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Volume 133 (2003)
- Issue 6 Pages 699-
- Issue 5 Pages 563-
- Issue 4 Pages 397-
- Issue 3 Pages 263-
- Issue 2 Pages 155-
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Volume 133, Issue 5
Displaying 1-18 of 18 articles from this issue
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Goro Kato, Shuichiro Maeda2003Volume 133Issue 5 Pages 563-569
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSc-Src-null mutants have not provided a full understanding of the cellular functions of c-Src, reflecting the functional redundancy among Src family members. c-Src is phosphorylated by cyclin-dependent kinase 1 (Cdkl) and Cdk5 at Ser75 in the unique amino terminal c-Src-specific domain. The specific roles of c-Src may be assessed by establishing mouse embryonic stem (ES) cells homozygous for a point mutation at Ser75. Mammalian homozygous cultured cells with a point mutation, however, have not yet been produced by gene targeting. Here we show an efficient procedure for producing ES cell clones bearing a homozygous Ser75 to Asp mutation in the c-src gene. This procedure was developed by combining two previously reported strategies: our procedure for introducing a point mutation into one allele with no exogenous sequence, and the high-geneticin (G418) selection procedure for introducing a mutation into both alleles. The mutant clones expressed the same levels of c-Src protein and autophosphorylation activity as wild-type cells, but the mutant c-Src was not phosphorylated on Ser75 during mitosis. This procedure is feasible for generating cells homozygous for a subtle mutation in most genes, and is expected to be applicable to other somatic cell lines.View full abstractDownload PDF (2680K) -
Hiroshi Oneda, Misa Shiihara, Kuniyo Inouye2003Volume 133Issue 5 Pages 571-576
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSInhibitory effects of green tea catechins and their derivatives on the matrilysin-catalyzed hydrolysis of a synthetic substrate, (7-methoxycoumarin-4-yl) acetyl-L-Pro-L-Leu-Gly-L-Leu-[N3-(2, 4-dinitrophenyl)-L-2, 3-diamino-propionyl]-L-Ala-L-Arg-NH2 [MOCAc-PLGL(Dpa)AR], were examined. The 10 catechins examined were classified into three groups according to their inhibition potency. Catechins with a galloyl group at the 3 position, including a major component of green tea catechin, (-)-epigallo-3-catechin gallate [(-)-EGCG], were the most potent inhibitors and inhibited matrilysin in a noncompetitive manner with Ki values of 0.47-1.65 μM. The inhibitory potency of (-)-EGCG was not influenced by the presence of an inhibitor, ZnC12, suggesting that the inhibitions of matrilysin by (-)-EGCG and by ZnC12 might be independent of each other. The inhibitory effects of green tea catechins suggest that a high intake of green tea might be effective for the prevention of tumor metastasis and invasion in which matrilysin is concerned.View full abstractDownload PDF (771K) -
Lei Zheng, Ulrich Baumann, Jean-Louis Reymond2003Volume 133Issue 5 Pages 577-581
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSFunctional expression of catalytic antibodies in the cytoplasm of E. coli is potentially of great interest in searching for new catalysts by genetic selection. Herein, a catalytic antibody single chain Fv (ScFv) 14D9, which catalyzes a highly enantioselective protonation, was expressed as a NusA fusion protein under the T7 promoter. A functional disulfide-containing ScFv fusion protein was obtained in the oxidizing environment of bacterial cytoplasm. The 14D9 ScFv could not be overexpressed alone without NusA fusion. The highly soluble NusA protein most likely retards aggregate formation of ScFv and indirectly supports correct folding and disulfide bridge formation in the fusion construct ScFv-NusA. The ScFv-NusA fusion product shows highly enantioselective, specific, hapten inhibited catalytic activity comparable to its parent monoclonal antibody, 14D9. The NusA fusion method might be generally helpful for functional antibody expression in vivo and for the new development of biocatalysts by genetic selection.View full abstractDownload PDF (1781K) -
Yuki Tsuchiya, Miki Nakajima, Tsuyoshi Yokoi2003Volume 133Issue 5 Pages 583-592
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSCytochrome P450 (CYP) 1B1 is known to be induced by polycyclic aromatic hydrocarbons including 2, 3, 7, 8-tetrachlorodibenzo-Ρ-dioxin (TCDD). The constitutive and TCDD-inducible transcriptional expression of human CYP1B1 is known to be cellspecific. In order to identify the cis-elements that cell-specifically regulate the constitutive and TCDD-inducible transcription of CYP1B1, we constructed luciferase reporter plasmids containing a series of deletions of the XRE core sequence in the 5'-flanking region of the human CYP1B1 gene. Luciferase assays were performed with MCF-7 (breast carcinoma), HepG2 (hepatocellular carcinoma), LS-180 (colon carcinoma), and OMC-3 (ovarian carcinoma) cells. Although there were large differences in the relative luciferase activity and inducibility between these four cell lines, the contribution of each reporter construct was similar. Constitutive expression increased with the regulatory elements that are present at -910 to -852 and -1652 to -1243. Potential enhancer elements for TCDD-induction were located from -1022 to -852 including three XREs, XRE3 at -853, XRE4 at -940, and XRE5 at -989. Gel shift analyses revealed binding of the AhR/ARNT heterodimer to XRE2 at -834, XRE3 at -853, XRE6 at -1024, and XRE7 at -1490. In addition, the binding of a nuclear transcriptional factor, Spl, near XRE2 and XRE8 was observed. It was suggested that mutual interaction of XRE2 and XRE3 is important for transcriptional regulation, and that the Sp1 binding to the Sp1-like motif (-824) enhances both the constitutive and inducible transcriptional activities of the human CYP1B1 gene.View full abstractDownload PDF (3963K) -
Ken-ichi Yoshioka, Yoshiko Yumoto-Yoshioka, Fabrice Fleury, Masayuki T ...2003Volume 133Issue 5 Pages 593-597
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSHuman HsRad51 protein assembles on a DNA molecule through cooperative binding and forms a long filament for homologous recombination. We have characterized the self-assembly of HsRad51 by measuring the fluorescence resonance energy transfer from the fluorescein-labeled protein to the rhodamine-labeled protein. Self-assembly quickly reached equilibrium and can be described by the head-to-tail polymerization of monomers, like that of its procaryotic homologue, RecA. It depended strongly on pH and was inhibited by high salt concentrations, indicating that ionic interactions between negatively and positively charged aminoacid residues are important. By contrast, neither ATP nor ADP significantly affected the reaction.View full abstractDownload PDF (647K) -
Satoshi Numazawa, Masahiko Watabe, Satoshi Nishimura, Masahiro Kurosaw ...2003Volume 133Issue 5 Pages 599-605
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSSB 203580 has been widely used to specifically shut down the p38 MAP kinasedependent pathway, although it is capable of inducing c-Raf kinase activity in cells. The present study demonstrates that SB 203580 activates members of the ERK cascade, c-Raf, MEK, and ERK, in human monocytic THP-1 cells. The activation of these kinases was sustained for at least 24h after SB 203580 treatment and was also observed in U937 cells, suggesting that c-Raf efficiently transduces the signal even in the presence of the inhibitor in these cells. However, the expression of ERK cascadedependent genes, such as c-fos and IL-1β, was extremely limitetd. Analysis of the cellular distribution of ERK in SB 203580-treated cells indicated that nuclear translocation of phosphorylated ERK was impaired. Also, nuclear translocation of ERK induced by 12-O-tetradecanoyl-phorbol-13-acetate (TPA) was inhibited by SB 239063, which does not associate with c-Raf and is highly selective for p38 MAP kinase. In addition, the forced expression of the dominant negative mutant of p38 MAP kinase suppressed serum responsive element-dependent transactivation induced by TPA. These results suggest that the steady-state level of p38 MAP kinase activity modulates ERK signaling.View full abstractDownload PDF (2756K) -
Fumiaki Yokoi, Hiroyuki Hiraishi, Kenji Izuhara2003Volume 133Issue 5 Pages 607-614
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSWe previously reported the isolation from bovine liver of a novel 56-kDa inorganic pyrophosphatase named phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPPase). It is a unique enzyme that hydrolyzes not only oxygen-phos-phorus bonds in inorganic pyrophosphate but also nitrogen-phosphorus bonds in phospholysine, phosphohistidine and imidodiphosphate in vitro. In this study, we determined the partial amino acid sequence of the purified bovine LHPPase. To investigate whether humans have the same enzyme, we isolated a cDNA clone from a HeLa cell cDNA library that encodes for the human homologue of LHPPase. Although its sequence does not include the consensus sequence of a typical inorganic pyro-phosphatase, it does contain a similar sequence of the active site in other phos-phatases such as protein-tyrosine phosphatase, dual-specific phosphatase and low molecular weight acid phosphatase. Human LHPPase was highly expressed in the liver and kidney, and moderately in the brain. The recombinant protein was pro-duced in E. coli. Its ability to hydrolyze oxygen-phosphorus bonds and nitrogen-phos-phorus bonds was confirmed. The enzymatic characteristics of this human protein were similar to those of purified bovine LHPPase. Thus, we concluded that the cDNA encoded the human counterpart of bovine LHPPase.View full abstractDownload PDF (2234K) -
Naoki Amada, Tomoyuki Tezuka, Akila Mayeda, Kazuaki Araki, Nobuyuki Ta ...2003Volume 133Issue 5 Pages 615-623
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe crooked neck (crn) gene of Drosophila melanogaster encodes a scaffold protein carrying multiple tetratricopeptide repeat (TPR) motifs, and its mutation results in a reduction in the number of neuroblasts and lethality during larval stages. Here, we isolated two structurally related genes from a rat embryonic brain cDNA library. One gene is the rat orthologue of crn, which encodes 690 amino acids including 16 copies of TPR. The other gene, ATH55, encodes an 855 amino acid protein including 21 TPR motifs, which presumably represents a rat crn homologue and an orthologue of human XAB2. Both genes are highly expressed in embryonic brain but their expres-sions decrease during development. ATH55-like immunoreactivity is present in the ventricular zone and newly formed cortical plate, while CRN-like immunoreactivity is more abundant in a younger ventricular zone. In agreement, both proteins were found to be enriched in cultured neural stem cells and to decrease in response to cell differentiation signals. As indicated for the yeast CRN-like protein, ATH55 and CRN immunoreactivities were both recovered in the nuclear fraction and detected in the splicing complex carrying pre-mRNA. These findings suggest that both TPR-motif-containing proteins are involved in RNA processing of mammalian neural stem cells and their immediate descendants.View full abstractDownload PDF (4403K) -
Kazutoyo Terada, Shota Ueno, Kentaro Yomogida, Tomoaki Imai, Hiroshi K ...2003Volume 133Issue 5 Pages 625-631
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe 34-kDa translocase of the outer mitochondrial membrane (Tom34) is a putative mammalian-specific factor involved in protein import into mitochondria. We ana-lyzed the genomic sequence of the mouse Tom34 gene and found it has two alternative initial exons. Using reverse transcription and the polymerase chain reaction (RT-PCR), we found that these two mRNAs differs only in the 5'-proximal sequences corre-sponding to the two initial exons (exon 1a and 1b). Tom34 mRNA with exon 1a (Tom34a) is expressed ubiquitously, while that with exon 1b (Tom34b) is expressed only in mature testicular germ cells. To explore the in vivo function of Tom34 pro-teins, we generated Tom34-deficient mice by targeted disruption. The Tom34-|- mice were viable and grew normally and had a normal Mendelian inheritance pattern. Male as well as female Tom34-|- mice were fertile. In vitro-preprotein import into iso-lated mitochondria showed no apparent difference between Tom34-|- and wild-type mice. These results indicate that Tom34 is dispensable for mouse growth and develop-ment under optimal conditions.View full abstractDownload PDF (3996K) -
Takayuki Furumatsu, Zheng Nan Shen, Akira Kawai, Keiichiro Nishida, Hi ...2003Volume 133Issue 5 Pages 633-639
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSVascular endothelial growth factor (VEGF)-mediated angiogenesis is essential for bone formation. However, the effect of VEGF on osteoblastic cells during osteoblast-ogenesis is still controversial. The aim of this study was to clarify the relationship between osteoblastic cells derived from human mesenchymal stem cells (MSCs) and VEGF in the early stage of osteoblastic differentiation. Continuous dexamethasone treatment with a low concentration stimulated osteoblastogenesis of MSCs and the expression of VEGF121 mRNA. The VEGF secretion from osteoblastic cells also increased along with osteoblastogenesis. Neuropilin-1, which mainly binds VEGF165, was detected at all stages during early osteoblastogenesis, but VEGF receptor-1 and -2 were not detected on RT PCR analyses. In this study, VEGF had no direct effect on the proliferation of osteoblastic cells. However, the secreted VEGF in the conditioned medium of osteoblastic cells exhibited high angiogenic power as to endothelial cell proliferation. Our findings indicated that VEGF121 principally acts as the main ang-iogenic factor in the early stage of human osteoblastogenesis. The present study also demonstrated the differential expression of VEGF121 during osteoblastogenesis. The increase of VEGF in the early stage might be a useful marker of induction of bone for-mation due to human MSCs.View full abstractDownload PDF (2971K) -
Mitsunori Fukuda2003Volume 133Issue 5 Pages 641-649
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSSynaptotagmins (Syts) represent a large family of putative membrane trafficking pro-teins found in various species from different phyla. In this study, I identified a novel class of Syt (named Syt XIV) conserved from Drosophila to humans and its highly related molecule, Strep14 (Syt X1V-related protein). Although both Syt XIV and Strep14 belong to the C-terminal-type (C-type) tandem C2 protein family, only Syt XIV has a single transmembrane domain at the N-terminus and a putative fatty-acylation site just downstream of the transmembrane domain. Biochemical analyses have indi-cated that Syt XIV is a Ca2+-independent Syt (e.g., Syts VIII, XII, and XIII) and that, like other Syt family proteins, it is capable of forming a Ca2+-independent oligomer. Unlike other Syt isoforms, however, expression of Syt XIV and Strep14 mRNA is highly restricted to mouse heart and testis and absent in the brain, where most other Syts are abundantly expressed, suggesting that Syt XIV and Strep14 may be involved in membrane trafficking in specific tissues outside the brain. I also identified all of the C-type tandem C2 proteins in humans, the mouse, the fruit fly, a nematode, a plant, and a yeast and discuss the molecular evolution of the C-type tandem C2 pro-tein families, including the Syt family, the Syt-like protein (Slp) family, and the Doc2 family.View full abstractDownload PDF (3264K) -
Naoki Nemoto, Tairo Oshima, Akihiko Yamagishi2003Volume 133Issue 5 Pages 651-657
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSWe purified a geranylgeranylglyceryl phosphate (GGGP) synthase from Thermoplasma acidophilum by several steps of chromatography. Based on the proteinase-fragment-mass-pattern analysis of the SDS-PAGE band of the partially purified pro-tein, the DNA sequence encoding the protein was identified from the whole genome sequence database of the species. The gene encoding GGGP synthase in T. acido-philum was cloned after PCR amplification of the gene from the genomic DNA. The recombinant enzyme was expressed in Escherichia coli and purified. A single band with a molecular mass of 27 kDa was obtained by SDS-PAGE analysis. The apparent native molecular mass of the enzyme was about 50 kDa based on gel filtration chro-matography, suggesting that the enzyme is active as a homodimer. As the GGGP syn-thase from Methanobacterium thermoautotrophicum has been reported as a pen-tamer, the enzymes of the two organisms have different oligomeric structures. Other characteristics, including substrate specificity, are similar for the GGGPs of these organisms.View full abstractDownload PDF (1781K) -
Yoko Keira, Satoru Noguchi, Nariiro Minami, Yukiko K. Hayashi, Ichizo ...2003Volume 133Issue 5 Pages 659-664
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSCalpain 3/p94, the skeletal muscle-specific isoform of the calpain large subunit family, is a protein product of the gene responsible for limb-girdle muscular dystrophy type 2A (LGMD2A). Through yeast two-hybrid experiments, calpain 3 has been shown to bind to titin in myofibrils [Sorimachi et al. (1995) J. Biol. Chem. 270, 31158-31162]. However, because of extensive autolysis activity, calpain 3 localization in skeletal muscle has been undefined. In this study, we generated a polyclonal antibody against an N-terminal 98-amino-acid calpain 3 fragment, which is not homologous to the corresponding regions of other conventional calpains. This antibody stained myofibrils with a unique repeated doublet-pattern. Confocal microscopic observation with marker antibodies confirmed that calpain 3 is localized in the N2 region of myofibrils. Furthermore, using this antibody, we examined the localization of calpain 3 in LGMD2A muscles.View full abstractDownload PDF (3842K) -
Yoshiya Ikawa, Kenta Sasaki, Hiroyuki Tominaga, Tan Inoue2003Volume 133Issue 5 Pages 665-670
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe P5 or P7 extensions in the group I intron ribozyme serve as “modular activator units” by stabilizing the conserved core of the ribozyme. The P5 extension of a group IC1 intron was introduced to a barely active group IA2 intron lacking its original P7 extension. The inserted P5 extension significantly activated the chimeric construct. Because the CYT-18 protein factor is also known to activate mutant group IA2 and IC1 introns lacking their P7 and P5 extensions, respectively, the RNA and protein activator units function in an analogous manner.View full abstractDownload PDF (2214K) -
Kohki Endo, Yutaka Hayashi, Takahiro Hibi, Kuniaki Hosono, Teruhiko Be ...2003Volume 133Issue 5 Pages 671-677
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSLaccase is an enzyme that catalyzes the oxidation of phenolic compounds by coupling the reduction of oxygen to water. While many laccases have been identified in plant and fungal species, enzymes of prokaryotic origin are poorly known. Here we report the enzymological characterization of EpoA, a laccase-like extracytoplasmic phenol oxidase produced by Streptomyces griseus. EpoA was expressed and purified with an Escherichia coli host-vector system as a recombinant protein fused with a C-terminal histidine-tag (rEpoA). Physicochemical analyses showed that rEpoA comprises a stable homotrimer containing all three types of copper (types 1-3). Various known laccase substrates were oxidized by rEpoA, while neither syringaldazine nor guaiacol served as substrates. Among the substrates examined, rEpoA most effectively oxidized N, N-dimethyl-p-phenylenediamine sulphate with a Km value of 0.42mM. Several metal chelators caused marked inhibition of rEpoA activity, implying the presence of a metal center essential for the oxidase activity. The pH and temperature optima of rEpoA were 6.5 and 40°C, respectively. The enzyme retained 40% activity after preincubation at 70°C for 60 min. EpoA-like activities were detected in cell extracts of 8/40 environmental actinomycetes strains, which suggests that similar oxidases are widely distributed among this group of bacteria.View full abstractDownload PDF (2799K) -
Hideo Iwahashi, Kazumasa Kumamoto, Tomihiro Hirai2003Volume 133Issue 5 Pages 679-685
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSMethemoglobin (MetHb), oxyhemoglobin (oxyHb), metmyoglobin (metMb), and oxymyoglobin (oxyMb) catalyze formation of the 7-carboxyheptyl and pentyl radicals from 13-hydroperoxy-9, 11-octadecadienoic acid. The relative HPLC-ESR peak height of the pentyl radical to the 7-carboxyheptyl radical was found to depend on the oxygen concentration in the reaction mixture. Under aerobic conditions, the 7-carboxyheptyl radical was predominant for the reaction mixture with ferrous ions (or cytochrome c, metHb, or metMb). On the other hand, under anaerobic conditions, the pentyl radical was predominant for the reaction mixture with ferrous ions (or cytochrome c), but the 7-carboxyheptyl radical was still predominant for the reaction mixture with metHb (or metMb), suggesting that metHb (or metMb) catalyzes the reaction through a mechanism different from that in the case of ferrous ions (or cytochrome c). In order to explain the above results, a mechanism, in which molecular oxygen is not involved, is proposed for the formation of the 7-carboxyheptyl radical in the reaction mixture of 13-HPODE with metHb (or metMb) under anaerobic conditions.View full abstractDownload PDF (747K) -
Mahito Kikumoto, Youiiro Tamura, Atsushi Ooi, Koshin Mihashi2003Volume 133Issue 5 Pages 687-691
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSWe determined the partial specific volume and partial specific adiabatic compressi-billity of either ATP-or ADP-bound monomeric actin in the presence of Ca2+ by meas-uring the density of and sound velocity in amonomeric astin solution at 18°C. The partial specific volume of ATP-bound monomeric actin, equla to 0.744 cm3/g, which is exceptionally high among globular proteins, was reduced to 0.727 cm3/g whe the tightly bound ATP was replaced wity ADP. Associated with this, the adiabatic com-pressibility of ATP-bound monomeric actin, equal to 8.8×10-12 cm2/dyne, decreased to 5.8×10-12 cm2/dyne, which is a common value for globular proteins. These results sug-gested that an extraordinarily soft global conformation of ATP-bound monomeric actin is packed into a compact mass associated with the hydrolysis of bound ATP. When monomeric actin was limitedly proteolyzed at subdomain 2 with subtilisin, the nucle-otide-dependent flexibility of the global conformation of monomeric actin waslost.View full abstractDownload PDF (623K) -
Nguyen Tien Huy, Satoshi Serada, Dai Thi Xuan Trang, Ryo Takano, Yoshi ...2003Volume 133Issue 5 Pages 693-698
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSPlasmodium falciparum histidine-rich protein 2 (PfHRP2) has been suggested to be an initiator of the polymerization of heme, which is produced as by-product on the digestion of hemoglobin, and a promoter of the H2O2-induced degradation of heme in food yacuoles of the malarial parasite. In this work, we have designed PfHRP2 model peptides, R18 and R27 (18 and 27 residues, respectively), and used them for optical and electron spin resonance spectroscopic measurements to confirm that the axial ligands of the heme-PfHRP2 complex are the nitrogenous donors derived from the imidazole moieties of histidine residues of PfHRP2. In addition, we revealed that the affinities of R18 and R27 for heme (Kd=2.21×10-6 M and 0.71×10-6M, respectively) might be as high as that of PfHRP2 (Kd=0.94×10-6M). The R27 peptide can remove heme from membrane-intercalated heme and inhibit heme-induced hemolysis. There-fore, we suggest another function of PfHRP2: it may play an important role in the neutralization of toxic heme in the parasite cytoplasm and infected erythrocytes by removing heme from heme-bound membranes or reducing heme-induced hemolysis.View full abstractDownload PDF (787K)
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