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134 巻, 6 号
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Yoshitaka Fukada2003 年 134 巻 6 号 p. 773-775
発行日: 2003年
公開日: 2008/06/30
ジャーナル フリーThe earth rotates on its own axis while orbiting around the sun. This regular movement of the solar system results in cyclic changes of the light condition of the earth with a period of 24h, although the lengths of daytime and nighttime depend on the latitude. The organisms living on the earth have evolved an internal time-measuring system called the “circadian clock, ” which ticks with a period of approximately 24h in order to adapt to the environment and to anticipate the next cycle. The fact that most of existing organisms retain the circadian clock suggests that the clock-owner-ship must have been advantageous over non-ownership during their evolution. Here I will introduce the background of the research field of circadian rhythm and present an outline of this Special Review series, which is composed of three articles that review recent research into the molecular mechanisms of the three types of circadian clock systems in vertebrates.抄録全体を表示PDF形式でダウンロード (1547K) -
Yasushi Isojima, Nobuaki Okumura, Katsuya Nagai2003 年 134 巻 6 号 p. 777-784
発行日: 2003年
公開日: 2008/06/30
ジャーナル フリーCircadian rhythms in behaviors and physiological phenomena of plants and animals have long been well known, but the frameworks of the molecular mechanism of circadian clocks have become clearer only within the last decade. A transcription-translation feedback loop has been shown to be an essential component of the clock, and this mechanism seems to be conserved over a wide range of species. The transcriptional activation by a Clock:Bmall heterodimer and the inhibition by Cryptochrome and Period are believed to provide the framework of the feedback loop in mammals. Posttranslational modifications such as phosphorylation, nuclear entry and degradation have also been demonstrated to be necessary for the oscillation. Complex auxiliary loops have also been found, and these are thought to contribute to the stabilization of the feedback loop. The molecular mechanisms by which the circadian clock is adjusted to external conditions such as daily light-dark cycles, and by which the oscillation of the feedback loop is transferred to the peripheral organs are also discussed.抄録全体を表示PDF形式でダウンロード (1109K) -
Yoshiki Tsuchiya, Eisuke Nishida2003 年 134 巻 6 号 p. 785-790
発行日: 2003年
公開日: 2008/06/30
ジャーナル フリーThe mammalian circadian system consists of multiple oscillators with basically hierarchical relationship, in which the hypothalamic suprachiasmatic nucleus (SCN) is the master pacemaker and the other oscillators in the periphery are subordinate. Although peripheral oscillators have been preceded by the SCN in circadian studies, accumulating data have revealed the importance and characteristics of peripheral oscillators. Cultured cell lines have also provided valuable information about intracellular mechanisms of circadian rhythms. This review outlines the properties of peripheral clocks in several perspectives such as the mechanisms of autonomous oscillations, the clock resetting, and the clock outputs, and describes the usefulness of immortalized cultured cells as a model system of mammalian circadian clocks by introducing some fruits of related works.抄録全体を表示PDF形式でダウンロード (2167K) -
Toshiyuki Okano, Yoshitaka Fukada2003 年 134 巻 6 号 p. 791-797
発行日: 2003年
公開日: 2008/06/30
ジャーナル フリーMany tissues in non-mammalian vertebrates contain both photoreceptors and circadian clock systems. Among these photosensitive clock structures, the chick pineal gland has been characterized in detail from cellular and molecular aspects of the clock oscillation and entrainment. Analyses of the pineal photic-input pathway revealed a phase-shifting mechanism mediated by activation of G11, one of the Gqtype G-proteins. A major photoreceptive molecule, pinopsin, likely triggers this pathway by transmitting the light signal to the circadian oscillator. In the chick pineal oscillator, the transcription/translation-based autoregulatory feedback loop is composed of positive and negative elements (clock gene products) that are homologous to those identified in mammals. In the molecular cycling, a CACGTG E-box located in the promoter region of the negative element genes plays a central role in the transcriptional regulation. The phase of the molecular cycling is modulated by many regulatory components, among which E4BP4 and extracellular signal-regulated kinase (ERK) are closely associated with the photic entrainment. A light-responsive element was found in the promoter region of the Pinopsin gene, and the element included a CACGTG E-box, suggesting a novel role of the E-box as a point of convergence of light and circadian signals. These observations together point to general and unique features of the chick pineal circadian system among animal clocks.抄録全体を表示PDF形式でダウンロード (3802K) -
Orawan Khow, Lawan Chanhome, Tamotsu Omori-Satoh, Yuko Ogawa, Ryohei Y ...2003 年 134 巻 6 号 p. 799-804
発行日: 2003年
公開日: 2008/06/30
ジャーナル フリーWe isolated the most lethal toxins in the venom of the Malayan krait (Bungarus candidus), one of the medically most important snake species in southeast Asia. Three β-BTx like basic neurotoxins, T1-1, T1-2, and T2, with PLA2 activity were isolated from pooled venom of eight B. candidus from southern Thailand by cation-exchange chromatography, followed by adsorption chromatography on hydroxylapatite and RPHPLC, with 14-, 16-, and 4-fold increases in toxicity compared to crude venom. The LDs50 determined in mice weighing 18-20g were 0.26, 0.22, and 0.84 μg per mouse with i. v injection. T1-1 and T1-2 possessed comparable lethal toxicities to those of β1-BTx, the most toxic neurotoxin in B. multicinctus venom, and the major neurotoxin in B. flaviceps venom. The apparent molecular weights of the native toxins were approximately 25-25.5 kDa. They consist of two polypeptide chains with apparent molecular weights of 15.5-16.5 and 8-8.5 kDa, respectively. The amino terminal sequences of the two chains of each of the toxins determined by Edman degradation exhibited considerable similarity with those of the A-chains and B-chains of β-BTxs in the venom of Bungarus multicinctus.抄録全体を表示PDF形式でダウンロード (1895K) -
Jiro Arima, Takashi Tamura, Hitoshi Kusakabe, Makoto Ashiuchi, Toshiha ...2003 年 134 巻 6 号 p. 805-812
発行日: 2003年
公開日: 2008/06/30
ジャーナル フリーL-Glutamate oxidase (LGOX) from Streptomyces sp. X-119-6 is a protein of 150 kDa that has hexamer structure α2β2γ2. The gene encoding LGOX was cloned and heterologously expressed in Escherichia coli. LGOX isolated from the E. coli transformant had the structure of a one chain polypeptide. Although the recombinant LGOX exhibited catalytic activity, it was inferior to the LGOX isolated from Streptomyces sp. X-119-6 in catalytic efficiency. The recombinant LGOX exhibited low thermostability compared to the LGOX isolated from Streptomyces sp. X-119-6 and was an aggregated form. Proteolysis of the recombinant LGOX with the metalloendopeptidase from Streptomyces griseus (Sgmp) improved its catalytic efficiency at various pH. Furthermore, the Sgmp-treated recombinant LGOX had a subunit structure of α2β2γ2. and nearly the same enzymological character as the LGOX isolated from Streptomyces sp. X-119-6. A higher molecular species observed for the recombinant LGOX was not detected for the Sgmp-treated recombinant LGOX. These results prove that proteolysis by Sgmp is involved in the stabilization of the recombinant LGOX.抄録全体を表示PDF形式でダウンロード (2305K) -
Deng Ning, Xiang Junjian, Wang Xunzhang, Chen Wenyin, Zhang Qing, Su K ...2003 年 134 巻 6 号 p. 813-817
発行日: 2003年
公開日: 2008/06/30
ジャーナル フリーAnti-HBs Fab fragment has considerable potential for use in the prevention and treatment of liver diseases by HBV. Here we established a high-level expression system to directly produce anti-HBs Fab fragment in Pichia pastoris. This was achieved by cointegration of the genes encoding the heavy and light chains both under the genome of the yeast cells. The Fab fragment was efficiently secreted into medium at a concentration of 50mg/liter. The authenticity of the Fab fragment was confirmed by immunoblot analysis, which yielded one band of _??_50 kDa under nonreducing conditions and two bands of _??_28 kDa under reducing conditions. The anti-HBs Fab fragment was prepared with a purity of 95% by affinity chromatography. The affinity activity of the recombinant Fab was detected by ELISA, which indicated that 1mg of recombinant Fab was equivalent to 40 IU HBIG (20 IU/mg). The results demonstrated that the recombinant Fab fragment could sufficiently neutralize the HBsAg.抄録全体を表示PDF形式でダウンロード (1551K) -
Yugesh Khare, Yuan-Wei Zhang, Masahiro Fujihashi, Kunio Miki, Tanetosh ...2003 年 134 巻 6 号 p. 819-826
発行日: 2003年
公開日: 2008/06/30
ジャーナル フリーUndecaprenyl diphosphate synthase catalyzes the sequential condensation of eight molecules of isopentenyl diphosphate (IPP) in the cis-configuration into farnesyl diphosphate (FPP) to produce undecaprenyl diphosphate (UPP), which is indispensable for the biosynthesis of the bacterial cell wall. This cis-type prenyltransferase exhibits a quite different mode of binding of homoallylic substrate IPP from that of trans-type prenyltransferase [Kharel Y. et al. (2001) J. Biol. Chem. 276, 28459-28464]. In order to know the IPP binding mode in more detail, we selected six highly conserved residues in Regions III, IV, and V among nine conserved aromatic residues in Micrococcus luteus B-P 26 UPP synthase for substitution by site-directed mutagenesis. The mutant enzymes were expressed and purified to homogeneity, and then their effects on substrate binding and the catalytic function were examined. All of the mutant enzymes showed moderately similar far-UV CD spectra to that of the wildtype, indicating that none of the replacement of conserved aromatic residues affected the secondary structure of the enzyme. Kinetic analysis showed that the replacement of Tyr-71 with Ser in Region III, Tyr-148 with Phe in Region IV, and Trp-210 with Ala in Region V brought about 10-1, 600-fold decreases in the kcat/Km values compared to that of the wild-type but the Km values for both substrates IPP and FPP resulted in only moderate changes. Substitution of Phe-207 with Ser in Region V resulted in a 13-fold increase in the Km value for IPP and a 1, 000-2, 000-fold lower kcat/Km value than those of the wild-type, although the Km values for FPP showed about no significant changes. In addition, the W224A mutant as to Region V showed 6-fold and 14-fold increased Km values for IPP and FPP, respectively, and 100-250-fold decreased kcat/Km values as compared to those of the wild-type. These results suggested that these conserved aromatic residues play important roles in the binding with both substrates, IPP and FPP, as well as the catalytic function of undecaprenyl diphosphate synthase.抄録全体を表示PDF形式でダウンロード (3116K) -
Tadashi Oshizawa, Teruhide Yamaguchi, Kazuhiro Suzuki, Yukio Yamamoto, ...2003 年 134 巻 6 号 p. 827-834
発行日: 2003年
公開日: 2008/06/30
ジャーナル フリーThe involvement of protein phosphatases in the activation of superoxide (O2-)- generating enzyme in human neutrophils was examined using calyculin A, an inhibitor of protein phosphatase type 1 and 2 A. Calyculin A inhibited the phorbol myristate acetate (PMA)- and opsonized zymosan (OZ)-activated O2- generation by human neutrophils. This inhibitory effect of calyculin A on PMA-activated O2- generation was reversed by the addition of KT5926, a specific inhibitor of myosin light chain kinase and Ca2+/calmodulin-dependent protein kinase II. These results suggest that the addition of calyculin A may cause hyperphosphorylation of some protein(s) that plays a crucial role in the PMA-dependent activation of O2- generating enzyme, and that this protein hyperphosphorylation may be evoked by a KT5926-sensitive kinase or its downstream kinase. Whereas two-dimensional analysis involving 32P revealed that calyculin A caused the hyperphosphorylation of many proteins, KT5926 mainly reduced the calyculin A-induced hyperphosphorylation of a 67 kDa protein in activated neutrophils, suggesting that the hyperphosphorylation of the 67 kDa protein might inhibit the PMA-dependent activation of NADPH oxidase. The 67 kDa cytosolic protein was moderately phosphorylated on the addition of PMA. On the other hand, in the absence of calyculin A, KT5926 inhibited both PMA-induced O2- generation and phosphorylation of the 67 kDa protein. Amino acid sequence analysis of peptides derived from the 67 kDa protein revealed that the 67 kDa protein was identical to L-plastin, an actin-bundling protein. We conclude that optimally phosphorylated L-plastin may play some crucial role in the activation of NADPH oxidase.抄録全体を表示PDF形式でダウンロード (1995K) -
Yasuzo Nishina, Kyosuke Sato, Haruhiko Tamaoki, Takeyuki Tanaka, Chiak ...2003 年 134 巻 6 号 p. 835-842
発行日: 2003年
公開日: 2008/06/30
ジャーナル フリーThe pKa value of a substrate analogue 3-thiaoctanoyl-CoA at αC-H is known to drop from ca. 16 in the free state to 5-6 upon binding to medium-chain acyl-CoA dehydrogenase (MCAD). The molecular mechanism underlying this phenomenon was investigated by taking advantage of artificial FADs, i.e., 8-CN-, 7, 8-Cl2-, 8-Cl-, 8-OCH3-, 8-NH2-, ribityl-2'-deoxy-8-CN-, and ribityl-2'-deoxy-8-Cl-FADs, reconstituted into MCAD. The stronger the electron-withdrawing ability of the substituent, the smaller the pKa value became [e. g., 7.4 (8-NH2-FAD) and 4.0 (8-CN-FAD)], suggesting that the flavin ring itself affects the pKa value of the ligand via a charge-transfer interaction with the ligand. The destruction of the hydrogen bond between the thioester C(1)=O and the ribityl-2'-OH of FAD raised the pKa by ca. 2.5 units. These results indicate that the interaction between the ligand and the flavin ring also serves to lower the pKa of the ligand, in addition to the hydrogen bonds at C(1)=O of the ligand.抄録全体を表示PDF形式でダウンロード (980K) -
Akira Hosono, Hiroyuki Mizuguchi, Hideyuki Hayashi, Masaru Goto, Ikuko ...2003 年 134 巻 6 号 p. 843-851
発行日: 2003年
公開日: 2008/06/30
ジャーナル フリーA subfamily I aminotransferase gene homologue containing an open reading frame encoding 381 amino acid residues (Mr=42, 271) has been identified in the process of the genome project of an extremely thermophilic bacterium, Thermus thermophilus HB8. Alignment of the predicted amino acid sequence using FASTA shows that this protein is a member of aminotransferase subfamily Iγ. The protein shows around 40% identity with both T. thermophilus aspartate aminotransferase [EC 2. 6. 1. 1] and mammalian glutamine:phenylpyruvate aminotransferase [EC 2. 6. 1. 64]. The recombinant protein expressed in Escherichia coli is a homodimer with a subunit molecular weight of 42, 000, has one pyridoxal 5'-phosphate per subunit, and is highly active toward glutamine, methionine, aromatic amino acids, and corresponding keto acids, but has no preference for alanine and dicarboxylic amino acids. These substrate specificities are similar to those described for mammalian glutamine: phenylpyruvate aminotransferase. This is the first enzyme reported so far that has the glutamine aminotransferase activity in non-eukaryotic cells. As the presence of aromatic amino acid:2-oxoglutarate aminotransferase [EC 2. 6. 1. 57] has not been reported in T. thermophilus, this enzyme is expected to catalyze the last transamination step of phenylalanine and tyrosine biosynthesis. It may also be involved in the methionine regeneration pathway associated with polyamine biosynthesis. The enzyme shows a strikingly high pKa value (9.3) of the coenzyme Schiff base in comparison with other subfamily I aminotransferases. The origin of this unique pKa value and the substrate specificity is discussed based on the previous crystallographic data of T. thermophilus and E. coli aspartate aminotransferases.抄録全体を表示PDF形式でダウンロード (3414K) -
Kazuya Yamaguchi, Akiro Kawamura, Hideyuki Ogawa, Shinnichiro Suzuki2003 年 134 巻 6 号 p. 853-858
発行日: 2003年
公開日: 2008/06/30
ジャーナル フリーA Cu-containing nitrous oxide reductase (HdN2OR) from a methylotrophic denitrifying bacterium, Hyphomicrobium denitrificans A3151, has been aerobically prepared and spectroscopically characterized. Purple and blue forms of HdN2OR have been isolated. Each form is a homodimer comprising monomers with a molecular mass of 65 kDa. The visible absorption spectrum of the purple form (designated as form A) exhibits three absorption bands at 480 nm, 540 nm, and 650 nm, with a shoulder near 780 nm, and that of the blue form (designated as form B) shows only one absorption band at 650 nm. Reversible spectral changes, between those of forms A and B, are observed on treatment of these forms with redox reagents. Forms A and B are oxidized and reduced forms, respectively. The 77-K EPR spectrum of form A indicates a seven-line copper hyperfine structure centered at g_??_ (g_??_=2.18, A_??_=4.5 mT), which is characteristic of a mixed-valence binuclear CuA site (Amv), and that of form B exhibits a broad featureless signal (g=2.06). The various spectral data of HdN2OR suggest that form A contains Amv, and a mixed-valence tetranuclear Cuz site (Zmv*), while form B includes reduced CuA (Ared) and Zmv*. The pH profiles of N2OR activity of the two forms are similar to each other, and the specific activity at optimum pH 8.8 was estimated to be 45±5 and 29±3 μunol•min-1•mg-1 for forms A and B, respectively.抄録全体を表示PDF形式でダウンロード (1638K) -
Yumiko Seike, Fusako Takeuchi, Motonari Tsubaki2003 年 134 巻 6 号 p. 859-867
発行日: 2003年
公開日: 2008/06/30
ジャーナル フリーCytochrome b561 from bovine adrenal chromaffin vesicles contains two heme B prosthetic groups. We verified that purified cytochrome b561 can donate electron equivalents directly to cytochrome c. The purified cytochrome b561 was successfully reconstituted into cholesterol-phosphatidylcholine-phosphatidylglycerol vesicles by a detergent-dialysis and extrusion method. When ascorbate-loaded vesicles with cytochrome b561 were mixed with ferricytochrome c, the intravesicular ascorbate was able to reduce external thiazole blue or cytochrome c. The reduction of thiazole blue or cytochrome c was dependent on the presence of cytochrome b561 in the vesicle membranes. Pre-treatment of cytochrome b561 with diethylpyrocarbonate suppressed the reduction of extravesicular cytochrome c significantly, confirming that the reduction was not due to leakage of ascorbate from the vesicles. The topology of the reconstituted cytochrome b561 in the vesicle membranes was examined by treatment with trypsin followed by SDS-PAGE and MALDI-TOF-MS analyses. Only one major cleav-age site at Lys191 was identified, indicating that cytochrome b561 was reconstituted into the membranes in an inside-out orientation irrespective of the modification with diethylpyrocarbonate. The addition of a soluble form of dopamine β-hydroxylase to the external medium resulted in the successful reconstitution of the hydroxylation activity towards tyramine, an analogue of dopamine, suggesting that a direct electron transfer via complex formation occurred. This activity was enhanced significantly upon the addition of ferricyanide as a mediator between cytochrome b561 and dopamine β-hydroxylase.抄録全体を表示PDF形式でダウンロード (2486K) -
Natsuko Horiguchi, Kei-ichiro Arimoto, Atsushi Mizutani, Yoko Endo-Ich ...2003 年 134 巻 6 号 p. 869-874
発行日: 2003年
公開日: 2008/06/30
ジャーナル フリーTo isolate cDNAs for molecules involved in cell adhesion to the extracellular matrix, expression cloning with non-adherent colon cancer Colo 201 cells was carried out. Four positive clones were isolated and, when sequenced, one was found to be galectin-1, a β-galactoside-binding protein. When cultured on fibronectin-, laminin-, and collagen-coated and non-coated dishes, the adherent galectin-1 cDNA-transfected Colo 201 cells increased and spread somewhat. Immunofluorescence staining revealed that galectin-1 was expressed inside and outside of Colo 201 cells. The adhesion was dependent on the carbohydrate-recognition domain of galectin-1 since lactose inhibited the adhesion and exogenously-added galectin-1 caused the adhesion. PD 58059, an inhibitor of mitogen-activated protein kinase, or LY 294002, a phosphoinositide 3-OH kinase inhibitor, decreased the adhesion. Furthermore, the expression of galectin-1 in Colo 201 cells induced apoptotic cell death, while exogenously-added galectin-1 did not cause apoptosis. These results indicate that galectin-1 plays a role in both cell-matrix interactions and the inhibition of Colo 201 cell proliferation, and suggest that galectin-1 expressed in cells could be associated with apoptosis.抄録全体を表示PDF形式でダウンロード (2817K) -
Mayumi Ikegami-Kawai, Ayako Suzuki, Ikuko Karita, Tomoko Takahashi2003 年 134 巻 6 号 p. 875-880
発行日: 2003年
公開日: 2008/06/30
ジャーナル フリーWe i nvestigated changes in renal hyaluronidase activity in streptozotocin (STZ)-induced diabetic rats during the progression of diabetes. Prior to the study, we characterized rat renal hyaluronidase activity to find that its optimum pH is 3.5 and that it consists of two isomers of 73 and 63 kDa, as detected by zymography. Hyaluronidase activity was traced in one whole kidney and in the cortex and medulla of the other kidney up to the 18 th week after STZ injection. Whole kidney hyaluronidase activity started to increase on day 3 and reached a maximum level 2.4 times that of the controls in the 3 rd week. Cortical hyaluronidase showed a similar tendency to that of whole kidney hyaluronidase, while medullary hyaluronidase activity continued to increase until the 8 th week, suggesting their different involvements in the progression of diabetic nephropathy. In zymography, the intensities of the two isomer bands increased with the progression of diabetes, but the intensity ratio did not change significantly and no new isomer band appeared. Renal HAase activity increased only in STZ-induced diabetic rats, but not in spontaneously diabetic Goto-Kakizaki rats still without remarkable renal disorder. Based on these findings, increased renal HAase activity may serve as a useful marker for diabetic nephropathy.抄録全体を表示PDF形式でダウンロード (2880K) -
Keiko Haga, Ryuta Kanai, Osamu Sakamoto, Masanobu Aoyagi, Kazuaki Hara ...2003 年 134 巻 6 号 p. 881-891
発行日: 2003年
公開日: 2008/06/30
ジャーナル フリーThe stacking interaction between a tyrosine residue and the sugar ring at the catalytic subsite -1 is strictly conserved in the glycoside hydrolase family 13 enzymes. Replacing Tyr100 with leucine in cyclodextrin glycosyltransferase (CGTase) from Bacillus sp. 1011 to prevent stacking significantly decreased all CGTase activities. The adjacent stacking interaction with both Phe 183 and Phe 259 onto the sugar ring at subsite +2 is essentially conserved among CGTases. F183L/F259L mutant CGTase affects donor substrate binding and/or acceptor binding during transglycosylation [Nakamura et al. (1994) Biochemistry 33, 9929-9936]. To elucidate the precise role of carbohydrate/aromatic stacking interaction at subsites -1 and +2 on the substrate binding of CGTases, we analyzed the X-ray structures of wild-type (2.0 Å resolution), and Y100L (2.2 Å resolution) and F183L/F259L mutant (1.9 Å resolution) CGTases complexed with the inhibitor, acarbose. The refined structures revealed that acarbose molecules bound to the Y100L mutant moved from the active center toward the side chain of Tyr195, and the hydrogen bonding and hydrophobic interaction between acarbose and subsites significantly diminished. The position of pseudo-tetrasaccharide binding in the F183L/F259L mutant was closer to the non-reducing end, and the torsion angles of glycosidic linkages at subsites -1 to +1 on molecule 1 and subsites -2 to -1 on molecule 2 significantly changed compared with that of each molecule of wild-type-acarbose complex to adopt the structural change of subsite +2. These structural and biochemical data suggest that substrate binding in the active site of CGTase is critically affected by the carbohydrate/aromatic stacking interaction with Tyr 100 at the catalytic subsite -1 and that this effect is likely a result of cooperation between Tyr 100 and Phe 259 through stacking interaction with substrate at subsite +2.抄録全体を表示PDF形式でダウンロード (5264K) -
Takayuki Tsukuba, Kuniaki Okamoto, Yoshiko Okamoto, Michiyo Yanagawa, ...2003 年 134 巻 6 号 p. 893-902
発行日: 2003年
公開日: 2008/06/30
ジャーナル フリーAtopic dermatitis (AD) is a pruritic inflammatory skin diseases associated with a family history of atropy. Here we show that mice lacking the endolysosomal aspartic proteinase cathepsin E spontaneously develop skin lesions similar to those of humans with AD when reared under conventional conditions but not under specific pathogenfree conditions. These mice showed the increase in the ratio of CD4+/CD8+ T cells, the strong polarization of naÏve T cells to T helper 2 cells, and the systemic accumulation of IL-18 and IL-1β accompanied by a marked increase in IL-4, IL-5, and IgE. The relative rates of degradation of IL-18 and IL-1β were significantly lower in cathepsin E-deficient mice than wild-type mice. These results strongly suggest that the development of AD in cathepsin E-deficient mice is initiated by systemic accumulation of IL-18 and IL-1β, mainly due to their reduced turnover rates. In addition, the reduced expression of cathepsin E was also observed in erythrocytes of both humans with AD and the AD mouse model NC/Nga. Cathepsin E deficiency might thus be responsible for the induction of AD in humans and mice.抄録全体を表示PDF形式でダウンロード (5336K) -
Yasuhiro Kambayashi, Shozo Tero-Kubota, Yorihiro Yamamoto, Masashi Kat ...2003 年 134 巻 6 号 p. 903-909
発行日: 2003年
公開日: 2008/06/30
ジャーナル フリーWe studied the mechanism of formation of oxygen radicals during ferrous ioninduced decomposition of linoleic acid hydroperoxide using the spin trapping and chemiluminescence methods. The formation of the superoxide anion (O2_??_-) was verified in the present study. The hydroxyl radical is also generated through Fenton type decomposition of hydrogen peroxide produced on disproportionation of O2_??_-. A carbon-centered radical was detected using 5-(diethoxyphosphoryl)-5-methyl-l-pyrroline N-oxide (DEPMPO) as a spin trap. Alkoxyl radical formation is essential for the conversion of linoleic acid hydroperoxide into the peroxyl radical by ferrous ion. It is likely that the alkoxyl radical [R1CH(O_??_)R2] is converted into the hydroxylcarbon radical [R1C_??_-(OH)R2] in water, and that this carbon radical reacts with oxygen to give the α-hydroxyperoxyl radical [R1R2C(OH)OO_??_], which decomposes into the carbocation [R1C+(OH)R2] and O2_??_-.抄録全体を表示PDF形式でダウンロード (864K) -
Jianglan Liu, Dongzhi Wei, Feng Qian, Yuxun Zhou, Jinzhi Wang, Yushu M ...2003 年 134 巻 6 号 p. 911-917
発行日: 2003年
公開日: 2008/06/30
ジャーナル フリーRecombinant antibodies, especially ScFv fragments, can be applied as detection reagents and even substitute for some reagents used in immunoassays such as antibodyenzyme conjugates. For ScFv fragments, there is no such universal system available up to now. A vector system was constructed based on pPIC9- Fc, in which the hinge, CH2 and CH3 domains (Fc fragment) of mouse IgG1 and His-tag were cloned into the Pichia expression vector pPIC9. A model ScFv was introduced into pPIC9-Fc, which can bind Glutathione-S-transferase (GST) from Schistosoma japonicum, to yield the expression cassette pPIC9-ScFv-Fc. Following fermentation in a 5-liter reactor, the fusion was expressed at high levels in the methylotrophic yeast Pichia Pastoris, secreted as a dimeric form in the culture, and purified by Ni2+-NTA column chromatography. The expression yield can reach 10-30mg/liter of culture medium. The ScFv-Fc fusion retains the biological binding ability of the parent ScFv, and can be applied as anti-GST antibodies for the detection of GST and GST fusion proteins. Furthermore, the successful expression and maintenance of the binding activity verify the efficacy of the vector system for use as detection reagents in vitro, by reacting with the specific antigens and being readily detected using general anti-mouse antibodies.抄録全体を表示PDF形式でダウンロード (3710K) -
Mana Nagita, Hiroki Inoue, Norihiro Nakamura, Hiroshi Kanazawa2003 年 134 巻 6 号 p. 919-925
発行日: 2003年
公開日: 2008/06/30
ジャーナル フリーWe previously showed that calcineurin B homologous protein 1 (CHP1) interacts with nuclear apoptosis-inducing protein kinase DRAK2, and that overexpression of DRAK2 induces the nuclear accumulation of CHP1, although CHP1 usually resides in the cytoplasm [Matsumoto et al. (2001) J. Biochem. 130, 217-225]. Here we show that CHP1 has two functional nuclear export signal (NES) sequences in its carboxyl-terminal region. Treatment of several cell lines with leptomycin B, a specific inhibitor of CRM1-dependent nuclear export, induces the nuclear accumulation of CHP1. Moreover, CHP1-GFP fusion proteins with deletions or point mutations affecting the two putative NES sequences accumulate in the nucleus to a greater extent than wild-type CHP1-GFP. Tagging glutathione S-transferase-GFP fusion protein with each NES sequence caused a shift in their intracellular localization from all over the cells to the cytoplasm. These results suggest that after CHP1 has entered the nucleus, it is exported to the cytoplasm in an NES-dependent manner.抄録全体を表示PDF形式でダウンロード (5395K) -
Sayaka Higuchi, Tadashi Tamura, Tatsuya Oda2003 年 134 巻 6 号 p. 927-933
発行日: 2003年
公開日: 2008/06/30
ジャーナル フリーRicin induced apoptotic nuclear morphological changes in mouse macrophage cell line RAW264.7 cells at concentrations sufficient to cause severe protein synthesis inhibition. Ricin also induced the release of tumor necrosis factor-α (TNF-α) from this cell line in a dose-dependent manner but the profile was bell-shaped. However, the isolated galactose-specific ricin B-chain had no such effects. These results suggest that the receptor-binding of ricin through the B-chain is not enough, and subsequent attack on the intracellular target, i.e., the 28 S ribosomal RNA (rRNA), by the A-chain of internalized ricin is required for the effects of ricin. Z-D-CH2-DCB, a caspase family inhibitor, showed potent inhibition of the release of TNF-α from RAW264.7 cells as well as blockage of the induction of apoptosis by ricin. Furthermore, SB202190, a specific P 38 mitogen-activated protein (MAP) kinase inhibitor that strongly inhibits the release of TNF-α, also showed significant inhibition of ricin-induced apoptosis. These results suggest that there may be cross-talk between the pathways leading to the release of TNF-α and apoptosis. Time course analysis revealed that the activation of p 38 MAP kinase started prior to the induction of TNF-α release and apoptosis. Since the activation of p 38 MAP kinase in ricin-treated RAW264.7 cells was not prevented by Z-D-CH2-DCB, the activation of p 38 MAP kinase may occur upstream of the caspase cascade. Among the other protein synthesis inhibitors examined, modeccin and anisomycin, which can trigger a ribotoxic stress response similar to ricin, induced the release of TNF-α, but emetine and cycloheximide did not. These results suggest that the specific attack on the 28 S ribosomal RNA and the resulting ribotoxic stress response may trigger the multiple signal transduction pathways through the activation of p 38 MAP kinase, which in turn leads to TNF-α release and apoptosis.抄録全体を表示PDF形式でダウンロード (2191K) -
Shigeo Tojo, Masayuki Matsunaga, Takashi Matsumoto, Choong-Min Kang, H ...2003 年 134 巻 6 号 p. 935-946
発行日: 2003年
公開日: 2008/06/30
ジャーナル フリーWe investigated the organization and expression of the Bacillus subtilis sigY operon, the first gene of which codes for σY, a member of the extracytoplasmic function (ECF) family of sigma factors. The sigY operon, comprising six genes (sigY, yxlC, D, E, F, and G), was induced upon nitrogen starvation; it was continuously transcribed from the 31 st base upstream of sigY to a neighboring convergent gene, yxlH, resulting in a 4.2-kb mRNA. The expression of the sigY operon was also positively autoregulated through σY, suggesting that its transcription is likely to be directed by σY. Deletion analysis of the sigY promoter, which was localized by primer extension, revealed the promoter region of sigY with the “-10” and “-35” sequences of CGTC and TGAACG, respectively. The latter sequence was distinct from those recognized by σW, σX, and σM. The σY-directed transcription of sigY was under negative regulation involving YxlD. sigY disruption affected sporulation induced by nitrogen starvation, but sigY induction upon nitrogen starvation was not associated with the sporulation process. The organization and function of the sigY operon are significantly conserved in several microorganisms living in adverse living environments.抄録全体を表示PDF形式でダウンロード (2678K)
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