巻号一覧
122 巻, 6 号
選択された号の論文の28件中1～28を表示しています
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Kazuhiro Sogawa, Yoshiaki Fujii-Kuriyama1997 年 122 巻 6 号 p. 1075-1079
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーThe aryl hydrocarbon receptor (AhR) is widely distributed in vertebrates and is known to be involved in metabolism of xenobiotics including man-made chemicals, most of which act as a ligand for the receptor, although no endogeneous ligand has yet been known. Upon binding a ligand, the receptor is activated to translocate to the nuclei, and during the nuclear translocation process, it is dissociated from the 90kDa heat shock protein (Hsp90) to form a heterodimer with Arnt (Ah receptor nuclear translocator). The heterodimer complex binds a DNA response element termed xenobiotic responsive element (XRE) localized upstream of the target genes of many drug-metabolizing enzymes including cytochrome P4501A1 and glutathione S-transferase to activate their transcription. Recent cDNA cloning has revealed that the AhR, like Arnt, possesses characteristic structural motifs of basic helix-loop-helix and PAS domains responsible for DNA recognition, heterodimerization, and ligand binding, and functions as a novel receptor-type transcription factor.抄録全体を表示PDF形式でダウンロード (697K) -
Yasuyuki Igarashi1997 年 122 巻 6 号 p. 1080-1087
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーThe signaling roles of ceramide and sphingosine produced through the degrading processes of membrane sphingolipids are currently receiving hot attention in the biochemical and biomedical research fields. For these 9 years at the Biomembrane Institute in Seattle, we have studied functional roles of various sphingolipids such as ceramide, sphingosine, methylsphingosines, and sphingosine 1-phosphate in a variety of biomedical systems. In this article, first, the recent conceptual developments on sphingolipid signaling pathways is outlined. Next, our recent findings on the functional roles of sphingolipids are described focusing on (i) functional roles of sphingosine 1-phosphate in cell motility regulation and platelet activation (ii) involvement of sphingosine in cell signaling (iii) effects of methylsphingosines in cancer cell apoptosis induction and in the regulation of inflammatory processes. Based upon these findings from our studies and others, the perspective of future sphingosine research (sphingology or sphingophysiology) is briefly discussed.抄録全体を表示PDF形式でダウンロード (1055K) -
Yasumasa Miyazaki, Osamu Tsunoka, Kazuo Shishido1997 年 122 巻 6 号 p. 1088-1091
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーThe 565 amino-acid PRIB protein with a Zn (II) 2Cys6 zinc-cluster DNA-binding motif is the expression product of the priB gene, which is most actively transcribed in an early stage of fruiting-body formation by the basidiomycete, Lentinus edodes. PRIB produced in Escherichia coli using the bacteriophage T7 expression system was purified by ion-exchange chromatographies and then subjected to random binding-site selection analysis using a pool of random 24-bp oligonucleotides with 13-bp PCR primer sites at each end. The oligonucleotides (50 bp) selected for PRIB binding were cloned into pUC19. A total of 303 cloned DNA fragments were picked randomly and sequenced. The PRIB binding sites could be grouped into 25 individual sequences, suggesting a consensus sequence of 16 bp, 5' GGGGGGGACAGGANCC 3'. Gel mobility-shift assaying of 10 randomly selected sequences all revealed a reasonable band shift. DNase I footprinting analysis of the 50-bp DNA fragment containing the sequence most similar to the consensus sequence showed that PRIB protects the entire 16-bp sequence from digestion by DNase I.抄録全体を表示PDF形式でダウンロード (2920K) -
Daisuke Tsuchiya, Takeshi Sekiguchi, Akio Takenaka1997 年 122 巻 6 号 p. 1092-1104
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーThe crystal structure of 3-isopropylmalate dehydrogenase from the moderate facultative thermophile Bacillus coagulans (BcIPMDH) has been determined by the X-ray method. BcIPMDH is a dimeric enzyme composed of two identical subunits, each of which takes an open α/β structure with 11 α-helices and 14 β-strands. The polypeptide is folded into two domains. The first domain is composed of residues 1-101 and 257-356, and the second domain, of residues 102-256. The latter domains of the two subunits are associated with one another by a dyad axis to make the dimer, locally forming a β-sheet and a four-helix bundle. As compared with the structure of the enzyme from the extreme thermophile Thermus thermophilus (TtIPMDH), a new short β-sheet (residues 329-330 and 340-341) absent in TtIPMDH is formed by the insertion of 5 residues in BcIPMDH. In terms of determinants for thermostabilization, both consistent and inconsistent changes were found between the two enzymes. The regions including inconsistent changes are formed by different usages of the determinants for stabilizing the loops at different levels. Those in BcIPMDH contain some structural redundancies in length of amino acid sequence and flexibility of residues, which seem to be unnecessary for the enzymatic reaction. Such redundancies are also found in the primary structure of the enzyme of the mesophile Bacillus subtilis, but these parts are more stabilized in BcIPMDH by hydrogen bonds and salt bridges. On the other hand, TtIPMDH is stabilized by reducing such redundant parts. This contrast suggests that different strategies may be preferred for thermostabilization, depending on temperature.抄録全体を表示PDF形式でダウンロード (3758K) -
Heimo Riedel, Jian Wang, Hans Hansen, Nasim Yousaf1997 年 122 巻 6 号 p. 1105-1113
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーInsulin stimulation results in a considerable spectrum of cellular responses, only part of which have been firmly correlated with the activation of established insulin receptor (IR) targets such as IRS-1, IRS-2, and Shc. Many responses may be transduced by alternative direct IR targets, some of which may still be unknown, may act in parallel to but independently of IRS-1, IRS-2, and Shc, and may be members of the growing family of SH2 domain-containing signaling adaptors. An SH2 domain-coding region of a protein termed PSM was cloned based on its interaction with an activated IR cytoplasmic fragment in a yeast two-hybrid screen. When used as a hybridization probe this region led to the isolation of a protein-coding cDNA which is expressed with a wide tissue distribution and exists in several variant forms. A pleckstrin homology domain and three Pro-rich regions including a putative SH3 domain binding site were identified in addition to the SH2 domain in the deduced 756 amino acid sequence. They imply a role of PSM in tyrosine kinase and phosphatase-mediated signaling pathways. A similar sequence termed SH2-B had been reported in an earlier study, which may represent the rat homolog of PSM. A role of PSM specifically in insulin action is suggested by the interaction of its SH2 domain with an activated but not with an inactive catalytic fragment of the IR in the yeast two-hybrid system in vivo, by the insulin-dependent association of a glutathione S-transferase (GST) PSM SH2 domain fusion protein with purified IR in vitro, and by the insulin-dependent association of GST PSM SH2 with the IR in cell extracts. In contrast, PSM was not found to associate with the established IR substrate IRS-1 under any conditions and appears to act independently of IRS-1. All of our findings are compatible with a putative role of PSM in insulin action.抄録全体を表示PDF形式でダウンロード (3530K) -
Mitsuhide Noshiro, Yuri Aoyama, Takeshi Kawamoto, Osamu Gotoh, Tadao H ...1997 年 122 巻 6 号 p. 1114-1121
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーThe structure of rat CYP51 gene encoding sterol 14-demethylase was examined. The CYP51 gene spanned about 18kb and contained 10 exons. The copy number of CYP51 in the rat genome was determined to be one. In addition, one CYP51 processed (intron-less) pseudogene covering the coding and ca. 600-bp 3'-noncoding sequences of CYP51 cDNA was found in the rat genome. Multiple transcription initiation sites were predicted by primer extension and 5'-RACE methods using poly(A)+ RNA from liver, ovary, and testis, and the major ones were located at 126 and 123 nucleotides upstream from the initiation ATG codon. The primer extension also showed several minor sites around the major ones. In addition to these sites, other minor initiation sites were also predicted at around 330 and 460 nucleotides upstream from the initiation ATG codon. No TATA box was found in the putative promoter region, but multiple GC boxes were found around the cap sites, supporting the previously inferred housekeeping nature of CYP51 gene and the existence of the multiple transcription initiation sites. A few consensus transcription regulatory elements such as CRE were found in the 5'- flanking region. Four polyadenylation signals were found in the 3'-noncoding region by the 3'-RACE method. Three of them were used to generate 3.1-, 2.7-, and 2.3-kb mRNAs in liver and ovary. The remaining one was used only in testis to generate 1.9-kb mRNA having an unusually short trailer sequence, suggesting a specific regulatory mechanism for generating CYP51 mRNA in testis different from that in liver and ovary.抄録全体を表示PDF形式でダウンロード (6138K) -
Yuzo Yoshida, Mitsuhide Noshiro, Yuri Aoyama, Takeshi Kawamoto, Tadao ...1997 年 122 巻 6 号 p. 1122-1128
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーPhylogenetic analyses based on protein sequence data indicated that sterol 14-demethylase P450 (CYP51) and bacterial CYP51-like protein were joined into a distinctive evolutionary cluster, CYP51 cluster, within the CYP protein superfamily. The most probable branch topology of the CYP51 phylogenetic tree was (bacteria, (plants, (fungi, mammals))), which is comparable to the phylogeny of major kingdoms of living matter, suggesting that CYP51 has been conserved from the era of prokaryotic evolution. This may be strong evidence supporting the prokaryotic origin of P450. Structure of flanking regions and the number and insertion sites of introns are quite different between mammalian and fungal CYP51s. This fact indicates that different mechanisms are operative in evolution of protein sequences and gene structures. CYP51 is the first example violating the well-documented rule that the basic structure of a gene, including intron insertion sites, is well conserved in each P450 family. One CYP51 processed a pseudogene was found in rat genome. Nonsynonymous nucleotide divergence observed between the pseudogene and CYP51 cDNA was less than one-fifth of the synonymous divergence. This unusually low rate of nonsynonymous nucleotide changes in the pseudogene suggests that it may be derived from another CYP51, which might have been active for a significant duration in the past.抄録全体を表示PDF形式でダウンロード (925K) -
Keiichi Takagaki, Toshiyuki Tazawa, Hidekazu Munakata, Toshiya Nakamur ...1997 年 122 巻 6 号 p. 1129-1132
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーHuman skin fibroblasts were cultured with a fluorogenic xyloside, 4-methylumbelliferyl-β-D-xyloside (Xyl-MU) as an initiator, and the effects of monensin, which destroys the normal structure of the Golgi complex, on the synthesis of Xyl-MU-initiated glycosaminoglycan (GAG-MU) and its linkage region oligosaccharides were investigated. When the cells were incubated with Xyl-MU in the presence of monensin, the synthesis of GAG-MU was inhibited. In addition, the synthesis of Galβ1-3Galβ1-4Xylβ1-MU as an intermediate of GAG-MU was inhibited, whereas the synthesis of Galβ1-4Xylβ1-MU, which is formed prior to Galβ1-3Galβ1-4Xylβ1-MU, was not. These results indicate that inhibition of GAG-MU synthesis by monensin occurs at the point where the second galactose is joined to Galβ1-4Xylβ1-MU.抄録全体を表示PDF形式でダウンロード (1722K) -
Yoshiaki Azuma, Takahisa Hachiya, Takeharu Nishimoto1997 年 122 巻 6 号 p. 1133-1138
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーNine monoclonal antibodies to RCC1, the guanine nucleotide-exchange factor on Ran GTPase, were obtained using recombinant RCC1 as the antigen. Epitopes of three monoclonal antibodies, which did not inhibit RCC1 function, were localized in the N-terminus outside the RCC1 repeat, while epitopes of the other 6 monoclonal antibodies were localized within the RCC1 repeat. Three of the latter 6 monoclonal antibodies, 2B6, 6C3, and 8D9, inhibited RCC1-stimulated nucleotide release. Two of them, 2B6 and 6C3, recognized the same amino acid residues in the N-terminus of the second RCC1 repeat, Tyr89, Ser90, Phe91, and G1y92, of which one, G1y92, is conserved in Saccharomyces cerevisiae and mutated in an ree1- strain, mtr1-2. The monoclonal antibody 8D9 recognized two amino acid residues, Arg320 and A1a321, downstream of Gly319 in the N-terminus of the 6th RCC1 repeat, which corresponds to Gly92 in the second RCC1 repeat. The monoclonal antibodies which inhibited RCC1 function bound to RCCI in homogenous solution and stained cellular RCC1. We propose that the N-terminus of the RCC1 repeat is exposed at the surface of RCC1 on the coated plate or in fixed cells, and is involved in the RCC1-stimulated nucleotide exchange on the Ran GTPase.抄録全体を表示PDF形式でダウンロード (2867K) -
Naoki Sato, Satoru Kawamura1997 年 122 巻 6 号 p. 1139-1145
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーS-Modulin is suggested to increase the light sensitivity of rods by inhibiting phosphorylation of light-activated rhodopsin (Rh*) at high Ca2+ concentrations. The inhibition of rhodopsin phosphorylation was almost constant over a wide range of the Rh*/S-modulin ratio (10-4-_??_101). A 125I-labeled cross-linker that had been conjugated with S-modulin interacted with a protein of 60kDa, a molecular mass close to that of frog rhodopsin kinase. These results suggested that the target molecule of S-modulin is rhodopsin kinase. To investigate the mechanism of the S-modulin action, we measured rhodopsin phosphorylation in the presence and absence of inhibition by S-modulin at various timings of ATP addition. The results suggested the following in situ mechanism of S-modulin action. After light-activation of rhodopsin kinase, the S-modulin/Ca2+ complex binds to the activated kinase and inhibits the phosphorylation of rhodopsin. The complex, however, does not affect the overall kinetics of the phosphorylation. The inhibition of the kinase by S-modulin is reversible in terms of the Ca2+ concentration. On the other hand, the kinase activity decreases as a function of time, probably via autophosphorylation.抄録全体を表示PDF形式でダウンロード (2291K) -
Shuji Wakatsuki, Shih-Ching Ho, Manabu Arioka, Makari Yamasaki, Katsuh ...1997 年 122 巻 6 号 p. 1146-1152
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーBy screening with monoclonal antibodies (mAbs) raised against growth cone membrane fraction from fetal porcine brains, we have identified a 230kDa antigen, termed p230. Western blot analysis of extracts from various tissues demonstrated that p230 is specifically expressed in brains, in which its expression is temporally restricted; it was especially prominent in the embryonic and the early postnatal stage, and decreased to subdetectable levels in the adult brain. Further characterization of p230 revealed that it is a peripherally-membrane associated, cell surface protein produced by astrocytes. Neurite outgrowth of E18 rat cerebral cortex neurons cultured on a monolayer of astrocytes was significantly reduced in the presence of anti-p230 polyclonal antibody. Partial amino acid sequences of p230 purified from fetal porcine brains were highly homologous to an extracellular matrix protein, tenascin-C. These lines of evidence suggest that p230, a tenascin-C-like molecule present in fetal porcine brains, plays important roles during early brain development, particularly in growth cone guidance.抄録全体を表示PDF形式でダウンロード (3248K) -
Yoshito Abe, Tadashi Ueda, Taiji Imoto1997 年 122 巻 6 号 p. 1153-1159
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーJones et al. have reported that the ε-carbons of methionine residues in myoglobin can be enriched with stable isotope (13C) in two steps, i.e., methylation of methionine residues with 13CH3I in the protein and thiolysis using dithiothreitol [Jones, W. C., Rothgeb, T. M., and Gurd, F. R. N. (1976) J. Biol. Chem. 251, 7452-7460]. Using their method, we failed to prepare active lysozyme in which the ε-carbons of methionine residues are enriched with 13C, because many side reactions took place under the thiolysis condition (pH 10.5, 37°C). When we employed 2-aminothiophenol as a reagent for thiolysis, the reduction proceeded under a weakly acidic condition to afford fully active lysozyme, in which the ε-carbons of two methionine residues were enriched with 13C, in a 30% yield. Analysis of the 13C-edited NOESY spectra of 13C-enriched methionine lysozyme in the absence and presence of a substrate analogue indicated the occurrence of conformational change around Met 105 in lysozyme.抄録全体を表示PDF形式でダウンロード (2835K) -
Kaoru Hirano, Azumi Tanaka, Kazuma Yoshizumi, Tamotsu Tanaka, Kiyoshi ...1997 年 122 巻 6 号 p. 1160-1166
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーThe properties of phospholipase A1 (PLA1) obtained from the white muscle of bonito, Euthynnus pelamis (Linnaeus), were examined. The PLA1 activity had a pH optimum from 6.5 to 7.0 for phosphatidylcholine (PC), and calcium ion was not required. The optimum temperature was from 20 to 30°C. When a fatty alcohol was used as an acceptor, a wax ester was produced by transferring a fatty acid at the sn-1 position of the donor's PC. The maximum production of lysophosphatidylcholine was shifted by 0.5pH units to the acidic side and the pH optimum of wax ester synthesis was from 6.0 to 6.5. The synthesis was independent of calcium ion and Coenzyme A. The transacylation was also observed when 1-lyso-2-acyl-sn-glycero-3-phosphocholine was used as an acceptor. Fatty acid at the sn-1 position of the donor PC was transferred to the unoccupied hydroxy group of the acceptor at the sn-1 position. When 2, 3-dipalmitoyl-sn-glycero-l-phosphocholine was used as the acyl donor, a similar amount of palmitic acid was transferred as in the case of 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine. However, l-acyl-2-lyso-sn-glycero-3-phosphocholine, a positional isomer, was a poor acceptor. These results indicate that the transacylation by the PLA1 from bonito muscle is not stereospecific, but is position-specific both for the acyl donor and acceptor.抄録全体を表示PDF形式でダウンロード (769K) -
Takayuki Kato, Kayo Hatanaka, Tomohiro Mega, Sumihiro Hase1997 年 122 巻 6 号 p. 1167-1173
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーEndo-β-N-acetylglucosaminidase from hen oviduct (Endo-HO) was purified to homogeneity by ammonium sulfate fractionation and then by column chromatographies on DEAE-Sephacel, hydroxyapatite, Octyl-Sepharose CL-413, Co2+-chelating Sepharose FF, and YMC-Pack Diol-200G. Partial purification of the enzyme was reported previously [Tarentino, A. L. and Maley, F. (1976) J. Biol. Chem. 251, 6537-6543]. The molecular weight was 54, 000 by gel filtration and 52, 000 by SDS-PAGE in the presence of 2-mercaptoethanol, indicating that Endo-HO is composed of a single polypeptide chain. The optimum pH was 6.5, and the Km value was 25μM when pyridylaminated Man6GlcNAc2 was used as a substrate. EDTA and metal cations tested, except Hg2+, had no effects on Endo-HO activity. Substrate specificity results using pyridylaminated N-linked sugar chains revealed that Endo-HO hydrolyzed oligomannose-type sugar chains faster than complex- and hybridtype chains, and that sugar chains containing the Manα1-2Manα1-3Manβ1-4GlcNAcβ1-GlcNAc structure were good substrates for the enzyme. These findings suggest that in cytosol the enzyme contributes to the production of a free oligosaccharide with one reducing end N-acetylglucosamine residue in cooperation with neutral α-mannosidase, an enzyme that specifically hydrolyzes oligosaccharides to Manα1-2Manα1-2Manα1-3(Manα1-6)Man-β1-4GlcNAc.抄録全体を表示PDF形式でダウンロード (1510K) -
Kazuya Yamashiro, Hiroyuki Itoh, Mai Yamagishi, Shunji Natsuka, Tomohi ...1997 年 122 巻 6 号 p. 1174-1181
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーNeutral α-mannosidase was purified to homogeneity from hen oviduct. The molecular mass of the enzyme was 480kDa on gel filtration, and the 110-kDa band on SDS-PAGE in the presence of 2-mercaptoethanol indicated that it is composed of four subunits. The activated enzyme hydrolyzed both p-nitrophenyl α-D-mannoside and high mannose-type sugar chains. This substrate specificity is almost the same as that reported for the neutral α-mannosidase from Japanese quail oviduct [Oku and Hase (1991) J. Biochem. 110, 982-989]. Manα1-6(Manα1-3)Manα1-6(Manα1-3)Manβ1-4GlcNAc (Km=0.44mM) was hydrolyzed four times faster than Manα1-6(Manα1-3)Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc, and Manα1-6(Manα1-2Manα1-2Manα1-3)Manβ1-4GlcNAc was obtained as the end product from Man9GlcNAc on digestion with the activated α-mannosidase. The enzyme was activated 24-fold on preincubation with Co2+. The activation with other metal ions, like Mn2+, Ca2+, Fe2+, Fe3+, and Sr2+, was less than 5-fold, and Zn2+, Cu2+, and Hg2+ inhibited the enzyme activity. The optimum pHs for both the enzyme activity and activation with Co2+ were around 7. The cobalt ion contents of the purified, EDTA-treated, and Co2+-activated enzymes were 1.5, 0.0, and 3.9, respectively, per molecule. Since the Co2+-activated enzyme gradually lost its activity on incubation with EDTA and the activity was restored promptly on the addition of Co2+, the binding of Co2+ to the enzyme seems to be essential for its activation. The results obtained with protease inhibitors together with those of the SDS-PAGE before and after activation, showed that the proteolytic cleavage reported for the activation of monkey brain α-mannosidase seems not to be involved.抄録全体を表示PDF形式でダウンロード (1985K) -
Kazuhisa Kishimoto, Tohru Yoshimura, Kenji Soda, Nobuyoshi Esaki1997 年 122 巻 6 号 p. 1182-1189
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーD-Amino acid aminotransferase is the only aminotransferase that catalyzes the transamination of D-amino acids. We studied the role of the binding site for the α-carboxyl group of substrates, which is presumably crucial for the unique stereospecificity of the enzyme. The site-directed mutagenesis of Arg98, which is the putative carboxyl-binding site, as judged on the basis of X-ray crystallographic studies [Sugio, S., Petsko, G. A., Manning, J. M., Soda, K., and Ringe, D. (1995) Biochemistry 34, 9661-9669], by replacement with methionine and lysine, resulted in decreases in the kmax, values and increases in the Kd values for both amino donors and amino acceptors. The introduction of another mutation, that of Tyr88, which is located near Arg98 in the spacial structure, by replacement with arginine, in addition to the above Arg98 mutation, resulted in increases in the kmax values but little change in the Kd values. These results suggest that Arg98 constitutes the carboxyl-binding site for the substrate, efficient catalysis by the enzyme being facilitated upon binding. The mutant enzymes are also relieved from inhibition by high concentrations of α-ketoglutarate, which is an inherent character of the wild-type enzyme. Therefore, Arg98 is also responsible for the inhibition by α-ketoglutarate.抄録全体を表示PDF形式でダウンロード (924K) -
Yukiko Kimura, Megumi Nakazawa, Naoto Tsuchiya, Shuichi Asakawa, Nobuy ...1997 年 122 巻 6 号 p. 1190-1195
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーThe OS-9 gene is frequently amplified in human sarcomas. We isolated and characterized an OS-9 genomic DNA from a human BAC library. Sequencing of the genomic DNA showed that the gene spanned approximately 30.4 kbp and had 15 exons. The 1, 010 by sequence of the 5' upstream region was also determined. The potential binding-sequence motifs TATA and CCAAT for general transcription factors were found in the 5' upstream region. Primer extension analysis revealed two putative transcription start sites. The significance of the 5' upstream sequence in the ubiquitous expression of the OS-9 gene in various tissues and culture cells is discussed.抄録全体を表示PDF形式でダウンロード (1964K) -
Misako Hayama, Satoshi Akiba, Misato Fukuzumi, Takashi Sato1997 年 122 巻 6 号 p. 1196-1201
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーThe stimulation of prostaglandin E2 (PGE2) production in mesangial cells exposed to a high glucose level was studied from the viewpoint of its implication in the glomerular hyperfiltration in diabetic nephropathy. The basal PGE2 synthesis apparently increased in the cells on incubation with a high glucose level (20mM) for 3-6h. Under these conditions, secretory phospholipase A2 activity was not detected in the incubation medium, but cytosolic phospholipase A2 (cPLA2) activity in the cells increased time-dependently up to 6h, compared with that with a normal glucose level (5mM). However, no difference in the cPLA2 protein content between the two glucose levels was observed on immunoblot analysis, suggesting that the increased cPLA2 activity under high glucose conditions is not due to stimulation of de novo synthesis. Stimulation with a calcium ionophore markedly enhanced arachidonic acid liberation and PGE2 production by cells exposed to the high glucose level. Furthermore, mitogen-activated protein kinase (MAPK) activity increased time-dependently under high glucose conditions, the rate of increase being consistent with those in cPLA2 activity and PGE2 production under the same conditions. These data suggest that glucose-induced cPLA2 activation through MAPK activation is responsible for the enhancement of PGE2 production in mesangial cells.抄録全体を表示PDF形式でダウンロード (1860K) -
Chisato Ushida, Shinpei Sasamura, Akira Muto1997 年 122 巻 6 号 p. 1202-1207
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーMCS4 RNA is one of the small stable RNAs found in Mycoplasma capricolum. Its function is unknown. The conformation of MCS4 RNA (125 nucleotides in length) in solution was investigated using chemical and enzymatic probes. Single and double-stranded regions were estimated by means of diethyl pyrocarbonate (DEPC) and by dimethyl sulfate (DMS) modifications. Ribonuclease V1 was also used to identify paired or stacked nucleotides. Based on these data, a secondary structure model for MCS4 RNA containing four stemstructures was constructed.抄録全体を表示PDF形式でダウンロード (4232K) -
Misato Kashiba-Iwatsuki, Keiko Kitoh, Emiko Kasahara, Hidenori Yu, Man ...1997 年 122 巻 6 号 p. 1208-1214
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーAlthough S-nitrosoglutathione (GS-NO) and other S-nitrosothiols (RS-NO) exhibit activity attributable to nitric oxide (NO), the dynamic aspects of their metabolism remain to be elucidated. To determine the fates and functions of RS-NO, the stability of GS-NO was analyzed in plasma, and various fractions of liver and kidney. GS-NO was fairly stable under physiological conditions in plasma and buffer solutions. However, GS-NO was rapidly decomposed in the presence of either homogenates of rat liver and kidney or their supernatant fractions. The ability of the supernatants to decompose GS-NO remained unchanged after the removal of proteins and large molecular weight compounds. Physiological levels of reducing agents, such as reduced glutathione (GSH), ascorbic acid (AsA), and cysteine, also enhanced the decomposition of RS-NO; the order of their potency was AsA>cysteine>GSH. Considering their intra-cellular concentrations and potency, AsA might principally be responsible for the enhanced decomposition of GS-NO. NO, GS-NO, and related RS-NO inhibited the respiration of Ehrlich ascites tumor cells. The inhibitory effect of GS-NO was enhanced by the reducing agents (cysteine>AsA>GSH). Intravenously administered GS-NO exhibited a depressor action through some ascorbic acid-enhancable mechanism. Thus, the metabolism and biological function of GS-NO and related RS-NO might be affected by AsA and other reducing agents.抄録全体を表示PDF形式でダウンロード (1579K) -
Yasuhiko Takahashi, Masamitsu Yamaguchi, Fumiko Hirose, Jun Kobayashi, ...1997 年 122 巻 6 号 p. 1215-1223
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーThe promoter region of the Bombyx mori gene encoding the proliferating cell nuclear antigen (PCNA), and its activating factor (s) were analyzed to ascertain similarities with Drosophila regulatory elements. Full promoter activity was established to reside within the region from -466 to +347 base pairs with respect to the transcription initiation site. Within this region, we found a sequence similar to the DNA replication-related element (DRE), 5'-TATCGATA, which is a promoter-activating sequence common to promoters of the Drosophila genes for DNA replication-related factors, including PCNA. A mutation in the DRE-like sequence of the B. mori PCNA gene promoter caused reduction of the promoter activity and also binding to the recombinant Drosophila DRE-binding factor (DREF). Furthermore, a factor (s) binding to the DRE sequence was detected in extracts of B. mori BmN4 cells. Monoclonal antibodies against Drosophila DREF inhibited the binding activity of the factor, as shown by gel mobility shift assays, and allowed specific detection of a 100kDa protein on immuno Western blot analysis. These results suggest that the B. mori DREF homolog binds to DRE to regulate transcription of the PCNA gene.抄録全体を表示PDF形式でダウンロード (3357K) -
Norihiro Sato, Shoko Fujiwara, Akihiko Kawaguchi, Mikio Tsuzuki1997 年 122 巻 6 号 p. 1224-1232
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーA gene for chloroplast ω6 desaturase, which catalyzes the desaturation of monoenoic to dienoic acids in chloroplasts, was isolated from Chlamydomonas reinhardtii. We first performed reverse transcriptase-polymerase chain reaction with oligonucleotide primers corresponding to regions conserved among plastid ω6 desaturases of higher plants and Δ12 desaturases of cyanobacteria, using C. reinhardtii poly (A)+ RNA. An amplified DNA fragment of 0.5kb, containing a frame for a protein homologous to these desaturases, was used as a probe for screening cDNA and genomic DNA libraries of C. reinhardtii. The cDNA clone of 2.2kb obtained contained an open reading frame encoding a protein of 424 amino acids with a putative molecular mass of 48.4kDa, the amino acid sequence of which showed 46-51% homology to those of higher plant plastid ω6 and cyanobacterial Δ12 desaturases. Introduction of the cloned genomic counterpart of this cDNA, designated as des6, into a Chlamydomonas mutant with defects in chloroplast ω6 desaturation and in the activities of photosystems I and II (PSI and PSII) complemented the desaturation mutation, indicating that the des6 gene codes for chloroplast ω6 desaturase. The complemented strains did not recover from the photosynthetic lesions, but showed lower PSII activity at 45°C than the desaturation mutant, proving that the photosynthetic lesions in hf-9 are not caused by the desaturation mutation, and that the lowered unsaturation level of chloroplast lipids in the mutant is responsible for the expression at this high temperature of PSII activity, one of the thylakoid membrane functions.抄録全体を表示PDF形式でダウンロード (2319K) -
Mizuho Kaneda, Ken-ichi Takeuchi, Keizo Inoue, Masato Umeda1997 年 122 巻 6 号 p. 1233-1240
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーIn this study, we demonstrated that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a phosphatidylserine (PS)-binding protein and localized the putative PS-binding site involved in the membrane fusion induced by this enzyme. In an attempt to identify the PS-binding proteins, we raised polyclonal antibodies against a 15-amino-acid synthetic peptide (amino acid residues 390-403 of phosphatidylserine decarboxylase), which was shown to bind specifically to PS. One polyclonal antibody, designated aPSD-2, crossreacted with GAPDH, and its binding to GAPDH was inhibited by PS but not by other phospholipids such as phosphatidylethanolamine and phosphatidylinositol. Kinetic analysis of GAPDH binding to phospholipid membranes by measuring surface plasmon resonance showed that GAPDH associated with the phospholipid membrane containing PS rapidly (kon=2.8×104M-1•s-1) and dissociated extremely slowly (koff=5.9×10-5s-1), giving a low dissociation constant (KD=2.6nM). GAPDH bound less effectively to membranes without PS with a dissociation constants of 0.2μM. GAPDH-induced vesicle fusion was also inhibited by aPSD-2, suggesting that this antibody recognizes the putative PS-binding site on GAPDH involved in the enzyme-induced membrane fusion. Chemical fragmentation of GAPDH with cyanogen bromide followed by separation and sequence analysis of the reactive peptide resulted in the identification of a single reactive peptide with the sequence of amino acid residues 45-103 of GAPDH. Analysis of aPSD-2 binding to synthetic peptides derived from the corresponding region localized the antibody-binding site to amino acid residues 70-94 of GAPDH. Both the 25-amino-acid synthetic peptide (amino acid residues 70-94 of GAPDH) and polyclonal antibody raised against this peptide inhibited GAPDH-induced membrane fusion, suggesting that these amino acid residues play a crucial role in this membrane fusion process.抄録全体を表示PDF形式でダウンロード (3549K) -
Wakano Ogawa, Takashi Kayahara, Masaaki Tsuda, Tohru Mizushima, Tomofu ...1997 年 122 巻 6 号 p. 1241-1245
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーL-Serine as well as L-valine inhibits the growth of Escherichia coli cells, and L-isoleucine releases this growth inhibition. We isolated an E. coli mutant (designated as WAT9) that was able to grow on lactate (or glucose) as a carbon source even in the presence of L-serine, the parent not being able to. Cells of WAT9 were not able to grow on L-serine as a carbon source even if L-isoleucine was present in the culture medium, while the parental cells grew. This mutant was shown to lack the principal L-serine transporter in E. coli, the Na+/serine symporter. This mutant is useful for analysis of the role (s) of the Na+/serine symporter in cell physiology and as a host for the cloning of L-serine transporter gene (s). In fact, we cloned a gene encoding a serine transporter from chromosomal DNA of E. coli using WAT9 as the host. The gene enabled the mutant cells to grow on L-serine. Transport activity for L-serine was restored in the mutant cells harboring a plasmid carrying the gene. We partially sequenced the gene and found that it was the tdcC gene. We showed that TdcC is an H+/serine symporter.抄録全体を表示PDF形式でダウンロード (666K) -
Koji Uchida, Yuri Hasui, Toshihiko Osawa1997 年 122 巻 6 号 p. 1246-1251
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーIt has been established that the covalent modification of proteins occurs in vivo as a consequence of reaction with reactive lipids such as 4-hydroxy-2-nonenal (HNE). The fact that HNE occurs in blood under physiological as well as pathophysiological conditions suggests that erythrocytes undergo modification by HNE. Here we describe the immunochemical characterization of HNE-treated erythrocytes by demonstrating the Michael-type HNE addition to both membrane and cytosolic proteins. Exposure of erythrocytes to HNE (0-0.5mM) for 2 h resulted in HNE labeling of multiple membrane proteins. Pretreatment of erythrocytes with a sulfhydryl reagent, N-ethylmaleimide (NEM), resulted in a significant decrease of HNE attached to the proteins, suggesting that HNE primarily reacts with the sulfhydryl groups of erythrocyte membrane proteins, whereas enhanced HNE labeling of the membrane proteins was observed when the erythrocytes were pretreated with H2O2 (0.1-5mM) for 15min. On the other hand, highly selective modification of a 30kDa protein was observed in the hemolysates of erythrocytes treated with HNE. The protein, which represents a major intracellular target of HNE in erythrocytes, was identified as carbonic anhydrase, based on the observations that (i) a reverse-phase HPLC analysis of the chloroform/ethanol extract of HNE-treated erythrocytes detected two major proteins, which cross-reacted with anti-carbonic anhydrase antibody as well as with the anti-HNE adducts antibody, (ii) the chloroform-ethanol extraction of authentic carbonic anhydrase gave a similar HPLC pattern, and (iii) the HNE treatment of erythrocytes resulted in the partial inhibition of the carbonic anhydrase activity.抄録全体を表示PDF形式でダウンロード (3213K) -
Long-Sen Chang, Yii-Cheng Chou, Shinne-Ren Lin, Bin-Nan Wu, Jordge Lin ...1997 年 122 巻 6 号 p. 1252-1259
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーA novel neurotoxin, cobrotoxin b, was isolated from Naja naja atra (Taiwan cobra) venom by successive chromatographies on gel filtration and SP-Sephadex C-25 columns. The yield of this novel toxin was 5% of that of cobrotoxin from the same venom. Its neurotoxicity determined as the inhibition of acetylcholine-induced muscle contractions was approximately 50% of that of cobrotoxin. Cobrotoxin b consists of 61 amino acid residues including 8 cysteine residues. Moreover, there are 12 amino acid substitutions between cobrotoxin b and cobrotoxin. The genomic DNA, with a size of 2, 386 bp, encoding the precursor of cobrotoxin b was isolated from the liver of N. naja atra. The gene consists of three exons separated by two introns. This exon/intron structure is essentially the same as that reported for the cobrotoxin gene. Moreover, the nucleotide sequences of the two neurotoxin genes exhibit 92% identity. These results highly suggest that the cobrotoxin b and cobrotoxin genes are derived from a common ancestor. Comparative analyses of cobrotoxin b and cobrotoxin precursors showed that the protein-coding regions of the exons are more diverse than introns, except for in the signal peptide domain. This indicates that the protein-coding regions may have arised via accelerated evolution. BLAST searches for sequence similarity in the GeneBank databases showed that intron 1 of the cobrotoxin b and cobrotoxin genes encodes a small nucleolar RNA (snoRNA). However, the snoRNA gene is absent from the gene encoding the Laticauda semifasciata erabutoxin c precursor (L. semifasciata and N. naja atra are sea and land snakes, respectively). Since previous studies suggested the potential mobility of snoRNA genes during evolution, we propose that intron insertions or deletions of snoRNA genes occurred with the evolutionary divergence between the sea snake and land snake neurotoxins.抄録全体を表示PDF形式でダウンロード (1018K) -
Keiichiro Suzuki, Masaharu Nakamura, Yutaka Hatanaka, Yoshiro Kayanoki ...1997 年 122 巻 6 号 p. 1260-1264
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーHuman vascular endothelial cells play a pivotal role in atherosclerotic changes but are resistant to apoptotic inducers such as Fas ligand and it has been difficult to induce apoptosis. We developed an experimental model for the apoptosis in the endothelial cells by using snake venom treatment. Snake venom was found to generate intracellular reactive oxygen species (ROS) in the endothelial cells, which leads to apoptosis as judged by electron microscopy as well as by DNA cleavage. Buthionine sulfoximine (BSO) and diethyldithiocarbamate (DDC) accelerated the apoptosis, indicating intracellular glutathione and superoxide levels play a critical role. Pretreatment with tumor necrosis factor (TNF) or phorbol ester (TPA), which increases the Mn-SOD level, prevented the apoptosis. These data suggest that intracellular ROS enhances apoptosis whereas several anti-oxidants are protective in human endothelial cells. The induction of apoptosis by ROS of endothelial cells may be related to initiation of atherosclerotic changes.抄録全体を表示PDF形式でダウンロード (1631K) -
Naoya Kojima, Yuriko Tachida, Shuichi Tsuji1997 年 122 巻 6 号 p. 1265-1273
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーWe previously cloned cDNAs encoding two different polysialic acid (PSA) synthases, STBSia II and IV, from mouse, and showed that both mouse ST8Sia II and IV can synthesize PSA on the neural cell adhesion molecule (NCAM) as well as other glycoproteins such as fetuin, at least in vitro (Kojima, N., Tachida, Y., Yoshida, Y., and Tsuji, S. (1996) J. Biol. Chem. 271, 19457-19463]. In the present study, to clarify how the two PSA synthases act differently in vivo, we first cloned PSA-expressing cell lines (N2a-II and N2a-IV) by stable transfection of the cDNA encoding either mST8Sia II or IV into mouse neuroblastoma Neuro2a cells, which do not express PSA but express NCAM, then compared the expression of the PSA and NCAM isoforms and de novo synthesis of PSA between N2a-II and N2a-IV. Western blotting with an anti-NCAM polyclonal antibody showed that NCAM was expressed as the polysialylated form in both ST8Sia II cDNA-transfected and STBSia IV cDNA-transfected Neuro2a cells, but that the polysialylated NCAMs expressed in ST8Sia IV cDNA-transfected clones migrated much slower on SDS-PAGE than those expressed in ST8Sia II cDNA-transfected clones. The slower migration of polysialylated NCAM of the ST8Sia IV cDNA-transfected clone (N2a-IV) than that of the ST8Sia II cDNA-transfected clone (N2a-II) was also observed when cells were metabolically labeled with [3H] glucosamine or pulse-chase labeled with [35S] methionine followed by immunoprecipitation with anti-PSA antibody or anti-NCAM monoclonal antibody. In addition, polysialylated N-glycans of PSA-carrying glycoproteins prepared from [3H] glucosamine-labeled N2a-IV by immunoprecipitation with anti-PSA monoclonal antibody were eluted at a much higher salt concentration than those from [3H]glucosamine-labeled N2a-II on an anion-exchange column. These results indicated that the degree of de novo polysialylation of NCAM by mST8Sia IV was much higher than that by mST8Sia II. In N2a-IV, NCAM-120, -140, and -180 were expressed as polysialylated forms, while polysialylation was restricted to NCAM-140 and -180, i.e., not NCAM-120, in N2a-ST8Sia II. Metabolic labeling of the cells with [3H] glucosamine, pulse-chase labeling with [35S] methionine followed by immunoprecipitation with anti-PSA antibody, and subsequent sialidase treatment revealed that NCAM-140 and -180 were specifically polysialylated in N2a-II, whereas not only NCAM but also other glycoproteins were de novo polysialylated in N2a-IV. The above results demonstrated that the two different PSA synthases, mST8Sia II and IV, synthesize PSA of different lengths on different substrate glycoproteins in vivo when the enzymes are expressed in neuroblastoma Neuro2a cells. These differences suggest that mST8Sia II and IV play different roles in the biosynthesis and expression of PSA.抄録全体を表示PDF形式でダウンロード (5348K)
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