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Volume 132, Issue 1
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Takashi Izumi, Takehiko Yokomizo, Hideru Obinata, Hideaki Ogasawara, T ...2002 Volume 132 Issue 1 Pages 1-6
Published: 2002
Released on J-STAGE: November 14, 2008
JOURNAL FREE ACCESSLeukotrienes (LTs) are potent pro-inflammatory mediators derived from arachidonic acid by the action of 5-lipoxygenase. There are two groups of LTs: LTB4 and cysteinyl LTs (LTC4, LTD4, and LTE4). Both of them play important roles in many inflammatory diseases and allergic responses. Recently, their G-protein coupled receptors have been cloned. The identification of these receptors enables us to analyze their gene structures, regulation of expression, and signal transduction in the cells, and it also leads to the development of useful antagonists. Some LT receptors have been disrupted by gene targeting. Such studies may reveal novel functions of leukotrienes, confirming deeper viewpoints for further research.View full abstractDownload PDF (1745K) -
Takayuki Sugiura, Keizo Waku2002 Volume 132 Issue 1 Pages 7-12
Published: 2002
Released on J-STAGE: November 14, 2008
JOURNAL FREE ACCESSΔ9-Tetrahydrocannabinol, a major psychoactive component of marijuana, has been shown to interact with specific cannabinoid receptors, thereby eliciting a variety of pharmacological responses in experimental animals and human. In 1990, the gene encoding a cannabinoid receptor (CB1) was cloned. This prompted the search for endogenous ligands. In 1992, N-arachidonoylethanolamine (anandamide) was isolated from pig brain as an endogenous ligand, and in 1995, 2-arachidonoylglycerol was isolated from rat brain and canine gut as another endogenous ligand. Both anandamide and 2-arachidonoylglycerol exhibit various cannabimimetic activities. The results of structure-activity relationship experiments, however, revealed that 2-arachidonoylglycerol, but not anandamide, is the intrinsic natural ligand for the cannabinoid receptor. 2-Arachidonoylglycerol is a degradation product of inositol phospholipids that links the function of cannabinoid receptors with the enhanced inositol phospholipid turnover in stimulated tissues and cells. The possible physiological roles of cannabinoid receptors and 2-arachidonoylglycerol in various mammalian tissues such as those of the nervous system are discussed.View full abstractDownload PDF (1851K) -
Keiko Tamiya-Koizumi2002 Volume 132 Issue 1 Pages 13-22
Published: 2002
Released on J-STAGE: November 14, 2008
JOURNAL FREE ACCESSEvidence has been accumulating that nuclear lipid metabolism is involved in the regula-tion of nuclear functions. Here I describe an autonomous nuclear lipid signaling that has been found to be associated with the metabolism of such lipids as phosphoinositides, choline phospholipids, and the acylation and deacylation cycle. Some lipid signals from the plasma membrane ultimately reach the nucleus and regulate the nuclear function. In this case, however, generated lipids and their metabolites may not directly act on the nuclear factors involved in nuclear function. The unique and direct effects of nuclear lipids and their metabolites on nuclear factors are also discussed.View full abstractDownload PDF (1257K) -
Dalia Dasgupta, Samit Adhya, Mukul Kumar Basu2002 Volume 132 Issue 1 Pages 23-27
Published: 2002
Released on J-STAGE: November 14, 2008
JOURNAL FREE ACCESSAn antisense oligonucleotide (20 mer) targeted to the parasite β-tubulin gene and encapsulated in cationic liposomes, was used to test its antileishmanial activity in vitro. Cationic liposomes containing dioleyl trimethyl ammonium propane (DOTAP) were found to have higher antileishmanial activity (88% at 4 μM oligonucleotide) compared to two other liposomes with stearyl amine (SA) and cetyl trimethyl ammonium bromide (CTAB) as cations. Dot-blot experiments were performed to analyse the expression of β-tubulin mRNA using β-tubulin-specific radiolabelled DNA as a probe. When compared with their respective controls, β-tubulin-specific gene expression was found to be diminished by treatment with a specific antisense oligonucleotide encapsulated in cationic liposomes (CTAB:DOPE) in a concentration-dependent manner. These experiments show that antisense oligonucleotides targeted to the β-tubulin gene of Leishmania donovani inhibit β-tubulin synthesis leading to the arrest of multiplication of intracellular para-sites.View full abstractDownload PDF (1463K) -
Chao-Tsai Liao, Hsuen-Chun Liao, Wei-Cheng Huang, Wen-Horng Wang, Ban- ...2002 Volume 132 Issue 1 Pages 29-36
Published: 2002
Released on J-STAGE: November 14, 2008
JOURNAL FREE ACCESSRegion 2.1 of the σ factor is once proposed to be involved in core binding, and certain bulky hydrophobic amino acids in region 2.1 are thought to make contact with the conserved isoleucine residues in the promoter -10 binding region on the same protein. To examine the roles of the contact between these two regions in σA structure and function, σA factor with L145A, I149A, or Y153A was created, and the effects of each substitution on the growth of Bacillus subtilis and on the structural and functional properties of σA were analyzed. Our data revealed that the growth potential of B. subtilis was signifi-cantly affected by each of the substitutions of σA at elevated temperature. The growth defect was most pronounced with the strain containing L145A-σA; it possessed a low growth potential even at 37°C. In parallel, changes in the structural stability and corebinding activity of at and in the promoter-binding and transcription activities of σA-RNA polymerase were observed for each of the substitutions, with the most drastic effects exerted by L145A. Clearly, region 2.1 of σA has extra functions, such as the binding of RNA polymerase to promoter DNA, other than the known core-binding ability. Moreover, the multiple effects of each of the substitutions on crA demonstrate that the contacts between the hydrophobic amino acids in region 2.1 and those in the promoter -10 binding region are critical to the maintenance of the functional o-A structure and that L 145 in region 2.1 plays an important role in this respect.View full abstractDownload PDF (4886K) -
Terumasa Tsuchiya, Yoshihiko Kominato, Masatsugu Ueda2002 Volume 132 Issue 1 Pages 37-44
Published: 2002
Released on J-STAGE: November 14, 2008
JOURNAL FREE ACCESSWe identified a human hypoxic signal transduction pathway acting through a signature motif in the carboxyl terminal of hepatocyte nuclear factor 4 (HNF-4), by functional comparison of the transcriptional and protein-protein interaction activities of the wild type and mutants. It was previously shown that HNF-4 functions as a tissue-specific and hypoxia-activated transcription factor for the erythropoietin (Epo) gene. Human HNF-4 (465 amino acid residues) has DNA-binding, ligand-binding, and transactivation domains. The deletion mutant without the carboxyl terminal transactivation domain (amino acids 369-465) has been shown to be a dominant-negative mutant that repressed Epo transcriptional activity in hypoxia. Further characterization of the hypoxia-responsive domain by site-directed mutagenesis indicated that a TKQE motif of the carboxyl terminal (amino acids 460-463) in HNF-4 was essential for hypoxia-inducible Epo gene expression. We also found, by means of immunoprecipitation and a mammalian twohybrid system, direct interactions between HNF-4 and hypoxia-inducible factor 1 (HIF-1), a heterodimer composed of α and β subunits. HNF-4 was observed to interact with HIF-1α and HIF-1β (arylhydrocarbon receptor nuclear translocator, ARNT) during hypoxia. In addition, the TKQE motif of HNF-4 was essential for protein-protein interac-tions with HIF-la and ARNT. These results indicate that the human hypoxic signal of IHF-1 is transduced through interactions with the signature TKQE motif of the carboxyl terminal of HNF-4 resulting in Epor gene expression as a response to hypoxia.View full abstractDownload PDF (2467K) -
Michele Caraglia, Monica Marra, Gaia Giuberti, Anna Maria D'Alessandro ...2002 Volume 132 Issue 1 Pages 45-52
Published: 2002
Released on J-STAGE: November 14, 2008
JOURNAL FREE ACCESSIt has been reported that theophylline induces growth inhibition and apoptosis in tumour cells. We report that theophylline induces growth inhibition and apoptosis of several human epithelial tumour cells with an IC:50 of 2.5 mM after 48 h of exposure. Moreover, 2.5 mM theophylline induces the accumulation of cancer cells in S-phase of the cell cycle with a concomitant reduction in the percentage of tumour cells in G1/G0 phase. These effects are paralleled by cytoskeletal remodelling with a consequent redistribution of actin fibers and shape change as demonstrated by fluorescence microscopy. The apoptotic death of tumour cells occcurs together with an increase in the expression and activity of the pro-apoptotic enzyme tissue transglutaminase (tTGase). All these effects are promptly antagonized by the specific PKA inhibitor KT5720, suggesting the involvement of cAMP intracellular elevation and, consequently, PKA activation. On the other hand, growth inhibition and tTGase expression and activity are potentiated by retinoic acid, a tTGase inducer. Therefore, a mechanistic model of theophylline action and anti-tumour strategies based on the concomitant use of theophylline and agents that potentiate tTGase activity can be hypothesized.View full abstractDownload PDF (3423K) -
Yuichi Niikura, Takashi Nonaka, Shinobu Imajoh-Ohmi2002 Volume 132 Issue 1 Pages 53-62
Published: 2002
Released on J-STAGE: November 14, 2008
JOURNAL FREE ACCESSWe generated and characterized novel antibodies specific for a cleavage site of human caspase-8/FLICE and its substrate, FLICE-like inhibitory protein (FLIP). The synthetic peptides used as immunogens were CQGDNYQKGIPVETD (#791) and VSEGQLEDSS-LLEVD (#1342), which corresponded to cleaved regions of N-terminal fragments of caspase-8 and FLIP generated by active caspase-8, respectively. Each antibody purified from rabbit antiserum reacted specifically with the immunogen but not with the peptide corresponding to the unproteolyzed form, as assessed by ELISA. In vitro cleavage of GST-FLIP by active caspase-8 generated an N-terminal fragment (GST-p 43) and a C-terminal one (p 12). Consistent with other in vivo data, the FLIP cleavage site follows the Asp residue, LEVD376 GPAMKNVEF, identified on N-terminal sequencing of the p 12 fragment. #1342-antibody (#1342-Ab) recognized the GST-p 43 fragment but not the uncleaved protein, thus confirming its specificity. When the antibodies were used for immunoblot-ting, flow cytometry, and confocal laser microscopy, the proteolysis of caspase-8 and FLIP, and the subcellular localization of their digests could be monitored in apoptotic U 937 cells. Interestingly, a significant increase in the percentage of cells exhibiting caspase-8 and FLIP cleavage was observed upon Fas stimulation in interferon-γ-treated U 937 cells, in which the susceptibility to Fas is extremely enhanced. In contrast, U 937 cells treated with vitamin D3 or all-trans retinoic acid showed Fas-resistance, and caspase-8 processing and FLIP cleavage were strongly inhibited. In conclusion, we established a system based on the cleavage site-directed antibodies to monitor the dynamics of caspase-8 processing and activation during apoptosis. Using this system, we found that Fas-susceptibility changes during U 937 differentiation occur upstream of caspase-8 processing/activation.View full abstractDownload PDF (5494K) -
Masami Ishida, Tairo Oshima2002 Volume 132 Issue 1 Pages 63-70
Published: 2002
Released on J-STAGE: November 14, 2008
JOURNAL FREE ACCESSTo overexpress broad kinds of GC-rich genes in Escherichia coli, we examined how the structures of leader open reading frames (leader ORFs) affect the expression of GC-rich genes, such as polA, trpA, and trpB, from Thermus thermophilus. When a leader ORF overlapped with the polA-initiation codon by 1. by in the TGATG motif, gene expression increased by more than 3-fold compared to when a leader ORF was several-bp distant from the initiation codon. A 4-bp overlap with the ATGA motif was more effective than a 1-bp overlap with the TGATG motif. When a 4-bp overlapping leader ORF was placed in front of the successive trpB and trpA genes, the trpA gene was poorly expressed whereas the trpB gene was overexpressed. Mutation analysis revealed that the expression of the trpA gene was strongly enhanced by replacing G and C in the translation termination region of the leader ORF with A and T. In contrast, other mutations, such as alterations between synonymous codons in the trpA-coding region, produced diminished gene expression. Using the most effective leader ORF obtained from these results, new expression vectors were constructed.View full abstractDownload PDF (4878K) -
Young-Mog Kim, Wakano Ogawa, Eiji Tamai, Teruo Kuroda, Tohru Mizushima ...2002 Volume 132 Issue 1 Pages 71-76
Published: 2002
Released on J-STAGE: November 14, 2008
JOURNAL FREE ACCESSA gene encoding Na+/serine symporter (SstT) of Escherichia coli has been cloned and sequenced in our laboratory [Ogawa et al. (1998) J. Bacterial. 180, 6749-6752]. In an attempt to overproduce the protein and purify it, we first constructed a plasmid pTSTH in which the modified sstT gene (sstT gene with 8 successive codons for His at the 3'-terminus) is located downstream from the trc promoter. Upon induction by IPTG, the Histagged SstT protein was overproduced (about 15% of total membrane proteins), and showed activity as high as the wild type SstT. The His-tagged SstT was solubilized with octylglucoside and purified to homogeneity using a nickel nitrilotriacetic acid (Ni2+-NTA) affinity resin. The N-terminal sequence (20 amino acid residues) of the purified protein showed that the sequence was identical to that deduced from the DNA sequence of the sstT gene and that the initiation methionine was excised. The purified His-tagged SstT was reconstituted into liposomes by the detergent dilution method. Reconstituted proteoliposomes mediated the transport of serine driven by an artificially imposed electrochemical Na+ gradient. The Km and the Vmax values for serine transport with the proteoliposomes were 0.82 μM and 0.37 nmol/min/mg protein, respectively. Serine transport was inhibited by L-threonine, but not by other amino acids. The purified protein was stable for at least 6 months at -80°C.View full abstractDownload PDF (2875K) -
Akira Saito, Masakatsu Usui, Youtao Song, Hiroyuki Azakami, Akio Kato2002 Volume 132 Issue 1 Pages 77-82
Published: 2002
Released on J-STAGE: November 14, 2008
JOURNAL FREE ACCESSThe secretion of N-linked glycosylated α-lactalbumin was much higher in the expression system of yeast Pichia pastoris carrying goat α-lactalbumin cDNA than in mammalian milk. This is possibly because of the presence of N-linked glycosylation signal sequences, Asn45-Asp46-Ser47 and Asn74-Ile75-Ser76, in wild-type α-lactalbumin. Attempts to elucidate the mechanism of the higher secretion of glycosylated α-lactalbumin in P. pastoris were made. Mutant N45D that deleted the N-linked glycosylation signal sequence at position 45 predominantly secreted nonglycosylated protein. On the other hand, mutant D46N with another N-glycosylation signal site at position 46 only secreted N-linked glycosylated α-lactalbumin, i.e. not the nonglycosylated protein. The total secreted amount of mutant N45D was greatly enhanced, while the secreted amounts of the wild-type and mutant D46N were very low, suggesting that the increase in the number of glycosylation sites greatly reduced the secretion of α-lactalbumin. It seems likely that the glycosylated α-lactalbumin may be degraded by the quality control system.View full abstractDownload PDF (3780K) -
Akiko Hirano, Mitsue Kurita-Taniguchi, Yuko Katayama, Misako Matsumoto ...2002 Volume 132 Issue 1 Pages 83-91
Published: 2002
Released on J-STAGE: November 14, 2008
JOURNAL FREE ACCESSCD 46, a complement regulatory protein widely expressed on human cells, serves as an entry receptor for measles virus (MV). We have previously shown that the expression of human CD 46 in mouse macrophages restricts MV replication in these cells and enhances the production of nitric oxide (NO) in the presence of gamma interferon (IFN-γ). In this study, we show that crosslinking human CD 46 expressed on the mouse macrophage-like cell line RAW 264.7 with purified C3b multimer but not monomer enhances NO production. The enhanced production of NO in response to IFN-γ was observed again with C3b multimer but not monomer. The augmentation of NO production is human CD46-dependent with a CYT1>CYT2 profile. Thus, the reported MV-mediated NO production, irrespective of whether it is IFN-γ-dependent or -independent, should be largely attributable to CD 46 signaling but not to MV replication. Similar CYT1-dependent augmentation of NO production was reproducible with two CD 46 ligating reagents, CD46-specific monoclonal antibodies (mAb) or their F(ab')2 and MV hemagglutinin (H) and fusion (F) glycoproteins. Co-cultivation of mouse macrophages bearing human CD 46 with Chinese hamster ovary (CHO) cells expressing MV H and F enhanced 1FN-γ-induced NO production. Yet, the NO levels induced by F(ab')2 against CD 46 or MV H/F on CHO cells were much lower than those induced by CD46-crosslinking mAb with Fc or MV infection. Removing the cytoplasmic tails of CD 46 abrogated the augmentation of NO production triggered by all three stimulators. Thus, the CD 46 CYT 1 and CYT 2 isoforms functionally diverge to elicit innate immune responses, which can be modulated by purified C3b multimer or anti-CD 46 mAbs.View full abstractDownload PDF (5854K) -
Chieko Kimura, Kayo Maeda, Yuichiro Maéda, Masao Miki2002 Volume 132 Issue 1 Pages 93-102
Published: 2002
Released on J-STAGE: November 14, 2008
JOURNAL FREE ACCESSTroponin T (TnT) is an essential component of troponin (Tn) for the Ca2+-regulation of vertebrate striated muscle contraction. TnT consists of an extended NH2-terminal domain that interacts with tropomyosin (Tm) and a globular COOH-terminal domain that interacts with Tm, troponin I (TnI), and troponin C (TnC). We have generated two mutants of a rabbit skeletal β-TnT 25-kDa fragment (59-266) that have a unique cysteine at position 60 (N-terminal region) or 250 (C-terminal region). To understand the spatial rearrangement of TnT on the thin filament in response to Ca2+ binding to TnC, we measured distances from Cys-60 and Cys-250 of TnT to Gln-41 and Cys-374 of F-actin on the reconstituted thin filament by using fluorescence resonance energy transfer (FRET). The distances from Cys-60 and Cys-250 of TnT to Gln-41 of F-actin were 39.5 and 30.0 Å, respectively in the absence of Ca2+, and increased by 2.6 and 5.8 Å, respectively upon binding of Ca2+ to TnC. The rigor binding of myosin subfragment 1 (S1) further increased these distances by 4 and 5 Å respectively, when the thin filaments were fully decorated with S1. This indicates that not only the C-terminal but also the N-terminal region of TnT showed the Ca2+- and S1-induced movement, and the C-terminal region moved more than N-terminal region. In the absence of Ca2+, the rigor S1 binding also increased the distances to the same extent as the presence of Ca2+ when the thin filaments were fully decorated with S1. The addition of ATP completely reversed the changes in FRET induced by rigor S1 binding both in the presence and absence of Ca2+. However, plots of the extent of S1-incuced conformational change vs. molar ratio of S1 to actin showed hyperbolic curve in the presence of Ca2+ but sigmoidal curve in the absence of Ca2+. FRET measurement of the distances from Cys-60 and Cys-250 of TnT to Cys-374 of actin showed almost the same results as the case of Gln-41 of actin. The present FRET measurements demonstrated that not only TnI but also TnT change their positions on the thin filament corresponding to three states of thin filaments (relaxed, Ca2+-induced or closed, and Sl-induced or open states).View full abstractDownload PDF (2460K) -
Jun Hirabayashi, Ko Hayama, Hiroyuki Kaji, Toshiaki Isobe, Ken-ichi Ka ...2002 Volume 132 Issue 1 Pages 103-114
Published: 2002
Released on J-STAGE: November 14, 2008
JOURNAL FREE ACCESSProtein glycosylation is a central issue for post-genomic (proteomic) sciences. We have taken a systematic approach for analyzing soluble glycoproteins produced in the nematode Caenorhabditis elegans. The approach aims at assigning (i) genes that encode glycoproteins, (ii) sites where glycosylation occurs, and (iii) types of attached glycan structures. A soluble extract of C. elegans, as a starting material, was applied first to a concanavalin A (ConA) colunm (specific for high-mannose type N-glycans), and then the flow-through fraction was applied to a galectin LEC-6 (GaL 6) column (specific for complex-type N-glycans). The adsorbed glycoproteins were digested with lysylendopepti-dase, and the resultant glycopeptides were selectively recaptured with the same lectin columns. The glycopeptides were separated by reversed-phase chromatography and then subjected to sequence determination. As a result, 44 and 23 glycopeptides captured by the ConA and GaL 6 columns, respectively, were successfully analyzed and assigned to 32 and 16 corresponding genes, respectively. For these glycopeptides, 49 N-glycosylation sites were experimentally confirmed, whereas 21 sites remained as potential sites. Of the identified genes, about 80% had apparent homologues in other species, as repre-sented by typical secreted proteins. However, the two sets of genes assigned for the ConA and GaL6-recognized glycopeptides showed only 1 overlap with each other. Proof of the practical applicability of the glyco-catch method to a model organism, C. elegans, directs us to explore more complex multicellular organismsView full abstractDownload PDF (3806K) -
Mitsuyoshi Motizuki, Takanori Satoh, Toshiaki Takei, Takehito Itoh, Sa ...2002 Volume 132 Issue 1 Pages 115-119
Published: 2002
Released on J-STAGE: November 14, 2008
JOURNAL FREE ACCESSWe previously showed that bovine apolipoprotein A-II (apoA-II) has antimicrobial activity against Escherichia coli in PBS, and its C-terminal residues 49-76 are responsible for the activity using synthetic peptides. In order to understand the structural requirements of peptide 49-76 for the antimicrobial activity, the N- or C-terminus was trun-cated and then the charged (Lys or Asp) or Ser residues were replaced by Ala. Deletion of the first or last three amino acids and replacement of Lys-54155 or 71/72 by Ala caused a substantial decreases in α-helical content in 50% TFE, showing the possible presence of helices in N- and C-terminal regions, respectively. The anti-Escherichia coli activity of the peptide correlated with its liposome-binding activity. Replacement of Lys-54/55 or 71/72 by Ala resulted in an almost complete loss of anti-E. con activity with a substantial decrease in liposome-binding activity. Moreover, deletion of the last three amino acids caused a reduction to 1/17 of the original anti-E. coli activity with a moderate decrease in liposome-binding activity. In contrast, replacement of Ser-65/66, Asp-59, or Asp-69 by Ma hardly affected the anti-E. coli activity. These findings suggest that Lys-54/55 and Lys-71/72 on the putative helices are critical for antimicrobial activity, and the C-terminal 3 amino acids are important for the structural integrity of the C-terminal region for effective antimicrobial activity.View full abstractDownload PDF (1753K) -
Manoj G. Pillai, Ateeque Ahmad, Toshihiro Yokochi, Toro Nakahara, Yasu ...2002 Volume 132 Issue 1 Pages 121-126
Published: 2002
Released on J-STAGE: November 14, 2008
JOURNAL FREE ACCESSThe incorporation of radiolabeled lipid precursors into triacylglycerol (TG) molecular species in Mortierella ramanniana var. angulispora, an oleaginous fungus, was studied to determine the biosynthetic pathways for TG molecular species. Radiolabeled TG molecular species were separated and quantified by reverse-phase high performance liquid chromatography with a radioisotope detector. The major TG molecular species labeled by [1-14C] oleic acid at 30°C were OOP, OOO, and OPP (TG molecular species designations represent three constituent acyl groups. G, γ-linolenic acid; L, linoleic acid; O, oleic acid; S, stearic acid; P, palmitic acid), which were abundant TG molecular species in this fungus. The incorporation of [1-14C] oleic acid at 15°C into these molecular species was the same, while that into most other species was decreased, suggesting that biosynthesis of major molecular species such as OOP, OOO, and OPP differs from that of other TG molecular species. [1-14C] Linoleic acid incorporation indicated that the major labeled molecular species were LOP and LOO, which may be due to acylation of oleoyl, palmitoyl-glycerol, or dioleoyl-glycerol by exogenous linoleic acid. This is basically the same mechanism as for OOP and OOO biosynthesis from exogenous oleic acid. [14C(U)] Glycerol incorporation suggested that TG molecular species containing palmitic acid such as OPP were more readily synthesized through the de novo pathway. Further experiments involving inhibitors such as sodium azide and cerulenin suggested that OOO biosynthesis included a mechanism differing from that in the cases of OOP and OPP. Trifluoperazine, which inhibits the conversion from phosphatidic acid to TG, decreased [1-14C] oleic acid incorporation into all molecular species, suggesting that the incorporation into all molecular species included the de novo TG biosynthetic pathway via phosphatidic acid. These results revealed that the biosynthetic pathways for TG molecular species can be classified into several groups, which exhibit different sensitivities to low temperature and inhibitors of lipid metabolism. This implies that the composition of TG molecular species is regulated through different biosynthetic pathways responsible for specific TG molecular species, providing a new insight into the biosynthesis of TG molecular species.View full abstractDownload PDF (2592K) -
Miwa Bando, Atsushi Matsuoka, Akihiko Tsuji, Yoshiko Matsuda2002 Volume 132 Issue 1 Pages 127-134
Published: 2002
Released on J-STAGE: November 14, 2008
JOURNAL FREE ACCESSThe differentiation of megakaryocytes into platelets is highly regulated by many cytokines and growth factors. PACE4 and furin are Ca2+-dependent serine endoproteases belonging to the subtilisin-like proprotein convertase (SPC) family. These enzymes are involved in the proteolytic activation of proteins that play essential roles in cell growth and differentiation. In this study, we examined the expression of PACE4 and furin during the differentiation of megakatyoblastic cell lines, Dami and HEL cells, induced by phorbol 12-myristate 13-acetate (PMA). PMA stimulates not only the expression of platelet-derived growth factor-B (PDGF-B) mRNA, but also PACE4 mRNA in these cell lines. The expression of PACE4 transcripts (both the PACE4A and PACE4C/CS isoforms) was upregulated more than 4-fold by PMA. Moreover, direct treatment with PDGF-BB also resulted in an increase in the level of PACE4 mRNA. Further, the effect of PDGF-BB on PACE4 expression was confirmed by promoter assay of the PACE4 gene. Although the furin mRNA level was increased by TGF-/β1 in Dami cells, it was not affected by PDGF-BB. These results indicate for the first time that PACE4 expression is specifically upregulated by PDGF-BB in differentiated megakaryoblasts, suggesting a unique role for PACE4 in platelet production.View full abstractDownload PDF (3951K) -
Hajime Julie Yuasa, Kazuo Kawamura, Hiroaki Yamamoto, Takashi Takagi2002 Volume 132 Issue 1 Pages 135-141
Published: 2002
Released on J-STAGE: November 14, 2008
JOURNAL FREE ACCESSThe organization of troponin I (TnI) genes from the ascidian Halocynthia roretzi have been determined. Halocynthia possesses roughly two types of TnI isoforms. One type is a single-copied adult TnI (adTnI) gene, which contains eight exons and seven introns. adTnI expresses two isoforms, the shorter body wall muscle TnI and the longer cardiac Tnl, through alternative splicing. The mRNAs of these TnI isoforms may undergo transsplicing of the 5'-leader sequences, like the Tnl mRNA of another ascidian species, Ciona intestinalis. The other type comprises multi-copied larval TnI (laTnl) genes. Halocyn-thia has at least three laTnIs (α, β, and γ), which are composed of five exons and four introns, and two of them (α and γ) are clustered in tandem. All laTnIs have B- and M-regions within their 5'-upstream regions, which have been discovered to be the regula-tory elements of Halocynthia larval actin genes. The expression of Halocynthia laTnIs and larval actins may be regulated in the same manner. It is known that Ciona does not possess a larva-specific TnI isoform. The phylogenetic tree of ascidian TnIs suggests that laTnIs might have only been generated within the Pleurogona lineage after Enterogona/Pleurogona divergence, and this scenario well agrees with the absence of laTnIs in Ciona.View full abstractDownload PDF (2201K) -
Alexei V. Cherepanov, Simon de Vries2002 Volume 132 Issue 1 Pages 143-147
Published: 2002
Released on J-STAGE: November 14, 2008
JOURNAL FREE ACCESSScanning mutagenesis is an attractive tool for protein structure-function correlation analysis. With one round of this method it is possible to obtain a library containing all possible single-residue mutants of the protein of interest. The practical application of this approach is currently limited by the large number and cost of the required 30-35mer oligonucleotides. As an alternative, we studied the ligation of shorter DNA oligonucleotides (6-11mer) containing a degenerate binding site and a desired mutation mismatch to a nested set of megaprimers annealed to the gene of interest. T4 DNA ligase was able to perform this task, and the obtained ligation products were elongated by DNA polymerase. The effectiveness of ligation depends on the length of the random binding site of the mutagenic oligonucleotide, on its molar excess over the templateprimer complex and on the position of the mismatching tri-nucleotide insert with respect to the joining site. The secondary structure of the DNA template close to the joining site also influences the ligation yield. Mismatching oligonucleotides, protected by a 3'-phosphate group, were joined to a nested set of megaprimers, the latter being obtained by a novel procedure called reversible chain termination, i.e., termination of the dsDNA synthesis with ddNTP followed by the subsequent removal of the incorporated ddNMP with exonuclease III. T7 sequenase 2.0 DNA polymerase elongated the ligation products after the 3'-phosphate protection group was removed with T4 polynucleotide kinase, resulting in the incorporation of a specific tri-nucleotide mismatch into dsDNA. This sequence of reactions serves as the basis for a novel scanning mutagenesis procedure.View full abstractDownload PDF (627K) -
Tomoko Kuwabara, Masaki Warashina, Kazunari Taira2002 Volume 132 Issue 1 Pages 149-155
Published: 2002
Released on J-STAGE: November 14, 2008
JOURNAL FREE ACCESSTo overcome obstacles to target site selection, we recently created a novel hybrid ribozyme that could access any chosen site by the recruitment of intracellular RNA helicases [Warashina et al. (2001) Proc. Natl. Acad. Sci. USA 98, 5572-5577; Kawasaki et al. (2002) Nat. Biotech. 20, 376-380]. We also demonstrated previously that pol III-driven maxizymes with two substrate-binding arms that were directed against two different sites within a target mRNA formed very active heterodimers in vivo [Kuwabara, et al. (2000) Trends Biotechnol. 18, 462-468; Tanabe et al. (2001) Nature 406, 473-474]. Despite the complicated dimerization process, all the maxizymes that we tested in cultured cells had greater catalytic activity than the parental ribozymes. To investigate the action of maxizymes in cells, we designed a specific maxizyme with two substrate-binding arms that was directed against endogenously expressed LTR-luciferase chimeric mRNA, where LTR refers to the long terminal repeat of HIV-1. One substrate-binding arm of the maxizyme was designed to bind to a site within HIV-1 TAR RNA that is known to form a stable stem structure that normally prevents binding of a ribozyme. The other substrate-binding arm was directed against a relatively accessible site within the luciferase gene. As expected, the conventional ribozyme failed to cleave the TAR region in vivo because of the latter's stable secondary structure. However, to our surprise, the maxizyme cleaved the TAR region within the stem with high efficiency in vivo. The enhanced cleavage in vivo by the maxizyme might have resulted from an entropically favorable, intramolecular, second binding process that occurred during the breathing of the stem structure of the target mRNA. Importantly, our data suggest that this maxizyme technology might be used as an alternative approach to the recruitment of RNA helicases in cleaving sites previously found to be inaccessible.View full abstractDownload PDF (3942K)
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