The Journal of Biochemistry
Online ISSN : 1756-2651
Print ISSN : 0021-924X
Volume 132, Issue 6
Displaying 1-22 of 22 articles from this issue
  • Ushio Kikkawa, Hidenori Matsuzaki, Toshiyoshi Yamamoto
    2002 Volume 132 Issue 6 Pages 831-839
    Published: 2002
    Released on J-STAGE: June 30, 2008
    JOURNAL FREE ACCESS
    Protein kinase C (PKC)δ was the first new/novel PKC isoform to be identified by the screening of mammalian cDNA libraries, based on the structural homology of its nucleotide sequences with those of classical/conventional PKC isoforms. PKCδ is expressed ubiquitously among cells and tissues. It is activated by diacylglycerol produced by receptor-mediated hydrolysis of membrane inositol phospholipids as well as by tumorpromoting phorbol ester through the binding of these compounds to the C1 region in its regulatory domain. It is also cleaved by caspase to generate a catalytically active fragment, and it is converted to an active form without proteolysis through the tyrosine phosphorylation reaction. Various lines of evidence indicate that PKCδ activated in distinct ways plays critical roles in cellular functions such as the control of growth, differentiation, and apoptosis. This article briefly summarizes the regulatory mechanisms of PKCδ activity and its functions in cell signaling.
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  • Amnon Altman, Martin Villalba
    2002 Volume 132 Issue 6 Pages 841-846
    Published: 2002
    Released on J-STAGE: June 30, 2008
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    The novel protein kinase C (PKC) isoform, PKCθ, is expressed in a relatively selective manner in T lymphocytes (and muscle). Recent analysis of this PKC isotype in T cells and the characterization of PKCθ-deficient mice revealed important clues about its function and regulation. PKCθ does not have an obvious role in T cell development, but it is essential for the activation of mature T cells. The requirement of PKCθ for T cell activation, proliferation and cytokine production reflects the essential role of this isotype in inducing signaling pathways leading to the activation of the transcription factors AP-1 and NF-κB in a T cell-specific manner. A unique feature of PKCθ is its highly selective translocation to the central region of the immunological synapse (IS) in antigen-stimulated T cells, a property apparently important for its proper signaling functions. This localization implies unique pathway(s) that regulate the translocation and/or activation of this enzyme. Our work suggests that sustained PKCθ membrane translocation and phosphorylation are relatively independent of phospholipase C (PLC) activation and diacylglycerol (DAG) production. Instead, a pathway that requires Vav, phosphatidylinositol 3-kinase (PI3-K), Racl and actin cytoskeleton reorganization mediates these events. Additionally, PKCθ provides an important survival signal to T cells. Nevertheless, several questions regarding the function and regulation of PKCθ and the identity of its immediate targets/substrates remain open. Resolution of these questions could open the way to the development of selective PKCθ inhibitors, which may have therapeutic potential in immunological diseases and in cancer.
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  • Yoshiko Akita
    2002 Volume 132 Issue 6 Pages 847-852
    Published: 2002
    Released on J-STAGE: June 30, 2008
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    Protein kinase C (PKC)-ε was first discovered among novel PKC isotypes by cDNA cloning, and characterized as a calcium-independent but phorbol ester/diacylglycerol-sensitive serine/threonine kinase. PKC-ε is targeted to a specific cellular compartment in a manner dependent on second messengers and on specific adapter proteins in response to extracellular signals that activate G-protein-coupled receptors, tyrosine kinase receptors, or tyrosine kinase-coupled receptors. PKC-ε then regulates various physiological functions including the activation of nervous, endocrine, exocrine, inflammatory, and immune systems. The controlled activation of PKC-ε plays a protective role in the development of cardiac ischemia and Alzheimer's disease, whereas its uncontrolled chronic activation results in severe diseases such as malignant tumors and diabetes. This review summarizes recent progress in our understanding of the unique structure and physiological and pathological roles of PKC-ε with a focus mainly on knockout, transgenic, and mutational studies.
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  • Mariko Kashiwagi, Motoi Ohba, Kazuhiro Chida, Toshio Kuroki
    2002 Volume 132 Issue 6 Pages 853-857
    Published: 2002
    Released on J-STAGE: June 30, 2008
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    The η isoform of protein kinase C (PKCη) is classified into the Cat2+-independent novel PKC subfamily and assigned to human chromosome 14 (14q22-23) and mouse chromosome 12 (12C3-D2). It is highly expressed in epithelial tissues especially in squamous epithelia. PKCη is unique in that it is specifically activated by cholesterol sulfate and sulfatide, sulfated metabolites of cholesterol and cerebroside, respectively. PKCη over-expression induces G1 arrest and differentiation in keratinocytes. PKCη-induced differentiation is accompanied by the transcriptional activation of transglutaminase I, a key enzyme in squamous differentiation, and involucrin, a precursor of cornified envelopes. In keratinocytes, PKCη associates with the cyclin E/cdk2/p21 complex and inhibits the cdk2-kinase activity, leading to G1 arrest. Cholesterol sulfate inhibits the promotional phase of skin carcinogenesis. Moreover, PKCηknockout mice show a much higher sensitivity to carcinogenesis, suggesting that PKCη is negatively involved in tumor promotion through stimulation of keratinocyte differentiation. In addition to epithelial cells, recent studies revealed that PKCη acts as a key regulator in early B-cell development.Although the functions of PKCη in other cell types are not yet fully elucidated, available evidence indicates that this particular isoform plays crucial roles in the signaling of cell differentiation in a cell-type-specific manner.
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  • Yousuki Okano, Tsuyoshi Inoue, Bruno Kilunga Kubata, Zakayi Kabututu, ...
    2002 Volume 132 Issue 6 Pages 859-861
    Published: 2002
    Released on J-STAGE: June 30, 2008
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    Prostaglandin F is a potent mediator of various physiological and pathological processes. Trypanosoma brucei prostaglandin F synthase (TbPGFS) catalyzes the NADPH-dependent reduction of 9, 11-endoperoxide PGH2 to PGF, and could thus be involved in the elevation of the PGF concentration during African trypanosomasis. In the present report, the purification and crystallization of recombinant TbPGFS are described. The active recombinant enzyme was crystallized by the hanging-drop vapor-diffusion method using ammonium sulfate as a precipitant. The crystal belonged to a tetragonal space group, P41212 or P43212, with unit-cell parameters of a=b=112.3 Å, and c=140.0 Å. Native data up to 2.6 Å resolution were collected from the crystal using our home facility.
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  • Hiroshi Abe, Kazunori Kawasaki, Hiroshi Nakanishi
    2002 Volume 132 Issue 6 Pages 863-874
    Published: 2002
    Released on J-STAGE: June 30, 2008
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    The conformational transition to a β-structure and the aggregation process of Alzheimer amyloid β-peptide (12-24) [abbreviated as Aβ12-24] were studied. The influence of sample dissolution methods for the aggregate structure was examined by electron microscopy (EM). The difference in the width of the aggregate of Aβ12-24 depended on the pH immediately after sample dissolution. Two types of sample dissolution methods, F and R, were employed. For dissolution method F, the peptide sample was immediately dissolved in water and then adjusted to pH 2.2 by adding buffer, while for dissolution method R, the peptide was directly dissolved in the buffer solution. In the latter case, the starting pH was 3.0. Slight fibrils (10-12 nm in diameter) were observed with method F, and wider ribbon-like aggregates (17-20 nm in diameter) with method R, despite the same pH range. A difference between methods F and R was also detected in the CD spectra, especially at pHs near 5.0. The CD intensity of the 214 nm band with method R changed with pH, with the highest value at pH 3.7, whereas that with method F was unchanged at pHs below 5.0. The temperature-dependent CD results showed that a thermostable aggregate of Aβ12-24 occurs at higher pHs than 3.0. NMR analysis showed that deprotonation of the C-terminal carboxylate group in Aβ12-24 triggered the aggregate formation, and the transition from a random coil to a β-conformation in the C-terminal region of V18-V24 was detected on analysis of the 3JαN, coupling constant in the pH range of 2.2 to 3.0.
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  • Masashi Sagara, Eri Takeda, Akiyo Nishiyama, Syunsaku Utsumi, Yoshiroh ...
    2002 Volume 132 Issue 6 Pages 875-879
    Published: 2002
    Released on J-STAGE: June 30, 2008
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    NPAT plays a role in S phase entry as a substrate of cyclin E-CDK2 and activation of histone gene transcription. Although analysis of its sequence indicates that NPAT contains typical nuclear localization signals (NLS) comprising segments of positively charged amino acids, there are currently no experimental data to show that these predictive NLS are functional. To investigate whether these sequences are effective for nuclear transport of NPAT, an NPAT-green fluorescent protein fusion (NP-GFP) was constructed. After transfection of the fusion gene containing the full coding region of NPAT into cultured cells, the NP-GFP product was found exclusively in the nucleus. As expected, some deletion mutants that retained the basic amino acid clusters at the carboxyl terminus also localize the fusion protein in the nucleus. However, other fusions that lacked one of the three basic amino acid-clusters were distributed throughout the nucleus and cytoplasm. Therefore all three clusters of basic residues are necessary for localization of NPAT to the nucleus. However, another sequence outside the carboxyl terminal region functions similarly to NLS. Construction of GFP fusions with a series of truncated forms of NPAT indicated that a short peptide sequence consisting of mainly hydrophobic amino acids near the central domain of NPAT also contributes to localizing the protein in the nucleus.
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  • Sachiko Tominaga, Minoru Morikawa, Takashi Osumi
    2002 Volume 132 Issue 6 Pages 881-889
    Published: 2002
    Released on J-STAGE: June 30, 2008
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    Reports vary on the role of growth hormone (GH) in adipocyte differentiation. In this study, we showed that GH exerted dual effects depending on the stage of differentiation, using a serum-free culture of 3T3-L1 preadipocytes. GH promoted the differentiation when added to the medium during differentiation-inducing treatment with a hormone cocktail, but apparently suppressed it when added after the treatment. Only the suppressive effect was observed in the presence of 10% fetal bovine serum (FBS). Immunodepletion study showed that GH contributes to the differentiation-promoting activity of FBS. Insulin-like growth factor-1 could not replicate either the stimulative or the suppressive effect of GH. Stimulation of differentiation by GH involved the enhanced expression of mRNA of middle to late adipocyte markers. Among the key regulators of adipogenesis, peroxisome proliferator-activated receptor (PPAR) γ and CCAAT/enhancer binding protein (C/EBP) α, but not C/EBPβ, were stimulated for mRNA expression by GH added during the treatment with hormone cocktail. The stimulation of adipogenesis by GH was indeed due to the increase in the ratio of differentiated cells, though GH also promoted cell growth.
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  • Takashi Ohkura, Akira Seko, Sayuri Hara-Kuge, Katsuko Yamashita
    2002 Volume 132 Issue 6 Pages 891-901
    Published: 2002
    Released on J-STAGE: June 30, 2008
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    Many reports show that N-glycans of glycoproteins play important roles in vectorial transport in MDCK cells. To assess whether structural differences in N-glycans exist between secretory glycoproteins and membrane glycoproteins, we studied the N-glycan structures of the glycoproteins isolated from MDCK cells. Polarized MDCK cells were metabolically labeled with [3H]glucosamine, and 3H-labeled N-glycans of four glycoprotein fractions, secretory glycoproteins in apical and basolateral media, and apical and basolateral membrane glycoproteins, were released by glycopeptidase F. The structures of the free N-glycans were comparatively analyzed using various lectin column chromatographies and sequential glycosidase digestion. The four samples commonly contained high-mannose-type glycans and bi- and tri-antennary glycans with a bisected or non-bisected trimannosyl core. However, secretory glycoproteins in both media predominantly contained (sialyl)LacdiNAc sequences, ±Siaα2→6GalNAcβ1→4G1cNAcβ1→R, which linked only to a non-bisected trimannosyl core. β1→4N-acetylgalactosaminyltransferase β4GalNAc-T) activity in MDCK cells preferred non-bisected glycans to bisected ones in accordance with the proposed N-glycan structures. This secretory glycoprotein-predominant LacdiNAc sequence was also found in the case of human embryonic kidney 293 cells. These results suggest that the secretory glycoprotein-specific (sialyl)LacdiNAc sequence and the corresponding β4GalNAc-T are involved in transport of secretory glycoproteins.
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  • Ryutaro Asano, Toshio Kudo, Yukihiro Nishimura, Koki Makabe, Hiroki Ha ...
    2002 Volume 132 Issue 6 Pages 903-909
    Published: 2002
    Released on J-STAGE: June 30, 2008
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    Recombinant fragments of the variable region of antibodies are useful in many experimental and clinical applications. However, it can be difficult to obtain these materials in soluble form after their expression in bacteria. Here, we report an efficient procedure for preparing several variable-domain fragments (Fv), single-chain Fv (scFv), and a diabody (the smallest functional bispecific antibody) of anti-carcinoembryonic antigen (CEA) antibody by overexpression in Escherichia coli in inclusion bodies, using a refolding system to obtain renatured proteins. Two types of refolded Fv were prepared: (i) Heavy and light chains of the immunoglobulin variable regions (VH and VL, respectively) were coexpressed with a dicistronic expression vector (designated Fv, co); (ii) VH and VL were expressed separately, mixed stoichiometrically, and refolded (designated Fvmix). All samples refolded with high efficiency; Fvco, Fvm;mix, scFv, and the bispecific diabody bound to several CEA-positive cell lines, exactly as did soluble Fv fragments secreted by E. coli (Fvsol) and the parent IgG. The refolded fragments inhibited binding of the parent IgG to CEA-positive cell lines, indicating that their epitope is identical to that of IgG. The bispecific diabody, which combined variable-region fragments of anti-CEA antibody with variable-region fragments of anti-CD3 antibody, was also prepared using the refolding system. This refolded diabody could bind to lymphokine-activated killer cells. In addition, its cytotoxicity toward human bile duct carcinoma TFK-1 and other several other CEA-positive cell lines was concentration-dependent. Taken together, our results suggest that a refolding procedure can be used to prepare various functional antibody fragments (Fv, scFv, and diabody).
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  • Masaki Kamakura
    2002 Volume 132 Issue 6 Pages 911-919
    Published: 2002
    Released on J-STAGE: June 30, 2008
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    A 57-kDa protein in royal jelly (RJ) was previously shown to stimulate hepatocyte DNA synthesis and prolongs the proliferation of hepatocytes as well as increasing albumin production [Kamakura, M., Suenobu, N., and Fukushima, M. (2001) Biochem. Biophys. Res. Commun. 282, 865-874]. In this study, I investigated the signal transduction mechanisms involved in the induction of hepatocyte DNA synthesis and the promotion of cell survival by this 57-kDa protein in primary cultures of adult rat hepatocytes. Hepatocyte DNA synthesis induced by the 57-kDa protein was not influenced by several α- and β-adrenoceptor antagonists, but was dose-dependently abolished by an inhibitor of a tyrosine-specific protein kinase, genistein. A phospholipase C inhibitor (U-73122) and a protein kinase C (PKC) inhibitor (sphingosine) inhibited 57-kDa protein-stimulated hepatocyte DNA synthesis, whereas a protein kinase A inhibitor (H-89) did not. The 57-kDa protein also activated PKC in rat hepatocytes. Various inhibitors of intracellular signal transduction elements (PD98059, p21 ras farnesyltransferase inhibitor, wortm nin and rapamycin) also blocked hepatocyte DNA synthesis induced by the 57-kDa protein. Fur-thermore, the 57-kDa protein activated mitogen-activated protein (MAP) kinase in rat hepatocytes. The activation of MAP kinase by the 57-kDa protein was inhibited by PD98059 and sphingosine. The 57-kDa protein also activated protein kinase B, which is a key regulator of cell survival. These results suggest that, like growth factors, the 57-kDa protein activates several important intracellular signaling factors involved in the stimulation of hepatocyte DNA synthesis and the protection of cells from apoptosis.
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  • Toshihiko Aki, Ken-ichi Yoshida, Tatsuya Fujimiya
    2002 Volume 132 Issue 6 Pages 921-926
    Published: 2002
    Released on J-STAGE: June 30, 2008
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    We have shown recently that phosphoinositide 3-kinase (PI 3-kinase) accelerates the hypoxia-induced necrotic cell death of H9c2, derived from rat cardiomyocytes, by enhancing metabolic acidosis. Here we show the downstream events of acidosis that cause hypoxic cell death. Hypoxia induces the proteolysis of fodrin, a substrate of calpain. Intracellular Ca2+ chelation by BAPTA, and the addition of SJA6017, a specific peptide inhibitor of calpain, also reduces cell death and fodrin proteolysis, indicating that Ca2+ influx and calpain activation might be involved in these events. The overexpression of wild type PI 3-kinase accelerates fodrin proteolysis, while dominant-negative PI 3-kinase reduces it. Both (N-ethyl-N-isopropyl) amiloride (EIPA), an inhibitor of the Na+/H+ exchanger, and KB-R7943, an inhibitor of the Na+/Ca2+ exchanger, reduce hypoxic cell death and fodrin proteolysis. The depletion of intracellular Ca2+ stores by thapsigargin, an inhibitor of endoplasmic reticulum Ca2+-ATPase, also reduces cell death and fodrin proteolysis, indicating that Ca2+ release from intracellular Ca2+ stores might be also involved. These results indicate that PI 3-kinase might accelerate hypoxic cell death by enhancing the calpain-dependent proteolysis of fodrin.
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  • S. Shanmuga Sundara Raj, Eiji Kibushi, Tatsuhiro Kurasawa, Atsuo Suzuk ...
    2002 Volume 132 Issue 6 Pages 927-933
    Published: 2002
    Released on J-STAGE: June 30, 2008
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    The Bowman-Birk trypsin inhibitor (BBI) from wheat germ (I-2b) consists of 123 amino acid residues with two inhibitory loops. The crystal structure of a bovine trypsin-wheat germ trypsin inhibitor (I-2b) complex (2:1) has been determined at 2.3 Å resolution to a final R-factor of 0.177. A distance of 37.2 Å between the contiguous contact loops allows them to bind and inhibit two trypsin molecules simultaneously and independently. Each domain shares the same overall fold with 8 kDa BBIs. The five disulfide bridges in each domain are a subset of seven disulfide bridges in the 8 kDa BBIs. I-2b consists of ten β-strands and the loops connecting these strands but it lacks α-helices. The conformations of the contiguous contact loops of I-2b are in a heart-like structure. The reactive sites in both domains, Arg 17 and Lys 76, are located on the loop connecting anti-parallel β-strands, β1/β2 and β6/β7. Strands β1 and β6 are in direct contact with trypsin molecules and form stable triple stranded β-sheet structures via hydrogen bonds.
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  • Masaki Fukuoka, Seiki Yamada, Shozo Miyoshi, Kenji Yamashita, Mamoru Y ...
    2002 Volume 132 Issue 6 Pages 935-943
    Published: 2002
    Released on J-STAGE: June 30, 2008
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    The roles of the D-ribosyl moiety and the bulky axial ligand of the nucleotide loop of adenosylcobalamin in coenzymic function have been investigated using two series of coenzyme analogs bearing various artificial bases. The 2-methylbenzimidazolyl trimethylene analog that exists exclusively in the base-off form was a totally inactive coenzyme for diol dehydratase and served as a competitive inhibitor. The benzimidazolyl trimethylene analog and the benzimidazolylcobamide coenzyme were highly active for diol dehydratase and ethanolamine ammonia-lyase. The imidazolylcobamide coenzyme was 59 and 9% as active as the normal coenzyme for diol dehydratase and ethanolamine ammonia-lyase, respectively. The latter analog served as an effective suicide coenzyme for both enzymes, although the partition ratio (kcat/kinact) of 630 for ethanolamine ammonialyase is much lower than that for diol dehydratase. Suicide inactivation was accompanied by the accumulation of a cob(II)amide species, indicating irreversible cleavage of the coenzyme Co-C bond during the inactivation. It was thus concluded that the bulkiness of a Co-coordinating base of the nucleotide loop is essential for both the initial activity and continuous catalytic turnovers. Since the kcat/kinact value for the imidazolyl-cobamide in diol dehydratase was 27-times higher than that for the imidazolyl trimethylene analog, it is clear that the ribosyl moiety protects the reaction intermediates from suicide inactivation. Stopped-flow measurements indicated that the rate of Co-C bond homolysis is essentially unaffected by the bulkiness of the Co-coordinating base for diol dehydratase. Thus, it seems unlikely that the Co-C bond is labilized through a ground state mechanochemical triggering mechanism in diol dehydratase.
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  • Yuko Muta, Kuniyo Inouye
    2002 Volume 132 Issue 6 Pages 945-951
    Published: 2002
    Released on J-STAGE: June 30, 2008
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    Alcohols inhibit the thermolysin-catalyzed hydrolysis of N-[3-(2-furyl)acryloyl]-Gly-L-Leu-NH2 and decrease the NaCl-induced activation of thermolysin in a concentration-dependent manner [K. Inouye et al. (1997) J. Biochem. 122, 358-364]. In this study, the inhibitory effects of alcohols on thermolysin activity were examined in detail using 10 different alcohols and a fluorescent 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. The inhibition by all alcohols examined is completely reversible, and thermolysin activity is recovered by dilution. The inhibitor constants (Ki) are in the range of 35-430 mM, and the order of the inhibitory effect is 1-pentanol, 1-propanol, 2-butanol, 2-methyl-l-pro-panol > 1-butanol > 2-propanol > ethanol, tert-amyl alcohol >> tert-butyl alcohol >> methanol. Linear and secondary alcohols whose mains chains consist of more than 3 carbons inhibit thermolysin effectively. Thermolysin activity is decreased by decreasing the dielectric constant, D, of the reaction medium containing the alcohol, and the decrease depending on the D value was almost the same manner for all alcohols except methanol, tert-butyl alcohol, and tert-amyl alcohol. Alcohols may inhibit thermolysin activity both by binding to the active site, most possibly to the S1' subsite, of thermolysin and by altering the electrostatic and hydrophobic environment around the thermolysin molecule.
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  • Hideki Ogino, Wataru Satou, Michihiko Fujii, Toshikazu Suzuki, Yuan He ...
    2002 Volume 132 Issue 6 Pages 953-959
    Published: 2002
    Released on J-STAGE: June 30, 2008
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    5-Bromodeoxyuridine (BrdU) immediately and clearly suppresses expression of the mouse Myod1 and human MYOD1 genes in myoblastic cells. Despite various studies, its molecular mechanism remains unknown. We failed to identify a BrdU-responsive element of the genes in experiments in which reporter constructs containing known regulatory sequences were transferred to mouse C2C12 myoblasts. Therefore, we transferred human chromosome 11 containing the MYOD1 gene to the cells by microcell-mediated chromosome transfer. In the resulting microcell hybrids, BrdU suppressed expression of the transgene, as determined by quantitative real-time RT PCR analysis. We then transfected human PAC clones containing the MYOD1 gene to the cells. In the resulting transfectants, BrdU suppressed the transgene similarly. Deletion analysis suggested that a BrdU-responsive element or chromatin structure exists between 24 and 47 kb upstream of the gene. These results are the first demonstrating BrdU-responsiveness of a trans-gene for the known BrdU-responsive genes and facilitating determination of its precise responsible structure.
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  • Yasushi Makino, Kayoko Yonezaki, Kaoru Omichi
    2002 Volume 132 Issue 6 Pages 961-966
    Published: 2002
    Released on J-STAGE: June 30, 2008
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    The linkage position of a glycosidic bond to the reducing-end residue of a pyridylamino (PA-) sugar can be determined sensitively by Smith degradation and HPLC [K. Omichi and S. Hase, (1994) J. Biochem. 115, 429-434]. With the aim of enhancing the sensitivity of this method of linkage position analysis to the fmol-level, use of the 7-(1, 3-disulfonaphtyl)amino (DSNA-) group instead of the PA-group as a fluorescent tag was examined. Smith degradation of DSNA-disaccharides with a DSNA-hexose, DSNA-N-acetylglu-cosamine, or DSNA-N-acetylgalactosamine reducing-end residue was carried out. HPLC and FAB-MS of the fluorogenic Smith degradation products showed that the DSNA-group was stable under the Smith degradation reaction conditions, and that the reaction proceeded in a manner similar to that using PA-disaccharides to give the predicted products. Fluorogenic Smith degradation products specific to the glycosidic linkage position were well separated by reversed-phase HPLC, and were easily assignable by comparing the HPLC elution positions with those of standard compounds. The method was successfully applied to analyzing the structure of an N-linked sugar chain.
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  • Joji Mima, Hidekazu Suzuki, Mieko Takahashi, Rikimaru Hayashi
    2002 Volume 132 Issue 6 Pages 967-973
    Published: 2002
    Released on J-STAGE: June 30, 2008
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    Carboxypeptidase Y (CPY) inhibitor, IC, a cytoplasmic inhibitor of vacuolar proteinases in yeast, Saccharomyces cerevisiae, was purified by means of a high-level expression system using a proteinase-deficient strain, BJ2168, and an expression vector with the promoter GAL1. The purified IC exists as a monomeric β-protein in solution with a molecular weight of 24, 398.4 as determined by gel filtration chromatography, MALDI-TOF mass spectrometry, and far-UV CD spectroscopy. The acetylated N-terminal methionine residue is the sole posttranslational modification. IC specifically inhibits both the peptidase and anilidase activities of CPY with inhibitor constants (Ki) of approximately 1.0×10-9 M. The chemical modification of IC with sulfhydryl reagents indicated that it lacks disulfide bonds and has two free SH groups, which are responsible, not for the inhibitory function, but, apparently, for the folding of the overall structure. The formation of a complex of IC with CPY was highly specific, as evidenced by no detectable interaction with pro-CPY. Chemical modification studies of the CPY-IC complex with specific reagents demonstrated that the catalytic Ser146 and S1 substrate-binding site of CPY are covered in the complex.
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  • Hiroko Hagiwara, Sumiko Kunihiro, Keiichi Nakajima, Motoaki Sano, Haru ...
    2002 Volume 132 Issue 6 Pages 975-982
    Published: 2002
    Released on J-STAGE: June 30, 2008
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    Phage display is a useful means of identifying and selecting proteins of interest that bind specific targets. In order to examine the potential of phage display for the genomewide screening of DNA-binding proteins, we constructed yeast genomic libraries using λfoo-based vectors devised in this work. After affinity selection using GAL4 UASG as a probe, phages expressing GAL4 were enriched approximately 5×105-fold from the library. Approximately 90% of polypeptides encoded in correct translation reading frames by the selected phages were known or putative polynucleotide-binding proteins. This result clearly indicates that the modified lambda phage display vector in combination with our enrichment technique has great potential for the enrichment of DNA-binding proteins in a sequence-specific manner.
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  • Hideji Yoshida, Yasushi Maki, Hisako Kato, Hisao Fujisawa, Kaori Izuts ...
    2002 Volume 132 Issue 6 Pages 983-989
    Published: 2002
    Released on J-STAGE: June 30, 2008
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    During the stationary growth phase, Escherichia coli 70S ribosomes are converted to 100S ribosomes, and translational activity is lost. This conversion is caused by the binding of the ribosome modulation factor (RMF) to 70S ribosomes. In order to elucidate the mechanisms by which 100S ribosomes form and translational inactivation occurs, the shape of the 100S ribosome and the RMF ribosomal binding site were investigated by electron microscopy and protein-protein cross-linking, respectively. We show that (i) the 100S ribosome is formed by the dimerization of two 70S ribosomes mediated by face-toface contacts between their constituent 30S subunits, and (ii) RMF binds near the ribosomal proteins S13, L13, and L2. The positions of these proteins indicate that the RMF binding site is near the peptidyl transferase center or the P site (peptidyl-tRNA binding site). These observations are consistent with the translational inactivation of the ribosome by RMF binding. After the “Recycling” stage, ribosomes can readily proceed to the “Initiation” stage during exponential growth, but during stationary phase, the majority of 70S ribosomes are stored as 100S ribosomes and are translationally inactive. We suggest that this conversion of 70S to 100S ribosomes represents a newly identified stage of the ribosomal cycle in stationary phase cells, and we have termed it the “Hibernation” stage.
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  • Masayuki Oda, Atsuo Tamura, Kenji Kanaori, Shuichi Kojima, Kin-ichiro ...
    2002 Volume 132 Issue 6 Pages 991-995
    Published: 2002
    Released on J-STAGE: June 30, 2008
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    Single amino acid mutations of Met103 in the hydrophobic core of a serine protease inhibitor, Streptomyces subtilisin inhibitor, caused little change in the inhibitory activity, as measured by the inhibitor constant, although some altered the thermodynamic stability of the protein considerably. 1H NMR investigations showed that the conformational stress caused by the replacement of Met103 with Gly, Ala, Val, and Ile, namely, the effects of the cavities generated by replacements with smaller side-chains and of the steric distortions generated by β-branched side-chains, caused considerable changes in the structural arrangement of the side-chains within the core. However, these structural changes were absorbed within the hydrophobic core, without distorting the structure of the reactive site essential for the protein function. These results provide an excellent example of the conformational flexibility of a protein core and the degree of its tolerance of an amino acid replacement. The results also reveal the crucially designed structural relationship between the core of the inhibitor and the enzyme-binding segment with the reactive site in a serine protease inhibitor.
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  • Hiroo Sawada, Tamayuki Shinomura, Koji Kimata, Jun Takeuchi, Takuo Tsu ...
    2002 Volume 132 Issue 6 Pages 997-1002
    Published: 2002
    Released on J-STAGE: June 30, 2008
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    6B6 is a monoclonal antibody raised against a purified small dermatan sulfate proteoglycan from human ovarian fibroma capsule. Although it has been widely used as an anti-decorin monoclonal antibody, its epitope has not yet been characterized at the molecular level. Here, we show that 6B6 is specific to decorin. The antibody recognized human, mouse, and bovine decorin core protein, but not biglycan. Using recombinant decorin domains, we determined that the epitope lies within the region of amino acid residues 50-65, termed the cysteine cluster region. Cross-reactivity among species further narrowed it down to a primary sequence of residues 57-65. We also established the conditions for immunostaining. 6B6 stained both frozen and fixed sections. Whereas the glycosaminoglycan chain of decorin inhibited access of the antibody in immunoblotting, pretreatment of tissue sections with chondrotinase ABC did not affect the intensity of staining, suggesting that the glycosanunoglycan chain is integrated and the Cys cluster region oriented outside of the collagen fibrils in the tissue. When 6B6 was applied to enzyme-linked immunosorbent assay, a concentration as low as 0.5μg/ml of decorin was detectable by either direct or sandwich ELISA. 6B6 is thus a sensitive and reliable antibody to study functions of decorin from various aspects.
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