The Journal of Biochemistry
Online ISSN : 1756-2651
Print ISSN : 0021-924X
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Showing 1-14 articles out of 14 articles from the selected issue
  • Akihiro Wada, Eiki Yamasaki, Toshiya Hirayama
    2004 Volume 136 Issue 6 Pages 741-746
    Published: December 01, 2004
    Released: June 30, 2008
    JOURNALS FREE ACCESS
    Pathogenic strains of Helicobacter pylori produce a potent exotoxin, VacA, which causes progressive vacuolation as well as gastric injury. Most H. pylori strains secrete VacA into the extracellular space. After exposure of VacA to acidic or basic pH, re-oligomerized VacA (mainly 6 monomeric units) at neutral pH is more toxic. Although themechanisms have not been defined, VacA induces multiple effects on epithelial and lymphatic cells, i.e., vacuolation with alterations of endo-lysosomal function, anionselective channel formation, mitochondrial damage, and the inhibition of primary human CD4+ cell proliferation. VacA binds to two types of receptor-like protein tyrosine phosphatases (RPTP), RPTPα and RPTPβ, on the surface of target cells. Oral administration of VacA to wild-type mice, but not to RPTPβ KO mice, results in gastric ulcers, suggesting that RPTPβ is essential for intoxication of gastric tissue by VacA. As the potential roles of VacA as a ligand for RPTPα and RPTPβ are only poor understood, further studies are needed to determine the importance of VacA in the pathogenisis of disease due to H. pylori infection.
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  • Yasushi Hanakawa, John R. Stanley
    2004 Volume 136 Issue 6 Pages 747-750
    Published: December 01, 2004
    Released: June 30, 2008
    JOURNALS FREE ACCESS
    Many children suffer from the bacterial skin diseases bullous impetigo and staphylococcal scalded skin syndrome (SSSS). Staphylococcus aureus, which produces exfoliative toxins (ETs), causes these diseases. Recently, it was proven that ETs cleave the cell adhesion molecule desmoglein (Dsg) 1, which plays an important role in maintaining the proper structure and barrier function of the epidermis. Surprisingly, Dsg1 is also the antibody target in the autoimmune disease pemphigus foliaceus. Skin biopsies from pemphigus foliaceus patients show the same pathology as those from bulbous impetigo and SSSS patients. The crystal structure of ET suggests that it is a serine protease with an inactive catalytic site, which may become activated when ET binds a specific receptor. This receptor binding is thought to cause a change in conformation that exposes the catalytic site. It has recently been shown that Dsg1 specifically binds and activates ET, which in turn cleaves the bound Dsg1 at only one peptide bond. This process is absolutely dependent on the calcium-dependent conformation of Dsg1. These data suggest that ETs have a very high specificity for human Dsg1, and that S. aureus uses ETs to disrupt the barrier of the human epidermis in order to survive and proliferate on the human body.
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  • Kazuhisa Nakayama
    2004 Volume 136 Issue 6 Pages 751-753
    Published: December 01, 2004
    Released: June 30, 2008
    JOURNALS FREE ACCESS
    The most outstanding feature of eukaryotic cells is compartmentalization by membranes, which enables them to achieve a broad spectrum of functions; some of them are common to all cell-types and others are specific to certain cell-types. Individual compartments, namely organelles, have unique sets of proteins that are specifically delivered from one compartment to another by membrane traffic. During the past several years, combinations of genomic, proteomic, structural and real-time imaging analyses with conventional genetical, biochemical and cell biological approaches have provided us with much new information not only about the intricate pathways and sophisticated regulatory mechanisms of membrane traffic but also about integration of membrane traffic with other cellular functions such as signaling and morphogenesis. This Minireview series composed of eight articles highlights the recent progress in this rapidly expanding research field.
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  • Ken Sato
    2004 Volume 136 Issue 6 Pages 755-760
    Published: December 01, 2004
    Released: June 30, 2008
    JOURNALS FREE ACCESS
    The coat protein complex II (COPII) generates transport vesicles that mediate protein transport from the endoplasmic reticulum (ER). Recent structural and biochemical studies have suggested that the COPII coat is responsible for direct capture of membrane cargo proteins and for the physical deformation of the ER membrane that drives the transport vesicle formation. The COPII-coated vesicle formation at the ER membrane is triggered by the activation of the Ras-like small GTPase Sar1 by GDP/GTP exchange, and activated Sar1 in turn promotes COPII coat assembly. Subsequent GTP hydrolysis by Sar1 leads to disassembly of the coat proteins, which are then recycled for additional rounds of vesicle formation. Thus, the Sar1 GTPase cycle is thought to regulate COPII coat assembly and disassembly. Emerging evidence suggests that the cargo proteins modulate the Sar1 GTP hydrolysis to coordinate coat assembly with cargo selection. Here, I discuss the possible roles of the GTP hydrolysis by Sar1 in COPII coat assembly and selective uptake of cargo proteins into transport vesicles.
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  • Hye-Won Shin, Kazuhisa Nakayama
    2004 Volume 136 Issue 6 Pages 761-767
    Published: December 01, 2004
    Released: June 30, 2008
    JOURNALS FREE ACCESS
    Small GTPases of the Arf family, by cycling between GDP-bound inactive and GTP-bound active states, play a crucial role not only in the regulation of membrane traffic and dynamics but also in rearrangement of actin cytoskeleton. The exchange of GDP for GTP on Arf is catalyzed by a family of guanine nucleotide-exchange factors (GEFs) containing a Sec7 domain. The Sec7 domain is a target of brefeldin A, which inhibits various trafficking processes and induces organelle disintegration. During the past few years, significant progress has been made in elucidating the structure and catalytic mechanism of the Sec7 domains and physiological functions of the Sec7 domain-containing Arf-GEFs. Here we review the structures and functions of Arf-GEFs by focusing on the regulation of membrane traffic.
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  • Lei Chun, Chang-Cheng Yin, Jing-Zhen Song, Ming-Xue Liu, Jin-Hua Piao, ...
    2004 Volume 136 Issue 6 Pages 769-776
    Published: December 01, 2004
    Released: June 30, 2008
    JOURNALS FREE ACCESS
    Secondary lymphoid tissue chemokine (SLC) is a CC chemokine that plays an important role in leukocytes homing to lymphoid tissues. The ability of SLC to co-localize both T cells and dendritic cells formed the rationale to evaluate its utility in cancer immunotherapy. The in vivo antitumor effect of murine SLC (mSLC) has been well documented, but little is known about that of human SLC (hSLC). To investigate the antitumor efficiency in vivo of hSLC, the hSLC gene was artificially synthesized and induced to express as a soluble form in Escherichia coli. After purification, the purity of the recombinant human SLC (rhSLC) protein was above 95% by SDS-PAGE analysis. The Kd of rhSLC binding to peripheral blood lymphocytes (PBLs) was 0.2186±0.02675 μM as assessed by FACS, and the maximal chemotactic index of rhSLC was 9.49 at 100 nM as assessed by in vitro chemotaxis assay. Then genomic sequences of hSLC and mSLC, and of human CCR7 (hCCR7) and murine CCR7 (mCCR7), the receptor for SLC, were aligned. It was found that hSLC and mSLC share 70.72% identity and hCCR7 and mCCR7share 86.77% identity. Furthermore, we found that rhSLC could chemoattract murine peripheral blood mononuclear cells (PBMCs) in vitro. On the basis of these facts, immune competent mice inoculated with S 180 sarcoma cells were chosen as an in vivo model. Intratumoral injections of rhSLC inhibited tumor growth and increased survival. These findings suggest that, despite its incapability to bind to either human or murine CXCR 3, which is related to angiostasis, rhSLC can induce an antitumor response in vivo by another route. This report proves that rhSLC has a potent tumor-inhibition ability that makes it a promising candidate agent in cancer immunotherapy.
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  • Kaori Sato, Shunji Hattori, Shinkichi Irie, Hiroyuki Sorimachi, Mitsus ...
    2004 Volume 136 Issue 6 Pages 777-785
    Published: December 01, 2004
    Released: June 30, 2008
    JOURNALS FREE ACCESS
    When skin fibroblasts were cultured on fibrillar collagen I gel, we observed rapid degradation of talin, fodrin and ezrin, which are well-known calpain substrates. The protease m-calpain was activated only in cells adhering to fibrillar collagen, whereas tscalpain was activated in cells adhering to monomeric or fibrillar collagen at the same level. The calpain inhibitor Z-Leu-Leu-aldehyde inhibited degradation of fodrin, but not talin. Degradation of fodrin, a-actinin and ezrin was prevented by over-expression of dominant negative m-calpain. However, over-expression of calpastatin, an endogenous calpain inhibitor, had no effect the degradation of these three proteins. These results suggest that m-calpain is responsible for degradation of their membrane proteins via adhesion to fibrillar collagen I gel.
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  • Duohong Sheng, Zhiguo Zheng, Bing Tian, Binghui Shen, Yuejin Hua
    2004 Volume 136 Issue 6 Pages 787-793
    Published: December 01, 2004
    Released: June 30, 2008
    JOURNALS FREE ACCESS
    The protein DRA0074 is suggested to be another LexA in Deinococcus radiodurans, having similar motifs and RecA-mediated cleavage activity to D. radiodurans LexA (dra0344). However, its function has not been studied. We disrupted the gene dra0074 and measured its effect on RecA induction using fusion translation, immunoblot, and proteomic analysis. Results showed that the product of gene dra0074 is not involved in RecA induction, but is a regulator of other metabolisms in D. radiodurans.
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  • Min Lin, Erin Trottier, John Pasick, Marta Sabara
    2004 Volume 136 Issue 6 Pages 795-804
    Published: December 01, 2004
    Released: June 30, 2008
    JOURNALS FREE ACCESS
    The structural glycoprotein Erns of classical swine fever virus (CSFV) is one of the major antibody targets upon infection of pigs with the virus. Molecular dissection of the structure of Erns would define the minimal immunodominant regions that induce antibody responses after infection and may thus help design an effective diagnostic reagent or vaccine. In this study, deletion analysis was made within amino acids (aa) 297 to 776 of the CSFV Alfort/187 polyprotein containing the large C-terminal portion of the Erns protein (aa 27 to 227), the entire E1 protein (aa 1 to 195), and the N-terminal portion of the E2 protein (aa 1 to 87). Various protein fragments with target deletions from N- or/and C-terminal ends were constructed with pET 30, expressed in Escherichia coli and probed on Western blots with antisera from pigs infected with CSFV. This has resulted in the identification within Erns of three overlapping antigenic regions: AR1(Ernsaa 65-145), AR2(Ernsaa 84-160) and AR3(Ernsaa109-220). N- or C-terminal deletions as small as 3 residues introduced into these regions disrupt their reactivity with antibodies, indicating that they are the minimum requirements for recognition by pig antibodies. The three minimal antigenic regions correlated well with the hydropathy profiles and the 3D structural model of Erns. Each individual region and a protein fragment containing AR1, AR2 and AR3 reacted equally well with pig anti-CSFV sera. Since variable and conserved sequences are present within the three overlapping antigenic regions of Erns of different pestiviruses, specific serological detection of CSFV infection or broad detection of pestivirus infections may be achieved with the use of a single Erns region or a combination of two or three Erns regions.
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  • Takeshi Sakuno, Yasuhiro Araki, Yuriko Ohya, Satoshi Kofuji, Shinya Ta ...
    2004 Volume 136 Issue 6 Pages 805-812
    Published: December 01, 2004
    Released: June 30, 2008
    JOURNALS FREE ACCESS
    Cleavage of the 5'-cap structure is involved in the major 5'-to-3' and nonsense-mediated mRNA decay pathways, and the protein complex consisting of Dcp1 and Dcp2 has been identified as the species responsible for the decapping reaction in Saccharomyces cerevisiae and human. Although in vitro studies indicate that Dcp2 is catalytically an active component, the role of Dcp1 in the decapping reaction remains to be explored in organisms other than budding yeast. To elucidate the Dcp1-dependent decapping mechanisms, we identified the homologues of S. cerevisiae Dcp1 (ScDcp1) in higher eukaryotes and analyzed their functions in the different species. The phenotypes of slow growth and mRNA stabilization induced by Scdcp1-gene disruption in budding yeast could be suppressed by the Shizosaccharomyces pombe SpDcp1 but not by the human homologue hDcp1. In contrast, the same phenotypes caused by Spdcp1-gene disruption in fission yeast were effectively complemented by hDcp1 and its partial sequence comparable to SpDcp1. These results indicate that not only Dcp2 but also Dcp1 plays an indispensable role in mRNA-decay pathway and that the characteristics of Dcp1-dependent decapping reaction in fission yeast hold an intermediate position in the evolution of mRNA-decay machinery from budding yeast to mammals.
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  • Masato Saito, Masaaki Kobayashi, Shin-ichiro Iwabuchi, Yasutaka Morita ...
    2004 Volume 136 Issue 6 Pages 813-823
    Published: December 01, 2004
    Released: June 30, 2008
    JOURNALS FREE ACCESS
    DNA condensation was only observed after the addition of Hoechst 33258 (H 33258) among various types of DNA binding molecules. The morphological structural change of DNA was found to depend on the H 33258 concentration. On comparison of fluorescence spectrum measurements with AFM observation, it was found that fluorescence quenching of DNA-H 33258 complexes occurred after DNA condensation. Additionally, we showed that DNA condensation by H 33258 was independent of sequence selectivity or binding style using two types of polynucleotides, i.e. poly (dA-dT)•poly (dA-dT) and poly (dG-dC)•poly (dG-dC). Moreover, it was concluded that the condensation was caused by a strong hydrophobic interaction, because the dissolution of condensed DNA into its native form on dimethyl sulfoxide (DMSO) treatment was observed. This study is the first report, which defines the DNA condensation mechanism of H 33258, showing the correlation between the single molecule scale morphology seen on AFM observation and the bulky scale morphology observed on fluorescence spectroscopy.
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  • Ascensión Prieto-Martín, Julio Montoya, Francisco Mart&i ...
    2004 Volume 136 Issue 6 Pages 825-830
    Published: December 01, 2004
    Released: June 30, 2008
    JOURNALS FREE ACCESS
    The molecular mechanisms involved in the regulation of the balance between rRNA and mRNA in mitochondria are poorly understood. The mitochondrial transcription termination factor (mTERF) was highlighted as a potential transcription-control point. In this study, rat mTERF has been expressed in vitro and in Escherichia coli. The mature protein, in addition to the expected specific DNA-binding capacity for the sequence required for termination, has a new DNA-binding activity, and is able to bind to rat mitochondrial promoter region. This finding suggests communication between transcription initiation and termination regions. However, the results of a competition experiment argue against the formation of a complex between rat mTERF and the termination probe and promoter probe simultaneously, although it remains to be investigated whether another factor (s) might be involved in this interaction. In addition, recombinant human mTERF is also able to bind to human mitochondrial promoter region.
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  • Wakako Koito, Tomohiro Araki, Seikoh Horiuchi, Ryoji Nagai
    2004 Volume 136 Issue 6 Pages 831-837
    Published: December 01, 2004
    Released: June 30, 2008
    JOURNALS FREE ACCESS
    Immunological strategies for the detection of Nε-(carboxymethyl)lysine (CML), one of the major antigenic structures of advanced glycation end products (AGE), are widely applied to demonstrate the contribution of CML to the pathogeneses of diabetic complications and atherosclerosis. Recent studies have indicated that methylglyoxal (MG), which is generated intracellularly through the Embden-Meyerhof and polyol pathways, reacts with proteins to form MG-derived AGE structures such as Nε-(carboxyethyl)lysine (CEL). In order to accurately measure the CML contents of the proteins by means of an immunochemical method, we prepared CML-specific antibodies since conventionally prepared polyclonal anti-CML antibody and monoclonal anti-CML antibody (6D12) cross-reacted with CEL. To prepare polyclonal CML-specific antibody, CML-keyhole limpet hemocyanin (CML-KLH) were immunized with rabbit and CEL-reactive antibody was removed by CEL-conjugated affinity chromatography. Monoclonal antibody specific for CML (CMS-10) was obtained by immunization with CML-KLH, followed by successive screening according to CML-bovine serum albumin (CML-BSA)-positive but CEL-BSA-negative criteria. Both polyclonal CML-specific antibody and CMS-10 significantly reacted with CML-proteins but not with CEL-proteins. It is likely therefore that these antibodies can recognize the difference of one methyl group between CML and CEL. Moreover, CMS-10 significantly reacted with BSA modified with several aldehydes and its reactivity was highly correlated with the CML content, which was determined by high performance liquid chromatography, whereas 6D12 showed a low correlation. These results indicate that CMS-10 can be used to determine the CML contents of modified proteins in a more specific way.
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  • Nobuhisa Umeki, Tsuyoshi Yoshizawa, Yasunobu Sugimoto, Toshiaki Mitsui ...
    2004 Volume 136 Issue 6 Pages 839-846
    Published: December 01, 2004
    Released: June 30, 2008
    JOURNALS FREE ACCESS
    The region containing reactive cysteines, Cys 707 (SH1)-Cys 697 (SH2), of skeletal muscle myosin is thought to play a key role in the conformational changes of the myosin head during force generation coupled to ATP hydrolysis. In the present study, we synthesized a photochromic crosslinker, 4, 4'-azobenzene-dimaleimide (ABDM), that undergoes reversible cis-trans isomerization upon ultra violet (UV) and visible (VIS) light irradiation resulting in a change in the crosslinking length from 5 to 17 Å. The reactive cysteines, SH1 and SH2, of myosin subfragment 1 (S1) were crosslinked with ABDM, yielding an ABDM-S1 complex. The changes in absorbance induced by UV/VIS light irradiation of the complex were similar to those of free ABDM indicating that the incorporation of ABDM at the SH1 and SH2 sites did not disrupt the isomerization of crosslinked ABDM. Small-angle synchrotron X-ray scattering analysis of the ABDM-S1 complex in solution suggested that the localized conformational changes resulting from the cis to trans isomerization on ABDM crosslinking of SH1 and SH2 induced a small but significant swing in the lever arm portion of S1 in the opposite direction from that induced by ATP.
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