Contents and Abstracts of Latest Issue of BBB

(Vol.66 No.3 2002)

Cloning, Sequencing, and Expression of a Gene Encoding the Monomeric
Isocitrate Dehydrogenase of the Nitrogen-fixing Bacterium,
Azotobacter vinelandii
Takehiko SAHARA,† Yasuhiro TAKADA,†† Yoji TAKEUCHI, Naoto YAMAOKA,
and Noriyuki FUKUNAGA p.489

Inhibition of Lymphocyte Kinase Lck and Phosphatidylinositol 3-kinase
by a Novel Immunosuppressant, Lymphostin
Hiroyuki NAGATA,1 Hiroshi YANO,1 Kimihito SASAKI,2 Soichiro SATO,2
Satoshi NAKANISHI,1 Isami TAKAHASHI,1 and Tatsuya TAMAOKI1 p.501

Purification and Characterization of an Endo-1,4-β-glucanase from Neisseria
sicca SB that Hydrolyzes β-1,4 Linkages in Cellulose Acetate
Kunihiko MORIYOSHI,† Takashi OHMOTO, Tatsuhiko OHE, and Kiyofumi SAKAI p.508

Active Form of Dipteran-Specific Insecticidal Protein Cry11A
Produced by Bacillus thuringiensis subsp. israelensis
Masashi YAMAGIWA,＊ Ruriko OGAWA,＊ Kohki YASUDA,＊ Hisako NATSUYAMA,＊
Kikuo SEN,＊＊ and Hiroshi SAKAI＊ p.516

A Novel Synthesis of Branched High-molecular-weight (C40＋)
Long-chain Alkanes
Hans-Joachim LEHMLER,† Robert G. BERGOSH,a Mark S. MEIER,a
and Robert M. K. CARLSONb p.523

Studies on the Antimicrobial Mechanisms of Capsaicin Using
Yeast DNA Microarray
Sakiko KURITA,2 Emiko KITAGAWA,2 Chang-Hwa KIM,2 Yuko MOMOSE,2
and Hitoshi IWAHASHI1,2† p.532

Polyoxygenated Cyclohexenes from the Chinese Tree, Uvaria Purpurea
Yoshifusa TAKEUCHI, Qing-Wen SHI, Takeyoshi SUGIYAMA, and Takayuki ORITANI p.537

Purification and Characterization of Pyridoxal 4-Dehydrogenase
from Aureobacterium luteolum
Yanee TRONGPANICH, Kinuyo ABE, Yasuo KANEDA, Tomotake MORITA,
and Toshiharu YAGI† p.543

Cysteine Synthase of an Extremely Thermophilic Bacterium,
Thermus thermophilus HB8
Yasuko MIZUNO,＊ Yoko MIYASHITA,＊＊ Shuzo YAMAGATA,＊,＊＊,†
Tomonori IWAMA,＊,＊＊ and Tsuyoshi AKAMATSU＊＊ p.549

Cloning and Expression of Bombyx mori Silk Gland Elongation
Factor 1γ in Escherichia coli
Katsuyoshi KAMIIE,1† Yoshitaka NOMURA,1 Satoru KOBAYASHI,2 Hideharu TAIRA,3
Kohmei KOBAYASHI,4 Tetsuro YAMASHITA,3 Shin-ichiro KIDOU,2 and Shin-ichiro EJIRI2
p.558

Biosynthesis of Rice Phytoalexin: Enzymatic Conversion
of 3β-Hydroxy-9β-pimara-7,15-dien-19,6β-olide to Momilactone A
Anotai ATAWONG,1 Morifumi HASEGAWA,1,2,3,† and Osamu KODAMA1,2,3 p.566

Erinacine Q, a New Erinacine from Hericium erinaceum,
and its Biosynthetic Route to Erinacine C in the Basidiomycete
Hiromichi KENMOKU,1 Takashi SHIMAI,2 Tomonobu TOYOMASU,1,2
Nobuo KATO,3 and Takeshi SASSA1,2,† p.571

Purification and Characterization of Malate Synthase from the
Glucose-grown Wood-rotting Basidiomycete Fomitopsis palustris
Erman MUNIR, Takefumi HATTORI, and Mikio SHIMADA† p.576

Syntheses of Racemic and Diastereomeric Mixtures of 3,7,11,15-
Tetramethylhentriacontane and 4,8,12,16-Tetramethyldotriacontane,
the Cuticular Tetramethylalkanes of the Tsetse Fly, Glossina brevipalpis†
Chi<Oh><0225><Wa>e SHIBATA, Ayako FURUKAWA, and Kenji MORI†† p.582

Purification and Characterization of Two α-Keto Ester Reductases
from Streptomyces thermocyaneoviolaceus IFO 14271＊
Hitomi YAMAGUCHI,1 Nobuyoshi NAKAJIMA,2 and Kohji ISHIHARA3,† p.588

Cloning and Expression in Escherichia coli of a Gene, hup,
Encoding the Histone-like Protein HU of Bifidobacterium longum
Akio TAKEUCHI, Hajime MATSUMURA, and Yasunobu KANO p.598

Rhamnose-binding Lectins from Steelhead Trout (Oncorhynchus mykiss)
Eggs Recognize Bacterial Lipopolysaccharides and Lipoteichoic Acid
Hiroaki TATENO,＊ Tomohisa OGAWA,＊ Koji MURAMOTO,＊,† Hisao KAMIYA,＊＊
and Mineo SANEYOSHI＊＊＊ p.604

Identification and Expression of a Rat Fatty Acid Elongase Involved
in the Biosynthesis of C18 Fatty Acids
Katsuya INAGAKI, Tsunehiro AKI,† Yoshihiro FUKUDA, Seiji KAWAMOTO, Seiko SHIGETA,
Kazuhisa ONO, and Osamu SUZUKI p.613

Biotransformation of L-Lysine to L-Pipecolic Acid Catalyzed by L-Lysine
6-Aminotransferase and Pyrroline-5-carboxylate Reductase
Tadashi FUJII,† Manabu MUKAIHARA, Hitosi AGEMATU, and Hiroshi TSUNEKAWA p.622

Note
Construction of Protease-deficient Candida boidinii Strains Useful
for Recombinant Protein Production: Cloning and Disruption
of Proteinase A Gene (PEP4) and Proteinase B Gene (PRB1)
Toshihiro KOMEDA,1 Yasuyoshi SAKAI,2 Nobuo KATO,2 and Keiji KONDO1 p.628

Note
Isolation and Characterization of cbbL and cbbS Genes Encoding Form I
Ribulose-1,5-bisphosphate Carboxylase/Oxygenase Large
and Small Subunits in Nitrosomonas sp. Strain ENI-11
Ryuichi HIROTA, Junichi KATO, Hiromu MORITA, Akio KURODA, Tsukasa IKEDA,
Noboru TAKIGUCHI, and Hisao OHTAKE p.632

Note
Enzymatic Synthesis of Oligosaccharide Containing Lex Unit
by Using Partially Purified Chicken Serum
Kazuhide TOTANI, Kaoru SHIMIZU,＊ Yoichiro HARADA,＊ Takeomi MURATA,＊
and Taichi USUI＊,† p.636

Note
Radical Scavenging Activities of Niacin-Related Compounds
Shin OGATA, Masayo TAKEUCHI, Shin TERADAIRA, Naokuni YAMAMOTO, Keiko IWATA,
Katsuzumi OKUMURA, and Hiroshi TAGUCHI† p.641

Note
Spectroscopic Evidence That Salicylic Acid Converts a Temporally
Inactivated Form of Horseradish Peroxidase (Compound III)
to the Irreversibly Inactivated Verdohemoprotein (P-670)
Tomonori KAWANO,＊,＊＊,† Shoshi MUTO,＊＊＊ Masaru ADACHI,＊＊＊＊ Hiroshi HOSOYA,＊＊＊＊＊
and Fr<Oh><0225><Wa>ed<Oh><0225><Wa>eric LAPEYRIE＊ p.646

Note
Spectroscopic Evidence in Support of Horseradish Peroxidase Compound
II-catalyzed Oxidation of Salicylic Acid but Not of Phenylethylamine
Tomonori KAWANO,＊,＊＊,† Shoshi MUTO,＊＊＊ Masaru ADACHI,＊＊＊＊
Hiroshi HOSOYA,＊＊＊＊＊ and Fr<Oh><0225><Wa>ed<Oh><0225><Wa>eric LAPEYRIE＊
p.651

Note
Myrsinoic Acids B, C and F, Anti-inflammatory Compounds
from Myrsine seguinii
Mitsuru HIROTA,† Shintaro MIYAZAKI, Tomomi MINAKUCHI, Tomoko TAKAGI,
and Hisao SHIBATA p.655

Note
Enantioselective Synthesis of Both Enantiomers of Dimethyl
cis-(4-Acetoxycyclopent-2-enyl)malonate, Key Intermediates
for Epijasmonate
Shinji TANIMORI,† Yuko TSUJI, and Mitsunori KIRIHATA p.660

Note
Identification of a Putative Vacuolar Serine Protease Gene
in the Rice Blast Fungus, Magnaporthe grisea
Satoru FUKIYA, Takao KUGE, Tomomi TANISHIMA, Teruo SONE, Takashi KAMAKURA,＊
Isamu YAMAGUCHI,＊ and Fusao TOMITA† p.663

Note
Statistical Analysis of the Relationship between Translation Initiation
AUG Context and Gene Expression Level in Humans
Hitoshi MIYASAKA,＊ Shozo KANAI,＊＊ Satoshi TANAKA,＊ Hideo AKIYAMA,＊＊
and Masahiko HIRANO＊＊ p.667

Note
Preparation of the Addition Products of α-Tocopherol
with Cholesteryl Linoleate-Peroxyl Radicals
Ryo YAMAUCHI,† Toshifumi KAMATANI, Makoto SHIMOYAMADA, and Koji KATO p.670

Note
Functional Expression in Aspergillus oryzae of p15, a Protein
with Potent Neurite-inducing Activity in PC12 Cells
Satoru NAKASHIMA, Manabu ARIOKA,† and Katsuhiko KITAMOTO p.674

Note
Isolation and Identification of a New Bifidogenic Growth Stimulator
Produced by Propionibacterium freudenreichii ET-3
Kakuhei ISAWA,＊ Kenichi HOJO,＊＊ Nobuo YODA,＊＊ Tomonori KAMIYAMA,＊＊
Seiya MAKINO,＊＊ Mizue SAITO,＊ Hitomi SUGANO,＊＊ Chinami MIZOGUCHI,＊＊
Saori KURAMA,＊＊ Mika SHIBASAKI,＊＊ Noriko ENDO,＊＊ and Yoshio SATO＊ p.679

Note
Requirement for C-terminal Extension to the RNA Binding Domain
for Efficient RNA Binding by Ribosomal Protein L2
Takeshi HAYASHI, Maino TAHARA, Kenta IWASAKI, Yoshiaki KOUZUMA,
and Makoto KIMURA† p.682

Note
Phytotoxic Sesterterpene, 11-Epiterpestacin, from Bipolaris
sorokiniana NSDR-011
Yoichiro NIHASHI,＊ Chi-Hwan LIM,＊＊ Chihiro TANAKA,＊＊＊ Hisashi MIYAGAWA,＊†
and Tamio UENO＊ p.685

Note
Luteolin, a Flavone, Does Not Suppress Postprandial Glucose Absorption
Through an Inhibition of α-Glucosidase Action
Toshiro MATSUI,† Mio KOBAYASHI, Sachiko HAYASHIDA, and Kiyoshi MATSUMOTO p.689

Note
A Simple Method for Enrichment of Uninucleate Conidia
of Aspergillus oryzae
Seiichi HARA, Ryohei F. TSUJI, Osamu HATAMOTO, and Tsutomu MASUDA p.693

Note
Synthesis of (1R,2S)-1-(3′-Chloro-4′-methoxyphenyl)-1,2-propanediol
(Trametol) and (1R,2S)-1-(3′,5′-Dichloro-4′-methoxyphenyl)-1,2-propanediol,
Chlorinated Fungal Metabolites in the Natural Environment†
Takeshi KOUSAKA1 and Kenji MORI1,2,†† p.697

Preliminary Communication
Milk Basic Protein (MBP) Increases Radial Bone Mineral Density
in Healthy Adult Women
Jun-ichi YAMAMURA,1 Seiichiro AOE,1 Yasuhiro TOBA,1 Mutsumi MOTOURI,1
Hiroshi KAWAKAMI,1 Masayoshi KUMEGAWA,2 Akira ITABASHI,3 and Yukihiro TAKADA1
p.702

-1-
Cloning, Sequencing, and Expression of a Gene Encoding the Monomeric
Isocitrate Dehydrogenase of the Nitrogen-fixing Bacterium,
Azotobacter vinelandii

Takehiko SAHARA,† Yasuhiro TAKADA,†† Yoji TAKEUCHI, Naoto YAMAOKA,
and Noriyuki FUKUNAGA

Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan

Received January 12, 2001; Accepted November 20, 2001
Isocitrate dehydrogenase (IDH: EC 1.1.1.42) of Azotobacter vinelandii was purified to an electrophoretically homogeneous state, and a gene (icd) encoding this enzyme was cloned and sequenced. The N-terminal amino acid sequence of the purified enzyme was consistent with that deduced from the nucleotide sequence of the icd gene. The deduced amino acid sequence of this gene showed high identity (62--66％) to those of the other bacterial monomeric IDHs. Expression of the icd gene in Escherichia coli was examined by measuring the enzyme activity and mRNA level. Primer extension analyses revealed that two species of mRNAs with different lengths of 5′-untranslated regions (TS-1 and TS-2) were present, of which the 5′-terminals (TS-1 and TS-2 sites) were cytosines located at 244 bp and 101 bp upstream of translational initiation codon, respectively. Conserved promoter elements were present at −35 and −10 regions from the TS-1 site, whereas no such a common motif was found in the upstream region of the TS-2 site. Deletion of the promoter elements upstream of the TS-1 site resulted in complete loss of IDH activity in the E. coli transformant. When the promoter elements upstream of the TS-1 site were intact, the levels of TS-1 and TS-2 were varied greatly by altering exogenous nutrients for growth. The cells grown in a nutrient-rich medium produced large amounts of TS-1 and had a low level of IDH activity. In a nutrient-poor medium, the cells contained large amounts of TS-2 and high levels of IDH activity.
Key words: isocitrate dehydrogenase gene; nitrogen fixing bacterium; Azotobacter vinelandii

-2-
Inhibition of Lymphocyte Kinase Lck and Phosphatidylinositol 3-kinase
by a Novel Immunosuppressant, Lymphostin

Hiroyuki NAGATA,1 Hiroshi YANO,1 Kimihito SASAKI,2 Soichiro SATO,2
Satoshi NAKANISHI,1 Isami TAKAHASHI,1 and Tatsuya TAMAOKI1

1Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., 3-6-6 Asahimachi, Machida-shi,
Tokyo 194-8533, Japan
2Pharmaceutical Research Institute, Kyowa Hakko Kogyo Co., Ltd., 1188 Shimotogari, Nagaizumi-cho,
Sunto-gun, Shizuoka-ken 411-8731, Japan

Received June 1, 2001; Accepted November 7, 2001
Lck is a Src-family tyrosine kinase that is expressed predominantly in T cells, where it plays important roles in T-cell activation. Lymphostin was isolated from Streptomyces sp. as an inhibitor of Lck. As previously reported, lymphostin inhibited Lck (IC50 0.05 μM) and the mixed lymphocyte reaction (IC50 0.009 μM). We have now examined the mechanism of inhibition by lymphostin. Lymphostin inhibited protein-tyrosine kinase activity in Jurkat T cells, demonstrating the effectiveness of the compound at the cellular level. Furthermore, lymphostin suppressed delayed-type hypersensitivity in mice. However, the inhibitory activity against Lck at the cellular level was weaker than that against the mixed lymphocyte reaction. Thus, we examined the effects of lymphostin on other kinases. Interestingly, lymphostin also inhibited phosphatidylinositol 3-kinase (IC50 0.001 μM). Consequently, we conclude that lymphostin inhibits the mixed lymphocyte reaction and delayed-type hypersensitivity not only through the blockade of Lck, but through the blockade of phosphatidylinositol 3-kinase as well.
Key words: Lck; phosphatidylinositol 3-kinase; immunosuppressant; T lymphocyte; protein tyrosine kinase

-3-
Purification and Characterization of an Endo-1,4-β-glucanase from Neisseria
sicca SB that Hydrolyzes β-1,4 Linkages in Cellulose Acetate

Kunihiko MORIYOSHI,† Takashi OHMOTO, Tatsuhiko OHE, and Kiyofumi SAKAI

Department of Biochemistry, Osaka Municipal Technical Research Institute, 1-6-50 Morinomiya, Joto-ku,
Osaka 536-8553, Japan

Received July 4, 2001; Accepted October 31, 2001
An enzyme catalyzing hydrolysis of β-1,4 bonds in cellulose acetate was purified 18.3-fold to electrophoretic homogeneity from a culture supernatant of Neisseria sicca SB, which can assimilate cellulose acetate as the sole carbon and energy source. The molecular mass of the enzyme was 41 kDa and the isoelectric point was 4.8. The pH and temperature optima of the enzyme were 6.0--7.0 and 60°C. The enzyme catalyzed hydrolysis of water-soluble cellulose acetate (degree of substitution, 0.88) and carboxymethyl cellulose. The Km and Vmax for water-soluble cellulose acetate and carboxymethyl cellulose were 0.242％ and 2.24 μmol/min/mg, and 2.28％ and 12.8 μmol/min/mg, respectively. It is estimated that the enzyme is a kind of endo-1,4-β-glucanase (EC 3.2.1.4) from the substrate specificity and hydrolysis products of cellooligosaccharides. The enzyme and cellulose acetate esterase from Neisseria sicca SB degraded water-insoluble cellulose acetate by synergistic action.
Key words: cellulose acetate; biodegradation; Neisseria sicca; endo-1,4-β-glucanase

-4-
Active Form of Dipteran-Specific Insecticidal Protein Cry11A
Produced by Bacillus thuringiensis subsp. israelensis

Masashi YAMAGIWA,＊ Ruriko OGAWA,＊ Kohki YASUDA,＊ Hisako NATSUYAMA,＊
Kikuo SEN,＊＊ and Hiroshi SAKAI＊

＊Department of Bioscience and Biotechnology, Okayama University, Okayama 700-8530, Japan
＊＊Department of Bioscience and Biotechnology, Shinshu University, Nagano 399-4598, Japan

Received August 9, 2001; Accepted November 23, 2001
The nucleotide sequence of the cry11A gene from Bacillus thuringiensis subsp. israelensis strain HD522 was analyzed and the molecular characterization of Cry11A toxin was done. The 70-kDa Cry11A protoxin was processed in vitro into 36- and 32-kDa fragments by trypsin and into 34- and 32-kDa fragments by gut proteases from C. pipiens. These two processed fragments are associated together to form the heterodimer. The results of the binding assay with BBMV and the bioassay toward C. pipiens larvae suggested that the heterodimer was biologically as active as the non-digested Cry11A toxin and the intramolecular cleavage did not promote the insecticidal activity. These results suggested that a probable complex of the 36- or 34-kDa and 32-kDa fragments was also one of the possible active forms of Cry11A, and that the biological functions of Cry11A was not essentially affected by the intramolecular cleavage of the 70-kDa protein.
Key words: Bacillus thuringiensis; Cry11A; Culex pipiens; insecticidal protein; activation process

-5-
A Novel Synthesis of Branched High-molecular-weight (C40＋)
Long-chain Alkanes

Hans-Joachim LEHMLER,† Robert G. BERGOSH,a Mark S. MEIER,a
and Robert M. K. CARLSONb

Graduate Center for Toxicology, University of Kentucky, Lexington, KY 40536, U.S.A.
aDepartment of Chemistry, University of Kentucky, Lexington, KY 40506, U.S.A.
bChevron Research ＆ Technology Company, P.O. Box 1627, Richmond, CA 94802, U.S.A.

Received August 20, 2001; Accepted October 30, 2001
Many biological and geochemical questions remain concerning the structures, functions, and properties of naturally occurring high-molecular-weight (C40＋) alkanes with various mid-chain alkylation patterns. Above C40, these alkanes are exceedingly difficult to separate and purify, and syntheses can be blocked by the low solubility of intermediates. To overcome these problems, a facile three-step synthesis employing the alkylation of 1,3-dithiane with a suitable α,ω-dibromoalkane was developed. Bisalkylation of the bis(dithianyl)alkane intermediate with the appropriate 1-bromoalkane and subsequent desulfurization with Raney nickel furnished the desired long-chain alkane. Long-chain alkanes modified at mid-chain and/or symmetrically near the chain termini (or unmodified, i.e., long-chain n-paraffins) are accessible by the selection of appropriate bromoalkanes. Nine mid-chain methylated (C38H78 to C53H108), one symmetrical terminal-chain
dimethylated (C40H82), and four linear (C44H90 to C58H118)
long-chain alkanes were synthesized by using this approach. High-temperature gas chromatography (HTGC) was found to have important advantages for evaluating the purity of the synthetic high-molecular-weight alkanes.
Key words: long-chain alkane; linear alkane; branched alkane; dithiane

-6-
Studies on the Antimicrobial Mechanisms of Capsaicin Using
Yeast DNA Microarray

Sakiko KURITA,2 Emiko KITAGAWA,2 Chang-Hwa KIM,2 Yuko MOMOSE,2
and Hitoshi IWAHASHI1,2†

1Human Stress Signal Research Center, and 2Research Institute of Biological Resources, National Institute
of Advanced Industrial Science and Technology, Central, 6 1-1-1 Higashi Tsukuba, Ibaraki 305-8566, Japan

Received August 27, 2001; Accepted October 29, 2001
Capsaicin is a pungent element in a variety of red peppers that are widely used as food additives and considered to be an antimicrobial factor. For our tests, we used yeast DNA micro-array methods to understand the mechanisms of inhibitory effects of capsaicin. The capsaicin treatment significantly induced 39 genes from approximately 6,000 genes. These induced genes were classified as multi-drug resistance transporter genes, membrane biosynthesis genes, genes encoding stress proteins, and uncharacterized genes. The growth abilities of the strains with the deletion of the induced genes suggest that capsaicin is pumped out of the yeast cells by the PDR5 transporter.
Key words: capsaicin; yeast; DNA micro-array; toxicity

-7-
Polyoxygenated Cyclohexenes from the Chinese Tree, Uvaria Purpurea

Yoshifusa TAKEUCHI, Qing-Wen SHI, Takeyoshi SUGIYAMA, and Takayuki ORITANI

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

Received August 27, 2001; Accepted November 5, 2001
Four new polyoxygenated cyclohexenes, 2-O-acetyl-6-O-methylzeylenol (2), 2-O-benzoyl-3-O-debenzoylzeylenone (5), 3-O-debenzoylzeylenone (6) and 3-O-debenzoylgrandiflorone (8), were isolated from the leaves of Uvaria purpurea. 2-O-acetyl-6-O-benzoylzeylenol (3) was also isolated for the first time as a natural product. Compounds 5--7 each showed inhibitory activity against the root growth of Lactuca sativa. Their structures were established by spectroscopic and chemical methods.
Key words: Uvaria purpurea; Annonaceae; leaf; polyoxygenated cyclohexene; plant growth activity

-8-
Purification and Characterization of Pyridoxal 4-Dehydrogenase
from Aureobacterium luteolum

Yanee TRONGPANICH, Kinuyo ABE, Yasuo KANEDA, Tomotake MORITA,
and Toshiharu YAGI†

Department of Bioresources Science, Faculty of Agriculture, Kochi University, Nankoku,
Kochi 783-8502, Japan

Received August 27, 2001; Accepted October 30, 2001
A pyridoxal dehydrogenase was purified to homogeneity from Aureobacterium luteolum, which can use pyridoxine as a carbon and nitrogen source, and characterized. The enzyme was a dimeric protein with a subunit molecular weight of 38,000. It had several properties distinct from those of the partially purified enzyme from Pseudomonas MA-1. The optimum pH (8.0--8.5) was 0.8--1.3 lower than that of the Pseudomonas enzyme. The Aureobacterium enzyme showed much higher and lower affinities for NAD＋ (Km, 0.140±0.008 mM) and pyridoxal (0.473±0.109 mM), respectively, than those of the Pseudomonas enzyme. The Aureobacterium enzyme could use NADP＋ as a substrate: the reactivity was 6.5％ of NAD＋. The enzyme was much more tolerant to metal-chelating agents. Irreversibility of the enzymatic reaction was shared by the two enzymes. No aldehyde dehydrogenase showed similarity to the amino-terminal amino acid sequence of the enzyme.
Key words: pyridoxal dehydrogenase; pyridoxine-degradation pathway; Aureobacterium luteolum; vitamin B6

-9-
Cysteine Synthase of an Extremely Thermophilic Bacterium,
Thermus thermophilus HB8

Yasuko MIZUNO,＊ Yoko MIYASHITA,＊＊ Shuzo YAMAGATA,＊,＊＊,†
Tomonori IWAMA,＊,＊＊ and Tsuyoshi AKAMATSU＊＊

＊United Graduate School of Agricultural Sciences, and ＊＊Department of Biotechnology,
Faculty of Agriculture, Gifu University, Gifu 501-1193, Japan

Received September 3, 2001; Accepted November 8, 2001
O-Acetyl-L-serine sulfhydrylase (EC 4.2.99.8) was first purified from an extremely thermophilic bacterium, Thermus thermophilus HB8, in order to ascertain that it is responsible for the cysteine synthesis in this organism cultured with either sulfate or methionine given as a sole sulfur source. Polyacrylamide gel electrophoreses both with and without SDS found high purity of the enzyme preparations finally obtained, through ammonium sulfate fractionation, ion exchange chromatography, gel filtration, and hydrophobic chromatography (or affinity chromatography). The enzyme activity formed only one elution curve in each of the four different chromatographies, strongly suggesting the presence of only one enzyme species in this organism. Molecular masses of 34,000 and 68,000 were estimated for dissociated subunit and the native enzyme, respectively, suggesting a homodimeric structure. The enzyme was stable at 70°C at pH 7.8 for 60 min, and more than 90％ of the activity was retained after incubation of its solution at 80°C with 10 mM dithiothreitol. The enzyme was also quite stable at pH 8--12 (50°C, 30 min). It had an apparent Km of 4.8 mM for O-acetyl-L-serine (with 1 mM sulfide) and a Vmax of 435 μmol/min/mg of protein. The apparent Km for sulfide was approximately 50 μM (with 20 mM acetylserine), suggesting that the enzyme can react with sulfide liberated very slowly from methionine. The absorption spectrum of the holo-enzyme and inhibition of the activity by carbonyl reagents suggested the presence of pyridoxal 5′-phosphate as a cofactor. The apo-enzyme showed an apparent Km of 29 μM for the cofactor at pH 8. Monoiodoacetic acid (1 mM) almost completely inactivated the enzyme. The meaning of a very high enzyme content in the cell is discussed.
Key words: cysteine synthesis; O-acetylserine sulfhydrylase; pyridoxal 5′-phosphate; extremophile; Thermus thermophilus

-10-
Cloning and Expression of Bombyx mori Silk Gland Elongation
Factor 1γ in Escherichia coli

Katsuyoshi KAMIIE,1† Yoshitaka NOMURA,1 Satoru KOBAYASHI,2 Hideharu TAIRA,3
Kohmei KOBAYASHI,4 Tetsuro YAMASHITA,3 Shin-ichiro KIDOU,2 and Shin-ichiro EJIRI2

1Department of Bioscience and Biotechnology, Faculty of Engineering, Aomori University,
Aomori 030-0943, Japan
2Cryobiosystem Research Center, Faculty of Agriculture, and
3Department of Bioscience and Technology Iwate University, Morioka 020-8550, Japan
4Japan Red Cross Hiroshima College of Nursing, Hatsukaichi 738-0052, Japan

Received September 3, 2001; Accepted October 29, 2001
Elongation factor 1 (EF-1) from the silk gland of Bombyx mori consists of α-, β-, γ-, and δ-subunits. EF-1α・GTP catalyzes the binding of aminoacyl-tRNA to ribosomes concomitant with the hydrolysis of GTP. EF-1βγδ catalyzes the exchange of EF-1α-bound GDP for exogenous GTP and stimulates the EF-1α-dependent binding of aminoacyl-tRNA to ribosomes.
EF-1γ cDNA, which contains an open reading frame (ORF) encoding a polypeptide of 423 amino acid residues, was amplified and cloned by PCR from a silk gland cDNA library. The calculated molecular mass and predicted pI of the product were 48,388 Da and 5.84, respectively. The silk gland EF-1γ shares 67.3％ amino acid identity with Artemia salina EF-1γ. The N-terminal domain (amino acid residues 1--211) of silk gland EF-1γ is 29.3％ identical to maize glutathione S-transferase. We demonstrated that silk gland EF-1γ bound to glutathione Sepharose, suggesting that the N-terminal domain of EF-1γ may have the capacity to bind to glutathione.
Key words: elongation factor 1γ; glutathione S-transferase (GST); glutathione; Bombyx mori

-11-
Biosynthesis of Rice Phytoalexin: Enzymatic Conversion
of 3β-Hydroxy-9β-pimara-7,15-dien-19,6β-olide to Momilactone A

Anotai ATAWONG,1 Morifumi HASEGAWA,1,2,3,† and Osamu KODAMA1,2,3

1United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology,
3-5-8 Saiwai-cho, Fuchu-shi, Tokyo 183-8509, Japan
2Laboratory of Phytochemical Ecology, College of Agriculture, Ibaraki University, 3-21-1 Chuo, Ami,
Ibaraki 300-0393, Japan
3Core Research for Evolutional Science and Technology of Japan Science and Technology Corporation, Japan

Received September 5, 2001; Accepted November 7, 2001
Momilactone A, a major rice diterpene phytoalexin, could be synthesized by dehydrogenation at the 3-position of 3β-hydroxy-9β-pimara-7,15-dien-19,6β-olide in rice leaves. The presence of 3β-hydroxy-9β-pimara-7,15-dien-19,6β-olide in UV-irradiated rice leaves was confirmed by comparing the mass spectra and retention times after a GC/MS analysis of the natural and synthetic compounds. The soluble protein fraction from UV-irradiated rice leaves showed dehydrogenase activity to convert 3β-hydroxy-9β-pimara-7,15-dien-19,6β-olide into momilactone A. The enzyme required NAD＋ or NADP＋ as a hydrogen acceptor. The optimum pH for the reaction was 8. The Km value to 3β-hydroxy-9β-pimara-7,15-dien-19,6β-olide was 36 μM when NAD＋ was supplied as a cofactor at a concentration of 1 mM. 3β-Hydroxy-9β-pimara-7,15-dien-19,6β-olide and its dehydrogenase activity were induced in a time-dependent manner by UV irradiation.
Key words: momilactone A; dehydrogenase; phytoalexin; diterpene; 3β-hydroxy-9β-pimara-7,15-dien-19,6β-olide

-12-
Erinacine Q, a New Erinacine from Hericium erinaceum,
and its Biosynthetic Route to Erinacine C in the Basidiomycete

Hiromichi KENMOKU,1 Takashi SHIMAI,2 Tomonobu TOYOMASU,1,2
Nobuo KATO,3 and Takeshi SASSA1,2,†
1The United Graduate School of Agricultural Sciences, Iwate University (Yamagata University),
Tsuruoka, Yamagata 997-8555, Japan

2Department of Bioresource Engineering, Faculty of Agriculture, Yamagata University, Wakaba-cho,
Tsuruoka, Yamagata 997-8555, Japan
3Institute of Advanced Material Study, Kyushu University, Kasuga, Fukuoka 816-8580, Japan

Received September 17, 2001; Accepted November 6, 2001
Erinacines as cyathane-xylosides are known to have potent stimulating activity for nerve-growth-factor synthesis. Our search for new cyathane metabolites from a liquid culture of Hericium erinaceum YB4-6237 resulted in the isolation of a new erinacine named erinacine Q (1). NMR spectrometry and a chemical derivation from erinacine P (2) determined the compound to be a derivative in which the formyl group of erinacine P had been reduced to the hydroxymethyl group. To clarify the biosynthetic relationship between erinacine Q and the others, [1′-13C]erinacine Q ([1′-13C]-1) was chemically derived from [1′-13C]erinacine P ([1′-13C]-2) which had been prepared by feeding [1-13C]-D-glucose to the basidiomycete. The biotransformation of labeled erinacine Q into [1′-13C]erinacine C ([1′-13C]-5) via [1′-13C]erinacine P in this basidiomycete was demonstrated by NMR spectrometry.
Key words: erinacine Q; erinacines P and C; Hericium erinaceum; biosynthesis of erinacines; [1-13C]-D-glucose

-13-
Purification and Characterization of Malate Synthase from the
Glucose-grown Wood-rotting Basidiomycete Fomitopsis palustris

Erman MUNIR, Takefumi HATTORI, and Mikio SHIMADA†

Wood Research Institute, Kyoto University, Uji, Kyoto 611-0011, Japan
Received September 25, 2001; Accepted November 13, 2001
Malate synthase (EC 4.1.3.2), the key enzyme of the glyoxylate cycle, was purified to a homogeneous protein from the wood-rotting basidiomycete Fomitopsis palustris grown on glucose. The purified enzyme, with a molecular mass of 520 kDa, was found to consist of eight 65-kDa subunits, and to have Km of 45 and 2.2 μM for glyoxylate and acetyl-CoA,

respectively. The enzyme activity was competitively inhibited by oxalate (Ki, 8.5 μM) and glycolate (Ki, 17 μM), and uncompetitively by coenzyme A (Ki, 100 μM). The potent inhibition of the activity by p-chloromercuribenzoate suggests that the enzyme has a sulfhydryl group at the active center. However, the enzyme was inhibited moderately by adenine nucleotides and weakly by some of the metabolic intermediates of glycolysis and tricarboxylic acid cycle. The enzyme was completely inactive in the absence of metal ions and was maximally activated by Mg2＋ (Km, 0.4 μM), which also served to significantly prevent enzyme inactivation during storage.
Key words: malate synthase; glyoxylate cycle; wood-rotting basidiomycetes; oxalate biosynthesis; Fomitopsis palustris

-14-
Syntheses of Racemic and Diastereomeric Mixtures of 3,7,11,15-
Tetramethylhentriacontane and 4,8,12,16-Tetramethyldotriacontane,
the Cuticular Tetramethylalkanes of the Tsetse Fly, Glossina brevipalpis†

Chi<Oh><0225><Wa>e SHIBATA, Ayako FURUKAWA, and Kenji MORI††

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

Received October 1, 2001; Accepted October 30, 2001
Cuticular hydrocarbons of the tsetse fly, Glossina brevipalpis, contain 3,7,11,15-tetramethylhentriacontane and 4,8,12,16-tetramethyldotriacontane as possible candidates for its contact sex pheromone. These were synthesized as racemic and diastereomeric mixtures starting from racemic citronellol and employing phenylsulfone-mediated chain-elongation as the key reaction.
Key words: cuticular hydrocarbon; Glossina brevipalpis; methyl-branched alkane; optically active alkane; pheromone

-15-
Purification and Characterization of Two α-Keto Ester Reductases
from Streptomyces thermocyaneoviolaceus IFO 14271＊

Hitomi YAMAGUCHI,1 Nobuyoshi NAKAJIMA,2 and Kohji ISHIHARA3,†

1Institute for Chemical Research, Kyoto University, Uji 611-0011, Japan
2Department of Nutritional Science, Graduate School of Health and Welfare,
Okayama Prefectural University, Soja, Okayama 719-1197, Japan
3Department of Chemistry, Kyoto University of Education, Fushimi-ku, Kyoto 612-8522, Japan
Received October 1, 2001; Accepted November 9, 2001
Two NADPH-dependent α-keto ester reductases (Streptomyces thermocyaneoviolaceus keto ester reductase, STKER-II and -III) were purified from S. thermocyaneoviolaceus IFO 14271, one of thermophilic actinomycetes. The molecular masses of native STKER-II and -III were estimated to be 60 kDa and 70 kDa by gel filtration chromatography,

respectively. These enzymes were both homodimers, with 29-kDa and 30-kDa subunit molecular masses based on SDS polyacrylamide gel electrophoresis. STKER-II and -III were stable from pH 7.0 to 10.0 and pH 5.5 to 9.0, respectively. Ethyl 3-methyl-2-oxobutanoate was reduced by both enzymes isolated to the corresponding (R)-hydroxy ester with excellent enantiomeric excess. STKER-III showed high stereoselectivity for the reduction of bulky substrates, while the selectivity of the STKER-II-catalyzed reduction was low except for ethyl 3-methyl-2-hydroxybutanoate. Both enzymes had small Km values toward aliphatic keto esters having a long alkyl chain.
Key words: thermophilic actinomycete; reduction; reductase; enzyme purification; α-hydroxy ester producing enzyme

-16-
Cloning and Expression in Escherichia coli of a Gene, hup,
Encoding the Histone-like Protein HU of Bifidobacterium longum

Akio TAKEUCHI, Hajime MATSUMURA, and Yasunobu KANO

Department of Molecular Genetics, Institute of Molecular and Cellular Biology for Pharmaceutical Sciences,
Kyoto Pharmaceutical University, 1 Shichono-cho, Misasagi, Yamashina-ku, Kyoto 607-8412, Japan

Received October 9, 2001; Accepted November 8, 2001
A genomic DNA library of Bifidobacterium longum ATCC15707 was transfected into an Escherichia coli strain deficient in both HU and IHF, the growth of which is cold-sensitive because of the deficiency in these proteins. Cold-resistant colonies were selected and the DNA was cloned and sequenced. A polypeptide consisted of 93 amino acids, a predicted molecular mass of 9983 Da with an isoelectric point of 10.35, was deduced from an orf in the middle of the DNA fragment. The amino acid sequence was highly similar to HU family proteins, and 26 aas of N terminal was identical to a histone-like protein, HBl, a HU family protein of B. longum. Incapabilities of Mu phage propagation in an E. coli mutant deficient in HU or IHF could be suppressed by DNA bearing this orf. These results showed that the orf is a gene hup encoding HBl, a histone-like protein HU of B. longum.
Key words: hup gene encoding histone-like protein HBl; Bifidobacterium longum; Escherichia coli; DNA-binding proteins HU and IHF; propagation of Mu phage

-17-
Rhamnose-binding Lectins from Steelhead Trout (Oncorhynchus mykiss)
Eggs Recognize Bacterial Lipopolysaccharides and Lipoteichoic Acid

Hiroaki TATENO,＊ Tomohisa OGAWA,＊ Koji MURAMOTO,＊,† Hisao KAMIYA,＊＊
and Mineo SANEYOSHI＊＊＊

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

Received October 12, 2001; Accepted November 12, 2001
The interaction between bacteria and three L-rhamnose-binding lectins, named STL1, STL2, and STL3, from steelhead trout (Oncorhynchus mykiss) eggs was investigated. Although STLs bound to most Gram-negative and Gram-positive bacteria, they agglutinated only Escherichia coli K-12 and Bacillus subtilis among the bacteria tested. The binding was inhibited by L-rhamnose. STLs bound to distinct serotypes of lipopolysaccharides (LPSs), and showed much higher binding activities to smooth-type LPSs of Escherichia coli K-12 and Shigella flexneri 1A than to their corresponding rough-type LPSs. STLs also bound to lipoteichoic acid (LTA) of Bacillus subtilis. These results indicate that STLs bound to bacteria by recognizing LPSs or LTA on the cell surfaces.
Key words: animal lectin; rhamnose-binding lectin; steelhead trout; lipopolysaccharide; lipoteichoic acid

-18-
Identification and Expression of a Rat Fatty Acid Elongase Involved
in the Biosynthesis of C18 Fatty Acids

Katsuya INAGAKI, Tsunehiro AKI,† Yoshihiro FUKUDA, Seiji KAWAMOTO, Seiko SHIGETA,
Kazuhisa ONO, and Osamu SUZUKI

Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter,
Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8530, Japan

Received October 18, 2001; Accepted November 13, 2001
A major part of the palmitic acid (C16:0) generated by fatty acid synthase is converted into stearic acid (C18:0) via carbon chain elongation. Here, we describe the cloning and expression of a rat hepatic enzyme, rELO2, responsible for the elongation of C16:0, presumably at the condensing reaction. Heterologous expression experiments in a yeast, Saccharomyces cerevisiae, demonstrated the elongation activity of rELO2 on C16:0 and to a lesser extent, C18:0 and fatty acids with low desaturation degree. This was distinct from that rELO1, a rat homolog of HELO1, which preferably catalyzed the elongation of mono- and polyunsaturated fatty acids of C16--C20. The Northern analysis showed that the expression of rELO2, but not rELO1, in hepatocytes was activated by the cycles of fasting and refeeding rats on a fat-free diet. Under these conditions, the rELO1 was expressed constitutively in various tissues but the rELO2 transcripts were detected predominantly in liver.
Key words: fatty acid chain elongation; liver; palmitic acid; stearic acid

-19-
Biotransformation of L-Lysine to L-Pipecolic Acid Catalyzed by L-Lysine
6-Aminotransferase and Pyrroline-5-carboxylate Reductase

Tadashi FUJII,† Manabu MUKAIHARA, Hitosi AGEMATU, and Hiroshi TSUNEKAWA

Bioresource Laboratories, Mercian Corp., 4-9-1, Johnan, Fujisawa 251-0057, Japan

Received November 7, 2001; Accepted November 9, 2001
The enzyme involved in the reduction of Δ1-piperideine-6-carboxylate (P6C) to L-pipecolic acid (L-PA) has never been identified. We found that Escherichia coli JM109 transformed with the lat gene encoding L-lysine 6-aminotransferase (LAT) converted L-lysine (L-Lys) to L-PA. This suggested that there is a gene encoding <Oh><0192><0192><Wa>P6C reductase′′ that catalyzes the reduction of P6C to L-PA in the genome of E. coli. The complementation experiment of proC32 in E. coli RK4904 for L-PA production clearly shows that the expression of both lat and proC is essential for the biotransformation of L-Lys to L-PA. Further, We showed that both LAT and pyrroline-5-carboxylate (P5C) reductase, the product of proC, were needed to convert L-Lys to L-PA in vitro. These results demonstrate that P5C reductase catalyzes the reduction of P6C to L-PA. Biotransformation of L-Lys to L-PA using lat-expressing E. coli BL21 was done and L-PA was accumulated in the medium to reach at an amount of 3.9 g/l after 159 h of cultivation. It is noteworthy that the ee-value of the produced pipecolic acid was 100％.
Key words: lat; L-pipecolic acid (L-PA); proC; Δ1-pyrroline-5-carboxylate (P5C) reductase; Δ1-piperideine-6-carboxylate (P6C)

-20-
Note
Construction of Protease-deficient Candida boidinii Strains Useful
for Recombinant Protein Production: Cloning and Disruption
of Proteinase A Gene (PEP4) and Proteinase B Gene (PRB1)

Toshihiro KOMEDA,1 Yasuyoshi SAKAI,2 Nobuo KATO,2 and Keiji KONDO1
1Central Laboratories for Key Technology, Kirin Brewery Co., Ltd., 1-13-5, Fukuura, Kanazawa-ku,

Yokohama-shi, Kanagawa 236-0004, Japan
2Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan

Received July 6, 2001; Accepted November 7, 2001
The yeast Candida boidinii PEP4 and PRB1 genes, encoding proteinase A (PrA) and proteinase B (PrB), respectively, have been cloned and their primary structures were analyzed. The open reading frames of the PEP4 gene (1263 bp encoding a protein of 420 amino acids) and the PRB1 gene (1683 bp encoding a protein of 560 amino acids) were found. The deduced amino acid sequences of PrA and PrB are very similar to Saccharomyces cerevisiae PrA and PrB (64％ and 61％ identities, respectively). Both PEP4 and PRB1 genes were disrupted in the C. boidinii genome by one-step gene disruption. The resultant pep4Δ and the pep4Δ prb1Δ strains lost protease activity when compared with the wild-type original strain. The constructed C. boidinii strains are expected to be useful hosts for heterologous protein production.
Key words: Candida boidinii; proteinase A; PEP4; proteinase B; PRB1

-21-
Note
Isolation and Characterization of cbbL and cbbS Genes Encoding Form I
Ribulose-1,5-bisphosphate Carboxylase/Oxygenase Large
and Small Subunits in Nitrosomonas sp. Strain ENI-11

Ryuichi HIROTA, Junichi KATO, Hiromu MORITA, 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 739-8527, Japan
Received July 30, 2001; Accepted November 7, 2001

The cbbL and cbbS genes encoding form I ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) large and small subunits in the ammonia-oxidizing bacterium Nitrosomonas sp. strain ENI-11 were cloned and sequenced. The deduced gene products, CbbL and CbbS, had 93 and 87％ identity with Thiobacillus intermedius CbbL and Nitrobacter winogradskyi CbbS, respectively. Expression of cbbL and cbbS in Escherichia coli led to the detection of RubisCO activity in the presence of 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG). To our knowledge, this is the first paper to report the genes involved in the carbon fixation reaction in chemolithotrophic ammonia-oxidizing bacteria.
Key words: Nitrosomonas; nitrification; CO<Oh>_<Wa>{2} fixation; ribulose-1,5-bisphosphate carboxylase/oxygenase

-22-
Note
Enzymatic Synthesis of Oligosaccharide Containing Lex Unit
by Using Partially Purified Chicken Serum

Kazuhide TOTANI, Kaoru SHIMIZU,＊ Yoichiro HARADA,＊ Takeomi MURATA,＊
and Taichi USUI＊,†

Science of Biological Resources, The United Graduate School of Agricultural Science, Gifu University,
Yanagido 1-1, Gifu 501-1193, Japan
＊Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, Ohya 836,
Shizuoka 422-8529, Japan

Received July 30, 2001; Accepted November 8, 2001
Animal sera were screened for an alternative enzyme source of α1,3-fucosyltransferase, and the highest activity was observed in chicken serum. A partially purified enzyme fraction almost devoid of coexisting glycosidases was prepared from the chicken serum, and used for the fucosylation of LacNAc compounds. The enzyme reaction was efficient enough to allow the one-pot preparation of designed Lex compounds such as LNFP III.
Key words: Lex; LNFP III; α1,3-fucosyltransferase; chicken serum

-23-
Note
Radical Scavenging Activities of Niacin-Related Compounds

Shin OGATA, Masayo TAKEUCHI, Shin TERADAIRA, Naokuni YAMAMOTO, Keiko IWATA,
Katsuzumi OKUMURA, and Hiroshi TAGUCHI†

Laboratory of Molecular and Cellular Biology, Department of Life Science, Faculty of Bioresources,
Mie University, Tsu, Mie 514-8507, Japan

Received August 8, 2001; Accepted November 2, 2001
We investigated whether niacin-related compounds had radical-scavenging activity by electron spin resonance methods. Many compounds, but not trigonelline, had radical-scavenging activity against hydroxyl radicals. However, for the nitric oxide radical and 1,1-diphenyl-2-picrylhydrazyl radical, only nicotinic acid hydrazide and isonicotinic acid hydrazide had scavenging activities. These results suggest that the moiety of hydrazide might have an important role in scavenging abilities of various radicals.
Key words: niacin; NAD; isonicotinic acid hydrazide; ESR; ROS

-24-
Note
Spectroscopic Evidence That Salicylic Acid Converts a TemporallyInactivated Form of Horseradish Peroxidase (Compound III)
to the Irreversibly Inactivated Verdohemoprotein (P-670)

Tomonori KAWANO,＊,＊＊,† Shoshi MUTO,＊＊＊ Masaru ADACHI,＊＊＊＊ Hiroshi HOSOYA,＊＊＊＊＊
and Fr<Oh><0225><Wa>ed<Oh><0225><Wa>eric LAPEYRIE＊

＊UMR INRA-UHP Interactions Arbre/Micro-Organismes, Centre Institut National de la Recherche
Agronomique de Nancy, F-54280 Champenoux, France
＊＊Present address: Department of Biological Science, Graduate School of Science, Hiroshima University,
Higashi-Hiroshima 739-8526, Japan
＊＊＊Nagoya University Bioscience Center, Chikusa-ku, Nagoya 464-8601, Japan
＊＊＊＊Department of Biochemistry, Miyazaki Medical College, Kiyotake, Miyazaki 889-2192, Japan
＊＊＊＊＊Department of Biological Science, Graduate School of Science and PRESTO-JST, Hiroshima University,
Higashi-Hiroshima 739-8526, Japan

Received August 13, 2001; Accepted November 16, 2001
We obtained spectroscopic evidence in support of salicylate-dependent inactivation of horseradish peroxidase-C. Addition of salicylate to the enzyme arrested at a temporal inactive state (Compound III) in the presence of H2O2, resulted in rapid and irreversible inactivation of the enzyme yielding verdohemoproteins (P-670). Multiple roles for salicylate in peroxidase-
catalyzed reactions are discussed.
Key words: Compound III; horseradish peroxidase; P-670; salicylic acid; verdohemoprotein

-25-
Note
Spectroscopic Evidence in Support of Horseradish Peroxidase Compound
II-catalyzed Oxidation of Salicylic Acid but Not of Phenylethylamine

Tomonori KAWANO,＊,＊＊,† Shoshi MUTO,＊＊＊ Masaru ADACHI,＊＊＊＊
Hiroshi HOSOYA,＊＊＊＊＊ and Fr<Oh><0225><Wa>ed<Oh><0225><Wa>eric LAPEYRIE＊

＊UMR INRA-UHP Interactions Arbre/Micro-Organismes, Centre Institut National de la Recherche
Agronomique de Nancy, F-54280 Champenoux, France
＊＊Present address: Department of Biological Science, Graduate School of Science, Hiroshima University,
Higashi-Hiroshima 739-8526, Japan
＊＊＊Nagoya University Bioscience Center, Chikusa-ku, Nagoya 464-8601, Japan
＊＊＊＊Department of Biochemistry, Miyazaki Medical College, Kiyotake, Miyazaki 889-2192, Japan
＊＊＊＊＊Department of Biological Science, Graduate School of Science and PRESTO-JST, Hiroshima University,
Higashi-Hiroshima 739-8526, Japan

Received August 15, 2001; Accepted October 29, 2001
Salicylic acid and phenylethylamine are putative substrates for naturally occurring reactions for generation of reactive oxygen species, which are catalyzed by plant peroxidases. Here, we used commercially available highly purified horseradish peroxidase-C (HRP-C) as a model enzyme for spectroscopic analysis, and obtained data suggesting that the Compound II form of HRP-C does not utilize phenylethylamine as substrate. In contrast, addition of salicylic acid to Compound II resulted in rapid conversion of Compound II to the native form.
Key words: Compound II; horseradish peroxidase; reactive oxygen species; phenylethylamine; salicylic acid

-26-
Note
Myrsinoic Acids B, C and F, Anti-inflammatory Compounds
from Myrsine seguinii

Mitsuru HIROTA,† Shintaro MIYAZAKI, Tomomi MINAKUCHI, Tomoko TAKAGI,
and Hisao SHIBATA

Department of Bioscience and Biotechnology, Faculty of Agriculture, Shinshu University,
8304 Minami-minowa, Kami-ina, Nagano 399-4598, Japan

Received August 20, 2001; Accepted October 29, 2001
The methanolic extract of Myrsine seguinii yielded three anti-inflammatory compounds, myrsinoic acids B, C and F, and their structures were elucidated from the spectroscopic data. These compounds suppressed the TPA-induced edema of mouse ear, myrsinoic acid F being the most active (IE 77％ at a dose of 0.56 μmol).
Key words: Myrsine seguinii; anti-inflammatory; TPA-induced edema

-27-
Note
Enantioselective Synthesis of Both Enantiomers of Dimethyl
cis-(4-Acetoxycyclopent-2-enyl)malonate, Key Intermediates
for Epijasmonate

Shinji TANIMORI,† Yuko TSUJI, and Mitsunori KIRIHATA

Department of Applied Biological Chemistry, Graduate School of Agriculture and Life Sciences,
Osaka Prefecture University, 1-1 Gakuen-cho, Sakai, Osaka 599-8531, Japan

Received August 27, 2001; Accepted October 17, 2001
Both enantiomers (3 and 4) of dimethyl cis-(4-acetoxycyclopent-2-enyl)malonate (3), known intermediates for the synthesis of epijasmonate, were prepared in an enantio-enriched form from common starting material 2 based on desymmetrization of the meso-character by palladium-catalyzed asymmetric allylic alkylation.
Key words: epijasmonoid; allylic alkylation; palladium catalyst; enantioselective synthesis

-28-
Note
Identification of a Putative Vacuolar Serine Protease Gene
in the Rice Blast Fungus, Magnaporthe grisea

Satoru FUKIYA, Takao KUGE, Tomomi TANISHIMA, Teruo SONE, Takashi KAMAKURA,＊
Isamu YAMAGUCHI,＊ and Fusao TOMITA†

Laboratory of Applied Microbiology, Graduate School of Agriculture, Hokkaido University,
Sapporo 060-8589, Japan
＊Microbial Toxicology Laboratory, The Institute of Physical and Chemical Research (RIKEN),
Wako, Saitama 351-0198, Japan

Received August 29, 2001; Accepted November 5, 2001
We identified and cloned a gene designated SPM1, encoding a serine protease from the rice blast fungus Magnaporthe grisea. SPM1 is a single-copy gene, encoding a subtilisin-like serine protease with 536 amino acids. Analyses of the deduced amino acid sequence of SPM1 suggested that SPM1 would be localized in a vacuole, an important organelle in pathogenicity.
Key words: Magnaporthe grisea; rice blast disease; gene cloning; vacuolar subtilisin-like serine protease; vacuole

-29-
Note
Statistical Analysis of the Relationship between Translation Initiation
AUG Context and Gene Expression Level in Humans

Hitoshi MIYASAKA,＊ Shozo KANAI,＊＊ Satoshi TANAKA,＊ Hideo AKIYAMA,＊＊
and Masahiko HIRANO＊＊

＊The Kansai Electric Power Co., Technical Research Center, 11-20 Nakoji 3-Chome, Amagasaki,
Hyogo 661-0974,Japan
＊＊Biological Sciences Department, Toray Research Center, Inc., Tebiro 1111, Kamakura 248-8555, Japan

Received September 5, 2001; Accepted November 5, 2001
The preference for the optimal nucleotide of the mammalian translation initiation AUG context [GCCGCC(A/G)CCaugG] is generally more pronounced in the highly expressed genes than in the transcription factor genes at the −9 through −1 positions in humans. The influence of amino acid preference on the nucleotide choice at the ＋4 position was also examined.
Key words: AUG context; translation initiation; consensus sequence; human; natural selection

-30-
Note
Preparation of the Addition Products of α-Tocopherol
with Cholesteryl Linoleate-Peroxyl Radicals

Ryo YAMAUCHI,† Toshifumi KAMATANI, Makoto SHIMOYAMADA, and Koji KATO

Department of Bioprocessing, Faculty of Agriculture, Gifu University, Gifu 501-1193, Japan

Received September 12, 2001; Accepted November 2, 2001
α-Tocopherol was reacted with cholesteryl linoleate hydroperoxides (Ch18:2-OOH) in the presence of an iron-chelate, Fe(III) acetylacetonate, at 37°C in benzene. The reaction products were isolated and identified as four positional isomers of cholesteryl (8a-dioxy-α-tocopherone)-epoxyoctadecenoates and two positional isomers of cholesteryl (8a-dioxy-α-tocopherone)-
octadecadienoates. The result indicates that the peroxyl radicals from Ch18:2-OOH react with the 8a-carbon radical of α-tocopherol to form the addition products.
Key words: α-tocopherol; antioxidant; cholesteryl ester hydroperoxide; lipid peroxidation

-31-
Note
Functional Expression in Aspergillus oryzae of p15, a Protein
with Potent Neurite-inducing Activity in PC12 Cells

Satoru NAKASHIMA, Manabu ARIOKA,† and Katsuhiko KITAMOTO

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

Received September 17, 2001; Accepted November 5, 2001
We previously reported that a fungal protein, p15, induces neurite outgrowth and differentiation of rat pheochromocytoma PC12 cells through the activation of the Ca2＋ signaling pathway. We report here the secretory production of p15 in Aspergillus oryzae. Analysis of culture supernatant of A. oryzae transformed with the gene encoding the p15 precursor tagged with a hemagglutinin (HA) epitope demonstrated that the transformant secreted a protein with an apparent molecular mass of 17.5 kDa, which is a little larger than the expected size of mature p15-HA. By heat denaturation and ion exchange chromatography, p15-HA was easily purified from the culture supernatant with sufficient abundance. Although purified p15-HA was less active than the native p15 obtained from the culture broth of a producing fungal strain, it had neurite-inducing activity in PC12 cells in a dose-dependent manner, providing a system to study the action mechanism of p15.
Key words: Aspergillus oryzae; neurite outgrowth; PC12; neuronal differentiation; fungal 15-kDa protein

-32-
Note
Isolation and Identification of a New Bifidogenic Growth Stimulator
Produced by Propionibacterium freudenreichii ET-3

Kakuhei ISAWA,＊ Kenichi HOJO,＊＊ Nobuo YODA,＊＊ Tomonori KAMIYAMA,＊＊
Seiya MAKINO,＊＊ Mizue SAITO,＊ Hitomi SUGANO,＊＊ Chinami MIZOGUCHI,＊＊
Saori KURAMA,＊＊ Mika SHIBASAKI,＊＊ Noriko ENDO,＊＊ and Yoshio SATO＊

＊Research ＆ Development Center, Meiji Dairies Corporation
＊＊Food Functionality Research Institute, Meiji Dairies Corporation, 540 Naruda Odawara,
Kanagawa 250-0862, Japan

Received September 18, 2001; Accepted November 14, 2001
We have found a new growth stimulator for bifidobacteria in the culture broth of Propionibacterium freudenreichii ET-3. The bifidogenic growth stimulator (BGS) was purified by Diaion HP-20 column chromatography and preparative HPLC. Spectroscopic methods including 1H-NMR, UV, and LC-ESI-MS experiments indicated that the chemical structure of the bifidogenic growth stimulator was 1,4-dihydroxy-2-naphthoic acid (DHNA). Approximately 10 mg/L of DHNA was found to be produced in the culture broth of P. freudenreichii ET-3.
Key words: 1,4-dihydroxy-2-naphthoic acid; bifidogenic growth stimulator; Propionibacterium freudenreichii

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Note
Requirement for C-terminal Extension to the RNA Binding Domain
for Efficient RNA Binding by Ribosomal Protein L2

Takeshi HAYASHI, Maino TAHARA, Kenta IWASAKI, Yoshiaki KOUZUMA,
and Makoto KIMURA†

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

Received September 19, 2001; Accepted October 31, 2001
Ribosomal protein L2 is a primary 23S rRNA binding protein in the large ribosomal subunit. We examined the contribution of the N- and C-terminal regions of Bacillus stearothermophilus L2 (BstL2) to the 23S rRNA binding activity. The mutant desN, in which the N-terminal 59 residues of BstL2 were deleted, bound to the 23S rRNA fragment to the same extent as wild type BstL2, but the mutation desC, in which the C-terminal 74 amino acid residues were deleted, abolished the binding activity. These observations indicated that the C-terminal region is involved in 23S rRNA binding. Subsequent deletion analysis of the C-terminal region found that the C-terminal 70 amino acids are required for efficient 23S rRNA binding by BstL2. Furthermore, the surface plasmon resonance analysis indicated that successive truncations of the C-terminal residues increased the dissociation rate constants, while they had little influence on association rate constants. The result indicated that reduced affinities of the C-terminal deletion mutants were due only to higher dissociation rate constants, suggesting that the C-terminal region primarily functions by stabilizing the protein L2-23S rRNA complex.
Key words: Bacillus stearothermophilus; protein-RNA interaction; surface plasmon resonance; ribosomal protein L2

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Note
Phytotoxic Sesterterpene, 11-Epiterpestacin, from Bipolaris
sorokiniana NSDR-011

Yoichiro NIHASHI,＊ Chi-Hwan LIM,＊＊ Chihiro TANAKA,＊＊＊ Hisashi MIYAGAWA,＊†
and Tamio UENO＊

＊Division of Applied Life Sciences, ＊＊＊Division of Science and Technology of Regional Environment,
Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan.
＊＊Department of Agricultural Chemistry, Chungnam National University, Taejon, 305-764, Korea.

Received September, 20, 2001; Accepted October 26, 2001
The structure of siccanol, a phytotoxic sesterterpene of fungal origin, was analyzed after chemical conversion by NMR spectroscopy. Siccanol was found to be an epimer of terpestacin that has been isolated from Arthrinium sp., and was thus renamed 11-epiterpestacin. Its stereochemistry was also identical with that of fusaproliferin, a structurally related mycotoxin from Fusarium proliferatum. Therefore, this sesterterpene may also be referred to as 24-deacetyl fusaproliferin. The phytotoxicity of 11-epiterpestacin was almost equal to that of terpestacin, but significantly higher than that of fusaproliferin.
Key words: 11-epiterpestacin; terpestacin; fusaproliferin; phytotoxin; sesterterpene

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Note
Luteolin, a Flavone, Does Not Suppress Postprandial Glucose Absorption
Through an Inhibition of α-Glucosidase Action

Toshiro MATSUI,† Mio KOBAYASHI, Sachiko HAYASHIDA, and Kiyoshi MATSUMOTO
Department of Bioscience and Biotechnology, Division of Bioresource and Bioenvironmental Sciences,
Faculty of Agriculture, Graduate School, Kyushu University, 6-10-1 Hakozaki, Higashi-ku,

Fukuoka 812-8581, Japan

Received September 25, 2001; Accepted October 26, 2001
In order to clarify the postprandial glucose suppression via α-glucosidase (AGH) inhibitory action by natural compounds, flavonoids were examined in this study. Among the flavonoids (luteolin, kaempferol, chrysin, and galangin), luteolin showed the potent maltase inhibitory activity with the IC50 of 2.3 mM, while less inhibitions were observed against sucrase. In addition, the effects of maltase inhibition by flavonoids were observed in the descending order of potency of luteolin＞kaempferol＞chrysin＞galangin. Apparently, the AGH inhibition power greatly increased with the replacement of hydroxyl groups at 3′ and 4′-position of the B-ring. However, the inhibitory power of luteolin was poorer than a therapeutic drug (acarbose: IC50; 430 nM). As a result of a single oral administration of maltose or sucrose (2 g/kg) in SD rats, no significant change in blood glucose level with the doses of 100 and 200 mg/kg of luteolin was observed. These findings strongly suggested that luteolin given at less than 200 mg/kg did not possess the ability to suppress the glucose production from carbohydrates through the inhibition of AGH action in the gut.
Key words: α-glucosidase; flavonoids; phenolic acids; noninsulin-dependent diabetes mellitus; luteolin

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Note
A Simple Method for Enrichment of Uninucleate Conidia
of Aspergillus oryza

Seiichi HARA, Ryohei F. TSUJI, Osamu HATAMOTO, and Tsutomu MASUDA

Noda Institute for Scientific Research, 399 Noda, Noda-shi, Chiba 278-0037, Japan

Received September 26, 2001; Accepted November 17, 2001
Aspergillus oryzae produces multinucleate conidia, which makes the obtaining of homokaryons labor-intensive. Analysis of conidia by flow cytometry clarified the relationship that conidia of lower nuclear number were smaller in size. Based on this, we have developed a simple way to enrich uninucleate conidia with a membrane filter. Our results also suggest that the method is useful for elimination of heterokaryons.
Key words: Aspergillus oryzae; uninucleate conidia; homokaryon; flow cytometry; membrane filtration

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Note
Synthesis of (1R,2S)-1-(3′-Chloro-4′-methoxyphenyl)-1,2-propanediol
(Trametol) and (1R,2S)-1-(3′,5′-Dichloro-4′-methoxyphenyl)-1,2-propanediol,
Chlorinated Fungal Metabolites in the Natural Environment†

Takeshi KOUSAKA1 and Kenji MORI1,2,††
1Department of Mathematics and Science Education, Graduate School of Science, and
2Department of Chemistry, Faculty of Science, Science University of Tokyo, Kagurazaka 1-3, Shinjuku-ku,

Tokyo 162-8601, Japan

Received October 1, 2000; Accepted November 2, 2001
(1R,2S)-1-(3′-Chloro-4′-methoxyphenyl)-1,2-
propanediol (Trametol, 3), a metabolite of the fungus Trametes sp. IVP-F640 and Bjerkandera sp. BOS55, was synthesized by employing Sharpless asymmetric dihydroxylation as the key step. Similarly, the (1R,2S)-isomer of 1-(3′,5′-dichloro-4′-methoxyphenyl)-1,2-propanediol (4), another metabolite of Bjerkandera sp. BOS55, was synthesized by asymmetric dihydroxylation.
Key words: asymmetric dihydroxylation; Bjerkandera sp.; chlorinated p-anisylpropanoid; optically active p-anisylpropanoid; white rot fungus

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Preliminary Communication
Milk Basic Protein (MBP) Increases Radial Bone Mineral Density
in Healthy Adult Women

Jun-ichi YAMAMURA,1 Seiichiro AOE,1 Yasuhiro TOBA,1 Mutsumi MOTOURI,1
Hiroshi KAWAKAMI,1 Masayoshi KUMEGAWA,2 Akira ITABASHI,3 and Yukihiro TAKADA1
1Technology and Research Institute, Snow Brand Milk Products Co. Ltd., 1-1-2 Minamidai,

Kawagoe, Saitama 350-1165, Japan
2Department of Oral Anatomy, Meikai University of Dentistry, 1-1 Keyakidai, Sakado,
Saitama 350-0248, Japan
3Central Clinical Laboratory, Saitama Medical School, 38 Morohongo, Moroyama,
Saitama 350-0495, Japan

Received September 26, 2001; Accepted December 4, 2001
We studied the effects of daily intake of milk basic protein (MBP) on radial bone mineral density (BMD) in healthy adult women. Thirty-three healthy women were randomly assigned to a 6-month trial with either placebo or MBP (40 mg per day). The radial BMD of each volunteer was measured at the beginning of and at six months after the trial. The mean BMD value at the 6th month in the MBP group increased significantly at both 1/6 and 1/10 portion from the distal end of the radius, whereas that in the control group did not. The BMD gain of each volunteer in the MBP group was significantly higher than that in the placebo group. Thus a daily MBP supplementation of 40 mg in healthy adult women can significantly increase radial BMD.
Key words: milk basic protein; MBP; bone mineral density; bone metabolism<QL>