Contents and Abstracts of Latest Issue of BBB

(Vol.64 No.5 2000)

Sang Ryeol PARK,1 Soo Jeong CHO,2 and Han Dae YUN1・

1Department of Agricultural Chemistry, Gyeongsang National University, Chinju 660-701, Korea
2Department of Microbiological Engineering, Chinju National University, Chinju 660-758, Korea

Received August 16, 1999; Accepted January 3, 2000
The phytopathogenic bacterium Erwinia chrysanthemi secretes multiple isozymes of plant cell wall disrupting enzymes such as pectate lyase and endoglucanase. We cloned genomic DNA from Erwinia chrysanthemi PY35. One of the E. coli XL1-Blue clones contained a 5.1-kb BamHI fragment and hydrolyzed carboxymethyl cellulose and polygalacturonic acid. By subsequent subcloning, we obtained a 2.9-kb fragment (pPY100) that contained the pel gene responsible for CMCase and pectate lyase activities. The pel gene had an open reading frame (ORF) of 1,278 bp encoding 425 amino acids with a signal peptide of 25 amino acids. Since the deduced amino acid sequence of this protein was very similar to that of PelL of E. chrysanthemi EC16, we concluded that it belonged to the pectate lyase family EC 4.2.2.2, and we designated it PelL1. Sequencing showed that the PelL1 protein contains 400 amino acids and has a calculated pI of 7.15 and a molecular mass of 42,925 Da. The molecular mass of PelL1 protein expressed in E. coli XL1-Blue, as analyzed by SDS-PAGE, appeared to be 43 kDa. The optimum pH for its enzymatic activity was 9, and the optimum temperature was about 40°C.
Key words: Erwinia chrysanthemi; pectate lyase gene; pelL1; CMCase

-2-
Purification and Characterization of Aspartic Proteinase from Sunflower Seeds

Hyekyeong PARK, Nanae YAMANAKA, Anita MIKKONEN,＊ Isao KUSAKABE,
and Hideyuki KOBAYASHI＊＊,・

Institute of Applied Biochemistry, University of Tsukuba, Ibaraki 305-0006, Japan
＊Research and Development Center of Kajaani, University of Oulu, FIN87101 Kajaani, Finland
＊＊National Food Research Institute, Ministry of Agriculture, Forestry and Fisheries, Ibaraki 305-8642, Japan

Received September 1, 1999; Accepted January 11, 2000
Aspartic proteinases were purified from sunflower seed extracts by affinity chromatography on a pepstatin A-EAH Sepharose column and by Mono Q column chromatography. The final preparation contained three purified fractions. SDS-PAGE showed that one of the fractions consisted of disulfide-bonded subunits (29 and 9 kDa), and the other two fractions contained non-covalently bound subunits (29 and 9 kDa). These purified enzymes showed optimum pH for hemoglobinolytic activity at pH 3.0 and were completely inhibited by pepstatin A like other typical aspartic proteinases. Sunflower enzymes showed more restricted specificity on oxidized insulin B chain and glucagon than other aspartic proteinases. The cDNA coding for an aspartic proteinase was cloned and sequenced. The deduced amino acid sequence showed that the mature enzyme consisted of 440 amino acid residues with a molecular mass of 47,559 Da. The difference between the molecular size of purified enzymes and of the mature enzyme was due to the fact that the purified enzymes were heterodimers formed by the proteolytic processing of the mature enzyme. The derived amino acid sequence of the enzyme showed 30--78％ sequence identity with that of other aspartic proteinases.
Key words: aspartic proteinase; sunflower seed; substrate specificity; primary structure; Helianthus annuus

-3-
Characterization of Acid-Stable Glucose Isomerase from Streptomyces sp.,
and Development of Single-Step Processes for High-Fructose
Corn Sweetener (HFCS) Production

Takahiro KANEKO,・ Saori TAKAHASHI, and Kyuichi SAITO

Department of Bioengineering, Akita Research Institute of Food and Brewing(ARIF), 4-26 Sanuki, Arayamachi, Akita 010-1623, Japan

Received October 4, 1999, Accepted December 10, 1999
The glucose isomerase from Streptomyces olivaceoviridis E-86 was purified by chromatographic procedures, showing one single protein band in the SDS-PAGE. The enzyme had high acid stability, and there was no loss in enzyme activity at pH 5.0 after incubation at 60°C for 30 hr. The enzyme had sufficients activity at 60°C, pH 5.5, (which is the reaction condition for a single-step process with a glucoamylase from A. niger), and at 58°C, pH 6.0, (condition with a glucoamylase from R. niveus). By using this acid-stable glucose isomerase, a single-step process to produce high-fructose corn sweetener (HFCS) from liquefied starch was formed without any reductant or other reagents for enzyme stabilization. The HFCS produced was about fifty percent fructose and less than 1.5％ unknown oligosaccharides.

-4-
Amino Acid Sequence of a Nuclease (Nuclease Le1) from Lentinus edodes

Hiroko KOBAYASHI,a Fumi KUMAGAI,a Tadashi ITAGAKI,a Norio INOKUCHI,a,・
Takashi KOYAMA,a Masanori IWAMA,b Kazuko OHGI,b and Masachika IRIEb

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

Received October 13, 1999; Accepted December 27, 1999
The fruit bodies of Lentinus edodes produce two acid nucleases, nucleases Le1 and Le3, both of which are thought to be candidates for the enzymes producing a tasty substance, 5?-GMP. To obtain the basic information on the mechanism of production of 5?-GMP, and structure-function relationship of these nucleases, the primary structure of nuclease Le1 was estimated by both protein chemistry and gene cloning. Nuclease Le1 is a glycoprotein and consists of 290 amino acid residues, and about 2 and 6 residues of hexosamine and neutral sugar, respectively. The nucleotide sequence of cDNA and genomic DNA encoding nuclease Le1 indicated the presence of 20 amino acid residues of a signal peptide.
Nuclease Le1 has 115 and 108 residues of identical amino acid residues with nucleases P1 and S, respectively. The amino acid residues concerning the coordination with Zn2+ in nuclease P1 are all conserved in nuclease Le1. Nuclease Le1 contains 8 half-cystine residues and 4 of them are located at the same places as those of nucleases P1 and S.
Key words: cDNA; genomic DNA; Lentinus edodes; nuclease; 3?-nucleotidase

-5-
Binding of Barley and Wheat α-Thionins to Polysaccharides

Shigeru OITA,1,・ Mayumi OHNISHI-KAMEYAMA,2 and Tadahiro NAGATA2

1Shikoku National Agricultural Experiment Station, Ministry of Agriculture, Forestry and Fisheries,
1-3-1 Senyu, Zentsuji, Kagawa 765-8508, Japan
2National Food Research Institute, Ministry of Agriculture, Forestry and Fisheries, 2-1-2 Kannondai,
Tsukuba, Ibaraki 305-8642, Japan

Received November 1, 1999; Accepted December 27, 1999
An antimicrobial peptide termed BCP-2 was purified from barley grain by chitin-affinity treatment and HPLC. The results of amino acid analysis and mass spectrometry of BCP-2 indicate that the peptide is very similar to barley α-thionin. BCP-2 and wheat α1-thionin were also bound to β-glucan but not to starch. The binding of BCP-2 to laminarin (β-1,3-1,6-glucan) and laminarioligosaccharides was supported by fluorescence polarization data. This is the first report on the binding of α-thionins to polysaccharide containing chitin and β-1,3-glucan, which construct fungal cell walls.
Key words: α-thionin; chitin; β-glucan; binding peptide; antifungal

-6-
Effect of Dietary Chitosans with Different Viscosity on Plasma Lipids and Lipid
Peroxidation in Rats Fed on A Diet Enriched with Cholesterol

Meng-Tsan CHIANG,・ Hsien-Tsung YAO, and Hsing-Chen CHEN

Department of Food Science, National Taiwan Ocean University, 2 Pei-Ning Road, Keelung,
Taiwan 202, R.O.C.

Received November 8, 1999; Accepted January 6, 2000
To investigate the effect of dietary chitosan on lipid metabolism, male SD (Sprague-Dawley) rats were fed a cholesterol-enriched diet containing 5％ cellulose (CE), 5％ chitosan (CCS; high viscosity), or 5％ chitosan (FCS; low viscosity) for 4 weeks. The two types of chitosan with a comparable degree of deacetylation had a different molecular weight and intrinsic viscosity. Significantly (p<0.05) lower plasma total cholesterol, LDL-cholesterol and VLDL-cholesterol concentrations were observed in the rats fed on the chitosan diets. In addition, chitosan significantly increased the fecal cholesterol and triglyceride contents. Although no significant difference in body weight was found among the dietary groups, the rats fed on the chitosan diets had lower relative liver weight when compared with those fed on the cellulose diet. Both of the chitosan groups had significantly lower liver total lipid and total cholesterol contents compared to the cellulose group, although the FCS group was less effective. The plasma and liver thiobarbituric acid reactive substances (TBAR) values were similar in the CE and FCS groups, while the CCS group had increased liver TBAR values. Although a significant increase in liver glucose-6-phosphate dehydrogenase activity was observed in the CCS group, no significant change was found in the FCS group. The observed influence of chitosans with different viscosity on the plasma lipid level, liver lipids and lipid peroxidation suggests that, while the hypocholesterolemic action of chitosans with different viscosity was similar, changes in the liver lipids and liver peroxidation status depended on their molecular weight when the deacetylation degree was comparable.
Key words: chitosan; rats; plasma lipids; liver lipids

-7-
End-Product Regulation and Kinetic Mechanism of Guanosine-Inosine
Kinase from Escherichia coli

Hisashi KAWASAKI, Megumi SHIMAOKA, Yoshihiro USUDA, and Takashi UTAGAWA

Applied Microbiology Laboratory, Fermentation and Biotechnology Laboratories, Ajinomoto Co., Inc.,
1-1 Suzuki-cho, Kawasaki-ku, Kawasaki-shi 210-8681, Japan

Received November 10, 1999; Accepted January 19, 2000
Escherichia coli guanosine-inosine kinase was overproduced, purified, and characterized. The native and subunit molecular weights were 85,000 and 45,000, respectively, indicating that the enzyme was a dimer. A pI of 6.0 was obtained by isoelectric focusing. In addition to ATP, it was found that deoxyadenosine 5?-triphosphate, UTP, and CTP could serve as phosphate donors. The phosphate acceptors were guanosine, inosine, deoxyguanosine and xanthosine, but not adenosine, cytidine, uridine, or deoxythymidine. Maximum activity was attained at an ATP/Mg＾{2+} concentration ratio of 0.5. In the presence of pyrimidine nucleotides, enzyme activity was slightly increased, while it was markedly inhibited by GDP and GTP. Initial velocity and product inhibition studies support an ordered Bi Bi mechanism in which guanosine was the first substrate to bind and GMP was the last product to be released. Guanosine kinase may be a regulatory enzyme that has a role in modulating nucleotide levels.
Key words: end-product regulation; kinetic mechanism; guanosine-inosine kinase; purine nucleotide; Escherichia coli

-8-
Transport of Pyruvate in Saccharomyces cerevisiae and Cloning
of the Gene Encoded Pyruvate Permease

Osamu AKITA,・ Chiharu NISHIMORI, Toshihiro SHIMAMOTO, Tsutomu FUJII,
and Haruyuki IEFUJI

National Research Institute of Brewing, 3-7-1 Kagamiyama, Higashi-Hiroshima 739-0046, Japan

Received November 11, 1999; Accepted January 13, 2000
Pyruvate uptake in Saccharomyces cerevisiae was not observed at 0°C and was prevented by the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP). The initial uptake rate of S. cerevisiae kyokai No. 901 was maximum at pH 6 and Km＝4.1 mM. It seemed that lactate inhibited the pyruvate uptake competitively from the results of the Lineweaver-Burk plots. The inhibition constant (K_{i}) in the presence of 3 mM lactate was 1.6 mM. The pyruvate uptake was inhibited by D-glucose and deoxyglucose, but not by L-glucose, acetate or ethanol.
Mutants of laboratory strain No. 5022 ((a) his(2,6), ura3) deficient in pyruvate uptake were isolated from fluoropyruvate resistant mutants. Transformation of the mutant with a yeast genomic library allowed the isolation of the gene JEN1 (YKL217w), which restored pyruvate uptake. Disruption of JEN1 abolished the uptake of pyruvate and gained the resistance against fluoropyruvate. The results indicate that no other monocarboxylate permease is able to efficiently transport pyruvate in S. cerevisiae.
Key words: Saccharomyces cerevisiae; pyruvate transporter; fluoropyruvate resistant mutant; pyruvate transport deficient mutant; JEN1 gene

-9-
Design of a Novel Membrane-Destabilizing Peptide Selectively Acting
on Acidic Liposomes

Sachiko MACHIDA,・ Setsuko NIIMI, Xiaohua SHI, Yoshiji ANDO,1 and Yong YU2

National Food Research Institute, Ministry of Agriculture, Forestry, and Fishery, 2-1-2 Kannondai,
Tsukuba, Ibaraki 305-8642, Japan
1National Institute of Animal Health, Ministry of Agriculture, Forestry, and Fishery, 3-1-1 Kannondai,
Tsukuba, Ibaraki 305-0852, Japan
2Leica K.K. 2450-2 Kamiyikoda, Tsukuba, Ibaraki 305-0854, Japan

Received November 12, 1999; Accepted December 28, 1999
The design of amphipathic peptides resulted in a novel peptide with a selective ability to destabilize lipid bilayers of acidic liposomes. The newly synthesized peptide, termed mast 21, is a 21-residue long amino acid chain and can only act effectively on acidic liposomes lacking cholesterol. Moreover, mast 21 killed Gram-positive and Gram-negative bacteria, and it had no hemolytic activity. The antimicrobial and hemolytic activities paralleled the results of membrane destabilizing activity using liposomes. Circular dichroism and Trp-fluorescence emission spectra showed changes in the peptide conformation and circumstances around the peptide during interaction with liposomes. These changes were consistent with an increased α-helical content and a less polar environment for the tryptophan residue of the peptide. Mast 21 was observed under dark-field microscopy in real time attacking liposomes. Acidic liposomes were attacked, which resulted in peeling of the lipid bilayer with its subsequent destruction.
Key words: membrane-destabilizing peptide; antimicrobial peptide; liposome; model membrane

-10-
The Pathway of Dephosphorylation of myo-Inositol Hexakisphosphate
by Phytases from Wheat Bran of Triticum aestivum L. cv. Nourin ・＃61

Tadao NAKANO,1 Toshio JOH,2 Kazumasa NARITA,2 and Toshiro HAYAKAWA2

1Graduate School of Science and Technology, Niigata University, Ikarashi 2-8050 Niigata,
Niigata 950-2181, Japan
2Department of Applied Biological Chemistry, Faculty of Agriculture, Niigata University,
Ikarashi 2-8050 Niigata, Niigata 950-2181, Japan

Received November 17, 1999; Accepted January 5, 2000
Phytases are the primary enzymes responsible for the hydrolysis of phytic acid, myo-inositol-1, 2, 3, 4, 5, 6-hexakisphosphate (InsP6). The pathway of hydrolysis of InsP6 by phytase from wheat bran of Triticum aestivum L. cv. Nourin ＃61 is proved in this study. Structures of the intermediates were established by a variety of nuclear magnetic resonance techniques (1H-, two-dimensional 1H-1H coupling-correlation spectra and two-dimensional 31P-1H correlation spectra), gas chromatography, and bioassay. On the basis of the structures identified, initial hydrolysis of the phosphate ester occurs at the D/L-4 position of InsP6 to yield D/L-Ins (1, 2, 3, 5, 6) P5. After the dephosphorylation, the pathway of dephosphorylation is divided into two routes. The main route proceeds via D/L-Ins (1, 2, 5, 6) P4, D/L-Ins (1, 2, 6) P3 and D/L-Ins (1, 2) P2, while the minor route proceeds via D/L-Ins (1, 2, 3, 6) P4, Ins (1, 2, 3) P3 and D/L-Ins (1, 2) P2. D/L-Ins (1, 2) P2 is hydrolyzed at the D/L-1 or 2-position, and finally myo-inositol is produced.
Key words: phytase; inositol phosphates; NMR; gas chromatography; dephosphorylation pathway

-11-
Induction of Oxidatively Modified Proteins in Skeletal Muscle by Electrical
Stimulation and Its Suppression by Dietary Supplementation
of (--)-Epigallocatechin Gallate

Takashi NAGASAWA,・ Hiromichi HAYASHI, Naoko FUJIMAKI, Naoyuki NISHIZAWA,
and David D. KITTS＊

Department of Bioscience and Technology, Faculty of Agriculture, Iwate University,
Morioka, Iwate 020-8550, Japan
＊Food, Nutrition and Health, Faculty of Agricultural Sciences, The University of British Columbia,
Vancouver, B.C. V6T 1W5, Canada

Received November 19, 1999; Accepted December 28, 1999
The oxidative stress produced by electrical stimulation-induced muscle contraction was examined in the skeletal muscle proteins of rats that had been fed on the dietary flavonoid, (--)-epigallocatechin gallate (EGCg). Electrical stimulation of the rat leg muscle every second day for a two-week period resulted in an increased (p<0.05) muscle weight and accumulation of oxidatively induced modified proteins. Similar stimulation conducted every day for only one week had no effect on the muscle weight or protein oxidation, although the rate of protein degradation increased. Rats fed on a 20％ casein diet supplemented with 0.1％ EGCg for 2 weeks responded to the electrical stimulation of muscle contraction by reducing the increased muscle protein carbonyl content when compared to their counterparts fed on a control diet. There was no change in activity of antioxidative enzymes in muscle tissue of the EGCg-fed rats receiving electrical stimulation. The results of this study show that the antioxidative property of EGCg was effective for suppressing oxidative modification of the skeletal muscle protein induced by electrical stimulation. This finding demonstrates that EGCg has a beneficial effect in vivo on the free radical-mediated oxidative damage to muscle proteins.
Key words: muscle; oxidatively modified protein; protein degradation; flavonoid; free radicals

-12-
Avoidance of Solidification of Sesame Oil at Low Temperature
by Self-interesterification with Immobilized Lipase

Hiroyuki SHIBASAKI1 and Tsuneo YAMANE2

1Fermentation and Food Experiment Station Kagawa Prefectural Government, 1351-1 Noma,
Uchinomi, Syozu, Kagawa 761-4421, Japan
2Laboratory of Molecular Biotechnology, Graduate School of Bio- and Agro-Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan

Received November 29, 1999; Accepted January 13, 2000
To reduce the freezing point of sesame oil, the lipase-catalyzed interesterification of sesame oil in a solvent free system was studied. The lipase was immobilized on Celite and refined sesame oil was used as the only substrate for the reaction. After interesterification, the oil did not solidify at 0°C even after 24 h, and even longer storage at 2--4°C did not result in solidification. The change of physical behavior was investigated with a differential scanning calorimeter and X-ray diffraction, and reduction in the thermodynamic and crystallographic stability of the interesterified oil was demonstrated. The change in triacylglycerol species composition after the reaction was analyzed, showing that content of trisaturated acylglycerol was decreased.
Key words: lipase; interesterification; sesame oil; DSC; X-ray diffraction

-13-
Alteration of the Self-incompatibility Phenotype in Brassica by Transformation
of the Antisense SLG Gene

Hiroshi SHIBA,a Nobuko KIMURA,a Seiji TAKAYAMA,a Kokichi HINATA,b
Akinori SUZUKI,c and Akira ISOGAIa,・

aGraduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama,
Ikoma 630-0101, Japan
bResearch Institute of Seed Production Co. Ltd., 6-6-3 Minamiyoshinari, Sendai 989-3204, Japan
cDepartment of Applied Biological Chemistry, Division of Agricultural Life Sciences, The University of Tokyo,
Tokyo 113-8657, Japan

Received November 29, 1999; Accepted December 28, 1999
Self-incompatible (SI) Brassica rapa (syn. B. campestris) was transformed with an antisense SLG gene by using SLG8 cDNA isolated from the B. campestris S8 homozygote. Two transformed lines were obtained and analyzed. Northern blot and Western blot analyses revealed that endogenous SLG and SRK were greatly reduced of the transcriptional and translational levels in the transformant. Pollination experiments confirmed that their SI phenotype had broken down. In addition, the progeny with the antisense SLG gene, resulting from self- or cross-pollination of the transgenic plant, also showed the self-compatible phenotype. The breakdown of SI in the tranformants was due to the change in property of the stigma and not of the pollen. These results provide strong evidence that SLG and/or SRK is implicated in the pollen-stigma recognition of SI and that they act only as stigmatic factors.
Key words: SLG; SRK; Brassica rapa; self-incompatibility; antisense RNA

-14-
Induction of N-Hydroxycinnamoyltyramine Synthesis and Tyramine
N-Hydroxycinnamoyltransferase (THT) Activity by Wounding
in Maize Leaves

Atsushi ISHIHARA,・ Naoki KAWATA, Tetsuya MATSUKAWA, and Hajime IWAMURA

Division of Applied Life Sciences, Graduate School of Kyoto University, Kyoto 606-8502, Japan
CREST, Japan Science and Technology Corporation (JST), Japan

Received December 13, 1999; Accepted January 14, 2000
Both N-p-coumaroyl- and N-feruloyltyramine accumulated in response to wounding in leaf segments of maize. The amount of N-hydroxycinnamoyltyramines started to increase 3--6 h after wounding and peaked at 12 h. Thereafter, the amount of N-p-coumaroyltyramine decreased rapidly, while the N-feruloyltyramine content remained at a high level. The accumulation of N-hydroxycinnamoyltyramines was accompanied by an increase in the tyramine N-hydroxycinnamoyltransferase (THT) activity. This increase was initially detected 3 h after wounding and reached a maximum at 36 h, the level of activity being 40 and 11 times that in the leaves before wounding and in the control leaves, respectively. Partial purification of THT from wounded leaves by (NH4)2SO4 precipitation and subsequent two steps of anion-exchange chromatography resulted in a 12.5-fold increase in specific activity. Kinetic studies with this partially purified enzyme revealed that the best substrates were tyramine and feruloyl-CoA, although tryptamine and sinapoyl-CoA also efficiently served as substrates. The apparent native molecular weight of the enzyme was determined by gel filtration as 40 kDa.
Key words: hydroxycinnamoyltyramine; tyramine N-hydroxycinnamoyltransferase; wound; Zea mays

-15-
Note
Application of a Bioluminescence Method for the Beer Industry: Sensitivity
of MicroStarTM-RMDS for Detecting Beer-spoilage Bacteria

Toshihiro TAKAHASHI,・ Yasukazu NAKAKITA, Junji WATARI, and Ken SHINOTSUKA

Brewing Research Laboratories, Sapporo Breweries, Ltd., 10 Okatome, Yaizu, Shizuoka 425-0013, Japan

Received July 14, 1999; Accepted January 18, 2000
We set up the original operating conditions of the MicroStarTM-Rapid Microbe Detection System (RMDS) to suppress false positives, which have kept this system from practical. The detection limit of our system was between 6.3×10--16 mol and 3.1×10--16 mol in terms of the amount of ATP, which is approximately equal to the ATP content of one yeast cell or 50 lactic acid bacteria cells. The detection time and the detection count were compared between the RMD method and the conventional plate count method (C.P.C. method) using 23 test samples of beer-spoilage Lactobacillus brevis. Judging from the detection time and detection count, 16--24 hours of cultivation for the RMD method corresponded to 40--96 hours of cultivation for the C.P.C. method. The RMD method reached a useful level for our practical use at the point of sensitivity.
Key words: MicroStarTM-RMDS; Lactobacillus brevis; rapid detection

-16-
Note
1,1-Diphenyl-2-picrylhydrazyl Radical-scavenging Compounds from Soybean
Miso and Antiproliferative Activity of Isoflavones from Soybean Miso toward
the Cancer Cell Lines

Akira HIROTA,＊ Shoji TAKI, Satoru KAWAII,＊＊ Masamichi YANO,＊＊ and Naoki ABE

Laboratory of Applied Microbiology, School of Food and Nutritional Sciences, University of Shizuoka,
52-1 Yada, Shizuoka 422-8526, Japan
＊＊National Institute of Fruit Tree Science, Okitsu-naka, Shimizu 424-0204, Japan

Received August 10, 1999; Accepted December 28, 1999
Guided by their DPPH radical-scavenging activity, nine compounds were isolated from soybean miso. Of these, 8-hydroxydaidzein, 8-hydroxygenistein and syringic acid had as high DPPH radical-scavenging activity as that of α-tocopherol. The antiproliferative activity of four of the isolated isoflavones toward three cancer cell lines was examined. 8-Hydroxygenistein showed the highest activity (IC50＝5.2 μM) toward human promyelocytic leukemia cells (HL-60).
Key words: soybean miso; 8-hydroxyisoflavone; DPPH radical-scavenging activity; antiproliferative activity; cancer cell

-17-
Note
α-Amylase Inhibitors from Roselle (Hibiscus sabdariffa Linn.) Tea

Chanida HANSAWASDI, Jun KAWABATA,・ and Takanori KASAI

Laboratory of Food Biochemistry, Division of Applied Bioscience, Graduate School of Agriculture,
Hokkaido University, Kita-ku, Sapporo 060-8589, Japan

Received September 27, 1999; Accepted January 14, 2000
A roselle (Hibiscus sabdariffa Linn.) tea extract was found to have high inhibitory activity against porcine pancreatic α-amylase. Hibiscus acid and its 6-methyl ester were respectively isolated as active principles from the 50％ methanol and acetone extracts of roselle tea. The activity of each isolate was compared to that of structurally related citric acid, a previously known inhibitor of fungal α-amylase.
Key words: porcine pancreatic α-amylase inhibitor; roselle; Hibiscus sabdariffa

-18-
Note
Identification of New Geometric Isomers of Methyl Linoleate Hydroperoxide
and Their Chromatographic Behavior

Masako TOKITA・ and Makio MORITA

Department of Food and Nutrition, Japan Women′s University, 2-8-1 Mejirodai, Bunkyo-ku,
Tokyo 112-8681, Japan

Received October 12, 1999; Accepted January 11, 2000
New geometric isomers, methyl (9Z,11Z)-13-hydroperoxy-9,11-octadecadienoate and methyl (10Z,12Z)-9-hydroperoxy-10,12-octadecadienoate, were proved to be present in methyl linoleate hydroperoxide produced by autoxidation. They were identified from their UV, MS, and 1H-NMR spectra after conversion to the corresponding oxo derivatives: methyl (9Z,11Z)-13-oxo-9,11-octadecadienoate and methyl (10Z,12Z)-9-oxo-10,12-octadecadienoate. Their chromatographic behavior is described.
Key words: methyl linoleate; hydroperoxide; autoxidation; oxooctadecadienoate

-19-
Note
Effect of Oxidized Cholesterol on Age-associated Changes to Immune
Parameters in Spleen Lymphocytes and Peritoneal Exudate
Cells Derived from Rats

Kyoichi OSADA,1,・ Kaori MINEHIRA,2 Shinichiro INOUE,2 Shingo NAKAMURA,1 Koji YAMADA,2
and Michihiro SUGANO3

1Laboratory of Applied Life Science, Faculty of Agriculture and Life Science, Hirosaki University,
Hirosaki 036-8152, Japan
2Laboratory of Food Science, Kyushu University, School of Agriculture, Fukuoka 812-8581, Japan
3Faculty of Environmental and Symbiotic Sciences, Prefectural University of Kumamoto,
Kumamoto 862-8502, Japan

Received October 18, 1999; Accepted January 8, 2000
The effects of oxidized cholesterol on immune parameters were examined by using spleen lymphocytes and peritoneal exudate cells (PEC) derived from 5-week- (Young) and 9-month-old (Adult) rats. The immunoglobulin (Ig) G and IgM production was inhibited by oxidized cholesterol in the rats of both ages when lymphocytes were exposed to 30 μg/ml of oxidized cholesterol for 24 hr. The intracellular IgA level was also lowered by 30 μg/ml of oxidized cholesterol, irrespective of age. In contrast, IgE production was significantly increased by the addition of 30 μg/ml of oxidized cholesterol in only young lymphocytes. Moreover, oxidized cholesterol enhanced the intracellular histamine accumulation in only adult PEC, although the total histamine level produced by PEC was similar in the rats of both ages. These results thus suggest the possibility that oxidized cholesterol can have different effects on the age-related modulation of immune functions such as Igs production and histamine release.
Key words: oxidized cholesterol; immunoglobulin; histamine; aging; rat

-20-
Note
Cloning and Expression of Bovine imc-415 cDNA from Mammary Gland

Seckho HA, Myunggi BAIK,＊ and Yunjaie CHOI・

Department of Animal Science ＆ Technology, School of Agricultural Biotechnology, Seoul National University,
Suweon 441-744, Korea
＊Department of Genetic Engineering and Institute of Biotechnology, Chonnam National University,
Kwangiu 500-757, Korea

Received October 18, 1999; Accepted January 12, 2000
Bovine imc-415 cDNA was cloned from mammary gland using RACE PCR; it coded for 245 amino acids. The deduced amino acid sequence of mouse and human showed about 94％ identity. Expression of bovine imc-415 increased about 40％ in involuted mammary tissues compared with lactating tissues.
Key words: inducible murine mast cell-415; mammary gland; involution

-21-
Note
Purification and Some Properties of an Aminopeptidase from the Seeds
of Cannabis sativa

Kazunari ARIMA,＊ Tetsuya UCHIKOBA,・ Masayuki SHIMADA, Hiroo YONEZAWA,
and Makoto KANEDA

Department of Chemistry, Faculty of Science, Kagoshima University, Korimoto, Kagoshima 890-0065, Japan
＊Plant Gene Expression Center, Department of Plant and Microbial Biology, University of California,
Berkeley, 800 Buchanan St. Albany, CA 94710 U.S.A.

Received October 19, 1999; Accepted January 11, 2000
An aminopeptidase (HSA) with a molecular mass of 78 kDa was purified from hemp (Cannabis sativa) seeds. The activity was inhibited by monoiodeacetic acid, p-chloromercuri-phenylsulfonic acid, and Zn2+ ion. The specificity of HSA was similar to that of a leucyl aminopeptidase [EC 3.4.11.1] from mammalian cytosol. However, other enzyme properties were different from these of leucyl aminopeptidase.
Key words: Cannabis sativa; hemp seed; aminopeptidase; substrate specificity

-22-
Note
Application of the Random Arbitrary Primed Polymerase Chain Reaction
Differential Display Method to Isolate Genes of Cholesterol
Metabolism-related Proteins from Rat Liver

Masao SATO,・ Susumu YOSHIDA, Koji NAGAO,＊ Shoko NISHIZONO,
Mihoko KUSABA, Mei-Chu HUNG, Ikuo IKEDA, and Katsumi IMAIZUMI

Laboratory of Nutrition Chemistry, Division of Bioresource and Bioenvironmental Sciences, Graduate School,
Kyushu University, Fukuoka 812-8581, Japan
＊Research Fellow of the Japan Society for the Promotion of Science

Received October 27, 1999; Accepted January 8, 2000
The random arbitrary primed (RAP) polymerase chain reaction (PCR) differential display (DD) method was applied to isolate genes related to cholesterol metabolism from exogenously hypercholesterolemic (ExHC) rats and the progenitor, SD rats. Forty-seven trials of RAP-PCR DD resulted in the isolation of 37 clones differing in strain, cholesterol supplementation or their interaction. Among their fingerprints, five clones gave reproducible patterns by a Northern blotting analysis. The sequence of two clones with lower mRNA abundance in ExHC rats than in SD rats was homologous to that of fatty acid synthase and oxalyl-CoA decarboxylase. Two other clones with higher mRNA on the ncholesterol diet were matrin F/G protein and the NMDA receptor glutamate-binding subunit. The other clone with higher mRNA abundance in ExHC rats on the cholesterol diet was myelodysplasai/myeloid leukemia factor 2. Fifteen trials of reverse transcriptase (RT)-PCR DD yielded 10 clones, but none of the fingerprints were reproduced by the Northern blotting analysis. These results indicate that RAP-PCR DD is an appropriate alternative to RT-PCR DD for isolating the genes involved in hypercholesterolemia.
Key words: differential display; hypercholesterolemia; random arbitrary primed polymerase chain reaction; reverse transcriptase polymerase chain reaction

-23-
Note
Inhibition of Fungal Cell Wall Synthesizing Enzymes by trans-Cinnamaldehyde

Kyu-Ho BANG, Dong-Won LEE, Hee-Moon PARK, and Young-Ha RHEE・

Department of Microbiology, College of Natural Sciences, Chungnam National University, Yusong,
Taejon 305-764, Korea

Received November 8, 1999; Accepted January 6, 2000
This study examined the inhibitory effects of trans-cinnamaldehyde (CA), an aromatic aldehyde derived from Cinnamomi Cortex, on Saccharomyces cerevisiae cell wall synthesizing enzymes in vitro. This compound was found to be a noncompetitive inhibitor of β-(1,3)-glucan synthase and a mixed inhibitor of chitin synthase 1 with 50％ inhibitory concentrations (IC50) of 0.84 and 1.44 mM, respectively. Chitin synthases 2 and 3 were less sensitive than chitin synthase 1 to CA. CA can be useful as a model compound of cell wall inhibitors for the development of effective antifungal agents.
Key words: β-(1,3)-glucan synthase inhibitor; chitin synthase inhibitor; noncompetitive and mixed inhibition; trans-cinnamaldehyde

-24-
Note
Substrate Specificity of Regiospecific Desaturation of Aliphatic Compounds
by a Mutant Rhodococcus Strain

Kenzo KOIKE,＃,・ Mikio TAKAIWA, Yoshiharu KIMURA, Shigeo INOUE, and Susumu ITO

Tochigi Research Laboratories of Kao Corporation, 2606, Akabane, Ichikai, Haga 321-3497, Japan

Received November 9, 1999; Accepted January 4, 2000
Substrate specificity of cis-desaturation of alipahtic compounds by resting cells of a mutant, Rhodococcus sp. strain KSM-MT66, was examined. Among substrates tested, the rhodococcal cells were able to convert n-alkanes (C13--C19), 1-chloroalkanes (C16 and C18), ethyl fatty acids (C14--C17) and alkyl (C1--C4) esters of palmitic acid to their corresponding unsaturated products of cis configuration. The products from n-alkanes and 1-chloroalkanes had a double bond mainly at the 9th carbon from their terminal methyl groups, and the products from acyl fatty acids had a double bond mainly at the 6th carbon from their carbonyl carbons.
Key words: Rhodococcus; desaturation; regiospecificity; alkene; alkenoic acid

-25-
Note
Purification of Alginate Oligosaccharides with Root Growth-promoting
Activity toward Lettuce

Ken-ichi IWASAKI・ and Yasuhito MATSUBARA＊

Food Research Institute, Kagawa Prefectural Government, 587-1 Goto, Takamatsu, Kagawa 761-8031, Japan
＊Kagawa Prefectural Fermentation and Food Experimental Station, Uchinomi, Shozu, Kagawa 761-4421, Japan

Received November 10, 1999; Accepted January 5, 2000
Sodium alginate was degraded by alginate lyase from Corynebacterium sp., and the product was purified by an ultrafiltration (UF) membrane module. The UF treatment was carried out at a transmembrane pressure of 0.15 MPa and a flow velocity of 0.6 m/s in the cross-flow mode, and non-degraded alginate was almost completely removed. The alginate oligosaccharide obtained was a mixture of di- to octasaccharides and had promoting activity toward lettuce root elongation (about 2-fold compared with the control) in the concentration range of 200--3000 μg/ml. The effect of the degree of polymerization on this activity was examined by using each oligosaccharide fractionated by gel chromatography. The tri-, tetra-, penta- and hexasaccharides were each found to have root growth-promoting activity in a lettuce bioassay.
Key words: alginate oligosaccharide; alginate lyase; ultrafiltration membrane; root growth-promoting activity; lettuce

-26-
Note
Syntheses of (・±)-Methyl 6?α-Demethyl-6?α-cyanoabscisate and (・±)-Methyl
6?α-Demethyl-6?α-methoxycarbonylabscisate

Takahiro INOUE 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 November 11, 1999; Accepted January 13, 2000
New abscisic acid analogs possessing a cyano or methoxycarbonyl group at the 6?α-position of methyl abscisate were synthesized by regioselective hydrocyanation. These compounds had weak activity in the rice second leaf sheath elongation test.
Key words: 6?α-cyano ・ABA; 6?α-methoxycarbonyl ABA; Michael reaction; rice seedling test

-27-
Note
Apoptosis Induced by the Flavonoid from Lemon Fruit
(Citrus limon BURM. f.) and Its Metabolites in HL-60 Cells

Shin OGATA,1 Yoshiaki MIYAKE,2 Kanefumi YAMAMOTO,2 Katsuzumi OKUMURA,1
and Hiroshi TAGUCHI1,・

1Laboratory of Molecular and Cellular Biology, Department of Life Science, Faculty of Bioresources,
Mie University, Tsu, Mie 514-8507, Japan
2Central Research Laboratory of Pokka Corporation Ltd., 45-2 Kumanosyo, Shikatsu-cho,
Nishikasugai-gun, Aichi 481-8515, Japan

Received November 11, 1999; Accepted January 18, 2000
The flavonoid from lemon fruit (Citrus limon BURM. f.) and its metabolites, particularly eriodictyol, 3,4-dihydroxyhydrocinnamic acid, and phloroglucinol had the function of DNA fragmentation in HL-60 cells when analyzed by flow cytometry. An apoptotic DNA ladder and chromatin condensation were observed in HL-60 cells when treated with these compounds. The caspase inhibitor prevented DNA fragmentation. These compounds are anticipated to be useful for medical purposes.
Key words: apoptosis; lemon fruit; flavonoids; eriodictyol; HL-60

-28-
Note
Use of Bacillus brevis for Synthesis and Secretion of Des-B30 Single-Chain
Human Insulin Precursor

Mamoru KOH, Hiroshi HANAGATA,＊ Shogo EBISU,＊ Kazuyuki MORIHARA,
and Hiroaki TAKAGI＊,・

Institute for Applied Life Science, University of East Asia, Graduate School, 2-1 Ichinomiya-Gakuen cho,
Shimonoseki-shi, Yamaguchi 751-0807, Japan
＊R ＆ D Department, Higeta Shoyu Co. Ltd., 2-8 Chuo-cho, Choshi-shi, Chiba 288-8680, Japan

Received November 15, 1999; Accepted January 18, 2000
A synthetic gene encoding a single chain human insulin precursor [B-chain (1-29)-A-chain] linked to the C-terminal lysine of human epidermal growth factor (1-28) (EGF-SCI) was constructed. This gene was expressed using Bacillus brevis. EGF-SCI was isolated from the supernatant of the culture broth. Treatment of EGF-SCI with lysyl endopeptidase resulted in the formation of des-B30 human insulin. The identification of the formed des-B30 human insulin was made by the measurement of molecular weight and amino acid analysis. The binding coefficient to anti-human insulin antibody was comparable to that of human insulin.
Key words: Bacillus brevis; single chain human insulin precursor; human epidermal growth factor (1-28); des-B30-human insulin; anti-human insulin antibody

-29-
Note
A Simple and Rapid Extraction of High Molecular Weight Chromosomal DNA
from Bacillus subtilis Protoplasts for Cosmid Cloning and Interspecific
Transformation

Takashi AKAMATSU,・ Hisataka TAGUCHI, and Hirosuke OKADA

Department of Applied Microbial Technology, Kumamoto Institute of Technology, Ikeda 4-22-1,
Kumamoto 860-0082, Japan

Received November 18, 1999; Accepted January 19, 2000
After conversion of Bacillus subtilis vegetative cells to protoplasts, a simple and rapid method for extracting high-molecular-weight chromosomal DNA was devised with the inclusion of bovine serum albumin and phenol-chloroform treatments. The DNA sample thus prepared was the size of 100--450 kb and could be used for cosmid cloning and interspecific transformation.
Key words: Bacillus subtilis; DNA; extraction; high-molecular-weight; transformation

-30-
Note
Identification of the UV-Responsive Sequence in the Human Tissue
Plasminogen Activator Gene

Jun FUJIWARA,1 Michihiko FUJII,2 Michiko SHIMODA,3 and Dai AYUSAWA2,・

1AISIN Cosmos R＆D CO., Ltd., Kazusa Incubation Ceter, 1698, Yana Kisarazu, Chiba, 292-0812 Japan
2Kihara Institute for Biological Research, Yokohama City University, Maioka-cho 641-12, Totsuka-ku,
Yokohama, Kanagawa 244-0813, Japan
3Department of Applied Biochemistry, Utsunomiya University, Minemachi 350,
Utsunomiya, Tochigi 321-0943, Japan

Received November 19, 1999; Accepted January 7, 2000
Functional analysis of regulatory elements in the human tissue plasminogen activator (tPA) gene showed that its promoter (--119/+169) is activated by UV irradiation in HeLa cells. We demonstrated here that the AP-2 like CCCCACCC sequence is involved in the UV-mediated activation and Sp1 binds to the sequence.
Key words: tissue plasminogen activator; UV; CAT assay; AP-2-like; Sp1

-31-
Note
Sodium ATPase and Sodium/proton Antiporter Are Not Obligatory
for Sodium Homeostasis of Enterococcus hirae at Acid pH

Miho IKEGAMI, Hiroko TAKAHASHI, Kazuei IGARASHI, and Yoshimi KAKINUMA＊

Faculty of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan

Received November 22, 1999; Accepted January 17, 2000
Enterococcus hirae grows in a broad pH range from 5 to 11. An E. hirae mutant 7683 lacking the activities of two sodium pumps, Na+-ATPase and Na+/H+ antiporter, does not grow in high Na+ medium at pH above 7.5. We found that 7683 grew normally in high Na+ medium at pH 5.5. Although an energy-dependent sodium extrusion at pH 5.5 was missing, the intracellular levels of Na+ and K+ were normal in this mutant. The Na+ influx rates of 7683 and two other strains at pH 5.5 were much slower than those at pH 7.5. These results suggest that Na+ elimination of this bacterium at acid pH is achieved by a decrease in Na+ entry and a normal K+ uptake.
Key words: Na+-ATPase; Na+/H+ antiporter; Na+ homeostasis; Enterococcus hirae

-32-
Note
Malvidin 3-Rutinoside as the Pigment Responsible for Bract Color
in Curcuma alismatifolia

Masayoshi NAKAYAMA,1 Mark S. ROH,2 Kenichi UCHIDA,3 Yuichi YAMAGUCHI,4
Keiko TAKANO,5 and Masaji KOSHIOKA1,・

1Department of Floriculture, National Research Institute of Vegetables, Ornamental Plants and Tea (NIVOT),
Ministry of Agriculture, Forestry and Fisheries, 360 Kusawa, Ano, Mie 514-2392, Japan
2Agricultural Research Service, USDA, Beltsville, MD 20705-2350, U.S.A.
3School of Science and Engineering, Teikyo University, 1-1 Toyosatodai, Utsunomiya, Tochigi 320-8551, Japan
4Department of Tea Processing Technology, NIVOT, 2769 Kanaya, Shizuoka 428-8501, Japan
5Floriculture and Ornamental Plants Section, Kochi Prefectural Agriculture Research Center, 1100 Hataeda,
Nangoku, Kochi 783-0023, Japan

Received November 24, 1999; Accepted January 18, 2000
Malvidin 3-rutinoside was the only anthocyanin identified from pink bracts of Curcuma alismatifolia cultivars. The concentration of malvidin 3-rutinoside in three cultivars increased as the intensity of the pink color in the bracts increased.
Key words: Curcuma alismatifolia; Zingiberaceae; pink bract; anthocyanin; malvidin 3-rutinoside

-33-
Note
Carotenoids in Human Blood Plasma after Ingesting Paprika Juice

Hideo ETOH,・ Yuji UTSUNOMIYA, Akiko KOMORI, Yoshiko MURAKAMI, Shunji OSHIMA,＊
and Takahiro INAKUMA＊

Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka 422-8529, Japan
＊Kagome Co. Ltd., 17 Nishitomiyama, Nishinasuno-cho, Nasu-gun, Tochigi 329-2762, Japan

Received December 1, 1999; Accepted January 17, 2000
We investigated the presence of different carotenoids in male human subject after the ingestion of paprika juice, and identified capsanthin, capsanthone, cucurbitaxanthin A, 11-cis-capsanthin, lutein and zeaxanthin in the human plasma. These results suggest that capsanthone and 11-cis-capsanthin might be as important as capsanthin for human health.
Key words: carotenoid; capsanthin; capsanthone; cucurbitaxanthin A; 11-cis-capsanthin

-34-
Note
Identification and Characterization of a Novel Gene, hos3+, the Function
of Which Is Necessary for Growth under High Osmotic Stress in Fission Yeast

Keisuke AOYAMA, Ryosuke KAWAURA, Hisami YAMADA, Hirofumi AIBA,・
and Takeshi MIZUNO

Laboratory of Molecular Microbiology, School of Agriculture, Nagoya University, Chikusa-ku,
Nagoya 464-8601, Japan

Received December 1, 1999; Accepted January 6, 2000
hos3 mutants of the fission yeast Schizosaccharomyces pombe showed the phenotype of high osmolarity sensitivity for growth. An S. pombe strain carrying the hos3-M26 allele cannot form colonies on agar plates containing 2 M glucose, but the parental strain can do so very well, as demonstrated previously. The hos3+ gene was cloned and identified as one that encodes a small protein of 94 amino acids, which shows no sequence similarity to any other proteins in the current databases. A hos3Δ strain, which we then constructed, had the phenotype of high osmolarity sensitivity, as in the case of the original hos3-M26 mutant. More interestingly, when these hos-- cells were grown in the non-permissive growth condition in the presence of 2 M glucose, we found that unusually many septated cells were accumulated after a prolonged incubation. A multicopy suppressor gene for hos-- mutations was also isolated and identified as the dsk1+ gene encoding a protein kinase, which was previously suggested to be implicated in a process of the mitotic regulation of S. pombe. The function of the hos3+ gene is discussed from these results.
Key words: osmoregulation; Schizosaccharomyces pombe; mitotic control

-35-
Note
Processing Glucosidase Inhibition by 1-Azafagomine

Makoto MUROI, Osamu ANDO, Mikael BOLS,＊ and Akira TAKATSUKI・

Animal and Cellular Systems Laboratory, RIKEN (The Institute of Physical and Chemical Research),
Wako-shi, Saitama 351-0198, Japan
＊Department of Organic Chemistry, The University of Aarthus, Aarthus C, DK-2800, Denmark

Received December 10, 1999; Accepted January 14, 2000
Natural azasugars have the ring oxygen substituted by nitrogen. They show potent inhibitory activity against glycosidases. The effect of substituting the ring carbon with nitrogen was examined with 1-azafagomine. 1-Azafagomine exhibited similar activity against processing glucosidase to that of fagomine.
Key words: 1-azafagomine; inhibitor; oligosaccharide; processing; glucosidase

-36-
Preliminary Commuunication
Identification of A New Gene Responsible for the Oxygen Tolerance
in Aerobic Life of Streptococcus mutans

Yuji YAMAMOTO,1 Masako HIGUCHI,1 Leslie B. POOLE,2 and Yoshiyuki KAMIO1,・

1Laboratory of Applied Microbiology, Department of Molecular and Cell Biology, Graduate School
of Agricultural Science, Tohoku University, Aoba-ku, Sendai 981-8555, Japan
2Department of Biochemistry, Wake Forest University Medical School, Winston-Salem,
North Carolina 27157, USA

Received January 14, 2000; Accepted February 18, 2000
Alkyl hydroperoxide reductase in Streptococcus mutans consists of two components, Nox-1 and AhpC. Deletion of nox-1 and ahpC in a double mutant as well as the wild-type of Streptococcus mutans can form colonies in the presence of air to the same extent. The evidence suggested the presence of some other antioxidant system(s) independent of the Nox-1/AhpC system in the bacterium. Here we identified a new antioxidant gene (dpr) and the gene product (Dpr) which complements the defect of peroxidase activity caused by the deletion of nox-1 and ahpC in S. mutans. The dpr-disruption mutant of S. mutans could form colonies anaerobically but not aerobically.
Key words: Dpr, Dps-like peroxide resistance gene; oxygen tolerance; NADH oxidase; alkyl hydroperoxide reductase; Streptococcus mutans