巻号一覧
125 巻, 6 号
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Hiroshi Hashimoto, Tomoya Matsumoto, Motomu Nishioka, Toru Yuasa, Shok ...1999 年 125 巻 6 号 p. 983-986
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーA hyperthermostable family B DNA polymerase from the hyperthermophilic archaeon, Pyrococcus kodakaraensis strain KOD1, has been crystallized by the hanging-drop vapor diffusion method at 293 K with 2-methyl-2, 4-pentanediol as the precipitant. The diffraction pattern of a crystal extends to 3.0 Å resolution, and two full sets of 3.0 Å resolution diffraction data for native crystals were successfully collected at 290 K and 100 K upon exposure to synchrotron radiation at KEK-PF, Japan. The crystals belong to the space group, 212121, with unit-cell dimensions of a=112.8, b=115.4, and c=75.4 Å at 290 K, and a=111.9, b=112.4, and c=73.9 at 100 K. Structural analysis by means of the multiple isomorphous replacement method is now in progress.抄録全体を表示PDF形式でダウンロード (1522K) -
Akira Watanabe, Yoichi Kurokawa, Tohru Yoshimura, Nobuyoshi Esaki1999 年 125 巻 6 号 p. 987-990
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーTyrosine 265 (Y265) of Bacillus stearothermophilus is believed to serve as a catalytic base specific to the L-enantiomer of a substrate amino acid by removing (or returning) an α-hydrogen from (or to) the isomer on the basis of the X-ray structure of the enzyme [Stamper, C. G., Morollo, A. A., and Ringe, D. (1998) Biochemistry 37, 10438-10443]. We found that the Y265→Ala mutant (Y265A) enzyme is virtually inactive as a catalyst for alanine racemization. We examined the role of Y265 further with β-chloroalanine as a substrate with the expectation that the Y265A mutant only catalyzes the α, β-elimination of the D-enantiomer of β-chloroalanine. However, L-β-chloroalanine also served as a substrate; this enantiomer was rather better as a substrate than its antipode. Moreover, the mutant enzyme was as equally active as the wild-type enzyme in the elimination reaction. These findings indicate that Y265 is essential for alanine racemization but not for β-chloroalanine elimination.抄録全体を表示PDF形式でダウンロード (414K) -
Motohiro Hirose, Hiroaki Kosugi, Koichi Nakazato, Toshihiko Hayashi1999 年 125 巻 6 号 p. 991-1000
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーAortic smooth muscle cells (A-SMC) undergo phenotypic transition to a synthetic and proliferative state and become fibroblast-like cells upon serial passage with culture on plastic dishes, especially in the presence of serum. Such fibroblast-like cells (M-SMC) derived from A-SMC may correspond to the cells identified pathologically as myofibro-blasts. We examined the effects of type IV collagen gels used as a culture substrate on the morphology and proliferation of M-SMC. The M-SMC underwent extreme elongation in shape when cultured on rigid type IV collagen gels, and eventually formed cell-to-cell junctions with the elongated processes. In contrast, M-SMC showed a spindle-like cell shape on dishes coated with a type IV collagen solution or type I collagen solution, or on type I collagen gels or fragile type IV collagen gels. Cell proliferation was totally repressed by culture on rigid type IV collagen gels for over 10 days, while the highest proliferative activity was seen for cells grown on dishes coated with type IV collagen solution. The expression of smooth muscle myosin heavy chains, specific markers for contractile A-SMC, was acquired by M-SMC cultured on rigid type IV collagen gels for 3 days, while M-SMC cultured on type IV collagen-coated dishes continued to show no expression. These results suggest that the quiescent and contractile phenotype of A-SMC might be restored in M-SMC by culture on rigid type IV collagen gels, even after they have become myofibroblastic.抄録全体を表示PDF形式でダウンロード (5257K) -
Shin-ichiro Uchiyama, Yoshitaka Miyazaki, Yoshihisa Amakasu, Hiroshi K ...1999 年 125 巻 6 号 p. 1001-1010
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーWe identified a unique phospholipase A (PLA) with relatively low heparin affinity, which was distinguishable from the heparin-binding secretory PLA2s, in rat, mouse, and bovine brains and testes. The partially purified enzyme was Ca2+-independent at neutral pH but Ca2+-dependent at alkaline pH. It predominantly hydrolyzed phosphatidic acid (PA) in the presence of Triton X-100 and phosphatidylethanolamine (PE) in its absence. When rat brain-derived endogenous phospholipids were used as a substrate, the enzyme released saturated fatty acids in marked preference to unsaturated ones. Consistent with this observation, the enzyme hydrolyzed sn-1 ester bonds in the substrates about 2, 000 times more efficiently than sn-2 ones, thereby acting like PLA1. The enzyme also exhibited weak but significant sn-1 lysophospholipase activity. On the basis of its limited tissue distribution, substrate head group specificity and immunochemical properties, this enzyme appears to be identical to the recently cloned PA-preferring PLA1.抄録全体を表示PDF形式でダウンロード (2309K) -
Yasuo Suda, Keiji Mori, Karyn Bird, Dalila Marques, Julie Ormsby, Sato ...1999 年 125 巻 6 号 p. 1011-1015
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーA new hetero-bifunctional photo crosslinking reagent, 2-(4-azidoanilyl)-4-(4-azabicyclo-[2, 2, 2] hexylammonio)-6-morpholino-1, 3, 5-triazine chloride, was designed to detect and isolate heparin-binding protein(s) that may act as heparin-receptor(s) on the platelet surface. In a preliminary study using ethanol as a model substrate, the reagent was shown to react with the alcoholic hydroxy group under mild conditions and its crosslinking photoreactivity was high. The reagent effectively formed similar covalent bonds with heparin, while preserving its anticoagulant anti-Xa activity. [3H] Heparin labeled with this reagent crosslinked to antithrombin III very specifically but not to ovalbumin, as analyzed by the Bio-imaging Analyzer System (BASTM, Fuji Photo Film, Tokyo). Affinity crosslinking of [3H] heparin was then used to detect heparin-binding proteins on the surface of intact platelets. Several discrete protein bands were detected by the BAS-imaging of SDS-PAGE.抄録全体を表示PDF形式でダウンロード (2450K) -
Terumichi Tanaka, Hiroshi Matsuzawa, Takahisa Ohta1999 年 125 巻 6 号 p. 1016-1021
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーAqualysin I is a bacterial subtilisin-related alkaline serine protease, originating in Thermus aquaticus YT-1. Based on computational analysis, we predicted that two residues, Ser102 and Gly131, form the S3 site of aqualysin I, and we proved that this prediction by site-directed mutagenesis. To alter the P3-specificity of the enzyme, we built a “wall” on the S3 site edge by introducing a bulky side chain at target sites. Six mutant proteins were prepared: S102H, S102K, S102E, G131H, G131K, and G131D. The mutant enzymes were examined with two kinetically typical peptides for aqualysin I, suc-X-Ala-Ala-pNA, where X is Ala or Phe. All mutations reduced the efficiency for the Phe-containing peptide, while they raised the kcat values for the Ala-containing peptide. Especially, the S102K mutant protein hydrolyzed the polyalanine peptide efficiently. The strategies we have adopted in this paper are applicable to all subtilisin-related enzymes.抄録全体を表示PDF形式でダウンロード (1639K) -
Takako Kitani, Atsuhiko Ishida, Sachiko Okuno, Masayuki Takeuchi, Isam ...1999 年 125 巻 6 号 p. 1022-1028
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーCalmodulin-dependent protein kinase (CaM-kinase) phosphatase dephosphorylates and concomitantly deactivates CaM-kinase II activated upon autophosphorylation, and CaM-kinases IV and I activated upon phosphorylation by CaM-kinase kinase [Ishida, I., Okuno, S., Kitani, T., Kameshita, I., and Fujisawa, H. (1998) Biochem. Biophys. Res. Commun. 253, 159-163], suggesting that CaM-kinase phosphatase plays important roles in the function of Ca2+ in the cell, because the three multifunctional CaM-kinases (CaM-kinases I, II, and IV) are thought to be the key enzymes in the Ca2+-signaling system. In the present study, cDNA for CaM-kinase phosphatase was cloned from a rat brain cDNA library. The coded protein consisted of 450 amino acids with a molecular weight of 49, 165. Western blot analysis showed the ubiquitous tissue distribution of CaM-kinase phosphatase. Immunocytochemical analysis revealed that CaM-kinase phosphatase is evenly distributed outside the nucleus in a cell.抄録全体を表示PDF形式でダウンロード (4479K) -
Elong Lin, Shain-Ren Liu, Alan Lin1999 年 125 巻 6 号 p. 1029-1033
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーOn limited trypsinization, eukaryotic ribosomes released sub-particles that comprised a 5S rRNA molecule and two peptides (a 32 kDa and a 14 kDa). By tryptic finger-printing and amino-terminal sequence analysis, these two peptides were determined to be derived from large subunit ribosomal protein L5 (rpL5). The 32 kDa peptide represents the rpL5 protein minus the amino terminal eight residues and the carboxyl terminal ends (approximately 21 residues), whereas the 14 kDa peptide comprised near the amino-terminal region. The time course of ribosome trypsinization revealed that the two peptides were released kinetically. The indicated that the amino and carboxyl terminal ends of rpL5 were the first to be hydrolyzed, suggesting that the two ends of the rpL5 protein were exposed on the surface of ribosomes. Exposure of the carboxyl-terminal end was confirmed by use of an anti-L5c antibody raised against the carboxyl terminal region of rpL5. The kinetic data also revealed that the neàrby amino terminal region of rpL5 (represented by the 14 kDa peptide) was the last part of rpL5 to be hydrolyzed, which was considered to be the 5S rRNA binding site.抄録全体を表示PDF形式でダウンロード (2857K) -
Gene Transfer Mediated by YKS-220 Cationic Particles: Convenient and Efficient Gene Delivery ReagentWei Yu, Atsuko Shimoyama, Takeshi Uneda, Satoshi Obika, Kazuyuki Miyas ...1999 年 125 巻 6 号 p. 1034-1038
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーA monocationic lipid, YKS-220, with a symmetrical and biodegradable structure can be used as an effective gene transfer vector in a cationic particle form (not a cationic liposome form), and is obtained by diluting an ethanol solution of YKS-220 and DOPE (1:5, molar ratio) with an aqueous medium. This preparation method is more convenient than that for cationic liposomes. YKS-220 cationic particles showed a heterogeneous large mean diameter of 4.4 μm. An obvious size change was not observed when plasmid DNA was added. The transfection activity of YKS-220 cationic particles was comparable to those of YKS-220 liposomes and DOSPA liposomes (LipofectAMINETM), and even higher than that of DOGS (TRNSFECTAM). Interestingly, the YKS-220 cationic particle/DNA complexes were resistant to the neutralizing effect of serum. All of these findings indicate that YKS-220 cationic particles are a convenient and efficient gene delivery reagent.抄録全体を表示PDF形式でダウンロード (557K) -
Chiemi Hine, Kei-ichi Enjyoji, Koichi Kokame, Shin Nakamur, Akira Take ...1999 年 125 巻 6 号 p. 1039-1047
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーIt has been reported that tissue factor pathway inhibitor (TFPI), a Kunitz-type protease inhibitor that regulates the extrinsic blood coagulation pathway, is not expressed in human, bovine, rabbit, or rat liver. Here, we found that TFPI is efficiently expressed in Macaque monkey liver. Monkey hepatocytes were identified as the expression cells by Northern blot analysis. The hepatocytes were stained with anti-human TFPI antibody, as were endothelial cells of the small vessels. We isolated and sequenced the 5'-flanking 1.4 kb regions of monkey and human TFPI genes, and found them to show 92.6% identity in their nucleotide sequences. We measured their transcriptional activities using a luciferase reporter gene and showed that the activity of the monkey TFPI gene is higher than that of the human gene in monkey primary hepatocytes. Although the binding motif of hepatocyte nuclear factor-1 is present only in the monkey gene, the site does not seem to be involved in the transcriptional activity. Mutagenetic analyses revealed that the region from -138 to +28 in the monkey gene is important for the expression of TFPI in hepatocytes. The present study indicates that the expression of the monkey TFPI gene is regulated by different mechanisms from the human TFPI gene.抄録全体を表示PDF形式でダウンロード (4302K) -
Kiyotaka Hitomi, Shinya Kanehiro, Koji Ikura, Masatoshi Maki1999 年 125 巻 6 号 p. 1048-1054
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーEpidermal-type TGase (TGase 3) is involved in the formation of the cornified cell envelope by cross-linking a variety of structural proteins in the epidermis. Unknown proteases activate this enzyme from the zymogen form by limited proteolysis during epidermal differentiation. It has been difficult to isolate sufficient quantities of native enzymes from tissues for biochemical studies of the properties of TGase 3. In this paper, we circumvented these problems by expressing recombinant full-length mouse TGase 3 in a baculovirus system, and purifying it to homogeneity by successive chromatography and HPLC. Treatment of the purified recombinant protein with dispase, a bacterial protease known to activate zymogens, produced activated TGase 3. The migration of TGase 3 zymogen in SDS-polyacrylamide gel electrophoresis was anomalous when the proTGase 3 was pre-incubated with calcium ion. GTP inhibited the enzymatic activity of recombinant TGase 3. Calpain, a calcium-dependent neutral protease, was a candidate protease, but had no effect on the activation of TGase 3 zymogen.抄録全体を表示PDF形式でダウンロード (3042K) -
Tsuyoshi Okagaki, Li-Hong Ye, Koichi Samizo, Takeshi Tanaka, Kazuhiro ...1999 年 125 巻 6 号 p. 1055-1060
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーThe catalytic domain of myosin light chain kinase (MLCK) not only exerts kinase activity to phosphorylate the 20 kDa light chain but also inhibits the actin myosin interaction. The site of action of this novel role of the domain has been suggested to be myosin [Okagaki et al. (1999) J. Biochem. 125, 619-626]. In this study, we have analyzed the amino acid sequences of MLCK and myosin that are involved in the inhibition. The ATP-binding peptide of Gly526-Lys548 of chicken gizzard MLCK exerted the inhibitory effect on the movement of actin filaments on a myosin-coated glass surface. However, the peptide that neighbors the sequence failed to inhibit the movement. The inhibition of the ATP-binding peptide was confirmed by measuring ATPase activities of the myosin. The inhibition by parent MLCK of the movement was relieved by the 20 kDa light chain, but not by the 17 kDa myosin light chain. The peptide of the 20 kDa light chain sequence of Ser1-Glu29 also relieved the inhibition. Thus, the interaction of the ATP-binding sequence with the 20 kDa light chain sequence should cause the inhibition of the actin-myosin interaction. Concerning the regulation of the inhibition, calmodulin relieved the inhibitory effect of MLCK on the movement of actin filaments. The calmodulin-binding peptide (Ala796 Ser815) prevented the relief, suggesting the involvement of this sequence. Thus, the mode of regulation by Ca2+ and calmodulin of the novel role of the catalytic domain is similar, but not identical, to the mode of regulation of the kinase activity of the domain.抄録全体を表示PDF形式でダウンロード (723K) -
Ryusuke Ohmiya, Chinatsu Kato, Hisami Yamada, Hirofumi Aiba, Takeshi M ...1999 年 125 巻 6 号 p. 1061-1066
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーAn inspection of the Schizosaccharomyces pombe genome database revealed that this eukaryotic microorganism possesses a gene that may encode a bacterial type of histidineto-aspartate (His-Asp) phosphorelay component, namely, a response regulator. The predicted gene, named prr1+ (S. p-ombe r-esponse regulator), encodes a protein that contains a typical phospho-accepting receiver domain, preceded by a mammalian heat shock factor (HSF)-like DNA-binding domain. Inactivation of this prr1+ gene resulted in mutant cells defective in some aspects of stress responses, including sensitivity to oxidative stress, cold-temperature, and heavy metal toxicity. It was also demonstrated that Prr1 is required for the transcription of some genes (e. g., trr1+, ctt1+), which are induced by oxidative stress. These results suggest that a His-Asp phosphorelay system may be involved in a stress-activated signaling pathway in S. pombe.抄録全体を表示PDF形式でダウンロード (2009K) -
Satoshi Miyata, Naohiko Koshikawa, Shouichi Higashi, Yohei Miyagi, Yoj ...1999 年 125 巻 6 号 p. 1067-1076
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーIt was recently found that overexpression of the trypsin gene in tumor cells stimulates their growth in culture and in nude mice. In the present study, expression of trypsin in various human cancer cell lines and tissues was studied by gelatin zymography and immunoblotting before and after enterokinase treatment and by immunohistochemistry. The analyses showed that many stomach, colon, and breast cancer cell lines secreted trypsinogens-1 and/or -2, as well as an unidentified serine proteinase of about 70 kDa, into culture medium. Lung cancer cell lines secreted 18- and 19-kDa unidentified trypsin-like proteins. Stomach cancer cell lines frequently secreted active trypsin, suggesting that they produced an endogenous activator of trypsinogen, most likely enterokinase. Active trypsin formed a complex with a soluble form of Alzheimer amyloid precursor protein (sAPP), a Kunitz-type trypsin inhibitor, which was secreted by all cell lines tested. This indicated that sAPP is a primary inhibitor of secreted trypsin. Immunohistochemical analysis showed that trypsin(ogen) was frequently expressed at high levels in stomach and colon cancers, but scarcely in breast cancers. In the stomach cancers, the trypsin immunoreactivity was higher in the malignant, non-cohesive type than in the cohesive type. These results support the hypothesis that tumor-derived trypsin is involved in the malignant growth of tumor cells, especially stomach cancer cells.抄録全体を表示PDF形式でダウンロード (9111K) -
Tatsuya Moriyama, Reiko Urade, Makoto Kito1999 年 125 巻 6 号 p. 1077-1085
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーDiacylglycerol lipase (DGL) was solubilized from human platelet microsomes with heptyl-β-D-thioglucoside, and purified to homogeneity on SDS-PAGE using a combination of chromatographic and electrophoretic methods. The molecular mass of the purified DGL was estimated to be 33 kDa. Its apparent pI was pH 6.0, as determined by Immobiline isoelectro-focusing. The enzymatic activity of the partially purified DGL was investigated in the presence of a variety of inhibitors and reagents, as well as its pH and calcium dependence. Thiol reagents such as p-chloromercurubenzoic acid (pCMB), N-ethylmaleimide (NEM), and HgCl2 inhibited the activity, while dithiothreitol (DTT) and reduced glutathione (GSH) enhanced it. In addition, the enzymatic activity was inhibited by two serine blockers, phenylmethylsulfonyl fluoride (PMSF) and diisopropyl fluorophosphate (DFP), and by a histidine modifying reagent, p-bromophenacyl bromide (pBPB). These results suggest that cysteine, serine and histidine residues are required for the enzymatic activity of DGL. DGL was optimally active in the pH range of 7-8 and its activity did not change significantly in the presence of various calcium concentrations, even in the presence of 2mM EGTA. This indicates that DGL can hydrolyze substrates with a basal cytosolic free Ca2+ level in the physiological pH range. A DGL inhibitor, RHC-80267, inhibited DGL activity in a dose-dependent manner with an IC50 (the concentration required for 50% inhibition) of about 5 μM. Unexpectedly, several phospholipase A2 (PLA2) inhibitors were potent inhibitors of DGL activity (IC50<5 μM), suggesting that the catalytic mechanisms of DGL and PLA2 may be similar. Finally, we show that DGL activity was inhibited by 2-monoacylglycerols (2-MGs), the reaction products of this enzyme. Among the three 2-MGs tested (2-arachidonoyl glycerol, 2-stearoyl glycerol, and 2-oleoyl glycerol), 2-arachidonoyl glycerol was the most potent inhibitor.抄録全体を表示PDF形式でダウンロード (2896K) -
Michael H. Wu, Roger A. Laine1999 年 125 巻 6 号 p. 1086-1093
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーThe nucleotide sequence of the gene encoding the cytoplasmic N, N'-diacetylchitobiase [EC 3. 2. 1. 14] from Vibrio parahaemolyticus (ATCC #27969) has been determined. The deduced peptide sequence of this unusual β-hexosaminidase surprisingly shows minimum evolutionary relationship to two other reported N, N'-diacetylchitobiases from vibrios, except in highly conserved regions which are also homologous to lysosomal β-hexosaminidases from eukaryotes including humans. In contrast, the two other β-hexosaminidases from vibrios with reported sequences are much more closely related to each other. This novel 85 kDa cytoplasmic glycosyl hydrolase with restricted specificity participates in the high level utilization of chitin-derived 2-deoxy-2-acetamido-D-glucose (GlcNAc) by vibrios as one of two parallel pathways for the metabolism of N, N'-diacetylchitobiose [Bassler, B. L., Yu, C., Lee, Y. C., and Roseman, S. (1991) J. Biol. Chem. 266, 24276-24286]. These pathways use chitin-binding proteins for the adherence of the bacterial chitinase to the substrate, and an extracellular chitinase and a periplasmic chitodextrinase to produce N, N'-diacetylchitobiose. The V. parahaemolyticus cytoplasmic N, N'-diacetyl-chitobiase reported herein appears to be a unique protein, lacking a signal sequence, and genetically distant from other known chitinoclastic β-N, N'-diacetyl-hexosaminidases. This is consistent with its limited substrate specificity to small GlcNAc terminated oligosaccharides and its cytoplasmic rather than periplasmic localization.抄録全体を表示PDF形式でダウンロード (894K) -
Michihiko Kobayashi, Eiji Sakuradani, Sakayu Shimizu1999 年 125 巻 6 号 p. 1094-1103
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーInformation on the amino acid sequences of the internal peptide fragments of cytochrome b5 from Mortierella hygrophila was used to prepare synthetic oligonucleotides as primers for the polymerase chain reaction. A 100-base DNA fragment was thus amplified, by using a genomic gene from Mortierella alpina 1S-4 as a template, which produced polyunsaturated fatty acids such as arachidonic acid. The amplified DNA fragment was used as the probe to clone both a 523-base cDNA fragment and a 2.1-kilobase Sal1-NruI genomic fragment coding for the whole M. alpina 1S-4 cytochrome b5. On the basis of nucleotide sequences of both cytochrome b5 genomic gene and cDNA, the genomic cytochrome b5 gene was found to consist of four exons and three introns. A novel type of RNA editing, in which the cDNA included either guanine insertion or adenine→guanine substitution at one base upstream of poly(A), was interestingly observed. The deduced amino acid sequence of M. alpina 1S-4 cytochrome b5 showed significant similarities with those of cytochrome b5s from other organisms such as rat, chicken, and yeast. The soluble form of the cytochrome b5 gene was expressed to 16% of the total soluble protein in Eseherichia coli. The holo-cytochrome b5 accounted for 8% of the total cytochrome b5 in the transformants. The purified cytochrome b5 showed the oxidized and reduced absorbance spectra characteristic of fungal microsomal cytochrome b5.抄録全体を表示PDF形式でダウンロード (3627K) -
Minoru Matsui, Walter C. Breau, Shiho Iwasaki, Sadahiro Hagiwara, Yosh ...1999 年 125 巻 6 号 p. 1104-1114
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーRetroviral genes are not usually expressed in mouse embryonal carcinoma (EC) cells, but they are readily expressed upon differentiation of these cells. We previously reported the isolation of EC cell lines that express a neomycin resistance (neo) gene introduced by a recombinant transducing Moloney murine leukemia virus from specific integration sites, Minta, Mintb, Mintc, or Mintd. In some of these clones, the entire 5' long terminal repeat (LTR) was deleted, and the neo gene was expressed by read-through transcription from upstream cellular promoters in a “promoter-trap” fashion. One such promoter (“promoter B” at the Mintb locus) was found in a CpG island, associated with an upstream enhancer (“enhancer B”). Although enhancer B caused expression of the neo gene in the transductant EC cell line, no endogenous transcription from promoter B was detected in the parental EC or NIH3T3 cells. In contrast, we found a strong counter-flow endogenous transcription unit (“R” for reverse), which apparently interfered with transcription from promoter B. Promoter R turned out to have a bidirectional activity in transfection assays. In normal tissues, promoter R activates gene R, which encodes an 800-residue protein that is highly homologous to the rat and human heterogeneous nuclear ribonucleoprotein U (hnRNP U). Northern and in situ hybridization analyses revealed that gene R was abundantly expressed in the testis, especially in the pachytene spermatocytes and round spermatids.抄録全体を表示PDF形式でダウンロード (5216K) -
Hiroshi Takeno, Seiji Yamamoto, Terumichi Tanaka, Yoshiyuki Sakano, Yo ...1999 年 125 巻 6 号 p. 1115-1119
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーRNA ligands (RNA aptamers) to a protease subtilisin were selected from pools of random RNA by SELEX (systematic evolution of ligands by exponential enrichment) and by use of a subtilisin-immobilized Sepharose column. After eight rounds of selection, RNA aptamers were isolated by cloning to a plasmid vector. We characterized one of the selected RNA molecules. This RNA aptamer displayed specific inhibition toward the subtilisin activity, even when the assay for subtilisin was performed using the chromogenic small peptide as substrate, and almost no inhibitory activity toward trypsin and chymotrypsin, although these enzymes are serine proteases similar to subtilisin. These findings indicate that this RNA can differentially recognize the surfaces of similar proteases. Kinetic analysis of the RNA aptamer revealed that the inhibition constant (K1) toward subtilisin was 2.5 μM.抄録全体を表示PDF形式でダウンロード (587K) -
Takuji Oyama, Masami Kusunoki, Yoji Kishimoto, Yoshiyuki Takasaki, Yas ...1999 年 125 巻 6 号 p. 1120-1130
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーThe crystal structure of β-amylase from Bacillus cereus var. mycoides was determined by the multiple isomorphous replacement method. The structure was refined to a final R-factor of 0.186 for 102, 807 independent reflections with F/σ(F)_??_2.0 at 2.2 Å resolution with root-mean-square deviations from ideality in bond lengths, and bond angles of 0.014 Å and 3.00°, respectively. The asymmetric unit comprises four molecules exhibiting a dimer-of-dimers structure. The enzyme, however, acts as a monomer in solution. The β-amylase molecule folds into three domains; the first one is the N-terminal catalytic domain with a (β/α)8 barrel, the second one is the excursion part from the first one, and the third one is the C-terminal domain with two almost anti-parallel β-sheets. The active site cleft, including two putative catalytic residues (Glu172 and Glu367), is located on the carboxyl side of the central β-sheet in the (β/α) barrel, as in most amylases. The active site structure of the enzyme resembles that of soybean β-amylase with slight differences. One calcium ion is bound per molecule far from the active site. The C-terminal domain has a fold similar to the raw starch binding domains of cyclodextrin glycosyltransferase and glucoamylase.抄録全体を表示PDF形式でダウンロード (7477K) -
Yoichi Yamazaki, Hideki Kandori, Tatsushi Mogit1999 年 125 巻 6 号 p. 1131-1136
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーCytochrome bd is a two-subunit ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli that does not belong to the heme-copper terminal oxidase superfamily. To explore unique protein structural changes associated with the reduction of the redox metal centers, we carried out Fourier-transform infrared and visible spectroscopic studies on cytochrome bd. For infrared measurements of a partially dehydrated thin sample solution, the air-oxidized enzyme was fully reduced by the intermolecular electron transfer of photo-excited riboflavin in the absence and presence of KCN, and redox difference spectra were calculated. Upon reduction, the bound cyanide was released from the heme b595-heme d binuclear center but remained in a protein pocket as a deprotonated form. Reduction of heme b558, heme b595 and heme d resulted in large changes in amide-I and protonated carboxylic CO-stretching vibrations and also a small change in the cysteine SH-stretching vibration. The location of the redox metal centers and the effects of cyanide suggest that these protein structural changes occur at the heme-binding pockets near the protein surface. Systematic site-directed mutagenesis and time-resolved FTIR studies on cytochrome bd will facilitate an understanding of the unique molecular mechanisms for dioxygen reduction and delivery of chemical protons to the active center at the atomic level.抄録全体を表示PDF形式でダウンロード (724K) -
Yoshiyuki Yasuda, Takashi Kageyama, Akifumi Akamine, Masahiro Shibata, ...1999 年 125 巻 6 号 p. 1137-1143
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーCathepsin E and cathepsin D are two major intracellular aspartic proteinases implicated in the physiological and pathological degradation of intra- and extracellular proteins. In this study, we designed and constructed highly sensitive synthetic decapeptide substrates for assays of cathepsins E and D based on the known sequence specificities of their cleavage sites. These substrates contain a highly fluorescent (7-methoxycoumarin-4-yl)acetyl (MOCAc) moiety and a quenching 2, 4-dinitrophenyl (Dnp) group. When the Phe-Phe bond is cleaved, the fluorescence at an excitation wavelength of 328 nm and emission wavelength of 393 increases due to diminished quenching resulting from the separation of the fluorescent and quenching moieties. The first substrate, MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(Dnp)γ-NH2, in which the Lys-Pro combination at positions P5 and P4 was designed for specific interaction with cathepsin E, is hydrolyzed equally well by cathepsins E and D (kcat/Km=10.9 μM-1•s-1 for cathepsin E and 15.6 μM-1•s-1 for cathepsin D). A very acidic pH optimum of 4.0 was obtained for both enzymes. The second substrate, MOCAc-Gly-Lys-Pro-Ile-Ile-Phe-Phe-Arg-Leu-Lys(Dnp) γ-NH2, in which the isoleucine residue at position P2 was meant to increase the specificity for cathepsin E, is also hydrolyzed equally by both enzymes (kcat/Km=12.2 μM-1•s-1 for cathepsin E and 16.3 μM-1•s-1 for cathepsin D). The kcat/Km values for both substrates are greater than those for the best substrates for cathepsins E and D described so far. Unfortunately, each substrate shows little discrimination between cathepsin E and cathepsin D, suggesting that amino acids at positions far from the cleavage site are important for discrimination between the two enzymes. However, in combination with aspartic proteinase inhibitors, such as pepstatin A and Ascaris pepsin inhibitor, these substrates enable a rapid and sensitive determination of the precise levels of cathepsins E and D in crude cell extracts of various tissues and cells. Thus these substrates represent a potentially valuable tool for routine assays and for mechanistic studies on cathepsins E and D.抄録全体を表示PDF形式でダウンロード (851K) -
Yasuo Sugiyama, Kiyoyasu Fujii, Yasuo Mukohata1999 年 125 巻 6 号 p. 1144-1150
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーCarboxyl group modification with DCCD and NCD-4 was employed to investigate the chemical environment of the side chains of archaeopsin-1 (aO-1) and bacterioopsin (bO). Some differences were observed between aO-1 and bO. Although DCCD or NCD-4 did not modify aO-1 in bleached membrane, they modified bO in bleached membrane and in mixed DMPC/CHAPS/SDS micelles at neutral pH, thereby affecting the opsin shift and the photocycle of the regenerated chromophore. On the contrary, after solubilization with SDS, aO-1 and bO were modified by DCCD and NCD-4, which decreased the chromophore regeneration. In particular, the reaction of aO- 1 in SDS with NCD-4 proceeded in a 1:1 ratio at neutral pH. The fluorescence and CD spectra indicated that the modified site was located in the hydrophobic, asymmetrical region. Lysyl-endopeptidase digestion of NCD-4 modified aO-1 produced a fluorescent fragment and amino acid sequence analysis showed that Asp85 or Asp96 in helix C is a probable candidate for the modified residue at present. Kinetic CD measurements revealed that the introduction of N-acylurea at an Asp residue in helix C did not affect the formation of the transient intermediate but inhibited the side chain packing during refolding.抄録全体を表示PDF形式でダウンロード (1832K) -
Shigeo Tsuchiya, Kenji Ogura, Hideki Hatanaka, Koji Nagata, Hiroaki Te ...1999 年 125 巻 6 号 p. 1151-1159
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリー1H, 13C, and 15N NMR resonances of the SH2 domain of Grb2/Ash in both the free form and the form complexed with a phosphotyrosine-containing peptide derived from the EGF receptor were assigned by analysis of multi-dimensional, double- and triple-resonance NMR experiments. From the chemical shift changes of individual residues upon peptide binding, the binding site for the peptide was mapped on the structure of Grb2/Ash SH2. The peptide was not recognized by the groove formed by the BG and EF loops, suggesting that the EGFR peptide does not bind to Grb2/Ash SH2 in an extended conformation. This was supported by analysis of the binding affinity of mutants where residues on the BG and EF loops were changed to alanine. The present results are consistent with the recently reported structures of Grb2/Ash SH2 complexed with BCR-Abl and Shc-derived phosphotyrosine containing peptides, where the peptide forms a turn conformation. This shows that the specific conformation of the phosphotyrosine-containing sequence is required for the SH2 binding responsible for downstream signaling.抄録全体を表示PDF形式でダウンロード (1732K) -
Jun You, Masamichi Kamihira, Shinji Iijima1999 年 125 巻 6 号 p. 1160-1167
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーWe have previously developed a simple gene transfection procedure mediated by cationic lipid vesicles for animal cells, in which a commercially available cationic surfactant, dimethyldioctadecyl ammonium bromide (DDAB), was used for making lipid vesicles. In the present study, we examined enhancement of transfection efficiency for this method by adding protamine to plasmid DNA solution before the formation of DNA/lipid vesicle complexes. Both free-base protamine and protamine sulfate provided enhanced transfection efficiency and expression level, but the optimal amount of the two protamines was different. The enhancement in transfection efficiency and expression level by protamines was observed in all the cell lines (COS-7, Hela, NIH3T3, MDCK, and BHK-21C13) and all the plasmids (pCMVβ, pmiwZ, and pCH110) tested. The enhancement in both transfection efficiency and expression level was at most 20-fold compared with that using only DDAB lipid vesicles. Protamines seemed to protect DNA from degradation by DNase and promote DNA delivery into a nucleus.抄録全体を表示PDF形式でダウンロード (3481K) -
Tomoyuki Harano, Nobuhiro Shimizu, Hidenori Otera, Yukio Fujiki1999 年 125 巻 6 号 p. 1168-1174
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーThe peroxisome biogenesis factor, peroxin Pex2p, is an integral membrane protein of peroxisomes [Tsukamoto, T., Miura, S., and Fujiki, Y. (1991) Nature 350, 77-81]. As a step toward elucidating the structure and biological function of Pex2p, we determined the transmembrane topology of Pex2p by expressing epitope-tagged rat Pex2p in COS-7 cells. Pex2p tagged with myc at the C-terminus was detected as a punctate staining pattern, when the cells were permeabilized with 50 μg/ml of digitonin, under which conditions intra-peroxisomal proteins such as PTS1-proteins are inaccessible to exogenous antibodies. N-terminally flag-tagged Pex2p was likewise detected upon the same treatment. These results strongly suggest that both the N- and C-terminal parts of Pex2p are exposed to the cytosol. The transmembrane orientation of Pex2p was also assessed by using rat liver peroxisomes and Pex2p region-specific antibodies. The two types of antibodies used, raised to the N-(amino acid residues 1-131) and C-terminal part (residues 226 to the C-terminus), respectively, specifically recognized Pex2p and immunoprecipitated intact, whole peroxisomes. Pex2p was not recognized by the antibodies when the peroxisomes were treated with Proteinase K. Furthermore, in situ crosslinking studies involving bifunctional reagents revealed an apparently dimeric form of Pex2p. Therefore, Pex2p is anchored to the peroxisomal membrane by two membrane-spanning segments, with its N- and C-terminal regions exposed to the cytosol.抄録全体を表示PDF形式でダウンロード (5480K) -
Hironobu Kimura, Isao Suetake, Shoji Tajima1999 年 125 巻 6 号 p. 1175-1182
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーIn vertebrates, DNA methylation plays an important role in the regulation of gene expression and embryogenesis. DNA methyltransferase, which catalyzes the introduction of a methyl group at the 5 th position of cytosine in the CpG sequence, is highly accumulated in mouse oocytes and is excluded from nuclei [Carlson et al. (1992) Genes Dev. 6, 2536-2541]. In this study, we examined the expression level and localization of Xenopus DNA methyltransferase in oocytes during oogenesis. The DNA methyltransferase protein was detectable in stage III oocytes and increased thereafter, until the oocytes had matured. The rate of DNA methyltransferase synthesis rapidly increased after stage IV oocytes. Different from in mouse oocytes, DNA methyltransferase was equally distributed in the nuclear and post-nuclear fractions, in stage VI oocytes. DNA methyltransferase translocated into nuclei was uniformly localized in the nuclear matrix, and the accumulated DNA methyltransferase in stage VI nuclei had DNA methylation activity.抄録全体を表示PDF形式でダウンロード (4225K) -
Tsutomu Tsuji, Seon Ae Han, Ken-ichi Takeuchi, Naoki Takahashi, Sen-it ...1999 年 125 巻 6 号 p. 1183-1188
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーIntegrin α3β1 (VLA-3) is an adhesion receptor for extracellular matrix proteins including various isoforms of laminin. We have isolated mouse genomic clones encoding the integrin α3 subunit and deduced the exon/intron organization. The mouse integrin α3 subunit gene is encoded by 26 exons spanning 40 kb. The exon/intron structure of the integrin α3 subunit gene resembles that of the integrin α6 subunit gene, but differs somewhat from those of other members of the integrin family. We have demonstrated that the cytoplasmic domain splicing variants of the α3 subunits (α3A and α3B) are generated by alternative exon usage. We also cloned the 5'-flanking region and performed a preliminary analysis of its promoter activity in various tumor cell lines with different degrees of integrin α3 expression. Following transfection, activity in the luciferase assay was found to be roughly correlated with the expression level of integrin α3 as measured by flow cytometry. Furthermore, the luciferase assay was performed with normal and SV-40- or polyoma virus-transformed fibroblasts. In mouse, human, and hamster fibroblasts, higher levels of luciferase expression were observed in transformed cells than in normal cells. This result is consistent with our previous finding that integrin α3 expression at both the protein and mRNA levels is enhanced upon oncogenic transformation of fibroblasts by tumor viruses.抄録全体を表示PDF形式でダウンロード (2650K) -
Yijian Fei, Suraporn Matragoon, Sylvia B. Smith, Paul A. Overbeek, Shi ...1999 年 125 巻 6 号 p. 1189-1199
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーThe essential control elements in the interphotoreceptor retinoid-binding protein gene (IRBP) promoter are located between -156 and + 19. The -156/-109 sequence contains a retina-specific DNAse I footprint and shows a positive regulatory activity in transiently transfected retinoblastoma cells. The -105/-85 sequence is G/C rich, shows a non-tissue specific DNAse I hypersensitivity, and a negative regulatory activity in retinoblastoma cells. The -76/-42 sequence shows a retinal-specific footprint and contains a “cone-rodhomeobox element” (CRXE) and a “photoreceptor conserved element” (PCE). IRBP promoter fragments with mutations in either CRXE, PCE or in both were linked to reporter genes and analyzed both by transient transfection and in transgenic mice. In retinoblastoma cells, the mutated CRXE-containing promoter shows a 60% repression of the CAT activity whereas the mutated PCE-containing promoter shows a 30% repression. In HeLa cells transfected with these promoters, co-transfection of a Crx expression vector with wild-type, but not with CRXE mutant promoter, activates CAT activity 20-fold over the background activity. Mutation of PCE alone or conversion of CRXE to PCE reduces this Crx-activated CAT activity to only 4-fold over the background activity. In the transgenic mouse experiments, none of the 12 lines with CRXE mutant promoter show significant expression of lacZ in the retina. In contrast, 9 of the 17 transgenic lines with PCE mutant promoter show photoreceptor-specific lacZ expression. Thus the Crx interaction with CRXE is essential for the photoreceptor-specific activity of the IRBP promoter in vivo. This interaction does not appear to require PCE, but is enhanced when PCE is present.抄録全体を表示PDF形式でダウンロード (6833K) -
Yan Li, Roger Aoki, J. Oliver Dolly1999 年 125 巻 6 号 p. 1200-1208
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーTetanus toxin, composed of a disulphide-linked heavy (HC) and light (LC) chain, preferentially blocks the release of inhibitory neurotransmitters in the spinal cord by Zn2+-dependent proteolytic cleavage of synaptobrevin. This intoxication involves binding via HC to ecto-acceptors on peripheral nerve endings, followed by internalisation and retrograde transportation to its prime site of action in central neurons. To facilitate exploitation of the toxin's unique activities, HC was expressed at a high level in Escherichia coli as a fusion with maltose binding protein; after cleavage by thrombin, free HC was isolated and its identity confirmed by Western blotting and N-terminal microsequencing. The expressed and native HC gave very similar circular dichroism spectra, excluding any gross differences in their folded structures. Recombinant HC antagonised the neuromuscular paralysing activity of the native toxin, by competing for binding to neuronal ecto-acceptors. The HC was reconstituted with bacterially-expressed LC to create disulphide-bridged dichain toxin that blocked neuromuscular transmission. The fully-recombinant toxin produced spastic paralysis in mice characteristic of the blockade of central inhibitory synapses, revealing that it undergoes axonal transport to the spinal cord, like the native toxin but with a reduced efficacy. This first report of the large-scale production of recombinant tetanus toxin in active form should facilitate studies on the use of engineered innocuous forms of the toxin as neuronal transport vehicles.抄録全体を表示PDF形式でダウンロード (3493K)
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