巻号一覧
131 巻, 6 号
選択された号の論文の22件中1～22を表示しています
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Yoh Takuwa, Noriko Takuwa, Naotoshi Sugimoto2002 年 131 巻 6 号 p. 767-771
発行日: 2002年
公開日: 2008/06/30
ジャーナル フリーSphingosine-l-phosphate (S1P) and lysophosphatidic acid (LPA) are blood-borne lysophospholipids with a wide spectrum of biological activities, which include stimulation of cell growth, prevention of apoptosis, regulation of actin cytoskeleton, and modulation of cell shape, cell migration, and invasion. Activated platelets appear to be a major source of both S1P and LPA in blood. Despite the diversity of their biosynthetic origins, they are considered to share substantial structural similarity. Indeed, recent investigation has revealed that S1P and LPA act via a single family of G protein-coupled receptors designated as Edg. Thus, the Edg isoforms, Edgl (also called S1P1), Edg5 (S1P2), Edg3 (S1P3), Edg6 (S1P4), and Edg8 (S1P5), are specific receptors for S1P (and SPC with a lower affinity), whereas Edg2 (LPA1), Edg4 (LPA2), and Edg7 (LPA3) serve as receptors specific for LPA. Each receptor isoform displays a unique tissue expression pattern and coupling to a distinct set of heterotrimeric G proteins, leading to the activation of an isoform-specific panel of multiple intracellular signaling pathways. Recent studies on knockout mice have unveiled non-redundant Edg receptor functions that are essential for normal development and vascular maturation. In addition, the Edg lysophospholipid signaling system may play a role in modulating cell motility under such pathological conditions as inflammation, tumor cell dissemination and vascular remodeling.抄録全体を表示PDF形式でダウンロード (735K) -
Zen-ichiro Honda, Satoshi Ishii, Takao Shimizu2002 年 131 巻 6 号 p. 773-779
発行日: 2002年
公開日: 2008/06/30
ジャーナル フリーPlatelet-activating factor (PAF) is a pro-inflammatory lipid mediator possessing a unique 1-O-alkyl glycerophospholipid (GPC) backbone (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholin). Cloned PAF receptor, which belongs to the G protein-coupled receptor superfamily, transduces pleiotropic functions including cell motility, smooth muscle contraction, and synthesis and release of mediators and cytokines via multiple heterotrimeric G proteins. Pharmacological studies have suggested that PAF functions in a variety of settings including allergy, inflammation, neural functions, reproduction, and atherosclerosis. Establishment of PAFR-/- mice confirmed that the PAF receptor is responsible for pro-inflammatory responses, but that its roles in other settings remain to be clarified.抄録全体を表示PDF形式でダウンロード (951K) -
Noriyuki Hatae, Yukihiko Sugimoto, Atsushi Ichikawa2002 年 131 巻 6 号 p. 781-784
発行日: 2002年
公開日: 2008/06/30
ジャーナル フリーProstaglandin (PG) E2 produces a broad range of physiological and pharmacological actions in diverse tissues through specific receptors on plasma membranes for maintenance of local homeostasis in the body. PGE receptors are divided into four subtypes, EP1, EP2, EP3, and EP4, which have been identified and cloned. These EP receptors are members of the G-protein coupled receptor family. Among these subtypes, the EP3 receptor is unique in its ability to couple to multiple G proteins. EP3 receptor signals are primarily involved in inhibition of adenylyl cyclase via Gi activation, and in Ca2+-mobilization through Gβγ from Gi. Along with Gi activation, the EP3 receptor can stimulate cAMP production via Gs activation. Recent evidence indicates that the EP3 receptor can augment Gs-coupled receptor-stimulated adenylyl cyclase activity, and can also be coupled to the G13 protein, resulting in activation of the small G protein Rho followed by morphological changes in neuronal cells. This article focuses on recent studies on the novel pathways of EP3 receptor signaling.抄録全体を表示PDF形式でダウンロード (548K) -
Yasunori Yokoyama, Masashi Sonoyama, Shigeki Mitaku2002 年 131 巻 6 号 p. 785-790
発行日: 2002年
公開日: 2008/06/30
ジャーナル フリーThe photo-intermediate state of bacteriorhodopsin is a metastable state that spontaneously transforms to the ground state over the energy barrier of a local minimum. As the recovery of the photocycle to the ground state and irreversible photobleaching to the denatured state may occur from the same local energy minimum, depending on the temperature, the structural stability of bacteriorhodopsin under illumination at high temperature was measured in order to study the intra- and inter-molecular interactions that contribute to the recovery of the ground state. Visible CD spectra of bacteriorhodopsin began to change at 60°C from a bilobed to positive type in accordance with an appearance of an absorption peak at 470 nm. Irreversible photobleaching, the lightinduced denaturation, also started to occur at 60°C, suggesting some correlation between irreversible photobleaching and the structural change to the high-temperature intermediate state. However, bacteriorhodopsin in the dark was stable up to 70°C, suggesting that there is some additional factor that lends structural stability to bacteriorhodopsin in the dark. The contribution of protein-protein interactions to stability is discussed on the basis of the difference in the denaturation behaviors between light and dark conditions.抄録全体を表示PDF形式でダウンロード (766K) -
Yuji Matsuda, Kei-ichiro Inamori, Tsukasa Osaki, Akane Eguchi, Azusa W ...2002 年 131 巻 6 号 p. 791-800
発行日: 2002年
公開日: 2008/06/30
ジャーナル フリーA cytochrome cb-type enzyme with cytochrome c-oxidase activity was purified from an aerobic phototrophic bacterium Roseobacter denitrificans. The enzyme was solubilized with sucrose monodecanoate from the membranes of R. denitrificans grown aerobically under light conditions, and purified to electrophoretic homogeneity. Absorption spectra of the purified enzyme showed peaks at 410nm and 530nm in the oxidized state, and peaks at 420, 522, and 551nm and a shoulder at around 560nm in the reduced state. The enzyme is composed of two subunits with apparent molecular weights on SDS-PAGE of 37, 000 and 18, 000, the latter positive to heme staining. The protein contains heme c, heme b, and copper in a 1:2:1 stoichiometry. The spectral properties indicated that the heme c and one heme b are in low-spin states, while the other heme b is in a high-spin state. The base sequences of the genes and the deduced amino acid sequences are similar to those of known NorB and NorC subunits of nitric oxide reductases from other bacterial species. The enzyme is similar to nitric oxide reductase, but differs in that it contains copper. Virtually no nitric oxide reductase activity was detected in the purified enzyme.抄録全体を表示PDF形式でダウンロード (2367K) -
Seiichi Taguchi, Hirofumi Nakamura, Tomohiro Hiraishi, Ichiro Yamato, ...2002 年 131 巻 6 号 p. 801-806
発行日: 2002年
公開日: 2008/06/30
ジャーナル フリーIn vitro evolution was applied to obtain highly active mutants of Ralstonia eutropha polyester synthase (PhbCRe), which is a key enzyme catalyzing the formation of polyhydroxybutyrate (PHB) from (R)-3-hydroxybutyryl-CoA (3HB-CoA). To search for beneficial mutations for activity improvement of this enzyme, we have conducted multi-step mutations, including activity loss and intragenic suppression-type activity reversion. Among 259 revertants, triple mutant E11S12 was obtained as the most active one via PCR-mediated secondary mutagenesis from mutant Ell with a single mutation (Ser to Pro at position 80), which exhibited reduced activity (as low as 27% of the wild-type level) but higher thermostability compared to the wild-type enzyme. Mutant E11S12 exhibited up to 79% of the wild-type enzyme activity. Mutation separation of E11S12 revealed that the replacement of Phe by Ser at position 420 (F420S), located in a highly conserved α/β hydrolase fold region, of the E11S12 mutant contributes to the improvement of the enzyme activity. A purified sample of the genetically engineered mutant, termed E11S12-1, with the F420S mutation alone was found to exhibit a 2.4-fold increase in specific activity toward 3HB-CoA, compared to the wild-type.抄録全体を表示PDF形式でダウンロード (3057K) -
Shunji Natsuka, Jiro Adachi, Masahumi Kawaguchi, Shin-ichi Nakakita, S ...2002 年 131 巻 6 号 p. 807-813
発行日: 2002年
公開日: 2008/06/30
ジャーナル フリーCaenorhabditis elegans is an excellent model for morphogenetic research. However, little information is available on the structure of cell-surface glycans in C. elegans, although several lines of evidence have suggested a role for these glycans in cell-cell interactions during development. In this study, we analyzed N-glycan structures. Oligosaccharides liberated by hydrazinolysis from a total membrane fraction were labeled by pyridylamination, and around 90% of the N-glycans were detected as neutral oligosaccharides. The most dominant structure was Manα1-6(Manαl-3)Manβ1-4GIcNAcβ14GIcNAc, which is commonly found in insects. Branching structures of major oligomannose-type glycans were the same as those found in mammals. Structures that had a core fucose or non-reducing end N-acetylglucosamine were also identified, but ordinary complex-type glycans with N-acetyllactosamine were not detected as major components.抄録全体を表示PDF形式でダウンロード (732K) -
Akm A. Sattar, Ramesh Boinpally, Marvin H. Stromer, Bhanu P. Jena2002 年 131 巻 6 号 p. 815-820
発行日: 2002年
公開日: 2008/06/30
ジャーナル フリーEarlier studies indicated that a Gi-like protein localized in pancreatic zymogen granule (ZG) membrane mediates vesicle swelling, and is a potentially important prerequisite for vesicle fusion at the cell plasma membrane (PM) [Jena et al. (1997) Proc. Natl. Acad. Sci. USA 94, 13317-13322]. In the present study, we demonstrate the presence of Gαi3 immunoreactivity in ZGs of rat exocrine pancreas using immunoblot assays, light and electron immunomicroscopy. Since GTP has been implicated in the fusion of isolated ZG with PM fractions [Nadin et al. (1989) J. Cell Biol. 109, 2801-2808], the potential role of ZG-associated Gαi3 was investigated. Immunoblot assays demonstrate an increase in Gαi3 protein in ZGs isolated from carbamylcholine stimulated pancreas. Thin layer chromatography shows an increase in GTP hydrolysis by GTPase in ZGs isolated from stimulated compared to resting pancreas. In vitro fusion assays demonstrate that ZGs isolated from carbamylcholine-stimulated pancreatic lobules fuse with the PM at a greater potency in the presence of GTP, mastoparan (G protein agonist) and its analogue mas7. Furthermore, Gαi3-specific a recombinant GAIP (G alpha interacting protein), potenti-ates ZG-PM fusion in the presence of GTP but not in presence of the non-hydrolyzable GTP analogue Gpp(NH)p. Our immunoblot analysis demonstrates the recruitment of Gαi3 immunoreactivity to ZG from stimulated acinar cells, and these isolated ZGs are more potent and efficient in fusing with plasma membrane fractions, suggesting the possible involvement of Gαi3 in ZG-PM fusion. The participation of ZG-associated Gαi3 in ZG-PM fusion is further confirmed by the influence of the Gαi3-specific GAIP, which is known to interact specifically with Gαi3, and not with Gαi2 or Gαq [DeVries et al. (1995) Proc. Natl. Acad. Sci. USA 92, 11916-11920]. Additionally, our data suggest that GTP hydrolysis is a requirement for ZG-PM fusion since GAIP in the presence of Gpp (NH) p shows little or no effect on fusion, whereas GAIP in the presence of GTP significantly potentiates ZG-PM fusion. Our studies suggest a possible role for ZG-associated Gαi3 in ZG-PM fusion.抄録全体を表示PDF形式でダウンロード (3565K) -
Shinya Kamauchi, Keiji Mitsui, Satoshi Ujike, Megumi Haga, Norihiro Na ...2002 年 131 巻 6 号 p. 821-831
発行日: 2002年
公開日: 2008/06/30
ジャーナル フリーGenes encoding the Na+/H+ antiporter (Nha1p) from Candida tropicalis (C. t.), Hansenula anomala (H. a.) (also named Pichia anomala), and Aspergillus nidulans (A. n.) were cloned, and the nucleotide sequences were determined. The deduced primary sequences revealed highly conserved hydrophobic regions and rather diverse hydrophilic regions. Among the seven known Nha1p sequences, Schizosaccharomyces pombe (S. p.) Nha1p is exceptional in lacking the hydrophilic region. Within the diverse hydrophilic regions, we found six conserved regions (C1-C6). Expression of C. t. Nha1p in Saccharomyces cerevisiae (S. c.) cells lacking NHA1 and ENA1 (Na+-ATPase) complemented the salinity-sen-sitive phenotype, suggesting that C. t. Nha1p is functionally related to S. c. Nha1p. Expression of various truncated forms of the C-terminal half of S. c. and C. t. Nha1p showed essentially the same phenotype for both species: deletion of the C4-C6 region caused cell growth to be more resistant to high salinity than the wild type, suggesting an inhibitory function of these domains on the antiporter activity. However, complete loss of C1-C6 caused a severe growth defect under conditions of high salinity, suggesting a defect in antiporter activity. The ΔC2-C6 form of C. t. Nha1p, containing only C1, restored the retarded cell growth at high salinity more than the control vector alone, but to a value lower than the wild type. These results suggest an essential role for C1 and an activating role of the C2-C3 region in the functional expression of Nhal. High expression of the ΔC2-C6 form of S. c. Nha1p was toxic for yeast cells, although low expression was not, suggesting that the overexpression of C1 is toxic. The results in this study suggest that the diverse hydrophilic region of yeast and fungal Nha1p has six con-served domains with conserved functions in terms of expression of Nha1p activity.抄録全体を表示PDF形式でダウンロード (4846K) -
Rimiko Koyanagi-Katsuta, Nobuyoshi Akimitsu, Hiroshi Hamamoto, Nagisa ...2002 年 131 巻 6 号 p. 833-837
発行日: 2002年
公開日: 2008/06/30
ジャーナル フリーWe report a lethal phenotype of mouse embryo with a disruption in the gene encoding p116, a subunit of the translation initiation factor, eIF3. The amino acid sequence of mouse p116, as deduced from the cDNA, shows high homology (97%) with human p116, and contains the conserved RNA binding sites, RNP1 and RNP2. The p116 mRNA is ubiquitously expressed in various organs, suggesting a house-keeping function of the p116 protein. To obtain genetic evidence for the essential role of the p116 protein in mouse cells, we constructed mice with a disruption in the p116 gene. Heterozygous p116+/- mice were intercrossed, and the genotypes of the offspring were determined. The results indicated no p116-/- pups among 84 neonates. Also, there were no p116-/- embryos 13.5 days postcoitum (d. p. c.). Among 77 embryos, there was only one p116-/- embryo at the blastocyst stage (3.5 d. p. c.). These results indicate that p116 plays an essential role in the early stages of mouse development.抄録全体を表示PDF形式でダウンロード (1671K) -
Terumichi Tanaka, Yasushi Kondo, Yoshiaki Hori, Yo Kikuchi2002 年 131 巻 6 号 p. 839-847
発行日: 2002年
公開日: 2008/06/30
ジャーナル フリーRecently, we revealed that the cloverleaf structure of some eukaryotic tRNAs is not always stable in vitro, and the denatured structures of these tRNAs are sometimes detected in bacterial RNase P reactions. We have designated the unusual internal cleavage reaction of these tRNAs as hyperprocessing. We have developed this hyperprocessing strategy as a useful tool for examining the stability of the tRNA cloverleaf structure. There are some common features in such unstable, hyperprocessible tRNAs, and the criteria for the hyperprocessing reaction of tRNA are extracted. Metazoan initiator methionine tRNAs and lysine tRNAs commonly fit the criteria, and are predicted to be hyperprocessible. The RNase P reactions of two metazoan lysine tRNAs from Homo sapiens and Caenorhabditis elegans, which fit the criteria, resulted in resistance to the internal cleavage reaction, while one bacterial lysine tRNA from Acholeplasma laidlawii, which also fits the criteria, was internally cleaved by the RNase P. The results showed that the metazoan lysine tRNAs examined are very stable without base modifications even under in vitro conditions. We also examined the 3'-half short construct of the human lysine tRNA, and the results showed that this RNA was internally cleaved by the enzyme. The results indicated that the human lysine tRNA has the ability to be hyperprocessed but is structurally stabilized in spite of lacking base modifications. A comparative study suggested, moreover, that the acceptor-stem bases should take part in the stabilization of metazoan lysine tRNAs. Our data strongly suggest that the cloverleaf shape of other metazoan lysine tRNAs should also be stabilized by means of similar strategies to in the case of human tRNA (Lys3).抄録全体を表示PDF形式でダウンロード (5439K) -
Taketoshi Uzawa, Akihiko Yamagishi, Tairo Oshima2002 年 131 巻 6 号 p. 849-853
発行日: 2002年
公開日: 2008/06/30
ジャーナル フリーPolypeptide synthesis at high temperature directed by single strand DNA as a messenger was investigated using cell-free extracts of an extremely thermophilic bacterium, Thermus thermophilus strain HB27, and a hyperthermophilic, acidophilic archaeon, Sulfolobus tokodaii strain 7. Aminoglycoside antibiotics enhanced the reaction; neomycin stimulated it most effectively when the extract of the thermophilic bacterium was used, and paromomycin was the best among the antibiotics tested for the extract of the hyperthermophilic archaeon. A common correlation was found between the stimulation of DNA-directed polypeptide synthesis and the misreading rate in RNA-directed polypeptide synthesis. Spermine stimulated the reaction directed by DNA like in the case of poly (Phe) synthesis directed by poly (rU). The cell-free systems can be used for direct production of proteins from genes in high throughput studies on the structural genomics of thermophilus.抄録全体を表示PDF形式でダウンロード (650K) -
Li-Ming Chen, Xing-Wen Yang, Jian-Guo Tang2002 年 131 巻 6 号 p. 855-859
発行日: 2002年
公開日: 2008/06/30
ジャーナル フリーTo investigate the role of C-peptide in the folding of insulin precursor, a series of C-pep-tide mutant proinsulin genes were constructed, overexpressed in Escherichia coli and the proteins purified. Correct disulfide linkages of these proteins were confirmed by both tryptic peptide mapping and insulin receptor binding analyses. In vitro refolding experiments were performed with the purified proteins and showed that mutations on the glycine-rich middle segment of C-peptide, GGGPGAG, and deletion of the C-terminal pentapeptide, EGSLQ, as well as mutations on the two pairs of dibasic residues at the two ends of C-peptide did not significantly affect the refolding yields. However, both alanine replacement mutation and deletion of three highly conserved acidic residues (EAED) at the N-terminus of the C-peptide resulted in serious aggregation during refolding. The results indicate that the highly conserved acidic N-terminal part of C-peptide is very important for insulin precursor folding, and that C-peptide may have some intramolecular chaperone-like function in the folding of insulin precursor.抄録全体を表示PDF形式でダウンロード (1967K) -
Masaki Ichikawa, Takanobu Kikuchi, Hisashi Tateiwa, Norimoto Gotoh, Ko ...2002 年 131 巻 6 号 p. 861-868
発行日: 2002年
公開日: 2008/06/30
ジャーナル フリーPTB-like protein (PTBLP) is a new homologue of pyrimidine tract binding protein (PTB), and has been cloned as a possible autoantigen in cancer-associated retinopathy. PTBLP has two functional domains, the nuclear localization signal and the RNA recognition motifs (RRMs). Full-length PTBLP (PTBLP-L) has four RRMs, and its alternative splicing product (PTBLP-S) lacks the third and fourth RRMs. Although PTBLPs are expressed in neuronal tissues, the function of PTBLPs has not been determined. We have studed whether PTBLP plays a role in neuronal differentiation using PC12 cells. During the process of nerve growth factor-induced neuronal differentiation of PC12 cells, PTBLP-L was down-regulated whereas PTBLP-S was up-regulated. Transfection of PTBLP-L into PC12 cells led to the suppression of neuronal differentiation. In PTBLP-S transfected cells, however, this suppression was not evident. When both PTBLP-L and PTBLP-S were co-transfected, the suppressive effect of PTBLP-L decreased. In differentiated cells, PTBLP-S localized in the nucleus and PTBLP-L was found dispersed throughout the cytoplasm and neuronal growth cone. These findings suggest that PTBLP-L acts as a negative regulator of neuronal differentiation and PTBLP-S acts as a competitor of PTBLP-L.抄録全体を表示PDF形式でダウンロード (4599K) -
Sang-Hee Park, Yun-Sil Lee, Yoshiaki Osawa, Misao Hachiya, Makoto Akas ...2002 年 131 巻 6 号 p. 869-875
発行日: 2002年
公開日: 2008/06/30
ジャーナル フリーExposure of cells to external stresses leads to the induction or activation of certain proteins. Expression of heat shock proteins (Hsps) is induced in response to these stresses. Hsps are known to have molecular chaperone activities; but recent studies have shown that Hsps have a variety of functions such as the triggering of proliferation, differentiation, and apoptosis of cells. Previously, we found that overexpression of a 25 kDa Hsp (Hsp25) induced expression of cell cycle inhibitory protein p21 (Waft/Cipl/Sdil) in murine fibroblastoid L929 cells. However, the mechanisms underlying the induction of p21 by Hsp25 are unknown. In the present study, we investigated the mechanisms underlying the regulation of p21 expression by Hsp25 in these cells. The introduction of Hsp25 cDNA stimulated the accumulation of p21 transcripts through transcriptional but not posttranscriptional regulation in these cells. We also found that overexpression of Hsp25 markedly increased the translational rate of p21 and stabilized the protein. Studies involving proteasome inhibitors and Western blot analysis for ubiquitination of p21 demonstrated that the stabilization of p21 is regulated through a ubiquitin-independent pathway. However, no direct association of Hsp25 with p21 was observed. These findings suggest that Hsp25 induces p21 expression through multiple mechanisms, and that transcriptional, translational, and post-translational regulation are important in the regulation of p21.抄録全体を表示PDF形式でダウンロード (4598K) -
Yasuko Sato, Hiroko Takita, Noboru Ohata, Masato Tamura, Yoshinori Kub ...2002 年 131 巻 6 号 p. 877-886
発行日: 2002年
公開日: 2008/06/30
ジャーナル フリーWe previously isolated pleiotrophin (PTN) from bovine bone as a protein and showed that it stimulated osteoblastic growth and differentiation. Further details of its function, however, have not been fully clarified. The aim of this paper was to elucidate the effects of PTN on bone morphogenetic protein (BMP)-induced ectopic osteogenesis. Recombinant human BMP (rhBMP)-2 (1.2μg) was combined with a fibrous glass membrane, which had been established as an effective carrier. Various amounts of the purified bovine PTN (5, 10, 50, and 100μg) or rhPTN (5 and 10μg) were added to the rhBMP-2/carrier composites and implanted into rats subcutaneously as reported. It was found that the amount of bone induced in the system increased with the addition of 10μg of either purified PTN or rhPTN. However, the amount of bone decreased with the addition of 50 or 100μg of purified PTN dose-dependently, as judged by both alkaline phosphatase activity and calcium content in the retrieved implants. It was concluded that purified PTN or rhPTN, at ratios of concentration of 10-100μg of PTN to 1.2μg of rhBMP-2 in the carrier, regulated the ectopic bone-inducing activity of rhBMP-2.抄録全体を表示PDF形式でダウンロード (5915K) -
Shinya Amino, Makoto Itakura, Hiroshi Ohnishi, Jun Tsujimura, Shinichi ...2002 年 131 巻 6 号 p. 887-894
発行日: 2002年
公開日: 2008/06/30
ジャーナル フリーNeurotrophins play important roles in the differentiation and survival of neurons during development, and in the regulation of synaptic transmission in adult brain. Brief treatment with nerve growth factor (NGF) enhances depolarization and ionomycin-in-duced dopamine and acetylcholine release from PC12 cells. The enhancing effect appears very quickly and reaches a plateau 10-15 min after application. NGF also enhances hypertonic solution-induced dopamine release, and increases the amount of dopamine released from membrane-permeabilized PC12 cells in the absence of MgATP, suggesting that NGF enhances neurotransmitter release by increasing the number of Ca2+ -responsive secretory vesicles. The activation of Trk receptors is essential for NGF action, since K252a abolishes the NGF-induced potentiation of dopamine release and brain-derived neurotrophic factor enhanced ionomycin-induced release only in TrkB-expressing cells. NGF-mediated potentiation of dopamine release is completely abolished by wortmannin, a PI 3-kinase inhibitor, and by U0126 and PD98059, MAP kinase kinase inhibitors, indicating that the activation of PI 3-kinase and MAP kinase pathways is essential for NGF action. These findings suggest that NGF regulates neurotransmitter release through the activation of TrkA receptors, possibly by increasing the number of secretory vesicles in a readily releasable pool.抄録全体を表示PDF形式でダウンロード (2271K) -
Yoshiro Chuman, Ayami Matsushima, Seiji Sato, Kenji Tomioka, Yoshiya T ...2002 年 131 巻 6 号 p. 895-903
発行日: 2002年
公開日: 2008/06/30
ジャーナル フリーPigment-dispersing factor (PDF) was recently reported to be a principal circadian neuromodulator involved in transmitting circadian rhythms of daily locomotion in insects. In Drosophila, PDF functions in some of the neurons expressing the clock genes period, timeless, Clock, and cycle, and those clock genes in turn regulate pdf gene expression. In the present study, we cloned a cDNA encoding PDF in the brain of a nocturnal insect, the cricket Gryllus bimaculatus, and found that an isolated clone (310 bp) codes for an extraordinarily short precursor protein with no definite signal sequence, but a nuclear localization signal (NLS)-like sequence instead. The cricket PDF exhibits high sequence identity (78-94%) and similarity (89-100%) to insect PDFs and also to crustacean β-PDH peptides. In the optic lobes of G. bimaculatus there are PDF-immunoreactive neurons in both the medulla and lamina neuropiles. Among the strongly immunoreactive lamina PDF neurons, on electron microscopy we identified cells exhibiting distinct staining that is not only cytoplasmic but also nuclear. When GFP-fused PDF precursor proteins were expressed in COS-7 cells, distinct translocation of the fusion protein into the nucleus was observed. This is the first finding of PDF peptide in the nucleus, which suggests a fundamental role of PDF peptide per se in the circadian clock system.抄録全体を表示PDF形式でダウンロード (3994K) -
Shinsaku Maruta, Yoshiaki Mizukura, Shigeru Chaen2002 年 131 巻 6 号 p. 905-911
発行日: 2002年
公開日: 2008/06/30
ジャーナル フリーA new fluorescent ribose-modified ATP analogue, 2'(3')-O-{6-(N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino)hexanoic}-ATP (NBD-ATP), was synthesized and its interaction with skeletal muscle myosin subfragment-1 (S-1) was studied. NBD-ATP was hydrolysed by S-1 at a rate and with divalent cation-dependence similar to those in the case of regular ATP. Skeletal HMM supported actin translocation using NBD-ATP and the velocity was slightly higher than that in the case of regular ATP. The addition of S1 to NBD-ATP resulted in quenching of NBD fluorescence. Recovery of the fluorescence intensity was noted after complete hydrolysis of NBD-ATP to NBD-ADP. The quenching of NBD-ATP fluorescence was accompanied by enhancement of intrinsic tryptophan fluorescence. These results suggested that the quenching of NBD-ATP fluorescence reflected the formation of transient states of ATPase. The formation of S-1•NBD-ADP•BeFn and S-1•NBD-ADP•AIF4- complexes was monitored by following changes in NBD fluorescence. The time-course of the formation fitted an exponential profile yielding rate constants of 7.38×10-2 s-1 for BeFn and 1.1×10-3 s-1 for AlF4-. These values were similar to those estimated from the intrinsic fluorescence enhancement of trp due to the formation of S-1•ADP•BeFn or AIF4- reported previously by our group. Our novel ATP analogue seems to be applicable to kinetic studies on myosin.抄録全体を表示PDF形式でダウンロード (847K) -
Heather A. Myler, Jennifer L. West2002 年 131 巻 6 号 p. 913-922
発行日: 2002年
公開日: 2008/06/30
ジャーナル フリーBasic fibroblast growth factor (bFGF) has been shown to play an instrumental role in the cascade of events leading to restenosis; however, the mechanisms of bFGF activation following vascular injury have remained elusive. We have demonstrated that heparanase and platelet factor-4 (PF4), released from activated platelets at the site of injury, liberate bFGF from the extracellular matrix (ECM) of vascular smooth muscle cells (SMC), resulting in the induction of SMC proliferation and migration. Increases in proliferation and migration were inhibited by treatment with a bFGF-neutralizing antibody, suggesting that proliferation and migration in response to heparanase or PF4 are mediated by bFGF activation. When platelets were seeded on top of SMCs, degranulation products were found to release bFGF from the ECM, increasing cell proliferation and cell migration. Again, these increases in SMC proliferation and migration were inhibited by treatment with an anti-bFGF antibody. Furthermore, these increases in proliferation were completely inhibited by treatment with an anti-heparanase antibody. Platelet degranulation products, such as heparanase and PF4, may liberate bFGF from extracellular sequestration, activating the growth factor and inducing the SMC proliferation and migration that contribute to the wound healing response following vascular injury.抄録全体を表示PDF形式でダウンロード (3525K) -
Tohru Kimura, Ayumi Yoshida, Yoshiaki Tabuchi, Akira Ikari, Noriaki Ta ...2002 年 131 巻 6 号 p. 923-932
発行日: 2002年
公開日: 2008/06/30
ジャーナル フリーStable cell lines expressing the gastric proton pump α- and/or β-subunits were constructed. The cell line co-expressing the α-and β-subunits showed inward Rb+ transport, which was activated by Rb+ in a concentration-dependent manner. In the α+β-expressing cell line, rapid recovery of intracellular pH was also observed after acid load, indicating that this cell line transported protons outward. These ion transport activities were inhibited by a proton pump inhibitor, 2-methyl-8-(phenylmethoxy)imidazo[1, 2-a]pyridine-3-acetonitrile (SCH 28080). In a membrane fraction of the α+β-expressing cell line, K+-stimulated ATPase (K+-ATPase) activity and the acylphosphorylation of the α-subunit were observed, both of which were also inhibited by SCH 28080. The specific activity and properties of the K+-ATPase were comparable to those found in the native gastric proton pump. In the stable cell lines, the α-subunit was retained in the intracellular compartment and was unstable in the absence of the β-subunit, but it was stabilized and reached the cell surface in the presence of the β-subunit. On the other hand, the β-subunit was stable and able to travel to the cell surface in the absence of the α-subunit. These cell lines are ideal for the structure-function study of ion transport by the gastric proton pump as well as for characterization of the cellular regulation of surface expression of the functional proton pump.抄録全体を表示PDF形式でダウンロード (5958K) -
Hironobu Kimura, Isao Suetake, Shoji Tajima2002 年 131 巻 6 号 p. 933-941
発行日: 2002年
公開日: 2008/06/30
ジャーナル フリーMouse DNA (cytosine-5) methyltransferases Dnmt3a and Dnmt3b are expected to be de novo-type DNA methyltransferases. In the present study, we found that exogenously expressed mouse Dnmt3a or Dnmt3b induced abnormal cell clusters at the gastrulation stage in Xenopus embryos. The abnormal cells were judged to be apoptotic from the positive staining with the TdT dUTP nucleotide end-labeling method and the rescue by hBcl-xL, a Bcl-2 homologue. On the other hand, neither bacterial DNA (cytosine-5) methyltransferase nor Dnmt3b3, one of the three isoforms of Dnmt3b that has no DNA methylation activity, induced apoptosis. In addition, mutant Dnmt3a and the other two Dnmt3b isoforms, Dnmt3b1 and Dnmt3b2, which have no DNA methylation activity due to a change of the cysteine residue in the catalytic center to an alanine residue, retained the ability to induce apoptosis. This indicates that the apoptosis was not induced by DNA methylation activity. The domain of Dnmt3b1 (3b2) responsible for the apoptosis is the catalytic domain in the carboxyl-terminal half.抄録全体を表示PDF形式でダウンロード (6405K)
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