(Vol.63 No.1 1999)
Review
Microbial Desulfurization of Organic Sulfur Compounds
in Petroleum
Takashi OHSHIRO and Yoshikazu IZUMI・
Characterization of
the Enantioselective Properties of the Quinohemoprotein Alcohol Dehydrogenase
of Acetobacter pasteurianus LMG 1635. 1. Different Enantiomeric Ratios of Whole
Cells and Purified Enzyme in the Kinetic Resolution of Racemic Glycidol
Sonia Salgueiro MACHADO, Ute WANDEL, Jaap A. JONGEJAN,・ Adrie J. J. STRAATHOF,
and Johannis A. DUINE
Purified Fusion Enzyme
between Rat Cytochrome P4501A1
and Yeast NADPH-Cytochrome P450 Oxidoreductase
Masayuki HARA,＊,＊＊,・ Jun MIYAKE,＊,＊＊ Yasuo ASADA,＊ and Hideo OHKAWA＊＊＊
Apolipoprotein A-I
of Hyperlipidemia Atherosclerosis Prone (LAP) Quail:
cDNA Sequence and Tissue Expression
Hironori IWASAKI, Hirosuke OKU, Takayoshi TODA,＊ Tetsuo NASU,＊＊
Koji ODA, Tsuyoshi MIYAGI, and Isao CHINEN
Structures of N-Linked
Oligosaccharides of Glycoproteins
from Tobacco BY2 Suspension Cultured Cells＊
Nirianne Q. PALACPAC,1 Yoshinobu KIMURA,2 Kazuhito FUJIYAMA,1
Toshiomi YOSHIDA, and Tatsuji SEKI1
Effect of Shellfish
Calcium on the Apparent Absorption of Calcium
and Bone Metabolism in Ovariectomized Rats
Takehito MIURA,＊ Yasuyoshi TAKAYAMA, and Masuo NAKANO・
Isolation and Characterization
of a New Quinoprotein Dehydrogenase,
L-Sorbose/L-Sorbosone Dehydrogenase
Akira ASAKURA and Tatsuo HOSHINO
Stabilization of L-Ascorbic
Acid by Superoxide Dismutase
and Catalase
Noriko MIYAKE, Miok KIM, and Tadao KURATA・
Cloning and Expression
of a cDNA Encoding the Laccase
from Schizophyllum commune
Osamu HATAMOTO,1,2 Hiroshi SEKINE,1 Eiichi NAKANO,2 and Keietsu ABE2
Purification and Properties
of a Low-molecular-weight, High-alkaline Pectate Lyase from an Alkaliphilic
Strain of Bacillus
Tohru KOBAYASHI,＊ Kenzo KOIKE, Tadashi YOSHIMATSU, Norihiko HIGAKI,
Atsushi SUZUMATSU, Tadahiro OZAWA, Yuji HATADA, and Susumu ITO
Purification and Properties
of Bacteriolytic Enzymes
from Bacillus licheniformis YS-1005 against Streptococcus mutans
So-Young KIM, Seung-Ho OHK, Dong-Hoon BAI,1 and Ju-Hyun YU
Protective Effect
of Dietary Tomato against Endothelial Dysfunction
in Hypercholesterolemic Mice
Hiroyuki SUGANUMA・ and Takahiro INAKUMA
Purification and Characterization
of Phospholipase B
from Kluyveromyces lactis, and Cloning of Phospholipase B gene
Hideki OISHI, Takahiro MORIMOTO, Yasuo WATANABE, and Youichi TAMAI・
Thermostability Reinforcement
through a Combination
of Thermostability-related Mutations of N-Carbamyl-D-Amino
Acid Amidohydrolase
Yasuhiro IKENAKA, Hirokazu NANBA, Kazuyoshi YAJIMA, Yukio YAMADA,
Masayuki TAKANO, and Satomi TAKAHASHI
Cloning of Bacillus
stearothermophilus ctaA and Heme A Synthesis
with the CtaA Protein Produced in Escherichia coli
Junshi SAKAMOTO,・ Akiko HAYAKAWA, Tomoko UEHARA,
Shunsuke NOGUCHI, and Nobuhito SONE
Increase of Hematopoietic
Responses by Triple or Single Helical Conformer
of an Antitumor (1→3)-β-D-Glucan Preparation, Sonifilan,
in Cyclophosphamide-induced Leukopenic Mice
Aiko TSUZUKI, Tomoko TATEISHI, Naohito OHNO, Yoshiyuki ADACHI,
and Toshiro YADOMAE＊
Effects of Docosahexaenoic
and Eicosapentaenoic Acid on Lipid Metabolism,
Eicosanoid Production, Platelet Aggregation and Atherosclerosis
in Hypercholesterolemic Rats
Yosef ADAN, Kenichi SHIBATA, Masao SATO, Ikuo IKEDA, and Katsumi IMAIZUMI・
Pectin Methylesterase
Gene (pmeA) from Aspergillus oryzae KBN616:
Its Sequence Analysis and Overexpression, and Characterization
of the Gene Product
Noriyuki KITAMOTO,・ Hideki OKADA,＊ Shoko YOSHINO, Kunio OHMIYA,＊＊
and Norihiro TSUKAGOSHI＊＊＊
Purification and Characterization
of Methylamine Oxidase Induced
in Aspergillus niger AKU 3302
Ivo FR´EBORT,＊ Kazunobu MATSUSHITA, Hirohide TOYAMA, Karel LEMR,＊＊
Mamoru YAMADA, and Osao ADACHI・
Dietary Effect of
EPA-rich and DHA-rich Fish Oils on the Immune Function
of Sprague-Dawley Rats
Pham HUNG,1 Shihoko KAKU,1 Shin-ichi YUNOKI,1 Ken-ichi OHKURA,1 Jiong-Yan
GU,1
Ikuo IKEDA,2 Michihiro SUGANO,3 Kazunaga YAZAWA,4 and Koji YAMADA1,・
Comparison of Base
Specificity and Other Enzymatic Properties
of Two Protozoan Ribonucleases from Physarum polycephalum
and Dictyostelium discoideum
Norio INOKUCHI,a,・ Shigeru SAITOH,a Hiroko KOBAYASHI,a Tadashi ITAGAKI,a
Takashi KOYAMA,a Saburo UCHIYAMA,b and Masachika IRIEc
Purification and Characterization
of Aspergillus ficuum Endoinulinase
Tai-Boong UHM,1,・ Mi Sun CHUNG,2 Sun Hee LEE,2 Fran・oise GOURRONC,3
Isabelle HOUSEN,4 Jong Hwa KIM,5 Josef Van BEEUMEN,6 Bernard HAYE,3
and Jean VANDENHAUTE4
Precursor of α-Methylene-γ-butyrolactone
Involved in the Insecticidal Activity
of Thunberg Spiraea, Spiraea thunbergii
Chul-Sa KIM,・ Probal Kanti DATTA, Tetsuro HARA, Eiji ITOH, and Michio HORIIKE
Cloning and Characterization
of Chemotaxis Genes
in Pseudomonas aeruginosa
Junichi KATO, Tetsuya NAKAMURA, Akio KURODA, and Hisao OHTAKE
Functional Analysis
of the Promoter of the Yeast SNQ2 Gene Encoding a
Multidrug Resistance Transporter that Confers the Resistance
to 4-Nitroquinoline N-Oxide
Zhifeng CUI, Dai HIRATA, and Tokichi MIYAKAWA
Purification and Characterization
of Serine Acetyltransferase
from Escherichia coli Partially Truncated at the C-Terminal Region
Koshiki MINO, Tsuyoshi YAMANOUE, Takaharu SAKIYAMA, Naoki EISAKI,＊
Asahi MATSUYAMA,＊ and Kazuhiro NAKANISHI・
Note
Insertion Analysis of Putative Functional Elements in
the Promoter Region
of the Aspergillus oryzae Taka-amylase A Gene (amyB) Using
a Heterologous Aspergillus nidulans amdS-lacZ Fusion Gene System
Yoji KANEMORI,1 Katsuya GOMI,2,＊,・ Katsuhiko KITAMOTO,2,＃
Chieko KUMAGAI,2,$ and Gakuzo TAMURA3
Note
Screening of Korean Forest Plants for Rat Lens Aldose
Reductase Inhibition
Hye-Young KIM1,・ and Jong Hwan OH2
Note
Identification of L-Bornesitol and Changes in Its Content
during
Flower Bud Development in Sweet Pea (Lathyrus odoratus L.)
Kazuo ICHIMURA,・ Katsunori KOHATA, Yuji MUKASA,・・ Yuichi YAMAGUCHI,
Rie GOTO, and Kenichi SUTO
Note
Identification of Endogenous Gibberellins in the Leaves
and Xylem Sap of Tea Plants
Naomi OYAMA,・ Tomoya NIKI, Kunio OKANO,＊ Toyomasa ANAN,＊＊
and Masaji KOSHIOKA
Note
An Improved Method for Isolating Yeasts in the Genus
Lipomyces
and Related Genera from Soil
Takafumi NAGANUMA,・ Ken KATSUMATA, Tsutomu ANDO, Hisayuki WATANABE,
Katsushi NISHIMURA, and Yasuyuki UZUKA
Note
Separation and Properties of Two Acetylacetoin Reductases
from Bacillus cereus YUF-4
Takeshi HOSAKA, Sadaharu UI, and Akio MIMURA
Note
Comparative Hypocholesterolemic Effects of Five Animal
Oils
in Cholesterol-fed Rats
Michihiro FUKUSHIMA, Tetsu OHASHI, Mitsuo SEKIKAWA,
and Masuo NAKANO・
Note
Cloning and Sequencing of β-Mannosidase Gene
from Aspergillus aculeatus No. F-50
Goro TAKADA, Takashi KAWAGUCHI, Tomoko KAGA,
Jun-ichi SUMITANI, and Motoo ARAI・
Note
A Simple Hydroponic Culture Method for the Development
of a Highly Viable Root System in Arabidopsis thaliana
Tomomi TODA,・ Hiroyuki KOYAMA and Tetsuo HARA
Note
Occurrence in Soybeans of a Novel Vitamin B6 Conjugate
that Liberates Pyridoxine by β-Glucosidase Action after Alkali Treatment
Kenjiro TADERA,・ Kazutaka ODA, and Chikako NAKAHARA
Note
Occurrence of Stereoisomers of 1-(2′-Pyrrolidinethione-3′-yl)-1,2,3,4-
tetrahydro-β-carboline-3-carboxylic Acid in Fermented Radish Roots
and Their Different Mutagenic Properties
Yoshio OZAWA,＊,・ Yasushi UDA,＊＊ Hiroki MATSUOKA,＊ Masako ABE,＊
Shunro KAWAKISHI,＊＊＊ and Toshihiko OSAWA＊＊＊＊
Note
The Amino Acid Sequence of Wood Duck Lysozyme
Tomohiro ARAKI・ and Takao TORIKATA
Note
Effect of Salt Addition on the Fractal Structure of
Aggregates Formed
by Heating Dilute BSA Solutions
Hitoshi KUMAGAI,＊ Tomohide MATSUNAGA, and Tomoaki HAGIWARA・
Note
Citric Acid Production from Xylan and Xylan Hydrolysate
by Semi-Solid Culture of Aspergillus niger
Kohtaro KIRIMURA,・ Taisei WATANABE, Tadahiro SUNAGAWA,
and Shoji USAMI
Note
Structural Identification of Sulfated Tyrosine in Human
Urine
J. G. Shirani RANASINGHE,1 Yoichi SAKAKIBARA,1 Miyu HARADA,1
Kazuo NISHIYAMA,1 Ming-Cheh LIU,2 and Masahito SUIKO1,・
Note
Detection of ALMB-toxin in the Larval Body of Myrmeleon
bore
by Anti-N-terminus Peptide Antibodies
Naofumi YOSHIDA, Hiroshi SUGAMA, Shintaro GOTOH, Kazuhiko MATSUDA,・
Keiichiro NISHIMURA,＊ and Koichiro KOMAI
Note
New Method of Screening for Pressure-sensitive Mutants
at High Hydrostatic Pressure
Noriaki MASUI・ and Chiaki KATO
Note
Palladium-catalyzed Substitution Reaction of Allylic
Derivatives
with Tinacetylene
Toshio NISHIKAWA and Minoru ISOBE
Note
A Novel Splicing Isoform of Mouse Sterol Regulatory
Element-binding Protein-1 (SREBP-1)
Jun INOUE and Ryuichiro SATO・
Preliminary Communication
A High-Mr Glycoprotein Fraction from Cow′s Milk Potent
in Inhibiting
Replication of Human Rotavirus in Vitro
Yoshihiro KANAMARU,1,・ Michiko ETOH,1 Xiang-Guang SONG,1 Takashi MIKOGAMI,2
Hirotoshi HAYASAWA,2 Takusaburo EBINA,3 and Nobuyuki MINAMOTO4
Review
Microbial Desulfurization of Organic Sulfur Compounds in Petroleum
Takashi OHSHIRO and Yoshikazu IZUMI・
Department of Biotechnology, Faculty of Engineering, Tottori University, Tottori 680-8552, Japan
Sulfur removal from petroleum is important from the standpoint of the global environment because the combustion of sulfur compounds leads to the production of sulfur oxides, which are the source of acid rain. As the regulations for sulfur in fuels become more stringent, the existing chemical desulfurizations are coming inadequate for the ``deeper desulfurization′′ to produce lower-sulfur fuels without new and innovative processes. Biodesulfurization is rising as one of the candidates. Several microorganisms were found to desulfurize dibenzothiophene (DBT), a representative of the organic sulfur compounds in petroleum, forming a sulfur-free compound, 2-hydroxybiphenyl. They are promising as biocatalysts in the microbial desulfurization of petroleum because without assimilation of the carbon content, they remove only sulfur from the heterocyclic compounds which is refractory to conventional chemical desulfurization. Both enzymological and molecular genetic studies are now in progress for the purpose of obtaining improved desulfurization activity of organisms. The genes involved in the sulfur-specific DBT desulfurization were identified and the corresponding enzymes have been investigated. From the practical point of view, it has been proved that the microbial desulfurization proceeds in the presence of high concentrations of hydrocarbons, and more complicated DBT analogs are also desulfurized by the microorganisms. This review outlines the progress in the studies of the microbial desulfurization from the basic and practical point of view.
dibenzothiophene; desulfurization; monooxygenase; Rhodococcus; petroleum
Characterization of the Enantioselective Properties of the Quinohemoprotein Alcohol Dehydrogenase of Acetobacter pasteurianus LMG 1635. 1. Different Enantiomeric Ratios of Whole Cells and Purified Enzyme in the Kinetic Resolution of Racemic Glycidol
Sonia Salgueiro MACHADO, Ute WANDEL, Jaap A. JONGEJAN,・Adrie J. J. STRAATHOF, and Johannis A. DUINE
Department of Microbiology and Enzymology, Kluyver Institute for Biotechnology,
Delft University of Technology, Julianalaan 67, 2628 BC Delft, The NetherlandsReceived December 22, 1997; Accepted July 6, 1998
Resting cells of Acetobacter pasteurianus LMG 1635 (ATCC 12874) show appreciable enantioselectivity (E＝16--18) in the oxidative kinetic resolution of racemic 2,3-epoxy-1-propanol, glycidol. Distinctly lower values (E＝7--9) are observed for the ferricyanide-coupled oxidation of glycidol by the isolated quinohemoprotein alcohol dehydrogenase, QH-ADH, which is responsible for the enantiospecific oxidation step in whole cells. The accuracy of E-values from conversion experiments could be verified using complementary methods for the measurement of enantiomeric ratios. Effects of pH, detergent, the use of artificial electron acceptors, and the presence of intermediate aldehydes, could be accounted for. Measurements of E-values at successive stages of the purification showed that the drop in enantioselectivity correlates with the separation of QH-ADH from the cytoplasmic membrane. It is argued that the native arrangement of QH-ADH in the membrane-associated complex favors the higher E-values. The consequences of these findings for the use of whole cells versus purified enzymes in biocatalytic kinetic resolutions of chiral alcohols are discussed.
Acetobacter pasteurianus; quinohemoprotein alcohol dehydrogenase; oxidation; enantioselectivity; kinetic resolution
Purified Fusion Enzyme between Rat
Cytochrome P4501A1
and Yeast NADPH-Cytochrome P450 Oxidoreductase
Masayuki HARA,＊,＊＊,・ Jun MIYAKE,＊,＊＊ Yasuo ASADA,＊ and Hideo OHKAWA＊＊＊
＊National Institute of Bioscience and Human-Technology, AIST, MITI, 1-1 Higashi, Tsukuba,
Ibaraki 305-8566, Japan
＊＊ National Institute for Advanced and Interdisciplinary Research, AIST, MITI, 1-1-4 Higashi,
Tsukuba, Ibaraki 305-8562, Japan
＊＊＊Department of Biological and Enviromental Science, Faculty of Agriculture, Kobe University,
1 Rokkodai-cho, Nada-ku, Kobe, 657-0013, JapanReceived April 17, 1998; Accepted September 11, 1998
A genetically engineered fusion enzyme between rat P4501A1 and yeast P450 reductase in the microsomal fraction of the recombinant yeast AH22/pAFCR1 was purified. The purified enzyme showed a typical CO-difference spectrum of P4501A1 and a single band with an apparent molecular weight of 125,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis. This agreed with the molecular weight of 131,202 calculated from the amino acid sequence. The purified enzyme showed both 7-ethoxycoumarin o-deethylase activity and horse heart cytochrome c reductase activity in the presence of NADPH. The 7-ethoxycoumarin o-deethylase activity depended on the species of lipid used for the reconstitution of the purified fusion enzyme although the purified enzyme showed the activity without reconstitution. The purified fusion enzyme had the Km value of 26 μM for 7-ethoxycoumarin and the maximal turnover rate of 29 mol product/min/mol enzyme at 30°C.
P4501A1; CYP1A1; NADPH P450 oxidoreductase; fusion enzyme
Apolipoprotein A-I of Hyperlipidemia
Atherosclerosis Prone (LAP) Quail:
cDNA Sequence and Tissue Expression
Hironori IWASAKI, Hirosuke OKU, Takayoshi TODA,＊ Tetsuo NASU,＊＊
Koji ODA, Tsuyoshi MIYAGI, and Isao CHINENLaboratory of Applied Biochemistry, Faculty of Agriculture, University of The Ryukyus,
Nishihara-Cho, Okinawa 903-0213, Japan
＊Department of Clinical Laboratory Medicine, School of Medicine, University of The Ryukyus,
Nishihara-Cho, Okinawa 903-0125, Japan
＊＊Department of Veterinary Anatomy, Faculty of Agriculture, Miyazaki University,
Miyazaki 889-2155, JapanReceived May 15, 1998; Accepted September 10, 1998
Apolipoprotein A-I (apo A-I) has an important role in the transport of cholesterol. This study describes the complete nucleotide and deduced amino acid sequence for apo A-I of LAP quail. A full length apo A-I cDNA clone for hyperlipidemia atherosclerosis prone (LAP) quail was isolated from a λgt10 liver cDNA library. The DNA sequence of LAP apo A-I cDNA was similar to that of normal Japanese quail. The deduced amino acid sequence of LAP apo A-I was hence identical to that of normal Japanese quail. LAP apo A-I mRNA is about 1.4 kilobases in length and expressed in a variety of tissues including small intestine, liver, lung, breast muscle, testis, and heart. Although the tissue distribution of apo A-I was similar between strains, LAP quail expressed more apo A-I mRNA than normal Japanese quail in all tissues examined. This tendency was pronounced with the small intestine. Although the concentration of serum apo A-I did not correlate with the tissue expression of mRNA, the observation may suggest that the increased apo A-I expression in LAP strain had some relevance to the susceptibility of this strain to the experimental atherosclerosis.
apolipoprotein A-I; quail; mRNA; sequence; expression level
Structures of N-Linked Oligosaccharides of Glycoproteins from Tobacco BY2 Suspension Cultured Cells＊
Nirianne Q. PALACPAC,1 Yoshinobu KIMURA,2 Kazuhito FUJIYAMA,1
Toshiomi YOSHIDA, and Tatsuji SEKI11The International Center for Biotechnology, Osaka University, Yamada-oka 2-1, Suita, Osaka 565, Japan
2Department of Bioresources Chemistry, Faculty of Agriculture, Okayama University, Tsushima-Naka 1-1-1, Okayama 700, Japan
Received May 20, 1998; Accepted September 21, 1998
The structures of N-linked sugar chains of glycoproteins expressed in tobacco BY2 cultured cells are reported. Five pyridylaminated (PA-) N-linked sugar chains were derived and purified from hydrazinolysates of the glycoproteins by reversed-phase HPLC and size-fractionation HPLC. The structures of the PA-sugar chains purified were identified by two-dimensional PA-sugar chain mapping, ion-spray MS/MS analysis, and exoglycosidase digestions. The five structures fell into two categories; the major class (92.5％ as molar ratio) was a xylose containing-type (Man3Fuc1Xyl1GlcNAc2 (41.0％), GlcNAc2Man3Fuc1Xyl1GlcNAc2 (26.5％), GlcNAc1Man3Fuc1Xyl1GlcNAc2 (21.7％), Man3Xyl1GlcNAc2 (3.3％)), and the minor class was a high-mannose type (Man5GlcNAc2 (7.5％)). This is the first report to show that α(1→3) fucosylation of N-glycans does occur but β(1→4) galactosylation of the sugar chains does not in the tobacco cultured cells.
N-glycans; sugar chain; plant glycoprotein; tobacco cultured cell; BY2 cell
Effect of Shellfish Calcium on the
Apparent Absorption of Calcium and Bone Metabolism in Ovariectomized Rats
Takehito MIURA,＊ Yasuyoshi TAKAYAMA, and Masuo NAKANO・
Department of Bioresource Science, Obihiro University of Agriculture and Veterinary Medicine,
Obihiro, Hokkaido 080-8555, Japan
＊United Graduate School of Agriculture Sciences, Iwate University,
Morioka, Iwate 020-8550, Japan
Recieved June 5, 1998; Accepted September 17, 1998
Fossil shellfish powder (FS) and Ezo giant scallop shell powder (EG) were rendered soluble with lactate and citrate under decompression (FSEx and EGEx, respectively) and we examined the effects of lactate-citrate solubilization of FS and EG on mineral absorption, tissue mineral contents, serum biochemical indices and bone mineral density (BMD) in ovariectomized (OVX) rats. The apparent absorption ratios of minerals tended to be high in the rats fed with the solubilized mineral sources, those in the FSEx group being significantly higher than in the FS group. There was no significant difference in the tibia mineral content among the OVX groups. BMD at the distal femoral diaphysis was significantly increased by FSEx and EGEx feeding. It is suggested that solubilization with lactate and citrate under decompression increased the solubility and bioavailability of calcium from such natural sources of shellfish calcium as FS and EG.
calcium; ovariectomized rat; osteoporosis; bone mineral density; bone metabolism
Isolation and Characterization of
a New Quinoprotein Dehydrogenase,
L-Sorbose/L-Sorbosone Dehydrogenase
Akira ASAKURA and Tatsuo HOSHINO
Department of Applied Microbiology, Nippon Roche Research Center, 200 Kajiwara,
Kamakura, Kanagawa 247-8530, JapanReceived June 8, 1998; Accepted September 28, 1998
Gluconobacter oxydans DSM 4025 effectively oxidizes L-sorbose to 2-keto-L-gulonic acid (2 KGA), an industrial precursor of vitamin C. From this microorganism, we purified the enzyme involved in this oxidation reaction. The enzyme is a unique quinoprotein dehydrogenase catalyzing not only the conversion of L-sorbose to L-sorbosone, but also that of L-sorbosone to 2 KGA. The molecular weight of the enzyme was about 135,000, consisting of two subunits with molecular weights of 64,500 and 62,500. As its prosthetic group, non-covalently bound PQQ was found. The dye-linked spectrophotometric enzyme assay showed that the optimum enzyme activity occurred in the pH range about 7.0-9.0, and the enzyme activity was inhibited by EDTA or EGTA. The enzyme showed extremely broad substrate specificity for primary and secondary alcohols, aldehydes, aldoses, ketoses, and other sugar alcohols, but not for methanol or formaldehyde. The cytochrome c obtained from the soluble fraction of this strain was found to act as a physiological electron acceptor of the enzyme.
Gluconobacter; 2-keto-L-gulonic acid; vitamin C; pyrroloquinoline quinone; dehydrogenase
Stabilization of L-Ascorbic Acid by
Superoxide Dismutase and Catalase
Noriko MIYAKE, Miok KIM, and Tadao KURATA・
Institute of Environmental Science for Human Life, Ochanomizu University,
Bunkyo-ku, Tokyo 112-8610, Japan
Received July 3, 1998; Accepted September 19, 1998
The effects of superoxide dismutase (SOD) and catalase on the autoxidation rate of L-ascorbic acid (ASA) in the absence of metal ion catalysts were examined. The stabilization of ASA by SOD was confirmed, and the enzyme activity of SOD, which scavenges the superoxide anion formed during the autoxidation of ASA, contributed strongly to this stabilization. The stabilization of ASA by catalase was observed for the first time; however, the specific enzyme ability of catalase would not have been involved in the stabilization of ASA. Such proteins as bovine serum albumin (BSA) and ovalbumin also inhibited the autoxidation of ASA, therefore it seems that non-specific interaction between ASA and such proteins as catalase and BSA might stabilize ASA and that the non-enzymatic superoxide anion scavenging ability of proteins might be involved.
L-ascorbic acid; autoxidation; superoxide dismutase; catalase; stabilization
Cloning and Expression of a cDNA Encoding the Laccase from Schizophyllum commune
Osamu HATAMOTO,1,2 Hiroshi SEKINE,1 Eiichi NAKANO,2 and Keietsu ABE2
1Noda Institute for Scientific Research, 399 Noda, Noda-shi, Chiba 278-0037, Japan
2Research and Development Division, Kikkoman Corporation, 399 Noda, Noda-shi, Chiba 278-0037, JapanReceived July 6, 1998; Accepted September 18, 1998
We cloned and analyzed the nucleotide sequence of a cDNA that encodes polyphenol oxidase (laccase) from the white-rot basidiomycete Schizophyllum commune. The nucleotide sequence of the full-length cDNA contained a 1554-base open reading frame that encoded a polypeptide of 518 amino acid residues, including a putative signal peptide of 16 residues. It contained four highly similar regions that are conserved in the deduced amino acid sequences of other laccases, including the region thought to be involved in copper binding. Aspergillus sojae strain 1860 (which has low protease levels) was transformed with the plasmid lacAL/pTPT, which contained the laccase gene under the control of the tannase promoter from Aspergillus oryzae. Laccase was secreted into the medium when transformants A1 and A2 were cultured in tannic acid-containing medium.
Schizophyllum commune; polyphenol oxidase; tannase; Aspergillus; laccase
Purification and Properties of a Low-molecular-weight, High-alkaline Pectate Lyase from an Alkaliphilic Strain of Bacillus
Tohru KOBAYASHI,＊ Kenzo KOIKE, Tadashi YOSHIMATSU, Norihiko HIGAKI,
Atsushi SUZUMATSU, Tadahiro OZAWA, Yuji HATADA, and Susumu ITOTochigi Research Laboratories of Kao Corporation, 2606 Akabane, Ichikai,
Haga, Tochigi 321-3497, JapanReceived July 13, 1998; Accepted September 18, 1998
A low-molecular-weight, high-alkaline pectate lyase (pectate transeliminase, EC 4.2.2.2) was found in an alkaline culture of Bacillus sp. strain KSM-P15, purified to homogeneity, and crystallized. The enzyme had a relative molecular weight of approximately 20,300 as measured by sedimentation equilibrium, with a sedimentation coefficient (s020,w) of 1.73S. It was a basic protein with an isoelectric point of pH 10.3, and the α-helical content was only 6.6％. In the presence of Ca2＋ ions, the enzyme degraded polygalacturonic acid in a random manner to yield 4,5-unsaturated oligo-galacturonides and had its optimal activity around pH 10.5 and 50--55°C. It also had a protopectinase-like activity on cotton fibers. The N-terminal amino acid sequences of the intact protein (28 amino acids) and its two lysyl endopeptidase-cleaved peptide fragments (8 and 12 amino acids) had very low sequence similarity with pectate lyases reported to date. These results strongly suggest that the pectate lyase of Bacillus sp. strain KSM-P15 may be a novel enzyme and belongs in a new family.
alkaliphile; Bacillus; pectate lyase; purification; trans-elimination
Purification and Properties of Bacteriolytic Enzymes from Bacillus licheniformis YS-1005 against Streptococcus mutans
So-Young KIM, Seung-Ho OHK, Dong-Hoon BAI,1 and Ju-Hyun YU
Department of Food and Biotechnology, and Bioproduct Research Center, Yonsei University,
Seoul 120-749, Korea
1Department of Food Engineering, Dankook University, Chunan 330-714, KoreaReceived July 21,1998; Accepted September 18,1998
To find a novel lytic enzyme against cariogenic Streptococci, strains showing strong lytic activity have been screened from soil using Streptococcus mutans. A strain identified as Bacillus licheniformis secreted two kinds of lytic enzymes, which were purified by methanol precipitation, CM-cellulose chromatography, gel filtration, and hydroxyapatite chromatography. The molecular weights of these two enzymes, L27 and L45, were 27,000 and 45,000, respectively. Optimum pH and temperature of both enzymes for lytic activity were pH 8 and 37°C. L27 and L45 digest the peptide linkage between L-Ala and D-Glu in peptidoglycan of Streptococcus mutans. The lytic activity was highly specific for Streptococcus mutans, suggesting their potential use as a dental care product.
bacteriolytic enzyme; Bacillus licheniformis; Streptococcus mutans
Protective Effect of Dietary Tomato against Endothelial Dysfunction in Hypercholesterolemic Mice
Hiroyuki SUGANUMA・ and Takahiro INAKUMA
Research Institute, Kagome Co. Ltd., Nishitomiyama-17, Nishinasuno-machi, Nasu-gun,
Tochigi 329-2762, JapanReceived July 22, 1998; Accepted September 29, 1998
The effects of dietary ingestion of tomato were studied in mice that had been made hypercholesterolemic by feeding atherogenic diets. Mice which had been fed on the atherogenic diet without tomato for 4 months had significantly increased plasma lipid peroxide, and the vaso-relaxing activity in the aorta induced by acetylcholine (ACh) was harmed when compared with mice fed on a common commercial diet. On the other hand, mice which had been fed on the atherogenic diet containing 20％ (w/w) lyophilized powder of tomato showed less increase in the plasma lipid peroxide level, and ACh-induced vaso-relaxation was maintained at the same level as that in normal mice. These results indicate that tomato has a preventive effect on atherosclerosis by protecting plasma lipids from oxidation.
atherosclerosis; endothelium; lipid peroxide; tomato
Purification and Characterization of Phospholipase B from Kluyveromyces lactis, and Cloning of Phospholipase B gene
Hideki OISHI, Takahiro MORIMOTO, Yasuo WATANABE, and Youichi TAMAI・
Department of Bioresources, Faculty of Agriculture, Ehime University,
Matsuyama, Ehime 790-8566, JapanReceived July 23, 1998; Accepted September 8, 1998
Phospholipase B (PLB) from the yeast Kluyveromyces lactis was purified to homogeneity from culture medium. The enzyme was highly glycosylated with apparent molecular mass of 160--250 kDa, and had two pH optima, at pH 2.0 and pH 7.5. At acidic pH the enzyme hydrolyzed all phospholipid substrates tested here without metal ion. On the other hand, at alkaline pH the enzyme showed substrate specificity for phosphatidylcholine and lysophosphatidylcholine and required Ca2＋, Fe3＋, or Al3＋ for the activity. The alkaline activity was increased more than 20-fold in the presence of Al3＋ compared to that in the presence of Ca2＋.
cDNA sequence of PLB (KlPLB) was analyzed by a combination of several PCR procedures. KlPLB encoded a protein consist of 640 amino acids and the deduced amino acid sequence showed 66.7％ similarity with the T. delbrueckii PLB. The amino acid sequence contained the lipase consensus sequence (G-X-S-X-G) and the catalytic aspartic acid motif. Replacement of Arg-112 or Asp-406 with alanine caused loss of the enzymatic actiivity at both pH. These results suggested that PLB activity are dependent on a catalytic mechanism similar to that of cytosolic phospholipase A2.
phospholipase B; Kluyveromyces lactis
Thermostability Reinforcement through a Combination of Thermostability-related Mutations of N-Carbamyl-D-Amino Acid Amidohydrolase
Yasuhiro IKENAKA, Hirokazu NANBA, Kazuyoshi YAJIMA, Yukio YAMADA,
Masayuki TAKANO, and Satomi TAKAHASHIFine Chemicals Research Laboratories, Kaneka Corporation, 1-8 Miyamae,
Takasago, Hyogo 676-8688, JapanReceived July 27, 1998; Accepted September 18, 1998
For the improvement of N-carbamyl-D-amino acid amidohydrolase (DCase), which can be used for the industrial production of D-amino acids, the stability of DCase from Agrobacterium sp. KNK712 was improved through various combinations of thermostability-related mutations. The thermostable temperature (defined as the temperature on heat treatment for 10 min that caused a decrease in the DCase activity of 50％) of the enzyme which had three amino acids, H57Y, P203E, and V236A, replaced was increased by about 19°C. The mutant DCase, designated as 455M, was purified and its enzymatic properties were studied. The enzyme had highly increased stability against not only temperature but also pH, the optimal temperature of the enzyme being about 75°C. The substrate specificity of the enzyme for various N-carbamyl-D-amino acids was changed little in comparison with that of the native enzyme. Enzymochemical parameters were also measured.
N-carbamyl-D-amino acid amidohydrolase; thermotolerant enzyme; D-amino acid
Cloning of Bacillus stearothermophilus ctaA and Heme A Synthesis with the CtaA Protein Produced in Escherichia coli
Junshi SAKAMOTO,・ Akiko HAYAKAWA, Tomoko UEHARA,
Shunsuke NOGUCHI, and Nobuhito SONEDepartment of Biochemical Engineering and Science, Kyushu Institute of Technology,
Kawazu 680-4, Iizuka, Fukuoka 820-8502, JapanReceived July 28, 1998; Accepted September 25, 1998
The Bacillus stearothermophilus ctaA gene, which is required for heme A synthesis, was found upstream of the ctaBCDEF/caaEABCD gene cluster as in B. subtilis and B. firmus. The deduced protein sequence indicate that CtaA is a 35-kDa intrinsic membrane protein with seven hydrophobic segments. Alignment of CtaA sequences showed conserved residues including histidines that may be involved in heme B binding and substrate binding. Expression of ctaA in E. coli resulted in increased formation of a membrane-bound b-type cytochrome, heme A production, and severe growth inhibition. Furthermore, B. stearothermophilus CtaA produced in E. coli was found to catalyze the conversion of heme O to heme A in vitro.
heme O; over-expression; protoheme; respiratory chain; thermophilic bacteria
Increase of Hematopoietic Responses by Triple or Single Helical Conformer of an Antitumor (1→3)-β-D-Glucan Preparation, Sonifilan, in Cyclophosphamide-induced Leukopenic Mice
Aiko TSUZUKI, Tomoko TATEISHI, Naohito OHNO, Yoshiyuki ADACHI,
and Toshiro YADOMAE＊Laboratory of Immunophamacology of Microbial Products, School of Pharmacy,
Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi,
Hachioji, Tokyo, 192-0392, JapanReceived July 29, 1998; Accepted October 9, 1998
It has been suggested that the immunopharmacological activity of soluble (1→3)-β-D-glucan depends on its conformation in mice. In this study, we examined the relationship between the conformation of Sonifilan (SPG) and hematopietic responses in cyclophosphamide (Cy)-induced leukopenic mice. SPG, a high molecular weight (1→3)-β-D-glucan, has a triple helical conformation in water, and it was changed by treatment with aqueous sodium hydroxide to the single helical conformer (SPG-OH). The effects of SPG or SPG-OH on hematopoietic responses in cyclophosphamide induced leukopenic mice were investigated by monitoring i) gene expression of cytokines by RT-PCR, ii) protein synthesis of interleukin 6 (IL-6) by ELISA and iii) colony formation of bone marrow cells (BMC). The mice administered Cy and SPG or SPG-OH expressed and produced higher levels of IL-6 mRNA and protein than the mice administered only Cy. Gene expression of NK1.1 was also induced by Cy/SPG (or SPG-OH) treatment. Induced gene expression of stem cell factor (SCF) and macrophage-colony stimulating factor (M-CSF) by SPG/SPG-OH were also found in in vitro culture of BMC from Cy treated mice. These results strongly suggested that conformation of the glucans, single and triple helix, are independent of the hematopietic response.
β-glucan; cytokine; hematopoietic response; cyclophosphamide; transplantation
Effects of Docosahexaenoic and Eicosapentaenoic Acid on Lipid Metabolism, Eicosanoid Production, Platelet Aggregation and Atherosclerosis in Hypercholesterolemic Rats
Yosef ADAN, Kenichi SHIBATA, Masao SATO, Ikuo IKEDA, and Katsumi IMAIZUMI・
Laboratory of Nutrition Chemistry, Department of Food Science and Technology,
Faculty of Agriculture (46-09), Kyushu University, Fukuoka 812--8581, JapanReceived July 31, 1998; Accepted October 5, 1998
Exogenously hypercholesterolemic (ExHC) rats were fed on an atherogenic diet supplemented with 1％ each of either ethyl ester docosahexaenoic acid [EE-DHA, 22:6(n-3)], ethyl ester eicosapentaenoic acid [EE-EPA, 20:5(n-3)] or safflower oil (SO) for 6 months. The rats fed on the diets containing EE-EPA or EE-DHA, compared with those fed on SO, had lower serum cholesterol and triacylglycerol levels, less aggregation of platelets and slower progress of intimal thickening in the ascending aorta. Relative to the SO-fed rats, both of the (n-3) fatty acid-fed rats had a significantly reduced proportion of arachidonic acid in the platelet and aortic phospholipids, and lower production of thromboxane A2 by platelets and of prostacyclin by the aorta. These results suggest that EPA and DHA are similarly involved in preventing atherosclerosis development by reducing hypercholesterolemia and modifying the platelet functions.
atherosclerosis; docosahexaenoic acid; eicosapentaenoic acid; hypercholesterolemia; eicosanoids
Pectin Methylesterase Gene (pmeA)
from Aspergillus oryzae KBN616:
Its Sequence Analysis and Overexpression, and Characterization of the Gene Product
Noriyuki KITAMOTO,・ Hideki OKADA,＊ Shoko YOSHINO, Kunio OHMIYA,＊＊
and Norihiro TSUKAGOSHI＊＊＊Food Research Institute, Aichi Prefectural Government, 2-1-1 Shinpukuji-cho, Nishi-ku,
Nagoya 451-0083, Japan
＊Shin Nihon Chemical Co. Ltd., 19-10 Showa-cho, Anjyo, Aichi 446-0063, Japan
＊＊Faculty of Bioresources, Mie University, 1515 Kamihama-cho, Tsu 514-8507, Japan
＊＊＊Department of Biological Mechanisms and Functions, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku, Nagoya 464-8601, JapanReceived August 10, 1998; Accepted October 1, 1988
A gene (pmeA) encoding pectin methylesterase was isolated from a shoyu koji mold, Aspergillus oryzae KBN616, and characterized. The structural gene comprised 1,370 bp with six introns. The PMEA protein consisted of 331 amino acids with a putative signal peptide of 17 amino acids. The deduced amino acid sequence was very similar to those of Aspergillus niger PMEA and Aspergillus aculeatus PME1. The pmeA gene was efficiently expressed under control of the A. oryzae TEF1 gene promoter for purification and characterization of the ezymatic properties. PMEA had a molecular mass of 38.5 kDa, a pH optimum of 5.0, and a temperature optimum of 55°C.
Aspergillus oryzae; gene cloning; pectin methylesterase; overexpression
Purification and Characterization
of Methylamine Oxidase Induced
in Aspergillus niger AKU 3302
Ivo FR´EBORT,＊ Kazunobu MATSUSHITA, Hirohide TOYAMA, Karel LEMR,＊＊
Mamoru YAMADA, and Osao ADACHI・Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University,
Yamaguchi 753-8515, Japan
＊＊Department of Analytical Chemistry, Faculty of Science, Palack´・ y University, T・・ r. Svobody 8,
CZ-771 46 Olomouc, Czech RepublicReceived August 10, 1998; Accepted September 25, 1998
Crude extract of Aspergillus niger AKU 3302 mycelia incubated with methylamine showed a single amine oxidase activity band in a developed polyacrylamide gel that weakly cross-reacted with the antibody against a copper/topa quinone-containing amine oxidase (AO-II) from the same strain induced by n-butylamine. Since the organism cannot grow on methylamine and the already known quinoprotein amine oxidases of the organism cannot catalyze oxidation of methylamine, the organism was forced to produce another enzyme that could oxidize methylamine when the mycelia were incubated with methylamine. The enzyme was separated and purified from the already known two quinoprotein amine oxidases formed in the same mycelia. The purified enzyme showed a sharp symmetric sedimentation peak in analytical ultracentrifugation showing S020,w of 6.5s. The molecular mass of 133 kDa estimated by gel chromatography and 66.6 kDa found by SDS-PAGE confirmed the dimeric structure of the enzyme. The purified enzyme was pink in color with an absorption maximum at 494 nm. The enzyme readily oxidized methylamine, n-hexylamine, and n-butylamine, but not benzylamine, histamine, or tyramine, favorite substrates for the already known two quinoprotein amine oxidases. Inactivation by carbonyl reagents and copper chelators suggested the presence of a copper/topa quinone cofactor. Spectrophotometric titration by p-nitrophenylhydrazine showed one reactive carbonyl group per subunit and redox-cyclic quinone staining confirmed the presence of a quinone cofactor. pH-dependent shift of the absorption spectrum of the enzyme-p-nitrophenylhydrazone (469 nm at neutral to 577 nm at alkaline pH) supported the identity of the cofactor with topaquinone. Nothern blot analysis indicated that the methylamine oxidase encoding gene is largely different from the already known amine oxidase in the organism.
Aspergillus niger; copper/topa quinone; methylamine oxidase; quinoprotein amine oxidase
Dietary Effect of EPA-rich and DHA-rich
Fish Oils on the Immune Function
of Sprague-Dawley Rats
Pham HUNG,1 Shihoko KAKU,1 Shin-ichi YUNOKI,1 Ken-ichi OHKURA,1 Jiong-Yan GU,1
Ikuo IKEDA,2 Michihiro SUGANO,3 Kazunaga YAZAWA,4 and Koji YAMADA1,・1Laboratory of Food Science and
2Laboratory of Nutrition Science, Department of Food Science and Technology,
Faculty of Agriculture, Kyushu University 46-09, 6-10-1 Hakozaki, Higashi-ku,
Fukuoka 812-8581, Japan
3Department of Human Life, Kumamoto Prefectural University, Kumamoto 862-8502, Japan
4Sagami Chemical Research Center, JapanReceived August 10, 1998; Accepted September 24, 1998
The dietary effect of fish oils (FOs) rich in eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) on the immune function of Sprague-Dawley rats was compared with that of safflower oil. After 3 weeks of feeding at the 10％ level of a dietary fat, the IgG and IgM production by splenocytes and IgG production by mesenteric lymph node (MLN) lymphocytes were significantly higher in the FO-fed rats, while no significant difference was found in IgA or IgE productivity by both the spleen and MLN lymphocytes. In the FO-fed rats, peritoneal exudate cells released a lower amount of LTB4, reflecting their lower arachidonic acid level, and a higher amount of LTB5, reflecting their higher EPA level in phospholipids. On these EPA-rich FO exerted a stronger effect than DHA-rich FO immune functions.
fish oil; fatty acid; histamine; immunoglobulin; leukotriene
Comparison of Base Specificity and Other Enzymatic Properties of Two Protozoan Ribonucleases from Physarum polycephalum and Dictyostelium discoideum
Norio INOKUCHI,a,・ Shigeru SAITOH,a Hiroko KOBAYASHI,a Tadashi ITAGAKI,a
Takashi KOYAMA,a Saburo UCHIYAMA,b and Masachika IRIEcaDepartment of Microbiology, College of Pharmacy, Nihon University, Narashinodai 7-7-1,
Funabashi, Chiba 274-0063, Japan
bDepartment of Biology, Dokkyo University School of Medicine, Mibu, Tochigi 321-0200, Japan
cDepartment of Microbiology, Hoshi College of Pharmacy, Ebara 2-4-41,
Shinagawa 142-0063, Tokyo, JapanReceived August 11, 1998; Accepted September 21, 1998
Base specificity and other enzymatic properties of two protozoan RNases, RNase Phyb from a true slime mold (Physarum polycephalum) and RNase DdI from a cellular slime mold (Dictyostelium discoideum), were compared. These two RNases have high amino acid sequence similarity (83 amino acid residues, 46％). The base specificities of two base recognition sites, The B1 site (base recognition site for the base at 5′-side of scissile phosphodiester bond) and the B2 site (base recognition site for the base at 3′-side of the scissile bond) of the both enzymes were estimated by the rates of hydrolysis of 16 dinucleoside phosphates. The base specificities estimated of B1 and B2 sites of RNase Phyb and RNase DdI were A, G, U>C and A≧G>C>U, and A≧G, U>C and G>U>A, C, respectively. The base specificities estimated from the depolymerization of homopolynucleotides and those from the releases of four mononucleotides upon digestion of RNA coincided well with those of the B2 sites of both enzymes. Thus, in these enzymes, the contribution of the B2 site to base specificity seems to be larger than that of the B1 site.
pH-stability, optimum temperature, and temperature stability, of both enzymes are discussed considering that RNase Phyb has one disulfide bridge deleted, compared to the RNase DdI with four disulfide bridges.
ribonuclease; base specificity; stability; Dictyostelium discoideum; Physarum polycephalum
Purification and Characterization of Aspergillus ficuum Endoinulinase
Tai-Boong UHM,1,・ Mi Sun CHUNG,2 Sun Hee LEE,2 Fran・oise GOURRONC,3
Isabelle HOUSEN,4 Jong Hwa KIM,5 Josef Van BEEUMEN,6 Bernard HAYE,3
and Jean VANDENHAUTE41Faculty of Biological Sciences and Institute for Molecular Biology and Genetics,
2Department of Food Science and Technology, Chonbuk National University, Chonju 561-756, Korea
3Laboratoire de Biochimie, Universit´・ e de Reims, UFR Sciences BP 1 039, 51687 Reims Cedex, France
4Laboratoire de G´・ en´・ etique Mol´・ eculaire, Facult´・ es Universitaires Notre-Dame de la Paix,
Rue de Bruxelles, 61, 5000 Namur, Belgium
5Department of Food Science and Engineering, Woo Suk University, Samrye 565-800, Korea
6Laboratorium voor Eiwitbiochimie en Eiwitengineering, University of Gent, Ledeganckstraat,
35, 9000 Gent, BelgiumReceived August 12, 1998; Accepted October 2, 1998
Endoinulinase from Aspergillus ficuum, which catalyzes the hydrolysis of inulin via an endo-cleavage mode, was purified by chromatography from Novozym 230 as a starting commercial enzyme mixture on CM-Sephadex and DEAE-Sepharose, and by preparative electrophoresis under native conditions. The enzyme was estimated to be pure on the basis of its I/S ratio, whose value was infinite in our assay conditions. Two forms separated by using this method. SDS gel electrophoresis showed the two purified forms to respectively exhibit molecular weights of 64,000±500 and 66,000±1,000. The results of deglycosylation indicated that the two forms were originally the same protein but with different sugar contents. A molecular weight of 54,800±1,500 was found by gel filtration of the native enzyme, indicating the native functional protein to be a monomer. The enzyme showed nearly absolute substrate specificity towards inulin and inulooligosaccharides, and acted via an endo-attack to produce mainly inulotriose during the late stage of the reaction. The apparent Km and Vmax values for inulin hydrolysis were 8.1±1.0 mM and 773±60 U/mg, respectively. The internal peptides of the enzyme showed sequence homology to the endoinulinase of Penicillium purpurogenum.
Aspergillus ficuum; endoinulinase; inulin
Precursor of α-Methylene-γ-butyrolactone
Involved in the Insecticidal Activity
of Thunberg Spiraea, Spiraea thunbergii
Chul-Sa KIM,・ Probal Kanti DATTA, Tetsuro HARA, Eiji ITOH, and Michio HORIIKE
Department of Bioresources Science, Faculty of Agriculture, Kochi University,
B200 Monobe, Nankoku 783-8502, JapanRecieved August 17, 1998; Accepted September 22, 1998
6-Tuliposide A {6--O--(4-hydroxy-α-methylenebutyryl)-D-glucopyranose} was isolated from thunberg spiraea (Spiraea thunbergii) leaves. Acid-hydrolysis of this compound generated tulipalin A (α-methylene-γ-butyrolactone). This compound is thus considered as a precursor of insecticidal tulipalin A.
insecticidal activity; α-methylene-γ-butyrolactone (tulipalin A); thunberg spiraea (Spiraea thunbergii); 6-tuliposide A {6--O--(4-hydroxy-α-methylenebutyryl)-D-glucopyranose}
Cloning and Characterization of Chemotaxis Genes in Pseudomonas aeruginosa
Junichi KATO, Tetsuya NAKAMURA, Akio KURODA, and Hisao OHTAKE
Department of Fermentation Technology, Hiroshima University, Higashi-Hiroshima,
Hiroshima 739-8527, JapanReceived August 21, 1998; Accepted September 21, 1998
Two chemotaxis-defective mutants of Pseudomonas aeruginosa, designated PC3 and PC4, were selected by the swarm plate method after N-methyl-N′-nitro-N-nitrosoguanidine mutagenesis. These mutants were not complemented by the P. aeruginosa cheY and cheZ genes, which had been previously cloned (Masduki et al., J. Bacteriol., 177, 948--952, 1995). DNA sequences downstream of the cheY and cheZ genes were able to complement PC3 but not PC4. Sequence analysis of a 9.7-kb region directly downstream of the cheZ gene found three chemotaxis genes, cheA, cheB, and cheW, and seven unknown open reading frames (ORFs). The predicted translation products of the cheA, cheB, and cheW genes showed 33, 36, and 31％ amino acid identity with Escherichia coli CheA, CheB, and CheW, respectively. Two of the unknown ORFs, ORF1 and ORF2, encoded putative polypeptides that resembled Bacillus subtilis MotA (40％ amino acid identity) and MotB (34％ amino acid identity) proteins, respectively. Although P. aeruginosa was found to have proteins similar to the enteric chemotaxis proteins CheA, CheB, CheW, CheY, and CheZ, the gene encoding a CheR homologue did not reside in the chemotaxis gene cluster. The P. aeruginosa cheR gene could be cloned by phenotypic complementation of the PC4 mutant. This gene was located at least 1,800 kb away from the chemotaxis gene cluster and encoded a putative polypeptide that had 32％ amino acid identity with E. coli CheR.
Pseudomonas aeruginosa; chemotaxis; genome mapping; che gene cluster
Functional Analysis of the Promoter of the Yeast SNQ2 Gene Encoding a Multidrug Resistance Transporter that Confers the Resistance to 4-Nitroquinoline N-Oxide
Zhifeng CUI, Dai HIRATA, and Tokichi MIYAKAWA
Department of Molecular Biotechnology, Faculty of Engineering, Hiroshima University,
Higashi-Hiroshima 739-8527, JapanReceived August 26, 1998; Accepted October 1, 1998
The yeast gene SNQ2, which encodes a multidrug resistance ABC superfamily protein, is required for resistance to the mutagen 4-nitroquinoline N-oxide (4-NQO). The expression of the SNQ2 gene is under the control of a regulatory network that involves the transcription factor Yrr1p, as well as Pdr1p/Pdr3p (Cui et al., Mol. Microbiol., 29, 1307--1315 (1998)). By 5′-deletion analysis of the promoter by using SNQ2-lacZ fusion constructs, four regions: －745 to －639 (region I), －639 to －578 (region II), －548 to －533 (region III) and －533 to －485 (region IV) were found to be important for SNQ2 expression. Genetic analysis suggested that the site in region IV was responsible for the Yrr1p-mediated SNQ2 expression. A consensus motif known for the binding of Pdr1p/Pdr3p (PDRE) was not found in region IV.
Saccharomyces cerevisiae; ABC transporter; multidrug resistance; transcriptional control; SNQ2
Purification and Characterization of Serine Acetyltransferase from Escherichia coli Partially Truncated at the C-Terminal Region
Koshiki MINO, Tsuyoshi YAMANOUE, Takaharu SAKIYAMA, Naoki EISAKI,＊ Asahi MATSUYAMA,＊ and Kazuhiro NAKANISHI・
Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University,
3-1-1 Tsushima-naka, Okayama 700-8530, Japan
＊Research and Development Division, Kikkoman Corporation, 399 Noda, Chiba 278-0037,
Japan/Research Institute of Innovative Technology for the Earth (RITE),
2-8-11 Nishi-shinbashi, Minato-ku, Tokyo 105-0003, JapanReceived September 9, 1998; Accepted September 17, 1998
Incubation of serine acetyltransferase (SAT) from Escherichia coli at 25°C in the absence of protease inhibitors yielded a truncated SAT. The truncated SAT was much less sensitive to feedback inhibition than the wild-type SAT. Analyses of the N- and C-terminal amino acid sequences found that the truncated SAT designated as SATΔC20 was a resultant form of the wild-type SAT cleaved between Ser 253 and Met 254, deleting 20 amino acid residues from the C-terminus. Based on these findings, we constructed a plasmid containing an altered cysE gene encoding the truncated SAT. SATΔC20 was produced using the cells of E. coli JM70 transformed with the plasmid and purified to be homogeneous on an SDS-polyacrylamide gel. Properties of the purified SATΔC20 were investigated in comparison with those of the wild-type SAT and Met-256-Ile mutant SAT, which was isolated by Denk and B¨ock but not purified (J. Gen. Microbiol., 133, 515--525 (1987)). SATΔC20 was composed of four identical subunits like the wild-type SAT and Met-256-Ile mutant SAT. Specific activity, optimum pH for reaction, thermal stability, and stability to reagents for SATΔC20 were similar to those for the wild-type SAT and Met-256-Ile mutant SAT. However, SATΔC20 did not form a complex with O-acetylserine sulfhydrylase-A (OASS-A), a counterpart of the cysteine synthetase and did not reduce OASS activity in contrast to the wild-type SAT and Met-256-Ile mutant SAT.
serine acetyltransferase; O-acetylserine sulfhydrylase-A; cysteine synthetase; truncated serine acetyltransferase; enzyme complex
Note
Insertion Analysis of Putative Functional Elements in the Promoter Region
of the Aspergillus oryzae Taka-amylase A Gene (amyB) Using a Heterologous Aspergillus
nidulans amdS-lacZ Fusion Gene System
Yoji KANEMORI,1 Katsuya GOMI,2,＊,・ Katsuhiko KITAMOTO,2,＃
Chieko KUMAGAI,2,$ and Gakuzo TAMURA31Research Institute of Gekkeikan Sake Co., Ltd., 24 Shimotoba-Koyanagi-cho,
Fushimi-ku, Kyoto 612, Japan
2National Research Institute of Brewing, 3-7-1 Kagamiyama, Higashi-Hiroshima 739, Japan
3Research Institute of Brewing Resources Co. Ltd., 1-54-18 Takinogawa, Kita-ku, Tokyo 114, JapanReceived May 20, 1998; Accepted October 2, 1998
Expression of the Taka-amylase A gene (amyB) of Aspergillus oryzae is induced by starch or maltose. The A. oryzae amyB gene promoter contains three highly conserved sequences, designated Regions I, II, and III, compared with promoter regions of the A. oryzae glaA encoding glucoamylase and the agdA encoding α-glucosidase. To identify the function of these sequences within the amyB promoter, various fragments containing conserved sequences in the amyB promoter were introduced into the upstream region of the heterologous A. nidulans amdS gene (encoding acetamidase) fused to the Escherichia coli lacZ gene as a reporter. Introduction of the sequence between －290 to －233 (the number indicates the distance in base pairs from the translation initiation point (＋1)) containing Region III significantly increased the expression of the lacZ reporter gene in the presence of maltose. The sequence between －377 to －290 containing Region I also increased the lacZ activity, but its maltose inducibility was less than that of Region III. The sequence between －233 to －181 containing Region II had no effect on the expression. These results indicated that Region III is most likely involved in the maltose induction of the amyB gene expression.
promoter; Aspergillus oryzae; Taka-amylase A; gene regulation
Note
Screening of Korean Forest Plants for Rat Lens Aldose Reductase Inhibition
Hye-Young KIM1,・ and Jong Hwan OH2
1Department of Food Chemistry and Biotechnology, Korea Food Research Institute, San 46-1 Baekhyun-dong, Bundang-gu, Sungnam-si, Kyonggi-do 463-420, Korea
2Department of Wood Chemistry, Forestry Research Institute, 207 Cheongyangni-dong, Dongdaemun-ku, Seoul 130-012, KoreaReceived May 28, 1998; Accepted September 19, 1998
Naturally occurring substances which can prevent and treat diabetic complications were sought by examining ethanol extracts prepared from Korean forest plants for their inhibitory effects on rat lens aldose reductase activity in vitro. Among the plants examined, Acer ginnala, Illicium religiosum and Cornus macrophylla exerted the most strong inhibitory activity on aldose reductase.
Korean forest plants; aldose reductase inhibitor; diabetic treatment
Note
Identification of L-Bornesitol and Changes in Its Content during Flower
Bud Development in Sweet Pea (Lathyrus odoratus L.)
Kazuo ICHIMURA,・ Katsunori KOHATA, Yuji MUKASA,・・ Yuichi YAMAGUCHI,
Rie GOTO, and Kenichi SUTONational Research Institute of Vegetables, Ornamental Plants and Tea, Ano, Mie 514-2392, Japan
Received June 15, 1998; Accepted September 28, 1998
An unidentified carbohydrate was isolated from sweet pea (Lathyrus odoratus L. cv. Diana) petals using HPLC. The isolated compound was identified as L-1-O-methyl-myo-inositol, called L-bornesitol, using 1H-NMR, 13C-NMR, and CI-MS. L-Bornesitol was distributed in all organs at high concentrations. L-Bornesitol concentration of petals gradually decreased during flower bud development, but the L-bornesitol content increased by about 5 times.
L-bornesitol; cut flower; soluble carbohydrate; sweet pea
Note
Identification of Endogenous Gibberellins in the Leaves and Xylem Sap of
Tea Plants
Naomi OYAMA,・ Tomoya NIKI, Kunio OKANO,＊ Toyomasa ANAN,＊＊
and Masaji KOSHIOKANational Research Institute of Vegetables, Ornamental Plants, and Tea (NIVOT),
Kusawa, Ano-cho, Age-gun, Mie 514-2392, Japan
＊NIVOT, Taketoyo-cho, Chita-gun, Aich 470-2351, Japan
＊＊NIVOT, Kanaya-cho, Haibara-gun, Shizuoka 428-3501, JapanReceived June 17, 1998; Accepted September 25, 1998
Endogenous gibberellins (GAs) in the young leaves and xylem sap of tea plants (Camellia sinensis L.) were analyzed by GC-MS. The following GAs were identified by comparing their mass spectra and KRIs with those of authentic specimens: GA9 and GA20 in the leaves; GA9, GA12, GA15, GA20, GA44, GA51 and GA53 in the xylem sap.
Camellia sinensis L.; xylem sap; gibberellins
Note
An Improved Method for Isolating Yeasts in the Genus Lipomyces and Related Genera
from Soil
Takafumi NAGANUMA,・ Ken KATSUMATA, Tsutomu ANDO, Hisayuki WATANABE,
Katsushi NISHIMURA, and Yasuyuki UZUKALaboratory of Biotechnology, Department of Applied Chemistry and Biotechnology,
Faculty of Engineering, Yamanashi University, Takeda, Kofu, Yamanashi 400-8511, JapanReceived July 2, 1998; Accepted September 16, 1998
An improved method for isolating soil yeasts in the genus Lipomyces and related genera was developed on the basis of their physiological properties. Liberation of yeast cells from soil, capture of the yeast cells by a membrane filter, and colony formation on an acidic (pH 3) nitrogen-free agar plate containing 0.1％ cycloheximide resulted in success.
soil yeast; Lipomyces yeast; cycloheximide; selective colony formation
Note
Separation and Properties of Two Acetylacetoin Reductases
from Bacillus cereus YUF-4
Takeshi HOSAKA, Sadaharu UI, and Akio MIMURA
Department of Applied Chemistry and Biotechnology, Faculty of Engineering Yamanashi University,
Kofu, Yamanashi 400-8511, JapanReceived July 6, 1998; Accepted September 24, 1998
The separation and purification of two kinds of acetylacetoin reductases (AACRs) from Bacillus cereus YUF-4 were examined. NADPH-linked AACR (AACR I) and NADH-linked AACR (AACR II) were separated from each other by ammonium sulfate fractionation, DEAE-cellulose chromatography, and hydroxyapatite chromatography. The former was purified 3.4-fold with a yield of 10.0％, and the latter was purified 29-fold with a yield of 15.6％. The two enzymes differ from each other in some enzymic properties such as substrate specificity.
acetylacetoin; acetylacetoin reductase; acetylbutanediol; 2,3-butanediol cycle
Note
Comparative Hypocholesterolemic Effects of Five Animal Oils in Cholesterol-fed
Rats
Michihiro FUKUSHIMA, Tetsu OHASHI, Mitsuo SEKIKAWA, and Masuo NAKANO・
Department of Bioresource Science, Obihiro University of Agriculture and Veterinary Medicine,
Obihiro, Hokkaido 080-8555, JapanReceived July 9, 1998; Accepted September 28, 1998
The hypocholesterolemic efficacy of various animal oils was compared in rats given a cholesterol-enriched diet. After acclimatization for one week, male F344 DuCrj rats (8 weeks of age) that had been fed with a conventional diet were assigned to diets containing 5％ of oil from emu (Dromaius), Japanese Sika deer (Cervus nippon yesoensis, Heude), sardine, beef tallow, or lard with 0.5％ cholesterol for 6 weeks. After this feeding period, the concentrations of serum total cholesterol and of very-low-density lipoprotein＋intermediate-density lipoprotein＋low-density lipoprotein-cholesterol in the sardine oil group were significantly lower than those in the other groups. The serum high-density lipoprotein-cholesterol concentration in the Japanese Sika deer oil group was significantly higher than that in the other groups. The atherosclerotic index and liver cholesterol concentration in the sardine oil and Japanese Sika deer oil groups were significantly lower than those in the other groups. The fecal cholesterol excretion by the Japanese Sika deer oil group was significantly higher than that of the other groups, except for the sardine oil group, and the fecal bile acid excretion by the sardine oil group was significantly higher than that of the other groups, except for the lard group. These results suggest that Japanese Sika deer oil reduced the atherosclerotic index and liver cholesterol concentration in the presence of excess cholesterol in the diet as well as sardine oil did by increasing the excretion of cholesterol from the intestines of rats.
animal oil; cholesterol; atherosclerotic index; HDL-cholesterol; rat
Note
Cloning and Sequencing of β-Mannosidase Gene from Aspergillus aculeatus
No. F-50
Goro TAKADA, Takashi KAWAGUCHI, Tomoko KAGA, Jun-ichi SUMITANI, and Motoo ARAI・
Department of Applied Biological Chemistry, and Research Institute for Advanced Science and Technology,
Osaka Prefecture University, Sakai 599-8531, JapanReceived July 10, 1998; Accepted August 19, 1998
The manB gene, coding for a unique β-mannosidase (MANB) of Aspergillus aculeatus, was cloned from genomic and cDNA libraries, and sequenced. The gene consists of 2,811 bp encoding a polypeptide of 937 amino acid residues with a molecular mass of 104,214 Da. The A. aculeatus MANB shared amino acid sequence identity with MANB of human (24％), goat (24％), bovine (24％), and Caenorhabditis elegans (22％). When the A. aculeatus MANB was compared with other related enzymes, a Glu residue corresponding to the active site identified by the Escherichia coli β-galactosidase and the human β-gluclonidase was conserved. This is the first fungal gene that encodes MANB.
Aspergillus aculeatus; β-mannosidase; β-galactosidase; β-guclonidase
Note
A Simple Hydroponic Culture Method for the Development of a Highly Viable
Root System in Arabidopsis thaliana
Tomomi TODA,・ Hiroyuki KOYAMA and Tetsuo HARA
Laboratory of Plant Cell Technology, Department of Biotechnology, Faculty of Agriculture,
Gifu University, 1-1 Yanagido, Gifu, Gifu, 501-1193, JapanReceived July 14, 1998; Accepted September 11, 1998
In the studies of nutritional absorption and metal toxicity in the root, it is important to grow plants without technical damage. We established a simple hydroponic culture system for Arabidopsis thaliana to obtain a healthy plant having a well-developed root system with many lateral roots. The phytotoxic effects of Cr, Cu, and Al ions were examined by FDA-PI staining using this culture system. The pattern of root inhibition varied with the ion, suggesting the usefulness of this culture system.
Arabidopsis thaliana; hydroponic culture; viability
Note
Occurrence in Soybeans of a Novel Vitamin B6 Conjugate that Liberates Pyridoxine
by β-Glucosidase Action after Alkali Treatment
Kenjiro TADERA,・ Kazutaka ODA, and Chikako NAKAHARA
Department of Biochemical Science and Technology, Faculty of Agriculture, Kagoshima University,
Korimoto 1-21-24, Kagoshima 890-0065, JapanReceived July 15, 1998; Accepted October 7, 1998
A type of vitamin B6 conjugates (B6X), which liberates free vitamin B6 by alkaline and successive β-glucosidase hydrolyses, is known to occur in rice bran and wheat bran. Conflicting experimental results, however, have been reported about the occurrence of B6X in soybeans. This study afforded evidence for B6X occuring in soybeans: certainly a highly purified B6X preparation from whole soybeans liberated pyridoxine when it was treated with alkali followed by β-glucosidase hydrolysis, and 5′-O-(β-D-glucopyranosyl)pyridoxine by alkali treatment alone. The B6X content varied with cultivars, of which a certain kind contained no B6X.
vitamin B6 conjugate; pyridoxine conjugate; derivative of pyridoxine glucoside; soybean; B6X
Note
Occurrence of Stereoisomers of 1-(2′-Pyrrolidinethione-3′-yl)-1,2,3,4-
tetrahydro-β-carboline-3-carboxylic Acid in Fermented Radish Roots and Their
Different Mutagenic Properties
Yoshio OZAWA,＊,・ Yasushi UDA,＊＊ Hiroki MATSUOKA,＊ Masako ABE,＊
Shunro KAWAKISHI,＊＊＊ and Toshihiko OSAWA＊＊＊＊＊Department of Food and Nutrition, Gunma Women′s Junior College, Takasaki 370-0033, Japan
＊＊Department of Bioproductive Sciences, Utsunomiya University, Utsunomiya 321-8505, Japan
＊＊＊Department of Food and Nutrition, Sugiyama Jogakuen University, Nagoya 464-8662, Japan
＊＊＊＊Laboratory of Food and Biodynamics, Nagoya University Graduate School of Bioagricultural Sciences, Nagoya 464-8601, JapanReceived July 23, 1998; Accepted October 3, 1998
Stereoisomers of the tetrahydro-β-carboline derivative,||1 - (2′ - pyrrolidinethione - 3′ - yl) - 1,2,3,4 - tetrahydro - β - carbo||line-3-carboxylic acid (PTCC), were formed from L-tryptophan with 4-methylthio-3-butenyl isothiocyanate, and their mutagenic properties and contents in different types of the radish products were studied. The isomers were identified as (1S＊, 3S＊, 3′R＊)- and (1R＊, 3S＊, 3′R＊)-PTCCs; the former was found as the major compound but had no mutagenic activity, while the latter was mutagenic toward Salmonella typhimurium TA 98 in the presence of a rat microsomal fraction. Both (1S＊, 3S＊, 3′R＊)- and (1R＊, 3S＊, 3′R＊)-PTCC were detected in a ratio of about 4:1 in a product fermented for 8 months, but only a trace was apparent in products manufactured within a few weeks.
tetrahydro-β-carboline derivative; stereoisomer; mutagenicity; fermented radish
Note
The Amino Acid Sequence of Wood Duck Lysozyme
Tomohiro ARAKI・ and Takao TORIKATA
Department of Biochemistry, School of Agriculture, Kyushu Tokai University,
Aso, Kumamoto 869-1404, JapanReceived July 28, 1998, Accepted October 2, 1998
The amino acid sequence of wood duck (Aix sponsa) lysozyme was analyzed. Carboxymethylated lysozyme was digested with trypsin and the resulting peptides were sequenced. The established amino acid sequence had the highest similarity to duck III lysozyme with four amino acid substitutions, and had eighteen amino acid substitutions from chicken lysozyme. The valine at position 75 was newly detected in chicken-type lysozymes. In the active site, Tyr34 and Glu57 were found at subsites F and D, respectively, when compared with chicken lysozyme.
lysozyme; amino acid sequence; egg white
Note
Effect of Salt Addition on the Fractal Structure of Aggregates Formed by
Heating Dilute BSA Solutions
Hitoshi KUMAGAI,＊ Tomohide MATSUNAGA, and Tomoaki HAGIWARA・
Department of Applied Biological Chemistry, Division of Agriculture and Agricultural Life Sciences,
The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, JapanReceived July 28, 1998; Accepted September 24, 1998
The fractal dimension, Df, of aggregates in a dilute BSA system with added salt was evaluated by static light scattering (SLS). A fractal structure was observed for the system with NaCl addition. The values of Df increased with increasing heating time and ionic strength. The values of Df were larger than those (Df＝1.8 or 2.1) predicted by the conventional cluster-cluster aggregation model, probably due to a ``restructuring′′ of aggregates during the aggregation process. On the other hand, a fractal structure was not apparent for the system with added CaCl2.
fractal; aggregate; light scattering; bovine serum albumin; metal salt
Note
Citric Acid Production from Xylan and Xylan Hydrolysate by Semi-Solid Culture
of Aspergillus niger
Kohtaro KIRIMURA,・Taisei WATANABE, Tadahiro SUNAGAWA,and Shoji USAMI
Department of Applied Chemistry, School of Science and Engineering, Waseda University,
Ohkubo 3-4-1, Shinjuku-ku, Tokyo 169-8555, JapanReceived July 29, 1998; Accepted October 5, 1998
Citric acid production from xylan and xylan hydrolysate was done by Aspergillus niger Yang no. 2 cultivated in a semi-solid culture using bagasse as a carrier. Yang no. 2 produced 72.4 g/l and 52.6 g/l of citric acid in 5 d from 140 g/l of xylose and arabinose, respectively. Yang no. 2 produced 51.6 g/l of citric acid in 3 d from a concentrated xylan hydrolysate prepared by cellulase treatment, containing 100 g/l of reducing sugars. Moreover, Yang no. 2 directly produced 39.6 g/l of citric acid maximally in 3 d from 140 g/l of xylan.
Aspergillus niger; citric acid; hemicellulose; xylan; xylose
Note
Structural Identification of Sulfated Tyrosine in Human Urine
J. G. Shirani RANASINGHE,1 Yoichi SAKAKIBARA,1 Miyu HARADA,1Kazuo NISHIYAMA,1 Ming-Cheh LIU,2 and Masahito SUIKO1,・
1Department of Biological Resource Sciences, Miyazaki University, Miyazaki 889-2192, Japan
2Department of Biochemistry, The University of Texas Health Center, Tyler, TX 75710 U.S.A.Received August 10, 1998; Accepted September 14, 1998
A reliable HPLC method was used for the identification of positional isomerism and stereoisomerism of sulfated tyrosine residues in human urine. Upon separation of human urine by ion-pair HPLC on a reverse-phase column, p-tyrosine-O-sulfate (p-TyrS) was identified. Differentiation of the L and D forms was done by using a column with a chiral stationary phase. It was concluded that L-p-tyrosine (L-p-Tyr) which is the predominant tyrosine isomer in the human body, was sulfated and excreted in human urine as a normal constituent. The sulfated forms of D-p-Tyr and m-Tyr could not be detected under these analytical conditions.
tyrosine sulfate; stereochemistry; human urine
Note
Detection of ALMB-toxin in the Larval Body of Myrmeleon bore
by Anti-N-terminus Peptide Antibodies
Naofumi YOSHIDA, Hiroshi SUGAMA, Shintaro GOTOH, Kazuhiko MATSUDA,・
Keiichiro NISHIMURA,＊ and Koichiro KOMAIDepartment of Agricultural Chemistry, Faculty of Agriculture, Kinki University,
3327-204 Naka-machi, Nara 631-8505, Japan
＊Research Institute for Advanced Science and Technology, Osaka Prefecture University,
1-2 Gakuen-cho, Sakai, Osaka 599-8570, JapanReceived August 11, 1998; Accepted September 9, 1998
Antibodies were raised against a synthetic antigen carrying the N-terminus peptide of ALMB-toxin, which had been isolated from the antlion, Myrmeleon bore, that exhibited high specificity to the toxin. Analyses with the antibodies showed the toxin to be present mainly at the larval stage and localized in a region from the thorax to abdomen of the larval body.
ALMB-toxin; antlion; Myrmeleon bore; antipeptide antibody; immunoblot
Note
New Method of Screening for Pressure-sensitive Mutants at High Hydrostatic
Pressure
Noriaki MASUI・ and Chiaki KATO
The DEEP STAR Group, Japan Marine Science and Technology Center, 2-15 Natsushima-cho, Yokosuka, Kanagawa 237-0061, Japan
Received August 12, 1998; Accepted September 30, 1998
We have developed an efficient method of screening to detect pressure-sensitive mutants of barophilic or barotolerant bacteria using conventional agar medium plates. By this new method, 75 colonies can be screened per plate under high hydrostatic pressure.
deep-sea; high hydrostatic pressure; barophilic bacteria; pressure-sensitive mutant; agar plate
Note
Palladium-catalyzed Substitution Reaction of Allylic Derivatives with Tinacetylene
Toshio NISHIKAWA and Minoru ISOBE
Laboratory of Organic Chemistry, School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan
Received August 14, 1998; Accepted September 19, 1998
The substitution reaction of allylic derivatives (acetate, carbonate, and chloride) with tinacetylene proceeded in the presence of palladium as a catalyst to give a product having a 1-ene-4-yne system.
allylic substitution; tinacetylene; allylic derivatives; palladium catalyst
Note
A Novel Splicing Isoform of Mouse Sterol Regulatory Element-binding Protein-1(SREBP-1)
Jun INOUE and Ryuichiro SATO・
Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences,
Osaka University, Suita, Osaka 565-0871, JapanReceived September 10, 1998; Accepted September 28, 1998
We cloned a cDNA encoding the NH2-terminal portion of mouse SREBP-1. The deduced amino acid sequence was 76％ and 90％ identical to human and hamster SREBP-1, respectively. We found out a novel splicing isoform of mouse SREBP-1 that lacks 42 amino acid residues composing a PEST sequence observed in unstable proteins. It has been reported that SREBP-1 is rapidly turned over in the nucleus. Although this isoform was not a dominant isoform, it might be possible that the produced protein functions differently from other isoforms including a complete PEST sequence.
SREBP; transcription factor; splicing
Preliminary Communication
A High-Mr Glycoprotein Fraction from Cow′s Milk Potent in Inhibiting Replication
of Human Rotavirus in Vitro
Yoshihiro KANAMARU,1,・ Michiko ETOH,1 Xiang-Guang SONG,1 Takashi MIKOGAMI,2
Hirotoshi HAYASAWA,2 Takusaburo EBINA,3 and Nobuyuki MINAMOTO41Department of Food Science and 4Division of Veterinary Science, Faculty of Agriculture, Gifu University,
Gifu 501-1193, Japan
2Nutritional Science Laboratory, Morinaga Milk Industry Co., Ltd., Zama, Kanagawa 228-8583, Japan
3Division of Immunology, Research Institute Miyagi Cancer Center, Natori, Miyagi 981-1293, JapanReceived September 4, 1998; Accepted Novermber 19, 1998
Rotavirus is the major cause of infectious diarrhea in infants and young children all over the world. We have found that a high-Mr glycoprotein fraction from cow′s milk is potent in inhibiting replication of human rotaviruses in vitro. Since the activity seems to be unique and specific, this fraction may be useful as a novel agent for treatment or prevention of rotavirus diarrhea.
cow′s milk; milk mucin; glycoprotein; human rotavirus; neutralization