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Volume 135 (2004)
- Issue 6 Pages 653-
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- Issue 4 Pages 455-
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Volume 135, Issue 6
Displaying 1-10 of 10 articles from this issue
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Keisuke Kuida, Diane M. Boucher2004 Volume 135 Issue 6 Pages 653-656
Published: June 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSMitogen-activated protein kinases (MAPKs) comprise a family of well-conserved serine/threonine kinases that control a vast array of physiological functions in a number of organisms ranging from yeast to mammals. Recently gene-targeting experiments have shed light on in vivo functions of MAPKs. In particular, embryos deficient in extracellular signal-regulated kinase (ERK) 2 lack mesoderm differentiation and placental angiogenesis. Knockout mice for c-Jun amino-terminal kinases have revealed roles for these kinases in neural apoptosis and activation/differentiation of T cells. Deletion of p38α MAPK results in angiogenic defects in the placenta and peripheral vessels. ERK5-deficient embryos are embryonic lethal due to defects in angiogenesis and cardiovascular development. Although these results have provided new insights for MAPK research, development and analysis of conditional knockout mice are required in order to investigate roles of MAPKs, especially, in other biological processes such as disease pathogenesis.View full abstractDownload PDF (608K) -
Katsuji Yoshioka2004 Volume 135 Issue 6 Pages 657-661
Published: June 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe mitogen-activated protein kinase (MAPK) signaling pathway, which is conserved from yeast to humans, is activated in response to a variety of extra- and intracellular stimuli, and plays key roles in multiple cellular processes, including proliferation, differentiation, and apoptosis. The MAPK pathway transmits its signal through the sequential phosphorylation of MAPK kinase kinase to MAPK kinase to MAPK. Specific and efficient activation of the MAPK cascades is crucial for proper cellular responses to stimuli. As shown in yeast, the mammalian MAPK signaling system may also employ scaffold proteins, in part, to organize the MAPK signaling components into functional MAPK modules, thereby enabling the efficient activation of specific MAPK pathways. This review article describes recent advances in the study of potential mammalian scaffold proteins that may help us understand the complex regulation, including the spatial and temporal control, of the mammalian MAPK signaling pathways.View full abstractDownload PDF (720K) -
Tatsuya Yamamoto, Shunsuke Izumi, Kunihiko Gekko2004 Volume 135 Issue 6 Pages 663-671
Published: June 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSTo address the effects of ligand binding on the structural fluctuations of Escherichia coli dihydrofolate reductase (DHFR), the hydrogen/deuterium (H/D) exchange kinetics of its binary and ternary complexes formed with various ligands (folate, dihydrofolate, tetrahydrofolate, NADPH, NADP+, and methotrexate) were examined using electrospray ionization mass spectrometry. The kinetic parameters of HID exchange reactions, which consisted of two phases with fast and slow rates, were sensitively influenced by ligand binding, indicating that changes in the structural fluctuation of the DHFR molecule are associated with the alternating binding and release of the cofactor and substrate. No additivity was observed in the kinetic parameters between a ternary complex and its constitutive binary complexes, indicating that ligand binding cooperatively affects the structural fluctuation of the DHFR molecule via longrange interactions. The local H/D exchange profile of pepsin digestion fragments was determined by matrix-assisted laser desorption/ionization mass spectrometry, and the helix and loop regions that appear to participate in substrate binding, largely fluctuating in the apo-form, are dominantly influenced by ligand binding. These results demonstrate that the structural fluctuation of kinetic intermediates plays an important role in enzyme function, and that mass spectrometry on H/D exchange coupled with ligand binding and protease digestion provide new insight into the structure-fluctuation-function relationship of enzymes.View full abstractDownload PDF (3804K) -
Eiko Yokoo, Yutaka Yatomi, Toshiro Takafuta, Makoto Osada, Yoshitaka O ...2004 Volume 135 Issue 6 Pages 673-681
Published: June 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSTo analyze the involvement in allergic reactions of platelets and sphingosine 1-phosphate (Sph-1-P), a lysophospholipid mediator released from activated platelets, the effects of Sph-1-P and a supernatant prepared from activated platelets on mast cell line RBL-2H3 were examined. Sph-1-P strongly inhibited the migration of both non-stimulated and fibronectin-stimulated RBL-2H3 cells, which was reversed by JTE-013, a specific antagonist of G protein-coupled Sph-1-P receptor SiP2; S1P2 was confirmed to be expressed in these cells. A similar anti-motility effect of Sph-1-P was observed in a phagokinetic assay. Consistent with these results, treatment of RBL-2H3 cells with Sph-1-P resulted in a rounded cell morphology, which was blocked by JTE-013. Under the present conditions, Sph-l-P failed to induce intracellular Ca2+ mobilization or histamine degranulation, responses postulated to be elicited by intracellular Sph-1-P. Importantly, the Sph-1-P effect, i.e., the regulation of RBL-2H3 cell motility, was mimicked by the supernatant (both with and without boiling) prepared from activated platelets, and this effect of the supernatant was also blocked by JTE-013. Our results suggest that the motility of mast cells can be regulated by Sph-1-P and also platelets (which release Sph-1-P), via cell surface receptor S1P2 (not through intracellular Sph-1-P actions, postulated previously in the same cells).View full abstractDownload PDF (4627K) -
Shigeharu Takiya, Yoshinori Nishita, Susumu Ishikawa, Kaoru Ohno, Taka ...2004 Volume 135 Issue 6 Pages 683-693
Published: June 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSA new member of the Y -box protein family of the silkworm Bombyx mori (BYB) was co-purified with the fibroin gene enhancer-binding protein FMBP-1, and stimulated the binding of FMBP-1 to its cognate DNA element. However, the stimulatory effect was not specific to FMBP-1, BYB also enhancing the binding of mammalian transcription factors OTF 2, SP 1 and AP 2 to their specific binding elements. Besides the above transcription regulatory factors, BYB facilitated the binding of basal transcription factor TBP, and enhanced transcription from the adenovirus 2 major late promoter in a reconstituted transcription system. Moreover, BYB stimulated the reactions of some restriction endonucleases under cold conditions. The C-terminal region of BYB was sufficient for these stimulatory effects, and the highly conserved cold shock domain (CSD) in the N-terminal region was dispensable. GST pull down experiments showed that the C-terminal region could interact with DNA independently of the CSD. The above results suggest that the C-terminal region of BYB causes the active interaction of various DNA binding proteins with their targets. Such a function of the C-terminal region of BYB may partly explain the functional diversity of Y-box proteins.View full abstractDownload PDF (8540K) -
Nancy S. Bae, Mark J. Swanson, Alex Vassilev, Bruce H. Howard2004 Volume 135 Issue 6 Pages 695-700
Published: June 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSHistone deacetylases are required for transcriptional repression in eukaryotes. Saccharomyces cerevisiae has several histone deacetylases, of which ySir2p is the most conserved throughout evolution. Currently, there is no report on the interacting protein partner of a human Sir2 homolog, SIRT 2. Here we show for the first time that SIRT 2 interacts with the homeobox transcription factor, HOXA 10, which was identified in a two-hybrid screen. Interactions were confirmed by co-immunoprecipitation from in vitro translations as well as in human cell-free extracts. Taken together with mouse knockout studies, our results raise the intriguing possibility that SIRT 2 plays a role in mammalian development.View full abstractDownload PDF (3675K) -
Kazufumi Takano, Minoru Saito, Masaaki Morikawa, Shigenori Kanaya2004 Volume 135 Issue 6 Pages 701-708
Published: June 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSIt is known that several naturally occurring substances known as osmolytes increase the conformational stability of proteins. Bolen and co-worker proposed the osmophobic theory, which asserts the osmolyte effect occurs because of an unfavorable interaction of osmolytes mainly with the protein backbone, based on the results on the transfer Gibbs energy of amino acids (Δg) [Bolen and Baskakov (2001) J. Mol. Biol. 310, 955-963]. In this paper, we report the effect of sarcosine on the conformational stability (ΔG) of RNase Sa (96 residues and one disulfide bond) and four mutant proteins. The thermal denaturation curves for RNase Sa in sarcosine fitted a two-state model on nonlinear least-squares analysis. All the RNase Sa proteins were stabilized by sarcosine. For example, the increase in stability of the wild-type protein in 4 M sarcosine due to the osmolyte effect (ΔoΔG) is 3.2 kcal/mol. Mutational analysis of the osmolyte effect indicated that the changed ΔoΔG values upon mutation (ΔmΔoΔG), as estimated from the Δg values, are similar to the experimental values. Structural-based analysis of the osmolyte effect was also performed using model denatured structures: (a) a fully extended model (single chain) with no disulfide bond, (b) two-part, unfolded models (two chains) with a disulfide bond constructed through molecular dynamic (MD) simulation, and (c) a two-part, folded model (two chains). The two-part, unfolded models were expected to be more suitable as denatured structures. The ΔoΔG values calculated using the two-part, unfolded models were more consistent with experimental values than those calculated using the fully extended and twopart, folded models. This suggests that MD simulation is useful for testing denatured structures. These results indicate that the osmophobic theory can explain the osmolyte effect on protein stability.View full abstractDownload PDF (2915K) -
Hiroshi Matsumoto, Satoru Noguchi, Kazuma Sugie, Megumu Ogawa, Kumiko ...2004 Volume 135 Issue 6 Pages 709-712
Published: June 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSFukuyama-type congenital muscular dystrophy and congenital muscular dystrophy 1C are congenital muscular dystrophies that commonly display reduced levels of glycosylation of α-dystroglycan in skeletal muscle. The genes responsible for these disorders are fukutin and fukutin-related protein (FKRP), respectively. Both gene products are thought to be glycosyltransferases, but their functions have not been established. In this study, we determined their subcellular localizations in cultured skeletal myocytes. FKRP localizes in rough endoplasmic reticulum, while fukutin localizes in the cis-Golgi compartment. FKRP was also localized in rough endoplasmic reticulum in skeletal muscle biopsy sample. Our data suggest that fukutin and FKRP may be involved at different steps in O-mannosylglycan synthesis of α-dystroglycan, and FKRP is most likely involved in the initial step in this synthesis.View full abstractDownload PDF (2986K) -
Mitsuyoshi Motizuki, Toshiaki Takei, Kachio Tasaka, Sadaki Yokota, Shu ...2004 Volume 135 Issue 6 Pages 713-719
Published: June 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSWe previously noted that bovine apolipoprotein A-II (apoA-II) had a bactericidal effect causing morphological changes in the cytoplasm. To determine whether and how apoA-II and apoA-I, which have acidic isoelectric points (pls), enter cells, we determined the rates of uptake of FITC-labeled proteins by fibroblast cells and found that they entered cells more easily at low pH than at neutral pH under conditions where endocytosis was inhibited. The enhanced uptake of proteins at low pH was also observed for other proteins examined regardless of the molecular weight (Mr) or pI in a time-dependent manner, although the efficiency of uptake varied among the proteins. Furthermore, a pH gradient was shown to be the main driving force for the translocation. As cells were viable above pH 4 for 2h at 4°C and internalized β-galactosidase was active under these conditions, we suggest that this procedure is applicable to the injection of proteins into cells without the use of an apparatus such as a microinjector.View full abstractDownload PDF (1952K) -
Tetsuo Ishida, Hiroyuki Tanaka, Kihachiro Horiike2004 Volume 135 Issue 6 Pages 721-730
Published: June 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSCatechol 2, 3-dioxygenase [EC 1. 13. 11. 2] from Pseudomonas putida mt-2 (Mpc) catalyzes the extradiol cleavage of catechol to produce 2-hydroxymuconate semialdehyde. The Km values for the catecholic substrate (KmA) and O2 (KmO2), and catalytic constants (kcat) were kinetically determined for eight C3/C4-substituted catechols at 25°C and pH 6.5 or 7.5. The first pKa values (pK1) were determined for eleven catechols (pK1=7.26-9.47), correlated with Hammett substituent constants, and electron-withdrawing substituents significantly stabilized the monoanionic species of free catechols. Mpc preferred catechols with non-ionic substituents at the C3 or C4 position. 3-Phenylcatechol, a biphenyl, was cleaved, while 4-tert-butylcatechol was not. The logarithm of kcat/KmA (substrate specificity constant) exhibited a good linear correlation with pK1 with the exception of those for 4-halocatechols. The logarithm of kcat/KmO2 showed a good linear correlation with pK1 with the exception of that of 3-phenylcatechol. These results demonstrate that catechol binding to the Mpc active site, the following O2 binding, and the activation of the bound O2 are all sensitive to electronic effects of the substituents. However, kcat did not correlate significantly with pK1. The present study distinguishes clearly between the electronic and the steric effects of catecholic substrates in the reactivity of Mpc, and provides important insight into the mechanistic basis for a vast range of substrate specificities of extradiol dioxygenases.View full abstractDownload PDF (1291K)
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