Contents and Abstracts of Latest Issue of BBB

(Vol.63 No.6 1999)

Masahiro KAMEKURA＊ and Morris KATES＊＊

＊Noda Institute for Scientific Research, 399 Noda, Noda, Chiba 278-0037, Japan
＊＊Department of Biochemistry, University of Ottawa, Ottawa, ON K1N 6N5, Canada

This minireview gives an updated and consolidated summary of taxonomic classification correlated with membrane phospholipid, glycolipid, and core lipid structural diversity within the family Halobacteriaceae. We also point out that the recently reported diversity in the membrane core lipid structure of a putative strain of Halobacterium (Halobacterium halobium strain IAM 13167) (Morita et al., Biosci. Biotechnol. Biochem., 62, 596--598, 1998) is not correct since the strain used by the authors has for some time been recognized not to be a member of the genus Halobacterium but a member of halobacteria group 2 (Grant and Larsen, Bergey′s Manual of Systematic Bacteriology, Vol.3, pp.2216--2233, 1989), which has recently been designated as a new genus, Natrinema (McGenity et al., Int. J. Syst. Bacteriol. 48, 1187--1196, 1998).
archaebacteria; diisopranylglycerol diethers; extremely halophilic bacteria; glycolipid structures; Halobacterium

-2-
Identification of the Major Antioxidative Metabolites in Biological Fluids
of the Rat with Ingested (＋)-Catechin and (−)-Epicatechin

Masami HARADA,・ Yukiko KAN,＊ Hideo NAOKI,＊ Yuko FUKUI, Norihiko KAGEYAMA,
Masaaki NAKAI, Wataru MIKI, and Yoshinobu KISO

Institute for Fundamental Research and ＊Institute for Bioorganic Research, Suntory Ltd. (SUNBOR), 1-1-1
Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka 618-8503, Japan

Received November 2, 1998; Accepted February 5, 1999
(＋)-Catechin and (−)-epicatechin are known to be biologically effective antioxidants present in the human diet, particularly in wine and tea. We studied the metabolism of these compounds to elucidate the truly active structures in biological fluids by their oral administration to rats. Without any treatment with β-glucuronidase and sulfatase, a pair of metabolites were detected at much higher concentrations in the plasma, bile, and urine than the originally ingested compounds. Each major metabolite found in the plasma at the highest concentration was excreted in both the bile and urine, and was purified from urine. Their chemical structures were established to be (＋)-catechin 5-O-β-glucuronide and (−)-epicatechin 5-O-β-glucuronide by MS and NMR analyses. These glucuronide conjugates exhibited high antioxidative activities as superoxide anion radical scavengers like their parent compounds. It is concluded that (＋)-catechin 5-O-β-glucuronide and (−)-epicatechin 5-O-β-glucuronide are the biologically active in vivo structures of the ingested polyphenolic antioxidants.
catechin; epicatechin; metabolite; β-glucuronide; antioxidant

-3-
Influences of Metabolic Inhibitors and Hydrolytic Enzymes on the Adhesion
of Appressoria of Pyricularia oryzae to Wax-coated Cover-glasses

Manabu OHTAKE, Hirotaka YAMAMOTO, and Takeo UCHIYAMA＊

Faculty of Agriculture, Niigata University, Ikarashi, Niigata 950-2102, Japan

Received Novenber 13, 1998; Accepted January 26, 1999
For discovering the components that contributed to the bonding strength of the glue substances produced by appressoria of Pyricularia oryzae on wax-coated cover-glasses, the influences of metabolic inhibitors and hydrolytic enzymes were investigated. The bonding strength of appressoria was assessed by the ratio of the remaining appressoria after sonication to the appressoria formed before sonication. Remaining appressoria decreased with increasing concentrations of cerulenin, an inhibitor of lipid synthesis, but isoprothiolane and compactin showed no influence on bonding strength. Tunicamycin, an inhibitor of glycoprotein synthesis, weakened the bonding strength of appressoria, but castanospermin had no effect. Of the hydrolytic enzymes tested, protease particularly weakened the bonding strength of appressoria. On the surfaces of substrata, the appressoria′s bonding strength was higher on the hydrophobic surfaces than on the hydrophilic. These results suggest that lipid components and glycoprotein were closely associated with appressoria bonding strength to the surface of wax-coated cover-glass.
Magnaporthe grisea; Pyricularia oryzae; glue substances; adhesion of appressorium; appressorium

-4-
Radical Scavenging Activity of Phenylpropanoid Glycosides
in Caryopteris incana

Jian-Jun GAO, Kiharu IGALASHI,＊ and Manabu NUKINA

Faculty of Agriculture, Yamagata University, Tsuruoka-shi, Yamagata 997-8555, Japan

Received November 26, 1998; Accepted February 16, 1999
In our screening program for antioxidants from traditional drugs and foodstuffs, one new phenylpropanoid glycoside, incanoside, was isolated together with four||known phenylpropanoid glycosides, verbascoside, isover||||bascoside, phlinoside A, and 6-O-caffeoyl-β-D-glucose from||||the whole plant of Caryopteris incana (Thunb.) Miq. On the||||basis of chemical evidence and spectral analysis data,||||the structure of incanoside was determined to be||||1 - O - (3,4 - dihydroxyphenyl)ethyl - O - β - D - glucopyranosyl||||(1→2) - α - L - rhamnopyranosyl(1→3) - 6 - O - caffeoyl - β - D - glu||copyranoside. The four phenylpropanoid glycosides exhibited potent radical scavenging activity against DPPH, hydroxyl (・OH), and superoxide anion (O2・−) radicals.
Caryopteris incana (Thunb.) Miq.; Verbenaceae; phenylpropanoid glycoside; incanoside; radical-scavenging activity

-5-
Reclassification of the Strains with Low G＋C Contents of DNA belonging
to the Genus Gluconobacter ASAI 1935 (Acetobacteraceae)

Machiko TANAKA,・ Shuichiro MURAKAMI,＊ Ryu SHINKE, and Kenji AOKI

Laboratory of Bio-functional Chemistry, Division of Bioscience, The Graduate School of Science and Technology,
Kobe University, Rokko, Kobe 657-8501, Japan
＊Laboratory of Applied Microbiology, Department of Bio-functional Chemistry, Faculty of Agriculture,
Kobe University, 1 Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan

Received November 30, 1998; Accepted January 25, 1999
Three species, Gluconobacter cerinus, G. asaii, and G. frateurii are reported to show lower G＋C contents than G. oxydans. Isolate 145 also showed a similar G＋C content to those of the three species. We try to reclassify the three species. G. frateurii including a type strain, isolate 145, G. cerinus IFO 3262, 3263, 3269, and G. asaii IFO 3265 formed acid from D-arabitol, ribitol, and L-arabitol. DNAs from G. cerinus IFO 3262, 3263, 3269, and G. asaii IFO 3265 showed 100--53％ sequence similarity with that from the type strain of G. frateurii, but showed 31--39％ and 34--42％ similarity with those from the type strains of G. cerinus and G. asaii, respectively. On the basis of these observations, G. cerinus IFO 3262, 3263, 3269 and G. asaii IFO 3265 were identified as G. frateurii. The type strains of G. cerinus and G. asaii formed acid from D-arabitol, but did not form acid from ribitol and L-arabitol. DNAs from the type strains of G. cerinus and G. asaii showed species-level similarity (95 and 88％) with each other. From these results, we concluded that G. asaii is conspesific and synonymous with G. cerinus.
Gluconobacter; Gluconobacter cerinus; Gluconobacter asaii; Gluconobacter frateuri; taxonomy

-6-
New Geometric Isomers of Oxooctadecadienoate in Copper-catalyzed
Decomposition Products of Linoleate Hydroperoxide

Masako TOKITA, Jyunko IWAHARA, and Makio MORITA

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

Received December 3, 1998; Accepted February 14, 1999
Methyl linoleate hydroperoxide produced by autoxidation was refluxed with 10−4 M Cu-naphthenate in benzene. Two new geometrical isomers of oxooctadecadienoate (compounds I and II) were found in addition to the four known isomers. They were isolated by a Sephadex LH-20 column chromatography with chloroform-hexane (2:1) and purified by HPLC on Nucleosil ・100-5 and Zorbax ODS columns. UV, IR, MS, and 1H-NMR spectra were measured. The geometry of conjugated dienes were assigned from the coupling constants of the olefinic protons. Compounds I and II were identified as 13-oxo-trans-9, cis-11- and 9-oxo-cis-10, trans-12-octadecadienoate, respectively. Each of them had a cis double bond adjacent to the oxo group. The hydroperoxides of the same geometry as compounds I and II were also detected in autoxidation products.
oxooctadecadienoate; linoleate; hydroperoxide; autoxidation

-7-
Cloning and Sequencing of a High-alkaline Pectate Lyase Gene
from an Alkaliphilic Bacillus Isolate＊

Yuji HATADA, Norihiko HIGAKI, Kazuhiro SAITO, Akinori OGAWA, Kazuhisa SAWADA,
Tadahiro OZAWA, Yoshihiro HAKAMADA, Tohru KOBAYASHI, and Susumu ITO＊＊

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

Received December 7, 1998; Accepted February 12, 1999
Alkaliphilic Bacillus sp. strain KSM-P103 was found to exoproduce a high-alkaline pectate lyase (pectate transeliminase, EC 4.2.2.2). The gene for this enzyme from the alkaliphile was cloned and sequenced for the first time. The structural gene contained a 1,038-bp open reading frame encoding 345 amino acids. The deduced amino acid sequence of the mature enzyme (302 amino acids, 33,312 Da), designated Pel-103, showed very low similarity to those of known pectate lyases with 28--36％ identity: the loop regions were very short and the amino acid usage in the parallel β-helix core structure was considerably different. Moreover, physicochemical and catalytic properties of Pel-103 were different from those of other enzymes reported so far. Pel-103 was a very basic protein with an isoelectric point close to pH 10.5 and had optimal activity at 60--65°C and at pH as high as 10.5. However, Pel-103 appeared to have a similar core and active site topology to the enzymes of known structure from Erwinia chrysanthemi and Bacillus subtilis. Expression of the gene for Pel-103 in B. subtilis resulted in high pectate lyase activity in the culture broth, concomitant with the appearance of a main protein band on an SDS gel at 33 kDa.
alkaliphile; Bacillus; trans-elimination; PCR; amino acid sequence

-8-
Serum Amyloid A, Cytokines, and Corticosterone Responses in Germfree
and Conventional Mice after Lipopolysaccharide Injection

Masamichi IKEDA,・ Kazuya HAMADA, Naomi SUMITOMO, Hiroshi OKAMOTO,＊
and Bunsaku SAKAKIBARA

Department of Nutritional Pathology, Faculty of Nutrition and ＊Department of pharmacology,
Faculty of Pharmaceutical Sciences, Kobe Gakuin University, Ikawadani-cho, Nishi-ku, Kobe 651-2180, Japan

Received December 8, 1998; Accepted February 4, 1999
To determine why germfree mice are less susceptible to lipopolysaccharide (LPS) than conventional mice, we studied serum levels of serum amyloid A (SAA), tumor necrosis factor (TNF), interleukin 1 (IL-1), IL-6, and corticosterone in mice after treatment with LPS. A single injection of LPS caused an elevation of SAA, an acute-phase protein in the mouse, in both conventional and germfree IQI mice, and the response was significantly less in germfree mice. LPS-induced elevations of serum TNF, IL-1, and IL-6 levels were also significantly less in germfree mice, while serum corticosterone levels were greater in germfree mice than in conventional mice. These results suggest that the lower susceptibility to LPS and a smaller response of SAA elevation by LPS in germfree mice may result from less elevation in serum of these cytokines in these mice, which are known to mediate the acute phase response of SAA. High levels of serum corticosterone in germfree mice may be partly responsible for the lower responsiveness of these inflammatory cytokines to LPS in these mice.
germfree mice; lipopolysaccharide; serum amyloid A; tumor necrosis factor; interleukin 1

-9-
Inhibitory Effect of the Conformation of Amylose as a Function
of I2 Concentration on Glucoamylase Activity

Hirofumi YAJIMA,1,・ Kazuo WATANABE,2 Tetsuo TAKEMURA,3 and Tadahiro ISHII1

1Department of Applied Chemistry, Faculty of Science, Science University of Tokyo,
1-3 Kagurazaka, Shinjuku-ku, Tokyo 162-8601, Japan
2Nippi Research Institute of Biomatrix, 1-1 Senju-Midoricho, Adachi-ku, Tokyo 120-0044, Japan
3Department of Chemistry, Faculty of Science, Science University of Tokyo,
1-3 Kagurazaka, Shinjuku-ku, Tokyo 162-8601, Japan

Received December 17, 1998; Accepted February 22, 1999
||In order to gain a better understanding of the adaptabil||||ity of glucoamylase to the conformational change in the||substrate related with the specificity of the enzyme action, the effect of the solution conformation of amylose (the substrate) as a function of the I2 concentration on enzyme activity was studied in the initial state by using amyloses with a wide range of average degree of polymerization (・・・DP・) in the presence of excess KI. The enzyme activity for the amylose oligomers in the ・・・DP・ range of 20 to 100 decreased monotonically with increasing I2 concentration. This inhibition of the enzyme activity is ascribed to the effect of the conformational change in amylose of the random coil or worm-like chain to a helix that is induced by the binding of I−3 ions responsible for inclusion complexation in combination with direct effect of free I−3 ions. In a rapidly mixed complex system for amylose with a large ・・・DP・ of 1,000, however, a significant two-step form of the inhibition of enzyme activity appeared with increasing I2 concentration, corresponding to the two-step conformational change of amylose in a random coil or worm-like chain to a helix and then to a rod-like compact structure.
amylose; amylose-iodine-iodide complex; conformation; glucoamylase; inhibition

-10-
Human Cysteine Dioxygenase Gene: Structural Organization, Tissue-specific
Expression and Downregulation by Phorbol 12-Myristate 13-Acetate

Nobuyo TSUBOYAMA-KASAOKA,1 Yu HOSOKAWA,2,・ Hiroyuki KODAMA,3 Akiyo MATSUMOTO,1
Jun OKA,4 and Masayuki TOTANI2

Division of 1Clinical Nutrition, 2Maternal and Child Health Science and 4Geriatric Health Science,
National Institute of Health and Nutrition, Shinjuku-ku, Tokyo 162-8636, Japan
3Department of Chemistry, Kochi Medical School, Nankoku-shi, Kochi 783-0043, Japan

Received December 21, 1998; Accepted February 8, 1999
The organization of the human cysteine dioxygenase (CDO) gene was found to be similar to its rat counterpart, and the location of the introns in the protein structure was identical to the rat CDO gene. The major transcription start site, identified by primer extension, was located 260 bp upstream from the ATG codon. The sequence of the 5′-immediate upstream region was highly conserved between the human and rat CDO genes. The putative promoter region contained a TATA-box-like sequence, and many putative cis-acting elements including HNF5, GRE, TRE, CRE, CArG box, ARE, MBS, and NF-kB. A Northern blot analysis revealed that CDO mRNA was strongly expressed in the liver and placenta, and weakly in the heart, brain and pancreas. CDO mRNA was also detected in human hepatoblastoma HepG2 cells. The CDO mRNA level in HepG2 cells was decreased after 2 h and reached a minimum 6 h--8 h after a phorbol 12-myristate 13-acetate (PMA) treatment, and then gradually returned to the basal level.
cysteine dioxygenase; gene organization; 5′-flanking sequence; tissue-specific expression; downregulation

-11-
Generation of Free Radicals during the Death of Saccharomyces cerevisiae Caused by Lipid Hydroperoxide

Hitoshi AOSHIMA,＊ Kiyoshi KADOYA,＊ Hitoshi TANIGUCHI,＊＊ Takumi SATOH,・,＊＊＊
and Hiroshi HATANAKA＊＊＊

＊Department of Physics, Biology and Informatics, Faculty of Science, Yamaguchi University,
1677-1 Yoshida, Yamaguchi 753-8512, Japan
＊＊Department of Veterinary Medicine, Faculty of Agriculture, Yamaguchi University,
1677-1 Yoshida, Yamaguchi 753-8515, Japan
＊＊＊Division of Protein Biosynthesis, Institute for Protein Research, Osaka University,
3-2 Yamadaoka, Suita, Osaka 565-0871, Japan

Received December 24, 1998; Accepted February 13, 1999
The exposure of Saccharomyces cerevisiae cells to 13-L-hydroperoxylinoleic acid (LOOH) caused their death, the degree of which was dependent on the growth phase of the cells. Pre-application of ethanol, hydrogen peroxide (H2O2) and LOOH to S. cerevisiae cells reduced the effect of LOOH on the cells, showing the transient cross adaptation to LOOH. Antioxidants such as N,N′,-diphenyl-p-phenylenediamine (DPPD), melatonin and vitamin E, and inhibitors of permeability transition of mitochondria, cyclosporin A and trifluoperazine, inhibited the LOOH-triggered cell death, while an inhibitor of glutathione synthetase, buthionine sulfoximine (BSO), enhanced the cell death by LOOH. Reactive oxygen species (ROS) were detected by flow cytometry, using the ROS-specific fluorescent indicator. A ferric iron chelator, deferoxamine, inhibited the LOOH-triggered cell death, and peroxyl radicals (LOO・) were detected by a spin trapping method. These reactive radicals possibly induced the death of S. cerevisiae cells. However, the DNA fragmentation characteristic of apoptosis was not observed in S. cerevisiae cells after exposure to LOOH, staurosporine, dexamethasone or etoposide, which have been reported to cause apoptosis in mammalian cells.
flow cytometry; lipid hydroperoxide; oxidative stress; Saccharomyces cerevisiae; spin trapping

-12-
The Incorporation and Accumulation of Supplemented Fatty Acids
in Mucor hiemalis HA-30

Hideyuki AOKI, Koshi NISHIOKA, Mitsumasa MANKURA, Yasushi ENDO,＊
and Kenshiro FUJIMOTO＊,・

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

Received January 11, 1999; Accepted February 19, 1999
Accumulation of free unsaturated fatty acids, added individually to the medium, into cellular triacylglycerol (TG) were examined using a fungus, Mucor hiemalis HA-30, which could produce the eicosapentaenoic acid (20:5n-3)-enriched TG. Linoleic (18:2n-6), γ-linolenic (18:3n-6), arachidonic acids (20:4n-6), and 20:5n-3 were favored for accumulation in TG and each fatty acid accumulated in TG at about 80％ purity. However, docosahexaenoic acid (22:6n-3) was not efficiently accumulated in TG and its content was about 60％.
Incorporation and accumulation of various fatty acids added to the cultivation medium were investigated using a model fatty acids mixture. Generally, unsaturated fatty acids were efficiently incorporated into cells more than saturated fatty acids. Fatty acids with odd-numbered double bonds such as 18:3n-3, 18:3n-6, and 20:5n-3 showed the higher incorporation efficiency compared with those with even-numbered double bonds such as 18:2n-6, 20:4n-6, and 22:6n-3. On the other hand, saturated and monounsaturated fatty acids of 16:0, 18:0, and 18:1 were preferentially accumulated in microbial cells over other fatty acids.
incorporation; accumulation; fatty acid; Mucor

-13-
Isolation and Some Properties of a Novel Killer Toxin-like Protein Produced
by Streptomyces sp. F-287

Kazumi HIRAGA, Suzuko HAYASHI, Mitsunori KITAZAWA, and Kohei ODA・

Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology,
Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan

Received January 11, 1999; Accepted February 15, 1999
A killer toxin-like protein was found in the culture supernatant of a strain isolated from soil. The strain was classified and designated as Streptomyces sp. F-287. The molecular weight of the purified killer toxin-like protein was estimated to be 9,500 by SDS-PAGE. The purified protein was heat stable (100°C, 5 min), pH stable (pH 6.0--9.0, 60°C, for 30 min), and had a relatively wide action spectra. The SKLP showed a cytocidal effect on both budding yeast, Saccharomyces cerevisiae W303 (IC50＝15.6 μg/ml) and on fission yeast, Schizosaccharomyces pombe SP870 (IC50＝20.0 μg/ml). The SKLP also caused morphological changes on some sensitive yeasts and filamentous fungi. These characteristics are apparently different from known killer toxins. These results suggest that this is a novel killer toxin-like protein from Streptomyces sp. strain F-287.
killer toxin-like protein; Streptomyces; yeast; cytocidal activity

-14-
Role of Carbohydrate Moiety in Carboxypeptidase Y: Structural Study
of Mutant Enzyme Lacking Carbohydrate Moiety

Hiroyuki SHIMIZU, Hiroshi UENO, and Rikimaru HAYASHI・

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

Received January 11, 1999; Accepted February 12, 1999
To study the roles of the carbohydrate moiety in the function of carboxypeptidase Y, asparagine residues at 13, 87, 168, and 368, the four-consensus N-linked glycosylation sites, were altered to alanine with site-directed mutagenesis. The mutant enzyme of 51 kDa completely lost the carbohydrate moiety which was present in the 61-kDa wild-type enzyme. Structural studies of the mutant enzyme showed that it maintained the native-like structure; hydrolytic activity, and substrate specificity of the mutant enzyme analogous to those of the wild-type enzyme. Susceptibility of the mutant enzyme toward proteolysis and pressure denaturation was reduced by 10--20％. It is concluded that the carbohydrate moiety functions to maintain the structural integrity of the enzyme under stressed.
carboxypeptidase Y; serine carboxypeptidase; site-directed mutagenesis; glycoprotein

-15-
Cloning and Expression in Escherichia coli of a Gene Coding for a Secondary
Alcohol Dehydrogenase from Candida parapsilosis

Hiroaki YAMAMOTO,・ Naoki KAWADA,＊ Akinobu MATSUYAMA, and Yoshinori KOBAYASHI

Tsukuba Research Center, Daicel Chemical Industries, Ltd., 27 Miyukigaoka, Tsukuba, Ibaraki 305-0841, Japan
＊Research Center, Daicel Chemical Industries, Ltd., 1275 Shinzaike, Himeji, Hyogo 671-1236, Japan

Received January 13, 1999; Accepted February 17, 1999
A gene encoding a stereo-specific secondary alcohol dehydrogenase (CpSADH) that catalyzed the oxidation of (S)-1,3-BDO to 4-hydroxy-2-butanone was cloned from Candida parapsilosis. This CpSADH-gene consisted of 1,009 nucleotides coding for a protein with Mr 35,964. A recombinant Escherichia coli JM109 strain harboring the expression plasmid, pKK-CPA1, produced (R)-1,3-BDO (93.5％ ee, 94.7％ yield) from the racemate without any additive to regenerate NAD＋ from NADH.
(S)-1,3-butanediol dehydrogenase; secondary alcohol dehydrogenase; Candida parapsilosis; (R)-1,3-butanediol; NAD＋ regeneration

-16-
Cloning, Nucleotide Sequence, and Disruption of Streptococcus mutans
Glutathione Reductase Gene (gor)

Yuji YAMAMOTO, Yoshiyuki KAMIO, and Masako HIGUCHI・

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

Received January 14, 1999; Accepted February 18, 1999
We cloned and sequenced the glutathione reductase gene (gor) of an oxygen-tolerant Streptococcus mutans, and constructed a gor-disruption mutant by homologous recombination. The gor gene consisted of 1,350 bp, coding for a protein of 450 amino acid residues. The deduced amino acid sequence of the S. mutans gor gene product showed extensive similarity with those of glutathione reductases from prokaryotes and eukaryotes. Although the mutant could grow aerobically, it showed no growth in the presence of 2 mM diamide, a thiol-specific oxidant. In contrast, growth of the wild-type strain was not significantly inhibited by 2 mM diamide, and glutathione reductase activity was increased 2.2-fold under these conditions. In addition, the level of glutathione reductase activity in the wild-type strain was increased 3.6-fold upon exposure to air, and the elevated level of the enzyme was retained throughout the aerobic growth. Thus, glutathione reductase may be important in protection of S. mutans against oxidative stress.
Streptococcus mutans; glutathione reductase; gor gene; diamide; oxidative stress

-17-
Novel Characteristics of Selenomonas ruminantium Lysine Decarboxylase
Capable of Decarboxylating Both L-Lysine and L-Ornithine

Yumiko TAKATSUKA,1 Motoko ONODA,1 Takeyoshi SUGIYAMA,2 Koji MURAMOTO,3
Toshio TOMITA,1 and Yoshiyuki KAMIO1,・

Laboratories of the Applied Microbiology,1 Applied Bioorganic Chemistry,2
and Applied Phytochemistry,3
Graduate School of Agricultural Science,
Tohoku University, 1-1 Tsutsumi-dori Amamiya-machi, Aoba-ku, Sendai 981-8555, Japan

Received January 19, 1999; Accepted February 12, 1999
Lysine decarboxylase (LDC; EC 4.1.1.18) of Selenomonas ruminantium is a constitutive enzyme and is involved in the synthesis of cadaverine, which is an essential constituent of the peptidoglycan for normal cell growth. We purified the S. ruminantium LDC by an improved method including hydrophobic chromatography and studied the fine characteristics of the enzyme. Kinetic study of LDC showed that S. ruminanitum LDC decarboxylated both L-lysine and L-ornithine with similar Km and the decarboxylase activities towards both substrates were competitively and irreversibly inhibited by DL-α-difluoromethylornithine, which is a specific inhibitor of ornithine decarboxylase (EC 4.1.1.17). We also showed a drastic descent of LDC activity owing to the degradation of LDC at entry into the stationary phase of cell growth.
Selenomonas ruminantium; lysine decarboxylase; ornithine decarboxylase; DL-α-difluoromethyllysine; DL-α-difluoromethylornithine

-18-
Guanidinated Casein Hydrolysate Stimulation of Cholecystokinin Release
via Pancreatic Enzyme- and Cholinergic-independent Mechanisms in Rats

Takashi NISHI, Hiroshi HARA, and Yoritaka AOYAMA

Department of Bioscience and Chemistry, Faculty of Agriculture, Hokkaido University, Sapporo 060-8589, Japan

Received January 25, 1999; Accepted February 16, 1999
We had demonstrated that a peptic hydrolysate of guanidinated casein that is made from casein by the conversion of lysine to homoarginine stimulated pancreatic exocrine secretion in rats with chronic bile-pancreatic juice (BPJ) diversion from the proximal small intestine. This modified protein also stimulated cholecystokinin (CCK) release from dispersed rat intestinal cells. In this study, we found that guanidinated casein hydrolysate stimulates CCK release in chronic BPJ-diverted rats with cholinergic control blocked by atropine. Intraduodenal guanidinated casein hydrolysate increased portal plasma CCK concentration and pancreatic secretion in atropine-treated BPJ-diverted rats. In contrast, the portal plasma CCK concentration was not increased by intact casein hydrolysate. We conclude that guanidinated casein hydrolysate directly stimulates CCK release from the intestine via some cholinergic-independent mechanism, and an increase of the pancreatic exocrine secretion is regulated by CCK released by guanidinated casein hydrolysate. A guanidyl residue is likely to be involved in this control.
cholecystokinin; pancreatic exocrine secretion; bile-pancreatic juice diversion; guanidyl residue; rat

-19-
Nystatin Effects on Vacuolar Function in Saccharomyces cerevisiae

M. Shah Alam BHUIYAN, Yuji ITO, Atsuko NAKAMURA, Naotaka TANAKA,
Kiyotaka FUJITA, Hiroshi FUKUI, and Kaoru TAKEGAWA＊

Department of Life Sciences, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa 761-0795, Japan

Received February 2, 1999; Accepted March 9, 1999
The effects of nystatin, a polyene antibiotic, was studied in Saccharomyces cerevisiae by isolating and characterizing nystatin-sensitive mutants. We isolated a number of nystatin-sensitive mutants by ethylmethane sulfonate mutagenesis. One of these mutants, the nss1 mutant, was characterized in detail. The mutant was sensitive to stresses such as high temperature or high concentrations of monovalent and divalent cations. The nss1 mutants showed severe vacuolar protein sorting and vacuolar morphology defects. The nss1 mutant was demonstrated to have a mutational lesion in the known VPS16 gene, which is essential for vacuolar protein sorting in S. cerevisiae. All of the vacuolar deficient mutants (vps11, vps16, vps18, and vps33) were sensitive to nystatin. Nystatin was found to cause extensive enlargement of the vacuole in wild-type S. cerevisiae cells. These results are discussed with special reference to the vacuolar function of S. cerevisiae.
nystatin; Saccharomyces cerevisiae; vacuole; protein sorting

-20-
Establishing the Independent Culture of a Strictly Symbiotic Bacterium
Symbiobacterium thermophilum from Its Supporting Bacillus Strain

Michiyo OHNO, Itsuho OKANO, Tomo-o WATSUJI, Teruhito KAKINUMA,
Kenji UEDA, and Teruhiko BEPPU・

Department of Applied Biological Sciences, College of Bioresource Sciences, Nihon University,
Kameino 1866, Fujisawa-shi, Kanagawa 252-8510, Japan

Received February 15, 1999; Accepted February 16, 1999
Symbiobacterium thermophilum is a strictly symbiotic thermophile, the growth of which is dependent on the coexistence of an associating thermophilic Bacillus sp., strain S. S. thermophilum grows only in mixed culture with the Bacillus strain in liquid media, and does not form visible colonies on solid media. To measure the growth of this symbiotic bacterium and to analyze its growth requirements, we developed a quantitative PCR method by using its specific sequences in a putative membrane translocator gene tnaT as primers. According to this method, independent growth of S. thermophilum was first confirmed in a dialyzing culture physically separated from Bacillus strain S with a cellulose membrane. Independent growth of S. thermophilum was also managed by adding conditioned medium prepared from the culture filtrate of the Bacillus strain, but the growth in the conditioned medium stopped at a very limited extent with appearance of filamentous cells, suggesting the uncoupling of cellular growth and cell division. Formation of micro-colonies of S. thermophilum was observed on the conditioned agar medium under both aerobic and anaerobic conditions, but the colony-forming efficiencies remained below 1％. Several other bacterial species, such as Bacillus stearothermophilus, Bacillus subtilis, Thermus thermophilus, and even Escherichia coli, were also found to support the growth of S. thermophilum. These results indicate that S. thermophilum essentially requires some ubiquitous metabolite(s) of low molecular weight produced by various bacterial species as growth factor(s) but coexistence of the living partner cells is still required, probably to maintain an effective level of the putative factor(s) in the medium.
microbial symbiosis; Symbiobacterium; quantitative PCR; growth factor

-21-
Note
Isolation and Characterization of the Actin Gene from the Cellulolytic Fungus
Humicola grisea and Analysis of Transcription Levels of Actin and
Cellulase Genes

Shou TAKASHIMA,・ Akira NAKAMURA, Makoto HIDAKA, Haruhiko MASAKI,・・
and Takeshi UOZUMI

Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo,
Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan

Received November 11, 1998; Accepted January 25, 1999
An actin gene was isolated from the cellulolytic fungus Humicola grisea. The gene structure, which has 5 introns in the coding region, is similar to those of the so far cloned fungal actin genes. But there are some differences in intron sizes and codon usage. Transcription levels of actin and cellulase genes were also investigated.
actin; cellulase; Humicola grisea

-22-
Note
Purification and Characterization of Recombinant Mortierella vinacea
α-Galactosidases I and II Expressed in Saccharomyces cerevisiae

Hajime SHIBUYA, Hideyuki KOBAYASHI,＊,・ Shigeki YOSHIDA, Satoshi KANEKO,＊
Gwi Gun PARK,＊＊ and Isao KUSAKABE

Institute of Applied Biochemistry, University of Tsukuba, Ibaraki 305-8572, Japan
＊National Food Research Institute, Ministry of Agriculture, Forestry, and Fisheries, Ibaraki 305-8642, Japan
＊＊Department of Food Engineering and Biotechnology, Kyungwon University, Kyunggi-do 461-701, Korea

Received November 12, 1998; Accepted February 1, 1999
The cDNAs coding for Mortierella vinacea α-galactosidases I and II were expressed in Saccharomyces cerevisiae under the control of the yeast GAL10 promoter. The recombinant enzymes purified to homogeneity from the culture filtrate were glycosylated, and had properties identical to those of the native enzymes except for improving the heat stability of α-galactosidase II and decreasing the specific activities of both enzymes.
α-galactosidase; Mortierella vinacea; expression; Saccharomyces cerevisiae

-23-
Note
Purification and Characterization of an Endo-Polygalacturonase
from a Mutant of Saccharomyces cerevisiae

Naoto HIROSE,・ Masao KISHIDA, Haruhiko KAWASAKI, and Takuo SAKAI・・

Department of Applied Biochemistry, Faculty of Agriculture, Osaka Prefecture University,
Gakuen-cho, Sakai, Osaka 599-8531, Japan
・・Department of Food Science, Faculty of Agriculture, Kinki University, Nakamachi, Nara 631-8505, Japan

Received November 18, 1998; Accepted February 12, 1999
An extracellular endo-polygalacturonase (PGase) produced by a mutant of Saccharomyces cerevisiae was isolated. The enzyme was regarded, immunologically, as a PGase belonging to the Kluyveromyces marxianus group. The enzyme had properties similar to the PGase from K. marxianus in heat and pH stability, and N-terminal amino acid sequence. However, the enzyme showed different properties in optimum pH and temperature, molecular weight, and reactivity in antiserum against PGase from K. marxianus, indicating that the enzyme has a different molecular structure from the PGase from K. marxianus.
polygalacturonase; Saccharomyces cerevisiae; mutant

-24-
Note
Dietary Effect of Conjugated Linoleic Acid on Lipid Levels
in White Adipose Tissue of Sprague-Dawley Rats

Masao YAMASAKI1, Keiko MANSHO1, Hiroko MISHIMA1, Masaaki KASAI2,
Michihiro SUGANO3, Hirofumi TACHIBANA1, and Koji YAMADA1,・

1Laboratory of Food Science, Department of Food Science and Technology, Faculty of Agriculture,
Kyushu University, Fukuoka 812-8581, Japan
2Nagoya Factory, Rinoru Oil Mills Co. Ltd., 37-15 Shiomi-cho, Minato-ku, Nagoya 455-0028, Japan
3Faculty of Human Life Sciences, Prefectural University of Kumamoto, Kumamoto 862-8502, Japan

Received November 30, 1998; Accepted February 2, 1999
We examined the effect of dietary conjugated linoleic acid (CLA) on lipid parameters in the liver, white adipose tissue (WAT) and brown adipose tissue (BAT) of Sprague-Dawley rats and found that it reduced the levels of triglycerides and non-esterified fatty acid in the liver and WAT without significant change in the BAT lipid levels. These results suggest that CLA has an obesity-preventing action.
conjugated linoleic acid; white adipose tissue; brown adipose tissue; liver

-25-
Note
Cloning and Sequencing of the β-Fructofuranosidase gene from Bacillus sp. V230

Masashi KURIMOTO, Keiji TSUSAKI, Michio KUBOTA,・ Shigeharu FUKUDA, and Yoshio TSUJISAKA

Hayashibara Biochemical Laboratories, Inc., 7-7 Amase-minami machi, Okayama 700-0834, Japan

Received December 2, 1998; Accepted Februaly 15, 1999
The β-fructofuranosidase gene (bff) from Bacillus sp. V230 has been cloned in Escherichia coli and its nucleotide sequence has been analyzed. The product of bff consists of a signal sequence of 32 amino acid (a.a.) residues for secretion and 455 a.a. residues of the extracellular β-fructofuranosidase. The a.a. sequence of the bff product has similarities with those of the Bacillus subtilis levanscrase (63.7％ identity), the Streptococcus mutans fructosyltransferase (33.7％), and the Zymomonas mobilis levanscrase and β-fructofuranosidase (15％).
β-fructofuranosidase; gene cloning; DNA sequence; amino acid homology

-26-
Note
Effect of Intracellular Glutathione on Heat-induced Cell Death
in the Cyanobacterium, Synechocystis PCC 6803

Katsuaki SUGINAKA,＊,＊＊ Keiko YAMAMOTO,＊ Hiroyuki ASHIDA,＊＊＊ Yoshihiro SAWA,＊
and Hitoshi SHIBATA＊,・

＊Department of Life Science and Biotechnology, Faculty of Life and Environmental Science,
Shimane University, Matsue, Shimane 690-8504, Japan
＊＊Institute of Industrial Science ＆ Technology of Shimane Prefecture, Higashiizumo-cho,
Shimane 690-0108, Japan
＊＊＊Research Institute for Molecular Genetics, Shimane University, Matsue, Shimane 690-8504, Japan

Received December 7, 1998; Accepted February 9, 1999
A correlation was found between the rate of cell death induced by heat and the GSH content of Synechocystis PCC 6803: cells accumulating GSH above the control level were more tolerant to heat than the control cells, and those containing a lower concentration of GSH were more sensitive. Lethal heating caused a decrease of GSH content, and a rapid intracellular oxidation in cells containing the decreased amount of GSH.
cyanobacteria; glutathione; lethal heat shock; oxidative stress

-27-
Note
Dephosphorylation of Specific Proteins during Induction of Senescence
in Immortal human Fibroblasts Expressing Thermolabile SV40 T Antigen

Michihiko FUJII, Atsuhiro JOGUCHI, Hideki OGINO, Kazuhiko NAKABAYASHI,
and Dai AYUSAWA・

Division of Biochemistry, Kihara Institute for Biological Research and Graduate School of Integrated Science,
Yokohama City University, Maioka-cho 641-12, Totsuka-ku, Yokohama 244-0813, Japan

Received December 15, 1998; Accepted February 2, 1999
Particular protein kinase inhibitors block a senescence-like phenomenon in SVts8 cells induced by a shift up in temperature. We characterized cellular proteins with affinity chromatography using one such inhibitor as a ligand. Two proteins of 56 and 100 kDa were found to be dephosphorylated specifically, probably due to induction of a protein phosphatase activity(s).
protein kinase inhibitor; protein phosphatase; H-9; senescence; human cell

-28-
Note
High-pressure Carbon Dioxide Effect on Kimchi Fermentation

Seok-In HONG＊ and Wan-Soo PARK

Korea Food Research Institute, San 46-1, Baekhyun, Bundang, Seongnam, Kyonggi 463-420, Korea

Received December 18, 1998; Accepted February 18, 1999
Baechu (Chinese cabbage) kimchi was treated with high-pressure carbon dioxide at 70 kg/cm2 for 24 h. The effect of this treatment on kimchi fermentation during storage at 10°C was investigated in terms of pH value, titratable acidity, lactic acid bacterial count, and sensory properties. Kimchi samples treated with pressurized CO2 had relatively a higher pH value, lower titratable acidity, and smaller lactic acid bacterial population than the untreated sample. A sensory evaluation showed that the sourness and overall acceptability of treated kimchi were better than those of the control. The color, flavor and texture were not significantly affected by the treatment. The results indicate that high-pressure CO2 treatment could be used as one of the applicable nonthermal processes for baechu kimchi preservation.
high-pressure CO2; baechu kimchi; nonthermal process

-29-
Note
Enantioselective Synthesis of Both the Enantiomers of Jasmine Ketolactone
and Its Epimer

Munenori INOUE, Tsunehiro NISHI, and Takeshi KITAHARA・

Department of Applied Biological Chemistry, Graduate School of Agricultural and Biological Sciences,
The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

Received December 24, 1998; Accepted February 15, 1999
The enantioselective synthesis of jasmine ketolactone 1, which was isolated as a component of Italian jasmine oil, and its epimer 2 is described. Lactones 1 and 2 were synthesized in 5 and 4 steps, respectively, by Yamaguchi′s macrolactonization method from alcohol 4.
jasmine ketolactone; Jasminum grandiflorum L.; macrolactonization; potato tuberization

-30-
Note
Indispensable Glutamic Acid Residue-139 of NtpK Proteolipid in the Reaction
of Vacuolar Na＋-Translocating ATPase in Enterococcus hirae

Kazuma TAKASE,・,・ Ichiro YAMATO,・ Kazuei IGARASHI,＊ and Yoshimi KAKINUMA＊

・Department of Biological Science and Technology, Science University of Tokyo, 2641 Yamazaki, Noda-shi,
Chiba 278-8510, Japan
＊Faculty of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan

Received December 25, 1998; Accepted March 3, 1999
Enterococcus hirae vacuolar ATPase catalyzes translocation of Na＋ or Li＋ coupled with ATP hydrolysis. It is suggested that the glutamic acid residue (Glu139) of NtpK proteolipid subunit of this multisubunit enzyme is the binding site of these ions for translocation. Here we established a complementation system for the ntpK gene with its deletion mutant, and found that the ATPase activity disappeared upon replacement of Glu139 by aspartic acid. The side-chain length of this acidic residue of NtpK is thus important for this ATPase reaction.
vacuolar ATPase; NtpK; complementation; Enterococcus hirae

-31-
Note
Antitumor Activity of Phenylahistin in Vitro and in Vivo

Kaneo KANOH,・ Shinkichi KOHNO, Jun KATADA, Yoshio HAYASHI, Michiko MURAMATSU,
and Isao UNO

Life Science Research Center, Advanced Technology Research Laboratories, Nippon Steel Corporation,
3-35-1 Ida, Nakahara-ku, Kawasaki 211-0035, Japan

Received January 11, 1999; Accepted February 11, 1999
Phenylahistin is a new cell cycle inhibitor produced by Aspergillus ustus. Since phenylahistin was produced as a scalemic mixture of (−)-phenylahistin and its enantiomer, we separated each enantiomer and evaluated their antitumor activity in vitro. (−)-Phenylahistin exhibited antitumor activity against 8 tumor cell lines with IC50 values ranging from 1.8×10−7 to 3.7×10−6, while (＋)-phenylahistin exhibited 33--100-fold less potent activity than (−)-phenylahistin did. (−)-Phenylahistin also showed antitumor activity against P388 leukemia and Lewis lung carcinoma cells in vivo.
phenylahistin; enantiomers; cell cycle inhibitor; antitumor activity

-32-
Note
Replication Origin Region of the Chromosome of Alkaliphilic
Bacillus halodurans C-125

Hideto TAKAMI・, Noriaki MASUI, Kaoru NAKASONE, and Koki HORIKOSHI

Deep-sea Microorganisms Research Group, Japan Marine Science and Technology Center,
2-15 Natsushima, Yokosuka, Kanagawa 237-0061, Japan

Received January 11, 1999; Accepted February 17, 1999
An 18.5-kb DNA fragment containing the oriC region of the chromosome of the alkaliphilic Bacillus halodurans C-125 was obtained by PCR and sequenced. Sixteen open reading frames (ORFs) were identified in this region. A sequencing similarity search using the BSORF database found that ORF1 to 13 all had significant similarities to gene products of Bacillus subtilis. Three other ORFs (ORF14-16) of unknown function were positioned downstream of gyrB instead of rrnO, which is found in the same region in the case of B. subtilis. The ORF organization from gidA to gyrA was the same as that of B. subtilis. The gene organization and the location of the DnaA-box region were also similar to those of the chromosomes of other bacteria, such as Escherichia coli and Pseudomonas putida. There were two DnaA-box clusters (Box-region C and R) with a consensus sequence TTATCCACA on both sides of the dnaA gene but another DnaA box cluster (Box-region L) which is found in the region between thdF and jag in B. subtilis was not found in the corresponding region in the case of alkaliphilic Bacillus halodurans C-125.
alkaliphilic Bacillus halodurans C-125; DNA replication; dnaA; DnaA box

-33-
Note
Purification of Chitinolytic Protein from Rehmannia glutinosa Showing

N-terminal Amino Acid Sequence Similarity to Thaumatin-Like Proteins
Cheol-Ho PAN, Eun-A LEE, Young-Am CHAE, and Su-Il KIM

Department of Agricultural Chemistry and Research Center for New Bio-Materials in Agriculture,
College of Agriculture and Life Sciences, Seoul National University, Suwon 441-744, Korea

Received January 12, 1999; Accepted February 18, 1999
We have purified a 21-kDa protein, designated as P1, from Rehmannia glutinosa to homogeneity by ammonium sulfate precipitation, anion exchange chromatography, hydrophobic interaction chromatography, and preparative native PAGE. The purified P1 had chitin degradation activity. The N-terminal amino acid sequence of P1 indicated that it is very similar to those of thaumatin and other reported thaumatin-like proteins.
chitinolytic protein; thaumatin-like protein; pathogenesis-related protein; Rehmannia glutinosa

-34-
Note
Microbial Synthesis of Coniferyl Alcohol by the Fungus Byssochlamys fulva V107

Hirotaka FURUKAWA,1 Marco WIESER,2 Hiroshi MORITA,1 and Toru NAGASAWA2,・

1Chisso Corporation, Yokohama Research Center, Kanazawa-ku, Yokohama 236-8605, Japan
2Department of Biomolecular Science, Faculty of Engineering, Gifu University, Gifu 501-1112, Japan

Received January 18, 1999; Accepted February 25, 1999
Coniferyl alcohol (123 mM＝21.9 g/l) was synthesized from eugenol with a yield of 94.6％ in a 36 h fed-batch bioconversion using resting cells of the fungus Byssochlamys fulva V107.
flavors; fragrances; ligninolysis; coniferyl alcohol; fungal oxidoreductase

-35-
Note
Tyrosine-83 of Human Renin Contributes to Biphasic pH Dependence
of the Renin-Angiotensinogen Reaction

Uddin Mohammad NASIR,＊ Fumiaki SUZUKI,＊＊,・ Takao NAGAI,＊＊＊ Tsutomu NAKAGAWA,＊＊＊
and Yukio NAKAMURA＊＊＊

＊United Graduate School of Agricultural Science, ＊＊Molecular Genetics Research Center, and
＊＊＊Department of Biotechnology, Faculty of Agriculture, Gifu University, 501-1193, Gifu Japan

Received February 9, 1999; Accepted March 3, 1999
The pH dependence of the reaction of human renin with sheep angiotensinogen had a couple of peaks at pH 6.5 and 8.7. The renin activity at pH 8.7 was 1.4 times higher than that at pH 6.5. After the substitutions of Phe, Ala, and His for Tyr83 of human renin, the peak at pH 6.5 could be observed but the peak at pH 8.7 disappeared. At pH 6.5, these substitutions reduced the kcat of human renin to 11.1, 1.31, and 3.49％ of that of wild-type renin, respectively, while their Km remained at similar levels to that of the wild type. These results indicate that Tyr83 of human renin contributes to the renin-angiotensinogen reaction at both pHs and it is essential for the catalytic reaction particularly at the basic pH.
renin; Tyrosine-83; catalytic mechanism; pH dependence

-36-
Preliminary Communication
Synthesis of 4′-C-Ethynyl-β-D-arabino- and 4′-C-Ethynyl-2′-deoxy-β-D-ribo-
pentofuranosyl Pyrimidines, and Their Biological Evaluation

Satoru KOHGO, Hiroko HORIE, and Hiroshi OHRUI＊

Department of Applied Biological Chemistry, Faculty of Agriculture, Tohoku University,
1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan

Received March 4, 1999; Accepted April 2, 1999
4′-C-Ethynyl-β-D-arabino-pentofuranosyl thymine (14) and cytosine (16), and 4′-C-ethynyl-2′-deoxy-β-D-ribo-pentofuranosyl thymine (25) and cytosine (27) were synthesized by properly protected 4′-C-hydroxy-methyl-3,5-di--O--benzyl-α-D-ribo-pentofuranose (1) from D-glucose. Among them, 2′-deoxy derivatives 25 and 27 exhibited antiviral activity, while cytidine derivatives 16 and 27 inhibited the growth of neoplastic cells.
4′-C-substituted nucleoside; ethynyl group; arabinosyl pyrimidine; 2′-deoxyribosyl pyrimidine; biological activity

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