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Volume 127 (2000)
- Issue 6 Pages 941-
- Issue 5 Pages 711-
- Issue 4 Pages 525-
- Issue 3 Pages 351-
- Issue 2 Pages 171-
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Volume 127, Issue 4
Displaying 1-25 of 25 articles from this issue
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Jiro Imanishi2000 Volume 127 Issue 4 Pages 525-530
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCytokines are very important in the host defense system, and play a critical role in pro-tection against bacterial and viral infections. Cytokines are also involved in the patho-genesis and development of symptoms in infections. In this article, Helicobacter pylori (H. pylori) infection as bacterial infection, and influenza virus infection, encephalomyo-carditis virus (EMCV) infection, and herpes simplex virus (HSV) infection as viral infec-tion are mentioned. In H. pylori infection, various chemokines, especially interleukin (IL)-8, induce inflammatory responses in the gastroduodenal mucosa. Furthermore, IL-6, IL-7, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ are involved in both protec-tion and pathogenesis. In influenza virus infection, IFN-α/β, IFN-γ, and IL-6 play protec-tive roles. In EMCV infection, IL-6 and TNF-α play important roles as a protective and exacerbative factor in acute myocarditis, respectively. Furthermore, in HSV infection, the production of inflammatory cytokines is closely correlated with the pathogenesis of herpetic keratitis, and IFN-γ plays an important role in enhancing viral clearance from the cornea and trigeminal ganglions.View full abstractDownload PDF (469K) -
Yasuo Komatsu, Ikuyo Kanzaki, Miho Shirai, Izumi Kumagai, Shigeko Yama ...2000 Volume 127 Issue 4 Pages 531-536
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFunctional structures of hairpin ribozymes have been investigated by constructing various chemically modified molecules. Domain-exchange and linker insertion experiments were performed to find active conformations of the RNA enzyme showing cleavage activity. Our experiments and other evidence suggest that the active structure has a bent conformation, and that domain-interactions are essential for the cleavage activity.View full abstractDownload PDF (480K) -
Hitoshi Uga, Hirofumi Komori, Kunio Miki, Chieko Wada2000 Volume 127 Issue 4 Pages 537-541
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have determined the nucleotide positions of an incC iteron essential for RepE binding by analyzing mutated incC iterons defective in exerting incompatibility towards mini-F plasmids. The mutations affecting this incompatibility occurred mostly at two positions within the incC iteron, i.e. an iteron conserved position and a mini-F specific position. Most of the iterons with a base-change at either of these two positions had lost the binding affinity for RepE. This agrees with the crystallographic structure of the RepE-iteron complex which showed that the N and C terminal domains of RepE interact with the two major grooves on one face of the iteron DNA. These grooves contain the iteron conserved and mini-F specific positions necessary for RepE binding. Thus the binding mode may be common to in the case of mini-F like plasmids.View full abstractDownload PDF (1599K) -
Osamu Satoh, Hirotaka Imai, Tetsuo Yoneyama, Tatsuo Miyamura, Hideo Ut ...2000 Volume 127 Issue 4 Pages 543-550
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSExpression of S protein, an envelope protein of hepatitis B virus, in the absence of other viral proteins, leads to the secretion of hepatitis B virus surface antigen (HBsAg) parti-cles that are formed by budding from the endoplasmic reticulum membranes. The HBsAg particles produced by mouse fibroblast cells show a unique lipid composition, with 1, 2-diacyl glycerophosphocholine being the dominant component. The lipid organi-zation of the HBsAg particles was studied by measuring electron spin resonance (ESR) using various spin-labeled fatty acids, and the results were compared with a parallel study on HVJ (Sendai virus) and vesicles reconstituted with total lipids of the HBsAg particles (HBs-lipid vesicles). HVJ and the HBs-lipid vesicles showed typical ESR spec-tra of lipids arranged in a lipid bilayer structure. In contrast, the ESR spectra obtained with the HBsAg particles showed that the movement of lipids in the particle is severely restricted and a typical immobilized signal characteristic of tight lipid-protein interac-tions was also evident. Phosphatidylcholine (PC) in the HBsAg particles was not ex-changeable by a PC-specific exchange protein purified from bovine liver, while phospho-lipase A2 from Ngja ngja vemon was able to hydrolyze all the PC in the particles. These analyses suggest that the lipids in the HBsAg particles are not organized in a typical lipid bilayer structure, but are located at the surface of the particles and are in a highly immobilized state. Based on these observations we propose a unique lipid assembly and membrane structure model for HBsAg particles.View full abstractDownload PDF (590K) -
Nobuhito Sone, Shuuji Koyanagi, Junshi Sakamoto2000 Volume 127 Issue 4 Pages 551-557
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe isolated a K17q8 mutant from K 17 mutant cells of Bacillus stearothermophilus which contain SoxB-type cytochrome bo3 as well as cytochrome bd but not SoxM-type cytochrome caa3, which is the main terminal oxidase in B. stearothermophilus K 1041. The respiration of K17q8 was highly sensitive to as little as 10 μM cyanide, indicating that the main terminal oxidase is cytochrome bo3. The aerobic growth yield of K17q8 was lower than that of wild-type K 1041, but higher than that of parental K 17. The H+/O ratio of K17q8 was about 5, i.e. a little lower than the 6.1-6.5 of K 1041, but higher than the 2.9-3.1 of K 17 [Sone et al. (1999) J. Biosci. Bioeng. 87, 495-499]. Analyses of membrane frag-ments indicated that K17q8 contains about 0.2 nmol cytochrome bo3 per mg membrane protein, and scarcely any subunits of cytochromes caa3 and bd. From the membrane fraction of K17q8, cytochrome bo3 was purified and shown to be composed of two subunits with apparent molecular masses of 56 and 19 kDa. The enzyme contained protoheme IX and heme O, as the main low-spin heme and high-spin heme. Analysis of the substrate specificity indicated that the high-affinity site is very specific to cytochrome c-551, a cytochrome c which is a membrane-bound lipoprotein of thermophilic Bacillus. The I50 of purified cytochrome bo3 was determined to be 4 μM, indicating that cyto-chrome bo3 among the three terminal oxidases in B. stearothermophilus was most susceptible to cyanide. The respiration of K17q8 was mostly inhibited by the addition of cyanide at this concentration.View full abstractDownload PDF (1238K) -
Hisaaki Mihara, Tatsuo Kurihara, Tohru Yoshimura, Nobuyoshi Esaki2000 Volume 127 Issue 4 Pages 559-567
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have purified three NifS homologs from Escherichia coli, CSD, CsdB, and IscS, that appear to be involved in iron-sulfur cluster formation and/or the biosynthesis of selenophosphate. All three homologs catalyze the elimination of Se and S from L-selenocysteine and L-cysteine, respectively, to form L-alanine. These pyridoxal 5'-phosphate enzymes were inactivated by abortive transamination, yielding pyruvate and a pyridox-amine 5'-phosphate form of the enzyme. The enzymes showed non-Michaelis-Menten behavior for L-selenocysteine and L-cysteine. When pyruvate was added, they showed Michaelis-Menten behavior for L-selenocysteine but not for L-cysteine. Pyruvate signifi-cantly enhanced the activity of CSD toward L-selenocysteine. Surprisingly, the enzyme activity toward L-cysteine was not increased as much by pyruvate, suggesting the pres-ence of different rate-limiting steps or reaction mechanisms for L-cysteine desulfuriza-tion and the degradation of L-selenocysteine. We substituted Ala for each of Cys 358 in CSD, Cys 364 in CsdB, and Cys 328 in Iscs, residues that correspond to the catalytically essential Cys 325 of Azotobacter vinelandii NifS. The enzyme activity toward L-cysteine was almost completely abolished by the mutations, whereas the activity toward L-sele-nocysteine was much less affected. This indicates that the reaction mechanism of L-cys-teine desulfurization is different from that of L-selenocysteine decomposition, and that the conserved cysteine residues play a critical role only in L-cysteine desulfurization.View full abstractDownload PDF (705K) -
Teruyuki Kobayashi, Masaki Ito, Kiyoshi Ikeda, Kiyoshi Tanaka, Megumi ...2000 Volume 127 Issue 4 Pages 569-575
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA membrane-bound sialidase in pig liver microsomes was solubilized with a nonionic detergent, IGEPAL CA 630, and purified to homogeneity by sequential chromatographies on SP-Toyopearl, Butyl-Toyopearl (1 st), SuperQ-Toyopearl, Hydroxyapatite, Butyl-Toyo-pearl (2 nd), GM1-Cellulofine affinity, and sialic acid-Cellulofine affinity columns. The molecular weight of the purified enzyme was estimated to be 57 kDa on SDS-PAGE. The pH optimum was 4.8 for the activity measured using 4-methylumbelliferyl-α-N-acetylneuraminic acid (4MU-Neu5Ac) as the substrate. The enzyme activity was inhibited by 2-deoxy-2, 3-dehydro-N-acetylneuraminic acid, iodoacetamide and p-chloromercuribenzoic acid. While the enzyme could effectively hydrolyze 4MU-Neu5Ac, it failed to significantly cleave a sialic acid residue(s) from sialyllactose, glycoproteins or gangliosides at pH 4. 8. These results suggest that the purified enzyme is a novel sialidase with a substrate spec-ificity distinct from those of known membrane-bound sialidases in mammalian tissues.View full abstractDownload PDF (1465K) -
Minoru Inagaki, Akiyoshi Tanaka, Ryoko Suzuki, Hirohito Wakashima, Tom ...2000 Volume 127 Issue 4 Pages 577-583
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe spike H protein of bacteriophage φX 174 was prepared as a hexa histidine-tagged fusion (HisH). On enzyme-linked plate assaying, HisH was found to bind specifically to the lipopolysaccharides (LPSs) of φX174-sensitive strains, Escherichia coli C and Salmonella typhimurium Ra chemotype, having the complete oligosaccharide sequence of the R-core on the LPSs. In sharp contrast, HisH bound weakly to the LPSs of ΦX174-insensitive strains, i.e. E. coli F 583 (Rd2) lacking some terminal saccharides and E. coli O111:B4 (smooth strain) having additional O-repeats on the R-core. The fluorescence spectra of HisH changed dose-dependently in the case of the LPS of E. coli C, the intensity increasing and the emission peak shifting to the shorter wavelength side, which was attributable to the hydrophobic interaction of HisH with the LPS. The binding equilibrium was analyzed by fluorometric titration to determine the dissociation constant Kd, 7.02±0.37 μM, and the Gibbs free energy change ΔG0, -29.1 kJ mol-1 (at 22°C, pH 7.4). Based on the temperature dependence of Kd in a van't Hoff plot, the standard enthalpy change ΔH0 and the entropy change. ΔS0 were calculated to be +23.7 kJ mol-1 and 179 J mol-1 K-1 at 22°C, respectively, and this binding was thereby concluded to be an entropy-driven reaction.View full abstractDownload PDF (1153K) -
Paxton Loke, Tiow-Suan Sim2000 Volume 127 Issue 4 Pages 585-589
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIsopenicillin N synthase (IPNS) is a key enzyme responsible for the catalytic conversion of δ-(L-α-aminoadipoyl)-L-cysteinyl-D-valine (ACV) to isopenicillin N in the β-lactam anti-biotic biosynthetic pathway. The Aspergillus nidulans IPNS crystal structure implicated amino acid residues tyrosine-189, arginine-279, and serine-281 in the substrate-binding of the valine carboxylate portion of ACV via hydrogen bonds. In previous reports, we provided mutational evidence for the critical involvement of the corresponding argin-ine-281 and serine-283, which constitute a conserved R-X-S motif, for the catalysis of Cephalosporium acremonium IPNS (cIPNS). In this study, we report the site-directed mutagenesis of the corresponding tyrosine-191 in cIPNS to four amino acids from differ-ent amino acid groups, namely, phenylalanine, serine, histidine, and aspartate. The mutants Y191F, Y191H, and Y191R respectively yielded specific activities at levels of 3, 8.6, and 18.8% relative to the wild-type when enzyme bioassays were performed using purified protein fractions. These results were surprising, as previous mutational analy-ses involving arginine-281 and serine-283 resulted in non-measurable specific activities, thus suggesting that tyrosine-191 is important but not critical for the activity of cIPNS due to its involvement in ACV binding. Hence, it is likely that tyrosine-191 is the least critical of the three residues involved in binding the ACV valine carboxylate moiety.View full abstractDownload PDF (1202K) -
Tadashi Okubo, Nobuaki Yanai, Sumiko Watanabe, Ken-ichi Arai, Masuo Ob ...2000 Volume 127 Issue 4 Pages 591-596
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBone marrow lineage-negative (Lin-) c-Kit+ Sca-1+ hematopoietic cells from human GM-CSF receptor gene transgenic mice were cultured on established bone marrow stromal cell (TBR59) layers and on semisolid medium. In the semisolid assay, an increasing num-ber of larger colonies were observed in the presence of hGM-CSF. By coculture with the stromal cells, cobblestones containing myeloid and lymphoid lineages of cells were formed from the stem cell enriched fraction, and addition of hGM-CSF strongly stimu-lated formation of the cobblestones containing both lineages. Repeating passages of the cobblestones on TBR 59 stromal cells in the presence of hGM-CSF gradually decreased cobblestone formation and inversely increased macrophages and granulocytes, while mast cells were generated when the cells derived from the semisolid assay were cul-tured in a liquid medium containing hGM-CSF. These results consistently suggest that cytokines such as GM-CSF may costimulate the immature hematopoietic cells at their stroma-dependent phase before lineage commitment, and after commitment that occurs by an intrinsic program of the cells, they may stimulate maintenance and maturation of rogenitor cells.View full abstractDownload PDF (2531K) -
Junko Tanaka, Takuya Nihira, Yasuhiro Yamada2000 Volume 127 Issue 4 Pages 597-601
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPseudomonas sp. 109 produces a unique lipase (LipL) which efficiently catalyzes intramolecular transesterification of ω-hydroxyesters to form macrocyclic lactones. The production of the enzymatically active LipL requires a specific molecular chaperon (LimL protein) together with a low-Mr lipase-activation-factor (LAF) of unknown structure. From 50g of Pseudomonas cells, 2.15mg of LAF was purified as a sulfobenzofurazanyl derivative after methanol extraction, derivatization, and C18 reverse-phase HPLC. One-dimensional and two-dimensional 600 MHz 1H-NMR and fast atom bombardment mass spectrometry (FAB-MS) revealed that LAF is glutathione. Because several SH com-pounds (L-cysteine and mercaptoethanol) were similarly effective to native LAF in the activation of LipL, and because only LipL contains two cysteinyl residues forming an intramolecular disulfide bond, it is concluded that the reduction of and reformation of the intramolecular disulfide bond of LipL is essential to liberate free and fully active LipL.View full abstractDownload PDF (958K) -
Chusuke Sato, Jun Nakamura, Yoshiaki Nakamaru2000 Volume 127 Issue 4 Pages 603-610
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMerocyanine 540 (MC540) is a widely used dye probe for membranous environments. However, fundamental knowledge of the spectral features of this dye in aqueous and hydrophobic environments is still lacking. Such knowledge is important because biomembranes involve a hydrophobic environment surrounded by a hydrophilic envi-ronment. Because many investigations so far have been performed based on indistinct spectral estimations, the interpretation of the data obtained using this dye as a fluores-cent transmembrane probe remains controversial. In order to determine the exact spec-tra in both aqueous and hydrophobic environments, we adopted principal factor analysis (PFA), a method of multivariate analysis. The PFA method can also determine the number of molecular species present in the reaction mixture, which is three in pure water and two in phospholipid suspension. Two of the species in both water and phos-pholipid suspension were the monomer and dimer. The third species in water was the trimer, but its amount was so small at 10 μM MC540 solution that the spectral data in water can be approximated neglecting this molecular species. The monomer spectrum changed its form markedly with a bathochromic shift when transferred from the water to phospholipid environment, whereas the dimer remained similar in its shape except for a remarkable red shift. In water, the dissociation constants, K1 and K2, for the assumed stacking-model reactions, M+M_??_M2 and M+M2_??_M3, were 3.1×10-4 M and 5.7×10-4 M, respectively. In the phospholipid environment, the dissociation constant K* for the assumed stacking-model reaction, M*+M*_??_M2*, was 1.9×10-5 M. The fluorescent intensities of MC 540 were also measured in both water and phospholipid environments. A comparison based on the absorption and fluorescence spectra suggested that the tem-poral increase in the amount of the monomer on the excitable membrane contributes to the fluorescent intensity change observed in the transmembrane potential change.View full abstractDownload PDF (578K) -
Lei Zhang, Akinori Ohta, Masamichi Takagi, Ryozo Imai2000 Volume 127 Issue 4 Pages 611-616
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo study functions of late embryogenesis abundant (LEA) proteins, which accumulate in plant cells under water deficit conditions, in vivo functional analyses were carried out using a yeast (Saccharomyces cerevisiae) heterologous expression system. Two lea genes, tomato le4 (group 2) and barley HVA1 (group 3), were expressed under the GAL1 pro-moter, and the gene products were detected using specific antisera. The growth of the transformants was scored and compared with a control strain to analyze the effect of these proteins on yeast cells under stress conditions. The yeast cells expressing HVA1 showed shorter lag period when transferred to a medium containing 1.2 M NaCl as com-pared to a control strain, while the cells expressing le4 did not show improved growth. Attenuated growth inhibition in a medium containing 1.2 M KCl was observed in the yeast cells expressing le4 and HVA1. No obvious growth improvement was observed in a high sorbitol medium in the cells expressing either le4 or HVA1. Increased freezing tol-erance was observed in both lea-expressing cells, while no effect on heat tolerance was observed. These results support the hypothesis that different LEA proteins play a dis-tinctive role in the protection against cellular dehydration.View full abstractDownload PDF (1702K) -
Hiroshi Takagi, Kazunori Hirai, Yoshimi Maeda, Hiroshi Matsuzawa, Shig ...2000 Volume 127 Issue 4 Pages 617-625
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe examined the effect of a novel disulfide bond engineered in subtilisin E from Bacil-lus subtilis based on the structure of a thermophilic subtilisin-type serine protease aqualysin I. Four sites (Ser163/Ser194, Lys170/Ser194, Lys170/Glu195, and Pro172/Glu195) in subtilisin E were chosen as candidates for Cys substitutions by site-directed mutagenesis. The Cys170/Cys195 mutant subtilisin formed a disulfide bond in B. subtilis, and showed a 5-10-fold increase in specific activity for an authentic peptide substrate for subtilisin, N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe p-nitroanilide, compared with the sin-gle-Cys mutants. However, the disulfide mutant had a 50% decrease in catalytic effi-ciency due to a smaller kcat and was thermolabile relative to the wild-type enzyme, whereas it was greatly stabilized relative to its reduced form. These results suggest that an electrostatic interaction between Lys 170 and Glu 195 is important for catalysis and stability in subtilisin E. Interestingly, the disulfide mutant was found to be more stable in polar organic solvents, such as dimethylformamide and ethanol, than the wild-type enzyme, even under reducing conditions; this is probably due to the substitution of uncharged Cys by charged surface residues (Lys 170 and Glu 195). Further, the amino-ter-minal engineered disulfide bond (Gly61Cys/Ser98Cys) and the mutation Ile31Leu were introduced to enhance the stability and catalytic activity. A prominent 3-4-fold increase in the catalytic efficiency occurred in the quintet mutant enzyme over the range of di-methylformamide concentration (up to 40%).View full abstractDownload PDF (2531K) -
Takashi Aoki, Rika Ami, Hitoshi Onagi, Hiroyoshi Fujino, Hiroyuki Wata ...2000 Volume 127 Issue 4 Pages 627-633
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe unique behavior of green fluorescent protein (GFP) on SDS-PAGE was applied to the detection of a single amino acid substitution in GFP-tagged polypeptides. This sim-ple detection method using SDS/urea gels was designated GFP-display. The N-terminal 18 or 37 amino acids of K-Ras was used as a model GFP-tagged polypeptide. K-ras exon 1 was fused to a gfp cDNA at each end and expressed in Escherichia coli. Amino acid num-ber 12 of K-Ras (wild type; Gly) was changed to Ser, Arg, Cys, Asp, Ala, or Val, and the mobility shift of the greenish fluorescent bands in the SDS/urea gel was analyzed. These mutants were easily detected by GFP-display; however, detection depended strongly on the urea concentration and electrophoresis temperature. Subsequently, GFP-display was applied to the 36 amino acids encoding human p53 exon 7. Amino acid number 248 (wild type; Arg) was changed to Gly, Trp, Gln, Pro, or Leu, and similar mobility shifts were observed. GFP-display could be coupled with an in vitro translation system. Fluo-rescent active GFP and GFP-Ras fusion proteins were synthesized within a few hours. GFP-display shows potential as a modern approach to gene mutation analysis at the protein level, and is a useful method for protein engineering studies.View full abstractDownload PDF (3048K) -
Atsushi Yamamoto, Koji Tomoo, Tadaoki Hara, Mitsuo Murata, Kunihiro Ki ...2000 Volume 127 Issue 4 Pages 635-643
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe crystal structure of the bovine spleen cathepsin B (BSCB)-CA074 complex was refined to R=0.152 using X-ray diffraction data up to 2.18 Å resolution. BSCB is charac-terized by an extra Cys148-Cys252 disulfide bridge, as compared with rat and human CBs. Although the crystal structures of these enzymes showed similar overall folding, a difference was observed in the occluding loop, a structural element specific only to CB. Comparison of the torsion angles indicated the different flexibilities of their loop struc-tures. The oxirane C6 atom of CA074 was covalently bonded to the Cys29 Sγ atom (C3-Sγ=1.81 Å), where the S-configuration was transformed to the R-form. Concerning the oxirane carbon atom that participates in the covalent bonding with the Cys residue, an acceptable rule has been proposed. The substrate specificities at the Sn (n=1-3) and Sn' (n=1 and 2) subsites of CB, together with the interaction features as to CA074, have been discussed in comparison with the crystal structure of the papain-CA028 (a CA074-related inhibitor) complex.View full abstractDownload PDF (659K) -
Tadataka Ishikawa, Masahiro Kai, Ikuo Wada, Hideo Kanoh2000 Volume 127 Issue 4 Pages 645-651
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSeveral isozymes of mammalian type 2, Mg2+-independent phosphatidic acid phos-phatase (PAP-2) have recently been cloned, and they are predicted to have their cata-lytic sites exposed at the cell surface membranes. We investigated the mode of utilization of extracellular lipid substrates by the human PAP-2b expressed in HEK293 cells as a green fluorescent fusion protein. We first confirmed the plasma membrane localization of the expressed PAP-2b. PAP-2b actively hydrolyzed exogenously added lysophosphatidic acid and short-chain phosphatidic acid. In the case of dephosphoryla-tion of lysophosphatidic acid, the reaction products, including inorganic phosphate and monoacylglycerol, were recovered exclusively in the extracellular medium. Interest-ingly, PAP-2b exhibited negligible activities toward long-chain phosphatidic acid either exogenously when added or generated within the membranes by treating the cells with bacterial phospholipase D. These findings indicate that PAP-2b acts at the outer leaflet of cell surface bilayers and can account for the ecto-PAP activities previously described for various types of cells.View full abstractDownload PDF (1060K) -
Yorishige Imamura, Toyohiko Migita, Yukie Uriu, Masaki Otagiri, Tadash ...2000 Volume 127 Issue 4 Pages 653-658
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe inhibitory effects of flavonoids (galangin, kaempferol, quercetin, myricetin, morin, and taxifolin) on rabbit heart carbonyl reductase (RHCR) were investigated using 4-ben-zoylpyridine (4BP) as the substrate. The stereochemical characteristics of the flavonoids were found to be a factor determining their inhibitory potencies toward RHCR. Furthermore, the lipophilicity, and the scavenging or antioxidative effects of the flavonoids were likely to complicate the structure-activity relationship of their inhibitory effects on RHCR. Quercetin inhibited RHCR uncompetitively with respect to NADPH and com-petitively with respect to 4BP, suggesting that it competes with 4BP at the substrate-binding site of RHCR. RHCR efficiently reduced benzoquinones (1, 4-benzoquinone and 2-methyl-1, 4-benzoquinone) and naphthoquinones (1, 4-naphthoquinone and menadi-one). Galangin was a potent inhibitor of RHCR when menadione was used as the sub-strate, and prevented the formation of the superoxide anion radical in the presence of RHCR, NADPH, and menadione. Flavonoids may be useful compounds for suppressing the cardiotoxicity of quinones by inhibiting RHCR.View full abstractDownload PDF (465K) -
Hanae Koiwai, Shuichi Akaba, Mitsunori Seo, Teruya Komano, Tomokazu Ko ...2000 Volume 127 Issue 4 Pages 659-664
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo investigate the biochemical and enzymatic properties of two aldehyde oxidase (AO) isoforms of Arabidopsis thaliana, we expressed AAO 1 and AAO 2 cDNAs in a heterologous yeast (Pichia pastoris) system and successfully obtained the proteins in active forms. The expressed AAO 1 and AAO 2 proteins gave activity bands with the same mobilities on native gel electrophoresis and exhibited the same substrate preferences on zymograms with 8 aldehydes as those of AOα and AOγ in Arabidopsis seedlings, respectively. Furthermore, anti-AAO 1 and anti-AAO 2 antibodies, which specifically recognize the seedling AOα and AOγ, respectively, reacted with the AAO 1 and AAO 2 proteins produced in P. pastoris, respectively. These results indicate that these AO proteins are accurately produced in the yeast system, as in Arabidopsis seedlings. Using AO preparations from P. pastoris, the enzymatic properties of Arabidopsis AOα and AOγ were investigated. AOα showed a relatively wide substrate specificity for 7 aldehydes tested, with high affinity to benzaldehyde and indole-3-aldehyde, while AOγ could most efficiently oxidize naphthaldehyde. AOα was strongly inhibited by iodoacetate and KCN, while AOγ was inhibited not only by iodoacetate and KCN but also by 2-mercaptethanol, dithiothreitol, menadion, and estradiol. AOα and AOγ showed the highest activity at around 65 and 50°C, respectively, and exhibited pH dependence around pH 8.0. These results indicate that the two AO isoforms in Arabidopsis seedlings have different enzymatic properties and may have different physiological roles in vivo.View full abstractDownload PDF (1787K) -
Toshiaki Oda, Chiharu Uchida, Satoshi Miura2000 Volume 127 Issue 4 Pages 665-671
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn the rat liver, two mRNAs for serine: pyruvate (or alanine: glyoxylate) aminotransferase are generated from a single gene by alternative transcription initiation. The longer mRNA encodes a precursor of a mitochondrial enzyme that has a mitochondrial targeting signal at the N-terminus and is translocated into mitochondria. The shorter mRNA encodes a peroxisomal enzyme of mature size that is imported into peroxisomes. We have been interested in the mechanism of selective targeting to mitochondria of the precursor protein that also contains a peroxisomal targeting signal in the molecule. In this study, we examined the effect of the mitochondrial targeting signal on the conformation of the protein and on the function of the peroxisomal targeting signal in the precursor molecule. The results suggest that the mitochondrial targeting signal causes the conformation of the protein to become unfolded and that this conformational change in turn causes repression of the putative peroxisomal targeting signal contained in the precursor protein.View full abstractDownload PDF (2728K) -
Ryutaro Asano, Shin-ichi Takemura, Kouhei Tsumoto, Naoki Sakurai, Atsu ...2000 Volume 127 Issue 4 Pages 673-679
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA bacterial expression system for the variable region fragments (Fvs) of the anti-MUC 1 tumor antigen antibody MUSE 11 has been constructed. The Fv fragment showed binding specificity toward TFK-1 cells, with slightly reduced affinity compared to its parent IgG. The single-chain Fv fragment was arranged in two orders, VH-linker-VL and VL-linker-VH. However, linking the regions with a flexible peptide linker (GGGGS), or with a shorter linker (GGGGS) led to a dramatic decrease in the biological activity toward the target antigen in both arrangements, suggesting that the MUSE 11 antibody loses its activity when the domains are linked with polypeptide linkers. These results indicate that the variable region domains of the anti-MUC 1 antibody MUSE 11 have specificity only in the Fv form, and that linking the domains strongly reduces the association with its target antigen. Gel filtration analysis indicates that the scFv has a dimeric structure, suggesting that the inactivation of MUSE 11 scFv is due to unfavorable intermolecular associations of the scFv chains. To our knowledge, this is the first report of a significant reduction in affinity caused by linking the variable domains in both arrangements, i.e., VH-VL and VL-VH.View full abstractDownload PDF (2451K) -
Ken-ichi Takahashi, Seiki Baba, Yojiro Hayashi, Yoshio Koyanagi, Naoki ...2000 Volume 127 Issue 4 Pages 681-686
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo positive-strand HIV-1 genomic RNAs form a dimer in virion particles through interaction of the dimerization initiation sites (DIS). The DIS RNA fragment spontaneously formed a “loose-dimer” and was converted into a “tight-dimer” by supplementation with nucleocapsid protein NCp 7. This two-step dimerization reaction requires the whole DIS sequence [Takahashi et al. (2000) RNA 6, 96-102]. In the present study, we measured imino proton resonances to investigate the secondary structures of the two types of dimers in a 39-mer RNA covering the entire DIS (DIS 39), including discrimination between intra- and inter-molecular base pairing. Both the presence and absence of intermolecular NOE between 15N-labeled and unlabeled DIS 39 were unambiguously detected in an equimolar mixture of 15N-labeled and unlabeled DIS 39. The stem-bulge-stem structures in both dimers were confirmed and found to be very close to each other from clear superimposition of the NMR spectra in the two dimeric states. Nevertheless, the modes of base paring in the stems of the loose- and tight-dimers were intra- and inter-molecular, respectively. Our results suggest a large structural alteration of genomic RNA occurs during virion maturation.View full abstractDownload PDF (1719K) -
Kyoung-Seok Ryu, Byong-Seok Choi, Seung-Wook Chi, Seung-Ho Kim, Hyoung ...2000 Volume 127 Issue 4 Pages 687-694
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe corticotropin-releasing factor (CRF) is a 41-amino acid peptide-amide hormone, which mediates a general stress-response. It has been reported that the substitution of His-32 in the ovine CRF (oCRF) with Ala brings about a 4.5-fold increase in activity [Kornreich et al. (1992) J. Med. Chem. 35, 1870-76]. Here, we have determined the secondary structure of this Ala-substituted ovine CRF ([Ala 32] oCRF) and compare it with that of oCRF using circular dichroism (CD) and NMR techniques in trifluoroethanol (TFE) solution, which is known to stabilize the α-helix formation. In contrast to an earlier report, it was observed the α-helical structure extends to the C-terminus of oCRF. By analyzing the C_??_H and NH chemical shifts, the properties of local structures of oCRF were elucidated. The oCRF and [Ala 32] oCRF have stable α-helical structures in the middle region, regardless of pH and temperature, and the α-helix initiation regions of these peptides are stabilized as the pH is decreased. However, the [Ala 32] oCRF has a more stable α-helical structure than oCRF in the vicinity of the substitution region, and it is thought that this is the cause of the increased activity of [Ala 32] oCRF.View full abstractDownload PDF (1258K) -
Keiichi Takagaki, Hidekazu Munakata, Mitsuo Majima, Ikuko Kakizaki, Ma ...2000 Volume 127 Issue 4 Pages 695-702
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA method was developed for the reconstruction of glycosaminoglycan (GAG) oligosaccharides using the transglycosylation reaction of an endo-β-N-acetylhexosaminidase, testicular hyaluronidase, under optimal conditions. Repetition of the transglycosylation using suitable combinations of various GAGs as acceptors and donors made it possible to custom-synthesize GAG oligosaccharides. Thus we prepared a library of chimeric GAG oligosaccharides with hybrid structures composed of disaccharide units such as GlcA-GlcNAc (from hyaluronic acid), GlcA-GalNAc (from chondroitin), GlcA-GalNAc 4 S (from chondroitin 4-sulfate), GlcA-GaINAc 6 S (from chondroitin 6-sulfate), IdoA-GalNAc (from desulfated dermatan sulfate), and GlcA-GalNAc 4, 6-diS (from chondroitin sulfate E). The specificity of the hyaluronidase from Streptococcus dysgalactiae (hyaluronidase SD) was then investigated using these chimeric GAG oligosaccharides as model substrates. The results indicate that the specificity of hyaluronidase SD is determined by the following restrictions at the nonreducing terminal side of the cleavage site: (i) at least one disaccharide unit (GIcA-GlcNAc) is necessary for the enzymatic action of hyaluronidase SD; (ii) cleavage is inhibited by sulfation of the N-acetylgalactosamine; (iii) hyaluronidase SD releases GlcA-GalNAc and IdoA-GalNAc units as well as GlcA-GlcNAc. At the reducing terminal side of the cleavage site, the sulfated residues on the N-acetylgalactosamines in the disaccharide units were found to have no influence on the cleavage. Additionally, we found that hyaluronidase SD can specifically and endolytically cleave the internal unsulfated regions of chondroitin sulfate chains. This demonstration indicates that custom-synthesized GAG oligosaccharides will open a new avenue in GAG glycotechnology.View full abstractDownload PDF (589K) -
Ge-Hong Sun-Wada, Shiro Manabe, Takao Yoshimizu, Chie Yamaguchi, Toshi ...2000 Volume 127 Issue 4 Pages 703-709
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe expression of murine transcription factor GATA 6 is restricted to tissues including the heart and gastrointestinal systems during embryogenesis, and is maintained throughout postnatal life. We have characterized the 5' upstream region (6.4 kb) of the mouse GATA 6 gene, and identified two closely spaced transcription initiation sites. The flanking sequence lacks a typical TATA-box, and is rich in guanine and cytosine. The role of the 5' upstream region was examined using the lacZ reporter gene in transgenic mice. A construct containing the 5' flanking sequence (4.9 kb), untranslated exon 1 and 1.3 kb intron 1 could drive the gene expression in the embryonic and adult heart regions. Weak expression was also observed in the stomach, liver, and bronchial arch in addition to the cardiac region. Deletion of the 5' upstream region (_??_1.2 kb) or intron 1 abolished all this expression, indicating that at least two cis-acting control elements are necessary for heart-specific expression of GATA 6 in vivo.View full abstractDownload PDF (692K)
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