The Journal of Biochemistry
Online ISSN : 1756-2651
Print ISSN : 0021-924X
Volume 127 , Issue 5
Showing 1-31 articles out of 31 articles from the selected issue
  • Hiroyuki Sasaki, Ko Ishihara, Reiko Kato
    2000 Volume 127 Issue 5 Pages 711-715
    Published: 2000
    Released: November 18, 2008
    JOURNALS FREE ACCESS
    The mouse Igf 2 and H 19 genes lie 70-kb apart on chromosome 7 and are reciprocally imprinted. Two regulatory regions are important for their parental allele-specific expression: a differentially methylated region (DMR) upstream of H 19 and a set of tissuespecific enhancers downstream of H 19. The enhancers specifically activate Igf 2 on the paternal chromosome and H 19 on the maternal chromosome. The interactions between the enhancers and the genes are regulated by the DMR, which works as a selector by exerting dual functions: a methylated DMR on the paternal chromosome inactivates adjacent H 19 and an unmethylated DMR on the maternal chromosome insulates Igf 2 from the enhancers. These processes appear to involve methyl-CpG-binding proteins, histone deacetylases and the formation of chromatin insulator complexes. The Igf 2/H 19 region provides a unique model in which to study the roles of DNA methylation and chromatin structure in the regulation of chromosome domains.
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  • Shigeaki Kato
    2000 Volume 127 Issue 5 Pages 717-722
    Published: 2000
    Released: November 18, 2008
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    Vitamin D has roles in a variety of biological actions such as calcium homeostasis, cell proliferation and cell differentiation to many target tissues. Most of these biological actions of vitamin D are now considered to be exerted through the nuclear vitamin D receptor (VDR)-mediated control of target genes. VDR belongs to the nuclear hormone receptor superfamily and acts as a ligand-inducible transcription factor. For the ligandinduced transactivation of VDR, coactivator complexes have recently been shown to be essential. The function of VDR as a ligand-induced transcription factor is overviewed, and the phenotype of VDR gene knock-out mice and the VDR-mediated transcriptional and negative regulation of the key enzyme in vitamin D biosynthesis are also described, based mainly on our recent findings, to gain a better understanding of the function of VDR in the transcriptional control of vitamin D target genes.
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  • Hikari Taka, Naoko Kaga, Tsutomu Fujimura, Reiko Mineki, Masamoto Imai ...
    2000 Volume 127 Issue 5 Pages 723-729
    Published: 2000
    Released: November 18, 2008
    JOURNALS FREE ACCESS
    The complete amino acid sequence of β-type parvalbumin (PA) from bullfrog Rana catesbeiana (pI 4.78) was determined by tandem mass spectrometry in combination with amino acid analysis and peptide sequencing following Arg-C and V8 protease digestion. The primary structure of the protein was compared with that of β-type PA from R. esculenta (pI 4.50), with which it is highly homologous. Compared with R. esculenta β-type PA 4.50, R. catesbeiana β-type parvalbumin (PA 4.78) differed in 15 out of 108 amino acid residues (14% displacement), PA 4. 78 had Cys at residue 64 and was acetylated at the amino terminus, but 25 residues of the carboxyl terminus were completely conserved. Several amino acid displacements were found between residues 51 and 80 (30% displacement), although the functionally important sequence of PA was completely conserved. The amino acids residues of putative calcium-binding sites were Asp-51, Asp-53, Ser-55, Phe-57, Glu-59, Glu-62, Asp-90, Asp-92, Asp-94, Lys-96, and Glu-101, which were conserved in all α and β-types of R. catesbeiana as well as other parvalbumins. In addition, Arg-75 and Glu-81, which are thought to form a salt bridge located in the interior of the molecule [Coffee, C. J. et al. (1976) Biochim. Biophys. Acta 453, 67-80], were also conserved in PA 4.78.
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  • Kuniho Nakata
    2000 Volume 127 Issue 5 Pages 731-737
    Published: 2000
    Released: November 18, 2008
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    Microbacterium sp. M 874 produced a glyceroglycolipid, di-O-12-methyl-tetradecanoyl-3-O-β-D-galactopyranosyl-sn-glycerol, at about the 50 μM level. Though the strain was highly resistant to tertiary-butyl hydroperoxide (tBHP) in a glycolipid-productive medium, the resistance was reduced in a nonproductive medium. Exogenous addition of the glycolipid to the nonproductive culture restored the resistance. This addition also increased the resistance to heat, ethanol, and 4-chloro-l-naphthol, in which oxygen radicals might participate. The parallel relationship found in strain M 874 mutants between glycolipid productivity and resistance to tBHP or heat suggested that the resistance was mainly caused by the glycolipid. On addition of the glycolipid to a glycolipid-nonproductive culture, it was immediately incorporated into the cells and functioned as an antioxygen radical reagent. Thereafter, its intracellular level remained largely unchanged for at least 5 h, even in the presence of tBHP, and its activity was maintained. The glycolipid at 142 μM was sufficient to prevent the cytotoxicity induced by 88.9mM tBHP. The glycolipid production was not induced by pretreatment with a low level of tBHP or a sublethal heat shock. In brief, the glycolipid might play an essential role in the prevention of damage by oxygen radicals in the glycolipid-producing bacterium.
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  • Masahide Uryu, Akiko Nakatomi, Michitoshi Watanabe, Rei Hatsuse, Michi ...
    2000 Volume 127 Issue 5 Pages 739-746
    Published: 2000
    Released: November 18, 2008
    JOURNALS FREE ACCESS
    Complementary DNAs encoding two subunits of scallop (Patinopecten yessoensis) testis calcineurin were cloned, and the nucleotide sequences of their coding regions were determined. The deduced amino acid sequences of the catalytic subunit, calcineurin A (486 amino acid residues, Mr 55, 005.91), and the regulatory subunit, calcineurin B (170 residues, Mr 19, 237.67), showed high similarity to those of mammalian calcineurins, especially to the brain-type ones rather than to the testis-specific isoforms. Northern blot analysis showed that only a single species for each subunit was expressed in testis and the expression of each subunit increased dramatically from January to March during the maturation stages of the one-year cycle. The period when the maximum amount of mRNAs for calcineurin was expressed corresponds to the one immediately after meiosis, that is, the maturation stage in which 20-80% of the average testis is occupied by spermatozoa. The result is consistent with the one as to the expression of the testis-specific isoform of calcineurin A in mouse, which occurs immediately after meiosis. This is the first report on the stage-specific expression of calcineurin in invertebrate testis and its sequence similarity to the mammalian brain-type isoforms may indicate that the mammalian testis-specific isoforms appeared in evolution after the divergence of mammals from the mollusks and then diverged rapidly for specific functions in testis.
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  • Keiichi Homma, Yuzo Yoshida, Akihiko Nakano
    2000 Volume 127 Issue 5 Pages 747-754
    Published: 2000
    Released: November 18, 2008
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    Cytochrome P 450 sterol 14-demethylase (P450-CYP51) is the enzyme that catalyzes 14α demethylation of lanosterol, a step in ergosterol biosynthesis, on the cytoplasmic side of the endoplasmic reticulum (ER) in Saccharomyces cerevisiae. To investigate its localization and the localization mechanism(s), we constructed a chimera by inserting a 30-residue segment, Leu283-Leu312 of P450-CYP51 containing a potential N-glycosylation site in the cytoplasmic region, into the N-terminus of the same protein and tagging the C-terminus with three repeats of a hemagglutinin epitope. This chimera complements gene disruption on a single-copy vector and undergoes N-glycosylation, showing that it functions normally in vivo. Indirect immunofluorescence microscopy revealed that this chimera is localized exclusively to the ER when it is expressed on either a single-copy or multicopy vector. We carried out pulse-chase experiments and found that this chimera, when expressed on a multicopy plasmid, gradually undergoes α1→6 glycosylation, a cis-Golgi-specific modification, but not α1→3 glycosylation, a medial Golgi-specific modification. In contrast, a single-copy expression of this chimera does not lead to the cis-Golgi specific modification. These findings suggest that, when expressed on a multicopy plasmid, a fraction of this chimera is transported from the ER to the cis-Golgi compartment and subsequently recycled to the ER, but when expressed on a single-copy plasmid, no significant transport of this protein from the ER takes place. We thus suggest the possibility that cytochrome P 450 is retained in the ER by a saturable static mechanism.
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  • Hiroo Yonezawa, Tsuyoshi Nonaka, Tetsuya Uchikoba, Shousaku Hattori, M ...
    2000 Volume 127 Issue 5 Pages 755-760
    Published: 2000
    Released: November 18, 2008
    JOURNALS FREE ACCESS
    Pepsinogen was isolated from the gastric mucosa of Trimeresurus flavoviridis (Habu snake) by DEAF-cellulose and DEAF-Sepharose ion-exchange chromatographies, and Sephacryl S-200 gel-chromatography. The yield calculated from the crude extract was 29% with 6.2-fold purification. The purified pepsinogen gave a single band on both native- and SDS-PAGE. As no other active enzyme was detected on the chromatographies, it was concluded that the Habu snake has one major pepsinogen. The molecular mass of the pepsinogen was estimated to be 38 kDa by SDS-PAGE. The sequence of the N-terminal 26 amino acid residues was determined and compared with those of other pepsinogens. The N-terminal structure of Habu snake pepsinogen was more homologous with those of mammalian pepsinogens C than those of mammalian pepsinogens A. The pepsinogen was rapidly converted to pepsin by way of an intermediate form induced by acidification. The optimum pH of Habu snake pepsin for bovine hemoglobin was 1.5-2.0, and it retained full activity at pH 6.2 and 30°C on incubation for 30min. The optimum temperature for the snake pepsin was 50°C and it was stable at 40°C on incubation for 10min. The proteolytic activity of the pepsin toward bovine hemoglobin was about two times higher than that of porcine pepsin A, however, the activity toward oxidized bovine insulin B-chain was lower than that of porcine pepsin A, and it did not hydrolyze oligopeptides. The specificity for oxidized bovine insulin B-chain of the pepsin was different from that of porcine pepsin A. Habu snake pepsin was inhibited by pepstatin A but not by serine, cysteine, or metallo protease inhibitors.
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  • Takashi Kageyama
    2000 Volume 127 Issue 5 Pages 761-770
    Published: 2000
    Released: November 18, 2008
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    Pepsinogens A and C, and prochymosin were purified from four species of adult New World monkeys, namely, common marmoset (Callithrix jacchus), cotton-top tamarin (Saguinus oedipus), squirrel monkey (Saimiri sciureus), and capuchin monkey (Cebus apella). The occurrence of prochymosin was quite unique since this zymogen is known to be neonate-specific and, in primates, it has been thought that the prochymosin gene is not functional. No multiple form has been detected for any type of pepsinogen except that two pepsinogen-A isozymogens were identified in capuchin monkey. Pepsins A and C, and chymosin hydrolyzed hemoglobin optimally at pH 2-2.5 with maximal activities of about 20, 30, and 15 units/mg protein. Pepsins A were inhibited in the presence of an equimolar amount of pepstatin, and chymosins and pepsins C needed 5- and 100-fold molar excesses of pepstatin for complete inhibition, respectively. Hydrolysis of insulin B chain occurred first at the Leu 15-Tyr16 bond in the case of pepsins A and chymosins, and at either the Leu 15-Tyr16 or Tyr16-Leu17 bond in the case of pepsins C. The presence of different types of pepsins might be advantageous to New World monkeys for the efficient digestion of a variety of foods. Molecular cloning of cDNAs for three types of pepsinogens from common marmoset was achieved. A phylogenetic tree of pepsinogens based on the nucleotide sequence showed that common marmoset diverged from the ancestral primate about 40 million years ago.
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  • Toshio Iwasaki, Ken Okamoto, Tomoko Nishino, Junko Mizushima, Hiroyuki ...
    2000 Volume 127 Issue 5 Pages 771-778
    Published: 2000
    Released: November 18, 2008
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    The sequence motif-specific assignment of the two distinct [2Fe-2S] clusters in rat xanthine oxidoreductase (XOR) was unequivocally established by site-directed mutagenesis of recombinant enzymes expressed in a baculovirus-insect cell system and electron paramagnetic resonance (EPR) spectroscopy. The conserved cysteine residues, including Cys-115, in the unusual C-terminal-Cys-Xaa2-Cys-//-Cys-Xaa1-Cys-motif serve as ligands to the Fe/S I center, which is probably located in close proximity to the Mopterin center. Other conserved cysteine residues, including Cys-43 and Cys-51, in the N-terminal plant ferredoxin-like motif serve as ligands to the Fe/S II center, which is distantly located from the Mo-pterin center. The present sequence motif-specific assignment of the Fe/S I and II centers is discussed in the light of the structural features of XOR.
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  • Tomoyasu Nishizawa, Akiko Ueda, Munehiko Asayama, Kiyonaga Fujii, Ken- ...
    2000 Volume 127 Issue 5 Pages 779-789
    Published: 2000
    Released: November 18, 2008
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    The peptide synthetase gene operon, which consists of mcyA, mcyB, and mcyC, for the activation and incorporation of the five amino acid constituents of microcystin has been identified [T. Nishizawa et al. (1999) J. Biochem. 126, 520-529]. By sequencing an additional 34 kb of DNA from microcystin-producing Microcystis aeruginosa K-139, we identified the residual microcystin synthetase gene operon, which consists of mcyD, mcyE, mcyF, and mcyG, in the opposite orientation to the mcyABC operon. McyD consisted of two polyketide synthase modules, and McyE contained a polyketide synthase module at the N-terminus and a peptide synthetase module at the C-terminus. McyF was found to exhibit similarity to amino acid racemase. McyG consisted of a peptide synthetase module at the N-terminus and a polyketide synthase at the C-terminus. The microcystin synthetase gene cluster was conserved in another microcystin-producing strain, Microcystis sp. S-70, which produces Microcystin-LR, -RR, and -YR. Insertional mutagenesis of mcyA, mcyD, or mcyE in Microcystis sp. S-70 abolished microcystin production. In conclusion, the mcyDEFG operon is presumed to be responsible for 3-amino-9-methoxy-2, 6, 8-trimethyl-10-phenyldeca-4, 6-dienoic acid (Adda) biosynthesis, and the incorporation of Adda and glutamic acid into the microcystin molecule.
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  • Junyi Ding, Tomoko Takano, Patrice Hermann, Sanyang Gao, Weihong Han, ...
    2000 Volume 127 Issue 5 Pages 791-796
    Published: 2000
    Released: November 18, 2008
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    Syk plays a crucial role in the transduction of oxidative stress signaling. In this paper, we investigated the roles of Src homology 2 (SH 2) domains of Syk in oxidative stress signaling, using Syk-negative DT 40 cells expressing the N- or C-terminal SH 2 domain mutant [mSH2(N) or mSH2(C)] of Syk. Tyrosine phosphorylation of Syk in cells expressing mSH2(N) Syk after H2O2 treatment was higher than that in cells expressing wildtype Syk or mSH2(C) Syk. The tyrosine phosphorylation of wild-type Syk and mSH2(C) Syk, but not that of mSH2(N), was sensitive to PP 2, a specific inhibitor of Src-family protein-tyrosine kinase. In oxidative stress, the C-terminal SH 2 domain of Syk was demonstrated to be required for induction of tyrosine phosphorylation of cellular proteins, phospholipase C (PLC)-γ2 phosphorylation, inositol 1, 4, 5-triphosphate (IP 3) generation, Ca2+ release from intracellular stores, and c-Jun N-terminal kinase activation. In contrast, in mSH2(N) Syk-expressing cells, tyrosine phosphorylation of intracellular proteins including PLC-γ2 was markedly induced in oxidative stress. The enhanced phosphorylation of mSH 2(N)Syk and PLC-γ2, however, did not link to Ca2+ mobilization from intracellular pools and IP3 generation. Thus, the N- and C-terminal SH 2 domains of Syk possess distinctive functions in oxidative stress signaling.
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  • Masayuki Ishihara, Katsuaki Ono, Keiichi Ishikawa, Hidemi Hattori, Yos ...
    2000 Volume 127 Issue 5 Pages 797-803
    Published: 2000
    Released: November 18, 2008
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    Heparin-carrying polystyrene (HCPS) consists of low-molecular-weight heparin chains enriched in trisulfated disaccharide structures linked to a polystyrene core. In this study, the interactions between HCPSs of various molecular weights and heparin-binding growth factors, VEGF165 FGF-2, and HGF, were compared to the interactions of the same factors with native heparin, periodate-oxidized heparin (IO4-heparin) and periodate-oxidized alkaline-degraded heparin (IO4-LMW-heparin). The binding of each growth factor to heparin-agarose beads (heparin-beads) was more strongly inhibited by HCPSs in a molecular weight-dependent manner than by native heparin or the modified heparins, indicating a stronger interaction between HCPS and these growth factors. HCPSs also inhibit heparin-binding growth factor-induced endothelial cell growth in a molecular weight-dependent manner much more strongly than the native or modified heparin. However, HCPSs did not inhibit the mitogenic activity of VEGF121, which has a non-heparin-binding nature. Thus, HCPSs exhibit enhanced abilities to interact with each of the heparin-binding growth factors studied and to inhibit heparin-binding growth factor-induced endothelial cell proliferation in a molecular weight-dependent manner. These effects might be ascribed to the heparin-clustering effect of HCPSs.
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  • Misaki Kojima, Takeya Morozumi, Akira Onishi, Tadayoshi Mitsuhashi
    2000 Volume 127 Issue 5 Pages 805-811
    Published: 2000
    Released: November 18, 2008
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    A cDNA coding sterol 14α-demethylase (CYP 51), which was isolated from a pig liver cDNA, contained a 1, 512 by open reading frame and a 758 by 3'-untranslated region. The deduced amino acid sequence was 94% identical to those of human and rat CYP51s. The pig CYP 51 gene spanned about 21 kb and was divided into 10 exons. The sites of exonintron junctions were completely identical to those in the human and rat CYP 51 genes. Five GC boxes, but not a TATA box, were found in the 5'-flanking region of the gene, and cyclic AMP and sterol responsive elements were also found in this region. The main transcription start site determined with the 5'-RACE method with poly (A)+ RNA from the liver and testis was located at 143 nucleotides upstream from the initiation codon in both tissues. Northern blot analysis revealed that an approximately 2.4 kb mRNA, which is produced through the use of a polyadenylation signal (AATAAA) located at 740 nucleotides downstream of the stop codon, was expressed in all the tissues examined in pigs: The mRNA levels were much higher in the liver and testis than in the kidney, lung, and epididymis. Furthermore, after the onset of spermatogenesis, a smaller size of mRNA (about 1.8 kb) was found in the testis but not in the epididymis. The 1.8 kb mRNA was produced through the use of an unusual polyadenylation signal (AAGAAA) located at 28 nucleotides downstream of the stop codon.
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  • Kuniho Nakata
    2000 Volume 127 Issue 5 Pages 813-819
    Published: 2000
    Released: November 18, 2008
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    An anti-oxygen radical reagent of a bacterial metabolite, M 874 monogalactoglycerolipid (di-O-12-methyl-tetradecanoyl-3-O-β-D-galactopyranosyl-sn-glycerol), was tested for its ability to protect two organisms against cellular lesions induced by 5-aminolevulinic acid (ALA) and light. In Corynebacterium flavescens ATCC 10340, extracellular uroporphyrin and coproporphyrin were the main porphyrin products. Although less than 2mM ALA increased porphyrin synthesis, ALA levels above 3mM inhibited the synthesis. Depending on the light intensity, the amount of porphyrin decreased and ALA-induced cytotoxicity increased. The lesion was more severe in the case of coproporphyrin than uroporphyrin. The porphyrin lesion produced in low intensity light (300 lx) was considerably reduced by 100 μM M 874 glycolipid, although the reduction in intense light (3, 000 lx) was restricted to a lower level. Similar results were obtained with radish (Raphanus sativus). The ALA concentration that inhibited porphyrin synthesis and stem growth was similar to that seen with C. flavescens. Although the exogenous addition of M 874 glycolipid to the radish did not prevent ALA-induced cellular injury, the co-culture of radish and a glycolipid producing bacterium (Microbacterium sp. M 874) resulted in a significant prevention of cellular injury. This was true only under enforced adhesion conditions through the action of a polysaccharide flocculant H 12. Some species of monogalactoglycerolipids were found in Corynebacterium and radish that showed prominent oxygen radical-protecting activities similar to that of M 874 glycolipid. These monogalactoglycerolipids might function in vivo as agents to prevent ALA-induced cytological lesions, although the concentrations were low in Corynebacterium and radish.
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  • M. Kanagawa, S. Watanabe, S. Kaya, K. Togawa, T. Imagawa, A. Shimada, ...
    2000 Volume 127 Issue 5 Pages 821-828
    Published: 2000
    Released: November 18, 2008
    JOURNALS FREE ACCESS
    H/K-ATPase preparations (the G 1 membrane) from pig stomach contain both kinases and phosphatases and show reversible phosphorylation of Tyr7, Tyr10, and Ser27 residues of the α-chain of H/K-ATPase. The Tyr-kinase is sensitive to genistein and quercetin and recognized by anti-c-Src antibody. The Ser-kinase is dependent on Ca2+ (K0.5=0.9 μM), sensitive to a PKC inhibitor, and recognized by antibodies against PKCα and PKCβII. The addition of 3-[(3-cholamidopropyl) dimethylammonio]-1-propane-sulfonic acid (CHAPS) caused a dramatic increase in the phosphorylation of added synthetic copolymer substrates and permitted the phosphorylation of maltose-binding proteins fused with the N-terminal domain of α-chains. The phosphotyrosine phosphatase was inhibited by vanadate. The phosphoserine phosphatase was inhibited by okadaic acid and by inhibitor-2. The presence of protein phosphatase-1 was immunologically detected. Column chromatographic separation of CHAPS-solubilized G 1 membrane and others indicate the apparent molecular weight of the Src-kinase to be _??_60 kDa, the PKCα and/or PKCβII to be _??_80 kDa, the Tyr-phosphatase to be 200 kDa, and PP-1 to be _??_35 kDa. These data show that these membrane-bound enzyme systems are in sufficiently close proximity to be responsible for reversible phosphorylation of Tyr7, Tyr10, and Ser27 of the catalytic subunit of membrane H/K-ATPase in parietal cells, the physiological role of which is unknown.
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  • Apollina Goel, Guy W. Beresford, David Colcher, Gabriela Pavlinkova, B ...
    2000 Volume 127 Issue 5 Pages 829-836
    Published: 2000
    Released: November 18, 2008
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    Single-chain variable fragments (scFvs) are tumor-recognition units that hold enormous potential in antibody-based therapeutics. Their clinical applications, however, require the large scale production and purification of biologically active recombinant scFvs. In the present study, we engineered and expressed divalent non-covalent [(scFv)2-His6] and covalent [sc (Fv)2-His6] scFvs of a tumor-associated monoclonal antibody (MAb) CC 49 in Pichia pastoris. The purity and immunoreactivity of the scFvs were analyzed by SDSPAGE, HPLC, and competitive ELISA. The binding affinity constant (KA), determined by surface plasmon resonance analysis using BlAcore, was 4.28×107, 2.75×107, and 1.14×108 M-1 for (scFv)2-His6, sc (Fv)2 His6, and CC 49 IgG, respectively. The expression of scFvs in P. pastoris was 30 to 40-fold higher than in Escherichia coli. Biodistribution studies in athymic mice bearing LS-174 T human colon carcinoma xenografts showed equivalent tumor-targeting of CC 49 dimers generated in yeast (scFv)2-His6 and bacteria (scFv)2 with 12.52% injected dose/gram (%ID/g) and 11.42%ID/g, respectively, at 6 h post-injection. Interestingly, the pharmacokinetic pattern of dimeric scFvs in xenografted mice exhibited a slower clearance of His-tagged scFvs from the blood pool than scFvs lacking the His-tag (0.1≥p≥0.05). In conclusion, improved yields of divalent scFvs were achieved using the P. pastoris expression/secretion system. The in vitro and in vivo properties of these scFv s suggest possible therapeutic applications.
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  • Yoshiyuki Ishii, Shuji Sonezaki, Yasushi Iwasaki, Yukako Miyata, Kazum ...
    2000 Volume 127 Issue 5 Pages 837-844
    Published: 2000
    Released: November 18, 2008
    JOURNALS FREE ACCESS
    The SulA protein is a cell division inhibitor in Escherichia coli, and is specifically degraded by Lon protease. To study the recognition site of SulA for Lon, we prepared a mutant SulA protein lacking the C-terminal 8 amino acid residues (SA 8). This deletion protein was accumulated and stabilized more than native SulA in lon+ cells in vivo. Moreover, the deletion SulA fused to maltose binding protein was not degraded by Lon protease, and did not stimulate the ATPase or peptidase activity of Lon in vitro, probably due to the much reduced interaction with Lon. A BIAcore study showed that SA 8 directly interacts with Lon. These results suggest that SA 8 of SulA was recognized by Lon protease. The SA 8 peptide, KIHSNLYH, specifically inhibited the degradation of native SulA by Lon protease in vitro, but not that of casein. A mutant SA 8, KA-HSNLYH, KIA-SNLYH, or KIHSNA-YH, also inhibited the degradation of SulA, while such peptides as KIHSNLYA- did not. These results show that SulA has the specified rows of C-terminal 8 residues recognized by Lon, leading to facilitated binding and subsequent cleavage by Lon protease both in vivo and in vitro.
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  • Nobuyuki Kurosawa, Shou Takashima, Mari Kono, Yuzuru Ikehara, Mio Inou ...
    2000 Volume 127 Issue 5 Pages 845-854
    Published: 2000
    Released: November 18, 2008
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    cDNA clones encoding mouse GalNAc α2, 6-sialyltransferase (ST 6 GalNAc I) were isolated from a mouse submaxillary gland cDNA library. The deduced amino acid sequence of cDNA clones is 526 amino acids in length and has highly conserved motifs among sialyl transferases, sialyl motifs L, S, and VS. The expressed recombinant enzyme exhibited similar substrate specificity to chicken ST 6 GalNAc I. The mouse ST 6 GalNAc I gene was expressed in submaxillary gland, mammary gland, colon, and spleen. The mouse ST 6 GalNAc I gene was also cloned from a mouse genomic library, which was divided into 9 exons spanning over 8 kilobases of genomic DNA. The genomic structure of the mouse ST 6 GalNAc I gene was similar to that of the mouse ST 6 GalNAc II gene. Unlike the ST 6 GalNAc II gene, however, which has a housekeeping gene-like promoter with GC-rich sequences, the ST 6 GalNAc I gene has two promoters and they do not contain GC-rich sequences but contain putative binding sites for tumor-associated transcription factors such as c-Myb, c-Myc/Max, and c-Ets. Analysis of the 5'-RACE PCR products suggested that the mouse ST 6 GalNAc I gene expression is regulated by these two promoters in tissue-specific manners.
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  • Akinori Matsushika, Takeshi Mizuno
    2000 Volume 127 Issue 5 Pages 855-860
    Published: 2000
    Released: November 18, 2008
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    The ArcB sensor plays a crucial role in the histidine to aspartate (His-to-Asp) phosphorelay signal transduction, which is involved in the transcriptional regulatory network that allows Escherichia coli cells to sense various respiratory growth conditions. ArcB is one of the best-studied hybrid His-kinases involved in the multi-step His-to-Asp phosphorelay. However, a major question that remains to be elucidated is: how does ArcB sense an anoxic signal? The N-terminal region of ArcB is considered to be a signal-input domain, which probably plays a role in such signal-perception. In this study, this N-terminal region of ArcB was dissected into three putative sub-domains, a “transmembrane domain, ” a “leucine-zipper-like domain, ” and a “PAS-like domain.” The importance of these structural domains was assessed in vivo and in vitro by systematically analyzing a number of arcB mutants, each of which encodes a mutant ArcB protein having an amino acid substitution or a deletion within one of these sub-domains. The results are discussed with special reference to the nature of the ArcB anaerobic sensor.
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  • Satoru Yamaguchi, Satoru Tuzi, Michikazu Tanio, Akira Naito, Janos K. ...
    2000 Volume 127 Issue 5 Pages 861-869
    Published: 2000
    Released: November 18, 2008
    JOURNALS FREE ACCESS
    We compared 13C NMR spectra of [3-13C] Ala- and [1-13C] Val-labeled bacterio-opsin (bO), produced either by bleaching bR with hydroxylamine or from a retinal-deficient strain, with those of bacteriorhodopsin (bR), in order to gain insight into the conformational changes of the protein backbone that lead to correct folding after retinal is added to bO. The observed 13C NMR spectrum of bO produced by bleaching is not greatly different from that of bR, except for the presence of suppressed or decreased peak-intensities. From careful evaluation of the intensity differences between cross polarization magic angle spinning (CP-MAS) and dipolar decoupled-magic angle spinning (DD-MAS) spectra, it appears that the reduced peak-intensities arise from reduced efficiency of cross polarization or interference of internal motions with proton decoupling frequencies. In particular, the E-F and F-G loops and some transmembrane helices of the bleached bO have acquired internal motions whose frequencies interfere with proton decoupling frequencies. In contrast, the protein backbone of the bO from the retinal-negative cells is incompletely folded. Although it contains mainly α-helices, its very broad 13C NMR signals indicate that its tertiary structure is different from bR. Importantly, this changed structure is identical in form to that of bleached bO from wild-type bR after it was regenerated with retinal in vitro, and bleached with hydroxylamine. We conclude that the binding of retinal is essential for the correct folding of bR after it is inserted in vitro into the lipid bilayer, and the final folded state does not revert to the partially folded form upon removal of the retinal.
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  • Tanweer Ahmed, Sharon M. Kelly, Nicholas C. Price, Anthony J. Lawrence
    2000 Volume 127 Issue 5 Pages 871-875
    Published: 2000
    Released: November 18, 2008
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    The acidic isoform of phospholipase A2 from Naja mossambica mossambica was activated by treatment with a molar equivalent of oleoyl imidazolide. Modification of the protein was accompanied by 50% quenching of tryptophan fluorescence and a significant red shift. The 3H (9, 10) labeled oleoyl residue was co-eluted with the enzyme during gel filtration in the presence of 20% 1-propanol or excess albumin, both of which remove free oleic acid from the enzyme. In contrast, the adduct was labile as to electrophoresis on SDS-PAGE and acid or alkali urea PAGE. The formation of a covalently linked adduct was demonstrated by electrospray mass spectrometry in the presence of 2% formic acid. No such adduct was formed by the phospholipase A3 isoform from Ndja ngja atra, which differs in sequence from the N. mossambica mossambica isoform by seven residues including 2 histidine residues and 1 lysine residue. We conclude that oleoyl imidazolide activates the N. mossambica mossambica enzyme by forming an acyl adduct which is unstable as to protein denaturation. The magnitude of tryptophan fluorescence quenching indicates that the site of acylation lies in the sequence WWHF.
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  • Takahiro Fujii, Yuko Doi, Hiroshi Ueno, Rikimaru Hayashi
    2000 Volume 127 Issue 5 Pages 877-881
    Published: 2000
    Released: November 18, 2008
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    An important role of C-terminal amino acid residues of bovine pancreatic ribonuclease A (RNase A) in the formation of the three-dimensional structure was previously implied. In this study, we replaced the C-terminal amino acid, Val 124, with amino acid residues with different properties by site-directed mutagenesis. The recombinant mutant enzymes were purified and subjected to a refolding study after being converted to a fully reduced and denatured state. There was a significant difference among the mutant enzymes in the rate of recovery of the activity when air oxidation was performed: the rate decreased in the order of V 124 E, V 124 L, V 124 G, V 124 K, V 124 A, and V 124 W. On the other hand, the recovery rates for all the mutant RNase A in the presence of GSH and GSSG were almost the same. The recovered activity of V 124 E after 24 h incubation reached approximately 90% of that of the wild type enzyme, followed by V 124 L 80%, V 124 A and V 124 W 65%, and V 124 K and V 124 G 50%. The duration of the initial lag phase became shorter in the order of V 124 W, V 124 A, V 124 K or V 124 G, V 124 E, or V 124 L. The results imply that the C-terminal amino acid significantly influences the formation of correct disulfide bonds during the refolding process and that the hydrophobic interaction of Val 124 is important for efficient packing of the RNase A molecule.
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  • Yoshiaki Osawa, Misao Hachiya, Shun-ichi Araki, Tomoko Kusama, Kouji M ...
    2000 Volume 127 Issue 5 Pages 883-893
    Published: 2000
    Released: November 18, 2008
    JOURNALS FREE ACCESS
    We tested the effect of IL-1 on the expression of p21WAF1 in human embryonic fibroblasts W138. Exposure to IL-1 caused induction of p21WAF1 protein in high-passage W138 cells but not in early-passage cells. However, IL-1 did not stimulate the transcription of a CAT-reporter gene having two copies of the p53-responsive element on its promoter or the p53-binding capacity of nuclear extracts, although it increased transcriptional rate of p21WAF1 in these high-passage cells. These results suggest that the induction of p21WAF1 by IL-1 occurs at the transcriptional level, but p53 function is not required in these cells. Further studies found that IL-1 did not cause cell-cycle arrest, and the overexpression of p21WAF1 resulted in only a slight delay of cell growth, while the level of p21WAF1 coprecipitated with cyclin-dependent kinase-2 (Cdk2) was increased by IL-1. Moreover, a kinase assay of Cdk2 immunoprecipitates showed that IL-1 did not reduce the kinase activity, and IL-1 did not affect the status of phosphorylation of the retinoblastoma gene product (Rb). These findings imply that despite the induction of p21WAF1, this cannot fully account for the growth arrest in high-passage W138 cells. Thus, IL-1 mediates p21WAF1 induction through a p53-independent pathway (s) in high-passage W138 cells, but the cell cycle is regulated independently of p21WAF1.
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  • Naoto Ohtani, Mitsuru Haruki, Ayumu Muroya, Masaaki Morikawa, Shigenor ...
    2000 Volume 127 Issue 5 Pages 895-899
    Published: 2000
    Released: November 18, 2008
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    Escherichia coli RNase HII is composed of 198 amino acid residues. The enzyme has been overproduced in an insoluble form, purified in a urea-denatured form, and refolded with poor yield [M. Itaya (1990) Proc. Natl. Acad. Sci. USA 87, 8587-8591]. To facilitate the preparation of the enzyme in an amount sufficient for physicochemical studies, we constructed an overproducing strain in which E. coli RNase HII is produced in a soluble form. The enzyme was purified from this strain and its biochemical and physicochemical properties were characterized. The good agreement in the molecular weights estimated from SDS-PAGE (23, 000) and gel filtration (22, 000) suggests that the enzyme acts as a monomer. From the far-UV circular dichroism spectrum, its helical content was calculated to be 23%. The enzyme showed Mn2+-dependent RNase H activity. Its specific activity determined using 41-labeled M13 RNA/DNA hybrid as a substrate was comparable to but slightly higher than that of the refolded enzyme, indicating that the enzyme overproduced and purified in a soluble form is more suitable for structural and functional analyses than the refolded enzyme.
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  • Yuki Nakajima, Shunji Natorri
    2000 Volume 127 Issue 5 Pages 901-908
    Published: 2000
    Released: November 18, 2008
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    We purified a novel protein with a molecular mass of 34 kDa from the Sarcophaga larval fat body. This protein, named AFP (anterior fat body protein), was restricted almost exclusively to the anterior fat body. The AFP content decreased after pupation on disintegration of the fat body tissue. cDNA analysis revealed that this protein consists of 306 amino acid residues and exhibits significant structural similarity with mammalian regucalcin (senescence marker protein-30), a calcium-binding liver protein. However, AFP did not seem to exhibit strong affinity with calcium. These results suggested that a seemingly uniform fat body tissue exhibits a regional difference in its function along the anterior-posterior axis.
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  • Yoshie Miura, Yutaka Yatomi, Ge Rile, Tsukasa Ohmori, Kaneo Satoh, Yuk ...
    2000 Volume 127 Issue 5 Pages 909-914
    Published: 2000
    Released: November 18, 2008
    JOURNALS FREE ACCESS
    Since sphingosine 1-phosphate (Sph-1-P) is stored in abundant amounts in blood platelets and released extracellularly upon stimulation, it is important to clarify the effects of this bioactive lysophospholipid on vascular endothelial cells from the viewpoint of platelet-endothelial cell interactions. In this study, we investigated the effects of Sph-l-P on the cytoskeletal remodeling of human umbilical vein endothelial cells (HUVECs). Of a focal adhesion kinase (FAK) family of non-receptor protein-tyrosine kinases, HUVECs were found to express FAK, but scarcely proline-rich tyrosine kinase 2. Sph-l-P induced FAK tyrosine phosphorylation, myosin light chain phosphorylation, and the formation of stress fibers in HUVECs. The specific Rho inactivator C3 transferase from Clostridium botulinum abolished all of these cytoskeletal responses induced by Sph-1-P, while pertussis toxin only partly inhibited FAK tyrosine phosphorylation, and hardly affected myosin light chain phosphorylation and stress fiber formation. In contrast, Sph-l-P?induced intracellular Ca2+ mobilization was suppressed by pertussis toxin, but not at all by C3 exoenzyme. Our results suggest that Sph-1-P, a bioactive lipid released from activated platelets, induces endothelial cell cytoskeletal reorganization, mainly through Rho-mediated signaling pathways.
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  • Misaki Asano-Miyoshi, Takashi Suda, Akihito Yasuoka, Syun-ichirou Osim ...
    2000 Volume 127 Issue 5 Pages 915-924
    Published: 2000
    Released: November 18, 2008
    JOURNALS FREE ACCESS
    Main olfactory receptor genes were isolated from a seawater fish, Fugu rubripes (pufferfish), and characterized. Two subfamilies of genes encoding seven transmembrane receptors were identified; one consists of five or more members, termed FOR1-1 to 5 of FOR1 subfamily, and the other appears to be a single copy gene, termed the FOR2 subfamily. FOR1 members show extremely high amino acid sequence similarities of about 95% to one another, and are distantly related to catfish-1 with the highest similarity of 37%. FOR2 shows 43% similarity to goldfish-A28. Phylogenically, both FOR members are categorized among pedigrees of the fish main olfactory receptor family outside the mammalian receptor family, although similarities between Fugu receptors and those of fresh-water fishes are lower than those among fresh-water fishes. In situ hybridization shows that both subfamilies of receptor genes are expressed randomly over the olfactory epithelium throughout all developmental stages, and no segregation of the signals was found. On the other hand, when three members of a vomeronasal olfactory receptor gene family, related to the Ca2+-sensing receptor, were used as probes, they were also randomly expressed over the same epithelium as the main olfactory receptors. This is in contrast to the expression profiles observed for zebrafish and goldfish, where the main or vomeronasal olfactory receptors are expressed in segregated patterns. It is thus suggested that the expression pattern of fish olfactory receptors varies depending on the species, although fish olfactory receptors are highly related to one another in their primary structures, and are phylogenically distinct from those of mammals.
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  • Norihiko Nakano, Shigeki Higashiyama, Kenichi Kajihara, Takeshi Endo, ...
    2000 Volume 127 Issue 5 Pages 925-930
    Published: 2000
    Released: November 18, 2008
    JOURNALS FREE ACCESS
    Neural-and thymus-derived activator for ErbB kinases (NTAK) is a recently described member of the neuregulin family that binds directly to ErbB3 and ErbB4 and transactivates ErbB2. Rat NTAK has at least five alternative-spliced isoforms: α1, α2a, α2b, β, and γ. In order to understand their biological properties, this study focused on the NTAKα2a and β isoforms, which have different EGF-like domains. The effect of these isoforms on cell growth and tyrosine phosphorylation in human breast cancer cells, MBA-ME-453 and T47D, was examined using the recombinant proteins. In terms of cell growth, NTAKα2a and NTAKβ preferentially stimulate T47D cells and MDA-MB-453 cells, respectively, in a dose-dependent manner. Although both NTAKs induce the highest level of tyrosine phosphorylation of ErbB2, NTAKo2a and NTAKβ preferentially induce ErbB3 and ErbB4 phosphorylation, respectively. Thus, NTAKα2a and NTAKβ stimulate cell growth in different ways, by means of different combinations of receptors.
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  • Yuko Asahara, Kyoko Atsuta, Ken Motohashi, Hideki Taguchi, Masafumi Yo ...
    2000 Volume 127 Issue 5 Pages 931-937
    Published: 2000
    Released: November 18, 2008
    JOURNALS FREE ACCESS
    FtsH of Escherichia coli is an essential membrane-integrated ATP-dependent protease. We cloned a gene for an FtsH homolog (T. FtsH) from Thermus thermophilus HB8, expressed it in E. coli, and purified the expressed protein. ATPase activity of T. FtsH was activated by proteins with unfolded structure (α-casein and pepsin), and T. FtsH digested these proteins in an ATP-, Zn2+-dependent manner. α-Lactalbumin was digested by T. FtsH when it was largely unfolded, but not in its native form. Analysis of the proteolytic products revealed that, in most cases, T. FtsH cleaved the C-terminal side of hydrophobic residues and produced a characteristic set of small peptides (<30 kDa) without releasing a large intermediate. Thus, T. FtsH recognizes the unfolded structure of the proteins and progressively digests them at the expense of ATP. A soluble domain of T. FtsH, which lacked the N-terminal two transmembrane helices, was also prepared but was found to retain neither ATPase nor protease activities. Thus, the membrane segment appeared to be indispensable for these activities of T. FtsH.
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  • 2000 Volume 127 Issue 5 Pages 939b
    Published: 2000
    Released: November 18, 2008
    JOURNALS FREE ACCESS
    Download PDF (53K)
  • 2000 Volume 127 Issue 5 Pages 939a
    Published: 2000
    Released: November 18, 2008
    JOURNALS FREE ACCESS
    Download PDF (53K)
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