Contents and Abstracts of Latest Issue of BBB

(Vol.63 No.10 1999)

Tanetoshi KOYAMA

Institute for Chemical Reaction Science, Tohoku University, Katahira 2-1-1, Aoba-ku,
Sendai 980-8577, Japan

Multiple alignments of primary structures of many kinds of prenyltransferases that participate in the most fundamental prenyl-chain backbone synthesizing process in isoprenoid biosynthesis showed seven conserved regions in the primary structures of (E)-prenyl diphosphate synthases. However, no information has been available about the structures of (Z)-prenyl diphosphate synthases until our recent isolation of the gene for the undecaprenyl diphosphate synthase of Micrococcus luteus B-P 26.
The amino acid sequence of the (Z)-prenyl diphosphate synthase is totally different from those of (E)-prenyl chain elongating enzymes. Protein data base searches for sequences similar to that of the undecaprenyl diphosphate synthase yielded many unknown proteins which have not yet been characterized. Two of the proteins have recently been identified as the undecaprenyl diphosphate synthase of Escherichia coli and the dehydrodolichyl diphosphate synthase of Saccharomyces cerevisiae, indicating that there are three highly conserved regions in the primary structure of (Z)-prenyl chain elongating enzymes.
prenyltransferase; isoprenoid biosynthesis; prenyl diphosphate synthase; polyprenol; farnesyl diphosphate

-2-
Enzymatic Synthesis of N-Acetylglucosaminyl-cyclodextrin
by the Reverse Reaction of N-Acetylhexosaminidase from Jack Bean

Kenichi HAMAYASU,a Koki FUJITA,a Kozo HARA,a Hitoshi HASHIMOTO,a
Toshiko TANIMOTO,b Kyoko KOIZUMI,b Hirofumi NAKANO,c and Sumio KITAHATAc

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

Received March 15, 1999, Accepted June 10, 1999
Novel heterobranched cyclodextrins (CDs), N-acetylglucosaminyl-cyclodextrins (GlcNAc-CD), were synthesized from a mixture of GlcNAc and α, β, or γCD by the reverse reaction of N-acetylhexosaminidase from jack bean. Optimum pH and temperature for the production of GlcNAc-αCD by N-acetylhexosaminidase were pH 4.9 and 50--70°C, respectively. The maximum yield of GlcNAc-αCD was 17.5％ ( mol/mol) at the concentration of 1 M GlcNAc and 0.25 M αCD. The reverse reaction product, GlcNAc-αCD, was separated into two peaks by HPLC analysis on the ODS column. Their structures were identified as 6-O-β-D-N-acetylglucosaminyl-αCD and 2-O-β-D-N-acetylglucosaminyl-αCD by FAB-MS and NMR spectroscopies. N-Acetylhexosaminidase from jack bean also synthesized N-acetylgalactosaminyl-αCD from N-acetylgalactosamine and αCD.
cyclodextrin; N-acetylglucosaminyl-cyclodextrin; N-acetylgalactosaminyl-cyclodextrin; N-acetylhexosaminidase; reverse reaction of jack bean N-acetylhexosaminidase

-3-
Structure-Activity Relationships of RGD Mimetics
as Fibrinogen-Receptor Antagonists

Masahiro MIYASHITA,1 Miki AKAMATSU,1,・ Hideki UENO,1 Yoshiaki NAKAGAWA,1
Keiichiro NISHIMURA,2 Yoshio HAYASHI,3 Yoshimi SATO,3 and Tamio UENO1

1Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
2Research Institute for Advanced Science and Technology, Osaka Prefecture University,
Sakai, Osaka 599-8570, Japan
3Life Science Research Center, Advanced Technology Research Laboratories, Nippon Steel Corporation,
Kawasaki 211-0035, Japan

Received April 5, 1999; Accepted June 30, 1999
The activities of a series of RGD mimetics, which contained a variety of cationic structures, for the inhibition of platelet aggregation and fibrinogen-receptor binding were measured. The stability of the coulombic ion-pairing complex of the model compounds with the acetate anion as a model for the receptor was calculated in terms of the ionic interaction energy. The results suggest that stability is one of the significant factors which govern the inhibitory potency of fibrinogen-receptor binding. The distance between cationic and anionic groups might also affect the potency. A compound which contained an amidinophenyl structure as the cationic moiety showed exceptionally high inhibitory activity, suggesting that some other factors, in addition to coulombic interaction and the distance, affect the potency.
Arg-Gly-Asp; fibrinogen antagonist; antithrombotic; peptide mimetic; GPIIb/IIIa

-4-
Effect of Several Solutions Including Mineral-encaging Zeolites on the Restoration
of Cell Motility of Tributyltin-intoxicated Euglena gracilis Z

Mari OHTA1, Kaoru NAKAMURA2, Hiroji TSUCHIYA3, Kozo TAKAMA1, and Tetsuya SUZUKI1＊

1Laboratory of Food Wholesomeness, Department of Marine Bioresources Chemistry, Faculty of Fisheries,
Hokkaido University, 3-1-1 Minato, Hakodate 041-8611, Japan
2Institute of Chemical Research, Kyoto University, Gokasho, Uji 611-0011, Japan
3Central Laboratory, Hamamatsu Photonics Co., 5000 Hiraguchi, Hamakita, Shizuoka 434-0041, Japan

Received April 7, 1999; Accepted June 14, 1999
To examine the detoxification effect of mineral-encaged zeolites on cells impaired by pollutant-intoxication, we used a bioassay system involving Euglena gracilis Z as the model organism and TBTCl as a pollutant. TBTCl exposure causes Euglena cells to quickly change shape from a spherical to spindle form, with the process being reversible by detoxification. Taking advantage of this morphological characteristic, we examined the restoration of motility by water containing zeolites encaging different minerals. TBTCl-intoxicated Euglena cells were incubated in processed water with different types of mineral-encaging zeolites for up to 3 hours. The restoration of motility was evaluated by observing the number of motile cells with a video microscope. Remarkable recovery was observed in the incubation systems with water containing Fe-, Zn-, and Mn-encaging zeolites. However, the effect was suppressed when the water species were treated with the chelator, Chelex-||100・. An equivalent concentration of FeCl3 to that in the||Fe-encaging zeolite processed water did not show significant restoration effect.
mineral-encaging zeolite; detoxification; organotin; endocrine disrupter; Euglena

-5-
Dietary Effect of Tocopherols and Tocotrienols on the Immune Function of Spleen and Mesenteric Lymph Node Lymphocytes in Brown Norway Rats

Jiong-Yan GU, Yoko WAKIZONO, Yousuke SUNADA, Phum HUNG,
Michiko NONAKA, Michihiro SUGANO,＊ and Koji YAMADA・

Laboratory of Food Chemistry, Department of Bioscience and Biotechnology, Division of Bioresource and Bioenvironmental Sciences, Kyushu University, Higashi-ku, Fukuoka 812-8581, Japan ＊Department of Living Environment Science, Prefectural University of Kumamoto, Kumamoto 862-0901, Japan

Received April 7, 1999; Accepted July 6, 1999
The immunoregulatory effects of dietary α-tocopherol (Toc) and tocotrienols (T-3) on humoral and cell-mediated immunity and cytokine productions were examined in Brown Norway rats. We found that the IgA and IgG productivity of spleen and mesenteric lymph node (MLN) lymphocytes was significantly enhanced in the rats fed on Toc or T-3, irrespective of concanavalin A (Con A) stimulation of the lymphocytes. On the contrary, the IgE productivity of lymphocytes from the rats fed on Toc or T-3 was less without Con A stimulation, but was greater in the presence of Con A, especially in the T-3 group. Toc or T-3 feeding significantly decreased the proportion of CD4＋||T cells and the ratio of CD4＋/CD8＋ in both spleen and||MLN lymphocytes of the rats fed on Toc or T-3. The interferon-γ productivity of MLN lymphocytes was higher in the rats fed on Toc or T-3 than in those fed on a control diet in the presence of Con A, while that of spleen lymphocytes was lower in the rats fed on Toc or T-3. In addition, T-3 feeding decreased the productivity of tumor necrosis factor-α of spleen lymphocytes, while it enhanced the productivity of MLN lymphocytes. These results suggest that oral administration of Toc and T-3 affects the proliferation and function of spleen and MLN lymphocytes.
α-tocopherol; tocotrienols; immunoglobulin production; IFN-γ; TNF-α

-6-
Site-Directed Mutagenesis of Restriction Endonuclease HindIII

Tang DAHAI, Shoji ANDO, Yozo TAKASAKI,・ and Jutaro TADANO＊

Chemistry Laboratory and ＊Department of Laboratory Medicine, Saga Medical School,
Saga City 849-8501, Japan

Received April 12, 1999; Accepted June 28, 1999
Site-directed mutagenesis by inverse PCR was done on the HindIII gene. Target residues to be mutated were chosen according to (i) the fact that a mutant obtained by sodium nitrite treatment showed almost no HindIII activity, where Asp-123 was replaced with Asn, and (ii) the model proposed by Stahl et al. (Stahl, F., Wende, W., Jeltsch, A. and Pingoud, A. Biol. Chem. 379, 467--473 (1998)). Seven||kinds of mutants were obtained by the PCR, and their en||||zymatic and biochemical properties were examined. Three||mutants, P50S, D108L, and D123N, showed fairly low HindIII activity. On the other hand, the other four, P84Q, E86K, V106E, and K125N, retained the activity. In particular, E86K showed higher activity than the wild enzyme. This fact was confirmed when activities of the purified wild and E86K enzymes were assayed. These results coincided fairly well with data using E. coli strains that carry the respective mutant plasmids, on their resistance to phage T7 and on growth rate. We conclude that the PE motif at residues 50 and 51, and DXK motif at residues 108--110, are responsible for the enzymic reaction of HindIII.
restriction enzyme; HindIII; PCR; site-directed mutagenesis

-7-
Purification and Characterization of an Esterase Involved
in Cellulose Acetate Degradation by Neisseria sicca SB

Kunihiko MORIYOSHI, Takashi OHMOTO, Tatsuhiko OHE, and Kiyofumi SAKAI・

Department of Biochemistry, Osaka Municipal Technical Research Institute, 1-6-50 Morinomiya,
Joto-ku, Osaka 536-8553, Japan

Received April 13, 1999; Accepted June 30, 1999
An esterase catalyzing the hydrolysis of acetyl ester moieties in cellulose acetate was purified 1,110-fold to electrophoretic homogeneity from the culture supernatant of Neisseria sicca SB, which can assimilate cellulose acetate as the sole carbon and energy source. The purified enzyme was a monomeric protein with a molecular mass of 40 kDa and the isoelectric point was 5.3. The pH and temperature optima of the enzyme were 8.0--8.5 and 45°C. The enzyme catalyzed the hydrolysis of acetyl saccharides, p-nitrophenyl esters of short-chain fatty acids, and was slightly active toward aliphatic and aromatic esters. The Km and Vmax for cellulose acetate (degree of substitution, 0.88) and p-nitrophenyl acetate were 0.0162％ (716 μM as acetyl content in the polymer) and 36.0 μM, and 66.8 and 39.1 μmol/min/mg, respectively. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate, which indicated that the enzyme was a serine esterase.
cellulose acetate; biodegradation; Neisseria sicca; esterase

-8-
Heterologous Expression and Characterization of Endoglucanase I (EGI)
from Trichoderma viride HK-75

Il KWON, Keisuke EKINO, Masatoshi GOTO, and Kensuke FURUKAWA・

Laboratory of Applied Microbiology, Department of Agricultural Chemistry, Kyushu University,
Hakozaki, Fukuoka 812-8581, Japan

Received April 15, 1999; Accepted June 23, 1999
Endoglucanase I (EGI) secreted from Trichoderma viride HK-75 has a unique transglycosylation activity. The genomic and cDNA clones encoding EGI (egl1) of T. viride HK-75 were isolated and characterized. The coding region of egl1, composed of 1392 bp, was found to encode a polypeptide of 464 amino acids that has extensive similarity (93.8％) with EGI of T. reesei. Expression of the egl1 gene in E. coli as a fusion protein (with N-terminal thioredoxin and C-terminal histidine tag) led to a large production of a nonglycosylated protein of 62.5 kDa. However, it formed an insoluble inclusion body. Upon denaturation with 8 M urea followed by dialysis and successive purification, the enzymatically active recombinant EGI (rEGI) was obtained at a level as high as 18.3 mg/l of 1,000 ml of culture. The rEGI had 67.8％ activity for carboxymethyl cellulose (CMC), compared to native EGI (nEGI). The optimum pH and optimum temperature of rEGI were lower than those of nEGI by 0.5 and 5°C, respectively. The rEGI also had narrower CMCase ranges than nEGI in pH and temperature stabilities. However, the catalytic and transglycosylation abilities against cellotriose of rEGI were comparable to those of nEGI. These results suggest that the glycosylation is important for the stabilities of EGI but not critical for the essential enzymatic capacity.
Trichoderma viride; endoglucanase; heterologous expression; transglycosylation

-9-
NAD＋-Dependent (S)-Specific Secondary Alcohol Dehydrogenase Involved
in Stereoinversion of 3-Pentyn-2-ol Catalyzed by Nocardia fusca AKU 2123

Sheng-Xue XIE, Jun OGAWA, and Sakayu SHIMIZU・

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

Received April 16, 1999; Accepted June 2, 1999
An NAD＋-dependent alcohol dehydrogenase was purified to homogeneity from Nocardia fusca AKU 2123. The enzyme catalyzed (S)-specific oxidation of 3-pentyn-2-ol (PYOH), i.e., part of the stereoinversion reaction for the production of (R)-PYOH, which is a valuable chiral building block for pharmaceuticals, from the racemate. The enzyme used a broad variety of secondary alcohols including alkyl alcohols, alkenyl alcohols, acetylenic alcohols, and aromatic alcohols as substrates. The oxidation was (S)-isomer specific in every case. The Km and Vmax for (S)-PYOH and (S)-2-hexanol oxidation were 1.6 mM and 53 μmol/min/mg, and 0.33 mM and 130 μmol/min/mg, respectively. The enzyme also catalyzed stereoselective reduction of carbonyl compounds. (S)-2-Hexanol and ethyl (R)-4-chloro-3-hydroxybutanoate in high optical purity were produced from 2-hexanone and ethyl 4-chloro-3-oxobutanoate by the purified enzyme, respectively. The Km and Vmax for 2-hexanone reduction were 2.5 mM and 260 μmol/min/mg. The enzyme has a relative molecular mass of 150,000 and consists of four identical subunits. The NH2-terminal amino acid sequence of the enzyme shows similarity with those of the carbonyl reductase from Rhodococcus erythropolis and phenylacetaldehyde reductase from Corynebacterium sp.
secondary alcohol dehydrogenase; stereoinversion; Nocardia fusca; acetylenic alcohol; stereospecific reduction

-10-
Enzymatic Quantification of L-Tartrate in Wines and Grapes by Using
the Secondary Activity of D-Malate Dehydrogenase

Tadayuki TSUKATANI＊,・ and Kiyoshi MATSUMOTO＊＊

＊Biotechnology and Food Research Institute, Fukuoka Industrial Technology Center,
1465-5 Aikawa-cho, Kurume, Fukuoka 839-0861, Japan
＊＊Department of Food Science and Technology, Faculty of Agriculture, Kyushu University,
6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan

Received April 19, 1999; Accepted June 17, 1999
L-Tartrate in wines and grapes was enzymatically quantified by using the secondary activity of D-malate dehydrogenase (D-MDH). NADH formed by the D-MDH reaction was monitored spectrophotometrically. Under the optimal conditions, L-tartrate (a 1.0 mM sample solution) was fully oxidized by D-MDH in 30 min. A linear relationship was obtained between the absorbance difference and the L-tartrate concentration in the range of a 0.02--1.0 mM sample solution with a correlation coefficient of 0.9991. The relative standard deviation from ten measurements was 1.71％ at the 1.0 mM sample solution level. The proposed method was compared with HPLC, and the values determined by both methods were in good agreement.
L-tartrate; D-malate dehydrogenase; enzymatic quantification; wine; grape

-11-
Three Domains Comprised in Thermostable Molecular Weight 54,000 Pullulanase of Type I from Bacillus flavocaldarius KP1228・

Shin-ichi KASHIWABARA, Sosaku OGAWA, Naoko MIYOSHI, Masayo ODA, and Yuzuru SUZUKI・・

Laboratory of Microbial Physiology and Applied Microbiology, Department of Applied Biochemistry,
Kyoto Prefectural University, Shimogamo, Sakyo-ku, Kyoto 606-8522, Japan

Received April 23, 1999; Accepted May 27, 1999
The gene that coded for a cellular pullulanase of type I (α-dextrin 6-glucanohydrolase, EC 3.2.1.41) in Bacillus flavocaldarius KP1228 (FERM-P9542) cells growing at 51 to 82°C was expressed in Escherichia coli MV1184. The enzyme had a half-life of 10 min at 107°C. Purification of the enzyme and its characterization showed that the enzyme was identical with the native one. Its primary structure of 475 residues with a molecular weight of 53,856 deduced from the gene was 15--21％ and 43％ identical to the corresponding C-terminal regions in the sequences of 2 plant and 6 bacterial pullulanases of type I, and of Bacillus stearothermophilus TRS40 neoplullulanase, respectively.
Sequence analysis showed that B. flavocaldarius pullulanase comprised 3 domains, i.e., one catalytic (β/α)8-barrel domain, one domain made of the region protruding from the barrel between the third β-strand and the third α-helix, and one β-stranded domain attached to the C-end of the barrel domain, but that the pullulanase lacked the β-stranded domain commonly found in addition to the 3 domains in the neopullulanase and all other pullulanases, and attached to the N-end of the barrel domain.
thermostable pullulanase type I, gene cloning; Bacillus flavocaldarius; multiple sequence alignments; three domains

-12-
High Fat Feeding of Lactating Mice Causing a Drastic Reduction in Fat
and Energy Content in Milk without Affecting the Apparent Growth
of Their Pups and the Production of Major Milk Fat Globule
Membrane Components MFG-E8 and Butyrophilin

Naohito AOKI,・ Yumiko YAMAGUCHI, Sachiyo OHIRA, and Tsukasa MATSUDA

Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University,
Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan

Received April 23, 1999; Accepted June 26, 1999
Lactating mice were fed either a low fat or a high fat diet. Milk samples were collected and the composition was examined. Triglyceride and free fatty acid contents were greatly reduced in the milks of high fat diet group, while protein and lactose contents were almost the same between both diet groups. Although the energy content of each component was also lower in milk of high fat diet group, there was apparently no significant difference in the growth of the pups raised by either diet group. This discrepancy might be in part explained by a hypothesis that the pups might monitor calorie content in milk and keep suckling until the energy intake reaches their satisfaction. Moreover, nearly the same amounts of major milk fat globule membrane proteins MFG-E8 and butyrophilin were shown to be present in the milks from both diet groups and gene expression of both proteins in the mammary glands were also indistinguishable, suggesting that production of major MFGM components is not simply related to fat production and secretion.
high fat diet; lactation; mammary gland; milk fat globule membrane

-13-
Structures and Expression Patterns of Two Tropinone Reductase Genes
from Hyoscyamus niger

Keiji NAKAJIMA, Yoko OSHITA, Masahiko KAYA, Yasuyuki YAMADA,
and Takashi HASHIMOTO・

Graduate School of Biological Sciences, Nara Institute of Science and Technology 8916-5 Takayama,
Ikoma, Nara 630-0101, Japan

Received April 26, 1999; Accepted July 8, 1999
In the biosynthesis of tropane alkaloids, two tropinone reductases (TRs) catalyze reduction of tropinone to different stereoisomers, tropine and pseudotropine. Two TRs from Hyoscyamus niger have 64％ of identical amino acids and hence a common evolutionary origin. In this study, genomic clones of TRs were isolated from H. niger. Their sequence comparison showed that although they have the same exon/intron organization, sequence similarity was restricted to the coding regions. In H. niger transgenic hairy roots, both TR promoters activated transcription of the reporter genes in endodermis and pericycle of the roots. A quantitative reporter assay and a nuclear run-on experiment indicated that the two genes are transcribed at a similar rate. The results indicate that although different activity levels have been observed for the TR enzymes in the H. niger root, the TR genes per se conserve similar tissue-specific expression pattern and transcriptional rate.
histochemical analysis; Hyoscyamus niger; reporter gene; tropane alkaloid; tropinone reductase

-14-
Polypeptide Requirement of Multicomponent Monooxygenase DsoABCDEF
for Dimethyl Sulfide Oxidizing Activity

Masae HORINOUCHI, Takako YOSHIDA, Hideaki NOJIRI, Hisakazu YAMANE,
and Toshio OMORI＊

Biotechnology Research Center, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan

Received May 6, 1999; Accepted June 14, 1999
In our previous study, the multicomponent monooxygenase DsoABCDEF in Acinetobacter sp. strain 20B was cloned based on its ability to oxidize dimethyl sulfide (DMS) to dimethyl sulfoxide (DMSO) in E. coli cells, which had high sequence similarity with phenol hydroxylase MopKLMNOP in Acinetobacter calcoaceticus NCIB8250, DmpKLMNOP in Pseudomonas sp. CF600 and some other multicomponent monooxygenases. In this study, DsoB, C, D, E, and F were found to be needed for DMS-oxidizing activity in polypeptide requirement experiments, while DsoA was not necessary for it. It was also found that complementation of the deletion mutants lacking DsoC or F with the corresponding Dmp polypeptides supported the DMS-oxidizing activity, while complementation of the deletion mutants lacking any of the oxygenase subunits (DsoB, D, or E) with the corresponding Dmp polypeptides reduced or nullified the activity.
dimethyl sulfide; dimethyl sulfoxide; multicomponent phenol hydroxylase

-15-
Absolute Configuration of Kelsoene and Prespatane

Kensuke NABETA,1,・ Masateru YAMAMOTO,1 Hiroyuki KOSHINO,2 Hiroki FUKUI,3
Yukiharu FUKUSHI,3 and Satoshi TAHARA3

1Department of Bioresource Science, Obihiro University of Agriculture and Veterinary Medicine,
Obihiro 080-8555, Japan
2The Institute of Physical and Chemical Research (RIKEN), Wako 351-0198, Japan
3Department of Applied Bioscience, Faculty of Agriculture, Hokkaido University, Sapporo 060-8589, Japan

Received May 7, 1999; Accepted June 14, 1999
The absolute configurations of the rare sesquiterpenes, kelsoene and prespatane, were determined to be (1S, 2R,||5S, 6S, 7R, 8R)-2,8-dimethyl-6-(1-methylethenyl)tricyclo||[5.3.0.02,5] decane (IUPAC name) and (1R, 2S, 5R, 6R, 7R, 8S)-1,5-dimethyl-8-(1-methylethenyl) tricyclo-[5.3.0.02,6] decane, respectively, on the basis of the observed chemical shifts and NOEs in NOESY and NOEDS experiments after conversion with the chiral reagent, 2′-methoxy-1,1′-binaphthalene-2-carbohydroximoyl chloride (MBCC). The absolute configurations of kelsoene and prespatane thus determined suggest that initial cyclization of FPP from the si-face at C-10 to form a 10R-germacradienyl cation leads to kelsoene, while that from the re-face leads to prespatane via the 10S-germacradienyl cation.
kelsoene; prespatane; absolute configuration; chiral reagent; 2′-methoxy-1,1′-binaphthalene-2-carbohydroximoyl chloride

-16-
Accumulation of Isohemigossypolone and Its Related Compounds
in the Inner Bark and Heartwood of Diseased Pachira aquatica

Mariko SHIBATANI,1 Yasuyuki HASHIDOKO,・,1,2 and Satoshi TAHARA1,2

1Department of Applied Bioscience, Faculty of Agriculture, Hokkaido University,
Kita-Ku, Sapporo 060-8589, Japan
2CREST, Japan Science and Technology Corporation, Honmachi 4-1-8, Kawaguchi 332-0012, Japan

Received May 12, 1999; Accepted June 22, 1999
Isohemigossypolone (1) and 2-O-methylisohemigossypolone (2), major fungitoxins of Pachira aquatica, were found to accumulate locally in the outer bark of the swollen trunk, whereas the inner bark and heartwood contained only a trace amount of them. From P. aquatica that was infected with a phytopathogenic bacterium, we detected significant amounts of 1 and 2 from browned inner tissues of the swollen trunk. According to a quantitative analysis by a gas-chromatograph, the concentration of 1 in the diseased inner tissues was calculated to be approximately 780 μg/g f.w., which was the same level as that in the outer bark of healthy individuals. These findings suggest that the inner tissues inducibly produced and accumulated antifungals 1 and 2 during infection events, as do many plants with phytoalexins. 11-Nor-2-O-methylisohemigossypolone (3), showing approximately equivalent fungitoxic activity to that of 1 and 2, was also isolated from the infected inner tissues. We screened pathogenic bacteria from the infected tissue, and isolated a rod-shaped bacterium that was tentatively identified as Pseudomonas sp. which promoted tissue-browning on sectioned disks of P. aquatica trunks.
Pachira aquatica; isohemigossypolone; cadinane sesquiterpenes; norcadinane; phytoalexin

-17-
Note
Isolation and Characterization of Basic Exochitinase from Leaf Extract
of Rehmannia glutinosa

Eun-A LEE, Cheol-Ho PAN, Jong-Mun SON, and Su-Il KIM

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

Received March 12, 1999; Accepted June 14, 1999
Rehmannia chitinases were extracted from the leaves of Rehmannia glutinosa under acidic conditions (pH 2.9). We purified a 28.6-kDa chitinase, designated as P2, from crude extract to homogeneity by (NH4)2SO4 precipitation, chromatography with regenerated chitin affinity and hydrophobic interaction column, and preparative native PAGE. Isolated P2 showed maximum chitinase activity at pH 5.0 and 60°C, and had a isoelectric point of 8.46. P2 produced only (GlcNAc)2 from (GlcNAc)4--6 and regenerated chitin. Based on these results, we arrived at the conclusion that P2 was a basic exochitinase.
basic chitinase; exochitinase; defense-related protein; Rehmannia glutinosa

-18-
Note
Stability of Lutein and Its Myristate Esters

Achmad SUBAGIO, Hiroko WAKAKI, and Naofumi MORITA・

Laboratory of Food Chemistry, College of Agriculture, Osaka Prefecture University,
Gakuencho 1-1, Sakai, Osaka 599-8531, Japan

Received April 5, 1999; Accepted June 17, 1999
Stabilities of lutein and its myristate esters against heat and UV light were studied by HPLC response. Free lutein (FL) was very unstable against heat; lutein monomyristate (LM), slightly stable; and lutein dimyristate (LD), very stable. Both LM and LD were more stable toward UV light than FL. These results suggest that esterification of the OH group with a fatty acid might stabilize lutein against heat and UV-light degradation.
carotenoids; fatty acid ester; lutein; stability

-19-
Note
Inhibition of Xanthine Oxidase by Flavonoids

Akihiko NAGAO,1 Michiko SEKI,2 and Hidetaka KOBAYASHI1

1National Food Research Institute, Ministry of Agriculture, Forestry and Fisheries,
Tsukuba, Ibaraki 305-8642, Japan
2Aomori Agricultural Products Processing Center, Yanagisawa 91, Rokunohe-machi,
Aomori 033-0071, Japan

Received April 5, 1999; Accepted June 9, 1999
Various dietary flavonoids were evaluated in vitro for their inhibitory effect on xanthine oxidase, which has been implicated in oxidative injury to tissue by ischemia-reperfusion. Xanthine oxidase activity was determined by directly measuring uric acid formation by HPLC. The structure-activity relationship revealed that the planar flavones and flavonols with a 7-hydroxyl group such as chrysin, luteolin, kaempferol, quercetin, myricetin, and isorhamnetin inhibited xanthine oxidase activity at low concentrations (IC50 values from 0.40 to 5.02 μM) in a mixed-type mode, while the nonplanar flavonoids, isoflavones and anthocyanidins were less inhibitory. These results suggest that certain flavonoids might suppress in vivo the formation of active oxygen species and urate by xanthine oxidase.
xanthine oxidase; flavonoid; inhibition; oxidative injury; gout

-20-
Note
Purification and Characterization of the Overexpressed Aspergillus oryzae
Xylanase, XynF1

Noriyuki KITAMOTO,・ Shoko YOSHINO, Kunio OHMIYA,＊ and Norihiro TSUKAGOSHI＊＊

Food Research Institute, Aichi Prefectural Government, 2-1-1 Shinpukuji-cho, Nishi-ku, Nagoya 451-0083, Japan
＊Faculty of Bioresources, Mie University, Tsu 514-8507, Japan
＊＊Department of Biological Mechanisms and Functions, Graduate School of Bioagricultural Sciences,
Nagoya University, Nagoya 464-8601, Japan

Received April 5, 1999; Accepted June 9, 1999
The Aspergillus oryzae xynF1 gene coding for a xylanase, XynF1, was successfully overexpressed under the strong A. oryzae TEF1 gene promoter. The high-XynF1-producing transformant secreted about 180 mg/l of XynF1 in the glucose-containing medium. The overexpressed XynF1 was purified by only one chromatographic step. The purified XynF1 had a molecular mass of 35.0 kDa, a pH optimum of 5.0, and a temperature optimum of 60°C.
Aspergillus oryzae; overexpression; xylanase

-21-
Note
Clerodendrin I, a New Neoclerodane Diterpenoid
from Clerodendron trichotomum

Kunji KAWAI,＊ Ritsuo NISHIDA,・ and Hiroshi FUKAMI＃

Laboratory of Chemical Ecology, Division of Applied Life Sciences, Graduate School of Agriculture,
Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
＃Faculty of Sciences and Engineering, Ritsumeikan University, Noji, Kusatsu 525-8577, Japan

Received April 7, 1999; Accepted June 23, 1999
A new neoclerodane diterpenoid, clerodendrin I, was isolated from leaves of Clerodendron trichotomum as a feeding stimulant of the turnip sawfly, Athalia rosae ruficornis, and characterized as a threo epimer of clerodendrin F in the 2,3-diacetoxy-2-methylbutanoyloxy moiety at the C-3β position.
clerodendrin; neoclerodane diterpene; Clerodendron trichotomum; Athalia rosae ruficornis; pharmacophagy

-22-
Note
Oxidation of Two Pregnane Steroids Catalyzed
by the Fungus Cephalosporium aphidicola

Afgan FAROOQ and J. R. HANSON

School of Molecular Sciences, University of Sussex, Falmer, Brighton BNI 9QJ, England

Received April 8, 1999; Accepted June 3, 1999
Two pregnane steroids, pregnane (1) and 3β-hydroxypregnane (2), were oxidized by fermentation with the fungus Cephalosporium aphidicola. The fermentation of pregnane (1) yielded 3β-hydroxypregnane (2) and 3β, 6β,11α-trihydroxypregnane (3), while that of 3β-hydroxypregnane (2) afforded 6β,11α-dihydroxypregn-3,20-dione (4), 3β,6β,15α-trihydroxypregn-20-one (5) and 3β,5α,11α-trihydroxypregn-20-one (6). The metabolites are characterized by detailed physical and spectroscopic studies.
pregnane steroid; oxidation; hydroxylation; Cephalosporium aphidicola; fermentation

-23-
Note
Identification of Trace Sterols in the Seeds of Foxtail Millet (Setaria italica Beauv.)

Yasuhisa NARUMI and Suguru TAKATSUTO・

Department of Chemistry, Joetsu University of Education, Joetsu-shi, Niigata 943-8512, Japan

Received April 9, 1999; Accepted June 22, 1999
Trace sterols in the seeds of foxtail millet (Setaria italica Beauv.) were investigated by GC-MS. Eleven of these trace sterols, i.e., brassicasterol, episterol, 24-methyllathosterol, 24-ethyllathosterol, avenasterol, 24-methylenecholesterol, fucosterol, isofucosterol, 24-methyl-5α-cholest-24(28)-en-3β-ol, 24-ethyl-5α-cholest-24(28)Z-en-3β-ol, and 24-ethyldesmosterol, were identified, suggesting that the previously reported high content of sitostanol was possibly contaminated with a small amount of isofucosterol.
foxtail millet; Setaria italica Beauv.; seed; sterol; sitostanol

-24-
Note
Stimulative Effect of Dietary Protein on the Phosphorylation of p70 S6 Kinase
in the Skeletal Muscle and Liver of Food-deprived Rats

Fumiaki YOSHIZAWA,・ Taketoshi KIDO,＊ and Takashi NAGASAWA＊

Department of Science of Living, Morioka Junior College, Iwate Prefectural University,
Takizawa, Iwate 020-0173, Japan
＊Department of Bioscience and Technology, Iwate University, Morioka, Iwate 020-8550, Japan

Received April 12, 1999; Accepted June 21, 1999
The effect of dietary protein on p70S6k phosphorylation was examined in rats starved for 18 h and then fed either a 20％ casein diet (20C) or a protein-free diet (0C). Refeeding the 20C diet, but not the 0C diet, increased p70S6k phosphorylation in both the skeletal muscle and liver. The plasma insulin concentrations were the same after refeeding the 20C or 0C diet, suggesting that a combination of dietary protein and insulin may be required to stimulate p70S6k phosphorylation.
protein synthesis; p70S6k; food intake; dietary protein; rat

-25-
Note
Molecular Analysis of the Corynebacterium glutamicum Transketolase Gene

Masato IKEDA,・ Nozomu KAMADA, Yutaka TAKANO, and Tetsuo NAKANO

Technical Research Laboratories, Hofu Plant, Kyowa Hakko Kogyo Co., Ltd., 1-1 Kyowa-machi,
Hofu, Yamaguchi 747-8522, Japan

Received April 13, 1999； Accepted June 3, 1999
Transketolase is important in production of the aromatic amino acids in Corynebacterium glutamicum. The complete nucleotide sequence of the C. glutamicum transketolase gene has been identified. The DNA-derived protein sequence is highly similar to the transketolase of Mycobacterium tuberculosis, taxonomically related to C. glutamicum. The alignment of the N-terminus regions between both transketolases showed TTG to be the most probable start codon. Potential ribosomal binding and promoter regions were situated upstream from the TTG. The deduced amino acid sequence consists of 700 residues with a calculated molecular mass of 75 kDa, and contains all amino acid residues involved in cofactor and substrate binding in the well-characterized yeast transketolase sequence.
Corynebacterium glutamicum; transketolase; tkt gene

-26-
Note
Identification of Zerumbone in Zingiber zerumbet Smith as a Potent Inhibitor
of 12-O-Tetradecanoylphorbol-13-acetate-induced Epstein-Barr Virus Activation

Akira MURAKAMI,1 Miyuki TAKAHASHI,1 Suratwadee JIWAJINDA,2 Koichi KOSHIMIZU,1
and Hajime OHIGASHI3,・

1Department of Biotechnological Science, Faculty of Biology-Oriented Science and Technology, Kinki University,
Iwade-Uchita, Wakayama 649-6493, Japan
2Environmental Science Unit, Central Laboratory and Greenhouse Complex, Kasetsart University, Thailand
3Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502

Received April 21, 1999; Accepted June 30, 1999
Zerumbone was isolated from the rhizomes of Zingiber zerumbet Smith as a potent inhibitor of tumor promoter 12-O-tetradecanoylphorbol-13-acetate-induced Epstein-Barr virus activation. The IC50 value of zerumbone (0.14 μM) is noticeably lower than those of the anti-tumor promoters we have hitherto obtained. Interestingly, α-humulene lacking the carbonyl group at the 8-position in zerumbone was inactive (IC50>100 μM), while 8-hydroxy-α-humulune was markedly active (IC50＝0.95 μM).
zerumbone; Epstein-Barr virus; anti-tumor promoter; chemoprevention; Zingiber zerumbet

-27-
Note
Chemical Characterization of Fucoidan from Commercially Cultured
Nemacystus decipiens (Itomozuku)

Masakuni TAKO,・ Taketo NAKADA and Fujiya HONGOU

Department of Bioscience and Biotechnology, Faculty of Agriculture, University of the Ryukyus,
Nishihara, Okinawa 903-0123, Japan

Received April 22, 1999; Accepted July 7, 1999
Fucoidan was isolated from Nemacystus decipiens KUCKUCK which is commercially cultured at the rate of about 2,000 t/year in fields of the Okinawa Islands, in Japan. The yield of fucoidan extracted by 0.1 M HCl was 0.5％ (W/W) based on the wet algae. The total carbohydrate, sulfuric acid, ash, and moisture in fucoidan was 65.8, 30.8, 22.3, and 3.8％, respectively. Fucose and galactose were identified by liquid chromatography. The molar ratio of the sugar residues in the fucoidan was estimated to be L-fucose:D-galactose:sulfuric acid＝1.0:0.05:1.0. The optical rotation of the fucoidan (0.4％) showed a value of −0.315° at 60°C, then it decreased a little with decreasing temperature. The infrared spectrum of isolated fucoidan is in agreement with that of standard fucoidan. The molecular mass of fucoidan was estimated to be 2.4×105. The 1H- and 13C-NMR spectral data suggest that fucoidan is mainly composed of L-fucopyranose, 2-mono-O-sulfo-L-fucopyranose, 4-mono-O-sulfo-L-fucopyranose, and 2,4-di-O-sulfo-L-fucopyranose.
fucoidan; Itomozuku; Nemacystus decipiens

-28-
Note
A 20-kDa Protein with the GTP-Binding and Trypsin Inhibitory Activities
from Glycine max

Toshifumi HIRATA,・ Shunsuke IZUMI, and Seiichiro TSUJI

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

Received April 26, 1999; Accepted June 18, 1999
A 20-kDa protein (p20) with a GTP binding activity was purified from the cultured cells of Glycine max (soybean). The amino acid sequence of p20 showed 65％ identity in a 23 amino acid overlap against the Kunitz-type trypsin inhibitor of soybean reported. Furthermore, it was found that a Kunitz-type soybean trypsin inhibitor of commercial origin also binds GTP.
GTP binding activity; trypsin inhibitory activity; 20-kDa protein; Glycine max; cultured cells

-29-
Note
Insight into the Molecular Evolution of Two Tropinone Reductases

Keiji NAKAJIMA, Yoko OSHITA, Yasuyuki YAMADA, and Takashi HASHIMOTO

Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama,
Ikoma, Nara 630-0101, Japan

Received April 26, 1999; Accepted July 8, 1999
In tropane alkaloid biosynthesis, two tropinone reductases produce different stereoisomers from a common substrate, tropinone. The two enzymes share 64％ of identical amino acids, and highly homologous proteins with variable substrate-binding residues have also been found in tropane alkaloid non-producing species. This exemplifies a simple evolutionary process that plants have taken to acquire a new secondary metabolic pathway.
molecular evolution; short-chain dehydrogenase/reductase; substrate specificity; tropane alkaloids; tropinone reductase

-30-
Note
Transcriptional Regulation of Tyrosine Phenol-lyase Gene Mediated through
TyrR and cAMP Receptor Protein

Takane KATAYAMA, Hideyuki SUZUKI, Kenji YAMAMOTO, and Hidehiko KUMAGAI

Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University,
Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan

Received April 30, 1999; Accepted June 2, 1999
Using a lac reporter system in Escherichia coli, we showed that the expression of E. herbicola tpl was regulated through TyrR and cAMP receptor protein. Three TyrR boxes upstream of tpl were essential for full expression. The results suggested that the tyrosine-mediated TyrR hexamerization was an important process. The DNA bending between two TyrR boxes, which is triggered by the binding of cAMP receptor protein, may facilitate the conformational change of TyrRs.
Erwinia herbicola; tyrosine phenol-lyase; TyrR-mediated activation

-31-
Note
Construction of a LukS-PV Mutant of a Staphylococcal Panton-Valentine
Leukocidin Component having a High LukS-like Function

Akiko SHIMATANI, Jun KANEKO, Toshio TOMITA, and Yoshiyuki KAMIO・

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

Received May 6, 1999; Accepted June 4, 1999
A 2-residue (D12I13) segment of LukS of a staphylococcal leukocidin component is an essential region for the hemolytic function of LukS towards rabbit erythrocytes in the presence of LukF. Here, we report that insertion of D, I, or AA residue(s) between A11 and E12 residues of LukS-PV, in which the 2-residue D12I13 segment in LukS was absent, confers the full LukS function on LukS-PV, which has only 4％ hemolytic activity of that of LukS towards rabbit erythrocytes.
staphylococcal leukocidin; Panton-Valentine leukocidin; LukF-PV; LukS-PV

-32-
Note
Effect of Optically Active Ethyl 2-Phthalimidooxypropionate
on the Growth of Cress, Lepidium sativum

Yukinori TAKEKIDA, Momotoshi OKAZAKI, and Yoshihiro SHUTO＊

Faculty of Agriculture, Ehime University, Tarumi, Matsuyama 790-8566, Japan

Received May 10, 1999; Accepted June 28, 1999
The effects of 2-phthalimidooxyalkanoic acid derivatives on the germination and root-growth of cress were examined. Since 2-phthalimidooxypropionates were most effective, the optically active ethyl esters were prepared. As the result of biological testing, the (S)-(−)-isomer exhibited stronger activity than the (R)-(＋)-isomer. This result is contrary to those from commercial herbicides with similar structures, phenoxy- and oxyphenoxy-propionate-type compounds, where the (R)-isomers are generally known to be the active principles.
||phthalimidooxy derivatives; 2-phthalimido||oxypropionate; optical activity; cress test; plant growth inhibition

-33-
Note
Synthesis of 2-(4-Hydroxyphenyl)naphthalene-1,8-dicarboxylic Anhydride,
a Phytoalexin Isolated from Unripe Banana (Musa acuminata)・

Hirosato TAKIKAWA, Masao YOSHIDA, and Kenji MORI・・

Department of Chemistry, Faculty of Science, Science University of Tokyo, Kagurazaka 1-3, Shinjuku-ku,
Tokyo 162-8601, Japan

Received May 13, 1999; Accepted June 10, 1999
2-(4-Hydroxyphenyl)naphthalene-1,8-dicarboxylic anhydride, a component of the phytoalexin that has been isolated from the peel of unripe banana (Musa acuminata), was synthesized from 3-bromoacenaphthene.
anhydride; arene; Colletotrichum musae; Musa acuminata; phytoalexin

-34-
Note
Consumption of a Buckwheat Protein Extract Retards 7,12-Dimethylbenz[α]
anthracene-Induced Mammary Carcinogenesis in Rats

Jun KAYASHITA,・ Iwao SHIMAOKA, Misao NAKAJOH, Naoko KISHIDA,
and Norihisa KATO＊,・・

Development, Health Care, Kissei Pharmaceutical Company, Matsumoto 399-8710, Japan
＊Department of Applied Biochemistry, Faculty of Applied Biological Science, Hiroshima University,
Higashi-Hiroshima 739-8528, Japan

Received May 18, 1999; Accepted June 17, 1999
Female rats were examined for the effects of feeding buckwheat protein extract (BWPE) on the development of mammary tumor caused by administration of 7,12-dimethylbenz[α]anthracene. The percentage of rats with palpable mammary tumors and serum estradiol were lower in the BWPE-fed animals than the casein-fed ones, implying that BWPE intake retarded the mammary carcinogenesis by lowering serum estradiol.
buckwheat protein; mammary carcinogenesis; estradiol; rat

-35-
Preliminary Communication
Saponin Stimulates Fruiting of the Edible Basidiomycete Pleurotus ostreatus

Yumi MAGAE

Forestry and Forest Products Research Institute, P.O. Box16, Tsukuba Norin, Ibaraki 305-8687, Japan

Received June 11, 1999; Accepted August 23, 1999
The addition of saponin from Quillaja to malt extract agar dramatically stimulated fruit body development of Pleurotus ostreatus. Higher concentration of Quillaja saponin added to the medium suppressed the growth of pilei of fruit bodies. The results indicate that the triterpenoid-saponin is a bioactive substance for fruiting of P. ostreatus.
Pleurotus ostreatus; saponin; fruit body; development

-36-
Preliminary Communication
Identification of the Amino Acid Residues Conferring Substrate Specificity
upon Selenomonas ruminantium Lysine Decarboxylase

Yumiko TAKATSUKA, Toshio TOMITA, and Yoshiyuki KAMIO＊

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

Received June 22, 1999; Accepted July 19, 1999
Lysine decarboxylase (LDC, EC 4.1.1.18) from Selenomonas ruminantium has decarboxylating activities towards both L-lysine and L-ornithine with similar Km and Vmax.Here, we identified four amino acid residues that confer substrate specificity upon S. ruminantium LDC and that are located in its catalytic domain. We have succeeded in converting S. ruminantium LDC to an enzyme with a preference in decarboxylating activity for L-ornithine when the four-residue of LDC were replaced by the corresponding residues of mouse ornithine decarboxylase (EC 4.1.1.17).
Selenomonas ruminantium; lysine decarboxylase; ornithine decarboxylase; substrate specificity; catalytic domain

-37-
Preliminary Communication
Preparation and Characterization of Fully Active Biotinylated Analogs
of Phytosulfokine-α

Yoshikatsu MATSUBAYASHI,＊ Yoshinori TAKAHATA, Akiko MORITA, Keisuke ATSUMI,
and Youji SAKAGAMI＊

Laboratory of Bioactive Natural Products Chemistry, Graduate School of Bio-Agricultural Sciences,
Nagoya University, Chikusa, Nagoya 464-8601, Japan

Received July 5, 1999; Accepted July 21, 1999
We report the preparation of biotinylated analogs of phytosulfokine-α (Tyr(SO3H)-Ile-Tyr(SO3H)-Thr-Gln; PSK-α), an endogenous peptide growth factor in plants. Because the modification of the N-terminal amino group leads to significant loss of the activities, a Lys residue was incorporated in the C-terminal region of PSK-α, and its e amino group was reacted with biotinylation reagent. Results of the binding assay showed that [Nε-(biotinyl)Lys5]PSK-α retained the same binding activity and mitogenic activity as that of native PSK-α. Insertion of a single or double 6-aminohexanoic acid spacer between the ε amino group of Lys5 and the carboxyl group of biotin did not significantly alter the activities of biotinylated [Lys5]PSK-α. Structure-activity information obtained here would be useful for the detection and isolation of PSK-α receptors.
phytosulfokine-α; binding assay; growth factor; biotin; receptor