Contents and Abstracts of BBB

(Vol.69 No.06 2005)

Production of Recombinant Protein Using the HeLa S3-Vaccinia Virus Expression System: Bioreactor Perfusion and Effects of Post-Infection Temperature
Nicole A. Bleckwenn,1,2,3 William E. Bentley,2,3 and Joseph Shiloach1, p.1065

Purification and Characterization of a Thermostable Raw Starch Digesting Amylase from a Streptomyces sp. Isolated in a Milling Factory
Takahiro Kaneko, Toshihisa Ohno, and Naganori Ohisa p.1073

IEC-6 Intestinal Cell Death Induced by Bovine Milk ±-Lactalbumin
Meiyu Xu, Yoshimasa Sugiura, Satoshi Nagaoka, and Yoshihiro Kanamaru p.1082

Copper-Dependent Production of a Pycnoporus coccineus Extracellular Laccase in Aspergillus oryzae and Saccharomyces cerevisiae
Hisashi Hoshida, Tsuneyasu Fujita, Kenji Murata, Kanako Kubo, and Rinji Akada p.1090

Representation of Individual Gene Expression in Completely Pooled mRNA Samples
Änne Glass,1, Jeannette Henning,2 Thomas Karopka,1 Thomas Scheel,1 Sven Bansemer,1 Dirk Koczan,3 Lothar Gierl,1,* Arndt Rolfs,2 and Ulrike Gimsa2 p.1098

Effect of Lactational Exposure to Tributyltin Chloride on Innate Immunodefenses in the F1 Generation in Mice
Kimiko Kimura,1 Kazuo Kobayashi,2 Hiroyuki Naito,1 Yoshihiko Suzuki,1 and Yoshiko Sugita-konishi2, p.1104

Regulation of Substrate Specificity of Plant ±-Mannosidase by Cobalt Ion: In Vitro Hydrolysis of High-Mannose Type N-Glycans by Co2+-Activated Ginkgo ±-Mannosidase
Kwan Kit Woo1 and Yoshinobu Kimura1,2, p.1111

Molecular Properties of Membrane-Bound FAD-Containing D-Sorbitol Dehydrogenase from Thermotolerant Gluconobacter frateurii Isolated from Thailand
Hirohide Toyama,1, Wichai Soemphol,1 Duangtip Moonmangmee,2 Osao Adachi,1 and Kazunobu Matsushita1 p.1120

Reconstitution of Photosynthetic Reaction Centers and Core Antenna-Reaction Center Complexes in Liposomes and Their Thermal Stability
Masayuki Kobayashi,1,2, Yoichiro Fujioka,1 Takayuki Mori,1 Minoru Terashima,1 Hiroaki Suzuki,1 Yuichiro Shimada,1 Takayuki Saito,3 Zheng-Yu Wang,1,4 and Tsunenori Nozawa1 p.1130

Rice Allergenic Proteins, 1416 kDa Albumin and ±-Globulin, Remain Insoluble in Rice Grains Recovered from Rice Miso (Rice-Containing Fermented Soybean Paste)
Masako Ito,1 Takeo Kato,1, and Tsukasa Matsuda2 p.1137

Molecular Phylogenetic Diversity of the Bacterial Community in the Gut of the Termite Coptotermes formosanus
Naoya Shinzato,1,3, Mizuho Muramatsu,1 Toru Matsui,3 and Yoshio Watanabe2 p.1145

Changes in the Composition of Xylem Sap during Development of the Spadix of Skunk Cabbage (Symplocarpus foetidus)
Yoshihiko Onda and Kikukatsu Ito p.1156

Engineering of Yeast Put4 Permease and Its Application to Lager Yeast for Efficient Proline Assimilation
Fumihiko Omura,1, Atsushi Fujita,1 Keiji Miyajima,1 and Nobuyuki Fukui2 p.1162

Two Tandemly Arranged Ferredoxin Genes in the Hydrogenobacter thermophilus Genome: Comparative Characterization of the Recombinant [4Fe4S] Ferredoxins
Takeshi Ikeda,1 Masahiro Yamamoto,1 Hiroyuki Arai,1 Daijiro Ohmori,2 Masaharu Ishii,1 and Yasuo Igarashi1, p.1172

Glucan-Binding Activity of Silkworm 30-kDa Apolipoprotein and Its Involvement in Defense against Fungal Infection
Minoru Ujita,1,2, Yosuke Katsuno,1 Ichiro Kawachi,1 Yoshinori Ueno,3 Yutaka Banno,3 Hiroshi Fujii,3 and Akira Hara1,2 p.1178

Note
Effects of Fenugreek Seed Extract in Obese Mice Fed a High-Fat Diet
Toshiaki Handa,1 Kohji Yamaguchi,1 Yoshikatsu Sono,2 and Kazunaga Yazawa1, p.1186

Note
Involvement of SDS-Stable Higher Mr Forms of Bovine Normal Milk ±-Lactalbumin in Inducing Intestinal IEC-6 Cell Death
Meiyu Xu, Yoshimasa Sugiura, Satoshi Nagaoka, and Yoshihiro Kanamaru p.1189

Note
Sequencing of Hemoglobin Gene 4 (dmhb4) and Southern Blot Analysis Provide Evidence of More Than Four Members of the Daphnia magna Globin Family
Frank Nunes, Désirée Spiering, Marc Wolf, Alexandra Wendler, Ralph Pirow, and Rüdiger J. Paul p.1193

Note
Purification and Characterization of a New Endo-1,4-²-D-glucanase from Beltraniella portoricensis
Yuko Baba,* Atsushi Shimonaka,* Jinichiro Koga, Koichiro Murashima, Hidetoshi Kubota, and Toshiaki Kono p.1198

Note
Quinolactacide, a New Quinolone Insecticide from Penicillium citrinum Thom F 1539*
Masaki Abe,1, Tetsuya Imai,1 Naoki Ishii,1 Makio Usui,1 Toru Okuda,2 and Toshikazu Oki2 p.1202

Note
Characterization of Refolded Hen Lysozyme Variant Lacking Two Outside Disulfide Bonds
Takatoshi Ohkuri,1 Taiji Imoto,2 and Tadashi Ueda1, p.1206

Note
ProteinProtein Interactions in the Subunits of Ribonuclease P in the Hyperthermophilic Archaeon Pyrococcus horikoshii OT3
Mayumi Kifusa, Hideo Fukuhara, Takeshi Hayashi, and Makoto Kimura p.1212

Note
Characterization of Halophilic Alkaline Phosphatase from Halomonas sp. 593, a Moderately Halophilic Bacterium
Matsujiro Ishibashi, Sayaka Yamashita, and Masao Tokunaga p.1213

Note
Characterization of Spore Germination of a Thermoacidophilic Spore-Forming Bacterium, Alicyclobacillus acidoterrestris
Hiroshi Terano,1, Kaori Takahashi,1 and Yutaka Sakakibara2 p.1217

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Production of Recombinant Protein Using the HeLa S3-Vaccinia Virus Expression System: Bioreactor Perfusion and Effects of Post-Infection Temperature

Nicole A. Bleckwenn,1,2,3 William E. Bentley,2,3 and Joseph Shiloach1

1Biotechnology Unit, NIDDK, National Institutes of Health, DHHS Bldg 14A Rm 173, MSC 5522, 9000 Rockville Pike, Bethesda, MD, 20892, U.S.A.
2Department of Chemical Engineering, University of Maryland, College Park, MD, U.S.A.
3Center for Biosystems Research, University of Maryland Biotechnology Institute, College Park, MD, U.S.A.

Adaptation of the vaccinia virus expression system to HeLa S3 suspension bioreactor culture for the production of recombinant protein was conducted. Evaluation of hollow fiber perfusion of suspension culture demonstrated its potential for increased cell density prior to infection. The hollow fiber was also used for medium manipulations prior to infection. Two process parameters, multiplicity of infection (MOI) and temperature during the protein production phase, were evaluated to determine their effect on expression of the reporter protein, enhanced green fluorescent protein (EGFP). An MOI of 1.0 was sufficient for infection and led to the highest level of intracellular EGFP expression. Reducing the temperature to 34 °C during the protein production phase increased production of the protein two-fold compared to 37 °C in spinner flask culture. Scaling up the process to a 1.5-liter bioreactor with hollow fiber perfusion led to an overall production level of 9.9 ¼g EGFP/106 infected cells, or 27 mg EGFP per liter.

Takahiro Kaneko, Toshihisa Ohno, and Naganori Ohisa

Department of Food Product Development, Akita Research Institute of Food and Brewing (ARIF), 4-26 Sanuki, Arayamachi, Akita 010-1623, Japan

A raw starch utilizing microbe was isolated from mud in a milling factory. The 16S ribosomal DNA (rDNA) sequencing and morphological properties of the strain indicated that it belongs to the genus Streptomyces. A strongly raw starch digesting amylase was purified from the culture supernatant of the strain by chromatographic procedures. The specific activity of the enzyme was 11.7 U/mg, molecular mass 47 kDa, optimum pH 6.0, and optimum temperature 50 to 60 °C. The enzyme showed sufficient activity even at 70 °C. It was activated by calcium, cobaltous, and magnesium ions, and inhibited by copper, nickel, zinc, and ferrous ions. It formed maltose mainly from raw and gelatinized starch, and glycogen. No products were formed from glucose, maltose, maltotriose, pullulan, or cyclodextrins (CDs). The enzyme digested raw wheat, rice, and waxy rice starch rapidly, and raw corn, waxy corn, sweet potato, tapioca, and potato starch normally.

Meiyu Xu, Yoshimasa Sugiura, Satoshi Nagaoka, and Yoshihiro Kanamaru

Department of Food Science, Faculty of Applied Biological Science, Gifu University, Gifu 501-1193, Japan

Potent inhibition of cell proliferation was found for commercial preparations of bovine ±-lactalbumin on cultured intestinal cell line IEC-6 albeit lot-dependent. The inhibition was irreversible and a single exposure to the culture medium containing ±-lactalbumin of an active lot for a period as short as 30 min was enough to provoke cell death, possibly through apoptosis. The oligomer fraction from size exclusion chromatography was significantly robust, while the monomer fraction remained totally inert, in inducing cell death. Incubation at 37 °C for 5 d with 30% trifluoroethanol in acetate, pH 5.5, in a slowly rotating test tube rendered the monomer fraction cytotoxic. Again, the resulting inhibitory activity was found in the oligomer fraction from size exclusion chromatography, with emergence of subtle peaks at 22- and 30-kDa. Furthermore, the occurrence of SDS-stable 30-kDa as well as 20-kDa bands on electrophoresis was a common feature for ±-lactalbumin with the activity inducing cell death. Thus, a certain dimeric state can be implicated in the cytotoxicity of bovine ±-lactalbumin.

Hisashi Hoshida, Tsuneyasu Fujita, Kenji Murata, Kanako Kubo, and Rinji Akada

Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering, Yamaguchi University, 2-16-1 Tokiwadai, Ube 755-8611, Japan

Laccase is a multicopper-containing enzyme that catalyzes the oxidation of phenolic compounds. lcc1 cDNA coding for a secretory laccase of Pycnoporus coccineus was expressed under the maltose inducible amyB promoter in Aspergillus oryzae and under the galactose inducible GAL10 promoter in Saccharomyces cerevisiae. Laccase activities, which were undetectable in the absence of copper, were observed by increasing copper concentrations in the media for both systems. The amounts of secreted laccase protein but not lcc1 mRNA increased in proportion to copper concentrations in A. oryzae. The extracellular activities of native A. oryzae amylase and recombinant RNase-T1 expressed from the same amyB promoter in A. oryzae were constant regardless of copper concentrations. Our results indicate that a high copper concentration is required for the production of active laccase in heterologous hosts and that the copper is required for a post-transcriptional process.

Änne Glass,1, Jeannette Henning,2 Thomas Karopka,1 Thomas Scheel,1 Sven Bansemer,1 Dirk Koczan,3 Lothar Gierl,1,* Arndt Rolfs,2 and Ulrike Gimsa2

1Institute for Medical Informatics and Biometry, University of Rostock, Medical Faculty, Rembrandtstr. 16/17, 18055 Rostock, Germany
2Department of Neurology, Neurobiological Laboratory, University of Rostock, Medical Faculty, Gehlsheimer Str. 20, 18147 Rostock, Germany
3Institute for Immunology, University of Rostock, Medical Faculty, Schillingallee 70, 18055 Rostock, Germany

Designing microarray experiments, scientists are often confronted with the question of pooling due to financial constraints, but discussion of the validity of pooling tends toward a sub-pooling recommendation. Since complete pooling protocols can be considered part of sub-pooling designs, gene expression data from three complete pooling experiments were analyzed. Data from complete pooled versus individual mRNA samples of rat brain tissue were compared to answer the question whether the pooled sample represents individual samples in small-sized experiments. Our analytic approach provided clear results concerning the Affymetrixregistered MAS 5.0 signal and detection call parameters. Despite a strong similarity of arrays within experimental groups, the individual signals were evidently not appropriately represented in the pooled sample, with slightly more than half of all the genes considered. Our analysis reveals problems in cases of small complete pooling designs with less than six subjects pooled.

Kimiko Kimura,1 Kazuo Kobayashi,2 Hiroyuki Naito,1 Yoshihiko Suzuki,1 and Yoshiko Sugita-konishi2

1Department of Veterinary Science, Azabu University, 1-17-1 Fuchinobe, Sagamihara-shi, Kanagawa 229-0006, Japan
2Division of Microbiology, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan

We examined the effect of lactational exposure to tributyltin on innate immunodefenses in the F1 generation using in vivo and in vitro experiments. Pregnant C57BL/6 mice were given drinking water containing 0, 15, or 50 ¼g/ml of tributyltin chloride (TBTCl) from parturition to weaning. At weaning time, offspring were inoculated with Escherichia coli K-12, and bacterial clearances from the peritoneal cavity and spleen were examined. In vivo infection experiments indicated that bacterial clearance was significantly depressed in offspring breast-fed by dams exposed to 15 ¼g/ml of TBTCl (15 ppm F1), but not in offspring by dams exposed to 50 ¼g/ml of TBTCl (50 ppm F1). In vitro functional assays revealed that the killing activity of neutrophils decreased significantly in 15 ppm F1, but not in 50 ppm F1. We suggest that lactational exposure to TBT impairs innate immunodefenses in the F1 generation against non-pathogenic bacterial infection.

Kwan Kit Woo1 and Yoshinobu Kimura1,2

1Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan
2Department of Bioresources Chemistry, Faculty of Agriculture, Okayama University, Okayama 700-8530, Japan

In our previous study (Woo, K. K., et al., Biosci. Biotechnol. Biochem., 68, 25472556 (2004), we purified an ±-mannosidase from Ginkgo biloba seeds; it was activated by cobalt ions and highly active towards high-mannose type free N-glycans occurring in plant cells. In the present study, we have found that the substrate specificity of Ginkgo ±-mannosidase is significantly regulated by cobalt ions. When pyridylamino derivative of Man\footnotesize 9GlcNAc\footnotesize 2 (M9A) was incubated with Ginkgo ±-mannosidase in the absence of cobalt ions, Man\footnotesize 5GlcNAc\footnotesize 2-PA (M5A) having no ±12 mannosyl residue was obtained as a major product. On the other hand, when Man\footnotesize 9GlcNAc\footnotesize 2-PA was incubated with ±-mannosidase in the presence of Co2+ (1 mM), Man\footnotesize 31GlcNAc2-PA were obtained as major products releasing ±13/6 mannosyl residues in addition to ±12 mannosyl residues. The structures of the products (Man\footnotesize 85GlcNAc\footnotesize 2-PA) derived from M9A by enzyme digestion in the absence of cobalt ions were the same as those in the presence of cobalt ions. These results clearly suggest that the trimming pathway from M9A to M5A is not affected by the addition of cobalt ions, but that hydrolytic activity towards ±13/6 mannosyl linkages is stimulated by Co2+. Structural analysis of the products also showed clearly that Ginkgo ±-mannosidase can produce truncated high-mannose type N-glycans, found in developing or growing plant cells, suggesting that ±-mannosidase might be involved in the degradation of high-mannose type free N-glycans.

Hirohide Toyama,1, Wichai Soemphol,1 Duangtip Moonmangmee,2 Osao Adachi,1 and Kazunobu Matsushita1

1Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan
2Department of Microbiology, Faculty of Science, King Mongkuts University of Technology Thonburi, Prachauthit Road., Tungkru, Bangkok 10140, Thailand

There are two types of membrane-bound D-sorbitol dehydrogenase (SLDH) reported: PQQSLDH, having pyrroloquinoline quinone (PQQ), and FADSLDH, containing FAD and heme c as the prosthetic groups. FADSLDH was purified and characterized from the PQQSLDH mutant strain of a thermotolerant Gluconobacter frateurii, having molecular mass of 61.5 kDa, 52 kDa, and 22 kDa. The enzyme properties were quite similar to those of the enzyme from mesophilic G. oxydans IFO 3254. This enzyme was shown to be inducible by D-sorbitol, but not PQQSLDH. The oxidation product of FADSLDH from D-sorbitol was identified as L-sorbose. The cloned gene of FADSLDH had three open reading frames (sldSLC) corresponding to the small, the large, and cytochrome c subunits of FADSLDH respectively. The deduced amino acid sequences showed high identity to those from G. oxydans IFO 3254: SldL showed to other FAD-enzymes, and SldC having three heme c binding motives to cytochrome c subunits of other membrane-bound dehydrogenases.

Masayuki Kobayashi,1,2, Yoichiro Fujioka,1 Takayuki Mori,1 Minoru Terashima,1 Hiroaki Suzuki,1 Yuichiro Shimada,1 Takayuki Saito,3 Zheng-Yu Wang,1,4 and Tsunenori Nozawa1

1Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aobayama 6-6-07, Aoba-ku, Sendai 980-8579, Japan
2Department of Chemical Science and Engineering, Ariake National College of Technology, 150 Higashihagio-machi, Omuta 836-8585, Japan
3Department of Industrial Chemistry, Hachinohe National College of Technology, Uenodaira 16-1, Tamonoki-aza, Hachinohe 039-1192, Japan
4Fuculty of Science, Ibaraki University, 2-1-1 Bunkyo, Mito 310-8512, Japan

Photosynthetic reaction centers (RCs) and their core light-harvesting complexes (LH1-RCs), purified from a thermophile, Thermochromatium (T.) tepidum, and a mesophile, Allochromatium (A.) vinosum, were reconstituted into liposomes. The RC and the LH1-RC in the reconstituted liposomes were found intact from the absorption spectra at about 4 and 40 °C respectively. The thermal stability of the RCs of T. tepidum in the liposome was dependent on whether they were surrounded directly by lipids or by the core light-harvesting complexes. The results show that the RC of T. tepidum gains its thermostability through interactions with the LH1. These results are consistent with the result that the thermal stability of the LH1 in T. tepidum is similar in both the reconstituted LH1-RC liposome and ICM. This is clearly different from the mesophilic bacterium, A. vinosum. The thermal stability of RC was also affected by its subunit constitution: the RC containing a cytochrome subunit was more thermostable than the cytochrome-detached RC. This suggests that the cytochrome subunit might play a role in protecting the special pair pigments from denaturation. The thermal denaturation showed a second-order reaction dependence on time. The interaction of the pigments with proteins and/or lipids might be the cause of the second-order reaction profile.

Masako Ito,1 Takeo Kato,1, and Tsukasa Matsuda2

1Aichi Industrial Technology Institute, Food Research Center, 2-1-1 Shinpukuji-cho, Nishi-ku, Nagoya 451-0083, Japan
2Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan

The rice grains (RG) and rice seed proteins remaining in rice miso were investigated with a view point to the potential allergenicity of rice miso. RG ranging from 36 to 180 mg dry weight per g dry miso were separated from several samples of commercially available rice miso. Scanning electron microscopy of the recovered RG indicated that starch granules disappeared almost completely while protein bodies remained intact in RG. Most of the major seed proteins were extracted from RG by heating with 1% SDS/2% 2-mercaptoethanol and detected by SDSpolyacrylamide gel electrophoresis. Major rice allergenic proteins, 1416 kDa albumin (Alb1416) and ±-globulin (±-Glb) were also detected by immunoblotting using the specific antisera, and their contents were estimated to be 1.7 to 9.0 and 1 to 7 mg protein per g dry RG respectively. However, the major rice proteins, including glutelin and prolamin, in RG were insoluble in salt, alcohol, and urea solutions, but soluble in 6 M guanidine hydrochloride (GuHCl). By immunoblotting and ELISA, no Alb1416 and only a slight amount of ±-Glb were detected even in the 6 M GuHCl fraction, indicating that these major allergenic proteins are denatured and are present in an insoluble form in rice miso.

Naoya Shinzato,1,3, Mizuho Muramatsu,1 Toru Matsui,3 and Yoshio Watanabe2

1Bioconsortia Program Laboratory, Tsukuba Central 6, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566, Japan
2Bioresource Laboratories, Mercian Corporation, Iwata, Shizuoka 438-0078, Japan
3Center of Molecular Biosciences, University of the Ryukyus, Nishihara-cho, Okinawa 903-0213, Japan

The phylogenetic diversity of the bacterial community in the gut of the termite Coptotermes formosanus was investigated using a 16S rRNA gene clone library constructed by PCR. After screening by restriction fragment length polymorphism (RFLP) analysis, 49 out of 261 clones with unique RFLP patterns were sequenced and phylogenetically analyzed. Many of the clones (94%) were derived from Bacteroidales, Spirochaetes, and low G+C content gram-positive bacteria consisting of Clostridiales, Mycoplasmatales, Bacillales, and Lactobacillales. In addition, a few clones derived from Actinobacteria, Proteobacteria, Planctomycetes, Verrucomicrobia, and the candidate phylum Synergistes were also found. The most frequently identified RFLP type, BCf1-03, was assigned to the order Bacteroideales, and it constituted about 70% of the analyzed clones. The phylogenetic analysis revealed that the representative clones found in this study tended to form some clusters with the sequences cloned from the termite gut in several other studies, suggesting the existence of termite-specific bacterial lineages.

Yoshihiko Onda and Kikukatsu Ito

Cryobiosystem Research Center, Faculty of Agriculture, Iwate University, Morioka, Iwate 020-8550, Japan

The spadix of skunk cabbage, Symplocarpus foetidus, is thermogenic and maintains an internal temperature of around 20 °C even when the ambient air temperature drops below freezing. This homeothermic heat production is observed only during the stigma stage, and thereafter ceases at the male stage when pollen is shed. To clarify the regulatory mechanism by which the stigma stage-specific heat production occurs in the spadix, sugars, organic acids, and amino acids in xylem sap were analyzed and compared with those of post-thermogenic plants. Interestingly, no significant difference was observed in the total volume of xylem sap per fresh weight of the spadix between thermogenic (31.2± 24.7 ¼l h-1g-1) and post-thermogenic (50.5± 30.4 ¼l h-1g-1) plants. However, concentrations of sugars (sucrose, glucose, and fructose), organic acids (malate and succinate), and amino acids (Asp, Asn, Glu, Gln, Gly, and Ala) in xylem sap decreased remarkably in post-thermogenic plants. Our results indicate that the composition of the xylem sap differs during the development of the spadix of S. foetidus.

Fumihiko Omura,1, Atsushi Fujita,1 Keiji Miyajima,1 and Nobuyuki Fukui2

1Institute for Beer and RTD Development, Suntory Limited, 1-1-1 Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka 618-8503, Japan
2Process Development Department, Suntory Limited, 5-2-5 Yamazaki, Shimamoto-cho, Mishima-gun, Osaka 618-0001, Japan

The Saccharomyces cerevisiae Put4 permease is significant for the transport of proline, alanine, and glycine. Put4p downregulation is counteracted by npi1 mutation that affects the cellular ubiquitination function. Here we describe mutant Put4 permeases, in which up to nine lysine residues in the cytoplasmic N-terminal domain have been replaced by arginine. The steady-state protein level of the mutant permease Put4-20p (Lys9, Lys34, Lys35, Lys60, Lys68, Lys71, Lys93, Lys105, Lys107 ’ Arg) was largely higher compared to that of the wild-type Put4p, indicating that the N-terminal lysines can undergo ubiquitination and the subsequent degradation steps. Proline is the only amino acid that yeast assimilates with difficulty under standard brewing conditions. A lager yeast strain provided with Put4-20p was able to assimilate proline efficiently during beer fermentations. These results suggest possible industrial applications of the mutant Put4 permeases in improved fermentation systems for beer and other alcoholic beverages based on proline-rich fermentable sources.

Takeshi Ikeda,1 Masahiro Yamamoto,1 Hiroyuki Arai,1 Daijiro Ohmori,2 Masaharu Ishii,1 and Yasuo Igarashi1

1Department of Biotechnology, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
2Department of Chemistry, School of Medicine, Juntendo University, Inba, Chiba 270-1695, Japan

A thermophilic, obligately chemolithoautotrophic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, assimilates carbon dioxide via the reductive tricarboxylic acid cycle. A gene encoding a ferredoxin involved in this cycle as an electron donor (HtFd1) was cloned and sequenced. Interestingly, another ferredoxin gene (encoding HtFd2) was found in tandem with the HtFd1 gene. These two ferredoxin genes overlapped by four bp, and transcriptional analysis revealed that they are co-transcribed as an operon. The deduced amino acid sequences of HtFd1 and HtFd2 were 42.9% identical and each contained four cysteine residues that serve as probable ligands to an iron-sulfur cluster. Spectroscopic analyses of the purified recombinant ferredoxins heterologously expressed in Escherichia coli indicated that each ferredoxin contains a single [4Fe4S]2+/1+ cluster.

Minoru Ujita,1,2, Yosuke Katsuno,1 Ichiro Kawachi,1 Yoshinori Ueno,3 Yutaka Banno,3 Hiroshi Fujii,3 and Akira Hara1,2

1Laboratory of Biological Chemistry, Department of Applied Biological Chemistry, Faculty of Agriculture, Meijo University, Tempaku-ku, Nagoya 468-8502, Japan
2Agricultural High-Tech Research Center, Meijo University, Tempaku-ku, Nagoya 468-8502, Japan
3Institute of Genetic Resources, Kyushu University, Higashi-ku, Fukuoka 812-8581, Japan

The silkworm Bombyx mori 30-kDa lipoproteins (6G1 and 19G1), major components of the hemolymph, were shown to bind to glucans. 6G1 apolipoprotein was expressed as a fusion protein with glutathione S-transferase in Escherichia coli and assayed for its binding activity. The purified recombinant 6G1 apolipoprotein specifically bound to ²-glucan, but not to chitin, mannan, peptidoglycan, or oligosaccharide chains on glycoproteins. The ²-glucan binding of the recombinant 6G1 was inhibited by laminaribiose and laminarin, a soluble glucan, but not by lipopolysaccharide or insect blood sugar, trehalose at physiological concentration. Furthermore, the recombinant 6G1 was shown to participate in the activation of prophenoloxidase cascade and to interfere with hyphal growth of the entomopathogenic fungus Paecilomyces tenuipes, injected into pupae of B. mori. These results suggest that 6G1 lipoprotein plays a role in the protection of B. mori against invading fungi.

Toshiaki Handa,1 Kohji Yamaguchi,1 Yoshikatsu Sono,2 and Kazunaga Yazawa1

1Laboratory of Nutraceuticals and Functional Foods Science, Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato-ku, Tokyo 108-8477, Japan
2Technical Sourcing International, Inc., 2-8-2 Nihonbashi Honcho, Chuo-ku, Tokyo 103-8448, Japan

It was found that fenugreek seed extract reduced the body weight gain induced by a high-fat diet in obese mice. The extract decreased plasma triglyceride gain induced by oil administration. The major component of the extract, 4-hydroxyisoleucine, also decreased plasma triglyceride gain. Consequently, fenugreek seed extract is expected to prevent the obesity induced by a high-fat diet.

Meiyu Xu, Yoshimasa Sugiura, Satoshi Nagaoka, and Yoshihiro Kanamaru

Department of Food Science, Faculty of Applied Biological Science, Gifu University, Gifu 501-1193, Japan

Monomeric 14-kDa bovine ±-lactalbumin was purified with a preparation of lower molecular weight whey protein concentrate from Holstein cow normal milk followed by size exclusion chromatography. The protein showed a stimulatory rather than an inhibitory effect on the proliferation of a cultured IEC-6 cell line from the rat small intestine. But incubation in 30% trifluoroethanol/acetate buffer (pH 5.5) at 37 °C for 5 d in a slowly rotating test tube rendered it highly cytotoxic with concomitant appearance of SDS-stable 20- and 30-kDa forms of ±-lactalbumin on electrophoresis. Furthermore, ±-lactalbumin obtained by a one-step purification procedure by affinity chromatography on an anti-±-lactalbumin antibody column from the lower molecular weight whey protein concentrate, which had been found to contain several SDS-stable higher Mr forms of ±-lactalbumin, exhibited potent inhibitory activity on IEC-6 cell growth. These results indicate the involvement of SDS-stable higher Mr forms of bovine normal milk ±-lactalbumin in inducing cell death on the intestinal IEC-6 cell line.

Frank Nunes, Désirée Spiering, Marc Wolf, Alexandra Wendler, Ralph Pirow, and Rüdiger J. Paul

Institut für Zoophysiologie, Westfälische Wilhelms-Universität Münster, Hindenburgplatz 55, D-48143 Münster, Germany

A genomic fragment containing the hemoglobin gene dmhb4 of Daphnia magna was cloned and its nucleotide sequence determined. Concerning induction under hypoxic conditions, dmhb4 was found to be expressed constitutively with similar mRNA quantities in D. magna bred in either normoxic or hypoxic medium. Southern blot analysis revealed at least six hemoglobin-like sequences in the genome of Daphnia magna.

Yuko Baba,* Atsushi Shimonaka,* Jinichiro Koga, Koichiro Murashima, Hidetoshi Kubota, and Toshiaki Kono

Food and Health R&D Laboratories, Meiji Seika Kaisha, Ltd., 5-3-1 Chiyoda, Sakado-shi, Saitama 350-0289, Japan

A new endoglucanase, designated BCE1, produced by Beltraniella portoricensis, was purified from the culture supernatant. The N-terminal amino acid sequence suggests that BCE1 belongs to family 45 glycoside hydrolase (family 45 endoglucanase). The molecular mass of BCE1 was found to be 40 kDa by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDSPAGE). The optimum pH for the carboxymethyl cellulase (CMCase) activity of BCE1 was 4.5, and the optimum temperature was 55 °C. Among family 45 endoglucanases, RCE1 and RCE2 from Rhizopus oryzae, PCE1 from Phycomyces nitens, and EGL3 and EGL4 from Humicola grisea, BCE1 was most resistant to anionic surfactant and oxidizing agent. These results indicate that BCE1 might prove to be a useful enzyme in the detergent industry.

Masaki Abe,1, Tetsuya Imai,1 Naoki Ishii,1 Makio Usui,1 Toru Okuda,2 and Toshikazu Oki2

1Naruto Research Center, Otsuka Chemical Co., Ltd., 615 Hanamen, Satoura-cho, Naruto, Tokushima 772-8601, Japan
2Mycology and Metabolic Diversity Research Institute, Tamagawa University Research Institute, 6-1-1 Tamagawa-Gakuen, Machida, Tokyo 194-8610, Japan

By a screening program searching for new pesticides from fungal sources, an insecticidal compound was isolated from Penicillium citrinum F 1539. The compound, named quinolactacide, was novel and showed 88% mortality against green peach aphids (Myzus persicae) at 250 ppm. Its structure was determined by spectroscopic techniques.

Takatoshi Ohkuri,1 Taiji Imoto,2 and Tadashi Ueda1

1Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
2Faculty of Engineering, Sojo University, 4-22-1 Ikeda, Kumamoto 860-0082, Japan

We characterized a refolded hen lysozyme variant containing only two SS-bonds, C64C80 and C76C94 (4CAHEL). From CD spectra and its activity, it was found that the refolded 4CAHEL has a structural topology analogous to wild-type lysozyme (WTHEL). Moreover, the refolded 4CAHEL showed no thermal transition, indicating that it had a character like a molten globule.

Mayumi Kifusa, Hideo Fukuhara, Takeshi Hayashi, and Makoto Kimura

Laboratory of Biochemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka 812-8581, Japan

Ribonuclease P (RNase P) is a ribonucleoprotein complex involved in the processing of the 5 leader sequence of precursor tRNA (pre-tRNA). RNase P in the hyperthermophilic archaeon Pyrococcus horikoshii OT3 consists of RNA and five protein subunits (Ph1481p, Ph1496p, Ph1601p, Ph1771p, and Ph1877p). In vivo interactions among five protein subunits of RNase P in P. horikoshii OT3 were examined using a yeast two-hybrid system. The analysis indicates that proteins Ph1481p and Ph1601p interact strongly with Ph1877p and Ph1771p respectively, whereas Ph1481p interacts moderately with Ph1601p. In contrast, no interaction was detected between Ph1496p and the other four proteins. Co-immunoprecipitation analysis confirmed the interactions obtained by yeast two-hybrid assay.

Matsujiro Ishibashi, Sayaka Yamashita, and Masao Tokunaga

Applied and Molecular Microbiology, Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan

A halophilic alkaline phosphatase was highly purified (about 510-fold with about 21% yield) from a moderate halophile, Halomonas sp. 593. The N-terminal 35 amino acid sequence of this enzyme was found to be more acidic than those previously isolated from Vibrio spp., and this enzyme was partially resistant to SDS. Several enzymatic properties demonstrated that it showed higher halophilicity than those enzymes from Vibrio spp.

Hiroshi Terano,1, Kaori Takahashi,1 and Yutaka Sakakibara2

1Faculty of Life Sciences, Toyo University, Itakura-machi, Ora-gun, Gunma 374-0193, Japan
2Suntory Limited, Shimamoto-cho, Mishima-gun, Osaka 618-8503, Japan

The germination behaviors of spores of Alicyclobacillus acidoterrestris, which has been considered to be a causative microorganism of flat sour type spoilage in acidic beverages, were investigated. The spores of A. acidoterrestris showed efficient germination and outgrowth after heat activation (80 °C, 20 min) in Potato dextrose medium (pH 4.0). Further, the spores treated with heat activation germinated in McIlvaine buffer (pH 4.0) in the presence of a germinative substance (L-alanine) and commercial fruit juices, although not in phosphate buffer (pH 7.0). Heat activation was necessary for germination. The spores of A. acidoterrestris, which easily survived the heat treatment in acidic conditions, lost their resistance to heat during germination. Our results suggest that the models obtained from spore germination of A. acidoterrestris might be beneficial to determine adequate thermal process in preventing the growth of potential spoilage bacteria in acidic beverages.