巻号一覧
127 巻, 3 号
選択された号の論文の22件中1～22を表示しています
- |<
- <
- 1
- >
- >|
-
Tamotsu Zako, Hidenobu Deguchi, Atsushi Kitayama, Hiroshi Ueda, Teruyu ...2000 年 127 巻 3 号 p. 351-354
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーThe renaturation yield of the denatured firefly luciferase decreased strongly with increasing protein concentration in a renaturation buffer, because of aggregation. In this study, firefly luciferase was immobilized on agarose beads at a high concentration. Although the protein concentration was extremely high (about 100-fold) compared to that of soluble luciferase, the renaturation yield was comparable with that for the soluble one. Thus, immobilization was shown to be effective for avoiding aggregation of firefly luciferase. It was also shown that the optimum buffer conditions for renaturation of the immobilized luciferase were the same as those for the renaturation in solution. Also, it was indicated that electrostatic interactions between a protein and the matrix have a negative effect on renaturation of the immobilized luciferase since the renaturation yield decreased at acidic pH only for the immobilized luciferase. These novel observations are described in detail in this paper.抄録全体を表示PDF形式でダウンロード (407K) -
Fumi Takahashi-Yanaga, Sachio Morimoto, Iwao Ohtsuki2000 年 127 巻 3 号 p. 355-357
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーIn order to determine the functional consequences of the Arg145Gly mutation in troponin I found in familial hypertrophic cardiomyopathy, human cardiac troponin I and its mutant were expressed in Escherichia coli and purified, and then their effects on the ATPase activity of porcine cardiac myofibrillar preparations from which both troponins C and I had been depleted were examined. Both the wild-type and mutant troponin Is suppressed the ATPase activity of the troponin C•I-depleted myofibrils, but the maximum inhibition caused by mutant troponin I was weaker than that by wild-type troponin I. In the Ca2+-activation profile of the myofibrillar ATPase activity after reconstitution with both troponins I and C, the Ca2+-sensitivity with mutant troponin I was higher than that with wild-type troponin I, whereas the maximum level of the ATPase activity with mutant troponin I was lower than that with wild-type troponin I. These findings strongly suggest that the Arg145Gly mutation in human cardiac troponin I modulates the Ca2+-regulation of contraction by impairing the interaction of troponin I with both actin-tropomyosin and troponin C.抄録全体を表示PDF形式でダウンロード (1061K) -
Makoto Fujiwara, Masayuki Ichinomiya, Takayuki Motoyama, Hiroyuki Hori ...2000 年 127 巻 3 号 p. 359-366
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーAlthough many chitin synthase genes have been identified in a broad range of fungal species, there have been only a few reports about their role in fungal morphogenesis. In most cases, single gene disruption or replacement did not reveal their function, possibly because of functional redundancy among them. We obtained null mutants of Aspergillus nidulans chsA and chsC genes encoding non-essential class II and class I chitin syntheses, respectively. The ΔAchsA ΔchsC mutant exhibited growth defects on media supplemented with sodium dodecyl sulfate (SDS), high concentration of salts, chitin-binding dyes, or chitin synthase competitive inhibitors, suggesting loss of integrity of hyphal wall. Moreover, remarkable abnormalities of the double mutant were observed microscopically during its asexual development. The conidiophore population was drastically reduced. Interestingly, secondary conidiophores were occasionally produced from vesicles of the primary ones. The morphology of these conidiophores was similar to those of the A. nidulans developmental mutants, medusa (medA), abacus (abaA), and some kinds of bristle (briA). In situ staining patterns suggested that chsA was mainly expressed in the metulae, phialides, and conidia, whereas chsC was expressed in hyphae as well as conidiophores. These results suggest that ChsA and ChsC share critical functions in hyphal wall integrity and differentiation.抄録全体を表示PDF形式でダウンロード (2758K) -
Yukie Koresawa, Shuji Miyagawa, Masahito Ikawa, Katsuyoshi Matsunami, ...2000 年 127 巻 3 号 p. 367-372
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーThe origin of the Cre recombinase gene is bacteriophage P1, and thus the codon usages are different from in mammals. In order to adapt this codon usage for mammals, we synthesized a “mammalian Cre recombinase gene” and examined its expression in Chinese hamster ovarian tumor (CHO) cells. Significant increases in protein production as well as mRNA levels were observed. When the recombination efficiency was compared using CHO cell transfectants having a cDNA containing loxP sites, the “mammalian Cre recombinase gene” recombined the loxP sites much more efficiently than the wild-type Cre recombinase gene.抄録全体を表示PDF形式でダウンロード (1849K) -
Kazuo Fujisawa, Kazuhiko Umesono, Yoshiharu Kikawa, Yosuke Shigematsu, ...2000 年 127 巻 3 号 p. 373-382
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーFructose-1, 6-bisphosphatase (FBPase) is a key gluconeogenic enzyme. The data herein show that both the enzyme activity and mRNA level of the human FBPase gene are enhanced by 9-cis retinoic acid (9cRA) and all-trans retinoic acid (atRA) as well as by 1, 25-dihydroxyvitamin D3 (VD3) in human promyelocytic UL60 cells and normal monocytes in peripheral blood, which were used as an alternative source to liver for the DNA diagnosis of FBPase deficiency. To understand the molecular mechanism of this enhancing action, the 2.4 kb 5'-regulatory region of the human FBPase gene was isolated and sequenced. Using luciferase reporter gene assays, a 0.5 kb FBPase basal promoter fragment was found to confer induction by VD3, 9cRA, and atRA that was mediated by the vitamin D3 receptor (VDR), retinoid X receptor (RXR), and retinoic acid receptor (RAR). Within this region, a direct repeat sequence, 5'-TAACCTttcTGAACT-3' (-340 to -326), which functions as a common response element for VD3, 9cFA, and atRA, was identified. The results of electrophoretic mobility shift assays indicated that VDR-RXR and RAR RXR heterodimers bind this response element. Collectively, these observations indicate that VD3 and RA are important modulators of the expression of the human FBPase gene in monocytic cells.抄録全体を表示PDF形式でダウンロード (3458K) -
Noriyuki Ishii, Keiko Haga, Kunio Yamane, Kazuaki Harata2000 年 127 巻 3 号 p. 383-391
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーThe product specificity of cyclodextrin glucanotransferase (CGTase) from alkalophilic Bacillus sp. #1011 is improved to near-uniformity by mutation of histidine-233 to asparagine. Asparagine 233-replaced CGTase (H233N-CGTase) no longer produces a-cyclodextrin, while the wild-type CGTase from the same bacterium produces a mixture of predominantly α-1, β-, and γ-cyclodextrins, catalyzing the conversion of starch into cyclic or linear α-1, 4-linked glucopyranosyl chains. In order to better understand the protein engineering of H233N-CGTase, the crystal structure of the mutant enzyme complexed with a maltotetraose analog, acarbose, was determined at 2.0 Å resolution with a final crystallographic R value of 0.163 for all data. Taking a close look at the active site cleft in which the acarbose molecule is bound, the most probable reason for the improved specificity of H233N-CGTase is the removal of interactions needed to form a compact ring like α-cyclodextrin.抄録全体を表示PDF形式でダウンロード (2800K) -
Satoko Isemura2000 年 127 巻 3 号 p. 393-398
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーThe nucleotide sequence of gene PBII encoding salivary proline-rich protein P-B was determined. PBII is 7.1 kb long and contains 3 exons. PBII exhibits considerable nucleotide sequence homology not only in exons but also in introns with PBI (accession number D89501), the gene whose nucleotide sequence was determined previously [lsemura and Saitoh (1997) J. Biochem. 121, 1025-1030]. PBI and II constitute a gene family distinct from that to which the majority of salivary proline-rich protein ones belong. The nucleotide sequence data reported in this paper will appear in the DDBJ, EM-BL, and GenBank nucleotide sequence databases under accession number ABO31740.抄録全体を表示PDF形式でダウンロード (596K) -
Shou Takashima, Nobuyuki Kurosawa, Yuriko Tachida, Mio Inoue, Shuichi ...2000 年 127 巻 3 号 p. 399-409
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーThe genomic organization of the genes encoding the mouse N-acetylgalactosamine α2, 6-sialyltransferase specific for Siaa2, 3Galβ1, 3GalNAc (ST6GalNAc III and IV) has been determined. The ST6GalNAc III gene spans over 120 kilobases of genomic DNA with 5 exons; on the other hand, the ST6GalNAc IV gene spans over 12 kilobases of genomic DNA with 6 exons. But the exon-intron boundaries of these genes are very similar. The 5'-flanking regions of these genes do not contain a TATA- or CAAT-box but have three putative Spl binding sites for each promoter. Transient transfection experiments demonstrated functional promoter activity in an ST6GalNAc III-expressing cell line, P19, for the ST6GalNAc III promoter, and in an ST6GalNAc IV-expressing cell line, NIH3T3, for the ST6GalNAc IV promoter. Mobility shift assaying and mutational analysis of the promoter region indicated that two of the three Spl binding sites are involved in the transcriptional regulation of the ST6GalNAc HI gene in P19 cells, while all three Spl binding sites are involved in the transcriptional regulation of the ST6GalNAc IV gene in NIH3T3 cells.抄録全体を表示PDF形式でダウンロード (4168K) -
Yoshikazu Ishimoto, Kazumasa Ohashi, Kensaku Mizuno, Toru Nakano2000 年 127 巻 3 号 p. 411-417
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーGas6, a ligand of receptor tyrosine kinases Axl, Sky, and Mer, potentiates cell proliferation and prevents cell death. It also contains γ-earboxylglutamic acid residues that mediate the interaction of some blood coagulation factors with negatively charged phospholipids. In our previous study, we demonstrated that Gas6 specifically binds to phosphatidylserine (PS) and links Axl-expressing cells to the PS-coated surface. In this study, to further understand the biological role of the interaction of Gas6 with PS, we examined the effect of Gas6 on the uptake of PS liposomes by macrophages. In vitro phagocytosis studies showed that Gas6 enhanced the uptake of PS liposomes approximately threefold and that the interaction of Gas6 with the surface of macrophages was essential for this enhancement. Analyses of the mechanism of the uptake of PS liposome suggested that Gas6 interacts with PS liposome via its N-terminal Gla domain and with macrophages via its C-terminal domain. Like that of PS liposomes, the uptake of apoptotic cells by macrophages was also enhanced, approximately twofold, in the presence of Gas6. These findings suggest that Gas6 may help phagocytic cells recognize cells with PS exposed on their surfaces, which is considered to be one of the mechanisms for clearing away dying cells. Thus, Gas6 may play a critical role in homeostasis by facilitating the clearance of PS-expressing cells.抄録全体を表示PDF形式でダウンロード (1755K) -
Shigeyuki Imamura, Shiro Kitaura2000 年 127 巻 3 号 p. 419-425
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーA thermostable monoacylglycerol lipase [MGLP, EC 3. 1. 1. 23] was purified for the first time from a cell-free extract of the moderately thermophilic Bacillus sp. H-257. The enzyme was purified 3, 028-fold to homogeneity by chromatography using Octyl Sepharose CL-4 B, Q-Sepharose FF, and Superose 12 columns. The molecular mass of the MGLP was estimated to be 25 kDa by gel filtration and 24 kDa by SDS-PAGE, suggesting a monomeric protein. The isoelectric point was determined to be 4.66 by isoelectric focusing. The MGLP retained its full activity upon incubation at 60°C for 10 min (pH 7.3), and was stable at pH 7-10. The optimal temperature for activity at pH 7.5 was 75°C, and the maximum activity was observed from pH 6-8. This enzyme hydrolyzes monoacylglycerols, with the highest activity occurring with 1-monolauroylglycerol. Di- and triacylglycerols, on the other hand, are essentially inert as substrates for the enzyme. The Km values for the hydrolysis of 1-monolauroylglycerol, 1-monooleoylglycerol, and 2-monooleoylglycerol were determined to be 140, 83 and 59 μM, respectively. The enzyme was not inhibited by cholate, but was slightly inhibited by Triton X-100 and deoxycholate. The amino acid sequence of the N-terminal region of the enzyme (16 residues) was also determined.抄録全体を表示PDF形式でダウンロード (1267K) -
Tomoko Nishiyama, Naoya Kimura, Yuki Jitsuhara, Makoto Uchida, Fumiko ...2000 年 127 巻 3 号 p. 427-433
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーIt was previously revealed [Yamaguchi, H., Nishiyama, T., and Uchida, M. (1999) J. Biochem. 126, 261-265] that N-glycans of both the high-mannose and complex types have binding affinity for aromatic amino acid residues. This study shows that free N-glycans protect proteins from protease digestion through their binding affinities for the aromatic amino acid residues exposed on protein molecules. Protease digestion of bovine pancreatic RNase A and bovine a-lactalbumin was depressed in solutions (1 mM or so) of free N-glycans of both the high-mannose and complex types. The increasing order of the protective effects of the N-glycans paralleled that of their affinities for aromatic amino acid residues; and the presence of aromatic amino acids practically abolished the protective effects of the N-glycans. The N-glycans also depressed the protease digestion of metallothionein, an aromatic amino acid-free protein, in agreement with the observation that the N-glycans also interact with the solvent-exposed aromatic amino acid residues of the proteases. Thus it seems probable that the N-glycans protect proteins from protease digestion by steric hindrance attributable to their binding affinity for the solvent-exposed aromatic amino acid residues of both substrate proteins and proteases.抄録全体を表示PDF形式でダウンロード (1057K) -
Junichiro Futami, Yoshiaki Tsushima, Hiroko Tada, Masaharu Seno, Hiden ...2000 年 127 巻 3 号 p. 435-441
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーWe investigated how the folding yield of disulfide-containing globular proteins having positive net charges from crude bacterial inclusion bodies was affected by additives in the folding buffer. In screening folding conditions for human ribonucleases and its derivative, we found that addition of salt (about 0.4 M) to a folding buffer increased the folding yield. This suggested that electrostatic interaction between polyanionic impurities such as nucleic acids and cationic unfolded protein led to the formation of aggregates under the low-salt conditions. Since inclusion bodies were found to contain nucleic acids regardless of the electrostatic nature of the expressed protein, the electrostatic interaction between phosphate moieties of nucleic acids and basic amino acid residues of a denatured protein may be large enough to cause aggregation, and therefore the addition of salt in a folding buffer may generally be useful for promotion of protein folding from crude inclusion bodies. We further systematically investigated additives such as glycerol, guanidium chloride, and urea that are known to act as chemical chaperons, and found that these additives, together with salt, synergistically improved folding yield. This study, suggesting that the addition of salt into the folding buffer is one of the crucial points to be considered, may pave the way for a systematic investigation of the folding conditions of disulfide-containing foreign proteins from crude bacterial inclusion bodies.抄録全体を表示PDF形式でダウンロード (1752K) -
Mitsuru Hashimoto, Eiji Majima, Takashi Hatanaka, Yasuo Shinohara, Mas ...2000 年 127 巻 3 号 p. 443-449
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーThe effect of the SH-reagent methyl methanethiosulfonate (MMTS) on the ADP/ATP carrier of bovine heart mitochondria was studied under various conditions. MMTS labeled predominately Cys56 in the first loop facing the matrix (loop Ml), and the labeling inhibited ADP transport via the carrier. The transport inhibition was found to be due to fixation of the carrier in the m-state conformation. MMTS labeling was suggested not to affect ADP binding to its major binding site. These features were the same as those of another commonly used SH-reagent, N-ethylmaleimide (NEM). Although the van der Waals volume of the non-hydrogen-bondable methylthio group of MMTS is much smaller than that of the ethylsuccinimide group of NEM, modification of Cys56 inhibited the interconversion between the m- and c-state conformation. The mechanism by which MMTS inhibited the transport activity is discussed in terms of stabilization of conformation of the loop M1.抄録全体を表示PDF形式でダウンロード (2729K) -
Suppression of Prostaglandin E2 mediated c-fos mRNA Induction by Interleukin-4 in Murine MacrophagesDarning Zhuang, Hiroo Kawajiri, Yoshitaka Takahashi, Tanihiro Yoshimot ...2000 年 127 巻 3 号 p. 451-456
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーWhen murine peritoneal macrophages were stimulated for 30 min with arachidonic acid, the growth-associated immediate early gene c-fos was induced in a concentrationdependent manner as assessed by Northern blot analysis. The arachidonic acid-induced c-fos mRNA expression was inhibited by a cyclooxygenase inhibitor, indomethacin, but not by a lipoxygenase inhibitor, nordihydroguaiaretic acid. Macrophages produced prostaglandin (PG) E2 from arachidonic acid as determined by an enzyme immunoassay. Northern blot analysis revealed the expression of PGE receptor EP2 and EP4 subtypes, but not EP1 and EP3 in murine macrophages. PGE2 brought about a marked elevation of cAMP, and c-fos mRNA expression was increased by PGE2 and dibutyryl cAMP in these cells. These results suggest that arachidonic acid is transformed to PGE2, which then binds to EP2 and EP4 receptors to increase intracellular cAMP and c-fos mRNA expression. Furthermore, the induction of c-fos by arachidonic acid, PGE2, and cAMP was suppressed by pretreatment with interleukin (IL)-4. We also showed that the tyrosine phosphorylation of a Janus kinase, JAK3, is enhanced by IL-4 treatment, suggesting that the PGEZ mediated c-fos mRNA induction is inhibited by IL-4 through the tyrosine phosphorylation of JAK3.抄録全体を表示PDF形式でダウンロード (2331K) -
Toshihiro Fujii, Hidenori Takagi, Makoto Arimoto, Hisataka Ootani, Tak ...2000 年 127 巻 3 号 p. 457-465
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーSmooth muscle basic calponin, a major actin-, tropomyosin-, and calmodulin-binding protein, has been examined for its ability to interact with desmin intermediate filaments from smooth muscle cells using sedimentation analysis, turbidity changes, chemical cross-linking, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOMS), and electron microscopic observations. Calponin interacted with desmin intermediate filaments in a concentration-dependent manner in vitro. The bind-ing of calponin to desmin produced dense aggregates at 30°C. The dense aggregates were observed by electron microscopy to be composed of large anisotropic bundles of desmin filaments, indicating that calponin forms bundles of desmin filaments. The addition of calmodulin or 5100 to the mixture of calponin and desmin caused the removal of calponin from the desmin filaments and inhibited bundle formation in the presence of Ca2+, but not in the presence of EGTA. Calponin-related proteins including G-actin, tropomyosin, and SM22, had little effect on the binding of calponin to desmin filaments, whereas tubulin weakly inhibited the binding. Desmin had little influence on the ealponin-actin and calponin-tubulin interactions using the zero-length cross-linker, EDC. Domain mapping with chymotryptic digestion showed that the binding site of calponin resides within the central a-helical rod domain of the desmin molecule. The chemical cross-linked products of calponin and synthetic peptides (TQ27, TNEKVELQELNDRFANYIEKVRFLEQQ; EE24, EEELRELRRQVDALTGQRARVEVE) derived from the rod domain were detected by MALDI TOF/MS. Furthermore, the calponin-desmin interaction was significantly inhibited by the addition of EE24, but only slightly by TQ27. These results suggest that calponin may act as a cross-linking protein between desmin filaments as well as among intermediate filaments, microfilaments and microtubules in smooth muscle cells.抄録全体を表示PDF形式でダウンロード (3771K) -
Kenji Watanabe, Hideaki Oikawa, Kenji Yagi, Satoshi Ohashi, Takashi Mi ...2000 年 127 巻 3 号 p. 467-473
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーMacrophoma commelinae isolated from spots on leaves of Commelina communis has the ability to transform 5-acetyl-4-methoxy-6-methyl-2-pyrone (1) to 4-acetyl-3-methoxy-5-methylbenzoic acid (macrophomic acid, 2). This biotransformation includes the condensation of the 2-pyrone ring with a C3-unit precursor to form a substituted benzoic acid. We optimized conditions for induction of enzyme activity in M. commelinae, identified oxalacetate as a C3-unit precursor with cell extract, and purified the novel enzyme, macrophomate synthase. Oxalacetate inhibited the enzyme activity at a concentration higher than 5mM, and magnesium chloride stimulated the enzyme activity. Kinetic analyses gave Km of 1.7mM for 1 at 5mM oxalacetate, Km of 1.2 mM for oxalacetate at 5mM 1, and kcat, of 0.46 s-1 per subunit. Pyruvate was a weak substrate, with Km of 35.2mM and kcat of 0.46 s-1 at 5mM 1. We cloned and sequenced a cDNA encoding the macrophomate synthase. The cDNA of 1, 225 bp contained an open reading frame that encoded a polypeptide of 339 amino acid residues and 36, 244 Da, the sequence of which showed no significant similarity with known proteins in a homology search with BLAST programs. Transformed E. coli cells carrying the cDNA encoding the mature protein of macrophomate synthase overproduced macrophomate synthase under the control of the T7 phage promoter induced by IPTG. The purified enzyme showed the same values of Km and optimum pH as the native macrophomate synthase.抄録全体を表示PDF形式でダウンロード (1647K) -
Tomohisa Okutsu, Yoshimi Kuroiwa, Fusako Kagitani, Masayuki Kai, Kohzo ...2000 年 127 巻 3 号 p. 475-483
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーA large imprinted gene cluster in human chromosome 11p15.5 has been implicated in Beckwith-Wiedemann syndrome and Wilms' tumor. We have identified a paternally expressed imprinted gene, PEG8/IGF2AS, in this locus. It is transcribed in the opposite direction to the IGF2 transcripts and some genomic regions are shared with the IGF2 gene, as in the case of the mouse imprinted Igf2as gene reported previously by T. Moore et al. As to the relationship between these genomic regions, the human and mouse genes are very similar but there is no homology in their middle parts. Interestingly, PEG8/ IGF2AS and IGF2 were found to be overexpressed in Wilms' tumor samples, at levels over ten and a hundred times higher than that in normal kidney tissues neighboring the tumors, respectively. These findings indicate that PEG8/IGF2AS is a good marker of Wilms' tumor and also suggest the possibility of PEG8/IGF2AS being one of the candidate Wilms' tumor genes.抄録全体を表示PDF形式でダウンロード (2516K) -
Masayuki Jimbo, Kiwamu Yamagishi, Toshiaki Yamaki, Katsuyuki Nunomura, ...2000 年 127 巻 3 号 p. 485-491
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーAnalogs of the potent inhibitor of glucosylceramide (GlcCer) synthase, D-threo-l-phenyl-2-pahnitoylamino-3-pyrrolidino-1-propanol (P4), based on substitutions in the palmitoyl group were made by means of a stereo-selective synthetic method in order to elucidate the role of the hydrophobic portion in both the inhibitory action toward the enzyme and the biological effects. While P4 strongly inhibited GlcCer synthase with an IC50, of 0.5 μM in vitro, it also inhibited cell growth by 50% at the concentration of 7 μM. The shorter N-acyl chain analogs including decanoyl, octanoyl, and hexanoyl groups showed similar IC50 values for GlcCer synthase (around 2 μM), but the hexanoyl analog exhibited only a slight inhibitory effect on cell growth, showing the dissociation between GlcCer depletion and cell growth. Several compounds which exhibit similar hydrophobicity to the hexanoyl analog of P4 were subsequently designed. We found that D-threo-1-phenyl-2-benzyloxycarbonylamino-3-pyrrolidino-1-propanol (PBPP) was a most potent inhibitor, showing an IC50 of 0.3 μM. In cultured cells, PBPP was able to deplete glycosphingolipids without affecting cell growth or the ceramide level.抄録全体を表示PDF形式でダウンロード (1371K) -
Ayako Okado-Matsumoto, Akio Matsumoto, Junichi Fujii, Naoyuki Taniguch ...2000 年 127 巻 3 号 p. 493-501
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーPeroxiredoxins (PRxs) play a role in protecting protein free thiol groups against oxidative damage and thioredoxin-dependent peroxidase activity. This report describes the characteristics of the fourth member of the mammalian PRxs, PRx IV. Rat PRx IV produced in Sf21 cells by a baculovirus expression system has two bands with different electrophoretic mobilities, 31 and 27 kDa [Matsumoto et al. (1999) FEBS Lett. 443, 246-250]. The 27-kDa PRx IV lacks the NH2-terminal 36 amino acids which correspond to a predicted leader peptide, which is required for secretion from cells. Thus, the 31-kDa form is probably a precursor form, and the 27-kDa form, a secretable form which is enzymatically active. Pulse-chase experiments of PRx IV-transfected COS-1 cells showed that PRx IV is processed within 10 min and released from cells. The secretable form contains both reduced and oxidized forms. The reduced form binds to both a heparin affinity column and human umbilical vein endothelial cells, while the oxidized form does not. The equilibrium dissociation constants, KD, for heparin and heparan sulfate as judged by surface plasmon resonance experiments were 19 and 870 nM, respectively. The secretable form corresponds to the major bands found in most tissues, as evidenced by immunoblot analysis. Within cells, secretable form was largely localized on the endoplasmic reticulum, as judged by colocalization with calreticulin. Moreover, PRx IV has glutathione-dependent peroxidase activity in addition to thioredoxin-dependent activity. These data indicate that PRx IV is a secretable protein and may exert its protective function against oxidative damage by scavenging reactive oxygen species in the extracellular space.抄録全体を表示PDF形式でダウンロード (3976K) -
Togo Ikuta, Taro Tachibana, Junko Watanabe, Minoru Yoshida, Yoshihiro ...2000 年 127 巻 3 号 p. 503-509
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーThe aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that acts in concert with the AhR nuclear translocator (ARNT), and alters gene expression in response to environmental contaminants such as 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TC-DD). We have previously shown that AhR contains both a nuclear localization signal (NLS), AhR (13-39), and a nuclear export signal (NES), AhR (55-75), in its N112 -terminal region. In this study, we obtained direct evidence for the nucleocytoplasmic shuttling of AhR and show the biological significance of the shuttling in terms of the transcriptional activation of its target gene, CYP1A1. When AhR (13-75) fused with glutathione S-transferase (GST)-green fluorescent protein (GFP) was microinjected into the nucleus of a polykaryotic of BHK21 cell, the GST-AhR (13-75)-GFP migrated from one nucleus to the other. This event, nucleocytoplasmic shuttling, was completely inhibited in the presence of leptomycin B (LMB). The interaction between chromosome region maintenance 1 (CRM1) and endogenous AhR was shown by immunoprecipitation with antibodies to AhR followed by imanunoblot analysis with antibodies to CRM1. The inhibition of the nuclear export of AhR by LMB repressed the transcriptional activation of the CYP1A1 gene. The findings suggest that nuclear-cytoplasmic shuttling of AhR is essential for the inducible expression of the CYP1A1 protein.抄録全体を表示PDF形式でダウンロード (3558K) -
Kiyosumi Takaishi, Toshiaki Ohtsuka, Satomi Tsuneyoshi, Noriko Maehara ...2000 年 127 巻 3 号 p. 511-516
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーCytokine-induced neutrophil chemoattractant (CINC)-1, a counterpart of the human growth-regulated gene product (GRO) of the interleukin-8 family, has been suggested to play critical roles as a mediator of inflammatory reactions with neutrophil infiltration in rats. NF-kB has been speculated to be involved in the production of CINC-1, since the NF-kB-binding domain is important for the CINC-1 promoter activity in several of our reporter assays. In the present study, we examined the effects of an overexpression of IkBα, a specific natural inhibitor of NF-kB, on CINC-1 production. For this purpose, we constructed two recombinant adenoviruses, AxCAIkBα and AxCAmutantIkBα, which express respectively wild IkBα and a mutated nondegradable IkBα in which serine residues 32 and 36 are replaced by alanine residues. Transfecting wild and mutant IkBα by these adenovirus vectors inhibited NF-kB activation and CINC-1 production, which were both caused by IL-1β stimulation in the normal rat kidney epithelial cell line NRK-52E. We also showed that the nondegradable mutant IkBα was approximately 30 times more potent than the wild type in inhibiting CINC-1 production. These findings demonstrate that CINC-1 production with NF-kB activation is primarily regulated by nonphosphorylated IkBα in the cytoplasm.抄録全体を表示PDF形式でダウンロード (2350K) -
Hideki Tsuiki, Kiyofumi Asai, Manami Yamamoto, Kaori Fujita, Yuichiro ...2000 年 127 巻 3 号 p. 517-523
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーWe isolated a rat glia maturation factor-γ (rGMFG) cDNA and examined the tissue distribution of GMFG in rat by Northern and Western blot analyses. Sequence analysis of the entire cDNA revealed an open reading frame of 426 nucleotides with a deduced protein of 142 amino acid residues. The deduced amino acid sequence of the putative product is highly homologous (78.9%) to rat glia maturation factor-β (rGMFB). Northern blot analysis indicated that a 0.9-kb mRNA is predominantly expressed in rat thymus, testis, and spleen. GMFG showed a different tissue distribution from GMFB, being present predominantly in proliferative and differentiative organs.抄録全体を表示PDF形式でダウンロード (2277K)
- |<
- <
- 1
- >
- >|