Contents and Abstracts of Latest Issue of BBB

(Vol.63 No.11 1999)

Role of Hydroxylysine on the Nodulation of Root Nodule Bacteria
Keiko SASAKI-IGAWA, Tetsuya SATO, Hiroshi MASUDA, and Takuji OHWADA

Characterization of a Thermostable Esterase Activity from the Moderate
Thermophile Bacillus licheniformis
Elisa ALVAREZ-MACARIE, Valerie AUGIER-MAGRO, and Jacques BARATTI＊

Involvement of Thioredoxin Peroxidase Type II (Ahp1p) of Saccharomyces
cerevisiae in Mn2＋ Homeostasis
Ileana Cornelia FARCASANU,1 Dai HIRATA, Eiko TSUCHIYA, Keiko MIZUTA,
and Tokichi MIYAKAWA2

Relationship between Polyploidy and Pollen Self-incompatibility Phenotype
in Petunia hybrida Vilm.
Tetsuyuki ENTANI, Seiji TAKAYAMA, Megumi IWANO, Hiroshi SHIBA,
Fang-Sik CHE, and Akira ISOGAI＊

Structure and Regulatory Expression of A Single Copy Alternative
Oxidase Gene from the Yeast Pichia anomala
Shigeru SAKAJO, Nobuko MINAGAWA, and Akio YOSHIMOTO

Purification and Characterization of A Novel Chitinase Isozyme from Yam Tuber
Yasuyuki ARAKANE1,2 and Daizo KOGA2,・

Transcriptional Regulation of the Bacillus ohbensis Cyclodextrin
Glucanotransferase Gene in B. subtilis
Tamotsu NISHIDA,＊ Akira NAKAMURA,・ Haruhiko MASAKI, and Takeshi UOZUMI＋

New Okaramine Congeners, Okamines J, K, L, M and Related Compounds,
from Penicillium simplicissimum ATCC 90288
Yoshihito SHIONO, Kohki AKIYAMA, and Hideo HAYASHI・

Polypeptide Compositions and NH2-terminal Amino Acid Sequences
of Proteins in Foxtail and Proso Millets
Keiko KOHAMA, Takashi NAGASAWA,＊ and Naoyuki NISHIZAWA＊

Purification and Properties of Extracellular Carboxyl Proteases
of Acid-tolerant Bacteria, Isolated from Compost
Toshihiko TAKEHANA,＊ Sakura INOUE, Ryo TAKEI,＊＊ Hiroyuki ITO,
Hirokazu MATSUI, and Mamoru HONMA

Total Syntheses of All Four Stereoisomers of Piscidic Acid via Catalytic
Asymmetric Dihydroxylation of (Z)- and (E)-trisubstituted Olefins
Hiroaki TOSHIMA,・ Masatoshi SAITO, and Teruhiko YOSHIHARA

Safety Assessment of Transgenic Potatoes with Soybean Glycinin
by Feeding Studies in Rats
Wataru HASHIMOTO,・ Keiko MOMMA, Hye-Jin YOON, Sachiko OZAWA, Yasunobu OHKAWA,＊
Teruo ISHIGE,＊ Makoto KITO, Shigeru UTSUMI, and Kousaku MURATA

Isolation and Characterization of a Mutant Strain of Chattonella marina
with Decreased Production of Superoxide Anion
Daekyung KIM,1 Tatsuya ODA,1,・ Atsushi ISHIMATSU,2 and Tsuyoshi MURAMATSU1

5-Hydroxy-4-oxo-L-norvaline Depletes Intracellular Glutathione:
a New Modulator of Drug Resistance
Motoyuki TAGASHIRA,1 Naoko NOZATO,1 Seiji ISONISHI,2 Aikou OKAMOTO,2
Kazunori OCHIAI,2 and Yasuyuki OHTAKE1

Purification and Characterization of a Novel Extracellular Lipase Catalyzing
Hydrolysis of Oleyl Benzoate from Acinetobacter nov. sp. Strain KM109
Kazuya MITSUHASHI,1 Midori YAMASHITA,1 Yeo Soo HWAN,1 Fumio IHARA,2
Takuya NIHIRA,1,・ and Yasuhiro YAMADA1

Structural Conversion from Non-native to Native form of Recombinant Human
Epidermal Growth Factor by Brevibacillus choshinensis
Akira MIYAUCHI,1,2 Makoto OZAWA,1 Makoto MIZUKAMI,1 Kouji YASHIRO,1
Shogo EBISU,1 Takashi TOJO,1 Takaaki FUJII,2 and Hiroaki TAKAGI1,2

Interaction Among the Subunits of Golgi Membrane Mannosyltransferase
Complexes of the Yeast Saccharomyces cerevisiae
Hidetoshi KOJIMA, Hitoshi HASHIMOTO, and Koji YODA・

Human Lysozyme Secretion Increased by Alpha-factor Pro-sequence
in Pichia pastoris
Chitoshi OKA,・ Masao TANAKA, Michiro MURAKI,＊ Kazuaki HARATA,＊
Katsunori SUZUKI,＊＊ and Yoshifumi JIGAMI＊

Note
Improving Effect of Dietary Taurine on Marked Hypercholesterolemia Induced
by a High-cholesterol Diet in Streptozotocin-induced Diabetic Rats
Hideki MOCHIZUKI, Jun TAKIDO, Hiroaki ODA,＊ and Hidehiko YOKOGOSHI・

Note
Effects of Methanol and Temperature on Enzyme Immunoassay
with Monoclonal Antibodies Specific to the Insecticide Etofenprox
Masanao KATAGIRI,・ Tomoko KADOYA, Kennichi MIYAKE, Fumihide ISHIBASHI,
and Hideo OHKAWA＊

Note
Resolution and Synthesis of (S)-1-(2-Naphthyl)ethanol
with Immobilized Pea Protein: As a New Biocatalyst
Hiroyuki NAGAOKA＊,＊＊＊ and Hiroshi KAYAHARA＊＊,・

Note
Isolation and Structure of a Novel Indophenol-reducing and
1,1-Diphenyl-2-picrylhydrazyl Radical-scavenging Compound from a Fungus
Chiaki ITO, Naoki ABE, and Akira HIROTA・

Note
Effects of Amino Acids, Sugars, and Ascorbic Acid on the Stability
of Linoleic Acid Hydroperoxide in the Water Phase
Tamako NISHIIKE,1 Jun ICHIKAWA,＊ Noriko KIKUGAWA,＊ Hitoshi TAKAMURA,＊
and Teruyoshi MATOBA

Note
The Trichothecene Biosynthesis Regulatory Gene from the Type B Producer
Fusarium Strains: Sequence of Tri6 and Its Expression in Escherichia coli・
Gentaro MATSUMOTO,1,2 Junko WUCHIYAMA,1 Yoshinori SHINGU,1,2 Makoto KIMURA,・・,1
Katsuyoshi YONEYAMA,2 and Isamu YAMAGUCHI・・・,1

Note
Effects of Dietary Protein Type on the Response of Lipid Metabolism
to Orotic Acid in Rats
Yoritaka AOYAMA・ and Mizuho WADA

Note
Kinetics for the Autoxidation of Conjugated Linoleic Acid
Hwan-Sook SEO, Yasushi ENDO,・ and Kenshiro FUJIMOTO

Note
Induction of GM-CSF Production of Macrophages by Advanced Glycation
End Products of the Maillard Reaction
Toshinori SASAKI,a,・ Seikoh HORIUCHI,b Masatoshi YAMAZAKI,a and Satoru YUIa

Note
Carotenogenesis Pathway of Novel Carotenoid Glucoside Mycolic Acid Esters
in Rhodococcus rhodochrous Using Carotenogenesis Mutants and Inhibitors
Shinichi TAKAICHI,1,・ Yasumori TAMURA,2,・・ Takeshi MIYAMOTO,1 Ken AZEGAMI,1
Yoko YAMAMOTO,2 and Jun-ichi ISHIDSU1

Note
Purification and Characterization of β-1,3-Xylanase from a Marine Bacterium,
Vibrio sp. XY-214
Toshiyoshi ARAKI, Shuji TANI, Keiko MAEDA, Shinnosuke HASHIKAWA,
Hiroki NAKAGAWA,＊ and Tatsuo MORISHITA

Note
The Subunit Structure of Nitrite Reductase Purified from the Denitrifier
Achromobacter cycloclastes
Ken-ichi INATOMI・

Note
Effects of a Feedback-Resistant Aspartokinase III Gene
on L-Isoleucine Production in Escherichia coli K-12
Ken-ichi HASHIGUCHI,＊ Hiroshi MATSUI,＊＊ and Osamu KURAHASHI＊

Note
Synthesis of Methyl (S)-(−)-6,8-Dihydroxyoctanoate as a Precursor
of (R)-(＋)-α-Lipoic Acid
Makoto GANAHA, Satoshi YAMAUCHI, and Yoshiro KINOSHITA・

Note
Lipids of Three Highly Migratory Fishes: Euthynnus affinis, Sarda orientalis,
and Elagatis bipinnulata
Hiroaki SAITO,＊ Rieko YAMASHIRO,・ Kenji ISHIHARA, and Changhu XUE・

Note
Further Characterization of Earthworm Serine Proteases:
Cleavage Specificity Against Peptide Substrates and on Autolysis
Nobuyoshi NAKAJIMA,1,・ Manabu SUGIMOTO,2 Kohji ISHIHARA,3 Kaoru NAKAMURA,4
and Hiroki HAMADA5

Preliminary Communication
Conversion of Bacillus subtilis 168: Natto Producing Bacillus subtilis
with Mosaic Genomes
Mitsuhiro ITAYA・ and Kuniko MATSUI

Preliminary Communication
New Cyclization Mechanism for Squalene: a Ring-expansion Step for the
Five-membered C-ring Intermediate in Hopene Biosynthesis
Tsutomu HOSHINO,＊ Masanori KOUDA, Takamasa ABE, and Shumi OHASHI

Preliminary Communication
Cloning and Characterization of a Drosophila melanogaster cDNA Encoding
a Glutamate Transporter
Tsuyoshi KAWANO,1,・ Kyoko TAKUWA,2 Hisato KUNIYOSHI,3 Naoto JUNI,3
Terumi NAKAJIMA,2 Daisuke YAMAMOTO,3 and Yasuo KIMURA1

-1-
Molecular Cloning and Characterization of a Cysteine-rich 16.6-kDa Prolamin
in Rice Seeds

Norihiro MITSUKAWA,＊＊ Ryoichi KONISHI, Masato UCHIKI, Takehiro MASUMURA,
and Kunisuke TANAKA＊

Laboratory of Genetic Engineering, Faculty of Agriculture Kyoto Prefectural University,
Shimogamo, Kyoto 606-8522, Japan Received February 15, 1999; Accepted July 26, 1999

Received February 15, 1999; Accepted july 26, 1999
An alcohol-soluble storage protein, a 16.6-kDa prolamin found in rice seeds, was purified from both the total protein body and purified type I protein body fractions. The partial amino acid sequences of three tryptic peptides generated from the purified polypeptide were analyzed. A part of the 16.6-kDa prolamin cDNA was amplified from developing seed mRNA by the reverse transcribed polymerase chain reaction using an oligo (dT) primer and a primer which was synthesized based on the partial amino acid sequence. The amplified product was used to isolate the full-length cDNA clone (λRP16) from a developing seed cDNA library. The cDNA has an open reading frame encoding a hydrophobic polypeptide of 149 amino acids. The polypeptide was rich in glutamine (20.0％), cysteine (10.0％), and methionine (6.9％). The cysteine content was higher than those of most other rice storage proteins. Messenger RNA of the 16.6-kDa prolamin was detected in seeds, but not in other aerial tissues.
seed maturation; rice prolamin; protein body; storage protein; sulfur-rich protein

-2-
Role of Hydroxylysine on the Nodulation of Root Nodule Bacteria

Keiko SASAKI-IGAWA, Tetsuya SATO, Hiroshi MASUDA, and Takuji OHWADA

Department of Bioresource Science, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho,
Obihiro City, Hokkaido, 080-8555, Japan

Received March 25, 1999; Accepted August 3, 1999
The growth of Bradyrhizobium japonicum as well as Rhizobium leguminosarum bv. phaseoli growing in minimal medium was repressed by the addition of hydroxylysine (Hyl), although the sensitivity of the former to Hyl seemed to be lower than that of the latter. The nodulation efficiency of both Glycine max (L.) inoculated with B. japonicum cells and Phaseolus vulgaris (L.) inoculated with R. leguminosarum bv. phaseoli cells was reduced in the presence of Hyl, concomitantly with the decrease in the elongation of roots. Besides, the Hyl contents in the seed (seedling) exudates tended to increase when the host plants were inoculated with an unfavorable strain for their nodulation. These results suggest that the Hyl plays a role in the effective symbiotic relationship by regulating the growth of the root nodule bacteria on the root surface and/or the elongation of the host plant′s roots.
hydroxylysine (Hyl); Rhizobium; root nodule bacteria

-3-
Characterization of a Thermostable Esterase Activity from the Moderate
Thermophile Bacillus licheniformis

Elisa ALVAREZ-MACARIE, Valerie AUGIER-MAGRO, and Jacques BARATTI＊

Mediterranee University, ESA CNRS 6111, Faculty of Sciences of Luminy, Case 901, 163 avenue de Luminy,
13288 Marseille cedex 9, France

Received April 19, 1999; Accepted June 22, 1999
A new esterase activity from Bacillus licheniformis was characterized from an Escherichia coli recombinant strain. The protein was a single polypeptide chain with a molecular mass of 81 kDa. The optimum pH for esterase activity||was 8--8.5 and it was stable in the range 7--8.5. The opti||mum temperature for activity was 45°C and the half-life was 1 h at 64°C. Maximum activity was observed on p-nitrophenyl caproate with little activity toward long-chain||fatty acid esters. The enzyme had a KM of 0.52 mM for p-||nitrophenyl caproate hydrolysis at pH 8 and 37°C. The enzyme activity was not affected by either metal ions or sulfydryl reagents. Surprisingly, the enzyme was only slightly inhibited by PMSF. These characteristics classified the new enzyme as a thermostable esterase that shared similarities with lipases. The esterase might be useful for biotechnological applications such as ester synthesis.
esterase; lipase; Bacillus licheniformis; specificity; caproate

-4-
Involvement of Thioredoxin Peroxidase Type II (Ahp1p) of Saccharomyces
cerevisiae in Mn2＋ Homeostasis

Ileana Cornelia FARCASANU,1 Dai HIRATA, Eiko TSUCHIYA, Keiko MIZUTA,
and Tokichi MIYAKAWA2

From the Department of Fermentation Technology, Faculty of Engineering, Hiroshima University,
Higashi-Hiroshima 739-8527, Japan

Received May 10, 1999 Accepted June 30, 1999
To identify new proteins involved in Mn2＋ homeostasis, we isolated Mn2＋-resistant mutants of Saccharomyces cerevisiae starting from a calcineurin-deficient, Mn2＋ hypersensitive strain (Δcmp1 Δcmp2). The mutations were found to lie in the PMR1 gene, known to encode a ``P-type′′ Ca2＋-ATPase that transports Ca2＋ and Mn2＋ from the cytosol to the Golgi apparatus. A second gene, AHP1, was cloned as a suppressor of the Mn2＋ tolerance of a Δcmp1 Δcmp2 pmr1 mutant. Ahp1p was recently described as a thioredoxin peroxidase type II, an antioxidant protein with alkyl hydroperoxide defense properties in yeast. AHP1 disruption in strain W303 decreased tolerance to Mn2＋ and H2O2. We found that a GFP-Ahp1p fusion construct was in the cytosol when cells were grown in glucose, and in the mitochondria when cells were grown in oleate. Based on Mn2＋ transport data, we concluded that Ahp1p is involved in cellular Mn2＋ homeostasis in trafficking of Mn2＋ from cytosol to mitochondria and from cytosol for export across the plasma membrane.
AHP1; manganese; organellar trafficking; Saccharomyces cerevisiae

-5-
Relationship between Polyploidy and Pollen Self-incompatibility Phenotype
in Petunia hybrida Vilm.

Tetsuyuki ENTANI, Seiji TAKAYAMA, Megumi IWANO, Hiroshi SHIBA,
Fang-Sik CHE, and Akira ISOGAI＊

Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama,
Ikoma, Nara 630-0101, Japan

Received May 17, 1999; Accepted July 2, 1999
Self-incompatibility in Solanaceae is controlled by a single multiallelic locus, the S-locus. The S-allele associated ribonucleases (S-RNases) in the pistil are involved in pollen rejection. In this work, we analyzed two newly isolated lines of Petunia hybrida, termed PB and PF. They both had the same set of S-RNases (SB1- and SB2-RNases), however the PB was a self-incompatible diploid while PF was a self-compatible tetraploid. Cross pollination tests between PB and PF indicated diploid pollen from PF lost the incompatibility phenotype. In order to clarify the effects of polyploidy on pollen phenotypic change, we artificially induced tetraploid plants from a diploid SB1SB2 heterozygote (＝PB) and a diploid SB1SB1 homozygote. The obtained SB1SB1SB1SB1 homoallelic tetraploid remained self-incompatible, whereas the SB1SB1SB2SB2 heteroallelic tetraploid became self-compatible. These data suggested that the diploid heteroallelic pollen lost the incompatibility phenotype and had the characteristics of self-compatibility with SB1SB2 style.
self-incompatibility; Petunia hybrida; S-RNase; polyploidy

-6-
Structure and Regulatory Expression of A Single Copy Alternative
Oxidase Gene from the Yeast Pichia anomala

Shigeru SAKAJO, Nobuko MINAGAWA, and Akio YOSHIMOTO

Department of Biochemistry, Niigata College of Pharmacy, Kamishin′ei-cho 5-13-2, Niigata 950-2081, Japan

Received May 19, 1999; Accepted July 6, 1999
To investigate the regulatory mechanism of alternative oxidase gene expression, genomic DNA was cloned from the yeast Pichia anomala. Genomic Southern blot analysis suggested that a single copy nuclear gene encoded an alternative oxidase in the yeast. The nucleotide sequence showed an uninterrupted coding region for the alternative oxidase protein. In the upstream region from the transcription initiation site found by primer extension analysis, CCAAT, TATAA, and UAS2-like elements were detected. The UAS2 is the element involved in transcriptional regulation by carbon source and the target site for the factor, HAP2/3/4/5 protein complex, in Saccharomyces cerevisiae. By a gel mobility shift assay, a specific retardation band was detected when a protein extract from cells grown on an inducing carbon source was incubated with a UAS2-containing probe. These results suggest that carbon source regulation of alternative oxidase gene expression is mediated by the UAS2-like element and a HAP-like factor in P. anomala.
alternative oxidase; cyanide-resistant respiration; fungi; mitochondria; nuclear gene

-7-
Purification and Characterization of A Novel Chitinase Isozyme from Yam Tuber

Yasuyuki ARAKANE1,2 and Daizo KOGA2,・

1Research Laboratory, Bankaku So-honpo Co. Ltd., Tokaishi, Aichi 476-8577, Japan
2Laboratory of Biochemistry, Department of Biological Science, Faculty of Agriculture, Yamaguchi University,
Yamaguchi 753-8515, Japan

Received May 20, 1999; Accepted August 17, 1999
A new chitinase isozyme (Chitinase A), which had only one optimum pH toward a long substrate, glycolchitin, was purified from the peel of yam tuber by CTAB (hexadecyl trimethyl ammonium bromide) treatment and ammonium sulfate fractionation, followed by column chromatography on DEAE-Cellulofine A-500, chromatofocusing, and gel filtration on Sephacryl S-100. The molecular weight was 28,000 by SDS-PAGE. The isoelectric point was 3.6. The optimum pH was 4.0 toward both a polymer substrate, glycolchitin, and an oligosaccharide substrate, GlcNAc5. The optimum temperature was 60°C. Chitinase A was stable between pH 6 and 11 and below 45°C. Kinetic analysis was done using a series of N-acetylchitooligosaccharides (GlcNAcn, n＝2 to 6) and glycolchitin as the substrates.||Chitinase A hydrolyzed N-acetylchitooligosaccharides in||||an endo/random fashion except the disaccharide, and ||||released the monosaccharide from all hydrolyzed oligosac||||charides. This enzyme preferred oligosaccharides with the||longer chain lengths. Chitinase A was inhibited 76％ by 55 μM allosamidin, which is known to be a specific inhibitor of insect chitinases.
chitinase; yam; N-acetylchitooligosaccharide

-8-
Transcriptional Regulation of the Bacillus ohbensis Cyclodextrin
Glucanotransferase Gene in B. subtilis

Tamotsu NISHIDA,＊ Akira NAKAMURA,・ Haruhiko MASAKI, and Takeshi UOZUMI＋

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

Received May 21, 1999; Accepted August 20, 1999
The expression of the cyclodextrin glucanotransferase (CGTase) gene (c・・t) of Bacillus ohbensis, when introduced into an α-amylase-defective strain of B. subtilis on a multicopy plasmid, pHY300PLK, was induced in the presence of starch and was subject to catabolite repression by glucose as well as in the original strain, B. ohbensis. We constructed a c・・t′::′lacZ translational fusion to study the expression in B. subtilis, and this construct was confirmed to be subject to both starch induction and catabolite repression. In order to define the region involved in the regulation of the c・・t gene, a series of c・・t′::′lacZ gene with various lengths of deletion in the promoter region was constructed on pHY300PLK. DNA regions responsible for starch induction and catabolite repression by glucose could be separated in the deletion experiment. Primer extension analysis showed that the catabolite repression was controlled at the initiation of transcription, while the starch induction is likely to be controlled by a transcriptional termination-antitermination mechanism.
cyclodextrin glucanotransferase; gene regulation; starch induction; catabolite repression; Bacillus subtilis

-9-
New Okaramine Congeners, Okamines J, K, L, M and Related Compounds,
from Penicillium simplicissimum ATCC 90288

Yoshihito SHIONO, Kohki AKIYAMA, and Hideo HAYASHI・

Department of Applied Biological Chemistry, College of Agriculture, Osaka Prefecture University,
1-1 Gakuen-cho, Sakai, Osaka 599-8531, Japan

Received May 24, 1999; Accepted July 16, 1999
Four new okaramine congeners, okaramines J, K, L and M, were isolated from okara fermented with Penicillium simplicissimum ATCC 90288, together with five biogenetically related compounds, cyclo (8a-(α,α-dimethylallyl)-L-Trp-8a-(α,α-dimethylallyl)-L-Trp), cyclo (N8-(α,α-dimethylallyl)-L-Trp-6a′-(α,α-dimethylallyl)-L-Trp), cyclo (L-Trp-L-Trp), cyclo (6a′-(α,α-dimethylallyl)-L-Trp-L-Trp), and cyclo (N8-(α,α-dimethylallyl)-L-Trp-L-Trp). Their structures were elucidated on the basis of spectroscopic methods. The new okaramines exhibited no insecticidal activity against silkworms.
Penicillium simplicissimum; okara; okaramines; insecticidal compound

-10-
Polypeptide Compositions and NH2-terminal Amino Acid Sequences
of Proteins in Foxtail and Proso Millets

Keiko KOHAMA, Takashi NAGASAWA,＊ and Naoyuki NISHIZAWA＊

Iwate Industrial Research Institute, Morioka, Iwate 020-0852, Japan and United Graduate School
of Agricultural Science, Iwate University
＊Department of Bioscience and Technology, Faculty of Agriculture, Iwate University,
Morioka, Iwate 020-8550, Japan

Received May 26, 1999; Accepted June 25, 1999
Seed protein of foxtail and proso millets were fractionated into polypeptides that were analyzed for their major protein, prolamin, and the NH2-terminal amino acid sequences of the proteins were determined. The proteins extracted from foxtail and proso millets were 64.1％ and 80.0％ prolamin, respectively. The polypeptides of the prolamins were classified into two groups. The major polypeptides of 27--19 kDa were rich in leucine and alanine, whereas the 17--14 kDa polypeptides were rich in methionine and cysteine. Glutelin-like proteins that were extracted with a reducing reagent were high in proline content, the major polypeptides being 17 and 20 kDa. The NH2-terminal amino acid sequence showed that the major polypeptides of prolamin were homologous to α-zein and a glutelin-like protein containing the Pro-Pro-Pro sequence, like the repetitive sequence of γ-zein. Although the prolamin consisted of a similar subunit to that of zein, polypeptides with various pI values were found among them.
millet; prolamins; polypeptide composition; NH2-terminal amino acid sequence; homology

-11-
Purification and Properties of Extracellular Carboxyl Proteases
of Acid-tolerant Bacteria, Isolated from Compost

Toshihiko TAKEHANA,＊ Sakura INOUE, Ryo TAKEI,＊＊ Hiroyuki ITO,
Hirokazu MATSUI, and Mamoru HONMA

Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University,
Sapporo 060-8589, Japan
＊＊Tsukuba Chemical R＆D Laboratory, Adeka Clean Aid Co., Ltd., 4-1 Koyodai, Ryugasaki,
Ibaraki 301-0852, Japan

Received May 27, 1999 Accepted July 23, 1999
Four strains of acid-tolerant and protein-using bacteria were isolated from compost. Two of them, Gram-negative strains MB8 and MB11, were identified as a new genus close to Stenotrophomonas species MB8 and Burkholderia species MB11, respectively. Both bacteria produced extracellular carboxyl proteases (CP) in acid-casein-starch medium. The enzymes, termed CP MB8 and CP MB11, purified through ion exchange and gel filtration chromatographies had molecular weights of 61,000 (CP MB8) and 36,000 (CP MB11) as estimated by SDS-PAGE, and showed optimum activities with hemoglobin as a substrate at pH 3.5 (CP MB8) and pH 3.7 (CP MB11) at 55°C. Both of the enzymes were not inhibited by pepstatin, DAN, or EPNP. These results suggest that both enzymes are members of the pepstatin-insensitive carboxyl proteinase family (EC 3.4.23.33), except for a larger molecular weight of the CP MB8 enzyme.
compost; Proteobacteria; Burkholderia; carboxyl protease; pepstatin

-12-
Total Syntheses of All Four Stereoisomers of Piscidic Acid via Catalytic
Asymmetric Dihydroxylation of (Z)- and (E)-trisubstituted Olefins

Hiroaki TOSHIMA,・ Masatoshi SAITO, and Teruhiko YOSHIHARA

Department of Bioscience and Chemistry, Faculty of Agriculture, Hokkaido University,
Sapporo 060-8589, Japan

Received May 31, 1999; Accepted July 7, 1999
All four stereoisomers of (2S, 3R)-(＋)-piscidic acid were synthesized with high optical purity via Sharpless catalytic asymmetric dihydroxylation of (Z)- and (E)-trisubstituted olefins in 6 steps from (4-hydroxyphenyl)pyruvic acid. The Wittig reaction of methyl (4-hydroxyphenyl)pyruvate with (carbomethoxymethylene)triphenylphosphorane gave (Z)- and (E)-trisubstituted olefins in a 3:1 ratio. After protecting the phenolic hydroxyl group as the tert-butyldimethylsilyl ether, the (Z)-olefin was subjected to asymmetric dihydroxylation by using the chiral ligand, dihydroquinidine 1,4-anthraquinonediyl diether, and the reaction proceeded with 89％ e.e. Desilylation and subsequent alkaline hydrolysis gave (2S, 3R)-(＋)-piscidic acid. The optical purity was increased to >99％ e.e. by recrystallization. The use of dihydroquinine 1,4-anthraquinonediyl diether enable (2R, 3S)-(−)-piscidic acid to be obtained. In the asymmetric dihydroxylation of the (E)-olefin, phthalazine ligands (dihydroquinidine and dihydroquinine 1,4-phthalazinediyl diethers) gave high e.e. values. Via the same deprotection procedure, (2S, 3S)-(＋)-3-epi-piscidic acid and (2R, 3R)-(−)-2-epi-piscidic acid were respectively obtained.
piscidic acid; hypnotic and narcotic drugs; phosphorous uptake; cough medicine constituent; catalytic asymmetric dihydroxylation

-13-
Safety Assessment of Transgenic Potatoes with Soybean Glycinin
by Feeding Studies in Rats

Wataru HASHIMOTO,・ Keiko MOMMA, Hye-Jin YOON, Sachiko OZAWA, Yasunobu OHKAWA,＊
Teruo ISHIGE,＊ Makoto KITO, Shigeru UTSUMI, and Kousaku MURATA

Research Institute for Food Science, Kyoto University, Uji, Kyoto 611-0011, Japan
＊National Institute of Agrobiological Resources, Tsukuba, Ibaraki 305-8056, Japan

Received June 8, 1999; Accepted August 9, 1999
Feeding studies of transgenic potatoes with native and designed soybean glycinins in rats were done for four weeks. The designed glycinin has four additional methioninyl residues in the middle of the glycinin molecule. Rats were divided into four groups fed (I) only a commercial diet, (II) the diet plus non-transgenic potatoes, (III) the diet plus transgenic potatoes with native glycinin, and (IV) the diet plus transgenic potatoes with designed glycinin. Rats were fed 2,000 mg/kg-weight potatoes every day by oral administration. During the period tested, rats in each group (groups II, III, and IV) grew well without marked differences in appearance, food intake, body weight, or in cumulative body weight gain. No significant differences were also found in blood count, blood composition, and in internal organ weights among the rats after feeding potatoes (groups II, III, and IV) for four weeks. Necropsy at the end of experiment indicated neither pathologic symptoms in all rats tested nor histopathological abnormalities in liver and kidney. Judging from these results, the transgenic potatoes with glycinins are confirmed to have nearly the same nutritional and biochemical characteristics as non-transgenic one.
animal feeding study; food safety; transgenic potato; soybean glycinin

-14-
Isolation and Characterization of a Mutant Strain of Chattonella marina
with Decreased Production of Superoxide Anion

Daekyung KIM,1 Tatsuya ODA,1,・ Atsushi ISHIMATSU,2 and Tsuyoshi MURAMATSU1

1Division of Biochemistry, Faculty of Fisheries, Nagasaki University, Nagasaki 852-8521, Japan
2Marine Research Institute, Nagasaki University, Nomozaki, Nagasaki 851-0505, Japan

Received June 9, 1999; Accepted July 9, 1999
Chattonella marina is well known as the causative organism of red tide, and is highly toxic to fish. However, the toxic mechanism of C. marina has not been established. Recent studies demonstrated that C. marina generates reactive oxygen species (ROS) such as O−2 and H2O2. In this study, we attempted to establish mutant strains of C. marina using a chemical mutagen, ethyl methane sulfonate (EMS). After 48 h of treatment with 1 mg/ml of EMS at 26°C, several viable cells were cloned, while more than 90％ of C. marina cells died under these conditions. Among the strains isolated, one strain (mutant C) was significantly decreased in the production of O−2, but its growth rate was indistinguishable from the parental C. marina. Furthermore, superoxide dismutase (SOD) had almost no effect on the proliferation of mutant C, while SOD suppressed the growth of parental C. marina. In accordance with the lower level of O−2 generation, mutant C was less toxic to Vibrio alginolyticus in a plankton/bacteria co-culture system as compared to parental C. marina. In contrast, no significant difference in H2O2 production between mutant C and parental C. marina was observed. These results may provide experimental evidence for a direct connection between the toxic effect of C. marina against V. alginolyticus and the production of superoxide anion. Although the detailed mechanism of the production of superoxide anion by C. marina is still unclear, our mutant strain isolated in this study may be a good tool for the clarification of the mechanism of O−2 generation by C. marina.
red tide plankton; chattonella marina; chattonella marina mutant strain; reactive oxygen species; toxicity

-15-
5-Hydroxy-4-oxo-L-norvaline Depletes Intracellular Glutathione:
a New Modulator of Drug Resistance

Motoyuki TAGASHIRA,1 Naoko NOZATO,1 Seiji ISONISHI,2 Aikou OKAMOTO,2
Kazunori OCHIAI,2 and Yasuyuki OHTAKE1

1Foods ＆ Pharmaceuticals Research ＆ Development Laboratory, Asahi Breweries Ltd., 1-1-21, Midori,
Moriya-machi, Kitasohma-gun, Ibaraki, 302-0106 Japan
2Department of Obstetrics ＆ Gynecology, The Jikei University School of Medicine, Tokyo 105-0003, Japan

Received June 11, 1999; Accepted August 4, 1999
To search for compounds that reverse the drug resistance induced by glutathione (GSH), an original screening system to detect intracellular GSH depleters was established. Among 8843 microbes derived from the soil samples tested, the extracts of two Streptomyces species, named KS6701 and KS8846, lowered the intracellular GSH level of Saccharomyces cerevisiae 5×47. From both the microbes, 5-hydroxy-4-oxo-L-norvaline (HON) was isolated as the active compound. At a concentration of 50--100 μg/ml, HON also decreased the GSH/protein level of the human ovarian tumor cell line, 2008/C13＊5.25 and reversed its resistance to cisplatin. We also investigated the mechanism of the depletion. HON had little effect on γ-glutamylcysteine synthetase (γ-GCS) or glutathione synthetase, but HON decreased the quantity of thiol substances when it was spontaneously reacted with them. This suggested that the GSH depletion by HON occurred through a mechanism different from that of buthionine sulfoximine, a selective γ-GCS inhibitor.
5-hydroxy-4-oxo-L-norvaline; glutathione; drug resistance; cisplatin; buthionine sulfoximine

-16-
Purification and Characterization of a Novel Extracellular Lipase Catalyzing
Hydrolysis of Oleyl Benzoate from Acinetobacter nov. sp. Strain KM109

Kazuya MITSUHASHI,1 Midori YAMASHITA,1 Yeo Soo HWAN,1 Fumio IHARA,2
Takuya NIHIRA,1,・ and Yasuhiro YAMADA1

1Department of Biotechnology, Graduate School of Engineering, Osaka University,
2-1 Yamadaoka, Suita, Osaka 565-0871, Japan
2Department of Plant Protection, National Institute of Fruit Tree Science, 2-1 Fujimoto,
Tsukuba, Ibaraki 305-8605, Japan

Received June 14, 1999; Accepted July 23, 1999
A new lipase (OBase) which efficiently hydrolyzes oleyl benzoate (OB) was found in the culture supernatant of Acinetobacter nov. sp. strain KM109, a new isolate growing in a minimum medium containing OB as the sole carbon source. OBase was purified to homogeneity with 213-fold purification and 0.8％ yield. The molecular weight was estimated to be 62,000±1,000 by SDS-PAGE under denatured-reduced conditions and to be 50,000±1,000 by gel-filtration HPLC under native conditions; these findings indicate that OBase is a monomeric enzyme. The optimum temperature and pH of OBase were about 45°C and pH 8. Temperature and pH stabilities were at or lower than 35°C and in a range of pH 6--8, respectively. Purified OBase preferentially hydrolyzed p-nitrophenyl benzoate (pNPB) over p-nitrophenyl acetate (pNPA) or p-nitrophenyl caproate (pNPC) [pNPB/pNPA＝20 and pNPB/pNPC＝5.4], indicating that OBase has a high affinity for benzoyl esters. Partial amino-acid sequences of OBase fragments obtained after lysyl endopeptidase treatment showed no similarity with known proteins.
lipase; Acinetobacter; oleyl benzoate

-17-
Structural Conversion from Non-native to Native form of Recombinant Human
Epidermal Growth Factor by Brevibacillus choshinensis

Akira MIYAUCHI,1,2 Makoto OZAWA,1 Makoto MIZUKAMI,1 Kouji YASHIRO,1
Shogo EBISU,1 Takashi TOJO,1 Takaaki FUJII,2 and Hiroaki TAKAGI1,2

1R ＆ D Department, Higeta Shoyu Co., Ltd., 2-8 Chuo-cho, Choshi-shi, Chiba 288-8680, Japan
2Department of Biotechnology, Graduate School of Science and Technology, Chiba University,
648 Matsudo, Matsudo, Chiba 271-8510, Japan

Received June 24, 1999; Accepted August 6, 1999
Brevibacillus choshinensis (Bacillus brevis) HPD31 is a very efficient producer of recombinant human epidermal growth factor (EGF). The produced EGF is secreted into the medium with high efficiency. However part of the EGF that accumulates in the medium, exists as multimeric forms which are biologically inactive. We found the bacterium has the activity to structurally convert multimeric forms to the monomeric, native ones. Optimal temperature and pH for the conversion were 40°C and pH 9, respectively. The reaction was promoted in the presence of reduced glutathione or cysteine. But the cells which had been sonicated or exposed to moderate heat treatment completely lost the activity. Thus, it was presumed that the activity might be due to the enzyme(s) that catalyze the protein disulfide exchanging reaction, and that they resides on the surface of viable cells.
Brevibacillus choshinensis; Bacillus brevis; disulfide exchange; recombinant human epidermal growth factor; protein secretion

-18-
Interaction Among the Subunits of Golgi Membrane Mannosyltransferase
Complexes of the Yeast Saccharomyces cerevisiae

Hidetoshi KOJIMA, Hitoshi HASHIMOTO, and Koji YODA・

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

Received June 25, 1999; Accepted August 4, 1999
Saccharomyces cerevisiae Mnn9 protein is a type II Golgi membrane protein which concerns in protein mannosylation. When solubilized by Triton X-100, it was recovered in two distinct complexes both having mannosyltransferase activity; one with Van1 protein (V-complex) and the other with Anp1, Hoc1, Mnn10, and Mnn11 proteins (A-complex). Characterization of the null mutants suggested that A-complex is also concerned in protein O-glycosylation. A-complex was more resistant than V-complex to dissociating conditions. Interaction between the lumenal domains of Van1 and Mnn9 was detected by a two-hybrid experiment. The anchor domain of Mnn9 protein could be replaced with other membrane anchors without losing the ability to form complexes similar to V- and A-complexes. Thus the lumenal domains are important to assemble these distinct complexes.
Golgi membrane; mannosyltransferase complex; glycosylation; Saccharomyces cerevisiae; MNN9

-19-
Human Lysozyme Secretion Increased by Alpha-factor Pro-sequence
in Pichia pastoris

Chitoshi OKA,・ Masao TANAKA, Michiro MURAKI,＊ Kazuaki HARATA,＊
Katsunori SUZUKI,＊＊ and Yoshifumi JIGAMI＊

Chiba Prefectural Industrial Research Institute, 889 Kasori, Wakaba, Chiba 264-0017, Japan
＊National Institute of Bioscience and Human-Technology, 1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan
＊＊Department of Biological Science, Faculty of Science, Hiroshima University,
1-3 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8526, Japan

Received June 28, 1999: Accepted August 9, 1999
To get high level secretion of human lysozyme in Pichia pastoris, the following three signal sequences and one prepro sequence were evaluated: chicken lysozyme signal peptide, leucine-rich artificial signal peptide, Saccharomyces invertase signal peptide, and Saccharomyces prepro sequence of alpha factor (MF-α Prepro).
Transformants harboring a lysozyme gene with MF-α Prepro secreted 20-fold more lysozyme than those harboring the lysozyme gene with any one of the other three signal sequences. Three mutant leader sequences derived from MF-α Prepro were constructed to discover the function of the pro region. The secretion was dramatically decreased by eliminating the pro region of MF-α Prepro. In contrast, MF-α Prepro with the EAEAEA sequence directed the secretion of an equivalent level of lysozyme having the extra amino acids (EAEAEA) in its N-terminus.
For the effective secretion of native human lysozyme, MF-α Prepro without any spacer sequences was most suitable. The secreted protein by MF-α Prepro construct was identical with the authentic human lysozyme, judging from N-terminal amino acid sequencing and molecular mass spectrometric and crystallographic analysis.
human lysozyme; secretion; alpha-factor; Pichia pastoris

-20-
Note
Improving Effect of Dietary Taurine on Marked Hypercholesterolemia Induced
by a High-cholesterol Diet in Streptozotocin-induced Diabetic Rats

Hideki MOCHIZUKI, Jun TAKIDO, Hiroaki ODA,＊ and Hidehiko YOKOGOSHI・

School of Food and Nutritional Sciences, The University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan
＊Department of Applied Biological Sciences, Nagoya University, Nagoya 464-8601, Japan

Received July 26, 1999; Accepted July 27, 1999
The effect of dietary taurine on hypercholesterolemia induced by a high-cholesterol diet in streptozotocin (STZ)-induced diabetic rats was investigated. The concentrations of serum and liver cholesterol were markedly elevated in STZ-diabetic rats fed on the cholesterol-containing diet, and dietary taurine significantly reduced this elevated level of cholesterol in the serum and liver. The gene expression of cholesterol 7α-hydroxylase (CYP7A1), which is the rate-limiting enzyme for cholesterol degradation, was induced by the supplementation of taurine to the high-cholesterol diet. It is suggested that one of the reasons for this hypocholesterolemic action by taurine might have been the enhancement of cholesterol degradation.
taurine; hypercholesterolemia; streptozotocin-induced diabetes; CYP7A1 (cholesterol 7α-hydroxylase); rat

-21-
Note
Effects of Methanol and Temperature on Enzyme Immunoassay
with Monoclonal Antibodies Specific to the Insecticide Etofenprox

Masanao KATAGIRI,・ Tomoko KADOYA, Kennichi MIYAKE, Fumihide ISHIBASHI,
and Hideo OHKAWA＊

Division of Natural Science, Osaka Kyoiku University, 4-Asahiga-oka, Kashiwara, Osaka 582-8582, Japan
＊Department of Biological and Environmental Science, Faculty of Agriculture, Kobe University,
Rokodai-cho, Nada-ku, Kobe, Hyogo 657-8501, Japan

Received February 8, 1999; Accepted Augast 9, 1999
We examined the effects of methanol and temperature on the reactivity of monoclonal antibodies specific to the insecticide etofenprox. When the antigen-antibody reaction was done at 4°C in 10％ methanol, the sensitivity in the enzyme immunoassay with each antibody was more than 10-fold higher than that measured at 37°C. Although in 10％ methanol one of the antibodies reacted equally with both etofenprox and the carbonate-derivative of etofenprox, in 50％ methanol the antibody reacted with etofenprox, but not with the derivative.
enzyme immunoassay; organic solvents; insecticide; antigen-antibody reaction

-22-
Note
Resolution and Synthesis of (S)-1-(2-Naphthyl)ethanol
with Immobilized Pea Protein: As a New Biocatalyst

Hiroyuki NAGAOKA＊,＊＊＊ and Hiroshi KAYAHARA＊＊,・

＊The United Graduate School of Agricultural Science, Gufu University, 1-1 Yanagido, Gufu 501-1193, Japan
＊＊Department of Bioscience and Biotechnology, Shinshu University, 8304 Minamiminowa, Kamiina,
Nagano 399-4598 Japan
＊＊＊Sanyo Shokuhin Co., Ltd., 555-4 Asakura, Maebashi, Gunma 371-0811, Japan

Received April 28, 1999; Accepted July 14, 1999
(S)-1-(2-Naphthyl)ethanol was yielded by immobilized pea (Pisum sativum L.) protein (IPP) from (R, S) 2-naphthyl ethanol (>99％ ee, yield; about 50％), in which the (R)-enantiomer was selectively oxidized to 2-acetonaphthone. IPP could be reused consecutively at least three times without any decrease of yield and optical purity.
immobilized pea protein; selective oxidation; biotransformation; (±) 2-naphthyl ethanol; (S)-1-(2-naphthyl)ethanol

-23-
Note
Isolation and Structure of a Novel Indophenol-reducing and
1,1-Diphenyl-2-picrylhydrazyl Radical-scavenging Compound from a Fungus

Chiaki ITO, Naoki ABE, and Akira HIROTA・

Laboratory of Applied Microbiology, School of Food and Nutritional Sciences, University of Shizuoka,
52-1 Yada, Shizuoka 422-8526, Japan

Received May 10, 1999; Accepted July 19, 1999
A novel indophenol-reducing and DPPH radical-scavenging compound (1) was isolated from a fungal metabolite. The structure of 1 was determined by a spectroscopic analysis, and the absolute configuration of 1 was determined by the modified Mosher′s method. Compound 1 showed slow-reacting DPPH radical-scavenging activity (ED50 value of 298 μM after 96 hr).
indophenol-reducing compound; antioxidant; DPPH radical-scavenging activity

-24-
Note
Effects of Amino Acids, Sugars, and Ascorbic Acid on the Stability
of Linoleic Acid Hydroperoxide in the Water Phase

Tamako NISHIIKE,1 Jun ICHIKAWA,＊ Noriko KIKUGAWA,＊ Hitoshi TAKAMURA,＊
and Teruyoshi MATOBA

Division of Human Life and Environmental Sciences, Graduate School of Human Culture,
Nara Women′s University, Kitauoya-Nishimachi, Nara 630-8506, Japan
＊Department of Food Science and Nutrition, Nara Women′s University,
Kitauoya-Nishimachi, Nara 630-8506, Japan

Received May 10, 1999; Accepted July 3, 1999
Although lipid hydroperoxides are known to decrease food quality and safety, the stability of hydroperoxides in foods has hardly been investigated. We examined HPOD decomposition by kinetic means with or without various food components. Most amino acids, especially lysine, arginine and tryptophan, stabilized HPOD, while cysteine and ascorbic acid accelerated its decomposition. Sugars has little effect. According to activation energy calculations, it was found that the HPOD decomposition mechanism in reaction systems with various food components was similar to that in water.
lipid oxidation; linoleic acid; hydroperoxide

-25-
Note
The Trichothecene Biosynthesis Regulatory Gene from the Type B Producer
Fusarium Strains: Sequence of Tri6 and Its Expression in Escherichia coli・

Gentaro MATSUMOTO,1,2 Junko WUCHIYAMA,1 Yoshinori SHINGU,1,2 Makoto KIMURA,・・,1
Katsuyoshi YONEYAMA,2 and Isamu YAMAGUCHI・・・,1

1Microbial Toxicology Laboratory, RIKEN (The Institute of Physical and Chemical Research),
2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan
2Plant Pathology Laboratory, Faculty of Agriculture, Meiji University, Higashimita 1-1-1, Tama-ku,
Kawasaki-shi, Kanagawa 214-8571, Japan

Received May 14, 1999; Accepted July 23, 1999
A genomic DNA fragment containing Tri6, a transcription activator gene of trichothecene biosynthesis, was cloned by vectorette PCR from Fusarium graminearum F15, which produces type B trichothecene, deoxynivalenol. The nucleotide sequence of the gene showed 84％ of identity to that of the type A trichothecene producer Fusarium sporotrichioides NRRL 3299, but the sequence around the initiation codon was not highly conserved between these producers. Based on the upstream and downstream sequences of the coding region of F. graminearum, Tri6 could be amplified by PCR from other type B trichothecene producers. Tri6 appeared to be expressed for only a limited period prior to the toxin production.
Fusarium mycotoxin; trichothecene biosynthesis; transcription regulatory gene; zinc finger motif; PCR-amplified gene

-26-
Note
Effects of Dietary Protein Type on the Response of Lipid Metabolism
to Orotic Acid in Rats

Yoritaka AOYAMA・ and Mizuho WADA

Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University,
Nishi-9, Kita-9, Kita-ku, Sapporo 060-8589, Japan

Received May 21, 1999; Accepted July 5, 1999
The effects of orotic acid supplementation to casein, egg protein, soy protein and wheat gluten diets on the lipids of liver and serum were compared. When orotic acid was added, the contents of total lipids and triacylglycerol in the liver of the casein group were significantly higher or tended to be higher than those of the other three dietary groups. Dietary orotic acid had no effect on the food intake. The liver weight, and liver total lipids, triacylglycerol, cholesterol and phospholipids were increased or tended to be increased by the addition of orotic acid. The serum triacylglycerol level was decreased by the addition of orotic acid to either the casein or soy protein diet. Thus, the response to liver lipid accumulation induced by orotic acid feeding depended on the dietary protein type.
orotic acid; fatty liver; serum lipid; dietary protein

-27-
Note
Kinetics for the Autoxidation of Conjugated Linoleic Acid

Hwan-Sook SEO, Yasushi ENDO,・ and Kenshiro FUJIMOTO

Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi,
Aoba, Sendai 981-8555, Japan

Received May 24, 1999; Accepted July 9, 1999.
The oxidation rates for conjugated linoleic acid (CLA), linoleic acid (LA), and their methyl and ethyl esters were measured in aqueous and solvent systems by the induction period method. In an aqueous system, the initiation rate was almost the same for all the samples, except for ethyl conjugated linoleate (ECL). The propagation rate, oxidizability, and kinetic chain length were different for the ester and free types of CLA and LA. CLA was the most stable, its oxidizability being half that of LA. However, ECL was very susceptible to free radical oxidation. In a solvent system, the CLA derivatives had higher or almost same oxidizability as the LA derivatives, although the propagation rates of CLA and LA were lower than those of their esters.
autoxidation; conjugated linoleic acid; kinetics; linoleic acid

-28-
Note
Induction of GM-CSF Production of Macrophages by Advanced Glycation
End Products of the Maillard Reaction

Toshinori SASAKI,a,・ Seikoh HORIUCHI,b Masatoshi YAMAZAKI,a and Satoru YUIa

aFaculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Tsukui-gun, Kanagawa 199-0195, Japan
bDepartment of Biochemistry, Kumamoto University School of Medicine, Honjo 2-2-1 Kumamoto 860-0811, Japan

Received May 24, 1999; Accepted August 3, 1999
We previously reported that AGEs can induce macrophage growth. In this paper, we examined whether advanced glycation end products (AGE) of protein induced GM-CSF production of macrophages. AGE of bovine serum albumin markedly stimulated not only the expression of GM-CSF mRNA, but also GM-CSF secretion in macrophage supernatant. Thus GM-CSF is suggested to be an endogenous signal for macrophage growth induction by AGEs.
GM-CSF; advanced glycation end products; macrophage growth

-29-
Note
Carotenogenesis Pathway of Novel Carotenoid Glucoside Mycolic Acid Esters
in Rhodococcus rhodochrous Using Carotenogenesis Mutants and Inhibitors

Shinichi TAKAICHI,1,・ Yasumori TAMURA,2,・・ Takeshi MIYAMOTO,1 Ken AZEGAMI,1
Yoko YAMAMOTO,2 and Jun-ichi ISHIDSU1

1Biological Laboratory, Nippon Medical School, Kosugi-cho 2, Nakahara, Kawasaki 211-0063, Japan
2Faculty of Agriculture, Meiji University, Higashimita, Tama, Kawasaki 214-8571, Japan

Received May 26, 1999; Accepted July 2, 1999
Carotenogenesis in Nocardioform actinomycete Rhodococcus rhodochrous was investigated using carotenogenesis mutants and inhibitors, and a postulated carotenogenesis pathway was proposed. At the end of the synthesis, fatty acid or mycolic acid was esterified by different esterases to the same C-6 hydroxyl group of β-D-glucoside.
carotenogenesis mutant; carotenogenesis pathway; carotenoid glucoside mycolic acid ester; Rhodococcus rhodochrous

-30-
Note
Purification and Characterization of β-1,3-Xylanase from a Marine Bacterium,
Vibrio sp. XY-214

Toshiyoshi ARAKI, Shuji TANI, Keiko MAEDA, Shinnosuke HASHIKAWA,
Hiroki NAKAGAWA,＊ and Tatsuo MORISHITA

Faculty of Bioresources, Mie University, Kamihama, Tsu, Mie 514-8507, Japan
＊Faculty of Agriculture, Saga University, Honjyo, Saga 840-8502, Japan

Received May 31, 1999; Accepted July 13, 1999
β-1,3-Xylanase was purified to gel electrophoretic homogeneity and 83-fold from a cell-free culture fluid of Vibrio sp. XY-214 by ammonium sulfate precipitation and successive chromatographies. The enzyme had a pl of 3.6 and a molecular mass of 52 kDa. The enzyme had the highest level of activity at pH 7.0 and 37°C. The enzyme activity was completely inhibited by Cu2＋, Hg2＋, and N-bromosuccinimide. The enzyme hydrolyzed β-1,3-xylan to produce mainly xylotriose and xylobiose but did not act on xylobiose, p-nitrophenyl-β-D-xyloside, β-1,4-xylan, β-1,3-glucan, or carboxymethyl cellulose.
β-1,3-xylanase; β-1,3-xylan; Vibrio; marine bacterium; Caulerpa racemosa

-31-
Note
The Subunit Structure of Nitrite Reductase Purified from the Denitrifier
Achromobacter cycloclastes

Ken-ichi INATOMI・

Advanced Technology R＆D Center, Mitsubishi Electric Corp., 811, Tsukaguchi,
Amagasaki, Hyogo, 661-6881, Japan

Received May 31, 1999; Accepted July 16, 1999
The copper-containing nitrite reductase of Achromobacter cycloclastes has been considered to be a homotrimer with three identical subunits both in the crystal and in solution. In this study, however, the enzyme was found to be a heterotrimer consisting of two subunits with molecular masses of 37 kDa and 36.2 kDa, and the 37 kDa subunit was 6 amino acid residues longer than the smaller subunit. Signal-peptide cleavage sites in its N-terminal region are discussed.
Achromobacter cycloclastes; nitrite reductase; copper enzyme; periplasmic protein; signal peptide

-32-
Note
Effects of a Feedback-Resistant Aspartokinase III Gene
on L-Isoleucine Production in Escherichia coli K-12

Ken-ichi HASHIGUCHI,＊ Hiroshi MATSUI,＊＊ and Osamu KURAHASHI＊

＊Fermentation ＆ Biotechnology Laboratories, and
＊＊Central Research Laboratories, Ajinomoto Co. Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki 210-8681, Japan

Received June 3, 1999; Accepted July 12, 1999
An L-isoleucine-overproducing recombinant strain of E. coli, TVD5, was also found to overproduce L-valine. The L-isoleucine productivity of TVD5 was markedly decreased by addition of L-lysine to the medium. Introduction of a gene encoding feedback-resistant aspartokinase III increased L-isoleucine productivity and decreased L-valine by-production. The resulting strain accumulated 12 g/l L-isoleucine from 40 g/l glucose, and suppression of L-isoleucine productivity by L-lysine was relieved.
L-isoleucine; overproduction; Escherichia coli; aspartokinase III

-33-
Note
Synthesis of Methyl (S)-(−)-6,8-Dihydroxyoctanoate as a Precursor
of (R)-(＋)-α-Lipoic Acid

Makoto GANAHA, Satoshi YAMAUCHI, and Yoshiro KINOSHITA・

Department of Agricultural Chemistry, Faculty of Agriculture, Ehime University,
Tarumi 3-5-7, Matsuyama, Ehime 790-8566, Japan

Received June 7, 1999; Accepted June 25, 1999
The synthesis of methyl (S)-(−)-6,8-dihydroxyoctanoate as a precursor of (R)-(＋)-α-lipoic acid was accomplished by using methyl (S)-(−)-(2-oxocyclohexyl)acetate, which had been obtained from baker′s yeast reduction, as a chiral starting material.
yeast reduction; γ-ketoester; asymmetric reduction; (R)-(＋)-α-lipoic acid

-34-
Note
Lipids of Three Highly Migratory Fishes: Euthynnus affinis, Sarda orientalis,
and Elagatis bipinnulata

Hiroaki SAITO,＊ Rieko YAMASHIRO,・ Kenji ISHIHARA, and Changhu XUE・

Laboratory of Lipid Chemistry, National Research Institute of Fisheries Science, Ministry of Agriculture,
Forestry and Fisheries, 2-12-4 Fuku-ura, Kanazawa-ku, Yokohama-shi 236-8648, Japan

Received June 7, 1999; Accepted July 21, 1999
The fatty acid composition of the total lipids in ordinary muscle and of the stomach contents of the three highly migratory fish species, Euthynnus affinis (Temminck et Schlegel), Sarda orientalis (Canter), and Elagatis bipinnulata (Quoy et Gaimard), was determined. DHA was the dominant fatty acid among the polyunsaturated fatty acids in the tissue lipids of the three fish species and the DHA levels in the lipids of the muscles were comparatively high, while those in the stomach contents originating from their prey were generally low.
chemoecology; docosahexaenoic acid; highly migratory fish; marine grazing food chain; n-3 polyunsaturated fatty acid

-35-
Note
Further Characterization of Earthworm Serine Proteases:
Cleavage Specificity Against Peptide Substrates and on Autolysis

Nobuyoshi NAKAJIMA,1,・ Manabu SUGIMOTO,2 Kohji ISHIHARA,3 Kaoru NAKAMURA,4
and Hiroki HAMADA5

1Department of Nutritional Science, Okayama Prefectural University, Soja, Okayama 719-1112, Japan
2Research Institute for Bioresources, Okayama University, Kurashiki, Okayama 710-0046, Japan
3Department of Chemistry, Kyoto University of Education, Fushimi-ku, Kyoto 612-8522, Japan
4Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan
5Department of Applied Science, Okayama University of Science, Ridai-cho, Okayama 700-0005, Japan

Received June 15, 1999; Accepted July 26, 1999
Cleavage specificity of two fibrinolytic enzymes from Lumbricus rubellus [Nakajima, N., et al., Biosci. Biotechnol. Biochem., 57, 1726--1730 (1993) and 60, 293--300 (1996)] was investigated using β-amyloid 1-40 and oxidized insulin B-chain as peptide substrates. The serine protease, F-III-2, cleaved the former substrate at six sites, and the latter at five sites. F-II digested them at six and ten, respectively. The cleavage specificity of F-III-2 resembled those of both trypsin and chymotrypsin. F-II had a broader specificity than F-III-2 and preferred also the bonds consisting neutral or hydrophobic amino acids. Furthermore, F-III-2 itself was digested initially on the site of Arg(144)-Tyr(145) to produce two peptide fragments, when it was autolyzed regularly by heating.
serine protease; earthworm; cleavage specificity; β-amyloid; fibrinolytic enzyme

-36-
Preliminary Communication
Conversion of Bacillus subtilis 168: Natto Producing Bacillus subtilis
with Mosaic Genomes

Mitsuhiro ITAYA・ and Kuniko MATSUI

Mitsubishi Kasei Institute of Life Sciences, 11 Minamiooya, Machida-shi, Tokyo 194-8511, Japan

Received May 24, 1999; Accepted August 31, 1999
Sequential replacement of sequences in the Bacillus subtilis 168 genome with DNA from Bacillus subtilis (natto) conferred the trait of the ability to ferment soybeans to B. subtilis 168 as its genome became mosaic. All mosaic strains retained competence, an intrinsic polygenic trait of the recipient B. subtilis 168.
Bacillus subtilis; Bacillus subtilis (natto); mosaic genome; horizontal transfer; transformation

-37-
Preliminary Communication
New Cyclization Mechanism for Squalene: a Ring-expansion Step for the
Five-membered C-ring Intermediate in Hopene Biosynthesis

Tsutomu HOSHINO,＊ Masanori KOUDA, Takamasa ABE, and Shumi OHASHI

Department of Applied Biological Chemistry, Faculty of Agriculture, and Graduate School of Science and
Technology, Niigata University, Ikarashi, Niigata 950-2181, Japan

Received June 23, 1999; Accepted August 9, 1999
Three triterpenes having the 6/6/5-fused tri- and 6/6/6/5-fused tetracyclic skeletons were isolated from an incubation mixture of the mutated F601A enzyme, these products being in accordance with a Markovnikov closure. Successful trapping of the tricyclic cationic intermediate by using the squalene analog having a highly nucleophilic hydroxyl group leads us to propose that the ring expansion process of the 5-membered C-ring is involved in the squalene cyclization cascade.
squalene; hopene; triterpene cyclase; 2,3-oxidosqualene

-38-
Preliminary Communication
Cloning and Characterization of a Drosophila melanogaster cDNA Encoding
a Glutamate Transporter

Tsuyoshi KAWANO,1,・ Kyoko TAKUWA,2 Hisato KUNIYOSHI,3 Naoto JUNI,3
Terumi NAKAJIMA,2 Daisuke YAMAMOTO,3 and Yasuo KIMURA1

1Department of Biological and Environmental Chemistry, Faculty of Agriculture, Tottori University,
Koyama-cho, Tottori City, Tottori 680-8553, Japan
2Suntory Institute for Bioorganic Research, Shimamoto-cho, Mishima-gun, Osaka 618-8503, Japan
3ERATO Yamamoto Behavior Genes Project, Minamiooya, Machida City, Tokyo 194-8511, Japan

Received June 29, 1999; Accepted August 11, 1999
A Drosophila cDNA encoding a glutamate transporter was cloned and examined. The predicted protein (479 amino acid residues) shows significant sequence identity with mammalian counterparts. The protein expressed in Xenopus oocytes had a glutamate transport activity. Northern blot analysis showed that the transcript increased in amount developmentally. This expression pattern is different from those of Drosophila glutamate receptors.
cDNA cloning; Drosophila; expression; glutamate transporter; insec