(Vol.61 No.2 1997)
Aminophosphonic and
Aminoboronic Acids as Key Elements of a Transition State Analogue Inhibitor
of Enzymes
Jun Hiratake and Jun'ichi Oda 211
Research Progress
in Production of Bacterial Cellulose by Aeration and Agitation Culture
and Its Application as a New Industrial Material
Fumihiro Yoshinaga, Naoto Tonouchi, and Kunihiko Watanabe 219
Potential D,L-Amino Acid Sequence
Analysis of Peptides from the C-Terminus
Hiroshi Ohrui, Eigo Itoh, Yoshihiro Nishida, Hiroko Horie, and Hiroshi
Meguro 392
Synthesis of 5-Deoxy-5-oxomilbemycin A4 5-Hydrazone
Derivatives and Their Activity against Tetranychus urticae
Yoshihisa Tsukamoto, Kazuo Sato, Keiji Tanaka, and Toshiaki Yanai 304
Cell Type- and Positionally Specific
Regulation of the Aldolase P Gene Expression in Rice Seedlings
Hisako Nakamura, Yasushi Tokairin, Seiko Tamayama, Saiichi Kon, Soh Hidaka,
Shin-ichiro Ejiri, and Ken-ichi Tsutsumi 256
Apolipoprotein A-1
of Japanese Quail: cDNA Sequence and Modulation of Tissue Expression by
Cholesterol Feeding
Hirosuke Oku, Takayoshi Toda, Junichi Nagata, Makoto Ishikawa, Kimihiko
Neyazaki, Choyu Shinjyo, and Isao Chinen 286
Cell Lysis Induced by Ricin
D and Ricin E in Various Cell Lines
Tatsuya Oda, Nobukazu Komatsu, and Tsuyoshi Muramatsu 291
Gypsophilin, a New Type 1 Ribosome-inactivating
Protein from Gypsophila elegans :
Purification, Enzymatic Characterization, and Subcellular Localization
Shigeo Yoshinari, Shigehiro Koresawa, Sadaki Yokota, Hiroshi Sawamoto,
Minoru Tamura, and Yaeta Endo 324
Inhibition by Hop
Bract Polyphenols of Cellular Adherence and Water-insoluble Glucan Synthesis
of Mutans Streptococci
Motoyuki Tagashira, Keiko Uchiyama, Tomoaki Yoshimura, Masayuki Shirota,
and Nobuo Uemitsu 332
Release
of Ectoenzymes from Small Intestine Brush Border Membranes of Mice by Phospholipases
Chisako Itami, Ryo Taguchi, Hiroh Ikezawa, and Toshikatsu Nakabayashi
336
Immunochemical Approach to Characterize
Post-translational Modification of Serum
Albumin Using Anti-glutaraldehyde-treated Serum Albumin Antibodies
Hiroyuki Ukeda, Toshinao Ishii, Yoshimitsu Shimizu, Masayoshi Sawamura,
and Hirozo Kusunose 341
Inhibition
of Arachidonate Lipoxygenase Activities by 2-(3,4-Dihydroxyphenyl)ethanol,
a Phenolic Compound from Olives
Noriko Kohyama, Tadahiro Nagata, Shin-ichi Fujimoto, and Keizo Sekiya 347
Sphingoid Base Composition of
Cerebrosides from Plant Leaves
Hiroyuki Imai, Masao Ohnishi, Kenmi Hotsubo, Mitiyuki Kojima, and Seisuke
Ito 351
Preparation of Dye-labeled
Alginate for the Assay of Alginate Lyase
Shigeki Yoshida, Sayaka Watanabe, Toshio Takeuchi, Katsumi Murata,
and Isao Kusakabe 357
Substrate Specificities of Ώ-Galactosidases
from Yeasts
Shigeki Yoshida, Chin Hui Tan, Tomoko Shimokawa, Hilkka Turakainen,
and Isao Kusakabe 359
Investigation of
Flavonoid Aglycones in Propolis Collected by Two Different Varieties
of Bees in the Same Region
Michel Hyun Koo and Yong Kun Park 367
Daidzein Stimulation
of Bone Resorption in Pit Formation Assay
Hiroyasu Tobe, Osamu Komiyama, Yoshiko Komiyama, and Hiromi B. Maruyama
370
Expression
of Alkaline Protease Gene in Bacillus subtilis Mutants That Lack Positive
Regulatory Genes degR, degQ, senS, tenA, and proB
Mitsuo Ogura and Teruo Tanaka 372
Amino Acid Sequence
and Characterization of Aldo-keto Reductase from Bakers' Yeast
Kaoru Nakamura, Shin-ichi Kondo, Yasushi Kawai, Nobuyoshi Nakajima, and
Atsuyoshi Ohno 375
44-Homooligomycin E,
a New Cytotoxic Macrolide Antibiotic from Streptomyces ostreogriseus
Hang Sub Kim, Hee Jae Bang, Sang Yong Lee, Ook Joon Yoo, Jin Cheol Yoo,
Young Ho Kim, and Jung Joon Lee 378
The Specificity of Peptide
Bond Cleavage of Acid Proteinase A from Aspergillus niger
var. macrosporus toward Oxidized Ribonuclease A
Kenji Takahashi 381
Purification and Characterization
of the NADP-Malic Enzyme from Bradyrhizobium japonicum A1017
Fan Chen, Youichi Okabe, Kaoru Osano, and Shigeyuki Tajima 384
Mutations within the Reactive-site Region
of Human Pancreatic Secretory Trypsin
Inhibitor Confer Ώ-Thrombin and Factor Xa Inhibitory Activities
Takaaki Katoh, Mikio Tamaki, Norihisa Kikuchi, Hiroshi Teraoka, Kiyoshi
Nagata, and Nobuo Yoshida 389
Effects of Nucleotides
on Cyanide-resistant Respiratory Activity in Mitochondria Isolated from
Antimycin A-treated Yeast Hansenula anomala
Shigeru Sakajo, Nobuko Minagawa, and Akio Yoshimoto 396
Prodigiosin 25-C
and Metacycloprodigiosin Suppress the Bone Resorption by Osteoclasts
Je-Tae Woo, Yasuo Ohba, Kahori Tagami, Koji Sumitani, Takao Kataoka,
and Kazuo Nagai 400
Effect of
Glutathione, Catechin, and Epicatechin on the Survival of Drosophila melanogaster
under Paraquat Treatment
Seok Joong Kim, Daeseok Han, Byung-Hak Ahn, and Joon Shick Rhee 225
Reduction of Renal
Transforming Growth Factor-ΐ Activity without Aggravation of Growth Retardation
in Nephritic Rats by a Methionine-threonine-supplemented Low-casein Diet
Kiyohito Fujisawa, Kazumi Yagasaki, Saori Ishizuka, Yutaka Miura, and
Ryuhei Funabiki 230
Stabilizing Effect of
Tropomyosin on Actin during Storage at Low Temperature
Hiroyuki Tanji, Yoshihide Ikeuchi, Ryosuke Shimizu, and Atsushi Suzuki
233
The Structures of Phosphoryl
Oligosaccharides Prepared from Potato Starch
Hiroshi Kamasaka, Kenji To-o, Kaname Kusaka, Takashi Kuriki, Takashi
Kometani, Hideo Hayashi, and Shigetaka Okada 238
Spice Constituents Scavenging
Free Radicals and Inhibiting Pentosidine Formation in a Model System
Tomoko Oya, Toshihiko Osawa, and Shunro Kawakishi 263
Polysaccharide from Aspalathus
linearis with Strong Anti-HIV Activity
Masatoshi Nakano, Yoshiko Itoh, Toshiaki Mizuno, and Hideki Nakashima
267
Characterization of a
Water-soluble Polysaccharide Fraction with Immunopotentiating Activity
from Bifidobacterium adolescentis M101-4
Akira Hosono , Jonghwa Lee , Akio Ametani, Midori Natsume, Masao Hirayama,
Takashi Adachi, and Shuichi Kaminogawa 312
Serum Cholesterol Reduction
and Cholesterol Absorption Inhibition in CaCo-2 Cells by a Soyprotein Peptic
Hydrolyzate
Satoshi Nagaoka, Takako Awano, Naoko Nagata, Motoki Masaoka, Goro Hori,
and Kei Hashimoto 354
Effect of Acetic Acid Bacterium on
Ethanol Oxidation in Vivo
Yukihiro Nomura, Masanori Yamamoto, and Tohru Fushiki 365
Occurrence of Acid-phosphatase Inhibitors in
Soybeans
Kenjiro Tadera, Yuji Minami, Masashi Uehune, Akira Nozaki, Fumio Yagi,
and Toshihiko Suganuma 387
Cloning, Sequencing, and Expression
of a Thermostable Cellulase Gene of Humicola grisea
Shou Takashima, Akira Nakamura, Haruhiko Masaki, and Takeshi Uozumi
245
Purification, Properties, and
N-Terminal Amino Acid Sequences of Guar Gum-degrading Enzyme from Bacillus
circulans K-1
Seiji Yosida, Yoshihiko Sako, and Aritsune Uchida 251
Cloning and Expression of
Purine Nucleoside Phosphorylase I Gene from Bacillus stearothermophilus
TH 6-2
Tomoki Hamamoto, Kiyoshi Okuyama, Toshitada Noguchi, and Yuichiro Midorikawa
272
Cloning of Purine Nucleoside
Phosphorylase II Gene from Bacillus stearothermophilus TH 6-2 and Characterization
of Its Gene Product
Tomoki Hamamoto, Toshitada Noguchi, and Yuichiro Midorikawa 276
Cloning and Expression
of an Intracellular Alkaline Protease Gene from Alkalophilic
Thermoactinomyces sp. HS682
Katsumi Tsuchiya, Ichiro Ikeda, Takashi Tsuchiya, and Tetsu Kimura 298
Microbilogical Aspects
of Acetate Oxidation by Acetic Acid Bacteria, Unfavorable
Phenomena in Vinegar Fermentation
Akihiko Saeki, Mariko Taniguchi, Kazunobu Matsushita, Hirohide Toyama,
Gunjana Theeragool, Napha Lotong, and Osao Adachi 317
A Gene Encoding Endo-1,4-ΐ-glucanase from
Bacillus sp. 22-28
Munetoshi Miyatake and Kiyohisa Imada 362
-1-
Review
Aminophosphonic and
Aminoboronic Acids as Key Elements of a Transition State Analogue Inhibitor
of Enzymes
Jun Hiratake and Jun'ichi Odaυ
Institute for Chemical Research, Kyoto University, Uji, Kyoto 611, Japan
@Amino acid analogues are of considerable interest as inhibitors of enzymes
involved in amino acid and peptide metabolism. In particular, Ώ-aminoalkylphosphonic
acids and Ώ-aminoalkylboronic acids, in which the carboxyl group of amino
acids is replaced by a phosphonic acid or boronic acid function, respectively,
constitute a unique class of amino acid mimics from which a number of potent
enzyme inhibitors have been prepared. The inhibitory activity mainly stems
from the fact that the tetrahedral phosphonic moiety or the tetrahedral
adduct of electrophilic boronic acid is a good mimic of the putative tetrahedral
transition state or intermediate encountered in the enzymatic hydrolysis
or formation of peptides. Since the peptide hydrolysis and formation invariably
involves the tetrahedral high energy species in the course of the reaction,
these amino acid mimics serve as a general key element for inhibitors of
a broad spectrum of proteases and peptide ligases. The transition state
analogy of aminophosphonic-
and aminoboronic acid-derived inhibitors also gives a clue to the detailed
reaction mechanisms of the enzymes by X-ray crystallographic and NMR analysis
of the enzyme-inhibitor complex.
Key words: aminoalkylphosphonic acid; aminoalkylboronic acid; transition-state
analogue inhibitor; proteases; synthetases
-2-
Review
Research Progress
in Production of Bacterial Cellulose by Aeration and Agitation Culture
and Its Application as a New Industrial Material
Fumihiro Yoshinaga, Naoto Tonouchi, and Kunihiko Watanabe
Bio-Polymer Research Co., Ltd., 3-2-1 Sakato, Takatsu-ku, Kawasaki-shi,
Kanagawa 213, Japan
@Some Acetobacter strains produce a cellulose called bacterial cellulose
in culture medium. This cellulose has unique structural and mechanical
properties compared with higher plant cellulose, and is expected to be
a commodity material in various fields of industry. For economical mass
production, it is essential to construct an aeration and agitation culture
process. To explore the industrial applications of the bacterial cellulose,
the structural features and physicochemical properties need to be understood
and potentially improved. This paper reviews recent progress in research
on this bacterial cellulose.
Key words: Acetobacter; cellulose; production; agitation culture; structure;
application;
physicochemical property
-3-
Effect of
Glutathione, Catechin, and Epicatechin on the Survival of Drosophila melanogaster
under Paraquat Treatment
Seok Joong Kim, Daeseok Han,υ Byung-Hak Ahn, and Joon Shick Rhee*
Korea Food Research Institute,Bundang, Songnam, Kyonggi 463-420, Korea
* Department of Biotechnology,Korea Advanced Institute of Science and Technology,
Taejon 305-701, Korea
Received December 6, 1995
@The biological effect of antioxidants which showed high superoxide-scavenging
(SOS) activity in an in vitro analysis was examined by using Drosophila
melanogaster. When the flies were exposed to paraquat as an endogenous
source of the superoxide anion, their survival rapidly decreased. Although
the SOS antioxidants did not have a preventive effect against paraquat
toxicity, a supplement of such SOS antioxidants as glutathione, (+)-catechin
and/or (-)-epicatechin to the diet had a reparative effect on flies damaged
by the superoxide anion.
@The survival ratio of flies fed on a diet enriched with SOS antioxidants
ranged from 77% to 87%, while that of the control group was 56%. When flies
were exposed to paraquat in the presence of hydrogen peroxide or iron,
each combination was more toxic than paraquat alone, since the two compounds
could accelerate the generation of reactive oxygen species in vivo. The
SOS antioxidants, however, allowed the flies to resist the combined toxicity
of paraquat and ferrous iron.
Key words: superoxide-scavenging activity; paraquat; Drosophila melanogaster;
antioxidant;
superoxide dismutase
-4-
Reduction of Renal
Transforming Growth Factor-ΐ Activity without Aggravation of
Growth Retardation in Nephritic Rats by a Methionine-threonine-supplemented
Low-casein Diet
Kiyohito Fujisawa, Kazumi Yagasaki,υ Saori Ishizuka, Yutaka Miura, and Ryuhei Funabiki
Department of Applied Biological Science, Tokyo Noko University, Fuchu,
Tokyo 183, Japan
Received January 8, 1996
@The effects of a low-casein diet fortified with methionine and threonine
on renal cortical and glomerular transforming growth factor (TGF)-ΐ activity
were studied in rats with nephritis induced by anti-rat kidney glomerular
basement membrane antiserum. Both normal and nephritic rats were fed experimental
diets for 10 days. An injection of nephrotoxic serum increased urinary
protein excretion and renal TGF-ΐ activity. A methionine-threonine-supplemented
8.5% casein diet, compared with a basal 20% casein diet, decreased these
two measurements without aggravating growth retardation in nephritic rats.
These results suggest that aggravation and alleviation of symptoms incident
to anti-GBM nephritis are relevant to elevation and reduction of TGF-ΐ
activity, respectively.
@The results also suggest that amino acid-balanced low-protein diets would
have beneficial effects on glomerulonephritis without causing severe protein
malnutrition.
Key words: nephritis; transforming growth factor-ΐ; proteinuria; low-casein
diet; methionine
-5-
Stabilizing Effect of
Tropomyosin on Actin during Storage at Low Temperature
Hiroyuki Tanji, Yoshihide Ikeuchi,*,υ Ryosuke Shimizu, and Atsushi Suzuki
Basic Research Department, Prima Meat Packers Co., Ltd., Tsuchiura,
Ibaraki 300, Japan
* Department of Applied Biological Chemistry, Faculty of Agriculture, Niigata
University, Niigata 950-21, Japan
Received June 3, 1996
@The effects of tropomyosin (TM) on the stability of actin with and without
heavy meromyosin (HMM) during storage at low temperature were studied by
the DNase I inhibition assay. The stabilizing effect of TM on actin without
ATP at 0C was associated with the extent of binding of TM to the actin
filaments that was dependent on the KCl concentrations: i.e., TM effectively
depressed the denaturation of actin at a KCl concentration ranging from
0.1 to 0.5 M whereby TM can be bound to F-actin. Stabilization by TM could
reduce the probability of the destabilization of actin due to a small amount
of HMM. A combination of TM and a large amount of HMM strongly stabilized
actin, even in a high salt concentration such as 0.6 M KCl. These results
indicate that tropomyosin played an important role in stabilizing actin
in an actomyosin complex (natural actomyosin) during treatment with salt
at a low temperature.
Key words: denaturation; F-actin; tropomyosin; HMM; DNase I inhibition
assay
-6-
The Structures of Phosphoryl
Oligosaccharides Prepared from Potato Starch
Hiroshi Kamasaka, Kenji To-o, Kaname Kusaka, Takashi Kuriki, Takashi Kometani, Hideo Hayashi,* and Shigetaka Okada
Biochemical Research Laboratory, Ezaki Glico Co., Ltd., 4-6-5 Utajima,
Nishiyodogawa-ku, Osaka 555, Japan
* Laboratory of Natural Products Chemistry, Department of Applied Biological
Chemistry, College of Agriculture,
Osaka Prefecture University, 1-1 Gakuen-cho, Sakai, Osaka 593, Japan Received
June 13, 1996
@In the previous study, we proposed that phosphoryl oligosaccharides (POs)
prepared from potato starch had an inhibitory effect on the formation of
a calcium phosphate precipitate in vitro. In this study, we investigated
the structures of the phosphoryl oligosaccharide-1 (PO-1) fraction that
was the main component of POs. By treating with bacterial saccharifying
Ώ-amylase (BSA) after glucoamylase (GA), the PO-1 fraction produced 32-phosphoryl
maltotriose from 3-phosphoryl oligosaccharides, and 63-phosphoryl maltotriose
from 6-phosphoryl oligosaccharides. These products were characterized spectrometrically
as well as chemically, including measurement of the amounts of the non-reducing-terminal
residue, reducing-terminal residue, and organic phosphate. A small amount
of 62-phosphoryl maltose was also detected after treating with GA alone,
indicating that 62-phosphoryl maltotriose existed in the PO-1 fraction.
According to the reaction specificities of GA and BSA, we conclude that
the PO-1 fraction was made up
of 3-phosphoryl oligosaccharides (33-phosphoryl maltotetraose and 34-phosphoryl
maltopentaose) and 6-phosphoryl oligosaccharides (63-phosphoryl maltotriose,
62-phosphoryl maltotriose, 63-phosphoryl maltotetraose, and 64-phosphoryl
maltopentaose).
Key words: phosphoryl oligosaccharides; potato starch
-7-
Cloning, Sequencing, and Expression
of a Thermostable Cellulase Gene of Humicola
griseaυ
Shou Takashima, Akira Nakamura, Haruhiko Masaki, and Takeshi Uozumi
* Department of Biotechnology, Faculty of Agriculture, The University
of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113, Japan
Received June 20, 1996
@The egl2 gene encoding a thermostable endoglucanase (EGL2) was cloned
from Humicola grisea. The DNA sequence of egl2 predicted two putative introns
in the coding region. The deduced amino acid sequence of EGL2 was 388 amino
acids in length and showed 99.5% identity with the H. insolens CMC 3. In
addition to TATA box and CAAT motifs, putative CREA binding sites were
observed in the 5' upstream region of the egl2 gene. The egl2 gene was
expressed in Aspergillus oryzae, and EGL2 was purified. EGL2 produced by
A. oryzae showed a high activity toward carboxymethyl cellulose. The optimal
temperature of EGL2 was 75C, and EGL2 had more than 80% residual activity
after heating up to 75C for 10 min. This is the first report of enzymatic
properties of the EGL2-type thermostable cellulase homologs from Humicola.
Key words: cellulase; Humicola grisea
-8-
Purification, Properties, and
N-Terminal Amino Acid Sequences of Guar Gum-degrading Enzyme from Bacillus
circulans K-1
Seiji Yosida, Yoshihiko Sako,* and Aritsune Uchida*
Research Institute of Technology, Konoike Construction Co., Ltd., 4-3-55
Denpo, Konohana-ku, Osaka 554, Japan
* Laboratory of Microbiology, Department of Fisheries, Faculty of Agriculture,
Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto 606, Japan
Received June 20, 1996
@A guar gum-degrading enzyme of the newly isolated Bacillus circulans
K-1 was purified to an electrophoretically homogeneous state. The molecular
weight of the purified enzyme was 62,000 by SDS-PAGE. The purified enzyme
was separated into at least six isozymes by isoelectric focusing and the
pI of these isozymes were 5.4, 5.5, 5.6, 5.8, 6.0, and 6.2, respectively.
The N-terminal amino acid sequences of the typical three of these proteins
were all the same, Ala-Ser-Gly-Phe-Tyr-Val-Ser-Gly-Thr-Lys-Leu-Leu-Asp-Ala-Thr-Gly-Gln-Pro-Phe-Val-Met-Arg.
The enzyme was most active at pH 6.9 and at 64C. The enzyme was activated
slightly by Al3+ and inhibited strongly by Sn2+ and Zn2+, N-bromosuccinimide,
2-mercaptoethanol, and ethylenediamine-tetraacetic acid.
Key words: guar gum-degrading enzyme; mannanase; N-terminal amino acid
sequence; isozyme;
isoelectric focusing
-9-
Cell Type- and Positionally Specific
Regulation of the Aldolase P Gene Expression in Rice Seedlings
Hisako Nakamura, Yasushi Tokairin,υ Seiko Tamayama,* Saiichi Kon,* Soh Hidaka,** Shin-ichiro Ejiri, and Ken-ichi Tsutsumiυυ
Institute for Cell Biology and Genetics, Faculty of Agriculture, Iwate
University, Ueda, Morioka, Iwate 020, Japan
* Department of Dermatology, Iwate Medical School, Uchimaru, Morioka, Iwate
020, Japan
** Department of Crop Breeding, Tohoku National Agricultural Experiment
Station, Shimo-kuriyagawa, Morioka, Iwate 020-01, Japan
Received June 24, 1996
@We describe here different regulation of the AldP gene, a nuclear gene
encoding chloroplast aldolase, in different tissues and growth ages of
rice seedlings. Expression of the AldP gene is mesophyll cell-specific,
and increases from the basal to the upper region in each leaf. The gene
expression is repressed in the dark-grown leaf blade, but is induced by
a short-term-exposure to light, to a level higher than that seen in the
normal leaf blade. However, the light-inducibility differs among the tissues,
and shows different patterns among leaf positions; i.e., the extent of
light-induction is higher in the third leaf blade as compared with the
earlier developed second leaf blade. Such positional differences in the
regulation are also seen in the leaf sheath. These responses are not accompanied
by changes of the cell type specificity in the expression.
Key words: aldolase P gene; mesophyll cell; differentiation; light-mediated
expression; Oryza sativa L.
-10-
Spice Constituents Scavenging
Free Radicals and Inhibiting Pentosidine Formation in a Model System
Tomoko Oya, Toshihiko Osawa, and Shunro Kawakishi
Laboratory of Food and Biodynamics, Department of Applied Biological
Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-01, Japan
Received June 27, 1996
@Many antioxidants have been found in spices and herbs, and some of them
are well known as strong scavengers of active oxygen radicals. We have
isolated active products, which markedly inhibited the formation of malondialdehyde
(MDA) from 2-deoxyribose and the hydroxylation of benzoate with the hydroxyl
radical, from methanol extracts of allspice and clove. Pimentol from allspice,
and biflorin and its isomer, abbreviated as clove3, from clove were identified
as the active principles. These revealed strong activity as hydroxyl radical
scavengers at a concentration of 2.0 ΚM. The antioxidative activities
in an in vitro model system involving the rabbit erythrocyte membrane ghost
were as strong as those of Ώ-tocopherol at 200 ΚM. Such advanced glycation
end products (AGE) as pentosidine are biomarkers of diabetes mellitus,
and active oxygens have been suggested to be involved in the formation
of AGE. The above-mentioned free radical scavengers effectively inhibited
the formation of pentosidine in a model system of N Ώ-t-butoxycarbonyl-fructoselysine
and N Ώ-t-butoxycarbonyl-arginine.
Key words: radical scavenger; antioxidant; spice; Maillard reaction; advanced
glycation end products
-11-
Polysaccharide from Aspalathus
linearis with Strong Anti-HIV Activity
Masatoshi Nakano,υ Yoshiko Itoh, Toshiaki Mizuno, and Hideki Nakashima*
Institute for Medical Science of Aging, Aichi Medical University, Nagakute,
Aichi 480-11, Japan
* Department of Microbiology, Yamanashi Medical University, 1110 Shimokato,
Tamaho, Nakakoma-gun, Yamanashi 409-38, Japan
Received June 27, 1996
@Polysaccharide that had been extracted with 1% sodium carbonate from
Rooibos leaves (Aspalathus linearis) showed strong anti-HIV activity. Du-Zhong
leaves also showed anti-HIV activity, although lower than the extract of
Aspalathus linearis, but Japanese tea leaves and a hot water extract of
Aspalathus linearis did not. The anti-HIV activity of the alkaline extract
from Aspalathus linearis was recovered mainly in the 25-75% ethanol-precipitated
fraction. The polysaccharide almost completely inhibited the binding of
HIV-1 to MT-4 cells. It is inferred from these results that the polysaccharide
from Aspalathus linearis is involved in the mechanism for virus binding
to T cells.
Key words: anti-HIV activity; polysaccharides; in vitro; alkaline extract;
Aspalathus linearis
-12-
Cloning and Expression of
Purine Nucleoside Phosphorylase I Gene from Bacillus stearothermophilus
TH 6-2
Tomoki Hamamoto, Kiyoshi Okuyama, Toshitada Noguchi, and Yuichiro Midorikawa
Research & Development Division, Yamasa Corporation, Choshi, Chiba
288, Japan Received July 2, 1996
@Bacillus stearothermophilus TH 6-2 has two kinds of purine nucleoside
phosphorylases (Pu-NPase I and Pu-NPase II). The Pu-NPase I is a functional
homolog of eukaryotic purine nucleoside phosphorylases that can catalyze
the phosphorolysis of inosine and guanosine, but not adenosine, the primary
substrate of Pu-NPase II. The Pu-NPase I gene of TH 6-2 has been cloned,
sequenced, and expressed in E. coli. The gene corresponded to an open reading
frame of 822 nucleotides that translates into a putative 274-amino acid
protein with a molecular weight of 29,637. The deduced amino terminus sequence
completely coincided with that found for the purified enzyme. The cloned
gene was overexpressed in E. coli by using the trc promoter to produce
an active enzyme in large quantities. The amino acid sequence of Pu-NPase
I shared 50% similarity with those of human and mouse purine nucleoside
phosphorylases.
Key words: Bacillus stearothermophilus; purine nucleoside phosphorylase;
gene cloning; sequence homology
-13-
Cloning of Purine Nucleoside
Phosphorylase II Gene from Bacillus stearothermophilus TH 6-2 and Characterization
of Its Gene Product
Tomoki Hamamoto, Toshitada Noguchi, and Yuichiro Midorikawa
Research & Development Division, Yamasa Corporation, Choshi, Chiba
288, Japan Received July 2, 1996
@Bacillus stearothermophilus TH 6-2 has two kinds of purine nucleoside
phosphorylases (Pu-NPases). The type I enzyme (Pu-NPase I) is a functional
and structural homolog of eukaryotic purine nucleoside phosphorylases that
catalyze the phosphorolysis of inosine, guanosine, and their derivatives.
The type II enzyme (Pu-NPase II) is a minor enzyme that efficiently phosphorolyzes
adenosine and its derivatives rather than other purine nucleosides like
Escherichia coli Pu-NPase. The gene coding for Pu-NPase II (punB gene)
has been cloned and sequenced from TH 6-2 strain. The deduced amino acid
sequence of Pu-NPase II shared 54% identity with that of E. coli enzyme,
while it had no significant homology to that of Pu-NPase I or eukaryotic
enzymes. By producing the Pu-NPase II in E. coli cells, the Pu-NPase II
has been purified and characterized.
Key words: Bacillus stearothermophilus; purine nucleoside phosphorylase;
gene cloning
-14-
Aqueous Oxidation
of Ethyl Linoleate, Ethyl Linolenate, and Ethyl Docosahexaenoate
Shinya Hirano, Kazuo Miyashita,υ Toru Ota, Masazumi Nishikawa,* Kazuki Maruyama,* and Suguru Nakayama*
Faculty of Fisheries, Hokkaido University, 3-1-1 Minatocho, Hakodate
041, Japan
* Central Research Institute, Maruha Co., 16-2 Wadai, Tsukuba, Ibaraki
300-42, Japan
Received July 3, 1996
@The oxidative stability of ethyl linoleate (LA), ethyl linolenate (LN),
and ethyl docosahexaenoate (DHA) in an aqueous solution was compared by
measuring the decrease in unoxidized substrate and the formation of total
hydroperoxides, conjugated dienes, and monohydroperoxides (MHPs). The highest
stability was shown by DHA, this being followed by LN and LA in both cases
when using Fe2+-ascorbic acid and 2,2'-azobis(2,4-dimethylvaleronitrile)
(AMVN) as a initiator, while the reverse order of oxidative stability was
obtained when the esters were oxidized in chloroform or ethanol with AMVN.
The stability of MHPs in an aqueous solution also increased with increasing
degree of unsaturation. HPLC analyses showed little difference in the positional
distribution of MHP isomers between aqueous oxidation and autoxidation
in the air. This result suggests no selective abstraction of hydrogen atoms
from the bisallylic positions of polyunsaturated fatty acid in an aqueous
phase. The product ratio of hydroperoxy epidioxides to MHPs in the aqueous
oxidation of LN was lower than that of autoxidized LN in the air, showing
the rapid decomposition of epidioxides in an aqueous solution.
Key words: aqueous oxidation; oxidative stability; DHA; oxidation products
-15-
Apolipoprotein A-1
of Japanese Quail: cDNA Sequence and Modulation of Tissue Expression by
Cholesterol Feeding
Hirosuke Oku,υ Takayoshi Toda,* Junichi Nagata, Makoto Ishikawa, Kimihiko Neyazaki, Choyu Shinjyo,** and Isao Chinen
Laboratory of Applied Biochemistry, Faculty of Agriculture, University
of The Ryukyus, Nishihara-cho, Okinawa 903-01, Japan
* Department of Clinical Laboratory Medicine, School of Medicine, University
of The Ryukyus, Nishihara-cho, Okinawa 903-01, Japan
** Laboratory of Plant and Animal Breeding, Faculty of Agriculture, University
of The Ryukyus, Nishihara-cho, Okinawa 903-01, Japan
Received July 10, 1996
@Apolipoprotein (apo) A-1 cDNA was amplified by the reverse-transcriptase-polymerase
chain reaction (RT-PCR). Primers were synthesized according to the nucleotide
sequence of chicken apo A-1, and the identity of apo A-1 cDNA was confirmed
by comparing with the N-terminal amino acid sequence. The open reading
frame of apo A-1 cDNA consists of 795 nucleotides, and it is capable of
coding a polypeptide of 264 amino acids. A comparison between quail and
chicken apo A-1 revealed 94.5% homology in the nucleotide sequence and
91.7% homology in the amino acid sequence. There was a similar 11- or 22-amino
acid repeat in quail apo A-1 as was the case for chicken apo A-1. Apo A-1
mRNA was evaluated to be 1.4 k in length and was expressed in various tissues
of Japanese quail: the liver, small intestine, lung, kidney, heart, and
muscle. A quantitative evaluation, however, revealed that the liver and
small intestine were the major organs for apo A-1 synthesis, accounting
for more than 90% of the total expression of apo A-1 mRNA. Besides apo
A-1 mRNA (1.4 k in length), a transcript of 4.1 k was detected in all the
tissues examined, with a magnitude ranging from 5 to 10% of the apo A-1
mRNA level. The effect of cholesterol level on the expression of apo A-1
mRNA was studied to address the physiological significance of apo A-1 in
the liver, small intestine, and muscle. The level of cholesterol in the
liver and breast muscle was increased by feeding with cholesterol and reached
a saturation level at day 7. There was also a temporal rise of cholesterol
level at day 7 in the small intestine. Dietary cholesterol increased the
expression of apo A-1 mRNA two fold in both the liver and small intestine.
This was not the case for breast muscle, in which the expression of apo
A-1 mRNA was not modulated by the cholesterol level.
Key words: apolipoprotein A-1; cDNA sequence; Japanese quail; tissue expression;
cholesterol feeding
-16-
Cell Lysis Induced by Ricin
D and Ricin E in Various Cell Lines
Tatsuya Oda, Nobukazu Komatsu, and Tsuyoshi Muramatsu
Division of Biochemistry, Faculty of Fisheries, Nagasaki University,
Bunkyo-machi, Nagasaki 852, Japan
Received July 15, 1996
@Ricin D, one of two isolectins from small castor beans showed stronger
cytotoxicity than another one, ricin E, based on the inhibition of colony
formation and the inhibition of protein synthesis. Both ricin D and ricin
E induced cell lysis to different extents in each cell line tested, albeit
ricin E was slightly less effective than ricin D. DNA fragmentation, a
characteristic feature of apoptosis, was also induced by ricin D and ricin
E in Vero cells. Scatchard plot analysis showed that ricin D binds to cells
with higher affinity than ricin E, while the number of binding sites per
cell was not much different, suggesting that the differences in the cytotoxicity
between ricin D and ricin E is mainly due to their differential binding
affinity to cells. In Vero cells, the cytolytic activities of ricin D and
ricin E were inhibited by brefeldin A (BFA), which is known to affect the
Golgi apparatus, but no significant effect of BFA was observed in a BFA-resistant
cell line, MDCK cells. These results suggest that the Golgi apparatus may
be involved in ricin-induced cell lysis.
Key words: ricin D; ricin E; cytolysis; apoptosis; brefeldin A
-17-
Cloning and Expression
of an Intracellular Alkaline Protease Gene from Alkalophilic Thermoactinomyces
sp. HS682υ
Katsumi Tsuchiya,* Ichiro Ikeda, Takashi Tsuchiya, and Tetsu Kimura
Faculty of Pharmaceutical Sciences, Josai University, Sakado, Saitama
350-02, Japan
Received July 15, 1996
@An intracellular alkaline serine protease gene of alkalophilic Thermoactinomyces
sp. HS682 was cloned and expressed in Escherichia coli. Sequence analysis
showed a putative promoter region, a putative transciptional termination
signal, and an open reading frame of 963 bases, coding for a polypeptide
of 321 amino acids. The protease expressed in E. coli was purified by DEAE-Toyopearl
650M and Sephadex G-75 chromatography. The N-terminal sequence (30 amino
acids) of the purified protein was coincident with Asp16-Val45 of the deduced
amino acid sequence of the ORF. Fifteen amino acids in the N-terminal region
were removed during the purification procedures. The deduced amino acid
sequence showed high similarity with microbial intracellular serine proteases.
The molecular mass of this enzyme was estimated to be 38 kDa by SDS-PAGE.
The enzyme was stable at pH 6.0-12.0 and below 60C in the presence of
Ca2+. The temperature and pH optima of the enzyme were 65C and pH 11.0,
respectively. The enzyme was inhibited by DFP and PMSF, but not by MIA
and EDTA.
Key words: intracellular alkaline serine protease; alkalophilic Thermoactinomyces
sp.; DNA sequence; amino acid sequence homology; protein purification
-18-
Synthesis of 5-Deoxy-5-oxomilbemycin A4 5-Hydrazone
Derivatives and Their Activity against Tetranychus urticae
Yoshihisa Tsukamoto, Kazuo Sato, Keiji Tanaka, and Toshiaki Yanai υ
Agroscience Research Laboratories, Sankyo Co., Ltd., 1041 Yasu, Yasu-cho,
Yasu-gun, Shiga 520-23, Japan
Received July 22, 1996
@5-Deoxy-5-oxomilbemycin A4 5-hydrazone derivatives were synthesized by
a condensation reaction of 5-deoxy-5-oxomilbemycin A4 (2) with hydrazines.
Acetic acid played an important role to give each hydrazone in a good yield
by regulating the reactivity of ketone 2 and the hydrazines. The acaricidal
activity of each synthesized compound was studied on the primary leaves
of plants of the Vigna sinensis Savi species infected with organic phosphate-sensitive
mites (Tetranychus urticae). Some of the synthesized derivatives exhibited
higher miticidal activity than that of milbemycin A4. Among them, 5-deoxy-5-oxomilbemycin
A4 5-N-(N ',N '-dimethylcarbamoyl)hydrazone (18) totally controlled the
mites at a concentration of 3 ppm.
Key words: milbemycin; 5-deoxy-5-oxomilbemycin A4 5-hydrazone derivatives;
Tetranychus urticae; two-spotted spider mite
-19-
Characterization of a
Water-soluble Polysaccharide Fraction with Immunopotentiating Activity
from Bifidobacterium adolescentis M101-4
Akira Hosono ,υ Jonghwa Lee υυ, Akio Ametani, Midori Natsume,* Masao Hirayama,* Takashi Adachi,* and Shuichi Kaminogawa
Department of Applied Biological Chemistry, The University of Tokyo,
Bunkyo-ku, Tokyo 113, Japan
* Bioscience Laboratories, Meiji Seika Kaisha Ltd., Sakado-shi, Saitama
350-02, Japan
Received July 24, 1996
@The soluble and insoluble fractions obtained after sonication and centrifugation
of Bifidobacterium adolescentis M101-4 cells were examined, and both of
these fractions exhibited mitogenic activity in an assay of murine splenocytes
and Peyer's patch cells in vitro. The soluble fraction was further treated
by a 6-step procedure involving proteinase K-treatment, ultrafiltration
with a 50-kDa cut-off molecular-sieving membrane, anion-exchange chromatography,
dialysis, ultrafiltration through a 6-kDa cut-off membrane filter, and
gel-filtration to yield a soluble high molecular weight fraction (SHF)
which was effective for stimulating the proliferation of murine splenocytes.
Almost three quarters of this fraction by weight was found to consist of
carbohydrates containing glucose and galactose as major constituents, and
the average molecular weight was estimated to be between 60,000 and 2,460,000,
with the main peak at 1,550,000 Da, by the retention time of gel permeation
chromatography. A structural analysis by 1H- and 13C-nuclear magnetic resonance
and methylation indicated that SHF contained polysaccharides consisting
of -4Galp1-, -4Glcp1-, and -6Glcp1- as the major residues, and Galf 1-
and -6Galf 1- as the minor residues. Immunopotentiating SHF was found to
contain galactofuranosyl residues as characteristic constituents which
had not been previously detected in other soluble fractions from Gram-positive
bacteria.
Key words: immunopotentiator; polysaccharide; Bifidobacterium adolescentis
-20-
Microbiological Aspects
of Acetate Oxidation by Acetic Acid Bacteria, Unfavorable
Phenomena in Vinegar Fermentationυ
Akihiko Saeki, Mariko Taniguchi,* Kazunobu Matsushita,* Hirohide Toyama,* Gunjana Theeragool,** Napha Lotong,** and Osao Adachi*,υυ
Department of Bioindustry, Industrial Technology Institute, Yamaguchi
Prefectural Government, Yamaguchi 753, Japan
* Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi
University, Yamaguchi 753, Japan
** Department of Microbiology, Faculty of Science, Kasetsart University,
Bangkok 10900, Thailand
Received July 29, 1996
@Several strains of acetic acid bacteria belonging to the genus Acetobacter,
showing strong acetate oxidation, were screened and their microbiological
aspects in acetate oxidation were investigated. When all available carbon
and energy sources were exhausted and only acetic acid remained in the
late stationary phase, the bacteria started to consume the acetic acid
that had been accumulated in the culture medium for vinegar fermentation.
They grew rapidly, showing the second stationary phase and a typical biphasic
growth curve was observed. The cells from the first growth phase were acid
tolerant, while the cells from the second growth phase turned over to become
acid sensitive. However, no distinct acetate oxidation took place when
oxidizable ethanol and other available carbon sources still remained in
the culture medium. Moreover, no apparent acetate oxidation was observed
in vinegar mash in which more than 4.5% of acetic acid was allowed to accumulate.
There was a threshold in acetate concentration since the most selected
strains oxidized acetate when the final concentration of acetic acid accumulated
was less than 3.7%. When only acetic acid was administrated as the sole
carbon and energy sources, the organisms finally used acetic acid after
a long lag time. The lag time was shortened by the addition of a small
amount of readily usable energy source, such as ethanol. From enzymatic
analysis, only acetyl-CoA synthetase increased much among the enzymes concerning
acetyl-CoA formation from acetate, while the enzyme activities of acetate
kinase and phosphotransacetylase were not changed significantly. The enzyme
activities of isocitrate lyase and malate synthase also increased significantly
in the cells when acetate was consumed. These results indicate that acetic
acid is converted to acetyl-CoA by acetyl-CoA synthetase to put acetate
into the TCA cycle as well as to the glyoxylate cycle allowing the bacteria
to grow rapidly on acetic acid after ethanol exhaustion. Taking together
with growth experiments and enzymatic data accumulated, it was strongly
suggested that cells different in physiological characteristics from the
first growth phase emerged in the second growth phase.
Key words: acetate oxidation; acetic acid bacteria; Acetobacter aceti;
acetyl coenzyme A synthetase; vinegar fermentation
-21-
Gypsophilin, a New Type 1 Ribosome-inactivating
Protein from Gypsophila elegans :
Purification, Enzymatic Characterization, and Subcellular Localization
Shigeo Yoshinari, Shigehiro Koresawa, Sadaki Yokota,* Hiroshi Sawamoto, Minoru Tamura, and Yaeta Endoυ
Department of Applied Chemistry, Faculty of Engineering, Ehime University,
Matsuyama 790, Japan
* Department of Anatomy, Yamanashi Medical College, Yamanashi 409-38, Japan
Received August 8, 1996
@Gypsophila elegans contains a new type 1 ribosome-inactivating protein,
which we named gypsophilin. The protein was purified to apparent homogeneity
by (NH4)2SO4 fractionation, ion-exchange chromatography, and adsorption
chromatography. The protein was found to have a molecular mass of 28.0
kDa and a pI of about 10.1. It does not contain glycosidic linkages. The
sequence of the N-terminal 22 amino acids of the protein shows a close
relationship to other RIPs. The enzyme strongly inhibits protein synthesis
in a rabbit reticulocyte lysate and depurinates 28S rRNA in rat liver ribosomes
in a manner identical to that of ricin A-chain and other RIPs. Using a
direct method for the measurement of the RNA N-glycosidase activity, the
substrate spcificity of gypsophilin was identified. EC50 of the protein
for ribosomes of rat liver, wheat germ, and E. coli was 39.8 pM, 0.24 nM,
and 0.82 ΚM, respectively. Gypsophilin may be one of the most active RNA
N-glycosidases among the RIPs known to date. Immunoelectron microscopic
localization of gypsophilin in the leaves shows that the protein is accumulated
densely in the intercellular spaces and is also distributed within vacuoles
in the cytoplasm.
Key words: ribosome-inactivating protein; RNA N-glycosidase; characterization;
subcellular localization; Gypsophila elegans
-22-
Inhibition by Hop
Bract Polyphenols of Cellular Adherence and Water-insoluble Glucan Synthesis
of Mutans Streptococci
Motoyuki Tagashira, Keiko Uchiyama, Tomoaki Yoshimura, Masayuki Shirota, and Nobuo Uemitsu
Bioscience Research & Development Laboratory, Asahi Breweries Ltd.,
2-13-1 Ohmori-Kita, Ohta-ku, Tokyo 143, Japan
Received August 22, 1996
@The inhibitory effect of hop bract polyphenols (HBP) on cariogenic streptococci
was investigated. It was found that the high molecular weight polyphenol
(estimated about 36,000-40,000) inhibited the cellular adherence of Streptococcus
mutans MT8148 (serotype C) and Streptococcus sobrinus ATCC 33478 (serotype
g) at much smaller concentrations than the polyphenols extracted from oolong
tea or green tea leaves. Furthermore, HBP also inhibited the action of
glucosyltransferase, which was involved in the water-insoluble glucan synthesis,
but did not suppress the growth and the acid production of the bacteria.
These results suggest that HBP would be a candidate to act against dental
caries caused by Mutans Streptococci.
Key words: hop bract polyphenol; cellular adherence; water-insoluble glucan
synthesis; glucosyltransferase; Mutans Streptococci
-23-
Release
of Ectoenzymes from Small Intestine Brush Border Membranes of Mice by Phospholipases
Chisako Itami, Ryo Taguchi,* Hiroh Ikezawa,* and Toshikatsu Nakabayashi υ
Laboratory of Biochemistry, Faculty of Pharmaceutical Sciences, Mukogawa
Women's University, 11-68 Koshien Kyuban-cho, Nishinomiya, Hyogo 663, Japan
* Department of Microbial Chemistry, Faculty of Pharmaceutical Sciences,
Nagoya City University, Mizuho-ku, Nagoya, Aichi 467, Japan
Received August 26, 1996
@This study investigated ectoenzyme release from small intestine brush
border membranes (duodenum and jejunum, Preparation A; ileum, Preparation
B) of mice by the action of phosphatidylinositol-specific phospholipase
C or glycosyl-phosphatidylinositol-specific phospholipase D. Most of the
alkaline phosphatase was solubilized from Preparation A, but about 60%
was released from Preparation B. As for alkaline phosphodiesterase I activity,
15 and 10% were released from Preparations A and B, respectively. With
Preparation B, octylglucoside treatment followed by phosphatidylinositol-specific
phospholipase C or glycosyl-phosphatidylinositol-specific phospholipase
D completely solubilized the alkaline phosphatase activity. However, this
treatment did not change the ratio of release of alkaline phosphodiesterase
I from Preparation A or B. These results indicate that the resistance to
alkaline phosphatase found in Preparation B is due to hindered accessibility
of the bonding splitting by phosphatidylinositol-specific phospholipase
C and not to a modified glycosyl-phosphatidylinositolanchor.
Key words: alkaline phosphatase; alkaline phosphodiesterase I;
glycosyl-phosphatidylinositol-anchored enzyme release;
glycosyl-phosphatidylinositol-specific phospholipase D;
phosphatidylinositol-specific phospholipase C
-24-
Immunochemical Approach to Characterize
Post-translational Modification of Serum
Albumin Using Anti-glutaraldehyde-treated Serum Albumin Antibodies
Hiroyuki Ukeda,υ Toshinao Ishii, Yoshimitsu Shimizu, Masayoshi Sawamura, and Hirozo Kusunose
Department of Bioresources Science, Faculty of Agriculture, Kochi University,
Monobe B-200, Nankoku 783, Japan
Received August 26, 1996
@Polyclonal antibodies against glutaraldehyde-treated rabbit serum albumin
(pRSA) and human serum albumin (pHSA) were prepared from rabbit and mouse,
respectively. Anti-pRSA antibody had the structural determinant depending
on the polymerization process of RSA and showed only a weak cross-reactivity
with the other glutaraldehyde-treated albumins. Anti-pHSA antibody (after
adsorption of anti-HSA antibody) recognized only pHSA, but not HSA and
the other treated albumins. The cross-reactivity of those antibodies was
examined with albumins treated by other methods such as modification of
glucose and fructose, carbodiimide, and transglutaminase. Among them, RSA
and HSA modified with glucose and fructose had an affinity for each antibody
and the reactivity depended on the extent of formation of the polymerized
albumin. The result suggests that functional groups involved in cross-linking
of albumin are important for formation of the cross-reactivity with the
antibody and that a definite structure immunochemically similar to glutaraldehyde-treated
albumin could be formed by the Maillard reaction.
Key words: serum albumin; glutaraldehyde; hepatitis B virus; post-translational
modification;
anti-glutaraldehyde-treated albumin antibody
-25-
Inhibition
of Arachidonate Lipoxygenase Activities by 2-(3,4-Dihydroxyphenyl)ethanol,
a Phenolic Compound from Olives
Noriko Kohyama, Tadahiro Nagata,* Shin-ichi Fujimoto, and Keizo Sekiyaυ
Shikoku National Agricultural Experiment Station, 1-3-1 Senyu-cho, Zentsuji,
Kagawa 765, Japan
* National Food Research Institute, 2-1-2 Kannondai, Tsukuba, Ibaraki 305,
Japan
Received August 27, 1996
@The effects of olive fruit extract on arachidonic acid lipoxygenase activities
were investigated using rat platelets and rat polymorphonuclear leukocytes
(PMNL). Olive extract strongly inhibited both 12-lipoxygenase (12-LO) and
5-lipoxygenase (5-LO) activities. One of the compounds responsible for
this inhibition was purified and identified as 2-(3,4-dihydroxyphenyl)ethanol
(DPE). DPE inhibited platelet 12-LO activity (IC(/)50, 4.2 ΚM) and PMNL
5-LO activity (IC(/)50, 13 ΚM) but not cyclooxygenase activity in cell-free
conditions. It also inhibited 12-LO activity in intact platelets (IC(/)50,
50 ΚM) and reduced leukotriene B4 production in intact PMNL stimulated
by A23187 (IC(/)50, 26 ΚM). The inhibition by DPE of both lipoxygenase
activities was stronger than that by oleuropein, caffeic acid, or 7 other
related phenolic compounds, especially in intact cells. These results suggest
that DPE is a potent specific inhibitor of lipoxygenase activities.
Key words: arachidonic acid; lipoxygenase; inhibitor; olive; phenolic compounds
-26-
Note
Sphingoid Base Composition of
Cerebrosides from Plant Leaves
Hiroyuki Imai,υ Masao Ohnishi,υυ Kenmi Hotsubo,υυυ Mitiyuki Kojima, and Seisuke Ito
Department of Bioresource Chemistry, Obihiro University of Agriculture
and Veterinary Medicine, Inada, Obihiro, Hokkaido 080, Japan
Received February 16, 1996
@Component sphingoid bases of cerebrosides from leaves of twenty-two plants
were analyzed by capillary GC as the corresponding fatty aldehydes. No
apparent difference in the ratios of 8-E-unsaturated sphingoid bases to
the 8-Z-forms was found between chilling-resistant and chilling-sensitive
plants. Nevertheless, most of the chilling-resistant plants analyzed had
more 8-Z-unsaturated trihydroxy base contents than those of the E-isomer.
Key words: cerebroside; sphingolipid; sphingoid base; leaf lipid; chilling-sensitive
plants
-27-
Note
Serum Cholesterol Reduction
and Cholesterol Absorption Inhibition in CaCo-2 Cells by a Soyprotein Peptic
Hydrolyzate
Satoshi Nagaoka,υ Takako Awano, Naoko Nagata, Motoki Masaoka, Goro Hori,* and Kei Hashimoto**
Department of Food Science, Faculty of Agriculture, Gifu University,
Gifu 501-11, Japan
* Tsukuba Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., Ibaraki 305,
Japan
** School of Food and Nutritional Sciences, University of Shizuoka, Shizuoka
422, Japan
Received May 17, 1996
@The serum cholesterol level in rats was significantly decreased in a
group fed on a soyprotein peptic hydrolyzate (SPH) when compared with a
group fed on a casein tryptic hydrolyzate (CTH). The fecal excretion of
total steroids was significantly greater with rats fed on the SPH diet
when compared with the CTH diet. The results of CaCo-2 studies clearly
suggest that the suppression of cholesterol absorption in the intestinal
epithelia is part of the mechanism for the hypocholesterolemic action induced
by SPH.
Key words: cholesterol; soyprotein; casein; CaCo-2; intestine
-28-
Note
Preparation of Dye-labeled
Alginate for the Assay of Alginate Lyase
Shigeki Yoshida, Sayaka Watanabe, Toshio Takeuchi,* Katsumi Murata,* and Isao Kusakabe
Institute of Applied Biochemistry, University of Tsukuba, 1-1-1 Tennoodai,
Tsukuba-shi, Ibaraki 305, Japan
* Research and Development Department, Kibun Food Chemifa Co., Ltd., 2-1-1
Irifune, Chuo-ku, Tokyo 104, Japan
Received June 3, 1996
@Dabsyl-alginate, the dye-labeled substrate of alginate lyase, was prepared.
Low-viscosity alginate, which was prepared by enzymatic degradation of
sodium alginate, and tetramethylenediamine were conjugated by reductive
amination. Then, dabsyl-Cl was coupled with the primary amino group of
the aminobutyl-alginate. The assay for endo-alginate lyase using dabsyl-alginate
was simple to use.
@The amount of dyed fragment released from the substrate increased with
the amount of the enzyme.
Key words: dabsyl-alginate; alginate lyase; chromogenic substrate; dye-releasing
assay
-29-
Note
Substrate Specificities of Ώ-Galactosidases
from Yeasts
Shigeki Yoshida, Chin Hui Tan, Tomoko Shimokawa, Hilkka Turakainen,* and Isao Kusakabe
Institute of Applied Biochemistry, University of Tsukuba, 1-1-1 Tennoodai,
Tsukuba-shi, Ibaraki 305, Japan
* Alkomohr Biotech Ltd., Biocenter, Viikinkaari 9C, FIN-00710 Helsinki,
Finland
Received August 8, 1996
@Twenty-nine strains of yeasts, which are capable of using galactose,
melibiose, or raffinose, were screened for Ώ-galactosidase production.
Among the strains, 5 produced intracellular and extracellular Ώ-galactosidases,
and 2 produced only intracellular enzyme. Substrate specificities of these
enzymes were explored using 63-Ώ-D-galactosyl-1,4-ΐ-D-mannotriose and
63-Ώ-D-galactosyl-1,4-ΐ-D-mannotetraose. All enzymes liberated the terminal
galactose from 63-Ώ-D-galactosyl-1,4-ΐ-D-mannotriose, but did not the
stub galactose from 63-Ώ-D-galactosyl-1,4-ΐ-D-mannotetraose.
Key words:Ώ-galactosidase; yeast; substrate specificity; galactomanno-oligosaccharide
-30-
Note
A Gene Encoding Endo-1,4-ΐ-glucanase from
Bacillus sp. 22-28
υ
Munetoshi Miyatake and Kiyohisa Imada
Department of Materials Science, Faculty of Engineering, Miyazaki University,
1-1 Gakuen Kibanadai Nishi, Miyazaki 889-21, Japan
Received June 11, 1996
@Clones of a gene encoding an endo-1,4-ΐ-glucanase (EC 3.2.1.4) were
obtained from Bacillus sp. 22-28,* and the nucleotides were sequenced.
A recombinant plasmid, pMK5, included a complete ORF of 2352 bp that encoded
783 amino acid residues. Another plasmid, pM3, which showed enzymatic activity
in E. coli JM109, was also obtained, and it included an incomplete ORF
of 1873 bp, which lacked the original stop codon and 479 bp of the C-terminal
region. The enzymes purified from both of the two types of transformants
have shown almost the same properties in comparison with that of the wild
type Bacillus sp. 22-28.
Key words: endo-1,4-ΐ-glucanase; Bacillus ; cloning; sequencing; carboxymethylcellulase
-31-
Note
Effect of Acetic Acid Bacterium on
Ethanol Oxidation in Vivo
Yukihiro Nomura, Masanori Yamamoto, and Tohru Fushiki *
Somatech Center, House Foods Co., Ltd., 1-5-7 Mikuriya-sakaemachi, Higashiosaka-shi,
Osaka 577, Japan
* Department of Food Science and Technology, Faculty of Agriculture, Kyoto
University, Kyoto 606-01, Japan
Received June 19, 1996
@We investigated the possible effects of acetic acid bacterium on ethanol
oxidation in vivo by monitoring the blood ethanol level after injecting
5% ethanol with (treated group) or without (control group) a freeze-dried
bacterial cell suspension directly into the stomach of anesthesized rats.
Paired comparison t-tests of the results indicate that the blood ethanol
concentration of the rats in the treated group was significantly ( p<0.07)
lower than that in the control group.
@When measured 10 min after administering ethanol into the gullet, the
concentration in the stomach of the rats that received acetic acid bacterium
simultaneously with ethanol was significantly ( p<0.10) lower than that
of the rats that received ethanol alone.
@We consider that freeze-dried cells of acetic acid bacterium oxidized
ethanol in the stomach and could be effective for reducing the blood ethanol
level after drinking.
Key words: acetic acid bacterium; ethanol oxidation; ethanol-oxidizing
enzyme activity
-32-
Note
Investigation of
Flavonoid Aglycones in Propolis Collected by Two Different Varieties of
Bees in the Same Region
Michel Hyun Koo and Yong Kun Park
State University of Campinas, College of Food Engineering (UNICAMP),
13081-970 Caixa Postal 6177, Campinas, S.P., Brazil
Received July 2, 1996
@The quality and quantity of flavonoids from two types of propolis collected
by two different varieties of Apis mellifera bee in the same region were
investigated. There was found a remarkable quantitative and qualitative
difference of flavonoids in propolis. These results indicate that the chemical
composition of propolis was dependent on the variety of the bee.
Key words: flavonoid; propolis; Apis mellifera; allozyme; Africanized bee
-33-
Note
Daidzein Stimulation
of Bone Resorption in Pit Formation Assay
Hiroyasu Tobe,υ Osamu Komiyama, Yoshiko Komiyama, and Hiromi B. Maruyama
Pharma Research and Development Division, Drug Discovery Research Laboratories,
Hoechst Japan Limited, 3-2 Minamidai 1-chome, Kawagoe-shi, Saitama 350-11,
Japan
Received July 15, 1996
@We found that daidzein stimulated bone resorption in the pit formation
assay at the concentration of 10-8-10-10 M. On the other hand, genistein
and ipriflavone at these concentrations did not have any effect on pit
formation. We speculated that daidzein had the ability to induce osteoclasts
directly or indirectly from their progenitors and might be a tool to study
osteoclast differentiation.
Key words: daidzein; genistein; ipriflavone; pit formation assay; casein
kinase II
-34-
Note
Expression
of Alkaline Protease Gene in Bacillus subtilis Mutants That Lack Positive
Regulatory Genes degR, degQ, senS, tenA, and proB
Mitsuo Ogura and Teruo Tanaka
Department of Marine Science, School of Marine Science and Technology,
Orido 3-20-1, Shimizu, Shizuoka 424, Japan
Received July 15, 1996
@In addition to the two-component regulatory system, DegS-DegU, five other
genes degR, degQ, senS, tenA, and proB positively regulate the production
of Bacillus subtilis exoproteases when they are present on a multicopy
plasmid. To study the extent of involvement of these genes in exoprotease
production in a single copy state and possible relationship among these
genes, strains carrying single or multiple disruption mutations in these
loci were constructed, and the expression of aprE '-'lacZ was analyzed.
From such studies, the following results were obtained with respect to
the regulation of aprE expression. i) Disruption mutations were divided
into two groups; one (degQ and degR) significantly reduced aprE expression,
while the other (senS, tenA, and proB) had little effect on it. ii) SenS
was involved in temporal regulation. iii) The combined effects of some
of the disruption mutations tested were not necessarily additive. iv) The
extent of the negative effects generated by the mutations depended on the
medium used.
Key words: Bacillus subtilis; degS-degU; degR; degQ; alkaline protease
-35-
Note
Amino Acid Sequence
and Characterization of Aldo-keto Reductase from Bakers' Yeast
Kaoru Nakamura, Shin-ichi Kondo,υ Yasushi Kawai, Nobuyoshi Nakajima,* and Atsuyoshi Ohno
Institute for Chemical Research, Kyoto University, Uji, Kyoto 611, Japan
* Department of Nutritional Science, Okayama Prefectural University, Soja,
Okayama 719-11, Japan
Received July 31, 1996
@One of the enzymes from bakers' yeast that catalyzes the reduction of
Ώ- and ΐ-keto esters has been studied. The N-terminal amino acid sequence
of the enzyme shows that the enzyme belongs to the aldo-keto reductase
superfamily. The substrate specificity of the enzyme is broad and resembles
those of other aldo-keto reductases. The enzyme catalyzes the reduction
of keto esters, aldehydes, and aldohexoses.
Key words: Saccharomyces cerevisiae ; bakers' yeast;
aldo-keto reductase; protein sequence;
stereoselectivity
-36-
Note
44-Homooligomycin E,
a New Cytotoxic Macrolide Antibiotic from Streptomyces ostreogriseus
Hang Sub Kim, Hee Jae Bang, Sang Yong Lee, Ook Joon Yoo,* Jin Cheol Yoo,** Young Ho Kim,υ and Jung Joon Lee υυ
KRIBB, Korea Institute of Science and Technology, P.O. Box 115, Yusung,
Taejon 305-606, Korea
* Department of Biological Science, KAIST, 371-1, Kusong-Dong, Yusung-Gu,
Taejon 305-701, Korea
** Department of Pharmacy, Choson University, Kwangju 501-759, Korea Received
July 31, 1996
@Homooligomycin E (1) was isolated from the culture broth of Streptomyces
ostreogriseus and its structure was analyzed on the basis of physicochemical
and spectroscopic data. It showed strong cytotoxicity against several human
tumor cell lines.
Key words: 44-homooligomycin E; macrolide antibiotic;
cytotoxicity; Streptomyces ostreogriseus
-37-
Note
The Specificity of Peptide
Bond Cleavage of Acid Proteinase A from Aspergillus niger var. macrosporus
toward Oxidized Ribonuclease A
υ
Kenji Takahashi*
Department of Biophysics and Biochemistry, Graduate School of Science,
The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan
Received August 19, 1996
@In order to investigate the specificity of peptide bond cleavage by acid
proteinase A from Aspergillus niger var. macrosporus (Aspergillopepsin
II), performic acid-oxidized bovine pancreatic ribonuclease A was digested
by the enzyme at pH 1.8 or 5.5, and the resulting peptides were separated
by HPLC and analyzed. Among the total 123 peptide bonds, approximately
thirty and thirteen bonds were cleaved at pH 1.8 for 2 h and at pH 5.5
for 20 h, respectively. Cleavages occurred fairly specifically at Tyr-X,
Phe-X, His-X, Asn-X, Asp-X, Gln-X, and Glu-X bonds.
Key words: acid proteinase A; aspergillopepsin II;
Aspergillus niger var. macrosporus;
oxidized ribonuclease A; substrate specificity
-38-
Note
Purification and Characterization
of the NADP-Malic Enzyme from Bradyrhizobium japonicum A1017
Fan Chen, Youichi Okabe, Kaoru Osano, and Shigeyuki Tajimaυ
Department of Bioresource Science, Faculty of Agriculture, Kagawa University,
Ikenobe 2393, Miki-cho, Kita-gun, Kagawa 761-07, Japan
Received August 20, 1996
@An NADP-malic enzyme [EC 1.1.1.40] was purified to homogeneity from Bradyrhizobium
japonicum A1017, and the molecular and physiological characteristics were
surveyed. The molecular mass of one subunit of the purified enzyme was
evaluated to be 77,600 Da by SDS-PAGE, and the native enzyme was a tetramer
in pH 7.0 and dimer in pH 8.0 conditions, showing complex oligomeric characteristics
corresponding to pH value.
Key words: Bradyrhizobium japonicum ; NAD; NADP;
malic enzyme; symbiotic nitrogen fixation
-39-
Note
Occurrence of Acid-phosphatase Inhibitors in
Soybeans
Kenjiro Tadera, Yuji Minami, Masashi Uehune, Akira Nozaki, Fumio Yagi, and Toshihiko Suganuma
Department of Biochemical Science and Technology, Faculty of Agriculture,
Kagoshima University, Korimoto 1-21-24, Kagoshima 890, Japan
Received August 22, 1996
@Inhibitors of acid phosphatase were found in soybeans. They were extracted
with hot-water, and partially separated from the other soybean components
by alcohol precipitation and ion-exchange chromatography. They seemed to
be a kind of acidic polysaccharide.
Key words: acid phosphatase; inhibitor; soybean;
acid phosphatase inhibitor
-40-
Note
Mutations within the Reactive-site Region
of Human Pancreatic Secretory Trypsin
Inhibitor Confer Ώ-Thrombin and Factor Xa Inhibitory Activities
Takaaki Katoh,υ Mikio Tamaki, Norihisa Kikuchi, Hiroshi Teraoka, Kiyoshi Nagata, and Nobuo Yoshida
Shionogi Research Laboratories, Shionogi & Co., Ltd., 12-4 Sagisu
5-chome, Fukushima-ku, Osaka 553, Japan
Received September 5, 1996
@Mutations were introduced into the reactive-site region of human pancreatic
secretory trypsin inhibitor (PSTI) to produce thrombin and/or factor Xa
inhibitors. All of five mutants showed trypsin inhibitory activity as strong
as wild-type PSTI. Moreover, the Arg (P1), Pro-Arg (P2-P1), and Pro-Arg-Ile-Tyr-Asn
(P2-P1-P1'-P2'-P3') (bold letters indicate replaced amino acids compared
to the wild type) mutants had additional inhibitory activities toward factor
Xa, both thrombin and factor Xa, and thrombin, respectively, at 1~10-5
M.
Key words: pancreatic secretory trypsin inhibitor;
reactive-site mutant; thrombin inhibitor;
factor Xa inhibitor
-41-
Rapid Paper
Potential D,L-Amino Acid Sequence
Analysis of Peptides from the C-Terminus
Hiroshi Ohrui,υ Eigo Itoh, Yoshihiro Nishida, Hiroko Horie, and Hiroshi Meguro
Department of Applied Biological Chemistry, Faculty of Agriculture,
Tohoku University, Tsutsumidori-Amamiyamachi 1-1, Aoba-ku, Sendai 981,
Japan
Received September 6, 1996
@A model tripeptide, Gly-L-Leu-L-Phe, was immobilized with activated aminomethyl
polystyrene, and its C-terminal was reduced to an alcohol. This peptidyl
alcohol was selectively hydrolyzed at the C-terminal amide bond to afford
a polymer-supported dipeptide (Gly-L-Leu) and amino alcohol (Phe-OH). The
amino alcohol, including its absolute configuration, was determined by
labelling with (+)-MNB-COOH, and the dipeptide was reused for a determination
of its C-terminal amino acids. The D,L-amino acids of the tripeptide were
sequentially determined from the C-terminus.
Key words: C-terminal; sequence analysis; d,l-amino acid; chiral amino
alcohol
-42-
Rapid Paper
Effects of Nucleotides
on Cyanide-resistant Respiratory Activity in Mitochondria Isolated from
Antimycin A-treated Yeast Hansenula anomala
Shigeru Sakajo,υ Nobuko Minagawa, and Akio Yoshimoto
Department of Biochemistry, Niigata College of Pharmacy, 5-13-2 Kamishin'ei-cho,
Niigata 950-21, Japan
Received September 20, 1996
@Mitochondria were isolated from Hansenula anomala induced for cyanide-resistant
respiration by antimycin A-treatment. Cyanide-resistant respiratory activity
in isolated mitochondria was stimulated by AMP, ADP, dAMP, and GMP, but
not by ATP, adenosine, cAMP, 2'(3')-AMP, CMP, and UMP. Effects of nucleotides
were also observed on cyanide-resistant and salicylhydroxamate-sensitive
decylubiquinol oxidase activity. Carbonylcyanide m-chlorophenylhydrazone,
oligomycin, and carboxyatractyloside did not affect activation of decylubiquinol
oxidase activity by AMP. It is suggested that purine nucleoside 5'-monophosphate
or diphosphate stimulates alternative oxidase activity from the outer surface
of the mitochondrial inner membrane with a mechanism different from respiratory
control. Alternative oxidase activity might be controlled by adenine nucleotides
posttranscriptionally in fungi.
Key words: cyanide-resistant respiration; alternative oxidase; mitochondria;
nucleotides;
Hansenula (Pichia) anomala
-43-
Short Communication
Prodigiosin 25-C
and Metacycloprodigiosin Suppress the Bone Resorption by Osteoclasts
Je-Tae Woo, Yasuo Ohba,* Kahori Tagami,* Koji Sumitani,* Takao Kataoka, and Kazuo Nagai υ
Department of Bioengineering, Tokyo Institute of Technology, 4259 Nagatsuta-cho,
Midori-ku, Yokohama-shi, Kanagawa 226, Japan
* Department of Orthodontics, School of Dentistry, The University of Tokushima,
Kuramoto-cho, Tokushima 770, Japan
Received August 14, 1996
@Prodigiosin 25-C and metacycloprodigiosin were found to suppress PTH-stimulated
pit formation by cultured osteoclasts on bone slices. They also inhibited
the acidification of vacuolar organelles in intact osteoclastic cells.
Since the acidic pH in these organelles is generated by the action of proton-pumping
ATPases of the organelle, these results indicate that the proton-pumping
activity of V-ATPase in osteoclastic cells is essential in bone resorption
and that the inhibition of the acidification of vacuolar organelles by
prodigiosins results in suppression of PTH-stimulated bone resorption.
Key words: osteoclast; bone resorption; V-ATPase, acidification; inhibitor