Contents and Abstracts of Latest Issue of BBB

(Vol.64 No.1 2000)

Mamoru WAKAYAMA, Harutaka YADA, Shun-ichi KANDA, Shin-ichi HAYASHI,
Yukinori YATSUDA, Kenji SAKAI, and Mitsuaki MORIGUCHI†a

Department of Applied Chemistry, Faculty of Engineering, Oita University, Oita 870-1192, Japan

Received May 24, 1999; Accepted September 24, 1999
D-Aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) was strongly inactivated by diethylpyrocarbonate (DEPC). An H67N mutant was barely active, with a kcat/Km 6.3×
104 times lower than that of the recombinant wild-type enzyme, while the H67I mutant lost detectable activity. The H67N mutant had almost constant Km, but greatly decreased kcat. These results suggested that His67 is essential to the catalytic event. Both H69N and H69I
mutants were overproduced in the insoluble fraction.
The kcat/Km of H250N mutant was reduced by a factor of 2.5×104-fold as compared with the wild-type enzyme. No significant difference between H251N mutant and wild-type enzymes in the Km and kcat was found. The Zn content of H250N mutant was nearly half of that of wild-type enzyme. These results suggest that the His250 residue might be essential to catalysis via Zn binding.
D-aminoacylase; Alcaligenes; histidine residues; Zn enzyme

-2-
Screening for the In Vitro Anti-tumor-promoting Activities
of Edible Plants from Malaysia

Akira MURAKAMI,1 Abdul Manaf ALI,2 Kamaruddin MAT-SALLEH,3
Koichi KOSHIMIZU,1 and Hajime OHIGASHI4,†a

1Department of Biotechnological Science, Faculty of Biology-Oriented Science and Technology,
Kinki University, Iwade-Uchita, Wakayama 649-6493, Japan
2Department of Biotechnology, Faculty of Food Science and Technology, Universiti Putra Malaysia,
43400 UPM Serdang, Selangor, Malaysia
3Department of Botany, Faculty of Life Sciences, Universiti Kebangsaan Malaysia,
46300 Bangi, Selangor, Malaysia
4Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University,
Kyoto 606-8502, Japan

Received June 7, 1999; Accepted September 16, 1999
A total of 114 methanol extracts from 42 plant families of edible Malaysian plants were screened for their inhibitory activities toward tumor promoter 12-O-hexadecanoylphorbol-13-acetate (HPA)-induced Epstein-Barr virus (EBV) activation in Raji cells. By testing at a concentration of 200 ?Eg/ml, 74� cancer chemoprevention is collectively discussed.
anti-tumor promotion; Epstein-Barr virus; Southeast Asia; Piper betle L.; Malaysia

-3-
Generation of Recombinant Human Large Latent Transforming Growth
Factor-?A1 and Monoclonal Antibodies to It

Haruhiko TSUMURA,1,3,†a Yasuyuki ISHII,2 Eiko SHIMIZU,3
Hideya OHASHI2 and Kazuhiro J. MORI1

1Department of Biology, Faculty of Science, Niigata University, Niigata 950-2102, Japan
2Pharmaceutical Research Laboratory, Kirin Brewery Co., Ltd., Miyahara 3, Takasaki,
Gunma 371-1295, Japan
3Pharmaceutical Development Laboratory, Kirin Brewery Co., Ltd., Soujya 1-2-2, Maebashi,
Gunma 371-0853, Japan

Received June 22, 1999; Accepted August 25, 1999
Transforming growth factor ?A1 (TGF-?A1) is a regulator of cell growth and differentiation. It is produced in various of cells and tissues as a biologically latent complex, whose significance is still unknown. We established a Chinese hamster ovary cells that produced recombinant human large latent TGF-?A1. The growth factor was purifeid from serum-free conditioned medium of the cell line was purified to apparent homogeneity by four steps of column chromatography. The purified protein gave a single band with the apparent molecular weight of 210,000 on SDS-PAGE, and had four subunits, of 12.5, 40, 53, and 150--190 kDa. These components were identical to TGF-?A1, the N-terminal remnant of pro-TGF-?A1, pro-TGF-?A1, and latent TGF-?A1 binding protein, respectively. The purified growth factor had biological activity similar to that of the growth factor purified from human platelets. We prepared four monoclonal antibodies by immunization of mice with the recombinant protein. In western blotting, two of the antibodies bound to latent TGF-?A1 binding protein. The two other antibodies reacted with the N-terminal remnant of pro-TGF-?A1. Recombinant large latent TGF-?A1 and its monoclonal antibodies could be used for detailed structural and functional studies of the large latent TGF-?A1 complex.
large latent transforming growth factor ?A1; monoclonal antibody; Chinese hamster ovary cell

-4-
Screening for Drosophila Proteins with Distinct Expression Patterns
during Development by use of Monoclonal Antibodies

Takeshi KUMAGAI, Hiroaki YOKOYAMA, Akira GOTO, Junko HIROSE,�-
Tatsuhiko KADOWAKI, Hiroshi NARITA,�- and Yasuo KITAGAWA†a

Graduate Program for Regulation of Biological Signals, Graduate School of Bioagricultural Sciences,
Nagoya University, Chikusa, Nagoya 464-8601, Japan
�-Department of Food and Nutrition, Kyoto Women�?s University, Higashiyamaku, Kyoto 605-8501, Japan

Received June 28, 1999; Accepted August 30, 1999
Kc 167 is a cell line established from Drosophila embryonic hemocytes and has been shown to express many extracellular matrix (ECM) and other proteins important during development. We have screened
monoclonal antibodies (mAbs) raised against heparin
affinity purified proteins from conditioned medium of Kc 167 cells to identify novel proteins with important roles for development. One mAb recognized a protein expressed with temporary and tissue specific patterns during Drosophila embryogenesis and larval development. This approach is an alternative to screening of Expression Sequence Tag (EST) clones by in situ hybridization to initiate reverse genetics. In addition, a number of mAbs recognizing ECM proteins were also identified. These mAbs will be useful for biochemical and cell biological analyses of Drosophila ECM proteins.
monoclonal antibody; Drosophila cell line; development; extracellular matrix proteins; reverse genetics

-5-
Gene Encoding a Dextransucrase-like Protein in Leuconostoc mesenteroides
NRRL B-512F�-

Kazumi FUNANE,†a Kouichi MIZUNO,�? Hidenari TAKAHARA1 and Mikihiko KOBAYASHI2

Carbohydrate Science Laboratory, National Food Research Institute, Ministry of Agriculture,
Forestry and Fisheries, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8642, Japan
1Department of Resource Biology, Faculty of Agriculture, Ibaraki University, Ami, Ibaraki 300-0393, Japan
2Akita Research Institute of Food and Brewing, 4-26 Sanuki, Araya, Akita-shi 010-1623, Japan

Received June 29, 1999; Accepted August 23, 1999
A gene, dsrT, encoding a dextransucrase-like protein was isolated from the genomic DNA libraries of Leuconostoc mesenteroides NRRL B-512F dextransucrase-like gene. The gene was similar to the intact open reading frames of the dextransucrase gene dsrS of L. mesenteroides NRRL B-512F, dextransucrase genes of strain NRRL B-1299 and streptococcal glucosyltransferase genes, but was truncated after the catalytic domain, apparently by the deletion of five nucleotides. dsrT mRNA was produced in this strain L. mesenteroides when cells were grown in a sucrose medum, but at a level of 20�
dextransucrase; glucosyltransferase; dextran; Leuconostoc mesenteroides

-6-
Induction and Repression of a Streptomyces lividans Chitinase Gene Promoter
in Response to Various Carbon Sources

Kiyotaka MIYASHITA,†a Takeshi FUJII, and Akihiro SAITO

National Institute of Agro-Environmental Sciences, 3-1-1 Kan-nondai, Tsukuba 305-8604, Japan

Received June 29, 1999; Accepted September 20, 1999
Induction and repression of a gene for chitinase (chiA) in Streptomyces lividans was investigated using a catechol 2,3-dioxygenase gene (xylE) as the reporter gene. Of various substrates examined, expression of the promoter (PchiA) was observed after a delay when colloidal chitin or small chitin-oligosaccharides were added to the medium. N-acetylglucosamine completely repressed the chiA promoter. The duration of the delay in expression of PchiA differed with the inducer used, with chitobiose inducing the activity most rapidly. The minimum concentration of chitobiose needed for induction was 1 ?EM. It appears, therefore, that an efficient inducer of the gene for chitinase in S. lividans is chitobiose.
Streptomyces lividans; chitinase gene; catabolite repression; chitobiose

-7-
Amino Acid Sequence of an Unique Ribonuclease with a C-Terminus rich
in O-Glycosylated Serine and Threonine from Culture Medium
of Lentinus edodes

Norio INOKUCHI,a,†a Hiroko KOBAYASHI,a Jyun HARA,a Tadashi ITAGAKI,a
Takashi KOYAMA,a Masanori IWAMA,b Kazuko OHGI,b and Masachika IRIEb

aDepartment of Microbiology, College of Pharmacy, Nihon University,
Narashinodai 7-7-1, Funabashi, Chiba 274-8555, Japan
bDepartment of Microbiology, Hoshi College of Pharmacy,
Ebara 2-4-41, Shinagawa-ku, Tokyo 142-8501, Japan

Received July 2, 1999; Accepted August 31, 1999
The mushroom Lentinus edodes produces three base-
non-specific and acid ribonucleases, RNases Le2, Le37, and Le45. The latter two are excreted from mycelia into the medium. The primary structure of RNase Le37, which had a molecular mass of 37 kDa, was sequenced. It was a member of the RNase T2 family, as is RNase Le2. RNase Le37 was some 30 amino acid residues longer at the C-terminal end than RNase Le2. The C-terminal region of RNase Le37 was rich in O-glycosylated serine and threonine. In fungal glucoamylases and chitinases, which hydrolyze raw-starch and chitin, respectively, have structures resembling the structure of the C-terminal of RNase Le37.
base-non-specific ribonuclease; Lentinus edodes; primary structure; ribonuclease; peptide rich in serine and threonine

-8-
Molecular Modeling Study of Highly Branching (1＝N3)-??-D-Glucan, a Model
Polysaccharide for Cariogenic Glucan, Using the N-H Mapping Method

Toshifumi YUI,†a Kimihiro GOTO, Yoshinobu KAWANO, and Kozo OGAWA�-

Faculty of Engineering, Miyazaki University, Nishi 1-1, Gakuen-kibanadai, Miyazaki,
Miyazaki 889-2192, Japan
�-Research Institute for Advanced Science and Technology (RIAST), Osaka Prefecture University,
1-2 Gakuen-cho, Sakai, Osaka 599-8570, Japan

Received July 5, 1999; Accepted September 16, 1999
A systematic search for possible regular helical structures of a highly branching (1＝N3)-??-D-glucan was done using the n-h mapping technique, combined with MM3-generated relaxed-residue energy map calculations with respect to the conformations of the backbone
glycosidic linkages. The ??-D-glucan, consisting of a
(1＝N3)-??-linked backbone with ??-D-glucose side
residues attaching to an O6 atom of every second backbone residue, was considered as a model polysaccharide of a branching part of the glucan produced by oral bacteria, which was known to be related to dental plaque formation and to contribute to dental caries. The potential energy surfaces of the trisaccharide repeating unit of the branching ??-D-glucan indicated that (1＝N6)-??-linked side residues did not appear to interfere significantly with the backbone stereochemistry, probably due to a further separation of the three-bond-linked side residue compared with an ordinary two-bond-linked residue. Based on the n-h maps of the branching ??-D-glucan, the side residues, when involved in a complete helix, mostly contributed additional stabilizations to particular helical structures. It was found by checking the typical helix models that formation of hydrogen bonds involving side residues was probably a major cause of the stabilization. This hydrogen bonding was expected to increase insolubility for the glucan chain---a typical, physical property observed for the bacterial ??-D-glucan---by introducing its backbone stereochemistry as an additional stiff feature.
cariogenic ??-D-glucan; relaxed-residue energy map; n-h mapping method; regular helix models

-9-
Structure of Ribulose 1,5-bisphosphate Carboxylase/Oxygenase Gene Cluster
from a Thermophilic Hydrogen-Oxidizing Bacterium, Hydrogenophilus
thermoluteolus, and Phylogeny of the Fructose 1,6-Bisphosphate
Aldolase Encoded by cbbA in the Cluster

Nobuhiro R. HAYASHI,1 Koichi TERAZONO,1 Tohru KODAMA,2 and Yasuo IGARASHI1,†a

1Department of Biotechnology, University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan
2Faculty of Textile Science and Technology, Shinshu University, Tokida 3-15-1, Ueda-City,
Nagano, 386-0018, Japan

Received July 8, 1999; Accepted September 1, 1999
Four genes, cbbO, cbbY, cbbA, and the pyruvate
kinase gene (pyk), were found downstream of ribulose
1,5-bisphosphate carboxylase/oxygenase (RubisCO) genes, cbbLS, from a thermophilic hydrogen-oxidizing bacterium, Hydrogenophilus thermoluteolus (formerly Pseudomonas hydrogenothermophila). cbbO was similar to norD in the denitrification gene cluster, and cbbY was similar to cbbY from other autotrophic bacteria. cbbA encoded fructose 1,6-bisphosphate aldolase (FBP aldolase); however, CbbA was little similar to other CbbA proteins. When CbbA was overexpressed in Escherichia coli, overproduction of CbbA was detected by SDS-PAGE. However, the cell extract had slightly higher activity than a cell extract of E. coli without cbbA. Phylogenetic analysis showed class II FBP aldolase divided into classes IIA and IIB, and that CbbA from H. thermoluteolus was in class IIA. Activities of RubisCO and FBP aldolase were examined under autotrophic, mixotrophic, and heterotrophic conditions. The activities of the two enzymes were regulated independently.
RubisCO; FBP aldolase; cbb gene; Hydrogenophilus thermoluteolus

-10-
Effects of Hydrostatic Pressure and Temperature on Growth and Lipid
Composition of the Inner Membrane of Barotolerant Pseudomonas sp.
BT1 Isolated from the Deep-sea

Hiroyuki KANEKO,†a Hideto TAKAMI, Akira INOUE, Koki HORIKOSHI

The DEEP STAR Group, Japan Marine Science and Technology Center, 2-15 Natsushima-cho,
Yokosuka 237-0061, Japan

Received July 9, 1999; Accepted October 1, 1999
A barotolerant member of the genus Pseudomonas was isolated from deep-sea sediment obtained from the Japan Trench, at a depth of 4418 m. The growth temperature was found to affect the hydrostatic pressure range in which the bacterium could grow; the optimum hydrostatic pressure for growth shifted to a higher pressure with increasing temperature. We examined the lipid composition of the inner membrane of cells grown at various hydrostatic pressures and temperatures. The fatty acid components of the inner membrane lipids were C16:0, C16:1, C18:0, and C18:1. The phospholipid components of the inner membrane were phosphatidylethanolamine, cardiolipin, phosphatidylglycerol, and phosphatidylserine. It is evident that the effects of elevated hydrostatic pressure are comparable to the effects of low temperature on both the fatty acid composition of the inner membrane lipids and the phospholipid composition of the inner membrane of this bacterium.
barotolerant bacterium; Pseudomonas; hydrostatic pressure; temperature; membrane composition

-11-
Sequence of egV and Properties of EgV, a Ruminococcus albus Endoglucanase
Containing a Dockerin Domain

Hiroki OHARA,1 Jyunko NOGUCHI,1 Shuichi KARITA,2,†a Tetsuya KIMURA,1
Kazuo SAKKA,1 and Kunio OHMIYA1

Faculty of Bioresources1 and Center for Molecular Biology and Genetics,2 Mie University, Tsu 514-8507, Japan

Received July 9, 1999; Accepted October 8, 1999
The Ruminococcus albus F-40 egV gene, encoding endoglucanase V (EGV), consists of an open reading frame of 1,833 nucleotides and encodes 611 amino acids with a deduced molecular weight of 67,103. The deduced EGV is a modular enzyme composed of a catalytic domain of family 5 of glycosyl hydrolases, a domain of unknown function, and a dockerin domain responsible for cellulosome assembly, suggesting that R. albus F-40 produces a cellulosome, and EGV is a component of the cellulosome. A truncated form of EGV with an apparent molecular weight of 42,000 was purified from a recombinant Escherichia coli and characterized since EGV suffered from partial proteolysis by E. coli protease(s). The truncated EGV was active toward carboxylmethyl cellulose, xylan, lichenan, and acid-swollen cellulose. The pH and temperature optima of the enzyme were 7.0 and 40�?C, respectively. By Western blot analysis using the antiserum raised against the truncated enzyme, EGV was detected in the whole cells but not in the culture supernatant of R. alubus F-40, suggesting that EGV was located on the cell surface.
Ruminococcus albus; endoglucanase; dockerin domain; cellulosome

-12-
Requirement for Lysine-19 of the Yeast Mitochondrial ATPase Inhibitor
for the Stability of the Inactivated Inhibitor-F1Fo Complex at Higher pH

Naoki ICHIKAWA,†a Ryoko FUJISAKA, and Reiko KURIBAYASHI

Department of Food and Nutrition, Faculty of Human Life Science, Osaka City University,
3-138 Sugimoto, Sumiyoshiku, Osaka 558-8585, Japan

Received July 13, 1999; Accepted September 22, 1999
The ATPase inhibitor is a regulatory subunit of mitochondrial ATP synthase. In this study, the role of Lys19 of the yeast ATPase inhibitor was examined by site-directed mutagenesis. Two amino acids (Gln and Glu) were substituted for the Lys19. The purified mutant inhibitor (Lys19＝NGln) had similar ATPase inhibitory activity to that of the wild-type inhibitor at pH 6.5, but was less active at pH 7.4. ATP synthesis in mutant mitochondria was normally activated by the addition of ADP and succinate, but the inactivated ATPase complex in the mutant mitochondria was activated more readily than that in control cells by raising pH. These results show that Lys19 of the yeast ATPase inhibitor is not essential for ATPase inhibitory activity,
but increases the stability of the inhibitor-F1Fo complex
at higher pH.
ATPase inhibitor (IF1); ATP synthase; F1Fo-ATPase; mitochondria

-13-
Purification and Characterization of a Thermostable Chitinase
from Streptomyces thermoviolaceus OPC-520 and Cloning
of the Encoding Gene

Hiroshi TSUJIBO,†a Naoya HATANO, Hiroshi ENDO, Katsushiro MIYAMOTO,
and Yoshihiko INAMORI

Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan

Received July 16, 1999; Accepted October 14, 1999
When Streptomyces thermoviolaceus OPC-520 was grown in a minimal medium with 1�
detected. Among them, Chi30 was purified from the culture filtrate of the strain. The molecular mass was estimated to be 30 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and its isoelectric point was 3.8. The optimum pH and temperature of Chi30 were 4.0 and 60�?C, respectively. Chi30 was stable at pH 6--8 up to 60�?C. The gene encoding Chi30 (chi30) was cloned and its nucleotides sequenced. The open reading frame of chi30 encoded a protein consisting of 347 amino acids with a calculated molecular weight of 35,621. The mature Chi30 consisted of only a catalytic domain and showed a significant similarity with ChiA from S. coelicolor and ChiA from S. lividans. The existence of a 12-bp direct repeat sequence in the promoter region of chi30 was detected, which have been suggested to be involved in both chitin induction and glucose repression.
chitinase; direct repeat sequence; Streptomyces thermoviolaceus

-14-
Okaramines N, O, P, Q and R, New Okaramine Congeners,
from Penicillium simplicissimum ATCC 90288

Yoshihito SHIONO, Kohki AKIYAMA, and Hideo HAYASHI†a

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

Received July 26, 1999; Accepted September 2, 1999
Five new okaramine congeners, okaramines N, O, P, Q, and R, were isolated from Penicillium simplicissimum ATCC 90288. Their structures were determined by an analysis of spectroscopic data. The insecticidal activity of these new okaramines was evaluated against silkworms.
Penicillium simplicissimum; okara; okaramines; insecticidal compound

-15-
New Paralytic Alkaloids, Asperparalines A, B and C, from Aspergillus
japonicus JV-23

Hideo HAYASHI,†a Yukifumi NISHIMOTO, Kohki AKIYAMA, and Hiroshi NOZAKI�-

Department of Applied Biological Chemistry, College of Agriculture, Osaka Prefecture University,
1-1 Gakuen-cho, Sakai, Osaka 599-8531, Japan �-Department of Biological Chemistry, Faculty of Science, Okayama University of Science,
Ridai-cho, Okayama 700-0005, Japan

Received July 28, 1999; Accepted September 27, 1999
New paralytic alkaloids, asperparalines A (1), B (2) and C (3), were isolated from okara (the insoluble residue of whole soybean) that had been fermented with Aspergillus japonicus JV-23. Their structures were elucidated by spectroscopic methods and X-ray crystallography. These asperparalines showed paralytic activity against silkworms.
paralytic compound; asperparaline; Aspergillus japonicus

-16-
High Molecular Weight Transglutaminase Inhibitor Produced
by a Microorganism (Streptomyces lavendulae Y-200)

Koji IKURA,†a Kazuo MINAMI, Chiemi OTOMO, Hiroyuki HASHIMOTO,
Shunji NATSUKA, Kohei ODA, and Katsuyoshi NAKANISHI

Department of Applied Biology, Kyoto Institute of Technology, Matsugasaki, Kyoto 606-8585, Japan

Received August 9, 1999; Accepted September 21, 1999
Transglutaminases catalyze the cross-linking and amine incorporation of proteins, and are implicated in various biological phenomena such as blood clotting, wound healing, apoptosis, and cell differentiation. Streptomyces lavendulae Y-200, isolated from soil, produced a substance that inhibited transglutaminases. The inhibitory substance was purified from the cultured medium by procedures of acid precipitation, deoxyribonuclease treatment, and gel filtration chromatography. The partially purified sample was dark brown. The inhibitory activity was stable under acidic, alkaline, and high temperature conditions, and resistant to the treatment with proteinases such as trypsin and Pronase. The molecular weight of the inhibitory substance was estimated to be between 104 and 105 from its permeability through ultrafilter membranes. The acid hydrolysate of the inhibitory substance contained amino acids and sugars. The inhibitory substance inhibited both calcium-dependent and calcium-independent transglutaminases in a competitive manner with a glutamine substrate. The extent of inhibition caused by the calcium-dependent transglutaminase increased with increasing calcium concentration. The results obtained here may help identify a novel regulatory substance of transglutaminase in biological systems.
transglutaminase; transglutaminase inhibitor

-17-
Aspirin and Salicylic Acid Do not Inhibit Methyl Jasmonate-inducible
Expression of a Gene for Ornithine Decarboxylase in Tobacco BY-2 Cells

Shunsuke IMANISHI,1,2 Makiko NAKAKITA,1 Kouji YAMASHITA, Ayami FURUTA,
Kaname UTSUNO,3 Nobuhiko MURAMOTO, Hisae KOJIMA, and Kenzo NAKAMURA†a

Laboratory of Biochemistry, Department of Biological Mechanisms and Functions, Graduate School of Bioagricultural Science, Nagoya University, Chikusa, Nagoya 464-8601, Japan

Received August 9, 1999; Accepted September 14, 1999
Similar to the prostanoid-mediated inflammatory response in mammals, jasmonate-mediated wound response in plant leaves is inhibited by salicylic acid (SA) or acetylsalicylate (aspirin). In tobacco BY-2 cells, expression of the gene for ornithine decarboxylase (ODC) involved in putrescine synthesis is rapidly inducible by methyl jasmonate (MeJA). A nuclear gene for ODC isolated from tobacco, gNtODC-1, was an intron-less gene and MeJA induced the expression of a GUS fusion gene with the gNtODC-1 promoter in transformed tobacco cells. Although SA alone did not induce the expression, 0.2 to 20 ?EM SA increased the MeJA-induced expression of the fusion gene to about two-fold. A similar increase was observed with aspirin but not with 3- or 4-hydroxybenzoic acids. SA at concentrations up to 200 ?EM did not inhibit the MeJA-induction of mRNAs for the GUS fusion gene and the endogenous gene for ODC.
jasmonate induction; salicylic acid; aspirin; ornithine decarboxylase gene of tobacco; tobacco cells

-18-
Enzymatic Synthesis of Stable, Odorless, and Powdered Furanone Glucosides
by Sucrose Phosphorylase

Satoshi KITAO,†a Takanao MATSUDO, Tsutomu SASAKI, Takuro KOGA�- and Mishio KAWAMURA�-�-

Research and Development Division, Kikkoman Corporation, 399 Noda, Noda, Chiba 278-0037, Japan
�-Noda Institute for Scientific Research, 399 Noda, Noda, Chiba 278-0037, Japan
�-�-Department of Biology, Osaka Kyoiku University, 4-698-1 Asahigaoka, Kashihara, Osaka 582-8582, Japan

Received August 9, 1999; Accepted September 9, 1999
Sucrose phosphorylase from Leuconostoc mesenteroides catalyzed transglucosylation from sucrose
to 4-hydroxy-3(2H)-furanone derivatives. When 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) and 2-ethyl-4-hydroxy-5-methyl-3(2H)-furanone or 5-ethyl-4-hydroxy-2-methyl-3(2H)-furanone (EHMF) were used as acceptors, their transfer ratios were more than 45�
identified as 2-ethyl-5-methyl-3(2H)-furanone 4-O-??-
D-glucopyranoside (2E5MF-G) and 5-ethyl-2-methyl-3(2H)-furanone 4-O-??-D-glucopyranoside (5E2MF-G) on the bases of spectrometric investigations. These glucosides were more stable than each aglycone. The glucosylated HDMF, DMF-G, was an odorless chemical, on the other hand, HDMF had a pineapple flavor. The glucosylated EHMF (EMF-G) were white odorless powders, though aglycone EHMF was a pale yellow syrup like a caramel with an intense sweet odor. Although DMF-G and EMF-G showed little radical-scavenging activity, hydrolyzates of these glucosides by an intestinal acetone powder from pigs had antioxidative activity as well as their aglycones. It was suggested that these glucosides improved some physical properties and may become prodrugs by glucosylation.
furanone; sucrose phosphorylase; transglucosylation; antioxidant

-19-
Molecular Characterization of a Novel Yeast Cell-wall Acid Phosphatase
Cloned from Kluyveromyces marxianus

Koji YODA,†a Jung-Hwan KO, Takuya NAGAMATSU, Ying LIN,
Chiaki KAIBARA, Tsuyoshi KAWADA, Nario TOMISHIGE, Hitoshi HASHIMOTO,
Yoichi NODA, and Makari YAMASAKI†a†a

Department of Biotechnology, the University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

Received August 9, 1999; Accepted September 9, 1999
A novel Kluyveromyces marxianus gene that encodes an acid phosphatase, Pho610, was cloned in Saccharomyces cerevisiae. The deduced amino acid sequence was distinct from S. cerevisiae phosphatases but similar to some fungal enzymes. A peculiar feature of the sequence is that it has hydrophobic stretches both at the N- and C-termini, which is a characteristic of
the precursors of glycosylphosphatidylinositol(GPI)-
anchored proteins. When the gene was expressed in S. cerevisiae, the active enzyme was recovered in the periplasmic fraction by glucanase digestion. The Pho610 polypeptide was highly glycosylated and a significant portion was covalently linked to the cell-wall glucan. The enzyme was secreted when the C-terminal region was truncated to remove the GPI signal. Therefore, Pho610 is a novel cell-wall protein having an enzyme activity.
GPI-anchor protein; cell wall; acid phosphatase; Saccharomyces cerevisiae; Kluyveromyces marxianus

-20-
Identification of a 14-3-3 Protein from Lentinus edodes That Interacts
with CAP (Adenylyl Cyclase-associated Protein), and Conservation
of This Interaction in Fission Yeast

Guo-Lei ZHOU, Takaharu YAMAMOTO, Fumiyo OZOE, Daisuke YANO,
Katsunori TANAKA, Hideyuki MATSUDA, and Makoto KAWAMUKAI†a

Department of Life Science and Biotechnology, Faculty of Life and Environmental Science,
Shimane University, Matsue, 690-8504, Japan

Received August 11, 1999; Accepted September 13, 1999
We previously identified a gene encoding a CAP (adenylyl cyclase-associated protein) homologue from the edible Basidiomycete Lentinus edodes. To further discover the cellular functions of the CAP protein, we searched for CAP-interacting proteins using a yeast two-hybrid system. Among the candidates thus obtained, many clones encoded the C-terminal half of an L. edodes 14-3-3 homologue (designated cip3). Southern blot analysis indicated that L. edodes contains only one 14-3-3 gene. Overexpression of the L. edodes 14-3-3 protein in the fission yeast Schizosaccharomyces pombe rad24 null cells complemented the loss of endogenous 14-3-3 protein functions in cell morphology and UV sensitivity, suggesting functional conservation of 14-3-3 proteins between L. edodes and S. pombe. The interaction between L. edodes CAP and 14-3-3 protein was restricted to the N-terminal domain of CAP and was confirmed by in vitro co-precipitation. Results from both the two-hybrid system and in vivo co-precipitation experiments showed the conservation of this interaction in S. pombe. The observation that a 14-3-3 protein interacts with the N-terminal portion of CAP but not with full-length CAP in L. edodes and S. pombe suggests that the C-terminal region of CAP may have a negative effect on the interaction between CAP and 14-3-3 proteins, and 14-3-3 proteins may play a role in regulation of CAP function.
CAP; 14-3-3; Lentinus edodes; two-hybrid; signal transduction

-21-
Effect of a Phosphorylated Guar Gum Hydrolysate on Increased Calcium
Solubilization and the Promotion of Calcium Absorption in Rats

Osamu WATANABE,1,2,†a Hiroshi HARA,1 and Takanori KASAI1

1Department of Bioscience and Chemistry, Faculty of Agriculture, Hokkaido University,
Kita 9, Nishi 9, Kita-ku, Sapporo 060-8589, Japan
2Hokkaido Food Processing Research Center, Midorimachi 589-4, Bunkyodai, Ebetsu 069-0836, Japan

Received August 18, 1999; Accepted September 24, 1999
The effect of a phosphorylated guar gum hydrolysate (P-GGH) on calcium solubilization and its influence on calcium absorption were studied in vitro and in vivo. P-GGH was prepared by chemically modifying a guar gum hydrolysate (GGH) with sodium metaphosphate. P-GGH inhibited the precipitation of calcium phosphate in vitro. The apparent calcium absorption and the amount of femur calcium were significantly higher in rats fed on the P-GGH diet (50 g/kg of diet) than in rats fed on the GGH diet (50 g/kg of diet) or the control diet. Moreover, the amount of soluble calcium in the ileal contents was significantly higher in the P-GGH-fed rats than in the GGH-fed rats. These results indicate that P-GGH may inhibit calcium phosphate formation in the lower part of the small intestine, and thus increase calcium absorption.
calcium; phosphorylation; guar gum hydrolysate; rats; solubility

-22-
Phosphatidylserine Synthesis Required for the Maximal Tryptophan Transport
Activity in Saccharomyces cerevisiae

Hidemitsu NAKAMURA, Keiji MIURA,�-,1 Yu FUKUDA,�-,2 Isao SHIBUYA,�-
Akinori OHTA,†a and Masamichi TAKAGI

Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
�-Department of Molecular Biology, Saitama University, Urawa 338-8570, Japan

Received August 26, 1999; Accepted September 28, 1999
Saccharomyces cerevisiae cho1/pss mutants, which are severely impaired in phosphatidylserine (PS) synthesis, do not have detectable amounts of PS in their lipid fractions. Their derivatives with mutations that cause defects in tryptophan synthesis grew poorly in a medium containing 5 ?Eg/ml of L-tryptophan, a concentration that met the requirements of tryptophan-auxotrophic CHO1/PSS strains. The rates of tryptophan uptake of trp1 cho1/pss mutants were low at low tryptophan concentrations. This defect in the use of tryptophan was restored either by expression of CHO1/PSS or by introduction of a gene encoding tryptophan transporter, TAT1 or TAT2. These results indicate that PS synthesis is required for the maximal tryptophan-transporting activity of S. cerevisiae at low tryptophan concentrations.
phosphatidylserine synthesis; phospholipid; Saccharomyces cerevisiae; tryptophan transporter

-23-
Note
Radical-capturing Reaction of 5,7,3�?,4�?-Tetramethylquercetin
with the AIBN Radical Initiator�-

Takahiro ISHIKAWA, Michiyo TAKAGI, Mamiko KANOU, Shingo KAWAI,
and Hideo OHASHI†a

Faculty of Agriculture, Gifu University, Yanagido, Gifu 501-1193, Japan

Received May 12, 1999; Accepted September 17, 1999
In order to clarify the mechanism for the radical-capturing reaction which is initiated at the C3-hydroxyl group of flavonols, 5,7,3�?,4�?-tetramethylquercetin (TMQ) was reacted with the 2,2�?-azobis-isobutyronitrile (AIBN) radical initiator in benzene. Six products, one depside and its two hydrolytic products, one nitrile adduct, and two others, were isolated from the reaction mixture, and their structures were determined by instrumental analyses. The quantitative change to the four main products against the reaction time was measured by an HPLC method. The radical-capturing reaction pathway for TMQ with AIBN is proposed from these products and their quantitative changes. The pathway dividing into two clearly reveals that one sub-path formed the depside and its hydrolytic products, while the other formed the nitrile adduct. The reactivity of each two sub-path was nearly the same, different from the case of TMQ and the 2,2�?-azobis-2,4-dimethylvaleronitrile (AMVN) radical initiator.
antioxidative activity; radical-capturing ability; quercetin; 2,2�?-azobis-isobutyronitrile (AIBN); reaction mechanism

-24-
Note
Ultraviolet Spectrometric Determination of Neutral Monosaccharides
by HPLC with Ethanolamine

Akihiro NAKAMURA,†a Chitoshi HATANAKA,�- and Yasunori NAGAMATSU�-

Hannan R｛D Center, Fuji Oil Co. Ltd., 1 Sumiyoshi-cho, Izumisano, Osaka 598-8540, Japan
�-Department of Applied Biochemistry, Faculty of Applied Biological Science, Hiroshima University, Higashi-Hiroshima, Hiroshima 739-8528, Japan

Received May 31, 1999; Accepted September 8, 1999
The reaction between ethanolamine and sugars in a borate solution gave intense UV absorption at 310 nm. By adding ethanolamine in the mobile phase, simple and sensitive ion-exchange HPLC of sugar-borate complexes was achieved. This method can be used to measure as little as 100 pmole of seven monosaccharides from pectic polysaccharides.
neutral monosaccharide; ethanolamine; UV detection; HPLC; pectin

-25-
Note
Purification of Collagenase and Specificity of Its Related Enzyme
from Bacillus subtilis FS-2

Hiroko NAGANO and Kim Anh TO�-

Faculty of Education, Gifu University, Gifu 501-1193, Japan
�-Center for Biotechnology, Hanoi University of Technology, Hanoi, Vietnam

Received Jane 7, 1999; Accepted September 20, 1999
A collagenase in the culture supernatant of B. subtilis FS-2, isolated from traditional fish sauce, was purified. The enzyme had a molecular mass of about 125 kDa. It degraded gelatin with maximum activity at pH 9 and a temperature of 50�?C. The purified enzyme was stable over a wide range of pH (5--10) and lost only 15�
collagenase; protease; Bacillus subtilis; traditional food; digestion site

-26-
Note
Inactivation of Human Type A and B Influenza Viruses by Tea-Seed Saponins

Kazuhiko HAYASHI,†a Yuko M. SAGESAKA, Takashi SUZUKI,�- and Yasuo SUZUKI�-

Central Research Institute, Ito En Ltd., 21 Mekami, Sagara-cho, Haibara-gun, Shizuoka 421-0516, Japan
�-Department of Biochemistry, School of Pharmaceutical Science, University of Shizuoka, 52-1 Yada, Shizuoka-shi, Shizuoka 422-8526, Japan

Received June 16, 1999; Accepted August 30, 1999
The effects of a mixture of tea-seed saponins obtained from the seeds of Camellia sinensis var. sinensis
on human influenza viruses types A and B were
investigated. At the concentrations of 60, 80, and
100 ?Eg/ml, respectively, the mixture inactivated
viruses A/Memphis/1/71 (H3N2), B/Lee/40, and A/PR/8/34 (H1N1) almost completely. The mixture also inactivated type A virus A/PR/8/34 after inoculation at concentrations of 1--30 ?Eg/ml dose-dependently.
saponin; tea; influenza virus; inactivation

-27-
Note
Phytotoxicity of Indole-3-acetic Acid Produced by the Fungus,
Pythium aphanidermatum

Atsumi SHIMADA,†a Sumiyo TAKEUCHI, Akira NAKAJIMA, Satoshi TANAKA,�-
Tsuyoshi KAWANO,�- and Yasuo KIMURA�-

Department of Environmental Chemistry, Faculty of Engineering, Kyushu Kyoritsu University,
1-8 Jiyugaoka, Yahatanishi, Kitakyushu 807-8585, Japan
�-Department of Agricultural Chemistry, Faculty of Agriculture, Tottori University,
Koyama, Tottori 680-8553, Japan

Received July 2, 1999; Accepted September 14, 1999
Pythium aphanidermatum causes the serious disease of Pythium red blight on bentgrass. IAA, one of the metabolites that has been isolated from this fungus, showed the same symptom of Pythium red blight on bentgrass at a concentration of 1,000 mg/l. The IAA content in the foliage of bentgrass infected by this fungus was about 200 times that of an untreated control. These results suggest that IAA produced by this fungus was the causal substance of Pythium red blight on bentgrass.
phytotoxicity; indole-3-acetic acid (IAA); Pythium aphanidermatum; bentgrass

-28-
Note
Kinetic and Thermodynamic Analyses of the Cyclodextrin-Allyl Isothiocyanate
Inclusion Complex in an Aqueous Solution

Yoshio OHTA,†a Kenichi TAKATANI,�- and Shunro KAWAKISHI�-�-

Hiroshima Prefectural Food Technology Research Center, Hijiyamahon-machi, Minami-ku,
Hiroshima 732-0816, Japan
�-�-Department of Food and Nutrition, School of Living Studies, Sugiyama Jogakuen University,
Hoshigaoka Motomachi, Chikusa-ku, Nagoya 464-8662, Japan

Received July 28, 1999; Accepted Septenber 24, 1999
The decomposition of allyl isothiocyanate (AITC) in an aqueous solution was depressed in the presence of cyclodextrin (CD), it�?s suppression effect increasing in the order of none<?A-CD<??-CD. The results of kinetic and thermodynamic analyses of the CD-AITC inclusion complexes showed that the inclusion process was mostly governed by an enthalpy change (?¢H�?) rather than by an entropy change (?¢S�?), and that Van der Waals forces played a primary role in the inclusion. Steric factors were important for the reaction activity of AITC inclusion into the CD cavity, especially significant being the stereospecificity between the size of the CD cavity and the AITC molecule which is the main factor concerning it�?s activity. Our results suggest that the association stability and activity of the included AITC molecule are important factors in the suppression mechanism for CDs. Therefore, both these factors would make an ??-CD-AITC system more advantageous than a ?A-CD-AITC system, and the marked suppression effect of ??-CD on the decomposition of AITC can be attributed to the formation of inclusion complexes in an aqueous solution.
kinetic and thermodynamic analyses; allyl isothiocyanate; cyclodextrin; inclusion complex; suppression mechanism

-29-
Note
Asymmetric Reduction of Ethyl 2-methyl 3-oxobutanoate by Fungi

Kenzo IWAMOTO,1 Takayuki KURAMOTO,1 Minoru IZUMI,2 Mitsunori KIRIHATA,3
Takaaki DOHMARU,1 and Fumiki YOSHIZAKO1,†a

1Research Institute for Advanced Science and Technology, Osaka Prefecture University,
1-2 Gakuen-cho, Sakai, Osaka 599-8570, Japan
2Department of Chemistry, Graduate School of Science, Osaka University,
1-1 Machikaneyama-cho, Toyonaka, Osaka 560-0043, Japan
3Department of Applied Biochemistry, College of Agriculture, Osaka Prefecture University,
1-1 Gakuen-cho, Sakai, Osaka 599-8531, Japan

Received August 9, 1999; Accepted September 21, 1999
Seven fungi, which are found to reduce ethyl 3-oxobutanoate in high yields, were tested for their reducing ability for ethyl 2-methyl 3-oxobutanoate. We obtained some interesting findings. In particular, Penicillium purpurogenum reduced ethyl 2-methyl 3-oxobutanoate to the corresponding alcohols with the
diastereomer (anti/syn) ratio of 93/7 with the enantiomeric excess of anti-(2S, 3S)- and syn-(2S, 3R)-hydroxy esters of 90 and >99 ee�
Penicillium purpurogenum; asymmetric reduction; ethyl 2-methyl 3-oxobutanoate; (2S, 3S)-ethyl 2-methyl 3-hydroxybutanoate; (2S, 3R)-ethyl 2-methyl 3-hydroxybutanoate

-30-
Note
Ovomucoid Rendered Insoluble by Heating with Wheat Gluten but not
with Milk Casein

Yasuko KATO,†a Hiroko WATANABE, and Tsukasa MATSUDA�-

Department of Clinical Nutrition, Medical Professions, Kawasaki University of Medical Welfare,
288 Matsushima, Kurashiki City, Okayama 701-0193, Japan
�-Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Science,
Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan

Received August 9, 1999; Accepted September 13, 1999
The effect of wheat gluten, soybean protein and milk casein on the heat-induced insolubilization of egg white ovomucoid was investigated by using ELISA inhibition and immunoblotting analyses. Heat treatment at 180�?C for 10 min of egg white mixed with wheat gluten specifically accelerated the heat-induced change in ovomucoid. Such an effect was weakly brought about by soybean protein, but not by casein.
insolubilization of ovomucoid; egg white; gluten; casein; soybean protein

-31-
Note
Effects of Glutathione-related Compounds on Increased Caspase-3
and Caspase-6-like Activities in Ricin-treated U937 Cells

Noriko SADAKATA, Tatsuya ODA,†a Nobukazu KOMATSU, and Tsuyoshi MURAMATSU

Division of Biochemistry, Faculty of Fisheries, Nagasaki University, Bunkyo-machi, Nagasaki 852-8521, Japan

Received August 9, 1999; Accepted September 9, 1999
Both caspase-3 and -6-like activities increased in the cytosolic extract from ricin-treated U937 cells that were inhibited by glutathione disulfide (GSSG) in a dose-dependent manner, but reduced glutathione (GSH) had no effect. Interestingly, caspase-6 like activity was more sensitive to GSSG than caspase-3 like activity. The IC50 of GSSG against caspase-3 and caspase-6-like activities were estimated to be 2.8 mM and 0.8 mM, respectively. Cystine but not cysteine also showed similar inhibitory effect on caspase-3-like activity. The inhibitory effect of GSSG on these caspase-like activities was prevented by the addition of DTT to the assay mixture. These results suggest that an intact disulfide portion of GSSG is required for the effective inhibition of caspase activity.
ricin; apoptosis; glutathione; caspases

-32-
Note
Efficiency of D-Tryptophan as Niacin in Rats

Katsumi SHIBATA,†a Mika SAWABE, Tsutomu FUKUWATARI, and Etsuro SUGIMOTO

Course of Food Science and Nutrition, Department of Life Style Studies, School of Human Cultures,
The University of Shiga Prefecture, 2500 Hassakacho, Hikone, Shiga 552-8533, Japan

Received August 11, 1999; Accepted September 14, 1999
L-Tryptophan is a very important precursor of niacin in mammals. Food preparation in which proteins are exposed to an alkali and/or high temperature for a long period generate appreciable amounts of D-amino acids from racemization. The efficiency of D-tryptophan as niacin was thus investigated by using weanling rats. The availability of D-tryptophan was almost the same as that in L-tryptophan as the precursor of niacin and was 1/6 as active as niacin.
D-tryptophan; niacin; D-amino acid; niacin activity

-33-
Note
Thymocyte Apoptosis by T-2 Toxin in Vivo in Mice Is Independent
of Fas/Fas Ligand System

M. Murshedul ALAM, Masahiro NAGASE, Takumi YOSHIZAWA,�- and Nobuo SAKATO†a

Department of Life Sciences, and �-Department of Biochemistry and Food Science, Faculty of Agriculture, Kagawa University, Ikenobe 2393, Miki-cho, Kita-gun, Kagawa 761-0795, Japan

Received August 30, 1999; Accepted October 4, 1999
To find whether Fas/Fas ligand (FasL) pathway is involved in T-2 toxin (T-2)-mediated thymocyte apoptosis, we used lpr/lpr (lpr) and gld/gld (gld) mice, whose Fas and FasL proteins, respectively, are functionally deficient. Based on the DNA fragmentation profile in gel electrophoresis and measurement of apoptotic cell percent by flow cytometry, the levels of thymocyte apoptosis in lpr and gld mice that had received T-2 showed that both lpr and gld mice had undergone apoptosis essentially to the same magnitude as those of corresponding wild type mice (＋/＋). These results strongly suggest that T-2-induced thymocyte apoptosis in vivo in mice is independent of the Fas/FasL pathway.
T-2 toxin; apoptosis; Fas; Fas ligand; thymocyte