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Volume 116 (1994)
- Issue 6 Pages 1187-
- Issue 5 Pages 939-
- Issue 4 Pages 719-
- Issue 3 Pages 471-
- Issue 2 Pages 229-
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Volume 116, Issue 4
Displaying 1-34 of 34 articles from this issue
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Makoto Komiyama, Takuya Inokawa1994 Volume 116 Issue 4 Pages 719-720
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA sequence-selective artificial ribonuclease was prepared by attaching ethylenediamine to the 5'-end of a DNA oligomer as the sequence-recognizing moiety. The hybrid, incorporating a 19-mer DNA which is complementary with the A44-A62 sequence of tRNAPhe, hydrolyzed the tRNA selectively at the 3'-side of C63.View full abstractDownload PDF (1547K) -
Takeo Matsuzawa, Tatsuhiko Kobayashi, Kazuhiro Tashiro, Masao Kasahara1994 Volume 116 Issue 4 Pages 721-727
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCompared with the activity obtained with a high-protein diet in rats, a low-protein diet doubled the activity of ornithine aminotransferase [EC 2. 6. 1. 13] (OAT), a key enzyme for citrulline synthesis, in the small intestine. The induction of ornithine aminotransferase in the small intestine by the low-protein diet and its suppression by the high-protein diet, and the converse in the liver, were immunohistochemically verified with anti-OAT antiserum. The immunohistochemical studies revealed that ornithine aminotransferase molecules localized in the villous surface epithelia, but not in the cryptic epithelia, were most responsive to the changes in dietary conditions, these results indicating that intestinal ornithine aminotransferase may be involved in the ornithine supply to the liver, with the reversal of the enzyme reaction occurring with a low-protein diet. Reconstituted model experiments on citrulline synthesis revealed that the addition of ornithine carbamoyltransferase and carbamoyl phosphate was essential to overcome the unfavorable equilibrium of the reverse reaction, and the further addition of glutamate dehydrogenase and ammonia resulted in a stimulating effect.View full abstractDownload PDF (2395K) -
Xiao-Feng Guo, Misako Nakaya, Shugo Watabe1994 Volume 116 Issue 4 Pages 728-735
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThree heavy chain isoforms of chymotryptic myosin subfragment-1 (S1) with different molecular sizes of 96 kDa (H1), 94 kDa (H2), and 92 kDa (H3), were detected in the fast skeletal muscle from thermally acclimated carp. In total, six S1 isoforms were present, including two S1 isoforms for each heavy chain due to associated A1 and A2 light chains. H1 heavy chain was dominant in the 10°C-acclimated carp and responsible for high acto-S1 Mg2+-ATPase activity and low thermostability. In contrast, H3 heavy chain predominating in the 30°C-acclimated carp showed low acto-S1 Mg2+-ATPase activity and high thermostability. H2 heavy chain was found in the 10- and 20°C-acclimated fish. H3 heavy chain featured three tryptic fragments with normal molecular masses of 25, 50, and 20 kDa in order from the N-terminus. However, H1 heavy chain contained an unusual, longer “20 kDa” peptide whose molecular size was estimated to be about 23 kDa.View full abstractDownload PDF (4010K) -
Yoshito Sakuma, Koichi Nishigaki1994 Volume 116 Issue 4 Pages 736-741
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA computer program which can predict general PCR products was developed and experimentally verified to be useful. This means that the approximation of solution structures of DNA based solely on Watson-Crick base pairing is effective. Intramolecular higher structures were shown to be responsible for the discrepancies between the predictions and the experimental results. This study provides a theoretical background to arbitrarily primed (or random) PCR. Other basic issues relating to general PCR are also discussed.View full abstractDownload PDF (1680K) -
Hidehiko Kikuchi, Taku Fujinawa, Futoshi Kuribayashi, Akira Nakanishi, ...1994 Volume 116 Issue 4 Pages 742-746
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCytochrome b558 composed of large and small subunits, and cytosolic 47- and 65-kDa proteins are important constituents of the superoxide (O2-) generating system in phago-cytes and B lymphocytes. In this paper, we describe changes in O2--generating activity and expression of O2--generating components during differentiation of human monoblastic leukemia U937 cells to macrophage-like cells. Undifferentiated U937 cells generated no O2-in response to a stimulation, although they expressed the three components other than the cytochrome b558 large subunit. When U937 cells were cultured with agents that induced the cell differentiation, such as vitamin D3, retinoic acid, interferon-γ, and tumor necrosis factor, O2--generating activity increased 5- to 200-fold depending on the agent used. Immunoblotting analysis revealed that the amounts of the four protein components essential for O2- generation increased, although their induction levels were significantly different between inducers. Among the four protein components, the cytochrome subunits were induced in low levels by all agents tested, which may explain why the O2--generating activity of differentiated U937 cells was much lower than that of neutrophils from peripheral blood.View full abstractDownload PDF (1771K) -
Yoshiaki Sohma, Takashi Suzuki, Hironobu Sasano, Hiroshi Nagura, Mahit ...1994 Volume 116 Issue 4 Pages 747-751
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn an effort to characterize mRNAs that are highly expressed during atherosclerosis, we employed differential hybridization screening of a cDNA library constructed from total RNA derived from the aorta of Watanabe heritable hyperlipidemic (WHHL) rabbits. Characterizing the cDNAs for mRNAs that are present in large amounts in WHHL rabbit aortae, we identified a positive clone encoding matrix γ-carboxyglutamic acid protein (MGP). The primary structure of rabbit MGP was deduced from nucleotide sequence analysis of the cDNA. Northern blot analysis of total RNA prepared from aortae of WHHL and normal rabbits of various ages indicated that the expression of MGP mRNA increased in proportion to the progression of atherosclerosis in WHHL rabbits. Analysis of MGP mRNA by in situ hybridization revealed that a significant amount of MGP mRNA is accumulated in atherosclerotic lesions of WHHL rabbits, suggesting that the expression of MGP mRNA is correlated with the progression of atherosclerosis.View full abstractDownload PDF (3248K) -
Toshiki Nakai, Yasuko Mera, Toshimasa Yasuhara, Akira Ohashi1994 Volume 116 Issue 4 Pages 752-758
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIntracellular protein degradation plays an important role in maintaining the stoichiometry of the different subunits of an oligomeric enzyme. In a Saccharomyces cerevisiae mutant defective in cytochrome c oxidase subunit 4 encoded in nuclear DNA, mitochondrialencoded subunits 2 and 3 cannot assemble normally [Dowhan et al. (1985) EMBO J. 4, 179-184]. In this study, we show that those unassembled forms of subunits 2 and 3 in this strain are eliminated rapidly by degradation. Reduction of the intracellular ATP level by inhibiting the glycolytic pathway, or inhibition of the entry of ATP into mitochondria by bongkrekic acid, both of which are expected to reduce the intramitochondrial ATP level in respiratory-deficient cells such as WD 1, significantly suppressed the degradation, suggesting that the degradation requires intramitochondrial ATP. The degradation was also inhibited by o-phenanthroline, a membrane-permeable metal chelator, and this inhibitory effect was suppressed by addition of an excess amount of Co2+, Mn2+, or Zn2+, but not by Ca2+ or Mg2+, suggesting a novel metal- dependence of the degradation of unassembled Cox II and Cox III which has not been reported previously for mitochondrial metabolic protein degradation systems. A potential advantage of using this strain for identifying the factor(s) involved by a genetical approach is discussed.View full abstractDownload PDF (4123K) -
Koichi Inano, Sylvia W. Curtis, Kenneth S. Korach, Saburo Omata, Tsune ...1994 Volume 116 Issue 4 Pages 759-766
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe previously showed that the 9 S estrogen receptor (ER) could be reconstituted from purified ER and purified heat shock protein 90 (hsp 90). So, we investigated the role of hsp 90 in the binding of purified ER to an estrogen responsive element (ERE) by using the reconstitution system. ER purified from calf uterus showed a very low binding capacity to an ERE from the vitellogenin A2 gene in the gel mobility shift assay. However, the binding was strongly stimulated by reconstitution with hsp 90 and was proportional to the amount of reconstituted 9 S ER. Hsp 70, a typical molecular chaperone and a component of some steroid receptors, did not cause similar stimulation. The equilibrium dissociation constants (Kd) of the occupied and unoccupied 9 S ER for the ERE were the same as each other, indicating that the binding of ER to the ERE was independent of the ligand. H222, a monoclonal antibody which binds to the hormone-binding domain (HBD) of ER, recovered the high affinity ER-ERE binding. The binding of hsp 90 to ER suppressed the Triton X-100 stimulated estradiol-dissociation from the ER. The sedimentation coefficients and Stokes' radii of the purified and unpurified cytosolic ER were compared, and it was shown that the purified ER was not unfolded and had a rather compact structure, similar to the cytosolic ER. From these results and others, the mechanism underlying stimulation of the ER-ERE binding by hsp 90 was speculated to be as follows: i) purified ER exists in a molten globule state, ii) hsp 90 recognizes the state and binds to the HBD, and iii) this binding changes the ER to a native form, and thus makes possible the binding of ER to the ERE.View full abstractDownload PDF (3464K) -
Hiroshi Itoh, Hisamitsu Ide, Hiroaki Kataoka, Masaki Tomita, Hiroyuki ...1994 Volume 116 Issue 4 Pages 767-772
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA cDNA encoding α1-microglobulin (α1mG)/inter-α-trypsin inhibitor light chain (ITI-LC) was cloned from mouse liver by reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends. Sequence analysis of the cDNA showed that the basic molecular structure of the proprotein was similar to that in other animals, so that two mature proteins, α1mG and ITI-LC, could be produced from the proprotein translated from the mRNA. Since ITI-LC is known as a positive acute phase reactant and since ITI-LC is genetically identical with mast cell proteinase inhibitor, trypstatin, we examined the mRNA level in the liver of parasite-infected mice showing extensive mastocytosis. The mRNA level was, however, not significantly changed during inflammatory processes, except for a slight increase on day 8 post-infection.View full abstractDownload PDF (2087K) -
Takuji Harano, Takeo Yamaguchi, Eiji Kimoto1994 Volume 116 Issue 4 Pages 773-777
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effect of intracellular Ca2+ on high pressure-induced hemolysis of human erythrocytes was examined. Red cells were incubated with Ca2+(0.01-1mM) in the presence of ionophore A23187. The Ca2+-loaded cells were subjected to a pressure of 200 MPa. Treatment with 0.1mM Ca2+ had the greatest suppressive effect on the hemolysis. On removal of intracellular Ca2+, red cells showed a morphological change from echinocytes to normal discocytes but the hemolysis remained unaltered. Measurement of intracellular K+ and viscosity demonstrated that the suppressive effect of Ca2+ on the hemolysis is irreversible and is largely associated with the increase of intracellular viscosity induced by K+ efflux.View full abstractDownload PDF (1603K) -
Shinya Yano, Ken-ichi Suzuki, Masao Katoh, Yuji Sugita, Seiji Kaku, Ko ...1994 Volume 116 Issue 4 Pages 778-786
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have developed two monoclonal antibodies (MAbs), B6A3 and C4G1. The whole molecules of the two MAbs inhibited in vitro human platelet aggregation induced by either ADP, collagen or thrombin, and their F(ab')2 fragments inhibited ex vivo platelet aggregation induced by ADP in monkey. The concentrations necessary for complete inhibition were 5 and 1 μg/ml for B6A3 and C4G1, respectively. The Fab fragment of C4G1 but not B6A3 inhibited platelet aggregation. B6A3 and C4G1 bound to activated platelets with dissociation constants of 0.25 and 0.82 nM, respectively. B6A3 recognized an epitope on β3, which was sensitive to reduction and alkylation of cystine residues, and C4G1 recognized a conformational epitope on the αIIbβ3 complex, which was sensitive to EDTA. The binding of fibrinogen to activated platelets was inhibited by both MAbs. However, the binding of fibrinogen to isolated αIIb/β3 was inhibited by the whole molecule of C4G1 but not B6A3, although both MAbs bound to the isolatedαIIb/β3. The binding of these MAbs to the isolated αIIb/β3 was not inhibited by either Arg Gly Asp Ser (RGDS) or fibrinogen γ-peptide. In addition, B6A3 but not C4G1 bound to human endothelial cells. These MAbs should contribute to the elucidation of the mechanism of platelet aggregation.View full abstractDownload PDF (2041K) -
Takashi Kumazaki, Shin-ichi Ishii, Hideyoshi Yokosawa1994 Volume 116 Issue 4 Pages 787-793
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA trypsin inhibitor from hemolymph of the solitary ascidian, Halocynthia roretzi, has been reported to be present in two forms, ATI-I and ATI-II. ATI-I consists of a single polypeptide chain with a unique sequence of 55 amino acid residues, while ATI-II has two chains that seem to be derived from ATI-I by cleavage at the Lys16-Met17 bond [Kumazaki, T., Hoshiba, N., Yokosawa, H., and Ishii, S. (1990) J. Biochem. 107, 409-413]. ATI-II (as modified inhibitor) was proved to be produced by incubation of ATI-I (as virgin inhibitor) with a catalytic amount of bovine trypsin. The tryptic hydrolysis at the Lys16-Metl7 bond in virgin inhibitor showed a maximum velocity at around pH 3.5. On the other hand, acid treatment of a complex prepared by mixing equimolar quantities of trypsin and the modified inhibitor yielded free trypsin and the virgin inhibitor. The results of chemical analyses indicated that the Lys16-Met17 bond that had been cleaved in ATI-II was resynthesized by the acid treatment. These findings strongly suggest that the Lys16-Met17 bond is the reactive site of ATI for trypsin.View full abstractDownload PDF (1622K) -
Shoutaro Tsuji, Kazuhiko Kaji, Shigeharu Nagasawa1994 Volume 116 Issue 4 Pages 794-800
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSComplement activation generally does not occur on homologous cells. We observed C3 deposition on cultured human umbilical vein endothelial cells (HUVEC) when those which had died of apoptosis were treated with human serum. The C3 deposition on apoptotic HUVEC required Mg2+ but not Ca2+. In addition, the incubation of apoptotic HUVEC with purified C3, B, and D in the presence of Mg2+ resulted in C3 deposition. These results indicated that the C3 deposition on apoptotic HUVEC is mediated by the activation of the alternative complement pathway. C3 contains an intrachain thioester bond in the α chain (110 kDa) and upon activation to C3b, binds with membrane molecules by forming an ester or amide bond. Western blotting of reduced C3b-membrane molecule complexes, isolated from serum-treated apoptotic HUVEC by immunoprecipitation with anti-C3, revealed the covalent binding of C3b to several membrane molecules. Most of the C3b-membrane molecule complexes were cleaved by hydroxylamine, suggesting covalent binding via an ester bond. The molecular mass of the major α' chain fragment released by hydroxylamine treatment was not 105 kDa but 68 kDa, which corresponds to the α1 fragment of iC3b. These results indicate that most of the C3b on HUVEC was cleaved at its α' chain to yield iC3b, which consists of three disulfide-linked polypeptide chains and is a ligand of the comple-ment receptor type 3 (CR3) of phagocytes. These results suggest that apoptotic HUVEC can activate the alternative pathway of the homologous complement and that the complement is related to the clearance of apoptotic cells by phagocytes.View full abstractDownload PDF (3127K) -
Ikuko Sagami, Hideaki Kikuchi, Shuntaro Ikawa, Minro Watanabe1994 Volume 116 Issue 4 Pages 801-810
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA genomic clone encoding the hamster CYP1A1 gene was isolated from a hamster EMBL-3 genomic library and characterized. The CYP1A1 gene contained seven exons including the noncoding first exon as determined for CYP1A1 of other species. DNA sequence analysis up to -2307 bp of the CYP1A1 gene revealed the occurrence of five consensus xenobiotic responsive elements (XREs) and one basal transcription element (BTE) in addition to the canonical TATA box. For functional analysis, transfection experiments were performed in human hepatoma HepG2 cells with reporter gene constructs consisting of fragments with various lengths of the 5'-flanking region of the CYP1A1 gene and bacterial chloramphenicol acetyltransferase (CAT) gene. External deletion of the upstream region from the reporter gene resulted in a stepwise decrease of the CAT activity, suggesting that XREs were responsible for inducible expression of CYP1A1 gene by 3-methylcholanthrene (MC). A negative regulatory element (NRE) was also identified in the 5'-flanking region at -833 to -642. Removal of the NRE from the CYP1A1-CAT fusion gene resulted in about 3-fold increase of MC-inducible CAT activity. Using gel retardation assays with HepG2 nuclear extract, we demonstrated the presence of a specific protein which bound to the NRE fragment. Further competition analysis and methylation interference assays revealed that the nuclear protein bound to a 22-base fragment (from -688 to -709) of the NRE region, whose sequences were conserved among hamster, human, and rat CYP1A1 genes.View full abstractDownload PDF (7384K) -
Su Chen, Ka Wan Li1994 Volume 116 Issue 4 Pages 811-817
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThis paper describes an approach for structurally analyzing aminophospholipids, including glycerophosphatidylethanolamine, glycerophosphatidylserine and their lyso analogues, and glycerophosphatidic acid by positive-ion liquid secondary-ion and four-sector tandem mass spectrometry. Polar head groups and the composition and position of the fatty acid chains in aminophospholipid species as well as diacyl and monoacyl phosphatidylethanolamine and their plasmalogen species can be characterized by collisionally induced dissociation of [M+H]+ ions of underivatized molecular species. Tandem mass spectrometry of [M+Na]+ and [M+2Na-H]+ ions of 9-fluorenylmethyloxycarbonyl-derivatized glycerophosphatidylethanolamine and glycerophosphatidylserine induces fragmentation processes, but product ions are generally less structurally informative. Collisionally induced dissociation of [M+2Na-H]+ ions of the molecular species of glycerophosphatidic acid not only yields information on the composition and position of fatty acid chains, but also allows the double bond location to be identified. This common positive-ion method is particularly effective in characterizing the molecular species of natural ethanolamine glycerophospholipids because of the advantage of a low detection limit. This approach also represents a useful alternative for the analysis of the molecular species from natural aminophospholipids by tandem mass spectrometry.View full abstractDownload PDF (802K) -
Ana Kitazono, Atsuko Kitano, Daisuke Tsuru, Tadashi Yoshimoto1994 Volume 116 Issue 4 Pages 818-825
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe Aeromonas sobria pap gene encoding prolyl aminopeptidase (PAP) was cloned. It consists of 425 codons and encodes a homotetrameric enzyme of 205 kDa. The purified enzyme showed an almost absolute specificity for amino-terminal proline. Proline and hydroxyproline residues from many peptide and amide substrates could be easily removed, while no activity was detected for substrates having other amino terminals. The enzyme was very similar to that from Bacillus coagulans in many aspects, such as the strong inhibition caused by PCMB and the weak or no inhibition caused by DFP and chelators, respectively. However, these enzymes show only 15% identity in their amino acid sequences. Differences were also observed in their molecular weight, stability and activity toward some peptide substrates. When aligning the deduced amino acid sequence with known sequences from other microorganisms, conserved sequences were found at the amino-terminal region; the significance of these conserved regions is discussed. Based on the results of this work, and on the studies available to date, the occurrence of at least two types of PAPs is postulated. One group would be formed by the Bacillus, Neisseria, and Lactobacillus enzymes, and the other by enzymes such as the Aeromonas PAP.View full abstractDownload PDF (3866K) -
Takashi Hibino, Byung Hyun Lee, Teruhiro Takabe1994 Volume 116 Issue 4 Pages 826-832
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPlastocyanin is a copper protein that functions as an electron carrier in the thylakoid lumen of the chloroplast. To characterize the transit peptide of plastocyanin and develop expression systems for it in Escherichia coli, three kinds of expression vectors which encode different size precursor plastocyanin molecules were constructed. Their expression, processing, and copper-binding activity have been examined. When the full-length cDNA encoding the precursor plastocyanin from Silene pratensis was expressed in E. coli, a large amount of precursor plastocyanin accumulated in insoluble aggregates. Its accumulation level was increased by the addition of copper ions. About six percent of precursor plastocyanin molecules were transported into the periplasmic space and processed to the mature protein. On the other hand, expression of the intermediate size cDNA, which contains the hydrophobic domain and basic amino acid of C-terminal transit peptide, caused exclusive translocation to the periplasmic space and correct processing to the mature size. The addition of copper ions increased the holo-protein content, but did not change the polypeptide content of mature plastocyanin, indicating that translocation and processing are independent of the incorporation of copper ions. The mature plastocyanin content corresponds to 8% (w/w) of the total E. coli protein content (123mg per liter of culture). The purified mature holo-protein showed almost the same spectroscopic and kinetic properties as those of purified spinach plastocyanin. Expression of the cDNA encoding the mature polypeptide and two preceeding amino acid residues caused the accumulation of only a small amount of plastocyanin. Since large quantities of precursor and mature plastocyanin are obtained, these expression systems will be useful for the study of assembly processes as well as for structural and functional studies of plastocyanin by site-directed mutagenesis.View full abstractDownload PDF (3122K) -
Tetsuo Nishikawa, Masao Omura, Masatoshi Noda, Sho Yoshida1994 Volume 116 Issue 4 Pages 833-837
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSOur previous investigations demonstrated that 5- and 15-lipoxygenase metabolites of arachidonic acid are synthesized in bovine adrenal fasciculata cells, although their exact role in the regulation of adrenal steroidogenesis is unknown. Thus we attempted to investigate their direct effects on cholesterol side-chain cleavage in bovine adrenal mitochondria. We also examined Ca2+ efflux in mitochondria, based on a reported correla-tion between pregnenolone formation and Ca2+ release in adrenal mitochondria. The present experiments showed that 5-HETE increased pregnenolone generation in the isolated intact mitochondria, but not in the inner mitochondrial membrane. Thus it is suggested that 5-HETE may activate cholesterol side-chain cleavage by inducing the translocation of cholesterol from the outer to the inner mitochondrial membrane. The present experiments also demonstrated that 5-HPETE, 5-HETE, 15-HPETE, and 15-HETE, but not leukotriene B4, activated cholesterol side-chain cleavage and Ca2+ efflux from mitochondria, suggesting that these substances may activate cholesterol side-chain cleavage by regulating Ca2+ movement in mitochondria. These effects were additively enhanced when mitochondria were stimulated simultaneously with these substances and GTP. Therefore, both GTP and lipoxygenase metabolites seem to play crucial roles in the regulation of pregnenolone generation. The direct effect of 5- and 15-lipoxygenase metabolites other than leukotriene B4 on the regulation of pregnenolone synthesis, which is known to be the rate-limiting step of steroidogenesis, were clearly observed in the present experiments.View full abstractDownload PDF (562K) -
Susumu Inamoto, Hirokazu Fukuda, Tatsuhiko Abo, Eiichi Ohtsubo1994 Volume 116 Issue 4 Pages 838-844
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe developed a purified system for reproducing the nicking reaction at the site 59 base pairs upstream of the TraY protein binding site, sbyA, in the oriT region of plasmid R100. Nicking at oriT occurred efficiently in the presence of the plasmid-encoded proteins, Tral and TraY, integration host factor (IHF), and Mg2+, but inefficiently in the presence of the Tral protein and Mg2+. The products were complex DNA molecules with a protein covalently linked with the 5' end of the nick in the strand, which is supposed to be transferred during conjugation. The same complex DNA molecules were formed in the presence of the Tral protein alone, indicating that the protein attached at the 5' end of the nick is the Tral protein. Stimulation of the nicking reaction by the TraY protein and by IHF, whose binding site has been mapped between the nicking site and sbyA, indicates that DNA bending is important in the formation of the complex including the Tral and TraY proteins at oriT.View full abstractDownload PDF (6424K) -
Masatsugu Ema, Natsuki Matsushita, Kazuhiro Sogawa, Takeshi Ariyama, J ...1994 Volume 116 Issue 4 Pages 845-851
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe isolated the human arylhydrocarbon receptor (AhR) cDNA from a human lung cDNA library, by using mouse AhR cDNA as a labeled probe. The nucleotide sequence of cloned human AhR cDNA is identical to the previously reported human AhR sequence [Dolwick et al. (1993), Mol. Pharmacol. 44, 911-917] from cell line HepG2. The overall amino acid identity with mouse AhR from cell line Hepa-1 is 72.5%. The human AhR expressed either in COS-7 cells or in a reticulocyte lysate in vitro translation system showed specific dioxin-binding activity and Arnt-dependent DNA-binding activity. Chromosomal localization of the AhR gene was determined to be chromosome 7p21 by fluorescent in situ hybridization and DNA blot hybridization using 23 human×mouse or Chinese hamster hybrid cell DNAs.View full abstractDownload PDF (3684K) -
Hidenori Yamada, Masaharu Seno, Ayumi Kobayashi, Takeshi Moriyama, Meg ...1994 Volume 116 Issue 4 Pages 852-857
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA novel S-alkylating reagent, N-(3-bromopropyl)-N, N, N', N', N'-pentamethyl-1, 3-propanedi(ammonium bromide) (TAP2-Br) which carries two positive charges in the molecule, was prepared to increase the solubility or to decrease the hydrophobicity of cysteinecontaining denatured proteins (or peptides). S-Alkylation with TAP2-Br introduces two positive charges per cysteine residue, which will effectively shift the net charge of a protein in the positive direction. Disulfide-containing proteins, such as hen egg-white lysozyme, RNase A, BSA, and soybean trypsin inhibitor (Kunitz type), were reduced and S-alkylated with TAP2-Br to evaluate the potential of this reagent compared with other S-alkylating reagents such as monoiodoacetic acid, bromosuccinic acid and (3-bromopropyl)trimeth-ylammonium bromide. The solubilities of these denatured proteins in the pH range of 2-10 indicated that S-alkylation with TAP2-Br effectively solubilized not only basic proteins (lysozyme and RNase) but also an acidic protein containing a fairly large number of cysteine residues (BSA). Moreover, the retentions of cysteine-containing tryptic peptides derived from lysozyme on reversed-phase HPLC were greatly reduced by S-alkylation with TAP2-Br. These results indicate that TAP2-Br is very useful to increase the solubility of some cysteine-containing denatured proteins and to decrease the hydrophobicity of peptides containing cysteine residue(s).View full abstractDownload PDF (685K) -
Xiaoying Wang, Kiyonao Sada, Shigeru Yanagi, Cheng Yang, Karim Rezaul, ...1994 Volume 116 Issue 4 Pages 858-861
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe previously demonstrated that thrombin-induced activation of p72syk was independent of intracellular Ca2+ elevation in platelets. However, our previous studies also demonstrated that activation of platelets by ionophore A23187 results in a dramatic increase in tyrosine phosphorylation of several cellular proteins. In the present study, we investigated the effect of Ca2+ elevation on the activity of p72syk. When washed porcine platelets were stimulated with ionophore A23187 and the activity of p72syk was assessed by means of an immunoprecipitation kinase assay, A23187 caused a time- and dose-dependent increase in the specific activity of p72syk. In addition, pretreatment of platelets with both aspirin and ADP scavengers or chelation of extracellular Ca2+ by EGTA had no effect on the A23187-induced activation of p72syk. These results indicate that A23187-induced activation of p72syk is independent of the formation of endoperoxide/thromboxane A2, released ADP and extracellular Ca2+, suggesting the existence of a novel pathway for activation of p72syk. Furthermore, evidence is presented which indicates a synergistic effect of A23187 and thrombin on the activation of p72syk, and an inhibitory effect of pretreatment with phorbol 12-myristate 13-acetate, a protein kinase C activator, on the activation of p72syk induced by either A23187 or thrombin.View full abstractDownload PDF (3646K) -
Yamato Kikkawa, Makoto Umeda, Kaoru Miyazaki1994 Volume 116 Issue 4 Pages 862-869
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSLadsin is a large cell-adhesive protein with potent cell-scattering activity, which was recently identified in the culture of a malignant human gastric carcinoma cell line [Miyazaki, K. et al. (1993) Proc. Natl. Acad. Sci. USA 90, 11767-11771]. It is a heterotri-meric protein, containing a 140-kDa subunit similar or identical to the laminin B2t chain. Ladsin is similar to the keratinocyte-derived matrix proteins, “epiligrin” and “kalinin.” In the present study, the cell-adhesion and cell-migration activities of ladsin were examined in comparison with those of three cell adhesion proteins, laminin, fibronectin, and vitronectin. Ladsin showed high cell-adhesion activity toward rat liver cell line BRL at concentrations 4-20-times lower than in the case of the other three proteins. In a monolayer culture, ladsin stimulated the migration of BRL cells about 2-times more strongly than the others, as compared at the minimal concentrations required for the maximal cell-adhesion activity. In Boyden chambers, ladsin stimulated both the chemotactic and chemokinetic migration of BRL cells. When the effect of anti-integrin antibodies on the adhesion of human fibrosarcoma cell line HT1080 was examined, the adhesion to ladsin was effectively inhibited by both the anti-integrin α3 and β1 antibodies, but not the anti-integrin α6 antibody, indicating that the primary receptor of ladsin is integrin α3β1. These results demonstrate that ladsin is a unique extracellular matrix component which may play a major role in cell migration.View full abstractDownload PDF (3391K) -
Shinobu Fujii, Takeshi Tani, Shuji Hada, Seiji Inoue, Kiyoshi Ikeda, S ...1994 Volume 116 Issue 4 Pages 870-876
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSEffects of Ca2+ on the kinetic parameters for the hydrolysis of monodispersed 1, 2-dihex-anoyl- sn-glycero-3-phosphorylcholine (diC6PC), catalyzed by Group I phospholipases A2 (PLA2s) from Pseudechis australis, Naja naja atra, and bovine pancreas and by Group II enzymes from Vipera russelli russelli, Agkistrodon halys blomhoffii, and Trimeresurus flavoviridis, were studied by the pH-stat assay method at 25°C, pH 7.5-8.2, and an ionic strength of 0.1 or 0.2 in the absence or presence of an amide-type substrate analog, 2-dodecanoyl-amino-1-hexanol-phosphoglycol. The binding of genuine substrate to the Group II enzymes and that of its analog to the Groups I and II enzymes were markedly facilitated by the binding of Ca2+ to the enzymes. On the other hand, the binding of genuine substrate to the Group I enzymes was found to be independent of the Ca2+ binding. The former result suggests that the structures of the Group II enzyme-genuine substrate complexes and both types of enzyme-analog complexes are generally stabilized by the Ca2+ binding, whereas the latter indicates that the structures of the Group I enzyme-genuine substrate complexes are already similar to those of their Ca2+ complexes and that, therefore, these enzyme-substrate interactions are independent of the Ca2+ binding.View full abstractDownload PDF (833K) -
Kenji Sugimoto, Hiroaki Yata, Yoshinao Muro, Michio Himeno1994 Volume 116 Issue 4 Pages 877-881
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMammalian centromere proteins (CENPs) can be divided into those that translocate from centromere to midzone in the progress of mitosis, and those that remain at the centromere throughout the cell cycle. The latter including CENP-A, CENP-B, and CENP-C is the candidate for DNA-binding protein. CENP-B has been shown previously to possess the specific DNA-binding activity to 17-base pair sequences dispersed on human centromeric alphoid repeats. In this study, we examinded DNA-binding property of CENP-C that is localized to inner kinetochore plate of the metaphase chromosome. We independently isolated a full-length cDNA encoding human CENP-C and expressed it as the polypeptide tagged with histidine oligomer in Escherichia coli. After affinity purification with Ni2+-chelated resin, DNA-binding activity of the recombinant CENP-C renatured on the membrane was demonstrated by using human genomic DNA and an alphoid subfamily in South-Western-type blotting analysis. By constructing a series of truncated products, the DNA-binding domain was located at an internal 101-amino-acid stretch with no apparent homology to any other DNA-binding proteins. This may suggest that CENP-C is directly involved in formation of kinetochore chromatin fibers.View full abstractDownload PDF (3324K) -
Bo Liu, Shigeru Nakashima, Hiroyuki Kanoh, Tomoko Takano, Takao Shimiz ...1994 Volume 116 Issue 4 Pages 882-891
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPlatelet-activating factor (PAF) activated phospholipase D (PLD) in WT-H cells, CHO cells stably expressing cloned guinea-pig PAF receptor. The PLD activation was found to be dependent on extracellular Ca2+, protein kinase C (PKC), and a currently unidentified protein tyrosine kinase (PTK). PTK inhibitors ST-638 and genistein inhibited PLD activation induced by PAF as well as phorbol myristate acetate, indicating that PTK acts downstream of PKC. Furthermore, activation of MAP (mitogen-activated protein) kinases, as assessed by their phosphorylation, was also dependent on Ca2+, PKC, and PTK. The correlation between PLD activity and MAP kinase activation, together with the previously observed MAP kinase activation associated with arachidonic acid release by cPLA2 [Honda et al. (1994) J. Biol. Chem. 269, 2307-2315], led us to examine the involvement of MAP kinase in PLD activation. The results indicate that PLD and MAP kinases are activated through the common pathway consisting of Ca2+, PKC, and the unidentified PTK, which act in parallel, but not in a linear sequence.View full abstractDownload PDF (3595K) -
Haruyoshi Yamada, Shingo Tajima, Takeji Nishikawa, Saood Murad, Sheldo ...1994 Volume 116 Issue 4 Pages 892-897
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSEffects of tranilast, N-(3, 4-dimethoxycinnamoyl)anthranilic acid, on collagen synthesis in cultured human skin fibroblasts were studied. Tranilast was found to inhibit collagen synthesis in a dose-dependent manner to a maximum of 55% at 300 μM during 48 h of treatment; the synthesis of type I and type III collagens was equally affected. Administered simultaneously or subsequently, tranilast reduced the stimulatory effect of transforming growth factor β1 (2.5 ng/ml) on collagen synthesis without affecting the accompanying stimulation of noncollagen protein synthesis. It did not affect prolyl or lysyl hydroxylase activity in vitro and in cells. The content of proα1(I) collagen mRNA was decreased 60% by tranilast. Tranilast prevented the TGF β1-mediated increase in pro α 1(I) collagen mRNA. These results indicate that tranilast specifically inhibits collagen production at a pretranslational level by interfering with TGFβ1 effects. Tranilast also inhibited collagen synthesis in scleroderma fibroblasts to the same extent and in keloid fibroblasts to a greater extent than in normal fibroblasts, attesting to its therapeutic potential as an antifibrotic drug.View full abstractDownload PDF (2656K) -
Satoshi Matsuyama, Keiko Ichihara-Tanaka, Kiyotoshi Sekiguchi1994 Volume 116 Issue 4 Pages 898-904
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA truncated form of fibronectin consisting of the N-terminal 70-kDa and C-terminal 37-kDa regions, referred to as “minifibronectin, ” retains the ability to assemble into the extracel-lular matrix, even though it lacks the central ≈ 120-kDa region containing most of the type III modules (Ichihara-Tanaka, K., Titani, K., and Sekiguchi, K., FEBS Lett. 299, 155-158, 1992). Taking advantage of the matrix assembly activity of minifibronectin, we developed a novel method to target non-matrix proteins to the extracellular matrix by inserting them between the N-terminal 70-kDa and the C-terminal 37-kDa regions of minifibronectin. Using the immunoglobulin-binding domain of Staphylococcal protein A as a model, we demonstrated that the bacterial protein expressed in mouse L cells as a chimeric protein with minifibronectin is secreted as disulfide-bonded dimers and successfully deposited onto the extracellular matrix of transfected cells. The chimeric protein retained the immunoglobulin-binding activity not only in solution but also after deposition at the matrix. This targeting strategy we developed will provide a means to manipulate the biological functions of the extracellular matrix through targeting of a wide variety of non-matrix proteins.View full abstractDownload PDF (4996K) -
Makoto Hase, Satohiko Araki, Kazuhiko Kaji, Hiroshi Hayashi1994 Volume 116 Issue 4 Pages 905-909
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe survival and death of human umbilical vascular endothelial cells in culture are affected by several factors, such as fibroblast growth factor (FGF), serum, phorbol ester (TPA), and vanadate. In order to identify common aspects of the various signal-transduction processes during the course of apoptotic or programmed cell death, we designed experiments to distinguish between these factors in terms of the pathway that is responsible for the processing of each stimulus. We found, for example, that the effect of removal of FGF was specifically overcome by the addition of the phorbol ester. Our results indicated that two distinct pathways were operative, one specific for signal transduction initiated by FGF and phorbol ester and another specific for signal transduction initiated by serum and vanadate. These two pathways merged down-stream of the individual signal-processing pathways.View full abstractDownload PDF (1378K) -
Masaru Hoshino, Yuji Goto1994 Volume 116 Issue 4 Pages 910-915
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMastoparan, a basic tetradecapeptide from wasp venom, has been considered to be unfolded under aqueous conditions. On the basis of the far-UV circular dichroism spectrum, we found that sodium perchlorate at molar concentrations stabilizes an α-helical structure of mastoparan. To understand the mechanism of the perchlorate-induced stabilization of the α-helical structure, we synthesized a dimeric form of mastoparan derivative, which was linked at the C terminal by a disulfide bond. The linkage decreased the concentration of perchlorate required to stabilize the α-helical structure by 30-fold. With the dimeric mastoparan derivative, we measured the effects of several salts such as sodium trichloro-acetate, sodium trifluoroacetate, and sodium chloride. The concentration of salts required to induce the conformational transition varied and the order of effectiveness of different salts was consistent with the electroselectivity series of anions toward anion-exchange resins, indicating that the anion binding to the positively charged amino groups is responsible for the transition. These results suggest that the salt-induced formation of the α-helical state of mastoparan can be explained by a mechanism similar to that proposed for the salt-induced conformational transition of melittin.View full abstractDownload PDF (754K) -
Hiroyuki Kakuda, Kiyoshi Hosono, Kazuya Shiroishi, Shigeyuki Ichihara1994 Volume 116 Issue 4 Pages 916-922
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe pta gene encoding phosphotransacetylase was cloned on a high copy plasmid with or without the ackA gene encoding acetate kinase in Escherichia coli. The acetate kinase and phosphotransacetylase were overproduced in cells harboring the plasmid possessing both genes. Nucleotide sequencing of the pta gene revealed that it is able to produce a polypeptide comprising 714 amino acid residues, which starts at 70 base pairs downstream from the stop codon of the ackA gene. The 77-kDa protein band of overproduced phosphotrans-acetylase was observed on SDS-polyacrylamide gel electrophoresis, of which the amino terminal sequence corresponds to that of the deduced polypeptide without the amino terminal methionine. Two transcripts of pta of different sizes were found in the cells. A 3, 700 nucleotide transcript, which covers the ackA and pta genes, seemed to be produced by the first promoter in the operon and a 2, 300 nucleotide transcript, which covers just pta, seemed to be produced by the second promoter. In a synthetic medium containing acetate as the sole carbon source, the growth of an ackA-pta double mutant was greatly impaired. Complementation analyses revealed that both the acetate kinase and phosphotransacetylase were required for the rapid growth in the acetate medium.View full abstractDownload PDF (1853K) -
Sachiko Okuno, Takako Kitani, Hitoshi Fujisawa1994 Volume 116 Issue 4 Pages 923-930
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCalmodulin-dependent protein kinase IV (CaM-kinase IV) kinase was recently discovered in the rat brain by its activity to activate the inactive recombinant CaM-kinase IV expressed in Escherichia coli [Okuno, S. and Fujisawa, H. (1993) J. Biochem. 114, 167-170]. In the present study, CaM-kinase IV kinase was purified approximately 2, 000-fold from rat cerebral cortex by purification procedures including calmodulin affinity chromatography, and its properties were examined. The highly purified CaM-kinase IV kinase gave one major protein band corresponding to a molecular weight of about 66, 000 upon SDS-PAGE. The purified CaM-kinase IV kinase phosphorylated and concomitantly activated CaM-kinase IV purified from rat brain as well as the recombinant kinase expressed in Escherichia coli in a Ca2+ /calmodulin-dependent manner. The phosphorylation of CaM-kinase IV by CaM-kinase IV kinase occurred on only serine residue(s). Among a number of proteins, including several known to be phosphorylated by the various protein kinases tested, CaM-kinase IV was the best substrate for CaM-kinase IV kinase. Since syntide-2, a synthetic peptide known to be a good peptide substrate for calmodulin-dependent protein kinase II (CaM-kinase II), was a fairly good substrate for CaM-kinase IV kinase, some kinetic properties of CaM-kinase IV kinase were examined using syntide-2 as a substrate. The Km value for the peptide substrate in the presence of Ca2+/calmodulin was almost two orders of magnitude lower than that in its absence, although the Vmax, value was almost the same in the presence and absence of Ca2+/calmodulin.View full abstractDownload PDF (5145K) -
Kunishige Kataoka, Harumi Takada, Katsuyuki Tanizawa, Tohru Yoshimura, ...1994 Volume 116 Issue 4 Pages 931-936
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPhenylalanine dehydrogenase of Thermoactinomyces intermedius acts preferentially on L-phenylalanine and L-tyrosine, whereas leucine dehydrogenase of Bacillus stearothermo-philus acts almost exclusively on L-leucine and some other branched-chain L-amino acids. The two enzymes share a sequence similarity (47%). Aiming at elucidation of the mechanism of substrate recognition by the two amino acid dehydrogenases, we have genetically constructed a chimeric enzyme consisting of an N-terminal domain of phenylalanine dehydrogenase containing the substrate-binding region and a C-terminal domain of leucine dehydrogenase containing the NAD+-binding region. The chimeric enzyme purified to homogeneity acted on phenylalanine with a specific activity of 6% of that of the parental phenylalanine dehydrogenase and showed a broad substrate specificity in the oxidative deamination, like phenylalanine dehydrogenase. However, it acted much more effectively than phenylalanine dehydrogenase on isoleucine and valine. Its Km values for L-phenylalanine and L-leucine were similar to those of phenylalanine dehydrogenase. The substrate specificity of the chimeric enzyme in the reductive amination was an admixture of those of the two parent enzymes. These results suggest that the two domains of phenylalanine dehydrogenase and leucine dehydrogenase probably can fold independently. Accordingly, their chimera forms a new active enzyme which consists of their N- and C-terminal domains containing the substrate- and coenzyme-binding regions, respectively. However, the two domains of chimeric enzyme interact and communicate with each other to form a new active site and consequently show the new substrate specificity.View full abstractDownload PDF (2005K) -
1994 Volume 116 Issue 4 Pages 937
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDownload PDF (18K)
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