Contents and Abstracts of Latest Issue of BBB

(Vol.64 No.6 2000)

Masao SATO,＋ Susumu YOSHIDA, Koji NAGAO, and Katsumi IMAIZUMI

Laboratory of Nutrition Chemistry, Division of Bioresource and Bioenvironmental Sciences,
Graduate School of Kyushu University, Fukuoka 812-8581, Japan

Received August 18, 1999; Accepted January 28, 2000
The exogenously hypercholesterolemic (ExHC) rat is a strain segregated from SD rats with a high response to dietary cholesterol. To understand the underlying mechanism(s) for this hypercholesterolemia, the interactive effects of dietary fatty acid and the susceptibility of rats to dietary cholesterol on the serum cholesterol concentration and hepatic mRNA abundance of the low-density lipoprotein (LDL) receptor, cholesterol 7α-hydroxylase (7α-hydroxylase) and 3-hydroxyl-3methyl-glutaryl (HMG) CoA reductase were examined. Both strains were fed on a diet supplemented with 10% each of olive, safflower or coconut oil with or without the addition of 1% cholesterol for one week. The ExHC rats fed on olive, safflower and coconut oil in combination with cholesterol respectively resulted in a 3.5-, 2.0- and 2.1-fold higher serum cholesterol concentration than that in the animals fed on the corresponding dietary fats without any supplementation of cholesterol (p<0.01 by dietary cholesterol or type of fat). The dietary cholesterol dependent-elevation of serum cholesterol in the SD rats was less than 1.5-fold (p<0.01) and there was no dietary fat effect. The ExHC rats fed on the safflower oil-containing diet supplemented with cholesterol resulted in a higher mRNA abundance of the LDL receptor and 7α-hydroxylase than in the corresponding fat-fed rats without cholesterol (p<0.05). There was no dietary cholesterol-dependent change of mRNA abundance in either strain fed on olive or coconut oil, except for a decreased abundance of HMG CoA reductase mRNA in the olive oil-fed ExHC rats and coconut oil-fed Sprague-Dawley (SD) rats (p<0.05). These results indicate that the hepatic mRNA abundance of the LDL receptor and of 7α-hydroxylase depended on the dietary combination of cholesterol and a fatty acid and suggest that a linoleic acid-rich diet may alleviate exogenous hypercholesterolemia by activating the process involved in the hepatic uptake and biliary excretion of serum cholesterol.
Key words: exogenous hypercholesterolemia; cholesterol 7α-hydroxylase mRNA; low density lipoprotein receptor mRNA; olive oil; safflower oil

Yasufumi KATAGIRI,1 Ragai K. IBRAHIM,2 and Satoshi TAHARA1,3,＋

1Department of Applied Bioscience, Faculty of Agriculture, Hokkaido University,
Kita-Ku, Sapporo 060-8589, Japan
2Plant Biochemistry Laboratory, Biology Department, Concordia University,
1455 De Maisonneuve Blvd. West, Montreal, Quebec, Canada H3G 1M8
3CREST, Japan Science and Technology Corporation, 4-1-8 Honmachi,
Kawaguchi 332-0012, Saitama, Japan

Received October 8, 1999; Accepted February 4, 2000
An investigation of the HPLC analytical conditions for simple isoflavones, prenylated isoflavones and some of their glucosyl derivatives resulted in reasonable separation and total elution in 35 min when using a reversed-phase C18 Lichrospher column and a gradient elution system of MeCN-THF-H2O. This method was successfully applied to quantify the changes in isoflavonoid constituents in white lupin (Lupinus albus L.) tissues: (a) young legumes (pods and seeds) during maturation, and (b) soaked, germinating seeds. In developing legumes, genistein and 2?-hydroxygenistein, as well as their prenylated derivatives, were present in the pods as the major components, together with minor amounts of glucosides, whereas only minute amounts of isoflavonoids were detectable in the ripening seeds. When soaked with water, mature lupin seeds which normally contain trace amounts of isoflavonoids, started rapidly to biosynthesize simple isoflavones and accumulate large amounts of genistein 7-O-glucoside and its 6!-O-malonyl derivative. These dynamic changes are discussed in relation to the role of isoflavonoids in the lupin defense system.
Key words: Lupinus albusLeguminosae; isoflavonoid; HPLC analysis; prenylisoflavones

Yu-Chi CHEN and Shih-Tong JENG＋

Department of Botany, National Taiwan University, Taipei 106, Taiwan, Republic of China

Received October 12, 1999; Accepted January 24, 2000
A promoter competition assay was used to measure the stability of T7 RNA polymerase with its promoter. When T7 RNA polymerase was incubated with GTP for 5 minutes before the elongation of transcription in either the supercoiled or linearized template, the half-lives of T7 RNA polymerase-DNA complexes were reduced. The transcription product increased when T7 RNA polymerase preincubated with GTP in the supercoiled DNA but not in the linearized DNA template. On the other hand, preincubation of ATP with T7 RNA polymerase decreased the stability of T7 RNA polymerase with the supercoiled DNA, but did not affect the stability of T7 RNA polymerase with the linearized DNA. Furthermore, the production of RNA transcript was increased when T7 RNA polymerase was incubated with ATP in either supercoiled or linearized template before transcription elongation. This study is important to understand the relationship between the transcription initiation and the stability of the ternary complex, and to produce large quantities of RNA transcript in vitro.
Key words: T7 RNA polymerase; binding affinity; DNA topology

Akinori OGAWA, Kazuhisa SAWADA, Kazuhiro SAITO, Yoshihiro HAKAMADA,
Nobuyuki SUMITOMO, Yuji HATADA, Tohru KOBAYASHI,** and Susumu ITO

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

Received October 14, 1999; Accepted January 26, 2000
A pectate lyase (Pel; pectate transeliminase: EC4.2.2.2.), designated Pel-15H, was found in an alkaline culture of Bacillus sp. strain KSM-P15 and purified to homogeneity by sequential column chromatographies. The molecular weight of the enzyme determined by SDS-polyacrylamide gel electrophoresis was approximately 70,000 and the pI was around pH 4.6. Pel-15H randomly trans-eliminated polygalacturonate in the presence of Ca＾{2+} ions, and the maximum activity was observed at pH 11.5 and at 55°C in glycine-NaOH buffer. The gene for Pel-15H was cloned and sequenced, and the structural gene contained a 2,031-bp open reading frame that encoded 677 amino acids including a possible 28-amino-acid signal sequence. The mature enzyme (649 amino acids, molecular weight 69,550) showed very low similarity to Pels from Bacillus with 12.7--18.2% identity. Interestingly, part of the amino acid sequence of Pel-15H had fairly high similarity only to an N-terminal half of PelL and a C-terminal half of PelX from Erwinia chrysanthemi 3937, and a C-terminal half of PelX from E. chrysanthemi EC16 (approximately 35% identity for all).
Key words: pectate lyase; trans-eliminase; cloning; alkaliphile; Bacillus

Shin OGATA,1 Masayo TAKEUCHI,1 Hiroaki FUJITA,1 Katsumi SHIBATA,2
Katsuzumi OKUMURA,1 and Hiroshi TAGUCHI1,*

1Laboratory of Molecular and Cellular Biology, Department of Life Science, Faculty
of Bioresources, Mie University, Tsu, Mie 514-8507, Japan
2Course of Food Science and Nutrition, Department of Life Style Studies, School of Human Cultures,
The University of Shiga Prefecture, Hikone, Shiga 522-8533, Japan

Received October 18, 1999; Accepted January 20, 2000
In our previous study, we found that niacin-related compounds induced apoptosis in human acute myelomonocytic leukemia cells, HL-60. We have investigated whether these compounds acted as inducers of apoptosis also in various other cell types. In human chronic myelogenous leukemia cells, K562, which are relatively resistant to various inducers of apoptosis, the apoptosis was induced by picolinic acid and dipicolinic acid in about 50% of the cells 5--10 mM via the caspase pathway, but was not at 1 mM. However, isonicotinamide did not induce apoptosis effectively in K562 cells. On the other hand, in normal human quiescent lymphocytes, the apoptosis was not induced by these compounds at the same concentrations. It is suggested that these compounds may induce apoptosis mainly in tumor cells. The change of intracellular peroxide levels was observed in the early phase of apoptosis induced by niacin-related compounds. We expect to make use of niacin-related compounds in the field of medicine.
Key words: apoptosis; niacin; K562 cells; normal human lymphocytes; antitumor drug

Hidefumi YOSHII,＋ Takeshi FURUTA, Jun KUDO, and Pekka LINKOa

Department of Biotechnology, Tottori University, Tottori 680-0945, Japan
aDepartment of Chemical Technology, Helsinki University of Technology,
FIN-02015 HUT (Espoo), Finland

Received October 22, 1999; Accepted January 12, 2000
Anhydrous sugars such as maltose and trehalose are useful for making dry powder of foods and liquids. The crystal-transformation rate of maltose and trehalose were investigated under humid conditions and by kneading. The enthalpy for solubilization was 7.0 kJ/mol for the anhydrous maltose. The crystal-transformation rate of anhydrous α-maltose to hydrous β-maltose depended on the temperature at 75% humidity. However, that of anhydrous trehalose did not depend on the temperature, and transformation was very rapid. An anomeric change to maltose and no such change to trehalose might have caused this. The activation energy of crystal transformation was 79 kJ/mol for maltose and zero for trehalose. The rate of crystal transformation of anhydrous maltose while kneading depended on the purity of the anhydrous α-maltose and the amount of water present. This crystal transformation rate fitted the Avrami equation.
Key words: maltose; trehalose; crystal transformation; kneading

Shoko NISHIZONO, Tadao HAYAMI, Ikuo IKEDA, and Katsumi IMAIZUMI*

Laboratory of Nutrition Chemistry, Division of Bioresource and Bioenvironmental Sciences,
Graduate School, Kyushu University, Fukuoka 812-8581, Japan

Received November 1, 1999, Accepted January 11, 2000
We determined whether an oral administration of the synthetic antioxidant, tert-butylhydroquinone (TBHQ), or the naturally occurring lipoxygenase inhibitor, curcumin, to rats would provide protection against the diabetogenic effect of streptozotocin (STZ). Male Sprague-Dawley rats were fed on an AIN-76TM-based purified diet containing 0.0028% TBHQ or on the purified diet with a daily intragastric administration of curcumin (200 mg/kg of body weight) for one week while receiving intravenously administered STZ. The rats fed on the TBHQ-containing diet were resistant to diabetes development when compared with the rats fed on the TBHQ-free diet and had a higher body weight gain and lower serum glucose concentration. Glucose-stimulated insulin secretion from the pancreatic islet in the rats that had received TBHQ was higher than that in the control rats. The rats receiving curcumin showed no beneficial effect on these diabetic symptoms. These findings provide direct evidence for the suggestion that dietary supplementation of an antioxidant may exert a preventive effect on the diabetogenic action of free-radical producers.
Key words: diabetes; tert-butylhydroquinone; curcumin; pancreatic islet; insulin release

Hidefumi YOSHII,* Takeshi FURUTA, Takahiro YONEHARA, Daisuke ITO,
Yu-Yen LINKO,** and Pekka LINKO**

Department of Biotechnology, Tottori University, Tottori 680-8552, Japan
**Department of Chemical Technology, Helsinki University of Technology. FIN-02015,
P.O.Box 6100 HUT, (Espoo), Finland

Received November 4, 1999; Accepted January 20, 2000
Refolding of reduced and denatured protein in vitro has been an important issue for both basic research and applied biotechnology. Refolding at low protein concentration requires large volumes of refolding buffer. Among various refolding methods, diafiltration is very useful to control the denaturant and red/ox reagents in a refolding solution. We constructed a refolding procedure of high lysozyme concentration (0.5--10 mg/ml) based on the linear reduction of the urea concentration during diafiltration under oxygen pressure. When the urea concentration in the refolding vessel was decreased from 4 M with a rate of 0.167 M/h, the refolding yields were 85% and 63% at protein concentrations, 5 mg/ml and 10 mg/ml, respectively, after 11 h. This method gave a high productivity of 40.1 μM/h of the refolding lysozyme. The change in refolding yields during the diafiltration could be simulated using the model of Hevehan and Clark.
Key words: diafiltration; protein refolding; lysozyme

Koji ISHIZUKA, Atsuhiro KANAYAMA, Hideo SATSU, Yusei MIYAMOTO,
Kazuo FURIHATA, and Makoto SHIMIZU＋

Department of Applied Biological Chemsitry, The University of Tokyo, Yayoi, Bunkyo-ku,
Tokyo 113-8657, Japan

Received November 9, 1999; Accepted February 14, 2000
An ethanol extract from sesame seeds inhibited the taurine uptake in human intestinal epithelial Caco-2 cells. The uptake of such α-amino acids as leucine and glutamic acid was not inhibited by the extract, indicating that this inhibition is specific to the taurine uptake. The unknown inhibitor in the sesame extract was purified by reversed-phase HPLC by monitoring the inhibitory effect on taurine uptake. The isolated substance was identified as lysophosphatidylcholine, linoleoyl (Lyso-PC), by NMR and MS analysis. Lyso-PC inhibited the taurine uptake in a dose-dependent manner with an IC50 value of approximately 200 μM. Although Lyso-PC is known to be a surface active and cell lytic compound, neither damage nor loss of integrity of the Caco-2 cell monolayer was apparent after treating with 200 μM Lyso-PC. Inhibition was observed by incubating cells with Lyso-PC for only 1 min prior to the uptake experiments. These results suggest the direct effect of Lyso-PC on the cell membrane to be the main mechanism for this inhibition. Lyso-PC may play a role in the regulation of certain intestinal transporters.
Key words: taurine; transporter; Caco-2; lysophosphatidylcholine

Vibhor SARASWAT＋ and Virendra S. BISARIA

Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology,
Delhi, Hauz Khas, New Delhi-110 016, India

Received November 18, 1999; Accepted January 25, 2000
The ascomycetous fungus Melanocarpus albomyces when grown on wheat straw produced seven extracellular xylanase isoenzymes, designated as Ia, Ib, Ic, IIa, IIb, IIc, and IId. All seven xylanases were purified to homogeneity by gel filtration and ion-exchange chromatography. The molecular mass (kDa) of Ia, Ib, Ic, IIa, IIb, IIc, and IId were estimated to be 22.9, 20.7, 18.6, 31.3, 25.4, 38.5, and 34.3, respectively by SDS-PAGE, and 23.7, 20.5, 17.1, 31.7, 25.1, 39.8, and 32.2, respectively by gel filtration. The isoelectric points of Ia, Ib, Ic, IIa and IIb were found to be 6.2, 6.3, 6.4, 3.7, and 4.4, respectively. The activity of the isoenzymes was dependent on the type of the xylan substrates; Xylanases Ia, Ib, and Ic showed highest specific activity toward larchwood xylan (an arabinoglucuronoxylan), IIa and IIc toward birchwood xylan (a glucuronoxylan), and IIb and IId toward beechwood xylan (a glucuronoxylan). Four isoenzymes Ia, Ib, Ic, and IIa had an arabinose-releasing property on larchwood xylan. Application of specific isoenzymes as prebleaching agents in paper manufacture is proposed.
Key words: xylanase; purification; characterization; isoenzyme; substrate specificity

Takaaki YANAI and Michikatsu SATO＋

Mercian Corporation, Wine & Spirits Research Institute, 9-1 Johnan 4-chome, Fujisawa 251-0057, Japan

Received November 30, 1999; Accepted February 3, 2000
An intracellular α-L-arabinofuranosidase from Pichia capsulata X91 was purified and characterized. The enzyme was purified to homogeneity from a cell-free extract by ammonium sulfate treatment, Concanavalin A-Sepharose, ion-exchange chromatography with DEAE Bio-Gel A agarose, arabinose-Sepharose 6B affinity chromatography, and hydroxyapatite column chromatography. The apparent molecular mass of the enzyme was estimated to be 250 kDa by native-PAGE. The enzyme molecule was suggested to be a tetramer with a subunit molecular mass of 72 kDa by SDS-PAGE. The enzyme had an isoelectric point at 5.1, and was most active at pH 6.0 and at around 50°C. The α-L-arabinofuranosidase was active at ethanol concentrations of wine. The enzyme was inhibited by Cu2＋, Hg2＋, and p-chloromercuribenzoate. The enzyme hydrolyzed beet arabinan and arabinogalactan, and efficiently released monoterpenols from an aroma precursor extracted from Muscat grape juice. A considerable amount of monoterpenols was produced in the Muscat wine coupled with the enzyme addition.
Key words: α-L-arabinofuranosidase; Pichia capsulataaroma enhancement; monoterpenol

Kenji MATSUI,＋ Chinatsu MIYAHARA, Jack WILKINSON,* Bill HIATT,* Vic KNAUF,*
and Tadahiko KAJIWARA

Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University,
Yamaguchi 753-8515, Japan
*Calgene, LLC, 1920 Fifth Street, Davis, CA 95616, USA

Received December 2, 1999; Accepted January 30, 2000
Fatty acid hydroperoxide lyase (HPL) is a member of a novel subfamily of cytochrome P450 and catalyzes a cleavage reaction of fatty acid hydroperoxides to form short-chain aldehydes and oxo-acids. A cDNA encoding tomato fruit HPL (LeHPL) was obtained. An active LeHPL was expressed in E. coli and purified. It showed highest activity against the 13-hydroperoxide of linolenic acid, followed by that of linoleic acid. 9-Hydroperoxides were poor substrates. The absorption spectrum of the purified LeHPL in the native form was similar to that of most P450s although a CO-adduct having a λmax at 450 nm could not be obtained. LeHPL activity is reversibly inhibited by nordihydroguaiaretic acid, while salicylic acid irreversibly inhibited it. LeHPL is kinetically inactivated by fatty acid hydroperoxides, especially 9-hydroperoxides. The inactivation is prevented by inhibitors of LeHPL. Thus, HPL catalytic activity is thought to be essential to its inactivation. During the inactivation, an abolition of the Soret band was evident, indicating that inactivation is caused mainly by degradation of the prosthetic heme in LeHPL.
Key words: fatty acid hydroperoxide lyase; cytochrome P450; tomato (Lycopersicon esculentum)

Kohji YAMAMOTO, Masatoshi YAKIYAMA,* Hiroshi FUJII,* Takahiro KUSAKABE,**
Katsumi KOGA,** Yoichi ASO, and Masatsune ISHIGURO＋

Laboratory of Protein Chemistry and Engineering, *Laboratory of Insect Genetic Resources
and **Laboratory of Sericultural Science, Kyushu University Graduate School of Bioresource
and Bioenvironmental Sciences, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan

Received December 6, 1999; Accepted February 17, 2000
Two clones encoding different prophenoloxidase isoforms were amplified by polymerase chain reaction of RNA from the hemocytes of an experimental strain of Bombyx mori. The nucleotide sequences of the clones and the deduced amino acid sequences were confirmed to be nearly identical to those of the orthologous clones previously obtained from a commercial race of B. mori. Northern blot hybridization using these clones as probes demonstrated that the prophenoloxidase mRNA in the hemocytes is expressed in a stage-specific manner during the final larval instar and pupal stage, showing a peak one day before pupation in males and on the day of pupation in females. A sexual difference was also observed when the content of prophenoloxidase protein in the hemolymph (including hemocytes) was measured by an enzyme-linked immunosorbent assay.
Key words: Bombyx morihemocytes; prophenoloxidase mRNA

Akinori KATO, Hiroe OHNISHI, Kaneyoshi YAMAMOTO, Eiji FURUTA,
Hiroyuki TANABE, and Ryutaro UTSUMI＋

Department of Agricultural Chemistry, Kinki University, 3327-204 Nakamachi, Nara 631-8505, Japan

Received December 6, 1999; Accepted February 16, 2000
Spontaneous mutations have been isolated in Escherichia coli that result in the constitutive expression of an emrKY promoter. These mutations were found to be single-nucleotide substitutions within the linker region of the sensor protein EvgS, which is part of a two-component regulatory system along with EvgA. In the linker mutants (evgS1 and evgS4), emrKY expression became constitutive and MIC against sodium deoxycholate was 20 mg/ml, eight-fold higher than in the wild type. Furthermore, the start site of transcription from the promoter of emrKY was identified; EvgA was shown to bind at the --52 to --84 region by the footprinting experiment.
Key words: drug efflux; two-component system; EvgA-EvgS; EmrK-EmrY

Sam-In KIM, Takahisa MIYAMOTO＋, Ken-ichi HONJOH, Masayoshi IIO, and Shoji HATANO*

Laboratory of Food Hygienic Chemistry, Division of Bioresource and Bioenvironmental Sciences,
Graduate School, Kyushu University, Fukuoka 812-8581, Japan
*Nishi-Kyushu University, Saga 842-8585, Japan

Received December 13, 1999; Accepted January 25, 2000
The gene of IMP dehydrogenase of Bacillus cereus ts-4, a temperature-sensitive mutant of B. cereus
JCM 2152, was subcloned and its sequence was analyzed. A B. cereus ts-4 DNA fragment of 2,065 bp containing the entire impdh gene and flanking regions was sequenced. The fragment contained an open reading frame of 1,527 bp encoding 509 amino acids with a calculated molecular mass of 55,390 Da. The impdh sequence of JCM 2152 was also analyzed by TA cloning using PCR products amplified with primers from B. cereus ts-4 impdh gene. The gene amplified by PCR was expressed in Escherichia coli using a pET17×b expression plasmid. The N-terminal amino acid sequence of the overproduced enzyme was identified as Met-Trp-
Glu - Ser - Lys - Phe - Val - Lys - Glu - Gly - Leu - Thr - Phe - Asp-
Asp-Val-Leu-Leu-Val-Pro. The overproduced enzyme was eluted at a molecular mass of about 225 kDa by gel filtration. The molecular mass of the subunit was estimated to be 56 kDa by SDS-PAGE. The overproduced enzyme was active against IMP, IDP, and ITP, and showed the highest activity at pH 9.5. These properties of the recombinant enzyme were almost identical to those of IMP dehydrogenase of B. cereus.
Key words: Bacillus cereusIMP dehydrogenase; sporulation; cloning; expression

Naoyuki YAMAMOTO,＋ Tadashi SHINODA, and Toshiaki TAKANO

R&D Center, Calpis Co., Ltd., 11-10, 5-Chome, Fuchinobe, Sagamihara, Kanagawa 229-0006, Japan

Received December 16, 1999 Accepted February 11, 2000
A 2.6-kilobase HaeIII DNA fragment corresponding to an extracellular proteinase gene (prtY) was cloned from chromosomal DNA of Lactobacillus helveticus CP790 in Escherichia coli using a pKK223-3 vector. The transformant expressed a 48-kDa protein that reacts with monoclonal antibodies specific to the proteinase and seemed to be a pre-proproteinase, but had no proteolytic activity. About 1.6 kilobases of the 2.6-kilobase DNA fragment, which contained the complete gene for the proteinase was sequenced. Sequence analysis found an open reading frame with a capacity to encode a protein of 449 amino acids. The coding region contained a Gram-positive-type signal peptide of 30 amino acids. The N-terminal sequences of the proproteinase and the mature proteinase have been observed in the polypeptide at position +31 and +38. The putative amino acid sequence showed a significant similarity to a surface layer protein of L. helveticus and Lactobacillus acidophilus in the amino terminal signal sequence and carboxyl terminus.
Key words: extracellular proteinase gene; Lactobacillus helveticussurface layer protein

Sumio KITAHATA,a,＋ Toshiko TANIMOTO,b Akiko IKUTA,b Keiko TANAKA,b Koki FUJITA,c Hitoshi HASHIMOTO,c Hiromi MURAKAMI,a Hirofumi NAKANO,a and Kyoko KOIZUMIb

aOsaka Municipal Technical Research Institute, 1-6-50, Morinomiya, Joto-ku, Osaka 536-0025, Japan
bSchool of Pharmaceutical Sciences, Mukogawa Women′s University, 11-68, Koshien, Kyuban-cho, Nishinomiya 663-8179, Japan
cBio Research Corporation of Yokohama, 13-46, Daikoku-cho, Tsurumi-ku, Yokohama 230-0053, Japan

Received December 16, 1999; Accepted February 4, 2000
From the mixture of 42-O-β-D-galactosyl-maltose (Gal-G2) and β-cyclodextrin (βCD), novel heterobranched βCDs, (Gal-G2)-βCD and (Gal-G2)2-βCDs, were synthesized by the reverse action of debranching enzyme. The optimum conditions for the production of
(Gal-G2)2-βCDs were examined. A mixture of (Gal-G2)2-
βCDs was produced in about 4% yield when Aerobacter aerogenes pullulanase (64 units per 1 g of Gal-G2) was incubated with 1.6 M Gal-G2 and 0.16 M βCD at 50°C for 4 days. The reaction products, (Gal-G2)2-βCDs, were separated into three peaks by HPLC analysis on a Hypercarb S column. Their structures were analyzed by fast atom bombardment mass spectroscopy and NMR spectroscopies, and confirmed by comparison of their hydrolyzates by β-galactosidase with the authentic (G2)2-βCDs. The structures of (Gal-G2)-βCD and three components of (Gal-G2)2-βCDs were identified as 6-O-(Gal-G2)-βCD, 61,62-, 61,63- and 61,64-di-O-(Gal-G2)2-βCD, respectively.
Key words: reverse action; pullulanase; heterobranched CD

Tetsuya KIMURA,1＋ Jun ITO,1 Akihiro KAWANO,1 Takashi MAKINO,1 Hideki KONDO,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 December 17, 1999 ; Accepted January 28, 2000
Penicillum sp. 40, which can grow in an extremely acidic medium at pH 2.0 was screened from an acidic soil. This fungus produces xylanases when grown in a medium containing xylan as a sole carbon source. A major xylanase was purified from the culture supernatant of Penicillium sp. 40 and designated XynA. The molecular mass of XynA was estimated to be 25,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. XynA has an optimum pH at 2.0 and is stable in pH 2.0--5.0. Western blot analysis using anit-XynA antibody showed that XynA was induced by xylan and repressed by glucose. Also, its production was increased by an acidic medium. The gene encoding XynA (xynA) was isolated from the genomic library of Penicillium sp. 40. The structural part of xynA was found to be 721 bp. The nucleotide sequence of cDNA amplified by RT-PCR showed that the open reading frame of xynA was interrupted by a single intron which was 58 bp in size and encoded 221 amino acids. Direct N-terminal amino acid sequencing showed that the precursor of XynA had a signal peptide composed of 31 amino acids. The molecular mass caliculated from the deduced amino acid sequence of XynA is 20,713. This is lower than that estimated by gel electrophoresis, suggesting that XynA is a glycoprotein. The predicted amino acid sequence of XynA has strong similarity to other family11 xylanases from fungi.
Key words: acidophilic; Penicilliumxylanase

Fumiki NAKATANI, Takashi KAWAGUCHI, Goro TAKADA, Jun-ichi SUMITANI,
Yasushi MORIYAMA,* and Motoo ARAI

Department of Applied Biological Chemistry, College of Agriculture, and Research Institute for Advanced Science and Technology, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan
*Fundamental Research Center, TOTO Ltd., Chigasaki, Kanagawa 253-8577, Japan

Received December 24, 1999; Accepted January 24, 2000
The egI gene, encoding a major endoglucanase (EGI) of Scopulariopsis brevicaulis TOF-1212, was cloned and sequenced. The egI gene consisted of 868 bp with one intron and encoded a protein of 229 amino acids with a calculated molecular mass of 22,392 daltons. The EGI was assigned to a family 45 of glycosyl hydrolases and showed high similarity with other fungal endoglucanases, especially with those of Humicola grisea and Fusarium oxysporum, on the basis of hydrophobic cluster analysis. The egI gene was expressed under the promoter of the phosphoglycerate kinase gene (PGK) in Saccharomyces cerevisiae. The transformed cells were able to secrete the enzyme efficiently in an active form.
Key words: Scopulariopsis brevicaulisendoglucanase; expression

Ryoji NAKAGAWA,* Daisuke YASOKAWA, Yukihiro OKUMURA, and Koji NAGASHIMA

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

Received January 7, 2000; Accepted February 17, 2000
Two lectins (designated as HTA I and HTA II) that seemed to be isolectins were found in Helianthus tuberosus callus. cDNA encoding HTA I was isolated from a ZAP Express expression library by immunoselection by using the anti-HTA antiserum. The sequence of this cDNA consisted of 432 bp nucleotides coding for a polypeptide of 143 amino acid residues (Mr, 15,314). When introduced into E. coli, the cDNA directed the synthesis of active HTA I as indicated by the hemagglutination activity. The deduced amino acid sequence showed homology with some lectins and jasmonate-induced proteins. When callus was cultured in the presence of methyl jasmonate (MeJA), the hemagglutination activity increased in a dose-dependent manner. The levels of expression of the HTA protein and of the corresponding mRNA also increased in the treated callus. In view of these results, HTA I is considered to be a jasmonate-induced protein.
Key words: callus; gene expression; Helianthus tuberosusjasmonate-induced protein; lectin

Michihiko KATAOKA,＋ Jun-ichi NOMURA, Makoto SHINOHARA, Kiyoshi NOSE,
Keiji SAKAMOTO,* and Sakayu SHIMIZU

Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University,
Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan
*Research Institute, Fuji Chemical Industries Ltd., 530 Chokeiji, Takaoka, Toyama 933-8511, Japan

Received January 14, 2000; Accepted February 18, 2000
A novel lactonohydrolase, catalyzing the stereospecific hydrolysis of L-pantoyl lactone to L-pantoic acid, was purified 2,400-fold to apparent homogeneity with a 1.96% overall recovery from Agrobacterium tumefaciens AKU 316 through a purification procedure including ammonium sulfate fractionation, and column chromatographies on DEAE-Sephacel, phenyl-Sepharose CL-4B, Sephacryl S-200, Mono-Q and alkyl-Superose. The relative molecular mass of the native enzyme estimated on high-pressure gel permeation chromatography was 62,000 Da, and the subunit molecular mass was estimated to 26,500 Da on SDS-polyacrylamide gel electrophoresis. The enzyme hydrolyzes several aromatic lactones, such as 3,4-dihydrocoumarin and homogentisic acid lactone, other than L-pantoyl lactone. The Km and Vmax for L-pantoyl lactone were 3.59 mM and 13.7 μmol/min/mg, respectively. The enzymatic activity was inhibited by several chelating reagents, Fe2+, Sn2+, Pb2+, and Fe3+.
Key words: lactonohydrolase; pantoyl lactone; aromatic lactone; Agrobacterium tumefaciens

Kei-ichi YOKOYAMA, Nami NAKAMURA,* Katsuya SEGURO,* and Kouji KUBOTA*

Central Research Laboratories, and *Food Research & Development Laboratories, Ajinomoto Co.
Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki, Kanagawa 210-0801, Japan

Received January 24, 2000; Accepted February 22, 2000
The Streptoverticillium transglutaminase (MTG) gene, synthesized previously for yeast expression, was modified and resynthesized for overexpession in E. coli. A high-level expression plasmid, pUCTRPMTG-02(+), was constructed. Furthermore, to eliminate the N-terminal methionine, pUCTRPMTGD2 was constructed. Cultivation of E. coli transformed with pUCTRPMTG-02(+) or pUCTRPMTGD2 yielded a large amount of MTG (200~300 mg/liter) as insoluble inclusion bodies. The N-terminal amino acid residue of the expressed protein was methionine or serine (the second amino acid residue of the mature MTG sequence), respectively. Transformed E. coli cells were disrupted, and collected pellets of inclusion bodies were solubilized with 8 M urea. Rapid dilution treatment of solubilized MTG restored the enzymatic activity. Refolded MTG, purified by ion-exchange chromatography, which had an N-terminal methionine or serine residue, showed activity equivalent to that of native MTG. These results indicated that recombinant MTG could be produced efficiently in E. coli.
Key words: transglutaminase; Escherichia colirefolding; texture

Masanori KAWAGUCHI,1 Hiroyuki IMAI,2,＋ Miyuki NAOE,2 Yoshihiro YASUI,1
and Masao OHNISHI2,＋＋

1Tokachi-Ikeda Research Institute for Viticulture and Enology, Ikeda, Hokkaido 083-0002, Japan
2Department of Bioresource Science, Obihiro University of Agriculture and Veterinary Medicine,
Inada, Obihiro, Hokkaido 080-8555, Japan

Received September 10, 1999; Accepted January 21, 2000
Cerebrosides from leaves of three grapevine species were analyzed in detail. The relative proportions of 8-E/Z isomers of 4-hydroxy-8-sphingenines [i.e. t18:1(8E) and (8Z)] differed amongst the species in respect to freezing tolerance. This suggests that the occurrence of high levels of t18:1(8Z) in cerebrosides is correlated with freezing tolerance in these species.
Key words: cerebroside; freezing tolerance; sphingoid base; sphingolipid; Vitis

Takao KANEKO,＋ Naomichi BABA,＋ and Mitsuyoshi MATSUO・

Tokyo Metropolitan Institute of Gerontology, 35-2 Sakaecho, Itabashi-ku, Tokyo 173-0015, Japan
＋Faculty of Agriculture, Okayama University, 1-1-1 Tsushimanaka, Okayamashi,
Okayama 700-0082, Japan
・Faculty of Science and High Technology Center, Konan University, 8-9-1 Okamoto,
Higashinada-ku, Kobe 658-0072, Japan

Received October 22, 1999; Accepted January 7, 2000
Phosphatidylcholine hydroperoxides show weak but distinct toxicity toward cultured human umbilical vein endothelial cells. The protective effect of phenolic antioxidants against the cytotoxicity of phosphatidylcholine hydroperoxides was examined. Probucol depressed the toxicity most effectively among the antioxidants studied under both pretreatment and concurrent treatment conditions. α-Tocopherol showed a protective effect in the case of concurrent treatment. Protection by phenolic antioxidants against the cytotoxicity of phosphatidylcholine hydroperoxides seems to depend on their incorporation rate into cells, their affinity for phospholipids, their antioxidative activity, and their orientation in membranes.
Key words: phosphatidylcholine hydroperoxide; toxicity; probucol; α-tocopherol; human umbilical vein endothelial cell

Hiromiki KUWAHARA, Takako FUNADA, Tomomitsu HATAKEYAMA,＋
and Haruhiko AOYAGI

Department of Applied Chemistry, Faculty of Engineering, Nagasaki University, Bunkyo-machi 1-14,
Nagasaki 852-8521, Japan

Received November 8, 1999; Accepted January 31, 2000
Effects of chemical modification of carboxyl groups in the hemolytic lectin CEL-III on its activities were investigated. When carboxyl groups were modified with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and glycine methyl ester, hemolytic activity of CEL-III decreased as the EDC concentration increased, accompanied by reduction of oligomerization ability and hemagglutinating activity. However, binding ability of CEL-III for immobilized lactose was retained fairly well after modification, suggesting that one of two carbohydrate-binding sites might be responsible for such inactivation of CEL-III.
Key words: lectin; hemolysis; carboxyl group; chemical modification

Kei SONOYAMA,＋ Reiko FUJIWARA, and Takanori KASAI

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

Received November 11, 1999; Accepted January 22, 2000
An intravenous infusion of hexamethonium, a ganglionic blocker, did not affect the increase in the apolipoprotein A-IV mRNA level in the residual ileum following a massive small bowel resection in unrestrained conscious rats. The result suggests that upregulation of the apolipoprotein A-IV gene in the residual ileum is not mediated by a neural pathway, including the nicotinic synapse route.
Key words: apolipoprotein A-IV; mRNA; small intestine; small bowel resection; hexamethonium

Tetsuya UCHIKOBA,＋ Hiroo YONEZAWA, Kazunari ARIMA,* and Makoto KANEDA

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

Received November 12, 1999; Accepted February 8, 2000
A small amount of peptidase activity could be detected using an amine derivatizing reagent, fluorescein isothiocyanate (FITC), which has been used to produce a fluorogenic peptide. The substrate produced, FITC-peptide, gave a clear spot on a silica gel sheet upon exposure to UV light. The peptidase activity of angiotensin-converting enzyme (ACE), trypsin, chymotrypsin, cucumisin, and that of some plant tissues were detected by using a fluorogenic angiotensin I. This showed that the substrate specificity of proteolytic enzymes can be distinguished from the others by this procedure.
Key words: FITC; peptidase activity; TLC; fluorogenic substrate; ACE

Megumi HADA,＋, ＋＋ Keisuke HINO,* G"unther BUCHHOLZ,** J"orn GOSS,**
Eckard WELLMANN,** and Masateru SHIN#

Department of Biology, Faculty of Science, Kobe University, Kobe 657-8501, Japan
*Department of Biology, Faculty of Science, Kohnan University, Kobe 658-8501, Japan
**Institute of Biology II, Freiburg University, 79104, Freiburg, Germany
#Teru Biolaboratory, Mino-shi, 562-0043 Osaka, Japan

Received November 22,1999; Accepted February 17, 2000
Spinach cyclobutane pyrimidine dimer (CPD)-specific DNA photolyase was successfully detected in leaf extracts by an assay system for plant photolyase using an improved enzyme-linked immunosorbent assay (ELISA) which was newly introduced by novel horseradish peroxidase (HRP)-linked CPD specific monoclonal antibodies. The assay system includes two main steps: a photorepair reaction of CPD introduced in substrate DNA and measurement of CPD remained after the photorepair by the improved ELISA. When CPD- induced salmon sperm DNA was used as a substrate, high CPD-photolyase activities were observed in the enzyme fraction prepared from whole spinach leaf extracts, but not from chloroplast extracts. This strongly suggests that spinach CPD-specific photolyases are localized in cell compartments other than chloroplasts.
Key words: DNA photolyase; cyclobutane pyrimidine dimer (CPD); photorepair; enzyme-linked immunosorbent assay (ELISA); spinach

Misao MIWA＋ and Yasuhisa HONGO*

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

Received November 22, 1999; Accepted January 21, 2000
Three cell lines (HL60, U937 and RAW264.7) were studied for their sensitivity against mutagens by using a single-cell gel electrophoresis (comet) assay. RAW264.7, the most sensitive one, was chosen to screen the antimutagenic activity in swine and bovine offal. Aqueous extracts of the swine stomach (0.2 mg/ml) and heart (10 mg/ml) were found to have antimutagenic activity against MeIQx (+S9mix)-treated cells.
Key words: comet assay; DNA damage; antimutagen; meat offal; MeIQx

Hiroya ISHIKAWA, Mitsuya SHIMODA,* Akiyoshi YONEKURA,* Keiko MISHIMA,*
Kiyoshi MATSUMOTO,* and Yutaka OSAJIMA**

Venture Business Laboratory, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan
*Laboratory of Food Biotechnology, Department of Bioscience and Biotechnology, Division of Bioresource
and Bioenviromental Science, Graduate School of Kyushu University, Fukuoka 812-8581, Japan
**School of Bioresources, Hiroshima Prefectural University, 562, Nanatsuka-cho, Shobara, Hiroshima 727-0023, Japan

Received November 29, 1999; Accepted January 21, 2000
The conformational changes in α-helical poly-L-glutamic acid caused by microbubbling supercritical CO2 were investigated with circular dichroism spectra. After microbubbling using a micropore filter at 35 and 30 MPa for 30 min, α-helix content decreased to 37%, while without the filter it was 68%. The α-helix structure was significantly decomposed by a high density of CO2. No important changes were observed in heating, autoclaving, or pH-lowering.
Key words: poly-L-glutamic acid; supercritical CO2microbubbles; unfolding

Aki KANEKO,1,2 Hidenori MIYADAI,2,3 Hirofumi DANBARA,1
and Kazuyoshi KAWAHARA2,3

1Department of Microbiology, School of Pharmaceutical Sciences, Kitasato University,
Minato-ku, Tokyo 108-8641, Japan
2Department of Bacteriology, The Kitasato Institute, Minato-ku, Tokyo 108-8642, Japan
3Kitasato University Graduate School of Fundamental Life Science, Sagamihara 228-8555,
Kanagawa, Japan

Received November 29, 1999; Accepted February 21, 2000
Mutants of Sphingomonas paucimobilis defective in a part of the carbohydrate moiety of the glycosphingolipid (GSL) were constructed by transposon (Tn5)-insertional mutagenesis. Defective mutants were selected by ELISA using the antibody recognizing the tetrasaccharide-type GSL (GSL-4A) of S. paucimobilis. Eight defective mutants were selected from about 8,000 kanamycin-resistant strains, and seven of them were found to lack the terminal mannose of GSL-4A. The chemical structure of the mutant GSL was investigated, and proved that the rest of the structure was not changed by the mutation.
Key words: glycosphingolipid; Tn5-insertional mutagenesis; Sphingomonas paucimobilis

Motoko MATSUFUJI,* Yasunori NAGAMATSU, and Akihiro YOSHIMOTO

*Central Research Laboratories, Mercian Corporation 9-1 Johnan 4-Chome, Fujisawa 251-0057, Japan
Faculty of Applied Biological Science, Hiroshima University, 4-4 Kagamiyama 1-Chome,
Higashi-Hiroshima 739-8526, Japan

Received December 1, 1999; Accepted February 18, 2000
Bacterial monogalactosyldiacylglycerol M874B (MGDAG), which protects against oxygen radicals, was found to increase the growth of the human promyelocytic leukemia cell HL60 when added to the cell culture, but suppresses the 12-O-tetradecanoyl phorbol-13-acetate-induced differentiation. Analogous MGDAG, S365B had weak, but similar effects. These activities were not observed with analogous plant glyceroglycolipids and diacylglycerol.
Key words: glyceroglycolipid; oxygen radical scavenger; HL60 cell; differentiation

Yoshiyuki ASHIDA, Akihito MATSUSHIMA, Yukari TSURU, Tomoko HIROTA,
and Toshifumi HIRATA＋

Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University,
1-3-1 Kagamiyama, Higashi-Hiroshima 739-8526, Japan

Receied December 3, 1999; Accepted January 21, 2000
The amino acid sequence of a novel trypsin inhibitor (p20) was completed by the molecular cloning of the protein in cultured soybean cells. The clone nucleotide contains an open reading frame encoding a polypeptide of 206 amino acids that shows 45--50% sequence homology to members of the Kunitz-type trypsin inhibitor family. The p20 transcript is expressed in the roots, stems and leaves of soybean seedlings. DNA gel blot analyses show that the p20 in soybean is encoded by a single gene, and that this gene may not contain an intron.
Key words: Glycine max (soybean); Leguminosae; molecular cloning; Kunitz-type trypsin inhibitor

Unang SUPRATMAN, Tomoyuki FUJITA, Kohki AKIYAMA, and Hideo HAYASHI＋

Department of Applied Biological Chemistry, College of Agriculture, Osaka Prefecture University,
1-1 Gakuen-cho, Sakai, Osaka 599-8531, Japan

Received December 6, 1999; Accepted January 31, 2000
Two insecticidal bufadienolides (1 and 2) were isolated from a methanol extract of the leaves of Kalanchoe pinnata by bioassay-guided fractionation. Compound 1 was identified as known bryophyllin A (bryotoxin C). The structure of new bufadienolide 2, named bryophyllin C, was determined by spectroscopic methods and the chemical transformation of 1. Compounds 1 and 2 showed strong insecticidal activity against third instar larvae of the silkworm (Bombyx mori), their LD50 values being evaluated as 3 and 5 μg/g of diet, respectively.
Key words: Kalanchoe pinnataCrassulaceae; bufadienolide; insecticidal activity; bryophyllin C

Takeshi YAMAGAMI,* Gunki FUNATSU, and Masatsune ISHIGURO

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

Received December 13, 1999; Accepted January 21, 2000
The positions of disulfide bonds of rye seed chitinase-a (RSC-a) were identified by the isolation of disulfide-containing peptides produced with enzymatic and/or chemical cleavages of RSC-a, followed by sequencing them. An unequivocal assignment of disulfide bonds in this enzyme was as follows: Cys3-Cys18, Cys12-Cys24, Cys15-Cys42, Cys17-Cys31, and Cys35-Cys39 in the chitin-binding domain (CB domain), Cys82-Cys144, Cys156-Cys164, and Cys282-Cys295 in the catalytic domain (Cat domain), and Cys263 was a free form.
Key words: chitinase; disulfide bond; rye seed

Tatsuya NARIKAWA,1 Hirofumi SHINOYAMA,1,* and Takaaki FUJII1,2

1Graduate School of Science and Technology, and 2Department of Bioproduction Science, Faculty
of Horticulture, Chiba University, 648 Matsudo, Matsudo-shi, Chiba 271-8510, Japan

Received January 12, 2000; Accepted March 27, 2000
A β-rutinosidase, which was specific for releasing the disaccharide rutinose from the flavonoid glycoside rutin, was purified from Penicillium rugulosum IFO 7242. This enzyme had the molecular weight of 245,000, a very low optimum pH of 2.2, and the remarkable specificity that the glycosidase did not hydrolyze any other substrates like 4-nitrophenyl β-glucoside and cellobiose, but only rutin and isoquercitrin.
Key words: rutin; β-rutinosidase; rutinase; flavonoid glycosidase; Penicillium rugulosum