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123 巻, 3 号
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So Iwata1998 年 123 巻 3 号 p. 369-375
発行日: 1998年
公開日: 2008/11/18
ジャーナル フリーThe crystal structure of cytochrome c oxidase from the soil bacterium Paracoccus denitrificans has been reported. This structure has provided a basis for understanding the mechanism of the redox-coupled transmembrane proton pump which is the key component of the respiratory chain in most aerobic organism. Over the past ten years, there have been many site-directed mutagenesis studies performed on bacterial oxidases. Structural features of Paracoccus oxidase have been summarized in the light of these mutagenesis studies and other structural works.抄録全体を表示PDF形式でダウンロード (2172K) -
Takashi Kuroha, Satoru Takahashi, Takuya Komeno, Ken Itoh, Toshiro Nag ...1998 年 123 巻 3 号 p. 376-379
発行日: 1998年
公開日: 2008/11/18
ジャーナル フリーMaf recognition elements (MAREs or NF-E2 binding sites) have been shown to be vital for erythroid- and megakaryocyte-specific gene expression. Transcription factor NF-E2 is composed of p45, a large subunit belonging to the CNC family proteins, and a small Maf subunit, and is thought to activate transcription through its binding to MAREs in both the erythroid and megakaryocytic cell lineages. While p45 gene knockout mice exhibit thrombocytopenia due to abnormal terminal differentiation of megakaryocytes, and the mutant mice die of massive bleeding within a week after birth, anemia is not apparent in these animals. Disruption of the nrf2 gene, encoding another CNC family protein, results in no hematological abnormalities. We have therefore tested the hypothesis that Nrf2 might compensate for the p45 deficiency in erythroid lineage cells of p45-knockout mice, thereby masking the anticipated anemia. However, we failed to detect any greater failure in either erythroid or megakaryocytic cell development in Nrf2 plus p45 compound mutant mice as compared to with either individual homozygous mutation. These data suggest that p45 and Nrf2 may both be dispensable for hematopoietic cell development, and that other factors regulate erythroid- and megakaryocyte-specific gene expression through their required MAREs.抄録全体を表示PDF形式でダウンロード (2898K) -
Peter Borowski, Lutz Kornetzky, Rainer Laufs1998 年 123 巻 3 号 p. 380-385
発行日: 1998年
公開日: 2008/11/18
ジャーナル フリーTreatment of A431 cell membranes with trypsin or Streptomyces griseus proteinase results in degradation of the EGF-R and the concomitant generation of an active kinase with a molecular mass of 42 kDa (42 kDa kinase). To investigate the biochemical properties of the 42 kDa kinase, the EGF-R was immunoaffinity-purified from the A431 cell membranes and the kinase proteolytically generated. The proteolysis of EGF-R changes both the Vmax and the Michaelis constants of substrates. These substrates determine the extent of the changes of the parameters. The 42 kDa kinase is less responsive to polyions as regulators of kinase activity and is less efficiently inhibited by genistein and tyrphostin. The experiments described here point to a role of the extracatalytic domains in determining the substrate specificity and regulation of kinase activity.抄録全体を表示PDF形式でダウンロード (3130K) -
Lawrence S. H. Wu, Lian-Du Wang, Peng-Wen Chen, Liang-Jwu Chen, Jason ...1998 年 123 巻 3 号 p. 386-391
発行日: 1998年
公開日: 2008/11/18
ジャーナル フリーOleosins are hydrophobic proteins localized abundantly in the oil bodies of plant seeds. Two distinct oleosin isoforms of molecular masses 18 and 16 kDa are present in rice oil bodies. These isoforms were found in similar ratio in rice embryos and aleurone layers. To survey potential DNA sequences involved in the activation of oleosin genes, a genomic clone of rice 18 kDa oleosin was sequenced, and its 5'-flanking region was compared with that of the known rice 16 kDa oleosin gene. Corresponding mRNAs of the two rice oleosin isoforms appeared seven days after pollination and vanished in mature seeds. Triacylglycerols and oleosins were accumulated concomitantly in maturing rice reeds in accord with the active assembly of oil bodies, and partly mobilized in postgerminative seedlings. Approximately 60% of the stored triacylglycerols in rice were not utilized: while the majority of oil bodies in embryos were mobilized in five days after imbibition, those in aleurone layers remained intact in postgerminative seedlings.抄録全体を表示PDF形式でダウンロード (4189K) -
Noboru Nakamichi, Douglas D. Rhoads, Jun-Ichi Hayashi, Yasuo Kagawa, T ...1998 年 123 巻 3 号 p. 392-398
発行日: 1998年
公開日: 2008/11/18
ジャーナル フリーThe mitochondrial regulatory region (mrr) located between the tRNAPhe and tRNAPro genes of mitochondrial DNA (mtDNA) is essential for regulation of replication and transcription of the mitochondrial genome. Polyadenylated short RNAs complementary to the L-strand of the mrr in human cells and similar RNAs (polyadenylation status unknown) in rat and mouse cells have been reported. We now report detection of ca. 0.2 kb polyadenylated mrrRNAs in cultured cells of Chinese hamster, African green monkey, mouse, rat, and human. We isolated a cDNA clone to a rat polyadenylated mrrRNA of 158 by in length excluding the polyadenyl tail, which spans the region from the light strand promoter (LSP) to the origin of heavy strand replication (OriH). This cDNA contains both an open reading frame encoding a 26 amino acid polypeptide and a 12 nucleotide sequence complementary to the 3'-terminus of rat mitochondrial 12S rRNA. A cDNA clone to a human HeLa cell polyadenylated mrrRNA also contains a 12 nucleotide region complementary to the human mitochondrial 12S rRNA. We used a mitochondrial genome-deficient HeLa cell line, ρ0 HeLa, and a derived cybrid cell line, HeEB, with a reconstituted mitochondrial genome, to demonstrate that the occurrence of the mrrRNA is dependent on the presence of a mitochondrial genome, and these polyadenylated mrrRNAs are transcribed from the mitochondrial genome. Our results further substantiate the common existence of polyadenylated mrrRNAs among mammals and support previously proposed hypotheses for the multi-functional nature of polyadenylated mrrRNA.抄録全体を表示PDF形式でダウンロード (3021K) -
Michiyo K. Sakamoto, Kuniaki Suzuki, Shigeharu Takiya, Yoshitaka Yoshi ...1998 年 123 巻 3 号 p. 399-407
発行日: 1998年
公開日: 2008/11/18
ジャーナル フリーThe cAMP-responsive element (CRE) binding protein/activating transcription factor (CREB/ATF) family plays a major role in the expression of skeletal-specific genes and skeletal tissue development. We analyzed the changes of the amount, degree of phosphorylation and binding activity of the CREB/ATF family in the course of development of the murine calvarial osteoblastic cell line MC3T3-El as an in vitro model system of bone formation. The amount of CREB in the whole-cell extract detectable by Western blot analysis was high through all stages of development and maximal in the proliferation stage. The degree of phosphorylation estimated with anti-phosphorylated CREB antibody changed greatly and reached high levels in the proliferation stage and early mineralization stage. The ratio of phosphorylated CREB to total CREB in the CREB-CRE complex was also examined by gel shift assay. Although the binding to the consensus/CRE probe reached almost equally high levels in the proliferation stage and early mineralization stage, the relative level of phosphorylated CREB in the CREB-CRE complex was different in these two stages. In the early mineralization stage, most CREB bound to consensus/CRE was phosphorylated, while both phosphorylated and unphosphorylated CREB were bound to consensus/CRE in the proliferation stage. ATF-1 was also detected as a minor component bound to the consensus/CRE probe. The alteration of the binding of CREB to consensus/CRE over the course of osteoblast development supports the hypothesis that CREB may regulate the expression of genes defining the developmental sequence of MC3T3-E1 cells.抄録全体を表示PDF形式でダウンロード (7582K) -
Nicolle Sitte, Wolfgang Dubiel, Peter-Michael Kloetzel1998 年 123 巻 3 号 p. 408-415
発行日: 1998年
公開日: 2008/11/18
ジャーナル フリーAn ATP-dependent protease in the intermembrane space of rat liver mitochondria, MISP I (mitochondrial intermembrane space protease), was partially purified and characterised. The protease complex has a molecular mass of 200 kDa and appears to be an oligomeric enzyme complex. The proteolytic activity of the enzyme can be stimulated up to 3-fold by Mg2+ATP. The Km for ATP is 200 μM. Nucleoside triphosphates, but not ADP, AMP, or nonhydrolysable ATP analogues, can substitute for ATP. The protease exhibits multicatalytic properties with chymotrypsin-like, peptidyl-glutamyl-hydrolysing, and trypsinlike activities. Of the latter the trypsin-like activity is not enhanced by ATP. In addition to the hydrolysis of fluorogenic peptide substrates the protease is able to degrade radiolabeled model proteins. The ATP-dependent mitochondrial protease was characterised as a cysteine protease sensitive to hemine. The cross reactivity of an anti-human-S4 antibody raised against an ATPase subunit of the PA700 complex with a component of MISP I indicated a structural relationship. Furthermore, ATP-agarose-binding assays revealed the connection of the peptide hydrolysing activity with an ATP binding domain. The data presented here and a comparison with known ATP-dependent mitochondrial proteases demonstrated that MISP I represents a novel ATP-dependent protease in the mitochondrial intermembrane space of rat liver.抄録全体を表示PDF形式でダウンロード (3259K) -
Yaw-Kuen Li, Hsuan-Shu Hsu, Li-Fen Chang, Genly Chen1998 年 123 巻 3 号 p. 416-422
発行日: 1998年
公開日: 2008/11/18
ジャーナル フリーSeries of 4-arylimidazoles, ω-N-acylhistamines and 4-(ω-phenylalkyl) imidazoles were synthesized in order to probe the active site topology of sweet almond β-glucosidase. These imidazole derivatives were shown to be very powerful competitive inhibitors. Among the 20 tested compounds, ω-N-benzoylhistamine and 4-(3'-phenylpropyl) imidazole are the most potent inhibitors of the enzyme, with pH-independent Ki values of 0.06 and 0.07 μM, respectively. The inhibition of 4-(ω-phenylalkyl) imidazoles exhibited an interesting trend as to Ki values: 4-phenylimidazole (6.6 μM)>4-benzylimidazole (1.4 μM)>4-(2'-phenylethyl) imidazole (0.82 μM)>4-(3'-phenylpropyl) imidazole (0.07 μM)<4-(4'-phenylbutyl) imidazole (0.13 μM)<4-(5'-phenylpentyl) imidazole (0.3 μM). This revealed that the imidazole and aryl binding sites (which result from favorable interactions within the corresponding glycone and aglycone binding subsites) are separated by the optimal distance equivalent to the length of a -CH2-CH2-CH2-group. Substitutions of the phenyl moieties of 4-phenylimidazole and 4-benzoylhistamine result in weaker inhibition. These classes of imidazoles are particularly powerful inhibitors of sweet almond β-glucosidase.抄録全体を表示PDF形式でダウンロード (741K) -
Toshihiko Ikeuchi, Atsushi Nakatani, Masashi Yamada, Naoya Itokazu, Ak ...1998 年 123 巻 3 号 p. 423-430
発行日: 1998年
公開日: 2008/11/18
ジャーナル フリーAlthough NGF (nerve growth factor) induces neuronal differentiation of PC12 cells, EGF (epidermal growth factor) acts as a mitogen for these cells. We have studied the effects of a synthetic pyrimidine derivative, MS-430, on the NGF and EGF actions on PC12h cells. We found that MS-430 accelerated NGF-induced neurite extension of PC12h cells and that, in the presence of MS-430, PC12h cells extended neurites in response to EGF. Next, we investigated the tyrosine phosphorylation of NGF receptor TrkA and the EGF receptor (EGFR) as well as mitogen-activated protein kinase (MAPK), which is a key protein in the intracellular signal transduction pathway. It was found that MS-430 prolonged the EGF-induced phosphorylation of EGFR and MAPK compared to that without MS-430. MS-430 also prolonged the NGF-induced phosphorylation of MAPK, but the phosphorylation of TrkA induced by NGF was not affected by MS-430. These results suggest that MS-430 influences the intracellular signal transduction pathway which causes the neuronal differentiation of PC12h cells.抄録全体を表示PDF形式でダウンロード (4256K) -
Kazuhiro Sasaki, Takako Hattori, Takuo Fujisawa, Kojiro Takahashi, Haj ...1998 年 123 巻 3 号 p. 431-439
発行日: 1998年
公開日: 2008/11/18
ジャーナル フリーWe recently reported that nitric oxide (NO), which is produced by chondrocytes treated with interleukin-1β (IL-1), releases basic fibroblast growth factor (bFGF) stored in the matrix of articular chondrocytes. To clarify the mechanism of the IL-1-induced bFGF release, we investigated the production and gene expression of bFGF, matrix metalloproteinases (MMPs), syndecan 3, and inducible NO synthase (iNOS) by IL-1-treated rabbit articular chondrocytes. IL-1 stimulated not only the release of bFGF but also the production of it. Gelatin and casein zymography revealed that IL-1 stimulated the production of not only MMP-9 but also MMP-3. The increase in the production of these MMPs preceded the IL-1-stimulated bFGF release. An MMP inhibitor partially suppressed the release of bFGF, indicating that matrix degradation is at least partially involved in the IL-1-stimulated bFGF release even if increased production of bFGF is related to the release. IL-1 sequentially stimulated mRNA expression of iNOS, membrane type 1-MMP, MMP-9 and -3, and bFGF, in that order. NG-Monomethyl-L-arginine, an inhibitor of NO production, inhibited gene expression of MMP-9 and bFGF. These findings suggest that elevation of the NO level via iNOS mRNA expression stimulated by IL-1 mediates gene expression and production of MMPs and bFGF, resulting in the release of bFGF, and also reveal molecular mechanisms implicating the degradation of articular cartilage followed by angiogenesis in the synovium in arthritic joints.抄録全体を表示PDF形式でダウンロード (3855K) -
Kenji Takase, Kiyoshi Hagiwara1998 年 123 巻 3 号 p. 440-444
発行日: 1998年
公開日: 2008/11/18
ジャーナル フリーα-Lactalbumin (αLA), a major milk protein, is the regulatory subunit of lactose synthase. To assess the production of recombinant αLA in plants, the cDNAs for human αLA with or without its own signal sequence were introduced into tobacco plants under the control of the cauliflower mosaic virus 35S promoter. The gene integration and expression at the mRNA level were confirmed in several regenerated plants, while the expression at the protein level could be confirmed only in a transgenic tobacco transformed with the gene containing the signal sequence. The tobacco-expressed αLA migrated in SDS-PAGE with identical mobility to αLA prepared from human milk, indicating that the signal peptide of human αLA was correctly processed to yield a mature protein in tobacco plants. The expressed αLA (ca. 5 μg/g of fresh leaves) was found in the soluble fraction and eluted from a DEAE-Sepharose column in the same salt concentration range as the milk aLA. The partially purified tobacco-αLA was fully active in the synthesis of lactose when combined with galactosyltransferase. Thus, the transgenic tobacco produces a fully active mature αLA in a soluble form.抄録全体を表示PDF形式でダウンロード (2952K) -
Michaela Stoltz, Roderich Brandsch1998 年 123 巻 3 号 p. 445-449
発行日: 1998年
公開日: 2008/11/18
ジャーナル フリーThe contribution of (8α)-(N3) histidyl bond formation to the conformation of covalently flavinylated proteins was investigated by trypsin treatment of wild type and mutant versions of a model enzyme, 6-hydroxy-D-nicotine oxidase (6-HDNO) of Arthrobacter nicotinovorans. In the absence of FAD, apo-6-HDNO exhibited a conformation exposing a protease accessible site. Holoenzyme formation through FAD-attachment to His71 induced a conformational change in the protein that shielded the trypsin recognition site. This conformational change, however, did not require FAD-histidyl bond formation since trypsin resistance was also exhibited by a 6-HDNO•Cys71 mutant protein which was unable to bind FAD covalently. Replacement of Arg67, an amino acid residue supposed to be essential in flavinylation, by Ala rendered the protein protease sensitive as did replacement of Pro73 by Ala. These amino acids apparently play an essential role in stabilizing the native protein conformation. The inability to reach the native conformation also prevented FAD attachment, indicating that a specific conformation of the protein is a prerequisite for FAD-histidyl bond formation. Deletion of Phe448 and Arg449 from the 458 amino acid residuescontaining enzyme resulted in complete protease sensitivity, demonstrating that flavinylation takes place posttranslationally.抄録全体を表示PDF形式でダウンロード (2887K) -
Tsutomu Mikawa, Ryoji Masui, Seiki Kuramitsu1998 年 123 巻 3 号 p. 450-457
発行日: 1998年
公開日: 2008/11/18
ジャーナル フリーThe single-stranded DNA-dependent ATPase activity of Escherichia coli RecA protein, especially its cooperativity for ATP, was investigated. To measure the ATPase activity in detail, the methods and reaction conditions for the ATPase assay were reexamined. Under conditions where RecA protein always showed a maximal rate of ATP hydrolysis, its poly (dT)-dependent ATPase activity was measured. At 25°C, increasing the concentration of RecA protein from 0.3 to 1.0 μM increased the turnover number (hat) from 0.16 to 0.19 s-1 and the Hill coefficient (nH) for ATP from 9.3 to 11.6. At 0.5 μM RecA protein, increasing the temperature from 25 to 37°C increased loot from 0.18 to 0.35 s- but decreased nH from 9.8 to 6.6. Interestingly, the ATPase activity of RecA protein measured in this study showed much higher cooperativity for ATP than those reported to date. Furthermore, the nH value of 11.6 for ATP obtained here was the highest of any ATPase reported so far. These results suggest that the binding of an ATP molecule to a RecA molecule within a nucleoprotein helical filament causes structural change of many other neighboring RecA molecules. This implies that ATP binding induces structural change of the whole nucleoprotein helical filament. Finally, we demonstrated that analysis of cooperativity is useful for revealing how a protein composed of many subunits functions as a whole.抄録全体を表示PDF形式でダウンロード (1873K) -
Taeko Nishiwaki, Nobuaki Maeda, Masaharu Noda1998 年 123 巻 3 号 p. 458-467
発行日: 1998年
公開日: 2008/11/18
ジャーナル フリーrotein tyrosine phosphatase ζ (PTP ζ/RPTPζ) is a receptor-like protein tyrosine phosphatase specifically expressed in the brain. Alternative splicing produces three isoforms of this molecule: PTPζ-A, the full-length form of PTPζ; PTPζ-B, the short form of PTPζ; and PTPζ-S, an extracellular variant. Here, we identified all these isoforms, including PTPζ-B, as chondroitin sulfate proteoglycans, and characterized their carbohydrate modification and expression profiles in the rat brain. The level of PTPζ-A expression was maintained during the prenatal period and decreased rapidly after birth. PTPζ-S was expressed in a similar manner, although the postnatal decrease was gradual. In contrast, relatively constant amounts of PTPC-B were observed from embryonic day 13 (E13) through adulthood. PTPζ-A and -S were constantly expressed only as proteoglycans during development, but a substantial amount of PTPζ-B was detected in a non-proteoglycan form at E13-15. Moreover, PTPζ-B did not contain Lex, HNK-1 carbohydrate, or keratan sulfate, although PTPζ-A and -S were generally modified with these carbohydrates. L cells transfected with PTPζ-A and -B cDNAs expressed these proteins as enzymatically active chondroitin sulfate proteoglycans. The PTPζ-A and -B in L cells showed essentially similar localizations in cell cortical structures on immunofluorescence microscopy, although immature or processed forms of PTPζ-A were accumulated additively in intracellular patchy structures. These results show that the three isoforms of PTPζ are differentially regulated during development, and that the extracellular deleted region in PTPζ-B is important for determination of carbohydrate modification.抄録全体を表示PDF形式でダウンロード (5437K) -
Yasuhiro Hashimoto, Minoru Suzuki, Paul R. Crocker, Akemi Suzuki1998 年 123 巻 3 号 p. 468-478
発行日: 1998年
公開日: 2008/11/18
ジャーナル フリーWe prepared a streptavidin-based neoglycoprotein which carries more than 140 GT1b oligosaccharides. GT1b oligosaccharides were covalently coupled to streptavidin by reductive amination, yielding a monomer form of streptavidin carrying 13 oligosaccharides. The monomer form of glycosylated streptavidin was polymerized with biotinylatedbovine serum albumin, which yielded a polymer carrying more than 140 oligosaccharides. Both the monomer and the polymer bound to Chinese hamster ovary cells expressing murine sialoadhesin. The relative binding potencies determined with the polymer, monomer, and free GT1b oligosaccharides were 3, 500, 83, and 1, respectively, indicating that an increase in the number of oligosaccharide ligands is critical for high avidity. The high avidity of the polymer enabled us to develop a sensitive and quantitative binding assay, and the assay was applied to characterization of the binding specificity of sialoadhesin. The polymer binding was inhibited by various gangliosides, the order of the inhibitory potencies being GM3 (IC50=40 μM)>GD1a (100 μM)>sialylparagloboside (120 μM)>GT1b (310 μM)>GM2 (640 μM)>GM4 (2, 100 μM)>GD1b>LacCer=GM1=paragloboside (no inhibition). These results indicate that the binding specificity is comparable to that reported, i.e. the determinant structure is NeuAcα2-3Galβ1-linked to either 3GalNAc, 3(4)GlcNAc, or 4Glc, and that the oligosaccharide structure on the polymer is properly presented to sialoadhesin on the cell surface. To determine the precise requirement of the NeuAc structure for binding, NeuAc of GM3 was converted into various derivatives, the inhibitory potencies of which were examined; i.e. GM3 containing NeuAc, IC50=40 μM; C7-or C8-aldehyde, 500 μM; C7-or C8-alcohol, 700 μM; C1-alcohol, 2, 000 μM; C1-amide, 2, 200 μM; and NeuGc, >3, 000 μM. These results confirmed the requirement of the hydroxyl group at C9 and/or C8, the carboxyl group at C1, and the methyl group of the N-acetyl residue of NeuAc in a quantitative manner. Thus, this streptavidin-based neoglycoprotein is a useful multivalent glycoprobe, which exhibits high affinity and specificity to murine sialoadhesin on the cell surface.抄録全体を表示PDF形式でダウンロード (3291K) -
Kouichi Yoshinari, Kiyoshi Nagata, Makoto Ogino, Ken-ichi Fujita, Tosh ...1998 年 123 巻 3 号 p. 479-486
発行日: 1998年
公開日: 2008/11/18
ジャーナル フリーA cDNA of amine sulfotransferase-RB1 (AST-RB1), which efficiently catalyzes 4-phenyl-1, 2, 3, 6-tetrahydropyridine (PTHP) sulfation, has been isolated by immunoscreening of a rabbit liver cDNA library. The cDNA consisted of 1, 117 base pairs and encoded a protein of 301 amino acids with a molecular weight of 35, 876. The deduced amino acid sequence matched at six positions those of peptide fragments obtained from purified AST-RB1 protein. The sequence had less than 38% identity at the amino acid level with cytosolic sulfotransferases in mammals, although high degrees of similarity were observed with regions conserved throughout mammalian sulfotransferases. These results indicate that AST-RB1, arbitrarily named sulfotransferase 3A1 (ST3A1), constitutes a new and third gene family of cytosolic sulfotransferases in mammals. ST3A1 expressed in Escherichia coli as a fused protein catalyzed sulfation of amines such as PTHP, aniline, 4-chloroaniline, 2-naphthylamine, and desipramine, but barely O-sulfation of typical aryl and hydroxysteroid sulfotransferase substrates. These data unequivocally demonstrate the existence of a cytosolic sulfotransferase showing a high selectivity for amine substrates, and indicate that multiple forms of sulfotransferase mediate sulfation of xenobiotics in mammalian livers.抄録全体を表示PDF形式でダウンロード (3257K) -
Takeshi Mikami, Motoi Kashiwagi, Keiko Tsuchihashi, Takumi Daino, Toyo ...1998 年 123 巻 3 号 p. 487-491
発行日: 1998年
公開日: 2008/11/18
ジャーナル フリーEquine brain gangliosides were isolated and their structures were characterized, to examine whether equine brain has N-glycolyl neuraminic acid in gangliosides, since other mammals predominantly possess N-acetyl neuraminic acid in brain gangliosides, and equine erythrocytes and organs except the brain have gangliosides exclusively containing N-glycolyl neuraminic acid. The gangliosides purified from the brain were identified by proton NMR spectroscopy and mass spectrometry, as well as GLC, resulting in their identification as GM4, GM3, GM2, GM1, GD1a, GD1b, and GT1b. Of these gangliosides, GM3 possessed N-glycolyl neuraminic acid as a minor component (18% of the total GM3), whereas other gangliosides exclusively contained N-acetyl neuraminic acid. The N-glycolyl neuraminic acid residue of the GM3 was confirmed by TLC immunostaining. The possibility of contamination of the GM3 by erythrocytes was eliminated based on the finding that the lipid compositions were characteristic of brain gangliosides. The presence, even as a minor component, of the N-glycolyl neuraminic acid in equine brain gangliosides is exceptional among the sialic acid species in mammalian central nervous system.抄録全体を表示PDF形式でダウンロード (3116K) -
Yoshihiko Kawarada, Naoyuki Miura, Toshihiro Sugiyama1998 年 123 巻 3 号 p. 492-498
発行日: 1998年
公開日: 2008/11/18
ジャーナル フリーOur previous study demonstrated that an antibody against single-stranded DNA could detect apoptotic cells [Naruse et al. (1994) Histochemistry 101, 73-78]. In this paper we describe the development of an improved method for the production of the antibody and investigations into the antigenic determinants of the antibody so that it could be of practical use for detecting apoptotic cells. Rabbits, hyperimmunized with complexes of alkaline-denatured calf thymus DNA and methylated bovine serum albumin, produced an IgG antibody to single-stranded DNA. Analysis by sandwich ELISA using various naturally occurring nucleic acids revealed that the antibody was specific to singlestranded DNA. Furthermore, using synthetic polymers in the assay, it was found that the antibody could recognize single-stranded DNA with various base sequences. Gel electrophoresis retardation assays, with synthetic oligodeoxynucleotides with differing lengths of single-stranded DNA, indicated that a hexadeoxynucleotide constituted the minimum size of the antigenic determinants, and suggested that the antibody probably consists of several antibodies which recognize hexadeoxynucleotides with various base sequences. Western blot analysis demonstrated that the antibody can recognize both a DNA ladder and oligonucleosomes prepared from rat liver nuclei with endogenous endonuclease. The present findings demonstrate that this antibody is a useful tool for detecting apoptotic cells.抄録全体を表示PDF形式でダウンロード (2828K) -
Naoto Tsuchiya, Daisuke Kamei, Akira Takano, Takaaki Matsui, Michiyuki ...1998 年 123 巻 3 号 p. 499-507
発行日: 1998年
公開日: 2008/11/18
ジャーナル フリーWe isolated a cDNA encoding a novel heterogeneous nuclear ribonucleoprotein (hnRNP)-like protein on DNA affinity screening of a K562 cDNA expression library with an oligodeoxynucleotide (JKT41) derived from intron 9 of the human myeloperoxidase gene. The cDNA has a 1, 305 by sequence that encodes a polypeptide of 301 amino acid residues. The protein, named JKTBP, contains two repeats of a putative RNA binding domain (RBD), each composed of canonical RNP-2 and RNP-1 motifs, and a glycine- and tyrosine-rich carboxyl terminus. The sequences of these two repeats are highly homologous with those of the 2×RBD-Gly rich group of hnRNPs. Northern blotting showed that two mRNAs of approximately 1.4 and 2.8 kb were present in most cultured cells examined. The recombinant protein expressed in Escherichia coli interacted with the double-stranded form of JKT41 as well as with its single-stranded form. This interaction was competitively inhibited by the same unlabeled JKT41 and to nearly the same extent by unrelated oligonucleotides. Moreover, the recombinant protein interacted with poly (G) and poly (A), but not with poly (U) or poly (C). Transient expression of the protein in SKM-1 cells repressed the expression of chloramphenicol acetyltransferase reporter genes located downstream of the intron 9 element of JKT41 or intron 7 element of FERE27. The implications of the protein in the biogenesis of mRNA are discussed.抄録全体を表示PDF形式でダウンロード (3435K) -
Masayasu Takada, Koichi Ogawa, Sanshirou Saito, Takeomi Murata, Taichi ...1998 年 123 巻 3 号 p. 508-515
発行日: 1998年
公開日: 2008/11/18
ジャーナル フリーWe performed chemo-enzymatic transformation of maltooligosaccharides into both endmodified oligosaccharidonolactones of potential use as substrate analogue inhibitors for mammalian α-amylases. Enzymatic modification of the non-reducing end glucosyl residue of the maltooligosaccharide was first performed by transglycosylation with β-D-galactosidase from Bacillus circulans. When maltotriose and maltotetraose were the acceptors, the enzyme regioselectively synthesized 43-O-β-D-galactosyl maltotriose (LG3) and 44-O-β-D-galactosyl maltotetraose (LG4) from lactose as a donor. LG4 was further selectively hydrolyzed with a specific a-amylase to afford 42-O-β-D-galactosyl maltose (LG2). The anomer hydroxyl groups of LG2 and LG3 were chemically oxidized to give the corresponding lactones, 42-O-β-D-galactosyl maltobionolactone (LG2O) and 43-O-β-D-galactosyl maltotrionolactone (LG3O), respectively. LG2O and LG3O, which are competitive inhibitors for mammalian α-amylases, exhibited Ki values of the order of 2.8-18.0 μM, with p-nitrophenyl α-maltopentaoside (G5P) as the substrate. On 1H-NMR analysis, these oligosaccharidonolactones were shown to be transformed into the corresponding aldonic acid forms with time in an aqueous solution. In this case, the lactone form was essential for the occurrence of the α-amylase inhibitor.抄録全体を表示PDF形式でダウンロード (750K) -
Ippei Nishimura, Makoto Uchida, Yusuke Inohana, Keiji Setoh, Kurumi Da ...1998 年 123 巻 3 号 p. 516-520
発行日: 1998年
公開日: 2008/11/18
ジャーナル フリーIt was previously revealed [Yamaguchi, H. and Uchida, M. (1996) J. Biochem. 120, 474-477] that both intra- and extramolecular high-mannose type Asn-glycans promote the renaturation of reductively denatured bovine pancreatic RNases A and B under oxidation conditions. To characterize the conformational changes of the polypeptides during the renaturation promoted by the intramolecular Asn-glycans, RNase B was compared with its nonglycosylated form, RNase A, as to the features of the regeneration from their reductively denatured species under Cu2+-catalyzed oxidation conditions. The refolding intermediates of RNase B, as compared with those of RNase A, seemed to contain much less impaired disulfide linkages. In agreement with this finding, the proper refolding of RNase B was much faster than that of RNase A, as revealed by the intrinsic fluorescence and 1-anilino-8-naphthalenesulfonate binding of the refolding intermediates. Such a promoting effect was also observed for extramolecular Asn-glycans of the complex as well as of the highmannose type. In contrast, common mono-, oligo-, and polysaccharides, but not yeast mannan, exhibited much lower stimulatory effects on the oxidative refolding of RNase A.抄録全体を表示PDF形式でダウンロード (490K) -
Yasuhiro Nakajima, Taketomo Fujiwara, Yoshihiro Fukumorit1998 年 123 巻 3 号 p. 521-527
発行日: 1998年
公開日: 2008/11/18
ジャーナル フリーA ferredoxin was purified from the hyperthermophilic archaeon, Pyrobaculum islandicum. EPR spectra and metal content analyses suggested that the ferredoxin molecule contained one [3Fe-4S] and one [4Fe-4S] cluster. The ferredoxin was rapidly reduced by 2-oxoglutarate: ferredoxin oxidoreductase purified from P. islandicum, indicating that it functions physiologically as an electron sink for the redox enzymes participating in glycolytic metabolism. Furthermore, the amino acid sequence of the P. islandicum ferredoxin was compared with those of several other bacterial ferredoxins.抄録全体を表示PDF形式でダウンロード (725K) -
Yoshiya Ikawa, Hideaki Shiraishi, Tan Inoue1998 年 123 巻 3 号 p. 528-533
発行日: 1998年
公開日: 2008/11/18
ジャーナル フリーThe Tetrahymena group I self-splicing intron contains peripheral domains P2-2.1. Mutant introns lacking these domains are hardly active. We found that if an independently prepared P2-2.1 RNA is added in trans, it efficiently enhances the catalytic activity of an intron lacking the domains. P2-2.1 RNA together with the previously identified activator, P5abc RNA, of the Tetrahymena intron can activate the intron lacking both of them. The trans-activation depends on the long-range interaction between P2.1 and P9.1 domains.抄録全体を表示PDF形式でダウンロード (2673K) -
Werner G. Siems, Alexej M. Pimenov, Hermann Esterbauer, Tilman Grune1998 年 123 巻 3 号 p. 534-539
発行日: 1998年
公開日: 2008/11/18
ジャーナル フリーThe metabolism of the aldehydic lipid peroxidation product, 4-hydroxynonenal (HNE), was studied in suspensions of mouse thymocytes. Thymocytes are characterized by low lipid peroxidation in comparison with other cell types notwithstanding their high content of arachidonic acid. In our study a very high capacity of HNE metabolism in thymocytes was observed: 27.7 nmol/mg w. w./min. That is about the same HNE degradation rate as determined in liver cells or small intestinal enterocytes, which are the cells with the by far highest capacity for the degradation of HNE and other aldehydic lipid peroxidation products in comparison with other cell types. The primary and secondary HNE metabolites in thymocytes were identified and quantified after the addition of 100 pM HNE to thymocyte suspensions: the glutathione-HNE conjugate, the hydroxynonenoic acid, the 1, 4-dihydroxynonene, water, and the glutathione-dihydroxynonene conjugate. Furthermore, the HNE binding to proteins was measured. The very rapid HNE degradation in thymocytes besides the high amounts of lipophilic chain-breaking antioxidants is postulated to be an important secondary antioxidative mechanism and the main factor for the low accumulation of lipid peroxidation products in these cells.抄録全体を表示PDF形式でダウンロード (3000K) -
Tomoko Ishino, Sumio Ohtsuki, Ko-ichi Homma, Shunji Natori1998 年 123 巻 3 号 p. 540-545
発行日: 1998年
公開日: 2008/11/18
ジャーナル フリーA cDNA for mouse prolyl endopeptidase (PEP) was cloned and its nucleotide sequence determined. The overall amino acid sequence identity between mouse and other mammalian PEPs was about 96%. A specific inhibitor of PEP, N-benzyloxycarbonyl-thioprolyl-thioprolinal-dimethylacetal (ZTTA), inhibited DNA synthesis by Swiss 3T3 cells. Mouse PEP was shown to be localized partly in restricted nuclear regions. These results suggest that PEP participates in mammalian DNA synthesis.抄録全体を表示PDF形式でダウンロード (2869K) -
Izumi Yoshino-Yasuda, Keiko Kobayashi, Masashi Akiyama, Hirotaka Itoh, ...1998 年 123 巻 3 号 p. 546-554
発行日: 1998年
公開日: 2008/11/18
ジャーナル フリーAs reported previously, caldecrin, a serum calcium-decreasing factor, from pancreas was found to be a serine protease, but the proteolytic activity was not necessary for its serum calcium decreasing activity. The caldecrin cDNA encoded a protease zymogen of the chymotrypsin/elastase superfamily consisting of a signal peptide, an activation peptide and a mature enzyme. On a homology search, we found that the sequence of rat caldecrin is almost identical to that of rat elastase IV (nucleotides: 99.3%, amino acids: 90.3%) with the exception of the central region. However, it is not known whether or not elastase IV is transcribed and translated in vivo, and has proteolytic activity. In the present study, we constructed a rat elastase IV cDNA by means of combinatorial PCR, and compared the recombinant elastase IV with the recombinant caldecrin synthesized in a baculovirus expression system. The recombinant caldecrin protein was expressed in the cells and secreted mainly into the medium. In contrast, in the case of elastase IV, no recombinant protein was immunologically or enzymatically detected in the medium, while an immunoreactive protein with much lower protease activity was found in the cells in an amount comparable to that of the caldecrin protein. Using the RT-PCR method to discriminate caldecrin mRNA from elastase IV mRNA, we detected caldecrin mRNA expression in rat pancreas, but no elastase IV mRNA expression in any tissues examined. PCR analysis of rat genomic DNA revealed the presence of caldecrin and the absence of elastase IV sequences. These results indicate that caldecrin is expressed in the pancreas, but that elastase IV is an artifact produced during cloning. Furthermore, we investigated the protein-chemical and enzymological properties of the rat and human caldecrins using their recombinant proteins. Both recombinant proteins were secreted into the medium as proforms and showed protease activity after trypsin treatment. Some differences were found in the activation process and stability between human and rat caldecrins; human caldecrin was more easily activated by trypsin, but was much more labile than rat caldecrin. Although both caldecrins were found to be chymotrypsin-type proteases, on the basis of their substrate and inhibitor specificities, they were not inhibited by TPCK, suggesting that caldecrin is a novel type of serine protease.抄録全体を表示PDF形式でダウンロード (3166K)
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