Contents and Abstracts of Latest Issue of BBB

(Vol.64 No.12 2000)

Sachiko ODAKE,*,*** Jacques P. ROOZEN,* and Jack J. BURGER**

*Wageningen Agricultural University, Bomenweg 2, 6703 HD Wageningen, The Netherlands
**Quest International Netherlands, Huizerstraatweg 28, 1411 GP Naarden, The Netherlands
***(at present) Yamanashi Women′s Junior College, Iida 5-11-1, Kofu, Yamanashi 400-0035, Japan

Received May 6, 1999; Accepted August 2, 2000
The release of volatile compounds from a cream style dressing, which consisted of a thickening agent dispersed in the water phase of an oil in water (o/w) type of emulsion, was studied by the purge-and-trap (PT), dynamic head space mastication (DHM) and dynamic headspace (DH) model systems for diacetyl and 2-heptanone as two volatile compounds. Big differences were detected in the quantity of volatiles released by the three models for both diacetyl and 2-heptanone: PT released the most, followed by DHM and DH. Nitrogen gas bubbling in PT and plunger up-and-down motion in DHM mimic mouth movements and promoted volatile release more than DH. The quantity of volatiles released depended on the nitrogen gas flow rate and isolation period with both the PT and the DHM model. Static headspace measurements indicated that no interaction occurred between the volatiles and the dispersion thickening agent, nor between the volatiles and protein of saliva.
Key words: flavor; flavor release; aroma; volatile; mastication

-2-
Gene Cloning and Characterization of α-Glucuronidase
of Bacillus stearothermophilus No. 236

Il-Dong CHOI, Hwa-Young KIM, and Yong-Jin CHOI・

Graduate School of Biotechnology, Korea University, Seoul 136-701, Korea

Received January 17, 2000; Accepted June 28, 2000
The α-glucuronidase gene of Bacillus stearothermophilus No. 236 was cloned, sequenced, and expressed in Escherichia coli. The gene, designated aguA, encoded a 691-residue polypeptide with calculated molecular weight of 78,156 and pI of 5.34. The α-glucuronidase produced by a recombinant E. coli strain containing the aguA gene was purified to apparent homogeneity and characterized. The molecular weight of the α-glucuronidase was 77,000 by SDS-PAGE and 161,000 by gel filtration; the functional form of the α-glucuronidase therefore was dimeric. The optimal pH and temperature for the enzyme activity were pH 6.5 and 40°C, respectively. The enzyme′s half-life at 50°C was 50 min. The values for the kinetic parameters of Km and Vmax were 0.78 mM and 15.3 U/mg for aldotriouronic acid ［2-O-α-
(4 - O - methyl - α - D - glucopyranosyluronic) - D - xylobiose］. The α-glucuronidase acted mainly on small substituted xylo-oligomers and did not release methylglucuronic acid from intact xylan. Nevertheless, synergism in the release of xylose from xylan was found when α-glucuronidase was added to a mixture of endoxylanase and β-xylosidase.
Key words: α-glucuronidase; aguA; Bacillus stearothermophilus; aldouronic acid; xylan

-3-
Modulation of Cholesterol Concentration in Caco-2 Cells by Incubation
with Different n-6 Fatty Acids

Kazunori KOBA,1 Jim-Wen LIU,2 Lu-Te CHUANG,2 Steven N. ANDERSON,2
Tammy BOWMAN,2 Emil BOBIK, Jr.,2 Michihiro SUGANO3, and Yung-Sheng HUANG2

1Siebold University of Nagasaki, 822 Yoshimuta-goh, Nagayo, Nagasaki 851-2195, Japan
2Ross Products Division Abbott Laboratories, 625 Cleveland Ave., Columbus, OH 43215-1724, USA
3Prefectural University of Kumamoto, 3-1-100 Tsukide, Kumamoto, 862-8502 Japan

Received March 21, 2000; Accepted July 27, 2000
Incorporation of exogenous cholesterol was compared in human adenocarcinoma colon cells (Caco-2) after incubation with 100 μM of either linoleic acid (LA, 18:2n-6), γ-linolenic acid (GLA, 18:3n-6), arachidonic acid (AA, 20:4n-6) or adrenic acid (or n-6 docosatetraenoic acid, DTA, 22:4n-6). In both cells 7 days after seeding and 14 days after confluency, incubation with LA significantly raised the proportion of 18:2n-6 but not its long-chain metabolites in cellular phospholipid. Incubation with GLA increased the levels of 18:3n-6, 20:3n-6, and 20:4n-6. Incubation with AA increased the levels of 20:4n-6 and 22:4n-6, and incubation with DTA increased the levels of 22:4n-6 as well as its retro-conversion metabolite, 20:4n-6. A subsequent addition of cholesterol (180 μM) to the medium significantly raised the cellular cholesterol level but less so in the cells 7 days after seeding incubated with GLA. The increase in cellular cholesterol level was generally greater in the cells of 7 days after seeding, particularly those incubated with long-chain highly unsaturated n-6 fatty acids, than in those of 14 days after confluency. These findings suggest that the cell growth and the extent of unsaturation in cell membrane phospholipid fatty acids modulate the incorporation of the exogenous cholesterol into the Caco-2 cells.
Key words: n-6 fatty acid; γ-linolenic acid; |||cholesterol; Caco-2 cells

-4-
Plasma Cholesterol-lowering Effect on Rats of Dietary Fiber Extracted
from Immature Plants

Naomichi NISHIMURA,・ Yuji TANIGUCHI,・・ and Shuhachi KIRIYAMA・・・

Laboratory of Nutritional Biochemistry, Department of Bioscience and Chemistry, Faculty of Agriculture, Hokkaido University, Kita-9 Nishi-9, Sapporo 060-0809, Japan

Received March 27, 2000; Accepted July 11, 2000
Crude dietary fiber samples were prepared from beet, cabbage, Japanese radish, onion and mung bean sprouts (BF, CF, RF, OF and MF, respectively). These samples contained total dietary fiber at the levels of 814, 699, 760, 693 and 666 g/kg, respectively. To examine the effect of these dietary fiber sources on the plasma cholesterol concentration, male Sprague-Dawley rats were fed on a fiber-free (FF) diet or on an FF diet supplemented with 5％ or 10％ dietary fiber. Dietary fiber extracted from vegetables, wood cellulose (CL), pectin (PE) and guar gum (GG) were used as the fiber sources. Compared with the rats fed on the FF diet, a significant reduction in the plasma cholesterol concentration was observed in the rats fed on BF, CF, RF, MF, PE or GG after a 21-d feeding period. Cecal acetate, n-butyrate and total short-chain fatty acids were significantly higher in the rats fed on these dietary fibers, except for CF, than in those fed on the FF diet. A negative correlation was apparent between the total dietary fiber content, hemicellulose content and pectin content of each dietary fiber source and the plasma cholesterol concentration. These results suggest that some vegetable fibers exert a plasma cholesterol-lowering effect through cecal fermentation of these fibers.
Key words: vegetable fiber; hypocholesterolemic activity; plasma cholesterol concentration; dietary fiber quality; rat

-5-
Time-dependent Structure and Activity Changes of α-Chymotrypsin
in Water/Alcohol Mixed Solvents

Makiko SATO, Toshiya SASAKI, Masami KOBAYASHI, and Hideo KISE・

Institute of Materials Science, University of Tsukuba, Tennoudai, Tsukuba, Ibaraki 305-8573, Japan

Received April 18, 2000; Accepted July 19, 2000
Secondary structure of α-chymotrypsin in water/ethanol was investigated by circular dichroic (CD) spectroscopy. The changes in catalytic activity were discussed in terms of structural changes of the enzyme. α-Chymotrypsin formed β-sheet structure in water/ethanol (50/50 by volume), but it was substantially less active as compared to that in water. At water/ethanol 10/90, α-chymotrypsin took on a native-like structure, which gradually changed to β conformation with concomitant loss of activity. Change of solvent composition from water/ethanol 50/50 to 90/10 or 10/90 by dilution with water or ethanol, respectively, led to partial recovery of native or native-like structure and activity. In water/methanol, α-chymotrypsin tended to form stable β-sheet structure at water/methanol ratios lower than 50/50, but the catalytic activity decreased with time. Change to α-helix structure with substantial loss in catalytic activity was observed when α-chymotrypsin was dissolved in water/2,2,2-trifluoroethanol with water contents lower than 50％. In water/2,2,2-trifluoroethanol 90/10, α-chymotrypsin initially had the CD spectrum of native structure, but it changed with time to that characteristic of β-sheet structure.
Key words: α-Chymotrypsin; water/alcohol mixed solvent; circular dichroic (CD) spectroscopy; time-dependent structure change; transesterification

-6-
Nematicidal Alkaloids and Related Compounds Produced by the Fungus
Penicillium cf. simplicissimum

Miyako KUSANO, Hiroyuki KOSHINO,* Jun UZAWA,* Shozo FUJIOKA,* Tsuyoshi KAWANO,
and Yasuo KIMURA・

Department of Agricultural Chemistry, Faculty of Agriculture, Tottori University, Tottori 680-8553, Japan
*The Institute of Physical and Chemical research (RIKEN), Wako-shi, Saitama 351-0198, Japan

Received April 19, 2000; Accepted June 30, 2000
A new nematicidal alkaloid, peniprequinolone (1), together with the known alkaloids penigequinolones A and B (2a, 2b), 3-methoxy-4-hydroxy-4-(4?-methoxyphenyl)quinolinone (3), and 3-methoxy-4,6-dihydroxy-4-(4?-methoxyphenyl)quinolinone (4), were isolated from Penicillium cf. simplicissimum (Oudemans) Thom. Cyclopenin (5) and a compound (6a/6b) structurally related to cyclopenin also were isolated from the fungus, and their structures were established by spectroscopic analysis. The biological activities of 1, 2, 3, 4, and 5 were examined by a bioassay with root-lesion nematodes.
Key words: Penicillium cf. simplicissimum; peniprequinolone; dihydroquinolinones; nematicidal activity

-7-
New Anthocyanins from Purple Pods of Pea (Pisum spp.)

Norihiko TERAHARA,・ Toshio HONDA,* Masahiro HAYASHI, and Kanji ISHIMARU**

Department of Food Science and Technology, College of Horticulture, Minami-Kyushu University,
Takanabe, Miyazaki 884-0003, Japan
*Institute of Medicinal Chemistry, Hoshi University, Shinagawa-ku, Tokyo 142-8501, Japan
**Department of Applied Biological Sciences, Faculty of Agriculture, Saga University, 1 Honjo,
Saga 840-8502, Japan

Received May 10, 2000; Accepted July 24, 2000
Two new anthocyanins were isolated from purple pods of pea (Pisum spp.). Their structures were identified as delphinidin 3-xylosylgalactoside-5-acetylglucoside and its deacetylated derivative by the usual chemical degradation methods and by spectroscopic methods such as UV-VIS, MS and NMR. Both pigments showed moderate stability and antioxidative activity in a neutral aqueous solution.
Key words: Pisum spp. L.; Leguminosae; purple pod; acetylated anthocyanin; antioxidative activity

-8-
DNA Repair Effect of Traditional Sweet Pepper Fushimi-togarashi:
Seen in Suppression of UV-induced Cyclobutane Pyrimidine Dimer
in Human Fibroblast

Yasushi NAKAMURA,・ Izumi TOMOKANE, Toshio MORI,* Atsuo TANAKA,** Jun KOUTANI,**
Tomoaki MATSUO,*** Shigehisa OKAMOTO,**** Kenji SATO, and Kozo OHTSUKI

Department of Food Sciences and Nutritional Health, Kyoto Prefectural University, Shimogamo,
Sakyo-ku, Kyoto 606-8522, Japan
*Radioisotope Center, Nara Medical University, Shijo, Kashihara, 634-8521, Japan
**Vegetable Division, Kyoto Prefectural Agricultural Research Institute, Amarube, Kameoka,
Kyoto 621-0806, Japan
***Departments of Biochemical Science and Technology and ****Agricultural Science, Kagoshima University,
Korimoto 1-21, Kagoshima 890-8580, Japan.

Received May 11, 2000, Accepted June 28, 2000
The aqueous fraction of Fushimi sweet pepper increased the repair effect of the solvent control against UV-induced cyclobutane pyrimidine dimers in human fibroblast to 150％, but ordinary sweet pepper did not have a statistically significant effect. When Fushimi sweet pepper was boiled, the activity of the aqueous fraction was elevated to 209％ of the control (p<0.05), while that of the grilled state was decreased to 125％ of the control. The repair activity of a dialyzate (MW<12,000) of the aqueous fraction from Fushimi sweet pepper showed 191％ of the control (p<0.05). The dialyzate was contained 1.9％ in the weight of the fresh fruit body of Fushimi sweet pepper, and the activity can be stable in its boiling state, and it might be therefore considered to be the worthy source for expecting the DNA repair activity in human diet.
Key words: sweet pepper; vegetable; antimutagen; pyrimidine dimer; excision repair

-9-
Analyses of Polyphenols in Cacao Liquor, Cocoa, and Chocolate
by Normal-Phase and Reversed-Phase HPLC

Midori NATSUME,1 Naomi OSAKABE,1 Megumi YAMAGISHI,1 Toshio TAKIZAWA,1
Tetsuo NAKAMURA,1 Haruka MIYATAKE,2 Tsutomu HATANO,2 and Takashi YOSHIDA,2

1Functional Foods R＆D Laboratory, Meiji Seika Kaisha Ltd. 5-3-1 Chiyoda Sakado-shi,
Saitama 350-0289, Japan
2Faculty of Pharmaceutical Sciences, Okayama University 1-1-1 Tsushima-naka, Okayama 700-8530, Japan

Received May 23, 2000; Accepted July 17, 2000
The antioxidant polyphenols in cacao liquor, a major ingredient of chocolate and cocoa, have been characterized as flavan-3-ols and proanthocyanidin oligomers. In this study, various cacao products were analyzed by normal-phase HPLC, and the profiles and quantities of the polyphenols present, grouped by molecular size (monomers~oligomers), were compared. Individual cacao polyphenols, flavan-3-ols (catechin and epicatechin), and dimeric (procyanidin B2), trimeric (procyanidin C1), and tetrameric (cinnamtannin A2) proanthocyanidins, and galactopyranosyl-ent-(-)-epicatechin (2α→7, 4α→8)-(-)-epicatechin (Gal-EC-EC), were analyzed by reversed-phase HPLC and/or HPLC/MS. The profile of monomers (catechins) and proanthocyanidin in dark chocolate was similar to that of cacao liquor, while the ratio of flavan-3-ols to the total amount of monomeric and oligomeric polyphenols in the case of pure cocoa powder was higher than that in the case of cacao liquor or chocolate.
Key words: cocoa; chocolate; polyphenol; HPLC analysis; proanthocyanidin

-10-
Dietary Effects of Eicosapentaenoic and Docosahexaenoic Acid Esters
on Lipid Metabolism and Immune Parameters in Sprague-Dawley Rats

Pham HUNG, Jiong-Yan GU, Shihoko KAKU, Shin-ichi YUNOKI, Ken-ichi OHKURA,
Ikuo IKEDA, Hirofumi TACHIBANA, Michihiro SUGANO,* Kazunaga YAZAWA,**
and Koji YAMADA

Department of Bioscience and Biotechnology, Division of Bioresource and Bioenvironmental Sciences, Graduate School of Kyushu University, Fukuoka 812-8581, Japan
*Department of Living Environment Science, Kumamoto Prefectural University, Kumamoto 862-8502, Japan
**Sagami Chemical Research Center, Sagamihara 229-0012, Japan

Received May 25, 2000; Accepted July 27, 2000
Sprague-Dawley rats were fed eicosapentaenoic (EPA) and docosahexaenoic acid (DHA) ethyl esters at the 2％ level for 3 weeks to clarify their effects on immune functions. In the rats fed EPA or DHA, serum cholesterol, triglyceride, and phospholipid (PL) levels were significantly lower than those in the rats fed safflower oil. In PL fractions of serum, liver, lung, splenocytes, and peritoneal exudate cells (PEC), increases in linoleic and dihomo-γ-linolenic acid contents and a decrease in arachidonic acid (AA) content were observed in the rats fed EPA or DHA. In addition, the EPA content increased in the rats fed EPA and DHA. In the rats fed EPA or DHA, a decrease of LTB4 productivity and an increase of LTB5 productivity were observed in the PEC, in response to the treatment with 5 mM calcium ionophore A23187 for 20 min. The changes in leukotriene production were more marked in EPA-fed rats than in DHA-fed rats. These results suggest that dietary EPA affects lipid metabolism and leukotriene synthesis more strongly than DHA.
Key words: DHA; EPA; fatty acid composition; LTB4・; LTB5

-11-
Effect of Soy and Milk Whey Protein Isolates and Their Hydrolysates
on Weight Reduction in Genetically Obese Mice

Toshiaki AOYAMA,・ Kensuke FUKUI,* Toshihiro NAKAMORI,* Yukio HASHIMOTO,*
Takashi YAMAMOTO,* Kiyoharu TAKAMATSU,* and Michihiro SUGANO**

*Novelty Materials Research Institute, Hannan R＆D Center, Fuji Oil Co. Ltd., 1 Sumiyoshi-cho,
Izumisano, Osaka 598-8540, Japan
**Study of Food and Health Environment, Faculty of Environmental and Symbiotic Sciences,
Prefectural University of Kumamoto, 3-1-100 Tsukide, Kumamoto 862-8502, Japan

Received May 25, 2000; Accepted July 13, 2000
The effect on genetically obese mice of a milk whey protein isolate (WPI) and soy protein isolate (SPI) and their hydrolysates (WPI-H, SPI-H) on the rate of body fat disappearance was investigated. Male yellow KK mice were made obese by feeding with a high-fat diet containing 30％ fat from 6 to 10 weeks of age. They were then fed with an energy-restricted low fat (5.0％) and high protein (35％ WPI, WPI-H, SPI or SPI-H) diet for 2 weeks at the 60％ level of energy intake by mice on laboratory feed. During the weight reduction period, the body weight of the WPI, WPI-H, SPI and SPI-H groups changed by --9.1, --9.1, --10.0 and --11.1 g/14 days, respectively, the reduction being significantly lower in the SPI-H group than in the WPI and WPI-H groups. The plasma total cholesterol level was significantly lower with the SPI diet, and the plasma glucose level was lower with the SPI and SPI-H diets than with the WPI and WPI-H diets. Although the body protein content was comparable in all the groups, the body fat content was significantly lower with the SPI diet than with the WPI diet, and was also significantly lower with the SPI-H diet than with the WPI and WPI-H diets. The weight of the perirenal fat pads was significantly lower with the SPI-H diet than with the WPI and WPI-H diets. These results indicate that SPI and SPI-H are suitable protein sources in an energy-restricted diet for treating obesity.
Key words: weight reduction; soy protein; protein hydrolysate; milk whey protein; genetically obese mice

-12-
Splice Isoforms of Transcription Factor Elf-1 Affecting Its Regulatory Function
in Transcription---Molecular Cloning of Rat Elf-1

Chiharu NISHIYAMA,*,** Kyoko TAKAHASHI,* Makoto NISHIYAMA,**** Ko OKUMURA,**,***
Chisei RA,**,*** Yasuyuki OHTAKE,* and Toyokazu YOKOTA*

*Foods ＆ Pharmaceuticals Research ＆ Development Laboratory, Asahi Breweries, Ltd., 1-1-21 Midori,
Moriya-machi, Kitasoma-gun, Ibaraki, 302-0106, Japan
**Allergy Research Center,*** Department of Immunology, Juntendo University School of Medicine,
2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421, Japan
****Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo,
113-8657, Japan

Received May 30, 2000; Accepted July 27, 2000
To elucidate the role of Elf-1 in FcεRI α chain expression, rat Elf-1 cDNAs were isolated and characterized. The rat Elf-1 cDNA of 2744 bp contained an open reading frame of 1848 bp. In addition to the full length rat Elf-1 cDNA (named type 1), two splice isoforms were isolated. One of the two isoforms lacked the amino acid residues from 85th to 120th (type 2), and the other from 85th to 175th (type 3). Similar isoforms were also observed in human tissue. Overexpression of rat Elf-1 (type 1) using a transient coexpression system inhibited of the α chain promoter activity. The inhibition activity was different between the isoforms; the inhibition activity of type 2 was lower than that of type 1, and type 3 did not have an inhibitory effect. This observation suggested that each Elf-1 isoform played a different role in the gene expression under its control.
Key words: Elf-1; cDNA; splice isoform; FcεRI

-13-
Comparison of Substrate Specificities of Transglutaminases Using Synthetic
Peptides as Acyl donors

Tomoko OHTSUKA, Masafumi OTA, Noriki NIO, and Masao MOTOKI

Food Research and Development Laboratories, Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kawasaki-ku,
Kawasaki-shi 210-8681, Japan

Received June 6, 2000; Accepted July 31, 2000
Transglutaminase (TGase) is an enzyme that catalyzes acyl transfer reactions between primary amines and Gln residues in proteins or peptides. Substrate specificities of TGase, Ca2+-independent microbial transglutaminase (MTGase), and Ca2+-dependent tissue type transglutaminase from guinea pig liver (GTGase) and fish, Red sea bream (Pagrus major), liver (FTGase), for acyl donors were investigated using synthetic peptides containing Gln residues and Gln analogues with different lengths of side chain. MTGase dose not recognize the Gln analogues as a substrate and has strict substrate specificities toward L-Gln. Substrate peptides with a variety of sequences around the Gln residue, GXXQXXG (X=G, A, S, L, V, F, Y, R, N, E, L) were synthesized and used as acyl donors. As an acyl acceptor, the fluorescent reagent monodancyl cadaverine was used and the reactions analyzed with RP-HPLC. Substitution of the C-terminal of a Gln residue with a hydrophobic amino acid accelerated the reaction by GTGase and FTGase. N-terminal substitution of Gln residues had similar effects on the reaction by MTGase.
Key words: transglutaminase; substrate specificity; acyl donor; synthetic peptide; ε-(γ-glutamyl)lysine bond

-14-
Cloning, Sequencing, and Expression of the Gene Encoding a Cell-bound
Multi-domain Xylanase from Clostridium josui, and Characterization
of the Translated Product

Jia-Xun FENG,1 Shuichi KARITA,2,・ Emi FUJINO,1 Tsuchiyoshi FUJINO,3 Tetsuya KIMURA,1
Kazuo SAKKA,1 and Kunio OHMIYA1

1Faculty of Bioresources and 2Center for Molecular Biology and Genetics, Mie University, Tsu 514-8507, Japan
3Nagoya Seiraku Co. Ltd., Tenpaku-ku, Nagoya 468-8588, Japan

Received June 12, 2000; Accepted July 27, 2000
The nucleotide sequence of the Clostridium josui FERM P-9684 xyn10A gene, encoding a xylanase Xyn10A, consists of 3,150 bp and encodes 1,050 amino acids with a molecular weight of 115,564. Xyn10A is a multidomain enzyme composed of an N-terminal signal peptide and six domains in the following order: two thermostabilizing domains, a family 10 xylanase domain, a family 9 carbohydrate-binding module (CBM), and two S-layer homologous (SLH) domains. Immunological analysis indicated the presence of Xyn10A in the culture supernatant of C. josui FERM P-9684 and on the cell surface. The full-length Xyn10A expressed in a recombinant Escherichia coli strain bound to ball-milled cellulose (BMC) and the cell wall fragments of C. josui, indicating that both the CBM and the SLH domains are fully functional in the recombinant enzyme. An 85-kDa xylanase species derived from Xyn10A by partial proteolysis at the C-terminal side, most likely at the internal region of the CBM, retained the ability to bind to BMC. This observation suggests that the catalytic domain or the thermostabilizing domains are responsible for binding of the enzyme to BMC. Xyn10A-II, the 100-kDa derivative of Xyn10A, was purified from the recombinant E. coli strain and characterized. The enzyme was highly active toward xylan but not toward p-nitrophenyl-β-D-xylopyranoside, p-nitrophenyl-β-D-cellobioside, or carboxymethylcellulose.
Key words: xylanase; Clostridium josui; carbohydrate-binding module; surface-layer homologous domain; thermostabilizing domain

-15-
Biogenic Amines in Drosophila virilis under Stress Conditions

Akinori HIRASHIMA,a,* Madina Jh. SUKHANOVA,b and Inga Yu. RAUSCHENBACHb

aDepartment of Applied Genetics and Pest Management, Faculty of Agriculture, Graduate School,
Kyushu University, Fukuoka 812-8581, Japan
bInstitute of Cytology and Genetics, Siberian Branch of Russian Academy of Sciences,
Novosibirsk 630090, Russia

Received June 12, 2000; Accepted July 19, 2000
The effect of heat stress (38°C) on the content of DL-β-(3,4-dihydroxyphenyl)alanine (DOPA), dopamine, tyramine, octopamine, and their precursor Tyr was studied in adults of two lines of Drosophila virilis contrasting in their stress response. In individuals of line 101 responding to stress by a hormonal stress reaction, the contents of DOPA, dopamine, octopamine, and Tyr were lower than those of line 147 that did not respond to the stress. However, heat stress caused an increase in the contents of DOPA, dopamine, octopamine, and Tyr in line 101, whereas the equivalent titers in line 147 remain unchanged.
Key words: Drosophila virilis; heat stress; biogenic amine; line 101; line 147

-16-
Prophage, fPV83-pro, Carrying Panton-Valentine Leukocidin Genes,
on the Staphylococcus aureus P83 Chromosome: Comparative Analysis
of the Genome Structures of fPV83-pro, fPVL, f11, and Other Phages

Dan ZOU, Jun KANEKO, Sachiko NARITA, and Yoshiyuki KAMIO・

Laboratory of Applied Microbiology, Department of Molecular and Cell Biology, Graduate School
of Agricultural Science, Tohoku University, 1-1 Tsutsumi-dori Amamiya-machi, Aoba-ku,
Sendai 981-8555, Japan

Received June 14, 2000; Accepted July 28, 2000
Staphylococcus aureus P83 has Panton-Valentine leukocidin (PVL)-like genes, lukM and lukF-PV. Here, lukM and lukF-PV genes were found on the genome of a prophage, which was designated as fPV83-pro. The precise genome size was 45,636 bp with att core sequences of 10 base pairs. Sixty-four ORFs were identified on the fPV83-pro genome, including two extra operons, lukM-lukF-PV and orfs63-64. The lukM-lukF-PV cluster was located 2.1 kb upstream of the attL site. The most striking feature of the fPV83-pro genome was a constituent of at least 4 regions from f11, fPVL, and other phages, i.e., (i) att sites identical with those of f11, (ii) a cos sequence and the genes encoding packaging and head proteins of fPVL (occupied half region of fPV83-pro), and (iii) the other two regions which showed no significant similarity with known phages (occupied about 40％ of fPV83-pro). Furthermore, two insertion sequences, ISSA1 and ISSA2 were integrated into attL site and orf44, respectively. fPV83-pro was not induced as phage particles from S. aureus P83 regardless of its treatment with mitomycin C. The insertion of ISSA1 into the attL site was one of the reasons of the failure of the induction of the phage particles by mitomycin C treatment of the strain P83.
Key words: Staphylococcus aureus P83; Panton-Valentine leukocidin; prophage φPV83-pro; phage genome structure; insertion sequence

-17-
Metabolism of Chiral Ionylideneacetic Acids on the Abscisic Acid Biosynthetic
Pathway in Cercospora

Hirotaka YAMAMOTO,・ Masahiro INOMATA, Shinobu TSUCHIYA, Makoto NAKAMURA,
and Takayuki ORITANI*

Faculty of Agriculture, Niigata University, Ikarashi, Niigata 950-2181, Japan
*Graduate School of Agricultural Science, Tohoku University, Aoba-ku, Sendai 981-8555, Japan

Received June 15, 2000; Accepted July 22, 2000
A Chiralcel OJ column was used to determine the absolute configuration of naturally occurring α-ionylideneacetic acid from Cercospora rosicola and γ-ionylideneacetic acid from C. cruenta as (R) enantiomers in accordance with their biosynthetic product, (S)-ABA. Both enantiomers of ［1, 2-13C2］-α and γ-ionylideneacetic acids were prepared and fed to C. rosicola and C. cruenta. Six combinations of feeding experiments comparatively and unequivocally demonstrated stereoselectivity in the biosynthetic conversions, including stepwise hydroxylation at C-1? and 4?. Enzymatic isomerization from the γ to α-intermediate was suggested not to be involved in ABA biosynthesis in C. rosicola.
Key words: abscisic acid; α-ionylideneacetic acid; γ-ionylideneacetic acid; Cercospora cruenta; Cercospora rosicola

-18-
Identification of a Compound in Chamaecyparis taiwanensis Inhibiting
the Ice-nucleating Activity of Pseudomonas fluorescens KUIN-1

Hidehisa KAWAHARA,1,2 Ken MASUDA,1 and Hitoshi OBATA1,2

1Department of Biotechnology, Faculty of Engineering and 2High technology Research Center,
Kansai University, 3-3-35, Yamate-cho, Suita, Osaka 564-8680, Japan

Received June 21, 2000; Accepted July 28, 2000
Inactivation of the ice-nucleating activity of Pseudomonas fluorescens KUIN-1 by compounds in the leaves from coniferous trees were investigated, and the inactivated material was identified. Intact cells of the strain KUIN-1 and the acetone or methanol extracts of leaves of various coniferous trees were allowed to react for 30 min at 18°C. Antinucleation compounds were obtained from Chamaecyparis taiwanensis. When the acetone extract from the leaves of coniferous trees was added to the cell suspension (about 10＾{6} cells/ml) in 50 mM potassium phosphate buffer (pH 7.0), the ice nucleating temperature, T50, was significantly decreased (T50<−5°C). This inhibitor was isolated by using TLC, then identified as hinokitiol based on UV-VIS, IR, and mass spectral data. When intact cells of the strain KUIN-1 were incubated with hinokitiol, limonene, and α-pinene of the principal constituent of the leaves of coniferous trees in 50 mM potassium phosphate buffer (pH 7.0), the ice-nucleating activity decreased, but not in α-terpinene. Furthermore, the ice-nucleating activities from other ice-nucleating bacteria also decreased in the presence of hinokitiol. This inhibition was proportional to the concentration of hinokitinol. The pH and thermal stabilities of the ice-nucleating activity of the cells were changed by the addition of hinokitiol (10 mM).
Key words: ice-nucleating bacterium; Pseudomonas fluorescens; hinokitiol

-19-
Ecdysteroids in Stress Responsive and Nonresponsive Drosophila virilis
Lines under Stress Conditions

Akinori HIRASHIMA,a,・ Inga Yu. RAUSCHENBACH,b and Madina Jh. SUKHANOVAb

aDepartment of Applied Genetics and Pest Management, Faculty of Agriculture, Graduate School,
Kyushu University, Fukuoka 812-8581, Japan
bInstitute of Cytology and Genetics, Siberian Branch of Russian Academy of Sciences,
Novosibirsk 630090, Russia

Received June 30, 2000; Accepted August 7, 2000
After exposure to thermal stress or a control temperature, the relative abundance of ecdysone (E) and 20-hydroxyecdysone (20E) was measured in a wild-type line of Drosophila virilis (101) that is stress responsive and in a mutant line (147) that is not stress responsive. In line 101, the 20E content was higher and E content lower in females than in males. The abundance of E and 20E in females of line 147 was significantly higher than that in females of line 101. Females of line 101 were found to respond to 60 min of heat stress (38°C) by an increase in the abundance of both E and 20E, while in males of this line the amount of 20E increased and that of E declined. A role of the ecdysteroids in the control of reproduction of D. virilis under stress is discussed.
Key words: ecdysone; 20-hydroxyecdysone; heat stress; Drosophila virilis

-20-
Novel Method for Producing Hypoallergenic Wheat Flour
by Enzymatic Fragmentation of the Constituent Allergens
and Its Application to Food Processing

Michiko WATANABE, Jun WATANABE,*,** Kei SONOYAMA,*,・ and Soichi TANABE

Faculty of Education, Tokyo Gakugei University, Koganei-shi, Tokyo 184-8501, Japan
*Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan
**Japan Society for the Promotion of Science, Chiyoda-ku, Tokyo 102-8471, Japan

Received July 5, 2000; Accepted July 25, 2000
A novel method is proposed to produce hypoallergenic wheat flour suitable for patients allergic to wheat. Wheat flour was mixed with a cellulase solution, and the mixture was incubated at 50°C for 1 h to hydrolyze the carbohydrate allergens. The hydrolysate was further incubated with actinase at 40°C for 1 h while gently stirring to decompose the proteinaceous allergens. The product was evaluated for its allergenicity by an enzyme-linked immunosorbent assay, the results of which suggested negative allergenicity in most cases. The product changed to a batter state that was difficult to process by the usual methods. Gelatinization of the starch in the product and the addition of a surfactant were beneficial for food processing.
Key words: hypoallergenic flour; wheat allergy; hypoallergenic food

-21-
A Cold Acclimation Protein with Refolding Activity
on Frozen Denatured Enzymes

Hidehisa KAWAHARA,1,2 Noriko KODA,1,2 Mika OSHIO,1 and Hitoshi OBATA1,2

1Department of Biotechnology, Faculty of Engineering and 2High Technology Research Center,
Kansai University, 3-3-35, Yamate-cho, Suita, Osaka 564-8680, Japan

Received July 12, 2000; Accepted August 14, 2000
We found that a cold acclimation protein from an
ice-nucleating bacterium, Patoea ananas KUIN-3, has refolding activity on frozen denatured protein. Based on a SDS-PAGE analysis, we confirmed that the cold shock-treated cells of strain KUIN-3 could produce some cold acclimation proteins that inhibit their syntheses by the addition of chloramphenicol during the cold acclimation. Among such proteins, Hsc25 had refolding activity similar to GroELS. Hsc25 was purified to apparent homogeneity by (NH4)2SO4 precipitation and some chromatographies. The purified Hsc25 was composed of 8 subunits of 25,000 each with a molecular mass of 200,000 and had refolding activity against denatured enzymes, which were denatured by heat-treatment at 100°C, cryopreservation at −20°C, or guanidine hydrochloride, in a manner similar to GroELS. The N-terminal sequence of Hsc25 was Met-Arg-Ala-Ser-Thr-Tyr-His-Ala-Ala-Arg-. Furthermore, Hsc25 had a high level of activity at low temperature (12°C). Also, the dissociation constants, KD (M) as the binding specificity for enolase, mutarotase, isocitrate dehydrogenase, and lactate dehydrogenase were 1.82×10−10, 4.35×10−9, 8.98×10−12, and 3.05×10−11, respectively. The affinity of Hsc25 for frozen danatured enzymes was higher than the affinity for heat denatured enzymes when compared with the affinity of GroEL. These results are the first report on the characterization of a purified chaperon that was induced by cold acclimation.
Key words: cold acclimation protein; chaperone; refolding activity; Pantoea ananas

-22-
Efflux System for Pyridoxine in Schizosaccharomyces pombe

Kumi HIROSE, Ruamsub CHUMNANTANA, Takashi NAKASHIMA, Makoto ASHIUCHI,
and Toshiharu YAGI

Department of Bioresources Science, Faculty of Agriculture, Kochi University, Monobe-Otsu 200,
Nankoku Kochi 783-8502, Japan

Received July 19, 2000; Accepted August 14, 2000
Pyridoxine-charged Schizosaccharomyces pombe released pyridoxine rapidly at 30°C: very low amounts of three other B6 vitamers were also released. The rate of efflux was temperature-dependent. The initial rate of efflux was dependent on the concentration of pyridoxine in the cells: the rate was almost zero at lower than 0.02 mM and became saturated at higher than 0.2 mM. Na+, sodium azide, and dinitrophenol increased the rate in both the presence and absence of D-glucose. Mg++, thiamine, and menadione inhibited the efflux. The intracellular concentration of ATP did not significantly affect the efflux rate. The system may be dependent on a membrane potential of the yeast cells. It was found that the fission yeast cells have a gate or carrier system for efflux of pyridoxine, which was distinct from that in Saccharomyces cerevisiae.
Key words: efflux; pyridoxine; vitamin B6・; Schizosaccharomyces pombe; yeast

-23-
Note
α-Oxidation of Long-chain Unsaturated Fatty Acids
in the Marine Green Alga Ulva pertusa

Yoshihiko AKAKABE,・ Kenji MATSUI, and Tadahiko KAJIWARA

Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8515, Japan

Received March 24, 2000; Accepted June 26, 2000
When long-chain unsaturated fatty acids such as oleic, linoleic, and linolenic acid were incubated with crude enzymes from the marine green alga Ulva pertusa, the corresponding (R)-2-hydroperoxy acids were formed with a high enantiomeric excess (>99％).
Key words: α-oxidation; long-chain fatty acid; 2-hydroperoxy acid; green alga

-24-
Note
cDNA Cloning of the Cry1Aa Receptor Variants from Bombyx mori
and Their Expression in Mammalian Cells

Satoshi IKAWA, Yoko TSUDA, Takashi FUKADA, Kenji SUGIMOTO, and Michio HIMENO

Laboratory of Applied Molecular Biology, Department of Applied Biochemistry, Osaka Prefecture University,
1-1 Gakuen-cho, Sakai, Osaka 599-8531, Japan

Received April 26, 2000; Accepted August 7, 2000
We cloned cDNA of three variants of BtR175, a putative Bombyx mori receptor for Bacillus thuringiensis Cry1Aa δ-endotoxin by PCR. These variants were likely to be allelic to BtR175. cDNA of BtR175b, the most distant variant from BtR175, was introduced into mammalian cells. BtR175b protein was expressed in the plasma membrane of the cells and showed binding activity to Cry1Aa.
Key words: Bacillus thuringiensis; δ-endotoxin; receptor; Bombyx mori; membrane protein

-25-
Note
Effects of Corticosterone on Connectin Content and Protein Breakdown
in Rat Skeletal Muscle

Kunioki HAYASHI,・ Osamu TADA, Kouji HIGUCHI, and Akira OHTSUKA

Department of Biochemical Science and Technology, Faculty of Agriculture, Kagoshima University,
Kagoshima 890-0065, Japan

Received May 15, 2000; Accepted June 26, 2000
We examined the effects of a glucocorticoid, corticosterone, on calpain activity, connectin content and protein breakdown in rat muscle. The results indicated that calpain activity was increased by corticosterone and thus breakdown of connectin was stimulated followed by increased breakdown of skeletal muscle protein.
Key words: connectin; corticosterone; calpain; muscle protein breakdown

-26-
Note
Stability and Bioavailability of Antioxidants in Garland
(Chrysanthemum coronarium L.)

Makiko TAKENAKA,・ Tadahiro NAGATA,* and Mitsuru YOSHIDA

National Food Research Institute, Ministry of Agriculture, Forestry, and Fisheries,
2-1-2 Kannondai, Tsukuba, Ibaraki 305-8642, Japan

Received May 16, 2000; Accepted July 6, 2000
The stability and bioavailability of the major antioxidants in garland (Chrysanthemum coronarium L.), chlorogenic acid, 3,5-dicaffeoylquinic acid and 4-
succinyl-3,5-dicaffeoylquinic acid, were investigated together with caffeic acid. These compounds were stable in artificial digestive juice, but more than 90％ of them disappeared from plasma within 30 min after intravenous injection into rats. When they were orally administered, only caffeic acid could be detected.
Key words: garland (Chrysanthemum coronarium L.); antioxidant; chlorogenic acid; artificial digestive juice; administration test

-27-
Note
Analysis of Catalytic Residues of Thermoactinomyces vulgaris R-47
α-Amylase II (TVA II) by Site-directed Mutagenesis

Kazuhiro ICHIKAWA, Takashi TONOZUKA, Takehiro YOKOTA, Yoichiro SHIMURA,
and Yoshiyuki SAKANO・

Department of Applied Biological Science, Faculty of Agriculture, Tokyo University of Agriculture
and Technology, 3-5-8, Saiwai-cho, Fuchu, Tokyo 183-8509, Japan

Received May 18, 2000; Accepted July 25, 2000
To confirm that the catalytic residues (Asp325, Glu354, and Asp421) are necessary for the hydrolysis of starch, pullulan, and cyclodextrins, we constructed TVA II mutated by site-directed mutagenesis. The mutated enzymes (D325N, E354Q, and D421N) had markedly reduced levels of activity, less than 0.006％ of the wild type, indicating that these three residues are the catalytic sites for these substrates. Even E354D had reduced levels of activity, less than 0.05％ of wild type. These four mutated enzymes retained a trace of activity. From the result of hydrolysis patterns for maltohexaose, in particular, D421N, unlike D325N and E354Q, catalyzed transglycosylation rather than hydrolysis. The results suggest that Asp421 could function to capture water molecules.
Key words: α-amylase; catalytic residue; transglycosylation

-28-
Note
Purification and Characterization of Cyclohexanone 1,2-Monooxygenase
from Exophiala jeanselmei strain KUFI-6N

Yoshie HASEGAWA,* Yuka NAKAI, Tai TOKUYAMA, and Hiroaki IWAKI

Department of Biotechnology, Faculty of Engineering and High Technology Research Center,
Kansai University, Yamate-cho, Suita 564-8680, Japan

Received May 29, 2000; Accepted August 9, 2000
Baeyer-Villiger cyclohexanone 1,2-monooxygenase (CHMO) was purified 17.1-fold from cell extracts of the fungus Exophiala jeanselmei grown on cyclohexanol to electrophoretically homogeneity by serial chromatographies. The molecular mass of the native enzyme was approximately 74 kDa by gel filtration and SDS-PAGE. Some enzymic characterizations were studied. The NH2-terminal amino acid residues were Ala-Lys-Ser-Leu-Asp-Val-Leu-Ile-Val-Gly-Ala-Gly-Phe-Gly-Gly-Ile-Tyr-Gln-Leu-, with similarity to the bacterial CHMOs of FAD-binding and NADPH-dependent type Baeyer-
Villiger monooxygenases.
Key words: Baeyer-Villiger oxidation; cyclohexanone monooxygenase; cyclohexanol-degradation; Exophiala jeanselmei

-29-
Note
Polygodial, a Potent Attachment-inhibiting Substance for the Blue Mussel,
Mytilus edulis galloprovincialis from Tasmannia lanceolata

Takahiro BAN, Inder Pal SINGH, and Hideo ETOH*

Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka 422-8529, Japan

Received May 31, 2000; Accepted July 29, 2000
A highly potent attachment-inhibitor, polygodial, was isolated from a hexane extract of the leaves of Tasmannia lanceolata. The attachment-inhibiting activity of polygodial against the blue mussel was increased 4-fold when used in combination with sorbic acid, anethole, and indole.
Key words: Tasmannia lanceolata; polygodial; Mytilus edulis galloprovincialis; attachment-inhibiting activity

-30-
Note
Synthesis and Potato Cell Expansion-inducing Activity of the Stereochemically
Restricted Bicyclic Analogue of 7-Epi-jasmonic Acid

Hiroaki TOSHIMA,・ Shinji NARA, Yumiko FUJINO, and Akitami ICHIHARA

Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University,
Sapporo 060-8589, Japan

Received June 5, 2000; Accepted July 11, 2000
The stereochemically restricted bicyclic analogue of 7-epi-jasmonic acid was synthesized from a known bicyclo［3.3.0］octane derivative. The enol triflate derived from the bicyclic compound was subjected to palladium-catalyzed coupling with allyltributyltin to give the desired carbon skeleton. Selective catalytic hydrogenation and subsequent acidic hydrolysis gave a new bicyclic analogue of 7-epi-jasmonic acid. The ACC conjugate of the bicyclic analogue was also synthesized. This ACC conjugate exhibited only slightly weaker potato cell expansion-inducing activity than that of the JA standard.
Key words: 7-epi-jasmonic acid; phytohormone; jasmonic acid analogue; bicyclo［3.3.0］octane

-31-
Note
Effects of a Fermented Milk Drink Containing Lactobacillus casei
Strain Shirota on the Immune System in Healthy Human Subjects

Fumiko NAGAO,1 Masafumi NAKAYAMA,1 Takashi MUTO,2 and Ko OKUMURA1

1Department of Immunology, 2Department of Public Health, Juntendo University School of Medicine,
2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan

Received June 5, 2000; Accepted July 27, 2000
Nine healthy volunteers drank fermented milk containing 4×1010 live cells of Lactobacillus casei strain Shirota daily for 3 weeks, and their NK activity and other immunological functions were measured. NK activity significantly increased (p<0.01) 3 weeks after the start of intake and remained elevated for the next 3 weeks. The effect was particularly prominent in low-NK-individuals.
Key words: fermented milk; immune system; NK cell activity; Lactobacillus casei strain Shirota

-32-
Note
Microbial Degradation of Disodium Terephthalate by Alkaliphilic
Dietzia sp. Strain GS-1

Daisuke SUGIMORI,1,・ Takayuki DAKE,1 and Satoshi NAKAMURA2

1Department of Chemistry and Biology Engineering, Fukui National College of Technology, Geshi-cho,
Sabae, Fukui 916-8507, Japan
2School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho,
Midori-ku, Yokohama 226-8501, Japan

Received June 5, 2000; Accepted July 27, 2000
An alkaliphilic Dietzia sp., strain GS-1, which degraded disodium terephthalate (DT), was isolated from soil. Strain GS-1 degraded 19.3 mM of DT in
168 h at pH 10. The maximum degradation velocity was 0.46 mM/h. The resting cells efficiently degraded 28.7 mM of DT in 51 h at 28°C and pH 10. The degradation velocity was 0.41 mM/(h g-wet cell).
Key words: alkaliphilic bacterium; Dietzia sp.; disodium terephthalate; microbial degradation; resting cell reaction

-33-
Note
Characterization of a Vitamin B12 Compound in the Edible Purple Laver,
Porphyra yezoensis

Fumio WATANABE,1,・ Shigeo TAKENAKA,2,・ Hiromi KATSURA,3 Emi MIYAMOTO,1
Katsuo ABE,1 Yoshiyuki TAMURA,2 Toshiyuki NAKATSUKA,4 and Yoshihisa NAKANO5

1Department of Health Science, Kochi Women′s University, Kochi 780-8515, Japan
2Laboratory of Nutrition and Food Science, Hagoromo-gakuen College, Sakai 592-8344, Japan
3Department of Health Science, Hiroshima Women′s University, Hiroshima,734-8558, Japan
4Department of Food Science, Shimane Women′s College, Matsue 690-0044, Japan
5Department of Applied Biological Chemistry, Osaka Prefecture University, Sakai 599-8531, Japan

Received June 6, 2000; Accepted July 6, 2000
The edible purple laver, Porphyra yezoensis, contained 51.49±1.51 μg of vitamin B12 compounds per 100 g dry weight of the laver (mean±SEM, n=4). A vitamin B12 compound was purified from the lyophilized purple laver and partially characterized. The silica gel 60 TLC and reversed-phase HPLC patterns of the purified pink-colored compound were identical to those of authentic vitamin B12, but not to those of vitamin B12 analogues inactive for humans.
Key words: vitamin B12; methylcobalamin; purple laver; Porphyra yezoensis; intrinsic factor

-34-
Note
Secretion of Hen Egg White Lysozyme from Kluyveromyces lactis

Ryoichi TANAKA, Matsujiro ISHIBASHI, Hiroko TOKUNAGA, and Masao TOKUNAGA・

Laboratory of Applied and Molecular Microbiology, Faculty of Agriculture, Kagoshima University,
1-21-24 Korimoto, Kagoshima 890-0065, Japan

Received June 9, 2000; Accepted August 11, 2000
Hen egg white (HEW) lysozyme was correctly processed and efficiently secreted from an alternative yeast, Kluyveromyces lactis. We constructed secretion vectors using PHO5, PGK, and LAC4 promoters, and found that the highest secretion was obtained under the direction of the PGK promoter in non-selective rich medium. K. lactis secreted HEW lysozyme with two-fold higher efficiency than S. cerevisiae, estimated by using a K. lactis-S. cerevisiae shuttle vector.
Key words: lysozyme; secretion; Kluyveromyces lactis; shuttle vector

-35-
Note
Specific Toxic Effect of Dinoflagellate Heterocapsa circularisquama
on the Rotifer Brachionus plicatilis

Daekyung KIM,1 Yoji SATO,1 Tatsuya ODA,1,・ Tsuyoshi MURAMATSU,1
Yukihiko MATSUYAMA,2 and Tsuneo HONJO3

1Division of Biochemistry, Faculty of Fisheries, Nagasaki University, Bunkyo-machi, Nagasaki 852-8521, Japan
2National Research Institute of Fisheries and Environment of the Inland Sea, Maruishi, Ohno, Saeki,
Hiroshima 739-0452, Japan
3Faculty of Agriculture Kyushu University, Hakozaki, Higashi, Fukuoka 812-8581, Japan

Received June 14, 2000; Accepted August 1, 2000
Heterocapsa circularisquama (Dinophyceae), a noxious red tide dinoflagellate, is known to have a specifically lethal effect on shellfish, especially bivalves such as pearl oyster (Pinctada fucata), but no detrimental effects of this alga on fishes have not been observed so far. In this study, we found that H. circularisquama was toxic to a microzooplankton, a rotifer (Brachionus plicatilis) in a cell concentration-dependent manner, while the cultured supernatant or ultrasonic ruptured H. circularisquama had no significant toxic effect on the rotifer. Since no such toxic effects on the rotifer were observed in Chattonella marina, Heterosigma akashiwo, or Cochlodinium polykrikoides, other species of harmful red tide plankton, H. circularisquama may have a strictly specific toxic mechanism against the rotifer as well as bivalves.
Key words: red tide; Heterocapsa circularisquama; rotifer; toxicity; dinoflagellate

-36-
Note
Synthesis of All Four Possible Stereoisomers of 5,9-Dimethylpentadecane,
the Major Sex Pheromone Component of the Coffee Leaf Miner Moth,
Perileucoptera coffeella

Shigefumi KUWAHARA,1,2,・ Ting LIANG,1 Walter Soares LEAL,3 Jiro ISHIKAWA,1
and Osamu KODAMA1

1Laboratory of Agricultural Chemicals, Faculty of Agriculture, Ibaraki University, Ami-machi,
Inashiki-gun, Ibaraki 300-0393, Japan
2Laboratory of Applied Bioorganic Chemistry, Division of Life Science, Graduate School of Agricultural
Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan
3Department of Entomology, University of California, Davis, CA 95616-8585, U.S,A.

Received June 20, 2000; Accepted July 21, 2000
All of the four possible stereoisomers of 5,9-dimethylpentadecane, the major sex pheromone component of the coffee leaf miner moth (Perileucoptera coffeella), were synthesized by using the methyl esters of (S)- and (R)-3-hydroxy-2-methylpropanoic acid as chiral sources for the purpose of determining the stereochemistry of the pheromone.
Key words: sex pheromone; Perileucoptera coffeella; 5,9-dimethylpentadecane

-37-
Note
Purification and Characterization of Nitrite-oxidizing Enzyme
from Heterotrophic Bacillus badius I-73, with Special Concern to Catalase

Kenji SAKAI,* Hiroto NISIJIMA, Yoshihito IKENAGA, Mamoru WAKAYAMA,**
and Mitsuaki MORIGUCHI

Department of Applied Chemistry, Faculty of Engineering, Oita University, Oita 870-1192, Japan

Received June 21, 2000; Accepted August 11, 2000
Nitrite-oxidizing enzyme I (NiOx I) was purified from a heterotrophic bacterium, Bacillus badius I-73. The enzyme was a homotetramer of a heme-containing peptide, and was similar to catalases from various sources in its N-terminal amino acid sequence. The purified enzyme also catalyzed H2O2 degradation. The nitrite oxidation reaction required ascorbic acid and oxygen. Successive H2O2 feeding could be substituted for ascorbic acid. These indicated that NiOx I is a catalase and nitrite was oxidized by a peroxidase-like reaction.
Key words: heterotrophic nitrification; nitrite oxidation; nitrite oxidoreductase; catalase; purification

-38-
Note
Introduction of Enterostatin (VPDPR) and a Related Sequence into Soybean
Proglycinin A1aB1b Subunit by Site-directed Mutagenesis

Yasuyuki TAKENAKA, Shigeru UTSUMI, and Masaaki YOSHIKAWA

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

Received June 23, 2000; Accepted July 25, 2000
Enterostatin (VPDPR), having anoretic and hypocholesterolemic activities, and its homologue LPYPR, a hypocholesterolemic peptide found in the glycinin A5A4B3 subunit, were introduced into the corresponding site (TNGPQ) of the proglycinin A1aB1b subunit by site-directed mutagenesis. Modified proglycinins were expressed in E. coli and recovered from the insoluble fraction. VPDPR and LPYPR were released by the action of chymotrypsin and trypsin as expected. The overall yields of purified VPDPR and LPYPR were 40％ and 62％, respectively.
Key words: anoretic activity; enterostatin; glycinin; hypocholesterolemic activity; soybean

-39-
Note
Molecular Cloning, Overexpression, and Purification
of a Major Xylanase from Aspergillus oryzae

Tetsuya KIMURA,1・ Hayato SUZUKI,1 Hirofumi FURUHASHI,1 Takeshi ABURATANI,1
Kenji MORIMOTO,1 Shuichi KARITA,2 Kazuo SAKKA,1 and Kunio OHMIYA1

1Faculty of Bioresources and 2Center for Molecular Biology and Genetics, Mie University,
Tsu, Mie 514-8507, Japan

Received June 23, 2000; Accepted July 31, 2000
The gene encoding xylanase G2 (xynG2) was isolated from a genomic library of Aspergillus oryzae KBN616, used for making shoyu koji. The structural part of xynG2 was found to be 767 bp. The nucleotide sequence of cDNA amplified by RT-PCR showed that the open reading frame of xynG2 was interrupted by a single intron which was 71 bp in size and encoded 232 amino acids. Direct N-terminal amino acid sequencing showed that the precursor of XynG2 had a signal peptide of 44 amino acids. The predicted amino acid sequence of XynG2 has strong similarity to other family 11 xylanases from fungi. The xynG2 gene was successfully overexpressed in A. oryzae and the overpexpressed XynG2 was purified. The molecular weight of XynG2 estimated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 21,000. This was almost the same as the molecular weight of 20,047 calculated from the deduced amino acid sequence. The purified XynG2 showed an optimum activity at pH 6.0 and 58°C. It had a Km of 5.1 mg/ml and a Vmax of 123 μmol/min/mg when birch wood xylan was used as a substrate.
Key words: Aspergillus oryzae; shoyu-koji; xylanase

-40-
Note
Enzymatic Preparation of Genuine Prosapogenin, 20(S)-Ginsenoside Rh1,
from Ginsenosides Re and Rg1

Sung-Ryong KO, Yukio SUZUKI,*,** Kang-Ju CHOI, and Young-Hoi KIM

Korea Ginseng and Tobacco Research Institute, 302 Shinsong-dong, Yusong-ku, Taejon, 305-345, Korea
*Research Institute for Bioresources, Okayama University, 2-20-1, Chuo, Kurashiki 710-0046, Japan

Received July 10, 2000; Accepted August 8, 2000
It was found that a lactase preparation from Penicillium sp. nearly quantitatively hydrolyzed ginsenosides Re and Rg1, which are major saponins in roots of Panax ginseng, to a minor saponin, 20(S)-ginsenoside Rh1 ［6-O-β-D-glucopyranosyl-20(S)-protopanaxatriol］. This is the first report on the enzymatic preparation of ginsenoside Rh1 with a high efficiency. This enzyme also readily hydrolyzed ginsenoside Rg2 to ginsenoside Rh1.
Key words: ginsenoside Re; ginsenoside Rg1; ginsenoside Rh1; ginsenoside Rg2; Penicillium sp. lactase

-41-
Preliminary Communication
Tyrosine72 Residue at the Bottom of Rim Domain in LukF Crucial
for the Sequential Binding of the Staphylococcal γ-Hemolysin
to Human Erythrocytes

Kenji YOKOTA and Yoshiyuki KAMIO・

Laboratory of Applied Microbiology, Department of Molecular and Cell Biology, Graduate School
of Agricultural Science, Tohoku University, 1-1 Tsutsumi-dori, Amamiya-machi, Aoba-ku,
Sendai 981-8555, Japan

Received August 15, 2000; Accepted September 12, 2000
Staphylococcal bi-component cytotoxins, leukocidin (Luk), Panton-Valentine leukocidin (PVL), and γ-hemolysin (Hlg) consist of LukF and LukS, LukF-PV and LukS-PV, and LukF and Hlg2, respectively, and Luk and Hlg share LukF. LukF-PV can not substitute for LukF for Hlg, despite 73％ identity in amino acid sequence and close similarity in the 3-dimensional structure between them. Here, we demonstrated that the absence of hemolytic activity of LukF-PV in cooperation with Hlg2 is due to the failure of the binding of LukF-PV to human erythrocytes. We identified Y72 residue at the bottom of rim domain in LukF as the crucial residue for its binding, which is a prerequisite to the subsequent binding of Hlg2 to human erythrocytes. The data obtained showed that a mutant of LukF-PV in which T71 residue was replaced by the corresponding residue of LukF, Y72, endowed LukF-PV with the binding capability to human erythrocytes which was accompanied by its hemolytic activity in the presence of Hlg2.
Key words: staphylococcal γ-hemolysin; LukF; LukF-PV; binding sites to erythrocytes; 3-dimensional structure