Bioscience, Biotechnology, and Biochemistry
Online ISSN : 1347-6947
Print ISSN : 0916-8451
Volume 66 , Issue 1
Showing 1-37 articles out of 37 articles from the selected issue
Review
  • Shin-ichiro EJIRI
    2002 Volume 66 Issue 1 Pages 1-21
    Published: 2002
    Released: May 01, 2003
    JOURNALS FREE ACCESS
      Eukaryotic polypeptide elongation factor EF-1 is not only a major translational factor, but also one of the most important multifunctional (moonlighting) proteins.

      EF-1 consists of four different subunits collectively termed EF-1αββ′γ and EF-1αβγδ in plants and animals, respectively. EF-1α•GTP catalyzes the binding of aminoacyl-tRNA to the A-site of the ribosome. EF-1ββ′γ (EF-1β and EF-1β′), catalyzes GDP/GTP exchange on EF-1α•GDP to regenerate EF-1α•GTP. EF-1γ has recently been shown to have glutathione S-transferase activity.

      EF-2 catalyzes the translocation of peptidyl-tRNA from the A-site to the P-site on the ribosome. Recently, molecular mimicry among tRNA, elongation factors, releasing factor (RF), and ribosome recycling factor (RRF) has been demonstrated and greatly improved our understanding of the mechanism of translation.

      Moreover, eukaryotic elongation factors have been shown to be concerned or likely to be concerned in various important cellular processes or serious diseases, including translational control, signal transduction, cytoskeletal organization, apoptosis, adult atopic dermatitis, oncogenic transformation, nutrition, and nuclear processes such as RNA synthesis and mitosis.

      This article aims to overview the recent advances in protein biosynthesis, concentrating on the moonlighting functions of EF-1.
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Organic Chemistry Regular Papers
  • Kensaku TAKARA, Daigo MATSUI, Koji WADA, Toshio ICHIBA, Yoko NAKASONE
    2002 Volume 66 Issue 1 Pages 29-35
    Published: 2002
    Released: May 01, 2003
    JOURNALS FREE ACCESS
      Nine compounds, 3-hydroxy-4,5-dimethoxyphenyl-β-D-glucopyranoside (1),β-D-fructfuranosyl-α-D-(6-vanilloyl)-glucopyranoside (2), β-D-fructfuranosyl-α-D-(6-syringyl)-glucopyranoside (3), 3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-2-[4-(3-hydroxy-1-(E)-propenyl)- 2-methoxyphenoxy]propyl-β-D-glucopyranoside(4), 3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-2-[4-(3- hydroxy-1-(E)-propenyl)-2,6-dimethoxyphenoxy] propyl-β-D-glucopyranoside (5), dehydrodiconiferyl alcohol-9′-β-D-glucopyranoside (6), 4-[ethane-2-[3-(4-hydroxy-3-methoxyphenyl)-2-propen]oxy]-2,6-dimethoxyphenyl-β-D-glucopyranoside (7), 4-[ethane-2-[3-(4-hydroxy-3-methoxyphenyl)-2-propen]oxy]-2-methoxyphenyl-β-D-glucopyranoside (8), and 3-hydroxy-1-(4-hydroxy-3,5-dimethoxyphenyl)-2-[4-(3-hydroxy-1-(E)-propenyl)-2,6-dimethoxyphenoxy]propyl-β-D-glucopyranoside (9), were isolated from Kokuto non- centrifuged cane sugar. Their structures were elucidated by spectroscopic evidence, mainly based on the NMR technique. Among them, seven new glycosides were identified. The 2-deoxyribose oxidation method was used to measure their antioxidative activity. All of these compounds showed antioxidative activities.
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  • Yoshihiro SOHTOME, Takashi TOKUNAGA, Katsuhiro UEDA, Shosuke YAMAMURA, ...
    2002 Volume 66 Issue 1 Pages 51-56
    Published: 2002
    Released: May 01, 2003
    JOURNALS FREE ACCESS
      Potassium (2R,3R)-2,3,4-trihydroxy-2-methylbutanoate (1) was identified as a leaf-closing substance in the nyctinastic plant, Leucaena leucocephala. Compound 1 showed strong leaf-closing activity toward L. leucocephala and was not effective against other nyctinastic plants. The potassium ion was indispensable for the bioactivity of 1. Compound 1 gradually lost its bioactivity because of the exchange of the counter cation during isolation. A leaf-opening substance was also observed in the same plant.
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  • Hirokazu TARUI, Naoki MORI, Ritsuo NISHIDA, Kimiko OKABE, Yasumasa KUW ...
    2002 Volume 66 Issue 1 Pages 135-140
    Published: 2002
    Released: May 01, 2003
    JOURNALS FREE ACCESS
      A new monoterpene lactone from the acarid mite, Schwiebea araujoae, was elucidated without its isolation by GC/FT-IR and GC/MS analyses to be 3-(4-methyl-3-pentenyl)-2(5H)-furanone (1) and tentatively named as α,α-acariolide. The structure of 1 was identified by its synthesis from α-bromo-γ-butyrolactone via 4 reaction steps. The synthesized compound gave the same GC/MS and GC/FT-IR spectra as those of the natural product.

    The other monoterpene lactone was likewise elucidated from the unidentified Rhizoglyphus mite to be 4-(4-methyl-3-pentenyl)-2(5H)-furanone (2) and named as α,β-acariolide; it was also identified by its synthesis in 5 reaction steps from the same butyrolactone as the starting material. GC/MS and GC/FT-IR spectra of the preparation were identical to those of the natural product.
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Organic Chemistry Notes
Biochemistry & Molecular Biology Regular Papers
  • Hiroshi NAGAI, Kyoko TAKUWA-KURODA, Masahiro NAKAO, Naomasa OSHIRO, Se ...
    2002 Volume 66 Issue 1 Pages 97-102
    Published: 2002
    Released: May 01, 2003
    JOURNALS FREE ACCESS
      The deadly box jellyfish (Sea Wasp, Habu-kurage in Japanese) Chiropsalmus quadrigatus Haeckel (Cubozoa) is distributed widely in the tropical Pacific region. In Japan, three fatal cases due to stings from this species have been reported officially. We successfully isolated C. quadrigatus toxin-A (CqTX-A, 44 kDa), a major proteinaceous toxin, for the first time, from the nematocysts of C. quadrigatus. CqTX-A showed lethal toxicity to crayfish when administered via intraperitoneal injection (LD50=80 μg/kg) and hemolytic activity toward 0.8% sheep red blood cells (ED50= 160 ng/ml). Furthermore, we sequenced the cDNA encoding CqTX-A. The deduced amino acid sequence of CqTX-A (462 amino acids) showed 25.2% and 21.6% sequence similarity to Carybdea rastoni toxins (CrTXs) and Carybdea alata toxin-A (CrTX-A), respectively, which are Cubozoan jellyfish toxins.
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  • Kenta IWASAKI, Shingo KIKUKAWA, Shunsuke KAWAMURA, Yoshiaki KOUZUMA, I ...
    2002 Volume 66 Issue 1 Pages 103-109
    Published: 2002
    Released: May 01, 2003
    JOURNALS FREE ACCESS
      Ribosomal protein L5, a 5S rRNA binding protein in the large subunit, is composed of a five-stranded antiparallel β-sheet and four α-helices, and folds in a way that is topologically similar to the ribonucleprotein (RNP) domain [Nakashima et al., RNA 7, 692–701, 2001]. The crystal structure of ribosomal protein L5 (BstL5) from Bacillus stearothermophilus suggests that a concave surface formed by an anti-parallel β-sheet and long loop structures are strongly involved in 5S rRNA binding. To identify amino acid residues responsible for 5S rRNA binding, we made use of Ala-scanning mutagenesis of evolutionarily conserved amino acids occurred at β-strands and loop structures in BstL5. The mutation of Lys33 at the β1-strand caused a significant reduction in 5S rRNA binding. In addition, the Arg92, Phe122, and Glu134 mutations on the β2-strand, the α3-β4 loop, and the β4-β5 loop, respectively, resulted in a moderate decrease in the 5S rRNA binding affinity. In contrast, mutation of the conserved residue Pro65 at the β2-strand had little effect on the 5S rRNA binding activity. These results, taken together with previous results, identified Lys33, Asn37, Gln63, and Thr90 on the β-sheet structure, and Phe77 at the β2-β3 loop as critical residues for the 5S rRNA binding. The contribution of these amino acids to 5S rRNA binding was further quantitatively evaluated by surface plasmon resonance (SPR) analysis by the use of BIAcore. The results showed that the amino acids on the β-sheet structure are required to decrease the dissociation rate constant for the BstL5-5S rRNA complex, while those on the loops are to increase the association rate constant for the BstL5-5S rRNA interaction.
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  • Il KWON, Keisuke EKINO, Takuji OKA, Masatoshi GOTO, Kensuke FURUKAWA
    2002 Volume 66 Issue 1 Pages 110-116
    Published: 2002
    Released: May 01, 2003
    JOURNALS FREE ACCESS
      Endoglucanase I (EGI) from Trichoderma viride HK-75 catalyzes not only hydrolysis but also transglycosylation reactions of cellooligosaccharides. In order to characterize the important amino acid residues in transglycosylation of EGI, three Tyr, one Leu, and two Glu residues of EGI were replaced by Trp or Asp. The seven resulting EGI, except for L200W, had reduced activities toward carboxymethyl-cellulose compared to that of wild type EGI. The results from the mutations in the catalytic residues of E196 and E201 indicate that the space just around the catalytic residues is not directly related to the transglycosylation reactions of EGI. Analyses of the enzymes with mutations in the substrate-binding residues showed that Y146, Y170, and L200 of EGI are closely involved in the mode of transglycosylation and that several amino acid residues within the active site are involved in the transglycosylation reaction of EGI.
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  • Kazuaki OKUNO, Masayuki YABUTA, Kouji KAWANISHI, Kazuhiro OHSUYE, Tosh ...
    2002 Volume 66 Issue 1 Pages 127-134
    Published: 2002
    Released: May 01, 2003
    JOURNALS FREE ACCESS
      Though OmpT has been reported to mainly cleave the peptide bond between consecutive basic amino acids, we identified more precise substrate specificity by using a series of modified substrates, termed PRX fusion proteins, consisting of 184 residues. The cleavage site of the substrate PRR was Arg140-Arg141 and the modified substrates PRX substituted all 19 natural amino acids at the P1´ site instead of Arg141. OmpT under denaturing conditions (in the presence of 4 M urea) cleaved not only between two consecutive basic amino acids but also at the carboxyl side of Arg140 except for the Arg140-Asp141, -Glu141, and -Pro141 pairs. In addition to Arg140 at the P1 site, similar results were obtained when Lys140 was substituted into the P1 site. In the absence of urea, an aspartic acid residue at the P1´ site was unfavorable for OmpT cleavage of synthetic decapeptides, the enzyme showed a preference for a dibasic site.
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  • Sompong THAMMASIRIRAK, Takao TORIKATA, Kazutoshi TAKAMI, Koichi MURATA ...
    2002 Volume 66 Issue 1 Pages 147-156
    Published: 2002
    Released: May 01, 2003
    JOURNALS FREE ACCESS
      The complete amino acid sequence of cassowary (Casuarius casuarius) goose type lysozyme was analyzed by direct protein sequencing of peptides obtained by cleavage with trypsin, V8 protease, chymotrypsin, lysyl endopeptidase, and cyanogen bromide. The N-terminal residue of the enzyme was deduced to be a pyroglutamate group by analysis with a LC/MS/MS system equipped with the oMALDI ionization source, and then confirmed by a glutamate aminopeptidase enzyme. The blocked N-terminal is the first reported in this enzyme group. The positions of disulfide bonds in this enzyme were chemically identified as Cys4-Cys60 and Cys18-Cys29. Cassowary lysozyme was proved to consist of 185 amino acid residues and had a molecular mass of 20408 Da calculated from the amino acid sequence. The amino acid sequence of cassowary lysozyme compared to that of reported G-type lysozymes had identities of 90%, 83%, and 81%, for ostrich, goose, and black swan lysozymes, respectively. The amino acid substitutions at PyroGlu1, Glu19, Gly40, Asp82, Thr102, Thr156, and Asn167 were newly detected in this enzyme group. The substituted amino acids that might contribute to substrate binding were found at subsite B (Asn122Ser, Phe123Met). The amino acid sequences that formed three α-helices and three β-sheets were completely conserved. The disulfide bond locations and catalytic amino acid were also strictly conserved. The conservation of the three α-helices structures and the location of disulfide bonds were considered to be important for the formation of the hydrophobic core structure of the catalytic site and for maintaining a similar three-dimensional structure in this enzyme group.
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  • Tomomitsu HATAKEYAMA, Noriaki MATSUO, Kouhei SHIBA, Shoichi NISHINOHAR ...
    2002 Volume 66 Issue 1 Pages 157-163
    Published: 2002
    Released: May 01, 2003
    JOURNALS FREE ACCESS
      CEL-I is one of the Ca2+-dependent lectins that has been isolated from the sea cucumber, Cucumaria echinata. This protein is composed of two identical subunits held by a single disulfide bond. The complete amino acid sequence of CEL-I was determined by sequencing the peptides produced by proteolytic fragmentation of S-pyridylethylated CEL-I. A subunit of CEL-I is composed of 140 amino acid residues. Two intrachain (Cys3-Cys14 and Cys31-Cys135) and one interchain (Cys36) disulfide bonds were also identified from an analysis of the cystine-containing peptides obtained from the intact protein. The similarity between the sequence of CEL-I and that of other C-type lectins was low, while the C-terminal region, including the putative Ca2+ and carbohydrate-binding sites, was relatively well conserved. When the carbohydrate-binding activity was examined by a solid-phase microplate assay, CEL-I showed much higher affinity for N-acetyl-D-galactosamine than for other galactose-related carbohydrates. The association constant of CEL-I for p-nitrophenyl N-acetyl-β-D-galactosaminide (NP-GalNAc) was determined to be 2.3×104 M1, and the maximum number of bound NP-GalNAc was estimated to be 1.6 by an equilibrium dialysis experiment.
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Biochemistry & Molecular Biology Notes
Food & Nutrition Science Regular Papers
  • Hsi-Huai LAI, Gow-Chin YEN
    2002 Volume 66 Issue 1 Pages 22-28
    Published: 2002
    Released: May 01, 2003
    JOURNALS FREE ACCESS
      Peroxynitrite, a potent oxidant formed in vivo from the reaction of nitric oxide with superoxide, can mediate low-density liprotein (LDL) oxidation which is thought to increase the risk of atherosclerosis. This study investigates the inhibitory effect of the isoflavones, genistein and daidzein, together with their glycosidic forms, genistin and daidzin, on the peroxynitrite-mediated LDL oxidation and nitration of tyrosine. Genistein and daidzein were observed to dose-dependently inhibit peroxynitrite-mediated LDL oxidation, while their glucoside conjugates showed less activity. Moreover, all the isoflavones used in this study were found to be potent peroxynitrite scavengers, preventing the nitration of tyrosine. The ability of the isoflavones at 50 μM to decrease the tyrosine nitration induced by peroxynitrite (1 mM) was in the ratios of genistein (49%), daidzein (40%), daidzin (41%) and genistin (42%) when compared to the control (tyrosine incubated only with peroxynitrite). These results suggest that an intake of isoflavones could contribute to protecting against cardiovascular diseases and chronic inflammatory diseases.
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  • Hiroyuki UKEDA, Yuko HASEGAWA, Yumi HARADA, Masayoshi SAWAMURA
    2002 Volume 66 Issue 1 Pages 36-43
    Published: 2002
    Released: May 01, 2003
    JOURNALS FREE ACCESS
      Glycolaldehyde, an intermediate of the Maillard reaction, and fructose, which is mainly derived from the polyol pathway, rapidly inactivate human Cu,Zn-superoxide dismutase (SOD) at the physiological concentration. We employed this inactivation with these carbonyl compounds as a model glycation reaction to investigate whether carnosine and its related compounds could protect the enzyme from inactivation. Of eight derivatives examined, histidine, Gly-His, carnosine and Ala-His inhibited the inactivation of the enzyme by fructose (p<0.001), and Gly-His, Ala-His, anserine, carnosine, and homocarnosine exhibited a marked protective effect against the inactivation by glycolaldehyde (p<0.001). The carnosine-related compounds that showed this highly protective effect against the inactivation by glycolaldehyde had high reactivity with glycolaldehyde and high scavenging activity toward the hydroxyl radical as common properties. On the other hand, the carnosine-related compounds that had a protective effect against the inactivation by fructose showed significant hydroxyl radical-scavenging ability. These results indicate that carnosine and such related compounds as Gly-His and Ala-His are effective anti-glycating agents for human Cu,Zn-SOD and that the effectiveness is based not only on high reactivity with carbonyl compounds but also on hydroxyl radical scavenging activity.
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  • Satoe YAGINUMA, Takuo SHIRAISHI, Hiroaki OHYA, Kiharu IGARASHI
    2002 Volume 66 Issue 1 Pages 65-72
    Published: 2002
    Released: May 01, 2003
    JOURNALS FREE ACCESS
      To assess effects of the environmental stress on polyphenol compounds (polyphenols) in plants, the polyphenol contents were investigated in the seedlings of safflower (Carthamus tinctrius L.) and cucumber (Cucumis sativus L.) grown under three types of growth conditions: control; light stress, irradiated with strong light in the visible wavelength range; and light/water stress, irradiated with strong visible light with a limited water supply. The total polyphenol contents and the amounts of the major polyphenols, especially luteolin 7-O- glucoside in safflower cotyledons, and luteolin 7-O-glucoside and luteolin in safflower foliage leaves, increased in response to both stresses. The polyphenol increasing effect of light/water stress was clearly observed in safflower compared to cucumber, suggesting that plants that are resistant to these stresses can accumulate substantial amounts of polyphenols compared to the plants which respond weakly to the stresses.
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  • Eiichiro ISHIDO, Shuji ADACHI, Yasumasa MINEMOTO, Ryuichi MATSUNO
    2002 Volume 66 Issue 1 Pages 73-77
    Published: 2002
    Released: May 01, 2003
    JOURNALS FREE ACCESS
      Two polyunsaturated fatty acids (PUFAs) or their esters were mixed, and their oxidation processes were measured at 65°C and ca. 0% relative humidity. Except when a PUFA ester was mixed with a free PUFA, the oxidation of the less-oxidative PUFA was promoted as its content in the mixture decreased, while the oxidation of the more-oxidative PUFA was delayed with a decrease in its content. A kinetic model is proposed whereby a PUFA acts as the diluent for another PUFA, and the oxidation rate of the PUFA is proportional to the product of the unoxidized PUFA concentration and the sum of the concentrations of the oxidized PUFA and the other oxidized PUFA. This model well expressed the oxidation processes of the PUFAs in their mixed system.
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  • Naoyuki NISHIZAWA, Daiki SATO, Yoshiaki ITO, Takashi NAGASAWA, Yasuko ...
    2002 Volume 66 Issue 1 Pages 92-96
    Published: 2002
    Released: May 01, 2003
    JOURNALS FREE ACCESS
      In this paper, we examined the effects of dietary protein from proso millet on liver injury induced by D-galactosamine or carbon tetrachloride in rats using serum enzyme activities as indices. D-galactosamine- induced elevations of serum activities of aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase were significantly suppressed by feeding the diet containing 20% protein of proso millet for 14 days as compared with those of rats fed a 20% casein diet, but not in the case of carbon tetrachloride. The results showed that proso millet protein is effective at lower dietary protein levels than that of dietary gluten reported previously. Therefore, the findings reported here may suggest that proso millet protein is considered to be another preventive food for liver injury.
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  • Yuichi OISHI, Zhengwei FU, Yoshinari OHNUKI, Hisanori KATO, Tadashi NO ...
    2002 Volume 66 Issue 1 Pages 117-126
    Published: 2002
    Released: May 01, 2003
    JOURNALS FREE ACCESS
      Protein malnutrition affects the status of dermal collagen, the major structural protein in the skin. However, the molecular mechanism underlying the alteration of collagen fibers in the skin by protein deficiency remains unknown. In the present study, the effect of dietary protein deprivation on collagen metabolism was studied by analyzing the status of the synthesis and degradation of collagen in the dorsal skin of rats. Feeding on a protein-free diet for 8 days caused a dramatic decrease in both types I and III tropocollagen with a concomitant decrease in their mRNA levels, with type III collagen being more severely affected. The active form of collagenase was significantly decreased by protein deprivation, whereas the latent form was not affected. The mRNA levels of collagenase and its inhibitors (TIMP-1 and 2) were also decreased by protein deprivation. These results suggest that both the synthesis and degradation of types I and III collagen were affected by protein deficiency.
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  • Masako IWAMOTO, Misaki KONO, Daisuke KAWAMOTO, Hiroko TOMOYORI, Masao ...
    2002 Volume 66 Issue 1 Pages 141-146
    Published: 2002
    Released: May 01, 2003
    JOURNALS FREE ACCESS
      Walnut oil (WO) is a good source of α-linolenic acid. We compared the effects of WO and high-linoleic safflower oil (HLSO) on the serum lipid level and atherosclerosis development in male and female apolipoprotein (apo) E-deficient mice. The WO diet resulted in a higher level of serum cholesterol than with HLSO. Female mice fed on the WO diet had a greater lesion area in the aortic root than did those on the HLSO diet. There was no diet-dependent difference in the level of cholesterol and its oxidation products in the abdominal and thoracic aorta. These results suggest that the unpleasant effects of the WO diet on apo E-deficient mice may be attributable to α-linolenic acid.
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Food & Nutrition Science Notes
  • Masaki KAMAKURA, Makoto FUKUSHIMA
    2002 Volume 66 Issue 1 Pages 175-178
    Published: 2002
    Released: May 01, 2003
    JOURNALS FREE ACCESS
      We have previously shown that 57-kDa protein in royal jelly (RJ) was specifically degraded in proportion to both storage temperature and storage period, and we suggested that it could be useful as a marker of freshness of RJ (Kamakura, M., Fukuda, T., Fukushima, M. and Yonekura, M., Biosci. Biotechnol. Biochem., 65, 277–284 (2001).). Here, we investigated the effects of various proteinase inhibitors on proteinase activity in RJ and on the specific degradation of 57-kDa protein during storage. Ethylenediaminetetraacetic acid (EDTA), but not other inhibitors, inhibited the proteinase activity in RJ, and dose-dependently suppressed storage-dependent degradation of 57-kDa protein. These results suggest that EDTA inhibits a specific proteinase activity in RJ, thereby suppressing the degradation of 57-kDa protein during storage at high temperature.
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  • Hiroshi MATSUZAKI, Junko OHDACHI, Masaaki FUCHIGAMI, Ritsuko MASUYAMA, ...
    2002 Volume 66 Issue 1 Pages 192-194
    Published: 2002
    Released: May 01, 2003
    JOURNALS FREE ACCESS
      To discover the details of the effects of magnesium (Mg) deficiency on kidney function, the course of changes in N-acetyl-β-D-glucosaminidase (NAG) activity in the urine and in urinary albumin excretion were examined in rats fed a Mg-deficient diet. NAG activity in the urine and urinary albumin excretion in rats fed the Mg-deficient diet significantly increased from 7 d until the end of the feeding period. We suggest that Mg-deficient diet rapidly induces kidney function insufficiency.
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  • Dong HAN, Masatomo MATSUNO, Yoshihide IKEUCHI, Atsushi SUZUKI
    2002 Volume 66 Issue 1 Pages 202-205
    Published: 2002
    Released: May 01, 2003
    JOURNALS FREE ACCESS
      The sera of bovine gamma globulin (BGG) positive beef allergic patients were used in this study in order to investigate changes in IgE-specific binding activity with regard to beef extract altered by heat or high-pressure treatment. In inhibition-ELISA, the sample treated at 60°C did not show any significant changes in the antigenicity of BGG, but the sample treated at 100°C showed a decrease of the antigenicity. In the case of the treatment with heating at 100°C, heat-coagulation occurred in the beef extract. The resulting supernatant and precipitate of the sample by centrifugation were analyzed by immunoblotting. Only the fraction of precipitate showed a specific binding activity with the sera. Based on this result, it was speculated that the persistent antigenicity found even after the treatment at 100°C in inhibition-ELISA remained principally in the heat-coagulated fraction, which indicated the importance of the method of handling the heat-coagulation in heat treatment. High-pressure treatments (200 MPa– 600 MPa) of beef extract did not show any significant changes in the binding with the sera.
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  • Mitsuru HIROTA, Alka MENTA, Keiko YONEYAMA, Naofumi KITABATAKE
    2002 Volume 66 Issue 1 Pages 206-210
    Published: 2002
    Released: May 01, 2003
    JOURNALS FREE ACCESS
      Citrinin is one of the mycotoxins produced by Penicillium citrinum. We examined the decomposition products after heating citrinin in water at 140°C and isolated a major product, citrinin H2 (3-(3,5-dihydroxy-2-methylphenyl)-2-formyloxy-butane). Citrinin H2 did not show significant cytotoxicity to HeLa cells up to a concentration of 200 μg/ml (% cytotoxicity: 39%) in 63 h of incubation, but citrinin showed severe toxicity at a concentration of 25 μg/ml (% cytotoxicity: 73%). HPLC analysis of citrinin after heating under various conditions indicates that citrinin H2 is mainly yielded from citrinin.
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Microbiology & Fermentation Technology Regular Papers
  • Takahisa MIYAMOTO, Md. Abu SAYED, Ryo SASAHARA, Kouji SUKIMOTO, Akiko ...
    2002 Volume 66 Issue 1 Pages 44-50
    Published: 2002
    Released: May 01, 2003
    JOURNALS FREE ACCESS
      The pbp3 gene encoding PBP3 of Bacillus cereus was cloned and sequenced. For this purpose, PBP3 was first purified from B. cereus ts-4, and N-terminal amino acid sequences of the peptides obtained from the protease digests of the protein were analyzed. The B. cereus ts-4 pbp3 gene consisted of an open reading frame of 1,986 bp encoding 662 amino acid residues with a calculated molecular mass of 73,044 Da. The active site-motifs SXXK, SXN, and KTG are present at the positions 393, 452, and 590, respectively, in the deduced amino acid sequence. The pbp3 structural gene was ligated into the pET17×b expression vector and pET-pbp3 was constructed. A protein was produced by the cells of E. coli carrying pET-pbp3. The produced protein migrated at about 75 kDa in SDS-polyacrylamide gel and strongly reacted with biotinylated ampicillin.
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  • Teruhide SUGISAWA, Tatsuo HOSHINO
    2002 Volume 66 Issue 1 Pages 57-64
    Published: 2002
    Released: May 01, 2003
    JOURNALS FREE ACCESS
      D-Sorbitol dehydrogenase was solubilized from the membrane fraction of Gluconobacter suboxydans IFO 3255 with Triton X-100 in the presence of D-sorbitol. Purification of the enzyme was done by fractionation with column chromatographies of DEAE-Cellulose, DEAE-Sepharose, hydroxylapatite, and Sephacryl HR300 in the presence of Triton X-100.

      The molecular mass of the enzyme was 800 kDa, consisting of homologous subunits of 80 kDa. The optimum pH of the enzyme activity was 6.0, and the optimum temperature was 30°C.

      Western blot analysis suggested the occurrence of the enzyme in all the Gluconobacter strains tested.
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  • Moriya OHKUMA, Satoko NODA, Yuichi HONGOH, Toshiaki KUDO
    2002 Volume 66 Issue 1 Pages 78-84
    Published: 2002
    Released: May 01, 2003
    JOURNALS FREE ACCESS
      Phylogenetically diverse clones of the partial 16S rDNA (ca. 850 bp) of bacteria belonging to the bacteroides subgroup of the cytophaga-flavobacter-bacteroides phylum were collected from the symbiotic microbial communities in the guts of six termite species without cultivation. Combined with the sequences reported previously, a total of thirty phylotypes of the subgroup were identified and classified into five phylogenetic clusters. One that was comprised of the phylotypes from a single termite species was related to the genus Rikenella. Two were clustered each with some cultured strains, genera of which have not been clearly defined yet. The remaining two clusters had no culturable representatives, suggesting the presence of yet- uncultivated genera within the termite guts. From these sequence data, we designed a specific primer for the bacteroides subgroup, which was successful in the terminal-restriction fragment length polymorphism analysis to detect the phylotypes of the subgroup in the termite gut.
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  • Hideshi YANASE, Keishi MORIYA, Norihiko MUKAI, Yasushi KAWATA, Kenji O ...
    2002 Volume 66 Issue 1 Pages 85-91
    Published: 2002
    Released: May 01, 2003
    JOURNALS FREE ACCESS
      The overexpression of fdm, which encodes the formaldehyde dismutase from Pseudomonas putida F61, resulted in the formation of inclusion bodies made up of aggregated enzyme, leaving little activity in the soluble fraction of the transformant cells. On the other hand, coexpression of groESL along with fdm facilitated in vivo solubilization of the enzyme protein in its active form. When coexpressed with groESL, formaldehyde dismutase purified from E. coli had the same crystalline form (i.e., a regular octahedron) as the native enzyme, and like the native enzyme, it bound 1 mol of NAD(H) and 2 mol of zinc in each subunit.
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Microbiology & Fermentation Technology Notes
Microbiology & Fermentation Technology Preliminary Communication
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