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141.
Sugawara A Torigoe T Tamura Y Kamiguchi K Nemoto K Oguro H Sato N 《Cell stress & chaperones》2009,14(2):133-139
Polyamine compound deoxyspergualin (DSG) is a potent immunosuppressive agent that has been applied clinically for protecting
graft rejection and treatment of Wegener's granulomatosis. Though DSG can bind to heat-shock proteins (HSPs) in cells, its
mechanism of immunosuppressive action remains unknown. It is widely accepted that extracellular HSPs are capable of stimulating
dendritic cells (DC) through cell surface receptors, leading to DC activation and cytokine release. In this study, we examined
if DSG analogs could inhibit HSP70-induced DC activation. Bone marrow derived immature mouse DCs and peripheral blood mononuclear
cell-derived immature human DCs were generated and incubated with Alexa 488-labeled Hsp70 in the presence of methoxyDSG (Gus-1)
that had comparable HSP70-binding affinity to DSG or DSG analog GUS-7, which had much more reduced binding affinity for HSP70.
The binding of HSP70 to immature DCs was analyzed by laser microscopy and flow cytometry. HSP70-induced DC activation was
assessed by TNF-α release by enzyme-linked immunosorbent assay. Binding of Hsp70 to the cell surface of immature DCs was inhibited
under the presence of Gus-1, but not under the presence of Gus-7. Immature DCs were activated and released TNF-α by the stimulation
with HSP70 for 12 hours; however, the HSP70-induced TNF-α release was suppressed under the presence of Gus-1, and partially
suppressed under the presence of Gus-7. Similar results were observed when immature human DCs were stimulated under the same
conditions. Immunosuppressive mechanism of DSG may be explained, at least in part, by the inhibition of extracellular HSP70-DC
interaction and HSP70-induced activation of immature DCs.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
142.
Christopher Looze David Yui Lester Leung Matthew Ingham Maryann Kaler Xianglan Yao Wells W. Wu Rong-Fong Shen Mathew P. Daniels Stewart J. Levine 《Biochemical and biophysical research communications》2009,378(3):433-438
Exosomes are nanovesicles that are released from cells as a mechanism of cell-free intercellular communication. Only a limited number of proteins have been identified from the plasma exosome proteome. Here, we developed a multi-step fractionation scheme incorporating gel exclusion chromatography, rate zonal centrifugation through continuous sucrose gradients, and high-speed centrifugation to purify exosomes from human plasma. Exosome-associated proteins were separated by SDS–PAGE and 66 proteins were identified by LC–MS/MS, which included both cellular and extracellular proteins. Furthermore, we identified and characterized peroxisome proliferator-activated receptor-γ (PPARγ), a nuclear receptor that regulates adipocyte differentiation and proliferation, as well as immune and inflammatory cell functions, as a novel component of plasma-derived exosomes. Given the important role of exosomes as intercellular messengers, the discovery of PPARγ as a component of human plasma exosomes identifies a potential new pathway for the paracrine transfer of nuclear receptors. 相似文献
143.
Young-Jun Park Kazuhiro Nemoto Tomotaro Nishikawa Kenichi Matsushima Mineo Minami Makoto Kawase 《Molecular breeding : new strategies in plant improvement》2010,25(4):623-635
Waxy strains raised by waxy mutation have been found for three amaranth grains. Three genes encoding waxy protein were isolated from Amaranthus caudatus (Wx-ca), A. cruentus (Wx-cr), and A. hypochondriacus (Wx-hy). Sequence analysis indicated that the Wx-ca, Wx-cr, and Wx-hy genes contained the same exon (13 exons) and intron (12 introns) structure. The lengths of the Wx-ca, Wx-cr, and Wx-hy genes were 3,236, 3,237, and 3,225 bp, respectively. The alignment of the coding sequence of the three Waxy genes showed 12 polymorphic sites, including 11 single nucleotide polymorphisms (SNPs) (in exons 10 and 12 and introns 1, 3, 4, 9, and 11) and 5 deletions or insertions (indels) (in introns 4, 9, and 11). In particular, major polymorphism was detected in 8- and 3-bp indels in intron 4. Moreover, the mutation in the waxy alleles (wx-ca, wx-cr, and wx-hy) of all three species was also isolated and characterized. Comparison of coding sequences of the three Waxy genes and their waxy alleles indicated one base insertion (wx-ca: insert of T base in exon 8) and a base substitution (wx-cr: a G-to-T base substitution in exon 10; wx-hy: a G-to-A base substitution in exon 6), which occurred as internal termination codon in the three Waxy genes, suggesting the involvement of a nonsense or frameshift mutation. Therefore, these different mutations in coding regions were considered to be the cause of the waxy (amylose-free) phenotype. 相似文献
144.
Matsuda H Yoshida K Miyagawa K Nemoto Y Asao Y Yoshikawa M 《Bioorganic & medicinal chemistry letters》2006,16(6):1567-1573
The methanolic extract and its alkaloid fraction from the rhizomes of Nuphar pumilum showed cytotoxic effects on human leukemia cell (U937), mouse melanoma cell (B16F10), and human fibroblast (HT1080). Dimeric sesquiterpene thioalkaloids with the 6-hydroxyl group (6-hydroxythiobinupharidine, 6,6'-dihydroxythiobinupharidine, 6-hydroxythionuphlutine B) showed substantial cytotoxic activity at a concentration of 10 microM, but dimeric sesquiterpene thioalkaloids lacking the 6-hydroxyl group (thiobinupharidine, thionuphlutine B, 6'-hydroxythionuphlutine B, neothiobinupharidine, thionuphlutine B beta-sulfoxide, and neothiobinupharidine beta-sulfoxide) and monomeric sesquiterpene alkaloids (nupharidine, 7-epideoxynupharidine, and nupharolutine) showed weak activity. Next, apoptosis-inducing activity of a principal active constituent, 6-hydroxythiobinupharidine, on U937 was examined using morphological observation and DNA fragmentation assay (TUNEL method). Apoptosis of U937 was immediately observed within 1 h after treatment of 6-hydroxythiobinupharidine at 2.5-10 microM. 相似文献
145.
A Tomiyama K Tachibana K Suzuki S Seino J Sunayama K-i Matsuda A Sato Y Matsumoto T Nomiya K Nemoto H Yamashita T Kayama K Ando C Kitanaka 《Cell death & disease》2010,1(7):e60
Recently developed heavy ion irradiation therapy using a carbon beam (CB) against systemic malignancy has numerous advantages. However, the clinical results of CB therapy against glioblastoma still have room for improvement. Therefore, we tried to clarify the molecular mechanism of CB-induced glioma cell death. T98G and U251 human glioblastoma cell lines were irradiated by CB, and caspase-dependent apoptosis was induced in both cell lines in a dose-dependent manner. Knockdown of Bax (BCL-2-associated X protein) and Bak (BCL-2-associated killer) and overexpression of Bcl-2 or Bcl-xl (B-cell lymphoma-extra large) showed the involvement of Bcl-2 family proteins upstream of caspase activation, including caspase-8, in CB-induced glioma cell death. We also detected the activation of extracellular signal-regulated kinase (ERK) and the knockdown of ERK regulator mitogen-activated protein kinase kinase (MEK)1/2 or overexpression of a dominant-negative (DN) ERK inhibited CB-induced glioma cell death upstream of the mitochondria. In addition, application of MEK-specific inhibitors for defined periods showed that the recovery of activation of ERK between 2 and 36 h after irradiation is essential for CB-induced glioma cell death. Furthermore, MEK inhibitors or overexpression of a DN ERK failed to significantly inhibit X-ray-induced T98G and U251 cell death. These results suggested that the MEK–ERK cascade has a crucial role in CB-induced glioma cell death, which is known to have a limited contribution to X-ray-induced glioma cell death. 相似文献
146.
We designed a new approach for selection of translation enhancer sequences that enables efficient protein synthesis in cell-free systems. The selection is based on a gel shift assay of a messenger RNA (mRNA)–protein fusion product that is synthesized in a cell-free translation system using an mRNA display method. A library of randomized 20-nt-long sequences, with all possible combinations of the four nucleotides, upstream of a coding region was screened by successive rounds of screening in which the translation time of the succeeding round was reduced compared with the previous round. An efficient translation enhancer sequence capable of more rapid initiation of cell-free protein synthesis, with a minimal translation time of 5 min, than a natural longer enhancer sequence (Xenopus β-globin 5′UTR) was selected using rabbit reticulocyte extract as a model cell-free translation system. Furthermore, a successful screening of cap-independent translation enhancer sequence and a significant sequence similarity of the selected candidates validated the efficiency of the combined mRNA display and gel shift assay method for the rapid development of advanced cell-free translation systems. 相似文献
147.
148.
Ichida M Yui Y Yoshioka K Tanaka T Wakamatsu T Yoshikawa H Itoh K 《FEBS letters》2011,585(24):4018-4024
We showed that the migration, morphology and adhesiveness of undifferentiated mesenchymal cells dramatically changed during osteogenic differentiation. The migration of these cells was transiently upregulated early in osteogenic differentiation. At a later stage, migration was decreased but adhesiveness was increased. Furthermore, Cdc42 and Rac1 Rho-family small GTPases were activated at early stages of differentiation and the phosphorylation level of FAK decreased as differentiation progressed. We also showed cell migration was promoted by inhibition of the Rho-ROCK-myosin signaling. Finally, using a mouse model of ectopic bone formation, we confirmed that treatment with ROCK inhibitor, Y-27632 increased cell movement into bone formation sites, resulting in enhanced osteogenesis. These results provide a new insight into the link between cell migration and osteogenic differentiation. 相似文献
149.
Ohara-Nemoto Y Shimoyama Y Kimura S Kon A Haraga H Ono T Nemoto TK 《The Journal of biological chemistry》2011,286(44):38115-38127
Porphyromonas gingivalis and Porphyromonas endodontalis, asaccharolytic black-pigmented anaerobes, are predominant pathogens of human chronic and periapical periodontitis, respectively. They incorporate di- and tripeptides from the environment as carbon and energy sources. In the present study we cloned a novel dipeptidyl peptidase (DPP) gene of P. endodontalis ATCC 35406, designated as DPP11. The DPP11 gene encoded 717 amino acids with a molecular mass of 81,090 Da and was present as a 75-kDa form with an N terminus of Asp(22). A homology search revealed the presence of a P. gingivalis orthologue, PGN0607, that has been categorized as an isoform of authentic DPP7. P. gingivalis DPP11 was exclusively cell-associated as a truncated 60-kDa form, and the gene ablation retarded cell growth. DPP11 specifically removed dipeptides from oligopeptides with the penultimate N-terminal Asp and Glu and has a P2-position preference to hydrophobic residues. Optimum pH was 7.0, and the k(cat)/K(m) value was higher for Asp than Glu. Those activities were lost by substitution of Ser(652) in P. endodontalis and Ser(655) in P. gingivalis DPP11 to Ala, and they were consistently decreased with increasing NaCl concentration. Arg(670) is a unique amino acid completely conserved in all DPP11 members distributed in the genera Porphyromonas, Bacteroides, and Parabacteroides, whereas this residue is converted to Gly in all authentic DPP7 members. Substitution analysis suggested that Arg(670) interacts with an acidic residue of the substrate. Considered to preferentially utilize acidic amino acids, DPP11 ensures efficient degradation of oligopeptide substrates in these Gram-negative anaerobic rods. 相似文献
150.