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Yasuyuki Endo Tomohiro Yoshimi Toru Iijima Yuko Yamakoshi 《Bioorganic & medicinal chemistry letters》1999,9(24):7958
Dicarba-closo-dodecaboranes (carboranes), which have spherical geometry and hydrophobicity, are applicable as a hydrophobic pharmacophore of biologically active molecules. We have designed and synthesized estrogenic antagonists based on the structure of the potent agonist 1-hydroxymethyl-12-(4-hydroxyphenyl)-1,12-dicarba-closo-dodecaborane, which we have developed. The compounds showed potent antagonistic activity in luciferase reporter gene assay using COS-1 cells transfected with rat ERα-expression plasmid and an appropriate reporter plasmid. 相似文献
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Recently, we demonstrated that octadecyl chains are important as alkyl chain moieties of polyamidoamine (PAMAM) dendron-bearing lipids for their serum-resistant transfection activity [Bioconjugate Chem.2007, 18, 1349-1354]. Toward production of highly potent vectors, we examined the influence of the generation of dendron moiety on transfection activity of PAMAM dendron-bearing lipids having two octadecyl chains. We synthesized dendron-bearing lipids with PAMAM G1, G2, and G3 dendrons, designated respectively as DL-G1-2C(18), DL-G2-2C(18), and DL-G3-2C(18). The DL-G2-2C(18) and DL-G3-2C(18) interacted with plasmid DNA effectively and formed stable lipoplexes with small sizes and spherical shape. However, DL-G1-2C(18) interacted with plasmid DNA less effectively and formed tubular-shaped lipoplexes with lower stability and larger size. Cells took up DL-G2-2C(18) and DL-G3-2C(18) lipoplexes efficiently, but cellular uptake of the DL-G1-2C(18) lipoplexes was less efficient. Nevertheless, DL-G1-2C(18) lipoplexes achieved 100-10?000 times higher levels of transgene expression, which was evaluated using luciferase gene as a reporter gene. Confocal scanning laser microscopic analysis of intracellular behaviors of the lipoplexes revealed that DL-G1-2C(18) lipoplexes generated free plasmid DNA molecules in the cytosol more effectively than other lipoplexes did. Moderate binding ability of DL-G1-2C(18) might be responsible for generation of lipoplexes which deliver plasmid DNA into cells, liberate it in the cytoplasm, and induce efficient transgene expression. 相似文献
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Adrenomedullin (AM) is a potent vasodilator peptide, which is ubiquitously expressed and has various biological actions, such as proliferative action and anti-oxidative stress action. AM expression is induced by various stresses, such as hypoxia and inflammatory cytokines, and during cell differentiation. The human AM gene promoter region (-70/-29) contains binding sites for stimulatory protein 1 (Sp1) and activator protein-2alpha (AP-2alpha), and has been shown to be important for the AM gene expression during cell differentiation to macrophages or adipocytes. We here show that Sp1 and AP-2alpha synergistically activate the AM gene promoter. Co-transfection of the reporter plasmid containing the AM promoter region (-103/-29) with Sp1 and AP-2alpha expression plasmids showed that Sp1 and AP-2alpha synergistically increased the promoter activity in HeLa cells. Sp1 or AP-2alpha alone caused only small increases in the promoter activity. EMSA showed that Sp1 bound to the promoter region (-70/-29), whereas AP-2alpha bound to a more upstream promoter region (-103/-71). Thus, the synergistic activation of the human AM gene promoter by Sp1 and AP-2alpha may be mediated by the binding of Sp1 to the promoter region (-70/-29) and the interaction with AP-2alpha, which binds to the promoter region (-103/-71). 相似文献
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Identification of posttranscriptionally active inhibitory sequences in human immunodeficiency virus type 1 RNA: novel level of gene regulation. 总被引:27,自引:19,他引:8
cis-acting inhibitory region (IR) sequences were identified within the gag/pol gene of the human immunodeficiency virus type 1 (HIV-1) by using a novel feedback-stimulated, rev-independent tat reporter gene to screen HIV-1 sequences in transient expression assays. Two regions, a 1,295-nucleotide segment in the gag gene (IR-1) and a 1,932-nucleotide segment of the pol gene (IR-2), each inhibited reporter gene expression 10- to 20-fold. IR-1 and IR-2 both contained subsequences which inhibited reporter gene expression. Introduction of IR sequences into a heterologous reporter plasmid, pCMV-CAT, resulted in decreased chloramphenicol acetyltransferase expression, suggesting that the inhibitory effect was not restricted to a reporter gene under the control of the HIV-1 promoter. The presence of HIV IR sequences in cis did not alter relative levels of reporter gene RNA; however, fractionation studies revealed IR-containing RNA accumulated in the nucleus. These findings demonstrate that IR sequences within the gag/pol region affect gene expression by altering the cellular distribution of viral RNA. 相似文献
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呼吸道黏蛋白5AC基因转录表达的顺式调控元件分析 总被引:1,自引:0,他引:1
目的:探讨呼吸道黏蛋白(mucin,MUC)5AC基因5'上游序列顺式调控元件在中性粒细胞弹力酶(neutrophil elastase , NE)诱导MUC5AC基因转录表达的调控机制。方法:应用DNA重组技术,构建含萤光素酶报告基因和MUC5AC启动子不同长度片段的嵌合质粒。采用定点突变技术,在嵌合质粒的基础上构建MUC5AC启动子区特殊蛋白(specificity protein)-1和核因子(nuclear factor, NF)-κB结合位点单独突变体,并测定NE刺激的转染细胞荧光素酶相对活性。结果:成功构建了4种含有不同长度MUC5AC基因启动子序列的荧光索酶报告基因质粒。含有启动子序列-1330bp、-689bp、-324bp的嵌合质粒荧光素酶相对光强度较对照组均显著增加,而含有启动子序列-64bp的嵌合质粒荧光素酶相对光强度与对照组相比差异无统计学意义。NE可诱导含有MUC5AC启动子区NF-кB结合位点单独突变体(pGL3E-MUC5AC-NF-кB-MU)荧光素酶相对光强度增加,而NE不能诱导Sp-l结合位点单独突变体(pGL3E-MUC5AC-SP-1-MU)荧光素酶表达增加。结论:MUC5AC 5'上游序列中-324~-64位点存在参与NE诱导MUC5AC基因表达的重要调控元件,位于此区域的顺式作用元件Sp-1位点在NE诱导MUC5AC基因表达机制中起重要作用,该位点可能作为靶向性基因治疗的关键调控元件。 相似文献
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Reduction of PTP1B by RNAi upregulates the activity of insulin controlled fatty acid synthase promoter 总被引:5,自引:0,他引:5
Xu J Li L Qian Z Hong J Shen S Huang W 《Biochemical and biophysical research communications》2005,329(2):538-543
Metabolic deregulation accompanying type II diabetes is characterized by insulin resistance in peripheral tissues (liver, muscle, and adipose), mediated by impairments in insulin receptor (IR) signaling. Protein tyrosine phosphatase 1B (PTP1B) has been shown to be a negative regulator of IR autophosphorylation and thus has been considered as a major therapeutic target for the treatment of type II diabetes. We use RNA interference technique to downregulate PTP1B expression in hepatoma cell line. A secretory HBV s-antigen was introduced as reporter and driven by mouse fatty acid synthase promoter, which is positively controlled by insulin signaling. Liver-targeted hydrodynamic injection in tail vein was introduced to transfer siRNA (or siRNA expression vector) and reporter plasmid into mouse liver. On fasted/refed and glucose stimulation condition, the HBV s-antigen in sera in RNAi group was higher than that in the negative group. Our results provided evidence that upregulation of insulin signaling by reducing PTP1B liver with RNAi can be a potent diabetes treatment method. 相似文献
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Recombinant human immunodeficiency virus type 1 genomes with tat unconstrained by overlapping reading frames reveal residues in Tat important for replication in tissue culture. 总被引:1,自引:0,他引:1
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Human immunodeficiency virus type 1 (HIV-1) Tat is essential for virus replication and is a potent trans activator of viral gene expression. Evidence suggests that Tat also influences virus infectivity and cytopathicity. Extensive structure-function studies of Tat in subgenomic settings with point mutagenesis and transient transfection readouts have been performed. These reporter assays have defined certain amino acid residues as being important for trans activation of reporter plasmids. However, they have not directly addressed functions related to virus replication. Here, we have studied Tat structure-function in the setting of replicating viruses. We characterized mutations that emerged in Tat during HIV-1 infections of T lymphocytes. To ensure that the selection pressure for change was directed toward protein function, we constructed HIV-Is in which the Tat reading frame was freed from constraints exerted by overlapping with the reading frames of vpr, rev, and env. When these recombinant viruses were passaged in T cells, 26 novel nucleotide changes in tat were observed from sequencing of 220 independently isolated clones. Recloning of these changes into a pNL4-3 molecular background allowed for the characterization of residues in Tat important for virus replication. Interestingly, many of the changes that affected replication when they were assayed in transient trans activation of plasmid reporters were found to be relatively neutral. We conclude that the structure-function of Tat in virus replication is incompletely reflected by activity measurements based only on subgenomic transient transfections. 相似文献
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Normalization of muscle plasmid uptake by Southern blot: application to SERCA1 promoter analysis 总被引:1,自引:0,他引:1
Mitchell-Felton Heather; Kandarian Susan C. 《American journal of physiology. Cell physiology》1999,277(6):C1269
Directinjection of plasmid DNA into muscle allows the study of promoters in aphysiological environment. Because of the variability of reporter geneactivity, attempts have been made to normalize activity to muscleplasmid uptake by coinjection of a second "control" plasmid whose reporter gene is constitutively expressed by a viral promoter. The purpose of this study was to evaluate the use of acontrol plasmid vs. Southern blot to normalize for differences inuptake of plasmids containing promoter fragments of the skeletal muscle-specific sarco(endo)plasmic reticulumCa2+-ATPase (SERCA1) gene. Resultsshowed that the correlation of luciferase activity from control vs.SERCA1 plasmids is poor and that normalization by a virally drivencontrol plasmid increased variability of SERCA1 luciferase activity. Inseveral cases, the presence of a control plasmid inhibited SERCA1reporter expression. When Southern blot analysis was used to normalizefor differences in plasmid uptake there was less variability than withcoinjection, and correlations between plasmid uptake and SERCA1luciferase activity were better. Moreover, there were no inhibitoryeffects of a control plasmid allowing for optimization of injectionconditions of the SERCA1 deletion constructs. The use of Southernanalysis is suggested to determine whether plasmid uptake isdifferentially affected by physiological stimuli, muscle types, orplasmid sizes under study. 相似文献
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Tanaka H Kamita SG Wolf NM Harris TR Wu Z Morisseau C Hammock BD 《Biochimica et biophysica acta》2008,1779(1):17-27
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Pick HM Meissner P Preuss AK Tromba P Vogel H Wurm FM 《Biotechnology and bioengineering》2002,79(6):595-601
Using transient expression, high amounts (>20 mg/mL) of secreted anti-human Rhesus-D IgG1 were produced in a suspension-adapted HEK293 EBNA cell line (Meissner et al., Biotechnol Bioeng 75: 197-203, 2001). Time of harvest was 3 days after transfection. For the estimation of transfection efficiencies, we routinely co-transfected EGFP reporter DNA. At higher reporter plasmid concentrations, >2% of total transfecting plasmid DNA, a substantial reduction of recombinant antibody synthesis, was observed. This phenomenon was investigated in detail by co-expressing various green fluorescent protein (GFP) reporter constructs, which were targeted at different subcellular locations. Enhanced and humanized GFPs targeted to either the endoplasmic reticulum, the cytosol, or the nucleus reduced recombinant antibody production by 30 to 40% when present at higher concentrations in the transfection solution. The most severe effects were observed when the co-transfected EGFP was targeted to the endoplasmic reticulum, leading to a reduction of up to 80% in the presence of only 5% of reporter DNA. Interestingly, one nuclear-targeted GFP variant that was not codon optimized for expression in human cell lines could be added, to up to almost half of the total amount of transfecting DNA, without adverse effect on antibody production. Although the minimum amount of this reporter DNA needed for fluorescence reading was 10 times higher than for the other variants, it provided a much broader quantity range within which the transfection process could be studied without being negatively affected. 相似文献
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We simultaneously tested the transfection efficiency of NT2/D1 and HeLa cells with Lipofectamine (Life Technologies) and Effectene (Qiagen) transfection reagents using the pCH110 eukaryotic assay vector, which contains the lacZ reporter gene. Under our culture conditions for NT2/D1 and HeLa cells, Effectene transfection efficiency could be augmented by simply increasing the amount of plasmid DNA 1.5-3 times above the recommended concentration without any visible cytotoxicity. With the Lipofectamine reagent, optimal transfection efficiency was obtained for both cell lines within the recommended concentrations, but at the top of the range. The results indicate that optimization of the transfection process should include plasmid DNA concentrations above the levels suggested by the manufacturers, in order to accomplish the highest transfection efficiency. 相似文献