巻号一覧
130 巻, 6 号
選択された号の論文の23件中1～23を表示しています
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Emi Hama, Keiro Shirotani, Hajime Masumoto, Yoko Sekine-Aizawa, Hiroyu ...2001 年 130 巻 6 号 p. 721-726
発行日: 2001年
公開日: 2008/11/18
ジャーナル フリーAmyloid β peptide (Aβ), the pathogenic agent of Alzheimer's disease (AD), is a physiolog-ical metabolite constantly anabolized and catabolized in the brain. We previously dem-onstrated that neprilysin is the major Aβ-degrading enzyme in rico. To investigate whether or not manipulation of neprilysin activity in the brain would be an effective strategy for regulating Aβ levels, we expressed neprilysin in primary cortical neurons using a Sindbis viral vector and examined the effect on Aβ metabolism. The correspond-ing recombinant protein, expressed in the cell bodies and processes, exhibited thior-phan-sensitive endopeptidase activity, whereas a mutant neprilysin with an amino acid substitution in the active site did not show any such activity. Expression of the wild-type neprilysin, but not the mutant, led to significant decreases in both the Aβ40 and 42 levels in the culture media in a dose-dependent manner. Moreover, neprilysin expres-sion also resulted in reducing cell-associated Aβ, which could be more neurotoxic than extracellular Aβ. These results indicate that the manipulation of neprilysin activity in neurons, the major source of Aβ in the brain, would be a relevant strategy for control-ling the All levels and thus the Aβ-associated pathology in brain tissues.抄録全体を表示PDF形式でダウンロード (1797K) -
Nobutoshi Ito, Osamu Nureki, Mikako Shirouzu, Shigeyuki Yokoyama, Fumi ...2001 年 130 巻 6 号 p. 727-730
発行日: 2001年
公開日: 2008/11/18
ジャーナル フリーAt the initiation of chromosomal DNA replication, DNA primases synthesize short RNA primers, which are subsequently elongated by DNA polymerases. To understand the structural basis for the primer synthesis by archaealkukaryotic-type primases, the gene of the DNA primase from hyperthermophilic archaeon Pyrococcus horihoshii was cloned and overexpressed in Escherichia coli as a fusion protein with a hexa-histidine tag at its amino terminus. The recombinant DNA primase was purified and crystallized by the hanging-drop vapor diffusion method at 293K, with polyethylene glycol 8000 as the pre-cipitant. The crystals belong to the P3221 space group with unit-cell parameters a =b=77.8, c=129.6 A, and α=β=90°, γ=120°. Crystals of the selenomethionine derivative were obtained by means of a cross-seeding method using native crystals. The data for the native and selenomethionine-substituted crystals were collected to 1.8 and 2.2 A res-olution, respectively, with synchrotron radiation at SPring-S under flash-frozen condi-tions at 100K. The four wavelength MAD data provided a phase to determine the structure of the primase at 2.2 A resolution.抄録全体を表示PDF形式でダウンロード (2416K) -
Shunji Natsuka, Ritsuko Takubo, Ryo Seki, Koji Ikura2001 年 130 巻 6 号 p. 731-735
発行日: 2001年
公開日: 2008/11/18
ジャーナル フリーFormation of cross-linking between proteins via a γ-glutamyl-ε-lysine residue is an important process in many biological phenomena including apoptosis. Formation of this linkage is catalyzed by the enzyme transglutaminase, which is widely distributed from bacteria to the animal kingdom. The simple multi-cellular organism Caenorhabditis elegans also possesses transglutaminase activity associated with apoptosis [Madi, A. et al. (1998) Eur. J. Biochem. 253, 583-590], but no gene with significant homology to verte-brate or bacterial transglutaminases has been found in the C. elegans genome sequence database. On the other hand, protein disulfide isomerases were recently recognized as a new family of transglutaminases [Chandrashekar, R. et al. (1998) Proc. Natl. Acad. Sci. USA 95, 531-536]. To identify the molecule with transglutaminase activity in C. elegans, we isolated from C. elegans a gene homologous to ERp57, which encodes a protein disulfide isomerase, expressed it in recombinant form, and characterized the transglutaminase and protein disulfide isomerase activities of the resultant protein. The C. elegans ERp57 protein had both enzyme activities, and the transglutaminase activity had similar characteristics to the activity in lysate of the whole worm. These results suggested that the ERp57 homologue was one of the substances with transglutaminase activity in C. elegans.抄録全体を表示PDF形式でダウンロード (1958K) -
Motoaki Wakiyama, Naoki Honkura, Kin-ichiro Miura2001 年 130 巻 6 号 p. 737-740
発行日: 2001年
公開日: 2008/11/18
ジャーナル フリーThe interaction between eukaryotic translation initiation factor 4G (eIF4G) and the poly(A)-binding protein (PABP) facilitates translational initiation of polyadenylated mRNAs. It was shown recently that the expression of an eIF4GI mutant defective in PABP binding in Xenopus oocytes reduces polyadenylated mRNA translation and dra-matically inhibits progesterone-induced oocyte maturation. These results strongly sug-gest that the eIF4G-PABP interaction plays a critical role in the translational control of maternal mRNAs during oocyte maturation. In the present work, we employed another strategy to interfere eIF4G-PABP interaction in Xenopus oocytes. The amino-terminal part of eIF4GI containing the PABP-binding site (4GNt-M1) was expressed in Xenopus oocytes. 4GNt-M1 could bind to PABP in oocytes, which suggests that 4GNt-M1 may evict PABP from the endogenous eIF4G. The expression of 4GNt-M1 resulted in reduc-tion of polyadenylated mRNA translation. Furthermore, 4GNt-M1 inhibited progester-one-induced oocyte maturation. In contrast, 4GNt-M2, in which the PABP-binding se-quences were mutated to abolish the PABP-binding activity, could not inhibit polyade-nylated mRNA translation or oocyte maturation. These results further support the idea that the eIF4G-PABP interaction is critical for translational regulation of maternal mRNAs in oocytes.抄録全体を表示PDF形式でダウンロード (1438K) -
Miki Shitashige, Masakazu Toi, Takeo Yano, Masao Shibata, Yoshinobu Ma ...2001 年 130 巻 6 号 p. 741-748
発行日: 2001年
公開日: 2008/11/18
ジャーナル フリーSerine 70 in the loop region of Bc1-2 is specifically phosphorylated by paclitaxel-treat-ment in tumor cells and BHK cells expressing Bc1-2. The phosphorylation of serine 70 of Bcl-2 (pS70-Bc1-2) peaks 24 to 48 h after paclitaxel treatment and accelerates apoptosis. Phosphorylation is effectively inhibited in the presence of actinomycin D or cycloheximide, which restore cell viability to the same level as control cells not expressing Bel-2. These results indicate that paclitaxel-induced kinase(s) and/or its activator(s) are syn-thesiz ed novo and play an important role in paclitaxel-induced apoptosis by phospho-rylating Bel-2. In binding assays using the phosphorylation-specific antibody against pS70-Bel-2, the induction of serine 70 phosphorylation 70 results in a loss of the binding ability of Bel-2 to Bax, a pro-apoptotic partner, and induces subsequent cell death. When the pS70-Bel-2 antibody was added to human breast cancer tissue, serine 70 phosphorylation was also detected, even prior to treatment with anticancer agents. Further study of breast cancers revealed 83% of tumors with high pS70-Bc1-2 expression responded to paclitaxel or docetaxel treatment, whereas 57% of those with low expression not respond. These findings suggest that pS70-Bel-2 might be a predictive factor for prognosis and sensitivity to paclitaxel treatment for breast cancer.抄録全体を表示PDF形式でダウンロード (3185K) -
Toshiaki Hara, Hiroaki Kodama, Yuichiro Higashimoto, Hiroshi Yamaguchi ...2001 年 130 巻 6 号 p. 749-755
発行日: 2001年
公開日: 2008/11/18
ジャーナル フリーAs models of ion channel proteins and naturally occurring pore-forming peptides, we designed a series of Aib rich peptides [Ac-(Aib-Xxx-Aib-Ala)5-NH2 (Xxx=Lys, Glu, Ser, and Gly: BXBA-20)] to investigate the effects of the side chains of the amino acid residues Lys, Glu, Ser, and Gly on the conformation and electrophysiological properties of ion channels. The conformation of peptides and their affinity for phospholipid membranes were evaluated by CD spectroscopy. Patch-clamp experiments revealed that all BXBA-20 peptides form ion channels in DPhPC bilayers exhibiting clearly resolved transitions between the open and closed states. The channel forming frequency was in the order BKBA-20⟩BEBA-20⟩BSBA-20⟩BGBA-20. In the case of BKBA-20 and BEBA-20, the self-assembled conductive oligomers expressed homogeneous and voltage-independent single channel conductances. In contrast, heterogeneous conductance was observed in BSBA-20 and BGBA-20 ion channels under similar experimental conditions. From these results, we conclude that peptides with a high degree of helical conformation, high amphipathicity, high affinity for lipid membranes, and self-associating characters in vesicles are most suitable for inducing ion channels with a high frequency of occurrence. Moreover, BEBA-20, BSBA-20, and BGBA-20 channels were cation-selective, whereas the BKBA-20 channel was non-selective.抄録全体を表示PDF形式でダウンロード (1146K) -
Youtao Song, Jin Sato, Akira Saito, Masakatsu Usui, Hiroyuki Azakami, ...2001 年 130 巻 6 号 p. 757-764
発行日: 2001年
公開日: 2008/11/18
ジャーナル フリーDisruption of the calnexin gene in Saccharomyces cerevisiae did not lead to gross effects on the levels of cell growth and secretion of wild-type hen egg white lysozymes (HEWL). To investigate the function of calnexin in relation to the secretion of glycoproteins, we expressed both stable and unstable mutant glycosylated lysozymes in calnexin-disrupted S. cerevisiae. The secreted amounts of stable mutant glycosylated lysozymes (G49N and S91T/G49N) were almost the same in both wild-type and calnexin-disrupted S. cerevisiae. In contrast, the secretion of unstable mutant glycosylated lysozymes (KI3D/G49N, C76A/G49N, and D66H/G49N) greatly increased in calnexin-disrupted S. cerevisiae, although their secretion was very low in the wild-type strain. This indicates that calnexin may act in the quality control of glycoproteins. We further investigated the expression level of the mRNA of the molecular chaperones BiP and PDI, which play a major role in the protein folding process in the ER, when glycosylated lysozymes were expressed in wild-type and calnexin-disrupted S. cerevisiae. The mRNA concentrations of BiP and PDI were evidently increased when the glycosylated lysozymes were expressed in calnexin-disrupted S. cerevisiae. This observation indicates that BiP and PDI may be induced by the accumulation of unfolded glycosylated lysozymes due to the deletion of calnexin.抄録全体を表示PDF形式でダウンロード (2415K) -
Norihiro Nakamura, Ge-Hong Sun-Wada, Akitsugu Yamamoto, Yoh Wada, Masa ...2001 年 130 巻 6 号 p. 765-771
発行日: 2001年
公開日: 2008/11/18
ジャーナル フリーSorting nexin 1 (SNX1) is a protein that binds to the cytoplasmic domain of plasma membrane receptors. We found that mouse sorting nexin 1 (SNX1) (521 amino acid residues) could partially rescue a yeast vam3 mutant defective in docking/fusion of vacuolar membranes. In mammalian cells, SNX1 is peripherally associated with membrane structures and localized immunochemically with EEA1, a marker protein of early endosomes. These results suggest that SNX1 regulates endocytic trafficking of plasma membrane proteins in early endosomes. Gel filtration of cell lysates and the purified recombinant protein, together with two-hybrid analysis, indicated that SNX1 self-assembles into a complex of _??_300 kDa.抄録全体を表示PDF形式でダウンロード (2771K) -
Atsushi Ikeda, Keiji Hasegawa, Megumi Masaki, Tetsuo Moriguchi, Eisuke ...2001 年 130 巻 6 号 p. 773-781
発行日: 2001年
公開日: 2008/11/18
ジャーナル フリーLeucine zipper-bearing kinase (LZK) is a novel member of the mixed lineage kinase (MLK) protein family, the cDNA of which was first cloned from a human brain cDNA library [Sakuma, H., Ikeda, A., Oka, S., Kozutsumi, Y., Zanetta, J. -P., and Kawasaki, T. (1997) J. Biol. Chem. 272, 28622-28629]. Several MLK family proteins have been proposed to function as MAP kinase kinase kinases in the c-Jun NH2 terminal kinase (JNK)/stressactivated protein kinase (SAPK) pathway. In the present study, we demonstrated that, like other MLKs, LZK activated the JNK/SAPK pathway but not the ERK pathway. LZK directly phosphorylated and activated MKK7, one of the two MAPKKs in the JNK/SAPK pathway, to a comparable extent to a constitutive active form of MEKK1 (MEKK1ΔN), suggesting a biological role of LZK as a MAPKKK in the JNK/SAPK pathway. Recent studies have revealed the essential roles of scaffold proteins in intracellular signaling pathways including MAP kinase pathways. JIP-1, one of the scaffold proteins, has been shown to be associated with MLKs, MKK7, and JNK [Whitmarsh, A. J., Cavanagh, J., Tournier, C., Yasuda, J., and Davis, R. J. (1998) Science 281, 1671-1674], suggesting the presence of a selective signaling pathway including LZK, MKK7, and JNK. Consistent with this hypothesis, we provided evidence that LZK is associated with the C-terminal region of JIP-1 through its kinase catalytic domain. In addition, LZK-induced JNK activation was markedly enhanced when LZK and JNK were co-expressed with JIP-1. These results constituted important clues for understanding the molecular mechanisms regulating the signaling specificities of various JNK activators under different cellular conditions.抄録全体を表示PDF形式でダウンロード (3521K) -
Keiko Kuzuya, Kuniyo Inouye2001 年 130 巻 6 号 p. 783-788
発行日: 2001年
公開日: 2008/11/18
ジャーナル フリーThermolysin is remarkably activated in the presence of high concentrations (1-5 M) of neutral salts [Inouye, K. (1992) J. Biochem. 112, 335-340]. The activity is enhanced 13-15 times with 4 M NaCI at pH 7.0 and 25°C Substitution of the active site zinc with other transition metals alters the activity of thermolysin [Holmquist, B. and Vallee, B. L. (1974) J. Biol. Chem. 249, 4601-4607]. Cobalt is the most effective among the transition metals and doubles the activity toward N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide. In this study, the effect of NaCI on the activity of cobalt-substituted thermolysin was examined. Cobalt-substituted thermolysin, with 2.8-fold increased activity compared with the native enzyme, is further activated by the addition of NaCl in an exponential fashion, and the activity is enhanced 13-15 times at 4M NaCl. The effects of cobalt-substitution and the addition of salt are independent of each other. The activity of cobalt-substituted thermolysin, expressed as kcat/Km, is pH-dependent and controlled by at least two ionizing residues with pKa, values of 6.0 and 7.8, the acidic pKa, being slightly higher compared to 5.6 of the native enzyme. These pKa, values remain constant in the presence of 4 M NaCl, indicating that the electrostatic environment of cobaltsubstituted thermolysin is more stable than that of the native enzyme, the acidic pKa, of which shifts remarkably from 5.6 to 6.7 at 4M NaCl. Zincov, a competitive inhibitor, binds more tightly to the cobalt-substituted than to native thermolysin at pH 4.9-9.0, probably because of its preference for cobalt in the fivefold coordination. The cobalt substitution has been shown to be a favorable tool with which to explore the active-site microenvironment of thermolysin.抄録全体を表示PDF形式でダウンロード (2258K) -
Kohji Takeuchi, Motonari Tsubaki, Junya Futagawa, Futoshi Masuya, Hiro ...2001 年 130 巻 6 号 p. 789-797
発行日: 2001年
公開日: 2008/11/18
ジャーナル フリーThe cholesterol side-chain cleavage reaction catalyzed by cytochrome P450scc com-prises three consecutive monooxygenase reactions (22R-hydroxylation, 20S-hydroxyla-tion, and C20-C22 bond scission) that produces pregnenolone. The electron equivalents necessary for the oxygen activation are supplied from a 2Fe-2S type ferredoxin, adreno-doxin. We found that 1:1 stoichiometric binding of oxidized adrenodoxin to oxidized cytochrome P450scc complexed with cholesterol or 25-hydroxycholesterol caused shifts of the high-spin EPR signals of the heme moiety at 5 K. Such shifts were not observed for the low-spin EPR signals. Ligation of CO or NO to the reduced heme of cytochrome P450scc complexed with reduced adrenodoxin and various steroid substrates did not cause any change in the axial EPR spectrum of the reduced iron-sulfur center at 77 K. These results are in remarkable contrast to those obtained for the cytochrome P450cam-d-camphor-putidaredoxin ternary complex, suggesting that the mode of cross talk between adrenodoxin and cytochrome P450sce is very different from that in the Pseudomonas system. The difference may be primarily due to the location of the charged amino acid residues of the ferredoxins important for the interaction with the partner cytochrome P450.抄録全体を表示PDF形式でダウンロード (2495K) -
Noriyuki Miyoshi, Yu Koyama, Yuko Katsuno, Sumio Hayakawa, Takashi Mit ...2001 年 130 巻 6 号 p. 799-805
発行日: 2001年
公開日: 2008/11/18
ジャーナル フリーEffects of rice bran agglutinin (RBA) on human monoblastic leukemia U937 cells wereexamined in comparison with those of wheat germ agglutinin (WGA) and Viscum album agglutinin (VAA). These lectins inhibit cell growth, and several lines of evidence indicatethat the growth inhibition is caused by the induction of apoptosis. We observed thatRBA induces chromatin condensation, externalization of membrane phosphatidylserine, and DNA ladder formation, features of apoptosis. DNA ladder formation was inhibitedby a general inhibitor against caspases, which are known to play essential roles in apoptosis. Flow cytometric analysis revealed that RBA and WGA cause G2IM. phase cell cyclearrest with increased expression of Wafl/p21, while cell cycle arrest was not observedfor VAA. These data indicate that RBA induces apoptosis associated with cell cyclearrest in U937 cells, and suggest that the induction mechanism for RBA is similar to thatfor WGA, but different from that for VAA.抄録全体を表示PDF形式でダウンロード (2515K) -
Hwan Young Lee, Kyu Bone Na, Hyun Min Koo, Yu Sam Kim2001 年 130 巻 6 号 p. 807-813
発行日: 2001年
公開日: 2008/11/18
ジャーナル フリーAcetyl-CoA synthetase (ACS) catalyses the activation of acetate to acetyl-CoA in the presence of ATP and CoA. The gene encoding Bradyrhyzobium japonicum ACS has been cloned, sequenced, and expressed in Escherichia coli. The enzyme comprises 648 amino acid residues with a calculated molecular mass of 71, 996 Da. The recombinant enzyme was also purified from the transformed E. coli. The enzyme was essentially indistinguishable from the ACS of B. japonicum bacteroids as to the criteria of polyacrylamide gel electrophoresis and biochemical properties. Based on the results of database analysis, Gly-263, Gly-266, Lys-269, and Glu-414 were selected for site-directed mutagenesis in order to identify amino acid residues essential for substrate binding and/or catalysis. Four different mutant enzymes (G263I, G266I, K269G, and E414Q) were prepared and then subjected to steady-state kinetic studies. The kinetic data obtained for the mutants suggest that Gly-266 and Lys-269 participate in the formation of acetyl-AMP, whereas Glu-414 may play a role in acetate binding.抄録全体を表示PDF形式でダウンロード (2525K) -
Saori Takahashi, Kazuyuki Hori, Keitaro Takahashi, Hironobu Ogasawara, ...2001 年 130 巻 6 号 p. 815-821
発行日: 2001年
公開日: 2008/11/18
ジャーナル フリーRenin-binding protein (RnBP) is an endogenous renin inhibitor originally isolated from porcine kidney as a complex of renin, so-called high molecular weight (HMW) renin. Our recent studies demonstrated that human RnBP is the enzyme N-acetyl-D-glucosamine (GlcNAc) 2-epimerase [Takahashi, S. et al. (1999) J. Biochem. 125, 348-353]. We have purified recombinant human, rat, and porcine RnBPs expressed in Escherichia coli JM 109 cells. The purified recombinant RnBPs existed as dimers and inhibited porcine renin activity strongly. On the other hand, porcine renin inhibited recombinant GIcNAc 2-epi-merase activities. The human GlcNAc 2-epimerase activity could not be detected in the absence of a nucleotide, whereas ATP, dATP, ddATP, ADP, and GTP enhanced the human GleNAc 2-epimerase activity. Other nucleotides had no effect on human GleNAc 2-epimerase activity. Rat and porcine GleNAc 2-epimerases were activated by several nucleotides. Nucleotides that enhance the activity of GIcNAc 2-epimerases protect these enzymes against degradation by thermolysin. These results indicate that mammalian RnBPs have GlcNAc 2-epimerase activity and that nucleotides are essential for formation of the catalytic domain of the enzyme.抄録全体を表示PDF形式でダウンロード (2220K) -
Eiko Sakai, Hisatsugu Miyamoto, Kuniaki Okamoto, Yuzo Kato, Kenji Yama ...2001 年 130 巻 6 号 p. 823-831
発行日: 2001年
公開日: 2008/11/18
ジャーナル フリーModifications occurring during the transformation of phagosomes into mature phagolysosomes were investigated in osteoclast-like cells (OCLs) and macrophages using latex beads as markers for the isolation of phagosomal compartments (LBC) at different time points after phagocytosis. In OCLs, newly formed LBC acquired cathepsin K, tartarateresistant phosphatase (TRAP), lysosome-associated membrane protein-1 (Lamp-1), and cathepsin D, and rapidly lost annexin II in a time-dependent manner. The levels of Rab7 and c-Src in OCLs initially increased and then gradually decreased during the transformation from early to late endosomal LBC or phagolysosomes. Receptor activator of NF-κB (RANKL) significantly increased the LBC levels of cathepsin K, TRAP, and c-Src, whereas calcitonin decreased the LBC levels of cathepsin K, TRAP, and Rab7, indicating that the transformation of early to late endosomal elements and lysosomes in OCLs is also regulated by osteoclastogenesis regulatory factors. On the other hand, changes in the LBC levels of Lamp-1, cathepsin D, and annexin II in macrophages were comparable to those in OCLs. However, contrary to osteoclastic LBC, Rab7 levels of macrophage LBC decreased in a time-dependent manner. Macrophage LBC were devoid of cathepsin K, TRAP, and c-Src in all transformation stages. These observations suggest that OCLs and macrophages have different phagosome maturation mechanisms that involve the specific and regulated acquisition of markers from endocytic organelles. The results also demonstrate that the use of LBC is a useful system in which to identify and characterize molecules involved in these different endocytic pathways.抄録全体を表示PDF形式でダウンロード (2520K) -
Masayuki Takeuchi, Atsuhiko Ishida, Isamu Kameshita, Takako Kitani, Sa ...2001 年 130 巻 6 号 p. 833-840
発行日: 2001年
公開日: 2008/11/18
ジャーナル フリーCalmodulin-dependent protein kinase phosphatase (CaMKP) dephosphorylates and concomitantly deactivates multifunctional Ca2+/calmodulin-dependent protein kinases (CaMKs), such as CaMKI, CaMKII, and CaMKIV. In the present study, a nuclear CaMKP-related protein, CaMKP-N, was identified. This protein consisted of 757 amino acid residues with a calculated molecular weight of 84, 176. Recombinant CaMKP-N dephosphorylated CaMKIV. The activity of CaMKP-N requires Mn2+ ions and is stimulated by polycations. Transiently expressed CaMKP-N in COS-7 cells was localized in the nucleus. This finding together with previous reports regarding localization of CaMKs indicates that CaMKP-N dephosphorylates CaMKIV and nuclear CaMKII, whereas CaMKP dephosphorylates CaMKI and cytosolic CaMKII.抄録全体を表示PDF形式でダウンロード (1992K) -
Akira Tsujimura, Kou Nunoue, Naokazu Inoue, Kyoko Shida, Mitsue Kurita ...2001 年 130 巻 6 号 p. 841-848
発行日: 2001年
公開日: 2008/11/18
ジャーナル フリーMurine CD46 (mCD46) is a type 1 membrane protein expressed predominantly in testicular germ cells, the distribution profile of which is in contrast to that of human CD46 showing a ubiquitous tissue distribution. We have identified an additional message of mCD46 that encodes a putative secretory form [Nomura et al. (1999) Immunogeneties 50, 245-254]. Here, we cloned three cDNAs encoding putative soluble CD46 from murine testis. These soluble form messages were yielded on insertion of unidentified nucleotide sequences, 77, 179, and 73 ntds, into the junctions between the SCR3 and SCR4 (variant 2), STc and UK (variant 3), and SCR4 and STc (variant 1) domains, respectively, the last one corresponding to the reported soluble form. The exons corresponding to these three inserts were identified in the murine CD46 genome, indicating that the alternative splicing of mRNA participates in the generation of these various CD46 messages. In normal mouse sera and cell lines, however, virtually no soluble CD46 was detected on immunoblotting. On Northern blotting analysis with specific probes, on the other hand, variant 1 was found to be predominantly expressed in the liver and heart. In addition, all variant messages were detected on PCR in all organs examined. When a rabbit cell line, RK13 cells, was transfected with cDNA of variant 1, protein synthesis was detected on immunoblotting. Although the mCD46 protein production was inefficient, this variant 1 exhibited factor I-cofactor activity as to inhibition of the complement cascade. Since the mCD46 protein was reported to be markedly up-regulated on infection of murine cells with mCMV, the soluble mCD46 proteins may act as a complement regulator that controls the systemic complement system under the conditions of a viral infection.抄録全体を表示PDF形式でダウンロード (2601K) -
Chika Yamashita, Yuri Aoyama, Mitsuhide Noshiro, Yuzo Yoshida2001 年 130 巻 6 号 p. 849-856
発行日: 2001年
公開日: 2008/11/18
ジャーナル フリーImmunohistochemical analysis showed that sterol 14-demethylase P450 (CYP51) is expressed in mature follicles and corpus lutea of rat ovaries. In follicles, CYP51 is expressed in granulosa and theca cells but not in oocytes. The ovarian CYP51 activity of hypophysectomized rats is very low and induced by pregnant mares' serum gonadotropin (PMSG) treatment together with ovarian growth. The expression of CYP51 first increases in growing follicles and then appears in the corpus lutea after luteinization. The former event may be due to the follicular-stimulating hormone action of PMSG, and the latter may be caused by the luteinizing hormone effect of PMSG. Sterol analysis indicated that the product of the CYP51-mediated lanosterol 14-demethylation, 4, 4-dimethylcholesta-8, 14, 24-trienol, which has been identified as a meiosis-activating steroid (MAS) in mammals [Byskov et al. (1995) Nature 374, 559-562], accumulates (about 10 pmol/mg of ovary) in mature rat ovaries, and the content is enough to activate the resumption of meiosis. These lines of evidence suggest that the expression of ovarian CYP51 is dependent on gonadotropins, and ovarian CYP51 activity is enough for accumulating MAS. Serum insulin does not affect the ovarian CYP51 level, although it is essential for hepatic CYP51 expression. These findings indicate that expression of CYP51 is regulated differently among organs.抄録全体を表示PDF形式でダウンロード (2527K) -
Mitsuhiro Kurata, Yoshimi Yamamoto, Shoji Watabe, Yumiko Makino, Kazuo ...2001 年 130 巻 6 号 p. 857-863
発行日: 2001年
公開日: 2008/11/18
ジャーナル フリーBombyx cysteine proteinase inhibitor (BCPI) is a novel cysteine proteinase inhibitor. The protein sequence is homologous to the proregions of certain cysteine proteinases. Here we report the mechanism of its inhibition of several cysteine proteinases. BCPI strongly inhibited Bombyx cysteine proteinase (BCP) activity with a Ki=5.9pM, and human cathepsin L with a Ki=6pM. The inhibition obeyedslow-binding kinetics. The inhibition of cathepsin H was much weaker (Ki=82nM), while inhibition of papain (Ki>1μm) and cathepsin B (Ki>4 μM) was negligible. Following incubation with BCP, BCPI was first truncated at the C-terminal end, and then gradually degraded over time. The truncation mainly involved twoC-terminal amino acid residues. Recombinant BCPI lacking the two C-terminal amino acid residues still retained substantial inhibitory activity. Our results indicate that BCPI is a stable and highly selective inhibitor of cathep-sin L-likecysteine proteinases.抄録全体を表示PDF形式でダウンロード (989K) -
Tetsuo Toraya, Masataka Eda, Takashi Kamachi, Kazunari Yoshizawa2001 年 130 巻 6 号 p. 865-872
発行日: 2001年
公開日: 2008/11/18
ジャーナル フリーCoenzyme B12 serves as a cofactor for enzymatic radical reactions. The essential steps in all the coenzyme B12-dependent rearrangements are two hydrogen abstraction steps: hydrogen abstraction of the adenosyl radical from substrates, and hydrogen back-abstraction (recombination) of a product-derived radical from 5'-deoxyadenosine. The energetic feasibility of these hydrogen abstraction steps in the diol dehyratase reaction was examined by theoretical calculations with a protein-free, simplified model at the B3LYP/6-311G_??_ level of density functional theory. Activation energies for the hydrogen abstraction and recombination with 1, 2-propanediol as substrate are 9. 0 and 15. 1 kcal/ mol, respectively, and essentially not affected by coordination of the substrate and the radical intermediate to K+. Since these energies can be considered to be supplied by the substrate-binding energy, the computational results with this simplified model indicate that the hydrogen abstraction and recombination in the coenzyme B12-dependent diol dehydratase reaction are energetically feasible.抄録全体を表示PDF形式でダウンロード (2614K) -
Yi-Min Lu, Kenichiro Miyazawa, Kou Yamaguchi, Kenichi Nowaki, Hiroyuki ...2001 年 130 巻 6 号 p. 873-878
発行日: 2001年
公開日: 2008/11/18
ジャーナル フリーT1 a mutant yeast lacking three regulatory proteins of F1 F0 ATPase, namely ATPase inhibitor, 9K protein and 15K protein, grew on non-fermentable carbon source at the same rate as normal cells but was less viable when incubated in water. During the incu-bation, the cellular ATP content decreased rapidly in the T1 cells but not in normal cells, and respiration-deficient cells appeared among the T1 cells. The same mutation was also induced in D26 cells lacking only the ATPase inhibitor. Overexpression of the ATPase inhibitor in YC63 cells, which were derived from the D26 strain harboring an expression vector containing the gene of the ATPase inhibitor, prevented the decrease of cellular ATP level and the mutation. Isolated T1 mitochondria exhibited ATP hydrolysis for maintenance of membrane potential when antimycin A was added to the mitochondria suspension, while normal and YC63 mitochondria continued to show low hydrolytic activity and low membrane potential. Thus, it is likely that deletion of the ATPase inhib-itor induces ATPase activity of F1 F0 ATPase to create a dispensable membrane potential under the non-nutritional conditions and that this depletes mitochondrial and cellular ATP. The depletion of mitochondrial ATP in turn leads to occurrence of aberrant DNA in mitochondria.抄録全体を表示PDF形式でダウンロード (1027K) -
Mayuko Okabe, Toshiharu Yakushi, Yukako Asai, Michio Homma2001 年 130 巻 6 号 p. 879-884
発行日: 2001年
公開日: 2008/11/18
ジャーナル フリーFour motor proteins, MotX, MotY, PomA, and PomB, have been identified as constituents of the Na+-driven flagellum of Vibrio species. In this study, the complete motX gene was cloned from Vibrio alginolyticus and shown to complement three mot mutations, motX94, motX115, and motX119, as well as a V. parahaemolyticus motX mutant. The motX94 mutant contains a frameshift at Va186 of MotX, while the motX115 and motX119 mutations comprise substitutions of Ala146 to Val and Gln 194 to amber, respectively. When MotX was overexpressed in Vibrio cells, the amount of MotY detected in the membrane fraction increased, and vice versa, suggesting that MotX and MotY mutually stabilize each other by interacting at the membrane level. When a plasmid containing the motX gene was introduced into motY mutants NMB117 (motY117) and V10542 (motY542), the mutations were suppressed. In contrast, motY could not cause the recovery of any swarm-defective motX mutants studied.Considering the above evidence, we propose that MotX is more directly involved than MotY in the mechanical functioning of the Na+-type flagellar motor, and thatMotY may stabilize MotX to support its interaction with other Mot proteins.抄録全体を表示PDF形式でダウンロード (2274K) -
Eri Kikkawa, Makiyo Hinata, Vincent W. Keng, Zaw Myint, Ayuko Sato, Ka ...2001 年 130 巻 6 号 p. 885-891
発行日: 2001年
公開日: 2008/11/18
ジャーナル フリーThe 5'-flanking region of the mouse Hex gene was examined in order to identify transcription factors regulating its expression in hepatocytes and haematopoietic cells. We have identified two further GC boxes (GC boxes 3 and 4 at nucleotide positions -149 to -140 and -79 to -70, respectively), i. e. in addition to the two previously determined ones (GC boxes 1 and 2 at nucleotide positions -197 to -188 and -176 to -167, respectively). Luciferase reporter assays revealed that all four GC boxes are transcriptionally active in both MH1, C1, rat hepatoma and K562 human chronic myelogenous leukemia cells. Electrophoretic mobility shift assays with specific competitors and antibodies showed that members of the Sp family, namely Spl and Sp3, bind to these GC boxes. Overexpression of Spl and Sp3 in Drosophila SL2 cells stimulated transcription of the Hex gene through interactions with GC boxes 1 to 4, Spl being a more potent activator than Sp3. Thus, we conclude that Spl and Sp3 stimulate transcription of the Hex gene in both MH1C1, and K562 cells.抄録全体を表示PDF形式でダウンロード (1112K)
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