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1.
Jun Iwaki Kunio Kikuchi Yoshiaki Mizuguchi Yutaka Kawahigashi Hiroshi Yoshida Eiji Uchida Toshihiro Takizawa 《PloS one》2013,8(7)
MicroRNA miR-376c was expressed in normal intrahepatic biliary epithelial cells (HIBEpiC), but was significantly suppressed in the HuCCT1 intrahepatic cholangiocarcinoma (ICC) cell line. The biological significance of the down-regulation of miR-376c in HuCCT1 cells is unknown. We hypothesized that miR-376c could function as a tumor suppressor in these cells. To test this hypothesis, we sought the targets of miR-376c, and characterized the effect of its down-regulation on HuCCT1 cells. We performed proteomic analysis of miR-376c-overexpressing HuCCT1 cells to identify candidate targets of miR-376c, and validated these targets by 3′-UTR reporter assay. Transwell migration assays were performed to study the migratory response of HuCCT1 cells to miR-376c overexpression. Furthermore, microarrays were used to identify the signaling that were potentially involved in the miR-376c-modulated migration of HuCCT1. Finally, we assessed epigenetic changes within the potential promoter region of the miR-376c gene in these cells. Proteomic analysis and subsequent validation assays showed that growth factor receptor-bound protein 2 (GRB2) was a direct target of miR-376c. The transwell migration assay revealed that miR-376c significantly reduced epidermal growth factor (EGF)-dependent cell migration in HuCCT1 cells. DNA microarray and subsequent pathway analysis showed that interleukin 1 beta and matrix metallopeptidase 9 were possible participants in EGF-dependent migration of HuCCT1 cells. Bisulfite sequencing showed higher methylation levels of CpG sites upstream of the miR-376c gene in HuCCT1 relative to HIBEpiC cells. Combined treatment with the DNA-demethylating agent 5-aza-2′-deoxycytidine and the histone deacetylase inhibitor trichostatin A significantly upregulated the expression of miR-376c in HuCCT1 cells. We revealed that epigenetic repression of miR-376c accelerated EGF-dependent cell migration through its target GRB2 in HuCCT1 cells. These findings suggest that miR-376c functions as a tumor suppressor. Since metastasis is the major cause of death in ICC, microRNA manipulation could lead to the development of novel anti-cancer therapy strategies for ICC. 相似文献
2.
S-100 Protein in Clonal Astroglioma Cells Is Released by Adrenocorticotropic Hormone and Corticotropin-Like Intermediate-Lobe Peptide 总被引:4,自引:1,他引:3
Fujiko Suzuki Kanefusa Kato Taiji Kato Nobuaki Ogasawara 《Journal of neurochemistry》1987,49(5):1557-1563
S-100 protein in clonal GA-1 and C6 rat glioma cell lines was released in serum-free medium supplemented with adrenocorticotropic hormone (ACTH). The induction of S-100 protein release by ACTH was dose-dependent, showing a half-maximal release at about 5 microM, and the S-100 protein concentration in the medium increased sharply within 3 min, but slightly during further incubation. The S-100 protein release was apparently accompanied by a decrease in the membrane-bound form of S-100 protein in the cell. The S-100 protein release was induced not by the ACTH1-24 fragment, which exhibits the known effects of ACTH, but by the ACTH18-39 fragment, which is designated as corticotropin-like intermediate-lobe peptide (CLIP). These results indicate that the C-terminal half of ACTH is responsible for the S-100 protein release. The enhancement of S-100 protein release by ACTH was also observed in normal rat glioblasts. The release induced by ACTH was apparently specific to S-100 protein, because little release of the cytoplasmic enzymes, creatine kinase, and enolase was observed under the same conditions. High concentrations (5 mM) of dibutyryl cyclic AMP or dibutyryl cyclic GMP were also found to induce S-100 protein release; however, catecholamines (epinephrine, norepinephrine, isoproterenol, and dopamine), acetylcholine, and glutamic acid did not enhance the release.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
3.
Phorbol myristate acetate inhibits increases in membrane fluidity induced by anti-IgM in B cells 总被引:3,自引:0,他引:3
J Mizuguchi N Utsunomiya M Nakanishi Y Arata 《Journal of immunology (Baltimore, Md. : 1950)》1988,140(8):2495-2499
Anti-IgM or anti-IgD stimulates B cells to induce increases in inositol phospholipid metabolism and intracellular free calcium concentration [( Ca2+]i). Anti-IgM also causes increases in membrane fluidity that occur more promptly than those in [Ca2+]i in resting B cells as well as BAL17 B lymphoma cells. However, other B cell activators such as LPS or PMA did not induce the membrane fluidity changes. Furthermore, sodium fluoride, which is considered to be an activator of the guanine nucleotide-binding protein, caused increases in membrane fluidity as well as increased [Ca2+]i or inositol phospholipid metabolism. Anti-IgM- or sodium fluoride-induced increases in membrane fluidity were inhibited by 20-min pretreatment of cells with PMA, but not by 24-h pretreatment. These results indicate that membrane fluidity changes are closely associated with increased [Ca2+]i after cross-linkage of membrane Ig and are regulated by protein kinase C in B cells. 相似文献
4.
Association of Lps gene with natural resistance of mouse macrophages against Legionella pneumophila 总被引:3,自引:0,他引:3
Shin-ichi Yoshida Yoshitaka Goto Hiroshi Miyamoto Hironobu Fujio Yasuo Mizuguchi 《FEMS microbiology letters》1991,89(1):51-56
We have cloned a 1.6-kb region of chromosomal DNA from Thermoplasma acidophilum into Escherichia coli using as a probe part of the Methanococcus vannielii fus-gene. The sequence of the clone was highly homologous to part of the corresponding Methanococcus vannielii gene. By chromosome walking, a 4.7-kb EcoRI fragment containing the complete gene was isolated. Nucleotide sequencing revealed an open reading frame of 2196 nucleotides. The deduced amino acid sequence contains the known peptide sequence around the ADP-ribosylation site of T. acidophilum elongation factor 2, which unequivocally confirms that the fus-gene has been cloned. The amino acid sequence was compared to that of hamster and E. coli, as well as to known archaebacterial EF-2 sequences. 相似文献
5.
Multiplication of Legionella pneumophila Philadelphia-1 in cultured peritoneal macrophages and its correlation to susceptibility of animals 总被引:13,自引:0,他引:13
Intracellular growth of Legionella pneumophila Philadelphia-1 strain in peritoneal macrophages (PMP) from various rodents was measured and its correlation to the level of susceptibility of the animal was examined. In guinea pig PMP, the organism grew well and the guinea pig was very susceptible to it (50% lethal dose, LD50 = 7.6 X 10(4)). On the other hand, the bacteria hardly multiplied in mouse PMP and the animal was resistant to infection (LD50 = 6.7 X 10(7)). Intracellular growth rate correlated well with susceptibility in these animals. In golden hamsters, a discrepancy between intracellular growth and susceptibility was found. The organism grew intracellularly as rapid as in guinea pig PMP, but the golden hamster was very resistant to infection (LD50 = 2.2 X 10(8)). In rat PMP, the organism did not grow intracellularly during a 24-h period of infection, but started to grow after that and the growth rate thereafter was as rapid as in guinea pig PMP. WKA rats were resistant and the LD50 in the animal was 1.9 X 10(7). In vivo natural resistance of rats and golden hamsters to the organism was considered to be a result of other factors than macrophages. 相似文献
6.
W E Paul J Mizuguchi M Brown K Nakanishi P Hornbeck E Rabin J Ohara 《Cellular immunology》1986,99(1):7-13
Lymphocyte growth and differentiation are controlled by signals resulting from the interaction of antigen and cellular products, such as lymphokines, with specific cell membrane receptors. Resting B lymphocytes can be activated by low concentrations (1-5 micrograms/ml) of antibodies to membrane IgM, which is the B-lymphocyte receptor for antigen. The binding of anti-IgM to B cells causes a rapid increase in intracellular free calcium concentration ([Ca2+]i), in inositol phosphate concentration, and in protein kinase activity. Moreover, the effects of anti-IgM on B cells are mimicked by the combined use of calcium ionophores and phorbol esters. Since phorbol esters activate protein kinase c, this suggests that the increase in [Ca2+]i and in phosphatidylinositol metabolism stimulated by anti-IgM are critical events in B-cell activation. The entry into S phase of B cells stimulated with anti-IgM depends on the action of a T-cell-derived factor designated B-cell stimulatory factor (BSF)-1. This is a 20,000-Da protein which is a powerful inducer of class II major histocompatibility complex molecules. Although an important cofactor for B-cell proliferative responses to anti-IgM, its major locus of action is on resting B cells. B cells stimulated with anti-IgM and BSF-1 do not synthesize secretory IgM. However, if two additional T-cell-derived factors, B151-TRF and interleukin-2, are added to cultures, a substantial proportion of stimulated B cells produce secretory IgM. BSF-1 has also been shown to participate in the "switch" in Ig class expression. Resting B cells cultured with lipopolysaccharide will switch to IgG1 secretion in the presence of purified BSF-1. 相似文献
7.
Mitsuaki Kameko Miho Ichikawa Tsutomu Katsuyama Masamitsu Kanai Michimasa Kato Taiji Akamatsu 《The Histochemical journal》1986,18(4):164-168
Summary This study was undertaken, employing the immunoenzyme method, to confirm the presence of retinol-binding protein in human pancreatic islets, and to compare its distribution with that of prealbumin, insulin, glucagon, somatostatin and pancreatic polypeptide. It was found that most islet cells contained retinol-binding protein, although centrally located cells showed stronger reactivity than those in the peripheral region. The distribution of each of the five polypeptides differed from that of retinolbinding protein, indicating that these peptides did not cross-react with anti-retinol-binding protein antibody. Islet cells which contained prealbumin, on the other hand, were mostly classified as A cells. Further studies are necessary to confirm whether the islet cells produce retinol-binding protein or only store it. 相似文献
8.
BSF-1 action on resting B cells does not require elevation of inositol phospholipid metabolism or increased [Ca2+]i 总被引:8,自引:0,他引:8
J Mizuguchi M A Beaven J Ohara W E Paul 《Journal of immunology (Baltimore, Md. : 1950)》1986,137(7):2215-2219
B cell stimulatory factor-1 (BSF-1) acts on resting B cells to increase expression of class II major histocompatibility complex (MHC) molecules and to prepare for more prompt entry into S phase in response to anti-IgM and lipopolysaccharide. It also acts as a costimulant, with low concentrations of anti-IgM, to cause resting B cells to synthesize DNA. Unlike anti-IgM, BSF-1 does not cause elevation in inositol phospholipid metabolism or in concentration of intracellular free calcium, nor does it enhance such biochemical responses to anti-IgM. Furthermore, increased expression of class II MHC molecules to BSF-1 is observed when essentially all extracellular calcium is chelated by EGTA, whereas lower concentrations of EGTA completely inhibit increases in class II molecules in response to anti-IgM. These results indicate that BSF-1 effects on resting B cells are not mediated by the inositol phospholipid metabolic pathway. 相似文献
9.
10.
Aminoacyl-tRNA exclusively in the 3'-isomeric form is bound to polypeptide chain elongation factor Tu 总被引:1,自引:0,他引:1
2' and 3'-O-(N-acetyl-L-phenylalanyl)adenosine (Ac-Phe-Ado) were chemically synthesized. These two isomers were clearly separated from each other by high-performance liquid chromatography (HPLC). From the two isomers of [3H]Phe-tRNA in equilibrium, Ac-[3H]Phe-Ado was prepared, without any change in the 2'/3'-isomer ratio, by acetylation of the phenylalanyl residue with acetic anhydride followed by digestion with pancreatic RNase A. By HPLC analysis of this preparation of Ac-[3H]Phe-Ado, the abundance ratio of the 2'-isomer and the 3'-isomer of [3H]Phe-tRNA was found to be 0.20:0.80. Further, [3H]Phe-tRNA was bound to Escherichia coli polypeptide chain elongation factor Tu (EF-Tu) with the ligand of GTP or guanosine 5'-[beta, gamma-imido]triphosphate (GMP-P(NH)P). The ternary complex was treated with phenol and acetic anhydride, and then digested with pancreatic RNase A. By HPLC analysis of Ac-[3H]Phe-Ado, the abundance ratio of the 2'-isomer and the 3'-isomer of [3H]Phe-tRNA was determined to be 0.07:0.93 in the complex with EF-Tu.GTP and 0.04:0.96 in the complex with EF-Tu.GMP-P(NH)P. These results clearly indicate that the 3'-isomer, rather than the 2'-isomer, of aminoacyl-tRNA is exclusively involved in the ternary complex. 相似文献