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Volume 136 (2004)
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- Issue 4 Pages 409-
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- Issue 2 Pages 123-
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Volume 136, Issue 2
Displaying 1-17 of 17 articles from this issue
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Hiroshi Nishina, Teiji Wada, Toshiaki Katada2004Volume 136Issue 2 Pages 123-126
Published: August 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSStress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) is activated by many types of cellular stresses and extracellular signals. Recent studies, including the analysis with knockout mice, have led to progress towards understanding the physiological roles of SAPK/JNK activation in embryonic development in addition to immune responses. SAPK/JNK activation plays essential roles in organogenesis during mouse development by regulating cell survival, apoptosis, and proliferation. Two SAPK/JNK activators, SEK1 and MKK7, are required for fetal liver formation and full activation of SAPK/JNK, which responds to various stimuli in an all-or-none manner. This article focuses on physiological roles of SAPK/JNK activation in fetal liver formation and in apoptosis regulation.View full abstractDownload PDF (3250K) -
Yuji Takahashi, Takashi Soyano, Michiko Sasabe, Yasunori Machida2004Volume 136Issue 2 Pages 127-132
Published: August 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSSeveral components of mitogen-activated protein kinase (MAPK) cascades have been identified in higher plants and have been implicated in cellular responses to a wide variety of abiotic and biotic stimuli. Our recent work has demonstrated that a MAP kinase cascade is involved in the regulation of cytokinesis in plant cells. The MAP kinase cascade in tobacco includes NPK1 MAPK kinase kinase, NQK1 MAPK kinase, and NRK1 MAPK, and its activation is triggered by the binding of NACK1/2 kinesinlike protein to the NPK1 MAPK kinase kinase at the late M-phase of the cell cycle. We refer to this cascade as the NACK-PQR pathway. In this review, we introduce a mechanism for the regulation of plant cytokinesis, focusing on the role of the NACK-PQR pathway.View full abstractDownload PDF (3340K) -
Maki Shiota, Masayuki Sano, Makoto Miyagishi, Kazunari Taira2004Volume 136Issue 2 Pages 133-147
Published: August 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSRibozymes are catalytic RNA molecules that cleave RNAs with high specificity. Since the discovery of these non-protein enzymes, the rapidly developing field of ribozymes has been of particular interest because of the potential utility of ribozymes as tools for reversed genetics. However, despite extensive efforts, the activity of ribozymes in vivo has not usually been high enough to achieve the desirable biological effects. Now, by the use of RNA polymerase III (pol III) promoters, the ribozyme activity in cells has been successfully improved by developing efficient transport systems for the transcripts to the cytoplasm. In addition, it is possible to cleave a specific target RNA in cells by using an allosterically controllable ribozyme or an RNA-protein hybrid ribozyme. These ribozymes are potentially applicable to molecular gene therapy and efficient gene discovery systems. Furthermore, the developed pol III expression system is applicable to the expression of small interfering RNAs (siRNAs). The advantage of such ribozymes over siRNAs is the high specificity of the ribozyme that would not cause interferon responses.View full abstractDownload PDF (4635K) -
Takeshi Kinpara, Ryuta Mizuno, Yuji Murakami, Masaaki Kobayashi, Shouh ...2004Volume 136Issue 2 Pages 149-154
Published: August 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe completion of human genome sequencing has shifted the focus of research from genes to proteins. In this regard, a protein library chip has become a useful tool for cell-free protein synthesis. In this study, we attempted to make a highly-integrated protein chip from a DNA library using in vitro protein synthesis on a microchamber array fabricated by using PDMS (polydimethyl siloxane), a hydrophobic surface, and glass, a hydrophilic bottom substrate. These structural properties prevented crosscontamination among the chambers. The minimum volume capacity of the smallest chamber was about 1 pl. The total number of chambers per chip was 10, 000 on one chip (capacity 150 pl) and 250, 000 on two others (1 and 5 pl). Next, we attempted in vitro protein synthesis using this microchamber array. The fluorescence of Green Fluorescent Protein (GFP) expressed on the chamber was rapidly detected (within just 1 h). GFP expression was also successful using immobilized DNA molecules on polymer beads. DNA immobilized beads were added as the source to each microchamber. Protein was successfully synthesized from DNA immobilized beads, which allowed easy handling of the DNA molecules.View full abstractDownload PDF (4488K) -
Ichiaki Ito, Nobuyoshi Mitsuoka, Junko Sobajima, Hiroko Uesugi, Shoich ...2004Volume 136Issue 2 Pages 155-162
Published: August 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSHMGB 1 and HMGB 2 are abundant nonhistone chromosomal proteins in eukaryotic organisms. Their respective primary sequences are highly conserved. Our previous studies showed that these proteins are novel autoantigens of anti-neutrophil cytoplasmic antibodies in sera from patients with ulcerative colitis (UC), rheumatic disease and autoimmune hepatitis (AIH). In the present paper, we showed that antiHMGB 1 and HMGB 2 antibodies in sera of patients with UC do not recognize HMGB 1 in neutrophils while they recognize the protein in lymphocytes. Anti-HMGB 2 mono-clonal antibody FBH 7, recognizing HMGB 1 in lymphocytes, showed a similar profile to the antibodies in the patients' sera. In order to elucidate the difference in immuno-reactivity to HMGB 1 between neutrophils and lymphocytes, we mapped the epitope for FBH 7 by means of several methods. The results showed that FBH 7 recognizes the intact conformation composed of 52-56 residues of HMGB 1 in lymphocytes. This suggested that HMGB 1 in neutrophils is conformationally changed in the epitope or the peripheral structure of the epitope from the protein in lymphocytes. The apparent conformational change of HMGB 1 between neutrophils and lymphocytes will be important for understanding the functional difference of HMGBl in these cells.View full abstractDownload PDF (4971K) -
Fuminori Katouno, Masashi Taguchi, Kengo Sakurai, Taku Uchiyama, Naoki ...2004Volume 136Issue 2 Pages 163-168
Published: August 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSChitinase B (ChiB) of S. marcescens has five exposed aromatic residues linearly aligned toward the catalytic cleft, Tyr481 and Trp479 in the C-terminal domain, and Trp252, Tyr240 and Phe190 in the catalytic domain. To determine the contribution of these residues to the hydrolysis of crystalline β-chitin, site-directed mutagenesis, to replace them by alanine, was carried out. The Y481A, W479A, W252A, and Y240A mutations all decreased the binding activity and hydrolyzing activity toward β-chitin microfibrils. Substitution of Trp residues affected the binding activity more severely than that of Tyr residues. The F190A mutation decreased neither the binding activity nor the hydrolyzing activity. None of the mutations decreased the hydrolyzing activity toward soluble substrates. These results suggest that ChiB hydrolyzes crystalline β-chitin via a mechanism in which four exposed aromatic residues play important roles, similar to the mechanism of hydrolysis by ChiA of this bacterium, although the directions of hydrolysis of the two chitinases are opposite.View full abstractDownload PDF (1828K) -
Jian-Feng Li, Long-Ding Liu, Shao-Hui Ma, Yan-Chun Che, Li-Chun Wang, ...2004Volume 136Issue 2 Pages 169-176
Published: August 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe interaction between virus and receptor is a process that mimics physiological ligand binding receptors and induces signal transduction. In the investigation of the interaction between HSV1 (Herpes Simplex virus 1) and human fibroblasts via virus binding to its receptor complex on cellular membranes, the HTRP (human transcription regulator protein), a protein encoded by an immediate-early gene of cellular response against the specific stimulation of HSV 1 binding, was cloned from a cDNA library established from early gene response mRNA. The localization of HTRP expressed as a fusion polypeptide with a fluorescent protein in HeLa cells was con-firmed to be the nucleus. The results of a yeast two-hybrid experiment indicated that HTRP is indeed involved in the interaction with the SAP (mSin 3-associate polypeptide) complex via SAP 30. A pull-down test and Western blotting in vitro, and immunoprecipitation in vivo also provided evidence in support of this result. The interaction of HTRP with SAP 30 in its conserved domain implies that this protein family, as the products of immediate-early genes, comprise functional molecules involved in the transcriptional regulation of cells, which might be related to the inhibition of some cell survival genes.View full abstractDownload PDF (3695K) -
Ilda Degirmentas, Nuran Deveci2004Volume 136Issue 2 Pages 177-182
Published: August 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSIn this study, the anaerobic treatment of high-strength antibiotic production wastewater and the development of a mathematical model for the treatment were attempted. Anaerobic treatability was investigated using synthetic solutions and original wastewater of which the initial chemical oxygen demand (COD) was deter-mined. Initial COD of solutions was increased from 3, 000 to 43, 000 mg O2/liter in an anaerobic bioreactor. The bioreactor pH was maintained at 6.5-7.5. The temperature was kept constant at 37±1°C. Raw materials and original wastewater containing penicillin antibiotics were obtained from Fako Pharmaceutical Factory (Fako) in Istanbul, Turkey. Anaerobic sludge used for treatment was obtained from Pakmaya Baker's Yeast Producing Factory (Pakmaya) in Izmit, Turkey and the Fako. A mathematical model based on substrate (total COD) concentration was developed assuming that only three consecutive reactions, namely, hydrolysis, acidogenesis and methanogenesis, are significant. From the experimental data, a model that can be used for COD calculation as a function of time was developed using the first- and the secondorder kinetic approaches. Making use of the developed model equation, it was proved that the anaerobic treatment of high strength (COD>25, 000mg O2/liter) antibiotic production wastewater fits the second-order kinetics.View full abstractDownload PDF (566K) -
Masayuki Takeuchi, Takanobu Taniguchi, Hitoshi Fujisawa2004Volume 136Issue 2 Pages 183-188
Published: August 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSCalmodulin-dependent protein kinase phosphatase (CaMKP) and CaMKP-N dephosphorylate and regulate multifunctional Ca2+/calmodulin-dependent protein kinases. The enzymatic properties of CaMKP-N and CaMKP resemble each other, whereas their localizations are different. CaMKP-N is localized in the nucleus, whereas CaMKP is localized in the cytosol. In the present study, the nuclear localization sig-nals (NLSs) of CaMKP-N were identified and characterized. CaMKP-N contains two NLSs, NLS 1 and NLS 2, at the C-terminus. A cluster of basic residues in the NLSs is important for their function. NLS1 and NLS 2 function independently, but mutagenesis analysis suggests that these NLSs interact with each other.View full abstractDownload PDF (4765K) -
Shinji Asahina, Chihiro Sato, Ken Kitajima2004Volume 136Issue 2 Pages 189-198
Published: August 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSPolysialoglycoprotein (PSGP) is a major cortical alveolus glycoprotein of rainbow trout eggs that is characterized by the attachment of a polysialic acid structure to its O-glycan chains. It has been demonstrated that the polysialic acid structure is synthesized by at least three sialyltransferases mostly localized in cortical alveoli. Here we have cloned a cDNA encoding the sialyltransferase, designated rtST6GaINAc, responsible for the transfer of the first sialic acid residue onto the O-glycan chain of PSGP. This enzyme belongs to the vertebrate ST6GaINAc II family, and is strongly expressed in ovaries. Of those O-glycoproteins tested as substrates, asialo-PSGP is the best substrate. These results indicate that rtST6GalNAc is the enzyme responsible for the sialylation of PSGP during oogenesis. Furthermore, the rtST6GalNAc mRNA is expressed throughout oogenesis, is down-regulated at the late yolk vesicle stage (May), and then up-regulated during vitellogenesis (until August). This developmental profile is highly similar to that of STL2, a cortical alveolus lectin, while it is quite different from that of PSGP, which is extensively expressed at the yolk vesicle stage and down-regulated at later stages. Thus, not all cortical alveolus components are transcribed concomitantly. This is the first description of a developmental change in the transcription of a glycosyltransferase during oogenesis.View full abstractDownload PDF (3588K) -
Umechiyo Tokumoto, Seiichi Kitamura, Keiichi Fukuyama, Yasuhiro Takaha ...2004Volume 136Issue 2 Pages 199-209
Published: August 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe assembly of iron-sulfur (Fe-S) clusters, a key step in the post-translational matu-ration of Fe-S proteins, is mediated by a complex apparatus. In E. coli, this process involves two independent systems called ISC and SUP encoded by the iscSUA-hscBA-fdx gene cluster and sufABCDSE operon, respectively. Another system, termed NIF (nitSU), is required for the maturation of nitrogenase in nitrogen-fixing bacteria. We have developed a novel genetic system to gain further insight into these multi-compo-nent systems, and to determine how ISC, SUP and NIF might differ in their roles in Fe-S assembly. We have constructed an E. coli mutant lacking both the isc and suf operons, and this strain can only survive in the presence of a complementing plasmid. Using the plasmid replacement technique, we examined the isc and suf operons, and identified the genes essential for the function. Additionally, we have found that nifSU-like genes cloned from Helicobacter pylori are functionally exchangeable with the isc and suf operons. Thus, the NIF-like system participates in the maturation of a wide variety of Fe-S proteins. An increased ability of NIF to complement isc and suf loss was seen under anaerobic conditions. This may explain why the NIF system is only found in a limited number of bacterial species, and most other organisms prefer the ISC and/or SUP systems. While the differences between ISC and SUF were small with respect to the complementing activity, the SUF system appears to be more advantageous for bacterial growth in the presence of hydrogen peroxide.View full abstractDownload PDF (5135K) -
Yoshiaki Omata, Hiroshi Sakamoto, Yuichiro Higashimoto, Shunsuke Hayas ...2004Volume 136Issue 2 Pages 211-220
Published: August 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe side-chain asymmetry of physiological porphyrins is produced by the coopera-tive action of hydroxymethylbilane synthase and uroporphyrinogen (uro'gen) III syn-thase. Although the role of uro'gen III synthase is essential for the chemistry of por-phyrin biosynthesis, many aspects, structural as well as mechanical, of uro'gen III synthase have yet to be studied. We report here an expression system in Escherichia coli and a purification procedure for human uro'gen III synthase. The enzyme in the lysate was unstable, but we found that glycerol prevents the activity loss in the lysate. The purified enzyme showed remarkable thermostability, particularly when kept in phosphate buffer containing DTT or EDTA, indicating that the enzyme activity may depend on its oxidation state. Examination of the relationship between the number of Cys residues that are accessible to 5, 5'-dithiobis(2-nitrobenzoic acid) and the remain-ing activity during heat inactivation showed that a particular Cys residue is involved in activity loss. From the crystal structure of human uro'gen III synthase [Mathews et al. (2001) EMBO J. 20, 5832-5839], this Cys residue was considered to be Cys73, which is buried deep inside the enzyme, suggesting that Cys73 of human uro'gen III synthase plays an important role in enzyme activity.View full abstractDownload PDF (2352K) -
Naoyuki Kondo, Seiki Kuramitsu, Ryoji Masui2004Volume 136Issue 2 Pages 221-231
Published: August 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe HD domain motif is found in a superfamily of proteins in bacteria, archaea and eukaryotes. A few of these proteins are known to have metal-dependant phosphohy-drolase activity, but the others are functionally unknown. Here we have character-ized an HD domain-containing protein, TT1383, from Thermus thermophilus HB 8. This protein has sequence similarity to Escherichia coli dGTP triphosphohydrolase, however, no dGTP hydrolytic activity was detected. The hydrolytic activity of the protein was determined in the presence of more than two kinds of deoxyribonucleo-side triphosphates (dNTPs), which were hydrolyzed to their respective deoxyribonu-cleosides and triphosphates, and was found to be strictly specific for dNTPs in the fol-lowing order of relative activity: dCTP>dGTP>dTTP>dATP. Interestingly, this dNTP triphosphohydrolase (dNTPase) activity requires the presence of dATP or dTTP in the dNTP mixture. dADP, dTDP, dAMP, and dTMP, which themselves were not hydrolyzed, were nonetheless able to stimulate the hydrolysis of dCTP. These results suggest the existence of binding sites specific for dATP and dTTP as positive modulators, distinct from the dNTPase catalytic site. This is, to our knowledge, the first report of a non-specific dNTPase that is activated by dNTP itself.View full abstractDownload PDF (2436K) -
Hiroshi Munakata, Ji-Ying Sun, Koji Yoshida, Tatsuya Nakatani, Eiko Ho ...2004Volume 136Issue 2 Pages 233-238
Published: August 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESS5-Aminolevulinate synthase (ALAS) is a mitochondrial enzyme that catalyzes the first step of the heme biosynthetic pathway. The mitochondrial import, as well as the synthesis, of the nonspecific isoform of ALAS (ALAS 1) is regulated by heme through a feedback mechanism. A short amino acid sequence, the heme regulatory motif (HRM), is known to be involved in the regulatory function of heme. To determine the role of the HRM in the heme-regulated transport of the nonspecific and erythroid forms of ALAS in vivo, we constructed a series of mutants of rat ALAS 1, in which the cysteine residues in the three putative HRMs in the N-terminal region of the enzyme were con-verted to serine ones by site-directed mutagenesis. The wild-type and mutant enzymes were expressed in quail QT6 fibroblasts through transient transfection, and the mitochondrial import of these enzymes was examined in the presence of hemin. Hemin inhibited the mitochondrial import of wild-type ALAS 1, but this inhibition was reversed on the mutation of all three HRMs in the enzyme, indicating that the HRMs are essential for the heme-mediated inhibition of ALAS 1 transport in the cell. By contrast, exogenous hemin did not affect the mitochondrial import of the erythroid-specific ALAS isoform (ALAS 2) under the same experimental conditions. These results may reflect the difference in the physiological functions of the two ALAS isoforms.View full abstractDownload PDF (3129K) -
Toshihiro Tsudzuki, Maki Tsujita2004Volume 136Issue 2 Pages 239-243
Published: August 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSRat brain synaptic vesicles were isoosmotically isolated and examined for Mg2+-ATPase [EC 3. 6. 1. 3.] and tyrosine hydroxylase [EC 1. 14. 16. 2.] associated with the syn-aptic vesicles. Synaptosomes in 0.32M sucrose were disrupted by freezing and thawing treatment, and the cytosol fraction was fractionated on a Sephacryl S-500 column with a mean exclusion size of 200 nm. Peak I at the void volume was a mixture of large vesicular membranes, small amounts of synaptic vesicles and coated vesicles, etc. Peak II consisted of non- and granulated synaptic vesicles of 35-40 nm diameter, and peak III of soluble proteins. The synaptic vesicles in peak II reacted with antibodies against the H+-ATPase A-subunit, vesicular acetylcholine transporter, and vesicular monoamine transporter. However, they showed little Mg2+-ATPase activity. Tyrosine hydroxylase was observed in either peak II or III on blotting with an anti-tyrosine hydroxylase antibody. These results imply that tyrosine hydroxylase exists in soluble and bound forms to synaptic vesicles in nerve terminals.View full abstractDownload PDF (3531K) -
Mitsunori Fukuda, Akitsugu Yamamoto2004Volume 136Issue 2 Pages 245-253
Published: August 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSSynaptotagmin IV (Syt IV) was originally described as an immediate early gene product induced by forskolin or membrane depolarization in PC 12 cells; however, nothing is known about the subeellular localization and transport of the newly translated Syt IV protein in PC 12 cells. In this study, we investigated the transport mechanism of Syt IV protein induced by forskolin and found that forskolin treatment dramatically increases the Syt IV protein level (approximately 10-fold, to a level comparable to that of Syt IX) and promotes the transport of Syt IV protein from the Golgi to the cell periphery by a microtubule-dependent motor (s). The expression levels and subcellular localizations of two major Syt isoforms (I and IX) in PC12 cells, on the other hand, were unaffected by such treatment. Immunoelectron microscopic analysis showed that some Syt IV signals are clearly associated with dense-core vesicles in forskolintreated PC12 cells, although the majority of the Syt IV molecules at the cell periphery were present on clear vesicular structures other than dense-core vesicles. An N-ter-minal antibody-uptake experiment indicated that Syt 1V-containing vesicles in for-skolin-treated PC12 cells undergo Ca2+-dependent exocytosis, because uptake of the anti-Syt IV -N antibody from the culture medium was slightly, but significantly, increased after forskolin treatment. Our results indicate that forskolin (or the increased cAMP level) is important for the transport of the Syt IV protein from the Golgi to the cell periphery, but not sufficient for the sorting of all Syt IV molecules to mature dense-core vesicles.View full abstractDownload PDF (7336K) -
Jian-Shan Ye, Xiao-Jing Zheng, King Wong Leung, Hueih Min Chen, Fwu-Sh ...2004Volume 136Issue 2 Pages 255-259
Published: August 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe anticancer activity of anti-bacterial cecropins makes them potentially useful as peptide anti-cancer drugs. We used the cell-attached patch to study the effect of cecropin B (CB; having one hydrophobic and one amphipathic α-helix) and its derivative, cecropin B 3 (CB 3; having two hydrophobic a-helices) on the membrane of Ags cancer cells. Application of 10-60 μM CB onto the membrane of the cancer cell produces short outward currents. Comparative study with CB 3, which induces no outward currents, shows that the amphipathic group of CB is necessary for the pore formation. The results provide a rationale to study the cell-killing activity of antimi-crobial peptides at the single cancer cell level.View full abstractDownload PDF (625K)
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