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Volume 134 (2003)
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Volume 134, Issue 3
Displaying 1-22 of 22 articles from this issue
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Shuh Narumiya2003 Volume 134 Issue 3 Pages 305-307
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSDownload PDF (430K) -
Hiroaki Miki, Tadaomi Takenawa2003 Volume 134 Issue 3 Pages 309-313
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSRapid reorganization of the actin cytoskeleton underlies morphological changes and motility of cells. WASP family proteins have received a great deal of attention as the signal-regulated molecular switches that initiate actin polymerization. The first member, WASP, was identified as the product of a gene of which dysfunction causes the human hereditary disease Wiskott-Aldrich syndrome. There are now five members in this protein family, namely WASP, N-WASP, WAVE/Scar 1, 2, and 3. WASP and N-WASP have functional and physical associations with Cdc 42, a Rho family small GTPase involved in filopodium formation. In contrast, there is evidence that links the WAVE/Scar proteins with another Rho family protein, Rac, which is a regulator of membrane ruffling. All WASP family members have a VCA domain at the C-terminus through which Arp2/3 complex is activated to nucleate actin polymerization. Analyses of model organisms have just begun to reveal unexpected functions of WASP family proteins in multicellular organisms.View full abstractDownload PDF (1940K) -
Haruhiko Bito2003 Volume 134 Issue 3 Pages 315-319
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSPolarization of the neuronal cell body and initiation of the first neuritic process represent the starting point of a series of dynamic metamorphic events by which the newly acquired identity of a group of neurons can be translated into a morphologically complex web of three-dimensional neuronal circuit. Despite the critical importance of these events, little is known about the molecular signaling mechanisms that either regulate the temporal sequence of these steps or ensure the accuracy and the spatial consistency of the resulting circuits. In this review, based on recent findings from our group and others, we present a working model on how the initial events in neuronal morphogenesis in the CNS may be controlled by multiple Rho pathways.View full abstractDownload PDF (3325K) -
Yuko Mimori-Kiyosue, Shoichiro Tsukita2003 Volume 134 Issue 3 Pages 321-326
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe generation of a polarized microtubule organization is critically important for proper cellular functions, such as cell division, differentiation and migration. Microtubules themselves are highly dynamic structures, and this dynamic property is temporally and spatially regulated within cells, especially at their plus ends. To explain how microtubules set up and make contacts with cellular structures, a “search-and-capture” mechanism has been proposed, in which the microtubule plus ends dynamically search for and capture specific sites, such as mitotic kinetochores and cell cortex. To date, several classes of proteins have been shown to be associated with microtubule plus ends in a wide range of organisms from fungi to humans and to play critical roles in the “search-and-capture” mechanism. In this review, we overview our current understanding of the “plus-end-binding proteins” (+TIPs), including APC (adenomatous polyposis coli) tumor suppressor protein, cytoplasmic linker proteins (CLIPs), CLIP-associating proteins (CLASPs), cytoplasmic dynein/dynactin, and EB1, an APC-interacting protein.View full abstractDownload PDF (3690K) -
Toshio Hakoshima, Toshiyuki Shimizu, Ryoko Maesaki2003 Volume 134 Issue 3 Pages 327-331
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSSmall GTPases of the Rho family serve as conformational switches in a wide variety of signal transduction pathways that regulate diverse cellular functions. The GTP-bound forms of Rho GTPases are capable of interacting with downstream effectors that control cytoskeletal rearrangements. Regulators that stimulate nucleotide exchange, the hydrolytic cycle and distribution between the membrane and cytosol control the switch. Detailed pictures of Rho GTPase switching, effector recognition and regulation by regulators have emerged from recent structural investigations. These include the most extensively studied Rho GTPases, RhoA, Rac1, 2 and Cdc42, and their complexes with effectors and regulators. These studies have revealed the general diversity of effector and regulator structures, and in particular the structural features concerning the specific interactions involved in Rho effector recognition and regulator interactions with Rho GTPase. These findings provide a critical insight into the nature of Rho GTPase activity and consequently allow for a detailed manipulation of signaling pathways mediated by these proteins.View full abstractDownload PDF (2729K) -
Yuka Eura, Naotada Ishihara, Sadaki Yokota, Katsuyoshi Mihara2003 Volume 134 Issue 3 Pages 333-344
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSMitochondria are dynamic organelles that undergo frequent fission and fusion or branching. Although these morphologic changes are considered crucial for cellular functions, the underlying mechanisms remain elusive, especially in mammalian cells. We characterized two rat mitochondrial outer membrane proteins, Mfn1 and Mfn2, with distinct tissue expressions, that are homologous to Drosophila Fzo, a GTPase involved in mitochondrial fusion. Expression of the GTPase-domain mutant of Mfn2 (Mfn2K109T) in HeLa cells induced mitochondrial fragmentation in which Mfn2K109T localized at the restricted domains. Immuno-electronmicroscopy revealed that Mfn2K109T was concentrated at the contact domains between adjacent mitochondria, suggesting that fusion of the outer membrane was arrested at some intermediate step. Mfn1 expression induced highly connected tubular network structures depending on the functional GTPase domain. The Mfn1-induced tubular networks were suppressed by co-expression with Mfn2. In vivo depletion of either isoform by RNA interference revealed that both are required to maintain normal mitochondrial morphology. The fusion of differentially-labeled mitochondria in HeLa cells subjected to depletion of either Mfn isoform and subsequent cell fusion by hemagglutinating virus of Japan revealed that both proteins have distinct functions in mitochondrial fusion. We conclude that the two Mfn isoforms cooperate in mitochondrial fusion in mammalian cells.View full abstractDownload PDF (9104K) -
Bert E. Johansson, Ian C. Brett2003 Volume 134 Issue 3 Pages 345-352
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSInfluenza virus N2 neuraminidases were chromatographically purified from several vaccine candidate strains from 1957 to 1994. Enzymatic kinetic parameters and immunogenicity were tested for each strain. For each NA tested, with ionic strength held constant, Ca2+ or Mg2+ increased the initial rate of enzymatic activity. Earlier N2-NA strains had the highest initial velocity, Vmax/Km and Vmax. There were significant differences among the influenza virus strains in enzymatic activity before and after addition of Ca2+ or Mg2+: Vmax/Km varied from 0.54M-1 s-1 to 0.88M-1 s-1 and Vmax varied from 2.45 s-1 to 4.3 s-1 before the addition of a divalent cation; and increased approxi-mately 2-fold each of these kinetic parameters for each strain after the addition of exogenous Ca2+ or Mg2+. Exhaustive dialysis with EDTA reduced the initial velocity of each strain with significant differences found among strains, with a range of 0.1% to 8% of original activity. Activity was partially restored by the addition of exogenous Ca2+ or Mg2+, varying from 8% to 60% of pre-dialysis levels, but original rates were not achieved. This reduction in enzymatic activity for the tested strains (i.e., A/Japan/57 and A/Johannesburg/94) was accompanied by a parallel decrease in NA-immunogenicity, with antibody response decreasing by as much as 76% as measured by NI titer, and ELISA titer decreasing by as much as 68%. The addition of Ca2+ or Mg2+ to the post-dialysis sample restored immunogenicity to as much as 80% of pre-dialysis NI titers and as much as 78% of pre-dialysis ELISA titers. Dialysis had the least effect on early strains as measured by enzymatic kinetic parameters and immunogenicity studies. Zn2+ had a slight inhibitory effect on the activity of all tested strains. Review of the nucleic acid sequence of each of these strains could not predict their enzymatic activity, immunogenicity or response to dialysis. If immunity against neuraminidase is desirable in vaccination against influenza, selection of vaccine candidate strains must include not only analysis of antigenic changes and sequence analysis but also enzymatic studies and determination of the requirement of divalent cations to maintain immunogenicity and activity during production.View full abstractDownload PDF (979K) -
Yoshinori Akagi, Atsuko Hashigasako, Patrick Degenaar, Shinichirou Iwa ...2003 Volume 134 Issue 3 Pages 353-358
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThis paper describes a method for imaging the endogenous release of glutamate from cerebral neurons. This method is based on the reactions of glutamate oxidase and peroxidase, and on the detection of hydrogen peroxide by a fluorescent substrate of peroxidase. Glutamate has been sensitively measured in vitro in the range of 20 nM to 1 μM. We used two types of Ca2+ channel inhibitors, MK-801 and ω-Conotoxin GVIA, which act to suppress Ca2+ transport at postsynaptic and presynaptic neurons, respectively. MK-801 did not inhibit the increase in glutamate release after KCl stimulation, while there was no increase in glutamate release after KCl stimulation when ω-Conotoxin GVIA was used, probably due to the inhibition of voltage-activated Ca2+ channels in the presynapse. Glutamate release and Ca2+ flow in the synaptic regions were imaged using a laser confocal fluorescence microscope. KCl-evoked glutamate release was localized around cell bodies linked to axon terminals. This procedure allows imaging that can be sensitively detected by the fluorometric enzymatic assay of endogenous glutamate release in synapses.View full abstractDownload PDF (2588K) -
Shigehito Nakashima, Yusuke Hiraku, Saeko Tada-Oikawa, Terutoshi Hishi ...2003 Volume 134 Issue 3 Pages 359-364
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSWe investigated the mechanism of apoptosis induced by bafilomycin A1, an inhibitor of vacuolar H+-ATPase. Bafilomycin A1 significantly inhibited the growth of MXN-1 human gastric cancer cells. Bafilomycin A1 induced apoptosis as demonstrated by DNA ladder formation and the TUNEL method. We designed a flow cytometric assay to detect the alteration in lysosomal pH using a fluorescent probe, fluorescein isothiocyanate-conjugated dextran. This assay revealed that bafilomycin A1 dramatically increased lysosomal pH. However, bafilomycin A1 induced neither significant decrease in mitochondrial transmembrane potential nor the release of mitochondrial cytochrome c into the cytoplasm. Western blotting showed that cathepsin D, but not cathepsin L, was released into the cytoplasm. The activity of caspase-3 was significantly increased by bafilomycin A1. However, cathepsin D did not directly cleave procaspase-3. These findings suggest that bafilomycin A1-induced apoptosis in MKN-1 cells is mediated by other proteases released after lysosomal dysfunction followed by activation of caspase-3 in a cytochrome c-independent manner. The present study showed that flow cytometric analysis of lysosomal pH can be useful to evaluate lysosomal protease-mediated apoptosis.View full abstractDownload PDF (1790K) -
Chao Yapeng, Gao Ningguo, Cheng Xiulan, Yang Jing, Qian Shijun, Zhang ...2003 Volume 134 Issue 3 Pages 365-371
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSA type of heparinase (heparin lysase, no EC number) was isolated from the periplasmic space of a novel species of Sphingobacterium by three-step osmotic shock. It was further purified to apparent homogeneity by a combination of SP-sepharose and Source 30 S chromatographies with a final specific activity of 17.6 IU/mg protein and purification factor of 13-fold. MALDI-TOF mass spectrum of the purified heparinase gave a molecular mass of 75, 674 Da of the native enzyme. Peptide mass spectrum showed poor homogeneity with the database in the peptide bank. Inhibition of the enzyme activity by N-acetylimidazole indicated that tyrosine residues were necessary for enzyme activity. Km and Vmax of the heparinase for de-o-sulfated-N-acetyl heparin were 42 μM and 166 μM/min/mg protein, respectively. The heparinase showed similiar activity on both heparin and heparan sulfate, except for the heparin from bovine lung. The heparinase exhibited only 8.3% of the activity when de-N-sulfated heparin was used as the substrate, but N-acetylation of the de-N-sulfated heparin restored the activity to 78.4%. Thus modification of N-site in heparin structure was favorable for heparinase activity. On the other hand, de-o-sulfation in heparin showed positive effects on the heparinase activity, since the enzyme activity for N-acetyl-de-o-sulfated heparin was increased by 150%. Based on the present findings, the sphingobacterial heparinase differed from flavobacterial and other reported heparinases in molecular mass, composition, charge properties, active site, substrate specificities and other important characteristics, suggesting that it a novel heparin lysase distinct from those from other sources.View full abstractDownload PDF (1439K) -
Kenjiro Sakaki, Kosuke Tashiro, Satoru Kuhara, Katsuyoshi Mihara2003 Volume 134 Issue 3 Pages 373-384
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe genome-wide expression pattern of budding yeast Saccharomyces. cerevisiae in response to mild heat treatment in a non-fermentable carbon source was analyzed using DNA microarrays. Of 5, 870 open reading frames (nuclear genome transcripts) examined, 104 genes were upregulated and 287 genes were downregulated upon shifting of the cells from 25°C to 37°C. Forty upregulated genes and 235 downregulated genes encoded localization-assigned proteins. Of 113 heat-repressible genes (excluding 122 heat-repressible ribosomal genes), 36 were mitochondria-related genes, whereas only 2 of 40 heat-inducible genes were mitochondria-related. In particular, 9 genes involved in the mitochondrial respiratory chain and 7 genes involved in mitochondrial protein translocation were significantly repressed, suggesting that mitochondrial respiratory function and biogenesis were downregulated. Consistent with these findings, the growth of yeast cells in a non-fermentable carbon source was repressed at 37°C and the mitochondria isolated from heat-stressed cells exhibited compromised preprotein-import activity compared with those from unstressed cells. In contrast, many genes involved in glycolysis and the metabolic pathway to produce glutamate via the tricarboxylic acid cycle, which is essential for biosynthetic reactions, were upregulated. Yeast cells might downregulate mitochondrial function to circumvent heat-induced oxidative stress, upregulate stress-related genes, and remodel genes for metabolic pathways in response to mild heat stress: an adaptive response at the expense of cell growth.View full abstractDownload PDF (9963K) -
Masayuki Ikuzawa, Shigeki Yasumasu, Tomofumi Inokuchi, Ken-ichiro Koba ...2003 Volume 134 Issue 3 Pages 385-394
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSCathepsin E (CE) was purified from the foregut of Xenopus laevis tadpoles as a mature dimeric form. The purified enzyme was a typical CE among aspartic proteinases with respect to pH dependence of proteolytic activity, susceptibility to pepstatin, and having N-linked high-mannose type oligosaccharide chains. We isolated two cDNAs for the CE (CE 1 and CE 2) from adult stomach. The amino acid sequence of the N-terminal region of the purified CE coincided with the corresponding sequence predicted from CE 1. Northern blot analysis and in situ hybridization were performed. The CE 1 mRNA was highly expressed in surface mucous cells and gland cells constituting the larval epithelium of the foregut of pro-metamorphic tadpoles. As metamorphosis began and progressed, CE 1 mRNA drastically decreased in amount, and subsequently both CE 1 and CE 2 mRNAs gradually increased. The increase in CE 2 mRNA was detected shortly after the increase in CE 1 mRNA. The decrease in CE 1 expression correlated with degeneration of the larval type epithelium, while the increases in both CE 1 and CE 2 expression correlated with formation of the adult type epithelium. Thus, cathepsin E gene expression was differentially regulated during metamorphosis-associated remodeling of the larval to adult type epithelium in stomach.View full abstractDownload PDF (4803K) -
Yoshiaki Kouzuma, Yota Suzuki, Masahiro Nakano, Kayo Matsuyama, Sumiki ...2003 Volume 134 Issue 3 Pages 395-402
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSCEL-III is a Ca2+-dependent, galactose/N-acetylgalactosamine (GalNAc)-specific lectin isolated from the marine invertebrate Cucumaria echinata. This lectin exhibits strong hemolytic activity and cytotoxicity through pore formation in target cell membranes. The amino acid sequence of CEL-III revealed the N-terminal two-thirds to have homology to the B-chains of ricin and abrin, which are galactose-specific plant toxic lectins; the C-terminal one-third shows no homology to any known proteins. To examine the carbohydrate-binding ability of the N-terminal region of CEL-III, the protein comprising Pyr1-Phe283 was expressed in Escherichia coli cells. The expressed protein showed both the ability to bind to a GalNAc-immobilized column as well as hemagglutinating activity for rabbit erythrocytes, confirming that the N-terminal region has binding activity for specific carbohydrates. Since the C-terminal region could not be expressed in E. coli cells, a fragment containing this region was produced by limited proteolysis of the native protein by trypsin. The resulting C-terminal 15 kDa fragment of CEL-III exhibited a tendency to self-associate, forming an oligomer. When mixed with erythrocytes, the oligomer of the C-terminal fragment caused hemagglutination, probably due to hydrophobic interaction with cell membranes, while the monomeric fragment did not. Chymotryptic digestion of the preformed CEL-III oligomer induced upon lactose binding also yielded an oligomer of the C-terminal fragment comprising six molecules of the 16 kDa fragment. These results suggest that after binding to cell surface carbohydrate chains, CEL-III oligomerizes through C-terminal domains, leading to the formation of ion-permeable pores by hydrophobic interaction with the cell membrane.View full abstractDownload PDF (5337K) -
Shigenobu Kimura, Takashi Iyanagi2003 Volume 134 Issue 3 Pages 403-413
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSA direct expression system for the solubilized catalytic domains of NADPH-cytochrome P-450 reductase (sCPR) from rat (RsCPR) and porcine (PsCPR) in Escherichia coli cells was constructed using the expression plasmid pCWori+. PsCPR was minimally expressed, whereas RsCPR was highly expressed. Replacement of the nucleotides encoding Thr60Ser61Ser62 in PsCPR with those for Ala60Pro61Pro62 in RsCPR markedly increased the expression level of the protein. The local secondary structures of the mRNAs, which were predicted with the prediction program GeneBee (http://www.genebee.msu.su), suggested that the intramolecular double strand between the ribosome binding site (RBS) and the Thr60Ser61Ser62 codons in PsCPR, and/or the base-pairing at the initiation codon of the mRNAs significantly affected protein expression. Silent mutations were systematically introduced into the codons for Thr58 and Thr60Ser61 in PsCPR to modulate the local secondary structure of the mRNA. The expression level of the silently mutated PsCPR suggests that the expression level of PsCPR depends on the stability of the local structure at the RBS in the mRNA. A high-level expression system for wild-type PsCPR was constructed by intro-ducing silent mutations at the codons for Thr60Ser61 in PsCPR. The purified PsCPR showed the characteristic absorption spectral changes of sCPR after reduction with NADPH. The yield of purified PsCPR from 1 liter of culture fluid was 45.8mg. These results substantiate that the introduction of silent mutations in the section of the gene encoding the N-terminal region of the protein based on the predicted local secondary structure of the mRNA at the RBS is a useful approach to control and increase the expression level of heterologous proteins in E. coli cells.View full abstractDownload PDF (5712K) -
Ryuichi Masaki, Keiichi Kameyama, Akitsugu Yamamoto2003 Volume 134 Issue 3 Pages 415-426
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSMicrosomal aldehyde dehydrogenase (msALDH) is a tail-anchored protein localized to the cytoplasmic face of the endoplasmic reticulum (ER). The carboxyl-terminal 35 amino acids of msALDH possess ER-targeting sequences in addition to a hydrophobic membrane-spanning domain. To study the mechanism for ER targeting of this protein in vivo, we took advantage of a green fluorescent protein-msALDH fusion protein containing the last 35 amino acids of msALDH [GFPALDH(35)]. When expressed from cDNA in COS-7 cells, the fusion protein was localized to the ER. We then prepared a recombinant fusion protein and injected it into the cytoplasm of COS-7 cells. The injected protein was correctly localized to the ER after a 30-min incubation at 37°C. However, a recombinant fusion protein that contained only the transmembrane domain of msALDH failed to be targeted to the ER. When the assay was carried out at 4°C, the recombinant GFPALDH (35) remained in the cytoplasm. Moreover, incubation of COS-7 cells under conditions of ATP depletion resulted in the cytoplasmic distribution of the injected protein. These results indicate that GFPALDH (35) is targeted to the ER post-translationally via an ATP-dependent pathway. This microinjection system worked effectively in different mammalian cell types, suggesting a common mechanism for ER targeting of the tail-anchored protein.View full abstractDownload PDF (10826K) -
Yoon-Hun Hong, Hyung-Sik Won, Hee-Chul Ahn, Bong-Jin Lee2003 Volume 134 Issue 3 Pages 427-432
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe conformational preferences and the solution structure of AnxIIN31, a peptide corresponding to the full-length sequence (residues 1-31) of the human annexin II N-terminal tail domain, were investigated by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. CD results showed that AnxIIN31 adopts a mainly α-helical conformation in hydrophobic or membrane-mimetic environments, while a predominantly random structure is adopted in aqueous buffer. In contrast to previous results of the annexin I N-terminal domain peptide [Yoon et al. (2000) FEBS Lett. 484, 241-245], calcium ions showed no effect on the structure of AnxIIN31. The NMR-derived structure of AnxIIN31 in 50% TFE/water mixture showed a horseshoe-like fold comprising the N-terminal amphipathic α-helix, the following loop, and the C-terminal helical region. Together, the results establish the first detailed structural data on the N-terminal tail domain of annexin II, and suggest the possibility of the domain to undergo Ca2+-independent membrane-binding.View full abstractDownload PDF (1813K) -
Shizuyo Koide, Ichiro Yoshida, Akihiko Tsuji, Yoshiko Matsuda2003 Volume 134 Issue 3 Pages 433-440
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSPACE 4 is a member of the mammalian subtilisin-like proprotein convertase (SPC) family, which contribute to the activation of transforming growth factor (TGF) β family proteins. We previously reported that PACE 4 is highly expressed in syncytiotro-phoblasts of human placenta [Tsuji et al. (2003) Biochim. Biophys. Acta 1645, 95-104]. In this study, the regulatory mechanism for PACE 4 expression in placenta was analyzed using a human placental choriocarcinoma cell line, BeWo cells. Promoter analysis indicated that an E-box cluster (E4-E9) in the 5'-flanking region of the PACE 4 gene acts as a negative regulatory element. The binding of human achaete-scute homologue 2 (Hash-2) to the E-box cluster was shown by gel mobility-shift assay. The over-expression of Hash-2 caused a marked decrease in PACE 4 gene expression. When BeWo cells were grown under low oxygen (2%) conditions, the expression of Hash-2 decreased, while that of PACE 4 increased. In both cases, other SPCs, such as furin, PC 5/6, and PC 7/8, were not affected. Further, PACE 4 expression was found to be developmentally regulated in rat placenta. By in situ hybridization, Mash-2 (mammalian achaete-scute homologue 2) mRNA was found to be expressed in the spongiotro-phoblast layer where PACE 4 was not expressed. In contrast, the PACE 4 mRNA was expressed mainly in the labyrinthine layer where Mash-2 was not detected. These results suggest that PACE 4 expression is down-regulated by Hash-2/Mash-2 in both human and rat placenta and that many bioactive proteins might be regulated by PACE 4 activity.View full abstractDownload PDF (5206K) -
Keishi Hata, Kazuyuki Hori, Saori Takahashi2003 Volume 134 Issue 3 Pages 441-445
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSWe examined the signaling mechanisms involved in the differentiation-inducing activity of lupeol toward B 16 2F2 melanoma cells. α-Melanocyte stimulating hormone (α-MSH), forskolin and dibutyryl cAMP, which are believed to be CAMP-elevating agents and analogues, enhanced lupeol-induced B 16 2F2 cell differentiation. However, H 89, an inhibitor of protein kinase A, completely abolished B 16-2F2 cell differentiation induced by lupeol. Furthermore, we studied the role of mitogen-activated protein kinases (MAPKs) in lupeol-induced B 16 2F2 cell differentiation. U0126, an inhibitor of MAPK kinases, induced B 16 2F2 cell differentiation and enhanced the cell differentiation induced by lupeol. However, SB203580, a selective inhibitor of p 38 MAPK, completely blocked lupeol-induced B 16 2F2 cell differentiation. Western blot analysis revealed that 10 μM lupeol transiently elevated the level of phosphorylation of p 38 MAPK. The phosphorylation of p 38 MAPK was detected on the addition of 1 μM lupeone, another lupane triterpene, but was not induced by 1 μM lupeol. These results suggested that lupeol induced B 16 2F2 cell differentiation through activation of p 38 MAPK, and that the structural differences at C-3 of lupane triterpenes played an important role in the activation of p 38 MAPK.View full abstractDownload PDF (2640K) -
Asako Furukohri, Noriko Sato, Hisao Masai, Ken-ichi Arai, Akio Sugino, ...2003 Volume 134 Issue 3 Pages 447-457
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSDbf4 is a regulatory subunit for the Cdc7 protein kinase that is required for the initiation of eukaryotic DNA replication, but the precise roles of Dbf4-Cdc7 remain to be determined. Here we identified a Xenopus homolog of Dbf4 (XDbf4) and characterized XDbf4 and Xenopus Cdc7 (XCdc7) in Xenopus egg extracts. XDbf4 formed a complex with XCdc7 in egg extracts and activated XCdc7 kinase activity in vitro. In contrast with Dbf4 in yeast and mammalian cultured cells, the XDbf4 levels in egg extracts did not change during the cell cycle progression. XDbf4 was a phosphoprotein in interphase extracts, and was apparently hyperphosphorylated in cytostatic factor (CSF)-mediated, metaphase-arrested extracts and in mitotic extracts. However, the hyperphosphorylation of XDbf4 did not seem to affect the level of kinase activation, or chromatin binding of the XDbf4-XCdc7 complex. Upon release from CSF-arrest, XDbf4 was partially dephosphorylated and bound to chromatin. Interestingly, XDbf4 was loaded onto chromatin before XCdc7 during DNA replication in egg extracts. These results suggest that the function of XDbf4-XCdc7 during the early embryonic cell cycle is regulated in a manner distinct from that during the somatic cell cycle.View full abstractDownload PDF (7168K) -
Hiroyuki Itabe, Masahiro Mori, Yasuyuki Fujimoto, Yusuke Higashi, Tats ...2003 Volume 134 Issue 3 Pages 459-465
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe oxidative modification of low-density lipoprotein (LDL) is involved in atherogenesis. Among a variety of modified LDLs mentioned in the literature, so-called minimally modified LDL (MM-LDL) was reported to have pro-atherogenic properties despite minimal changes in its oxidative measures. After treatment of LDL with 1 μM FeSO4 at 4°C for 96h, the resulting MM-LDL showed a slight increase in thiobarbituric acid-reactive substances (TBARS) and little association with macrophages. On the other hand, heavily oxidized LDL, which was prepared by copper-induced oxidation of LDL at 37°C, showed a sharp increase in TBARS and strong association with macrophages. By introducing a fluorometric procedure to detect aldehyde-containing phosphatidylcholines (aldehyde-PCs), we examined the amounts of aldehyde-PCs in modified LDL preparations. Aldehyde-PCs increased to 23.4 pmol/μg protein in MM-LDL, which was more than four-fold higher than in the heavily oxidized LDL. We conclude that MM-LDL is a unique type of oxidized LDL enriched with aldehyde-PCs.View full abstractDownload PDF (1934K) -
Ryoko Iijima, Koichi J. Homma, Shunji Natori2003 Volume 134 Issue 3 Pages 467-471
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSWe examined the effects of various protease substrates on Xenopus laevis embryogenesis. Thirty-three peptidyl-MCA substrates were added to the culture medium in which Xenopus embryos were developing. Five of the 33 substrates were found to inhibit embryogenesis at the early gastrula stage or much earlier ones. These results suggest that proteases that hydrolyze these substrates are involved in embryonic development. We found that the developmental stage of embryos is crucial for these substrates to inhibit their development. We purified a protease that hydrolyzes Pyr-Arg-Thr-Lys-Arg-MCA, a substrate that inhibits embryogenesis, from Xenopus embryos. This protease turned out to be a component of proteasomes. We found that 4 of the 5 substrates that inhibit embryogenesis are among the proteasome substrates. Thus, we concluded that proteasomes play a crucial role in the development of Xenopus embryos. Possibly, various catalytic subunits in proteasomes function independently, in stage-specific manners.View full abstractDownload PDF (2943K) -
Yusuke Hoshino, Shigeru Nakamori, Hiroshi Takagi2003 Volume 134 Issue 3 Pages 473-478
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSStreptomyces albulus IFO 14147 produces ε-poly-L-lysine, which exhibits antimicrobial activity. It is necessary for its molecular breeding to develop host-vector systems. We recently found a novel cryptic plasmid, pNO 33, in this strain. As part of a search for a selectable marker gene for pNO 33, we report here the isolation and analysis of the β-lactamase gene of this strain, which can grow on ampicillin-containing plates. It was shown that the β-lactamase production in S. albulus was induced by ampicillin. By introducing a genomic library of S. albulus into Escherichia coli, a 3.6-kbp fragment was identified as the region involved in ampicillin resistance. It contained three open reading frames, all of which are highly homologous to the β-lactamase (the blaL product) and its regulatory proteins (the blaA and blaB products) of S. cacaoi. The growth phenotypes and enzyme assaying of E. coli and S. lividans showed that the blaL homologue (blaSa) encodes a β-lactamase required for ampicillin resistance. The β-lactamse gene can be utilized as a selectable marker in a cloning vector of S. albulus. However, the β-lactamase activity was decreased in E. coli and repressed in S. lividans by the blaA and blaB homologues (blaASa and blaBSa). It appears as if the blaASa product is a repressor of blaSa instead of an activator as in S. cacaoi.View full abstractDownload PDF (1929K)
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