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Volume 131, Issue 1
Displaying 1-20 of 20 articles from this issue
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Futoshi Shibasaki, Ulrika Hallin, Hiroyuki Uchino2002 Volume 131 Issue 1 Pages 1-15
Published: 2002
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSLong recognized as an important regulatory mechanism in biosignal processes, modulation of the phosphorylation state of proteins has emerged as the most important mechanism for understanding signal transduction. In contrast to the multitude of protein kinases and the clear signal transduction pathways, relatively few protein phosphatases are known and their regulation is unclear. Among them, calcineurin, a calcium/ealmodulin-dependent phosphatase (PP2B), is the best enzyme to unveil the phosphatase function, because it was shown to be the direct target for immunosuppressants CsA and FK506, which are powerful tools for understanding this function in diseases as well as in several tissues and organs. Although calcineurin has been found in the highest concentrations in brain, it has also been detected in many other mammalian tissues. Well characterized in T cell activation by analysing the transcription factor NFAT, the function of calcineurin, however, was less well understood in other tissues and organs. Since the mid-1990s, several novel functions of this phosphatase have been reported, revealing that it plays important roles as a multifunctional regulator under the direct regulation of calcium signaling.View full abstractDownload PDF (3274K) -
Youhei Murakami, Shin Kamiyama, Zakir H. Howlader, Tokuhisa Yamato, Mi ...2002 Volume 131 Issue 1 Pages 17-19
Published: 2002
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe tried to confirm the antioxidative capability of lecithin: cholesterol acyltransferase (LCAT) reported by Vohl et al. [Biochemistry (1999) 38, 5976-5981]. The enzyme solution protected LDL against oxidation. However, this protection was not due to LCAT enzyme, but to some unknown low-molecular-weight substance(s) in the solution; LCAT itself exerted little protective effect against LDL oxidation.View full abstractDownload PDF (1166K) -
Ali M. Khorrami, Amit Choudhury, Mahefatiana Andrianifahanana, Grish C ...2002 Volume 131 Issue 1 Pages 21-29
Published: 2002
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPancreatic mucins consist of core proteins that are decorated with carbohydrate structures. Previous studies have identified at least two physically distinct populations of mucins produced by a pancreatic adenocarcinoma cell line (HPAF); one is the MUC1 core protein, which includes an oligosaccharide structure identified by a monoclonal antibody (MAb) recognizing the DU-PAN-2 epitope. In this study, we purified and characterized a second mucin fraction, which also shows reactivity with the DU-PAN-2 antibody, but which has an amino acid composition that is not consistent with the MUC1 core protein. This new mucin was purified by ammonium sulfate precipitation, molecular sieve chromatography, and density gradient centrifugation. It eluted in the void volume of a Sepharose 4B column together with an associated low molecular weight protein, which could be further resolved. The mucin is highly polyanionic due to numerous sulfated and sialylated saccharide chains. Carbohydrate analyses of the purified mucin showed the presence of galactose, glucosamine, galactosamine, and sialic acid, but no mannose, glucose, or uronic acid. The purified and deglycosylated mucin shows no reactivity with anti-MUC1 apomucin antibody, but reacts with antiserum against deglycosylated tracheal mucins and antiserum against the MUC4 tandem repeat peptide. Analysis of mucin expression in HPAF cells revealed high levels of MUC1 and MUC4 mRNA, and moderate levels of MUC5AC and MUC5B mRNA. The amino acid composition of the purified mucin shows a high degree of similarity to the MUC4 core protein.View full abstractDownload PDF (2411K) -
Yukihiro Yabuta, Ezhilkani Subbian, Hiroshi Takagi, Ujwal Shinde, Masa ...2002 Volume 131 Issue 1 Pages 31-37
Published: 2002
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSubtilisin is produced as a precursor that requires its N-terminal propeptide to chaperone the folding of its protease domain. Once folded, subtilisin adopts a remarkably stable conformation, which has been attributed to a high affinity Ca2+ binding site. We investigated the role of the metal ligand in the maturation of pro-subtilisin, a process that involves folding, autoprocessing and partial degradation. Our results establish that although Ca2+ ions can stabilize the protease domain, the folding and autoprocessing of pro-subtilisin take place independent of Ca2+, ion. We demonstrate that the stabilizing effect of calcium is observed only after the completion of autoprocessing and that the metal ion appears to be responsible for shifting the folding equilibrium towards the native conformation in both mature subtilisin and the autoprocessed propeptide:subtilisin complex. Furthermore, the addition of active subtilisin to unautoprocessed pro-subtilisin in trans does not facilitate precursor maturation, but rather promotes rapid autodegradation. The primary cleavage site that initiates this autodegradation is at Gln19 in the N-terminus of mature subtilisin. This corresponds to the loop that links α-helix-2 and β-strand-1 in mature subtilisin and has indirect effects on the formation of the Ca2+ binding site. Our results show that the N-terminus of mature subtilisin undergoes rearrangement subsequent to propeptide autoprocessing. Since this structural change enhances the proteolytic stability of the precursor, our results suggest that the autoprocessing reaction must be completed before the release of active subtilisin in order to maximize folding efficiency.View full abstractDownload PDF (2683K) -
Fumihiko Tanaka, Naohiro Hori, Kenzo Sato2002 Volume 131 Issue 1 Pages 39-44
Published: 2002
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRat hepatoma Fao is a well-differentiated cell line that expresses numerous liver-specific enzymes and liver-enriched transcription factors, whereas the C2 cell line derived from Fao exhibits dedifferentiated phenotypes and fails to express these differentiated traits. Hence, to identify the gene related to the differentiation status in these cells, we analyzed differentially expressed genes between Fao and C2 cells using a microarray of 1, 000 identified cDNA clones, and isolated a cDNA clone from subtractive library constructed by suppression subtractive hybridization. D6.1A, a member of the tetraspanin protein superfamily, was identified as a gene enhanced in C2 cells compared with Fao cells. Interestingly, expression of this gene was also induced following liver injury by CCl4 and in liver regeneration following partial hepatectomy. These results suggest that the D6.1A gene is related to stimulation of cell proliferation and differentiation status in C2 hepatoma cells.View full abstractDownload PDF (2010K) -
Yumiko Ohhashi, Yoshihisa Hagihara, Gennady Kozhukh, Masaru Hoshino, K ...2002 Volume 131 Issue 1 Pages 45-52
Published: 2002
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSβ2-Microglobulin (β2M), the light chain of the type I major histocompatibility complex, is a major component of dialysis-related amyloid fibrils. β2M in the native state has a typical immunoglobulin fold with a buried intrachain disulfide bond. The conformation and stability of recombinant β2M in which the intrachain disulfide bond was reduced were studied by CD, tryptophan fluorescence, and one-dimensional NMR. The conformation of the reduced β2M in the absence of denaturant at pH 8.5 was similar to that of the intact protein unless the thiol groups were modified. However, reduction of the disulfide bond decreased the stability as measured by denaturation in guanidine hydrochloride. Intact β2M formed amyloid fibrils at pH 2.5 by extension reaction using sonicated amyloid fibrils as seeds. Under the same conditions, reduced β2M did not form typical amyloid fibrils, although it inhibited fibril extension competitively, suggesting that the conformation defined by the disulfide bond is important for amyloid fibril formation of β2M.View full abstractDownload PDF (1823K) -
Hideki Masuda, Toshio Uchiumi, Michiko Wada, Tetsuroh Ichiba, Akira Ha ...2002 Volume 131 Issue 1 Pages 53-58
Published: 2002
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSEach of the 10 proline residues of the inorganic pyrophosphatase (PPase) subunit of thermophilic bacterium PS-3 (PS-3) was replaced with alanine by the PCR-mutagenesis method. The variants were classified into three groups according to the effects of the replacements on their catalytic activities in 20mM Tris-HCI, pH 7.8, containing 5mM MgCl2: the catalytic activity was (i) slightly affected (P39A and P69A), (ii) considerably reduced (P14A, P43A, P59A, and P116A), and (iii) completely or almost completely abolished (P72A, P100A, P104A, and P146A). HPLC-gel chromatography in the presence of 5mM MgCl2 revealed the following subunit assembly of the variants: group (i), a hexamer; group (ii), a hexamer or a mixture of a hexamer and a trimer, although the hexamer was predominant; and group (iii), a trimer or a monomer. The thermostability of the variant PPases depended upon the amount of hexamer remaining in the presence of Mg2+ at high temperature. The results indicated that the hexamer state formed through protomer-protomer and trimer-trimer interactions is necessary for the PS-3 PPase to retain the correct structure for full catalytic activity and thermostability.View full abstractDownload PDF (1550K) -
Chiaki Setoyama, Yasuzo Nishina, Haruhiko Tamaoki, Hisashi Mizutan, Ik ...2002 Volume 131 Issue 1 Pages 59-69
Published: 2002
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAccording to the three-dimensional structure of a porcine kidney D-amino acid oxidasesubstrate (D-leucine) complex model, the G313 backbone carbonyl recognizes the substrate amino group by hydrogen bonding and the side-chain hydroxyl of T317 forms a hydrogen bond with C(2)=O of the flavin moiety of FAD [Miura et al. (1997) J. Biochem. 122, 825-833]. We have designed and expressed the G313A and T317A mutants and compared their enzymatic and spectroscopic properties with those of the wild type. The G313A mutant shows decreased activities to various D-amino acids, but the pattern of substrate specificity is different from that of the wild type. The results imply that the hydrogen bond between the G313 backbone carbonyl and the substrate amino group plays important roles in substrate recognition and in defining the substrate specificity of D-amino acid oxidase. The T317A mutant shows a decreased affinity for FAD. The steady-state kinetic measurements indicate diminished activities of T317A to substrate D-amino acids. The transient kinetic parameters measured by stopped-flow spectroscopy revealed that T317 plays key roles in stabilizing the purple intermediate, a requisite intermediate in the oxidative half-reaction, and in enhancing the release of the product from the active site, thereby optimizing the overall catalytic process of D-amino acid oxidase.View full abstractDownload PDF (1927K) -
Masayuki Furukawa, Takayuki Okubo, Makoto Ogino, Torn Yamazaki, Miki S ...2002 Volume 131 Issue 1 Pages 71-78
Published: 2002
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe use of cultured mammalian cells and artificial promoters for analyses of gene regulation gives results that are sometimes inconsistent with in vivo events and thus inconclusive. To understand the in vivo mechanism of chemically mediated CYP3A4 gene activation, we have used a natural promoter of the CYP3A4 gene and an adenovirus as a reporter vector. The adenovirus reporter vector (AdCYP3A4-362) was constructed with a proximal promoter region (-362 to +11 nt) of the CYP3A4 gene and a luciferase-reporter gene. AdCYP3A4-362 was then infected into mice, and both the reporter and mouse CYP3A activities were measured. Clear increases in the reporter activity were observed in livers of all mice treated with chemicals. The profile of the CYP3A4 gene activation with chemicals was in good agreement with that of endogenous mouse CYP3A-mediated testosterone 6β-hydroxylase. Introduction of nucleotide mutations in the receptor-binding region (ER-6) of the CYP3A4 promoter resulted in diminished reporter activity. These results indicate the advantage of the adenovirus-mediated in vivo system over the currently available in vitro systems for gene transcriptional activation.View full abstractDownload PDF (746K) -
Togo Ikuta, Junko Watanabe, Kaname Kawajiri2002 Volume 131 Issue 1 Pages 79-85
Published: 2002
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that acts in concert with the AhR nuclear translocator (ARNT). Subcellular localization and transcriptional activation of target genes are mainly regulated by ligands such as 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD). We have previously reported that AhR migrates in cells as a nucleocytoplasmic shuttling protein mediated by its nuclear localization and export signals. A short sequence motif LxxLL (L is leucine and x is any amino acid) found in transcriptional co-activators has been reported to mediate the binding to liganded nuclear receptors. The role of the two LxxLL motifs, AhR [50-54] and [224-228], has now been analyzed by determining the localization of AhR and its transcriptional activity with Leu to Ala mutations in full-length AhR. Immunocytostaining revealed that mutation of the motif at AhR [50-54] promotes the efficiency of nuclear localization in the absence of ligand without altering HSP90 and ARA9 binding or nuclear export activity. Furthermore, this mutation decreases the transcriptional activity of the AhR/ARNT system, which is likely due to the suppression of AhRJARNT/XRE complex formation. Another LxxLL motif at AhR [224-227] affects neither the subcellular localization nor transcriptional activity. These results indicate that the LxxLL motif at AhR [50-54] is important for the regulation of AhR activity.View full abstractDownload PDF (2691K) -
Kumiko Shimono, Kazutaka Shigeru, Akiho Tsuchiya, Noriko Itou, Yukari ...2002 Volume 131 Issue 1 Pages 87-96
Published: 2002
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSChitosanase is the glycolytic enzyme that hydrolyzes the glucosamine GlcN-GlcN bonds of chitosan. To determine the catalytically important residues of chitosanase A (ChoA) from Matsuebacter chitosanotabidus 3001, we performed both site-directed and random mutagenesis of choA, obtaining 31 mutants. These mutations indicated that Glu-121 and Glu-141 were catalytically important residues, as mutation at these sites to Ala or Asp drastically decreased the enzymatic activity to 0.1-0.3% of that of the wild type enzyme. Glu-141 mutations remarkably decreased kinetic constant kcat for hydrolysis of chitosan, meanwhile Glu-121 mutations decreased the activities to undeterminable levels, precluding parameter analysis. No hydrolysis of (GlcN)6 was observed with the purified Glu-121 mutant and extremely slow hydrolysis with the Glu-141 mutant. We also found that Asp-139, Asp-148, Arg-150, Gly-151, Asp-164, and Gly-280 were important residues for enzymatic activities, although they are not directly involved in catalysis. In addition, mutation of any of the six cysteine residues of ChoA abrogated the enzymatic activity, and Cys-136 and Cys-231 were found to form a disulfide bond. In support of the significance of the disulfide bond of ChoA, chitosanase activity was impaired on incubation with a reducing agent. Thus, ChoA from M. chitosanotabidus 3001 uses two glutamic acid residues as putative catalytic residues and has at least one disulfide bond.View full abstractDownload PDF (2399K) -
Kuniyo Inouye, Iori Ueno, Shin-ichi Yokoyama, Toshiyuki Sakaki2002 Volume 131 Issue 1 Pages 97-105
Published: 2002
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA synchronous enzyme-reaction system using water-soluble formazan and a non-enzymatic electron mediator was developed and applied to an enzyme immunoassay (EIA). The reaction system consists of four steps: (I) dephosphorylation of NADP+ to produce NAD+ by alkaline phosphatase (ALP), (II) reduction of NAD+ to produce NADH with oxidation of ethanol to yield acetaldehyde by alcohol dehydrogenase (ADH), (III) reduction of water-soluble tetrazolium salt (WST-1) to produce formazan by NADH via 1-methoxy-5-methyl-phenazinium methyl sulfate (PMS), and (IV) re-reduction of NAD+ to produce NADH by ADH. During each cycle, one molecule of tetrazolium is converted to one molecule of formazan. The concentration of formazan during the reaction was given by second-order polynomials of the reaction time. Kinetic studies strongly suggested that the synchronous enzyme-reaction system had the potential to detect an analyte at the attomole level in EIA. On the basis of the kinetic studies, optimal conditions for EIA incorporating the synchronous system were examined. NADP+ was purified thoroughly to remove minor traces of NAD+ in the preparation, and an ADH preparation contaminated with the lowest level of ALP activity was used. When the synchronous system was applied to a sandwich-type EIA for human C-reactive protein, the protein was detected with a sensitivity of 50 attomole per well of a micro-titer plate (0.1ml) in a 1-h reaction. In addition, ETA with water-soluble formazan showed a more quantitative and sensitive result than that with insoluble formazan. These findings indicated that the (WST-1)-PMS system introduced in this study has a great potential for highly sensitive enzyme immunoassay.View full abstractDownload PDF (881K) -
Jichun Ma, Akio Ito2002 Volume 131 Issue 1 Pages 107-111
Published: 2002
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMonoamine oxidase is a flavin-containing enzyme which is located at the mitochondrial outer membrane and catalyzes the oxidative deamination of amines. To investigate the role of tyrosine residues near the FAD-binding site, Cys-406, of monoamine oxidase A, the tyrosine residues at positions 402, 407, and 410 were individually replaced with alanine or phenylalanine and the effects of the mutations on catalytic activity, FAD binding, and enzyme structure were examined. Half or fewer of the mutant proteins incorporated FAD. The mutation of Tyr-407 to alanine led to an almost completely loss of catalytic activity for serotonin, phenylethylamine, tyramine, and tryptamine. A substantial decrease in the catalytic activity was also observed with the enzymes mutated at Tyr-402 and Tyr-410 to alanine, although the effect of the latter mutation was much less. All these mutants were sensitive to trypsin treatment of the purified enzyme, while the wild type enzyme was resistant to treatment. On the other hand, substitution of Tyr-402 or Tyr-407 with phenylalanine had little effect on these properties. Taken together, we conclude that tyrosine residues near Cys-406 may form a pocket to facilitate FAD incorporation, the catalytic center, and a stable conformation, probably through interactions among the aromatic rings of the tyrosine residues and FAD.View full abstractDownload PDF (2253K) -
Yasushi Hasegawa, Takahiro Araki2002 Volume 131 Issue 1 Pages 113-119
Published: 2002
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe isolated a cDNA encoding a novel unconventional myosin from scallop mantle tissue (sc-allop un-conventional m-yosin: ScunM) and determined the nucleotide sequence. It comprises 2, 739 bp with 5' and 3'-noncoding sequences and has an open reading frame of 2, 334 bp that encodes 778 amino acids. While ScunM has a motor domain and a short tail domain without having light chain-binding IQ motifs like myosin XIV, the deduced amino acid sequence exhibits low homology, 30-36%, to known myosins. Phylogenetic analysis of the motor domain suggested that ScunM belongs to a novel unconventional myosin class. ScunM has an insertion of 67 amino acids in the putative actin-binding site (loop2 site). Western blot analysis with an antibody produced against the N-terminal region revealed that ScunM was strongly expressed in the mantle and mantle pallial cell layer of scallop.View full abstractDownload PDF (1303K) -
Katsuya Satoh, Issay Narumi, Masahiro Kikuchi, Shigeru Kitayama, Tadas ...2002 Volume 131 Issue 1 Pages 121-129
Published: 2002
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRecA protein is considered to be the most important participant in the radiation resistance of Deinococcus radiodurans. However, it is still unclear how RecA contributes to the resistance. In this study, we identified a new recA mutation (recA424) in the DNA-repair deficient mutant strain KI696, the phenotype of which is remarkably different from mutant strain rec30 carrying recA670. The properties of the gene products from the recA mutants were compared. recA424 could not complement the deficiency in Escherichia coli RecA, as found for recA670. In vitro, neither RecA424 nor RecA670 could promote DNA strand exchange under conditions in which wild-type RecA promoted the reaction, indicating that both RecA424 and Rec670 are defective in recombination activity. RecA424 promoted the autocleavage reaction of LexA in vitro, whereas RecA670 did not. The intracellular LexA level in KI696 was decreased following γ-irradiation. However, the LexA level in strain rec30 was constant irrespective of irradiation. These results indicate that RecA424 retains co-protease activity, whereas RecA670 does not. While strain rec30 is extremely radiation sensitive, strain KI696 is only slightly sensitive. Together, these observations suggest that the co-protease activity rather than the recombination activity of RecA contributes to radiation resistance in D. radiodurans.View full abstractDownload PDF (3230K) -
Katsuya Ota, Alexander G. Yakovlev, Asako Itaya, Masanori Kameoka, Yas ...2002 Volume 131 Issue 1 Pages 131-135
Published: 2002
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBrain and liver extracts of rats at different stages after birth were examined for cytochrome c/dATP-dependent caspase (DEVDase)-activation (mitochondria pathway) in vitro. The caspase-activating activity in the brain extracts rapidly decreased after birth, reaching approximately 50 and 5%, at 1 and 2 weeks, respectively, of that in a 3-days-newborn sample, and essentially no caspase-activation was detected in the adult rat brain extracts. Such a dramatic change was not detected in the liver samples, suggesting that the observed abrogation of the cytochrome c-dependent mitochondria pathway after birth is a brain-specific event. In order to determine the factor(s) lacking in adult brain, we separately measured Apaf-1, procaspase 9, and pro-DEVDase activities using a supplementation assay. In adult brain, Apaf-1 activity was scarcely detected, while the tissue retained low but significant amounts of procaspase 9 (16% of that in the fetal tissue) and a pro-DEVDase (3.4%). In contrast, adult liver extracts retained relatively high levels of all of these factors. Immunoblot analyses clearly indicated that the expression of Apaf-1 and procaspase 3 is markedly suppressed within 4 weeks after birth in brain tissue while they are even expressed in adult liver. Considering these results together, we propose that, in the brain, the cytochrome c-dependent mitochondria pathway, which is essential for the programmed cell death during normal morphogenesis, is abrogated within 2-4 weeks after birth, whereas the pathway is still active in other adult tissues such as liver.View full abstractDownload PDF (1540K) -
Shuichiro Ito, Tomoko Takayama, Hiroyuki Hanzawa, Kimihisa Ichikawa, J ...2002 Volume 131 Issue 1 Pages 137-143
Published: 2002
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBinding of Fas ligand to Fas induces apoptosis. The Fas-Fas ligand system plays important roles in many biological processes, including the elimination of autoreactive lymphoid cells. The mouse anti-human Fas monoclonal antibody HFE7A (m-HFE7A), which induces apoptosis, has been humanized based on a structure predicted by homology modeling. A version of humanized HFE7A is currently under development for the treatment of autoimmune diseases such as rheumatoid arthritis. For a deeper understanding of the protein engineering aspect of antibody humanization, for which information on the three-dimensional structure is essential, we determined the crystal structure of the m-HFE7A antigen-binding fragment (Fab) by X-ray crystallography at 2.5 Å resolution. The main-chain conformation of the five loops in the six complementarity-determining regions (CDRs) was correctly predicted with root-mean-square deviations of 0.30-1.04 Å based on a comparison of the crystal structure with the predicted structure. The CDRH3 conformation of the crystal structure, which was not classified as one of the canonical structures, was completely different from that of the predicted structure but adopted the conformation which followed the “H3-rules.” The results of charge distribution analysis of the antigen-binding site suggest that electrostatic interactions may be important for its binding to Fas.View full abstractDownload PDF (3325K) -
Erika Fujihira, Norihisa Tamura, Akihito Yamaguchi2002 Volume 131 Issue 1 Pages 145-151
Published: 2002
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAcrA/B in Escherichia coli is a multicomponent system responsible for intrinsic resistance to a wide range of toxic compounds, and probably cooperates with the outer membrane protein TolC. In this study, acrAB genes were cloned from the E. coli W3104 chromosome. To determine the topology of the inner membrane component AcrB, we employed a chemical labeling approach to analyse mutants of AcrB in which a single cysteine residue had been introduced. The cysteine-free AcrB mutant, in which the two intrinsic Cys residues were replaced by Ala, retained full drug resistance. We constructed 33 cysteine mutants in which a single cysteine was introduced into each putative hydrophilic loop region of the cysteine-free AcrB. The binding of [14C]N-ethylmaleimide (NEM) to the Cys residue and the competition of NEM binding with the binding of a membrane-impermeant maleimide, 4-acetamide-4'-maleimidylstilbene-2, 2'-disulfonic acid (AMS), in intact cells were investigated. The results revealed that the N- and C-terminals are localized on the cytoplasmic surface of the membrane and the two large loops are localized on the periplasmic surface of the membrane. The results supported the 12-membrane-spanning structure of AcrB. Three of the four short periplasmic loop regions were covered by the two large periplasmic loop domains and were not exposed to the water phase until one of the two large periplasmic loops was removed.View full abstractDownload PDF (3005K) -
Yasuyuki Nakamura, Mitsuhiro Shimizu, Michiteru Yoshida2002 Volume 131 Issue 1 Pages 153-160
Published: 2002
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBMGB2 (HMG2) protein binds with DNA duplex in a sequence-nonspecific manner, then bends and unwinds the DNA. In DNA cyclization analyses for the bending activity of HMGB2, two unidentified bands, denoted α and β, were observed in addition to monomer circular DNA (1C) on the gel. Re-electrophoresis and proteinase K digestion revealed that α and β are complexes of circularized probe DNA (seeming 1C) with HMGB2 (Kd_??_10-10M). The DNA components of α and β (α- and β-DNA) showed higher affinities to HMGB2 than did the linear probe DNA (Kd_??_10-7M). The DNAs have distorted structures containing partial single-stranded regions. Nicked circular molecules presumably due to severe DNA distortion by HMGB2 were observed in α- and β-DNA, in addition to closed circular double-stranded molecules. The α and β bands were not formed in the presence of sole DNA binding regions which are necessary for DNA bending, indicating that the acidic C-tail in the HMGB2 molecule is necessary for inducing the peculiar distorted structures of higher affinity to HMGB2. HMGB2 binds with linker DNA and/or the entry and exit of nucleosomes fixed at both ends likewise mini-circles similar to α-DNA and β-DNA. Thus, the distorted structures present in α-DNA and β-DNA should be important in considering the functional mechanisms in which HMGB2 participates.View full abstractDownload PDF (4732K) -
Zang Hee Lee, Shee Eun Lee, Chang-Woo Kim, Sang Ho Lee, Si Wouk Kim, K ...2002 Volume 131 Issue 1 Pages 161-166
Published: 2002
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSOsteoclasts, cells that resorb bone, die once fully differentiated. Several factors including interleukin-1 (IL-1) have been shown to regulate the survival of mature osteoclasts. However, information on the mechanism underlying the regulation of osteoclast survival has been limited. In this study, we investigated the mechanism for the IL-1-stimulated survival of osteoclasts. Treatment of purified osteoclasts with IL-1α led to activation of the serine-threonine kinases Akt and ERK. Blocking the activation of Akt with LY 294002, a specific inhibitor of the Akt up-stream molecule PI 3-kinase, or with an adenoviral vector for a dominant-negative form of Akt prevented the stimulation of osteoclast survival by IL-1α. PD 98059, a specific inhibitor of the ERK-activating kinase MEK 1, also abolished the effects of IL-1α on ERK activation and osteoclast survival. IL-1α reduced the apoptosis of osteoclasts by reducing caspase 3 activity. The IL-1α-mediated suppression of apoptosis was abolished by the PI 3-kinase/Akt or MEK1/ERK pathway inhibitor. These findings implicate the PI 3-kinase/Akt and ERK signaling pathways in the promotion of osteoclast survival by IL-1α.View full abstractDownload PDF (1968K)
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