Contents and Abstracts of Latest Issue of BBB

(Vol.66 No.7 2002)

Growth-promoting Activity of Pyrazinoic Acid, a Putative Active
Compound of Antituberculosis Drug Pyrazinamide, in Niacin-deficient
Rats through the Inhibition of ACMSD Activity
Tsutomu FUKUWATARI,† Etsuro SUGIMOTO, and Katsumi SHIBATA p.1435

Thermal Inactivation and Product Inhibition of Aspergillus terreus CECT
2663 α-L-Rhamnosidase and Their Role on Hydrolysis
of Naringin Solutions
Fernando SORIA and Guillermo ELLENRIEDER† p.1442

2-[3-(2-Thioxopyrrolidin-3-ylidene)methyl]-tryptophan, a Novel Yellow
Pigment in Salted Radish Roots
Hiroki MATSUOKA,1,† Asaka TAKAHASHI,2 Yoshio OZAWA,1 Yoichi YAMADA,3
Yasushi UDA,4 and Shunro KAWAKISHI5 p.145

Changes in Activity Coefficient γw of Water and the Foaming Capacity
of Protein during Hydrolysis
Hitomi KUMAGAI,1 Hirotaka SETO,1 Yuko NORIMATSU,1 Kenji ISHII,1
and Hitoshi KUMAGAI2,† p.1455

Isolation and Characterization of Dibenzofuran-degrading Actinomycetes:
Analysis of Multiple Extradiol Dioxygenase Genes
in Dibenzofuran-degrading Rhodococcus Species
Toshiya IIDA,1,† Yuki MUKOUZAKA,1,2 Kaoru NAKAMURA,1 Isamu YAMAGUCHI,3
and Toshiaki KUDO1,4,5 p.1462

Phenotype of Hepatocyte Spheroids in Arg-Gly-Asp (RGD) Containing
a Thermo-Reversible Extracellular Matrix
Keun-Hong PARK1,† and You Han BAE2 p.1473

Relationship between Estrogen Receptor-Binding and Estrogenic Activities
of Environmental Estrogens and Suppression by Flavonoids
Dal-Ho HAN,1,† Michael S. DENISON,1 Hirofumi TACHIBANA,2 and Koji YAMADA2 p.1479

Foreign Insect Hormones Stimulating the Transcription of the ie-1 Promoter
of Bombyx mori Nuclear Polyhedrosis Virus in Vivo and in Vitro
Yajing ZHOU,1,2 Qingli XIAO,1 Zhifang ZHANG,1,† Jialu HE,1 and Yuanxing ZHANG2
p.1488

Synthesis of 1,2-Oxygenated 6-arylfurofuran Lignan: Stereoselective
Synthesis of (1S,2S,5R,6S)-1-hydroxysamin
Satoshi YAMAUCHI,† Satoshi BANDO, and Yoshiro KINOSHITA p.1495

Recognition of a Cysteine Substrate by E. coli γ-Glutamylcysteine Synthetase
Probed by Sulfoximine-based Transition-state Analogue Inhibitors
Jun HIRATAKE,† Takayuki IRIE, Nobuya TOKUTAKE, and Jun′ichi ODA†† p.1500

Guanosine 5′-diphosphate 3′-diphosphate (ppGpp) Synthetic Activities
on Escherichia coli SpoT Domains
Chizuko FUJITA, Akiko NISHIMURA,＊ Ryoko IWAMOTO, and Kenji IKEHARA† p.1515

Structural Analysis of an Extracellular Polysaccharide
Bioflocculant of Klebsiella pneumoniae
Takayoshi KOBAYASHI,1 Yasuyuki TAKIGUCHI,1† Yuuki YAZAWA,1 Kuniho NAKATA,2
Tatsuaki YAMAGUCHI,1 and Ryuichiro KURANE1＊ p.1524

Synthesis of (2S,2′R,3S,4R)-2-(2′-Hydroxy-21′-methyldocosanoylamino)-
1,3,4-pentadecanetriol, the Ceramide Sex Pheromone of the Female
Hair Crab, Erimacrus isenbeckii†
Yui MASUDA,1 Masao YOSHIDA,1 and Kenji MORI2,†† p.1531

Significance of the 20-kDa Subunit of Heterodimeric 2-Deoxy-scyllo-inosose
Synthase for the Biosynthesis of Butirosin Antibiotics in Bacillus circulans
Hideyuki TAMEGAI, Eriko NANGO, Ayumi KOIKE-TAKESHITA,
Fumitaka KUDO, and Katsumi KAKINUMA† p.1538

Structural Analysis of the Extracellular Polysaccharide Produced
by Sphaerotilus natans
Minoru TAKEDA,† Shogo NOMOTO, and Jun-ichi KOIZUMI p.1546

Note
α-Glucosidase Inhibitory Activity of a 70％ Methanol Extract
from Ezoishige (Pelvetia babingtonii de Toni) and Its Effect
on the Elevation of Blood Glucose Level in Rats
Tomoki OHTA,1,† Shigefumi SASAKI,1 Tadashi OOHORI,1 Shuji YOSHIKAWA,1
and Hideyuki KURIHARA2 p.1552

Note
Production of Long-chain Levan by a sacC Insertional Mutant
from Bacillus subtilis 327UH
Toshio SHIDA, Kohsuke MUKAIJO, Shu ISHIKAWA, Hiroki YAMAMOTO,
and Junichi SEKIGUCHI† p.1555

Note
Effect of Tea Catechins on Cellular Lipid Peroxidation
and Cytotoxicity in HepG2 Cells
Chikako MURAKAMI, Yuki HIRAKAWA, Hiroshi INUI,＊ Yoshihisa NAKANO,＊
and Hiromi YOSHIDA† p.1559

Note
Total DNA Preparation Excluding Extracellular Acidic Polysaccharide
from Lipomyces Yeasts and its Application to Taxonomic Studies
Katsushi NISHIMURA,1,† Koji SHIMADA,2 Hiroki IWASAWA,2 Takafumi NAGANUMA,2
and Yasuyuki UZUKA2 p.1563

Note
Structure of Genes for Hsp30 from the White-rot fungus Coriolus versicolor
and the Increase of their Expression by Heat Shock and Exposure
to a Hazardous Chemical
Yosuke IIMURA† and Kenji TATSUMI p.1567

Note
Isolation and Measurement of Quercetin Glucosides in Flower Buds
of Japanese Butterbur (Petasites japonicus subsp. gigantea Kitam.)
Hideyuki MATSUURA,1,＊,† Midori AMANO,1 Jun KAWABATA,2 and Junya MIZUTANI3
p.1571

Note
α-Glucosidase Inhibitor from the Seeds of Balsam Pear (Momordica charantia)
and the Fruit Bodies of Grifola frondosa
Hideyuki MATSUURA,1,＊,† Chikako ASAKAWA,1 Masanori KURIMOTO,1
and Junya MIZUTANI2 p.1576

Note
Microbial Degradation of Lipid by Acinetobacter sp. Strain SOD-1
Daisuke SUGIMORI,† Masatoshi NAKAMURA, and Yuma MIHARA p.1579

Note
DnaK Chaperone Machine and Trigger Factor are Only Partially
Required for Normal Growth of Bacillus subtilis
Dindo Y. REYES and Hirofumi YOSHIKAWA† p.1583

Note
Cloning and Expression of the Exo-β-D-1,3-glucanase Gene (exgS)
from Aspergillus saitoi††
Ken ODA,† Shin KASAHARA, Youhei YAMAGATA, Keietsu ABE, and Tasuku NAKAJIMA
p.1587

Note
Synthesis of the (17R)- and (17S)-Isomers of Volicitin, an Elicitor of Plant
Volatiles Contained in the Oral Secretion of the Beet Armyworm
Seiji ITOH,1,2 Shigefumi KUWAHARA,2,† Morifumi HASEGAWA,1 and Osamu KODAMA1
p.1591

Note
Isolation of Enterococcus hirae Mutant Deficient in Low-affinity
Potassium Uptake at Alkaline pH
Miyuki KAWANO,1 Kazuei IGARASHI,1 and Yoshimi KAKINUMA2,† p.1597

Note
A Highly Sensitive Assay for Proteases Using Staphylococcal Protein A
Fused with Enhanced Green Fluorescent Protein
Hiroyoshi FUJINO,1,2 Takashi AOKI,1,† and Hiroyuki WATABE1 p.1601

Note
Effects of Dietary Eritadenine on Δ6-Desaturase Activity and Fatty
Acid Profiles of Several Lipids in Rats Fed Different Fats
Yasuhiko SHIMADA, Tatsuya MORITA, and Kimio SUGIYAMA† p.1605

Preliminary Communication
Suppressive Effect of Polysaccharides from the Edible and Medicinal
Mushrooms, Lentinus edodes and Agaricus blazei,
on the Expression of Cytochrome P450s in Mice
Takashi HASHIMOTO,1 Yuji NONAKA,2 Ken-ichiro MINATO,3 Sachiko KAWAKAMI,1,4
Masashi MIZUNO,1 Itsuko FUKUDA,2 Kazuki KANAZAWA,1 and Hitoshi ASHIDA1,2,†
p.1610

Preliminary Communication
Binding Selectivity of Conformationally Restricted Analogues
of (−)-Indolactam-V to the C1 Domains of Protein Kinase C Isozymes
Akiko MASUDA, Kazuhiro IRIE,† Yu NAKAGAWA, and Hajime OHIGASHI p.1615

-1-
Growth-promoting Activity of Pyrazinoic Acid, a Putative Active
Compound of Antituberculosis Drug Pyrazinamide, in Niacin-deficient
Rats through the Inhibition of ACMSD Activity

Tsutomu FUKUWATARI,† Etsuro SUGIMOTO, and Katsumi SHIBATA

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

Received September 27, 2001; Accepted March 7, 2002
We have recently reported that the antituberculosis drug, pyrazinamide (PZA), caused a significant increase in the conversion ratio of tryptophan to niacin in rats. In the present work, we investigated whether or not pyrazinoic acid (POA), a putative metabolite of PZA, increased the conversion ratio of tryptophan to niacin. Weaning rats were fed with a niacin-free and tryptophan-limited diet (negative control diet), or with the negative control diet supplemented with 0.003％ nicotinic acid (positive control diet) or 1％ POA (test diet) for 27 days. The growth rate was almost same between the groups fed on the positive control diet and the test diet. Dietary POA significantly increased the conversion ratio of tryptophan to niacin. Although POA did not directly inhibit the activity of α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD), the rate-limiting enzyme in the tryptophan-niacin pathway, liver ACMSD activity was only not detected in the test diet group. These results suggest that a derivative of POA metabolized by rats inhibited the ACMSD activity.
Key words: pyrazinamide; pyrazinoic acid; tryptophan metabolism; nicotinamide; tryptophan-niacin conversion ratio

-2-
Thermal Inactivation and Product Inhibition of Aspergillus terreus CECT
2663 α-L-Rhamnosidase and Their Role on Hydrolysis
of Naringin Solutions

Fernando SORIA and Guillermo ELLENRIEDER†

Chemical Industry Research Institute (INIQUI), National University of Salta-CONICET,
Buenos Aires 177-4400 Salta, Argentina

Received October 26, 2001; Accepted March 8, 2002
The kinetics of thermal inactivation of A. terreus α-rhamnosidase was studied using the substrate p-nitrophenyl α-L-rhamnoside between 50°C and 70°C. Up to 60°C the inactivation of the purified enzyme was completely reversible, but samples of crude or partially purified enzyme showed partial reversibility. The presence of the product rhamnose, the substrate naringin, and other additives reduced the reversible inactivation, maintaining in some cases full enzyme activity at 60°C. A mechanism for the inactivation process, which permitted the reproduction of experimental results, was proposed. The products rhamnose (inhibition constant, 2.1 mM) and prunin (2.6 mM) competitively inhibited the enzyme reaction. The maximum hydrolysis of supersaturated naringin solution, without enzyme inactivation, was observed at 60°C. Hydrolysis of naringin reached 99％ with 1％ naringin solution, although the hydrolysis degree of naringin was only 40％ due to products inhibition when the initial concentration of flavonoid was 10％. The experimental results fitted an equation based on the integrated Michaelis-Menten′s, including competitive inhibition by products satisfactorily.
Key words: α-L-Rhamnosidase; Rhamnose; thermal inactivation; product inhibition

-3-
2-[3-(2-Thioxopyrrolidin-3-ylidene)methyl]-tryptophan, a Novel Yellow
Pigment in Salted Radish Roots

Hiroki MATSUOKA,1,† Asaka TAKAHASHI,2 Yoshio OZAWA,1 Yoichi YAMADA,3
Yasushi UDA,4 and Shunro KAWAKISHI5

1Department of Health and Nutrition, Takasaki University of Health and Welfare, Nakaorui-cho,
Takasaki 370-0033, Japan
2Department of Food Hygiene, Nagano Prefectural College, Miwa, Nagano 380-8525, Japan
3Faculty of Education, Utsunomiya University, Utsunomiya 321-8505, Japan
4Department of Bioproductive Sciences, Utsunomiya University, Utsunomiya 321-8505, Japan
5Department of Food and Nutrition, Sugiyama Jogakuen University, Nagoya 464-8662, Japan

Received November 14, 2001; Accepted March 19, 2002
The structure of the yellow pigment found in salted radish roots was studied. It was found that 1-(2-thioxopyrrolidin-3-yl)-1,2,3,4-tetrahydro-β-carboline-3-
carboxylic acid (TPCC) was unstable under neutral pH, and was easily converted into the yellow pigment. The yellow pigment was isolated and identified as 2-[3-(2-thioxopyrrolidin-3-ylidene)methyl]-tryptophan (TPMT) by IR, MS, 1H-, and 13C-NMR spectroscopy. In addition, we proved that this compound was the main yellow pigment in salted radish roots. This compound induced no mutagenicity in Salmonella typhimurium TA98 and TA100, either with or without prior activation.
Key words: yellow pigment; tryptophan derivative; tetrahydro-β-carboline derivative; salted radish roots; 4-methylthio-3-butenyl isothiocyanate

-4-
Changes in Activity Coefficient γw of Water and the Foaming Capacity
of Protein during Hydrolysis

Hitomi KUMAGAI,1 Hirotaka SETO,1 Yuko NORIMATSU,1 Kenji ISHII,1
and Hitoshi KUMAGAI2,†

1Department of Agricultural and Biological Chemistry, Nihon University, 1866 Kameino, Fujisawa-shi,
Kanagawa 252-8510, Japan
2Department of Applied Biological Chemistry, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku,
Tokyo 113-8657, Japan

Received November 16, 2001; Accepted March 11, 2002
The changes in the interaction between food proteins and water and in their surface functional property during enzymatic hydrolysis were investigated. Ovalbumin, a soy protein isolate (SPI), and casein were hydrolyzed with trypsin, and the degree of hydrolysis, water activity aw, and foaming capacity of each hydrolysate were measured. Ovalbumin showed the minimum value for aw, and the values for SPI and casein progressively decreased during hydrolysis. Therefore, the activity coefficient of water, γw (＝aw/xw, where xw is the mole fraction of water) was obtained to remove the influence of mole change and to examine the interaction of protein hydrolysates with water. In order to calculate xw in a sample during protein hydrolysis, a method for roughly estimating the number of moles of the protein hydrolysate in a solution was developed. The strategy was to modify the TNBS (2, 4, 6-trinitrobenzenesulfonic acid) method and to combine this method with the modified Ellman method and the determination of lysine by an amino acid analyzer. During enzymatic hydrolysis, each protein sample showed a minimum γw value and maximum foaming capacity.
Key words: water activity; activity coefficient of water; protein; trypsin; foaming capacity

-5-
Isolation and Characterization of Dibenzofuran-degrading Actinomycetes:
Analysis of Multiple Extradiol Dioxygenase Genes
in Dibenzofuran-degrading Rhodococcus Species

Toshiya IIDA,1,† Yuki MUKOUZAKA,1,2 Kaoru NAKAMURA,1 Isamu YAMAGUCHI,3
and Toshiaki KUDO1,4,5

1Microbiology Laboratory, RIKEN (The Institute of Physical and Chemical Research), Wako,
Saitama 351-0198, Japan
2Graduate School of Science and Engineering, Saitama University, Saitama, Saitama 338-0825, Japan
3Environmental Plant Research Group, RIKEN (The Institute of Physical and Chemical Research),
Wako, Saitama 351-0198, Japan
4Bio-Recycle Project, Japan Science and Technology Corporation (JST), Wako, Saitama 351-0198, Japan
5Science of Biological Supramolecular Systems, Graduate School of Integrated Science, Yokohama City
University, Suehiro, Tsurumi-ku, Yokohama 230-0045, Japan

Received November 21, 2001; Accepted March 11, 2002
Sixteen actinomycetes capable of utilizing dibenzofuran as a sole source of carbon and energy were isolated, including Rhodococcus, Microbacterium, and Terrabacter genera. Heretofore, no dibenzofuran-utilizing strain belonging to the genus Microbacterium has been reported. Five extradiol dioxygenase genes (dfdB, and edi1 to 4) of the strain Rhodococcus sp. YK2 were cloned and analyzed. The nucleotide sequence of dfdB gene was almost identical to the bphC1 gene of Terrabacter sp. DPO360, which was involved in dibenzofuran metabolism in this strain. Southern and Northern hybridization analyses using these extradiol dioxygenase genes as probes suggest that the dfdB gene in YK2 was conserved in diverse dibenzofuran-utilizing actinomycetes; also, the dfdB gene was the only expressed gene among five extradiol dioxygenase genes in the medium containing DF as a sole carbon source. These results suggest that the dfdB gene is important for dibenzofuran metabolism not only in the strain YK2, but also in diverse dibenzofuran-degrading actinomycetes.
Key words: dibenzofuran; Rhodococcus; Microbacterium; Terrabacter; extradiol dioxygenase

-6-
Phenotype of Hepatocyte Spheroids in Arg-Gly-Asp (RGD) Containing
a Thermo-Reversible Extracellular Matrix

Keun-Hong PARK1,† and You Han BAE2

1Department of Oral and Maxillofacial Surgery, College of Dentistry, Seoul National University,
28-2 Yeongun-dong Jongro-ku, Seoul 110-749, Korea
2Department of Materials Science ＆ Engineering, Kwangju Institute of Science and Technology,
1 Oryong-dong Puk-gu, Kwangju 500-712, Korea

Received December 10, 2001; Accepted March 8, 2002
The spheroid of specific cells is often regarded as the better form in artificial organs and mammalian cell bioreactors for improved cell-specific functions. In this study, freshly harvested primary rat hepatocytes, which had been cultivated as spheroids and entrapped in a synthetic thermo-reversible extracellular matrix, were examined for differentiated morphology and enhanced liver-specific functions as compared to a control set (hepatocytes in single-cell form). A copolymer of N-isopropylacrylamide (98 mole ％ in the feed) and acrylic acid (poly(NiPAAm-co-AAc)), and the adhesion molecule, an Arg-Gly-Asp (RGD)-incorporated thermo-reversible matrix, were used to entrap hepatocytes in the form of either spheroids or single cells. In a 28-day culture period, the spheroids in the RGD-incorporated gel maintained higher viability and produced albumin and urea at constant rates, while there was lower cell viability and less albumin secretion by the spheroids in p(NiPAAm-co-AAc). Hepatocytes cultured as spheroids in the RGD-incorporated gel would constitute a potentially useful three-dimensional cell system for application in a bio-artificial liver device.
Key words: artificial liver; thermo-reversible extracellular matrix; hepatocytes; spheroids

-7-
Relationship between Estrogen Receptor-Binding and Estrogenic Activities
of Environmental Estrogens and Suppression by Flavonoids

Dal-Ho HAN,1,† Michael S. DENISON,1 Hirofumi TACHIBANA,2 and Koji YAMADA2

1Department of Environmental Toxicology, University of California, Davis, CA 95616-8588, USA
2Laboratory of Food Chemistry, Department of Bioscience and Biotechnology, Division of Bioresource
and Bioenvironmental Science, Graduate School, Kyushu University, Fukuoka 812-8581, Japan

Received December 14, 2001; Accepted February 4, 2002
In this study, we investigated the estrogenic activity of environmental estrogens by a competition binding assay using a human recombinant estrogens receptor (hERβ) and by a proliferation assay using MCF-7 cells and a sulforhodamine-B assay. In the binding assay, pharmaceuticals had a stronger binding activity to hERβ than that of some phytoestrogens (coumestrol, daidzein, genistein, luteolin, chrysin, flavone, and naringenin) or industrial chemicals, but phytoestrogens such as coumestrol had a binding activity as strong as pharmaceuticals such as 17α-ethynylestradiol (EE), tamoxifen (Tam), and mestranol. In the proliferation assay, pharmaceuticals such as diethylstilbestrol, EE, Tam, and clomiphene, and industrial chemicals such as 4-nonylphenol, bisphenol A, and 4-dihydroxybiphenyl had a proliferation-stimulating activity as strong as 17β-estradiol (ES). In addition, we found that phytoestrogens such as coumestrol, daidzein, luteolin, and quercetin exerted a proliferation stimulating activity as strong as ES. Furthermore, we examined the suppression of proliferation-stimulating activity, induced by environmental estrogen, by flavonoids, such as daidzein, genistein, quercetin, and luteolin, and found that these flavonoids suppressed the induction of the proliferation-stimulating activity of environmental estrogens. The suppressive effect of flavonoids suggests that these compounds have anti-estrogenic and anti-cancer activities.
Key words: 17β-estradiol; environmental estrogens; flavonoids

-8-
Foreign Insect Hormones Stimulating the Transcription of the ie-1 Promoter
of Bombyx mori Nuclear Polyhedrosis Virus in Vivo and in Vitro

Yajing ZHOU,1,2 Qingli XIAO,1 Zhifang ZHANG,1,† Jialu HE,1 and Yuanxing ZHANG2

1Key Laboratory of Silkworm Biotechnology, Ministry of Agriculture, Sericultural Research Institute,
Chinese Academy of Agricultural Sciences, Zhenjiang, 212018, Jiangsu, China
2State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology,
Shanghai 200237, China

Received January 7, 2002; Accepted March 8, 2002
Via a transient expression assay system, an experimental study was undertaken to characterize the effects of insect ecdysone and juvenile hormone analogue on the transient expression of the luciferase gene under the control of the immediate-early gene (ie-1) promoter of Bombyx mori nuclear polyhedrosis virus. The results demonstrated that the transcriptional activity of the ie-1 promoter was increased to a certain extent by different insect hormone treatments in uninfected insect cells or fifth instar silkworm larvae transfected with a plasmid containing a luciferase gene driven by the ie-1 promoter. By ecdysone treatment alone, an increase of 5--7 fold was reached in Bm-N, or Bm-5 cells, or in the early developmental stage of fifth instar larvae. By treatment with juvenile hormone analogue alone, about 2-fold, in Bm-N, Bm-5, and Sf-21 cells, or about 5-fold increase in the middle developmental stage of larvae was given, respectively. By co-treatment with ecdysone and juvenile hormone analogue, the incease was given between that of ecdysone and juvenile hormone analogue treatment alone. In addition, the synergistic effects of foreign/endogenous hormones on the activity of ie-1 promoter are discussed.
Key words: Bm-N cell line; ecdysone; ie-1 promoter; juvenile hormone analogue; silkworms

-9-
Synthesis of 1,2-Oxygenated 6-arylfurofuran Lignan: Stereoselective
Synthesis of (1S,2S,5R,6S)-1-hydroxysamin

Satoshi YAMAUCHI,† Satoshi BANDO, and Yoshiro KINOSHITA

College of Agriculture, Ehime University, Tarumi 3-5-7, Matsuyama, Ehime 790-8566, Japan

Received January 9, 2002; Accepted March 5, 2002
(1S,2S,5R,6S)-6-(3,4-Methylenedioxyphenyl)-3,7-
dioxabicyclo[3.3.0]octan-1,2-diol ((＋)-1-hydroxysamin 1) was synthesized, starting from olefin 8. Stereoselective α-hydroxylation was achieved after converting 8 to aldehyde 13. Resulting unstable α-hydroxy aldehyde 14 was then transformed to (＋)-1-hydroxysamin (1). This is a new efficient synthetic route to 1,2-oxygenated 6-arylfurofuran lignans.
Key words: lignan; furofuran lignan

-10-
Recognition of a Cysteine Substrate by E. coli γ-Glutamylcysteine Synthetase
Probed by Sulfoximine-based Transition-state Analogue Inhibitors

Jun HIRATAKE,† Takayuki IRIE, Nobuya TOKUTAKE, and Jun′ichi ODA††

Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan

Received January 15, 2002; Accepted February 26, 2002
A series of sulfoximine-based transition-state analogue inhibitors with a varying alkyl side chain was synthesized to probe the recognition of a Cys substrate by E. coli γ-glutamylcysteine synthetase (γ-GCS). The sulfoximines with a small alkyl group (H, methyl, ethyl, propyl, butyl and CH2OH) each served as a slow-binding inhibitor, the sulfoximine with an ethyl being by far the most potent inhibitor to cause facile and irreversible enzyme inhibition. As the size of the side chain changed from an ethyl, the inhibition potency markedly decreased to reduce the overall affinity with concomitant loss in the inactivation rate and with facile enzyme reactivation by dilution. The sulfoximine without a side chain inhibited the enzyme with almost the same potency as that of L-buthionine-(SR)-sulfoximine (BSO). The free energy difference calculated from the inhibition constants indicates that the side chain of Cys was recognized by its size through hydrophobic interaction and contributed almost equally or even more than the carboxy group to the overall binding of Cys in the transition state.
Key words: E. coli γ-glutamylcysteine synthetase; sulfoximine; transition-state analogue inhibitor; substrate specificity; transition-state stabilization

-11-
Guanosine 5′-diphosphate 3′-diphosphate (ppGpp) Synthetic Activities
on Escherichia coli SpoT Domains

Chizuko FUJITA, Akiko NISHIMURA,＊ Ryoko IWAMOTO, and Kenji IKEHARA†

Department of Chemistry, Faculty of Science, Nara Women′s University, Kita-uoya-nishi-machi,
Nara 630-8506, Japan
＊National Institute for Genetics of Japan, Mishima, Shizuoka 411-8540, Japan

Received January 16, 2002; Accepted March 5, 2002
Escherichia coli SpoT protein, with 702 amino acid residues, is a bifunctional enzyme catalyzing both guanosine 5′-diphosphate 3′-diphosphate (ppGpp) degradation and its synthesis. First, we investigated how many domains are included in SpoT protein, by limited hydrolysis of the protein with serine proteases, α-chymotrypsin, and elastase. Based on the results, we deduced that SpoT protein is composed of two major domains, an N-terminal half domain from Met1 to Phe373 and a C-terminal half domain from Glu374 to Asn702 (C-terminal end). In addition, by a further α-chymotrypsin digestion, two cleaved sites were found at Arg196 in the N-terminal half domain (D12) and at Lys475 in the C-terminal half domain (D34), to produce four minor domains, D1, D2, D3, and D4. Next, plasmids expressing the two major domains (D12 and D34) and four minor domains (D1, D2, D3, and D4) were constructed. Consequently, the deduced SpoT minor domains as well as the major domains were expressed as stable protein units, except for D4. D4 may also be folded into a stable protein in E. coli cells, since high expression of D4 from a plasmid results in host cell lethality. E. coli relA−, spoT− double null strains expressing D1, D2, and D12 recovered cell growth in M9 minimal medium, but the transformants of D3, D4, and D34 did not grow in the minimal medium. This indicates that ppGpp synthetic activities could be restricted in the N-terminal half domain (D12, D1, and D2).
Key words: stringent response; ppGpp synthesis; SpoT protein; protease digestion; domain structure

-12-
Structural Analysis of an Extracellular Polysaccharide
Bioflocculant of Klebsiella pneumoniae

Takayoshi KOBAYASHI,1 Yasuyuki TAKIGUCHI,1† Yuuki YAZAWA,1 Kuniho NAKATA,2
Tatsuaki YAMAGUCHI,1 and Ryuichiro KURANE1＊

1Department of Industrial Chemistry, Faculty of Engineering, Chiba Institute of Technology, 2-17-1,
Tsudanuma, Narashino 275-0016, Japan
2Japan Bioindustry Association, Grande Bldg. 8F, 2-26-9, Hatchobori, Chuo-ku, Tokyo 104-0032, Japan

Received January 21, 2002; Accepted March 29, 2002
The glycoside composition and sequence of an extracellular polysaccharide flocculant of Klebsiella pneumoniae H12 was analyzed. GC and HPLC analysis of the acid-hydrolysate identified its constituent monosaccharides as D-Glc, D-Man, D-Gal, and D-GlcA in an approximate molar ratio of 3.9:1.0:2.3:3.6. To analyze the glycoside sequence, the polysaccharide was partially hydrolyzed by acid and enzyme treatment. GC, HPLC, TLC, MALDI-TOF/MS, and 1H- and 13C- NMR spectroscopy characterized the obtained oligosaccharides.
The results clarified the partial structure of H12 polysaccharide as a linear polymer of a unit of pentasaccharide with a side chain of one D-GlcA to D-Glc moiety (see below). Although the existence of other sequences or other constituent glycosides could not be fully excluded, H12 polysaccharide must be a novel types as such a complicated unit for a polymer has not so far been reported. The partial structure of a H12 polysaccharide flocculant is also discussed in this report.
→4)-α-D-Glcp-(1→2)-α-D-Manp-(1→3)-4,6-Pyr-β-D-
Galp-(1→4)-β-D-Galp-(1→→4)-α-D-G3
↓
1
β-D-GlcpA
Key words: Klebsiella pneumoniae; polysaccharide; oligosaccharides; flocculant

-13-
Synthesis of (2S,2′R,3S,4R)-2-(2′-Hydroxy-21′-methyldocosanoylamino)-
1,3,4-pentadecanetriol, the Ceramide Sex Pheromone of the Female
Hair Crab, Erimacrus isenbeckii†

Yui MASUDA,1 Masao YOSHIDA,1 and Kenji MORI2,††

1Department of Chemistry, Graduate School of Science, Science University of Tokyo, Kagurazaka 1-3,
Shinjuku-ku, Tokyo 162-8601, Japan
2Insect Pheromone and Traps Division, Fuji Flavor Co. Ltd., Midorigaoka 3-5-8, Hamura-City,
Tokyo 205-8503, Japan

Received January 22, 2002; Accepted March 1, 2002
The ceramide sex pheromone [(2S,2′R,3S,4R)-2-
(2′-hydroxy-21′-methyldocosanoylamino)-1,3,4-pen-
tadecanetriol (1)] of the female hair crab (Erimacrus isenbeckii) was synthesized by starting from (S)-serine and 12-bromo-1-dodecanol.
Key words: ceramide; crab; Erimacrus isenbeckii; pheromone; sphingolipid, synthesis

-14-
Significance of the 20-kDa Subunit of Heterodimeric 2-Deoxy-scyllo-inosose
Synthase for the Biosynthesis of Butirosin Antibiotics in Bacillus circulans

Hideyuki TAMEGAI, Eriko NANGO, Ayumi KOIKE-TAKESHITA,
Fumitaka KUDO, and Katsumi KAKINUMA†

Department of Chemistry, Tokyo Institute of Technology, 2-12-1 O-okayama, Meguro-ku,
Tokyo 152-8551, Japan

Received January 24, 2002; Accepted March 20, 2002
A gene (btrC2) encoding the 20-kDa subunit of 2-deoxy-scyllo-inosose (DOI) synthase, a key enzyme in the biosynthesis of 2-deoxystreptamine, was identified from the butirosin-producer Bacillus circulans by reverse genetics. The deduced amino acid sequence of BtrC2 closely resembled that of YaaE of B. subtilis, but the function of the latter has not been known to date. Instead, BtrC2 appeared to show sequence similarity to a certain extent with HisH of B. subtilis, an amidotransferase subunit of imidazole glycerol phosphate synthase. Disruption of btrC2 reduced the growth rate compared with the wild type, and simultaneously antibiotic producing activity was lost. Addition of NH4Cl to the medium complemented only the growth rate of the disruptant, and both the growth rate and antibiotic production were restored by addition of yeast extract. In addition, a heterologous co-expression system of btrC2 with btrC was constructed in Escherichia coli. The simultaneously over-expressed BtrC2 and BtrC constituted a heterodimer, the biochemical features of which resembled those of DOI synthase from B. circulans more than those of the recombinant homodimeric BtrC. Despite the similarity of BtrC2 to HisH the heterodimer showed neither aminotransfer nor amidotransfer activity for 2-deoxy-scyllo-inosose as a substrate. All the observations suggest that BtrC2 is involved not only in the secondary metabolism, but also in the primary metabolism in B. circulans. The function of BtrC2 in the butirosin biosynthesis appears to be indirect, and may be involved in stabilization of DOI synthase and in regulation of its enzyme activity.
Key words: butirosin-binsynthesis; 2-deoxy-scyllo-inosose synthase; 20-kDa subunit

-15-
Structural Analysis of the Extracellular Polysaccharide Produced
by Sphaerotilus natans

Minoru TAKEDA,† Shogo NOMOTO, and Jun-ichi KOIZUMI

Division of Materials Science and Chemical Engineering, Faculty of Engineering, Yokohama National
University, Yokohama, Kanagawa 240-8501, Japan

Received February 13, 2002; Accepted March 18, 2002
An extracellular polysaccharide (EPS) was recovered and purified from the culture fluid of a sheathed bacterium, Sphaerotilus natans. Glucose, rhamnose, and aldobiouronic acid were detected in the acid hydrolysate of EPS by thin-layer chromatography (TLC). The aldobiouronic acid was found to be composed of glucuronic acid and rhamnose by TLC and gas-liquid chromatography analyses of the corresponding neutral disaccharide. The structure of EPS was identified by methylation linkage analysis and nuclear magnetic resonance. Additionally, partial acid hydrolysates of EPS were prepared and put through fast atom bombardment-mass spectrometry to determine the sugar sequence of EPS. The resulting data showed that EPS produced by S. natans is a new gellan-like polysaccharide constructed from a tetrasaccharide repeating unit, as shown below.
→4)-α-D-Glcp-(1→2)-β-D-GlcAp-(1→2)-α-L-Rhap-
(1→3)-β-L-Rhap-(1→
Key words: Sphaerotilus natans; extracellular polysaccharide; structural analysis

-16-
Note
α-Glucosidase Inhibitory Activity of a 70％ Methanol Extract
from Ezoishige (Pelvetia babingtonii de Toni) and Its Effect
on the Elevation of Blood Glucose Level in Rats

Tomoki OHTA,1,† Shigefumi SASAKI,1 Tadashi OOHORI,1 Shuji YOSHIKAWA,1
and Hideyuki KURIHARA2

1Hokkaido Food Processing Research Center, Bunkyoudai-midorimachi, Ebetsu, Hokkaido 069-0836, Japan
2Graduate School of Fisheries Sciences, Hokkaido University, Minato, Hakodate, Hokkaido 041-8611, Japan

Received September 20, 2001; Accepted February 19, 2002
The 70％ methanol extract from ezoishige (Pelvetia babingtonii de Toni) inhibited the rat-intestinal α-glucosidase, sucrase and maltase activities, with IC50 values of 2.24 and 2.84 mg/ml. Sucrose was orally administered with or without the extract to rats at 1000 mg/kg. The postprandial elevation in the blood glucose level at 15 and 30 min after the administration of sucrose with the extract was significantly suppressed when compared with the control. These results suggest that the extract from ezoishige has potent α-glucosidase inhibitors and would be effective for suppressing postprandial hyperglycemia.
Key words: inhibitor; α-glucosidase; Pelvetia babingtonii de Toni

-17-
Note
Production of Long-chain Levan by a sacC Insertional Mutant
from Bacillus subtilis 327UH

Toshio SHIDA, Kohsuke MUKAIJO, Shu ISHIKAWA, Hiroki YAMAMOTO,
and Junichi SEKIGUCHI†

Department of Applied Biology, Faculty of Textile Science and Technology, Shinshu University,
3-15-1 Tokida, Ueda, Nagano 386-8567, Japan

Received October 2, 2001; Accepted March 21, 2002
A hyper extracellular protein producer, Bacillus subtilis 327UH, produced large amounts of levan in a medium containing 20％ sucrose, and the yield of levan after 10 hours was more than 60％, when based on the fructose amount of sucrose. After transformation of 327UH with a levanase-deficient 168SC (sacC::Cmr) chromosomal DNA, a Cmr transformant 327UHSC (sacC::Cmr degSU(Hy)) produced 3 times longer levan than that of the wild type.
Key words: levan; levanase; sacC; degU-hyper strain; Bacillus subtilis

-18-
Note
Effect of Tea Catechins on Cellular Lipid Peroxidation
and Cytotoxicity in HepG2 Cells

Chikako MURAKAMI, Yuki HIRAKAWA, Hiroshi INUI,＊ Yoshihisa NAKANO,＊
and Hiromi YOSHIDA†

Faculty of Nutrition, Kobe Gakuin University, Arise 518, Ikawadani-cho, Nishi-ku, Kobe 651-2180, Japan
＊Faculty of Applied Biological Chemistry, Osaka Prefecture University, Gakuencho 1-1, Sakai,
Osaka 599-8531, Japan

Received November 12, 2001; Accepted February 20, 2002
Tea catechins inhibited TBARS accumulation in HepG2 cells, the order of effectiveness being (−)-epigallocatechin gallate (EGCG)＞(−)-epigallocatechin (EGC)≧(−)-epicatechin gallate (ECG)＞(−)-epicatechin (EC). EGCG and EGC protected the depletion of α-tocopherol in the cells, and the glutathione content was enhanced by all four catechins. Moreover, all four catechins suppressed the formation of glutathione disulfide and the activation of glutathione peroxidase induced by tert-butylated hydroperoxide.
Key words: tea catechin; antioxidant; lipid peroxidation

-19-
Note
Total DNA Preparation Excluding Extracellular Acidic Polysaccharide
from Lipomyces Yeasts and its Application to Taxonomic Studies

Katsushi NISHIMURA,1,† Koji SHIMADA,2 Hiroki IWASAWA,2 Takafumi NAGANUMA,2
and Yasuyuki UZUKA2

1Department of Materials and Applied Chemistry, College of Science and Technology, Nihon University,
1-8-14 Kanda Surugadai, Chiyoda-ku, Tokyo 101-8308, Japan
2Department of Applied Chemistry and Biotechnology, Faculty of Engineering, Yamanashi University,
4-3-11 Takeda, Kofu, Yamanashi 400-8511, Japan

Received November 27, 2001; Accepted March 13, 2002
A preparation method of total DNA from Lipomyces yeasts was improved in order to exclude extracellular acidic polysaccharide thoroughly. The method combined an ultracentrifuge and polyethylene glycol precipitation with the usual method. The total DNAs obtained were analyzed for G＋C content and by DNA-DNA hybridization. The results all agreed almost completely with literature data. All the DNA samples prepared using this method were pure enough for these taxonomic analyses and could also be used as templates of PCR for amplification of small subunit ribosomal DNA and the internal transcribed spacer region.
Key words: Lipomyces yeast; polysaccharide; polyethylene glycol; DNA-DNA hybridization; taxonomic study

-20-
Note
Structure of Genes for Hsp30 from the White-rot fungus Coriolus versicolor
and the Increase of their Expression by Heat Shock and Exposure
to a Hazardous Chemical

Yosuke IIMURA† and Kenji TATSUMI

Institute for Environmmental Management Technology, National Institute of Advanced Industrial Science,
16-1 Onogawa, Tsukuba, Ibaraki 305-8569, Japan

Received December 11, 2001; Accepted March 14, 2002
We isolated and analysed two genomic DNAs that encode the heat-shock protein Hsp30 from Coriolus versicolor. The amino acid sequences substitute only three amino acid substitutions. The promoter regions contain the consensus heat-shock element, a xenobiotic-response element, a stress-response element, and a metal-response element. The levels of mRNAs for Hsp30 increased markedly after exposure of C. versicolor to pentachlorophenol and levels were higher than those after heat shock.
Key words: white-rot fungi; heat shock protein; HSE; XRE

-21-
Note
Isolation and Measurement of Quercetin Glucosides in Flower Buds
of Japanese Butterbur (Petasites japonicus subsp. gigantea Kitam.)

Hideyuki MATSUURA,1,＊,† Midori AMANO,1 Jun KAWABATA,2 and Junya MIZUTANI3

1Northern Advancement Center for Science ＆ Technology, Kita-21, Nishi-12, Sapporo 001-0021, Japan
2Laboratory of Food Biochemistry, Division of Applied Bioscience, Research School of Agriculture,
Hokkaido University, Kita-ku, Sapporo 060-8589, Japan
3Plant Ecochemicals Research Center, R＆BP Center Bldg. 3F, E-310, 3-1-1, Megumino-kita, Eniwa,
Hokkaido 061-1374, Japan

Received December 12, 2001; Accepted March 15, 2002
Three quercetin glucosides were isolated from flower buds of Japanese butterbur (Petasites japonicus subsp. gigantea Kitam.) together with caffeic acid as the ingredients that had DPPH radical scavenging activity, using the DPPH-HPLC method for measuring the radical scavenging activity. These quercetin glucosides were identified as quercetin 3-O-β-D-glucoside, quercetin 3-O-β-D-6″-O-acetylglucoside, and rutin, and the amounts of the glucosides in flower buds were also examined by HPLC. The flower buds were harvested from four different sites, the total amount of quercetin glucosides in each site was 100--170 mg/100 g fr. wt., and there were no great differences of the amounts between growing fields
Key words: Petasites japonicus; Japanese butterbur; quercetin glucoside; edible wild plant

-22-
Note
α-Glucosidase Inhibitor from the Seeds of Balsam Pear (Momordica charantia)
and the Fruit Bodies of Grifola frondosa

Hideyuki MATSUURA,1,＊,† Chikako ASAKAWA,1 Masanori KURIMOTO,1
and Junya MIZUTANI2

1Northern Advancement Center for Science ＆ Technology, Kita-21, Nishi-12, Sapporo 001-0021, Japan
2Plant Ecochemicals Research Center, R＆BP Center Bldg. 3F, E-310, 3-1-1, Megumino-kita, Eniwa,
Hokkaido 061-1374, Japan

Received December 25, 2001; Accepted February 14, 2002
α-Glucosidase inhibitory activities were found in aqueous methanol extracts of the seeds of Momordica charantia and the fruit bodies of Grifola frondosa. An active principle against the enzyme prepared from rat small intestine acetone powders was isolated and characterized. The structure of the isolated compound was identified as D-(＋)-trehalose by FDMS, 1H-, 13C-NMR, and [α]D measurements. The inhibitory activity of trehalose was compared with 1-deoxynojirimycin. Trehalose showed 45％ inhibitory activity at the concentration of 2×10−3 M, but 1-deoxynojirimycin had 52％ inhibitory activity at 1×10−7 M.
Key words: Momordica charantia; Grifola frondosa; α-glucosidase inhibitor; trehalose

-23-
Note
Microbial Degradation of Lipid by Acinetobacter sp. Strain SOD-1

Daisuke SUGIMORI,† Masatoshi NAKAMURA, and Yuma MIHARA

Department of Chemistry and Biology Engineering, Fukui National College of Technology,
Geshi, Sabae, Fukui 916-8507, Japan

Received January 7, 2002; Accepted March 13, 2002
Acinetobacter sp. strain SOD-1, capable of rapidly degrading salad oil, was isolated from soil. Strain SOD-1 showed good growth and degraded 68.7±2.7 and 83.0％ of an initial 3000 ppm salad oil suspension in 24 h at 20°C and pH 7.0 and at 35°C and pH 8.0, respectively. The degradation rate depended on pH, temperature, phosphate concentration, and initial cell density.
Key words: Acinetobacter sp.; lipid degradation; lipid-degrading bacterium; microbial degradation; wastewater treatment

-24-
Note
DnaK Chaperone Machine and Trigger Factor are Only Partially
Required for Normal Growth of Bacillus subtilis

Dindo Y. REYES and Hirofumi YOSHIKAWA†

Department of Bioscience, Tokyo University of Agriculture, Sakuragaoka, Setagaya-ku,
Tokyo 156-8502, Japan

Received January 7, 2002; Accepted March 11, 2002
While dnaK and tig are the essential components for nascent polypeptide folding in E. coli, deletion did not confer synthetic lethality in B. subtilis, suggesting that under normal growth conditions, another system or mechanism with a specific role prevails. Likewise, survival at high temperature suffered dramatically, resulting from deletion of several sets of heat shock genes, thus during sudden stress various heat shock genes act synergistically to protect the proteins.
Key words: molecular chaperones; dnaK; nascent chain polypeptides; heat-shock response; Bacillus subtilis

-25-
Note
Cloning and Expression of the Exo-β-D-1,3-glucanase Gene (exgS)
from Aspergillus saitoi††

Ken ODA,† Shin KASAHARA, Youhei YAMAGATA, Keietsu ABE, and Tasuku NAKAJIMA

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

Received January 8, 2002; Accepted March 7, 2002
A gene of exo-1,3-β-D-glucanase (exgS) was cloned from a koji mold, Aspergillus saitoi, genomic DNA using PCR. The exgS has an ORF comprising 2832 bp, which contains one intron of 45 bp, and encodes 945 amino acids. The deduced amino acid sequences showed that the ExgS has a non-homologous linker region consisting of 180 amino acids, which encompassed highly conserved regions observed in Exg homologues from filamentous fungi. A recombinant protein (ExgS) has been recovered from the cultural filtrate of an Aspergillus oryzae strain that carried an expression vector containing full length of the exgS. The N-terminal amino acid sequences of the recombinant exo-1,3-β-D-glucanase (ExgS) were identical to that of native ExgS from A. saitoi.
Key words: exo-1,3-β-D-glucanase gene (exgS); Aspergillus saitoi; gene cloning; DNA sequence; gene expression

-26-
Note
Synthesis of the (17R)- and (17S)-Isomers of Volicitin, an Elicitor of Plant
Volatiles Contained in the Oral Secretion of the Beet Armyworm

Seiji ITOH,1,2 Shigefumi KUWAHARA,2,† Morifumi HASEGAWA,1 and Osamu KODAMA1

1Laboratory of Agricultural Chemicals, Faculty of Agriculture, Ibaraki University, Ami-machi,
Inashiki-gun, Ibaraki 300-0393, Japan
2Laboratory of Applied Bioorganic Chemistry, Division of Life Science, Graduate School of Agricultural
Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan

Received January 23, 2002; Accepted February 18, 2002
Both the (17R)- and (17S)-isomers of volicitin, which is contained in the oral secretion of the beet armyworm and induces corn seedlings to emit a blend of volatile compounds to attract the natural enemy of the herbivore, were synthesized via the semi-hydrogenation of an intermediary diyne and (Z)-selective olefination as the key steps. They were both obtained as crystalline compounds.
Key words: volicitin; elicitor; Spodoptera exigua; 17-hydroxylinolenic acid

-27-
Note
Isolation of Enterococcus hirae Mutant Deficient in Low-affinity
Potassium Uptake at Alkaline pH

Miyuki KAWANO,1 Kazuei IGARASHI,1 and Yoshimi KAKINUMA2,†

1Graduate School of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Inage-ku,
Chiba 263-8522, Japan
2Department of Applied Chemistry, Muroran Institute of Technology, 27-1 Mizumoto-cho,
Muroran 050-8585, Japan

Received January 23, 2002; Accepted March 20, 2002
We here isolated an Enterococcus hirae mutant unable to grow well at pH 10. The influx rate calculated from steady-state 42K＋/K＋ exchange and the intracellular K＋ concentration of the mutant were reduced to 53 and 55％ of those of the wild-type, respectively. The activities of two high-affinity K＋ uptake systems, KtrI and KtrII, were normal in the mutant, but the kinetics of net K＋ uptake at pH 10 indicated that a low-affinity K＋ uptake with a Km of about 20 mM (Kawano, M, Abuki, R, Igarashi, K, Kakinuma, Y. (2001) Arch. Microbiol. 175: 41--45), which were seen in the wild-type, was deficient in this mutant.
Key words: K＋ transport; KtrI; KtrII; low-affinity; Enterococcus hirae

-28-
Note
A Highly Sensitive Assay for Proteases Using Staphylococcal Protein A
Fused with Enhanced Green Fluorescent Protein

Hiroyoshi FUJINO,1,2 Takashi AOKI,1,† and Hiroyuki WATABE1

1Department of Biochemistry, Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido,
Ishikari-Tobetsu, Hokkaido 061-0293, Japan
2Katayama Chemical Industries Co., 3-26-22 Higashi-Nanba, Amagasaki 660-0892, Japan

Received January 28, 2002; Accepted February 26, 2002
Enhanced green fluorescent protein (EGFP) was fused with staphylococcal protein A (SpA) and used as a substrate for proteases. An SpA-EGFP assay was done in three steps: (i) digestion of SpA-EGFP by proteases, (ii) addition of rabbit IgG immobilized on Sepharose beads, and (iii) measurement of the fluorescence intensity of supernatant. The assay was sensitive enough to measure picogram levels of trypsin and chymotrypsin, and may be applicable to various other proteases as one of the most sensitive methods.
Key words: enhanced green fluorescent protein; protein A; protease assay

-29-
Note
Effects of Dietary Eritadenine on Δ6-Desaturase Activity and Fatty
Acid Profiles of Several Lipids in Rats Fed Different Fats

Yasuhiko SHIMADA, Tatsuya MORITA, and Kimio SUGIYAMA†

Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University,
Suzuoka 422-8529, Japan

Reviced February 8, 2002; Accepted March 13, 2002
Effects of dietary eritadenine on liver microsomal Δ6-desaturase activity and the fatty acid profile of phosphatidylcholine, cholesteryl esters, and triglycerides of liver microsomes or plasma were investigated in rats fed different fats (palm oil, olive oil, and safflower oil). The activity of Δ6-desaturase was influenced by both dietary fat types and eritadenine. In rats fed control diets, Δ6-desaturase activity was higher in the order of the palm oil, olive oil, and safflower oil groups. In rats fed eritadenine-supplemented diets, the enzyme activity was markedly decreased to a constant level irrespective of dietary fat type. The 20:4n-6/18:2n-6 ratio of phosphatidylcholine and cholesteryl esters, as compared with triglycerides, was highly sensitive to eritadenine. The results suggest that the activity of Δ6-desaturase is regulated by dietary fats and eritadenine independently, and that the effect of eritadenine is stronger than that of dietary fats.
Key words: eritadenine; Δ6-desaturase; linoleic acid metabolism; dietary fat; rat

-30-
Preliminary Communication
Suppressive Effect of Polysaccharides from the Edible and Medicinal
Mushrooms, Lentinus edodes and Agaricus blazei,
on the Expression of Cytochrome P450s in Mice

Takashi HASHIMOTO,1 Yuji NONAKA,2 Ken-ichiro MINATO,3 Sachiko KAWAKAMI,1,4
Masashi MIZUNO,1 Itsuko FUKUDA,2 Kazuki KANAZAWA,1 and Hitoshi ASHIDA1,2,†

1Division of Life Science, Graduate School of Science and Technology, Kobe University, Rokkodai-cho 1,
Nada-ku, Kobe 657-8501, Japan
2Department of Biofunctional Chemistry, Faculty of Agriculture, Kobe University, Rokkodai-cho 1,
Nada-ku, Kobe 657-8501, Japan
3Miyagi Agricultural College, 2-2-1 Hatatate, Taihaku-ku, Sendai 982-0215, Japan
4Tottori Mycological Institute, 211 Kokoge, Tottori 689-1125, Japan

Received February 18, 2002; Accepted April 15, 2002
To investigate the effects of lentinan from Lentinas edodes and polysaccharides from Agaricus blazei (ABPS) on the expression of cytochrome P450s (CYPs), lentinan (10 mg/kg/day) or ABPS (200 mg/kg/day) was administered to female BALB/c mice four times every other day by intraperitoneal injection. Lentinan and ABPS suppressed both the constitutive and 3-methylcholanthrene-induced CYP1A expression and ethoxyresorufin-O-deethylation activity in the liver.
Key words: lentinan; Lentinas edodes; Agaricus blazei polysaccharides; cytochrome P450; tumor necrosis factor-α (TNF-α)

-31-
Preliminary Communication
Binding Selectivity of Conformationally Restricted Analogues
of (−)-Indolactam-V to the C1 Domains of Protein Kinase C Isozymes

Akiko MASUDA, Kazuhiro IRIE,† Yu NAKAGAWA, and Hajime OHIGASHI

Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University,
Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan

Received March 11, 2002; Accepted April 15, 2002
Two conformationally restricted analogues of (−)-indolactam-V (1) (cis and trans amides) were examined for their binding selectivity to the synthetic C1 peptides of all protein kinase C (PKC) isozymes. Although the binding constants of the cis amide-restricted analogue (2) were equal to those of 1, the trans amide-restricted analogue (3) bound significantly only to the novel PKC (δ, ε, η, θ) isozymes.
Key words: (−)-indolactam-V; isozyme selective binding; phorbol ester; protein kinase C; tumor promoter