Bioscience, Biotechnology, and Biochemistry
Online ISSN : 1347-6947
Print ISSN : 0916-8451
Volume 64 , Issue 10
Showing 1-44 articles out of 44 articles from the selected issue
Organic Chemistry Regular Papers
Organic Chemistry Notes
Biochemistry & Molecular Biology Regular Papers
  • Noriaki SHIOTA, Susumu KODAMA, Hideyuki INUI, Hideo OHKAWA
    2000 Volume 64 Issue 10 Pages 2025-2033
    Published: 2000
    Released: February 03, 2005
    JOURNALS FREE ACCESS
      Among 11 isoforms of the human cytochrome P450 enzymes metabolizing xenobiotics, CYP 1A1 and CYP 1A2 were major P450 species in the metabolism of the herbicides chlortoluron and atrazine in a yeast expression system. CYP1A2 was more active in the metabolism of both herbicides than CYP1A1. The fused enzymes of CYP1A1 and CYP1A2 with yeast NADPH-cytochrome P450 oxidoreductase were functionally active in the microsomal fraction of the yeast Saccharomyces cerevisiae and showed increased specific activity towards 7-ethoxyresorufin as compared to CYP1A1 and CYP1A2 alone. Then, both fused enzymes were each expressed in the microsomes of tobacco (Nicotiana tabacum cv. Samsun NN) plants. The transgenic plants expressing the CYP1A2 fusion enzyme had higher resistance to the herbicide chlortoluron than the plants expressing the CYP1A1 fusion enzyme did. The transgenic plants expressing the CYP1A2 fused enzyme metabolized chlortoluron to a larger extent to its non-phytotoxic metabolites through N-demethylation and ring-methyl hydroxylation as compared to the plants expressing the CYP1A1 fused enzyme. Thus, the possibility of increasing the herbicide resistance in the transgenic plants by the selection of P450 species and the fusion with P450 reductase is discussed.
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  • Takafumi HIRATA, Mitsuaki WATANABE, Shinji MIURA, Katsushi IJICHI, Mas ...
    2000 Volume 64 Issue 10 Pages 2047-2052
    Published: 2000
    Released: February 03, 2005
    JOURNALS FREE ACCESS
      The high rate of glycolysis despite the presence of oxygen and mitochondria in tumor cells implies an important role for this process in cell division. The rate of glycolysis is assumed to be dependent on the cellular concentration of fructose 2,6-bisphosphate, the concentration of which in turn depends on a bifunctional enzyme and the ratio of this enzyme’s 6-phosphofructo-2-kinase versus its fructose 2,6-bisphosphatase activities. To prove the hypothesis that inhibition of glycolysis in tumor cells by 6-phosphofructo-2-kinase inhibitors would cause inhibition of tumor cell proliferation, ten N-bromoacetylethanolamine phosphate analogues were designed, synthesized, and tested. They were screened for their activities against various human tumor cell lines to study the effects of inhibition of glycolysis on cell proliferation. The relationship between the structure of these compounds and their inhibitory activity on cell proliferation was also discussed. It was found that the activity of N-(2-methoxyethyl)-bromoacetamide, N-(2-ethoxyethyl)-bromoacetamide, and N-(3-methoxypropyl)-bromoacetamide was comparable to that of the positive control AraC. These three inhibitors showed in vivo anticancer effects in P388 transplant BDF1 mice.
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  • Kazuko OHGI, Sanae KUDO, Mitsuaki TAKEUCHI, Masanori IWAMA, Masachika ...
    2000 Volume 64 Issue 10 Pages 2068-2074
    Published: 2000
    Released: February 03, 2005
    JOURNALS FREE ACCESS
      To investigate the role of Phe101, a component of a base recognition site (B2 site) of a base-nonspecific RNase Rh from Rhizopus niveus, we prepared several enzymes mutated at this position, F101W, F101L, F101I, F101A, F101Q, F101R, and F101K, and their enzymatic activities towards RNA, 16 dinucleoside phosphates, and 2′, 3′-cyclic pyrimidine nucleotides were measured. Enzymatic activity toward RNA of F101W, F101L, and F101I were about 7, 20, and 3.8% of the native enzyme, respectively, and those of the other mutants were less than 1% of the RNase Rh. Similar results were also obtained with GpG as substrate. Thus, it was concluded that Phe101 is a very important residue as a component of the B2 site of RNase Rh, and its role could be replaced by Leu, then Trp and Ile, though in less effectively. The results suggested that some kind of interaction between B2 base and the side chain of amino acid residue at the 101th position, such as π/π or CH/π interaction is very important for the enzymatic activity of RNase Rh. The mutation of Phe101 markedly affected the enzymatic activity toward dinucleoside phosphates and polymer substrates, but only moderately the rate of hydrolysis of cyclic nucleotides, indicating the presence of secondary effect of the mutation on B1 site.
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  • Tsunashi KAMO, Nobuhiro HIRAI, Mitsuya TSUDA, Daie FUJIOKA, Hajime OHI ...
    2000 Volume 64 Issue 10 Pages 2089-2098
    Published: 2000
    Released: February 03, 2005
    JOURNALS FREE ACCESS
      Changes in the phytoalexin content in unripe fruit of banana, Musa acuminata, were analyzed after various treatments. The results show that level of hydroxyanigorufone started to increase 1-2 day after either wounding or inoculation with conidia of Colletotrichum musae. Inoculation followed by wounding induced the formation of many other phenylphenalenones. The accumulation of hydroxyanigorufone decreased, after its transient maximum, on ripening by exposure of the wounded fruit to ethylene. The level of production of hydroxyanigorufone in ripe fruit treated by wounding and/or by inoculation was much lower than that in unripe fruit. 2-Aminooxyacetic acid, an inhibitor of phenylalanine ammonia-lyase (PAL), inhibited the accumulation of hydroxyanigorufone in wounded fruit, and the PAL activity increased after wounding and ethylene treatment, respectively. Feeding experiments with [1-13C] and [2-13C]cinnamic acids, and [2-13C]malonate show that two molecules of cinnamic acid and one of malonate were incorporated into each molecule of hydroxyanigorufone. The phytoalexins isolated from fruit to which deuterated hydroxyanigorufone and irenolone had been administered revealed that 2-(4′-hydroxyphenyl)-1,8-naphthalic anhydride was biosynthesized from hydroxyanigorufone rather than from irenolone.
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  • Hiroo YONEZAWA, Hideki KAIZUKA, Tetsuya UCHIKOBA, Kazunari ARIMA, Mako ...
    2000 Volume 64 Issue 10 Pages 2104-2108
    Published: 2000
    Released: February 03, 2005
    JOURNALS FREE ACCESS
      The substrate specificity of cucumisin [EC 3.4.21.25] was identified by the use of the synthetic peptide substrates Leum-Pro-Glu-Ala-Leun (m=0-4, n=0-3). Neither Pro-Glu-Ala-Leu (m=0) nor Leu-Pro-Glu-Ala (n=0) was cleaved by cucumisin, however other analogus peptides were cleaved between Glu-Ala. The hydrolysis rates of Leum-Pro-Glu-Ala-Leu increased with the increase of m=1 to 2 and 3, but was however, essentially same with the increase of m=3 to 4. Similarly, the hydrolysis rates of Leu-Leu-Pro-Glu-Ala-Leun increased with the increase of n=0 to 1 and 2, but was essentially same with the increase of n=2 to 3. Then, it was concluded that cucumisin has a S5-S3′ subsite length. In order to identify the substrate specificity at P1 position, Leu-Leu-Pro-X-Ala-Leu (X; Gly, Ala, Val, Leu, Ile, Pro, Asp, Glu, Lys, Arg, Asn, Gln, Phe, Tyr, Ser, Thr, Met, Trp, His) were synthesized and digested by cucumisin. Cucumisin showed broad specificity at the P1 position. However, cucumisin did not cleave the C-terminal side of Gly, Ile, Pro, and preferred Leu, Asn, Gln, Thr, and Met, especially Met. Moreover, the substrates, Leu-Leu-Pro-Glu-Y-Leu (Y; Gly, Ala, Ser, Leu, Val, Glu, Lys, Phe) were synthesized and digested by cucumisin. Cucumisin did not cleave the N-terminal side of Val but preferred Gly, Ser, Ala, and Lys especially Ser. The specificity of cucumisin for naturally occurring peptides does not agree strictly with the specificity obtained by synthetic peptides at the P1 or P1′ position alone, but it becomes clear that the most of the cleavage sites on naturally occurring peptides by cucumisin contain suitable amino acid residues at P1 and (or) P1′ positions. Moreover, cucumisin prefers Pro than Leu at P2 position, indicating that the specificity at P2 position differs from that of papain.
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  • Yoshinobu KIMURA, Chiyoko MIYAGI, Mariko KIMURA, Teruhiko NITODA, Nobu ...
    2000 Volume 64 Issue 10 Pages 2109-2120
    Published: 2000
    Released: February 03, 2005
    JOURNALS FREE ACCESS
      The structures of N-glycans of total glycoproteins in royal jelly have been explored to clarify whether antigenic N-glycans occur in the famous health food. The structural feature of N-glycans linked to glycoproteins in royal jelly was first characterized by immunoblotting with an antiserum against plant complex type N-glycan and lectin-blotting with Con A and WGA. For the detail structural analysis of such N-glycans, the pyridylaminated (PA-) N-glycans were prepared from hydrazinolysates of total glycoproteins in royal jelly and each PA-sugar chain was purified by reverse-phase HPLC and size-fractionation HPLC. Each structure of the PA-sugar chains purified was identified by the combination of two-dimensional PA-sugar chain mapping, ESI-MS and MS/MS analyses, sequential exoglycosidase digestions, and 500 MHz 1H-NMR spectrometry.
      The immunoblotting and lectinblotting analyses preliminarily suggested the absence of antigenic N-glycan bearing β1-2 xylosyl and/or α1-3 fucosyl residue(s) and occurrence of β1-4GlcNAc residue in the insect glycoproteins.
      The detailed structural analysis of N-glycans of total royal jelly glycoproteins revealed that the antigenic N-glycans do not occur but the typical high mannose-type structure (Man9~4GlcNAc2) occupies 71.6% of total N-glycan, biantennary-type structures (GlcNAc2Man3GlcNAc2) 8.4%, and hybrid type structure (GlcNAc1Man4GlcNAc2) 3.0%. Although the complete structures of the remaining 17% N-glycans; C4, (HexNAc3Hex3HexNAc2: 3.0%), D2 (HexNAc2Hex5HexNAc2: 4.5%), and D3 (HexNAc3Hex4HexNAc2: 9.5%) are still obscure so far, ESI-MS analysis, exoglycosidase digestions by two kinds of β-N-acetylglucosaminidase, and WGA blotting suggested that these N-glycans might bear a β1-4 linkage N-acetylglucosaminyl residue.
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  • Ken-ichi NISHIYAMA, Hirofumi SUZUKI, Hajime TOKUDA
    2000 Volume 64 Issue 10 Pages 2121-2127
    Published: 2000
    Released: February 03, 2005
    JOURNALS FREE ACCESS
      SecE is an essential component of the protein translocation machinery of Escherichia coli and has three transmembrane stretches. An N-terminal region (SecE-N) encompassing the first two transmembrane stretches is dispensable for protein translocation but a SecE derivative (SecE-C) lacking this region is very unstable. We show here that FtsH, the AAA (ATPases associated with diverse cellular activities) family protease, causes the instability of SecE-C. SecE-C became stable when SecE-N was co-expressed. Deletion of the N-terminal region of SecE also rendered the SecE-SecY-SecG complex unstable. In spite of these alterations, the N-terminal region of SecE had little stimulatory effect on protein translocation in vivo or in vitro.
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  • Kiyotaka HITOMI, Yoshihisa YAMAGIWA, Koji IKURA, Kiyofumi YAMANISHI, M ...
    2000 Volume 64 Issue 10 Pages 2128-2137
    Published: 2000
    Released: February 03, 2005
    JOURNALS FREE ACCESS
      Transglutaminase 1 (TGase 1) is required for the formation of a cornified envelope in stratified squamous epithelia. Recombinant human TGase 1 expressed in baculovirus-infected cells was purified in a soluble form at the molecular mass of 92 kDa. Recombinant TGase 1 was susceptible to limited proteolysis by both μ- and m-calpains, the calcium-dependent intracellular cysteine proteases. Although the proteolysis did not induce the elevation of the specific enzyme activity of TGase 1, the requirement of calcium ion in the enzymatic reaction was reduced. Furthermore, the effects of GTP, nitric oxide, and sphingosylphosphocholine, known as regulatory factors for tissue-type isozyme (TGase 2), on the enzymatic activity of TGase 1 were investigated.
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  • Kyoko TAKAHASHI, Toshifumi YUUKI, Toshiro TAKAI, Chisei RA, Ko OKUMURA ...
    2000 Volume 64 Issue 10 Pages 2138-2144
    Published: 2000
    Released: February 03, 2005
    JOURNALS FREE ACCESS
      Binding of allergen-IgE complexes to the high affinity IgE receptor (FcεRI) on mast cells and basophils leads to the release of various mediaters such as histamine. Fab fragments prepared by the papain digestion of humanized antibody against human FcεRI inhibited the release of histamine from human basophils. Here we established an expression system to directly produce Fab fragments of the humanized anti-human FcεRI antibody in methylotropic yeast, P. pastoris. Fab fragments were efficiently secreted into the medium at a concentration of 10-40 mg/L using a signal sequence from the P. pastoris phosphatase gene. They were consisted of disulfide-linked light and heavy chains correctly starting from the first amino acid residues by proper cleavage of the signal peptides. The obtained Fab fragments inhibited the binding between IgE and FcεΡΙ as efficiently as the counterpart prepared by papain digestion of the whole antibody.
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  • Mitsuaki KOMODA, Shin-ichi INOMATA, Akihiko ONO, Hayaki WATANABE, Tets ...
    2000 Volume 64 Issue 10 Pages 2145-2151
    Published: 2000
    Released: February 03, 2005
    JOURNALS FREE ACCESS
      Virgin females of M. confusa, A. peponis, and C. eriosoma secrete (Z)-7-dodecenyl acetate as a common main pheromone component. Their pheromone titers decreased after decapitation, and increased in the decapitated females after injection of a synthetic hormone, pheromone biosynthetic activating neuropeptide (PBAN) of Bombyx mori. In addition, an extract of brain-subesophageal ganglion complexes of each Plusiinae species activated pheromone biosynthesis in decapitated females of not only the corresponding species, but also that of Mamestra brassicae. These results indicate that pheromone biosynthesis of the three Plusiinae species is also controlled by a PBAN-like substance. However, the Plusiinae females exceptionally contained remarkable amounts of the pheromone even 1 day after decapitation. Since it has been reported that pheromones completely disappear at least 1 day after decapitation in females of many other lepdidoptran species including B. mori and M. brassicae, a different mechanism is likely regarding the regulation of the studied Plusiinae pheromone biosynthesis. Furthermore, an incorporation experiment with a labeled pheromone precursor, D9-(Z)-7-dodecenoic acid, showed that moderate biosynthesis still proceeded in the pheromone glands of M. confusa females 1 day after decapitation, providing an evidence why complete disappearance of the pheromone was not observed in the females which otherwise lacked a source of the pheromonotropic neuropeptide.
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  • Lina ZHANG, Mei ZHANG, Qi ZHOU, Jinghua CHEN, Fanbo ZENG
    2000 Volume 64 Issue 10 Pages 2172-2178
    Published: 2000
    Released: February 03, 2005
    JOURNALS FREE ACCESS
      Four fractions of a water-insoluble α-(1→3)-D-glucan GL extracted from fruiting bodies of Ganoderma lucidum were dissolved in 0.25 M LiCl/DMSO, and then reacted with sulfur trioxide-pyridine complex at 80°C to synthesize a series of water-soluble sulfated derivatives S-GL. The degree of substitution of DS was measured by using IR infrared spectra, elemental analysis, and 13C NMR to be 1.2-1.6 in the non-selective sulfation. Weight-average molecular weight Mw and intrinsic viscosity [η] of the sulfated derivatives S-GL were measured by multi-angle laser light scattering and viscometry. The Mw value (2.4×104) of sulfated glucan S-GL-1 was much lower than that (44.5×104) of original α-(1→3)-D-glucan GL-1. The Mark-Houwink equation and average value of characteristic ratio C for the S-GL in 0.2 M NaCl aqueous solution at 25°C were found to be: [η]=1.32×10-3Mw1.06 (cm3 g-1) and 16, respectively, in the Mw range from 1.1×104 to 2.4×104. It indicated that the sulfated derivatives of the α-(1→3)-D-glucan in the aqueous solution behave as an expanded chain, owing to intramolecular hydrogen bonding or interaction between charge groups. Interestingly, two sulfated derivatives synthesized from the α-(1→3)-D-glucan and curdlan, a β-(1→3)-D-glucan, all had significant higher antitumor activity against Ehrlich ascites carcinoma (EAC) than the originals. The effect of expanded chains of the sulfated glucan in the aqueous solution on the improvement of the antitumor activity could not be negligible.
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  • Takaaki YANAI, Michikatsu SATO
    2000 Volume 64 Issue 10 Pages 2179-2185
    Published: 2000
    Released: February 03, 2005
    JOURNALS FREE ACCESS
      An intracellular α-L-rhamnosidase from Pichia angusta X349 was purified to homogeneity through four chromatographic steps. The α-L-rhamnosidase appeared to be a monomeric protein with a molecular mass of 90 kDa. The enzyme had an isoelectric point at 4.9, and was optimally active at pH 6.0 and at around 40°C. The Ki for L-rhamnose inhibition was 25 mM. The enzyme was inhibited by Cu2+, Hg2+, and p-chloromercuribenzoate. The α-L-rhamnosidase was highly specific for α-L-rhamnopyranoside and liberated rhamnose from naringin, rutin, hesperidin, and 3-quercitrin. The α-L-rhamnosidase was active at the ethanol concentrations of wine. It efficiently released monoterpenols, such as linalool and geraniol, from an aroma precursor extracted from Muscat grape juice.
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  • Kiyotaka OSHIKAWA, Ken-ichi AOKI, Yuuki YOSHINO, Shigeyuki TERADA
    2000 Volume 64 Issue 10 Pages 2186-2192
    Published: 2000
    Released: February 03, 2005
    JOURNALS FREE ACCESS
      A peptidase was purified from seeds of Canavalia ensiformis by extraction with water, ammonium sulfate precipitation, and successive chromatographies on DEAE-Toyopearl 650M, butyl-Toyopearl 650M, and G-3000 SW columns. The enzyme has an apparent molecular weight of 41,000. Activity is maximal at pH 9 and 60°C. The enzyme hydrolyzed synthetic substrates at Arg-X and Lys-X bonds more rapidly than bovine trypsin did, and did not cleave protein or ester substrates. The enzyme was inhibited by alkylamines and several serine protease inhibitors such as diisopropylfluorophosphate, chymostatin, leupeptin, and benzamidine. Cysteine protease-, metalloprotease-, and proteinous trypsin inhibitors were ineffective. Inhibition by alkylamines was dependent on length of the alkyl chains. From the substrate specificity and susceptibility to chemicals, the enzyme is a unique peptidase with trypsin-like specificity.
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  • Toshiya KAWAMURA, Kiyofumi KIKUNO, Tatsuya ODA, Tsuyoshi MURAMATSU
    2000 Volume 64 Issue 10 Pages 2193-2200
    Published: 2000
    Released: February 03, 2005
    JOURNALS FREE ACCESS
      Dipeptidyl carboxypeptidase (DCP) from the polychaete Neanthes virens, resembling mammalian angiotensin I converting enzyme (ACE), was studied to discover some of its molecular and inhibitory properties, as the first evidence of these in a marine invertebrate. Amino acid and carbohydrate contents were analyzed. The N-terminal amino acid sequence of N. virens DCP was (NH2)D-E-E-A-G-R-Q-W-L-A-E-Y-D-L-R-N-Q-T-V-L-. Peptide maps of N. virens DCP from lysyl endopeptidase digestion were different from rabbit p-ACE. The far-ultraviolet circular dichroic spectra of N. virens DCP indicated that the secondary structure of this enzyme seemed to be an α-helical structure and was similar to that of rabbit p-ACE, but the near-ultraviolet circular dichroic spectra of N. virens DCP indicated that the aromatic amino acid residue circumambience of this enzyme was different from rabbit p-ACE. The effects of several reagents for chemical modification of amino acids on the activity of N. virens DCP were tested. Arg, Tyr, Glu, and/or Asp, His, Trp, and Met caused loss of the activity. In addition, the IC50 and Ki values for a well-known ACE inhibitor, Val-Tyr, which was a competitive inhibitor of N. virens DCP, were 263 and 20 μM, respectively. These results suggested that N. virens DCP is different from mammalian ACE in the molecular and inhibitory properties, although the same substrate specificity was demonstrated in a previous paper.
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Biochemistry & Molecular Biology Notes
  • Qin ZENG, Andrew J. WOOD
    2000 Volume 64 Issue 10 Pages 2221-2224
    Published: 2000
    Released: February 03, 2005
    JOURNALS FREE ACCESS
      A Tortula ruralis cDNA, Rpl15, encoding a predicted polypeptide with significant similarity to the large-subunit ribosomal protein L15 (RPL15) was isolated from a desiccated gametophyte cDNA library, and is the first L15 homologue identified from a bryophyte. The deduced 203 amino acid polypeptide is approximately 24.1 kDa, with a predicted pI of 11.1, and shares extensive identity with rat RPL15 deduced polypeptide (>57%). RNA blot hybridization using total RNA demonstrated that Rpl15 is constitutively expressed in moss gametophytes during a desiccation-rehydration cycle and that Rpl15 mRNA transcripts are maintained in desiccated gametophytes as conserved mRNAs.
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  • Mijoung LEE, Sanghyun NA, Deahyun JEON, Hyungha KIM, Yunjaie CHOI, Myu ...
    2000 Volume 64 Issue 10 Pages 2225-2228
    Published: 2000
    Released: February 03, 2005
    JOURNALS FREE ACCESS
      To understand the molecular mechanisms of mammary gland involution, an involution-induced clone was identified from a cDNA library of mouse mammary gland by differential screening. Characterization of a clone by sequencing and northern analysis showed that expression of the osteopontin gene was induced during involution of mouse mammary gland. But induction of the osteopontin gene was not observed in apoptotic HC11 mammary epithelial cells under serum starvation. In HCll cells, dexamethasone treatment from the seeding stage showed five-fold induction of osteopontin gene expression, but the expression was not changed when dexamethasone was added to confluent cells.
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  • Norihiro FUTAMURA, Hiroshi KOUCHI, Kenji SHINOHARA
    2000 Volume 64 Issue 10 Pages 2232-2235
    Published: 2000
    Released: February 03, 2005
    JOURNALS FREE ACCESS
      We did in situ hybridization to identify the sites at which DnaJ homologs (SGJ1 and SGJ3) and Hsp70 were expressed in male and female flowers of the Japanese willow, Salix gilgiana Seemen. Transcripts of SGJ1, SGJ3, and Hsp70 were detected in microspores and in the tapetal layers of immature anthers, in the inner integument of ovules, and in the funicular epidermal layers of ovaries. Transcripts of SGJ3 and Hsp70 were also detected in the nectaries of male and female flowers, in which transcripts of SGJ1 were undetectable.
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  • Hyungha KIM, Yountae CHI, Kichul CHUNG, Kwanghyun KIM, Yunjaie CHOI, M ...
    2000 Volume 64 Issue 10 Pages 2240-2242
    Published: 2000
    Released: February 03, 2005
    JOURNALS FREE ACCESS
      To understand the molecular mechanism for intramuscular fat deposition, the expression of the obese gene was examined in response to fasting. Food deprivation for 48 h induced a decrease in the level of obese mRNA in pooled adipose tissues (abdominal, perirenal, subcutaneous, intermuscular and intramuscular). The expression of obese mRNA was examined for individual adipose tissue from several fat depots. It was highly expressed in perirenal adipose tissue, but fasting did not affect its expression level in this tissue. Moderate levels were detected in subcutaneous and intermuscular adipose tissues, and a fasting-induced decrease in obese mRNA was apparent in these tissues. The expression level of the obese gene in intramuscular adipose tissue was very low and did not respond to fasting.
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  • Hisashi ASHIDA, Kenji YAMAMOTO, Hidehiko KUMAGAI
    2000 Volume 64 Issue 10 Pages 2266-2268
    Published: 2000
    Released: February 03, 2005
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      The effects of bovine fetuin O-glycans on its trypsin inhibitory activity were examined. De-sialylated (asialo-) and de-O-glycosylated fetuin were prepared from native fetuin using Arthrobacter neuraminidase and the mixture of it and Bacillus endo-α-N-acetylgalactosaminidase, respectively. De-sialylation and de-O-glycosylation from fetuin were confirmed with SDS-PAGE followed by western blotting using anti-human Thomsen-Friedenreich antigen (T antigen) antibody which recognizes O-linked galactosyl β1,3 N-acetylgalactosamine (Galβ1→3GalNAc). Native fetuin completely inhibited the trypsin activity at about a 1:1 molar ratio. In contrast, the trypsin inhibitory activity of asialo- and de-O-glycosylated fetuin decreased about a half and one-third of that of native fetuin, respectively.
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  • Sung-Soo PARK, Toshihisa SUMI, Hideki OHBA, Osamu NAKAMURA, Makoto KIM ...
    2000 Volume 64 Issue 10 Pages 2272-2275
    Published: 2000
    Released: February 03, 2005
    JOURNALS FREE ACCESS
      Three major serine proteinase inhibitors (SBI-1, -2, and -3) were purified from the seeds of white sword bean (Canavalia gladiata) by FPLC and reversed-phase HPLC. The sequences of these inhibitors were established by automatic Edman degradation and TOF-mass spectrometry. SBI-1, -2, and -3 consisted of 72, 73, and 75 amino acid residues, with molecular masses of 7806.5, 7919.8, and 8163.4, respectively. The sequences of SBI-1 and -2 coincided with those of CLT I and II [Terada et al. (1994) Biosci. Biotech. Biochem., 58, 376-379] except only N- or C-terminal amino acid residues. Analysis of the amino acid sequences showed that the active sites of the inhibitors contained a Lys21-Ser22 against trypsin and Leu48-Ser49 against chymotrypsin, respectively. Further, it became apparent that about seven disulfide bonds were present. These results suggest that sword bean inhibitors are members of the Bowman-Birk proteinase inhibitor family.
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Biochemistry & Molecular Biology Preliminary Communication
Food & Nutrition Science Regular Papers
  • Ulala FUNADA, Masahiro WADA, Tetsunori KAWATA, Nobuo TANAKA, Tadahiro ...
    2000 Volume 64 Issue 10 Pages 2053-2058
    Published: 2000
    Released: February 03, 2005
    JOURNALS FREE ACCESS
      Cyanocobalamin, deoxyadenosylcobalamin and methylcobalamin were separately administered to mice sensitized with the ovalbumin antigen to investigate the allergic response. The serum IgE and pulmonary histamine concentrations were significantly lower in all cobalamin (Cbl)-administered groups. The production of interleukin (IL)-2 and IL-4 in splenocytes was also lower in all Cbl-administered groups. These results show that Cbls were effective in mitigating allergic reactions and IL-2 production. CD3+CD28+ (CD28 is an accessory molecule related to IL-2 production) and CD4+CD28+ in splenocytes were higher in all the Cbl-administered groups. However, CD3+CD28-, CD4+CD28- and CD5+CD25- (CD25: IL-2 Rα/p55) were lower in the Cbl-administered groups. In addition, Cbl specifically inhibited the cellular phosphorylation of tyrosine induced by ovalbumin sensitization. These results indicate that the signal in a cell by CD 28 was restrained by Cbl. We infer that Cbl administration significantly reduced the IL-2 concentration, and secondarily the IL-4, IgE and histamine concentrations.
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  • Masao YAMASAKI, Koji KISHIHARA, Keiko MANSHO, Yuuya OGINO, Masaaki KAS ...
    2000 Volume 64 Issue 10 Pages 2159-2164
    Published: 2000
    Released: February 03, 2005
    JOURNALS FREE ACCESS
      The dietary effects of conjugated linoleic acid (CLA) on Ig production of Sprague-Dawley rats were examined at various doses such as 0 (control), 0.05, 0.10, 0.25, and 0.50%. CLA increased IgG and IgM production of spleen lymphocytes in a dose-dependent manner, and these levels reached a plateau at 0.25%. IgA production was not detected in the control group, while it was detected in all CLA-fed groups and IgA productivity of spleen lymphocytes increased in a dose-dependent manner at the doses from 0.05 to 0.50%. Dietary CLA did not affect serum Ig levels. The major fatty acid composition of spleen lymphocytes was not affected by dietary CLA, which itself was hardly incorporated into the cells. In an in vitro assay, the effects of CLA and its oxidative derivatives, furan type fatty acids, on Ig productivity were also examined. As a result, 100 μM CLA suppressed Ig production of spleen lymphocytes and the degree was as follows IgA>IgG>IgM. Each CLA isomer and the furan type fatty acids also suppressed Ig production but the degree was weaker than the mixture of CLA isomers. In this result, dietary CLA increased Ig productivity of spleen lymphocytes in vivo.
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  • Yukiko YAMAMOTO, Ikuyo SOGAWA, Akiko NISHINA, Shigeru SAEKI, Naoki ICH ...
    2000 Volume 64 Issue 10 Pages 2165-2171
    Published: 2000
    Released: February 03, 2005
    JOURNALS FREE ACCESS
      This study describes the effects of mixtures of xanthan gum and galactomannan, guar gum, or locust bean gum, on the lipids in plasma and liver in non-diabetic and diabetic rats. Non-diabetic rats were fed cholesterol-free diets with 3% guar gum, locust bean gum, or xanthan gum (3G, 3L, and 3X), or a mixture of xanthan gum and guar gum or locust bean gum (1:2, w/w) (2G1X, 2L1X) for 2 weeks. Rats fed diets not containing these polysaccharides were used as controls. The total cholesterol in plasma and the triacylglycerol in liver were significantly lowered in rats fed the 2G1X diet. The 3G, 3X, 3L, and 2L1X diets showed no significant effect on the total cholesterol and triacylglycerol in plasma and liver. In the streptozotocin-induced (STZ) diabetic rats, the total cholesterol in plasma was lowered in rats fed the 3G, 3X or 2G1X diet for 4 weeks, and the 2G1X diet was more effective than the 3G and 3X diets. The triacylglycerol in plasma in STZ diabetic rats was also significantly lowered by the 2G1X diet. These results showed that a mixture of xanthan gum and guar gum has an improved hypolipidemic effect on non-diabetic and STZ diabetic rats. The effects of the 2G1X diet on the diabetic symptoms in STZ diabetic rats, suppression of food and water intakes, decrease in glucose in urine, and lowering of plasma glucose, were also observed.
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  • Yasushi KAWAI, Boku SAITOH, Osamu TAKAHASHI, Haruki KITAZAWA, Tadao SA ...
    2000 Volume 64 Issue 10 Pages 2201-2208
    Published: 2000
    Released: February 03, 2005
    JOURNALS FREE ACCESS
      A broad-spectral bacteriocin, named gassericin T, produced by Lactobacillus gasseri SBT 2055 (from human feces) was isolated to homogeneity from the culture supernatant by hydrophobic chromatography. By SDS-PAGE and in situ activity assay, the purified gassericin T migrated as a single band with bacteriocin activity and molecular size of 5,400. A 2.9-kbp HindIII-HindIII fragment of chromosome DNA was hybridized with the oligonucleotide probe designed from the partial N-terminal amino acid sequence of gassericin T and was cloned. Six ORFs including the structural gene of gassericin T were deduced by computer analysis and the data bases. The structural gene of gassericin T (gatA) was identified as the fourth ORF, which encoded a protein composed of 75 amino acids that included the GG motif of the cleavage site. Chemical sequencing analysis of the complete amino acid sequence showed that gassericin T (57 amino acids) had a disulfide bond in the molecule and no modified amino acid residues, making it a class II bacteriocin. The gassericin T had 60% sequence similarity to mature LafA (57 amino acids, lactacin F, bacteriocins produced by L. johnsonii VPI11088), and the sequences around the processing site and C-terminal area were well conserved. The fifth ORF was designated as gatX, encoded as a peptide composed of 65 amino acids containing the GG motif of the putative cleavage site, however mature GatX and its antibacterial activity were not detected in the culture supernatant. GatX has higher similarity with LafX than with lactobin A (50 amino acids) belonging to the first lactacin F-family. These results indicated that gassericin T belongs to the hydrophobic class II bacteriocins and the most vicinal lactacin F-family.
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Food & Nutrition Science Notes
Microbiology & Fermentation Technology Regular Papers
  • Kohtaro KIRIMURA, Masashi YODA, Hideki SHIMIZU, Shuji SUGANO, Makoto M ...
    2000 Volume 64 Issue 10 Pages 2034-2039
    Published: 2000
    Released: February 03, 2005
    JOURNALS FREE ACCESS
      In Aspergillus niger, a cyanide (CN)- and antimycin A-insensitive and salicylhydroxamic acid (SHAM)-sensitive respiratory pathway exists besides the cytochrome pathway and is catalyzed by the alternative oxidase (AOX). In this study, A. niger WU-2223L, a citric acid-producing strain, was cultivated in a medium containing 120 g/l of glucose, which is the concentration usually needed for citric acid production, and the effects of 2% (v/v) methanol, an inducer of citric acid, 2 μM antimycin A, and 1 mM SHAM on AOX activities and citric acid production were investigated. The AOX activity, measured as duroquinol oxidase, was localized in the purified mitochondria regardless of the presence of any additives. When WU-2223L was cultivated with antimycin A or methanol, both citric acid production and citric acid productivity, shown as the ratio of production per mycelial dry weight, increased with the increase of both the activity of AOX and the rate of CN-insensitive and SHAM-sensitive respiration. On the other hand, when WU-2223L was cultivated with SHAM, an inhibitor of AOX, the CN-insensitive and SHAM-sensitive respiration was not detected and the citric acid production and the productivity drastically decreased, although mycelial growth was not affected. These results clearly indicated that the CN-insensitive and SHAM-sensitive respiration catalyzed by AOX, localized in the mitochondria, contributed to citric acid production by A. niger.
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  • Kuniho NAKATA, Takayoshi KOBAYASHI, Yasuyuki TAKIGUCHI, Tatsuaki YAMAG ...
    2000 Volume 64 Issue 10 Pages 2040-2046
    Published: 2000
    Released: February 03, 2005
    JOURNALS FREE ACCESS
      A polysaccharide flocculant of Klebsiella pneumoniae H12 has been suggested to mediate microbe-plant interactions with the aid of Ca2+ [K. Nakata et al., Biosci. Biotechnol. Biochem., 64, 459-465, 2000]. Here, two-way regulation of polysaccharide-mediated interactions between K. pneumoniae and Raphanus sativus was studied using organic acids. Namely, 10 mM equivalents of organic acids promoted production of the polysaccharide by the bacterium, but inhibited flocculation of bacterial cells by the polysaccharide. These phenomena were counterbalanced by equi-molar equivalents of Ca2+, suggesting competition for Ca2+ between the carboxylic residues of the polysaccharide and those of the aliphatic acids. By electron microscopy observations, bacterial cell aggregates were sparsely distributed over the main roots and root hairs, had various sizes, and seemed to tightly adhere to root tissues. Their shapes seemed to be distorted and abundant in cavities. In brief, these microscopical observations may be explained by a two-way regulation system of bacterial adhesion to a plant by organic acids.
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  • Takao INOUE, Kazuo KAMIMURA, Tsuyoshi SUGIO
    2000 Volume 64 Issue 10 Pages 2059-2067
    Published: 2000
    Released: February 03, 2005
    JOURNALS FREE ACCESS
      An iron-oxidizing bacterium strain, OKM-9, isolated from mud obtained from the bottom of a pond, Minamikata Ohike, in Okayama prefecture, Japan, grew well in an FeSO4•7H2O (3%)-medium (pH 2.5) with 0.03% yeast extract. However, the strain could not grow either in an FeSO4•7H2O (3%)-medium without yeast extract or in a yeast extract (0.03%)-medium (pH 2.5) without Fe2+. The strain did not use elemental sulfur as an energy source and did not have the activity to fix carbon dioxide. Strain OKM-9 could grow in an FeSO4•7H2O (3%)-medium with twenty different L-amino acids instead of yeast extract. Incorporation of [U-14C] glutamic acid into the cells was dependent on the energy produced by the oxidation of Fe2+. Strain OKM-9 did not grow heterotrophically using amino acids and hexoses as a sole energy and carbon source. The results that strain OKM-9 absolutely required ferrous iron (Fe2+) as a sole energy source and yeast extract or L-amino acids as a carbon source for growth strongly suggest that the strain is a mixotrophic iron-oxidizing bacterium. Strain OKM-9 was a Gram-negative and rod-shaped bacterium (0.4-0.6×1.6-2.2 μm) and the mean G+C content of the DNA of the bacterium was 59.6 mol%. The optimum temperature and pH for growth were 30°C and 2.1, respectively. However, the strain could not grow at temperatures above 45°C. Iron-oxidizing activities of strain OKM-9 measured with intact cells and the plasma membrane were 14.3 and 5.7 μl O2 uptake/mg protein/min, respectively. The pyridine ferrohemochromes prepared from the plasma membrane of this strain showed absorption peaks characteristic of α-bands of heme a and b, but not heme c, at 587 and 557 nm, respectively. The results suggest that the cytochromes composing an iron-oxidation system of strain OKM-9 are different from those of the well-known mesophilic iron-oxidizing bacteria Thiobacillus ferrooxidans and Leptospirillum ferrooxidans.
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  • Shin-ichi ETOH, Kayoko ASAMURA, Azumi OBU, Kenji SONOMOTO, Ayaaki ISHI ...
    2000 Volume 64 Issue 10 Pages 2083-2088
    Published: 2000
    Released: February 03, 2005
    JOURNALS FREE ACCESS
      Natural rubber serum powder, which is a by-product obtained in the production of latex rubber, has a strong growth-stimulating activity for Bifidobacterium bifidum JCM 1254. The retained fraction obtained by ultrafiltration (molecular weight cutoff 1000) showed a growth- stimulating activity in a dose-dependent manner on B12 assay medium with ammonium sulfate. One of the growth stimulators was purified from the retained fraction by acetone precipitation, solid-phase extraction with a hydrophobic pretreatment column, and multi-stage reversed-phase HPLC. An increase of 53-fold in the specific activity, and a recovery of 1.3% were obtained. The amino acid composition and N-terminal sequence analysis of this growth stimulator provided the structure of Ala-Thr-Pro-Glu-Lys-Glu-Glu-Pro-Thr-Ala. The molecular mass was 1075 by MALDI-TOF MS analysis. These results showed that this growth stimulator was a decapeptide with the sequence shown above. This is the first report that clarified the structure of an active peptide for the growth of Bifidobacterium.
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  • Kohji ISHIHARA, Hitomi YAMAGUCHI, Noriko ADACHI, Hiroki HAMADA, Nobuyo ...
    2000 Volume 64 Issue 10 Pages 2099-2103
    Published: 2000
    Released: February 03, 2005
    JOURNALS FREE ACCESS
      The stereocontrolled reduction of α- and β-keto esters using micro green algae was accomplished by a combination of the cultivation method and the introduction of an additive. The reduction of ethyl pyruvate and ethyl benzoylformate by the photoautotrophically cultivated Chlorella sorokiniana gave the corresponding alcohol in high e.e. (>99% e.e. (S) and >99% e.e. (R), respectively). In the presence of glucose as an additive, the reduction of ethyl 3-methyl-2-oxobutanoate by the heterotrophically cultivated C. sorokiniana afforded the corresponding (R)-alcohol. On the other hand, the reduction in the presence of ethyl propionate gave the (S)-alcohol. Ethyl 2-methyl-3-oxobutanoate was reduced in the presence of glycerol by the photoautotrophically cultivated C. sorokiniana or the heterotrophically cultivated C. sorokiniana to the corresponding syn-(2R,3S)-hydroxy ester with high diastereo- and enantiomeric excess (e.e.). Some additives altered the stereochemical course in the reduction of α- and β-keto esters.
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  • Gen NONAKA, Tomoki ISHIKAWA, Tin-tin LIU, Harushi NAKAJIMA, Katsuhiko ...
    2000 Volume 64 Issue 10 Pages 2152-2158
    Published: 2000
    Released: February 03, 2005
    JOURNALS FREE ACCESS
      Even though most fungal hydrolytic enzymes have been successfully secreted in S. cerevisiae cells by expression of corresponding cDNA, overexpression of A. oryzae RNase T1 causes severe growth inhibition in yeast. We observed that yeast strains carrying RNase T1 cDNA under control of the GAL1 promoter with a single-copy vector were able to grow on galactose medium while those with a multi-copy vector were not. It was found that overexpression of three mutated versions of RNase T1 with low enzymatic activity did not affect the growth. We also observed that expression of RNase T1 without a signal sequence severely inhibited growth of the transformant even on the single-copy plasmid. Subcellular fractionation showed that overexpressed myc-tagged RNase T1 was localized in the membrane fraction. In the yeast secretory pathway, while the mutants defective in translocation into the ER, ER-Golgi trafficking and vacuole formation had severe growth inhibition during expression of RNase T1 from the single-copy plasmid. These results suggest that a mislocalization of active RNase T1 in cytosol by overflow from the secretory apparatus has toxic effects on the host cells.
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Microbiology & Fermentation Technology Notes
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