巻号一覧
131 巻, 4 号
選択された号の論文の17件中1～17を表示しています
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Takehiko Sasaki, Akira Suzuki, Junko Sasaki, Josef M. Penninger2002 年 131 巻 4 号 p. 495-501
発行日: 2002/04/01
公開日: 2008/06/30
ジャーナル フリーPhosphoinositide 3-kinases (PI3Ks) constitute a family of evolutionarily conserved lipid kinases that phosphorylate the D3 position of the inositol ring of phosphoinositides and produce PI(3)P, PI(3, 4)P2, and PI(3, 4, 5)P3. Intense in vitro research over the last decade has unequivocally demonstrated that PI3Ks, in particular those belonging to class I, regulate a vast array of fundamental cellular responses. Given the pleiotropic roles of PI3Ks and the lipid product PI(3, 4, 5)P3 in plethora of cellular responses, it is pertinent to explore the significance of PI3K signaling in vivo. In the past two or three years, the components of this signaling pathway have been genetically manipulated in mouse. This review briefly summarizes the immunological significance of PI3K signaling as revealed by the study of gene-targeted “knockout” mice.抄録全体を表示PDF形式でダウンロード (988K) -
Yasunori Kanaho, Teruhiko Suzuki2002 年 131 巻 4 号 p. 503-509
発行日: 2002/04/01
公開日: 2008/06/30
ジャーナル フリーPhosphoinositide kinases comprise a unique family of enzymes that catalyze the phosphorylation of phosphatidylinositol and its phosphorylated metabolites to produce seven phosphoinositides. Recent advances have revealed that these phosphoinositides have specific physiological functions, such as actin cytoskeletal reorganization, membrane transport, cell proliferation and survival, in eukaryotic cells and that each phosphoinositide kinase is differently and precisely regulated. Here we describe the diverse regulation and physiological functions of phosphoinositide kinases involving their products.抄録全体を表示PDF形式でダウンロード (888K) -
Teruko Toyoda, Tsutomu Arakawa, Haruki Yamaguchi2002 年 131 巻 4 号 p. 511-515
発行日: 2002/04/01
公開日: 2008/06/30
ジャーナル フリーHuman erythropoietin (EPO) produced in Chinese hamster ovary cells is a hydrophobic protein highly stabilized by multibranched complex-type N-glycans. To reveal the molecular basis of the interaction between the N-glycans and the EPO protein, complex-type N-glycans of different structures were analyzed as to their binding affinity for Escherichia coli-expressed EPO by means of the surface plasmon resonance technique. It appears well established that complex-type N-glycans, particularly multibranched ones, have hydrophobic regions that extensively stretch across the plane holding acetylamino groups and that N-glycan-protein hydrophobic interactions characterized by a slow rate of dissociation stabilize the protein conformation.抄録全体を表示PDF形式でダウンロード (1747K) -
Masaru Koma, Shuji Miyagawa, Koichi Honke, Rie Nakai, Shinichirou Miyo ...2002 年 131 巻 4 号 p. 517-522
発行日: 2002/04/01
公開日: 2008/06/30
ジャーナル フリーGlycoprotein-3-sulfotransferase (GP3ST) is a key enzyme in downregulating the expression of Galαl, 3Galβ1, 4GlcNAc-R (the α-Gal epitope), via enzymatic competition with an α1, 3 galactosyltransferase (α1, 3GT), such as α2, 6 sialyltransferase (α2, 6ST). In this study, we report the dominance of GP3ST over α1, 3GT using transfected pig endothelial cell (PEC) lines. The introduction of the GP3ST gene into PEC suppresses its antigenicity with respect to normal human pooled serum (NHS), including the α-Gal epitope and the Hanganutziu-Deicher (H-D) antigen, and, in addition, reduces the susceptibility to NHS in complement-mediated cell lysis. Western and lectin blot analyses of the products of parental PEC and its transfectants indicated that proteins smaller than 66 kDa have a diminished reactivity with NHS and the IB4 lectin. The levels of the α-Gal epitope in neutral glycosphingolipids were also decreased in the GP3ST transfectants as detected in thin layer chromatography by immunostaining. These data indicate that GP3ST is very effective in reducing xenoepitope levels.抄録全体を表示PDF形式でダウンロード (2147K) -
Hirofumi Nakajima, Tetsuo Ishida, Hiroyuki Tanaka, Kihachiro Horiike2002 年 131 巻 4 号 p. 523-531
発行日: 2002/04/01
公開日: 2008/06/30
ジャーナル フリーAn enzymatic method for measuring the O2 concentrations of aqueous solutions was developed by involving 4-chlorocatechol and catechol 2, 3-dioxygenase from Pseudomonas putida. With this system, the amount of O2 in a sample solution can be measured as the amount of 5-chloro-2-hydroxymuconate semialdehyde formed through the enzyme reaction. The product was stable and its anion exhibited strong absorption around 380nm (molar absorption coefficient of 4.3×104 M-1 cm-1, pK value of 5.4). A sensitive HPLC method involving a BioAssist Q column was developed to individually quantify the products derived from 4-chlorocatechol and catechol. When the O2 concentration in a sample solution sealed in a vial was lowered from the air-saturation level by means of the amount enzymatically reacted with a known amount of catechol, the concentration of remaining O2 could be successfully measured by the HPLC method. We developed devices through which reagents could be added to solutions sealed in cuvettes or the vessel of an oxygen electrode system under a flow of argon. By applying these devices, the submicromolar O2 concentration of an anoxic solution and the back diffusion of O2 from the atmosphere could be directly determined for the first time. The Km values of the dioxygenase and an ascorbate oxidase for oxygen were also determined to be 7.2 (at pH 7.5) and 114 μM (at pH 6.5), respectively, at 25°C.抄録全体を表示PDF形式でダウンロード (1269K) -
Rafiqul Islam, Chun Fan, Yoshito Fujii, Li Jun Hao, Shoichi Suzuki, At ...2002 年 131 巻 4 号 p. 533-540
発行日: 2002/04/01
公開日: 2008/06/30
ジャーナル フリーGp91phox is a key component of the phagocyte NADPH oxidase. Mutations of its promoter found in patients with chronic granulomatous disease cause deficient binding of PU. 1 and HAF-1. Because the two factors bind to the same site (Pu box) of the promoter, we attempted to clarify their relative in vivo contributions to activation of the gp91phox promoter in monocytically differentiated PLB-985 cells using a dual luciferase reporter assay and a gel shift competition assay. We found that the activity of a series of singlepoint-mutated promoters increases or decreases according to an increase or decrease, respectively, in the affinity of the promoters to PU. 1 but not to HAF-1. Two of 7 mutants showing weak binding affinity to PU.l exhibited moderate promoter activity and normal binding affinity for HAF-1. These results suggest PU. 1 is the dominant activator and HAF-1 is supplementary. The increased promoter activity of single-, double-, and triplepoint-mutated constructs with sequences closer to that of the Ets-binding element correlates with their binding affinity to PU. 1 but not to HAF-1, supporting that PU. 1 is a more efficient activator than HAF-1. In contrast to co-expressed wild-type PU. 1, dominantnegative PU. 1 significantly inhibited the activity of a PU. 1-optimised gp91phox promoter construct. Therefore, we conclude that PU. 1 and HAF-1 binding to the Pu box is dominant and supplementary, respectively, for activation of the gp91phox promoter in human monocytic cells.抄録全体を表示PDF形式でダウンロード (2315K) -
Jong-Ryul Lee, Hitomi Takenaka, Naoko Takahashi, Masaki Makise, Yoshih ...2002 年 131 巻 4 号 p. 541-546
発行日: 2002/04/01
公開日: 2008/06/30
ジャーナル フリーOrigin Recognition Complex (ORC) is a candidate initiator of chromosomal DNA replication in eukaryotes. We recently reported that cardiolipin inhibits the interaction of ORC with origin DNA, as is the case of DnaA, the initiator of chromosomal DNA replication in prokaryotes. We report here that another acidic phospholipid, phosphatidylglycerol (PG), also inhibits the interaction. Synthetic PG with only unsaturated fatty acids inhibits ORC-binding to origin DNA more strongly than PG with only saturated fatty acids. On the other hand, phosphatidylcholine (neutral phospholipid) does not affect the ORC-origin interaction, regardless of the presence of saturated or unsaturated fatty acids. These results suggest that an acidic moiety and unsaturated fatty acids are important factors for the inhibitory effect of phospholipids on ORC binding to origin DNA, as is the case for DnaA. The inhibitory effect of cardiolipin on ORC binding to origin DNA was more apparent at 30°C than at 4°C. Furthermore, chlorpromazine restored the ORC-origin interaction in the presence of cardiolipin. Since the presence of unsaturated fatty acids, low incubation temperatures, and the addition of chlorpromazine all decrease membrane fluidity, these results suggest that membrane fluidity is important for the inhibitory effect of acidic phospholipids on ORC-binding to origin DNA, as is the case for DnaA.抄録全体を表示PDF形式でダウンロード (2974K) -
Masatoshi Beppu, Kenji Ohishi, Masaaki Kasahara, Kengo Kizaki, Yuko In ...2002 年 131 巻 4 号 p. 547-555
発行日: 2002/04/01
公開日: 2008/06/30
ジャーナル フリーThe involvement of intracellular protein phosphorylation in macrophages in the binding and uptake of oxidized low density lipoprotein (oxLDL) was investigated. The treatment of fibronectin-unstimulated and stimulated mouse thioglycolate-induced macrophages with inhibitors of myosin light chain kinase, protein kinase C and protein tyrosine kinase resulted in decreased macrophage binding of oxLDL, macrophage foam cell formation, and whole intracellular protein phosphorylation. The treatment of fibronectin-unstimulated and stimulated macrophages with inhibitors of protein serine/threonine and tyrosine phosphatases caused enhanced macrophage binding of oxLDL, macrophage foam cell formation, and whole intracellular protein phosphorylation. Fibronectin, which stimulates macrophage activity, enhanced macrophage intracellular protein phosphorylation. Myosine light chain phosphorylation may be involved in the fibronectin stimulation of macrophages. Treatment of fibronectin-unstimulated and stimulated macrophages with thiophosphate, which forms thiophosphate esters of intracellular proteins that are not so susceptible to protein phosphatases, enhanced macrophage binding of oxLDL. The above results indicate that intracellular protein phosphorylation maintains and enhances macrophage binding and the uptake of oxLDL.抄録全体を表示PDF形式でダウンロード (2465K) -
Chiye Sasaki, Ai Yokoyama, Yoshifumi Itoh, Masayuki Hashimoto, Takeshi ...2002 年 131 巻 4 号 p. 557-564
発行日: 2002/04/01
公開日: 2008/06/30
ジャーナル フリーHydrolytic mechanisms of family 18 chitinases from rice (Oryza sativa L.) and Bacillus circulans WL-12 were comparatively studied by a combination of HPLC analysis of the reaction products and theoretical calculation of reaction time-courses. All of the enzymes tested produced β-anomers from chitin hexasaccharide [(G1cNAc)6], indicating that they catalyze the hydrolysis through a retaining mechanism. The rice chitinases hydrolyzed predominantly the fourth and fifth glycosidic linkages from the nonreducing end of (G1cNAc)6, whereas B. circulans chitinase Al hydrolyzed the second linkage from the nonreducing end. In addition, the Bacillus enzyme efficiently catalyzed trans-glycosylation, producing significant amounts of chitin oligomers larger than the initial substrate, but the rice chitinases did not. The time-courses of (G1cNAc)6 degradation ob-tained by HPLC were analyzed by theoretical calculation, and the subsite structures of the rice chitinases were identified to be (-4) (-3) (-2) (-1) (+1) (+2). From the HPLC profile of the reaction products previously reported [Terwisscha van Scheltinga et al. (1995) Biochemistry 34, 15619-15623], family 18 chitinase from rubber tree (Hevea brasiliensis) was estimated to have the same type of subsite structure. Theoretical analysis of the reaction time-course for the Bacillus enzyme revealed that the enzyme has (-2) (-1) (+1) (+2) (+3) (+4)-type subsite structure, which is identical to that of fungal chitinase from Coccidioides immitis [Fukamizo et al. (2001) Biochemistry 40, 2448-2454]. The Bacillus enzyme also resembled the fungal chitinase in its transglycosylation activity. Minor structural differences between plant and microbial enzymes appear to result in such functional variations, even though all of these chitinases are classified into the identical family of glycosyl hydrolases.抄録全体を表示PDF形式でダウンロード (962K) -
Kunio Yumino, Ikuo Kawakami, Mamoru Tamura, Takaaki Hayashi, Masao Nak ...2002 年 131 巻 4 号 p. 565-570
発行日: 2002/04/01
公開日: 2008/06/30
ジャーナル フリーNADPH-menadione reductase activity by rat brain microsomes (Ms) was decreased 40-50% by 10 μM dicumarol, a potent inhibitor of DT-diaphorase, whereas no change in NADPH-paraquat (PQ) and -diquat (DQ) reductase activity was observed. NADPH-DQ reductase activity in brain Ms was 2.5-fold higher than NADPH-PQ reductase activity. The formation of PQ and DQ radicals was verified optically and observed directly by ESR spectroscopy in the NADPH-PQ and -DQ reductase reactions by brain Ms under anaerobic conditions. PQ- and DQ-induced superoxide formation was confirmed by the detection of DMPO-OOH ESR signals and followed by chemiluminescence (CL) of a Cypridina luciferin analogue (CLA). The kinetics and intensity of the CL were consistent with the observations that the reduction in DQ is faster than that in PQ. Thiobarbituric acid reactive substances (TBARS) and phospholipid hydroperoxides in brain Ms increased in the presence of NADPH and Fe3+. The generation of both lipid peroxidation products derived from brain Ms decreased with increasing concentrations of PQ and DQ. The inhibitory effect of DQ is more pronounced than that of PQ. The formation of PQ- and DQ-induced reactive oxygen species was not associated with lipid peroxidation in rat brain Ms.抄録全体を表示PDF形式でダウンロード (729K) -
Magorzata Giel-Pietraszuk, Mirosawa Z. Barciszewska2002 年 131 巻 4 号 p. 571-578
発行日: 2002/04/01
公開日: 2008/06/30
ジャーナル フリーXenopus transcription factor IIIA (TFIIIA) binds 5S rRNA and the 5S rRNA gene, these interactions being mediated by nine zinc fingers. To determine the contribution of each finger to the binding to 5S rRNA we prepared a series of peptides containing different numbers of zinc fingers and analyzed their interactions with RNA. The topography of these complexes was analyzed with the specific RNase and hydroxyl radical footprinting methods. Our results show the direct contribution of each zinc finger (ZF) peptide to the specific recognition of 5S rRNA. These data clearly suggest that total binding of TFIIIA with 5S rRNA is the sum of the specific interactions of the individual zinc fingers with RNA and that they have an additive character.抄録全体を表示PDF形式でダウンロード (5888K) -
Hiromasa Uchimura, Hitoshi Enjoji, Takafumi Seki, Ayako Taguchi, Naoki ...2002 年 131 巻 4 号 p. 579-586
発行日: 2002/04/01
公開日: 2008/06/30
ジャーナル フリーDissimilatory nitrate reductase (Nar) was solubilized and partially purified from the large particle (mitochondrial) fraction of the denitrifying fungus Fusarium oxysporum and characterized. Many lines of evidence showed that the membrane-bound Nar is distinct from the soluble, assimilatory nitrate reductase. Further, the spectral and other properties of the fungal Nar were similar to those of dissimilatory Nars of Escherichia coli and denitrifying bacteria, which are comprised of a molybdoprotein, a cytochrome b, and an iron-sulfur protein. Formate-nitrate oxidoreductase activity was also detected in the mitochondrial fraction, which was shown to arise from the coupling of formate dehydrogenase (Fdh), Nar, and a ubiquinone/ubiquinol pool. This is the first report of the occurrence in a eukaryote of Fdh that is associated with the respiratory chain. The coupling with Fdh showed that the fungal Nar system is more similar to that involved in the nitrate respiration by Escherichia coli than that in the bacterial denitrifying system. Analyses of the mutant species of F. oxysporum that were defective in Nar and/or assimilatory nitrate reductase conclusively showed that Nar is essential for the fungal denitrification.抄録全体を表示PDF形式でダウンロード (2178K) -
Hideo Miyake, Chikako Otsuka, Shigenori Nishimura, Yasunori Nitta2002 年 131 巻 4 号 p. 587-591
発行日: 2002/04/01
公開日: 2008/06/30
ジャーナル フリーThe hydrolytic activity of β-amylase from Bacillus cereus var. mycoides was lost on replacement of either of the catalytic residues (Glu172 or G1u367) with an alanyl residue. When maltopentaose and 2 M azide existed together mutant, E367A cleaved the glucosidic linkage of maltopentaose and produced maltose at pH 7.0 and 25°C, but the other mutants (E172A and double mutant E172A/E367A) did not. This indicates that azide acts as a general base instead of E367 and Glu172 acting as general acids, and that the hydroxide ion generated from a water molecule activated by azide attacks a reactive pyranose nucleophilically so that β-maltose is produced.抄録全体を表示PDF形式でダウンロード (2852K) -
Hidenori Ito, Keiko Kamei, Ikuko Iwamoto, Yutaka Inaguma, Rafael Garc& ...2002 年 131 巻 4 号 p. 593-603
発行日: 2002/04/01
公開日: 2008/06/30
ジャーナル フリーMolecular chaperones and the ubiquitin-proteasome pathway are known to participate in the quality control of proteins in cells. In this study, we examined the responses of small heat shock proteins to proteasome inhibitors to clarify their roles under conditions where misfolded proteins are abnormally accumulated. HSP27 and αB-crystallin accumulated in both soluble and, more prominently, insoluble fractions after exposure to MG-132, a proteasome inhibitor. Enhanced expression of mRNAs for HSP27 and αBcrystallin was observed, suggesting transcriptional activation. Phosphorylation of HSP27 and αB-crystallin in cells treated with MG-132 was enhanced concomitantly with activation of p38 and p44/42 MAP kinase pathways. Immunofluorescence analysis revealed that exposure to proteasome inhibitors induced the formation of aggresomes in U373 MG cells, to which HSP27 and αB-crystallin were recruited. However, phosphorylation was not required for this accumulation in aggresomes. Thus, HSP27 and αBcrystallin are increased, phosphorylated and localized in aggresomes when proteasome activity is inhibited.抄録全体を表示PDF形式でダウンロード (8381K) -
Saori Takahashi, Hironobu Ogasawara, Keitaro Takahashi, Kazuyuki Hori, ...2002 年 131 巻 4 号 p. 605-610
発行日: 2002/04/01
公開日: 2008/06/30
ジャーナル フリーRenin binding protein (RnBP), a cellular renin inhibitor, has been identified as the enzyme N-acetyl-D-glucosamine (G1cNAc) 2-epimerase. Our recent studies demonstrated that rat G1cNAc 2-epimerase has a ten-times higher affinity for ATP, dATP, and ddATP than the human enzyme [Takahashi, S. et al. (2001) J. Biochem. 130, 815-821]. To identify the domain conferring nucleotide binding to GlcNAc 2-epimerase, we constructed a series of chimeric enzymes successively replacing the three domains of the human enzyme (N-terminal, middle, and C-terminal domains) with the corresponding domains of the rat enzyme. Chimeras were expressed in Escherichia coli JM109 cells under the control of the Taq promoter. The purified chimeric enzymes had GlcNAc 2-epimerase activity and inhibited renin activity in a dose-dependent manner. The recombinant human and rat enzymes required catalytic amounts of ATP with apparent Km values of 73 and 5.5 μM, respectively. Chimeric enzymes of HHR, RHH, and RHR (H, human type domain; R, rat type domain) had nearly the same nucleotide specificity as the human G1cNAc 2-epimerase. On the other hand, HRR, HRH, and RRH chimeras had the same nucleotide specificity as the rat enzyme. These results indicate that the middle domain of the GlcNAc 2-epimerase molecule participates in the specificity for and binding of nucleotides, and that nucleotides are essential to form the catalytic domain of the enzyme.抄録全体を表示PDF形式でダウンロード (1621K) -
Hiromi Sakai, Yohei Masada, Shinji Takeoka, Eishun Tsuchida2002 年 131 巻 4 号 p. 611-617
発行日: 2002/04/01
公開日: 2008/06/30
ジャーナル フリーHemoglobin-vesicles (HbV) have been developed for use as artificial O2 carriers in which a purified Hb solution is encapsulated within a phospholipid bilayer membrane. In this study, bovine Hb (BHb) was tested as a source of HbV instead of human Hb (HHb). We compared the preparation process and characteristics of BHbV with those of HHbV. The purification of BHb was effectively performed simply with an ultrafiltration system including a process for removing virus and scrapie agent. The removal ratio of the phospholipid components of bovine red blood cells was over 99.99%, and the protein purity was over 99.9%. The deoxygenated and carbonylated BHb showed denaturation transition temperatures at 83 and 87°C, respectively, which are higher than those of HHb (80 and 78°C, respectively), and resistant to pasteurization (60°C, 10h). The purified BHb was concentrated to over 40g/dl, and encapsulated in a phospholipid bilayer membrane to form BHbV with a diameter of about 280nm. The O2 affinity (P50) of the BHbV was regulated by coencapsulation of an appropriate amount of Cl- (as NaC1), which binds to BHb as an allosteric effector, in the range 16-28 Torr, comparable to human blood (P50=28 Torr). This is quite simple in comparison with HHb which requires phosphate derivatives such as pyridoxal 5'-phosphate as a replacement for 2, 3-diphoshoglyceric acid. The viscosity and colloid osmotic pressure of the BHbV when suspended in 5% human serum albumin are 3.5cP and 20 Torr, respectively, comparable to those of human blood. In conclusion, BHb can be used as a source for the production of HbV, not only because of its abundance in the cattle industry, but also because of the physicochemical advantages of the purification process, thermal stability, and regulation of 02 affinity in comparison with HHb.抄録全体を表示PDF形式でダウンロード (2012K) -
Takayuki Furumatsu, Noriko Yamaguchi, Keiichiro Nishida, Akira Kawai, ...2002 年 131 巻 4 号 p. 619-626
発行日: 2002/04/01
公開日: 2008/06/30
ジャーナル フリーEndostatin derived from collagen XVIII is a potent endogenous anti-angiogenic factor that induces regression of various tumors of epithelial origin. Endostatin has been shown to inhibit endothelial cell functions, however, its effect remains controversial. We first attempted here to apply the inhibitory effect of recombinant human endostatin on chondrosarcomas, which originate from the mesenchyme, in nude mice. Endostatin induced reduction of chondrosarcoma growth and tumor angiogenesis in vivo. However, endostatin showed no effect on the proliferation and migration of chondrosarcoma cells in vitro. Next, we investigated the interactions between endostatin and endothelial cells in detail. Endostatin inhibited the migration on and attachment to collagen I but did not affect the proliferation of endothelial cells. Although the migration of endothelial cells was stimulated by angiogenic factors such as basic fibroblast growth factor and vascular endothelial growth factor, endostatin showed similar inhibitory effects on it in the presence and absence of the stimulants. Moreover, the inhibitory effect against endothelial cell attachment to collagen I was attenuated or modulated in the presence of neutralizing antibodies of α2, α5β1, and αvβ3 integrins but not that of α1 integrin. Our results suggest that endostatin might suppress the α2β1 integrin function of endothelial cells via α5β1 or αvβ3 integrin. We propose here that endostatin might be effective for anti-angio-genic therapy for human chondrosarcomas through the suppression of α2α1 integrin functions in endothelial cells.抄録全体を表示PDF形式でダウンロード (2508K)
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