and Katsumi AJISAKA＊
Meiji Milk Products Co., Ltd., Institute of Health Science, 540 Naruda, Odawara 250-0862, Japan

Received December 24, 1997
A recombinant β-galactosidase, which was obtained from the β-galactosidase C gene of Bacillus circulans and cleaves the non-reducing end galactosyl residue of β(1→3)-linkages selectively, was immobilized using CNBr-Sepharose. Although the effect of pH was not changed by the immobilization, the thermostability and stability in the presence of DMF were increased. Optimization of the transglycosylation using para-nitrophenyl β-D-galactopyranoside as a donor and benzyl-α-D-N-acetylgalactosaminide as an acceptor afforded a β(1→3)-linked disaccharide derivative with 62％ molar yield in a gram scale synthesis. Using the methyl-analogue as an acceptor, 53％ of the acceptor was converted to the respective β(1→3)-disaccharide.
β-galactosidase; Bacillus circulans; immobilization; transglycosylation; Gal-β(1→3)-GalNAc

-9-
Asparagine-linked Glycosylation of the Rat Leukemia Inhibitory Factor Expressed by Simian COS7 Cells

Jun-ichi AIKAWA,1, Ei-ichiro SATO,2, Shigeru KYUWA,3 Eimei SATO,3,Ken SASAI,2 Kunio SHIOTA,2 and Tomoya OGAWA1,2
1Laboratory of Synthetic Cellular Chemistry, RIKEN (the Institute of Physical and Chemical Research),
Hirosawa 2-1, Wako-shi, Saitama 351-0198, Japan
2Department of Animal Resource Science, Graduate School of Agricultural Life Science,
the University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan
3Department of Reproductive and Developmental Biology, Institute of Medical Science,
the University of Tokyo, Shirokanedai 4-6-1, Minato-ku, Tokyo 108-0071, Japan

Received December 24, 1997
The leukemia inhibitory factor (LIF) is a secretory glycoprotein and a pluripotent growth factor which acts in diverse cell systems. LIF has been reported to be heavily glycosylated. In this paper, we examine the transient expression of rat LIF (rLIF) in COS7 cells and its glycosylation by a PNGaseF treatment and lectin blot. rLIF expression in COS7 cells resulted in seven molecular species being produced with zero to six N-glycosyl moieties. Mutated rLIF proteins with substitutions at the seven possible N-glycosylation sites were also expressed. An analysis of the molecular weight of the mutated rLIF confirmed the six N-glycosylation sites. Bioassays of mouse leukemia cell lines were performed to analyze the contribution of the glycosyl moieties to their functions. We found that the glycosyl moieties at each of the N-glycosylation sites were not essential to their function of the protein, but the reduced functions to promote the proliferation of DA-1a cells that had been observed for some mutants suggests a biochemical role for the in vitro function.
leukemia inhibitory factor (LIF); asparagine-linked glycosylation (N-glycosylation); site-directed mutagenesis; COS7 cells; DA-1a cells

-10-
Characterization of Truncated Human Mannan-Binding Protein (MBP) Expressed in Escherichia coli

Souji EDA,1 Yasuhiko SUZUKI,1 Takao KAWAI,2 Katsuki OHTANI,1
Tetsuo KASE,3 Takashi SAKAMOTO,4 and Nobutaka WAKAMIYA4＊
Department of 1Pathology, 2Food Microbiology, and 3Virology, Osaka Prefectural Institute of Public Health, Higashinari, Osaka 537-0025, Japan
4Department of Viral Infections, Research Institute for Microbial Diseases, Osaka University,
Suita, Osaka 565-0871, Japan

Received December 26, 1997
Mannan-binding protein (MBP) is a calcium-dependent mammalian serum lectin important in first-line host defense. MBP belongs to the collectin family, which is characterized by an NH2-terminal cysteine-rich domain, a collagen-like domain, a neck domain, and a carbohydrate recognition domain (CRD). We have expressed a recombinant human MBP, consisting of the short collagen region (two repeats of Gly-Xaa-Yaa amino acid sequences), the neck domain, and the CRD, in Escherichia coli. The truncated MBP was capable of forming trimers by association of the neck domain and could bind sugar with a specificity similar to that of the native form. Results of hemagglutination inhibition (HI) assay of influenza A virus showed that the truncated MBP inhibited hemagglutination less strongly, although the native MBP induced the HI phenomenon. These results suggest that an oligomeric structure is an advantage for MBP to have full biological activity against influenza A virus.
mannan-binding protein; c-type lectin; expression; influenza virus

-11-
Anomer-Selective Glucosylation of l-Menthol by Yeast α-Glucosidase

Hiroyuki NAKAGAWA, Masaaki YOSHIYAMA,＊ Susumu SHIMURA,＊ Kohtaro KIRIMURA,and Shoji USAMI
Department of Applied Chemistry, School of Science and Engineering, Waseda University,
Ohkubo 3-4-1, Shinjuku-ku, Tokyo 169-8555, Japan
＊Central Laboratory, Lotte Co., Ltd, Numakage 3-1-1, Urawa, Saitama 336-0027, Japan

Received December 26, 1997
l-Menthol was glucosylated by the α-glucosidase (EC 3.2.1.20) of Saccharomyces cerevisiae using maltose as the glucosyl donor. When 50 mg of l-menthol and 1.6 M maltose in 10 mM citrate-phosphate buffer (pH 5.5) were incubated at 45°C, l-menthyl α-D-glucopyranoside (α-MenG) was α-anomer-selectively formed as a product. The specificity of the α-linkage was confirmed by 13C-NMR analysis. In the reaction mixture after 2 h, α-MenG was mainly accumulated in a crystalline form and the concentration of dissolved α-MenG was constant at 1.4 mM. The molar conversion yield of α-MenG produced based on the supplied l-menthol was maximally 30.7％ at 48 h of reaction.
α-glucosidase; menthol; transglucosylation; Saccharomyces cerevisiae

-12-
Purification and IgE-Binding Epitopes of a Major Allergen in the Gastropod Turbo cornutus

Masaru ISHIKAWA, Masami ISHIDA, Kuniyoshi SHIMAKURA, Yuji NAGASHIMA,
and Kazuo SHIOMI
Department of Food Science and Technology, Tokyo University of Fisheries,
Konan, Minato-ku, Tokyo 108-8477, Japan

Received January 9, 1998
The major allergen (Tur c 1) in the muscle of the gastropod, Turbo cornutus, was isolated by Sephacryl S-300, Mono Q HR 5/5 and TSKgel Phenyl-5PW RP column chromatography. ELISA showed Tur c 1 to react strongly with sera from three individuals sensitive to both mollusks and crustaceans. SDS-PAGE showed Tur c 1 to produce a major band corresponding to a molecular mass of 35 kDa under the reduced condition. Its amino acid composition was characterized by the abundance of Glx, followed by Leu, Ala and Lys in decreasing abundance, and the absence of Trp. In addition to these properties, the determined partial amino acid sequence identified Tur c 1 to be a tropomyosin, as in the case of the known mollusk and crustacean allergens. However, the results of competitive ELISA inhibition experiments suggest that Tur c 1 has an IgE-binding epitope in the C-terminal region which is dissimilar to those proposed for Cra g 1 (the oyster Crassostrea gigas allergen) and Pen i 1 (the shrimp Penaeus indicus allergen).
allergen; gastropod; IgE-binding epitope; tropomyosin

-13-
A Novel Fungal Endo-β-N-acetylglucosaminidase that Specifically Acts on Plant Glycoproteins

Yoko SAKAMOTO, Yoshiro KURIMURA, and Yasunobu TSUJI
Research Laboratory of Higashimaru Shoyu Co. Ltd., 100-3, Tominaga, Tatsuno, Hyogo 679-4193, Japan

Received January 9, 1998
An endo-β-N-acetylglucosaminidase specific for plant glycoprotein oligosaccharides was purified from the culture fluid of a fungus. The Mr of the purified enzyme was 89,000. This enzyme was stable at pH 5.5--7.0, up to 30°C, and showed the highest activity at pH 6.0. Among sugar chains tested, xylose-containing sugar chains (M3X, M3FX, and M2FX) were the most favored substrates. Oligomannose type (M3, M5, and M9) and hybrid type (GNM3) sugar chains were hydrolyzed much more slowly than xylose-containing sugar chains, and a complex type sugar chain (GN2M3) was not hydrolyzed at all by the enzyme. Moreover, the enzyme released sugar chains from native horseradish peroxidase and stem bromelain, which were not hydrolyzed by other endo-β-N-acetylglucosaminidases (Endo H, D, and F). The enzyme could transfer the xylose-containing sugar chain from bromelain to DNS-Asn-GlcNAc-Fuc.
endo-β-N-acetylglucosaminidase; N-linked oligosaccharide; plant glycoprotein

-14-
Three Different Types of α-Amylases from Aspergillus awamori KT-11: Their Purifications, Properties, and Specificities

Trisanti ANINDYAWATI, Ruth MELLIAWATI,＊ Kazuo ITO, Masaru IIZUKA,
and Noshi MINAMIURA
Faculty of Science, Osaka City University, Sumiyoshi, Osaka 558-8585, Japan
＊R＆D Centre for Biotechnology, Indonesian Institute of Sciences (LIPI), Cibinong 16911, Bogor, Indonesia

Received January 9, 1998
Three forms of α-amylases, designated Amyl I, Amyl II, and Amyl III were purified to a homogenous state by several column chromatographies from a koji culture in wheat bran of a strain of black mold, which was isolated in Indonesia and identified as Aspergillus awamori. They have molecular weights of 49,000, 63,000, and 97,000 by SDS-PAGE, respectively, and the optimum pHs were 4.0 for Amyl I and 5.5 for both Amyl II and Amyl III on soluble starch. Amyl I hydrolyzed malto-tetraose, -pentaose, -hexaose, -heptaose, and β- and γ-cyclodextrin to produce maltose and maltotriose as major products but not or hardly hydrolyzed maltose, isomaltose, maltotriose, isomaltotriose, α-cyclodextrin, or raw corn starch. On the other hand, both Amyl II and Amyl III hydrolyzed maltotriose as well as all the maltooligosaccharides described above and α-, β-, and γ-cyclodextrin, and even raw corn starch as well as heat-gelatinized corn starch to produce maltose as a major product and glucose and maltotriose as minor products, but they did not hydrolyze maltose, isomaltose, or isomaltotriose. The limit hydrolyses of soluble starch with three kinds of enzymes were 33％ for Amyl I, 35％ for Amyl II, and 38％ for Amyl III, the reaction products had α-anomeric forms by NMR analysis, and the blue color reaction with I2 disappeared completely at about 18％ of hydrolysis of the starch for all enzymes.
Aspergillus awamori KT-11; α-amylase; action mode; raw starch degradation

-15-
Isolation of Genomic DNA Containing a Cytosolic Ascorbate Peroxidase Gene (ApxSC) from the Strawberry (Fragaria x ananassa)

In-Jung KIM and Won-Il CHUNG＊
Department of Biological Sciences, Korea Advanced Institute of Science and Technology,
373-1, Kusong-dong, Yusong-ku, Taejon 305-701, Korea

Received January 13, 1998
We isolated a genomic DNA harboring a cytosolic ascorbate peroxidase gene (ApxSC) from a genomic library of the strawberry (Fragaria x ananassa). Restriction mapping and sequence analyses showed that the DNA is composed of 2.5 kb of the full-length ApxSC gene, 3.7 kb of the 5′-upstream region, and 0.5 kb of the 3′-downstream region. The ApxSC genomic DNA contains 10 exons and 9 introns, which is similar to the structure of pea ApxI. A primer extension analysis suggested that the transcription of ApxSC gene was started at three start sites with different degrees. The promoter region of ApxSC gene contains a sequence or structure distinct from other reported plant ascorbate peroxidase genes, though with several known functional elements such as a TATA box.
cytosolic ascorbate peroxidase; genomic DNA; promoter; strawberry (Fragaria x ananassa)

-16-
New Monoterpentriols from the Fruiting Body of Flammulina velutipes

Yuichi HIRAI, Michimasa IKEDA＊, Tetsuya MURAYAMA, and Toshiharu OHATA
Department of Bioproduction, Faculty of Agriculture, Yamagata Uiversity,
1-23 Wakabamati, Tsuruoka, Yamagata 997-0037, Japan

Received January 14, 1998
Two monoterpentriols were isolated from the stipe segment without either the pileus or apical growth zone of the harvested fruiting body of F. velutipes. The structures of these monoterpentriols were elucidated as (1R, 2R, 4R, 8S)-(−)-p-menthane-2,8,9-triol (1) and its 8-epimer (2) based on a spectroscopic analysis and stereoselective chemical transformation from an authentic monoterpene alcohol. These monoterpentriols showed growth-promoting activities on the excised stipe with the pileus segment, which had been excised from a fruiting body just under the growth zone before the middle stage of the rapid growth period, at concentrations below 10 ppm, while the growth of the segment was inhibited at concentrations of 100 ppm and above.
monoterpentriol; p-menthane-2,8,9-triol; stipe elongation; Flammulina velutipes; fruiting body constituent

-17-
Effects of Dietary Shrimp, Squid and Octopus on Serum and Liver Lipid Levels in Mice

Kazunari TANAKA,・ Tadashi SAKAI,＊ Ikuo IKEDA,＊＊ Katsumi IMAIZUMI,＊＊
and Michihiro SUGANO＊＊＊
Laboratory of Nutrition, Nagasaki Prefectural Women′s Junior College, Nagasaki 850-0011, Japan ＊Applied Biochemistry Division, Faculty of Agriculture, Miyazaki University, Miyazaki 889-2155, Japan
＊＊Laboratory of Nutrition Chemistry, Faculty of Agriculture, Kyushu University, Fukuoka 812-0053, Japan
＊＊＊Faculty of Human Life Sciences, Prefectural University of Kumamoto, Kumamoto 862-0920, Japan

Received January 23, 1998
The effects of three seafoods, shrimp, squid and octopus, on lipid metabolism were investigated in mice fed on 0.1％ and 1.0％ cholesterol-supplemented diets in the first experiment. One of each of these seafoods and casein were added to the basal diet at levels of 15％ and 5％, respectively, as proteins. Casein served as the sole protein source of the control diet. The serum cholesterol concentration was significantly lower in the mice fed on shrimp and squid in the 0.1％ cholesterol diet and on any seafood in the 1.0％ cholesterol diet when compared with that in the mice fed on the control diet. The liver cholesterol concentration was significantly lower in all seafood groups given the 0.1％ cholesterol diet, and in the squid and octopus groups given the 1.0％ cholesterol diet. In the second experiment, the effect of these seafoods on lipid metabolism was compared with that of their defatted products in mice fed on a 0.2％ cholesterol diet. Defatting resulted in an increase in the serum cholesterol and triglyceride levels in the shrimp and squid groups. The hepatic cholesterol concentration in all the seafood groups was significantly lower than that in the control group, and defatting did not influence the liver cholesterol concentration. Fecal total steroid excretion was higher in all the seafood groups when compared with that in the control group, and was not modified by the removal of fats. Thus, shrimp, squid and octopus exerted hypolipidemic activity; the serum cholesterol-lowering activity of shrimp and squid was attributed to their lipid fraction, whereas the non-lipid fraction of shrimp, squid and octopus contributed to a reduction of hepatic cholesterol and an increase of fecal steroid excretion.
shrimp; squid; octopus; cholesterol; triglyceride

-18-
Functional Interaction of Isr1, a Predicted Protein Kinase,with the Pkc1 Pathway in Saccharomyces cerevisiae

Kohji MIYAHARA1, Dai HIRATA, and Tokichi MIYAKAWA
Department of Molecular Biotechnology, Faculty of Engineering, Hiroshima University,
Higashi-Hiroshima 739-8527, Japan

Received February 2, 1998
Staurosporine is a potent inhibitor of protein kinase C. To identify the genes that functionally interact with the Pkc1 pathway of the yeast Saccharomyces cerevisiae, we screened for the genes that cause induced staurosporine sensitivity when overexpressed from a galactose-inducible promoter. The novel gene ISR1 encodes a predicted protein kinase with the highest sequence similarity to mammalian Raf in the kinase domain. Drug sensitivity induced by ISR1 overexpression is specific to staurosporine. Although ISR1 disruption causes no obvious phenotype, it does exacerbate the phenotypes of a temperature-sensitive allele (stt1-1) of PKC1, but not of the mpk1 and bck1 mutants of the Mpk1 MAP kinase pathway. These results suggest that Isr1 functions in an event important for growth in a manner redundant with a Mpk1-independent branch of the Pkc1 signalling pathways.
PKC signalling pathway; protein kinase; Saccharomyces cerevisiae; staurosporine

-19-
Analysis of the Dielectric Relaxation of a Gelatin Solution

Satosi IWAMOTO and Hitoshi KUMAGAI
Department of Applied Biological Chemistry, Division of Agriculture and Agricultural Life Sciences,
The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

Received February 2, 1998
The behavior of the dielectric properties of gelatin in the frequency range from 103 Hz to 107 Hz was investigated and compared with that of the globule protein, bovine serum albumin (BSA), desalted gelatin and BSA being used. Dielectric relaxation was observed for both the gelatin and BSA solutions. The relaxation data were fitted well by the Cole-Cole equation; the Cole-Cole parameter (β) and the relaxation time (τ) were obtained. For the BSA solutions, τ was proportional to the solution viscosity (η) at 40°C and 25°C, and the values of β at 40°C were similar to those at 25°C. For gelatin solution, τ was proportional to η at 40°C, but was not proportional to η at 25°C. In addition, the values of β at 25°C were smaller than those at 40°C. These results indicate that the rotation of gelatin and/or polarization of submolecular groups in the coil state greatly contributed to the dielectric relaxation at 40°C; on the other hand, the formation of cross-linking junctions consisting of helix structures would have affected the dielectric relaxation at 25°C.
dielectric relaxation; relaxation time; gelatin; viscosity

-20-
Breeding of a 5-Fluorouridine-resistant Mutant with Increased Cellulose Production from Acetobacter xylinum subsp. nonacetoxidans

Atsushi ISHIKAWA,＊, Naoto TONOUCHI, Takayasu TSUCHIDA, and Fumihiro YOSHINAGA
Bio-Polymer Research Co., Ltd., KSP R＆D B-1015, 3-2-1 Sakato, Takatsu-ku, Kawasaki 213-0012, Japan

Received February 6, 1988
UDP-glucose (UDP-G), the direct precursor of cellulose, is known to be produced from UTP and glucose-1-phosphate. In an attempt to increase UTP biosynthesis, 5-fluorouridine (5-FUR: a pyrimidine analog)-resistant mutants were obtained using Acetobacter xylinum subsp. nonacetoxidans 757 as the parent strain. One of the 5-FUR-resistant mutants, FUR-35, showed about 40％ higher cellulose productivion compared to the parent strain. Intracellular levels of UTP and UDP-G in FUR-35 was found to be higher than those in the parent strain. The carbamyl phosphate synthetase II (CPS) activity of FUR-35 was higher than that of the parent strain and the feedback inhibition of CPS by UTP in FUR-35 had been released compared with that in the parent strain. These results suggest that the increased cellulose production of FUR-35 was attributable to its higher of intracellular UDP-G level resulting from increased UTP biosynthesis.
cellulose; Acetobacter; 5-fluorouridine; UDP-glucose; carbamyl phosphate synthetase II

-21-
Primary Structure of Streptomyces griseus Metalloendopeptidase II

Shuichi KOJIMA, Takashi KUMAZAKI,1 Shin-ichi ISHII,1 and Kin-ichiro MIURA
Institute for Biomolecular Science, Gakushuin University, Mejiro, Tokyo 171-8588, Japan
1Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo 060-0812, Japan

Received February 17, 1998
Streptomyces griseus metalloendopeptidase II (SGMPII) is a unique protease, since it shows anomalous susceptibility to the proteinaceous ``serine protease inhibitors′′ produced by Streptomyces, such as Streptomyces subtilisin inhibitor (SSI) and its homologous proteins. In this study, we analyzed the amino acid sequence of SGMPII by analyzing various peptide fragments produced enzymatically. The sequence of SGMPII, which is composed of 334 amino acids, showed no extensive similarity to SSI-insensitive metalloproteases produced by other species of Streptomyces, except for the amino acid residues essential for catalysis and zinc binding. However, SGMPII is 35--41％ similar to thermolysin and its related metalloproteases, which are not inhibited by SSI, and the residues presumed to be critical for catalysis and zinc-binding are well conserved in SGMPII. Glu137 in a ``His-Glu-Xaa-Xaa-His′′ motif of SGMPII was identified as the residue modified by ClCH2CO-DL-(N--OH)Leu-Ala-Gly-NH2, an active-site-directed irreversible inhibitor of thermolysin-like metalloproteases. Based on the sequence comparison of SGMPII and other bacterial metalloproteases, we discuss the structural basis for the differences in substrate specificity and stability between SGMPII and other thermolysin-like proteases. A possible SSI-binding locus of SGMPII is also proposed.
primary structure; sequence alignment; Streptomyces griseus metalloendopeptidase II

-22-
Stimulation by Histamine of TPA-dependent Hepatocyte Growth Factor Production in Promyelocytic Leukemia HL-60 Cells

Mitsuru SUZUKI,1 Kunio MATSUMOTO,2 Toshikazu NAKAMURA,2 and Kiwao NAKANO1
1Nagoya University Bioscience Center, Chikusa, Nagoya 464-8601, Japan
2Division of Biochemistry, Biomedical Research Center, Osaka University Medical Center,
Suita, Osaka 565-0871, Japan

Received February 20, 1998
Hepatocyte growth factor (HGF) is most likely a physiological hepatotrophic factor that triggers regeneration of the injured liver. Histamine may also be important in the pathophysiology of the injured liver. Previously we showed that histamine production was increased in liver macrophages of mice injected with CCl4, a well-known hepatotoxin. Therefore, it is likely that the biological actions of histamine in repairing processes of the injured liver are mediated by HGF. This study was aimed at examining the effects of histamine on production of HGF using, as a model, the human promyelocytic leukemia cells, HL-60. 12-o-Tetradecanoylphorbol-13-acetate (TPA) markedly stimulated HGF production and release from the cells; the maximal amount of HGF was released at a concentration of 3 ng/ml of TPA. Histamine significantly stimulated the TPA-induced HGF production and release in these cells, depending on incubation time and its dose. These actions of histamine were abrogated by a H2 receptor antagonist, ranitidine.
histamine; hepatocyte growth factor; HL-60 Cells; CCl4-hepatotoxin

-23-
Molecular Cloning and Expression in Saccharomyces cerevisiae of Tobacco NADPH-Cytochrome P450 Oxidoreductase cDNA

Takashi YAMADA, Hiromasa IMAISHI, Atsuhiro OKA,＊ and Hideo OHKAWA
Department of Biological and Environmental Science, Faculty of Agriculture, Kobe University,
Rokkodai-cho 1-1, Nada-ku, Kobe, 657-8501, Japan
＊Laboratory of Molecular Genetics, Institute for Chemical Research, Kyoto University,
Gokasho, Uji, Kyoto, 611-0011, Japan

Received February 25, 1998
We obtained information on the full length tobacco NADPH-cytochrome P450 oxidoreductase (P450 reductase) by a combination of the cDNA clone pCTR1 and the genomic DNA clone pGTR1. The deduced primary structure consisting of 713 amino acid residues contained sequences corresponding to FMN, FAD, and NADPH-binding regions. Based on this information, we prepared the full-length cDNA pFTR of tobacco P450 reductase by RT-PCR and expressed it in the yeast Saccharomyces cerevisiae. The transformed yeast cells carrying pFTR produced the corresponding mRNA and protein, and had increased cytochrome c reductase activity in the microsomes. An in vitro reconstitution system of the yeast microsomal fractions expressed tobacco P450 reductase and rat P450 1A1 showed an increased 7-ethoxycoumarin O-deethylase activity. These results indicated that tobacco P450 reductase expressed in the yeast microsomes coupled with rat P450 1A1 resulting in an increased monooxygenase activity.
NADPH-cytochrome P450 oxidoreductase; tobacco; cDNA cloning; expression

-24-
Note
Inhibition of Icosanoid Production in MC/9 Mouse Mast Cells by n-3 Polyunsaturated Fatty Acids Isolated from Edible Marine Algae

Kenji ISHIHARA, Masakazu MURATA, Masaki KANENIWA, Hiroaki SAITO,
Kazuki SHINOHARA,＊ and Mari MAEDA-YAMAMOTO＊＊
Marine Biochemistry Division, National Research Institute of Fisheries Science, 2-12-4 Fukuura, Kanazawa-ku, Yokohama 236-8648, Japan
＊National Food Research Institute, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-0856, Japan
＊＊National Research Institute of Vegetables, Ornamental Plants and Tea, 2769 Kanaya, Shizuoka 428-8501, Japan

Received November 25, 1997
The effects of two polyunsaturated fatty acids, 18:4n-3 and 16:4n-3 purified from the marine algae, Undaria pinnatifida and Ulva pertusa, on icosanoid production in MC/9 mouse mast cells were assessed. Both fatty acids suppressed the production of leukotriene B4 (LTB4), leukotriene C4 (LTC4), and 5-hydroxyeicosatetraenoic acid (5-HETE). The order of the suppressive activity for the two marine algae-derived fatty acids and three other common polyunsaturated fatty acids was as follows; 22:6n-3＝18:4n-3＝18:3n-3>20:5n-3＝16:4n-3 for LTB4; 22:6n-3＝18:4n-3＝18:3n-3>16:4n-3>20:5n-3 (no suppression) for LTC4; 22:6n-3＝18:4n-3>18:3n-3>20:5n-3＝16:4n-3 for 5-HETE.
Mast cell, Octadecatetraenoic acid, Hexadecatetraenoic acid, Icosanoid, Marine alga

-25-
Note
Purification and Characterization of Cysteine Protease from Pleurotus ostreatus

Hyun-Hee SHIN and Hye-Seon CHOI
Department of Microbiology, University of Ulsan, Ulsan 680-749, Korea

Received December 2, 1997
Cysteine protease activity in mycelial culture increased 7.7-fold after fruit body formation in Pleurotus ostreatus, using the Leu pNA (LPNA) cleavage assay. The enzyme was purified from fruit bodies and its Mr was 97,000 by gel filtration and 48,500 by SDS-PAGE, indicating that it is a dimer. The enzyme was sensitive to iodoacetic acid, p-chloromercuribenzoate, N-ethylmaleimide, and HgCl2. The sequence of the first 9 N-terminal amino acids of cysteine protease was ASGLXXAIL.
cysteine protease; Leucine pNA cleavage activity; fruit body; differentiation; Pleurotus ostreatus

-26-
Note
Possible Formation of Dehydro-L-ascorbic Acid from 2,3-Diketo-L-gulonic Acid in an Aqueous Solution

Noriko MIYAKE and Tadao KURATA
Institute of Environmental Science for Human Life, Ochanomizu University, Bunkyo-ku, Tokyo 112-8610, Japan

Received December 15, 1997
The reaction of 2,3-diketo-L-gulonic acid (DKG), which is one of the important intermediate products in the degradation of L-ascorbic acid (ASA) in both food and biological systems, in an aqueous solution was studied. The formation of a small amount of the γ-lactone, dehydro-L-ascorbic acid (DASA), from DKG was observed. This strongly suggests the chemical possibility of a reverse reaction in DASA hydrolysis which has been long believed to be irreversible.
2,3-diketo-L-gulonic acid; dehydro-L-ascorbic acid; L-ascorbic acid; lactonization

-27-
Note
Occurrence of Bound Salt in Freeze-concentrated Soy Sauce

Michiko WATANABE, Soichi TANABE, Kazuo FURIHATA,＊ Akira OKUBO,＊
and Soichi ARAI＊＊
Food Science Laboratory, Faculty of Education, Tokyo Gakugei University, 4-1-1 Nukuikita-machi,
Koganei-shi, Tokyo 184-8501, Japan
＊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
＊＊Department of Nutrition, Tokyo University of Agriculture, Sakuragaoka, Setagaya-ku, Tokyo 156-0054, Japan

Received December 18, 1997
Part of the salt in soy sauce formed no eutectic| |H2O・2NaCl crystals at sub-zero temperatures and remained| |in a freeze-concentrated product. NMR line width of 23Na was broader in the concentrated soy sauce than in the material. A broad line width of 23Na was also observed in an aqueous solution of NaCl and a non-diffusible soy sauce fraction. The data indicate that part of the salt in soy sauce interacted with its non-diffusible fraction and that such bound salt formed no eutectic crystals.
bound salt; soy sauce; freeze concentration

-28-
Note
Suppression of the SOS-Inducing Activity of Trp-P-1 and Aflatoxin B1 by Meso-dihydroguaiaretic Acid from Machilus thunbergii in the Salmonella typhimurium TA1535/pSK1002 umu Test

Mitsuo MIYAZAWA,1 Yoshiharu OKUNO,1 Kayo OSHIRO,1 Hiroyuki KASAHARA,1
Hideo SHIMAMURA,1 Sei-ichi NAKAMURA,2 and Hiromu KAMEOKA1
1Department of Applied Chemistry, Faculty of Science and Engineering, Kinki University Kowakae,
Higashiosaka-shi, Osaka 577-8502, Japan
2Osaka Prefectural Institute of Public Health, Nakamichi-1, Higashinari-ku, Osaka 537-0025, Japan

Received December 19, 1997
The methanol extract from Machilus thunbergii showed a suppressive effect on umu gene expression of the SOS response in Salmonella typhimurium TA1535/pSK1002 against the mutagen, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), which requires liver metabolizing enzymes. The methanol extract from M. thunbergii was successively re-extracted with chloroform, butanol and water. A suppressive compound in the chloroform extract fraction was isolated by SiO2 column chromatography and identified as meso-dihydroguaiaretic acid by GC-MS, and 1H- and 13C-NMR spectroscopy.
� Meso-dihydroguaiaretic acid inhibited of the SOS-inducing activity of Trp-P-1 in the umu test. Gene expression was suppressed by 62％ at less than 0.18 μmol/ml, the ID50 value being 0.08 μmol/ml. Compound 1 was also assayed with aflatoxin B1 (AfB1) and showed a suppressive effect.
Machilus thunbergii; Lauraceae; meso-dihydroguaiaretic acid; SOS response; umu test

-29-
Note
β-Glucosidase Activity in the Rat Small Intestine toward Quercetin Monoglucosides

Kana IOKU, Yutana PONGPIRIYADACHA,＊ Yotaro KONISHI,＊＊ Yoko TAKEI,
Nobuji NAKATANI,＊＊ and Junji TERAO＊＊＊
Osaka Kyoiku University, 4-698-1 Asahigaoka, Kashiwara, Osaka 582-8582, Japan
＊＊Faculty of Human Life Science, Osaka City University, 3-3-138, Sugimoto, Sumiyoshi, Osaka, 558-8585, Japan
＊＊＊School of Medicine, Tokushima University, 3 Kuramoto, Tokushima, 770-8503, Japan

Received December 24, 1997
In order to evaluate the positional specificity for a glucoside group in the hydrolysis of flavonoid glucosides in the rat small intestine, β-glucosidase activity was measured with the quercetin monoglucosides, quercetin-3-O-β-D-glucopyranoside (Q3G), quercetin-4′-O-β-D-glucopyranoside (Q4′G) and quercetin-7-O-β-D-glucopyranoside (Q7G), as well as with quercetin-3-O-rutinoside (rutin) and p-nitrophenyl-β-D-glucopyranoside (NPG) by using the HPLC technique. Enzymes were prepared from rat small intestinal mucosa of the duodenum, jejunum and ileum, among which the enzyme activity of the jejunum was highest for all the glycosides tested. Q4′G was the richest substrate for a β-glucosidase solution among these glycosides, while rutin and NPG were both poor substrates. This suggests that dietary flavonoid glucosides are primarily hydrolyzed and liberated aglycones in the jejunum.
flavonoid glucosides; quercetin; β-glucosidase

-30-
Note
Ornatipolide, a Novel Phenolic Metabolite from the Basidiomycete, Boletus ornatipes

Hisao SHIBATA,＃ Takashi FUKUDA, Tomonari WADA, Yasufumi MORITA,＊
Toshihiro HASHIMOTO,＊＊ and Yoshinori ASAKAWA＊＊
Department of Bioscience and Biotechnology, Faculty of Agriculture, Shinshu University,
8304 Minami-minowa-mura, Nagano 399-4598, Japan
＊Department of Chemical and Biochemical Engineering, Toyama National College of Technology,
13 Hongo, Toyama 939-8045, Japan
＊＊Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima 770-8055, Japan

Received January 9, 1998
A novel phenolic metabolite, ornatipolide, was isolated from the Boletus ornatipes fungus. Its structure was established by a combination of spectroscopic and chemical methods, and by an X-ray crystallographic analysis.
ornatipolide; macrolide phenolic compound; Basidiomycete; Boletus ornatipes

-31-
Note
Lipase-catalyzed Kinetic Resolution of (±)-cis,cis-Spiro[4.4]nonane-1,6-diol

Yoshitaka MATSUSHIMA,＊ Takeshi KITAHARA, and Kenji MORI＊＊
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 January 9, 1998
(±)-cis,cis-Spiro[4.4]nonane-1,6-diol (1), which has axial chirality, was efficiently resolved by lipase-catalyzed enantioselective esterification in diisopropyl ether.
lipase-catalyzed kinetic resolution; cis,cis-spiro[4.4]nonane-1,6-diol; axial chirality; enantioselective esterification

-32-
Note
Increase of Lipid Hydroperoxides in the Rat Liver and Kidney after Administering Ferric Nitrilotriacetate

Kazumi IKEDA,＊ Fang SUN,＊ Kyoko TANAKA,＊ Sadako TOKUMARU,and Shosuke KOJO＊,＊＊
＊Department of Food Science and Nutrition, Nara Women′s University, Nara 630-8506 Japan
Joetsu University of Education, Joetsu, Niigata 943-8512, Japan

Received January 14, 1998
Two hours after an intraperitoneal injection of ferric nitrilotriacetate to rats at a dose of 15 mg of Fe/kg of body weight, the level of lipid hydroperoxides, which was determined by a specific method involving chemical conversion into 1-naphthyldiphenylphosphine oxide and HPLC, had increased significantly in the liver (from 190.1±58.8 of the control to 467.1±175.8 pmol/mg of protein) and kidney (from 181.8±52.3 of the control to 405.9±22.7 pmol/mg of protein). These results demonstrate that oxidative stress was transiently increased by an iron overload in these tissues.
ferric nitrilotriacetate; hydroperoxide; lipid peroxidation; radical reaction; iron overload

-33-
Note
Comparison of Volatile Compounds from Chungkuk-Jang and Itohiki-Natto

Tadayoshi TANAKA,1 Kanako MURAMATSU,2 Haeng-ran KIM,2, Tazuko WATANABE,1
Michiyo TAKEYASU,2 Yukiko KANAI,2 and Kan KIUCHI2
1Department of the Science of Living, Kyoritsu Womens′ Junior College,
2-2-1 Hitotsubashi, Chiyoda-ku, Tokyo 101-8433, Japan
2Department of Food Science and Nutrition, Faculty of Home Economics, Kyoritsu Women′s University,
2-2-1 Hitotsubashi, Chiyoda-ku, Tokyo 101-8433, Japan

Received January 26, 1998
Volatile compounds from two South-East Asian fermented soybean foods, Chungkuk-jang (CKJ) and Itohiki-natto (natto), were analysed by gas chromatography-mass spectrometry (GC-MS), gas chromatography (GC), and GC-sniffing. A total of 112 compounds were identified. A large amount of ethanol was detected from CKJ, while acetone and methyl isobutyrate were major components of natto. The characteristic odor compounds of CKJ were some ethyl esters of short chain fatty acids, diallyl disulfide, and several natto-like odor compounds were identified as ammonia, 2,5-dimethylpyrazine, and 2-methylbutanoic acid.
Chungkuk-jang; Itohiki-natto; volatile compounds; gas chromatograph-mass spectrometry; gas chromatograph-sniffing

-34-
Note
Application of Two-dimensional Mapping for an Analysis of Galactosyllactoses in Yogurt

Tadao SAITO,＊ Tomoya TSUJI, Haruki KITAZAWA, Yasushi KAWAI,
and Takatoshi ITOH
Laboratory of Animal Products Chemistry, Graduate School of Agricultural Science,
Tohoku University, Aoba-ku, Sendai 981-8555, Japan

Received February 2, 1998
A two-dimensional mapping analysis was performed by HPLC for 4 kinds of standard galactosyllactoses (GLs, trisaccharide) which were assumed to be produced from lactose (galactopyranosylβ1→4 glucopyranose) in yogurt during the fermentation of lactic acid bacteria. After the pyridylamination of GLs, they were analyzed by HPLC in the reverse-phase (RP) and anion-exchange (AE) modes. The retention times of each peak obtained were converted to glucose units (GU) in RP mode for the pyridylaminated isomaltooligosaccharides (G1-3) and to relative retention time (RRT) in AE mode against pyridylaminated-isomaltotriose, and then the address data [GU, RRT] were plotted on a graph. This two-dimensional mapping method was found useful for a rapid qualitative evaluation of the chemical structure of trisaccharides formed in yogurt.
galactosyllactose; HPLC; two-dimensional mapping; pyridylamination

-35-
Note
Effect of Jasmonates and Related Compounds on Seed Germination of Orobanche minor Smith and Striga hermonthica (Del.) Benth

Koichi YONEYAMA, Masaru OGASAWARA, Yasutomo TAKEUCHI, Makoto KONNAI,
Yukihiro SUGIMOTO,＊ Hideharu SETO,＊＊ and Shigeo YOSHIDA＊＊
Weed Science Center, Utsunomiya University, Utsunomiya 321-8505, Japan
＊Arid Land Research Center, Tottori University, Tottori 680-0001, Japan
＊＊The Institute of Physical and Chemical Research (RIKEN), Wako 351-0198, Japan

Received February 3, 1998
Jasmonates and related compounds were found to elicit the seed germination of the important root parasites, clover broomrape (Orobanche minor Smith) and witchweed [Striga hermonthica (Del.) Benth]. The stimulation of seed germination by the esters was more effective than by the corresponding free acids, and methyl jasmonate (MJA) was the most active stimulant among the compounds tested.
cucurbic acid; jasmonic acid; Orobanche minor Smith; seed germination; Striga hermonthica (Del.) Benth

-36-
Note
Acetylation of Bacterial Cellulose: Preparation of Cellulose Acetate Having a High Degree of Polymerization

Mari TABUCHI, Kunihiko WATANABE,＊ Yasushi MORINAGA, and Fumihiro YOSHINAGA
Bio-Polymer Research Co., Ltd., KSP R＆D B-1015, 3-2-1 Sakato, Takatsu, Kawasaki 213-0012, Japan

Received February 16, 1998
Cellulose triacetate prepared from bacterial cellulose of Acetobacter xylinum subsp. sucrofermentans BPR3001A showed a higher degree of polymerization and higher mechanical strength than that from the cotton linter. The fine fibrils of bacterial cellulose required only a short time for acetylation which preserved the high degree of polymerization.
bacterial cellulose; acetylation; cellulose acetate; degree of polymerization; mechanical properties

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Note
ATP-Dependent Inactivation of Escherichia coli γ-Glutamylcysteine Synthetase by L-Glutamic Acid γ-Monohydroxamate

Makoto KATOH, Jun HIRATAKE, and Jun′ichi ODA
Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan

Received February 17, 1998
Incubation of Escherichia coli γ-glutamylcysteine synthetase with L-glutamic acid γ-monohydroxamate and ATP caused slow but irreversible inhibition of the enzyme, and more than 90％ activity was lost in three days. The enzyme was not inactivated when ATP was absent or L-aspartic acid β-monohydroxamate was substituted for L-glutamic acid γ-monohydroxamate, suggesting that the inactivation process reflected a mechanism-based reaction of L-glutamic acid γ-monohydroxamate and ATP.
γ-glutamylcysteine synthetase; ADP-forming peptide ligase; ATP dependence; enzyme inactivation; L-glutamic acid γ-monohydroxamate

-38-
Note
Suppression of Senescence in Normal Human Fibroblasts by Introduction of ominant-Negative p53 Mutants or Human Papilloma Virus Type 16 E6 Protein

Akiko IDE, Michihiko FUJII, Kazuhiko NAKABABASHI, 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 February 26, 1998
Transfection of nearly senesced human fibroblasts with plasmids encoding HPV16 E6 protein or dominant-negative p53 mutants greatly increased their colony-forming ability. Isolated colonies with these plasmids showed extension of lifespan compared to those with a control plasmid. These data demonstrate that p53 plays a major role in senescence in normal human fibroblasts.
p53; senescence; DNA tumor virus; telomere; lifespan

�-39-
Preliminary Communication
Safety Assessment of Genetically Engineered Food: Detection and Monitoring of Glyphosate-Tolerant Soybeans

Nobuaki SHIRAI,＊ Keiko MOMMA, Sachiko OZAWA, Wataru HASHIMOTO, Makoto KITO,
Shigeru UTSUMI, and Kousaku MURATA
Research Institute for Food Science, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan
＊Industrial Research Center of Shiga Prefecture, 232 Kamitoyama, Rittoh, Kurita, Shiga 520-3004, Japan

Received March 10, 1998
A detection technique for the genetically engineered food, glyphosate-tolerant soybean (GTS), was designed. Commercial soybeans imported from North America were cultured in pots and genomic DNA was isolated from their leaves. To detect the genes, promoter and terminator, involved in the expression of glyphosate tolerance, PCR was done using the genomic DNA and chemically synthesized primers specific to the genes. DNAs with predicted sizes were amplified and confirmed by DNA sequencing to be the genes responsible for the expression of glyphosate tolerance. Glyphosate-tolerant soybeans were found to form approximately 1.1％ of the commercial soybeans, when commercially available soybeans were cultivated and number of soybeans resistant to glyphosate was found. This level is somewhat lower than an estimated value announced officially on the basis of the cultivation area of the glyphosate-tolerant soybeans.
genetically engineered food; glyphosate-tolerant soybeans; monitoring; PCR

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Preliminary Communication
An N-terminal Region of LukF of Staphylococcal Leukocidin/γ-Hemolysin Crucial for the Biological Activity of the Toxin

Jun KANEKO, Ma. Anita L. MASCARENAS, Md. Nazmul HUDA, Toshio TOMITA,
and Yoshiyuki KAMIO＊
Department of Applied Biological Chemistry, Faculty of Agriculture, Tohoku University,
1-1 Tsutsumi-dori Amamiya-machi, Aoba-ku, Sendai 981-8555, Japan

Received April 6, 1998
The two staphylococcal bi-component toxins, leukocidin and γ-hemolysin share LukF [Kamio et al, FEBS Lett., 321, 15--18 (1993)]. This report identifies the pivotal amino acid residues in the N-terminal region of LukF for the leukocytolytic and hemolytic activities in the presence of LukS and Hlg2, respectively, measuring the toxin activiy of a series of LukF mutants with truncated N-terminals. The data obtained showed that the LukF mutant TF21, lacking 20 amino acid residues at the N-terminus of LukF, failed to have any hemolytic activity and had less 10％ leukocytolytic activity than that of the intact LukF, while 16-residue truncations retained both toxin activities without loss. The LukF mutants lacking 18- through 19-residue segments from the N-terminus showed low toxin activity on both target cells. All mutants having no toxin activity were also not capable of binding to the human erythrocytes. It can thus be concluded that the 3-residue segment, L＾{18}Y＾{19}K＾{20} of LukF is crucial for the biological activity of the toxin.
Staphylococcus aureus; LukF; leukocidin; γ-hemolysin; N-terminal region

-41-
Preliminary Communication
Stereochemical Difference in Secoisolariciresinol Formation between Cell-free Extracts from Petioles and from Ripening Seeds of Arctium lappa L.

Shiro SUZUKI, Toshiaki UMEZAWA,＊ and Mikio SHIMADA
Wood Research Institute, Kyoto University, Uji, Kyoto 611-0011, Japan

Received April 9, 1998
Cell-free extracts from ripening seeds of Arctium lappa L. catalyzed the enantioselective formation of (−)-pinoresinol, (−)-lariciresinol and (−)-secoisolariciresinol from achiral coniferyl alcohol in the presence of NADPH and H2O2. The enantioselectivity of the lignan formation was opposite to that of the (＋)-secoisolariciresinol formation catalyzed by cell-free extracts from petioles of the same plant species.
Arctium lappa; ripening seeds; enantioselective; lignan; biosynthesis

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