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Volume 135 (2004)
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Volume 135, Issue 3
Displaying 1-20 of 20 articles from this issue
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Tsuyoshi Inoue, Yousuke Okano, Yuji Kado, Kousuke Aritake, Daisuke Iri ...2004 Volume 135 Issue 3 Pages 279-283
Published: March 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSHematopoietic prostaglandin (PG) D synthase (H-PGDS) is responsible for the production of PGD2 as an allergy or inflammation mediator in mast and Th2 cells. We determined the X-ray structure of human H-PGDS complexed with an inhibitor, 2-(2'-benzothiazolyl)-5-styryl-3-(4'-phthalhydrazidyl) tetrazolium chloride (BSPT) at 1.9 Å resolution in the presence of Mg2+. The styryl group of the inhibitor penetrated to the bottom of the active site cleft, and the tetrazole ring was stabilized by the stacking interaction with Trp 104, inducing large movement around the α 5-helix, which caused the space group of the complex crystal to change from P21 to P1 upon binding of BSPT. The phthalhydrazidyl group of BSPT exhibited steric hindrance due to the cofactor, glutathione (GSH), increasing the IC50 value of BSPT for human H-PGDS from 36.2 μM to 98.1 μM upon binding of Mg2+, because the Km value of GSH for human H-PGDS was decreased from 0.60 μM in the presence of EDTA to 0.14 μM in the presence of Mg2+. We have to avoid steric hindrance of the GSH molecule that was stabilized by intracellular Mg2+ in the mM range in the cytosol for further development of structure-based anti-allergic drugs.View full abstractDownload PDF (3030K) -
Masato Yonezawa, Nobuhide Doi, Toru Higashinakagawa, Hiroshi Yanagawa2004 Volume 135 Issue 3 Pages 285-288
Published: March 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSIn vitro display technologies are powerful tools for screening peptides with desired functions. We previously proposed a DNA display system in which streptavidin-fused peptides are linked with their encoding DNAs via biotin labels in emulsion compartments and successfully applied it to the screening of random peptide libraries. Here we describe its application to functional and folded proteins. By introducing peptide linkers between streptavidin and fused proteins, we achieved highly efficient (>95%) formation of DNA-protein conjugates. Furthermore, we successfully enriched a glutathione-S-transferase gene by a factor of 20-30-fold per round on glutathione-coupled beads. Thus, DNA display should be useful for rapidly screening or evolving proteins based on affinity selection.View full abstractDownload PDF (3258K) -
Hiroyuki Okano, Masayo Asakawa, Yoshikazu Ohya2004 Volume 135 Issue 3 Pages 289-295
Published: March 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSCalmodulin (CaM) performs essential functions in cell proliferation in Saccharomyces cerevisiae. Previously, we isolated fourteen temperature-sensitive Phe-to-Ala mutations of the CaM-encoding gene CMD1. These mutations were classified into four intragenic complementation groups, suggesting that each group represents a loss of CaM interaction with its specific essential target protein. Nuf1p/Spc110p, one of the essential targets, is a spindle pole body component that is required for proper mitosis. We investigated which intragenic complementation group of CaM represents the malfunction of Nuf1p. Immunoprecipitation analysis showed that two cmd1 mutations belonging to two distinct intragenic complementation groups had the most severely impaired complex formation with Nuf1p at the restrictive temperature. The temperature-sensitive growth of these cmd1 mutants was suppressed by a CaM-independent dominant allele of NUF1. Additionally, these mutants displayed characteristic mitotic defects: an increased ratio of artificial chromosome loss, which could be suppressed by the CaM-independent dominant allele of NUF1, and aberrant microtubule structures. These results indicate that these cmd1 mutants display the temperature-sensitive growth due to the compromised interaction with Nuf1p. However, the interaction was restored in a heterozygous diploid of the two cmd1 alleles, suggesting that intragenic complementation between these cmd1 alleles occurs by a novel mechanism, whereby co-presence of both mutant proteins rescues the interaction with Nuf1p.View full abstractDownload PDF (4301K) -
Weidong Pan, Xianghui Liu, Feng Ge, Jie Han, Tao Zheng2004 Volume 135 Issue 3 Pages 297-304
Published: March 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSA novel antimicrobial peptide was isolated and partially characterized from the homogenate of an Asian marine clamworm, Perinereis aibuhitensis Grube. This novel peptide, named Perinerin, was purified to homogeneity by heparin-affinity column and reverse-phase HPLC, and biologically tested with a MTS-PMS colorimetric assay. Perinerin consists of 51 amino acid residues and structurally appears to be highly basic and hydrophobic. It shows marked activity in vitro against both Gram-negative and Gram-positive bacteria and fungi, which indicates a bactericidal effect as well. Perinerin appears to be constitutively present and its sequence is novel among all other known antimicrobial peptides. These results suggest that Perinerin has the potential to serve as a convenient “evaluation marker” for studying alterations in the biochemistry of the host, particularly with respect to environmental changes. In addition, the MTS-PMS colorimetric assay examination of antimicrobial activity appears to be superior to existing methods and may offer more general application in the search for new antibiotic molecules.View full abstractDownload PDF (3344K) -
Kouhei Ohnishi, Akiko Okuta, Jiansong Ju, Tohru Hamada, Haruo Misono, ...2004 Volume 135 Issue 3 Pages 305-317
Published: March 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESS3-Chlorobiphenyl is known to be mineralized by biphenyl-utilizing bacteria to 3-chlorobenzoate, which is further metabolized to 3-chlorocatechol. An extradiol dioxygenase, 2, 3-dihydroxybiphenyl 1, 2-dioxygenase (DHB12O; EC 1.13. 11.39), which is encoded by the bphC gene, catalyzes the third step of the upper pathway of 3-chlorobiphenyl degradation. In this study, two full-length bphCs and nine partial fragments of bphCs fused to the 3' end of bphC in Pseudomonas pseudoalcaligenes KF707 were cloned from different biphenyl-utilizing soil bacteria and expressed in Escherichia coli. The enzyme activities of the expressed DHB12Os were inhibited to varying degrees by 3-chlorocatechol, and the E. coli cells overexpressing DHB12O could not grow or grew very slowly in the presence of 3-chlorocatechol. These sensitivities of enzyme activity and cell growth to 3-chlorocatechol were well correlated, and this phenomenon was employed in screening chimeric BphCs formed by family shuffling of bphC genes isolated from Comamonas testosteroni KF704 and C. testosteroni KF712. The resultant DHB12Os were more resistant by a factor of two to 3-chlorocatechol than one of the best parents, KF707 DHB12O.View full abstractDownload PDF (5760K) -
Yuichi Hashimoto, Takahiko Matsuda, Yoshiharu Matsuura, Tatsuya Haga, ...2004 Volume 135 Issue 3 Pages 319-329
Published: March 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe α-subunit of rod photoreceptor G protein transducin (Gt1α) is heterogeneously modified at the N-terminus by a mixture of acyl groups, laurate (C12:0), myristate (C14:0), and two unsaturated fatty acids (C14:1 and C14:2). Although the N-fatty acylation of Gt1α plays important roles in protein-protein and protein-membrane interactions in light signaling, the biological significance of the heterogeneous acylation remains unclear due to the difficulty in isolating each Gt1α isoform from the retinal rod cells. Here we found that Gt1α/Gi1α chimera (Gt/iα) expressed in Sf9 cells is also heterogeneously modified by myristate (_??_90%) and laurate (_??_10%), raising the possibility that the N-acyl group of recombinant Gt/iα may be manipulated by modifying culture media. In fact, addition of myristic acid to the medium decreased the relative content of lauroylated Gt/iα to an undetectable level, whereas exogenously added lauric acid significantly increased the relative content of lauroylated Gt/iα in a concentrationdependent manner. By culturing the Gt/iα-virus infected Sf9 cells with fatty acids, we obtained four different preparations of N-acylated Gt/iα, in which the relative abundance of lauroylated isoform was 0%, 20%, 33% and _??_70%, respectively. Functional analysis of these proteins showed that an increase in the relative content of the lauroylated isoform remarkably slowed down the steady-state GTP hydrolysis rate of Gt/iα; the steady-state GTPase activity of the lauroylated isoform was estimated to be one order of magnitude lower than that of the myristoylated isoform. These results suggest that the retinal Gt1α is composed of isoforms having functionally heterogeneous signaling properties.View full abstractDownload PDF (5504K) -
Hebok Song, Sin-Il Kim, Myoung Seok Ko, Hyo Jeong Kim, Jin Chul Heo, H ...2004 Volume 135 Issue 3 Pages 331-335
Published: March 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSDRG2, a member of the DRG subfamily in the GTP-binding protein superfamily, was identified as a repressed gene product in fibroblasts transformed by SV40. The significance of this down-regulation and the cellular role of DRG2 has not been understood in the past. To investigate the function of DRG 2 we made a Jurkat cell line, Jurkat-LNCX2-DRG2, stably transfected with pLNCX2-DRG2 to overexpress human DRG2. Cell cycle distribution analysis revealed an increased accumulation of G2/M phase cells in Jurkat-LNCX2-DRG2 cells, indicating a retardation of cell-cycle progression. In addition, an overexpression of DRG2 reduced the sensitivity of Jurkat cells to the mitotic poison nocodazole. Our data suggest that overexpression of DRG2 in Jurkat cells affects genes regulating cell-cycle arrest and apoptosis, and that these molecular changes may be important in the growth or differentiation of cells.View full abstractDownload PDF (1996K) -
Sung Jean Park, Ji-Sun Kim, Woo-Sung Son, Bong Jin Lee2004 Volume 135 Issue 3 Pages 337-346
Published: March 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSAcyl carrier protein (ACP) is a small acidic protein and its primary structure is highly conserved in various bacterial sources. Despite its small size, it interacts with diverse proteins associated with many biosynthetic pathways. The three-dimensional structure of H. pylori ACP and its structural characteristics were clarified using NAM and CD spectroscopy. H. pylori ACP consists of four helices connected by different sized loops. The helices correspond to residues L3-Q14 (αI), S36-G50 (αII), D56-E60 (αIII), and V65-K76 (αVI). The size of each helix differs slightly from that of homologous ACPs. However, H. pylori ACP showed a distinct pH-dependent conformational characteristic: at neutral pH, it adopts a partially unfolded structure, while it has a tight fold at pH 6. The chemical shift perturbation and 1H-15N steady state NOE analysis at both pH 6 and 7 showed that the local change of structural components occurred mainly around loop II, and this change was reflected by the changes of the residues Ile 54 and Asp 56. Examination of the structure showed that the network of Glu 47, Ile 54, Asn 75, and Lys 76 is very important for the structural stability. The pH-dependent folding process shows a kind of cooperativity, since all the residues involved in the conformational transitions are contiguous and in spatial proximity.View full abstractDownload PDF (3390K) -
Masanao Minato, Satoshi Kubota, Harumi Kawaki, Takashi Nishida, Akira ...2004 Volume 135 Issue 3 Pages 347-354
Published: March 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSConnective tissue growth factor/hypertrophic chondrocyte specific gene product 24 (CTGF/Hcs24/CCN2) shows diverse functions in the process of endochondral ossification. It promotes not only the proliferation and differentiation of chondrocytes and osteoblasts in vitro, but also angiogenesis in vivo. The ctgf gene is a member of the gene family called CCN, and it encodes the characteristic 4-module structure of this family, with the protein containing IGFBP, VWC, TSP and CT modules. We raised several monoclonal antibodies and polyclonal antisera against CTGF, and located the epitopes in the modules by Western blotting. For mapping the epitopes, Brevibacillu-sproduced independent modules were utilized. As a result, at least 1 antibody or antiserum was prepared for the detection of each module in CTGF. Western blotting with these antibodies is expected to be useful for the analysis of CTGF fragmentation. Moreover, we examined the effects of these monoclonal antibodies on the biological functions of CTGF. One out of 3 humanized monoclonal antibodies was found to neutralize efficiently the stimulatory effect of CTGF on chondrocytic cell proliferation. This particular antibody bound to the CT module. In contrast, surprisingly, all of the 3 antibodies recognizing IGFBP, VWC and CT modules stimulated proteoglycan synthesis in chondrocytic cells. Together with previous findings, these results provide insight into the structural-functional relationships of CTGF in executing multiple functions.View full abstractDownload PDF (2223K) -
Zhengmao Ye, Hideo Miyake, Maki Tatsumi, Shigenori Nishimura, Yasunori ...2004 Volume 135 Issue 3 Pages 355-363
Published: March 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSIn the previous X-ray crystallographic study, it was found that β-amylase from Bacillus cereus var. mycoides has three carbohydrate-binding sites aside from the active site: two (Site2 and Site3) in domain B and one (Site1) in domain C. To investigate the roles of these sites in the catalytic reaction and raw starch-binding, Site1 and Site2 were mutated. From analyses of the raw starch-binding of wild-type and mutant enzymes, it was found that Sitel contributes to the binding affinity to raw-starch more than Site2, and that the binding capacity is maintained when either Site1 or Site2 exists. The raw starch-digesting ability of this enzyme was poor. From inhibition studies by maltitol, GGX and α-CD for hydrolyses of maltopentaose (G5) and amylose (-DPn=16) catalyzed by wild-type and mutant enzymes, it was found that α-CD is a competitive inhibitor, while, maltitol behaves as a mixed-type or competitive inhibitor depending on the chain length of the substrate and the mutant enzyme. From the analysis of the inhibition mechanism, we conclude that the bindings of maltitol and GGX to Site2 in domain B form an abortive ESI complex when amylose (-DPn=16) is used as a substrate.View full abstractDownload PDF (2327K) -
Hideharu Ochiai, Kazunari Higa, Hiroshi Fujise2004 Volume 135 Issue 3 Pages 365-374
Published: March 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSKCC1 cDNA was cloned in dog erythroblasts that had differentiated from peripheral mononuclear cells. The size of the cDNA was 3, 258 bp, the same as in pigs, but 3 bp longer than in humans and rodents. The dog KCC1 cDNA encodes for 1, 086 amino acid residues with a calculated molecular mass of 120 kDa. The 560 bp cDNA fragment from position 679 to 1, 238 in the full length cDNA from the dog erythroblasts was 100% identical to that in the kidney. Hydropathy analysis showed that the structure of dog KCC1 was similar to in other species; 12 trans membrane domains, four glycosylation sites in loop 5, and 17 consensus phosphorylation sites in the cytosol. However, there were variations in dog KCC1 compared to in other species; there was one CK2 phosphorylation site that was found only in dog KCC1. There were also substitutions of amino acids that affect pH sensitivity (His) and change acidic/basic residues or charged residues. In HEK 293 cells transfected with dog KCC1 cDNA (HEK-dKCC1), the Rb influx, which was ouabain-resistant, Cl-dependent, N-ethyl maleimide (NEM)-stimulative and Na-independent, was measured as for K-Cl cotransport, and the influx was found to be increased _??_3 fold in HEK-dKCC1 compared to in the control. This ouabain-resistant Cl-dependent Rb influx was also volume-sensitive in hyposmotic medium, and the volume-sensitive component was inhibited by furosemide. Thus, the KCC1 cDNA cloned in dog erythroblasts encodes a volume-sensitive K-Cl cotransporter.View full abstractDownload PDF (4993K) -
Kenji Fukui, Ryoji Masui, Seiki Kuramitsu2004 Volume 135 Issue 3 Pages 375-384
Published: March 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe mismatch repair system (MMR) recognizes and corrects mismatched or unpaired bases caused mainly by DNA polymerase, and contributes to the fidelity of DNA replication in living cells. In Escherichia coli, the MutHLS system is known to function in MMR, and homologues of MutS and MutL are widely conserved in almost all organisms. However, the MutH endonuclease has not been found in the majority of organisms. Such organisms, including Thermus thermophilus HB8, often possess the socalled MutS2 protein, which is highly homologous to MutS but contains an extra Cterminal stretch. To elucidate the function of MutS2, we overexpressed and purified T. thermophilus MutS2 (ttMutS2). ttMutS2 demonstrated the ability to bind doublestranded (ds) DNA, but, unlike ttMutS, ttMutS2 showed no specificity for mismatched duplexes. ttMutS2 ATPase activity was also detected and was stimulated by dsDNA. Our results also showed that ttMutS2 incises dsDNA. ttMutS2 incises not only oligo dsDNA but also plasmid DNA, suggesting that ttMutS2 possesses an endonuclease activity. At low concentrations, the incision activity was not retained, but was promoted by T. thermophilus MutL.View full abstractDownload PDF (7241K) -
Emi Mizuno, Kensuke Kawahata, Aya Okamoto, Naomi Kitamura, Masayuki Ko ...2004 Volume 135 Issue 3 Pages 385-396
Published: March 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSMembers of the STAM family of proteins, STAM1 and STAM2, are associated with Hrs through their coiled-coil regions. Both Hrs and STAM bind ubiquitin and are involved in endosomal sorting of ubiquitinated cargo proteins for trafficking to the lysosome. Here we examined the biological significance of STAM binding to Hrs. Endogenous STAM1 and STAM2 were mostly localized on the early endosome, suggesting that they are resident endosomal proteins. A STAM2 mutant that lacks the coiled-coil region and does not bind Hrs, in contrast, mislocalized to the cytoplasm. Deletion of a region located N-terminal to the coiled-coil region and conserved among STAM proteins also severely affected Hrs binding and the endosomal localization of STAM2, suggesting that this region is also involved in these activities. Depletion of endogenous Hrs by RNA interference similarly caused the mislocalization of exogenously expressed STAM2 to the cytoplasm. These results indicate that STAM is localized to the early endosome by binding to Hrs on the target membrane. In addition, the expression level of endogenous STAM proteins was drastically reduced in Hrs-depleted cells, suggesting that STAM is stabilized by binding to Hrs. Finally, STAM2 mutants lacking the Hrs-binding activity were defective in causing the enlargement of early endosomes, accumulating ubiquitinated proteins on this aberrant organelle, and inhibiting the degradation of ligand-activated epidermal growth factor receptors, suggesting that the association with Hrs is a prerequisite for STAM function.View full abstractDownload PDF (9220K) -
Liang Cai, Changyun Hu, Shuiyuan Shen, Weirong Wang, Weida Huang2004 Volume 135 Issue 3 Pages 397-403
Published: March 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSDNA ligases of bacteriophage T4 and T7 have been widely used in molecular biology for decades, but little is known about bacteriophage T3 DNA ligase. Here is the first report on the cloning, expression and biochemical characterization of bacteriophage T3 DNA ligase. The polyhistidine-tagged recombinant T3 DNA ligase was shown to be an ATP-dependent enzyme. The enzymatic activity was not affected by high concentration of monovalent cations up to 1 M, whereas 2mM ATP could inhibit its activity by 50%. Under optimal conditions (pH 8.0, 0.5mM ATP, 5mM DTT, 1mM Mg2+ and 300mM Na+), 1 fmol of T3 DNA ligase could achieve 90% ligation of 450 fmol of cohesive dsDNA fragments in 30min. T3 DNA ligase was shown to be over 5-fold more efficient than T4 DNA ligase for ligation of cohesive DNA fragments, but less active for bluntended DNA fragments. Phylogenetic analysis showed that T3 DNA ligase is more closely related to T7 DNA ligase than to T4 DNA ligase.View full abstractDownload PDF (2189K) -
Koichi Matsuo, Ryuta Yonehara, Kunihiko Gekko2004 Volume 135 Issue 3 Pages 405-411
Published: March 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe vacuum ultraviolet circular dichroism (VUVCD) spectra of 15 globular proteins (myoglobin, hemoglobin, human serum albumin, cytochrome c, peroxidase, α-lactalbumin, lysozyme, ovalbumin, ribonuclease A, β-lactoglobulin, pepsin, trypsinogen, α-chymotrypsinogen, soybean trypsin inhibitor, and concanavalin A) were measured in aqueous solutions at 25°C in the wavelength region from 260 to 160 nm under a high vacuum, using a synchrotron-radiation VUVCD spectrophotometer. The VUVCD spectra below 190 nm revealed some characteristic bands corresponding to different secondary structures. The contents of α-helices, β-strands, turns, and unordered structures were estimated using the SELCON3 program with VUVCD spectra data on the 15 proteins. Prediction of the secondary-structure contents was greatly improved by extending the circular dichroism spectra to 165 nm. The numbers of α-helix and β-strand segments calculated from the distorted α-helix and β-strand contents did not differ greatly from those obtained from X-ray crystal structures. These results demonstrate that synchrotron-radiation VUVCD spectroscopy is a powerful tool for analyzing the secondary structures of proteins.View full abstractDownload PDF (812K) -
Nobuhiro Ijichi, Naomi Tsujimoto, Toru Iwaki, Yasuyuki Fukumaki, Akiko ...2004 Volume 135 Issue 3 Pages 413-420
Published: March 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSαB-Crystallin, a member of the small heat shock protein (sHSP) family, is expressed in various tissues including lens, heart, and skeletal muscle. Previously we identified the gene of HSPB2, another member of the sHSP family, located 1-kb upstream of the αB-crystallin gene in a head-to-head manner. In the present study, we found a highly conserved region of 220 by approximately 2.4-kb upstream of the all-crystallin gene and examined its role in expression of the αB-crystallin gene. Transgenic mice containing 3 kb of the upstream sequence of the αB-crystallin gene showed lacZ reporter gene expression in the lens as well as the myotome and heart on embryonic day 12.5. Deletion analysis revealed that the -2656/-2267 region including the conserved region with four putative Sox binding elements (E1-E4) exhibits lens enhancer activity toward the αB-crystallin promoter. Gel shift assays showed that the Sox1 and Sox2 proteins preferentially bound to E2 and E4. Moreover, disruption of E2 and E4 abolished the reporter gene expression in the lens. These results indicate that the newly identified enhancer with Sox elements activates the αB-crystallin promoter in the lens, although they are separated by the entire HSPB2 gene.View full abstractDownload PDF (4643K) -
Hiroshi Oneda, Seungjae Lee, Kuniyo Inouye2004 Volume 135 Issue 3 Pages 421-427
Published: March 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe inhibitory effect of 0.19 α-amylase inhibitor (0.19 AI) from wheat kernel on the porcine pancreas α-amylase (PPA)-catalyzed hydrolysis of p-nitrophenyl-α-D-maltoside (pNP-G2) was examined. 0.19 AI is a homodimer of 26.6 kDa with 13.3-kDa subunits under the conditions used. The elution behaviors in gel filtration HPLC of PPA and 0.19 AI indicated that a PPA molecule bound with a 0.19 AI molecule (homodimer) at a molar ratio of 1:1. 0.19 AI inhibited PPA activity in a competitive manner with an inhibitor constant, Ki, of 57.3 nM at pH 6.9, 30°C, and the binding between them was found to be endothermic and entropy-driven. The activation energy for the thermal inactivation of 0.19 AI was determined to be 87.0 kJ/mol, and the temperature, T50, giving 50% inactivation in a 30-min incubation at pH 6.9 was 88.1°C. The high inhibitory activity of 0.19 AI against PPA and its high thermal stability suggest its potential for use in the prevention and therapy of obesity and diabetes.View full abstractDownload PDF (1807K) -
Joseph L. Dynes, Shuping Xu, Sarah Bothner, Jill M. Lahti, Roderick T. ...2004 Volume 135 Issue 3 Pages 429-438
Published: March 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe protein complex Selectivity Factor 1, composed of TBP, TAFI48, TAFI63 and TAFI110, is required for rRNA transcription by RNA polymerase I in the nucleolus. The steps involved in targeting Selectivity Factor 1 will be dependent on the transport pathways that are used and the localization signals that direct this trafficking. In order to investigate these issues, we characterized human TAFI48, a subunit of Selectivity Factor 1. By domain analysis of TAFI48, the carboxyl-terminal 51 residues were found to be required for the localization of TAFI48, as well as sufficient to direct Green Fluorescent Protein to the nucleus and nucleolus. The carboxyl-terminus of TAFI48 also has the ability to associate with multiple members of the β-karyopherin family of nuclear import receptors, including importin β (karyopherin β1), transportin (karyopherin β2) and RanBP5 (karyopherin β3), in a Ran-dependent manner. This property of interacting with multiple β-karyopherins has been previously reported for the nuclear localization signals of some ribosomal proteins that are likewise directed to the nucleolus. This study identifies the first nuclear import sequence identified within the TBP-Associated Factor subunits of Selectivity Factor 1.View full abstractDownload PDF (7986K) -
Koji Shimoke, Hisayuki Amano, Soichiro Kishi, Hitoshi Uchida, Motoshig ...2004 Volume 135 Issue 3 Pages 439-446
Published: March 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSFollowing endoplasmic reticulum (ER) stress, which occurs via inhibition of the glyc-osylation of newly synthesized proteins, caspase family proteins are activated to promote ER stress-mediated apoptosis. Here we report that nerve growth factor (NGF) suppressed the ER stress-mediated apoptosis in tunicamycin-treated PC12 cells through an extensive decrease of the caspase-3/-9/-12 activity. Detailed analysis of the mechanism underlying the NGF-mediated cell survival revealed that the activities of all seriate caspases were reduced through the phosphatidylinositol 3-kinase (PI3-K) signaling pathway induced by NGF. Moreover, we found that the activity of c-Jun N-terminal kinase (JNK) was not essential for the tunicamycin-induced apoptosis of PC 12 cells. These results demonstrate that the inactivation of caspase-12 via the NGFmediated PI3-K signaling pathway leads to inactivation of the caspase cascade including caspase-3 and -9.View full abstractDownload PDF (5649K) -
Shogo Misumi, Yukimi Morikawa, Mitsunori Tomonaga, Kouichi Ohkuma, Nob ...2004 Volume 135 Issue 3 Pages 447-453
Published: March 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSOur previous study suggested that the p2gag peptide, AEAMSQVTNTATIM, inhibits human immunodeficiency virus type 1 (HIV-1) protease (PR) activity in vitro. In this study, Ala substitutions (Met4Ala and Thr8Ala) and deletion of amino acid Asn9 within the nona p2gag peptide (AEAMSQVTN) were found to decrease the inhibitory effect on HIV-1 PR activity. Furthermore, treatment of PMA-activated latently infected T lymphocytes, ACH-2 cells, with the p2gag peptide (100 and 250 μM) resulted in a decrease in the amount of p24gag in the resultant viral lysates derived from the cell-free supernatant. In addition, the HIV-1-Tat-p2gag fusion peptide was synthesized to effectively deliver the p2gag peptide into the cells. The fusion peptide was incorporated into chronically infected T lymphocytes, CEM/LAV1 cells, as detected on indirect immunofluorescence analysis using anti-p2gag peptide monoclonal antibodies, which recognize the nona peptide (AEAMSQVTN) derived from the N-terminus of the p2gag peptide, and cleaved by HIV-1 PR in vitro. Treatment of CEM/LAV-1 cells with the fusion peptide also resulted in a decrease in the amount of p24gag in the resultant viral lysate derived from the cell-free supernatant. Taken together, these data suggest that the p2gag peptide consequently blocks the autolysis of HIV-1 virions for the conservation of viral species.View full abstractDownload PDF (3872K)
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