Contents and Abstracts of Latest Issue of BBB

(Vol.66 No.6 2002)

S-RNases from Self-incompatible and -compatible Apple Cultivars:
Purification, Cloning, Enzymic Properties, and Pollen Tube
Growth Inhibitory Activity
Naoki KATOH1, Kazunori GOTO, Junpei ASANO, Kiyoe FUKUSHIMA, Kenji YAMADA,
Aya KASAI, Tian Zhong LI, Makoto TAKANOHA, Kazuo MIYAIRI, and Toshikatsu OKUNO†
p.1185

Effects of Fatty Liver Induced by Niacin-free Diet with Orotic Acid
on the Metabolism of Tryptophan to Niacin in Rats
Tsutomu FUKUWATARI†, Yuko MORIKAWA, Etsuro SUGIMOTO, and Katsumi SHIBATA p.1196

3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) Is Incorporated
into Rat Splenocytes, Thymocytes, and Hepatocytes Through
Monoamine Transporters and Induces Apoptosis
Takashi HASHIMOTO,1 Takashi FURUYASHIKI,2 Takashi SANO,2 Wakana ITO,2
Gen-ichi DANNO,1,2 Kazuki KANAZAWA,1 and Hitoshi ASHIDA1,2,† p.1205

Design of a Highly Potent Anti-hypertensive Peptide Based on Ovokinin(2-7)
Yuko YAMADA,† Nobuyuki MATOBA, Hachiro USUI, Kunihiko ONISHI,
and Masaaki YOSHIKAWA p.1213

Relationship between the Electrical and Rheological Properties
of Potato Tuber Tissue after Various Forms of Processing
Petr DEJMEK† and Osato MIYAWAKI†† p.1218

Monoamine-dependent Production of Reactive Oxygen Species
Catalyzed by Pseudoperoxidase Activity of Human Hemoglobin
Tomonori KAWANO,1,† Reinhard PINONTOAN,2 Hishoshi HOSOYA,1 and Shoshi MUTO2
p.1224

Effect of Dietary Fiber on Morphine-induced Constipation in Rats
Takashi NIWA,1 Makoto NAKAO,1 Seiko HOSHI,2 Kiyofumi YAMADA,1
Kazuhiro INAGAKI,3 Mikio NISHIDA,3 and Toshitaka NABESHIMA1,† p.1233

Increase of Immunoglobulin Productivity of Human-Human
Hybridoma HB4C5 Cells by Histone
Takeaki OKAMOTO, Takuya SUGAHARA,＊ Hirofumi TACHIBANA, and Koji YAMADA† p.1241

Cloning and Structural Analysis of bglM Gene Coding for the Fungal Cell
Wall-lytic β-1,3-Glucan-hydrolase BglM of Bacillus circulans IAM1165
Takeshi ASANO, Jiro TAKI, Mami YAMAMOTO, and Rikizo AONO† p.1246

Cloning and Expression of Fructosyl-amino Acid Oxidase Gene
from Corynebacterium sp. 2-4-1 in Escherichia coli
Ryoichi SAKAUE,† Minoru HIRUMA, Naoki KAJIYAMA, and Yasuji KOYAMA p.1256

Stereochemical Diversity in Lignan Biosynthesis of Arctium lappa L.
Shiro SUZUKI, Toshiaki UMEZAWA,† and Mikio SHIMADA p.1262

Endophytic Bacillus sp. Isolated from the Interior of Balloon Flower Root
Soo Jeong CHO,1 Sang Ryeol PARK,2 Min Keun KIM,1 Woo Jin LIM,1
Sung Kee RYU,1 Chang Long AN,1 Su Young HONG,1 Young Han LEE,3
Seon Gi JEONG,3 Yong Un CHO,4 and Han Dae YUN1,2,† p.1270

Increased Hydrophobicity of Carboxymethyl Starch Film
by Conjugation with Zein
Koji TAKAHASHI,† Atsushi OGATA, Wen-Hong YANG, and Makoto HATTORI p.1276

Sodium-23 and Lanthanum-139 Nuclear Magnetic Resonance Studies of
Cation Binding to Aqualysin I, a Thermostable Serine Protease
Kyoko SUEFUJI,1,††† Shie-Jea LIN,1,††† Takayoshi WAKAGI,1 Hiroshi MATSUZAWA,1,††
and Etsuro YOSHIMURA2,† p.1281

Induction of Oral Tolerance in Mice by Continuous Feeding
with β-Lactoglobulin and Milk
Koko MIZUMACHI1 and Jun-ichi KURISAKI2 p.1287

Characterization of Partially Truncated Human Midkine
Expressed in Pichia pastoris
Akira MURASUGI† and Yumiko TOHMA-AIBA p.1295

Structural Studies by Stepwise Enzymatic Degradation of the Main Backbone
of Soybean Soluble Polysaccharides Consisting of Galacturonan
and Rhamnogalacturonan
Akihiro NAKAMURA,† Hitoshi FURUTA, Hirokazu MAEDA, Toshifumi TAKAO,1
and Yasunori NAGAMATSU2 p.1301

Two Low-temperature-inducible Chlorella Genes for Δ12 and ω-3 Fatty
Acid Desaturase (FAD): Isolation of Δ12 and ω-3 fad cDNA Clones,
Expression of Δ12 fad in Saccharomyces cerevisiae, and Expression
of ω-3 fad in Nicotiana tabacum
Koushirou SUGA,1,2 Ken-ichi HONJOH,3 Naoki FURUYA,1 Hideyuki SHIMIZU,1
Koutarou NISHI,1 Fuminori SHINOHARA,1 Yoshie HIRABARU,1 Isao MARUYAMA,2
Takahisa MIYAMOTO,3 Shoji HATANO,4 and Masayoshi IIO3,† p.1314

Cloning, Characterization, and Expression of cDNA Encoding
a Lipase from Kurtzmanomyces sp. I-11
Koji KAKUGAWA,1,2,3,† Megumi SHOBAYASHI,2 Osamu SUZUKI,1 and Tokichi MIYAKAWA1
p.1328

Transcriptome Analysis of Acetate Metabolism in Corynebacterium
glutamicum Using a Newly Developed Metabolic Array
Mikiro HAYASHI, Hiroshi MIZOGUCHI, Norihiko SHIRAISHI, Masaya OBAYASHI,
Satoshi NAKAGAWA, Jun-ichi IMAI,＊ Shinya WATANABE,＊ Toshio OTA,
and Masato IKEDA† p.1337

Amino Acid Sequence and Characterization of a Nuclease (Nuclease Le3)
from Lentinus edodes
Hiroko KOBAYASHI, Fumi KUMAGAI, Tadashi ITAGAKI, Takashi KOYAMA,
Norio INOKUCHI,† Masanori IWAMA,＊ Kazuko OHGI,＊ and Masachika IRIE＊ p.1345

Distribution and Molecular Evolution of Rhamnose-binding Lectins
in Salmonidae: Isolation and Characterization of Two Lectins
from White-spotted Charr (Salvelinus leucomaenis) Eggs
Hiroaki TATENO,1 Tomohisa OGAWA,1 Koji MURAMOTO,1,† Hisao KAMIYA,2
and Mineo SANEYOSHI3 p.1356

Note
Cloning and Characterization of Extracellular Metal Protease Gene
of the Algicidal Marine Bacterium Pseudoalteromonas sp. Strain A28
Sun-Og LEE, Junichi KATO,† Katsunori NAKASHIMA, Akio KURODA,
Tsukasa IKEDA, Noboru TAKIGUCHI, and Hisao OHTAKE p.1366

Note
Casein Digestion by Debaryomyces hansenii Isolated from Cheese
Haruto KUMURA,† Kentaro TAKAGAKI, Teruo SONE,1 Maki TSUKAHARA,
Tetsuya TANAKA, and Kei-ichi SHIMAZAKI p.1370

Note
Stabilization of Flavobacterium meningosepticum Glycerol Kinase by
Introduction of a Hydrogen Bond
Shin-ichi SAKASEGAWA,1,† Hideki TAKEHARA,2 Issei YOSHIOKA,1 Hideo MISAKI,1
Haruhiko SAKURABA,3 and Toshihisa OHSHIMA3 p.1374

Note
Cloning and Expression of an Endo-1,6-β-D-glucanase Gene (neg1)
from Neurospora crassa＊
Seiji OYAMA, Youhei YAMAGATA, Keietsu ABE, and Tasuku NAKAJIMA† p.1378

Note
Characterization of S-Sulfokeratein from Pig Hair and Its Use
as a Modifier of Type I Collagen Gel
Yoshihiro NOMURA,† Hidetoshi NAKATO, Yasuhiro ISHII, and Kunio SHIRAI p.1382

Note
Peroxynitrite Scavenging Activities of Aromatic Compounds Isolated
from Konnyaku, Amorphophallus konjac K.Koch.
Tatsuya NIWA, Kazushi MURAKAMI,1 Tetsuro OHTAKE,1 Hideo ETOH,1,†
Atsushi SHIMIZU,2 Yasuo SHIMIZU,2 Yoji KATO,3 and Hitoshi TANAKA,4 p.1386

Note
Synthesis of (R)- and (S)- Muscone
Satoko FUJIMOTO,1 Keisuke YOSHIKAWA,2 Masamichi ITOH,2 and Takeshi KITAHARA1,†
p.1389

Note
Increased Proteolytic Susceptibility of Carboxypeptidase Y Caused
by Modification of the Disulfide Zipper
Toshihiko MAKI, Hajime KOZAWA, Joji MIMA,† Hiroshi UENO,†† and Rikimaru HAYASHI
p.1393

Note
Purification and Identification of Flavonoids from the Yellow Green
Cocoon Shell (Sasamayu) of the Silkworm, Bombyx mori
Akira KURIOKA† and Masayoshi YAMAZAKI p.1396

Note
Antimutagenic Effect of Amino Acids on the Mutagenicity
of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG)
Molay Kumar ROY, Yuji KUWABARA,＊ Kazusi HARA,＊ Yasuo WATANABE,
and Youichi TAMAI† p.1400

Note
Molecular Cloning and Characterization of a Novel β-1,3-Glucanase
Gene from Rice
Tomoya YAMAGUCHI, Katsuhiro NAKAYAMA, Takami HAYASHI,
Yoshiyuki TANAKA,＊ and Setsuo KOIKE† p.1403

Note
Synthesis of Milbemycins β9 and β10 from Milbemycins A3
and A4 and Their Biological Activities
Takahiro TSUKIYAMA,† Ayako KINOSHITA, Reiji ICHINOSE,＊ and Kazuo SATO p.1407

Note
Inhibition of Transglutaminase by Synthetic Tyrosine Melanin
Koji IKURA,† Chiemi OTOMO, Shunji NATSUKA, Akira ICHIKAWA,
Kazumasa WAKAMATSU,＊ Shosuke ITO,＊ and Shin-ichi TAOGUCHI p.1412

Note
Synthesis of the Racemate of (Z)-exo-α-Bergamotenal, a Pheromone
Component of the White-spotted Spined Bug, Eysarcoris parvus Uhler
Babak Heidary ALIZADEH, Shigefumi KUWAHARA,† Walter Soares LEAL,1 and
Hong-Chao MEN2,＃ p.1415

Note
Two Novel Oligosaccharides Formed by 1F-Fructosyltransferase Purified
from Roots of Asparagus (Asparagus officinalis L.)
Akira YAMAMORI, Shuichi ONODERA, Masanori KIKUCHI, and Norio SHIOMI† p.1419

Note
Transglutaminase Activity of Human ER-60
Hirokazu OKUDO, Makoto KITO,† Tatsuya MORIYAMA, Tadashi OGAWA,
and Reiko URADE†† p.1423

Note
Tissue-specific Expression of Rhamnose-binding Lectins
in the Steelhead Trout (Oncorhynchus mykiss)
Hiroaki TATENO,1 Yasushi SHIBATA,2 Yoshitaka NAGAHAMA,2 Toshiaki HIRAI,3
Mineo SANEYOSHI,3 Tomohisa OGAWA,1 Koji MURAMOTO,1,† and Hisao KAMIYA4 p.1427

Preliminary Communication
Identification of a 22-kDa Protein Required for the Degradation
of Selenomonas ruminantium Lysine Decarboxylase
by ATP-dependent Protease
Yoshihiro YAMAGUCHI, Yumiko TAKATSUKA, and Yoshiyuki KAMIO† p.1431

-1-
S-RNases from Self-incompatible and -compatible Apple Cultivars:
Purification, Cloning, Enzymic Properties, and Pollen Tube
Growth Inhibitory Activity

Naoki KATOH1, Kazunori GOTO, Junpei ASANO, Kiyoe FUKUSHIMA, Kenji YAMADA,
Aya KASAI, Tian Zhong LI, Makoto TAKANOHA, Kazuo MIYAIRI, and Toshikatsu OKUNO†

Laboratory of Biochemistry, Faculty of Agriculture and Life Science, Hirosaki University,
Bunkyo-cho 3, Hirosaki 036-8561, Japan

Received August 2, 2001; Accepted February 1, 2002
Four S-RNases (RNase associated with self-incompatibility) were purified from the styles of two apple cultivars (Malus domestica), a self-incompatible cv., Starking Delicious (SD), and a self-compatible cv., Megumi (MG). Each cultivar produced two S-RNases and their enzymatic properties such as specific activity, pH optimum, thermal stability, and molecular mass, were characterized. The four S-RNases inhibited the tube growth of apple pollen in an in vitro bioassay at 25 μg/ml (1.0 μM), but did not distinguish self from non-self pollen. The cDNAs of four S-RNases were cloned, and the nucleotide and deduced amino acid sequences were analyzed. The nucleotide sequence of SD-Se RNase was a new one and the other was identical to that of Sc-RNase of cv. Fuji. In MG one was identical to the sequence of SD-Sc RNase and the other to that of Sa-RNase of cv. Golden Delicious except for one base. From results of the isolation amounts and the Western blot analysis for stylar crude extracts the amount of S-RNases in MG was apparently less than that in SD.
Key words: S-RNase; apple; self-incompatibility; pollen growth inhibition

-2-
Effects of Fatty Liver Induced by Niacin-free Diet with Orotic Acid
on the Metabolism of Tryptophan to Niacin in Rats

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

Laboratory of Food Science and Nutrition, Department of Life Style Studies, School of Human Cultures,
The University of Shiga Prefecture, 2500 Hassakacho, Hikone, Shiga, 522-8533, Japan

Received September 5, 2001; Accepted January 25, 2002
The effects of dietary orotic acid on the metabolism of tryptophan to niacin in weaning rats was investigated. The rats were fed with a niacin-free, 20％ casein diet containing 0％ (control diet) or 1％ orotic acid diet (test diet) for 29 d. Retardation of growth, development of fatty liver, and enlargement of liver were observed in the test group in comparison with the control group. The concentrations of NAD and NADP in liver significantly decreased, while these in blood did not decrease compared to the control group. The formation of the upper metabolites of tryptophan to niacin such as anthranilic acid, kynurenic acid, and 3-hydroxyanthranilic acid were not affected, but the quinolinic acid and beyond, such as nicotinamide, N1-methylnicotinamide, N1-methyl-2-pyridone-5-carboxamide, and N1 -methyl-4-pyridone-3-carboxamide, were significantly reduced by the administration of orotic acid. Therefore, the conversion ratio of tryptophan to niacin significantly decreased in the test group in comparison with the control group.
Key words: orotic acid; tryptophan; niacin; NAD; fatty liver

-3-
3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) Is Incorporated
into Rat Splenocytes, Thymocytes, and Hepatocytes Through
Monoamine Transporters and Induces Apoptosis

Takashi HASHIMOTO,1 Takashi FURUYASHIKI,2 Takashi SANO,2 Wakana ITO,2
Gen-ichi DANNO,1,2 Kazuki KANAZAWA,1 and Hitoshi ASHIDA1,2,†

1Division of Life Science, Graduate School of Science and Technology, Kobe University, Rokkodai-cho 1, Nada-ku, Kobe 657-8501, Japan
2Department of Biofunctional Chemistry, Faculty of Agriculture, Kobe University, Rokkodai-cho 1,
Nada-ku, Kobe 657-8501, Japan

Received September 5, 2001; Accepted February 8, 2002
3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), which is a tryptophan pyrolysate formed during cooking, induces apoptosis in rat splenocytes, thymocytes, and hepatocytes. In this study, we investigated whether Trp-P-1 is transported into these cells and causes apoptosis. Trp-P-1 was immediately incorporated into rat splenocytes, thymocytes, and hepatocytes in a dose- and time-dependent manner. Dopamine and serotonin significantly competed with the uptake of Trp-P-1 into these cells, and nomifensine and indatraline, which are inhibitors of dopamine- and serotonin-transporters, respectively, markedly suppressed the uptake of Trp-P-1. On the other hand, amino acids including tryptophan did not compete with Trp-P-1. Inhibition of monoamine transporters using nomifensine and indatraline partially suppressed Trp-P-1-induced cell death in these cells. In hepatocytes, the inhibition of transporters prevented Trp-P-1-induced morphological changes and activation of caspase-3. These results demonstrated that Trp-P-1 is incorporated into the cells through monoamine transporters and induces apoptosis.
Key words: 3 - amino - 1, 4 - dimethyl - 5H - pyrido [4, 3 -b]indole (Trp-P-1); apoptosis; monoamine transporter; immunocytes; hepatocytes

-4-
Design of a Highly Potent Anti-hypertensive Peptide Based on Ovokinin(2-7)

Yuko YAMADA,† Nobuyuki MATOBA, Hachiro USUI, Kunihiko ONISHI,
and Masaaki YOSHIKAWA

Division of Food Bioscience and Biotechnology, Graduate School of Agriculture,
Kyoto University, Uji, Kyoto 611-0011, Japan

Received September 26, 2001; Accepted January 31, 2002
Ovokinin(2-7) (RADHPF), an orally active anti-hypertensive peptide derived from ovalbumin, lowers blood pressure in SHRs at a dose of 10 mg/kg. Attempts were made to potentiate its anti-hypertensive activity by replacing the amino acid residues in [Pro2, Phe3]-ovokinin(2-7), which was previously reported to have 33-fold stronger activity than ovokinin(2-7). The anti-hypertensive activity of [Pro2, Phe3]-ovokinin(2-7) was improved by replacement of the C-terminal Phe residue with Trp. Then, the best amino acid residues at other positions for the anti-hypertensive effect were selected. RPLKPW, the most potent derivative obtained, showed significant anti-hypertensive activities at a dose of 0.1 mg/kg after oral administration in spontaneously hypertensive rats (SHRs). Thus, RPLKPW showed 100-fold more potent anti-hypertensive activity than ovokinin(2-7).
Key words: ovokinin(2-7); anti-hypertensive; blood pressure; spontaneously hypertensive rat (SHR)

-5-
Relationship between the Electrical and Rheological Properties
of Potato Tuber Tissue after Various Forms of Processing

Petr DEJMEK† and Osato MIYAWAKI††

Department of Applied Biological Chemistry, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku,
Tokyo 113-8657, Japan

Received October 5, 2001; Accepted February 12, 2002
The impedance at frequencies of 1-1000 kHz and dynamic bending storage modulus measured by the vibrating reed method were compared for potato tuber tissue, which had been processed by various methods. Raw potato tuber tissue strips were either heated for 30 min up to 100°C or frozen-thawed. Some samples were osmotically dehydrated in a mannitol solution up to a concentration of 0.7 mol/l. The electrical reactance correlated well with the storage modulus of heated or frozen-thawed potato tissues, but not with the storage modulus of the mannitol-treated tissue. The storage modulus appeared to be strongly dependent on the turgor pressure of the cells which was drastically decreased by the heating, freezing-thawing, and osmotic treatments. The electrical properties reflect the cell integrity, and a large difference was observed between the change in impedance after heating or freezing-thawing, and that after the osmotic treatment. A significant change in the electric properties was also observed for a starch suspension at the gelatinization temperature. However, the contribution due to gelatinization did not appear to play an important role in the change of electrical properties of potato tissue by heating.
Key words: impedance; storage modulus; vibrating reed; heat processing; osmotic dehydration

-6-
Monoamine-dependent Production of Reactive Oxygen Species
Catalyzed by Pseudoperoxidase Activity of Human Hemoglobin

Tomonori KAWANO,1,† Reinhard PINONTOAN,2 Hishoshi HOSOYA,1 and Shoshi MUTO2

1Department of Biological Science, Graduate School of Science, Hiroshima University,
Higashi-Hiroshima 739-8526, Japan
2Nagoya University Bioscience Center, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan

Received October 17, 2001; Accepted January 30, 2002
Hemoglobin (Hb) solution-based blood substitutes are being developed as oxygen-carrying agents for the prevention of ischemic tissue damage and low blood volume-shock. However, the cell-free Hb molecule has intrinsic toxicity to the tissue since harmful reactive oxygen species (ROS) are readily produced during autoxidation of Hb from the ferrous state to the ferric state, and the cell-free Hb also causes distortion in the oxidant/antioxidant balance in the tissues. There may be further hindering dangers in the use of free Hb as a blood substitute. It has been reported that Hb has peroxidase-like activity oxidizing peroxidase substrates such as aromatic amines. Here we observed the Hb-catalyzed ROS production coupled to oxidation of a neurotransmitter precursor, β-phenylethylamine (PEA). Addition of PEA to Hb solution resulted in generation of superoxide anion (O2<09-25>-). We also observed that PEA increases the Hb-catalyzed monovalent oxidation of ascorbate to ascorbate free radicals (Asc<09-25>). The O2<09-25>- generation and Asc<09-25> formation were detected by O2<09-25>--specific chemiluminescence of the Cypridina lucigenin analog and electron spin resonance spectroscopy, respectively. PEA-dependent O2<09-25>- production and monovalent oxidation of ascorbate in the Hb solution occurred without addition of H2O2, but a trace of H2O2 added to the system greatly increased the production of both O2<09-25>- and Asc<09-25>. Addition of GSH completely inhibited the PEA-dependent production of O2<09-25>- and Asc<09-25> in Hb solution. We propose that the O2<09-25>- generation and Asc<09-25> formation in the Hb solution are due to the pseudoperoxidase activity-dependent oxidation of PEA and resultant ROS may damage tissues rich in monoamines, if the Hb-based blood substitutes were circulated without addition of ROS scavengers such as thiols.
Key words: hemoglobin; methemoglobin; monoamine; reactive oxygen species; superoxide

-7-
Effect of Dietary Fiber on Morphine-induced Constipation in Rats

Takashi NIWA,1 Makoto NAKAO,1 Seiko HOSHI,2 Kiyofumi YAMADA,1
Kazuhiro INAGAKI,3 Mikio NISHIDA,3 and Toshitaka NABESHIMA1,†

1Department of Neuropsychopharmacolgy and Hospital Pharmacy, Nagoya University Graduate School
of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya 466-8560, Japan
2Department of Nutritional Research, Nutrition Science Institute, Meiji Milk Products Co. Ltd,
540 Naruda, Odawara 250-0862, Japan
3Department of Clinical Pharmacy, Faculty of Pharmacy, Meijo University, 150 Yagotoyama,
Tempaku-ku, Nagoya 468-8503, Japan

Received October 19, 2001; Accepted February 2, 2002
Morphine is used to alleviate chronic cancer pain. However, constipation is a major adverse effect that often detracts from the patient′s quality of life. In this study, we investigated the effectiveness of dietary fiber on morphine-induced constipation. Rats were fed on a normal diet or one containing either 10％ or 20％ apple fiber for two weeks before morphine was administered. In the control diet group, the fecal number and dry weight were decreased by treating with morphine in a dose-dependent manner. Moreover, the motility of the small and large intestines was reduced. The fecal number and weight were increased and the colon motility was promoted by dietary fiber, regardless of whether morphine was being administered. The dietary fiber increased the concentration of short-chain fatty acids (SCFAs) in the cecum. These results suggest that dietary fiber has a preventative effect on morphine-induced constipation by increasing SCFAs in the cecum, and thereby promoting colon motility in rats.
Key words: constipation; dietary fiber; morphine; SCFAs

-8-
Increase of Immunoglobulin Productivity of Human-Human
Hybridoma HB4C5 Cells by Histone

Takeaki OKAMOTO, Takuya SUGAHARA,＊ Hirofumi TACHIBANA, and Koji YAMADA†

Laboratory of Food Chemistry, Department of Bioscience and Biotechnology, Division of Bioresource
and Bioenvironmental Sciences, Graduate School of Kyushu University 46-09, 6-10-1 Hakozaki,
Higashi-ku, Fukuoka 812-8581, Japan
＊Faculty of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama, Ehime 790-8566, Japan

Received October 22, 2001; Accepted February 12, 2002
A histone mixture (H1, H2A, H2B, H3, and H4) derived from calf thymus stimulated IgM production by human-human hybridoma HB4C5 cells. On the contrary, the histone mixture did not increase IgM production by the human Burkitt′s lymphoma cell line NAT-30, IgG production by the human B lymphoblastoid cell line HMy-2, and IgE production by the human myeloma cell line U266. The immunoglobulin production-stimulating activity of the histone mixture was inactivated by trypsin or chymotrypsin digestion. In addition, confocal laser microscopic analysis had shown that HB4C5 cells incorporated a lot of histone but other cell lines did not incorporate it as much.
These facts strongly suggest that histone acts as an immunoglobulin production-stimulating factor (IPSF) after internalization into the human B cell lines and the native structure of histone is required for the IPSF activity.
Key words: histone; human B cell line; immunoglobulin production stimulating factor (IPSF); serum-free culture

-9-
Cloning and Structural Analysis of bglM Gene Coding for the Fungal Cell
Wall-lytic β-1,3-Glucan-hydrolase BglM of Bacillus circulans IAM1165

Takeshi ASANO, Jiro TAKI, Mami YAMAMOTO, and Rikizo AONO†

Department of Biological Information, Graduate School of Bioscience and Biotechnology,
Tokyo Institute of Technology, Nagatsuta 4259, Midori-ku, Yokohama 226-8501, Japan

Received October 25, 2001; Accepted January 10, 2002
Bacillus circulans IAM1165 produces isoforms of β-1,3-glucan-hydrolases. Of these enzymes, the 42-kDa enzyme BglM degrades Aspergillus oryzae cell walls the most actively. A gene coding for a BglM precursor consisting of 411 amino acid residues was cloned. The 27 N-terminal amino acid sequence of the precursor is a signal peptide. The 141 C-terminal amino acid sequence showed a motif of carbohydrate-binding module family 13. This domain bound to pachyman, lichenan, and A. oryzae cell walls. The central domain showed a bacterial β-1,3-glucan-hydrolase motif belonging to glycosyl hydrolase family 16. By removal of the C-terminal domain in the IAM1165 culture, mature BglM was processed to several 27-kDa fragments that hydrolyze a soluble β-1,3-glucan.
Key words: Bacillus circulans; β-1,3-glucanase; 1,3-(1,3;1,4)-β-D-glucan 3(4)-glucanohydrolase; fungal cell wall-binding domain; fungal cell wall lytic enzyme

-10-
Cloning and Expression of Fructosyl-amino Acid Oxidase Gene
from Corynebacterium sp. 2-4-1 in Escherichia coli

Ryoichi SAKAUE,† Minoru HIRUMA, Naoki KAJIYAMA, and Yasuji KOYAMA

Research and Development Division, Kikkoman Corporation, Noda, Chiba 278-0037, Japan

Rceived November 13, 2001; Accepted February 4, 2002
The gene encoding the fructosyl-amino acid oxidase (fructosyl-α-L-amino acid: oxygen oxidoreductase (defructosylating); EC 1.5.3) of Corynebacterium sp. 2-4-1 was cloned and expressed in Escherichia coli. The gene consists of 1,116 nucleotides and encodes a protein of 372 amino acids with a predicted molecular mass of 39,042. The open reading frame was confirmed as the gene of the fructosyl-amino acid oxidase by comparison with the N-terminal amino acid sequence of the purified fructosyl-amino acid oxidase from Corynebacterium sp. 2-4-1. The sequence of the AMP-binding motif, GXGXXG, was found in the deduced N-terminal region. The amino acid sequence of the fructosyl-amino acid oxidase showed no similarity to that of fungal fructosyl-amino acid oxidases. In addition, substrate specificities of this fructosyl-amino acid oxidase were different from those of other fructosyl-amino acid oxidases. The fructosyl-amino acid oxidase of Corynebacterium sp. 2-4-1 is an enzyme that has unique substrate specificity and primary structure in comparison with fungal fructosyl-amino acid oxidases.
Key words: diabetes; HbA1c; Amadori compound; fructosyl-amino acid oxidase

-11-
Stereochemical Diversity in Lignan Biosynthesis of Arctium lappa L.

Shiro SUZUKI, Toshiaki UMEZAWA,† and Mikio SHIMADA

Wood Research Institute, Kyoto University, Uji, Kyoto 611-0011, Japan

Received November 15, 2001; Accepted January 29, 2002
The stereochemistry of lignan biosynthesis in Arctium lappa L. is regulated organ-specifically. (＋)-Secoisolariciresinol [81％ enantiomeric excess (e.e.)] was isolated from A. lappa petioles. In sharp contrast, lignans whose predominant enantiomers have the opposite absolute configuration to that of (＋)-secoisolariciresinol [i.e., (−)-matairesinol (＞99％ e.e.), (−)-arctigenin (＞99％ e.e.), and (−)-secoisolariciresinol (65％ e.e.)] were isolated from seeds of the species. The stereochemical diversity of secoisolariciresinol was demonstrated with enzyme preparations from A. lappa petioles and seeds. Thus, a petiole enzyme preparation catalyzed the formation of
(＋)-pinoresinol (33％ e.e.), (＋)-lariciresinol (30％ e.e.),
and (＋)-secoisolariciresinol (20％ e.e.) from achiral coniferyl alcohol in the presence of NADPH and H2O2, whereas that from ripening seeds catalyzed the formation of (−)-pinoresinol (22％ e.e.), (−)-lariciresinol (＞99％ e.e.), and (−)-secoisolariciresinol (38％ e.e.) under the same conditions. In addition, the ripening seed enzyme preparation mediated the selective formation of the optically pure (＞99％ e.e.) (−)-enantiomer of matairesinol from racemic (±)-secoisolariciresinols in the presence of NADP.
These results indicate that the stereochemical mechanism for lignan biosynthesis in A. lappa varies with organs, suggesting that multiple lignan-synthesizing isozymes are involved in the stereochemical control of lignan formation in A. lappa.
Key words: Arctium lappa; secoisolariciresinol; enantioselective; lignan; biosynthesis

-12-
Endophytic Bacillus sp. Isolated from the Interior of Balloon Flower Root

Soo Jeong CHO,1 Sang Ryeol PARK,2 Min Keun KIM,1 Woo Jin LIM,1
Sung Kee RYU,1 Chang Long AN,1 Su Young HONG,1 Young Han LEE,3
Seon Gi JEONG,3 Yong Un CHO,4 and Han Dae YUN1,2,†

1Division of Applied Life Science and 2Research Institute for Life Science, Gyeongsang National University,
Chinju 660-701, Korea
3Gyeongnam Agricultural Research and Extension Services, Chinju 660-307, Korea
4Department of Microbiological Engineering, Chinju National University, Chinju 660-758, Korea

Received November 19, 2001; Accepted February 4, 2002
A bacterial strain, designated CY22, was isolated from the interior of balloon flower (Platycodon grandiflorum) root in the Republic of Korea. The isolate coproduced an iturin-like antifungal compound and a surfactin-like potent biosurfactant. Analysis of the 16S-rDNA of strain CY22 showed that the isolate was a member of Bacillus. High similarities were observed between strain CY22 and Bacillus sp. TKSP 24, and between strain CY22 and B. subtilis 168. Phylogenetic analysis based on 16S-rDNA sequences showed that strain CY22 was closely related to Bacillus sp. The main whole-cell fatty acids were anteiso-C15:0 (37％), C17:0 (5.1％), and iso-C15:0 (27.7％). DNA G＋C content was 54 mol％. Based on phylogenetic inference, phenotypic and chemotaxonomic characteristics, this endophytic strain Bacillus sp. CY22 was assigned to the genus Bacillus.
Key words: Bacillus sp.; Platycodon grandiflorum; endophytes

-13-
Increased Hydrophobicity of Carboxymethyl Starch Film
by Conjugation with Zein

Koji TAKAHASHI,† Atsushi OGATA, Wen-Hong YANG, and Makoto HATTORI

Department of Applied Biological Science, Faculty of Agriculture, Tokyo University
of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan

Received November 21, 2001; Accepted February 12, 2002
A carboxymethyl starch (CMS) film was prepared by a process in which gelatinized CMS was dried, and subsequently treated with water-soluble carbodiimide in the presence of zein in 70％ ethanol or 70％ acetone to form acid-amide cross-linkages in order to increase the hydrophobicity of the surface of the film. A small amount of zein protein was found to be present on the surface of the zein-CMS conjugate (Zein-CMS) film, resulting in its insolubility in hot water, low water vapor permeability, and resistance to digestion with α-amylase and β-amylase. Digestion of the Zein-CMS film with protease rendered the film readily water-soluble, suggesting conjugation with zein as an effective means of increasing the hydrophobicity of biodegradable starch-based articles.
Key words: biodegradable material; starch; zein; conjugation

-14-
Sodium-23 and Lanthanum-139 Nuclear Magnetic Resonance Studies of
Cation Binding to Aqualysin I, a Thermostable Serine Protease

Kyoko SUEFUJI,1,††† Shie-Jea LIN,1,††† Takayoshi WAKAGI,1 Hiroshi MATSUZAWA,1,††
and Etsuro YOSHIMURA2,†

1Department of Biotechnology, The University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
2Department of Applied Biological Chemistry, The University of Tokyo, Yayoi, Bunkyo-ku,
Tokyo 113-8657, Japan

Received December 3, 2001; Accepted February 4, 2002
Aqualysin I has at least two Ca2＋-binding sites that have different affinities for Ca2＋. The binding of various metal ions to aqualysin I was studied using 23Na- and 139La-NMR spectrometry. Evidence is presented that Ca2＋, La3＋, and Na＋ bind to the low-affinity Ca2＋-binding site of aqualysin I, but Mg2＋ does not. Our results confirm that binding of metals at the low-affinity Ca2＋-binding site is essential for thermostabilization, since the addition of Mg2＋ did not result in thermostabilization. La3＋ was found to bind to both the low-affinity Ca2＋-binding site and an additional metal ion-binding site that can also be involved in the thermostabilization of aqualysin I.
Key words: aqualysin I; 139La-NMR; 23Na-NMR; thermostable serine protease

-15-
Induction of Oral Tolerance in Mice by Continuous Feeding
with β-Lactoglobulin and Milk

Koko MIZUMACHI1 and Jun-ichi KURISAKI2

1Department of Animal Products Research, National Institute of Livestock and Grassland Science,
Ikenodai 2, Kukizaki, Inashiki, Ibaraki 305-0901, Japan
2Genetic Diversity Department, National Institute of Agrobiological Sciences, Kannondai 2-1-2,
Tsukuba, Ibaraki 305-8602, Japan

Received December 7, 2001; Accepted January 29, 2002
We examined the immune response of mice to β-lactoglobulin (BLG), a potent milk allergen, after continuous feeding of a BLG solution or milk instead of drinking-water. Strong suppression of the anti-BLG antibody response and antigen-specific T cell response were observed in mice fed BLG and milk. Although the profile of the antibody specificity to BLG peptides in mice fed BLG or milk was different from the control, the dominant determinants were still recognized and a limited number of new recognition sites appeared by BLG or milk feeding. These findings suggest that continuous feeding with BLG or milk not only induced tolerance in the periphery, but also priming. In terms of the antigen-specific IgG subclass response, the production of both IgG1 and IgG2a was significantly reduced. The cytokine secretion of interleukin (IL)-2, IFN-γ and IL-10 was also reduced in the culture supernatants of lymph node cells from the mice fed BLG. The results indicate that the continuous feeding of BLG or milk induces suppression of both Th1- and Th2-dependent responses. This may reflect a state of oral tolerance induced by food ingestion.
Key words: β-lactoglobulin; oral tolerance; synthetic peptide; epitope

-16-
Characterization of Partially Truncated Human Midkine
Expressed in Pichia pastoris

Akira MURASUGI† and Yumiko TOHMA-AIBA

Meiji Cell Technology Center, Meiji Milk Products Co., Ltd., 540 Naruda, Odawara,
Kanagawa 250-0862, Japan

Received December10, 2001; Accepted February 12, 2002
Recombinant human midkine (rh-midkine) was expressed under the control of the AOX1 gene promoter in Pichia pastoris. Approximately 640 mg of rh-midkine was secreted into one liter of medium of the high cell-density fermentation. The protein processing of the rh-midkine was done efficiently and correctly in P. pastoris, and O-mannosylation was not detected in the purified rh-midkine. However, only about 30％ of the purified rh-midkine was intact. The other ones lost 5--12 amino acid residues from the amino-termini, provably by proteolysis. Even the mixture of these truncated midkines could promote CHO cell proliferation as well as the authentic rh-midkine.
Key words: truncated human midkine; high level expression; non-glycosylation; precursors; Pichia pastoris

-17-
Structural Studies by Stepwise Enzymatic Degradation of the Main Backbone
of Soybean Soluble Polysaccharides Consisting of Galacturonan
and Rhamnogalacturonan

Akihiro NAKAMURA,† Hitoshi FURUTA, Hirokazu MAEDA, Toshifumi TAKAO,1
and Yasunori NAGAMATSU2

Tsukuba R＆D Center, Fuji Oil Co., Ltd., 4-3 Kinunodai, Yawara, Tsukuba-gun, Ibaraki 300-2497, Japan
1Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan
2Department of Applied Biochemistry, Faculty of Applied Biological Science, Hiroshima University,
1-4-4 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8528, Japan

Received December 13, 2001; Accepted February 21, 2002
Soybean soluble polysaccharides (SSPS) extracted from soybean cotyledons are acidic polysaccharides and have a pectin-like structure. The results of a structural analysis of SSPS by using polygalacturonase (PGase) and rhamnogalacturonase (RGase) clarified that the main backbone consisted of galacturonan (GN) and rhamnogalacturonan (RG), which were composed of the diglycosyl repeating unit, -4)-α-D-GalpA-(1→2)-α-L-Rhap-(1-. The side chains of β-1,4-galactans, branched with fucose and arabinose residues, were linked to the C-4 side of rhamnose residues in the RG regions. The degree of polymerization (dps) of GN, which linked the RG regions together, was estimated to be about 4--10 residues, and some were modified with xylose residues on the C-3 side of the galacturonates. The dps of GN at the reducing end of SSPS was estimated to be about 7--9 residues. Moreover, the fragment of the basic structure of the RG region, -[4)-α-D-GalpA-(1→2)-α-L-Rhap-
(1-]2-, some of which had long-chain β-1,4-galactans branched on the C-4 side of rhamnose residues, were liberated from SSPS by the RGase treatment. The dps of the galactan side chain was estimated to be about 43--47 residues by an analysis of the digestion products from the β-galactosidase treatment.
Key words: soybean; polysaccharides; pectin

-18-
Two Low-temperature-inducible Chlorella Genes for Δ12 and ω-3 Fatty
Acid Desaturase (FAD): Isolation of Δ12 and ω-3 fad cDNA Clones,
Expression of Δ12 fad in Saccharomyces cerevisiae, and Expression
of ω-3 fad in Nicotiana tabacum

Koushirou SUGA,1,2 Ken-ichi HONJOH,3 Naoki FURUYA,1 Hideyuki SHIMIZU,1
Koutarou NISHI,1 Fuminori SHINOHARA,1 Yoshie HIRABARU,1 Isao MARUYAMA,2
Takahisa MIYAMOTO,3 Shoji HATANO,4 and Masayoshi IIO3,†

1Division of Bioresource and Bioenvironmental Sciences, Graduate School, Kyushu University,
Fukuoka 812-8581, Japan
2Chlorella Industry Co. Ltd., 1343 Hisatomi, Chikugo, Fukuoka 833-0056, Japan
3Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University,
Fukuoka 812-8581, Japan
4Graduate School of Health and Social Welfare Science, Nishikyushu University, 4490-9 Ooazaozaki,
Kanzaki-machi, Kanzaki-gun, Saga 842-8585, Japan

Received December 19, 2001; Accepted February 20, 2002
In an attempt to clarify the involvement of fatty acid desaturases (FADs) in the freezing tolerance of Chlorella vulgaris IAM C-27, developed by hardening, we have isolated cDNA clones for two types of FADs from the Chlorella strain, based on the sequence information of genes for Δ12 and ω-3 FADs, respectively desaturating oleic acid (18:1) to linoleic acid (18:2) and linoleic acid (18:2) to linolenic acid (18:3). The deduced amino acid sequence of the first clone, designated CvFad2, showed about 66％ similarity to the microsomal Δ12 FADs from several higher plants and this gene had Δ12 FAD activity when expressed in Saccharomyces cerevisiae. The predicted protein encoded by a second gene, designated CvFad3, showed about 60％ similarity to the microsomal and plastidial ω-3 FADs from several higher plants. The features of the amino acid sequences of the C- and N-terminal regions of CvFAD3 and fatty acid analysis of polar lipids in transgenic tobacco plant expressing the CvFad3 gene suggested that this gene encodes the microsomal ω-3 FAD. Southern blot analysis showed that both genes were single-copy genes in the genome of the Chlorella strain. Different transcriptional patterns were observed with the two genes during hardening in Northern blot analysis.
Key words: cDNA cloning; fatty acid desaturase; Chlorella vulgaris C-27; freezing tolerance; hardening induced gene

-19-
Cloning, Characterization, and Expression of cDNA Encoding
a Lipase from Kurtzmanomyces sp. I-11

Koji KAKUGAWA,1,2,3,† Megumi SHOBAYASHI,2 Osamu SUZUKI,1 and Tokichi MIYAKAWA1

1Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter,
Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8530, Japan
2Hiroshima Prefectural Institute of Industrial Science and Technology, 3-10-32 Kagamiyama,
Higashi-Hiroshima, Hiroshima 739-0046, Japan
3Hiroshima Prefectural Food Technology Research Center, 12-70 Hijiyama-honmachi, Minami-ku,
Hiroshima 732-0816, Japan

Received December 25, 2001; Accepted January 31, 2002
A cDNA clone of the lipase secreted by Kurtzmanomyces sp. I-11 was isolated from a cDNA library of this yeast by PCR screening using oligonucleotide primers designed on the basis of the partial amino acid sequence of the lipase. The cloned cDNA (lip1) encoded a hydrophobic protein of 484 amino acids, where the first 20 amino acids and the following 6 amino acid sequences were predicted to be the signal sequence for secretion and a pro-sequence, respectively. The deduced amino acid sequence of the Kurtzmanomyces lipase was most similar to Candida antarctica DSM 3855 lipase A (74％ identity) and weakly to other lipases. The consensus pentapeptide (-Gly-X-Ser-X-Gly-) that forms a part of the interfacial lipid recognition site in lipases was conserved. A high level of lipase was produced by Pichia pastoris transformed with the lip1 cDNA, indicating that the cloned cDNA indeed encodes a lipase.
Key words: lipase; mannosylerythritol lipid; Kurtzmanomyces sp. yeast; cDNA

-20-
Transcriptome Analysis of Acetate Metabolism in Corynebacterium
glutamicum Using a Newly Developed Metabolic Array

Mikiro HAYASHI, Hiroshi MIZOGUCHI, Norihiko SHIRAISHI, Masaya OBAYASHI,
Satoshi NAKAGAWA, Jun-ichi IMAI,＊ Shinya WATANABE,＊ Toshio OTA,
and Masato IKEDA†

Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., Asahi-machi, Machida, Tokyo 194-8533, Japan
＊Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639, Japan

Received January 9, 2002; Accepted February 4, 2002
Following the determination of the whole-genome sequence of Corynebacterium glutamicum, we have developed a DNA array to extensively investigate gene expression and regulation relevant to carbon metabolism. For this purpose, a total of 120 C. glutamicum genes, including those in central metabolism and amino acid biosyntheses, were amplified by PCR and printed onto glass slides. The resulting array, designated a <Oh><0192><0192><Wa>metabolic array′′, was used for hybridization with fluorescently labeled cDNA probes generated by reverse transcription from total RNA samples. As the first demonstration of transcriptome analysis in this industrially important microorganism, we applied the metabolic array to study differential transcription profiles between cells grown on glucose and on acetate as the sole carbon source. The changes in gene expression observed for the known acetate-regulated genes (aceA, aceB, pta, and ack) were well consistent with the literature data of northern analyses and enzyme assays, indicating the utility of the metabolic array in transcriptome analysis of C. glutamicum. In addition to the known responses, many previously unrecognized co-regulated genes were identified. For example, several TCA cycle genes, such as gltA, sdhA, sdhB, fumH, and mdh, and the gluconeogenic gene pck were up-regulated in the acetate medium. On the other hand, a few genes involved in glycolysis and the pentose phosphate pathway, as well as many amino acid biosynthetic genes, were down-regulated in acetate. Furthermore, two gap genes, gapA and gapB, were found to be inversely regulated, suggesting the presence of a new regulatory step for carbon metabolism between glycolysis and gluconeogenesis.
Key words: DNA array; transcriptome; gene expression; acetate metabolism; Corynebacterium glutamicum

-21-
Amino Acid Sequence and Characterization of a Nuclease (Nuclease Le3)
from Lentinus edodes

Hiroko KOBAYASHI, Fumi KUMAGAI, Tadashi ITAGAKI, Takashi KOYAMA,
Norio INOKUCHI,† Masanori IWAMA,＊ Kazuko OHGI,＊ and Masachika IRIE＊

Department of Microbiology, College of Pharmacy, Nihon University, Narashinodai 7-7-1, Funabashi,
Chiba 274-8555, Japan
＊Department of Microbiology, Hoshi College of Pharmacy, Ebara 2-4-41, Shinagawa, Tokyo 142-8501, Japan

Received January 15, 2002; Accepted February 18, 2002
The fruit body of shiitake (Lentinus edodes) produces two acid nucleases, nuclease Le1 and nuclease Le3, both of which are thought to be candidates for the enzyme that produces a flavorful substance, 5′-GMP, and the primary structure of one of the nucleases, nuclease Le1, has been analyzed by both protein chemistry and gene cloning [Biosci. Biotechnol. Biochem. 64, 948--957 (2000)].
In this study the amino acid sequence of nuclease Le3 was analyzed by protein chemistry and gene cloning. Nuclease Le3 is a glycoprotein that contains 280 amino acid residues, and the molecular mass of the protein moiety of nuclease Le3 is 31,045. The nucleotide sequence of the cDNA and genomic DNA encoding nuclease Le3 revealed the presence of an 18-residue putative signal peptide.
Nuclease Le3 contains 170, 108, and 98 amino acid residues that are identical to residues of nuclease Le1, nuclease P1, and nuclease S, respectively. The amino acid residues involved in coordination with Zn2＋ atoms in nuclease P1 are all conserved in nuclease Le3. Nuclease Le3 contains 9 half-cystine residues, and 7 of them are located in the same positions as in nuclease Le1.
Key words: cDNA; Lentinus edodes; nuclease; 3′-nucleotidase; primary structure

-22-
Distribution and Molecular Evolution of Rhamnose-binding Lectins
in Salmonidae: Isolation and Characterization of Two Lectins
from White-spotted Charr (Salvelinus leucomaenis) Eggs

Hiroaki TATENO,1 Tomohisa OGAWA,1 Koji MURAMOTO,1,† Hisao KAMIYA,2
and Mineo SANEYOSHI3

1Department of Biological Resource Sciences, Graduate School of Agricultural Science,
Tohoku University, Sendai 981-8555, Japan
2School of Fisheries Sciences, Kitasato University, Sanriku, Iwate 022-0101, Japan
3Department of Biological Sciences, Teikyo University of Science and Technology, Uenohara,
Yamanashi 409-0193, Japan

Received January 17, 2002; Accepted February 16, 2002
L-Rhamnose-binding lectins were isolated from white-spotted charr (Salvelinus leucomaenis) eggs to understand the distribution and molecular evolution of the lectins in Salmonidae. Only two L-rhamnose-binding lectins, named WCL1 and WCL3, were isolated from white-spotted charr eggs, though three lectins, named STL1, STL2, and STL3, had been obtained from steelhead trout (Oncorhynchus mykiss) eggs. The cDNAs of WCL1 and WCL3 included 1,245 and 838 bp nucleotides with open reading frames of 933 and 651 nucleotides, respectively, and encoded for the complete amino acid sequences of mature proteins consisted of 288 (WCL1) and 195 (WCL3) residues, and signal sequences of 23 and 22 residues, respectively. WCLs were composed of three (for WCL1) or two (for WCL3) tandemly repeated homologous domains, which consisted of about 95 amino acid residues, and showed 91 and 93％ sequence identities to STL1 and STL3, respectively. The mRNAs of WCL1 and WCL3 were detected exclusively in liver and ovary, respectively, however, neither a protein nor mRNA corresponding to STL2 could be identified in white-spotted charr. The phylogenetic tree of the 16 sequences encoding carbohydrate recognition domains of 7 lectins from 4 species shows 5 functional clusters and their evolutional process. These results indicate that multiple L-rhamnose-binding isolectins have diverged by gene duplication and exon shuffling to play various biological roles in each species.
Key words: rhamnose-binding lectin; Salmonidae; fish egg; white-spotted charr; molecular evolution

-23-
Note
Cloning and Characterization of Extracellular Metal Protease Gene
of the Algicidal Marine Bacterium Pseudoalteromonas sp. Strain A28

Sun-Og LEE, Junichi KATO,† Katsunori NAKASHIMA, Akio KURODA,
Tsukasa IKEDA, Noboru TAKIGUCHI, and Hisao OHTAKE

Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter,
Hiroshima University, Higashi-Hiroshima, Hiroshima 739-8530, Japan

Received October 15, 2001; Accepted January 26, 2002
The gene (empI) encoding an extracellular metal protease was isolated from a Pseudoalteromonas sp. strain A28 DNA library. The recombinant EmpI protein was expressed in E. coli and purified. Paper-disk assays showed that the purified protease had potent algicidal activity. A skim milk-polyacrylamide gel electrophoresis protease assay showed that the 38-kDa band of protease activity, which co-migrated with purified EmpI and was sensitive to 1,10-phenathroline, was detected in the extracellular supernatant of A28.
Key words: Pseudoalteromonas; algicidal marine bacterium; extracellular metal protease; red tide algae

-24-
Note
Casein Digestion by Debaryomyces hansenii Isolated from Cheese

Haruto KUMURA,† Kentaro TAKAGAKI, Teruo SONE,1 Maki TSUKAHARA,
Tetsuya TANAKA, and Kei-ichi SHIMAZAKI

Laboratory of Dairy Science, Research Group of Animal Product Science, Division of Bioresources
and Product Science, 1Laboratory of Applied Microbiology, Research Group of Molecular Bioscience,
Division of Applied Bioscience, The Graduate School of Agriculture, Hokkaido University,
Kita 9 Nishi 9, Sapporo 060-8589, Japan

Received November 8, 2001; Accepted January 29, 2002
To understand the possible proteolytic contribution of yeast during cheese ripening, Debaryomyces hansenii 212 was isolated from commercial blue-veined cheese and incubated in a medium containing casein. Growth and casein degradation were recognized at the cheese-ripening temperature. Proteolytic activity was found in the intracellular fraction, and the enzyme, which was attached to the cell wall, primarily acted on β-casein. The cytosol contained more than 90％ of the total proteolytic activity which was responsible for the degradation of both αs- and β-casein. These results suggest that the contribution of yeast to cheese ripening would depend on the susceptibility to cell lysis in addition to its proteolytic activity.
Key words: Debaryomyces hansenii; protease; cheese

-25-
Note
Stabilization of Flavobacterium meningosepticum Glycerol Kinase by
Introduction of a Hydrogen Bond

Shin-ichi SAKASEGAWA,1,† Hideki TAKEHARA,2 Issei YOSHIOKA,1 Hideo MISAKI,1
Haruhiko SAKURABA,3 and Toshihisa OHSHIMA3

1Asahi Kasei Corporation, Ohito-Cho, Tagata-gun, Shizuoka 410-2321, Japan
2Molecular Gene Technics, Genetic Resource Technology, Kyushu University, Fukuoka 812-8581, Japan
3Department of Biological Science and Technology, Faculty of Engineering, The University
of Tokushima, Tokushima 770-8506, Japan

Received November 12, 2001; Accepted February 26, 2002
The thermostability of Flavobacterium meningosepticum glycerol kinase was increased by the change from Ser329 to Asp [Protein Eng., 14, 663--667 (2001)]. Based on a three-dimensional structure model of the mutant, we have postulated that a new charged-neutral hydrogen bond was formed between Asp329 and Ser414, and the formation of the hydrogen bond contributed to the stabilization of the tertiary structure and increased thermostability of the mutant enzyme. If the postulation is the case, FGK thermostabilization would be possible similarly by the single amino acid substitution from Ser414 to another amino acid which could form the hydrogen bond with Ser329. We did a single amino acid substitution of the wild-type enzyme from Ser414 to Asn. As we expected, S414N showed comparable thermostability to that of S329D. On the other hand, a difference in kinetic properties for ATP between S414N and S329D was observed.
Key words: Flavobacterium meningosepticum; site-directed mutagenesis; glycerol kinase; thermostability; hydrogen bond

-26-
Note
Cloning and Expression of an Endo-1,6-β-D-glucanase Gene (neg1)
from Neurospora crassa＊

Seiji OYAMA, Youhei YAMAGATA, Keietsu ABE, and Tasuku NAKAJIMA†

Laboratory of Enzymology, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555,
Japan

Received November 26, 2001; Accepted January 28, 2002
A gene (neg1) encoding an endo-1,6-β-D-glucanase from Neurospora crassa was cloned. The putative neg1 was 1443-bp long and encoded a mature endo-1,6-β-D-glucanase protein of 463 amino acids and signal peptide of 17 amino acids. The purified recombinant protein (Neg1) obtained from Escherichia coli showed 1,6-β-D-glucanase activity. No genes similar in sequence were found in yeasts and fungi.
Key words: fungal β-1,6-glucanase; Neurospora crassa; β-1,6-glucanase gene; recombinant β-1,6-glucanase; fungal cell wall

-27-
Note
Characterization of S-Sulfokeratein from Pig Hair and Its Use
as a Modifier of Type I Collagen Gel

Yoshihiro NOMURA,† Hidetoshi NAKATO, Yasuhiro ISHII, and Kunio SHIRAI

Faculty of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8, Saiwai-cho, Fuchu,
Tokyo 183-8509, Japan

Received November 28, 2001; Accepted February 1, 2002
S-sulfokeratein is prepared through S-sulfonation after the cleavage of disulfide bonds in keratin using ditiothreitol in urea. S-sulfokeratein is composed of two fractions, matrix and microfibril components, and S-sulfokeratein from the matrix component (Bs) can regenerate disulfide bonds. In this study, the effects of Bs and partially reduced Bs on type I collagen self-assembly and properties of reconstructed Bs- or partially reduced Bs-collagen gel were investigated. It was proved that collagen self-assembly was accelerated by the increased amount of added Bs, but partially reduced Bs with 10 mg DTT/100 mg Bs (Bs-10) did not affect the ratio of collagen self-assembly. The mechanical strength of Bs-collagen gel proved to be lower than control, but that of Bs-10-collagen gel was times higher than that of control.
Key words: type I collagen; S-sulfokeratein; regeneration of disulfide bound; collagen self-assembly; mechanical strength

-28-
Note
Peroxynitrite Scavenging Activities of Aromatic Compounds Isolated
from Konnyaku, Amorphophallus konjac K.Koch.

Tatsuya NIWA, Kazushi MURAKAMI,1 Tetsuro OHTAKE,1 Hideo ETOH,1,†
Atsushi SHIMIZU,2 Yasuo SHIMIZU,2 Yoji KATO,3 and Hitoshi TANAKA,4

The United Graduate School of Agricultural Sciences, Gifu University (Shizuoka University),
Shizuoka 422-8529, Japan
1Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka 422-8529, Japan
2Toa Co. Ltd., 2-8-14 Toyota, Shizuoka 422-8027, Japan
3School of Humanities for Environmental Policy and Technology, Himeji Institute of Technology,
Himeji 670-0092, Japan
4Faculty of Pharmacy, Meijo University, Tempaku, Nagoya 468-8503, Japan

Received December 3, 2001; Accepted February 9, 2002
(±)-5,5′-Dimethoxysesamin, erythrinasinate, indole-3-carbaldehyde, (7R,8S)-dihydrodehydrodiconiferyl alcohol 9-O-β-D-glucopyranoside, cis- and trans-N-(p-coumaroyl)serotonin, serotonin, 3,4-dihydroxybenzoic acid, and 3,4-dihydroxybenzaldehyde have been found in tobiko, a food by-product, and evaluation of their peroxynitrite scavenging activities has been done. Among these compounds, serotonin, trans-N-(p-coumaroyl)serotonin, 3,4-dihydroxybenzaldehyde, and 3,4-dihydroxybenzoic acid showed stronger activities than that of BHT (butylated hydroxytoluene) at 200 μM.
Key words: konnyaku; tobiko; peroxynitrite; antioxidative compounds; neolignan

-29-
Note
Synthesis of (R)- and (S)- Muscone

Satoko FUJIMOTO,1 Keisuke YOSHIKAWA,2 Masamichi ITOH,2 and Takeshi KITAHARA1,†

1Department 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
2Technical Research Center, T. Hasegawa Co. Ltd., 335 Kariyado, Nakahara-ku, Kawasaki-shi 211-0022,
Japan

Received December 7, 2001; Accepted February 4, 2002
(R)-(−)-Muscone (3-methylcyclopentadecanone, 1) the key perfumery component isolated from the male musk deer, Moschus moschiferus,＊ was synthesized from the easily available chiral building block, (R)-3-tert-butoxycarbonyl-2-methylpropanoic acid (2), by employing ring-closing olefin metathesis (RCM). Antipode (＋)-1 was also synthesized in a similar manner from tert-butyl (S)-3-methoxycarbonylbutanoate (10).
＊(a) Walbaum, H. J. J. Prakt. Chem., 73, 488 (1906); (b) Ruzicka, L., Further considerations on the constitution of muscone. Helv. Chim. Acta, 9, 715, 1008--1017 (1926).
Key words: cyclic ketones; cycloalkanes; metathesis; ruthenium

-30-
Note
Increased Proteolytic Susceptibility of Carboxypeptidase Y Caused
by Modification of the Disulfide Zipper

Toshihiko MAKI, Hajime KOZAWA, Joji MIMA,† Hiroshi UENO,†† and Rikimaru HAYASHI

Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku,
Kyoto 606-8502, Japan

Received December 10, 2001; Accepted January 31, 2002
To investigate the structural importance of a <Oh><0192><0192><Wa>disulfide zipper′′ motif of carboxypeptidase Y, disulfide-deficient mutant enzymes were expressed in two strains of Saccharomyces cerevisiae. The mutant enzymes were rapidly degraded into fragments by intracellular proteases. Thus, it is concluded that the disulfide zipper is essential in maintaining the structural integrity of CPase Y against proteolytic susceptibility.
Key words: carboxypeptidase Y; disulfide zipper; pro-carboxypeptidase Y; protease susceptibility; structural motif

-31-
Note
Purification and Identification of Flavonoids from the Yellow Green
Cocoon Shell (Sasamayu) of the Silkworm, Bombyx mori

Akira KURIOKA† and Masayoshi YAMAZAKI

Silk Science Research Institute, Hyakuninncho 3-25-1, Shinjuku-ku, Tokyo 169-0073, Japan

Received December 18, 2001; Accepted February 7, 2002
Three quercetin glycosides, quercetin 5-O-β-D-glucoside, quercetin 7-O-β-D-glucoside, and quercetin 4′-O-β-D-glucoside, and two kaempferol glycosides, kaempferol 5-O-β-D-glucoside and kaempferol 7-O-β-D-glucoside, along with their aglycones, quercetin and kaempferol, were isolated from an ethanolic extract of Sasamayu cocoon shells. The chemical structures were characterized by chemical and spectroscopic methods including UV spectrometry and HPLC-ESI-MS. The five flavonol glycosides of the shell are different structurally from those of the leaves of mulberry (Morus alba). It was suggested that potent antioxidative activity in the cocoon is mainly due to flavonoid compounds since free radical scavenging activity was found in the cocoon flavonoids identified here.
Key words: flavonoid; ethanolic cocoon shell extract; free radical scavenging activity; Bombyx mori

-32-
Note
Antimutagenic Effect of Amino Acids on the Mutagenicity
of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG)

Molay Kumar ROY, Yuji KUWABARA,＊ Kazusi HARA,＊ Yasuo WATANABE,
and Youichi TAMAI†

Department of Bioresource, Faculty of Agriculture, Ehime University, Matsuyama, Ehime 790-8566, Japan
＊Shikoku Milk Product Co. Ltd., Kawauchi, Ehime 791-0397, Japan

Received December 21, 2001; Accepted February 15, 2002
The antimutagenic activity of protein-constituting amino acids except histidine on the mutagenicity of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) was investigated in vitro using Salmonella typhinurium TA-100 as an indicator bacterium (Ames test), and concentrations (IC50) of amino acids that inhibit 50％ of the mutagenecity were measured. Cysteine was found to be most active and glycine, tryptophan, lysine, and arginine were strong antimutagenic amino acids. Other amino acids showed moderate or weak antimutagenic activities, depending on the amino acids. The results indicate that amino acids play a substantial role in chemoprevention of N-nitroso amine-induced mutagenicity.
Key words: antimutaganic activity; MNNG; amino acid; cysteine

-33-
Note
Molecular Cloning and Characterization of a Novel β-1,3-Glucanase
Gene from Rice

Tomoya YAMAGUCHI, Katsuhiro NAKAYAMA, Takami HAYASHI,
Yoshiyuki TANAKA,＊ and Setsuo KOIKE†

Plant Physiology Laboratory, National Agricultural Research Center for Tohoku Region,
Shimo-kuriyagawa, Morioka 020-0198, Japan
＊Plant Physiology Department, National Institute of Agrobiological Sciences, Kannondai,
Tsukuba 305-8602, Japan

Received December 27, 2001; Accepted February 20, 2002
Beta-1,3-glucanases are referred to as pathogenesis-related proteins and they are also involved in several developmental processes. We isolated a cDNA for β-1,3-glucanase from rice anther and named it Oryza sativa glucanase 1 (Osg1). Phylogenetic analysis showed that Osg1 belonged to monocotyledonous endo-β-1,3-glucanase subgroup A. RT-PCR analysis revealed that Osg1 transcripts were present in leaves, roots, and anthers.
Key words: β-1,3-glucanase gene; pathogenesis; rice anther

-34-
Note
Synthesis of Milbemycins β9 and β10 from Milbemycins A3
and A4 and Their Biological Activities

Takahiro TSUKIYAMA,† Ayako KINOSHITA, Reiji ICHINOSE,＊ and Kazuo SATO

Agroscience Research Laboratories, Sankyo Co. Ltd., 1041 Yasu, Yasu-cho, Yasu-gun,
Shiga 520-2342, Japan
＊Crop Protection Department, Sankyo Co. Ltd., 7-12 Ginza 2-Chome, Chuo-ku, Tokyo 104-8113, Japan

Received December 27, 2001; Accepted February 8, 2002
Chemical derivation methods were used to prepare milbemycins β9 and β10 from milbemycins A3 and A4. Their acaricidal activities were also assessed against the organophosphorus-sensitive two-spotted spider mite (Tetranychus urticae) on primary leaves of cowpea plants (Vigna sinesis Savi species) by spraying.
Key words: milbemycin; acaricide

-35-
Note
Inhibition of Transglutaminase by Synthetic Tyrosine Melanin

Koji IKURA,† Chiemi OTOMO, Shunji NATSUKA, Akira ICHIKAWA,
Kazumasa WAKAMATSU,＊ Shosuke ITO,＊ and Shin-ichi TAOGUCHI

Department of Applied Biology, Kyoto Institute of Technology, Matsugasaki, Kyoto 606-8585, Japan
＊School of Health Sciences, Fujita Health University, Toyoake 470-1192, Japan

Received January 7, 2002; Accepted January 30, 2002
Transglutaminases catalyze the cross-linking and amine incorporation of proteins, and are implicated in various biological phenomena. Previously, we found a high molecular mass transglutaminase-inhibitory substance produced by Streptomyces lavendulae Y-200 that appeared to be a melanin substance. Here, we report that synthetic tyrosine melanin inhibited various types of transglutaminases. Tyrosine melanin inhibited tissue-type transglutaminase in a competitive manner with a glutamine substrate, and also inhibited the cross-linking of casein catalyzed by a tissue-type transglutaminase. The melanized hemolymph of the silkworm and melanin solutions prepared from melanin precursors inhibited tissue-type transglutaminase. These results suggested that the melanin substances generally inhibit transglutaminases.
Key words: transglutaminase; transglutaminase inhibitor; melanin-like substance

-36-
Note
Synthesis of the Racemate of (Z)-exo-α-Bergamotenal, a Pheromone
Component of the White-spotted Spined Bug, Eysarcoris parvus Uhler

Babak Heidary ALIZADEH, Shigefumi KUWAHARA,† Walter Soares LEAL,1 and
Hong-Chao MEN2,＃

Laboratory of Applied Bioorganic Chemistry, Division of Life Science, Graduate School of Agricultural
Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan
1Honorary Maeda-Duffey Laboratory, Department of Entomology, University of California Davis,
1 Shields Ave., Davis, CA 95616-8585, USA
2Laboratory of Chemical Prospecting, National Institute of Sericultural and Entomological Science,
1-2 Ohwashi, Tsukuba 305-8634, Japan

Received January 7, 2002; Accepted February 4, 2002
The racemate of (Z)-exo-α-bergamotenal, a sex pheromone component of the white-spotted spined bug, was synthesized from racemic exo-α-bergamotene by a five-step sequence involving regioselective epoxidation and (Z)-selective Wittig olefination reactions. The 1H- and 13C-NMR spectra of the synthetic sample were identical with those of the natural material.
Key words: bergamotenal; sesquiterpene; pheromone; white-spotted spined bug; Eysarcoris parvus

-37-
Note
Two Novel Oligosaccharides Formed by 1F-Fructosyltransferase Purified
from Roots of Asparagus (Asparagus officinalis L.)

Akira YAMAMORI, Shuichi ONODERA, Masanori KIKUCHI, and Norio SHIOMI†

Department of Food Production and Utility Development, Graduate School of Dairy Science Research,
Rakuno Gakuen University, Ebetsu 069-8501, Japan

Received January 15, 2002; Accepted January 30, 2002
Two novel oligosaccharides, tetra-and penta-saccharides were synthesized by fructosyl transfer from 1-kestose to 4G-β-D-galactopyranosylsucrose with a purified 1F-fructosyltransferase of asparagus roots and identified as 1F-β-D-fructofuranosyl-4G-β-D-galactopyranosylsucrose, O-β-D-fructofuranosyl-(2→1)-β-D-fructofurano-
syl-O-[β-D-galactopyranosyl-(1→4)]-α-D-glucopyranoside and 1F(1-β-D-fructofuranosyl)2-4G-β-D-galactopyranosylsucrose, [O-β-D-fructofuranosyl-(2→1)]2-β-D-fructofuranosyl-O-[β-D-galactopyranosyl-(1→4)]-α-D-glucopyranoside, respectively. Both oligosaccharides
were scarcely hydrolyzed by carbohydrase from rat small intestine.
Human intestinal bacterial growth by 1F-β-D-fructofuranosyl-4G-β-D-galactopyranosylsucrose was compared with that by the tetrasaccharides, stachyose and nystose. Bifidobacteria utilized 1F-β-D-fructofuranosyl-4G-β-D-galactopyranosylsucrose to the same extent as stachyose or nystose. On the other hand, the unfavorable bacteria, Clostridium perfringens, Escherichia coli and Enterococcus faecalis, that produce mutagenic substances did not use the synthetic oligosaccharide.
Key words: fructosyloligosaccharide; indigestible oligosaccharide; 1F-fructosyltransferase; Bifidobacterium

-38-
Note
Transglutaminase Activity of Human ER-60

Hirokazu OKUDO, Makoto KITO,† Tatsuya MORIYAMA, Tadashi OGAWA,
and Reiko URADE††

Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan

Received January 15, 2002; Accepted February 4, 2002
Human recombinant ER-60 was confirmed to have transglutaminase activity by a microtiter plate assay. Transglutaminase activity of ER-60 did not require calcium and was inhibited by cystamine, a substrate analogue. In addition, the transglutaminase activity of ER-60 was not inhibited by SH-blocking reagents. These results suggest that the properties of the transglutaminase activity of ER-60 are different from those in the cases of known mammalian transglutaminases of which the active site includes a cysteine residue.
Key words: transglutaminase; protein disulfide isomerase

-39-
Note
Tissue-specific Expression of Rhamnose-binding Lectins
in the Steelhead Trout (Oncorhynchus mykiss)

Hiroaki TATENO,1 Yasushi SHIBATA,2 Yoshitaka NAGAHAMA,2 Toshiaki HIRAI,3
Mineo SANEYOSHI,3 Tomohisa OGAWA,1 Koji MURAMOTO,1,† and Hisao KAMIYA4

1Department of Biological Resource Sciences, Graduate School of Agricultural Science, Tohoku University,
Sendai 981-8555, Japan
2Laboratory of Reproductive Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan
3Department of Biological Sciences, Teikyo University of Science and Technology, Uenohara,
Yamanashi 409-0193, Japan
4School of Fisheries Sciences, Kitasato University, Sanriku, Iwate 022-0101, Japan

Received January 17, 2002; Accepted February 22, 2002
Tissue-specific expression of three L-rhamnose-binding lectins, named STL1, STL2, and STL3, in the steelhead trout (Oncorhynchus mykiss) was investigated. STL2 and STL3 mRNAs were restricted in the oocytes. In contrast, STL1 mRNA was detected only in the liver. The transcription of STL2 and STL3 started in previtellogenic oocytes. These results showed distinct expression profiles of rhamnose-binding lectins in the fish.
Key words: animal lectin; lectin; rhamnose-binding lectin; steelhead trout

-40-
Preliminary Communication
Identification of a 22-kDa Protein Required for the Degradation
of Selenomonas ruminantium Lysine Decarboxylase
by ATP-dependent Protease

Yoshihiro YAMAGUCHI, Yumiko TAKATSUKA, and Yoshiyuki KAMIO†

Laboratory of Applied Microbiology, Department of Molecular and Cell Biology, Graduate School
of Agricultural Science, Tohoku University, 1-1 Tsutsumi-dori Amamiya-machi,
Aoba-ku, Sendai 981-8555, Japan

Received February 8, 2002; Accepted February 27, 2002
In Selenomonas ruminantium, a strictly anaerobic, Gram-negative bacterium isolated from sheep rumen, a rapid degradation of lysine decarboxylase (LDC) occurred on entry into the stationary phase of cell growth. Here, we identified a 22-kDa protein as a stimulating factor for the degradation of LDC, which was catalyzed by ATP-dependent protease(s) in S. ruminantium. The purified 22-kDa protein preparation itself had no degradation activity towards LDC but it was required for the degradation of LDC by ATP-dependent proteases in a cell-free system. The 22-kDa protein had similar biochemical and biophysical characteristics to those of antizyme, the regulator for the degradation of mammalian ODC, which had been reported only in mammalian cells. From the sequencing data of the N-terminal 30 amino acid residues of the 22-kDa protein preparation, 22-kDa protein was found to be a new protein which was distinguished from antizyme. This is the first report of the presence of an antizyme-like regulator protein in a prokaryote.
Key words: Selenomonas ruminantium; regulator protein; degradation of lysine decarboxylase (LDC); ATP-dependent protease; antizyme<EL>