Contents and Abstracts of BBB

(Vol.69 No.03 2005)

Review
AdpA, a Central Transcriptional Regulator in the A-Factor Regulatory Cascade That Leads to Morphological Development and Secondary Metabolism in Streptomyces griseus
Yasuo Ohnishi, Haruka Yamazaki, Jun-ya Kato, Ayami Tomono, and Sueharu Horinouchi p.431

Synthesis of Lipophilic Poly-Lauroyl-(+)-Catechins and Radical-Scavenging Activity
Guangzhi Jin and Hisashi Yoshioka p.440

Potential Ability of Hot Water Adzuki (Vigna angularis) Extracts to Inhibit the Adhesion, Invasion, and Metastasis of Murine B16 Melanoma Cells
Tomohiro Itoh1, Hayato Umekawa2, and Yukio Furuichi2 p.448

The N-Terminal Regions of β and γ Subunits Lower the Solubility of Adenosylcobalamin-Dependent Diol Dehydratase p.455
Takamasa Tobimatsu, Masahiro Kawata, and Tetsuo Toraya

Purification and Characterization of Inducible Cephalexin Synthesizing Enzyme in Gluconobacter oxydans
Chia-Yang Shiau3, Shun-Chung Pai1, Wen-Po Lin1, Dar-Der Ji2, and Yu-Tien Liu1 p.463

Effect of Culture Conditions on Production of 5-Aminolevulinic Acid by Recombinant Escherichia coli
Dae-Hee Lee1, Woo-Jin Jun2, Dong-Hoon Shin1, Hong-Yon Cho1, and Bum-Shik Hong1 p.470

Cloning and Heterologous Expression of the Antibiotic Peptide (ABP) Genes from Rhizopus oligosporus NBRC 8631
Osamu Yamada1, Kazutoshi Sakamoto1, Mihoko Tominaga1, Tasuku Nakayama2, Takuya Koseki1, Akiko Fujita1, and Osamu Akita1 p.477

Lipid Peroxidation of a Human Hepatoma Cell Line (HepG2) after Incorporation of Linoleic Acid, Arachidonic Acid, and Docosahexaenoic Acid
Mina Araseki1, Hidetaka Kobayashi1,2, Masashi Hosokawa1, and Kazuo Miyashita1 p.483

A Series of Crystal Structures of a meta-Cleavage Product Hydrolase from Pseudomonas fluorescens IP01 (CumD) Complexed with Various Cleavage Products
Shinya Fushinobu1, So-Young Jun1, Masafumi Hidaka1, Hideaki Nojiri2, Hisakazu Yamane2, Hirofumi Shoun1, Toshio Omori3, and Takayoshi Wakagi1 p.491

The Effect of Protozoa on the Composition of Rumen Bacteria in Cattle Using 16S rRNA Gene Clone Libraries
Yuhei Ozutsumi1, Kiyoshi Tajima2, Akio Takenaka2, and Hisao Itabashi1 p.499

Superoxide Dismutase from the Silkworm, Bombyx mori: Sequence, Distribution, and Overexpression
Kohji Yamamoto1, Pingbo Zhang1, Yutaka Banno1, Hiroshi Fujii1, Fumio Miake2, Nobuhiro Kashige2, and Yoichi Aso3 p.507

FR207944, an Antifungal Antibiotic from Chaetomium sp. No. 217 I. Taxonomy, Fermentation, and Biological Properties
Motoo Kobayashi, Ryuichi Kanasaki, Ikuko Sato, Fumie Abe, Kumiko Nitta, Masami Ezaki, Kazutoshi Sakamoto, Michizane Hashimoto, Akihiko Fujie, Motohiro Hino, and Yasuhiro Hori p.515

Purification and Characterization of a Novel α-Glucuronidase from Aspergillus niger Specific for O-α-D-Glucosyluronic Acid α-D-Glucosiduronic Acid
Takaaki Kiryu, Hirofumi Nakano, Taro Kiso, and Hiromi Murakami p.522

Purification and Characterization of Hydantoin Racemase from Microbacterium liquefaciens AJ 3912
Shun’ichi Suzuki, Norimasa Onishi, and Kenzo Yokozeki p.530

Static Suppression of Tomato Bacterial Wilt by Bacterial Coagulation Using a New Functional Polymer That Coagulates Bacterial Cells and Is Highly Biodegradable
Nariyoshi Kawabata, Wataru Sakakura, and Yoshihiro Nishimura p.537

Highly Active Mutants of Carbonyl Reductase S1 with Inverted Coenzyme Specificity and Production of Optically Active Alcohols
Souichi Morikawa1, Takahisa Nakai1, Yoshihiko Yasohara2, Hirokazu Nanba2, Noriyuki Kizaki2, and Junzo Hasegawa3 p.544

Structure of β-Glucan Oligomer from Laminarin and Its Effect on Human Monocytes to Inhibit the Proliferation of U937 Cells
Zhongcun Pang1, Kodo Otaka1, Takashi Maoka2, Kumi Hidaka2, Sumio Ishijima1, Masayuki Oda1, and Masatake Ohnishi1 p.553

Effect of Bread Containing Resistant Starch on Postprandial Blood Glucose Levels in Humans
Yuji Yamada1, Seio Hosoya1, Shigeru Nishimura1, Takashi Tanaka1, Yoshitaka Kajimoto2, Akira Nishimura2, and Osami Kajimoto3 p.559

Strain and Age-Related Changes in the Localization of Intestinal CD161+ Natural Killer Cells and CD8+ Intraepithelial Lymphocytes along the Longitudinal Crypt Axis in Inbred Rats
Hong Xu1, Syunsuke Imanishi1, Kazuhiko Yamada2, Hiroshi Hara1, and Satoshi Ishizuka1 p.567

Identification of rpoBC Genes Encoding for β and β’ Subunits of RNA Polymerase in a Deep-Sea Piezophilic Bacterium, Shewanella violacea Strain DSS12
Hiroaki Kawano1,2, Kaoru Nakasone3, Fumiyoshi Abe1, Chiaki Kato1, Yasuhiko Yoshida2, Ron Usami2, and Koki Horikoshi1 p.575

Transepithelial Transport of Rosmarinic Acid in Intestinal Caco-2 Cell Monolayers
Yutaka Konishi1 and Shoko Kobayashi2 p.583

Functional Analysis of Two Solanesyl Diphosphate Synthases from Arabidopsis thaliana
Kazutake Hirooka1, Yoshikazu Izumi1, Chung-Il An1, Yoshihisa Nakazawa2, Ei-ichiro Fukusaki1, and Akio Kobayashi1 p.592

Cloning and Expression of a Bacillus circulans KA-304 Gene Encoding Chitinase I, Which Participates in Protoplast Formation of Schizophyllum commune
Shigekazu Yano, Nopakarn Rattanakit, Mamoru Wakayama, and Takashi Tachiki p.602

Preparation of Functional Single-Chain Antibodies against Bioactive Gibberellins by Utilizing Randomly Mutagenized Phage-Display Libraries
Yoshihito Suzuki, Shinsaku Ito, Kaori Otsuka, Eriko Iwasawa, Masatoshi Nakajima, and Isomaro Yamaguchi p.610

Note
Protective Effect of Epigallocatechin Gallate and Esculetin on Oxidative DNA Damage Induced by Psoralen Plus Ultraviolet-A Therapy
Shoichi Tahara1, Naomichi Baba2, Mitsuyoshi Matsuo3, and Takao Kaneko1 p.620

Note
Effects of Serum Deprivation on Expression of Proteolytic-Related Genes in Chick Myotube Cultures
Kazuki Nakashima, Makoto Yamazaki, and Hiroyuki Abe p.623

Note
Biosynthesis of the Marine Antibiotic Pentabromopseudilin. 2. The Pyrrole Ring
Jörg D. Peschke1, Ulf Hanefeld1,2, and Hartmut Laatsch1 p.628

Note
Effect of Molecular Mass on Antitumor Activity of Heteropolysaccharide from Poria cocos
Lina Zhang1, Li Chen1, Xiaojuan Xu1, Fanbo Zeng2, and Peter Chi Keung Cheung3 p.631

Note
Production of Heterogeneous Dimer Lignostilbenedioxygenase II from lsdA and lsdB in Escherichia coli Cells
Shigehiro Kamoda2, Tamami Terada1, and Yoshimasa Saburi1 p.635

Note
Molecular Monitoring of the Developmental Bacterial Community in the Gastrointestinal Tract of Japanese Infants
Prapa Songjinda1, Jiro Nakayama1, Yumiko Kuroki1, Shigemitsu Tanaka1, Sanae Fukuda2, Chikako Kiyohara3, Tetsuro Yamamoto2,4, Kunio Izuchi5, Taro Shirakawa2, and Kenji Sonomoto1 p.638

Note
Amino Acid Sequence and Antimicrobial Activity of Chitin-Binding Peptides, Pp-AMP 1 and Pp-AMP 2, from Japanese Bamboo Shoots (Phyllostachys pubescens)
Masatoshi Fujimura1, Mineo Ideguchi1, Yuji Minami1, Keiichi Watanabe2, and Kenjiro Tadera1 p.642

Note
Microtubule-Cyclodextrin Conjugate: Functionalization of Motile Filament with Molecular Inclusion Ability
Kazuaki Kato1, Rie Goto1, Kaoru Katoh2, and Motonari Shibakami1 p.646

Note
Cloning and Sequence Analysis of the Micronuclear and Macronuclear Gene Encoding Rab Protein of Euplotes octocarinatus
Wei Wang, Hui Zhi, Baofeng Chai, and Aihua Liang p.649

Communication
Anti-Invasive Activity of Niacin and Trigonelline against Cancer Cells
Nobuhiro Hirakawa, Rieko Okauchi, Yutaka Miura, and Kazumi Yagasaki p.653

Communication
Microbial Production of L-Ascorbic Acid from D-Sorbitol, L-Sorbose, L-Gulose, and L-Sorbosone by Ketogulonicigenium vulgare DSM 4025
Teruhide Sugisawa1, Taro Miyazaki2, and Tatsuo Hoshino3 p.659


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Review
AdpA, a Central Transcriptional Regulator in the A-Factor Regulatory Cascade That Leads to Morphological Development and Secondary Metabolism in Streptomyces griseus

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

A-factor (2-isocapryloyl-3R-hydroxymethyl-γ-butyrolactone) is a microbial hormone that triggers aerial mycelium formation and secondary metabolism in Streptomyces griseus. A-factor produced in a growth-dependent manner switches on the transcription of adpA encoding a transcriptional activator by binding to the A-factor receptor protein (ArpA), which has bound the adpA promoter, and dissociating the DNA-bound ArpA from the DNA. AdpA then activates a number of genes with various functions required for morphological development and secondary metabolism, forming an AdpA regulon. AdpA, which contains a ThiJ/PfpI/DJ-1-like dimerization domain at its N-terminal portion and an AraC/XylS-type DNA-binding domain at its C-terminal portion, is a representative of a large subfamily of the AraC/XylS family. AdpA binds various positions with respect to the transcriptional start points of the target genes and recruits RNA polymerase to the specific promoter region, and facilitates the isomerization of the RNA polymerase-DNA complex into an open complex competent for transcriptional initiation. The AdpA-binding consensus sequence is 5’-TGGCSNGWWY-3’ (S: G or C; W: A or T; Y: T or C; N: any nucleotide). The DNA-binding specificity of AdpA in conjunction with that of other AraC/XylS family members is also discussed.

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Synthesis of Lipophilic Poly-Lauroyl-(+)-Catechins and Radical-Scavenging Activity

Guangzhi Jin and Hisashi Yoshioka

Institute for Environmental Sciences, University of Shizuoka, 52-1 Yada, Shizuoka-shi 422-8526, Japan

Lipophilic catechins were synthesized to improve absorption into living bodies and obtain new antioxidants effective in lipid bilayers. The hydroxyl (OH) groups of (+)-catechin was acylated randomly using lauroyl chloride. The mixture was separated by preparative HPLC, and 3-lauroyl-, 3’,4’-dilauroyl- and 3,3’,4’-trilauroyl-catechins (3-LC, 3’,4’-LC, and 3,3’,4’-LC) were obtained, their structures being determined by 1H NMR. Their radical scavenging activity was measured in a ethanol solution using the 1,1-diphenyl-2-picrylhydrazyl radical, and was compared with that of (+)-catechin. The activity of 3-LC was almost same as that of (+)-catechin, but those of 3’,4’-LC and 3,3’,4’-LC were small, showing that the blocking of phenolic OH groups in the B ring lowered the activity. The scavenging activity on lipophilic radicals in a liposome system was also measured, and the activities were in the order of 3-LC > 3’,4’-LC = (+)-catechin. These results suggested that radical scavenging activity in the lipid membrane depended not only on the number and the relative positions of phenolic OH groups of catechins but also on affinity to the membrane.

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Potential Ability of Hot Water Adzuki (Vigna angularis) Extracts to Inhibit the Adhesion, Invasion, and Metastasis of Murine B16 Melanoma Cells

Tomohiro Itoh1, Hayato Umekawa2, and Yukio Furuichi2

1Imuraya Confectionary Co., Ltd., Takachaya 7-1-1, Tsu, Mie 514-8530, Japan
2Faculty of Bioresources, Mie University, Kamihama 1515, Tsu, Mie 514-8507, Japan

The 40% ethanol eluent of the fraction of hot-water extract from adzuki beans (EtEx.40) adsorbed onto DIAION HP-20 resin has many biological activities, for example, antioxidant, antitumorigenesis, and intestinal α-glucosidase suppressing activities. This study examined the inhibitory effect of EtEx.40 on experimental lung metastasis and the invasion of B16-BL6 melanoma cells. EtEx.40 was found significantly to reduce the number of tumor colonies. It also inhibited the adhesion and migration of B16-BL6 melanoma cells into extracellular matrix components and their invasion into reconstituted basement membrane (matrigel) without affecting cell proliferation in vitro. These in vivo data suggest that EtEx.40 possesses a strong antimetastatic ability, which might be a lead compound in functional food development.

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The N-Terminal Regions of β and γ Subunits Lower the Solubility of Adenosylcobalamin-Dependent Diol Dehydratase

Takamasa Tobimatsu, Masahiro Kawata, and Tetsuo Toraya

Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, Tsushima-naka, Okayama 700-8530, Japan

Adenosylcobalamin-dependent diol dehydratase is one of essential components of carboxysome-like polyhedral bodies. It exists as a heterohexamer (αβγ)2, and its activity is recovered in a precipitant fraction of Klebsiella oxytoca and overexpressing Escherichia coli cells. Limited proteolysis of the enzyme with trypsin converted the enzyme into a highly soluble form without loss of enzyme activity. The N-terminal amino acid sequencing of the enzyme thus solubilized indicated that the N-terminal 20 and 16 amino acid residues had been removed from the β and γ subunits, respectively. Mutant enzymes with the same N-terminal truncations of either or both of the β and γ subunits were expressed on a high level in E. coli cells. All the mutant enzymes obtained were expressed in a soluble, active form. These results indicate that the N-terminal regions of the β and γ subunits lower the solubility of diol dehydratase. The mutant enzyme with the N-terminal truncations of both β and γ subunits was essentially indistinguishable in catalytic properties from recombinant wild-type enzyme or the enzyme purified from K. oxytoca in a soluble form.

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Purification and Characterization of Inducible Cephalexin Synthesizing Enzyme in Gluconobacter oxydans

Chia-Yang Shiau3, Shun-Chung Pai1, Wen-Po Lin1, Dar-Der Ji2, and Yu-Tien Liu1

1Institute of Microbiology and Immunology, National Defense Medical Center, PO Box 90048, Taipei, Taiwan, Republic of China
2Institute of Preventive Medicine, National Defense Medical Center, PO Box 90048, Taipei, Taiwan, Republic of China
3Institute of Medical Science, National Defense Medical Center, PO Box 90048, Taipei, Taiwan, Republic of China

Cephalexin synthesizing enzyme (CSE) of Gluconobacter oxydans ATCC 9324 was purified up to about 940-fold at a yield of 12%. CSE biosynthesis in G. oxydans was found inducible in the presence of D-phenylglycine but not its substrate phenylglycine methyl ester. The purified enzyme was shown homogeneous on SDS–PAGE and exhibited a specific activity of 440 U per mg protein. The apparent molecular mass of the native enzyme was estimated to be 70 kDa over a Superdex 200 gel filtration column and 68 kDa on SDS–PAGE, indicating that the native enzyme is a monomer. Its isoelectric focusing point is 7.1, indicating a neutral character. The enzyme had maximal activity around pH 6.0 to 6.5, and this activity was thermally stable up to 40 °C. Synthesis of cephalexin from D-phenylglycine methyl ester and 7-amino-3-deacetoxycephalosporanic acid (7-ADCA) by the purified CSE was demonstrated. Its L-enantiomer was not accepted by CSE. Apart from cephalexin, ampicillin was also synthesized by the purified CSE from its acyl precursors and 6-aminopenicillanic acid (6-APA). Substrate specificity studies indicated that the enzyme required a free α amino group and an activated carboxyl group as a methyl ester of D-form phenylglycine. Interestingly, the purified enzyme did not catalyze hydrolysis of its products, e.g., cephalexin, cephradine, and ampicillin, in contrast to enzymes from other strains of Pseudomonadaceae.

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Effect of Culture Conditions on Production of 5-Aminolevulinic Acid by Recombinant Escherichia coli

Dae-Hee Lee1, Woo-Jin Jun2, Dong-Hoon Shin1, Hong-Yon Cho1, and Bum-Shik Hong1

1Department of Food Science and Technology, Graduate School of Biotechnology, Korea University, Anam-Dong, Sungbuk-Ku, Seoul 136-701, Korea
2Department of Food and Nutrition, Chonnam National University, Yongbong-Dong, Book-Ku, Gwangju 500-757, Korea

Aminolevulinic acid (ALA) is formed by the enzyme ALA synthase (hemA gene). Then ALA is converted to Porphobilinogen (PBG) by the ALA dehydratase (hemB gene). For the overproduction of ALA, we used an Escherichia coli BL21(DE3) containing a hemA gene from Bradyrhzobium japonicum, which was created in our previous work. The effects of pH on the ALA synthase and ALA dehydratase were investigated. The ALA synthase and ALA dehydratase activities were dependent on the pH of the medium, with maximal activities occurring at pH 6.5 and 8.0 respectively. At pH 6.5, extracellular ALA reached 23 mM in a jar-fermenter. In addition, the effects of some nutritional factors, such as nitrogen source and the ratio of carbon to nitrogen (C/N) on the fermentative production of ALA were investigated. The highest ALA production was found with 8:1 of C/N ratio. Among various nitrogen sources, the tryptone might be a useful one for ALA production.

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Cloning and Heterologous Expression of the Antibiotic Peptide (ABP) Genes from Rhizopus oligosporus NBRC 8631

Osamu Yamada1, Kazutoshi Sakamoto1, Mihoko Tominaga1, Tasuku Nakayama2, Takuya Koseki1, Akiko Fujita1, and Osamu Akita1

1National Research Institute of Brewing, 3-7-1 Kagamiyama, Higashi-Hiroshima 739-0046, Japan
2Faculty of Engineering, Hiroshima University, 1-4-1 Kagamiyama, Higashi-Hiroshima 739-8527, Japan

We carried out protein sequencing of purified Antibiotic Peptide (ABP), and cloned two genes encoding this peptide as abp1 and abp2, from Rhizopus oligosporus NBRC 8631. Both genes contain an almost identical 231-bp segment, with only 3 nucleotide substitutions, encoding a 77 amino acid peptide. The abp gene product comprises a 28 amino acid signal sequence and a 49 amino acid mature peptide. Northern blot analysis showed that at least one of the abp genes is transcribed in R. oligosporus NBRC 8631. A truncated form of abp1 encoding only the mature peptide was fused with the α-factor signal peptide and engineered for expression in Pichia pastoris SMD1168H. Culture broth of the recombinant Pichia displayed ABP activity against Bacillus subtilis NBRC 3335 after induction of heterologous gene expression. This result indicates that mature ABP formed the active structure without the aid of other factors from R. oligosporus, and was secreted.

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Lipid Peroxidation of a Human Hepatoma Cell Line (HepG2) after Incorporation of Linoleic Acid, Arachidonic Acid, and Docosahexaenoic Acid

Mina Araseki1, Hidetaka Kobayashi1,2, Masashi Hosokawa1, and Kazuo Miyashita1

1Laboratory of Biofunctional Material Chemistry, Division of Marine Bioscience, Graduate School of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido 041-8611, Japan
2National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan

Lipid peroxidation of human heptoma cell line, HepG2, after incorporation of linoleic acid (LA), arachidonic acid (AA), and docosahexaenoic acid (DHA) was measured with a fluorescent probe and gas chromatography–mass spectrometry (GC–MS) analysis. The analysis with a fluorescent probe showed that incorporation of each polyunsaturated fatty acid (PUFA) enhanced the cellular lipid peroxidation level, but there was little difference in the effect of LA, AA, or DHA on the enhancement of cellular lipid peroxidation. The fluorescent analysis also showed that the addition of H2O2 (0.5 mM) enhanced the cellular lipid peroxidation levels in LA and AA supplemented cells as compared with those without H2O2. However, the enhancement of lipid peroxidation by H2O2 was not observed in DHA-supplemented cells. The same result was obtained in the GC–MS analysis of total amounts of monohydroperoxides (MHP) formed in the cellular phospholipid oxidation. In this case, the main source for MHP was LA in LA-, AA-, and DHA-supplemented cells. A significant amount of AA–MHP and a small amount of DHA–MHP were observed in AA- and DHA-supplemented cells respectively. GC–MS analysis also indicated the specific positional distribution of DHA–MHP isomers. The isomers were formed only by hydrogen abstraction at the C-18 (16-MHP + 20-MHP; 46.5%), C-6 (4-MHP + 8-MHP; 38.5%), and C-12 (10-MHP + 14-MHP; 15.1%) positions, but not at the C-9 or C-15 positions.

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A Series of Crystal Structures of a meta-Cleavage Product Hydrolase from Pseudomonas fluorescens IP01 (CumD) Complexed with Various Cleavage Products

Shinya Fushinobu1, So-Young Jun1, Masafumi Hidaka1, Hideaki Nojiri2, Hisakazu Yamane2, Hirofumi Shoun1, Toshio Omori3, and Takayoshi Wakagi1

1Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
2Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
3Department of Industrial Chemistry, Shibaura Institute of Technology, 3-9-14 Shibaura, Minato-ku, Tokyo 108-8548, Japan

Meta-cleavage product hydrolase (MCP-hydrolase) is one of the key enzymes in the microbial degradation of aromatic compounds. MCP-hydrolase produces 2-hydroxypenta-2,4-dienoate and various organic acids, according to the C6 substituent of the substrate. Comprehensive analysis of the substrate specificity of the MCP-hydrolase from Pseudomonas fluorescens IP01 (CumD) was carried out by determining the kinetic parameters for nine substrates and crystal structures complexed with eight cleavage products. CumD preferred substrates with long non-branched C6 substituents, but did not effectively hydrolyze a substrate with a phenyl group. Superimposition of the complex structures indicated that benzoate was bound in a significantly different direction than other aliphatic cleavage products. The directions of the bound organic acids appeared to be related with the kcat values of the corresponding substrates. The Ile139 and Trp143 residues on helix α 4 appeared to cause steric hindrance with the aromatic ring of the substrate, which hampers base-catalyzed attack by water.

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The Effect of Protozoa on the Composition of Rumen Bacteria in Cattle Using 16S rRNA Gene Clone Libraries

Yuhei Ozutsumi1, Kiyoshi Tajima2, Akio Takenaka2, and Hisao Itabashi1

1United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, Fuchu-shi, Tokyo 183-8509, Japan
2National Institute of Livestock and Grassland Science, Tsukuba-shi, Ibaraki 305-0901, Japan

The effect of the presence of protozoa on the composition of rumen bacteria was investigated in cattle. Seven castrated Holstein cattle were divided into two groups: four faunated and three unfaunated, and 16S ribosomal RNA gene (rDNA) clonal libraries were constructed. A total of 312 clones were sequenced across 1,500 bp. The 151 sequences of the faunated cattle were classified into 98 operational taxonomic units (OTUs) having at least 97% similarity. The sequences derived from the faunated cattle were classified into Firmicutes (59.7%), Bacteroidetes (34.4%), Spirochaetes (2.6%), Actinobacteria (2.0%), and Proteobacteria (1.3%). Bacteroides and Prevotella (34.4%) were the major groups in the faunated cattle. The 161 sequences in the unfaunated cattle were classified into 72 OTUs. The sequences derived from the unfaunated libraries were classified into Firmicutes (65.7%), Bacteroidetes (31.1%), Proteobacteria (1.9%), and Spirochaetes (1.2%). The Clostridium botulinum group and its relatives (36.0%) were the major groups in the unfaunated cattle.
An analysis by the computer program LIBSHUFF clarified that the presence of ruminal protozoa markedly affected the composition of rumen bacteria.

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Superoxide Dismutase from the Silkworm, Bombyx mori: Sequence, Distribution, and Overexpression

Kohji Yamamoto1, Pingbo Zhang1, Yutaka Banno1, Hiroshi Fujii1, Fumio Miake2, Nobuhiro Kashige2, and Yoichi Aso3

1Laboratory of Insect Genetic Resources, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan
2Microbiology Laboratory, Faculty of Pharmaceutical Sciences, Fukuoka University, Fukuoka 814-0181, Japan
3Laboratory of Genetic and Protein Engineering, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan

Superoxide dismutase (SOD) is an enzyme facilitating the removal of superoxide anions from living organisms. This study focused on SOD from the silkworm, Bombyx mori (bmSOD). cDNA encoding bmSOD was amplified by reverse transcriptase–polymerase chain reaction. The deduced amino acid sequence of bmSOD indicated that the residues forming the Cu/Zn binding site are conserved and that the sequence is in 60% identity to that of the Drosophila melanogaster. B. mori SOD was also close to the D. melanogaster SOD in a phylogenetic tree. The bmSOD mRNA and the enzyme activity were widely distributed in diverse tissues. bmSOD functionally overexpressed in Escherichia coli in a soluble form was purified, and its stability was examined. bmSOD at 4 °C retained almost all of its original activity after incubation at pH 4–11 for 24 h. Incubation (pH 7) for 30 min at temperatures below 40 °C also affected activity insignificantly.

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FR207944, an Antifungal Antibiotic from Chaetomium sp. No. 217 I. Taxonomy, Fermentation, and Biological Properties

Motoo Kobayashi, Ryuichi Kanasaki, Ikuko Sato, Fumie Abe, Kumiko Nitta, Masami Ezaki, Kazutoshi Sakamoto, Michizane Hashimoto, Akihiko Fujie, Motohiro Hino, and Yasuhiro Hori

Fermentation Research Laboratories, Fujisawa Pharmaceutical Co., Ltd., 5-2-3 Tokodai, Tsukuba, Ibaraki 300-2698, Japan

An antifungal antibiotic, FR207944, was isolated from the culture broth of a fungal strain Chaetomium sp. no. 217. FR207944 is a triterpene glucoside with antifungal activity against Aspergillus fumigatus and Candida albicans. Specifically, FR207944 exhibits in vitro and in vivo antifungal activity against A. fumigatus. The effects of FR207944 on the morphology of A. fumigatus were shown to be similar to those of FR901379, a known 1,3-β-glucan synthase inhibitor. The MECs of FR207944 against A. fumigatus FP1305 and C. albicans FP633 in micro-broth dilution test were 0.039 and 1.6 μg/ml respectively. FR207944 showed good potency by subcutaneous injection and oral administration against A. fumigatus in a murine systemic infection model, with ED50s of 5.7 and 17 mg/kg respectively.

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Purification and Characterization of a Novel α-Glucuronidase from Aspergillus niger Specific for O-α-D-Glucosyluronic Acid α-D-Glucosiduronic Acid

Takaaki Kiryu, Hirofumi Nakano, Taro Kiso, and Hiromi Murakami

Department of Biochemistry and Materials for Human Life, Osaka Municipal Technical Research Institute, 6-50, Morinomiya 1-chome, Joto-ku, Osaka 536-8553, Japan

A new α-glucuronidase that specifically hydrolyzed O-α-D-glucosyluronic acid α-D-glucosiduronic acid (trehalose dicarboxylate, TreDC) was purified from a commercial enzyme preparation from Aspergillus niger, and its properties were examined. The enzyme did not degrade O-α-D-glucosyluronic acid α-D-glucoside, O-α-D-glucosyluronic acid β-D-glucosiduronic acid, O-α-D-glucosyluronic acid-(1→ 2)-β-D-fructosiduronic acid, p-nitrophenyl-O-α-D-glucosiduronic acid, methyl-O-α-D-glucosiduronic acid, or 6-O-α-(4-O-α-D-glucosyluronic acid)-D-glucosyl-β-cyclodextrine. Furthermore, it showed no activity on α-glucuronyl linkages of 4-O-methyl-D-glucosyluronic acid-α-(1→ 2)-xylooligosaccharides, derived from xylan, a supposed substrate of α-glucuronidases.
The molecular mass of the enzyme was estimated to be 120 kDa by gel filtration and 58 kDa by SDS–PAGE suggesting, the enzyme is composed of two identical subunits. It was most active at pH 3.0–3.5 and at 40 °C. It was stable in pH 2.0–4.5 and below 30 °C. It hydrolyzed O-α-D-glucosyluronic acid α-D-glucosiduronic acid to produce α- and β-anomers of D-glucuronic acid in an equimolar ratio. This result suggests that inversion of the anomeric configuration of the substrate is involved in the hydrolysis mechanism.

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Purification and Characterization of Hydantoin Racemase from Microbacterium liquefaciens AJ 3912

Shun’ichi Suzuki, Norimasa Onishi, and Kenzo Yokozeki

AminoScience Laboratories, Ajinomoto Co., Inc., Kawasaki-ku, Kawasaki, Kanagawa 210-8681, Japan

A hydantoin racemase that catalyzed the racemization of 5-benzyl-hydantoin was detected in a cell-free extract of Microbacterium liquefaciens AJ 3912, a bacterial strain known to produce L-amino acids from their corresponding DL-5-substituted-hydantoins. This hydantoin racemase was purified 658-fold to electrophoretic homogeneity by serial chromatography. The N-terminal amino acid sequence of the enzyme showed homology with known hydantoin racemases from other microorganisms. The apparent molecular mass of the purified enzyme was 27 kDa on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and 117 kDa on gel-filtration in the purification conditions, indicating a homotetrameric structure. The purified enzyme exhibited optimal activity at pH 8.2 and 55 °C, and showed a chiral preference for L-5-benzyl- rather than D-5-benzyl-hydantoin.

-15-
Static Suppression of Tomato Bacterial Wilt by Bacterial Coagulation Using a New Functional Polymer That Coagulates Bacterial Cells and Is Highly Biodegradable

Nariyoshi Kawabata, Wataru Sakakura, and Yoshihiro Nishimura

Department of Materials Science, Faculty of Engineering, University of Shiga Prefecture, Hassaka-cho, Hikone, Shiga 522-8533, Japan

Tomato bacterial wilt by Ralstonia solanacearum was suppressed by coagulation of bacterial cells without disinfection using a copolymer of methyl methacrylate with N-benzyl-4-vinylpyridinium chloride in a molar ratio of 3:1 (PMMA-co-BVP) as a polymeric coagulant for bacterial cells. When 10 mg/kg of PMMA-co-BVP was added to soil before transplanting of tomato seedlings, and 2 mg/kg was supplemented once a week after transplanting, a 51% reduction of appearance and a 54% reduction of index of symptoms were observed. PMMA-co-BVP did not exhibit bactericidal activity against R. solanacearum, and coagulation of the bacterial cells appeared to reduce the opportunity for infectious contact of roots of tomato with cells of R. solanacearum, and resulted in disease suppression. PMMA-co-BVP was shown to be highly biodegradable, and the half-life was 5.1 d when treated with activated sludge in soil.

-16-
Highly Active Mutants of Carbonyl Reductase S1 with Inverted Coenzyme Specificity and Production of Optically Active Alcohols

Souichi Morikawa1, Takahisa Nakai1, Yoshihiko Yasohara2, Hirokazu Nanba2, Noriyuki Kizaki2, and Junzo Hasegawa3

1Life Science Research Laboratories, Kaneka Corporation, 1-8 Miyamae-machi, Takasago-cho, Takasago, Hyogo 676-8688, Japan
2Fine Chemicals Research Laboratories, Kaneka Corporation, 1-8 Miyamae-machi, Takasago-cho, Takasago, Hyogo 676-8688, Japan
3Life Science RD Center, Kaneka Corporation, 1-8 Miyamae-machi, Takasago-cho, Takasago, Hyogo 676-8688, Japan

A wild type NADPH-dependent carbonyl reductase from Candida magnoliae (reductase S1) has been found not to utilize NADH as a coenzyme. A mutation to exchange the coenzyme specificity in reductase S1 has been designed by computer-aided methods, including three-dimensional structure modeling and in silico screening of enzyme mutants. Site-directed mutagenesis has been used to introduce systematic substitutions of seven or eight amino acid residues onto the adenosine-binding pocket of the enzyme according to rational computational design. The resulting S1 mutants show NADH-dependency and have lost their ability to utilize NADPH as a coenzyme, but retain those catalytic activities. Kinetic parameter Vmax and Km values of those mutants for NADH are 1/3- to 1/10-fold those of the wild type enzyme for NADPH. As a model system for industrial production of optically active alcohols, the S1 mutants can be applied to an asymmetric reduction of ketones, cooperating with a coenzyme-regeneration system that uses an NAD-dependent formate dehydrogenase.

-17-
Structure of β-Glucan Oligomer from Laminarin and Its Effect on Human Monocytes to Inhibit the Proliferation of U937 Cells

Zhongcun Pang1, Kodo Otaka1, Takashi Maoka2, Kumi Hidaka2, Sumio Ishijima1, Masayuki Oda1, and Masatake Ohnishi1

1Graduate School of Agriculture, Kyoto Prefectural University, 1-5 Shimogamo Nakaragi-cho, Sakyo-ku, Kyoto 606-8522, Japan
2Research Institute for Production Development, 15 Shimogamo Morimoto-cho, Sakyo-ku, Kyoto 606-0805, Japan

We analyzed the human monocyte-stimulating ability of laminarin from Eisenia bicyclis, lichenan from Cetraria islandica, and their oligomers depolymerized with endo-1,3-β-glucanase from Arthrobacter sp. The respective β-glucan oligomers with different degrees of polymerization (DP) were fractionated from hydrolytic products of laminarin and lichenan using gel-filtration chromatography. The monocyte-conditioned medium pre-cultured in the presence of a fraction of β-glucan oligomer (DP≥ 8) from laminarin exhibited inhibitory activity against the proliferation of human myeloid leukemia U937 cells, while those pre-cultured with other β-glucan oligomers and the original laminarin and lichenan showed little or no activity. NMR analysis indicated that the β-glucan oligomer (DP≥ 8) has an average DP value of 13, and its ratio of β-1,3- to β-1,6-linkages in glucopyranose units was estimated to be 1.3:1. These results indicate that the β-1,3-glucan oligomer with a higher content of β-1,6-linkage stimulates monocytes to inhibit the proliferation of U937 cells.

-18-
Effect of Bread Containing Resistant Starch on Postprandial Blood Glucose Levels in Humans

Yuji Yamada1, Seio Hosoya1, Shigeru Nishimura1, Takashi Tanaka1, Yoshitaka Kajimoto2, Akira Nishimura2, and Osami Kajimoto3

1Central Institute, Yamazaki Baking Co., Ltd., 3-5-16 Chitose, Sumida-ku, Tokyo 130-0025, Japan
2Soiken Inc., Senri Life Science Center, 1-4-2 Shinsenri-higashimachi, Toyonaka, Osaka Prefecture 560-0082, Japan
3Center for Health Care, Osaka University of Foreign Studies, 8-1-1 Aomadani-higashi, Mino, Osaka Prefecture 562-8558, Japan

We examined the inhibitory effect of a single ingestion of bread containing resistant starch (bread containing about 6 g of resistant starch derived from tapioca per 2 slices) (test food) on the postprandial increase in blood glucose in male and female adults with a fasting blood glucose level between 100 and 140 mg/dl. Bread not containing resistant starch (placebo) was used as the control.
The study was conducted in 20 subjects (9 men and 11 women with a mean age of 50.5± 7.5 years) using the crossover method, with a single ingestion of either bread containing resistant starch or the placebo. Blood glucose and insulin were measured before ingestion, and at 0.5, 1, 1.5, and 2 h after ingestion. The blood glucose level before ingestion was stratified into a borderline group (blood glucose level ≥ 111 mg/dl) and a normal group (blood glucose level ≤ 110 mg/dl), with the upper limit of the normal range defined as 110 mg/dl. Postprandial increases in both blood glucose and blood insulin were significantly inhibited in subjects in the borderline group who took the test food in comparison with the placebo group (blood glucose: p<0.05 and p<0.01 at 1 and 1.5 h after ingestion respectively; insulin: p<0.05, p<0.01 and p<0.05 at 1, 1.5, and 2 h after ingestion respectively).
These results indicate that bread containing resistant starch is useful for prevention of lifestyle-related diseases such as diabetes mellitus, and as a supplementary means of dietetic therapy.

-19-
Strain and Age-Related Changes in the Localization of Intestinal CD161+ Natural Killer Cells and CD8+ Intraepithelial Lymphocytes along the Longitudinal Crypt Axis in Inbred Rats

Hong Xu1, Syunsuke Imanishi1, Kazuhiko Yamada2, Hiroshi Hara1, and Satoshi Ishizuka1

1Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan
2Division of Food Analysis Research, National Institute of Health and Nutrition, Shinjuku, Tokyo 162-8636, Japan

Using a cell position approach, this study indicates that the frequency of CD161+ natural killer (NK) cells in the epithelia of DA rats was greater than that of WKAH and F344 rats. We further divided the epithelia into proliferating and differentiated regions according to the localization of BrdU-incorporated cells. Comparison between the different regions indicates that a majority of CD161+ NK cells were located in the proliferating region. With age, a decline in the number of CD161+ NK cells and CD8+ intraepithelial lymphocytes (IELs) was observed in the distal colon, especially in the proliferating region of all three strains. Taken together with our previous report that DA rats have far stronger resistance in the colon to preneoplastic lesion than do other strains, these results indicate that CD161+ NK cells play an important role in immune-surveillance at the bottom of the crypt.

-20-
Identification of rpoBC Genes Encoding for β and β’ Subunits of RNA Polymerase in a Deep-Sea Piezophilic Bacterium, Shewanella violacea Strain DSS12

Hiroaki Kawano1,2, Kaoru Nakasone3, Fumiyoshi Abe1, Chiaki Kato1, Yasuhiko Yoshida2, Ron Usami2, and Koki Horikoshi1

1Extremobiosphere Research Center (XBR), Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2-15 Natsushima-cho, Yokosuka, Kanagawa 237-0061, Japan
2Department of Applied Chemistry, Faculty of Engineering, Toyo University, 2100 Kujirai, Kawagoe, Saitama 350-0852, Japan
3Department of Biotechnology and Chemistry, School of Engineering, Kinki University, 1 Umenobe Takaya, Higashihiroshima, Hiroshima 739-2116, Japan

RNA polymerase from cells of the deep-sea bacterium Shewanella violacea DSS12 was purified using three chromatographic steps. An in vitro transcription assay indicated that the purified enzyme was σ70 containing RNA polymerase. The enzyme activity was inhibited in the presence of rifampicin when the sensitive domain was targeted. The rpoBC genes encoding for the β and β’ subunits of RNA polymerase were cloned and their nucleotide sequences determined. Expression plasmids, designated pQSVB and pQSVC, to overproduce these proteins were constructed, and the proteins were purified using a Ni2+ affinity column. In vitro reconstitution using all proteins for the holoenzyme (α, β, β’, σ70) was carried out and the activity of the recombinant RNA polymerase was detected.

-21-
Transepithelial Transport of Rosmarinic Acid in Intestinal Caco-2 Cell Monolayers

Yutaka Konishi1 and Shoko Kobayashi2

1Applied Bioresearch Center, Research and Development Department, Kirin Brewery Co., Ltd., 3 Miyahara-cho, Takasaki-shi, Gunma 370-1295, Japan
2Department of Food and Life Science, Takasaki University of Health and Welfare, Takasaki 370-0033, Japan

The absorption characteristics of rosmarinic acid (RA) were examined by measuring permeation across Caco-2 cell monolayers using an HPLC-electrochemical detector (ECD) fitted with a coulometric detection system. RA exhibited nonsaturable transport even at 30 mM, and the permeation at 5 mM in the apical-to-basolateral direction, Jap→bl, was 0.13 nmol/min/mg of protein. This permeation rate is nearly the same as that of 5 mM chlorogenic acid (CLA) and gallic acid, which are paracellularly transported compounds. Almost all of the apically loaded RA was retained on the apical side, and Jap→bl was inversely correlated with paracellular permeability. These results indicate that RA transport was mainly via paracelluar diffusion, and the intestinal absorption efficiency of RA was low. Furthermore, RA appeared to be unsusceptible to hydrolysis by mucosa esterase in Caco-2 cells. These results, together with our previous work (J. Agric. Food Chem., 52, 2518–2526 (2004), J. Agric. Food Chem., 52, 6418–6424 (2004)) suggest that the majority of RA is further metabolized and degraded into m-coumaric and hydroxylated phenylpropionic acids by gut microflora, which are then efficiently absorbed and distributed by the monocarboxylic acid transporter (MCT) within the body. The potential of orally administered RA in vivo will be further investigated.

-22-
Functional Analysis of Two Solanesyl Diphosphate Synthases from Arabidopsis thaliana

Kazutake Hirooka1, Yoshikazu Izumi1, Chung-Il An1, Yoshihisa Nakazawa2, Ei-ichiro Fukusaki1, and Akio Kobayashi1

1Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita, Yamadaoka 2-1, Osaka 565-0871, Japan
2Hitachi Zosen Corporation, Innoshima, Habu 2264-1, Hiroshima 722-2393, Japan

Solanesyl diphosphate (SPP) is regarded as the precursor of the side-chains of both plastoquinone and ubiquinone in Arabidopsis thaliana. We previously analyzed A. thaliana SPP synthase (At-SPS1) (Hirooka et al., Biochem. J., 370, 679–686 (2003)). In this study, we cloned a second SPP synthase (At-SPS2) gene from A. thaliana and characterized the recombinant protein. Kinetic analysis indicated that At-SPS2 prefers geranylgeranyl diphosphate to farnesyl diphosphate as the allylic substrate. Several of its features, including the substrate preference, were similar to those of At-SPS1. These data indicate that At-SPS1 and At-SPS2 share their basic catalytic machinery. Moreover, analysis of the subcellular localization by the transient expression of green fluorescent protein-fusion proteins showed that At-SPS2 is transported into chloroplasts, whereas At-SPS1 is likely to be localized in the endoplasmic reticulum in the A. thaliana cells. It is known that the ubiquinone side-chain originates from isopentenyl diphosphate derived from the cytosolic mevalonate pathway, while the plastoquinone side-chain is synthesized from isopentenyl diphosphate derived from the plastidial methylerythritol phosphate pathway. Based on this information, we propose that At-SPS1 contributes to the biosynthesis of the ubiquinone side-chain and that At-SPS2 supplies the precursor of the plastoquinone side-chain in A. thaliana.

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Cloning and Expression of a Bacillus circulans KA-304 Gene Encoding Chitinase I, Which Participates in Protoplast Formation of Schizophyllum commune

Shigekazu Yano, Nopakarn Rattanakit, Mamoru Wakayama, and Takashi Tachiki

Department of Bioscience and Biotechnology, Faculty of Science and Engineering, Ritsumeikan University, Kusatsu 525-8577, Japan

KA-prep, a culture filtrate of Bacillus circulans KA-304 grown on a cell-wall preparation of Schizophyllum commune, has an activity to form protoplasts from S. commune mycelia, and a combination of α-1,3-glucanase and chitinase I, isolated from KA-prep, brings about the protoplast-forming activity.
The gene of chitinase I was cloned from B. circulans KA-304 into pGEM-T Easy vector. The gene consists of 1,239 nucleotides, which encodes 413 amino acids including a putative signal peptide (24 amino acid residues). The molecular weight of 40,510, calculated depending on the open reading frame without the putative signal peptide, coincided with the apparent molecular weight of 41,000 of purified chitinase I estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The C-terminal domain of the deduced amino acid sequence showed high similarity to that of family 19 chitinases of actinomycetes and other organisms, indicating that chitinase I is the first example of family 19 chitinase in Bacillus species.
Recombinant chitinase I without the putative signal peptide was expressed in Escherichia coli Rosetta-gami B (DE 3). The properties of the purified recombinant enzyme were almost the same as those of chitinase I purified from KA-prep, and showed the protoplast-forming activity when it was combined with α-1,3-glucanase from KA-prep. Recombinant chitinase I as well as the native enzyme inhibited hyphal extension of Trichoderma reesei.

-24-
Preparation of Functional Single-Chain Antibodies against Bioactive Gibberellins by Utilizing Randomly Mutagenized Phage-Display Libraries

Yoshihito Suzuki, Shinsaku Ito, Kaori Otsuka, Eriko Iwasawa, Masatoshi Nakajima, and Isomaro Yamaguchi

Department of Applied Biological Chemistry, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

Screening randomly mutagenized proteins displayed on a phage surface by biopanning is a powerful strategy to obtain evolved clones with improved properties such as higher stability and functionality. We utilized this method to overcome the problem that functional single-chain antibodies against active gibberellins, a class of plant hormones, can not be prepared by some of the conventional methods. Single-chain antibody libraries with random mutations were constructed from two independent anti-bioactive gibberellin monoclonal antibody lines in a phagemid vector, so that the mutagenized scFvs were expressed in a phage-displayed form upon helper phage infection. From both libraries, scFv clones with binding activity to GA4 were successfully obtained by successive rounds of biopanning against BSA-GA4, the original immunogen. The results are highly suggestive that this approach might be a general solution when a single-chain antibody does not show binding activity. We found further that a ribosomal frameshift to complement a nonsense mutation frequently occurred in an amber suppressor strain of E. coli TG1, resulting in the display of a functional antibody, while such a nonsense mutant failed to produce a soluble antibody in a non-amber suppressor strain. This result explains at least partly why single-chain antibodies are sometimes functional only in a phage-displayed form, not in a soluble form.

-25-
Note
Protective Effect of Epigallocatechin Gallate and Esculetin on Oxidative DNA Damage Induced by Psoralen Plus Ultraviolet-A Therapy

Shoichi Tahara1, Naomichi Baba2, Mitsuyoshi Matsuo3, and Takao Kaneko1

1Redox Regulation Research Group, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo 173-0015, Japan
2Faculty of Agriculture, Okayama University, 1-1-1 Tsushimanaka, Okayama-shi, Okayama 700-0082, Japan
3Department of Biology, Faculty of Science, Konan University, 8-9-1 Okamoto, Higashinada-ku, Kobe, Hyogo 658-8501, Japan

We examined antioxidants exhibiting no effects on DNA cross-linking, which is the basis of psoralen and ultraviolet-A therapy for skin diseases, and suppressing oxidative DNA damage incidental to the therapy. Epigallocatechin gallate and esculetin effectively suppressed oxidative DNA damage with little effect on the formation of DNA cross-linking. These antioxidants might be useful in suppressing the adverse reaction induced by this therapy.

-26-
Note
Effects of Serum Deprivation on Expression of Proteolytic-Related Genes in Chick Myotube Cultures

Kazuki Nakashima, Makoto Yamazaki, and Hiroyuki Abe

Nutrient Function Laboratory, Department of Animal Physiology and Nutrition, National Institute of Livestock and Grassland Science, 2 Ikenodai, Tsukuba, Ibaraki 305-0901, Japan

We previously reported that serum deprivation stimulates myofibrillar proteolysis in chick myotubes. In the present study, we examined the effect of serum deprivation on expression of the proteolytic-related genes (ubiquitin, proteasome, calpains, and cathepsin B) by real-time PCR of cDNA in chick myotubes. Myotubes were incubated with serum-free medium for 24 h. Ubiquitin and proteasome subunits (C1 and C2) and calpains (m-, μ-, and p94/calpain-3) but not cathepsin B mRNA expression were increased by serum deprivation. These results indicate that serum deprivation stimulates ubiquitin-proteasome and calpain proteolytic pathways, resulting in an increase in myofibrillar proteolysis in chick myotubes.

-27-
Note
Biosynthesis of the Marine Antibiotic Pentabromopseudilin. 2. The Pyrrole Ring

Jörg D. Peschke1, Ulf Hanefeld1,2, and Hartmut Laatsch1

1Institut für Organische und Biomolekulare Chemie der Georg-August-Universität, Tammannstr. 2, D-37077 Göttingen, Germany
2Department of Chemistry, Campus Box 351700, University of Washington, Seattle, Washington 98195-1700, U.S.A.

The biosynthesis of the potent marine antibiotic, pentabromopseudilin (1), was investigated. Feeding studies with Alteromonas luteoviolaceus were performed on a defined medium. D,L-[5-13C]proline was incorporated symmetrically, demonstrating that the pyrrole ring of pentabromopseudilin is derived from proline.

-28-
Note
Effect of Molecular Mass on Antitumor Activity of Heteropolysaccharide from Poria cocos

Lina Zhang1, Li Chen1, Xiaojuan Xu1, Fanbo Zeng2, and Peter Chi Keung Cheung3

1Department of Chemistry, Wuhan University, Wuhan 430072, China
2Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China
3Department of Biology, Chinese University of Hong Kong, Hong Kong

A water-soluble heteropolysaccharide ac-PCM0 from Poria cocos was successfully fractionated using a preparative size exclusion chromatography (SEC) column, and their weight-average molecular mass (Mw) was characterized by analytical SEC combined with laser light scattering (SEC-LLS). The results indicate that the fractions having relatively high Mw exhibited higher inhibition ratio in vivo antitumor activity than those having Mw below 3.29× 104. However, the relatively low molecular mass was beneficial to the in vitro antitumor activity. Moreover, ac-PCM0 has a significantly higher enhancement ratio of the body weight than 5-fluorouracil, and its 50% lethal dose is above 1250 mg/kg, indicating a nontoxic nature.

-29-
Note
Production of Heterogeneous Dimer Lignostilbenedioxygenase II from lsdA and lsdB in Escherichia coli Cells

Shigehiro Kamoda2, Tamami Terada1, and Yoshimasa Saburi1

1Department of Biomaterial Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan
2University Forest in Hokkaido, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yamabe, Furano-shi, Hokkaido 079-1561, Japan

The lignostilbenedioxygenase isozyme I and III genes, lsdA and lsdB, were coexpressed within the same Escherichia coli cells. The lignostilbenedioxygenase isozymes produced were separated on QAE anion-exchange column chromatography. Three parts of active fractions corresponding to αα, αβ , and ββ dimers were detected. The purified isozyme from second active fractions corresponded to the native heterogeneous isozyme II.

-30-
Note
Molecular Monitoring of the Developmental Bacterial Community in the Gastrointestinal Tract of Japanese Infants

Prapa Songjinda1, Jiro Nakayama1, Yumiko Kuroki1, Shigemitsu Tanaka1, Sanae Fukuda2, Chikako Kiyohara3, Tetsuro Yamamoto2,4, Kunio Izuchi5, Taro Shirakawa2, and Kenji Sonomoto1

1Laboratory of Microbial Technology, Division of Microbial Science and Technology, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan
2Department of Health Promotion and Human Behavior, Kyoto University Graduate School of Public Health, Yoshida-Konoe cho, Sakyo-ku, Kyoto 606-8501, Japan
3Department of Preventive Medicine, Division of Social Medicine, Graduate School of Medical Sciences, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan
4Total Technology Consultant, 1-20-2 Ebisunishi, Shibuya-ku, Tokyo 150-0021, Japan
5Izuchi Hospital, 4-15-6 Yakuin, Chuo-ku, Fukuoka 810-0022, Japan

The dynamics of the developmental bacterial community in the Japanese neonatal gastrointestinal tract were examined by monitoring 16S ribosomal RNA gene (rDNA) diversity in fecal samples by PCR and denaturing gradient gel electrophoresis (DGGE). The results showed a certain pattern common in infants without antibiotic treatment, in which aerobes, e.g., Pseudomonas, appeared first and were then immediately replaced by facultative anaerobe, Enterococcus, Streptococcus, and Enterobacteriaceae through the first month, and finally strictly anaerobic Bifidobactrerium appeared.

-31-
Note
Amino Acid Sequence and Antimicrobial Activity of Chitin-Binding Peptides, Pp-AMP 1 and Pp-AMP 2, from Japanese Bamboo Shoots (Phyllostachys pubescens)

Masatoshi Fujimura1, Mineo Ideguchi1, Yuji Minami1, Keiichi Watanabe2, and Kenjiro Tadera1

1Department of Biochemical Science and Technology, Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan
2Department of Applied Biological Sciences, Faculty of Agriculture, Saga University, Honjo-machi, Saga 840-8502, Japan

Two novel chitin-binding peptides, designated Pp-AMP 1 and Pp-AMP 2, which had antimicrobial activity against pathogenic bacteria and fungi, were purified from Japanese bamboo shoots (Phyllostachys pubescens) by a simple procedure based on chitin affinity chromatography. They had the common structural features of the plant defensin family, but they could not be grouped in any type of that family. They showed a high degree of homology to mistletoe toxins.

-32-
Note
Microtubule-Cyclodextrin Conjugate: Functionalization of Motile Filament with Molecular Inclusion Ability

Kazuaki Kato1, Rie Goto1, Kaoru Katoh2, and Motonari Shibakami1

1Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 5, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan
2Neuroscience Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan

A microtubule-β-cyclodextrin conjugate was prepared on a kinesin-adsorbed glass surface by chemical and biochemical means. Fluorescence microscope observation and a motility assay indicated that the conjugate simultaneously expressed an inherent motor function and an inclusion property.

-33-
Note
Cloning and Sequence Analysis of the Micronuclear and Macronuclear Gene Encoding Rab Protein of Euplotes octocarinatus

Wei Wang, Hui Zhi, Baofeng Chai, and Aihua Liang

Institute of Biotechnology, Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Shanxi University, Taiyuan 030006, P. R. China

The DNA in a micronucleus undergoes remarkable rearrangements when it develops into a macronucleus after cell mating in the hypotrichous ciliate. A Rab gene was isolated from the macronuclear plasmid mini-library of Euplotes octocarinatus. A micronuclear version of the Rab gene was amplified by polymerase chain reaction (PCR). The macronuclear DNA molecule carrying the Rab gene is 767 bp long and shows characteristics typical of macronuclear chromosomes of hypotrichous ciliates. Three of the five cysteines are encoded by the opal codon UGA. The deduced protein is a 207-amino acid (aa) with a molecular mass of 23 kDa. The protein shares 36% identity with Rab 1 protein of Plasmodium and yeast. Analysis of the sequences indicated that the micronuclear version of the Rab gene contains two internal eliminated sequences, internal eliminated sequence (IES)1 and IES2. IES1 is flanked by a pair of hepta-nucleotide 5’-AAATTTT-3’ direct repeats, and IES2 is flanked by 5’-TA-3’ direct repeats.

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Communication
Anti-Invasive Activity of Niacin and Trigonelline against Cancer Cells

Nobuhiro Hirakawa, Rieko Okauchi, Yutaka Miura, and Kazumi Yagasaki

Department of Applied Biological Science, Tokyo Noko University, Saiwai-cho 3-5-8, Fuchu, Tokyo 183-8509, Japan

The effects of niacin, namely, nicotinic acid and nicotinamide, and trigonelline on the proliferation and invasion of cancer cells were studied using a rat ascites hepatoma cell line of AH109A in culture. Niacin and trigonelline inhibited the invasion of hepatoma cells at concentrations of 2.5–40 μM without affecting proliferation. Hepatoma cells previously cultured with a reactive oxygen species (ROS)-generating system showed increased invasive activity. Niacin and trigonelline suppressed this ROS-potentiated invasive capacity through simultaneous treatment of AH109A cells with the ROS-generating system. The present study indicates for the first time the anti-invasive activities of niacin and trigonelline against cancer cells.

-35-
Communication
Microbial Production of L-Ascorbic Acid from D-Sorbitol, L-Sorbose, L-Gulose, and L-Sorbosone by Ketogulonicigenium vulgare DSM 4025

Teruhide Sugisawa1, Taro Miyazaki2, and Tatsuo Hoshino3

1Biotechnology R&D, DSM Nutritional Products Ltd., Bldg 203/854, CH-4002 Basel, Switzerland
2Department of Genome Antibody Product Research, Chugai Pharmaceutical Co., Ltd., Fuji Gotemba Research Labs, 1-135 Komakado, Gotemba, Shizuoka 412-8513, Japan
3Tamagawa University Research Institute, 6-1-1 Tamagawa-Gakuen, Machida, Tokyo 194-8610, Japan

Ketogulonicigenium vulgare DSM 4025, known as a 2-keto-L-gulonic acid producing strain from L-sorbose via L-sorbosone, surprisingly produced L-ascorbic acid from D-sorbitol, L-sorbose, L-gulose, and L-sorbosone as the substrate under a growing or resting condition. As the best result, K. vulgare DSM 4025 produced 1.37 g per liter of L-AA from 5.00 g per liter of L-sorbosone during 4 h incubation time at 30 °C under the resting cell condition having 5.70 g per liter of wet cells. The precursor of L-AA formation from D-sorbitol and L-sorbose, except for L-gulose, was thought to be the putative furanose form of L-sorbosone. This is the first time it is reported that bacteria can produce vitamin C via L-sorbosone.