Contents and Abstracts of BBB

(Vol.67 No.8 2003)

Small Scale Rat Hepatocyte Primary Culture with Applications for Screening Hepatoprotective Substances
Ryusuke NAKAGIRI,1,† Hiroaki ODA,2 and Toshikazu KAMIYA1 p.1629

Purification, Characterization, and Sequencing of a Novel Type of Antimicrobial Peptides, Fa-AMP1 and Fa-AMP2, from Seeds of Buckwheat (Fagopyrum esculentum Moench.)
Masatoshi FUJIMURA,1,† Yuji MINAMI,1 Keiichi WATANABE,2 and Kenjiro TADERA1
p.1636

Polymerization and Calcium Binding of the Tubulin-colchicine Complex in the GDP State
Hiroshi DOI,1,† Makiko KAWAGUCHI,1 and Serge N. TIMASHEFF2 p.1643

Purification, Amino Acid Sequence, and cDNA Cloning of Trypsin Inhibitors from Onion (Allium cepa L.) Bulbs
Masanobu DESHIMARU, Akira WATANABE, Keiko SUEMATSU, Maki HATANO, and Shigeyuki TERADA
p.1653

Polar Organic Solvent Added to an Aqueous Solution Changes Hydrolytic Property of Lipase
Wakako TSUZUKI, Akemi UE, and Akihiko NAGAO p.1660

MpFAE3, a β-Ketoacyl-CoA Synthase Gene in the Liverwort Marchantia polymorpha L., Is Preferentially Involved in Elongation of Palmitic Acid to Stearic Acid
Masataka KAJIKAWA,1 Katsuyuki T. YAMATO,1 Hiroyuki KANAMARU,2 Eiji SAKURADANI,2 Sakayu SHIMIZU,2 Hideya FUKUZAWA,1 Yasuyoshi SAKAI,3 and Kanji OHYAMA1,†, p.1667

Vitamin C Activity in Guinea Pigs of 6-O-Acyl-2-O-α-D-glucopyranosyl-L- ascorbic Acids with a Branched-acyl Chain
Akihiro TAI, Daisuke KAWASAKI, Satomi GOTO, Eiichi GOHDA, and Itaru YAMAMOTO
p.1675

Bottle-choice Tests in Sprague-Dawley Rats Using Liquid Diets That Differ in Oil and Sucrose Contents
Fumiko KIMURA, Risa OKADA, Yasushi ENDO, and Kenshiro FUJIMOTO p.1683

Developmental Transition of the Flavonoid Contents in Safflower Leaves during Stress-loaded Cultivation
Satoe YAGINUMA,1,† Takuo SHIRAISHI,1 and Kiharu IGARASHI2 1691

Enhanced Methionine and Cysteine Levels in Transgenic Rice Seeds by the Accumulation of Sesame 2S Albumin
Tiger T. T. LEE,1 Miki M. C. WANG,1 Rolis C. W. HOU,1 Liang-Jwu CHEN,2 Ruey-Chih SU,3 Chang-Sheng WANG,4 and Jason T. C. TZEN1, p.1699

Effect of Dietary Pectin on the Production of Immunoglobulins and Cytokines by Mesenteric Lymph Node Lymphocytes in Mouse Colitis Induced with Dextran Sulfate Sodium
Beong Ou LIM,1,2,† Sun Hee LEE,1 Dong Ki PARK,2 and Ryo Won CHOUE1 p.1706

Absorbability of Calcium from Calcium-bound Phosphoryl Oligosaccharides in Comparison with That from Various Calcium Compounds in the Rat Ligated Jejunum Loop
Kenji TO-O,1, Hiroshi KAMASAKA,1 Takahisa NISHIMURA,1 Takashi KURIKI,1 Shigeru SAEKI,2 and Yukihiro NAKABOU3 p.1713

Inhibition of β-Fructofuranosidases and α-Glucosidases by Synthetic Thio-fructofuranoside
Taro KISO,1,† Kenichi HAMAYASU,2 Koki FUJITA,2 Kozo HARA,2 Sumio KITAHATA,1 and Hirofumi NAKANO1 p.1719

rDNA ITS Sequence of Rhizopus oryzae: Its Application to Classification and Identification of Lactic Acid Producers
Ayumi ABE,1 Teruo SONE,1,† I Nengah SUJAYA,1 Katsuichi SAITO,2 Yuji ODA,2 Kozo ASANO,1 and Fusao TOMITA1 p.1725

Simple Synthesis of Mite Pheromone β-Acaridial and Its Analogs in the Secretion of Caloglyphus polyphyllae (Acari: Acaridae)
Nobuhiro SHIMIZU, Naoki MORI, and Yasumasa KUWAHARA p.1732

Functional Roles of the Tissue Inhibitor of Metalloproteinase 3 (TIMP-3) during Ascorbate-induced Differentiation of Osteoblastic MC3T3-E1 Cells
Hironao SUZUKI,1 Yuko NEZAKI,1 Eriko KUNO,1 Ikuko SUGIYAMA,1 Akifumi MIZUTANI,2,† and Norihiro TSUKAGOSHI3 p.1737

Synthesis of (4R,6S,7R)-7-Hydroxy-4,6-dimethyl-3-nonanone and (3R,5S,6R)- 6-Hydroxy-3,5-dimethyl-2-octanone, the Pheromone Components of the Bostrychid Beetle, Dinoderus bifoveolatus
Yui MASUDA,1 Ken FUJITA,1 and Kenji MORI2,† p.1744

Primary Structure of Inorganic Polyphosphate/ATP-NAD Kinase from Micrococcus flavus, and Occurrence of Substrate Inorganic Polyphosphate for the Enzyme
Shigeyuki KAWAI, Shigetarou MORI, and Kousaku MURATA p.1751

Synthesis of an Unnatural N-Glycan-linked Dolichyl Pyrophosphate Precursor
Yuko NAKAHARA, Tomoharu NONAKA, Hironobu HOJO, and Yoshiaki NAKAHARA p.1761

RNase ES of Streptomyces coelicolor A3(2) Can Complement the rne and rng Mutations in Escherichia coli
Takabumi INAGAWA,1 Susumu OKAMOTO,1 Masaaki WACHI,2 and Kozo OCHI1,† p.1767

Isolation of Suppressor Mutants of Phosphatidylinositol 3-Phosphate 5-kinase Deficient Cells in Schizosaccharomyces pombe
Masayuki ONISHI,1 Yoko NAKAMURA,1 Takako KOGA,1, Kaoru TAKEGAWA,2 and Yasuhisa FUKUI1,† p.1772

Identification and Purification of Sulfotransferases for 20-Hydroxysteroid from the Larval Fat Body of a Fleshfly, Sarcophaga peregrina
Eiko MATSUMOTO, Michio MATSUI, and Hiro-omi TAMURA p.1780

Heterogeneous Nuclear Ribonucleoprotein K Interacts with and Is Proteolyzed by Calpain in vivo
Eiichi KIMURA,1 Keiko ABE,1 Koichi SUZUKI,2 and Hiroyuki SORIMACHI1,3,† p.1786

Note
Isolation of Actinomycetes from Termites' Guts
Yoshio WATANABE,1,† Naoya SHINZATO,2 and Takema FUKATSU2 p.1797

Note
Flagellum-independent Trail Formation of Escherichia coli on Semi-solid Agar
Hajime FUKUOKA,1,2,† Michio HOMMA,3 and Shigeyuki ICHIHARA1,2 p.1802

Note
Effect of γ-Aminobutyric Acid-enriched Tempeh-like Fermented Soybean (GABA-Tempeh) on the Blood Pressure of Spontaneously Hypertensive Rats
Hideyuki AOKI,1,† Yuji FURUYA,1 Yasushi ENDO,2 and Kenshiro FUJIMOTO2 p.1806

Note
Organic Solvent Tolerance of Halophilic Archaea
Ron USAMI, Tadamasa FUKUSHIMA, Toru MIZUKI, Akira INOUE, Yasuhiko YOSHIDA, and Koki HORIKOSHI p.1809

Note
Neuroprotective Activity of p-Terphenyl Leucomentins from the Mushroom Paxillus panuoides
In-Kyoung LEE,1 Bong-Sik YUN,1 Jong-Pyung KIM,1 In-Ja RYOO,1 Young-Ho KIM,2 and Ick-Dong YOO1,† p.1813

Note
Two Distinct Curved DNAs Upstream of the Light-responsive psbA Gene in a Cyanobacterium
Ganesh Kumar AGRAWAL, Munehiko ASAYAMA, and Makoto SHIRAI p.1817

Note
Stimulated Accumulation of Lectin mRNA and Stress Response in Helianthus tuberosus Callus by Methyl Jasmonate
Ryoji NAKAGAWA, Yukihiro OKUMURA, Makoto KAWAKAMI, Daisuke YASOKAWA, and Koji NAGASHIMA p.1822

Note
Comparative Analyses of Hairpin Substrate Recognition by Escherichia coli and Bacillus subtilis Ribonuclease P Ribozymes
Tomoaki ANDO, Terumichi TANAKA, and Yo KIKUCHI p.1825

Note
Comparison of Acylated Plant Pigments: Light-resistance and Radical-scavenging Ability
Nobuyoshi NAKAJIMA,1,† Manabu SUGIMOTO,2 Hiroshi YOKOI,3 Hideaki TSUJI,1 and Kohji ISHIHARA4, p.1828

Note
Peptide Mapping and Assessment of Cryoprotective Activity of 26/27-kDa Dehydrin from Soybean Seeds
Michiko MOMMA, Shigenobu KANEKO, Kazutomo HARAGUCHI, and Ushio MATSUKURA p.1832

Note
Expression of CD38 in Human Promyelocytic Leukemia HL-60 Cell Line during Differentiation by Niacin-related Compounds
Keiko IWATA, Shin OGATA, Katsuzumi OKUMURA, and Hiroshi TAGUCHI p.1836

Note
Interaction between Acidic Polysaccharides and Proteins
Kenjiro TADERA, Yuji MINAMI, and Miki CHOHCHI p.1840

Note
Paradoxical Effect of Synthetic Hydroxy Isothiocyanates on Antimicrobial Action of Aminoglycosides
Hirokuni TAJIMA,1,† Hisashi KIMOTO,2 and Akira TAKETO3 p.1844

Communication
Absorptive Activity of Calcium in the Isolated Cecal Epithelium Adaptively Increased by 2 Week's Feeding of Difructose Anhydride III in Rats
Hitoshi MINEO,1,4 Midori AMANO,1,2 Hideyuki CHIJI,2 Norihiro SHIGEMATSU,3 Fusao TOMITA,4 and Hiroshi HARA4,† p.1847

Communication
First Evidence for Occurrence of Galβ1-3GlcNAcβ1-4Man Unit in N-Glycans of Insect Glycoprotein: β1-3Gal and β1-4GlcNAc Transferases Are Involved in N-Glycan Processing of Royal Jelly Glycoproteins
Yoshinobu KIMURA,1,† Kazunori TSUMURA,1 Mariko KIMURA,2 Kiyoshi OKIHARA,3 Hiroyuki SUGIMOTO,3 and Hideo YAMADA3 p.1852

-1-
Small Scale Rat Hepatocyte Primary Culture with Applications for Screening Hepatoprotective Substances

Ryusuke NAKAGIRI,1,† Hiroaki ODA,2 and Toshikazu KAMIYA1

1Kyowa Hakko Kogyo Co., Ltd., Tsukuba Research Laboratories, 2 Miyukigaoka, Tsukuba-shi, Ibaraki 305--0841, Japan 2Laboratory of Nutritional Biochemistry, Department of Applied Biological Sciences, Nagoya University, Furo-cho, Chigusa-ku, Nagoya-shi, Aichi 464--8601, Japan

Received November 1, 2002; Accepted May 12, 2003
Isolated hepatocytes are known to maintain their physiological functions for over a week when cultured on Matrigel, artificially reconstituted from basement membrane components. Although this culture technique has been frequently used in research on hepatocyte functions, there has been a limitation on its application for small scale experiments due to some technical problems. By using micro-culture plates with 96 round-bottom wells, we succeeded in coating the wells uniformly with Matrigel. When the cultured hepatocytes were treated with either 10 mM, 15 mM, or 20 mM of acetaminophen or 1 mM, 10 mM, or 20 mM of D-galactosamine, the viability of the hepatocytes became 91.1％, 75.3％, 64.7％, and 79.0％, 43.8％, 26.2％ of the non-treated control at 48 hours, respectively. Fractionated extracts of Glycyrrhiza glabra L. and Schisandra chinensis Baillon inhibited the action of acetaminophen or D-galactosamine in this model. From these results, we concluded that the microculture system presented here is capable of maintaining the in vivo characteristics of hepatocytes and is suitable for the screening of hepatoprotective substances.
Key words: hepatocyte; hepatoprotective; screening; acetaminophen; D-galactosamine

-2-
Purification, Characterization, and Sequencing of a Novel Type of Antimicrobial Peptides, Fa-AMP1 and Fa-AMP2, from Seeds of Buckwheat (Fagopyrum esculentum Moench.)

Masatoshi FUJIMURA,1,† Yuji MINAMI,1 Keiichi WATANABE,2 and Kenjiro TADERA1

1Department of Biochemical Science and Technology, Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan 2Department of Applied Biological Sciences, Faculty of Agriculture, Saga University, Honjo-machi, Saga 840-8502, Japan

Received December 18, 2002; Accepted May 9, 2003
Novel antimicrobial peptides (AMP), designated Fa-AMP1 and Fa-AMP2, were purified from the seeds of buckwheat (Fagopyrum esculentum Moench.) by gel filtration on Sephadex G75, ion-exchange HPLC on SP COSMOGEL, and reverse-phase HPLC. They were basic peptides having isoelectric points of over 10. Fa-AMP1 and Fa-AMP2 had molecular masses of 3,879 Da and 3,906 Da on MALDI-TOF MS analysis, and their extinction coefficients in 1％ aqueous solutions at 280 nm were 42.8 and 38.9, respectively. Half of all amino acid residues of Fa-AMP1 and Fa-AMP2 were cysteine and glycine, and they had continuous sequences of cysteine and glycine. The concentrations of peptides required for 50％ inhibition (IC50) of the growth of plant pathogenic fungi, and Gram-positive and -negative bacteria were 11 to 36 μg/ml. The structural and antimicrobial characteristics of Fa-AMPs indicated that they are a novel type of antimicrobial peptides belonging to a plant defensin family.
Key words: antimicrobial peptide (AMP); Fa-AMP; pathogenic related protein; buckwheat; Fagopyrum esculentum

-3-
Polymerization and Calcium Binding of the Tubulin-colchicine Complex in the GDP State

Hiroshi DOI,1,† Makiko KAWAGUCHI,1 and Serge N. TIMASHEFF2

1Department of Food Science and Nutrition, Mukogawa Women's University, Nishinomiya, Hyogo 663-8558, Japan 2Graduate Department of Biochemistry, Brandeis University, Waltham, MA 02254, USA

Received December 27, 2002; Accepted May 1, 2003
The tubulin-colchicine complex instead of tubulin was used in an imidazole buffer throughout experiments. The interaction with calcium was examined, especially in the GDP state. The high affinity sites of calcium took part in the polymerization of the complex in the GTP state, while the low ones participated in the depolymerization. The complex had 2 high affinity sites with the dissociation constant of 11.5×10−6 M, and 16 low affinity sites with the dissociation constant of 2.27×10−4 M in the GTP state. In the case of GDP state, the dissociation constant of the high affinity site was 7.2×10−6 M, and the low affinity site was not observed. The ultracentrifugal experiment indicated a little compact structure in the GTP state compared with the GDP state. This agreed with the results of calcium binding.
Key words: tubulin; colchicine; calcium binding; guanosine 5'-diphosphate (GDP) state

-4-
Purification, Amino Acid Sequence, and cDNA Cloning of Trypsin Inhibitors from Onion (Allium cepa L.) Bulbs

Masanobu DESHIMARU, Akira WATANABE, Keiko SUEMATSU, Maki HATANO, and Shigeyuki TERADA

Department of Chemistry, Faculty of Science, Fukuoka University, 8-19-1 Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan

Received January 16, 2003; Accepted May 9, 2003
Three protease inhibitors (OTI-1--3) have been purified from onion (Allium cepa L.) bulbs. Molecular masses of these inhibitors were found to be 7,370.2, 7,472.2, and 7,642.6 Da by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), respectively. Based on amino acid composition and N-terminal sequence, OTI-1 and -2 are the N-terminal truncated proteins of OTI-3. All the inhibitors are stable to heat and extreme pH. OTI-3 inhibited trypsin, chymotrypsin, and plasmin with dissociation constants of 1.3×10−9 M, 2.3×10−7 M, and 3.1×10−7 M, respectively. The complete amino acid sequence of OTI-3 showed a significant homology to Bowman-Birk family inhibitors, and the first reactive site (P1) was found to be Arg17 by limited proteolysis by trypsin. The second reactive site (P1) was estimated to be Leu46, that may inhibit chymotrypsin. OTI-3 lacks an S-S bond near the second reactive site, resulting in a low affinity for the enzyme. The sequence of OTI-3 was also ascertained by the nucleotide sequence of a cDNA clone encoding a 101-residue precursor of the onion inhibitor.
Key words: amino acid sequence; cDNA cloning; onion bulbs; proteinase inhibitor; purification

-5-
Polar Organic Solvent Added to an Aqueous Solution Changes Hydrolytic Property of Lipase

Wakako TSUZUKI, Akemi UE, and Akihiko NAGAO

National Food Research Institute, Kannondai 2-1-12, Tsukuba, Ibaraki 305-8642, Japan

Received January 16, 2003; Accepted April 21, 2003
For developing further uses of lipase as a biocatalyst, its hydrolytic activity toward some esters was investigated in a miscible solution composed of a buffer and a polar organic solvent. Twenty percent dimethylformamide, 35％ dimethylsulfoxide, 15％ 1,4-dioxane, 15％ dimethoxyethane, and 2％ diethoxyethane promoted the hydrolysis by a lipase from Rhizomucor miehei toward some hydrophobic substrates, 4-methylumbelliferyl oleate, 4-methylumbelliferyl palmitate, and monoolein. While hydrolysis by this lipase toward the substrates with a relatively weak hydrophobicity (4-metylumbelliferyl heptanoate and 4-methylumbelliferyl nanoate) was suppressed by these solvents. A fluorometric analysis showed that the polar organic solvent in the buffer induced some conformational change around a tryptophan residue of R. miehei lipase. In addition to the influence of the miscible solvent on the solubility of the substrates, the conformational change of the protein induced by the miscible solvent would also affect the reactive properties of the lipase. Adding a polar organic solvent to an aqueous solution will be an efficient method for changing hydrolytic performance of lipases.
Key words: lipase; hydrolysis; polar organic solvent

-6-
MpFAE3, a β-Ketoacyl-CoA Synthase Gene in the Liverwort Marchantia polymorpha L., Is Preferentially Involved in Elongation of Palmitic Acid to Stearic Acid

Masataka KAJIKAWA,1 Katsuyuki T. YAMATO,1 Hiroyuki KANAMARU,2 Eiji SAKURADANI,2 Sakayu SHIMIZU,2 Hideya FUKUZAWA,1 Yasuyoshi SAKAI,3 and Kanji OHYAMA1,†,

1Laboratory of Plant Molecular Biology, Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan 2Laboratory of Fermentation Physiology and Applied Microbiology, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan 3Laboratory of Microbial Biotechnology, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan

Received January 16, 2003; Accepted April 17, 2003
Fatty acid chain elongation is a crucial step in the biosynthesis of long chain fatty acids. An essential reaction in the elongation process is condensation of malonyl-CoA with acyl-CoA, which is catalyzed by β-ketoacyl-CoA synthase (KCS) in plants. We have isolated and characterized the MpFAE3 gene, one of the KCS gene family in the liverwort Marchantia polymorpha. Transgenic M. polymorpha plants overexpressing MpFAE3 accumulate fatty acids 18:0, 20:0, and 22:0. In these plants, the amount of 16:0 is reduced to 50％ of wild type. In a heterologous assay, transgenic methylotrophic yeast expressing the MpFAE3 gene accumulates fatty acid 18:0 and generates several longer fatty acids which are not detectable in the control, accompanied by a decrease of 16:0. These observations indicate that the MpFAE3 protein is preferentially involved in the elongation of 16:0 to 18:0 and also in the subsequent steps of 18:0 to 20:0 and 20:0 to 22:0 in M. polymorpha.
Key words: fatty acid chain elongation; heterologous expression; overexpression; bryophyte; β-ketoacyl-CoA synthase (KCS)

-7-
Vitamin C Activity in Guinea Pigs of 6-O-Acyl-2-O-α-D-glucopyranosyl-L- ascorbic Acids with a Branched-acyl Chain

Akihiro TAI, Daisuke KAWASAKI, Satomi GOTO, Eiichi GOHDA, and Itaru YAMAMOTO

Department of Immunochemistry, Faculty of Pharmaceutical Sciences, Okayama University, Okayama 700-8530, Japan

Received January 17, 2003; Accepted May 1, 2003
A series of novel acylated ascorbic acid derivatives, 6-O-acyl-2-O-α-D-glucopyranosyl-L-ascorbic acids with a branched-acyl chain (6-bAcyl-AA-2G) were recently developed in our laboratory as stable and lipophilic ascorbate derivatives. In this study, the bioavailability of 6-bAcyl-AA-2G was investigated in guinea pigs. Various tissue homogenates from guinea pigs hydrolyzed 6-bAcyl-AA-2G to give ascorbic acid (AA), 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G), and 6-O-acyl AA. The releasing pattern of the three hydrolysates suggested that 6-bAcyl-AA-2G was hydrolyzed via 6-O-acyl AA to AA as a main pathway and via AA-2G to AA as a minor pathway. The former pathway seems to be of advantage, because 6-O-acyl AA, as well as AA, can have vitamin C activity. In addition, we found that a derivative with an acyl chain of C12, 6-bDode-AA-2G, had a pronounced therapeutic effect in scorbutic guinea pigs by its repeated oral administrations. These results indicate that 6-bAcyl-AA-2G is a readily available source of AA in vivo, and may be a promising antioxidant for skin care and treatment of diseases associated with oxidative stress.
Key words: 6-O-acyl-2-O-α-D-glucopyranosyl-L-ascorbic acids (6-Acyl-AA-2G); lipophilic ascorbate; stable ascorbate; antiscorbutic activity; metabolism

-8-
Bottle-choice Tests in Sprague-Dawley Rats Using Liquid Diets That Differ in Oil and Sucrose Contents

Fumiko KIMURA, Risa OKADA, Yasushi ENDO, and Kenshiro FUJIMOTO

Laboratory of Food and Biomolecular Science, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan

Received January 22, 2003; Accepted May 7, 2003
Bottle choice tests using liquid diets were done with Sprague-Dawley (SD) rats. SD rats ingested more oil-and-sucrose-enriched milk (hi-fat) and less oil-enriched milk (hi-fat-no-carb) than sucrose-enriched (hi-carb) milk by two-bottle choice tests after they were habituated to liquid diets for 4 days. Chronic food restriction didn't increase hi-fat ingestion but hi-fat-no-carb. Rats ingested less without habituation, and overnight food deprivation increased intake. This increment was maintained after rats were free-fed. The difference in fat content of the maintenance diet had little effect on fat preference. These results showed SD rats prefer a sweet and fatty liquid diet than a sweet and lean liquid diet. Habituation and food restriction were more important than the composition of the maintenance diet to demonstrate a clear preference for the fatty liquid diet.
Key words: food preference; optional food; bottle choice test; rats; high fat diet

-9-
Developmental Transition of the Flavonoid Contents in Safflower Leaves during Stress-loaded Cultivation

Satoe YAGINUMA,1,† Takuo SHIRAISHI,1 and Kiharu IGARASHI2

1JST Regional Joint Research Project of Yamagata Prefecture, 2-2-1 Matsuei, Yamagata, Yamagata 990-2473, Japan 2Department of Bioresource Engineering, Faculty of Agriculture, Yamagata University, 1-23 Wakaba-machi, Tsuruoka, Yamagata 997-8555, Japan

Received January 28, 2003; Accepted April 9, 2003
We have previously reported that strong visible light with limited water caused a significant increase in the polyphenol contents of safflower seedlings (Carthamus tinctrius L.), suggesting that the appropriate stress loading could be applied to effectively cultivate flavonoid-rich plants. In this present study, we investigated in detail the time-dependent transition in the flavonoid contents of safflower leaves during the stress-loaded cultivation. In the cotyledons, the light/water stress continuously increased the content of luteolin 7-O-glucoside, which is a strong antioxidant, whereas the content of acacetin 7-O-glucuronide, a weak antioxidant, generally remained unchanged. In the foliage leaves under the stress condition, the contents of the flavonoid glucosides (luteolin 7-O-glucoside and quercetin 7-O-glucoside) markedly increased on the 2nd day and then decreased to the level before stress loading on the 5th day. These results indicate that appropriate selection of the time for stress loading could provide more flavonoid-rich plants during the practical cultivation of vegetables.
Key words: safflower (Carthamus tinctrius L.); stress; polyphenol; flavonoid; developmental transition

-10-
Enhanced Methionine and Cysteine Levels in Transgenic Rice Seeds by the Accumulation of Sesame 2S Albumin

Tiger T. T. LEE,1 Miki M. C. WANG,1 Rolis C. W. HOU,1 Liang-Jwu CHEN,2 Ruey-Chih SU,3 Chang-Sheng WANG,4 and Jason T. C. TZEN1,

1Graduate Institute of Biotechnology, National Chung-Hsing University, Taichung, Taiwan 40227, ROC 2Institute of Molecular Biology, National Chung-Hsing University, Taichung, Taiwan 40227, ROC 3Department of Life Science, Fu Jen University, Taipei, Taiwan 24205, ROC 4Department of Agronomy, Taiwan Agricultural Research Institute, Taichung, Taiwan

Received January 29, 2003; Accepted April 18, 2003
A chimeric gene encoding a precursor polypeptide of sesame 2S albumin, a sulfur-rich seed storage protein, was expressed in transgenic rice plants under the control of the glutelin promoter with the aim of improving the nutritive value of rice. Rice grains harvested from the first generation of ten different transformed lines inherited the transgene, and the accumulated sesame 2S albumin was presumably processed correctly as its mature form in sesame seed. This transgene was specifically expressed in maturing rice seeds with its encoded sesame 2S albumin exclusively accumulated in the seeds. The crude protein content in rice grains from five putative homozygous lines was increased by 0.64--3.54％, and the methionine and cysteine contents of these transgenic rice grains were respectively elevated by 29--76％ and 31--75％ compared with those of wild-type rice grains.
Key words: methionine; sulfur amino acid; sesame 2S albumin; transgenic rice

-11-
Effect of Dietary Pectin on the Production of Immunoglobulins and Cytokines by Mesenteric Lymph Node Lymphocytes in Mouse Colitis Induced with Dextran Sulfate Sodium

Beong Ou LIM,1,2,† Sun Hee LEE,1 Dong Ki PARK,2 and Ryo Won CHOUE1

1Graduate School of East-West Medical Science, Department of Medical Nutrition, Kyung Hee University, 1 Hoeki-Dong, Dongdaemoon-ku, Seoul 130-701, Korea 2Cell Activation Research Institute, Konkuk University, Seoul 143-701, Korea

Received February 4, 2003; Accepted April 25, 2003
The present study explores the dietary effect of pectin on the MLN lymphocyte functions of mice with dextran sulfate sodium (DS)-induced colitis. We found that the immunoglobulin (Ig)A level in mesenteric lymph node (MLN) lymphocytes was high, while the IgE level was lower, in mice fed with pectin than in those fed with cellulose. Interestingly, the fecal IgA concentration of the pectin-fed mice was significantly higher than that of the cellulose-fed mice. The concentrations of interferon-γ and interleukin (IL)-2 treated with concanavalin A (ConA) were significantly higher in the pectin-fed group than in the cellulose-fed group. Although dietary pectin did not affect the IL-4 and IL-10 levels, the activation-induced IL-4 and IL-10 secretion was lower in MLN cells of the pectin-fed mice than of the cellulose-fed mice following DS-induced colitis. Based on these findings, we propose that the effect of dietary pectin on mice with DS-induced colitis is mediated by the manipulation of Th1 cells. Furthermore, the inhibitory effect of IL-4 and IL-10 by dietary pectin may play an important role in promoting a change in Th1/Th2 balance toward Th1-dominant immunity.
Key words: inflammatory bowel disease; dietary pectin; mesenteric lymph node; dextran sodium sulfate; fecal IgA

-12-
Absorbability of Calcium from Calcium-bound Phosphoryl Oligosaccharides in Comparison with That from Various Calcium Compounds in the Rat Ligated Jejunum Loop

Kenji TO-O,1, Hiroshi KAMASAKA,1 Takahisa NISHIMURA,1 Takashi KURIKI,1 Shigeru SAEKI,2 and Yukihiro NAKABOU3

1Biochemical Research Laboratory, Ezaki Glico Co., Ltd., Osaka 555-8502, Japan 2Department of Food and Nutrition, Graduate School of Human Life Science, Osaka City University, Osaka 558-8585, Japan 3Faculty of Human Environmental Science, Kyoto Prefectural University, Kyoto 606-8522, Japan

Received February 7, 2003; Accepted April 16, 2003
Calcium-bound phosphoryl oligosaccharides (POs-Ca) were prepared from potato starch. Their solubility and in situ absorbability as a calcium source were investigated by comparing with the soluble calcium compounds, calcium chloride and calcium lactate, or insoluble calcium compounds, calcium carbonate and dibasic calcium phosphate. The solubility of POs-Ca was as high as that of calcium chloride and about 3-fold higher than that of calcium lactate. An in situ experiment showed that the intestinal calcium absorption rate of POs-Ca was almost comparable with that of the soluble calcium compounds, and was significantly higher (p＜0.05) than that of the insoluble calcium groups. Moreover, the total absorption rate of a 1:1 mixture of the calcium from POs-Ca and a whey mineral complex (WMC) was significantly higher (p＜0.05) than that of WMC alone. These results suggest that POs-Ca would be a useful soluble calcium source with relatively high absorption in the intestinal tract.
Key words: phosphoryl oligosaccharides; POs-Ca; calcium; rat; ligated loop

-13-
Inhibition of β-Fructofuranosidases and α-Glucosidases by Synthetic Thio-fructofuranoside

Taro KISO,1,† Kenichi HAMAYASU,2 Koki FUJITA,2 Kozo HARA,2 Sumio KITAHATA,1 and Hirofumi NAKANO1

1Osaka Municipal Technical Research Institute, 1-6-50 Morinomiya, Joto-ku, Osaka 536-8553, Japan 2Bio Research Corporation of Yokohama, 1-1-1 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan

Received February 21, 2003; Accepted April 21, 2003
A synthetic β-thio-fructofuranoside of mercaptoethanol inhibited not only β-fructofuranosidases but also α-glucosidases. The compound was hardly hydrolyzed by the glycosidases. The thio-fructoside competitively inhibited β-fructofuranosidases from Aspergillus niger, Candida sp., and Saccharomyces cerevisiae, but not Arthrobacter β-fructofuranosidase at all. Sucrase activity of rat intestinal sucrase/isomaltase complex was also suppressed in the presence of the thio-fructoside. The thio-fructoside showed noncompetitive inhibition toward maltase activity of the rat intestinal enzyme complex and Saccharomyces sp. α-glucosidase. Inhibition against the Bacillus stearothermophilus α-glucosidase, Rhizopus glucoamylase, and porcine kidney trehalase were more slight than that against these two α-glucosidases.
Key words: β-fructofuranosidase; α-glucosidase; inhibition; thio-fructoside

-14-
rDNA ITS Sequence of Rhizopus oryzae: Its Application to Classification and Identification of Lactic Acid Producers

Ayumi ABE,1 Teruo SONE,1,† I Nengah SUJAYA,1 Katsuichi SAITO,2 Yuji ODA,2 Kozo ASANO,1 and Fusao TOMITA1

1Laboratory of Applied Microbiology, the Graduate School of Agriculture, Hokkaido University, Kita-9 Nishi-9, Kita-ku, Sapporo 060-8589, Japan 2Department of Upland Agriculture, National Agricultural Research Center for Hokkaido Region, Memuro, Hokkaido 082-0071, Japan

Received February 26, 2003; Accepted May 10, 2003
Rhizopus oryzae is an important organism for its production of organic acids such as lactic acid, fumaric acid, etc. To date, there were no easy methods to classify strains according to their acid production. The sequences of the ribosomal RNA-encoding DNA (rDNA) internal transcribed spacer (ITS) region of 64 strains of R. oryzae were analyzed and found to conserve mutations correspond to acid production. We have devised a way to use these mutations for a novel method to identify lactic-acid-producing Rhizopus oryzae, by designing specific polymerase chain reaction (PCR) primers on them. Touch down PCR using these primers amplified the ITS DNA of lactic acid producers specifically. By this method, we could isolate lactic acid producing strains from Indonesian fermented foods.
Key words: Rhizopus oryzae; ribosomal RNA-encoding DNA (rDNA); internal transcribed spacer (ITS); selective polymerase chain reaction (PCR); lactic acid fermentation

-15-
Simple Synthesis of Mite Pheromone β-Acaridial and Its Analogs in the Secretion of Caloglyphus polyphyllae (Acari: Acaridae)

Nobuhiro SHIMIZU, Naoki MORI, and Yasumasa KUWAHARA

Laboratory of Chemical Ecology, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan

Received February 27, 2003; Accepted April 17, 2003
A simple synthesis of β-acaridial [(E)-1], the active principle of the sex, alarm and aggregation pheromone among astigmatid mites, was achieved in 5 steps from 1,2,4-butanetriol 2 in a 19％ overall yield. Its analog, β-acariolal 8, was also prepared in a 63％ yield by oxidation of the intermediate, β-acaridiol [(E)-7], with pyridinium dichromate (PDC). This synthetic route also gave β-(Z)-acaridiol [(Z)-7] by using a Z-selective base in the Wittig reaction. (Z)-7 was oxidized to give a new monoterpene, β-(Z)-acaridial [(Z)-1], which was detected as a trace component in the secretion of Caloglyphus polyphyllae, together with 8.
Key words: Caloglyphus polyphyllae; Astigmata; β-acaridial; β-acariolal; β-(Z)-acaridial

-16-
Functional Roles of the Tissue Inhibitor of Metalloproteinase 3 (TIMP-3) during Ascorbate-induced Differentiation of Osteoblastic MC3T3-E1 Cells

Hironao SUZUKI,1 Yuko NEZAKI,1 Eriko KUNO,1 Ikuko SUGIYAMA,1 Akifumi MIZUTANI,2,† and Norihiro TSUKAGOSHI3

1Department of Biological Mechanisms and Functions, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan 2Department of Physiology and Biophysics, University of Medicine and Dentistry of New Jersey---Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854, USA 3Aichi Study Center, The University of the Air, 101-2 Yagoto-honmachi, Showa, Nagoya 466-0825, Japan

Received March 4, 2003; Accepted April 28, 2003
The tissue inhibitor of the metalloproteinase-3 (TIMP-3) gene was isolated as a gene involved in the process of ascorbate-induced differentiation of mouse MC3T3-E1 cells by the differential display method. The functional roles of TIMP-3 were characterized by establishing stable cell lines, which constitutively expressed the TIMP-3 gene. The TIMP-3 transfectants produced type I collagen at the same level as that of normal cells in response to ascorbic acid 2-phosphate (AscP). However, the expression of the other osteoblastic marker proteins such as alkaline phosphatase (ALPase), osteopontin (OP), osteocalcin (OC), osteonectin (ON) and matrix metalloproteinases (MMPs) remained at a low level even in the presence of AscP. Furthermore, no mineralization of the extracellular matrix (ECM) occurred with the transfectants. Remodeling ECM through TIMPs and MMPs is concluded to be required for osteoblastic differentiation.
Key words: tissue inhibitor of metalloproteinase-3 (TIMP-3); matrix metalloproteinases (MMPs); ascorbate induced osteoblastic differentiation; extracellular matrix (ECM)

-17-
Synthesis of (4R,6S,7R)-7-Hydroxy-4,6-dimethyl-3-nonanone and (3R,5S,6R)- 6-Hydroxy-3,5-dimethyl-2-octanone, the Pheromone Components of the Bostrychid Beetle, Dinoderus bifoveolatus

Yui MASUDA,1 Ken FUJITA,1 and Kenji MORI2,†

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

Received March 7, 2003; Accepted April 23, 2003
(4R,6S,7R)-7-Hydroxy-4,6-dimethyl-3-nonanone and (3R,5S,6R)-6-hydroxy-3,5-dimethyl-2-octanone, the pheromone components of the bostrychid beetle, Dinoderus bifoveolatus, as well as their (4R,6S,7S)- and (3R,5S,6S)-isomers were synthesized from (2R,4S,5R)- and (2R,4S,5S)-2,4-dimethyl-5-heptanolide, respectively.
Key words: bostrychid beetle; nor-serricornin stereoisomer; pheromone; serricornin stereoisomer

-18-
Primary Structure of Inorganic Polyphosphate/ATP-NAD Kinase from Micrococcus flavus, and Occurrence of Substrate Inorganic Polyphosphate for the Enzyme

Shigeyuki KAWAI, Shigetarou MORI, and Kousaku MURATA

Department of Basic and Applied Molecular Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan

Received March 11, 2003; Accepted April 28, 2003
The gene encoding an inorganic polyphosphate/ATP-NAD kinase was cloned from Micrococcus flavus, and its primary structure was analyzed. Alignment of the primary structure with those of other characterized NAD kinases revealed candidate amino acid residues, mainly charged ones, that would be related to inorganic polyphosphate use. The alignment also showed that the primary structure found carried a protruding C-terminal polypeptide. Although the C-terminal polypeptide was demonstrated to be dispensable for the kinase activities, and was proposed to be removed in M. flavus, the entire primary structure including the C-terminal polypeptide was homologous with that of the ATP synthase β chain. The inorganic polyphosphate used by the inorganic polyphosphate/ATP-NAD kinase as a phosphoryl donor was isolated from cells of M. flavus, suggesting that the ability of the enzyme to use inorganic polyphosphate is of physiological significance and is not an evolutionary trait alone.
Key words: inorganic polyphosphate; NAD kinase; Micrococcus flavus; ATP synthase; divergent evolution

-19-
Synthesis of an Unnatural N-Glycan-linked Dolichyl Pyrophosphate Precursor

Yuko NAKAHARA, Tomoharu NONAKA, Hironobu HOJO, and Yoshiaki NAKAHARA

Institute of Glycotechnology, Department of Applied Biochemistry, Tokai University, Kitakaname 1117, Hiratsuka, Kanagawa 259-1292, Japan

Received March 12, 2003; Accepted April 24, 2003
An unnatural α-D-mannopyranose-linked chitobiosyl dolichyl pyrophosphate, a stereoisomer of the N-glycan biosynthesis intermediate, was synthesized. The protected trisaccharide, α-D-Man-(1→4)-β-D-GlcNAc-(1→4)-D-GlcNAc, carrying a 4-methylbenzoyl group was prepared for the convenience of a TLC analysis. 1-O-Phosphorylation, condensation with dolichyl phosphate, and subsequent deprotection afforded the title compound.
Key words: synthesis; glycosylation; N-glycan biosynthesis; unnatural N-glycan; dolichyl pyrophosphate precursor

-20-
RNase ES of Streptomyces coelicolor A3(2) Can Complement the rne and rng Mutations in Escherichia coli

Takabumi INAGAWA,1 Susumu OKAMOTO,1 Masaaki WACHI,2 and Kozo OCHI1,†

1National Food Research Institute, Tsukuba, Ibaraki 305-8642, Japan 2Department of Bioengineering, Tokyo Institute of Technology, Midori-ku, Yokohama 226-8501, Japan

Received March 13, 2003; Accepted May 8, 2003
The Streptomyces coelicolor gene SCC88.10c encodes a protein (RNase ES) which is homologous to endoribonucleases in the RNase E/G family. We expressed S. coelicolor RNase ES as a 6×His-tagged protein in an Escherichia coli mutant carrying a rng (which encodes RNase G) or a rne (which encodes RNase E) mutation to study whether S. coelicolor RNase ES is able to complement these mutations in host E. coli cells. The results clearly indicated that the S. coelicolor RNase ES can partially abrogate either the rng::cat or rne-1 mutation, as measured by the ability to suppress the several aberrant phenotypes resulting from the rng or rne mutation. Thus, S. coelicolor RNase ES appears to have the dual ability to supplant the functions of both RNase G and RNase E in E. coli.
Key words: RNase ES; Streptomyces coelicolor A3(2); Escherichia coli

-21-
Isolation of Suppressor Mutants of Phosphatidylinositol 3-Phosphate 5-kinase Deficient Cells in Schizosaccharomyces pombe

Masayuki ONISHI,1 Yoko NAKAMURA,1 Takako KOGA,1, Kaoru TAKEGAWA,2 and Yasuhisa FUKUI1,†

1Laboratory of Biological Chemistry, Graduate School of Agricultural and Life Science, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan 2Department of Life Sciences, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa 761-0795, Japan

Received March 25, 2003; Accepted April 22, 2003
The ste12＋ gene of Schizosaccharomyces pombe codes for a phosphatidylinositol (PI) 3-phosphate 5'-kinase, which is required for efficient mating. Suppressor mutants for sterility of ste12Δ cells were screened for. Most of the mutant genes turned out to be recessive. Six genes were cloned and the open reading frames responsible for the suppressor activity were identified. They included genes coding for proteins with domains homologous to calcium transporters, casein kinase II, UBC13, AMSH, Vps23p, and Vps27p of Saccharomyces cerevisiae. Disruption of these genes resulted in suppression of the defects of the ste12Δ cells, including low mating efficiency and formation of large vacuoles. Since many of these gene products are homologous to the proteins involved in vesicle transport, sterility caused by inactivation of ste12 may be due to a disordered vesicle transport system.
Key words: Schizosaccharomyces pombe; conjugation; phosphatidylinositol 3-phosphate 5-kinase; vacuolar protein sorting; suppressor mutation

-22-
Identification and Purification of Sulfotransferases for 20-Hydroxysteroid from the Larval Fat Body of a Fleshfly, Sarcophaga peregrina

Eiko MATSUMOTO, Michio MATSUI, and Hiro-omi TAMURA

Kyoritsu College of Pharmacy, Minato-ku, Tokyo 105-8512, Japan

Received April 7, 2003; Accepted May 12, 2003
Sulfotransferase (ST) activity for 20-hydroxyecdysone (20E) was identified in a larval fat body lysate of the fleshfly, Sarcophaga peregrina, but not in the hemolymph. The activity was highly sensitive to 2,6-dichloro-4-nitrophenol (DCNP) (IC50＝0.61 μM), a specific inhibitor of phenol ST (P-ST), but insensitive to triethylamine, a hydroxysteroid ST inhibitor. These results suggest that 20E-specific ST enzymes belong to the P-ST family, despite the fact that 20E is a hydroxysteroid. In addition to 20E ST activity, a relatively high level of 2-naphthol ST activity was detected in the fat body lysate. The ST activity for both substrates transiently decreased to the 50％ of maximal levels, 6 hrs after induction of pupation. The ST enzymes were separated on a DEAE-cellulose column. The 20E-ST enzymes were eluted around 50 mM KCl as two separate peaks of close proximity and the P-ST was eluted at 0.1 M KCl. The 20E ST enzymes were further purified using 3'-phosphoadenosine 5'-phosphate (PAP)-agarose affinity column chromatography. Both of the eluted active fractions demonstrated 43-kDa proteins on SDS-polyacrylamide gel. Photoaffinity labeling with [35S]-3'-phosphoadenosine 5'-phosphosulfate (PAPS) showed 43-kDa bands in the fat body lysate, as well as in the purified fractions. These results suggest that the 43-kDa proteins catalyze 20E sulfation within the fat body of S. peregrina.
Key words: 20-hydroxyecdysone; sulfotransferase; fly; Sarcophaga peregrina

-23-
Heterogeneous Nuclear Ribonucleoprotein K Interacts with and Is Proteolyzed by Calpain in vivo

Eiichi KIMURA,1 Keiko ABE,1 Koichi SUZUKI,2 and Hiroyuki SORIMACHI1,3,†

1Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan 2Tokyo Metropolitan Institute of Gerontology, Tokyo 173-0015, Japan 3CREST, JST, Kawaguchi 332-0012, Japan

Received April 8, 2003; Accepted May 9, 2003
Calpain is a cytosolic <Oh><0192><0192><Wa>modulator protease'' that modulates cellular functions in response to Ca2＋. To identify in vivo substrates of calpain, yeast two-hybrid screening was done using the 5-EF-hand (penta-EF-hand; PEF) domain of the μ-calpain large subunit (domain IV), since several possible in vivo substrates for calpain have been previously reported to bind to the 5-EF-hand domains. Other than the regulatory subunit of calpain, which binds to the domain IV, heterogeneous nuclear ribonucleoproteins (hnRNP) K and R were identified, and shown to be proteolyzed by μ-calpain in vitro. When expressed in COS7 cells, hnRNP K and μ-calpain co-localized in the cytosol, and Ca2＋-ionophore stimulation of the cells resulted in proteolysis of hnRNP K, indicating that hnRNP K is an in vivo substrate for calpain. Now, hnRNP K is considered to function as a scaffold protein for its binding proteins, such as PKCδ and C/EBPβ, which were reported to be calpain substrates, suggesting that hnRNP-K is a scaffold for calpain to proteolyze these proteins.
Key words: calpain; proteolysis; heterogeneous nuclear ribonucleoprotein K; Ca2＋; yeast two-hybrid

-24-
Note
Isolation of Actinomycetes from Termites' Guts

Yoshio WATANABE,1,† Naoya SHINZATO,2 and Takema FUKATSU2

1Bioresource Laboratories, Mercian Corporation, 1808 Nakaizumi, Iwata, Shizuoka 438-0078, Japan 2Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology, Tsukuba Central 6-4A, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan

Received June 17, 2002; Accepted April 29, 2003
Actinomycetes could be isolated efficiently on a defatted wood powder medium from the guts of various species of termites. The actinomycete flora in the termites' guts depended largely on the area in which the termites naturally occur. In termites from the same area, the actinomycete flora changed depending on the taxonomic difference between termite species. Some actinomycetes isolated from termites' guts grew satisfactorily on lignin-related media, and the others grew on cellulose-related media.
Key words: actinomycetes; termite gut; wood powder medium; isolation method

-25-
Note
Flagellum-independent Trail Formation of Escherichia coli on Semi-solid Agar

Hajime FUKUOKA,1,2,† Michio HOMMA,3 and Shigeyuki ICHIHARA1,2

1The Agricultural High-Tech Research Center, Meijo University, Tenpaku-Ku, Nagoya 468-8502, Japan 2Laboratory of Microbiology, School of Agriculture, Meijo University, Tenpaku-Ku, Nagoya 468-8502, Japan 3Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-Ku, Nagoya 464-8602, Japan

Received November 25, 2002; Accepted April 25, 2003
Escherichia coli can form linear trails and move in a flagellum-independent manner on semisolid agar containing carbon sources. Trail formation seemed to correlate with the growth speed and/or carbon metabolism. Cell morphology in linear trails changed into larger cell sizes. We speculate that the flagellum-independent trail formation is a new mechanism for migration of E. coli cells.
Key words: Escherichia coli; cell morphology; flagella; migration; growth

-26-
Note
Effect of γ-Aminobutyric Acid-enriched Tempeh-like Fermented Soybean (GABA-Tempeh) on the Blood Pressure of Spontaneously Hypertensive Rats

Hideyuki AOKI,1,† Yuji FURUYA,1 Yasushi ENDO,2 and Kenshiro FUJIMOTO2

1Research Laboratory, Ikeda Tohka Industries Co., Ltd., 95-7 Minooki-cho, Fukuyama, Hiroshima 721-0956, Japan 2Department of Science of Biological Function, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Sendai 981-8555, Japan

Received January 6, 2003; Accepted April 17, 2003
GABA-enriched tempeh-like fermented soybean (GABA-tempeh) was supplemented to the AIN-76 diet and fed for 2 months to spontaneously hypertensive rats (SHRs), an animal model of spontaneously developed hypertension, to compare the antihypertensive activity with that of authentic GABA. The elevation of systolic blood pressure in SHRs was significantly retarded in the GABA-tempeh group as well as that with authentic GABA when compared with the controls, and the effect lasted for two months of the feeding period. The blood urea nitrogen level tended to be higher in the control group than in the GABA-supplemented groups. On the other hand, no effect was apparent on the plasma levels of cholesterol, triacylglycerol and glucose, or on the urinary excretion of Na and K.
Key words: γ-aminobutyric acid; fermented soybean; GABA; SHR; tempeh

-27-
Note
Organic Solvent Tolerance of Halophilic Archaea

Ron USAMI, Tadamasa FUKUSHIMA, Toru MIZUKI, Akira INOUE, Yasuhiko YOSHIDA, and Koki HORIKOSHI

Department of Applied Chemistry, Faculty of Engineering, Toyo University, 2100 Kujirai, Kawagoe-shi, Saitama 350-8585, Japan

Received January 9, 2003; Accepted April 29, 2003
Organic solvent tolerance was tested in type strains of type species of the sixteen genera of Halobacteriaceae, the halophilic archaea. Most of the strains were observed to grow in the presence of hexylether (log Pow＝5.1), but none grew in the presence of n-octane (log Pow＝4.9) except Halogeometricum borinquense JCM 10706T and Halorubrum saccharovorum JCM 8865T. On the other hand, two strains, Haloarcula spp. OHF-1 and 2 isolated from a French solar salt were found to show stronger tolerance even to isooctane (log Pow＝4.8). Growth of some strains was retarded by the presence of n-decane but reached to the same cell densities at late stationary phase. Final cell densities of some strains were greatly repressed by the presence of the solvent.
Key words: halophilic archaea; strains OHF-1 and OHF-2, and log Pow value

-28-
Note
Neuroprotective Activity of p-Terphenyl Leucomentins from the Mushroom Paxillus panuoides

In-Kyoung LEE,1 Bong-Sik YUN,1 Jong-Pyung KIM,1 In-Ja RYOO,1 Young-Ho KIM,2 and Ick-Dong YOO1,†

1Korea Research Institute of Bioscience and Biotechnology, Yuseong, Daejeon 305-600, Korea 2College of Pharmacy, Chungnam National University, Yuseong, Daejeon 305-764, Korea

Received January 30, 2003; Accepted May 6, 2003
The neuroprotective mechanism of p-terphenyl leucomentins from the mushroom Paxillus panuoides was studied. Leucomentins showed potent inhibition of lipid peroxidation and H2O2 neurotoxicity, but free from any role as reactive oxygen species (ROS) scavengers. Iron-mediated oxidative damage has been implicated in these processes, as a provider of ROS via iron. Leucomentins can chelate iron when DNA is present with iron and H2O2, and so inhibiting DNA single strand breakage. These results suggest that the neuroprotective action of leucomentins is dependent on their ability to chelate iron.
Key words: mushroom; Paxillus panuoides; leucomentins; glutamate neurotoxicity; iron chelator

-29-
Note
Two Distinct Curved DNAs Upstream of the Light-responsive psbA Gene in a Cyanobacterium

Ganesh Kumar AGRAWAL, Munehiko ASAYAMA, and Makoto SHIRAI

Laboratory of Molecular Genetics, School of Agriculture, Ibaraki University, Ami, Inashiki, Ibaraki 300-0393, Japan

Received February 3, 2003; Accepted April 26, 2003
A functional intrinsic DNA curvature, CIT, and potential DNA-binding factors for the basal transcription of psbA2 have been reported in a cyanobacterium, Microcystis aeruginosa K-81 (Asayama et al., Nucleic Acids Res., 30, 4658--4666 (2002)). In this article, we found another novel curved DNA, which was induced by RNA polymerases binding to the promoter region. Circular permutation analyses showed that the curved center of RNA polymerase-induced DNA bending (RIB) lies at approximately the ＋10 site, referring to the transcription start point as ＋1, in the RNA polymerase-DNA complex. Regions containing the curved center of RIB and CIT contributed to the basal transcription in vivo and in vitro. These results indicate that the region upstream of K-81 psbA2 has two distinct curved DNAs, CIT (sequence-directed type) and RIB (protein-induced type).
Key words: transcription; RNA polymerase; sigma factor; intrinsic curvature; protein-induced DNA bending

-30-
Note
Stimulated Accumulation of Lectin mRNA and Stress Response in Helianthus tuberosus Callus by Methyl Jasmonate

Ryoji NAKAGAWA, Yukihiro OKUMURA, Makoto KAWAKAMI, Daisuke YASOKAWA, and Koji NAGASHIMA

Hokkaido Food Processing Research Center, 589-4 Bunkyodai Midori-Machi, Ebetsu 069-0836, Japan

Received February 6, 2003; Accepted May 8, 2003
Callus from Helianthus tuberosus expresses a mannose-specific lectin (HTA). The level of HTA mRNA significantly increased one hour after treatment of the callus with 20 mg/l methyl jasmonate. Following this, fragmentation of the callus DNA at regular intervals was observed together with strong self-fluorescence emission in the callus cells.
Key words: Helianthus tuberosus; jasmonate; lectin; DNA fragmentation; self-fluorescence

-31-
Note
Comparative Analyses of Hairpin Substrate Recognition by Escherichia coli and Bacillus subtilis Ribonuclease P Ribozymes

Tomoaki ANDO, Terumichi TANAKA, and Yo KIKUCHI

Division of Bioscience and Biotechnology, Department of Ecological Engineering, Toyohashi University of Technology, Tempaku-cho, Toyohashi, Aichi 441-8580, Japan

Received February 12, 2003; Accepted May 12, 2003
Previously, we reported that the substrate shape recognition of the Escherichia coli ribonuclease (RNase) P ribozyme depends on the concentration of magnesium ion in vitro. We additionally examined the Bacillus subtilis RNase P ribozyme and found that the B. subtilis enzyme also required high magnesium ion, above 10 mM, for cleavage of a hairpin substrate. The results of kinetic studies showed that the metal ion concentration affected both the catalysis and the affinity of the ribozymes toward a hairpin RNA substrate.
Key words: transfer RNA; specificity; ribonuclease P; Bacillus; ribozyme

-32-
Note
Comparison of Acylated Plant Pigments: Light-resistance and Radical-scavenging Ability

Nobuyoshi NAKAJIMA,1,† Manabu SUGIMOTO,2 Hiroshi YOKOI,3 Hideaki TSUJI,1 and Kohji ISHIHARA4,

1Department of Nutritional Science, Okayama Prefectural University, Soja, Okayama 719-1197, Japan 2Research Institute for Bioresources, Okayama University, Kurashiki, Okayama 710-0046, Japan 3Department of Applied Science, Okayama University of Science, Ridai-cho, Okayama 700-0005, Japan 4Department of Chemistry, Kyoto University of Education, Fushimi-ku, Kyoto 612-8522, Japan

Received February 19, 2003; Accepted April 22, 2003
The acylated plant pigments synthesized by lipase-catalyzed transesterification with aromatic acids were compared in respect of their light-resistance and radical-scavenging ability. With both the flavonols and anthocyanins, their acylated derivatives were more stable against illumination with fluorescent light than their non-acylated glucosides. Their radical-scavenging ability partially decreased or was retained by acylation to the glucoside molecules.
Key words: plant pigment; flavonoid; anthocyanin; light-resistance; radical-scavenging ability

-33-
Note
Peptide Mapping and Assessment of Cryoprotective Activity of 26/27-kDa Dehydrin from Soybean Seeds

Michiko MOMMA, Shigenobu KANEKO, Kazutomo HARAGUCHI, and Ushio MATSUKURA

National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan

Received February 24, 2003; Accepted May 7, 2003
To characterize the molecular weight diversity of seed dehydrin among soybean cultivars, 26/27-kDa soybean dehydrins were purified and compared in peptide mapping patterns, partial amino acid sequences, and cryoprotective activity on enzyme. In reverse phase chromatograms of their trypsin digests, we detected several distinctive peaks, one of which was attributed to a part of the internal glycine-rich region. Partial amino acid sequences of peptide fragments from trypsin and S. aureus V8 protease cleavage were found to be identical to the Mat9 translation. The CP50 of purified 26/27-kDa dehydrins were estimated to be 0.30 and 0.11 μM, respectively.
Key words: dehydrin; group2 LEA protein; soybean seed protein; cryoprotective activity

-34-
Note
Expression of CD38 in Human Promyelocytic Leukemia HL-60 Cell Line during Differentiation by Niacin-related Compounds

Keiko IWATA, Shin OGATA, Katsuzumi OKUMURA, and Hiroshi TAGUCHI

Laboratory of Molecular and Cellular Biology, Department of Life Science, Faculty of Bioresources, Mie University, Tsu, Mie 514-8507, Japan

Received March 14, 2003; Accepted May 1, 2003
It was found that three niacin-related compounds, isonicotinic acid, nicotinamide, and nicotinamide N-oxide, induced granulocytic differentiation in HL-60 cells. We investigated the expression of CD38, which catalyzes the synthesis of cyclic ADP-ribose, a Ca2＋ mobilizer, during differentiation by niacin-related compounds. It was found that CD38 was induced by isonicotinic acid, whereas nicotinamide and nicotinamide N-oxide containing an amino group did not induce it. The difference in expression of CD38 may provide some useful information for the elucidation of the mechanisms of cell differentiation.
Key words: CD38; niacin; isonicotinic acid; cell differentiation; HL-60

-35-
Note
Interaction between Acidic Polysaccharides and Proteins

Kenjiro TADERA, Yuji MINAMI, and Miki CHOHCHI

Department of Biochemical Science and Technology, Faculty of Agriculture, Kagoshima University, Korimoto 1-21-24, Kagoshima 890-0065, Japan

Received March 24, 2003; Accepted May 13, 2003
There was an ionic interaction between acidic polysaccharides (APS) and proteins at the pH range in which APS were negatively charged and proteins were positively charged, and in enzymes the interaction was detected as a change in the enzyme activity. At pH 4.7, acid phosphatase (pI, 5.4), α-glucosidase (pI, 5.7), and β-glucosidase (pI, 7.3) were inhibited by APS to various extents. On the other hand, α-glucosidase and alkaline phosphatase (pI, 4.5) were not inhibited by APS at pH 6.8 and 9.8, respectively, most of these two enzymes being negatively charged at the respective pHs. Sulfated polysaccharides combined with hemoglobin (pI, 6.8<10-48>7.0) by an ionic bond at pH 2 to make hemoglobin unsusceptible to proteolysis by pepsin, but polyuronides which were not charged at this pH did not affect hydrolysis of hemoglobin.
Key words: interaction; acidic polysaccharide; protein; inhibition; vitamin B6

-36-
Note
Paradoxical Effect of Synthetic Hydroxy Isothiocyanates on Antimicrobial Action of Aminoglycosides

Hirokuni TAJIMA,1,† Hisashi KIMOTO,2 and Akira TAKETO3

1Fukui Research Laboratory, Rengo Co., Ltd., 10--8--1 Jiyugaoka, Kanazu-cho, Sakai-gun, Fukui 919--0604, Japan 2Department of Biochemistry I, Fukui Medical School, Matsuoka, Fukui 910--1103, Japan 3Department of Applied Physics and Chemistry, Fukui University of Technology, Fukui 910--8505, Japan

Received April 11, 2003; Accepted May 12, 2003
Hydroxy isothiocyanates, especially 2-(4-hydroxyphenyl)ethyl isothiocyanate (hITC), were examined for antimicrobial synergism with streptomycin (SM) against Escherichia coli. On the course of those experiments, a peculiar suppression of SM by a low concentration of hITC was observed, besides the antibacterial synergism of hITC with SM. Further, bactericidal activity of SM in physiological saline was reduced by addition of hITC. Time course experiments proved that the antagonistic effect of hITC occurred in an early stage after exposure of bacterial cells to SM.
Key words: hydroxy isothiocyanate; 2-(4-hydroxyphenyl)ethyl isothiocyanate; antagonism; synergism; streptomycin

-37-
Communication
Absorptive Activity of Calcium in the Isolated Cecal Epithelium Adaptively Increased by 2 Week's Feeding of Difructose Anhydride III in Rats

Hitoshi MINEO,1,4 Midori AMANO,1,2 Hideyuki CHIJI,2 Norihiro SHIGEMATSU,3 Fusao TOMITA,4 and Hiroshi HARA4,†

1Northern Advancement Center for Science and Technology, Colabo-Hokkaido, Sapporo 001-0021, Japan 2Department of Food Science and Human Nutrition, Faculty of Human Life Science, Fuji Women's University, Ishikari 061-3204, Japan 3Central Research Center, Fancl Corporation, Yokohama 244-0806, Japan 4Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan

Received February 5, 2003; Accepted June 27, 2003
We compared net Ca absorption and Lucifer Yellow (LY), a paracellular passage dye, permeability in the epithelium isolated from the rat small intestine, cecum, and colon after feeding with control and difructose anhydride (DFA) III diets for 14 days using the Ussing chamber system. Feeding of DFA III increased net Ca transport and LY passage in the cecal but not in small intestinal or colonic epithelium. Ability of paracellular Ca passage via Tight-junction (TJ) in the cecum was changed adaptively by feeding of DFA III. Changes in microbial fermentation may affect the functional changes of Ca transport in cecal epithelium itself.
Key words: Ca absorption; indigestible saccharide; Ussing chamber; intestinal epithelium; Tight junction

-38-
Communication
First Evidence for Occurrence of Galβ1-3GlcNAcβ1-4Man Unit in N-Glycans of Insect Glycoprotein: β1-3Gal and β1-4GlcNAc Transferases Are Involved in N-Glycan Processing of Royal Jelly Glycoproteins

Yoshinobu KIMURA,1,† Kazunori TSUMURA,1 Mariko KIMURA,2 Kiyoshi OKIHARA,3 Hiroyuki SUGIMOTO,3 and Hideo YAMADA3

1Department of Bioresources Chemistry, Faculty of Agriculture, Okayama University, Tsushima-Naka 1-1-1, Okayama 700-8530, Japan 2Faculty of Food Culture, Department of Food System, Kurashiki Sakuyo University, Nagao-Tamashima, Kurashiki 710-0292, Japan 3Yamada Apiculture Center Inc., Ichiba 194, Kagamino-Cho, Tomada-Gun, Okayama 708-0393, Japan

Received May 26, 2003; Accepted June 30, 2003
On a way of structural analysis of total N-glycans linked to glycoproteins in royal jelly (Kimura, Y. et al., Biosci. Biotechnol. Biochem., 64, 2109--2120 (2000), Kimura, M. et al., Biosci. Biotechnol. Biochem., 66, 1985--1989 (2002)), we found that some complex type N-glycans containing a β1-3galactose residue occur on the insect glycoproteins. Up to date, it has been considered that naturally occurring insect glycoproteins do not bear the galactose-containing N-glycans, therefore, in this report we describe the structural analysis of the complex type N-glycans of royal jelly glycoproteins. By a combination of endo- and exo-glycosidase digestions, IS-MS analysis, and ＾{1}H-NMR spectroscopy, the structures of the β1-3 galactose-containing N-glycan were identified as the following; GlcNAcβ1-2Manα1-6[GlcNAcβ1-2(Galβ1-3GlcNAcβ1-4)Manα1-3]Manβ1-4GlcNAcβ1-4GlcNAc, Manα1-3Manα1-6[GlcNAcβ1-2(Galβ1-3GlcNAcβ1-4)Manα1-3]Manβ1-4GlcNAcβ1-4GlcNAc, and Manα1-6(Manα1-3)Manα1-6[GlcNAcβ1-2(Galβ1-3GlcNAcβ1-4)Manα1-3]Manβ1-4GlcNAcβ1-4GlcNAc. To our knowledge, this is the first report showing that the Galβ1-3GlcNAcβ1-4Man unit occurs in N-glycans of insect glycoproteins, indicating a β1-3 galactosyl transferase and β1-4GlcNAc transferase (GNT-IV) are expressed in the honeybee cells.
Key words: N-glycan; insect glycoprotein; β1-3galactosyl transferase; royal
jelly; Apis mellifera