Contents and Abstracts of Latest Issue of BBB

(Vol.64 No.7 2000)

Katsuichi SAITO and Fusao TOMITA＋

Laboratory of Applied Microbiology, Graduate School of Agriculture, Hokkaido University,
Sapporo 060-8589, Japan

Difructose anhydrides (DFAs) are the smallest cyclic disaccharides consisting of two fructose residues, and are expected to have novel physiological functions from their unique structures and properties.
For mass-production of α-D-fructofuranose-β-D-
fructofuranose-2?,1:2,3?-dianhydride (DFA III) and β-D - fructofuranose - β - D - fructofuranose - 2?,6:2,6? - dianhydride (DFA IV), Arthrobacter sp. H65-7 and A. nicotinovorans GS-9 were selected as the best producers of inulase II, which produced DFA III from inulin and LFTase, which produced DFA IV from levan. The enzymes were purified and their genes were subsequently cloned and expressed in E. coli at higher levels than in the original bacteria. Thus, it became possible to provide a large amount of DFA III and DFA IV for evaluating their physiological properties.
DFA III and DFA IV have half the sweetness of sucrose, but cannot be digested by the digestive system of rats. Their use by the intestinal microorganisms was observed in vivo even though their assimilation could not be detected in vitro. This implied that they were degraded by an unknown system in the intestine. It was also found that they affected calcium absorption mainly in the small intestine through mechanisms different from the known stimulants such as fructooligosaccharides and raffinose.
Key words: difructose anhydride; DFA III; DFA IV; inulin; levan

Masaaki HIROSE

The Research Institute for Food Science, Kyoto University, Uji, Kyoto 611-0011, Japan

Transferrins are a group of iron-binding proteins that control the levels of iron in the body fluids of vertebrates by their ability to bind two Fe3+ and two CO2--3. The transferrin molecule, with a molecular mass of about 80 kDa, is folded into two similarly sized homologous N- and C-lobes that are stabilized by many intrachain disulfides. As observed by X-ray crystallography, each lobe is further divided into two similarly sized domains, domain 1 and domain 2, and an Fe3+-binding site is within the interdomain cleft. Four of the six Fe3+ coordination sites are occupied by protein ligands (2 Tyr residues, 1 Asp, and 1 His) and the other two by a bidentate CO2--3. Upon uptake and release of Fe3+, transferrins undergo a large-scale conformational change dependng on a common structural mechanism: domains 1 and 2 rotate as rigid bodies around a rotation axis that passes through the two antiparallel β-strands linking the domains. The extent of the rotaion is, however, variable for different transferrin species and lobes. As a Fe3+ release mechanisms at low pH from the N-lobes of serum transferrin and ovotransferrin, the structral evidence for `dilysine trigger mechanism′ is shown. A structural mechanism for the Fe3+ release in presence of a non-synergistic anion is proposed on the basis of the sulfate-bound apo crystal structure of the ovotransferrin N-lobe. Domain-opened structures with the coordinated Fe3+ by the two tyrosine residues are demonstrated in fragment and intact forms, and their functional implications as a possible intermediate for iron uptake and release are discussed.
Key words: transferrin; ovotransferrin; lactoferrin; structural mechanism; iron uptake and release

Masaru NAGAI, Akira OZAWA,* Tohoru KATSURAGI,** and Takuo SAKAI＋

Department of Food Science and Nutrition, Faculty of Agriculture, Kinki University,3327-204 Naka-machi,
Nara 631-8505, Japan
*Department of Applied Biological Chemistry, College of Agriculture, Osaka Prefecture University,
1-1 Gakuen-cho, Sakai City, Osaka 599-8531, Japan
**Graduate School of Biological Sciences, Nara Institute of Science and Technology,
Takayama Science Town, Ikoma, Nara 630-0101, Japan
Received September 10, 1999; Accepted February 24, 2000


Aspergillus awamori IFO 4033 produced an acid-stable protopectinase in solid-state fermentation using wheat bran as the medium. The enzyme was purified to a homogeneous preparation with anion-exchange, hydrophobic, and size-exclusion chromatography. The enzyme was a monomeric protein of 52 kDa, by SDS-PAGE analysis, with an isoelectric point of pH 3.7. The optimum pH for enzyme activity was 2.0, and it was most active at 50°C (at pH 2.0) and was stable up to 50°C (at pH 2.0). The enzyme showed pectin-releasing activity toward protopectins from various origins, especially on lemon protopectin. An outstanding characteristic of the enzyme was its extreme stability in acidic conditions: the enzyme activity was not lost after incubating at pH 2.0 and 37°C for 24 h.
Key words: polygalacturonase; protopectinase; Aspergillus awamori; solid-state fermentation; acid-stable enzyme

Hiroki FUKUI*, Yukiharu FUKUSHI*,**,＋ and Satoshi TAHARA*,**

*Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University,
Kita-ku, Sapporo 060-8589, Hokkaido, Japan
**CREST, Japan Science and Technology Corporation, 4-1-8 Honmachi, Kawaguchi 332-0012,
Saitama, Japan

Received November 1, 1999; Accepted February 24, 2000
Both the enantiomers of the axially chiral reagent, 2?-methoxy-1,1?-binaphthalene-2-carbohydroximoyl chloride (MBCC), were used to convert igalan, an α-exo-methylene-γ-lactone, to yield 4,5-dihydroisoxazoles. The absolute configuration of igalan was determined to be (3aR,5R,6R,7aR)-6-ethenylhexahydro-6-methyl-3-methylene - 5 - (1 - methylethenyl) - 2(3H) - benzofuranone (IUPAC name) on the basis of NOE correlations in these derivatives. The absolute configurations of other α-exo-methylene-γ-lactones can be determined by the same method.
Key words: igalan; α-exo-methylene-γ-lactone; NOE; absolute configuration; MBCC

Koji KISHI,*,＋ Yoshifumi WATAZU,* Yoshiaki KATAYAMA,**
and Hiroaki OKABE***

*International Reagents Corp., 1-1-2, Murotani, Nishi-ku, Kobe 651-2241, Japan
**Department of Clinical Chemistry, National Cardiovascular Center Hospital,
5-7-1, Fujishiro-dai, Suita 565-0873, Japan
***Department of Laboratory Medicine, School of Medicine, Kumamoto University,
1-1-1 Honjo, Kumamoto 860-8556, Japan

Received November 8, 1999; Accepted February 24, 2000
Mass production of an r-CDH derived from Nocardia species was made possible by gene technology. (Horinouchi et al., Applied and Environmental Microbiology, 57, 1386--1393 (1991)). However, the characteristics of the r-CDH have not been studied in detail and have not been improved enough for industrial use. We accordingly characterized both the native-CDH and the r-CDH prepared from Streptomyces lividans. Both CDHs were monomers with molecular masses of 37 kDa. The Km of r-CDH was 2.50×10--3 M for cholesterol and 2.33×10--4 M for NAD. The activators of CDHs were TritonX-100 and cholate. TritonX-405, Ag+, and Zn2+ inhibited both enzymes. The residual activity of native CDH after heat treatment was 32% (37°C, 60 min), while the r-CDH showed a residual activity of 87% (37°C, 60 min). The r-CDH is an enzyme with high substrate specificity for cholesterol as well as native CDH and higher thermal stability than native CDH.
We have developed a novel serum cholesterol assay using the r-CDH, which permits the direct measurement of cholesterol by measuring NADH reaction products. We conclude that this r-CDH enzyme is useful and can be used to measure cholesterol in a clinical chemistry setting.
Key words: cholesterol dehydrogenase; cholesterol oxidase; recombinant enzyme; serum

Ken YAGI,1 Taku MATSUMOTO,1,2 Tetsuya CHUJO,1 Hideaki NOJIRI,1 Toshio OMORI,1
Kiwamu MINAMISAWA,3 Makoto NISHIYAMA,1 and Hisakazu YAMANE1＋

1Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku,
Tokyo 113-8657, Japan
2Laboratory for Biological Resources Research, Lead Discovery Research Laboratories,
Sankyo Co. Ltd., 33 Miyukigaoka, Tsukuba-shi, Ibaraki 305-0841, Japan
3Center of Genetic Ecology, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai,
Miyagi 980-8577, Japan

Received November 24, 1999; Accepted February 19, 2000
We isolated 11 low-indole-3-acetic acid (IAA)-producing mutants of Bradyrhizobium elkanii by Tn5 mutagenesis. The amount of IAA produced by each mutant was 2.2--13.6% of that of the wild-type. It was found by resting cell reactions that the biosynthetic step to convert indole-3-pyruvic acid to indole-3-acetaldehyde was blocked in all the mutants.
Key words: Bradyrhizobium elkanii; biosynthesis of indole-3-acetic acid; indole-3-pyruvic acid decarboxylase; Tn5 mutagenesis

Koji TAKAHASHI,＋ Hiroshi KONDO, Hiroshi KUROIWA, Yoshiko YOKOTE,ffl
and Makoto HATTORI

Department of Applied Biological Science, Faculty of Agriculture, Tokyo University
of Agriculture and Technology, Tokyo 183-8509, Japan
fflDepartment of Chemistry, Faculty of Science, Josai University, Saitama 350-0248, Japan

Received December 13, 1999; Accepted March 9, 2000
The reversible thermal transition of soluble branched starch chains prepared from slightly acid-treated potato starch granules (ATS) was investigated. Potato starch was immersed in 15% sulfuric acid to obtain ATS with a 1% hydrolysis rate. About half of the molecules of ATS, which spontaneously formed large aggregates with a mass of a few million daltons in aqueous solution, was fractionated and soluble branched starch chains with a relative molecular weight (Mr) of 8.91×104 were obtained. Structural analysis indicated that the soluble branched starch chains consisted of three unit chains with Mr 7,900 and 21 unit chains with Mr 2,700. DSC and FT-IR measurements showed that the soluble branched starch chains underwent a reversible thermal transition, which is considered to be a helix-coil transition, during heating and cooling, but a debranched sample and β-limit dextrins showed substantially different thermal behavior, indicating the contribution of the ordered structure of the branched chains.
Key words: acid-treated starch; thermal transition; DSC; FT-IR

Mingzhou SUN, Hiroyuki SAKAKIBARA, Hitoshi ASHIDA, Gen-ichi DANNO,
and Kazuki KANAZAWA＋

Department of Life Science, Graduate School of Science and Technology, Kobe University,
Rokkodai, Nada-ku, Kobe 657-8501, Japan

Received December 13, 1999; Accepted February 18, 2000
We have earlier found that flavones and flavonols in vegetables specifically inhibited one of the carcinogenesis-related enzymes, cytochrome P450 (CYP) 1A1, and subsequently suppressed the mutagenicity of food-derived carcinogens. In this study, we explored other candidates for the enzyme inhibitor in Chinese medicinal plants. Some of them were antimutagenic toward 3-amino-1-methyl-5H-pyrido[4, 3-b]indole (Trp-P-2). For example, Rheum officinale contained large amounts of anthraquinones as the active compounds, 3.4 mg of emodin, 2.1 mg of chrysophanol and 1.8 mg of rhein in 10 g of dry matter. Anthraquinones showed similar IC50 values for antimutagenicity against Trp-P-2 to those for inhibition of the N-hydroxylation activity of CYP1A1 toward Trp-P-2, indicating that the antimutagenicity was attributable to CYP inhibition. The structure-activity relationships were then examined with 14 commercial chemicals, and it was found that the interaction with an enzyme required three rings and an oxygen group in the side ring. This characteristic is similar to that of flavones and flavonols.
Key words: anthraquinone; antimutagen; CYP inhibition; phytochemical; Chinese medicinal plant

Shin-ichi KASHIWABARA, Sayuri AZUMA, Masao TSUDUKI, and Yuzuru SUZUKI＋＋

Laboratory of Microbial Physiology and Applied Microbiology, Department of Applied Biochemistry,
Kyoto Prefectural University, Shimogamo, Sakyo-ku, Kyoto 606-8522, Japan

Received December 15, 1999; Accepted March 6, 2000
The gene that coded for the subunit of an molecular weight (Mr) 540,000 homohexameric α-glucosidase II (α-D-glucoside glucohydrolase, EC 3.2.1.20) produced by Bacillus thermoamyloliquefaciens KP1071 (FERM-P8477) growing at 30 to 66°C was expressed in Escherichia coli HB101. The resulting homohexameric enzyme had a half-life of 10 min at 80°C. Its purification and characterization showed that the enzyme was identical with the native one except for the latter deleting 7 N-terminal residues found in the former. The primary sequence of the subunit with 787 residues and an Mr of 91,070 deduced from the gene was 24--34% identical to the corresponding sequences of 15 α-glucosidases in the glycosyl hydrolase family 31 from 14 eukaryotic origins and the archaeon Sulfolobus solfataricus 98/2.
From the sequence analysis by the neural network method of Rost and Sander [Rost, B. and Sander, C., Proteins: Struct. Funct. Genet., 19, 55--72 (1994)], we inferred that α-glucosidase II might make each subunit of 3 secondary structural regions, i.e., one N-terminal β region, one central α/β region with two catalytic residues Asp407 and Asp484, and one C-terminal β region.
Key words: thermostable homohexameric α-glucosidase; gene cloning; Bacillus thermoamyloliquefaciens; glycosyl hydrolase family 31; multiple sequence alignments

Takeshi YAMAGAMI,* Kazuki TSUTSUMI, and Masatsune ISHIGURO

Laboratory of Protein Chemistry and Engineering, Faculty of Agriculture, Kyushu University,
Fukuoka 812-8581, Japan

Received December 15, 1999; Accepted March 1, 2000
A cDNA encoding tulip bulb chitinase-1 (TBC-1) was cloned using a combination of immunoscreening from a λ ZAP cDNA library with anti-TBC-1 antiserum and the 5? rapid amplification of cDNA end (RACE) method, and sequenced. The cDNA consists of 1,106 nucleotides and included an open reading frame encoding a polypeptide of 314 amino acids. Comparison of the deduced amino acid sequence and the determined protein sequence indicated the presence of a signal peptide and an extra peptide composed of 26 and 13 amino acids at the N- and C-termini, respectively. The deduced sequence of TBC-1 had 10--20% and 63% sequence similarities to plant class III chitinases and gladiolus bulb class IIIb chitinase (GBC-a), respectively. The cDNA encoding mature TBC-1 was amplified by polymerase chain reaction (PCR), ligated into the expression vector pET-22b, and expressed in Escherichia coli BL21(DE3). The recombinant TBC-1 (rTBC-1) expressed in E. coli was purified by gel filtration followed by ion-exchange chromatography. Specific activity of the rTBC-1 was almost same as the authentic TBC-1 toward glycolchitin. This is the first report on the cDNA cloning of a class III chitinase having C-terminal extra peptide.
Key words: chitinase; cDNA cloning; expression

Akiko NISHII,＋ Miho TAKEMURA, Hidetomo FUJITA, Masahito SHIKATA,
Akiho YOKOTA, and Takayuki KOHCHI*

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

Received January 11, 2000; Accepted February 4, 2000
By differential screening of an arrayed normalized cDNA library from the inflorescence apex in Arabidopsis, a cDNA clone having a deduced amino acid sequence with a motif for a zinc finger was isolated as one of the genes expressed specifically in the reproductive phase. The deduced protein has a modular structure with a putative single C2-C2 zinc-finger motif distantly related to a GATA-1-type finger, a basic region with a sequence resembling a nuclear localization signal, and an acidic region. The gene seemed to have been formed by the exon-shuffling during its molecular evolution, since individual domains are encoded by discrete exons. RNA gel blot analysis showed its expression in shoot apex and flowers in the reproductive phase. The gene was named ZIM for Zinc-finger protein expressed in
Inflorescence Meristem. The nuclear localization of ZIM was detected using GFP as a reporter. These results suggest that ZIM is a putative transcription factor involved in inflorescence and flower development.
Key words: Arabidopsis thaliana; differential screening; inflorescence; transcription factor; zinc-finger protein

Hisayuki TSUJIMORI＋＋ and Kenji MORI＋＋＋

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

Received January 17, 2000; Accepted February 23, 2000
(+)-Hongoquercin B (1), a weakly antibacterial fungal metabolite, was synthesized by starting from (S)-3-hydroxy-2,2-dimethylcyclohexanone, and its absolute configuration was determined as depicted by structure 1.
Key words: configuration determination; heterocycles; hongoquercin B; terpenoids; total synthesis

Takafumi KUBODERA, Nobuo YAMASHITA, and Akira NISHIMURA

Research & Development Department, Hakutsuru Sake Brewing Co. Ltd., 4-5-5,
Sumiyoshiminami-machi, Higashinada-ku, Kobe 658-0041, Japan

Received January 19, 2000; Accepted February 29, 2000
A pyrithiamine (PT) resistance gene (ptrA) was cloned from a genomic DNA library prepared from a PT resistant mutant of Aspergillus oryzae. It conferred high resistance to PT on an A. oryzae industrial strain as well as A. nidulans. Nucleotide sequence analysis showed that the ptrA gene contained one intron (58-bp) and encodes 327 amino acid (aa) residues. Additionally, the deduced aa sequence has 72% and 63% identity to Fusarium solani sti35 encoding a stress-inducible protein and Saccharomyces cerevisiae THI4 encoding an enzyme involved in thiamine biosynthesis, respectively, indicating that ptrA is a mutated allele of a gene belonging to the THI4 family. The mutation point was identified in the conserved motif in 5?-flanking region of these three THI4 homologous genes (ptrA, sti35, and THI4). The introduction of the ptrA gene allowed an A. oryzae industrial strain to grow on the minimum medium containing PT (0.1 mg/l) on which an untransformed strain did not grow. This result indicates that the ptrA is applicable as a dominant selectable marker for transformation of A. oryzae.
Key words: drug resistance gene; pyrithiamine; A. oryzae; dominant selectable marker

Naoko HASEGAWA,1 Yoshinobu KIMURA,2 Tatsuya ODA,1,* Nobukazu KOMATSU,1
and Tsuyoshi MURAMATSU1

1Division of Biochemistry, Faculty of Fisheries, Nagasaki University, Bunkyo-machi,
Nagasaki 852-8521, Japan
2Department of Bioresources Chemistry, Faculty of Agriculture, Okayama University,
Okayama 700-8530, Japan

Received January 21, 2000 Accepted March 3, 2000
We have previously reported that ricin, a toxic lectin that inhibits protein synthesis induced apoptotic cell death. In this study, we have found that isolated ricin CM-B-chain, which has no effect on cellular protein synthesis, induced DNA fragmentation in U937 cells in a dose- and time-dependent manner, albeit it required a longer incubation time and higher concentration than those of holotoxin ricin. Z-Asp-CH2-DCB, a caspase family inhibitor and serine protease inhibitor, 3,4-dichloroisocoumarine (DCI) effectively inhibited the CM-B-chain-mediated DNA fragmentation as well as in ricin. Thus, like ricin, multiple proteases with different substrate specificity may also be involved in the CM-B-chain-mediated apoptotic pathway. Furthermore, BFA inhibited both ricin- and CM-B-chain-mediated DNA fragmentation, suggesting an intracellular vesicle transport system through the Golgi complex may be involved in the apoptotic induction by these proteins as a common feature. On the other hand, cycloheximide (CHA) strongly increased the CM-B-chain-mediated DNA fragmentation, but inhibited ricin-mediated DNA fragmentation. The opposite effects of CHA may reflect the difference in the apoptotic mechanism between ricin and CM-B-chain. In conclusion, our results suggest that ricin-B-chain can induce apoptosis through its lectin activity, but the underlying mechanism may be distinct from that of ricin in which the A-chain contributes profoundly to the apoptotic induction.
Key words: ricin; ricin B-chain; cytotoxicity; lectin; apoptosis

Yoshihiko YASOHARA,＋ Noriyuki KIZAKI, Junzo HASEGAWA, Masaru WADA,*,＋＋
Michihiko KATAOKA,* and Sakayu SHIMIZU*

Fine Chemical Research Laboratories, Kaneka Corporation, 1-8 Miyamae-machi, Takasago-cho,
Takasago 676-8688, Japan
*Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University,
Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan

Received January 21, 2000; Accepted March 7, 2000
An NADPH-dependent carbonyl reductase (S1) isolated from Candida magnoliae catalyzed the reduction of ethyl 4-chloro-3-oxobutanoate (COBE) to ethyl (S)-4-chloro-3-hydroxybutanoate (CHBE), with a 100% enantiomeric excess, which is a useful chiral building block for the synthesis of pharmaceuticals. The gene encoding the enzyme was cloned and sequenced. The S1 gene comprises 849 bp and encodes a polypeptide of 30,420 Da. The deduced amino acid sequence showed a high degree of similarity to those of the other members of the short-chain alcohol dehydrogenase superfamily. The S1 gene was overexpressed in Escherichia coli under the control of the lac promoter. The enzyme expressed in E. coli was purified to homogeneity and had the same catalytic properties as the enzyme from C. magnoliae did. An E. coli transformant reduced COBE to 125 g/l of (S)-CHBE, with an optical purity of 100% enantiomeric excess, in an organic solvent two-phase system.
Key words: carbonyl reductase; stereoselective reduction; Candida magnoliae; ethyl 4-chloro-3-hydroxybutanoate

Noritaka MATSUO,＋,* Koji YAMADA, Mitsuo MORI, Kentaro SHOJI, Takashi UEYAMA,
Shinichi YUNOKI, Kouhei YAMASHITA, Makoto OZEKI,** Michihiro SUGANO***

Laboratory of Food Chemistry, Department of Bioscience and Biotechnology, Division
of Bioresource and Bioenvironmental Science, Graduate School Kyushu University,
Fukuoka 812-8581, Japan
*Department of Biochemistry, Oita Medical University, Oita 879-5593, Japan
**Research & Development Central Research Laboratories, Taiyo Kagaku Co. Ltd.,
Yokkaichi, Mie 510-0844, Japan
***Department of Living Environment Science, Prefectural University of Kumamoto,
Kumamoto 862-8502, Japan

Received January 24, 2000; Accepted March 6, 2000
Male Wistar rats were given purified diets containing safflower (SAF), perilla (PER), or palm (PAL) oils with or without 1% tea polyphenols (TP) for 3 weeks, and chemical mediator releasing activity from rat peritoneal exudate cells (PEC) was measured. Histamine releasing activity was not influenced by TP, while histamine release and intracellular histamine content were significantly increased in the PAL-fed group. On the contrary, leukotriene B4 (LTB4) release was significantly lower in rats fed PER than in those fed SAF and PAL, and TP significantly decreased the release in all fat groups. TP also significantly inhibited the release of LTB5, which was generated only in rats fed PER. TP significantly decreased the proportion of arachidonic acid (AA) in PEC in the SAF-fed group and that of eicosapentaenoic acid (EPA), the precursor of LTB5 in the PER-fed group, but did not influence that of AA in the PAL- and PER-fed group. These results suggest that ingestion of TP improves type I allergic symptom through the inhibition of LT release though the inhibition by TP could not be totally explained by the reduction of substrate fatty acid.
Key words: dietary fats; tea polyphenols; histamine; leukotriene; peritoneal exudate cells

Ryou OKAZAKI, Hiromasa KIYOTA,＋ and Takayuki ORITANI

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

Received January 27, 2000; Accepted March 6, 2000
Lipase-catalyzed optical resolution of (±)-epoxy-β-cyclogeraniol (1), a key synthetic intermediate for epoxy-β-ionylideneacetic acid, was achieved in high enantiomeric purity. Transesterification with vinyl acetate by using lipase P (Nagase) made enriched (--)-1, while hydrolysis of the corresponding acetate by using lipase P (Amano) afforded (+)-1 with a high E value (E＝1600).
Key words: lipase; epoxy-β-cyclogeraniol; epoxy-β-ionylideneacetic acid; abscisic acid; transesterification

Nobuhiro HIRAI,1,＋ Ryuji YOSHIDA,2 Yasushi TODOROKI,1 and Hajime OHIGASHI1

1Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University,
Kyoto 606-8502, Japan
2Department of Agriculture Technology, Toyama Prefectural University, Toyama 939-0311, Japan

Received January 28, 2000; Accepted March 2, 2000
The biosynthetic pathways to abscisic acid (ABA) were investigated by feeding [1-13C]-D-glucose to cuttings from young tulip tree shoots and to two ABA-producing phytopathogenic fungi. 13C-NMR spectra of the ABA samples isolated showed that the carbons at 1, 5, 6, 4?, 7? and 9? of ABA from the tulip tree were labeled with 13C, while the carbons at 2, 4, 6, 1?, 3?, 5?, 7?, 8? and 9? of ABA from the fungi were labeled with 13C. The former corresponds to C-1 and -5 of isopentenyl pyrophosphate, and the latter to C-2, -4 and -5 of isopentenyl pyrophosphate. This finding reveals that ABA was biosynthesized by the non-mevalonate pathway in the plant, and by the mevalonate pathway in the fungi. 13C-Labeled β-carotene from the tulip tree showed that the positions of the labeled carbons were the same as those of ABA, being consistent with the biosynthesis of ABA via carotenoids. Lipiferolide of the tulip tree was also biosynthesized by the non-mevalonate pathway.
Key words: abscisic acid; β-carotene; non-mevalonate pathway; Liriodendron tulipifera; lipiferolide

Shinya NAGAFUCHI,＋,*,** Mamoru TOTSUKA,** Satoshi HACHIMURA,** Masao GOTO,**
Takeshi TAKAHASHI,* Takaji YAJIMA,* Tamotsu KUWATA,* and Shuichi KAMINOGAWA**

*Nutrition Science Institute, Meiji Milk Products Co., Ltd., 540 Naruda, Odawara,
Kanagawa 250-0862, Japan
**Department of Applied Biological Chemistry, The University of Tokyo, 1-1-1 Yayoi,
Bunkyo-ku, Tokyo 113-8657, Japan

Received January 28, 2000; Accepted March 6, 2000
We have investigated the effects of dietary nucleotides on intraepithelial lymphocytes (IEL) and intestinal epithelial cells (IEC) in weanling mice. The proportion of T-cell receptor (TCR) γδ+ IEL in BALB/c mice fed a diet supplemented with nucleotides (NT(+) diet) was significantly higher than that in mice fed the nucleotide-free diet, while the proportion of TCRαβ+ IEL in NT(+) diet-fed mice was significantly decreased. The change of the TCRαβ+/TCRγδ+ ratio was mainly observed in a CD8αα+ subset of IEL. IEC from NT(+) diet-fed mice produced a higher level of IL-7, which is important in the development of TCRγδ+ IEL, than those from control diet-fed mice. The expression levels of IL-7 and IL-2 receptors on IEL were not different between the two dietary groups. Our findings suggest that the increased population of a TCRγδ+ IEL subset by feeding nucleotides may be caused by the increased production of IL-7 by IEC.
Key words: dietary nucleotides; intraepithelial lymphocytes; TCRγδ+ T cells; interleukin-7; intestinal epithelial cells

Miho KOIE, Miyuki TANAKA, Kazutoshi HAYASE,＋＋ and Hidehiko YOKOGOSHI*

Department of Home Economics, Aichi University of Education, Igaya-cho, Kariya,
Aichi 448-8542, Japan
*Laboratory of Nutritional Biochemistry,School of Nutritional Sciences,
The University of Shizuoka, Yada, Shizuoka 422-8526, Japan

Received January 31, 2000; Accepted March 15, 2000
The purpose of this study was to find whether the addition of dietary lysine affected the rate of brain protein synthesis in aged rats fed on a gluten diet. Experiments were done on two groups of aged rats (30 wk) given the diets containing 5% gluten or 5% gluten+0.3% lysine for 10 d. The fractional rates of protein synthesis in brain, liver, and kidney increased with an addition of dietary lysine. In brain, liver, and kidney, the RNA activity [g protein synthesized/(g RNA・d)] was significantly correlated with the fractional rate of protein synthesis. The RNA concentration (mg RNA/g protein) was not related to the fractional rate of protein synthesis in any organ. The results suggest that the addition of the limiting amino acid for the low quality protein elevates the rate of protein synthesis in the brain of aged rats, and that RNA activity is at least partly related to the fractional rate of brain protein synthesis.
Key words: dietary protein quality; age; brain protein synthesis; limiting amino acids; rats

Hirofumi NAKANO,1) Hiromi MURAKAMI,1) Motohiro SHIZUMA,1) Taro KISO,1)
Teomar Lustosa de ARAUJO,2) and Sumio KITAHATA1)

1)Osaka Municipal Technical Research Institute, 6-50 Morinomiya 1-chome,
Joto-ku, Osaka 536-8553, Japan
2)Federal University of Pernambuco, Keizo Asami Laboratory of Imunopatology Cidad Universitaria,
Recife, PE, 50670-901, Brazil

Received February 3, 2000; Accepted March 15, 2000
β-Fructofuranosidase fructosylated not only the hydroxyl group but also the thiol group of 2-mercaptoethanol in a transfer reaction using sucrose as a donor substrate. The enzymes from Candida utilis and Saccharomyces cerevisiae (bakers′ yeast) were effective catalysts for the thio-fructofuranosylation. The thio-fructosylation product was isolated by activated carbon chromatography and its structure was confirmed by Fab-mass spectrometry and NMR spectroscopy. The thio-fructofuranoside was synthesized effectively at around 3.0 M for the acceptor concentration. The product increased with the sucrose concentration at least up to 1.9 M. O-Fructofuranoside was simultaneously synthesized at an early stage of the reaction, although it was hydrolyzed on further incubation. On the contrary, the thio-furctofuranoside accumulated efficiently after synthesis, indicating it was very stable against the hydrolytic action of the β-fructofranosidase.
Key words: β-fructofuranosidase; thiol compound; transfructofuranosylation; thio-fructofuranoside

Kazuhisa HATAKEYAMA, Makoto GOTO, Miki KOBAYASHI,＋ Masato TERASAWA,
and Hideaki YUKAWA＋＋

Biochemicals Laboratory, Tsukuba Research Center, Mitsubishi Chemical Corporation,
8-3-1 Chuo, Ami, Inashiki, Ibaraki 300-0332, Japan

Received February 9, 2000; Accepted March 15, 2000
The maleate cis-trans isomerase gene (maiA) from Serratia marcescens IFO3736 was cloned and sequenced. Serratia MaiA has 62.4% amino acid identity with Alcaligenes faecalis IFO13111 MaiA and 64.9% with Bacillus stearothermophilus MI-102 MaiA. All known ten amino acid sequences of MaiA had significant conserved regions containing cysteine residues, which were previously suggested to be involved in an active site of the enzyme. The maiA gene was expressed in Escherichia coli, and expressed products MaiA was purified and characterized. The purified enzyme of strain IFO3736 showed high activity at room temperature and high heat stability. It also showed higher activity in the presence of high concentration of aspartic acid than the enzyme of A. faecalis IFO13111, but it was also sensitive to chemical oxidation. By amino acid composition analysis, cysteine, methionine, and tyrosine residues were suggested to be oxidized to inactivate the enzyme by chemical oxidation. To investigate the mechanism of chemical oxidation of the enzyme, six methionine residues in the conserved regions of S. marcescens MaiA were replaced with cysteine residues by site-directed mutagenesis. The analysis of the constructed mutants suggested that the Met201 residue near the Cys198 residue is involved in the sensitivity of the enzyme to chemical oxidation.
Key words: maleate cis-trans isomerase; stability to the chemical oxidation; Serratia marcescens

Yuji FURUYA,1,2 Hidemi SAWADA,1 Toshikatsu HIRAHARA,2 Kazue ITO,2
Takashi OHSHIRO,1 and Yoshikazu IZUMI1,*

1Department of Biotechnology, Faculty of Engineering, Tottori University, Koyama-cho,
Tottori, Tottori 680-8552, Japan
2Research Laboratory, Ikeda Food Research Co. Ltd., 95-7 Minooki-cho, Fukuyama,
Hiroshima 721-8558, Japan

Received February 10, 2000; Accepted March 21, 2000
A basidiomycete, Coprinus sp. SF-1, was found to produce an L-Trp-oxidizing enzyme by screening from the culture collection of our laboratory. After solubilization by 1 M NaSCN from the particulate fraction of disrupted cells of the strain, the enzyme was purified about 76-fold to essential homogeneity. The enzyme had a molecular mass of about 420 kDa and the subunit molecular mass was 68 kDa. The enzyme contained 1 mol of non-covalently bound FAD per mol of the subunit. It catalyzed the simultaneous reactions of oxidative deamination and oxygenative decarboxylation of L-Trp to form indolepyruvic acid and indole-3-acetamide, the former of which was further oxidized to indole-3-acetic acid. The molar ratio of the respective reaction products was about 9:1. The enzyme specifically oxidized L-Trp, and slightly acted on L-Phe and L-Tyr. The Km for L-Trp was about 0.5 mM in both oxidase and oxygenase reactions. Thus, the enzyme is a novel one and was tentatively designated ``L-Trp oxidase (deaminating and decarboxylating)′′. The optimum pHs of oxidase and oxygenase activities were 7.0 and 9.0, respectively. The optimum temperatures of both activities were 50°C. The enzyme was stable at pH 6.0--10.5 and below 50°C, and at 4°C for 1 year.
Key words: L-tryptophan oxidase; oxidative deamination; oxygenative decarboxylation; basidiomycete; Coprinus sp.

Hideki SATO,1 Kazuhiro NAKAJIMA,2 Yukio MAENO,2 Takashi KAMAISHI,2
Tatsuo KAMATA,1 Hajime MORI,1 Kaeko KAMEI,1 Ryo TAKANO,1
Ken-ichi KUDO,3 and Saburo HARA1

1Department of Applied Biology, Kyoto Institute of Technology, Kyoto 606-8585, Japan
2National Research Institute of Aquaculture, Fisheries Agency, Mie 516-0193, Japan
3Sanwa Cornstarch Co., Ltd, Kashihara, Nara 630-8585, Japan

Received February 10, 2000; Accepted March 21, 2000
Yellowtail ascites virus (YAV) is a member of the family Birnaviridae and causes viral ascites among juvenile yellowtail (Seriola quinqueradiata). We have reported the cloning and expression of two viral cDNAs, the first being segment A encoding a polyprotein of viral capsid proteins (VP2 and VP3) and a protease (NS), and the second being VP2-epitope encoding serotype-specific epitope region on VP2, using a baculovirus expression system. Another viral cDNA encoding a polyprotein of NS and VP3 was cloned and expressed in this study. For the expression of NS/VP3 (YAV nt 1626 to 3066) in insect cells a 31-kDa protein, corresponding to VP3 was detected, indicating an appropriate posttranslational processing of NS/VP3 polypeptide by NS protease itself. The analysis of the N-terminal amino acid sequence of this protein showed that NS protease may cleave an Ala-Ser bond. A study of the potential for vaccination of yellowtail fry by injection of insect cell lysates infected with baculovirus, containing either cDNA of segment A, VP2-epitope, or NS/VP3 was undertaken. Only a vaccination with cell lysates infected with a recombinant virus carrying the full length of YAV segment A gene demonstrated approximately the same effect as that of inactivated YAV. This result suggested that all proteins VP2, VP3, and NS are required for an effective vaccination.
Key words: YAV; segment A; baculovirus; gene expression; vaccination

Keisuke NAKASHIMA＋ and Jun-ichi AZUMA

Division of Environmental Science and Technology, Graduate School of Agricultural Science,
Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan

Received February 14, 2000; Accepted March 16, 2000
A multi-enzyme distribution of endo-β-1,4-glucanase activity was found in the digestive system of a worker caste of the lower termite Coptotermes formosanus (Shiraki) by zymogram analysis. Its distribution analysis demonstrated that about 80% of this activity was localized in salivary glands from where only one component (EG-E) was secreted into the digestive tract.
EG-E was isolated by a combination of chromatographic and electrophoretic techniques. Its molecular mass, optimal pH and temperature, isoelectric point, and Km were 48 kDa, 6.0, 50°C, 4.2, and 3.8 (mg/ml on carboxymethylcellulose), respectively. EG-E hydrolyzed cellooligosaccharides with a degree of polymerization of 4 and larger, and had low activity on crystalline cellulose. Main reaction products from low molecular weight cellulose were cellobiose and cellotriose. The N-terminal amino acid sequence of EG-E has similarity with fungal endo-β-1,4-glucanases and cellobiohydrolases of the glycosyl hydrolase family 7 rather than the other insect endo-β-1,4-glucanases of family 9.
Key words: cellulase; endo-β-1,4-glucanase; Coptotermes formosanus; lower termite; salivary glands

Badr A. ALBASSAM

Department of Biochemistry, College of Science, King Saud University, P.O.Box 2455,
Riyadh 11451, Saudi Arabia

Received August 30, 1999; Accepted February 8, 2000
Phenolic acids inhibited the activities of wheat leaf nitrate reductase depending on phenolic structure and concentration. Possible conformational change(s) in the enzyme induced by hydrogen bonding and/or hydrophobic interactions might be the cause of the enzyme inhibition. NADH:cytochrome C reductase partial activity was unaffected, which indicates that terminal nitrate-reducing domain of NR may be the site of polyphenol binding.
Key words: wheat; nitrate reductase; inhibition kinetics; tannic acid; phenolic acids

LIU Ji-Kai,# MA Yun-Bao, WU Da-Gang, LU Yang,* SHEN Zhi-Qiang,**
ZHENG Qi-Tai,* and CHEN Zhi-He**

Department of Phytochemistry, Kunming Institute of Botany, The Chinese Academy of Sciences,
Kunming 650204, P.R. China
*Institute of Materia Medica, The Chinese Academy of Medical Sciences,
Beijing 100050, P.R. China
**Yunnan Pharmacological Laboratory of Natural Products, Kunming Medical College,
Kunming 650031, P.R. China

Received October 18, 1999; Accepted February 29, 2000
A novel monoterpenoid-derived metabolite, paeonilide, was isolated from the roots of Paeonia delavayi. Its structure was established by a combination of spectroscopic and X-ray crystallographic analyses. It showed an anti-PAF effect with an IC50 value of ca. 8 mg/ml.
Key words: paeonilide; monoterpenoid; anti-PAF; Paeonia delavayi

Zhi-Hong ZHAO,＋ Shin-Ichi KUSAKARI,1 Kyotsugu OKADA,1 Akio MIYAZAKI,1
and Tetsuya OSAKA2

＋Satosen Co., Ltd., 2-20-65 Tamadenishi, Nishinari-ku, Osaka 557-0025, Japan
1Osaka Prefectural Agricultural and Forestry Research Center, 442 Shakudo,
Habikino-shi 583-0862, Japan
2Department of Applied Chemistry, School of Science and Engineering, Waseda University,
3-4-1 Okubo, Shinjuku-ku, Tokyo 169-0072, Japan

Received November 8, 1999; Accepted February 23, 2000
Silver-coated cloth (SCC) effectively controlled root rot that was caused by Pythium aphanidermatum in hydroponically grown cucumber plants. The presence of SCC in the hydroponic solution reduced the root rot from 100% to 10% 20 days after inoculation with zoospores of P. aphanidermatum. It was suggested that the inhibition of SCC was caused not only by the silver ion dissolved from SCC, but also by the metallic silver and silver compounds formed on the surface of the root.
Key words: hydroponically grown cucumber; Pythium aphanidermatum; root rot; zoospore; silver-coated cloth

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 November 15, 1999; Accepted March 2, 2000
Four degraded okaramine B (2) products, 4?,5?-dihydrookaramine B (3), two azetidine ring-opened compounds (4 and 5) and 1?,2?,4?,5?-tetrahydrookaramine B (6), were prepared and their insecticidal activity was examined. Neither compounds 4 nor 5 showed such activity against silkworms, indicating that the azetidine ring moiety played an important role in the insecticidal activity. Moreover, both compounds 3 and 6 exhibited lower activity than 2, which means that the azocine ring moiety was indispensable to form the active conformation.
Key words: Penicillium simplicissimum; okaramines; insecticidal compound

Toshio SHIDA, Hiromi HATTORI, Fuminori ISE, 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 November 19, 1999; Accepted March 13, 2000
N-acetylmuramoyl-L-alanine amidase CwlC of Bacillus subtilis was overproduced in Escherichia coli and purified 21-fold. The amidase hydrolyzed type A cell walls such as B. subtilis. The amidase bound slightly to the Microbacterium lacticum cell wall (type B), but did not entirely hydrolyze it. The presence of calcium or magnesium ion increased the resistance of the amidase to heat denaturation.
Key words: Bacillus subtilis; N-acetylmuramoyl-L-alanine amidase; CwlC; substrate specificity; circular dichroism

Myungil CHUNG,1 Seckho HA,1 Soonseog JEONG,2 Jinduck BOK,1
Kwangkeun CHO,1 Myunggi BAIK,3 and Yunjaie CHOI1,＋

1Department of Animal Science & Technology, School of Agricultural Biotechnology,
College of Agriculture & Life Science, Seoul National University, Suweon 441-744, Korea
2Department of Biochemistry, College of Medicine, University of Saskatchewan,
Saskatoon, SK, S7N 5E5, Canada
3Department of Genetic Engineering and Institute of Biotechnology, Chonnam National University,
Kwangju 500-757, Korea

Received December 13, 1999; Accepted Feburary 24, 2000
Two overlapping cDNA clones have been isolated from bovine adipose tissue by the reverse-transcription-polymerase chain reaction (RT-PCR) method. The combined sequence of the two clones was 1039 bp in length and encoded 345 amino acids. The deduced amino acid sequence of the clones showed 96.5% similarity to that of sheep SCD and more than 88% similarities to other mammalian SCD1s, indicating that the clones are the cDNAs for the bovine SCD1. The transcript size of the bovine SCD1 was about 4.9 kb, the message was detected in the bovine adipose tissues but not in the liver. Female cattle expressed threefold higher levels of SCD1 mRNA than male animals.
Key words: stearoyl CoA desaturase; cDNA; mRNA expression; bovine adipose tissues

Masaki TERAHARA,＋ Sachiko NISHIDE, and Tsutomu KANEKO

Food Functionality Research Institute, Meiji Milk Products Co. Ltd., 1-21-3 Sakae-Cho,
Higashimurayama, Tokyo 189-8530, Japan

Received January 7, 2000; Accepted March 3, 2000
We examined the 3-amino-1,4-dimethyl-5H-Pyrido-[4,3-b]indole (Trp-P-1) concentration in the blood after administering Trp-P-1 (0.25 mg/ml) with or without Streptococcus thermophilus 1131 cells (10 mg/ml) to rats. The Trp-P-1 concentration in the blood from the portal vein was significantly lower in the rats that had been administered with Trp-P-1 with the strain 1131 cells than without them. However, there was no difference in the Trp-P-1 concentration in the blood taken from the abdominal aorta of these rats.
Key words: portal vein; abdominal aorta; blue rayon

Kenji TO-O, Hiroshi KAMASAKA, Takashi KURIKI, and Shigetaka OKADA

Biochemical Research Laboratory, Ezaki Glico Co., Ltd. 4-6-5, Utajima, Nishiyodogawa-ku,
Osaka 555-8502, Japan

Received January 7, 2000; Accepted March 21, 2000
The intracellular acid phosphatase II (ACPase II) produced by Aspergillus niger KU-8 preferentially dephosphorylates C-6 phosphate groups rather than C-3 phosphate groups of phosphoryl oligosaccharides. In this study, the kinetic parameters of ACPase II were measured. 32-phosphoryl maltotriose and 62-phosphoryl maltotriose, which differ only in the binding position of the phosphate group, were prepared and used as the substrates. The Km for both substrates were similar. However, the kcat value for the 62-phosphoryl maltotriose was about three-fold of that for the 32-phosphoryl maltotriose.
Key words: acid phosphatase; Aspergillus niger; phosphoryl oligosaccharides; kinetic; substrate selectivity

Hideki HYODO,* Miho TAKEMURA,* Akiho YOKOTA,* Kanji OHYAMA,*,**
and Takayuki KOHCHI*,＋

*Graduate School of Biological Sciences, Nara Institute of Science and Technology,
8916-5 Takayama, Ikoma, Nara 630-0101, Japan
**Laboratory of Plant Molecular Biology, Division of Integrated Life Science,
Graduate School of Biostudies, Kyoto University, Kyoto, 606-8502, Japan

Received January 11, 2000; Accepted February 1, 2000
To analyze plant meristem functions, we systematically isolated thirteen cDNA clones for transcripts that are highly expressed in the inflorescence apices of Arabidopsis by differential screening, using an equalized cDNA library with the clones arrayed on membranes. Their deduced amino acid sequences indicate important functions for proteins in determining the cell wall architecture, translation capacity and mitotic activity in the shoot apex during reproductive growth.
Key words: Arabidopsis thaliana; differential screening; equalized cDNA library; gene expression; shoot apex

Ken SHIMUTA, Naoko ODA-UEDA, Masahiro WASHIO, Hiroshi OYAMA,*
Kohei ODA,* and Daisuke TSURU＋

Department of Applied Microbial Technology, sojo University,
Ikeda 4-22-1, Kumamoto 860-0082, Japan
*Faculty of Textile Science, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku,
Kyoto 606-0962, Japan

Received January 17, 2000; Accepted March 21, 2000
An expression and secretion system for scytalidopepsin B, an acid protease from Scytalidium lignicolum, was constructed in yeast. Saccharomyces cerevisiae AH22 was transformed with an yeast-E. coli shuttle vector, pAM82, in which an yeast invertase signal segment and the cDNA encoding the pro- and mature enzyme regions were inserted. The transformant was found to secret a pepstatin-insensitive acid protease, when cultured aerobically in a low phosphate (Pi) medium. Amino terminal amino acid sequencing analysis indicated that the recombinant acid protease was accurately processed and secreted as a mature form.
Key words: pepstatin-insensitive acid protease; scytalidopepsin B; yeast invertase signal segment; shuttle vector

Takashi AOKI,1,＋ Mamiko MIYASHITA,1 Hiroyoshi FUJINO,1,2 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 21, 2000; Accepted March 6, 2000
A green fluorescent protein mutant (S147P GFP) was fused with protein A and expressed in Escherichia coli. This fusion protein (PA-GFP147) was used in immunoblotting studies as a new detection system, designated as ``flexible single-step detection (FSSD)′′. In FSSD, the detection of blotted antigen was done in one step, and the antigen-antibody reaction can be monitored by UV-irradiation in real time. The reaction time, therefore, can be flexibly controlled by monitoring the green fluorescence.
Key words: green fluorescent protein; protein A; fusion protein; immunoblotting