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Sample records for antisense oligodeoxynucleotide inhibition

  1. Inhibition of Leukemic Cell Telomerase Activity by Antisense Phosphorothioate Oligodeoxynucleotides

    Institute of Scientific and Technical Information of China (English)

    HEDongmei; ZHANGYuan

    2002-01-01

    Objective To evaluate the effect of human telomerase reverse transcriptase(hTERT) gene antisense oligodeoxynucleotide (ASON) on telomerase activity in K562 cells.Methods Telomerase activity was detemined by polymerase chain reaction enzyme-linked immunoassay (PCR-ELISA) in K562 cells treated with ASODN and hTERTmRNA expression was detected by reverse transcriptase polymerase chain reaction (RT-PCR). Results The hTERTmRNA level was decreased,and telomerase activity was significantly inhibited when the K562 cells were treated with ASODN for 48 h. Conclusion It is suggested that hTETR ASODN might specifically inhibit telomrase activity of K562 cells at translation level,and it is further proved that hTERT gene has significant correlation with telopmerase activity.

  2. Antisense oligodeoxynucleotides targeting the serine/threonine kinase Pim-2 inhibited proliferation of DU-145 cells

    Institute of Scientific and Technical Information of China (English)

    Jin-ming DAI; Shu-qun ZHANG; Wei ZHANG; Ru-xian LIN; Zong-zheng JI; Sheng-qi WANG

    2005-01-01

    Aim: To investigate the effect of antisense oligodeoxynucleotides (ASODN) targeting Pim-2 on cell proliferation of DU-145 cells. Methods: Three ASODN targeting Pim-2 were designed and synthesized. After transfection with ASODN, cell proliferation was analyzed using an MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] assay. In addition, Pim-2 mRNA, protein levels, and cell cycles were examined. Results:The ASODN designed and synthesized by our laboratory significantly reduced Pim-2 mRNA level and protein content in DU-145 cells. After transfection with ASODN for 48 h, a marked reduction in cell viability was observed in DU- 145 cells in a dose-dependent manner. No remarkable apoptosis occurred in cells treated with ASODN compared with control cells. However, it should be noted that G1 phase arrest was clearly observed in ASODN-treated cells. Conclusion: ASODN targeting Pim-2 resulted in a marked reduction in DU-145 cell proliferation, and induction of G1 phase cell cycle arrest is one of the important mechanisms for ASODN to reduce cell growth. Moreover, antisense inhibition of Pim-2 expression provides a new promising therapy target for prostate cancer.

  3. Antisense oligodeoxynucleotide inhibition as a potent diagnostic tool for gene function in plant biology

    Energy Technology Data Exchange (ETDEWEB)

    Jansson, Christer; Sun, Chuanxin; Ghebramedhin, Haile; Hoglund, Anna-Stina; Jansson, Christer

    2008-01-15

    Antisense oligodeoxynucleotide (ODN) inhibition emerges as an effective means for probing gene function in plant cells. Employing this method we have established the importance of the SUSIBA2 transcription factor for regulation of starch synthesis in barley endosperm, and arrived at a model for the role of the SUSIBAs in sugar signaling and source-sink commutation during cereal endosperm development. In this addendum we provide additional data demonstrating the suitability of the antisense ODN technology in studies on starch branching enzyme activities in barley leaves. We also comment on the mechanism for ODN uptake in plant cells. Antisense ODNs are short (12-25 nt-long) stretches of single-stranded ODNs that hybridize to the cognate mRNA in a sequence-specific manner, thereby inhibiting gene expression. They are naturally occurring in both prokaryotes and eukaryotes where they partake in gene regulation and defense against viral infection. The mechanisms for antisense ODN inhibition are not fully understood but it is generally considered that the ODN either sterically interferes with translation or promotes transcript degradation by RNase H activation. The earliest indication of the usefulness of antisense ODN technology for the purposes of molecular biology and medical therapy was the demonstration in 1978 that synthetic ODNs complementary to Raos sarcoma virus could inhibit virus replication in tissue cultures of chick embryo fibroblasts. Since then the antisense ODN technology has been widely used in animal sciences and as an important emerging therapeutic approach in clinical medicine. However, antisense ODN inhibition has been an under-exploited strategy for plant tissues, although the prospects for plant cells in suspension cultures to take up single-stranded ODNs was reported over a decade ago. In 2001, two reports from Malho and coworker demonstrated the use of cationic-complexed antisense ODNs to suppress expression of genes encoding pollen

  4. Antisense oligodeoxynucleotide inhibits vascular endothelial growth factor expression in U937 foam cells

    Institute of Scientific and Technical Information of China (English)

    YANGPeng-Yuan; RUIYao-Cheng; JINYou-Xin; LITie-Jun; QIUYan; ZHANGLi; WANGJie-Song

    2003-01-01

    AIM:To study the expression of vascular endothelial growth factor (VEGF) induced by oxidized low density liprotein (ox-LDL) and the inhibitory effects of antisense oligodeoxynucleotide (asODN) on the levels of VEGF protein and mRNA in the U937 foam cells. METHODS: U937 cells were incubated with ox-LDL 80 mg/L for 48h, then ,the foam cells were treated with asODN (0,5,10, and 20μmol/L). The VEGF concentration in the media was determined by ELISA. The VEGF protein expression level in cells was measured by immuohistochemistry; the positive ratio detected by a morphometrical analysis system was used as the amount of the VEGF expression level. The VEGF mRNA level was examined by Northern blotting. RESULTS: After U937 cells were incubated with ox-LDL, VEGF expression level increased greatly both in the cells and in the media. asODN markeldy inhibited the increase of VEGF. After treatment with asODN 20μmol/L, the VEGF protein concentration in the media decreased by 45.0%, the VEGF positive ratio detected by immuohistochemistry in cells decreased by 64.9%, and the VEGF mRNA level decreased by 47.1%. CONCLUSION: The expression of VEGF in U937 foam cells was strong. asODN inhibited VEGF expression significantly in U937 foam cells in vitro.

  5. [Knock-down of apollon gene by antisense oligodeoxynucleotide inhibits the proliferation of Lovo cells and enhances chemo-sensitivity].

    Science.gov (United States)

    He, Jin-hua; Zhang, Xiao-ying; Wu, Feng-yun; Liao, Xiao-li; Wang, Wei; Jiang, Jian-wei

    2011-02-01

    In this study, the effects of apollon antisense oligodeoxynucleotide (ASODN) on the proliferation and apoptosis of human Lovo cells in vitro were investigated. Apollon ASODN was incubated with human colorectal Lovo cells for 48 h, the proliferation inhibition and the clone forming rates were detected by WST method and clone formation assay, respectively. The expression of apollon mRNA was analyzed by real time fluorescent quantitative reverse transcription polymerase chain reaction. The percentage of apoptotic cells and cell cycle distribution were determined by flow cytometry. The morphology of apoptotic cells was examined by fluorescence microscope. Lovo cells incubated with apollon ASODN combined with 5-fluorouracil (5-FU), cisplatin (DDP) or epirubicin (EPI) of different concentrations, cell proliferation inhibition rates were detected with WST method and IC50 was calculated. It was found that ASODN targeting apollon gene could all suppress the growth of Lovo cells and induce apoptosis of these cells significantly (P Lovo cells treated with apollon ASODN for 48 hours, the expression of the apollon mRNA level was suppressed significantly. And a marked concentration-dependent decline of cell proliferation and clone forming, increasing of cell apoptosis levels were observed. The percentage of G0/G1 phage cells was abated and that of S phage cells was increased and the Lovo cells arrested at S phage of the cell cycle detected with flow cytometry. Many Lovo cells stained with Hoechst 33258 exhibited apoptotic morphology such as cell shrinkage, nuclear condensation and nuclear fragmentation. Cell proliferation inhibition was detected and their chemo-therapeutic effects of 5-FU, DDP and EPI on Lovo cells combined with apollon ASODN (0.08 micromol x L(-1)) were enhanced independently compared with single 5-FU, DDP and EPI groups, and the sensitivity enhanced about 2.58, 4.47, and 5.33 times respectively. It can be concluded that ASODN targeting apollon can suppress the

  6. Focal adhesion kinase antisense oligodeoxynucleotides inhibit human pulmonary artery smooth muscle cells proliferation and promote human pulmonary artery smooth muscle cells apoptosis

    Institute of Scientific and Technical Information of China (English)

    LIN Chun-long; ZHANG Zhen-xiang; XU Yong-jian; NI Wang; CHEN Shi-xin

    2005-01-01

    Background Pulmonary artery smooth muscle cell (PASMC) proliferation plays an important role in pulmonary vessel structural remodelling. At present, the mechanisms related to proliferation of PASMCs are not clear. Focal adhesion kinase (FAK) is a widely expressed nonreceptor protein tyrosine kinase. Recent research indicates that FAK is implicated in signalling pathways which regulate cytoskeletal organization, adhesion, migration, survival and proliferation of cells. Furthermore, there are no reports about the role of FAK in human pulmonary artery smooth muscle cells (HPASMCs). We investigated whether FAK takes part in the intracellular signalling pathway involved in HPASMCs proliferation and apoptosis, by using antisense oligodeoxynucleotides (ODNs) to selectively suppress the expression of FAK protein.Methods Cultured HPASMCs stimulated by fibronectin (40 μg/ml) were passively transfected with ODNs, sense FAK, mismatch sense and antisense-FAK respectively. Expression of FAK, Jun NH2-terminal kinase (JNK), cyclin-dependent kinase 2 (CDK 2) and caspase-3 proteins were detected by immunoprecipitation and Western blots. Cell cycle and cell apoptosis were analysed by flow cytometry. In addition, cytoplasmic FAK expression was detected by immunocytochemical staining.Results When compared with mismatch sense group, the protein expressions of FAK, JNK and CDK 2 in HPASMCs decreased in antisense-FAK ODNs group and increased in sense-FAK ODNs group significantly. Caspase-3 expression upregulated in HPASMCs when treated with antisense ODNs and downregulated when treated with sense ODNs. When compared with mismatch sense ODNs group, the proportion of cells at G1 phase decreased significantly in sense ODNs group, while the proportion of cells at S phase increased significantly. In contrast, compared with mismatch sense ODNs group, the proportion of cells at G1 phase was increased significantly in antisense-FAK ODNs group. The level of cell apoptosis in antisense-FAK group

  7. The Effects of Aerosolized STAT1 Antisense Oligodeoxynucleotides on Rat Pulmonary Fibrosis

    OpenAIRE

    Wang, Wenjun; Liao, Bin; Zeng, Ming; Zhu, Chen; Fan, Xianming

    2009-01-01

    Previous study showed that aerosolized signal transducer and activator of transcription-1 (STAT1) antisense oligodeoxynucleotide (ASON) inhibited the expression of STAT1 and ICAM-1 mRNA and protein in alveolar macrophages (AMs) and decreased the concentrations of TGF-β, PDGF and TNF-α in bronchioalveolar lavage fluid (BALF) in bleomycin (BLM)-induced rat pulmonary fibrosis. Administration of STAT1 ASON ameliorated alveolitis in rat pulmonary fibrosis. However, further investigations are neede...

  8. Cathepsin B antisense oligodeoxynucleotide suppresses invasive potential of MG-63 cells

    Institute of Scientific and Technical Information of China (English)

    He Maolin; Xiao Zengming; Li Shide; Chen Anmin

    2008-01-01

    Objective To study the biological effects of cathepsin B phosporotbioated antisense oligodeoxynucleotide on human osteosarcoma cell line MG-63 after transfection. Methods A 18-mer phosphorothioate antisense oligodeoxynucleotide (ASODN) targeted against the cathepsin B mRNA was transfected into the human osteosarcoma cell line MG-63 by lipofectamine 2000. The sense and nonsense oligodeoxynucleotides to cathepsin B and blank vector were used as controls. The expression of cathepsin B mRNA was examined by RT-PCR and the expression of cathepsin B was examined by Western blot. The invasive capability of MG-63 cells was evaluated by the boydern chamber assay. Results The expression of cathcpsin B was obviously inhibited in antlsense oligodeoxynucleotide treated cells compared with the control cells. The number of invading MG-63 cells was significantly lower in the ASODN-treated groups than that in the control groups. Conclusion The cathepsin B ASODN significantly inhibits the expression of cathepsin B and invasive ability of MG-63 cell in osteosarcoma.

  9. Cathepsin B antisense oligodeoxynucleotide suppresses invasive potential of MG-63 cells

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Objective To study the biological effects of cathepsin B phosporothioated antisense oligodeoxynucleotide on human osteosarcoma cell line MG-63 after transfection.Methods A 18-mer phosphorothioate antisense oligodeoxynucleotide(ASODN)targeted against the cathepsin B mRNA was transfected into the human osteosarcoma cell line MG-63 by lipofectamine 2000.The sense and nonsense oligodeoxynucleotides to cathepsin B and blank vector were used as controls.The expression of cathepsin B mRNA was examined by RT-PCR an...

  10. Sustained Release of Cx43 Antisense Oligodeoxynucleotides from Coated Collagen Scaffolds Promotes Wound Healing.

    Science.gov (United States)

    Gilmartin, Daniel J; Soon, Allyson; Thrasivoulou, Christopher; Phillips, Anthony R J; Jayasinghe, Suwan N; Becker, David L

    2016-07-01

    Antisense oligodeoxynucleotides targeting the mRNA of the gap junction protein Cx43 promote tissue repair in a variety of different wounds. Delivery of the antisense drug has most often been achieved by a thermoreversible hydrogel, Pluronic F-127, which is very effective in the short term but does not allow for sustained delivery over several days. For chronic wounds that take a long time to heal, repeated dosing with the drug may be desirable but is not always compatible with conventional treatments such as the weekly changing of compression bandages on venous leg ulcers. Here the coating of collagen scaffolds with antisense oligonucleotides is investigated and a way to provide protection of the oligodeoxynucleotide drug is found in conjunction with sustained release over a 7 d period. This approach significantly reduces the normal foreign body reaction to the scaffold, which induces an increase of Cx43 protein and an inhibition of healing. As a result of the antisense integration into the scaffold, inflammation is reduced with the rate of wound healing and contracture is significantly improved. This coated scaffold approach may be very useful for treating venous leg ulcers and also for providing a sustained release of any other types of oligonucleotide drugs that are being developed. PMID:27253638

  11. Effect of C-myc Antisense Oligodeoxynucleotides on Hypoxia-induced Proliferation of Pulmonary Vascular Pericytes

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    To study the effect of c-myc antisense oligodeoxynucleotides (ODNs) on proliferation of pulmonary vascular pericytes (PC) induced by hypoxia, cell culture, dot hybridization using probe of digoxigenin-11-dUTP-labeled cDNA,3H-thymidine incorporation, immunocytochemical technique and image analysis methods were used to observe the effect of c-myc antisense ODNs on expression of c-myc gene and proliferating cell nuclear antigen (PCNA), and 3H-thymidine incorporation of PC induced by hypoxia. The results showed that hypoxia could significantly enhance the expression of c-myc and PCNA (P<0.01), and elevate 3H-thymidine incorporation of PC (P<0.01), but antisense ODNs could significantly inhibit the expression of c-myc and PCNA (P<0.05), and 3H-thymidine incorporation of PC (P<0.01). It was suggested that hypoxia could promote the proliferation of PC by up-regulating the expression of c-myc gene, but c-myc antisense ODNs could inhibit hypoxia-induced proliferation of PC by downregulating the expression of c-myc gene.

  12. Inhibition of replication of fresh HIV type 1 patient isolates by a polypurine tract-specific self-complementary oligodeoxynucleotide

    OpenAIRE

    Jendis, J; Strack, B; Volkmann, S; Böni, J; Mölling, K.

    1996-01-01

    A previously described self-complementary oligodeoxynucleotide termed triplex-forming oligodeoxynucleotide (TFO A), 54 bases in length, designed against the polypurine tract of HIV-1 RNA, inhibited viral replication at a 1 to 3 microM concentration in acutely infected cells, whereas antisense and scrambled sequence oligodeoxynucleotides were ineffective. Three HIV-1 viral isolates from patients of clinical categories A1, B, and C3 were transmitted to peripheral blood mononuclear cells and tes...

  13. Effect of c- erbB2 Antisense Oligodeoxynucleotides on Radiosensitivity of Human Ovarian Cancer Cell Line

    Institute of Scientific and Technical Information of China (English)

    RENQing-Lan

    2003-01-01

    Object To explore tile effect of lipofectin - c - erbB2 antisense oligodeoxynucleotides on radiosensitivity of human ovarian cancer cell llne. Methods The expression of c - erbB2 was detected by means of RT - PCR, cellular response to irradiation was evaluated by tile colony forming assay. Results Lipofectin- c - erbB2 antisense oligodeoxynucleotides(AS- ODN) could suppress the expression of c - erbB2 , and significantly decreased the colony forming rate of human ovarian cancer cells after ionizing irradiation (P 0.05 ). Condusion c - erbB2 antisense oligodeoxynueleotides sensitized the SKOV3 to ionizing irradiation through decreasing the expression of e - erbB2 , which might be the result of the fact that c - erbB2 antisense oligodeoxynueleotides inhibit the eelluar signal transductionpathway relating to the radiation- resistant phenotype.

  14. Effects of phosphorothioate anti-sense oligodeoxynucleotides on colorectal cancer cell growth and telomerase activity

    Institute of Scientific and Technical Information of China (English)

    Xi-Shan Wang; Kuan Wang; Xue Li; Song-Bin Fu

    2004-01-01

    AIM: To investigate the inhibitory effect of phosphorothioate anti-sense oligodeoxynucleotides (PASODN) on colorectal cancer LS-174T cells in vitro and the mechanism of inhibition of telomerase activity in these cells.METHODS: PASODN were used to infect LS-174T cells and block human telomerase RNA (hTR) through anti-sense technology. The inhibitory effect of PASODN was evaluated by colony-forming inhibition assay and growth curve. Changes of telomerase activity in LS-174T cells were detected by polymerase chain reaction-enzyme-linked immunosorbent assay (PCR-ELISA), and the level of apoptosis was analyzed by flow cytometry (FCM) assay.RESULTS: PASODN showed a dose and time-dependent inhibition of cell proliferation. The optimal dosage of PASODN was 10 μmol/L. The colony-forming efficiency was 10.3% in PASODN group after 10 d, whereas that in phosphorothioate mis-sense oligodeoxynucleotides (PMSODN) group with the same concentration and in PBS group (blank control) was 49.1% and 50.7%, respectively. PCR-ELISA results indicated that telomerase activity in the PASODN group was obviously inhibited in comparison with in the control groups (P<0.01,t = 3.317 and 3.241, t0.01 (20) = 2.845). Meanwhile, before the number of cells was decreased, the morphological changes were observed in the cells of PASODN group. The cells in PASODN group showed the apoptotic peak at 72 h after infection, whereas the control group did not show.CONCLUSION: Specific sequence oligonucleotides can inhibit telomerase activity and lead to cell apoptosis,suggesting a novel treatment strategy for malignant tumors induced by telomerase.

  15. Effect of cisplatin and c-myb antisense phosphorothioate oligodeoxynucleotides combination on a human colon carcinoma cell line in vitro and in vivo.

    OpenAIRE

    Del Bufalo, D; Cucco, C.; C. Leonetti; Citro, G.; D'Agnano, I; M. Benassi; Geiser, T; Zon, G; Calabretta, B; Zupi, G.

    1996-01-01

    We investigated the effect of c-myb antisense phosphorothioate oligodeoxynucleotides [(S)ODNs] and cisplatin (CDDP) combination on the human colon carcinoma cell line LoVo Dx both in vitro and in nude mice bearing LoVo Dx solid tumour. We show that antisense (S)ODN treatment decreases c-myb mRNA and protein expression, induces growth arrest in the G1 phase of the cell cycle, and inhibits cell proliferation. In vivo treatment with c-myb antisense (S)ODNs results in a reduction in tumour growth...

  16. Antisense phosphorothioate oligodeoxynucleotides inhibit the expression of genes causing drug resistance in Escherichia coli producing ESBLs%反义寡核苷酸抑制产ESBLs大肠埃希菌耐药基因表达的研究

    Institute of Scientific and Technical Information of China (English)

    张劲丰; 吴英; 苏荣; 杨烨建; 庄健海; 何娴; 谢小梅

    2013-01-01

    目的 探讨反义寡核苷酸(AS-ODNs)对产超广谱β-内酰胺酶(ESBLs)大肠埃希菌耐药基因CTX-M表达的影响. 方法 用脂质体包裹AS-ODNs(W086)后导人目的细菌B052,通过平板克隆形成试验计数菌落数(CFU);通过RT-PCR法检测B052耐药基因CTX-M的表达变化. 结果 ZT-AS-ODNs各剂量组B052的CFU与空白对照组比较显著减少(P<0.05),且具有浓度依赖性抑制效应;单纯脂质体组、单纯AS-ODNS组及对照链组与空白对照组比较差异无统计学意义(P>0.05).W086能显著抑制B052 CTX M的表达. 结论 AS-ODNs能与CTX-M mRNA特异性结合并阻断其表达,为预防细菌耐药提供了一个新的研究策略.%Objective To investigate the effects of antisense phosphorothioate oligodeoxynucleotide (AS-ODN) W086on expression of CTX-M,a gene responsible for drug resistance in Escherichia coli producing extended-spectrum β-lactamases (ESBLs).Methods AS-ODN liposomes were introduced into the target strain,B052.The total number of colony-forming units (CFU) was counted.Inhibition of the expression of CTX-M by AS-ODN was observed in B052 using RT-PCR.Results The number of B052 colonies significantly decreased in all groups treated with ZT-AS-ODN in a concentration-dependent manner (P<0.05) while the CFU of B052 was not affected in the group administered liposomes alone,AS-ODN alone,or control ODNs(P>0.05).The expression of CTX-M was significantly inhibited.Conclusion AS-ODNs can bind specifically to CTX-M mRNA and block expression.This indicates that AS-ODNs offer a new strategy to control antibiotic resistance.

  17. The Effects of Aerosolized STAT1 Antisense Oligodeoxynucleotides on Rat Pulmonary Fibrosis

    Institute of Scientific and Technical Information of China (English)

    Wenjun Wang; Bin Liao; Ming Zeng; Chen Zhu; Xianming Fan

    2009-01-01

    Previous study showed that aerosolized signal transducer and activator of transcription-1 (STAT1) antisense oligodeoxynucleotide (ASON) inhibited the expression of STATI and ICAM-1 mRNA and protein in alveolar macrophages (Ams) and decreased the concentrations of TGF-β, PDGF and TNF-α in bronchioalveolar lavage fluid (BALF) in bleomycin (BLM)-induced rat pulmonary fibrosis. Administration of STAT1 ASON ameliorated alveolitis in rat pulmonary fibrosis. However, further investigations are needed to determine whether there is an effect from administration of STAT1 ASON on fibrosis. This study investigated the effect of aerosolized STAT1 ASON on the expressions of inflammatory mediators, hydroxyproline and type Ⅰ and type Ⅲ collagen mRNA in BLM-induced rat pulmonary fibrosis. The results showed that STAT1 ASON applied by aerosolization could ameliorate alveolitis and fibrosis, inhibit the expressions of inflammatory mediators, decrease the content of hydroxyproline, and suppress the expressions of type Ⅰ and type Ⅲ collagen mRNA in lung tissue in BLM-induced rat pulmonary fibrosis. These results suggest that aerosolized STAT1 ASON might be considered as a promising new strategy in the treatment of pulmonary fibrosis.

  18. ENHANCEMENT OF RADIATION-INDUCED APOPTOSIS IN RAJI CELL LINE BY BC1-2 ANTISENSE OLIGODEOXYNUCLEOTIDE

    Institute of Scientific and Technical Information of China (English)

    HE Dong-mei; ZHANG Huan

    2005-01-01

    Objective: To investigate whether the Bc1-2 antisense oligonucleotide(ASODN) may enhance radiation-induced apoptosis in Raji cell line. Methods: Cell surviving fraction was determined using the trypan blue dye exclusion assay. The expression level of bc1-2 protein was assayed by immunofluorescence using fluoresce isothiocyanate label. Apoptosis was detected by Giemsa staining and flow cytomertric cell cycle analysis. Results: It was found that Bc1-2 ASODN combined with radiation had significantly reduced the number of viable cells (P<0.05). There was no difference on cell survival between mismatch Bc1-2 oligodeoxynucleotide/radiation combination and radiation-treated cells alone. Bc1-2 ASODN combined with radiation could significantly inhibit expression of Bc1-2 protein in Raji cells (P<0.05). Cells treated with Bc1-2 ASODN combined with radiation at 72 h displayed classic apoptotic changes. Apoptosis rates of Raji cells treated with Bc1-2 oligodeoxynucleotide/radiation combination and radiation-treated cells alone, respectively. Conclusion: Bc1-2 antisense oligonucleotide can enhance radiation-induced apoptosis in Raji cell line.

  19. HCV 5′NCR调控抑制活性的反义寡核苷酸的筛选%Specific regulatory inhibition of transfected HepG2 with HCV 5'NCR by antisense phosphorothioate oligodeoxynucleotides

    Institute of Scientific and Technical Information of China (English)

    王升启; 管伟; 王小红; 李梦东

    2000-01-01

    目的:寻找高效特异的新型抗HCV药物, 探讨针对HCV基因的硫代修饰反义寡核甘酸(S-ASODNs)最佳作用靶序列。 方法:参考HCV 5′NCR计算机预测的二级结构图,设计合成了15条S-ASODN、3条正义寡核苷酸及1条随机序列,采用HCV 5′NCR调控荧光素酶基因的瞬时表达系统,将S-ASODN与pHCV-neo4共转染,通过荧光素酶活性表达高低,反映S-ASODN对HCV调控基因的抑制活性。结果:5条S-ASODN即HCV65、HCV279、HCV363、HCV349及HCV352在25~100nmol/L浓度范围内,对HCV调控基因具有特异性的剂量依赖性抑制活性。当浓度为100nmol/L时,这些ASODN的抑制率可达80%以上,IC50值提示HCV363的抑制活性最强。通过阴性对照实验包括正义寡核苷酸、随机序列及不含HCV序列的靶载体pGL3提示ASODN仅存在轻度非特异性作用。此外,实验结果还提示不同作用靶位的ASODN之间具有一定的协同作用。 结论:HCV 5′NCR第二茎环结构Ⅱa、第三茎环结构Ⅲd和翻译起始区可能是ASODN作用的重要靶序列。%Objective: To screen efficient and specific new drugs against hepatitis C virus (HCV) and find the best target sequence of antisense oligodeoxynucleotides (ASODNs) targeting at HCV gene. Methods: Fifteen S-ASODNs targeting at the 5'NCR and start AUG of HCV RNA were designed and synthesized according to the predicted RNA secondary structure of the HCV 5'NCR and the adjacent AUG region. HepG2 cells were co-transfected with pHCV-neo4 and S-ASODN. The inhibitory effects of S-ASODNs on gene expression controlled by HCV 5'NCR were determined by the assay of luciferase activity. Results: Five S-ASODNs, i.e. HCV65, HCV279, HCV363, HCV349 and HCV352, showed sequence-specific and dose-dependent inhibitory activities with an inhibition rate of more than 80% at a concentration of 100 nmol/L. According to 50% inhibitory concentrations, the inhibitory activity of HCV363 was the best. On the

  20. Inhibitory effects of antisense phosphorothioate oligodeoxynucleotides on pancreatic cancer cell Bxpc-3 telomerase activity and cell growth in vitro

    Institute of Scientific and Technical Information of China (English)

    Yun-Feng Wang; Ke-Jian Guo; Bei-Ting Huang; Yong Liu; Xiao-Yun Tang; Jian-Jun Zhang; Qiang Xia

    2006-01-01

    AIM: To investigate the effect of telomerase hTERT gene antisense oligonucleotide (hTERT-ASO) on proliferation and telomerase activity of pancreatic cancer cell line Bxpc-3.METHODS: MTT assay was used to detect the effect of different doses of hTERT-ASO on proliferation of Bxpc-3 cell for different times. To study the anti-tumor activity,the cells were divided into there groups: Control group (pancreatic cancer cell Bxpc-3); antisense oligonucleotide (hTERT-ASO) group; and nosense oligonucleotide group decorated with phosphorothioate. Telomerase activity was detected using TRAP-PCR-ELISA. Cell DNA distribution was examined using flow cytometry assay.Cell apoptosis was observed by transmission electron microscope in each group.RESULTS: After treatment with 6 mmol/L hTERTASO, cell proliferation was inhibited in dose- and timedependent manner. The telomerase activity decreased after treatment with hTERT-ASO for 72 h. Flow cytometry showed the cell number of G0/G1 phase increased from 2.7% to 14.7%, the cell number of S phase decreased from 72.7% to 51.0%, and a sub-G1 stage cell apoptosis peak appeared in front of G1 stage.CONCLUSION: Telomerase antisense oligodeoxynucleotide can inhibit the proliferation of pancreatic cancer cell line Bxpc-3 and decrease the telomerase activity and increase cell apoptosis rate in vitro.

  1. Telomerase activity and cell apoptosis in colon cancer cell by human telomerase reverse transcriptase gene antisense oligodeoxynucleotide

    Institute of Scientific and Technical Information of China (English)

    Ying-An Jiang; He-Sheng Luo; You-Yuan Zhang; Li-Fang Fan; Chong-Qing Jiang; Wei-Jin Chen

    2003-01-01

    AIM: To evaluate the effect of human telomerase reverse transcriptase (hTERT) gene antisense oligodeoxynudeotide (As-ODN) on telomerase activity and cell apoptosis in colon cancer cell line SW480.METHODS: As-ODN was transfected into cells SW480 by liposomal transfection. Cultured cells were divided into three groups: ASODN (5′GGAGCGCGCGGCATCGCGGG-3), sense oligodeoxynucleotide (5′-CCCGCGATGCCGCGCGCTCC-3; SODN) and control. The concentration of oligodeoxynucleotide and lipsome was 10 μmol/L and 16 mg/L, respectively. The activity of telomerase was examined by telomeric repeat amplification protocol (TRAP)-enzyme-linked immunosorbent assay (ELISA), and cell apoptosis was observed by morphology and flow cytometry in each group.RESULTS: Telomerase activity began to be down-regulated or inhibited when cells SW480 were treated with As-ODN for 72 h, and cell apoptosis was induced.CONCLUSION: It is suggested that hTERT As-ODN might specially inhibit the activity of telomerase in colon cancer cells and it is further proved that the hTERT gene has a significant correlation with telomerase activity. Further evidence is needed to prove whether hTERT As-ODN is a potential tool for the treatment of colon cancer.

  2. Effect of antisense oligodeoxynucleotide of telomerase RNA on telomerase activity and cell apoptosis in human colon cancer

    Institute of Scientific and Technical Information of China (English)

    Ying-An Jiang; He-Sheng Luo; Li-Fang Fan; Chong-Qing Jiang; Wei-Jin Chen

    2004-01-01

    AIM: To explore the effect of antisense oligodeoxynucleotide (As-ODN) of telomerase RNA on telomerase activity and cell apoptosis in human colon cancer.METHODS: As-ODN waS transfected into SW480 cells by liposomal transfection reagent. Telomerase activity of SW480 cells was examined by telomeric repeat amplification protocol (TRAP) and enzyme-linked immunosorbent assay (ELISA).Apoptosis was analyzed by morphology and flow cytometry.RESULTS: The telomerase activity in SW480 cells trandected with 1.0 μmol/L of As-ODN for 2-5 days, was significantly decreased in a time-dependent manner, and the cells underwent apoptosis. The missense ODN (Ms-ODN) and the control group transfected with SW480 cells did not show these changes.CONCLUSION: As-ODN can specifically inhibit the telomerase activity of SW480 cells and induce apoptosis.

  3. Lipolysis and apoptosis of adipocytes induced by neuropeptide Y—Y5 receptor antisense oligodeoxynucleotides in obese rats

    Institute of Scientific and Technical Information of China (English)

    GONGHai-Xia; GUOXi-Rong; FEILi; GUOMei; LIUQian-Qi; CHENRong-Hua

    2003-01-01

    AIM:To investigate the influence of central administration of neuropeptide Y-Y5 receptor antisense oligodeoxynucleotides(ODN) on the body weight and fat pads of high-energy diet-induced obese rats, and the effects on white adipocyte lipolysis and apoptosis. METHODS: Y5 receptor antisense, sense, mismatched oligodeoxynucleotides (ODN) or vehicle were intracerebroventricularly injected, and average adipocyte area was calculated. DNA ladders were measured to evaluate adipocyte apoptosis, and RT-PCR was used to analyze the expression of bcl-2 and bax gene. RESULTS: (1) Central administration of Y5 receptor antisense ODN significantly decreased body weight, fat pads, and average adipocyte area. (2) DNA fragmentation was presented after electrophoresis at both epididymal and retroperitoneal adipose tissue. (3) The expression of bcl-2 gene was downregulated, while the expression of bax was upregulated. CONCLUSION:Lipolysis and adipocyte apoptosis may be important reasons for Y5 receptor antisense therapy.

  4. Inhibition of HIV-1 replication by chimeric phosphorothioate oligodeoxynucleotides applied in free solution

    DEFF Research Database (Denmark)

    Lund, O S; Hansen, J E

    1998-01-01

    Oligodeoxynucleotides (ODNs) containing a variable number of 3' and 5' terminal phosphorothioate linkages were applied in free solution to cells infected by HIV-1. ODNs of 28 nt length were applied at up to 5 microM concentration. The ODNs were found to inhibit HIV-1 infection in a dose dependent...... manner, which correlated with the number of modified linkages (4, 8 and 12, respectively). A target sequence in the HIV-1 rev mRNA, previously reported as sensitive to antisense inhibition by full length phosphorothioate ODNs, only revealed non-sequence dependent inhibition of HIV-1, when tested by these...

  5. Effects of Antisense Oligodeoxynucleotide to Follicle-stimulating Hormone Receptor on the Cell Proliferation and Apoptosis in Cells Derived from Human Ovarian Mucinous Cystadenocarcinoma in Vitro

    Institute of Scientific and Technical Information of China (English)

    LI Shuang; MA Ding; ZHU Changhong

    2007-01-01

    The human ovarian mucinous cystadenocarcinoma (hOMC) cells were co-cultured with antisense oligodeoxynucleotide (antisense ODN), nonsense ODN, and follicle-stimulating hormone (FSH) at different concentrations for the purpose of observing the effects of antisense ODN to FSH receptor (FSHR) on the proliferation and apoptosis of cultured hOMC cells in vitro. The inhibitory rates of growth were measured by using MTT method on the 2nd, 4th, 6th, 8th and 10th days after the interference of antisense ODN, nonsense ODN, and FSH, respectively. The apoptotic rates and the cell cycles were determined by means of flow cytometry, the apoptosis indexes were detected by using TUNEL, and the expression of caspase-3 was measured by using SP immunohistochemistry. Compared with that in the control group, the proliferative activity of hOMC cells was increased obviously in FSH groups (P<0.05 or P<0.01), decreased distinctly in antisense ODN groups (P<0.05 or P<0.01), and unchanged in nonsense ODN groups, respectively. Meanwhile, antisense ODN could significantly antagonize the FSH-promoted cell proliferative activity (P<0.01). Compared with those in the control group, the apoptotic rates and the expression of caspase-3 were dramatically increased in the mid- and high-dose antisense ODN groups (P<0.05 or P<0.01), while the number of cells in G1/G0 phase was significantly decreased and that in S phase distinctly increased (P<0.01). There was no change in nonsense ODN groups (P>0.05). It was suggested that FSH may improve the development of hOMC cells. However, antisense ODN could inhibit proliferative activity and the FSH-promoted proliferative activity in hOMC cells, at the same time, antisense ODN could inhibit hOMC cell growth by inducing apoptosis.

  6. Intrathecal PLC(β3) oligodeoxynucleotides antisense potentiates acute morphine efficacy and attenuates chronic morphine tolerance.

    Science.gov (United States)

    Quanhong, Zhou; Ying, Xue; Moxi, Chen; Tao, Xu; Jing, Wang; Xin, Zhang; Li, Wang; Derong, Cui; Xiaoli, Zhang; Wei, Jiang

    2012-09-01

    Morphine is a mainstay for chronic pain treatment, but its efficacy has been hampered by physical tolerance. The underlying mechanism for chronic morphine induced tolerance is complicated and not well understood. PLC(β3) is regarded as an important factor in the morphine tolerance signal pathway. In this study, we determined intrathecal (i.t.) administration of an antisense oligodeoxynucleotide (ODN) of PLC(β3) could quicken the on-set antinociceptive efficacy of acute morphine treatment and prolong the maximum effect up to 4h. The antisense could also attenuate the development of morphine-induced tolerance and left shift the ED50 after 7 day of coadministration with morphine. These results probably were contributed by the PLC(β3) antisense ODN as they successfully knocked down protein expression levels and reduced activity of PLC(β3) in spinal cord in rats. The mismatch group had no such effects. The results confirmed the important involvement of PLC(β3) in both acute morphine efficacy and chronic morphine tolerance at spinal level in rats. This study may provide an idea for producing a novel adjuvant for morphine treatment.

  7. Effect of TGF-β1 antisense oligodeoxynucleotide on renal function in chronic renal failure rats

    Institute of Scientific and Technical Information of China (English)

    Law Chung HIONG; Kiew Lik VOON; Nor Azizan ABDULLAH; Munavvar A SATTAR; Nazarina AbduRAHMAN; Abdul Hye KHAN; Edward James JOHNS

    2008-01-01

    Aim:The aim of the present study was to investigate the effectiveness of trans-forming growth factor (TGF)-β1 antisense oligodeoxynucleotides (ODN) in ame-liorating deteriorated kidney function in rats with puromycin-induced chronic renal failure (CRF). Methods:Saline, puromycin, puromycin+TGF-β1 antisense ODN or puromycin+scrambled ODN were administered to unilaterally nephrecto-mized rats. Renal hemodynamic and excretory measurements were taken in the anaesthetized rats that had undergone surgical procedure. Results:It was ob-served that in the CRF rats, there was a marked reduction in the renal blood flow (RBF), glomerular filtration rate (GFR), severe proteinuria, and almost 6-fold in-creased fractional excretion of sodium (FE Na+) as compared to that in the control rats (all P<0.05). It was further observed that in the CRF rats, the treatment with TGF-β1 antisense, but not scrambled ODN, markedly attenuated the reduction of RBF, GFR, and proteinuria and markedly prevented the increase of the FE Na+ (all P<0.05). In addition, the renal hypertrophy in the CRF group (P<0.05 vs non-renal failure control) was markedly attenuated after treatment with TGF-1 antisense ODN (P<0.05). Focal segmental glomerulosclerosis was evident only in the un-treated and scrambled ODN-treated CRF groups. An interesting observation of this study was that in the CRF rats, although there was marked attenuating and preventive effects of the TGF-β1 antisense ODN on the deteriorated renal functions, the antisense treatment did not cause any marked change in the renal expression of TGF-β1 at the protein level. Conclusion:Collectively, the data obtained sug-gests that TGF-β1 antisense ODN possesses beneficial effects in puromycin-induced chronic renal failure and that the deterioration in morphology and im-paired renal function in this pathological state is in part dependent upon the action of TGF-β1 within the kidney.

  8. Survivin Antisense Oligodeoxy-Nucleotid Induces Apoptosis in Leukaemia Cell Line K562

    Institute of Scientific and Technical Information of China (English)

    Lijun Chen; Qiuyue Jin; Hong Xie; Ruimin Wang; Li Yao

    2006-01-01

    OBJECTIVE To investigate the effects of survivin antisense oligodeoxynucleotid (ASODN) on proliferation and apoptosis in the chronic myeloid leukemia cell line K562.METHODS Different concentrations of an antisense oligodeoxy-nucleotid and control sequence (scrambled ODN) targeting the survivin gene were transferred into K562 by a lipofectin reagent. The MTT assay was used to measure the growth inhibitory rate, IC50, and to observe the cytotoxicity of survivin ASODN in the K562 cells. The morphologic changes in the nucleus and the apoptotic rate were observed by Hoechst33342/PI staining.Caspase-3 activity was evaluated by a kinase activity assay. The changes of survivin protein expression after transfection were detected by Western blots.RESULTS Eight hours after transfection, fluorescence in the K562 cells was well distributed. Treatment of the cells for 44 h with different concentrations of survivin ASODN produced a IC50 of 800 nmol/L. The growth inhibitory rate with 200, 400, 600 and 1000 nmol/L of survivin ASODN was 15.8±1.6%, 23.8±5.9%, 37.1±5.6% and 77.3±2.5% respectively. After 36 h of of survivin ASODN treatment, distinct morphologic changes characteristic of cell apoptosis such as karyopyknosis and conglomeration were observed by Hoechst33342/PI staining. Caspase-3 activity increased significantly after treatment of the cells with different concentrations of survivin ASODN (P<0.01)and following treatment with 800 nmol/L survivin ASODN, survivin expression decreased significantly.CONCLUSION Survivin ASODN exerts an anti-cancer effect by inducing apoptosis in K562 leukaemia cells. Up-regulated expression of caspase3 may play a role in this process.

  9. STUDY ON THE INHIBITORY EFFECT OF ANTISENSE ETAR OLIGODEOXYNUCLEOTIDES ON THE PROLIFERATION OF VASCULAR SMOOTH CELLS

    Institute of Scientific and Technical Information of China (English)

    张岚; 张柏根; 张纪蔚; 钱济先; 张皓; 黄晓钟

    2002-01-01

    Objective To study the inhibitory effect of antisense endothelin receptor A (ETAR) on the proliferation of the vascular smooth muscle cells. Methods The sense, antisense and mismatched ODNs for ETAR were designed and synthetized. The study was carried out using MTT method and binding assays.Results ETAR-ODNs could move successfully across VSMC membranes. Photo-absorption in the MTT test was reduced significantly (P<0.05) in the antisense group at 5μmol/L; the reduction of CPM also occurred in the 125I-ET-1 specific binding assay; and the sense and mismatched ODNs groups did not show this reduction. Conclusion Our study suggested that the antisense oligomers inhibited the proliferation of VSMCs by hindering the translation of target mRNA and by reducing the production of related protein.

  10. Irradiation of Human Prostate Cancer Cells Increases Uptake of Antisense Oligodeoxynucleotide

    International Nuclear Information System (INIS)

    Purpose: To investigate whether irradiation before antisense Bcl-2 oligodeoxynucleotide (ODN) administration enhances tissue uptake, and whether periodic dosing enhances cellular uptake of fluorescently labeled ODN relative to constant dosing. Methods and Materials: PC-3-Bcl-2 cells (prostate cancer cell line engineered to overexpress Bcl-2) were subjected to increasing doses of irradiation (0-10 Gy) with or without increasing concentrations of fluorescently labeled antisense Bcl-2 ODN (G4243). The fluorescent signal intensity was quantified as the total grain area with commercial software. In addition, PC-3-Bcl-2 subcutaneous xenograft tumors were treated with or without irradiation in combination with various dosing schemas of G4243. The uptake of fluorescent G4243 in tumors was quantitated. Results: The uptake of G4243 was increased in prostate cancer cells exposed to low doses of irradiation both in vitro and in vivo. Irradiation before G4243 treatment resulted in increased fluorescent signal intensity in xenograft tumors compared with those irradiated after G4243 treatment. A single weekly dose of G4243 produced higher G4243 uptake in xenograft tumors than daily dosing, even when the total dose administered per week was held constant. Conclusions: These findings suggest that ionizing radiation increases the uptake of therapeutic ODN in target tissues and, thus, has potential to increase the efficacy of ODN in clinical applications

  11. EFFECTS OF ANTISENSE OLIGODEOXYNUCLEOTIDES ON EXPRESSION OF CASPASE-3 IN Γ-RADIATION INDUCED APOPTOTIC HL-60 CELLS

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Objective To study the inhibitory effects of caspase-3 mRNA antisense oligodeoxynucleotides (ASODN) on expressions of caspase-3 and it's mRNA in γ-radiation induced apoptotic HL-60 cells, and screen the effective ASODN. Methods ASODN-1 and ASODN-2 targeting 5′-noncoding region and initial translation region of caspase-3 mRNA were respectively designed, synthesized and introduced into HL-60 cells by means of liposome-mediated transfection followed by 10Gy γ-radiation exposures. TUNEL assay was conducted to investigate the morphologic change and apoptotic percentage of HL-60 cells 18 h later. Immunocytochemical staining and one step RT-PCR were respectively performed to detect the expressions of caspase-3 and it's mRNA. Mismatched oligodeoxynucleotide (MODN) transfected and un-transfected HL-60 cells were taken as control. Results TUNEL assay found that the apoptotic percentages in ASODN-1 and ASODN-2 groups were significantly reduced compared with the control groups (P<0.01) when the final concentration of both ASODNs was ≥3μmol/L. Immunocytochemistry showed that caspase-3 positive cell percentages were reduced but the average gray values increased significantly compared with the control groups (P<0.01). RT-PCR showed expressions of caspase-3 mRNA was decreased after ASODN transfection. Furthermore, ASODN-1 proved more effective in inhibiting HL-60 cell apoptosis than ASODN-2 (P<0.01). Conclusion Caspase-3 mRNA ASODNs can prevent HL-60 cells from apoptosis induced by γ-radiation and reduce expression of caspase-3 and its mRNA. These effects are dose dependent in a certain range.

  12. Anti-angiogenesis effect of generation 4 polyamidoamine/vascular endothelial growth factor antisense oligodeoxynucleotide on breast cancer in vitro

    Institute of Scientific and Technical Information of China (English)

    Shan-zhi GU; Xin-han ZHAO; Ling-xiao ZHANG; Li LI; Zhi-yu WANG; Min MENG; Gai-li AN

    2009-01-01

    Objective: To study the effects of the generation 4 polyamidoamine/vascular endothelial growth factor antisense oligodeoxynucleotide (G4PAMAMNEGFASODN) compound on the expressions of vascular endothelial growth factor (VEGF) and its mRNA of breast cancer cells and on the inhibition of vascular endothelial cells. Methods: We examined the morphology of G4PAMAM/VEGFASODN compound and its pH stability, in vitro transfection efficiency and toxicity, and the expressions of VEGF and its mRNA. Methyl thiazolyl tetrazolium assay was used to detect the inhibitory function of the compound on vascular endothelial cells. Results: The compound was about 10 nm in diameter and was homogeneously netlike. From pH 5 to 10, it showed quite a buffered ability. The 48-h transfection rate in the charge ratio of 1:40 was 98.76%, significantly higher than that of the liposome group (P<0.05). None of the transfection products showed obvious toxicity on the cells. The expressions of both VEGF protein and its mRNA after G4PAMAM/VEGFASODN transfection decreased markedly. Conclusion: With a low toxicity, high safety, and high transfection rate, G4PAMAMNEGFASODN could be a promising gene vector. Specifically, it inhibits VEGF gene expression efficiently, laying a basis for further in vivo animal studies.

  13. In vivo treatment of human leukemia in a scid mouse model with c-myb antisense oligodeoxynucleotides.

    OpenAIRE

    Ratajczak, M Z; Kant, J A; Luger, S M; Hijiya, N.; Zhang, J; Zon, G; Gewirtz, A. M.

    1992-01-01

    The c-myb protooncogene encodes proteins that are critical for hematopoietic cell proliferation and development. Disrupting c-myb function might, therefore, prove an effective therapeutic strategy for controlling leukemic cell growth. Antisense oligodeoxynucleotides have been utilized for this purpose in vitro, but their in vivo efficacy has not been reported. We therefore established human leukemia-scid mouse chimeras with K562 cells and treated diseased animals with phosphorothioate-modifie...

  14. Water-absorbent polymer as a carrier for a discrete deposit of antisense oligodeoxynucleotides in the central nervous system.

    Science.gov (United States)

    Bannai, M; Ichikawa, M; Nishimura, F; Nishihara, M; Takahashi, M

    1998-09-01

    One of the problems of introducing antisense oligodeoxynucleotides (ODN) into the central nervous system (CNS) is their rapid disappearance from the target site due to their dispersion and diffusion, which results in poor uptake and/or retention in cells (M. Morris, A.B. Lucion, Antisense oligonucleotides in the study of neuroendocrine systems, J. Neuroendocrinol. 7 (1995) 493-500; S. Ogawa, H.E. Brown, H.J. Okano, D.W. Pfaff, Cellular uptake of intracerebrally administrated oligodeoxynucleotides in mouse brain, Regul. Pept. 59 (1995) 143-149) [2,5]. Recently, we adapted a new method using water-absorbent polymer (WAP; internally cross-linked starch-grafted-polyacrylates) as a carrier for antisense ODN. The polymer forms a hydro-gel after absorbing water which is chemically and biologically inert. In these studies, the polymer (powder-form) is fully swollen by physiological saline containing antisense ODN (0.2 micromol/ml) to make 80-fold volume gel. Hydro-gel (1 microliter) is injected into the target site, and water solutes are assumed to be diffused stoichiometrically into CNS from the surface of the gel. Histological studies indicate that 24 h after the injection, antisense ODN (5'biotinylated-S-oligos of 15 mer) are distributed to within 800 micrometer from the edge of the area where the gel is located and then gradually disappear from this area within days, but still remain within 300-micrometer distance 7 days later. Antisense ODN are effectively incorporated by all the cell types examined, i.e., neurons, astrocytes and microglias, and suppress the synthesis of the target protein. This method can be adapted to slow delivery of antisense ODN and other water soluble substances into the CNS. PMID:9767125

  15. Apoptosis induction with polo-like kinase-1 antisense phosphorothioate oligodeoxynucleotide of colon cancer cell line SW480

    Institute of Scientific and Technical Information of China (English)

    Yu Fan; Shu Zheng; Ze-Feng Xu; Jia-Yi Ding

    2005-01-01

    AIM: To investigate the effects of polo-like kinase-1 (PLK1) antisense phosphorothioate oligodeoxynucleotide (ASODN) on apoptosis and cell cycle of human colon cancer cell line 5W480.METHODS: After SW480 colon cancer cells were transfected with PLK1 ASODN, Northern and Western blot analyses were used to examine PLK1 gene expression in cancer cells. We studied apoptosis using terminal uridine deoxynucleotidyl nick end labeling. Apoptosis and cell cycle of SW480 cells were examined by fluorescence-activated cell sorter scan.RESULTS: The levels of PLK1 mRNA and protein were greatly inhibited by PLK1 ASODN in SW480 cancer cells transfected with PLK1 ASODN. Apoptosis index (AI) induced PLK1 ASODN in a time- and dose-dependent manner.Results from FLM showed that sub-2N DNA content of transfected cancer cells was significantly increased and arrested at G2/M compared with control groups.CONCLUSION: PLK1 ASODN can induce apoptosis of human colon cancer cell line SW480.

  16. Antisense oligonucleotide for tissue factor inhibits hepatic ischemic reperfusion injury.

    Science.gov (United States)

    Nakamura, Kenji; Kadotani, Yayoi; Ushigome, Hidetaka; Akioka, Kiyokazu; Okamoto, Masahiko; Ohmori, Yoshihiro; Yaoi, Takeshi; Fushiki, Shinji; Yoshimura, Rikio; Yoshimura, Norio

    2002-09-27

    Tissue factor (TF) is an initiation factor for blood coagulation and its expression is induced on endothelial cells during inflammatory or immune responses. We designed an antisense oligodeoxynucleotide (AS-1/TF) for rat TF and studied its effect on hepatic ischemic reperfusion injury. AS-1/TF was delivered intravenously to Lewis rats. After 10 h, hepatic artery and portal vein were partially clamped. Livers were reperfused after 180 min and harvested. TF expression was studied using immunohistochemical staining. One of 10 rats survived in a 5-day survival rate and TF was strongly stained on endothelial cells in non-treatment group. However, by treatment with AS-1/TF, six of seven survived and TF staining was significantly reduced. Furthermore, we observed that fluorescein-labeled AS-1/TF was absorbed into endothelial cells. These results suggest that AS-1/TF can strongly suppress the expression of TF and thereby inhibit ischemic reperfusion injury to the rat liver. PMID:12270110

  17. Antisense oligodeoxynucleotides targeting ATM strengthen apoptosis of laryngeal squamous cell carcinoma grown in nude mice

    Directory of Open Access Journals (Sweden)

    Zhao Yongsheng

    2011-04-01

    Full Text Available Abstract Background To conserve laryngeal function and elevate living quality of laryngeal squamous cell carcinoma (LSCC patients, we designed antisense oligodeoxynucleotides (AS-ODNs to reduce expression of ATM and to enhance the apoptosis of hep-2 (Human epidermoid laryngeal carcinoma cells to radiation in vitro and in vivo. Methods The expression of ATM mRNA and protein in hep-2 cells were examined by real-time quantitative PCR and western blotting respectively. Clonogenic survival assay was carried out to detect the survival ability of hep-2 cells after irradiation, and analyzed the cell apoptosis by flow cytometry. The volume of solid tumors was measured, while TUNEL assay and western blotting used to analyze cell apoptosis and protein expression after irradiation. Results The relative ATM mRNA and protein expression in hep-2 cells treated with ATM AS-ODNs were decreased to 11.03 ± 2.51% and 48.14 ± 5.53% of that in untreated cells respectively (P Conclusion AS-ODNs of ATM reduce ATM expression and enhance hep-2 cells apoptosis to radiation in vitro and in vivo.

  18. Effect of MUC2 Antisense Oligodeoxynucleotide on Cell Proliferation, Adhesion, and Proteolytic Enzyme in Human Gastric Carcinoma in vitro

    Institute of Scientific and Technical Information of China (English)

    YANG Ya-ying; YI yong-fen; ZHANG Xiao-yan; XIAO Chun-wei; LIN Xiao; ZHOU Wen-wen

    2007-01-01

    Objective: To investigate the effect of MUC2 antisense oligodeoxynucleotide (ASODN) on cell proliferation, adhesion and proteolytic enzyme in human gastric carcinoma cell line (SGC7901). Methods: Phosphorothioate MUC2 ASODN was synthesized and packaged by lipofectin, and then transfected to SGC7901 cells. The expression of MUC2 mRNA and protein after transfection was detected by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical method respectively,and the effect of MUC2 ASODN on cell proliferation,adhesion and proteolytic enzyme was determined by flow cytometry(FCM), MTT method, Rose Bengal and immunohistochemical method. Results: Compared with the blank control group, ASODN efficiently downregulated the expression of MUC2 mRNA and protein in SGC7901 cells 48h after transfection(P<0.01). Various concentrations of ASODN could significantly inhibit the growth of SGC7901, and the inhibition peaked at the 48th hour after transfection(P<0.05). The apoptosis rate of the experimental group was about 4.38%, and the percentage of S-phase cells rose while G0/G1-phase cells fell because most of them were blocked at S-phase. In addition, cells treated with MUC2 ASODN showed lower adhesion ability with matrix and endothelial cells than control cells in vitro(P<0.01). By immunohistochemical method, the upregulation of E-cadherin proteins and the downregulation of MMP2 and cathepsinD proteins were also observed(P<0.05). Conclusion: MUC2 ASODN could efficiently inhibit SGC7901 cell proliferation, reduce cell adhesion ability and downregulate the expression levels of proteolytic enzyme in vitro.

  19. Inhibitory effects of antisense oligodeoxynucleotide on replication and expression of HDV genome in vitro and in vivo

    Institute of Scientific and Technical Information of China (English)

    MAO Qing; LI Qi-fen; LI Hong-wen; WANG Sheng-qi; ZHENG Hong; DING Jian; WU Chun-qing; ZHU Bao-zhen

    2001-01-01

    Objective: To study the inhibitory effects of antisense oligodeoxynucleotide (ASODN) and its thiophosphate (S-ASODN) on the replication and expression of hepatitis D virus (HDV) in H1δ9 cells and in tupaia body. Methods:After 15mer-ASODN and S-ASODN were synthesized, different concentrations of ASODN and S-ASODN were added to the culture medium of H1δ9 cell line and then HDAg in the supernatant of the culture was examined with ELISA and HDV-RNA in the cells determined with dot blot hybridization. Sixteen tupaiae were successfully infected with HDV and mg every other day for 7 times and the control group were injected with sime volume of normal saline. On the 5th, 10th,15th and 20th day after the administration, samples of blood and liver tissues were examined with immunohistochemical method for HDAg and dot blot hybridizaton and in situ hybridization for HDV-RNA. Results: Twenty-four hours after the addition ofa tinal concentration of 6 μmol/L of S-ASODN, the replication of HDV-RNA and the release of HDAg in th e H1δ9 cells were suppressed by 84.5% and 76.14% respectvely. The inhibition was dose-dependent when the final concentrations of 2, 4 and 6 μmol/L of S-ASODN were given. When ASODN and S-ASODN were administered in the same dosage, the inhibition showed no significantly difference between the 2 agents. On the last day of the administration of S-ASODN, 7 out of the 8 tupaiae of the treated group showed negative HDAg and HDV-RNA in the liver tissue while only 1 out of the 8 tupaiae of the congrol group was negative. Ten days after the cessation of drug administration, 3 tupaiae of the treated group and 7 of control were positive of HDAg and HDV-RNA. Conclusion: Our findings show that SASODN efficiently inhibites the replication and expression of HDV gene in H1 δ9 cells and in the body of tupaia, which provides an experimental basis for the anti-HDV application of antisense oligonucleotides.

  20. Effects of suppressing glucose transporter-1 by an antisense oligodeoxynucleotide on the growth of human hepatocellular carcinoma cells

    Institute of Scientific and Technical Information of China (English)

    Tian-QiLiu; JunFan; LinZhou; Shu-SenZheng

    2011-01-01

    BACKGROUND: The glucose transporter-1 (Glut-1), a key rate-limiting factor in the transport and metabolism of glucose in cancer cells, is over-expressed in many human cancer cells and this over-expression is correlated with poor biological behavior. The increased levels of Glut-1 expression in hepatocellular carcinoma (HCC) cells functionally affect tumorigenicity. This study was undertaken to investigate effects of suppressing Glut-1 by an antisense oligodeoxynucleotide (AS-ODN) on the growth of human hepatocellular carcinoma (HepG-2) cells. METHODS: We used AS-ODN targeting against the Glut-1 gene in a HepG-2 cell line. There were four experimental groups:empty pcDNA3.1 vector (mock transfection), pcDNA3.1-anti-Glut (+), pcDNA3.1-Glut (+), and non-transfected HepG-2 cells. The Glut-1 mRNA expression was detected by RT-PCR and the Glut-1 protein expression by Western blotting after cell culture, and the glucose uptake was detected after glucose stimulation in each group. RESULTS:  Compared with non-transfected HepG-2 or Glut-1 pcDNA3.1, a down-regulation of Glut-1 mRNA in HepG-2 cells transfected with anti-Glut-1 pcDNA3.1 was noted (P CONCLUSIONS: The application of Glut-1 AS-ODN can down-regulate the expression of Glut-1 at mRNA and protein, and inhibit glucose uptake partially in HepG-2 cells. The Glut-1 gene maybe a potential therapeutic target for HCC.

  1. Effect of anti-sense oligodeoxynucleotides homeobox B2 on the proliferation and expression of primary human umbilical vein endothelial cells

    Institute of Scientific and Technical Information of China (English)

    刘旭盛; 张晓

    2002-01-01

    Objective: To explore the effect of homeobox B2 (HOXB2) antisense oligodeoxynucleotides (asodn) on the proliferation and expression of primary human umbilical vein endothelial cells (HUVECs).   Methods: Various concentrations of HOXB2 asodn modified by thiophosphate transfected the induction of liposome into HUVECs. MTT and RT-PCR methods were employed to determine the effect of different concentrations of asodn on the endothelial proliferation and the expression level of HOXB2 mRNA.   Results: After the transfection of HOXB2 asodn, the endothelial proliferation was inhibited in a dose-dependent fashion. Simultaneously, the expression of HOXB2 mRNA decreased significantly.   Conclusions: HOXB2 plays an important role in the proliferation of endothelia.

  2. 30. Knockdown of IGF-IR by Antisense Oligodeoxynucleotide auguments the sensitivity of bladder cancer cells to MMC

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    play key roles in autocrine mechanisms of bladder cancer cells remain to be definite and their physiological mechanism remain to be fully understood. More are still needed to know about the role of IGF1R signaling in bladder cancers. The following problems remain to be investigated in bladder cancer cells, about which the present studies are concerned: ①Is IGFs/IGF-IR signaling pathway involved in autocrine growth of human bladder cancer cells and how does bladder instillation drugs such as MMC affect the autocrine expression of bladder cancer cells? ②Can targeting against IGF1R gene can significantly enhance drug sensitivity of urinary bladder cancer cells to chemotherapy? ③What potential intracellular signaling mechanisms are involved in IGF1R blockage? ④May IGF1R self-stablized antisense ODN serves as a potential therapeutic approach to bladder cancer? To investigate whether IGF-1R was involved in drug resistance of bladder cancer cells. METHODS: RT-PCR was used to detect the mRNA expression of IGF-I, IGF-Ⅱ, and IGF-IR in T24 cells and normal urothelial cells. Flow cytometry and MTT tests were used to assess the effect of antisense oligodeoxynucleotide (ODN) on drug sensitivities and apoptosis of T24 cells to mitomycin (MMC). Western blot was used to analyze the effect of ODN on expression of IGF-IR protein. RESULTS: mRNA of IGF-I, IGF-Ⅱ, and IGF-IR were strongly expressed in serum-free cultured T24 cell line, whereas normal urothelial cells did not express these factors/receptors or only in trace levels; knockdown of IGF1R by antisense ODN significantly inhibited the growth of bladder cancer cells and enhanced sensitivity and apoptosis of T24 cells to MMC. CONCLUSION: These results suggested that blockage of IGF-IR signaling might potentially contribute to the treatment of bladder cancer cells which are insensitive to chemotherapy.

  3. 比较和分析NtGNL1的反义寡聚核苷酸抑制在烟草三种培养体系中的效果%Comparison and Analysis of Effects of Antisense Oligodeoxynucleotide Inhibition of NtGNL1 in Three Culture Systems of Tobacco

    Institute of Scientific and Technical Information of China (English)

    廖芳蕾; 王鲁; 辛可行; 陈文荣; 郭卫东

    2014-01-01

    为探讨反义寡聚核苷酸抑制(antisense oligodeoxynucleotides inhibition)技术在植物材料上的应用,以已知功能的NtGNLl (Nicotiana tabacum GNOM-Like1)为目标基因,寻找反义寡聚核苷酸抑制最适作用体系,并进一步分析NtGNL1的具体作用.根据目标基因mRNA序列设计反义寡聚核苷酸序列,商业合成后并将其添加到烟草胚珠、种子和花粉管离体培养的培养基中,以抑制NtGNL1的表达.结果表明,将反义寡聚核苷酸引入离体培养系统,短时间内抑制了目标基因mRNA的表达,但对胚胎发育过程的影响和种子萌发的抑制都不明显.在花粉离体萌发系统中,反义寡聚核苷酸抑制能够高效地引起目标基因mRNA的下调.对FM4-64染色的花粉管显微缩时观察发现,NtGNL1的下调能够引起囊泡分布和运输方向的改变.对花粉管膜流相关的5个基因的半定量分析也显示反义寡聚核苷酸进入花粉管后,导致3个基因的表达下调,暗示NtGNLJ抑制表达会影响花粉管囊泡运输的多个节点.

  4. Effects of Antisense Oligodeoxynucleotide to Follicle-stimulating Hormone Receptor on the Expression of Proliferating Cell Nuclear Antigen and Vascular Endothelial Growth Factor in Primary Culture Cells Derived from Human Ovarian Mucinous Cystadenocarcino

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    The effects of antisense oligodeoxynucleotide (antisense ODN) to follicle-stimulating hormone receptor (FSHR) and follicle-stimulating hormone (FSH) on the expression of proliferating cell nuclear antigen (PCNA) and vascular endothelial growth factor (VEGF) were studied in primary culture cells derived from human ovarian mucinous cystadenocarcinoma (OMC). The prlmary OMC cells were cultured with the enzyme digestion method, and the expression of pan Keratin protein and FSHR mRNA was detected for identification of the cells. OMC cells were co-cultured with antisense ODN, nonsense ODN and FSH with different concentrations for 48 h and 72 h. The expression of PCNA and VEGF was detected by using SP immunohistochemistry. Compared with that in the control group, the PCNA and VEGF expression was increased obviously in FSH groups (P<0.05 or P< 0.01), while decreased significantly in antisense ODN groups (P<0. 05 or P<0.01) and unchanged in nonsense ODN groups, respectively. Meanwhile, antisense ODN could antagonize the increased expression of PCNA and VEGF caused by FSH significantly (P<0.01). It was suggested that FSH might promotethe development of OMC to some extent. Antisense ODN could inhibit the proliferative activity of OMC cells and the promoting proliferative activity enhanced by FSH.

  5. Aerosolized STAT1 Antisense Oligodeoxynucleotides Decrease the Concentrations of Inflammatory Mediators in Bronchoalveolar Lavage Fluid in Bleomycin-Induced Rat Pulmonary Fibrosis

    Institute of Scientific and Technical Information of China (English)

    Ming Zeng; Bin Liao; Chen Zhu; Wenjun Wang; Xiaoqin Zhan; Xianming Fan

    2008-01-01

    It has been demonstrated that alveolar macrophages (AMs) play a key role in the pathogenesis of pulmonary fibrosis by releasing a variety of cytokines and inflammatory mediators. In addition, abnormal signal transducer and activator of transcription-1 (STAT1) activation in AMs may play a pivotal role in the process of alveolitis and pulmonary fibrosis. In this study, we transfected STAT1 antisense oligodeoxynucleotide (ASON) into rats by aerosolization, and then investigated the effect of STAT1 ASON on inflammatory mediators such as TGF-β, PDGF and TNF-α in bronchoalveolar lavage fluid (BALF) from rats with bleomycin (BLM)-induced rat pulmonary fibrosis. Our results showed that STAT1 ASON by aerosolization could enter into lung tissues and AMs. STAT1 ASON could inhibit mRNA and protein expressions of STATI and ICAM-1 in AMs of rat with pulmonary fibrosis, and had no toxic side effect on liver and kidney. Aerosolized STAT1 ASON could ameliorate the alveolitis through inhibiting the secretion of inflammatory mediators in BLM-induced rat pulmonary fibrosis. These results suggest that aerosolized STAT1 ASON might be considered as a promising new strategy in the treatment of pulmonary fibrosis. Cellular & Molecular Immunology. 2008;5(3):219-224.

  6. Local Delivery of C-myc Antisense Oligodeoxynucleotide by Gelatin Coated Platinium-Iridium Stent to Prevent Restenosis in a Normal Rabbit Carotid Artery

    Institute of Scientific and Technical Information of China (English)

    Zhang Xinxia; Wei Wenbin; Duan Wen; Xu Xiangguang; Hu Xuesong

    2005-01-01

    Objectives To investigate the feasibility and effect of local deliveryof c-myc antisense oligodeoxynucleotide (ASODN) by gelatin coated Platinium-Iridium stent to prevent restenosis in a normal rabbit carotid artery. Methods Gelatin coated Platinium-Iridium stent were implanted in the right carotid arteries of 32 rabbits under vision. Animals were randomized to the control group and the treated group receiving c-myc ASODN (n=16 respectively).7,14,30,90 days following the stenting procedure,morphometry for caculation of neointimal area and mean neointimal thickness were performed.The expression of c-myc protein was detected by immunohistochemical methods. Results 32 stents were successfully implanted into the right carotid arteries in 32 animals. Morphometric analysis showed that neointimal area and mean neointimal thickness siginificantly increased continuously up to 12 weeks after stent implantation,and at each time point ,neointimal area and mean neointimal thickness were siginificantly smaller in the treated group than control group. (P<0.001 ,respectively).c-myc protein expression was weak positive or negative in treated group and positive in control group. Conclusions Gelatin coated Platinium-Iridium stent mediated local delivery of c-myc ASODN is feasibility , and it can inhibit neointimal hyperplasia to prevent restenosis in a normal rabbit carotid artery.

  7. Inhibition of hTERT antisense oligodeoxynucleotide on proliferation and telomerase activity in keloid fibroblasts%hTERT反义寡核苷酸对瘢痕疙瘩成纤维细胞增殖及端粒酶活性的影响

    Institute of Scientific and Technical Information of China (English)

    黄勇; 林立新; 王鹏; 王学明; 王玉婷; 姜蕾

    2012-01-01

    目的 研究hTERT基因反义寡核苷酸(ASODN)对瘢痕疙瘩成纤维细胞生长增殖及端粒酶活性的抑制作用,探讨其在瘢痕疙瘩治疗中的作用.方法 应用ASODN封闭瘢痕疙瘩成纤维细胞hTERT基因的表达,不同浓度不同时间以ASODN作用于体外培养的瘢痕疙瘩成纤维细胞,细胞计数和MTT法检测成纤维细胞生长增殖情况,端粒酶PCR-ELISA法检测不同处理浓度和时间成纤维细胞端粒酶活性.结果 ASODN作用于瘢痕疙瘩成纤维细胞72 h后,成纤维细胞生长受抑,增殖能力减弱,并具有浓度、时间依赖性.端粒酶活性检测显示:ASODN 0.5、1.0和1.5 μmol/L作用组OD450-690值分别为0.612±0.038、0.535±0.027、0.423±0.023,与空白对照组(0.898±0.058)比较,差异有统计学意义(P<0.05);正义寡核苷酸1.0 μmol/L作用组(OD450-690值为0.893±0.042)与空白对照组比较,差异无统计学意义(P>0.05).结论 hTERT基因ASODN能抑制瘢痕疙瘩成纤维细胞的生长增殖,降低成纤维细胞端粒酶活性是其重要作用机制之一.通过抑制端粒酶活性进行抗瘢痕疙瘩治疗可能是一个新途径.%Objective To investigate the inhibition of hTERT antisense oligodeoxynucleotide (ASODN)on the proliferation and telomerase activity in keloid fibroblasts and to explore its anti-keloid effect.Methods Keloid fibroblasts in culture medium in vitro were given different concentrations of hTERT ASODN and were observed in different time.After treated by hTERT ASODN the proliferation of the fibroblasts was measured by cell count and MTT assay method,the changes of telomerase activity of keloid fibroblasts treated with hTERT ASODN was determined with PCR-ELISA method.The data was analyzed by statistical software (SPSS 11.5).Results The results showed that after sealing hTERT gene with ASODN for 72 hours,the fibroblasts growth was repressed and the ability of proliferation decreased,and the effect was specific in sequence and

  8. 反义寡核苷酸抑制缺氧/再给氧时内皮细胞细胞间粘附分子-1的表达%Inhibition of ICAM- 1 expression on endothelial cells inhypoxia/reoxygenation by antisense oligodeoxynucleotides

    Institute of Scientific and Technical Information of China (English)

    夏斌; 徐洪实; 邵福源; 陈蕾

    2000-01-01

    目的:探讨反义寡核苷酸(As-0DN)对缺氧/再给氧(H/R)时内皮细胞细胞间粘附分子-1(ICAM-1)表达的影响。方法:流式细胞仪测定肾小球血管内皮细胞在缺氧、再给氧及加入AS-0DN后ICAM-1表达的阳性百分率。结果:缺氧10 h,肾小球血管内皮细胞ICAM-1的表达与对照组无显著性差异,再给氧6 h ICAM-1的表达明显高于正常,加入As-ODN后,ICAM-1阳性细胞的百分率下降40.6%。结论:AS-ODN可以降低H/R时内皮细胞ICAM-1的表达。%AIM: To investigate the effect of antisense oligodeoxynucleotides (AS - ODN) on the intercellsular adhesion molecule- 1 (ICAM- 1 ) expression on endothelial cells in hypoxia/reoxygenation(H/R). METHODS: With cultured glomerular vascular endothelial cell in H/R, the positive percentage of ICAM - 1 expression was measured by flow cytometry before and after giving AS - ODN. RESULTS: The ICAM - 1 expression did not increase on glomerular vascular endothelial cell in 10 hours hypoxia compared to control group, it increased in 6 hours reoxygenation, and decreased by 40.6 % after giving AS- OND. CONCLUSION: AS - ODN may decrease the expression of ICAM- 1 on endothelial cells in H/R.

  9. EFFECT OF STENT ABSORBED c-myc ANTISENSE OLIGODEOXYNUCLEOTIDE ON SMOOTH MUSCLE CELLS APOPTOSIS IN RABBIT CAROTID ARTERY

    Institute of Scientific and Technical Information of China (English)

    张新霞; 崔长琮; 李江; 崔翰斌; 徐仓宝; 朱参战

    2002-01-01

    Objective To investigate the effect of gelatin coated Platinium-Iridium stent absorbed c-myc antisense oligodeoxynucleotide (ASODN) on smooth muscle cells apoptosis in a normal rabbit carotid arteries. Methods Gelatin coated Platinium-Iridium stents were implanted in the right carotid arteries of 32 rabbits under vision. Animals were randomly divided into control group and treated group receiving c-myc ASODN (n=16, respectively). On 7, 14, 30 and 90 days following the stenting procedure ,morphometry for caculation of neointimal area and mean neointimal thickness were performed.The expression of c-myc protein was detected by immunohistochemical method. Apoptotic smooth muscle cells was detected by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL). Results At 7 and 14 days after stenting,there were no detectable apoptotic cells in both groups. The apoptotic cells occurred in the neointima 30 and 90 days after stenting, and the number of apoptotic cells at 30 days were less [4.50±1.29 vs 25.75±1.89 (number/0.1mm2)] than that at 90 days [13.50±1.91 vs 41.50±6.46 (number/0.1mm2)]. Meanwhile c-myc ASODN induced more apoptotic cells than the control group(P<0.0001). c-myc protein expression was weak positive or negative in treated group and positive in control group.Conclusion c-myc ASODN can induce smooth muscle cells apoptosis after stenting in normal rabbit carotid arteries,and it can be used to prevent in-stent restenosis.

  10. Antibodies and antisense oligodeoxynucleotides to μ-opioid receptors, selectively block the effects of μ-opioid agonists on intestinal transit and permeability in mice

    OpenAIRE

    Pol, Olga; Valle, Lluís; Sánchez-Blázquez, Pilar; Garzón, Javier; Puig, Margarita M.

    1999-01-01

    We have studied the effects of μ and δ opioids on intestinal function (permeability, PER; gastrointestinal transit, GIT), and their antagonism after the intracerebroventricular (i.c.v.) administration of specific antibodies (ABs) or antisense oligodeoxynucleotides (ODN) to μ-receptors (OR). Central versus peripheral site/s of action of subcutaneous (s.c.) μ-opioids, were also assessed.Male Swiss CD-1 mice were used. GIT was measured with charcoal and PER by the passage of 51Cr-EDTA from blood...

  11. Inhibition of Telomerase Activity of Lymphoblastic Leukemic Cells by hTERT Antisense

    Institute of Scientific and Technical Information of China (English)

    ZHANGYuan; LIWenyu

    2004-01-01

    To investigate the effect of antisense, human telomerase reverse transcriptase (hTERT) mRNA oligodeoxynucleotide on telomerase activity of lymphoblastic leukemic cells. Methods: Telomerase activity was measured by the telomerase PCR ELISA assay kit (TRAP), hTERT protein by immunochemistry and flowcytometry, hTERT mRNA expression by reverse transcription polymerase chain reaction (RT-PCR) assay and gel-image system. Results: Incubation of lymphoblastic leukemic cells (Jurkat, Raji and CEM cell lines) with 10 μmol/L AS PS-ODN could significantly decline the mRNA and hTERT after 72 h, and the telomerase activity was significantly down-regulated or inhibited. Conclusion: The hTERT AS PS-ODN was an excellent inhibitor for telomerase activity of lymphoblastic leukemic cells.

  12. Cationic liposomes loaded with pro-apoptotic peptide D-(KLAKLAK)2 and bcl-2 antisense oligodeoxynucleotide G3139 for enhanced anti-cancer therapy

    Science.gov (United States)

    Ko, Young Tag; Falcao, Claudio; Torchilin, Vladimir P.

    2009-01-01

    The treatment of cancer using macromolecular therapeutics such as oligonucleotides or peptides requires efficient delivery systems capable of intracellular penetration and may also benefit from use of a combination of therapeutics with different mechanisms of action. With this possibility in mind, we constructed cationic liposome loaded with the proapoptotic peptide, D-(KLAKLAK)2 and the bcl-2 antisense oligodeoxynucleotide, G3139, and determined whether the combination of the proapoptotic macromolecules in a single cationic liposome can enhance antitumor efficacy. Advantage was taken of alternating charge interaction to entrap macromolecules of opposite charge. The polycationic pepetide D-(KLAKLAK)2 was first condensed with the polyanionic oligodeoxynucleotide G3139 to obtain overall negatively charged peptide/oligodeoxynucleotide complexes. The complexes were then entrapped into DOTAP/DOPE cationic liposomes (CL). This sequential charge interaction ensured efficient entrapment of D-(KLAKLAK)2 and G3139 with a high loading efficiency (50 %) and capacity (7.5 wt%). In vitro treatment of mouse melanoma B16(F10) with CL loaded with D-(KLAKLAK)2/G3139 led to significantly enhanced antitumor efficacy, mediated by stimulated induction of apoptotic (Caspase 3/7) activity, when compared to CL loaded with G3139 alone. Intratumoral injection of CL loaded with D-(KLAKLAK)2/G3139 in B16(F10) mice xenograft also led to suppressed tumor growth associated with enhanced apoptotic activity. Thus, the combination of proapoptotic peptide D-(KLAKLAK)2 and antisense oligonucleotide G3139 in a cationic liposome led to enhanced apoptotic/antitumor efficacy and may provide a promising tool for cancer treatment. PMID:19317442

  13. 硫代磷酸化修饰的反义 TGF-β1寡核苷酸抑制血管损伤后内膜增殖%Phosphorothioate-modified antisense TGF-β1 oligodeoxynucleotide inhibits neointimal hyperplasia after vascular balloon injury in rats

    Institute of Scientific and Technical Information of China (English)

    林志鸿; 谢良地; 吴可贵; 李庚山; 蔺佩鸿

    2014-01-01

    [ABSTRACT]AIM:ToevaluatetheeffectsofantisenseTGF-β1oligodeoxynucleotide(ASTGF-β1)ontheex-pression of TGF-β1 , deposition of extracellular matrix ( ECM) and the neointima formation in the arteries after balloon inju-ry.METHODS:The unmodified and phosphorothioate-modified AS TGF-β1 which containing 15 bases and surrounding the initiation codon region (ATG) of rat TGF-β1 complementary DNA (cDNA) were designed.At the same time, sense TGF-β1 oligodeoxynucleotide ( S TGF-β1 ) with the base sequence complement to AS TGF-β1 was synthesized as a control . The oligodeoxynucleotides were introduced into in vivo and in vitro experiments , respectively .RESULTS:The AS TGF-β1 significantly inhibited the protein expression of TGF-β1 in a concentration-dependent manner , and S TGF-β1 did not have the same effect.Furthermore, no effect of the AS TGF-β1 on the mRNA expression of TGF-β1 in injured VSMCs was ob-served.Moreover, for the injured VSMCs, AS TGF-β1 significantly and concentration-dependently inhibited the basal DNA synthesis.Both AS TGF-β1 and S TGF-β1 did not exhibit dose-dependent effects on DNA synthesis in uninjured VSMCs . Fibronectin ( FN) mRNA expression in injured VSMCs was significantly decreased by AS TGF-β1 in a concentration (0.01~1 μmol/L)-dependent manner .AS TGF-β1 significantly increased the mRNA expression of contractile marker SM 22α, and decreased the mRNA expression of synthetic markers osteopontin and matrix Gla , especially at the concentration of 0.01μmol/L and 0.1 μmol/L.After treatment with AS TGF-β1 (90 μg· kg-1 · d-1 ) for 28 d, the neointima formation was significantly inhibited , and the area ratio of intima/media was markedly decreased by 68% compared with untreated group , but S TGF-β1 had no effect on neointimal formation .CONCLUSION:The AS TGF-β1 specifically inhibits the pro-tein expression of TGF-β1 in the VSMCs derived from injured arteries .Moreover , it significantly inhibits DNA synthesis and cell

  14. Efficient down-regulation of PKC-α gene expression in A549 lung cancer cells mediated by antisense oligodeoxynucleotides in dendrosomes.

    Science.gov (United States)

    Movassaghian, Sara; Moghimi, Hamid R; Shirazi, Farshad H; Koshkaryev, Alexander; Trivedi, Malav S; Torchilin, Vladimir P

    2013-01-30

    The completion of human genome project has increased our knowledge of the molecular mechanisms of many diseases, including cancer, thus providing new opportunities for gene therapy. Antisense oligodeoxynucleotides (AsODN) possess great potential as sequence-specific therapeutic agents, which in contrast to classic treatments provide more efficient and target-specific approach to modulate disease-related genes. To be therapeutically effective, sufficient concentrations of intact AsODN must bypass membrane barriers and access the site of action. In this study, a dendrosome delivery strategy was designed to improve the encapsulation of AsODN in non-cationic liposomes to target PKC-α in lung cancer cells in vitro. Subcellular trafficking of fluorescently labeled AsODN was visualized using confocal microscopy. Uptake and expression of mRNA and target protein after AsODN delivery was measured by flow cytometry, qRT-PCR and Western blot analysis, respectively. Dendrosomes showed favorable physicochemical parameters: high encapsulation efficiency and uptake in serum-containing medium with no apparent cytotoxicity. AsODN encapsulated in dendrosome efficiently and specifically suppress the target gene at both mRNA and protein levels. Additional in vivo studies on the application of dendrosome as a delivery system for nucleic acid molecules may lead to improvement of this technology and facilitate the development of therapeutic antisense techniques. PMID:23262426

  15. Changes of Tc-99m sestamibi uptake in P-glycoprotein expressing leukaemia cells treated in vivo with antisense oligodeoxynucleotide complementary to mdr1 mRNA

    International Nuclear Information System (INIS)

    We examined the feasibility of Tc-99m sestamibi to monitor changes of mRNA expression of MDRl/P-glycoprotein (Pgp) following antisense oligodeoxynucleotide (AS-ODN) treatment in vivo. Three days after the intraperitoneal inoculation of murine leukaemia P388/R cells expressing MDR1/P-gp in CDFI mice, 15-mer phosphorothioate ASODN to the initiation codon of mouse mdr1 mRNA was administered intraperitoneally at 10 mg/kg daily for 3 or 4 days. Cells collected from ascites were suspended in medium for Tc-99m sestamibi uptake studies. To know the duration of antisense effects, cells were harvested 2 days later after the 3-day treatment. AS-ODN treatment increased Tc-99m sestamibi uptake. Effects of 3-day treatment and 4-day treatment were the same. Treatment effects were not detected when uptake was observed 2 days after 3-day treatment. Based on the results it was concluded that in vivo treatment with AS-ODN specific to the coding portion of mdr1 mRNA increased Tc-99m sestamibi uptake in leukaemia cells possessing MDR function. (author)

  16. In vivo distribution of c-myc antisense oligodeoxynucleotides local delivered by gelatin-coated platinmn-iridium stents in rabbits and its effect on apoptosis

    Institute of Scientific and Technical Information of China (English)

    张新霞; 崔长琮; 许香广; 胡雪松; 方卫华; 邝碧娟

    2004-01-01

    Background Post-stenting restenosis is a significant clinical problem, involving vascular smooth muscle cells(VSMCs) proliferation and apoptosis. It is reported that c-myc antisense oligodeoxynucleotides (ASODNs) local delivered by catheter can inhibit VSMCs proliferation. This study was designed to assess tissue distribution of c-myc ASODN local delivered using gelatin-coated platinum-iridium (Pt-Ir) stents, and its effect on apoptosis of VSMCs. Methods Gelatin-coated Pt-Ir stents that had absorbed caroboxyfluorescein-5-succimidyl ester (FAM) labeled c-myc ASODNs (550 μg per stent) were implanted into the right carotid arteries of 6 rabbits. Tissue samples were obtained at 45 minutes, 2 hours, and 6 hours. Tissue distribution of c- myc ASODNs was assessed by fluorescence microscopy. In addition, 32 rabbits were randomly divided into two groups. Rabbits in the control group (n=16) were implanted with gelatin-coated Pt-Ir stents, and those in the treatment group (n=16) were implanted with gelatin-coated stents that had absorbed c-myc ASODNs. 7, 14, 30, or 90 days (n=4, respectively, for each group) after the stenting procedure, the stented segments were harvested, and histopathological examinations were performed to calculate neointimal area and mean neointimal thickness. The expression of c-myc was assessed using in situ hybridization (ISH) and immunohistochemical methods. Apoptotic VSMCs were detected using terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) and transmission electron microscope (TEM). Results According to fluorescence microscopic results, FAM-labeled c-myc ASODNs were concentrated in the target vessel media at the 45 minutes time point, and then dispersed to the adventitia. Morphometric analysis showed that neointimal area and mean neointimal thickness increased continuously up to 90 days after stent implantation, but that total neointimal area and mean neointimal thickness were less in the treatment group than in the

  17. Inhibitory effect of antisense oligodeoxynucleotide to p44/p42 MAPK on angiotensin II-induced hypertrophic response in cultured neonatal rat cardiac myocyte

    Institute of Scientific and Technical Information of China (English)

    Shi-qinZHANG; BoDING; Zhao-guiGUO; Yun-xiaLI

    2004-01-01

    AIM: To explore the inhibitory effect of antisense oligonucleotide (ODN) to mitogen activated protein kinase(MAPK) on cardiomyocyte hypertrophy induced by angiotensin Ⅱ (Ang Ⅱ). METHODS: A 17-mer phosphorothioate-protected antisense ODN directed against the initiation of translation sites of the p42 and p44 MAPK isoforms byliposomal transfection was applied to inhibit the translation of p44/p42 MAPK mRNA. The sense and random ODNs to p44/p42MAPK were used as sequence controls. Neonatal cardiac myocytes were exposed to Ang Ⅱ (10nmol/L) for 5 min and then harvested in lysis buffer for the measurement of the activity and the phosphorylated protein content of p44/p42MAPK that were tested by P-81 phosphocellulose filter paper method and Western blotting, respectively. The rate of protein synthesis by [3H]leucine incorporation and the diameter of cell were measured after exposure to Ang Ⅱ for 24 h and 72 h, respectively. RESULTS: In cardiac myocyte Ang Ⅱ increased p44/p42MAPK activity and phosphorylated protein content by 140 % and 699 %, and also increased [3H]leucine incorporation and cell diameter by 40 % and 27 %. c-fos and c-myc mRNAs were induced significantly after exposure to Ang Ⅱ. Antisense ODN to p44/p42MAPK (0.2 μmol/L) reduced Ang Ⅱ-induced MAPK activity by 30 %,and phophorylated MAPK protein expression by 59 % in cardiac myocyte, and inhibited c-fos and c-myc mRNA expression induced by Ang Ⅱ by 44 % and 43 %, respectively. The diameter and the rate of protein synthesis of cardiac myocyte induced by Ang Ⅱ were decreased by 16 % and 22 % after pretreatment with antisense ODN to p44/p42MAPK. CONCLUSION: Antisense ODN to p44/p42 MAPK inhibited the increase of rate of protein synthesis,and the augmentation of cell diameter and expression of c-fos and c-myc mRNA induced by Ang Ⅱ in culturedcardiac myocytes, p44/p42 MAPK played a critical role in the hypertrophic response induced by Ang Ⅱ in cultured neonatal rat cardiac myocytes.

  18. Effect of Antisense Oligodeoxynucleotide Directed to NF-κB-RelA on Bcl-xL mRNA in Extended Drug Resistance Leukemia Cell Line HL-60/E6

    Institute of Scientific and Technical Information of China (English)

    曹文静; 张瑶珍; 张东华; 邹萍

    2001-01-01

    To explore the effect of NF-κB on bcl-x gene transcription in extended drug resistance leukemia cell line HL-60/E6, drug-resistant subline HL-60/E6 was derived by intermittently exposing HL-60 cells to 6 ng/ml epirubicin. Indirect immunofluorescence was used to demonstrate the location of NF-κB-RelA in HL-60/E6 cells. FCM analysis and RT-PCR were used to detect the efficiency of liposome-mediated ODN transfection and the change of bcl-xL mRNA levels after 5 μmol/L phosphorothioate (PS)-derivatized antisense (AS) oligodeoxynucleotide (ODN) directed to RelA was transferred into HL-60/E6 cells. The results showed that RelA remained persistently active and located at the nuclei of HL-60/E6 cells,but in the cytoplasm of HL-60 cells, the efficiency of liposome-mediated ODN transfection was significantly higher than that of null ODN (P<0.01 in 4 h, 6 h, 12 h, 24 h). Exposure of HL-60/E6 cells to 5 μmol/L AS-PS-ODN directed to RelA led to a maximal 40 % decline of bcl-xL mRNA levels within 8 h. The inhibition rate of bcl-xL mRNA was (15±1.79) %, (28±2.34) %, (40±3.47) %, (20±1.54) % in 4 h, 6 h, 8 h, 15 h, respectively, but it was less than 15 % in control group. It was concluded that NF-κB was involved in regulating bcl-x transcription. It was suggested that NF-κB was an important factor for drug resistance in leukemia cells.

  19. Antisense angiopoietin-1 inhibits tumorigenesis and angiogenesis of gastric cancer

    Institute of Scientific and Technical Information of China (English)

    Jun Wang; Kai-Chun Wu; De-Xin Zhang; Dai-Ming Fan

    2006-01-01

    AIM: To investigate the effect of angiopoietin-1 (Ang-1)on biological behaviors in vitro and tumorigenesis and angiogenesis in vitro of human gastric cancer cells.METHODS: Human full-length Ang-1 gene was cloned from human placental tissues by RT-PCR method.Recombinant human Ang-1 antisense eukaryotic expression vector was constructed by directional cloning,and transfected by lipofectin method into human gastric cancer line SGC7901 with high Ang-1 expression level.Inhibition efficiency was confirmed by semi- quantitive PCR and Western blot method. Cell growth curve and cell cycle were observed with MTT assays and flow cytometry, respectively. Nude mice tumorigenicity test was employed to compare in vitro tumorigenesis of cells with Ang-1 suppression. Microvessel density (MVD) of implanted tumor tissues was analyzed by immunohistochemistry for factor Ⅷ staining.RESULTS: Full-length Ang-1 gene was successfully cloned and stable transfectants were established,namely 7Ang1- for antisense, and 7901P for empty vector transfected. 7Ang1- cells showed down-regulated Ang-1 expression, while its in vitro proliferation and cell cycle distribution were not significantly changed.In contrast, xenograft of 7Ang1- cells in nude mice had lower volume and weight than those of 7901P after 30 days' implantation (P<0.01, 293.00±95.54 mg vs. 624.00±77.78 mg) accompanied with less vessel formation with MVD 6.00±1.73 compared to 7901P group 8.44±1.33 (P<0.01).CONCLUSION: Ang-1 may play an important role in tumorigenesis and angiogenesis of gastric cancer, and targeting its expression may be beneficial for the therapy of gastric cancer.

  20. NFkappaB decoy oligodeoxynucleotides ameliorates osteoporosis through inhibition of activation and differentiation of osteoclasts.

    Science.gov (United States)

    Shimizu, H; Nakagami, H; Tsukamoto, I; Morita, S; Kunugiza, Y; Tomita, T; Yoshikawa, H; Kaneda, Y; Ogihara, T; Morishita, R

    2006-06-01

    The transcription factor, nuclear factor-kappa B (NFkappaB), is believed to play a pivotal role in osteoclast formation. In this study, we focused on NFkappaB decoy oligodeoxynucleotides (ODN) as a new therapeutic strategy to attenuate osteoporosis. Tartrate-resistant acid phosphatase (TRAP)-positive multinuclear osteoclasts formed in mononuclear cells including osteoclast precursors from neonatal rabbit bone marrow were increased in the presence of 1,25-dihydroxyvitamin D3, whereas transfection of NFkappaB decoy ODN decreased the number of TRAP-positive cells and attenuated RANKL and M-CSF-induced osteoclast formation. NFkappaB decoy ODN also inhibited the activity of osteoclasts, as assessed by pit formation. In rat ovariectomized model of estrogen deficiency, continuous administration of NFkappaB decoy ODN attenuated the increase of TRAP activity, accompanied by a significant increase in calcium concentration in tibia and femur and decrease in urinary deoxypyridinoline. In additional osteoporosis model using vitamin C-deficient rat, inhibition of NFkappaB by decoy ODN dramatically improved the bone length, weight, density as assessed by dual-energy X-ray absorptiometry. Overall, inhibition of NFkappaB by decoy strategy prevented osteoporosis through the inhibition of bone resorption. Targeting of NFkappaB might be potential therapy in various bone metabolic diseases.

  1. Antisense inhibition of ATM gene enhances the radiosensitivity of head and neck squamous cell carcinoma in mice

    Directory of Open Access Journals (Sweden)

    Zheng Xuelian

    2008-10-01

    Full Text Available Abstract Background Treatment failure after radiotherapy of head and neck squamous cell carcinoma (HNSCC could be a significant problem. Our objective is to sensitize SCCVII cells to ionizing radiation in vitro and in vivo through inhibiting ATM expression using antisense oligodeoxynucleotides (AS-ODNs, and investigate the potential mechanism of radiosensitization. Methods We designed and synthesized AS-ODNs that target ATM mRNA to reduce the ATM expression. The influence on the expression of ATM mRNA and protein in SCCVII cells were analysed by real-time quantitative PCR and western blotting respectively. Clonogenic survival assay was performed to detect the survival ability of SCCVII cells after irradiation, while flow cytometry used to analyse the cell cycle and apoptosis. The volume of solid tumors generated with SCCVII cells was measured, and cell apoptosis was analysed by TUNEL assay after irradiation. Results The relative ATM mRNA and protein expression in SCCVII cells treated with ATM AS-ODNs were decreased to 25.7 ± 3.1% and 24.1 ± 2.8% of that in untreated cells respectively (P P P P P Conclusion Inhibition of ATM expression sensitized SCCVII cells to ionizing radiation in vitro and in vivo. The potential mechanism should be the defective G2/M cell cycle checkpoint control and enhanced radiation-induced apoptosis.

  2. 一种针对mdr1基因的寡核苷酸-阿霉素偶联物:对KB-A-1细胞的毒性及其抑制P-gp蛋白表达的效果%A novel antisense oligodeoxynucleotide-doxorubicin conjugate against the multidrug resistance gene mdr1:inhibition to KB-A-1 cell and P-glycoprotein expression

    Institute of Scientific and Technical Information of China (English)

    詹晓云; 任宇红; 张强; 卢艳花; 魏东芝; 刘建文

    2004-01-01

    Over-expression of P-glycoprotein(P-gp) on the surface of tumor cells that works as a membrane pump enhancing the drug efflux is regarded as a main candidate mechanism of multidrug resistance(MDR). And mdr1 is one of the two main genes that encode P-gp in human cells. In this study, a novel conjugate made of mdr 1-anti-sense oligodeoxynucleotide(ODN) and a potent anticancer drug doxorubicin was constructed. The cytotoxicity of the conjugate as well as the effect of the conjugate on the modulation of P-gp-mediated MDR in KB-A-1 cell lines was studied. The molecular mechanism of its regulation effect was also investigated. METHODS: Multidrug resistant KB-A-1 cell lines were used. MTF was applied to measure the cytotoxicity of the conjugate and doxorubicin to KB-A-1 cells.Effect of the conjugate on intracellular doxorubicin accumulation was determined by HPLC. RT-PCR and Western blot were used to determine the expression of mdr 1 gene and P-glycoprotein in KB-A-1 cells. RESULRTS: The results showed that the conjugate exhibited more cytotoxicity than ODN, and also it could promote the cytotoxicity of doxorubicin to KB-A-1 cells. The IC50 of doxorubicin dropped from 24 to 18 μg·ml-1. The results of RT-PCR and Western blot analysis illuminated that conjugate might down-regulate the P-gp expression. CONCLUSION: All the findings suggest that the conjugate is more potential for efficient reversal of MDR phenomenon than free ODN.The study indicated that such a conjugate should be a new feasible and efficient anti-sense reagent both in lab research and further in clinical therapy.%目的:癌细胞膜上P-gp糖蛋白的过量表达是肿瘤多药抗性的主要机制.人体内编码P-gp糖蛋白的基因中仅有mdr1涉及多药抗性.本研究中设计了针对mdr1的反义核酸与阿霉素的偶联物,并且对其细胞毒性进行了考察.同时对偶联物对人表皮癌细胞株KB-A-1内的P-gp蛋白的表达也做了分子水平上的研究.方法:使

  3. Experimental study on the inhibitory effect on coxsackievirus B3 pathogenicity by antisense oligodeoxynucleotide%反义寡核苷酸抑制柯萨奇病毒 B3 致病作用的实验研究

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    目的:观察在细胞培养系统和小鼠体内反义寡核苷酸 (AODN) 抑制柯萨奇病毒 B3(CVB3)致病作用。方法:观察细胞病变效应(CPE),常规微量法滴定病毒的 50%组织细胞感染量(TCID50)以及观察感染小鼠心肌组织的病理改变。结果:1.AODN 可推迟和减轻 CVB3 感染细胞 CPE,且随 AODN 浓度增高,对 CPE 的抑制作用增强;培养上清中病毒滴度随 AODN 浓度升高而降低。2.随 AODN 剂量增加,感染小鼠心肌组织病毒滴度逐渐降低;心肌细胞病变程度逐渐减轻;心肌炎发病率也逐渐降低。结论:AODN 可能通过抑制 CVB3 复制来保护 Vero 细胞和小鼠心肌组织抵抗感染。%Objective: To investigate the inhibitory effect of antisense oligodeoxynucleotide(AODN) as a specific inhibitor of CVB3 replication on CVB3 pathogenicity in vero cells and in Balb/C mice. Methods: Cytopathic effect(CPE) was investigated, 50% tissue culture infection dose(TCID50) was titrated on vero cells monolayer and cardiac histological lesions were investigated. Results: The specific AODN could significantly inhibit CPE of CVB3-infected vero cells and decrease viral titres in culture supernatant in dose dependent manner. The specific AODN could also significantly decrease viral titres of myocardial tissue, lighten cardiac lesions and reduce incidence rate of myocarditis in mice in dose dependent manner. Conclusion: AODN may be protect vero cells and myocardial tissue against CVB3 infection by inhibiting CVB3 replication.

  4. Inhibition of telomerase with human telomerase reverse transcriptase antisense enhances tumor necrosis factor-a-induced apoptosis in bladder cancer cells

    Institute of Scientific and Technical Information of China (English)

    GAO Xiao-dong; CHEN Yi-rong

    2007-01-01

    Background Telomerase activity is found in 85%-90% of all human cancers but not in their adjacent normal cells.Human telomerase reverse transcriptase (hTERT) is an essential component in the telomerase complex that plays an important role in telomerase activity. This study investigated the effect of the telomerase inhibition with an hTERT antisense oligodeoxynucleotide (ODN) in bladder cancer cells (T24) on tumor necrosis factor-o (TNF-α)-induced apoptosis.Methods Antisense phosphorothioate oligodeoxynucleotide (AS PS-ODN) was synthesized and purified. Telomerase activity was measured by polymerase chain reaction enzyme-linked immunoassay (PCR-ELISA). hTERT mRNA expression was measured by reverse transcription polymerase chain reaction (RT-PCR) assay and a gel-image system.hTERT protein was detected by immunochemistry and flow cytometry. Cell viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2, 5-Diphenyltetrazolium (MTT) assay. Cell apoptosis was observed by a morphological method and determined by flow cytometry.Results AS PS-ODN significantly inhibited telomerase activity and decreased the levels of hTERT mRNA which preceded the decline in the telomerase activity. AS PS-ODN significantly reduced the percentage of positive cells expressing hTERT protein following the decline of hTERT mRNA levels. There was no difference seen in the telomerase activity, hTERT mRNA expression or the protein levels between the sense phosphorothioate oligodeoxynucleotide (SPS-ODN) and the control group. AS PS-ODN treatment significantly decreased the cell viability and enhanced the apoptotic rate of T24 cells in response to TNF-α while there was no difference in cell viability and apoptotic rate between the S PS-ODN and the control group.Conclusions AS PS-ODN can significantly inhibit telomerase activity by downregulating the hTERT mRNA and protein expression. Treatment with AS PS-ODN may be a potential and most promising strategy for bladder cancer with telomerase

  5. Inhibition of retroviral replication by anti-sense RNA.

    OpenAIRE

    To, R Y; Booth, S C; Neiman, P E

    1986-01-01

    We tested the effect of anti-sense RNA on the replication of avian retroviruses in cultured cells. The replication of a recombinant retrovirus carrying a neomycin resistance gene (neor) in the anti-sense orientation was blocked when the cells expressed high steady-state levels of RNA molecules with neor in sequence in the sense was blocked when the cells expressed high steady-state levels of RNA molecules with neor sequences in the sense orientation, i.e., complementary to the viral sequence....

  6. Induction of apoptosis and inhibition of proliferation in Hep-2 by antisense survivin RNA in vitro

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Objective: To study induction of apoptosis and inhibition of proliferation in Hep-2 by antisense survivin RNA. Methods: Antisense survivin RNA expression vector was constructed and then was transfected to human laryngeal carcinoma cell line Hep-2 by lipofectamine. HpEGFP/survivin cells (transfected with the combinant of antisense survivin RNA) were obstained by using G418. The levels of survivin protein before and after transfection were determined by Western-blot. Proliferation activity was measured by MTT assay. The experiment of colony formation in soft agar was carried out for assessing ability of proliferation of Hep-2 cell. Apoptosis was assessed by flow cytometry and acrdine orange(AO).Results:After antisense survivin RNA plasmids were transfected, the level of survivin protein was inhibited in Hep-2. ComPared with control, proliferation of HpEGFP/survivin cells were suppressed significantly. The experiment of colony formation in soft agar showed the ability of colony formation decreased in HpEGFP/survivin cells compared to control (P<0.05). Apoptosis rate increased about 1.81 folds compared with control. Conclusion: The antisense survivin RNA can partly inhibit the level of survivin protein expression in Hep-2 and can induce apoptosis and inhibit the proliferation of Hep-2 by down-regulating the expression of endogenous survivin in vitro.

  7. Inhibition of Inositol-1-phosphate Synthetase in Mycobacterium Tuberculosis by Chitosan-antisense Nanoparticles

    Institute of Scientific and Technical Information of China (English)

    LI Yuanyuan; CHEN Zhifei; ZHANG Hongling; LI Xiaobo; SHEN Jie; LU Shi; XU Shunqing

    2009-01-01

    Oligodeoxynucleotides (ODNs) were combined with the biodegradable polymer chitosan to form chitosan-ODN nanoparticles by complex coacervation, in order to improve the sta-bility and intracellular penetration. The diameter of the nanoparticles was light strength size and ranged between 60 and 219 nm with a mean value of 132 nm, while zeta potential was between +12 and + 20 mV at pH 5.5. The chitosan-ODN nanoparticles could partially protect the encapsulated ODN from nuclease degradation. Moreover, chitosan-ODN nanoparticles were much more effective in inhibiting the proliferation of M.tuberculosis than free ODN.

  8. BTEB2 antisense RNA inhibits intimal hyperplasia in a rat carotid balloon injury model

    Institute of Scientific and Technical Information of China (English)

    LI De; HE Guo-xiang; TANG Bing; TANG Bo

    2006-01-01

    Objective: To investigate the effects of basic transcriptional element binding protein-2(BTEB2) antisense RNA on vascular smooth muscle cells (VSMCs) proliferation and the neointimal formation after carotid balloon injury in rats. Methods: The cultured VSMCs were transfected with an adenoviral vector containing BTEB2 antisense gene, Ad ASBTEB2. Effects of BTEB2 antisense RNA on the expression of BTEB2 were investigated by Western blot analysis. The cell cycle was analyzed using flow cytometry. Ad ASBTEB2, control adenoviral vector Ad. LacZ or PBS was transduced into the rat carotid artery after balloon injury. The expression of BTEB2 at 7, 14, and 21 d after gene transfer was detected by immunohistochemistry and neointima-to-media (I/M) area ratio at these time points was calculated. Results: The cell cycle was arrested in G0/G1 phase and the expression of BETB2 was downregulated after transfection with Ad ASBTEB2. Ad ASBTEB2 treatment reduced I/M area ratios on day 7, 14, and 21 after injury by 45%, 50% and 53% respectively, whereas the Ad LacZ treatment did not significantly alter these ratios compared with control group. Conclusion: BTEB2 antisense RNA mediated by adenoviral vector inhibits proliferation of VSMCs and significantly reduces neointimal hyperplasia in the rat carotid balloon injury model. BTEB2 antisense RNA is a potential therapeutic approach to preventing neointimal formation after balloon injury.

  9. Enhanced sensitivity of BIU87 cells to cisplatin by bcl-2 antisense oligodeoxynucleotides%bcl-2反义寡核苷酸增强膀胱癌细胞株BIU87对顺铂的敏感性

    Institute of Scientific and Technical Information of China (English)

    王志华; 曾星; 胡志全; 叶章群; 王冀; 李有元; 刘双林; 鲁海洋; 邓康俐

    2011-01-01

    Objective To observe the influence of bcl-2 antisense oligodeoxynucleotides (ASON)on reversal of cisplatin resistance in BIU87 cell line of bladder carcinoma. Methods Cisplatin resistance bladder carcinoma cells served as models. The bcl-2 antisense oligodeoxynucleotides were transfected to the cells by lipofectamine and electroporation. The sense-and random-oligodeoxynucleotides were used as controls. The expression level of bcl-2 mRNA was detected by using semi-quantitative reverse transcription polymerase chain reaction ( RT-PCR) . The apoptosis of bladder cancer cell lines was examined by DNA ladder assay. The protein level and 50% inhibitory concentration (IC50) were measured by immunofluorescence technique and methyl thiazol tetrazolium (MTT) assay, respectively. Results Transfecting BIU87/DDP cells with antisense oligonucleotides statistically reduced IC50, values of BID/DDP cells from (66. 000 ±4. 637) to (20. 400 ±2. 590) mg/L. Twelve h after transfection by lipofectamine, 30% cells had apoptotic or necrotic changes under the inverted phase contrast microscopy, and DNA " Ladder" was observed with agrose gel electrophoresis in the antisense-transfected cells. Forty-eight h after transfection by electroporation followed by treatment of 10 mg/L cisplatin, RT-PCR showed the expression of bcl-2 mRNA in the transfected cells was significantly lower (0. 72 ± 0. 07 ) than before transfection ( 2. 94 ± 0. 09 ) , and that of bcl-2 protein was decreased, as well as the IC50. Conclusion bcl-2 antisense oligodeoxynucleotides can reverse the resistance to cisplatin in BIU87/DDP cells by inhibiting the expression of bcl-2 protein and increasing the apoptosis.%目的 观察bcl-2反义寡核苷酸对膀胱癌顺铂(DDP)耐药细胞耐药性的逆转并增强其敏感性的作用.方法 以膀胱癌顺铂耐药细胞株BIU87/DDP为模型,用脂质体转染和电转染的方法,将bcl-2反义寡核苷酸(bcl-2-AODN)转染BIU87/DDP细胞,同时以转染bcl-2

  10. Aurora A反义寡核苷酸对肺癌细胞A549的作用和对紫杉醇化疗敏感性的影响%The effect of antisense oligodeoxynucleotides targeting Aurora A kinase on cell proliferation and chemosensitivity to paclitaxel in human lung cancer cell line A549

    Institute of Scientific and Technical Information of China (English)

    Rui Meng; Gang Wu; Jing Cheng; Tao Wang

    2007-01-01

    Objective:Aurora A kinase representing a family of evolutionarily conserved mitotic serine/threonine kinases has been found elevated in human lung adenocarcinoma cell line A549.It is suggested that the overexpression of Aurora A contributes to the carcinogenesis,chromosomal instability (CIN),and de-differentiation of lung cancers.To address its possibility as a therapeutic target for lung cancer,we employed the antisense oligodeoxynucleotide (ASODN) technique to inhibi Aurora A expression and investigate its effects on tumor growth and cell cycle of A549.as well as the chemosensitivilty to paclitaxel.Methods:Aurora A ASODN was synthesized and transfected into A549 cells by lipofectAMINE 2000.Aurora A mRNA and protein expression were examined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot respectively.Cell cycle distribution was observed by flow cytometer.MTT assay was used to evaluate cell inhibition ratio before and after transfection.Results:The proliferation of the A549 cell swas inhibited by Aurora AASODN dose and time dependently.It was also observed thal the IC50 of A549 cells after 48 hours'treatmenl of ASODN was about 300 nmol/L and under such circumstances,the Aurora A mRNA and protein expression significantly decreased(P<0.05),along with the induction of accumulation of cells in S phase and the G2-M transition.Furlhermore.cell inhibition ratio of the combination of Aurora AASODN and paclitaxel was higher significantly than paclitaxel(P<0.05)or Aurora AASODN alone (P<0.05).Conclusion:Inhibition of Aurora A expression can result in the suppression of cell growth and chemosensilizina activity to paclitaxel in human lung cancer cell line A549.

  11. Effect of antisense oligodeoxynucleotides of type α1(Ⅰ) precollagen gene on the biological characteristics of human hypertrophic scar fibroblasts%α1(Ⅰ)型前胶原基因反义寡聚脱氧核苷酸对人增生性瘢痕成纤维细胞生物学特性的影响

    Institute of Scientific and Technical Information of China (English)

    邢帮荣; 利天增; 谢举临; 徐盈斌; 祁少海

    2013-01-01

    Objective To study the effect of antisense oligodeoxynucleotides of type α1 (Ⅰ) precollagen gene on growth and the collagen synthesis of human hypertrophic scar fibroblasts. Methods The HF were divided into 3 groups according to the transfection liquids: the experimental group with lipofectAMINETM and type α1 (Ⅰ) precollagen gene antisense oligodeoxynucleotides, the control group 1 with only LipofectAMINETM and the control group 2 with only DMEM culture medium as the control group. At 0. 5, 1, 3, 5 and 7 days after transfection, different measures were adopted to evaluate the biological characteristics of hypertrophic scar fibroblasts, such as the fibroblast growth curve protracted by the cell count technique, the ultra-structure observed by transmission eletron microscopy (TEM) , total RNA of the hypertrophic scar fibroblasts were extracted and the expression level of type α1 (Ⅰ) precollagen mRNA was detected by RT-PCR,the type I collagen synthesis of hypertrophic scar fibroblasts was tested by the western-bloting and compared with the control group. Results ①The fibroblast proliferation was not inhibited by antisense oligodeoxynucleotides.②The organelles for the protein synthesis in the experimental group was not in active state. ③Compared with the control group, the expression level of type α1 (Ⅰ) precollege mRNA and protein synthesis of hypertrophic scar fibroblasts in the experimental group were obviously decreased. Conclusion The anti-sense oligodeoxynucleotides inhibits availably the expression level of type α1 (Ⅰ) precollagen mRNA and the protein synthesis of Ⅰ collagen of hypertrophic scar fibroblasts cultured in vitro, which can inhibint the scar hyperplasia.%目的 探讨应用α1(Ⅰ)型前胶原基因反义寡聚脱氧核苷酸对增生性瘢痕成纤维细胞生长、胶原合成等生物学特性的影响.方法 根据转染液分3组,实验组为脂质体加α1(Ⅰ)型前胶原基因反义寡聚脱氧核苷酸;对照组1

  12. Inhibiting effect of antisense hTRT on telomerase activity of human liver cancer cell line SMMC-7721

    Institute of Scientific and Technical Information of China (English)

    牟娇; 李晓冬; 杨庆; 贾凤岐; 卫立辛; 郭亚军; 吴孟超

    2003-01-01

    Objective: To induce changes in biological character of human liver cancer cell line SMMC-7721 by blocking the expression of telomerase genes hTRT and to explore its value in cancer gene therapy. Methods: The vehicle for eukaryotic expression of antisense hTRT was constructed and then transfected into SMMC-7721 cells. The effects of antisense hTRT gene on telomerase activity, cancer cell growth and malignant phenotypes were analyzed. Results: The obtained transfectants that could express antisense hTRT gene stably showed marked decrease in telomerase activity; the shortening of telomere was obvious; cells presented contact growth inhibition; in nude mice transplantation, the rate of tumor induction dramatically decreased. Conclusion: Antisense hTRT gene expression can significantly inhibit telomerase activity of cancer cells and decrease malignant phenotypes in vitro and in vivo. Therefore, as a telomerase inhibitor, antisense hTRT gene may be a new pathway for cancer therapy.

  13. Inhibition of Gene Expression in Escherichia coli by Antisense Phosphorodiamidate Morpholino Oligomers

    OpenAIRE

    Geller, B L; Deere, J. D.; Stein, D A; Kroeker, A. D.; Moulton, H. M.; Iversen, P. L.

    2003-01-01

    Antisense phosphorodiamidate morpholino oligomers (PMOs) were tested for the ability to inhibit gene expression in Escherichia coli. PMOs targeted to either a myc-luciferase reporter gene product or 16S rRNA did not inhibit luciferase expression or growth. However, in a strain with defective lipopolysaccharide (lpxA mutant), which has a leaky outer membrane, PMOs targeted to the myc-luciferase or acyl carrier protein (acpP) mRNA significantly inhibited their targets in a dose-dependent respon...

  14. Inhibition of Proliferation of Human Osteosarcoma Cells Transfected with PIN1 Antisense Gene

    Institute of Scientific and Technical Information of China (English)

    XIONG Wenhua; CHEN Anmin; GUO Fengjin

    2006-01-01

    Objective: To evaluate the inhibition of proliferation of human osteosarcoma cells transfected with Pin1 anti-sense gene. Methods: Different doses of antisense Pin1 gene (0, 20, 50, 100, 200, 250μL) were transfected into osteosarcoma MG-63 cells. The cells and culture supernatant before and after transfection were collected. The curve of cell growth was made by MTT method. The cell growth cycle and apoptosis were detected by FCM. The expression of Pin1 was detected by Western-blot and that of Pin1 mRNA by polymerase chain reaction (RT-PCR) respectively. Results: MTT and FCM assays indicated that the transfection by antisense Pin1 gene could inhibit MG-63 proliferation and induce apoptosis. Western-blot assays revealed that the antisense Pin1 gene-transfected MG-63 cells had weaker staining than those without transfected with antisense Pin1 gene, and staining intensity was negatively related with doses. The cells transfected by different doses of gene (0, 20, 50, 100, 200, 250μL) had different absorbance rate: 0.854±0.136, 0.866±0.138, 0.732±0.154, 0.611±0.121, 0.547±0.109, 0.398±0.113,0.320±0.151 respectively, with the difference being significant by F and q test (P<0.05). The expression of Pin1 mRNA had the similar results and its absorbance rate was 0.983±0.125, 0.988±0.127, 0.915±0.157,0.786±0.125, 0.608±0.124, 0.433±0.130, 0.410±0.158 respectively (P<0.05). Conclusion: The expression of Pin1 mRNA in MG-63 cells could be inhibited by antisense Pin1 gene, so to reduce the expression of Pin1 and depress the proliferation of human osteosarcoma cells MG-63.

  15. hTERT反义核酸对淋巴白血病细胞端粒酶活性的抑制%Inhibition of Telomerase Activity of Lymphoblastic Leukemic Cells by hTERT Antisense

    Institute of Scientific and Technical Information of China (English)

    张洹; 李文瑜

    2004-01-01

    Objective: To investigate the effect of antisense, human telomerase reverse transcriptase (hTERT) mRNA oligodeoxynucleotide on telomerase activity of lymphoblastic leukemic cells. Methods:Telomerase activity was measured by the telomerase PCR ELISA assay kit (TRAP), hTERT protein by immunochemistry and flowcytometry, hTERT mRNA expression by reverse transcription polymerase chain reaction (RT-PCR) assay and gel-image system. Results: Incubation of lymphoblastic leukemic cells (Jurkat, Raji and CEM cell lines) with 10 μmol/L AS PS-ODN could significantly decline the mRNA and hTERT after 72 h, and the telomerase activity was significantly down-regulated or inhibited. Conclusion:The hTERT AS PS-ODN was an excellent inhibitor for telomerase activity of lymphoblastic leukemic cells.

  16. Internalization of oligodeoxynucleotide antisense to type-1 plasminogen activator inhibitor mRNA in endothelial cells: a three-dimensional reconstruction by confocal microscopy.

    Science.gov (United States)

    Wyroba, E; Pawlowska, Z; Kobylanska, A; Pluskota, E; Maszewska, M; Stec, W J; Cierniewski, C S

    1996-01-01

    A three-dimensional reconstruction analysis of localization of phosphodiester and phosphorothioate oligonucleotide antisense to type-1 plasminogen activator inhibitor (PAI-1) mRNA within endothelial cells is described. When EA.hy 926 cells were incubated with fluorescently labelled phosphodiester (PO-16) or phosphorothioate (PS-16) oligonucleotides at low, not cytotoxical concentrations, the relative brightness composition of the images of the particular samples was much higher for PS-16 than PO-16 and dependent upon the extracellular concentration and the incubation time. The 3-D reconstructions based on the series of optical sections of the samples, spaced every 1.5 microns, showed the punctuate accumulation of the oligonucleotides and a striking difference in a spatial distribution between PO-16 and PS-16 within the cytoplasm. Even after 24 h incubation of endothelial cells with 2.5 microM of PO-16 and PS-16 oligonucleotides, there was a predominant oligonucleotide localization within the cytoplasm and only traces of oligonucleotides could be seen in the cell nucleus and/or perinuclear organelles.

  17. Antisense MMP-9 RNA inhibits malignant glioma cell growth in vitro and in vivo

    Institute of Scientific and Technical Information of China (English)

    Cuiyun Sun; Qian Wang; Hongxu Zhou; Shizhu Yu; Alain R.Simard; Chunsheng Kang; Yanyan Li

    2013-01-01

    The matrix-degrading metalloproteinases (MMPs),particularly MMP-9,play important roles in the pathogenesis and development of malignant gliomas.In the present study,the oncogenic role of MMP-9 in malignant glioma cells was investigated via antisense RNA blockade in vitro and in vivo.TJ905 malignant glioma cells were transfected with pcDNA3.0 vector expressing antisense MMP-9 RNA (pcDNA-ASMMP9),which significantly decreased MMP-9 expression,and cell proliferation was assessed.For in vivo studies,U251 cells,a human malignant glioma cell line,were implanted subcutaneously into 4-to 6-week-old BALB/c nude mice.The mice bearing well-established U251 gliomas were treated with intratumoral pcDNA-AS-MMP9-Lipofectamine complex (AS-MMP-9-treated group),subcutaneous injection of endostatin (endostatin-treated group),or both (combined therapy group).Mice treated with pcDNA (empty vector)-Lipofectamine served as the control group.Four or eight weeks later,the volume and weight of tumor,MMP-9 expression,microvessel density and proliferative activity were assayed.We demonstrate that pcDNA-AS-MMP9 significantly decreased MMP-9 expression and inhibited glioma cell proliferation.Volume and weight of tumor,MMP-9 expression,microvessel density and proliferative activity in the antisense-MMP-9-treated and therapeutic alliance groups were significantly lower than those in the control group.The results suggest that MMP-9 not only promotes malignant glioma cell invasiveness,but also affects tumor cell proliferation.Blocking the expression of MMP-9 with antisense RNA substantially suppresses the malignant phenotype of glioma cells,and thus can be used as an effective therapeutic strategy for malignant gliomas.

  18. Inhibition, Escape, and Attenuated Growth of Severe Acute Respiratory Syndrome Coronavirus Treated with Antisense Morpholino Oligomers†

    OpenAIRE

    Neuman, Benjamin W.; Stein, David A.; Kroeker, Andrew D.; Churchill, Michael J.; Kim, Alice M.; Kuhn, Peter; Dawson, Philip; Moulton, Hong M.; Bestwick, Richard K.; Iversen, Patrick L.; Michael J Buchmeier

    2005-01-01

    The recently emerged severe acute respiratory syndrome coronavirus (SARS-CoV) is a potent pathogen of humans and is capable of rapid global spread. Peptide-conjugated antisense morpholino oligomers (P-PMO) were designed to bind by base pairing to specific sequences in the SARS-CoV (Tor2 strain) genome. The P-PMO were tested for their capacity to inhibit production of infectious virus as well as to probe the function of conserved viral RNA motifs and secondary structures. Several virus-targete...

  19. Efficient inhibition of human telomerase activity by antisense oligonucleotides sensitizes cancer cells to radiotherapy

    Institute of Scientific and Technical Information of China (English)

    Xue-mei JI; Cong-hua XIE; Ming-hao FANG; Fu-xiang ZHOU; Wen-jie ZHANG; Ming-sheng ZHANG; Yun-feng ZHOU

    2006-01-01

    Aim: To investigate the effect of the antisense oligonucleotides (ASODN) specific for human telomerase RNA (hTR) on radio sensitization and proliferation inhibition in human neurogliocytoma cells (U251). Methods: U251 cells were transfected with hTR ASODN or nonspecific oligonucleotides (NSODN). Before and after irradiation of 60Co-γray, telomerase activity was assayed by telomeric repeat amplification protocol (TRAP-PCR-ELISA), and DNA damage and repair were examined by the comet assay. The classical colony assay was used to plot the cell-survival curve, to detect the D0 value. Results: hTR antisense oligonucleotides could downregulate the telomerase activity, increase radiation induced DNA damage and reduce the subsequent repair. Furthermore, it could inhibit the proliferation and decrease the D0 value which demonstrates rising radiosensitivity. However, telomere length was unchanged over a short period of time. Conclusion: These findings suggest that an ASODN-based strategy may be used to develop telomerase inhibitors, which can efficiently sensitize radiotherapy.

  20. Proliferative response of human prostate cancer cell to hormone inhibited by androgen receptor antisense RNA

    Institute of Scientific and Technical Information of China (English)

    江军; 王洛夫; 方玉华; 靳风烁; 靳文生

    2004-01-01

    Background The failure of endocrine treatment for advanced prostate cancer might be related to aberrant activation of androgen receptor (AR). Prostate cancer cell line LNCaP contains AR that can be activated by androgen, estrogen and progesterone. This study was set to investigate the effects of antisense AR RNA on growth of LNCaP cultured in medium containing varied concentrations of R1881, 17β-estradiol, and progesterone, respectively. Methods LNCaP cells transfected with antisense AR RNA retroviral vector pL-AR-SN were designated as LNCaPas-AR. LNCaP cells containing empty vector pLXSN served as LNCaPNeo. LNCaP and LNCaPNeo were taken as controls. In vitro cell growth assay, proliferative cells of LNCaP and tranfected LNCaPs were counted by typan staining when they cultured with synthetic androgen R1881, 17β-estradiol, and progesterone, respectively. Results Growth of LNCaPas-AR was inhibited significantly (P<0.05) compared with that of LNCaP and LNCaPNeo at 1 nmol/L R1881, 10 nmol/L 17β-estradiol, and 1 nmol/L progesterone, respectively. No difference was seen between LNCaP and LNCaPNeo(P>0.05). Microscopic observation showed that LNCaP and LNCaPNeo cells grew well, but only few LNCaPas-AR cells were alive. Conclusions Our observations indicate that antisense AR RNA retroviral vector pL-AR-SN could change androgen-independent characteristics of LNCaP cells, which might shed some novel insights into the treatment of androgen-independent prostate cancer.

  1. Inhibition of HSP70 Gene Expression by Modified Antisense and Its Effects on Embryonic Sensitivity to Heat Shock

    Institute of Scientific and Technical Information of China (English)

    TIAN Wen-ru; DU Li-yin; HE Jian-bin; LI Shou-jun

    2004-01-01

    Experiments were performed to evaluate the efficiency of inhibition of HSP70 gene expression by antisense oligonucleotides complementary to the mRNA of HSP70 and to test the effects of inhibition of HSP70 gene expression on subsequent embryonic sensitivity to heat shock. The results showed that transfection of pre-implantation embryos at 4-cell stage with 5 μM antisense oligo had no effect on in vitro blastocyst development. However, transfection with 10 to 40 μM antisense oligo had reduced in vitro blastocyst development to 15, 10% and 0; For the embryos which exposed to 40 μM As arrested at the 16-cell stage, there was no blastocyst formation within the heat shock groups. In contrast, transfection had no effect on embryonic sensitivity to heat shock, above 25% of embryos developed to blastocyst stage in control groups.

  2. Growth inhibiting effects of antisense eukaryotic expression vector of proliferating cell nuclear antigen gene on human bladder cancer cells

    Institute of Scientific and Technical Information of China (English)

    童强松; 曾甫清; 林晨; 赵军; 鲁功成

    2003-01-01

    Objective To explore the growth inhibiting effects on human bladder cancer by antisense RNA targeting the proliferating cell nuclear antigen (PCNA) gene. Methods The eukaryotic expression vector for antisense PCNA cDNA was constructed and transferred into a bladder cancer EJ cell line. The PCNA expression in the cancer cells was detected by RT-PCR and Western blotting assays. The in vitro proliferation activities of the transferred cells were observed by growth curve, tetrazolium bromide (MTT) colorimetry, tritiated thymidine (3H-TdR)incorporation, flow cytometry and clone formation testing, while its in vivo anti-tumor effects were detected on nude mice allograft models.Results After the antisense vector, pLAPSN, was transferred, cellular PCNA expression was inhibited at both protein and mRNA levels. The growth rates of EJ cells were reduced from 27.91% to 62.07% (P<0.01), with an inhibition of DNA synthesis rate by 52.31% (P<0.01). Transferred cells were blocked at G0/G1 phases in cell-cycle assay, with the clone formation ability decreased by 50.81% (P<0.01). The in vivo carcinogenic abilities of the transferred cancer cells were decreased by 54.23% (P<0.05). Conclusions Antisense PCNA gene transfer could inhibit the growth of bladder cancer cells in vitro and in vivo, which provided an ideal strategy for gene therapy of human cancers.

  3. Heat shock protein 70 antisense oligonucleotide inhibits cell growth and induces apoptosis in human gastric cancer cell line SGC-7901

    Institute of Scientific and Technical Information of China (English)

    Zhi-Gang Zhao; Wen-Lu Shen

    2005-01-01

    AIM: Heat shock protein (HSP)70 is over-expressed in human gastric cancer and plays an important role in the progression of this cancer. We investigated the effects of antisense HSP70 oligomer on human gastric cancer cell line SGC-7901, and its potential role in gene therapy for this cancer.METHODS: Human gastric cancer cell line SGC-7901 was treated in vitro with various concentrations of antisense HSP70 oligonucleotides at different intervals. Growth inhibition was determined as percentage by trypan blue dye exclusion test. Extracted DNA was electrophoresed on agarose gel, and distribution of cell cycle and kinetics of apoptosis induction were analyzed by propidium iodide DNA incorporation using flow cytometry, which was also used to detect the effects of antisense oligomer pretreatment on the subsequent apoptosis induced by heat shock in SGC-7901 cells. Proteins were extracted for simultaneous measurement of HSP70 expression level by SDS-PAGE Western blotting.RESULTS: The number of viable cells decreased in a doseand time-dependent manner, and ladder-like patterns of DNA fragments were observed in SGC-7901 cells treated with antisense HSP70 oligomers at a concentration of 10 μmol/L for 48 h or 8 μmol/L for 72 h, which were consistent with inter-nucleosomal DNA fragmentation. Flow cytometric analysis showed a dose- and time-dependent increase in apoptotic rate by HSP70 antisense oligomers. This response was accompanied with a decrease in the percentage of cells in the G1 and S phases of the cell cycle, suggesting inhibition of cell proliferation. In addition, flow cytometry also showed that pretreatment of SGC-7901 cells with HSP70 antisense oligomers enhanced the subsequent apoptosis induced by heat shock treatment. Western blotting demonstrated that HSP70 antisense oligomers inhibited HSP70 expression, which preceded apoptosis, and HSP70 was undetectable at the concentration of 10 μmol/L for 48 h or 8 μmol/L for 72 h.CONCLUSION: Antisense HSP70 oligomers

  4. Characterization of the oligodeoxynucleotide-mediated inhibition of interferon-gamma-induced major histocompatibility complex class I and intercellular adhesion molecule-1.

    Science.gov (United States)

    Ramanathan, M; Lantz, M; MacGregor, R D; Garovoy, M R; Hunt, C A

    1994-10-01

    The major histocompatibility complex (MHC) Class I and II genes and intercellular adhesion molecule-1 (ICAM-1) are regulated by interferon-gamma in a variety of cell types. We have previously shown that the oligodeoxynucleotide 5'-GGG GTT GGT TGT GTT GGG TGT TGT GT-RNH2 (oligo I) inhibits the interferon-gamma-mediated enhancement of MHC Class I and ICAM-1 proteins in the K562 cell line. We have now investigated the mechanism of action of oligo I and report that it acts by inhibiting the binding of interferon-gamma to cells. We also show that the dose-response curves, the selectivity profile, and the kinetics of oligo I are consistent with this novel mechanism of action. The dose-response curves for oligo I, obtained using antibodies against the MHC Class I heavy chain, beta 2-microglobulin, or ICAM-1, are almost superimposable at each observation time. MHC Class I induction by 6400 units/ml interferon-alpha or interferon-beta or ICAM-1 enhancement by 800 units/ml tumor necrosis factor-alpha is not inhibited by oligo I. However, the synergistic induction of MHC Class I by mixtures of tumor necrosis factor-alpha and interferon-gamma is inhibited. Oligo I belongs to a class of active oligodeoxynucleotides that inhibits interferon-gamma-induced MHC Class I and ICAM-1 in K562 cells. The activity and potency is sequence-dependent, but remarkably different sequences can have comparable effects. The activity of oligo I in the HeLa S3 cell line inhibits the interferon-gamma-mediated enhancement of both ICAM-1 and MHC Class II DR and the interferon-gamma-mediated reduction in transferrin receptor expression. Thus, oligo I appears to specifically inhibit interferon-gamma-induced changes in protein expression, which is consistent with oligo I acting at an early step(s) in the induction process. Taken together, our results show that oligo I exerts its effects by inhibiting the association of interferon-gamma with the cell surface, which is a novel mechanism of action for

  5. BC047440 antisense eukaryotic expression vectors inhibited HepG2 cell proliferation and suppressed xenograft tumorigenicity

    International Nuclear Information System (INIS)

    The biological functions of the BC047440 gene highly expressed by hepatocellular carcinoma (HCC) are unknown. The objective of this study was to reconstruct antisense eukaryotic expression vectors of the gene for inhibiting HepG2 cell proliferation and suppressing their xenograft tumorigenicity. The full-length BC047440 cDNA was cloned from human primary HCC by RT-PCR. BC047440 gene fragments were ligated with pMD18-T simple vectors and subsequent pcDNA3.1(+) plasmids to construct the recombinant antisense eukaryotic vector pcDNA3.1(+)BC047440AS. The endogenous BC047440 mRNA abundance in target gene-transfected, vector-transfected and naive HepG2 cells was semiquantitatively analyzed by RT-PCR and cell proliferation was measured by the MTT assay. Cell cycle distribution and apoptosis were profiled by flow cytometry. The in vivo xenograft experiment was performed on nude mice to examine the effects of antisense vector on tumorigenicity. BC047440 cDNA fragments were reversely inserted into pcDNA3.1(+) plasmids. The antisense vector significantly reduced the endogenous BC047440 mRNA abundance by 41% in HepG2 cells and inhibited their proliferation in vitro (P < 0.01). More cells were arrested by the antisense vector at the G1 phase in an apoptosis-independent manner (P = 0.014). Additionally, transfection with pcDNA3.1(+) BC047440AS significantly reduced the xenograft tumorigenicity in nude mice. As a novel cell cycle regulator associated with HCC, the BC047440 gene was involved in cell proliferation in vitro and xenograft tumorigenicity in vivo through apoptosis-independent mechanisms

  6. Inhibition of luciferase expression in transgenic Aedes aegypti mosquitoes by Sindbis virus expression of antisense luciferase RNA.

    Science.gov (United States)

    Johnson, B W; Olson, K E; Allen-Miura, T; Rayms-Keller, A; Carlson, J O; Coates, C J; Jasinskiene, N; James, A A; Beaty, B J; Higgs, S

    1999-11-01

    A rapid and reproducible method of inhibiting the expression of specific genes in mosquitoes should further our understanding of gene function and may lead to the identification of mosquito genes that determine vector competence or are involved in pathogen transmission. We hypothesized that the virus expression system based on the mosquito-borne Alphavirus, Sindbis (Togaviridae), may efficiently transcribe effector RNAs that inhibit expression of a targeted mosquito gene. To test this hypothesis, germ-line-transformed Aedes aegypti that express luciferase (LUC) from the mosquito Apyrase promoter were intrathoracically inoculated with a double subgenomic Sindbis (dsSIN) virus TE/3'2J/anti-luc (Anti-luc) that transcribes RNA complementary to the 5' end of the LUC mRNA. LUC activity was monitored in mosquitoes infected with either Anti-luc or control dsSIN viruses expressing unrelated antisense RNAs. Mosquitoes infected with Anti-luc virus exhibited 90% reduction in LUC compared with uninfected and control dsSIN-infected mosquitoes at 5 and 9 days postinoculation. We demonstrate that a gene expressed from the mosquito genome can be inhibited by using an antisense strategy. The dsSIN antisense RNA expression system is an important tool for studying gene function in vivo. PMID:10557332

  7. Electroporation-based delivery of cell-penetrating peptide conjugates of peptide nucleic acids for antisense inhibition of intracellular bacteria.

    Science.gov (United States)

    Ma, Sai; Schroeder, Betsy; Sun, Chen; Loufakis, Despina Nelie; Cao, Zhenning; Sriranganathan, Nammalwar; Lu, Chang

    2014-10-01

    Cell penetrating peptides (CPPs) have been used for a myriad of cellular delivery applications and were recently explored for delivery of antisense agents such as peptide nucleic acids (PNAs) for bacterial inhibition. Although these molecular systems (i.e. CPP-PNAs) have shown ability to inhibit growth of bacterial cultures in vitro, they show limited effectiveness in killing encapsulated intracellular bacteria in mammalian cells such as macrophages, presumably due to difficulty involved in the endosomal escape of the reagents. In this report, we show that electroporation delivery dramatically increases the bioavailability of CPP-PNAs to kill Salmonella enterica serovar Typhimurium LT2 inside macrophages. Electroporation delivers the molecules without involving endocytosis and greatly increases the antisense effect. The decrease in the average number of Salmonella per macrophage under a 1200 V cm(-1) and 5 ms pulse was a factor of 9 higher than that without electroporation (in an experiment with a multiplicity of infection of 2 : 1). Our results suggest that electroporation is an effective approach for a wide range of applications involving CPP-based delivery. The microfluidic format will allow convenient functional screening and testing of PNA-based reagents for antisense applications.

  8. Adenovirus-mediated Expression of both Antisense Ornithine Decarboxylase and S-adenosylmethionine Decarboxylase Inhibits Lung Cancer Cell Growth

    Institute of Scientific and Technical Information of China (English)

    Hui TIAN; Xianxi LIU; Bing ZHANG; Qifeng SUN; Dongfeng SUN

    2007-01-01

    Polyamine biosynthesis is controlled primarily by ornithine decarboxylase (ODC) and Sadenosylmethionine decarboxylase (AdoMetDC). Antisense sequences of ODC and AdoMetDC genes were cloned into an adenoviral vector (named Ad-ODC-AdoMetDCas). To evaluate the effects of recombinant adenovirus Ad-ODC-AdoMetDCas that can simultaneously express both antisense ODC and AdoMetDC,the human lung cancer cell line A-549 was infected with Ad-ODC-AdoMetDCas or the control vector.Viable cell counting, determination of polyamine concentrations, cell cycle analysis, and Matrigel invasion assays were carried out to assess the properties of tumor growth and invasiveness. Our study showed that adenovirus-mediated antisense ODC and AdoMetDC expression inhibits tumor cell growth through blocking the polyamine synthesis pathway. Tumor cells were arrested at the G1 phase after gene transfer and the invasiveness was reduced. It suggested that the recombinant adenovirus Ad-ODC-AdoMetDCas might be a new anticancer reagent in the treatment of lung cancers.

  9. Inhibition of human immunodeficiency virus type 1 multiplication by antisense and sense RNA expression.

    OpenAIRE

    Joshi, S; Van Brunschot, A; Asad, S.; van der Elst, I; Read, S. E.; Bernstein, A

    1991-01-01

    Human immunodeficiency virus type 1 (HIV-1) primarily infects CD4+ lymphocytes and macrophages and causes AIDS in humans. Retroviral vectors allowing neomycin phosphotransferase (npt) gene expression were engineered to express 5' sequences of HIV-1 RNA in the antisense or sense orientation and used to transform the human CD4+ lymphocyte-derived MT4 cell line. Cells expressing antisense or sense RNA to the HIV-1 tat mRNA leader sequence, as part of the 3' untranslated region of the npt mRNA, r...

  10. Survivin antisense compound inhibits proliferation and promotes apoptosis in liver cancer cells

    Institute of Scientific and Technical Information of China (English)

    De-Jian Dai; Cai-De Lu; Ri-Yong Lai; Jun-Ming Guo; Hua Meng; Wei-Sheng Chen; Jun Gu

    2005-01-01

    AIM: To evaluate the effects of survivin on cell proliferation and apoptosis in liver cancer.METHODS: MTT assay was used to generate and optimize phosphorothioate antisense oligonucleotides (ODNs)LipofectamineTM2000 (LiP) compound by varying ODNs (μg):LiP (μL) ratios from 1:0.5 to 1:5. Then, liver cancer cells (HepG2) were transfected with the compound. By using RT-PCR and Western blot, the expression levels of survivin mRNA and proteins were detected in HepG2 cells treated with antisense compounds (ODNs:LiP = 1:4), and compared with those treated with sense compounds (1:4) as control.MTT assay was applied to the determination of cell proliferation in HepG2 cells. Active caspase-3 was evaluated by flow cytometric analysis. The morphological changes were assessed by electron microscopy. Laser scanning confocal microscopy was performed to detect the subcellular localization of survivin proteins in treated and untreated cells.RESULTS: Antisense compounds (1:4) down-regulated survivin expression (mRNA and protein) in a dose-dependent manner with an IC50 of 250 nmol/L. Its maximum effect was achieved at a concentration of 500 nmol/L, at whichmRNA and protein levels were down-regulated by 80%.The similar results were found in MTT assay. Antisense compound (1:4)-treated cells revealed increased caspase3-like protease activity compared with untreated cells.Untreated cells as control were primarily negative for the presence of active-caspase-3. As shown by transmission electron microscopy, treated cells with antisense compounds (1:4) resulted in morphological changes such as blebbing and loss of microvilli, vacuolization in the cytoplasm,condensation of the cytoplasm and nuclei, and fragmented chromatin. Immunofluorescence analysis confirmed the presence of survivin protein pool inside the cytoplasm in untreated cells. Labeled-FITC immunofluorescence staining of survivin clearly showed that survivin was distributed mainly in a spotted form inside the cytoplasm. Whereas

  11. Inhibition of lipoxygenase in lentil protoplasts by expression of antisense RNA

    NARCIS (Netherlands)

    Vliegenthart, J.F.G.; Maccarrone, M.; Hilbers, M.P.; Finazzi Agrò, A.

    1995-01-01

    A number of plasmids were constructed containing chimeric genes consisting of fragments of antisense-oriented lentil lipoxygenase cDNA. The different constructs were tested for their ability to lower lipoxygenase activity in lentil protoplasts. Plasmids containing a full length lentil lipoxygenase c

  12. The effects of integrin β1 antisense oligodeoxynucleotide on the expression of F-actin in ECV-304 cells exposed to infrasound%整合素β1反义寡核苷酸对次声诱导ECV-304细胞F-actin表达的影响

    Institute of Scientific and Technical Information of China (English)

    王冰水; 陈景藻; 郭国祯; 王斌; 袁华; 刘静; 陈丹

    2009-01-01

    Objective To study the effect of integrin β1 antisense oligodeoxynucleotide(ASODN)on the expression of F-actin in ECV-304 ceils exposed to infrasound,and to explore the pathway of infrasound signal transfer into the cells.Methods integrin β1 oligodeoxynucleotides were embedded in cationic liposome lipofectamine 2000 reagent and transfected into ECV-304 cells.Laser scanning confoeal microscopv was used to observe F-actin's expression when the cells were exposed to 16 Hz infrasound at 130 dB for 2 h.The cells' average fluorescence was examined using spectrofluorimetric quantification after immunofluorescent staining. Results Among the three false exposure groups,most fluorescein-labelled substances in the cells were in a diffuse state.There were few actin filaments,and they were tenuous and short, without direction.The cells' fluorescence intensities were almost the same.In a group exposed to infrasound,F-actin was thick and long with a longitudinal arrangement.Fluorescence intensities were strong.However,less F-actin expression was observed in the ceils of the ASODN group exposed to infrasound compared with the others similarly exposed.Correspondingly.F-actin expression in the ASODN group exposed to infrasound was almost the same as in the other exposed group.Conclusion F-actin expression can be increased in ECV-304 cells by infrasound exposure,but integrin β1 ASODN may partially inhibit its expression.The extraeellular matrix-integrin-cytoskeleton may be one of the transduetion pathways for infrasound signals into the cells.%目的 研究整合素β1反义寡核苷酸(ASODN)对次声诱导人脐血管内皮细胞(ECV-304)骨架微丝F-aetin表达的影响,探讨次声信号向细胞内传导的途径.方法 应用脂质体转染试剂介导整合素β1正义寡核苷酸(SODN)和ASODN转染ECV-304,并将细胞分为次声假暴露组、次声假暴露+ASODN组(转染整合紊β1反义寡核苷酸片断)、次声假暴露+SODN组(转染整合素β1正义寡核苷

  13. Short hairpin-loop-structured oligodeoxynucleotides reduce HSV-1 replication

    Directory of Open Access Journals (Sweden)

    Heinrich Jochen

    2009-04-01

    Full Text Available Abstract The Herpes simplex virus (HSV is known as an infectious agent and widespread in the human population. The symptoms of HSV infections can range from mild to life threatening, especially in immune-compromised individuals. HSV infections are commonly treated with the guanosine analogue Aciclovir, but reports of resistance are increasing. Efforts are made to establish single-stranded antisense oligodeoxynucleotides (as and small interfering ribonucleic acids (siRNAs for antiviral treatment. Recently, another class of short interfering nucleic acids, partially double-stranded hairpin loop-structured 54 mer oligodeoxynucleotides (ODNs, was shown to allow hydrolysis of HIV RNA by binding to the viral RNA. This leads to a substrate for the viral RNase H. To assess the potential of such ODNs for inhibition of HSV-1 replication, five partially double-stranded ODNs were designed based on the sequences of known siRNAs against HSV-1 with antiviral activity. Three of them are directed against early and two against leaky late genes. Primary human lung fibroblasts, MRC-5, and African green monkey kidney cells, Vero, were transfected with ODNs and subsequently infected. The effect on HSV-1 replication was determined by analyzing the virus titer in cell culture supernatants by quantitative PCR and plaque assays. An inhibitory effect was observed with all five selected ODNs, with two cases showing statistical significance in both cell types. The observed effect was sequence-specific and dose dependent. In one case the ODN was more efficient than a previously described siRNA directed against the same target site in the mRNA of UL5, a component of the helicase/primase complex. HSV-1 virions and ODNs can be applied simultaneously without transfection reagent, but at a 50-fold higher concentration to Vero cells with similar efficiencies. The results underline the potential of partially double-stranded hairpin loop-structured ODNs as antiviral agents.

  14. INHIBITION OF APOPTOSIS BY bcr-abl FUSION GENE IN K562 CELLS

    Institute of Scientific and Technical Information of China (English)

    WANG Chun-hong; SUN Bing-zhong; YUAN Yue-chuan

    1999-01-01

    Objective: To investigate the effect of bcr-abl fusion gene on CML cell apoptosis. Methods: Apoptosis of exvivo cultured K562 cells were observed after exposure to synthetic 18 mer antisense oligodeoxynucleotide complementary to the bcr-abl junction (b3a2). Results: Apoptosis of K562 cells was significantly increased associated with inhibition of bcr-abl expression. Conclusion: bcr-abl fusion gene formation due to chromosome translocation may be the major mechanism of CML via inhibition of apoptosis.

  15. Design, synthesis, and evaluation of mitomycin-tethered phosphorothioate oligodeoxynucleotides.

    Science.gov (United States)

    Huh, N; Rege, A A; Yoo, B; Kogan, T P; Kohn, H

    1996-01-01

    separate from 22. In the second procedure, phosphorothioate oligodexynucleotides that contained a hexylamino spacer at the 5'termini were coupled to 10-des(carbamoyloxy)-10-isothiocyanatoporfiromycin (9). Compound 9 was prepared in four steps from 11. Mesylation (methanesulfonyl chloride/pyridine) of 11 gave the C(10) mesylate 13, which was then treated with NaN3 (dimethylformamide, 90 degrees C) to give 10-des(carbamoyloxy)-10-azidoporfiromycin (14). Catalytic reduction (PtO2, H2) of 14 in pyridine afforded C(10) amine 15. Treatment of 15 with di-2-pyridyl thionocarbonate provided the desired 10-des(carbamoyloxy)-10-isothiocyanatoporfiromycin (9). Compound 9 readily coupled with 17 and base in both methylene chloride and aqueous buffered solutions to give 25. Use of the 5'hexylaminophosphorothioate oligodeoxynucleotides 32-35 in place of 17 gave the conjugated adducts 28-31, respectively, in a 12% to near-quantitative yield. The products were purified by semipreparative HPLC. Antisense agents 28-31 were designed to target a 30-base-long region from the coding region of the human FGFR1 gene. One adduct, 29, reduced the number of FGFR1 receptors in human aortic smooth cells for bFGF on the cell surface, which suggested down-regulation of FGFR1 gene expression. Further, 29 inhibited cultured human aortic smooth muscle cell proliferation and was less cytotoxic than porfiromycin (2). The biological assay data suggest that the phosphorothioate oligodexynucleotide porfiromycin conjugates may be more target selective and less toxic than either mitomycin or porfiromycin and thus be promising therapeutic agents. PMID:8950485

  16. Down-regulation of Survivin by Antisense Oligonucleotides Increases Apoptosis, Inhibits Cytokinesis and Anchorage-Independent Growth

    Directory of Open Access Journals (Sweden)

    Jun Chen

    2000-05-01

    Full Text Available Survivin, a member of the inhibitor of apoptosis protein (IAP family, is detected in most common human cancers but not in adjacent normal cells. Previous studies suggest that survivin associates with the mitotic spindle and directly inhibits caspase activity. To further investigate the function of survivin, we used a survivin antisense (AS oligonucleotide to downregulate survivin expression in normal and cancer cells. We found that inhibition of survivin expression increased apoptosis and polyploidy while decreasing colony formation in soft agar. Immunohistochemistry showed that cells without survivin can initiate the cleavage furrow and contractile ring, but cannot complete cytokinesis, thus resulting in multinucleated cells. These findings indicate that survivin plays important roles in a late stage of cytokinesis, as well as in apoptosis.

  17. Inhibition on the production of collagen type Ⅰ, Ⅲ of activated hepatic stellate cells by antisense TIMP-1 recombinant plasmid

    Institute of Scientific and Technical Information of China (English)

    Wen-Bin Liu; Chang-Qing Yang; Wei Jiang; Yi-Qing Wang; Jing-Sheng Guo; Bo-Ming He; Ji-Yao Wang

    2003-01-01

    AIM: To investigate the inhibition effects on the productionof collagen type I, Ⅲ secreted by activated rat hepatic stellatecells (rHSCs) by antisense tissue inhibitors of metalloproteinase1 (TIMP-1) recombinant plasmid through elevating interstitialcollagenase activity.METHODS: rHSCs were extracted from normal rat liverby pronase and collagenase digestion and purified bycentrifugal elutriation, and were cultured on plastic dishesuntil they were activated to a myofibroblastic phenotypeafter 7-10 days. RT-Nest-PCR and gene recombinanttechniques were used to construct the rat antisense TIMP-1 recombinant plasmids which can express in eucaryoticcells. The recombinant plasmid and the pcDNA3 emptyplasmid were transfected in rHSCs by Effectene (QIAGEN)separately. Cells were selected after growing in DMEMcontaining 400 μg/ml G418 for 2-3 weeks. Expression ofexogenous gene was assessed by Northern blot, andexpression oflIMP-1 in rHSCs was determined by Northernblot and Western blot. We tested the interstitial collagenaseactivity with FITC-labled type I collagen as substrate.Ultimately, we quantified the type Ⅰ, Ⅲ collagen byWestern blot.RESULTS: The exogenous antisense TIMP-1 recombinantplasmid could be expressed in rHSCs well, which couldblock the expression of TIMP-1 greatly, the ratio of TIMP-1/GAPDH was 0.67, 2.41, and 2.97 separately at mRNAlevel (P<0.05); the ratio of TIMP-1/β-actin was 0.31, 0.98and 1.32 separately at protein level (P<0.05); It mightelevate active and latent interstitial collagenase activity,the collagenase activity was 0.3049, 0.1411 and 0.1196respectively. (P<0.05), which led to promotion thedegradation of type Ⅰ, Ⅲ collagen, the ratio of collagen I/β-actin was 0.63, 1.78 and 1.92 separately (P<0.05); andthe ratio of collagen Ⅲ/β-actin was 0.59, 1.81 and 1.98separately (P<0.05).CONCLUSION: These data shows that the antisense TIMP-1 recombinant plasmid has the inhibitory effects on theproduction of type Ⅰ, Ⅲ collagens

  18. Oncolytic adenovirus-mediated transfer of the antisense chk2 selectively inhibits tumor growth in vitro and in vivo.

    Science.gov (United States)

    Chen, G; Zhou, J; Gao, Q; Huang, X; Li, K; Zhuang, L; Huang, M; Xu, G; Wang, S; Lu, Y; Ma, D

    2006-10-01

    Screening and identifying molecules target to checkpoint pathways has fostered the development of checkpoint-based anticancer strategies. Among these targets, inhibition of chk2 may induce cell death for tumors whose growth depends on enhanced chk2 activity. However, improvement of the potency and specificity of such therapeutics remains a major challenge. To resolve this problem, we constructed M3, a novel recombinant adenovirus with a 27-bp deletion in E1A CR2 region by which to realize tumor-specific replication, and an 829-bp of antisense chk2 fragment inserted into the E3 coding region. In this design, M3 exploited the native adenovirus E3 promoters to express antisense chk2 cDNA in a viral replication-dependent fashion, and preferentially silenced the chk2 gene in tumor cells. In vitro and in vivo assays confirmed that downregulated chk2 expression induced by M3 infection was tumor-specific and virus replication-dependent. Furthermore, systemic administration of M3 combined with a low dose of cisplatin cured 75% (9/12) of orthotopic hepatic carcinoma mouse models that were otherwise resistant to cisplatin. Our results indicated that the upcoming development in this field would improve the antitumor efficacy and maximize the synergistic effect of oncolytic viruses administered with traditional chemotherapy or radiotherapy. PMID:16741520

  19. Regulation of S-like ribonuclease levels in Arabidopsis. Antisense inhibition of RNS1 or RNS2 elevates anthocyanin accumulation

    Energy Technology Data Exchange (ETDEWEB)

    Bariola, P.A.; MacIntosh, G.C.; Green, P.J. [Michigan State Univ., East Lansing, MI (United States). Plant Research Lab.

    1999-01-01

    The S-like ribonucleases (RNases) RNS1 and RNS2 of Arabidopsis are members of the widespread T{sub 2} ribonuclease family, whose members also include the S-RNases, involved in gametophytic self-incompatibility in plants. Both RNS1 and RNS2 mRNAs have been shown previously to be induced by inorganic phosphate (Pi) starvation. In this study the authors examined this regulation at the protein level and determined the effects of diminishing RNS1 and RNS2 expression using antisense techniques. The Pi-starvation control of RNS1 and RNS2 was confirmed using antibodies specific for each protein. These specific antibodies also demonstrated that RNS1 is secreted, whereas RNS2 is intracellular. By introducing antisense constructs, mRNA accumulation was inhibited by up to 90% for RNS1 and up to 65% for NS2. These plants contained abnormally high levels of anthocyanins, the production of which is often associated with several forms of stress, including Pi starvation. This effect demonstrates that diminishing the amounts of either RNS1 or RNS2 leads to effects that cannot be compensated for by the actions of other RNases, even though Arabidopsis contains a large number of different RNase activities. These results, together with the differential localization of the proteins, imply that RNS1 and RNS2 have distinct functions in the plant.

  20. Inhibition of allergic airway inflammation by antisense-induced blockade of STAT6 expression

    Institute of Scientific and Technical Information of China (English)

    TIAN Xin-rui; TIAN Xin-li; BO Jian-ping; LI Shao-gang; LIU Zhuo-la; NIU Bo

    2011-01-01

    Background The signal transducer and activator of transcription 6 (STAT6) expression in lung epithelial cells plays a pivotal role in asthma pathogenesis. Activation of STAT6 expression results in T helper cell type 2 (Th2) cell differentiation leading to Th2-mediated IgE production, development of allergic airway inflammation and hyperreactivity. Therefore,antagonizing the expression and/or the function of STAT6 could be used as a mode of therapy for allergic airway inflammation.Methods In this study, we synthesized a 20-mer phosphorothioate antisense oligonucleotide (ASODN) overlapping the translation starting site of STAT6 and constructed STAT6 antisense RNA (pANTI-STAT6), then transfected them into murine spleen lymphocytes and analyzed the effects of antagonizing STAT6 function in vitro and in a murine model of asthma.Results In vitro, we showed suppression of STAT6 expression and interleukin (IL)-4 production of lymphocytes by STAT6 ASODN. This effect was more prominent when cells were cultured with pANTI-STAT6. In a murine model of asthma associated with allergic pulmonary inflammation in ovalbumin (OVA)-sensitized mice, local intranasal administration of fluorescein isothiocyanate (FITC)-labeled STAT6 ASODN to DNA uptake in lung cells was accompanied by a reduction of intracellular STAT6 expression. Such intrapulmonary blockade of STAT6 expression abrogated signs of lung inflammation, infiltration of eosinophils and Th2 cytokine production.Conclusion These data suggest a critical role of STAT6 in the pathogenesis of asthma and the use of local delivery of STAT6 ASODN as a novel approach for the treatment of allergic airway inflammation such as in asthma.

  1. Effects of Endothelin- 1 mRNA Antisense Oligodeoxynucleotide on the Uitrastructure of Myocardium in Diabetic Rats%内皮素-1反义脱氧寡核苷酸对糖尿病大鼠心肌超微结构的影响

    Institute of Scientific and Technical Information of China (English)

    崔岫; 李玉香; 张焱; 朱万平; 余建强; 袁文俊

    2011-01-01

    目的 研究内皮素-1反义脱氧寡核苷酸(ET -1 AS - ODN)对链脲佐菌素(streptozotocin,STZ)诱导的糖尿病大鼠心肌超微结构的影响.方法 一次性腹腔注射STZ(60mg.kg-1),建立大鼠糖尿病模型,并饲养5周后,造成糖尿病心肌损伤大鼠模型,ET -1 AS - ODN干预,采用BL - 420生物信号采集系统检测大鼠心功能,透射电镜观察大鼠心肌超微结构.结果 ET -1 AS - ODN各剂量组均可使LVEDP、+dp/dtmax、- dp/dtmax表现出一定的改善(P<0.05,P<0.01);电镜观察可见ET -1 AS - ODN各剂量组心肌细胞超微结构均明显改善,表现心肌细胞内肌节结构清晰、排列基本正常,线粒体增多、肿胀减轻、内嵴结构趋于清晰,排列整 齐.结论 内皮素-1反义寡核苷酸可减轻STZ诱导的糖尿病大鼠的心肌损伤,对心肌组织具有保护作用.%Objective To investigate effects of endothelin - lmRNA Antisense Oligodeoxynucleotide on the ultrastructure of myocardium in streptozotocin - induced diabetic rats. Methods Diabetic rats were induced by intraperitoneal administration of streptozotocin ( STZ) at 60mg ? Kg-1. Through 5 weeks , the diabetic myocardiopathy rats model was established successfully. ET - 1 AS - ODN was administrated to the intervention groups . Bio - tech system BL - 420 was employed to investigate the cardiac function and electroscope to observe the ultrastructure of myocardial cells of the rats Results The value of LVEDP、 + dp/dtmax、- dp/dt-max showed significantly different degree of improvements in ET - 1 AS - ODN intervention sub - groups ( P < 0.05,P<0.01) ; significantly improvements of the ultrastructure of myocardial cells in t rats were found among ET - 1 AS - ODN groups with different dosage under electroscope with neat arranged, clear structured of myocardial sarcomere, increased mitochondrial which was slight swollen but the neat structure of mitochondria cristae could be clearly seen. Conclusion Endothelin - lmRNA antisense

  2. Electroporation increases antitumoral efficacy of the bcl-2 antisense G3139 and chemotherapy in a human melanoma xenograft

    Directory of Open Access Journals (Sweden)

    Baldi Alfonso

    2011-07-01

    Full Text Available Abstract Background Nucleic acids designed to modulate the expression of target proteins remain a promising therapeutic strategy in several diseases, including cancer. However, clinical success is limited by the lack of efficient intracellular delivery. In this study we evaluated whether electroporation could increase the delivery of antisense oligodeoxynucleotides against bcl-2 (G3139 as well as the efficacy of combination chemotherapy in human melanoma xenografts. Methods Melanoma-bearing nude mice were treated i.v. with G3139 and/or cisplatin (DDP followed by the application of trains of electric pulses to tumors. Western blot, immunohistochemistry and real-time PCR were performed to analyze protein and mRNA expression. The effect of electroporation on muscles was determined by histology, while tumor apoptosis and the proliferation index were analyzed by immunohistochemistry. Antisense oligodeoxynucleotides tumor accumulation was measured by FACS and confocal microscopy. Results The G3139/Electroporation combined therapy produced a significant inhibition of tumor growth (TWI, more than 50% accompanied by a marked tumor re-growth delay (TRD, about 20 days. The efficacy of this treatment was due to the higher G3139 uptake in tumor cells which led to a marked down-regulation of bcl-2 protein expression. Moreover, the G3139/EP combination treatment resulted in an enhanced apoptotic index and a decreased proliferation rate of tumors. Finally, an increased tumor response was observed after treatment with the triple combination G3139/DDP/EP, showing a TWI of about 75% and TRD of 30 days. Conclusions These results demonstrate that electroporation is an effective strategy to improve the delivery of antisense oligodeoxynucleotides within tumor cells in vivo and it may be instrumental in optimizing the response of melanoma to chemotherapy. The high response rate observed in this study suggest to apply this strategy for the treatment of melanoma patients.

  3. Curcumin synergistically augments bcr/abl phosphorethieate antisense oligonucleotides to inhibit growth of chronic myelogenous leukemia cells

    Institute of Scientific and Technical Information of China (English)

    Kun-zhong ZHANG; Jian-hua XU; Xiu-wang HUANG; Li-xian WU; Yu SU; Yuan-zhong CHEN

    2007-01-01

    Aim: To investigate the growth inhibition effect of the combination of bcr/abl phosphorothioate antisense oligonucleotides (PS-ASODN) and curcumin (cur), and the possible mechanisms of cur on the chronic myelogenous leukemia cell line K562. Methods: The K562 cell line was used as a P210bcr/abl-positive cell model in vitro and was exposed to different concentrations of PS-ASODN (0-20 μmol/L), cur (0-20 μmol/L), or a combination of both. Growth inhibition and apoptosis of K562 cells were assessed by MTT assay and AO/EB fluorescent staining, respec-tively. The expression levels of P210bct/abl, NF-κB and heat shock protein 90 (Hsp90) were assessed by Western blot. Results: Exposure to cur (5-20 μmol/L) and PS-ASODN (5-20 μmol/L) resulted in a synergistic inhibitory effect on cell growth.Growth inhibition was associated with the inhibition of the proliferation and in-duction of apoptosis. Western blot analysis showed that the drugs synergisti-cally downregulated the level of P210bcr/abl and NF-κB. Cur downregulated Hsp90,whereas no synergism was observed when cur was combined with PS-ASODN.Conclusion: PS-ASODN and cur exhibited a synergistic inhibitory effect on the cell growth of K562. The synergistic growth inhibition was mediated through different mechanisms that involved the inhibition of P210bcr/abl.

  4. p44/p42 MAPK反义寡脱氧核苷酸对血管紧张素Ⅱ诱导的培养乳鼠心肌细胞肥大反应的抑制作用%Inhibitory effect of antisense oligodeoxynucleotide to p44/p42 MAPK on angiotensin Ⅱ-induced hypertrophic response in cultured neonatal rat cardiac myocyte

    Institute of Scientific and Technical Information of China (English)

    张世勤; 丁波; 郭兆贵; 李云霞

    2004-01-01

    AIM: To explore the inhibitory effect of antisense oligonucleotide (ODN) to mitogen activated protein kinase (MAPK) on cardiomyocyte hypertrophy induced by angiotensin Ⅱ (Ang Ⅱ). METHODS: A 17-mer phosphorothioateprotected antisense ODN directed against the initiation of translation sites of the p42 and p44 MAPK isoforms by liposomal transfection was applied to inhibit the translation of p44/p42 MAPK mRNA. The sense and random ODNs to p44/p42MAPK were used as sequence controls. Neonatal cardiac myocytes were exposed to Ang Ⅱ (10 nmol/L) for 5 min and then harvested in lysis buffer for the measurement of the activity and the phosphorylated protein content of p44/p42MAPK that were tested by P-81 phosphocellulose filter paper method and Western blotting, respectively. The rate of protein synthesis by [3H]leucine incorporation and the diameter of cell were measured after exposure to Ang Ⅱ for 24 h and 72 h, respectively. RESULTS: In cardiac myocyte Ang Ⅱ increased p44/p42MAPK activity and phosphorylated protein content by 140 % and 699 %, and also increased [3H]leucine incorporation and cell diameter by 40 % and 27 %. c-fos and c-myc mRNAs were induced significantly after exposure to Ang Ⅱ. Antisense ODN to p44/p42MAPK (0.2 μmol/L) reduced Ang Ⅱ-induced MAPK activity by 30 %,and phophorylated MAPK protein expression by 59 % in cardiac myocyte, and inhibited c-fos and c-myc mRNA expression induced by Ang Ⅱ by 44 % and 43 %, respectively. The diameter and the rate of protein synthesis of cardiac myocyte induced by Ang Ⅱ were decreased by 16 % and 22 % after pretreatment with antisense ODN to p44/p42MAPK. CONCLUSION: Antisense ODN to p44/p42 MAPK inhibited the increase of rate of protein synthesis,and the augmentation of cell diameter and expression of c-fos and c-nyc mRNA induced by Ang Ⅱ in cultured cardiac myocytes, p44/p42 MAPK played a critical role in the hypertrophic response induced by Ang Ⅱ in cultured neonatal rat cardiac myocytes.

  5. Bioresponsive antisense DNA gold nanobeacons as a hybrid in vivo theranostics platform for the inhibition of cancer cells and metastasis

    Science.gov (United States)

    Bao, Chenchen; Conde, João; Curtin, James; Artzi, Natalie; Tian, Furong; Cui, Daxiang

    2015-07-01

    Gold nanobeacons can be used as a powerful tool for cancer theranostics. Here, we proposed a nanomaterial platform based on gold nanobeacons to detect, target and inhibit the expression of a mutant Kras gene in an in vivo murine gastric cancer model. The conjugation of fluorescently-labeled antisense DNA hairpin oligonucleotides to the surface of gold nanoparticles enables using their localized surface plasmon resonance properties to directly track the delivery to the primary gastric tumor and to lung metastatic sites. The fluorescently labeled nanobeacons reports on the interaction with the target as the fluorescent Cy3 signal is quenched by the gold nanoparticle and only emit light following conjugation to the Kras target owing to reorganization and opening of the nanobeacons, thus increasing the distance between the dye and the quencher. The systemic administration of the anti-Kras nanobeacons resulted in approximately 60% tumor size reduction and a 90% reduction in tumor vascularization. More important, the inhibition of the Kras gene expression in gastric tumors prevents the occurrence of metastasis to lung (80% reduction), increasing mice survival in more than 85%. Our developed platform can be easily adjusted to hybridize with any specific target and provide facile diagnosis and treatment for neoplastic diseases.

  6. Conjugated agent insulin-antisense-c-myb-PS-ODN enhances the inhibitory effect on proliferation of rat aortic artery smooth muscle cells

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    AIM:Vascular smooth muscle cell (SMC) proliferation and migration from the arterial wall media into the intima are believed to play a critical role in the pathogenesis of restenosis. Several studies have demonstrated that phosphothioate (PS) oligodeoxynucleotides targeted against genes involved in SMC proliferation inhibits in vitro SMC proliferation and migration. However, the therapeutic effect of antisense ODN on the individual who receives the treatment of delivery of the agent depends on the efficacy of this agent in great degree. We investigated the inhibition effect of a novel agent, insulin-antisense-c-myb-PS-ODN on SMC proliferation in vitro. METHODS:The rat aortic artery SMCs were cultured in Dulbecco's modified Eagel's medium. The passage 8 to 13 were used as the experiment. Cell surface receptor binding assay was quantified through counting gamma particles emitted from 125    I labeled insulin. SMC rapid proliferation was brought by stimulation of high concentration of fetal bovine serum (FBS). The novel agent of insulin conjugated to the antisense-c-myb-PS-ODN was obtained via incubation of both in condition of certain reagents, pH, temperature, and ion concentration. The characterization and purification of the agent was performed through HPLC. Inhibition of SMC proliferation was reflected by incorporation rate of trillium labeled thymidine deoxyribonucleotide.RESULTS:The binding efficacy of insulin to the receptor was remarkably increased in SMC cultured in supplement of 20% FBS. The inhibition effect of conjugator insulin-c-myb-antisense-PS-ODN was stronger than that of the simple c-myb-antisense-PS-ODN. The inhibition rate of conjugator and simple form on SMC proliferation were 48.34% and 29.54%, respectively. CONCLUSION:The binding efficacy and specificity of c-myb-antisense-PS-ODN to SMC may be enhanced by the insulin receptor mediation through the insulin-insulin receptor interaction. The insulin-receptor targeted method may be a

  7. Suppression of intracranial glioma tumorigenesis with vascular endothelial growth factor antisense oligonucleotide in rats

    Institute of Scientific and Technical Information of China (English)

    李维方; 张光霁; 朱诚; 金由辛; 卢亦成

    2003-01-01

    Objective: To observe the inhibition of intracranial glioma tumorigenesis by vascular endothelial growth factor (VEGF) antisense oligodeoxynucleotide (ODN) in rats. Methods: Totally 20 μl Hank's liquid containing 1×106 C6 glioma cells was seeded into rat right caudate putamen in high-flow microinfusion with stereotactic technique. VEGF antisense ODN was simultaneously used with glioma cell. Each rat of the treated groupⅠ and the treated group Ⅱ was treated with 1 000 μmol/L VEGF antisense ODN. Each rat of the treated group Ⅲ and the treated group Ⅳ was treated with 2 000 μmol/L VEGF antisense ODN. The experimental periods of the treated group Ⅰ, the treated group Ⅲ and the control group Ⅰ were 2 weeks, those of the treated group Ⅱ, the treated group Ⅳ and the control group Ⅱ were 3 weeks. Before sacrifice, MRI was performed on each rat. Tumor magnitude and pathologic examination were detected after samples were dissected. Results: The survival state of all treated rats was better, and that of the control rats was in severe danger. The tumor volumes of the treated group Ⅰ and the treated group Ⅱ were remarkably lessened. Tumor tissue could not be found macroscopically in the brain samples of the treated group Ⅲ and the treated group Ⅳ, but tumor nest could be found with microscopy. Tumors of the treated groupⅠand the treated group Ⅱ had weak expressions of VEGF mRNA and VEGF, while normal brains and the samples of the treated group Ⅲ and the treated group Ⅳ had negative expressions, but tumors of the control groups had strong expressions. Conclusion: VEGF antisense ODN used early in situ can suppress angiogenesis and growth of rat intracranial glioma to retard tumorigenesis.

  8. CpG Oligodeoxynucleotide Inhibits Cockroach-Induced Asthma via Induction of IFN-γ+ Th1 Cells or Foxp3+ Regulatory T Cells in the Lung

    OpenAIRE

    Kim, Do-Hyun; Sohn, Jung-Ho; Park, Hong-Jai; Lee, Jae-Hyun; Park, Jung-Won; Choi, Je-Min

    2015-01-01

    Purpose CpG oligodeoxynucleotide (CpG-ODN), a TLR9 agonist, activates innate immunity and induces Th1 response. Although the immune modulatory effect of CpG-ODN has been extensively studied, its function in cockroach extract-induced allergic asthma has not been studied. Here, we investigated the inhibitory function of CpG-ODN in cockroach extract-induced asthma in mice with different treatment schemes. Methods Scheme 1: BALB/C mice were intra-nasally co-administered by cockroach extract and C...

  9. Inhibition of PCNA Antisense Oligonucleotides Mediated by Liposome on mRNA Expression and Proliferation of h-RPE Cells

    Institute of Scientific and Technical Information of China (English)

    CHEN Jianbin; XIANG Nan; XU Lili; ZENG Shuiqing

    2006-01-01

    The proliferating cell nuclear antigen (PCNA) gene expression was blocked and retinal pigment epithelium (RPE) proliferation was inhibited by using antisense oligonucleotides (AS-ODN)mediated by liposome, to find a new genetic therapy of proliferative vitreoretinopathy (PVR). RPE cells cultured in vitro were transfected with synthetic fluorescence labled AS-ODN mediated by liposome-Lipofectamine, and the intracellular distribution and persistence time of AS-ODN were dynamically observed. AS-ODN (0.07, 0.28 and 1.12 μ mol/L and sense oligonucleotides (S-ODN with the same concentrations as AS-ODN) mediated by liposome were delivered to the RPE cells cultured in vitro, and CPM values were measured by 3H-TdR incorporation assay and analyzed statistically by variance by comparison with blank control group.Expression of PCNA mRNA in RPE cells was detected by in situ hybridization after the treatment of different concentrations of PCNA AS-ODN and S-ODN, and the average optic density (AOD) was measured by image analysis system and was subjected to q-test and correlation analysis with CPM.Our results showed that AS-ODN mediated by liposome could quickly aggregate in cellular plasma and nuclei in 30 min and 6 h, and stayed for as long as 6 days. AS-ODN (0.28 and 1.12 μ mol/L) markedly suppressed proliferation of RPE cells in a dose-dependent manner with the difference being statistically significant (P<0.05 and P<0.01,repectively) as compared with blank control group. AOD was well correlated with CPM (r=0.975). It is concluded that liposome could increase transfection efficiency of AS-ODN in RPE cells, and AS-ODN could sequence-specifically suppress PCNA mRNA expression and proliferation of human RPE cells.

  10. Adenovirus-mediated expression of both antisense ODC and AdoMetDC inhibited colorectal cancer cell growth in vitro

    Institute of Scientific and Technical Information of China (English)

    Bing ZHANG; Xian-xi LIU; Yan ZHANG; Chun-ying JIANG; Qing-shan TENG; Hai-yan HU; Wei WANG; Lei GONG

    2006-01-01

    Aim: To construct a recombinant adenovirus that can simultaneously express both antisense ornithine decarboxylase (ODC) and adenosylmethionine decarboxylase (AdoMetDC) and detect its inhibitory effect on the intracellular polyamine pool and colorectal cancer cell growth. Methods: A 205-bp cDNA of AdoMetDC was reverse-inserted into recombinant pAdTrack-ODCas vectors and recombined with pAdEasy-1 vectors in AdEasy-1 cells. Positive clones were selected and transfected into the packaging cell HEK293 after they were linearized by Pad. Green fluorescent protein expression was used to monitor the process of adenovirus packaging. The ODC and AdoMetDC protein levels were identified by western blotting, and intracellular polyamine content was detected by reverse-phase high performance liquid chromatography. A viable cell count was used to determine the growth of HT-29 cells with or without exogenous polyamine. Results: Sequencing confirmed that AdoMetDC cDNA was successfully ligated into the pAdTrack-ODCas vector. GFP expression in 293 cells during virus packing and amplification was observed by fluorescence microscopy. Western blotting demonstrated that both ODC and AdoMetDC were downregulated by Ad-ODC-AdoMetDCas, and consequently 3 kinds of polyamine (putrescine, spermidine and spermine) were reduced to very low levels. HT-29 cell growth was significantly inhibited as compared with control conditions, and growth arrest was not reversed by exogenous putrescine. Conclusion: The successfully constructed recombinant adenovirus, Ad-ODC-AdoMetDCas, blocked polyamine synthesis and has therapeutic potential for treating colorectal cancer in vitro.

  11. Increased cell survival by inhibition of BRCA1 using an antisense approach in an estrogen responsive ovarian carcinoma cell line

    International Nuclear Information System (INIS)

    We tested the hypothesis that BRCA1 may play a role in the regulation of ovarian tumor cell death as well as the inhibition of ovarian cell proliferation. Introduction of BRCA1 antisense retroviral constructs into BG-1 estrogen-dependent ovarian adenocarcinoma cells resulted in reduced BRCA1 expression. BRCA1 antisense pooled populations and derived subclones were able to proliferate in monolayer culture without estrogen, whereas control cells began to die after 10 days of estrogen deprivation. In addition, both populations and subclones of BRCA1 antisense infected cells demonstrated a growth advantage in monolayer culture in the presence of estrogen and were able to proliferate in monolayer culture without estrogen, while control cells did not. Furthermore, clonal studies demonstrated that reduced levels of BRCA1 protein correlated with growth in soft agar and greater tumor formation in nude mice in the absence of estrogen. These data suggest that reduction of BRCA1 protein in BG-1 ovarian adenocarcinoma cells may have an effect on cell survival during estrogen deprivation both in vitro and in vivo. Germline mutations in the breast and ovarian cancer susceptibility gene BRCA1, which is located on chromosome 17q21, are associated with a predisposition to the development of cancer in these organs [1,2]. No mutations in the BRCA1 gene have been detected in sporadic breast cancer cases, but mutations have been detected in sporadic cases of ovarian cancer [3,4]. Although there is debate regarding the level of cancer risk associated with mutations in BRCA1 and the significance of the lack of mutations in sporadic tumors, it is possible that alterations in the function of BRCA1 may occur by mechanisms other than mutation, leading to an underestimation of risk when it is calculated solely on the basis of mutational analysis. Such alterations cannot be identified until the function and regulation of BRCA1 are better understood. The BRCA1 gene encodes a 220-kDa nuclear

  12. Translational inhibition of CTX M extended spectrum β-lactamase in clinical strains of Escherichia coli by synthetic antisense oligonucleotides partially restores sensitivity to cefotaxime.

    Directory of Open Access Journals (Sweden)

    John Benedict Readman

    2016-03-01

    Full Text Available Synthetic antisense oligomers are DNA mimics that can specifically inhibit gene expression at the translational level by ribosomal steric hindrance. They bind to their mRNA targets by Watson Crick base pairing and are resistant to degradation by both nucleases and proteases. A 25 mer phosphorodiamidate morpholino oligomer (PMO and a 13 mer polyamide (peptide nucleic acid (PNA were designed to target mRNA (positions -4 to +21, and –17 to –5 respectively close to the translational initiation site of the extended spectrum β lactamase resistance genes of CTX M group 1. These antisense oligonucleotides were found to inhibit β lactamase activity by up to 96% in a cell free translation transcription coupled system using an expression vector carrying a blaCTX-M-15 gene cloned from a clinical isolate. Despite evidence for up regulation of CTX-M gene expression, they were both found to significantly restore sensitivity to cefotaxime in E. coli AS19, an atypical cell wall permeable mutant, in a dose dependant manner (0 - 40 nM. The PMO and PNA were covalently bound to the cell penetrating peptide (KFF3K and both significantly (P<0.05 increased sensitivity to cefotaxime in a dose dependent manner (0 - 40 nM in field isolates harbouring CTX-M group 1 β-lactamases. Antisense oligonucleotides targeted to the translational initiation site and Shine Dalgarno region of blaCTX-M-15 inhibited gene expression, and when conjugated to a cell penetrating delivery vehicle, partially restored antibiotic sensitivity to both field and clinical isolates.

  13. Translational Inhibition of CTX-M Extended Spectrum β-Lactamase in Clinical Strains of Escherichia coli by Synthetic Antisense Oligonucleotides Partially Restores Sensitivity to Cefotaxime.

    Science.gov (United States)

    Readman, John B; Dickson, George; Coldham, Nick G

    2016-01-01

    Synthetic antisense oligomers are DNA mimics that can specifically inhibit gene expression at the translational level by ribosomal steric hindrance. They bind to their mRNA targets by Watson-Crick base pairing and are resistant to degradation by both nucleases and proteases. A 25-mer phosphorodiamidate morpholino oligomer (PMO) and a 13-mer polyamide (peptide) nucleic acid (PNA) were designed to target mRNA (positions -4 to +21, and -17 to -5, respectively) close to the translational initiation site of the extended-spectrum β-lactamase resistance genes of CTX-M group 1. These antisense oligonucleotides were found to inhibit β-lactamase activity by up to 96% in a cell-free translation-transcription coupled system using an expression vector carrying a bla CTX-M-15 gene cloned from a clinical isolate. Despite evidence for up-regulation of CTX-M gene expression, they were both found to significantly restore sensitivity to cefotaxime (CTX) in E. coli AS19, an atypical cell wall permeable mutant, in a dose dependant manner (0-40 nM). The PMO and PNA were covalently bound to the cell penetrating peptide (CPP; (KFF)3K) and both significantly (P < 0.05) increased sensitivity to CTX in a dose dependent manner (0-40 nM) in field and clinical isolates harboring CTX-M group 1 β-lactamases. Antisense oligonucleotides targeted to the translational initiation site and Shine-Dalgarno region of bla CTX-M-15 inhibited gene expression, and when conjugated to a cell penetrating delivery vehicle, partially restored antibiotic sensitivity to both field and clinical isolates. PMID:27047482

  14. Antisense RNA of Survivin Gene Inhibits the Proliferation of Leukemia Cells and Sensitizes Leukemia Cell Line to Taxol-induced Apoptosis

    Institute of Scientific and Technical Information of China (English)

    Wenhan LI; Xiaojuan WANG; Ping LEI; Qing YE; Huifen ZHU; Yue ZHANG; Jinfang SHAO; Jing YANG; Guanxin SHEN

    2008-01-01

    The effectS of survivin antisense RNA on proliferation of leukemia cell line HL-60 and taxol.induced chemotherapy was explorcd.A cDNA fragment of survivin obtained by RT-PCR was inserted into a plamid vector named pcDNA3 in the reverse direction.The vector encoding antisense RNA of survivin was confirmed by restriction enzyme digestion and DNA sequencing.The recombi-nant plasmid was delivered into HL-60 cells by electroporation.Growth curves were plotted based on cell counting.Trypan blue dye exclusion assay and MTT assay were carried out after the cells were incubated with taxol.DNA gel electrophoresis and nuclear staining were performed for cell apoptosis assay.The correct construction of the recombinant plasmid has been identificd bv restriction enzy.me digestion and DNA sequencing.A stable down.regulation has been achieved in HL-60 SVVas cells after G418 selection.Compared tO HL-60 cells.the proliferation of HL-60 SVVaS cells was signifi.cantly inhibited(P<0.05).Cytotoxicity assays indicated that IC50 of HL-60 SVVas for taxol was rela-tively lower than controls(P<0.01).Apoptosis assays revealed that taxol-induced apoptosis was de-tected in HL-60 sVVas cells incubated with 50 ng/ml taxol for 12 h,while in HL-60 cells incubated with 100 ng/ml taxol for 72 h.It was suggested that Survivin antisense RNA could inhibit the prolif-eration of HL-60 cells and enhance taxol-induced apoptosis in HL-60 cells.which may lay an ex-perimental foundation for further research on gene therapy in leukemia.

  15. Long-term stable expression of antisense cDNA of cyclin B1 profoundly inhibits the proliferation of tumor cells and suppresses tumorigenicity in implanted mice

    Institute of Scientific and Technical Information of China (English)

    ZHANG Tao; SU Xiao-mei; ZHANG Ling; LI Ji-cheng; WEI Dong; WEI Yu-quan; ZHANG Ru; CHENG Peng; CHEN Xian-cheng; LIU Huan-yi

    2008-01-01

    Background Cyclin B1 (CLB1) is necessary for mitotic initiation in mammalian cells and plays important roles in cancer development. Therefore, a potential strategy in cancer therapy is to suppress the activity of CLB1 by delivering antisense constructs of CLB1 into tumor cells. In previous CLB1 studies, antisense constructs with a short half life were often used and these constructs might not persistently inhibit CLB1.Methods We successfully created a recombinant plasmid encoding the full-length antisense cDNA of mouse cyclin B1 (AS-mCLB1) and transfected this construct to the murine Lewis lung carcinoma (LL/2) and CT-26 colon carcinoma (CT-26) cells. We isolated clones of LL/2 and CT-26 transfectants with stable expression of AS-mGLB1. Reverse transcriptional polymerase chain reaction (RT-PCR) and Western blot were applied to detect the expression of the mRNA and protein levels of CLB1. To further test the efficacy of this strategy in vivo, AS-mCLBl-expressing LL/2 and CT-26 transfectants were implanted into mice.Results We found the expression of the mRNA and protein levels of CLB1 decrease in these trensfectants. The inhibition of CLB1 caused prominent G1 arrest, abnormal morphology, retarded cell growth and an increase in apoptosis. In AS-mCLB1-expressing LL/2 and CT-26 transfectants implanted mice, tumorigenicity was effectively suppressed compared with the controls. In addition, the expression of AS-mCLB1 also significantly increases the survival duration of implanted animals.Conclusion AS-mCLB1 is likely to be useful in future cancer therapy, which may be associated with its ability to down-regulate the expression of CLB1 and then induce G1 arrest and apoptosis in tumor cells.

  16. Preparation of oligodeoxynucleotide encapsulated cationic liposomes and release study with models of cellular membranes

    OpenAIRE

    Tamaddon AM.; Hosseini-Shirazi F.; Moghimi HR

    2007-01-01

    Cationic liposomes are used for cellular delivery of antisense oligodeoxynucleotide (AsODN), where release of encapsulated AsODN is mainly controlled by endocytosis and fusion mechanisms. In this investigation, it was tried to model such a release process that is difficult to evaluate in cell culture. For this purpose, an AsODN model (against protein kinase C-α) was encapsulated in a DODAP-containing cationic liposome and evaluated for size, zeta-potential, encapsulation and ODN stab...

  17. hPOT1 antisense-nucieic acids inhibit the proliferation of gastric cancer cell line SGC7901

    Institute of Scientific and Technical Information of China (English)

    帖君

    2006-01-01

    Objective To investigate the effect of hPOT1 (human protection of telomeres,hPOT1) on the growth and proliferation of human gastric cancer cell line SGC7901. Methods The constructed sense and antisense hPOT1 gene eukaryotic expressing vectors were transfected into SGC7901 cells respectively, and positive clones were selected by G418. Changes of hPOT1 protein expression,

  18. Effect of a antisense oligonucleotide to noggin on the expression of nestin and GFAP in the hippocampus of adult rats%反义Noggin基因对成年大鼠海马内Nestin及GFAP表达的影响

    Institute of Scientific and Technical Information of China (English)

    徐海伟; 范晓棠

    2005-01-01

    目的探讨Noggin基因对成年大鼠海马内Nestin及GFAP表达的影响.方法反义寡核苷酸技术封闭内源性Noggin基因的表达,免疫组化法检测成年大鼠海马内Nestin与GFAP的表达.结果侧脑室连续4 d注射Noggin基因的反义寡核苷酸后,可见海马齿状回(dentate gyrus,DG)内Nestin阳性细胞数与GFAP阳性细胞数较对照组显著增加;室下区GFAP阳性细胞数亦明显增加.结论Noggin对成年海马干细胞的分化有重要作用,内源性Noggin基因的表达可使神经干细胞向神经元方向分化.%Objective To examine the role of noggin on the expression of nestin and glial fibrillary acidic protein (GFAP) in the hippocampus of adult rats. Methods Antisense oligodeoxynucleotide (ASODN) technique was employed to inhibit endogenous noggin expression and immunohistochemistry was used to detect the expressions of Nestin and GFAP in the hippocampus of adult rats. Results It was observed that the number of nestin and GFAP immunoreactive cells in the dentate gyrus (DG) of hippocampus was increased in adult rats treated with antisense oligodeoxynucleotide to noggin. Moreover, the number of GFAP immunoreactive cells was increased in the subventricular zone of the rats treated with antisense oligodeoxynucleotide to noggin. Conclusion The results in the present study indicates that noggin may play a role in the differentiation of neural stem cells in the adult hippocampus, and it promotes the differentiation of neural stem cells in the DG to neuronal fate.

  19. Antisense to the glucocorticoid receptor in hippocampal dentate gyrus reduces immobility in forced swim test

    NARCIS (Netherlands)

    Korte, S.M.; de Kloet, E.R.; Buwalda, B; Bouman, S.D.; Bohus, B

    1996-01-01

    Immobility time of rats in the forced swim test was reduced after bilateral infusion of an 18-mer antisense phosphorothioate oligodeoxynucleotide targeted to the glucocorticoid receptor mRNA into the dentate gyrus of the hippocampus. Vehicle-, sense- and scrambled sequence-treated animals spent sign

  20. 22. Proteomic Analysis of Differential Protein Expression in vero Cell with Antisense Blocking of Relevant Gene Involved in inhibition of Nontargeted Mutagenesis

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective: Recent studies have demonstrated that cells exposed to ionizing radiation or alkylating agents can develop prolonged genetic instability. But its mechanism is still unknown. A cDNA fragment (fragment 9) has been isolated in MNNG-exposed vero cell by mRNA differential display in this lab. After antisense blocking the expression of its relevant gene (fragment 9 related gene, FNR gene), we found that nontargeted mutation frequency induced by MNNG was enhanced significantly. which implicated that the product of the blocked gene may be involved in the inhibition of nontargeted mutation. In order to elucidate the functional mechanism of the FNR gene, we try to separate the proteins from the established cell line expressing antisense fragment 9 to find out the FNR gene-coded protein. Method: The total cellular proteins of MNNG-exposed vero cell transfected with antisense RNA expression plasmid (vero-pM-amp--9-) and those with vector DNA (vero-pM-amp-) were separated by two-dimensional gel electrophoresis, and the resulting maps were analyzed with 2-D analysis software packages to find out the differentially expressed protein spots. Then the related 2-D PAGE database (http://biobase.dk/cgi-bin/celis/) was searched according to the protein spots information obtained from 2-DE including the position in the gel, isoelectric point (pl) and molecular weight (Mr). Result: Twelve proteins were specifically expressed only in vero-pM-amp-, and 2 proteins in vero-pM-amp--9-. In addition, there were 24 proteins expressed in higher level in vero-pM-amp--9- as compared with vero-pM-amp- (P<0.05), among them the expression of 7 proteins were enhanced by greater than 5 folds. On the other hand, no sequence similarity was found by homology analysis in GenBank through comparing the fragment 9 with the cDNA sequences of those proteins found in this study. Conclusion: Gene expression alterations bave occurred after antisense blocking of the FNR gene expression as demonstrated by

  1. Anti-sense RNA Inhibits the Expression of Synaptotagmin Ⅱ in RBL-2H3 and Enhances the Exocytosis of Lysosomes in RBL-2H3

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    The expression of synaptotagmin Ⅱ (Syt2) in RBL-2H3 (RBL) and its role during exocytosis of RBL was investigated. The expression of Syt2 in RBL was detected by western blot and Syt2 gene was amplified by PCR. The anti-sense full length Syt2 cDNA expression vector was constructed with pEGFP-N1 and transfected into RBL by electroporation, and stable transfectants were selected by using G418. To analyze the role of Syt2 during exocytosis of RBL, the release of cathepsin D was assayed by immunoblotting. The results showed that Syt2 was expressed in RBL.The anti-sense expression vector pEGFP-N1-Syt2-AS was constructed and the sequence of insertion was completely consistent with rat Syt2 (accession number in GeneBank: NM012665). The stable transfectants (RBL-Syt2-AS) were obtained. Western blot showed that RBL-Syt2-AS expressed a lower level of Syt2 (8 % and 10 % of control cells), indicating that the expression of Syt2 in RBLSyt2-AS was markedly down-regulated by anti-RNA. Compared with control, the release of cathepsin D by RBL-Syt2-AS was increased. It was concluded that Syt2 expressed in RBL and could inhibit exocytosis of lysosomes in RBL.

  2. EFFECT OF TWO NEW BCL-2 ANTISENSES ON DRUG-SENSITIVITY OF CELLS FROMN LEUKEMIA PATIENTS

    Institute of Scientific and Technical Information of China (English)

    LEI Xiao-yong; ZHANG Huan

    2005-01-01

    Objective:To investigate the effect of two antisense oligonucleotides on cell surviving, bcl-2 expression and apoptosis of leukemia cells. Methods: The experimental assays were performed with cell culture, immunochemistry and flowcytometry. Results: The two antisense oligodeoxynucleotides, combined with Vp16 or Ara-c or DNR, were able to decline the survival rate of myeleukemic cells, downregulate bcl-2 gene expression and induce apoptosis of leukemic cells significantly, as compared with Vp16 or Ara-c or DNR alone. Conclusion: It is possible for the two new bcl-2 antisenses to be developed into clinical trials for leukemia and tumor with bcl-2 gene overexpression.

  3. Combination of telomerase antisense oligonucleotides simultaneously targeting hTR and hTERT produces synergism of inhibition of telomerase activity and growth in human colon cancer cell line

    OpenAIRE

    FU, XIAO-HUA; Zhang, Jian-Song; Zhang, Na; Zhang, Yang-de

    2005-01-01

    AIM: To investigate synergism of inhibition of telomerase activity and proliferation of human colon cancer cells by combination of telomerase antisense oligonucleotides (ASODNs) simultaneously targeting human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT) in vitro.

  4. Antisense Phosphorodiamidate Morpholino Oligomer Length and Target Position Effects on Gene-Specific Inhibition in Escherichia coli

    OpenAIRE

    Deere, Jesse; Iversen, Pat; Geller, Bruce L.

    2005-01-01

    Phosphorodiamidate morpholino oligomers (PMOs) are synthetic DNA analogs that inhibit gene expression in a sequence-dependent manner. PMOs of various lengths (7 to 20 bases) were tested for inhibition of luciferase expression in Escherichia coli. Shorter PMOs generally inhibited luciferase greater than longer PMOs. Conversely, in bacterial cell-free protein synthesis reactions, longer PMOs inhibited equally or more than shorter PMOs. Overlapping, isometric (10-base) PMOs complementary to the ...

  5. Studies of Liposomal bcl-2 Antisense Oligode-oxynucleofide Induction of Apoptosis in Raji Cells

    Institute of Scientific and Technical Information of China (English)

    DongmeiHe; HuanZhong

    2004-01-01

    OBJECTIVE To explore the effect of liposomal G3139 and transfected antisense phosphorothioate oligodeoxynucleotides directed against the coding region of the bcl-2 messenger RNA and the translation site on apoptosis in Raji cells.METHODS Cytotoxic effects were measured by use of the MTT method; The expression levels of Bcl-2 protein were assayed by immunofiuorescence using a fluoresce isothiocyanate label. Apoptosis was determined by morphological observation and flow cytometric analysis.RESULTS The 2 antisense oligonucleotides and G3139 can reduce Bcl-2 protein levels and Raji cell viability (IC50=4.54, 4.72 and 4.26 μmol/L, respectively), and induce apoptosis. A scrambled sequence control oligonucleotide and empty liposomes did not alter cell viability, Bcl-2 protein expression or apoptosis rates. There was no difference in reducing Bcl-2 protein levels and apoptosis rates found among the 3 antisense oligonucleotides.CONCLUSION The 2 antisense oligodeoxynucleotides of bcl-2 messenger RNA can effectively induce apoptosis of Raji cells. The 2 antisense sequences and G3139 have a similarity in their antisense effect.

  6. Inhibition of the intrinsic coagulation pathway factor XI by antisense oligonucleotides: a novel antithrombotic strategy with lowered bleeding risk

    NARCIS (Netherlands)

    H. Zhang; E.C. Löwenberg; J.R. Crosby; A.R. Macleod; C. Zhao; D. Gao; C. Black; A.S. Revenko; J.C.M. Meijers; E.S. Stroes; M. Levi; B.P. Monia

    2010-01-01

    Existing anticoagulants effectively inhibit the activity of coagulation factors of the extrinsic and common pathway but have substantial limitations and can cause severe bleeding complications. Here we describe a novel therapeutic approach to thrombosis treatment. We have developed and characterized

  7. Inhibition of tumor growth and metastasis with antisense oligonucleotides(Cantide) targeting hTERT in an in situ human hepatocellular carcinoma model

    Institute of Scientific and Technical Information of China (English)

    Ru-xian LIN; Chao-wei TUO; Qiu-jun L(u); Wei ZHANG; Sheng-qi WANG

    2005-01-01

    Aim: To evaluate the in vivo antitumor effects of Cantide and the combined effect with 5-fluorouracil. Methods: An in situ human hepatocellular carcinoma model was established in mice livers orthotopically. Drugs were administered intravenously and tumor sizes were monitored with calipers. Plasma alpha-fetoprotein (AFP) were detected by radiation immunoassay. Morphology of tumors was evaluated by hematoxylin-eosin (H&E) staining of histological sections. Human telomerase reverse transcriptase (hTERT) protein levels were detected by Western blotting. Results: Cantide significantly inhibit in situ human hepatocellular compared to the saline group in a dose-dependent manner, which included injectthe tumor in liver. Cantide was also found to prevent tumor recurrence in the liver and metastasis in the lung, showing a dose-dependent response. When Cantide was administered by iv combined with 5-fluorouracil, it resulted in a significant reduction in tumor growth compared to either agent alone treatment group. After the treatment with Cantide alone or combined with 5-fluorouracil, plasma AFP concentration decreased in a dose-dependent manner. Conclusion: These results demonstrated that Cantide was an effective antitumor antisense oligonucleotide in vivo and has the potential to be developed into a clinical anti-cancer drug.

  8. Prostate Specific Antigen Promoter-Driven Adenovirus-Mediated Expression of Both ODC and AdoMetDC Antisenses Inhibit Prostate Cancer Growth

    Institute of Scientific and Technical Information of China (English)

    Wei Li; Hui Xiong; Yi-lin Hong; Chun-hua Zhang; Chang-chun Liu

    2010-01-01

    Objective:To generate recombinant adenovirus that could simultaneously express ornithine decarboxylase(ODC)and S-adenosylmethionine decarboxylase(AdoMetDC)antisenses specifically in prostate cancer cells,and evaluate its inhibitory effect on prostate cancer in vivo.Methods:Fragments of ODC and AdoMetDC genes were generated by PCR,cloned into the pPGL-PSES,and then recombined with pAdEasy-1 vectors in AdEasy-1 cells.Ad-PSES-ODC-AdoMetDCas virus was produced in HEK293 cells.Following transfection with Ad-PSES-ODC-AdoMetDCas,the levels of ODC or AdoMetDC were determined by RT-PCR and western blot assays.The effect of Ad-PSES-ODC-AdoMetDCas treatment on tumor formation and growth was evaluated in xenograft models of prostate cancers in vivo.Results:The plasmid pAdEasy-PSES-ODC-AdoMetDCas was successfully constructed and the recombinant Ad-PSES-ODC-AdoMetDCas adenovirus was produced.Transfection with Ad-PSES-ODC-AdoMetDCas adenovirus significantly inhibited the expression of ODC and AdoMetDC genes specifically in prostate DU145cells,but not H1299,HT29 and HepG2 cancer cells,and disrupted the ability of DU145 cells to form solid prostate cancer in vivo.Intratumoral treatment with Ad-PSES-ODC-AdoMetDCas adenovirus significantly inhibited the growth of engrafted prostate tumors in vivo.Conclusion:The recombinant Ad-PSES-ODC-AdoMetDCas adenovirus specifically reduces the expression of both ODC and AdoMetDC genes in prostate cells and may be used for treatment of prostate cancers at the clinic.

  9. Prostate Specific Antigen Promoter-Driven Adenovirus-Mediated Expression of Both ODC and AdoMetDC Antisenses Inhibit Prostate Cancer Growth

    Institute of Scientific and Technical Information of China (English)

    Wei Li; Hui Xiong; Yi-lin Hong; Chun-hua Zhang; Chang-chun Liu

    2011-01-01

    Objective: To generate recombinant adenovirus that could simultaneously express ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase(AdoMetDC) antisenses specifically in prostate cancer cells,and evaluate its inhibitory effect on prostate cancer in vivo.Methods: Fragments of ODC and AdoMetDC genes were generated by PCR,cloned into the pPGL-PSES,and then recombined with pAdEasy-1 vectors in AdEasy-1 cells.Ad-PSES-ODC-AdoMetDCas virus was produced in HEK293 cells.Following transfection with Ad-PSES-ODC-AdoMetDCas,the levels of ODC or AdoMetDC were determined by RT-PCR and western blot assays.The effect of Ad-PSES-ODC-AdoMetDCas treatment on tumor formation and growth was evaluated in xenograft models of prostate cancers in vivo.Results: The plasmid pAdEasy-PSES-ODC-AdoMetDCas was successfully constructed and the recombinant Ad-PSES-ODC-AdoMetDCas adenovirus was produced.Transfection with Ad-PSES-ODC-AdoMetDCasadenovirus significantly inhibited the expression of ODC and AdoMetDC genes specifically in prostate DU145 cells,but not H1299,HT29 and HepG2 cancer cells,and disrupted the ability of DU145 cells to form solid prostate cancer in vivo.Intratumoral treatment with Ad-PSES-ODC-AdoMetDCas adenovirus significantly inhibited the growth of engrafted prostate tumors in vivo.both ODC and AdoMetDC genes in prostate cells and may be used for treatment of prostate cancers at the clinic.

  10. NCYM, a Cis-antisense gene of MYCN, encodes a de novo evolved protein that inhibits GSK3β resulting in the stabilization of MYCN in human neuroblastomas.

    Directory of Open Access Journals (Sweden)

    Yusuke Suenaga

    2014-01-01

    Full Text Available The rearrangement of pre-existing genes has long been thought of as the major mode of new gene generation. Recently, de novo gene birth from non-genic DNA was found to be an alternative mechanism to generate novel protein-coding genes. However, its functional role in human disease remains largely unknown. Here we show that NCYM, a cis-antisense gene of the MYCN oncogene, initially thought to be a large non-coding RNA, encodes a de novo evolved protein regulating the pathogenesis of human cancers, particularly neuroblastoma. The NCYM gene is evolutionally conserved only in the taxonomic group containing humans and chimpanzees. In primary human neuroblastomas, NCYM is 100% co-amplified and co-expressed with MYCN, and NCYM mRNA expression is associated with poor clinical outcome. MYCN directly transactivates both NCYM and MYCN mRNA, whereas NCYM stabilizes MYCN protein by inhibiting the activity of GSK3β, a kinase that promotes MYCN degradation. In contrast to MYCN transgenic mice, neuroblastomas in MYCN/NCYM double transgenic mice were frequently accompanied by distant metastases, behavior reminiscent of human neuroblastomas with MYCN amplification. The NCYM protein also interacts with GSK3β, thereby stabilizing the MYCN protein in the tumors of the MYCN/NCYM double transgenic mice. Thus, these results suggest that GSK3β inhibition by NCYM stabilizes the MYCN protein both in vitro and in vivo. Furthermore, the survival of MYCN transgenic mice bearing neuroblastoma was improved by treatment with NVP-BEZ235, a dual PI3K/mTOR inhibitor shown to destabilize MYCN via GSK3β activation. In contrast, tumors caused in MYCN/NCYM double transgenic mice showed chemo-resistance to the drug. Collectively, our results show that NCYM is the first de novo evolved protein known to act as an oncopromoting factor in human cancer, and suggest that de novo evolved proteins may functionally characterize human disease.

  11. Progress toward therapy with antisense-mediated splicing modulation

    OpenAIRE

    Du, Liutao; Gatti, Richard A.

    2009-01-01

    Antisense oligonucleotides (AO) or antisense RNA can complementarily bind to a target site in pre-mRNA and regulate gene splicing, either to restore gene function by reprogramming gene splicing or to inhibit gene expression by disrupting splicing. These two applications represent novel therapeutic strategies for several types of diseases such as genetic disorders, cancers and infectious diseases. In this review, the recent developments and applications of antisense-mediated splicing modulatio...

  12. Antisense Therapy in Neurology

    OpenAIRE

    Lee, Joshua J.A.; Toshifumi Yokota

    2013-01-01

    Antisense therapy is an approach to fighting diseases using short DNA-like molecules called antisense oligonucleotides. Recently, antisense therapy has emerged as an exciting and promising strategy for the treatment of various neurodegenerative and neuromuscular disorders. Previous and ongoing pre-clinical and clinical trials have provided encouraging early results. Spinal muscular atrophy (SMA), Huntington’s disease (HD), amyotrophic lateral sclerosis (ALS), Duchenne muscular dystrophy (DMD)...

  13. Antisense approaches in prostate cancer.

    Science.gov (United States)

    Chi, Kim N; Gleave, Martin E

    2004-06-01

    Patients with hormone refractory prostate cancer have limited treatment options and new therapies are urgently needed. Advances in the understanding of the molecular mechanisms implicated in prostate cancer progression have identified many potential therapeutic gene targets that are involved in apoptosis, growth factors, cell signalling and the androgen receptor (AR). Antisense oligonucleotides are short sequences of synthetic modified DNA that are designed to be complimentary to a selected gene's mRNA and thereby specifically inhibit expression of that gene. The antisense approach continues to hold promise as a therapeutic modality to target genes involved in cancer progression, especially those in which the gene products are not amenable to small molecule inhibition or antibodies. The current status and future direction of a number of antisense oligonucleotides targeting several genes, including BCL-2, BCL-XL, clusterin, the inhibitors of apoptosis (IAP) family, MDM2, protein kinase C-alpha, c-raf, insulin-like growth factor binding proteins and the AR, that have potential clinical use in prostate cancer are reviewed. PMID:15174974

  14. Inhibition of metastasis to lung of a human nasopharyngeal carcinoma cell line CNE-2L2 transfected with pRc/CMV-antisense 6A8 cDNA in nude mice

    Institute of Scientific and Technical Information of China (English)

    张立新; 刘玉琴; 马凤蓉; 顾蓓; 史耕先; 赵雪梅; 李波; 高进; 赵方萄; 张淑珍; 李国燕; 王讯; 朱立平

    1999-01-01

    The growth of CNE-2L2 cell, a cloned line of human nasopharyngeal carcinoma with a high potentiality of metastasis to lung was inhibited to a certain extent after transfection with a recombinant antisense expression vector of a cDNA encoding a human α-mannosidase (pRc/CMV-antisense 6A8 cDNA)( the Genbank accession number of 6A8 cDNA is U37248) in comparison with that of the cell transfected with the Mock and of the wild cell. Two months after a subcutaneous inoculation of CNE-2L2 cell into the axilla of nude mice metastatic lesions in the lung were observed in 9/10 mice (90%) with grade Ⅲ in 8 mice and grade Ⅱ in one mouse in the wild cell group, in 6/8 mice (75%) with grade Ⅲ in one mouse, grade Ⅱ in 2 mice and grade Ⅰ in 3 mice in the Mock-transfection group, in only 3/10 mice (30%) with all grade Ⅰ in pRc/CMV-antisense 6A8 cDNA-transfection group.

  15. Inhibiting the growth of methicillin-resistant Staphylococcus aureus in vitro with antisense peptide nucleic acid conjugates targeting the ftsZ gene

    Directory of Open Access Journals (Sweden)

    Shumei Liang

    2015-01-01

    Conclusion: Our results demonstrate that the potent effects of PNAs on bacterial growth and cell viability were mediated by the down-regulation or even knock-out of ftsZ gene expression. This highlights the utility of ftsZ as a promising target for the development of new antisense antibacterial agents to treat MRSA infections.

  16. Development of Antisense Therapeutic and Imaging Agents to Detect and Suppress Inducible Nitric Oxide Synthase (iNOS) Expression in Acute Lung Injury (ALI)

    Science.gov (United States)

    Shen, Yuefei

    antisense PNA-YR9·oligodeoxynucleotide (ODN) hybrid and a cationic shell-crosslinked knedel-like nanoparticle (cSCK) to specifically target and image iNOS mRNA toward the diagnosis of ALI. The Y (tyrosine) residue was used for 123I radiolabeling while the R9 (arginine9) peptide was used to facilitate endosomal, lysosomal, and cellular escape of untargeted PNA probe. Complete binding of the antisense PNA-YR9·ODN hybrid to the cSCK was achieved at an 8:1 cSCK amine to ODN phosphate (N/P) ratio. The antisense PNA-YR9·ODN*cSCK nanocomplexes efficiently entered RAW 264.7 cells, while the PNA-YR9 ·ODN alone was not taken up. Low concentrations of 123 I-labeled antisense PNA-YR9·ODN complexed with cSCK, showed significantly higher retention of radioactivity in iNOS-induced RAW 264.7 cells when compared to a mismatched PNA. The fourth effort was the study of a degradable polyphosphoester-based cationic nanoparticle (dg-cSCK), which itself demonstrated efficient iNOS inhibition without further loading of any other therapeutic drugs. It appeared that spontaneous hydrolytic degradation and/or assisted by enzymes caused the particle to quickly release degraded small fragments. One of the expected degradation products showed dose-dependent iNOS inhibition, and might serve as a novel inhibitor that could explain the behavior of dg-cSCK. Dg-cSCK showed much more efficient iNOS inhibition than the degradation product, probably due to higher cellular uptake on the nanoparticle precursor than the degradation product. Dg-cSCK also led to the decrease of iNOS mRNA level, suggesting that the inhibition might be taking place upstream of iNOS. (Abstract shortened by UMI.)

  17. 反义寡核苷酸沉默乙酰肝素酶对食管鳞癌细胞株TE-13侵袭能力的影响%Inhibitory Effect of Silencing Heparanase Expression with Antisense Oligodeoxynucleotide on Invasive Capability of Human Esophageal Squamous Cancer Cell Line TE-13

    Institute of Scientific and Technical Information of China (English)

    朱辉; 王士杰; 王顺祥; 孟宪利; 王永军

    2005-01-01

    背景与目的:乙酰肝素酶(heparanase,Hpa)为降解硫酸乙酰肝素多糖的内糖苷酶,在多种恶性肿瘤的侵袭转移中发挥重要作用.本研究旨在探讨Hpa在食管鳞癌细胞株TE-13中的表达和转染Hpa反义寡核苷酸(antisense oligodeoxynucleotide,ASODN)对TE-13细胞的影响.方法:脂质体法转染人工合成的Hpa ASODN片段,应用RT-PCR、Western blot和免疫细胞化学方法检测转染前后Hpa基因和蛋白表达的变化,Matrigel侵袭实验观察转染ASODN后TE-13细胞侵袭行为的改变.结果:在TE-13细胞中,RT-PCR扩增得到大小为585 bp的Hpa基因条带,Western blot检测到50 ku大小的Hpa蛋白,免疫细胞化学染色表明Hpa主要定位于胞浆和胞膜.转染不同浓度Hpa ASODN后,RT-PCR、Western blot和细胞免疫染色均证实TE-13细胞Hpa基因和蛋白表达下调,且随Hpa ASODN的浓度增高,基因和蛋白表达量逐渐降低(P<0.05),不同浓度Hpa ASODN组间有显著性差异(P<O.05).Matrigel侵袭实验中,转染不同浓度Hpa ASODN后,侵袭至下室面的TE-13细胞数均下降(P<0.05).随Hpa ASODN浓度的增高,侵袭至下室面的TE-13细胞个数逐渐减少(P<0.05),不同浓度Hpa ASODN组间有显著性差异(P<0.05).结论:TE-13细胞存在Hpa基因的表达.Hpa ASODN能显著降低Hpa基因表达量,并且使TE-13细胞侵袭力明显下降.

  18. Peptide nucleic acid (PNA) antisense effects in Escherichia coli

    DEFF Research Database (Denmark)

    Good, L; Nielsen, P E

    1999-01-01

    Antisense peptide nucleic acid (PNA) can be used to control cell growth, gene expression and growth phenotypes in the bacteria Escherichia coli. PNAs targeted to the RNA components of the ribosome can inhibit translation and cell growth, and PNAs targeted to mRNA can limit gene expression with gene...... and sequence specificity. In an E. coli cell extract, efficient inhibition is observed when using PNA concentrations in the nanomolar range, whereas micromolar concentrations are required for inhibition in growing cells. A mutant strain of E. coli that is more permeable to antibiotics also is more susceptible...... to antisense PNAs than the wild type. This chapter details methods for testing the antisense activities of PNA in E. coli. As an example of the specific antisense inhibition possible, we show the effects of an anti-beta-galactosidase PNA in comparison to control PNAs. With improvements in cell uptake...

  19. Advanced In vivo Use of CRISPR/Cas9 and Anti-sense DNA Inhibition for Gene Manipulation in the Brain

    OpenAIRE

    Walters, Brandon J.; Azam, Amber B.; Gillon, Colleen J.; Josselyn, Sheena A; Zovkic, Iva B.

    2016-01-01

    Gene editing tools are essential for uncovering how genes mediate normal brain–behavior relationships and contribute to neurodegenerative and neuropsychiatric disorders. Recent progress in gene editing technology now allows neuroscientists unprecedented access to edit the genome efficiently. Although many important tools have been developed, here we focus on approaches that allow for rapid gene editing in the adult nervous system, particularly CRISPR/Cas9 and anti-sense nucleotide-based techn...

  20. Inhibition of flower formation by antisense repression of mitochondrial citrate synthase in transgenic potato plants leads to a specific disintegration of the ovary tissues of flowers.

    OpenAIRE

    Landschütze, V; Willmitzer, L.; Müller-Röber, B

    1995-01-01

    The tricarboxylic acid (TCA) cycle constitutes a major component of the mitochondrial metabolism of eucaryotes, including higher plants. To analyze the importance of this pathway, we down-regulated mitochondrial citrate synthase (mCS; EC 4.1.3.7), the first enzyme of the TCA cycle, in transgenic potato plants using an antisense RNA approach. Several transformants were identified with reduced citrate synthase activity (down to approximately 6% of wild-type activity). These plants were indistin...

  1. In vitro inhibition of promyelocytic leukemia/retinoic acid receptor-alpha (PML/RARalpha) expression and leukemogenic activity by DNA/LNA chimeric antisense oligos.

    Science.gov (United States)

    Caprodossi, Sara; Galluzzi, Luca; Biagetti, Simona; Della Chiara, Giulia; Pelicci, Pier Giuseppe; Magnani, Mauro; Fanelli, Mirco

    2005-01-01

    Acute promyelocytic leukemia (APL) is a subtype of myeloid leukemia characterized by the chromosomal translocation t(15:17) that leads to the expression of promyelocytic leukemia/retinoic acid receptor-alpha (PML/ RARalpha) oncofusion protein. The block of differentiation at the promyelocytic stage of the blasts and their increased survival induced by PML/RARalpha are the principal biological features of the disease. Therapies based on pharmacological doses of retinoic acid (RA, 10(-6) M) are able to restore APL cell differentiation in most cases, but not to achieve complete hematological remission because retinoic acid resistance occurs in many patients. In order to elaborate alternative therapeutic approaches, we focused our attention on the use of antisense oligonucleotides as gene-specific drug directed to PML/RARalpha mRNA target. We used antisense molecules containing multiple locked nucleic acid (LNA) modifications. The LNAs are nucleotide analogues that are able to form duplexes with complementary DNA or RNA sequences with highly increased thermal stability and are resistant to 3'-exonuclease degradation in vitro. The DNA/LNA chimeric molecules were designed on the fusion sequence of PML and RARalpha genes to specifically target the oncofusion protein. Cell-free and in vitro experiments using U937-PR9-inducible cell line showed that DNA/LNA oligonucleotides were able to interfere with PML/RARalpha expression more efficiently than the corresponding unmodified DNA oligo. Moreover, the treatment of U937-PR9 cells with these chimeric antisense molecules was able to abrogate the block of differentiation induced by PML/RARalpha oncoprotein. These data suggest a possible application of oligonucleotides containing LNA in an antisense therapeutic strategy for APL.

  2. Electroporation increases antitumoral efficacy of the bcl-2 antisense G3139 and chemotherapy in a human melanoma xenograft

    OpenAIRE

    Baldi Alfonso; D'Angelo Carmen; Scarsella Marco; De Mori Roberta; Biroccio Annamaria; Spugnini Enrico P; Leonetti Carlo

    2011-01-01

    Abstract Background Nucleic acids designed to modulate the expression of target proteins remain a promising therapeutic strategy in several diseases, including cancer. However, clinical success is limited by the lack of efficient intracellular delivery. In this study we evaluated whether electroporation could increase the delivery of antisense oligodeoxynucleotides against bcl-2 (G3139) as well as the efficacy of combination chemotherapy in human melanoma xenografts. Methods Melanoma-bearing ...

  3. Nucleic acid-based approaches to STAT inhibition.

    Science.gov (United States)

    Sen, Malabika; Grandis, Jennifer R

    2012-10-01

    Silencing of abnormally activated genes can be accomplished in a highly specific manner using nucleic acid based approaches. The focus of this review includes the different nucleic acid based inhibition strategies such as antisense oligodeoxynucleotides, small interfering RNA (siRNA), dominant-negative constructs, G-quartet oligonucleotides and decoy oligonucleotides, their mechanism of action and the effectiveness of these approaches to targeting the STAT (signal transducer and activator of transcription) proteins in cancer. Among the STAT proteins, especially STAT3, followed by STAT5, are the most frequently activated oncogenic STATs, which have emerged as plausible therapeutic cancer targets. Both STAT3 and STAT5 have been shown to regulate numerous oncogenic signaling pathways including proliferation, survival, angiogenesis and migration/invasion. PMID:24058785

  4. Effects of antisense oligonucleotides targeting VEGF on radio sensitivity of uterine cervix cancer Hela cells%血管内皮生长因子反义核酸对宫颈癌Hela细胞的放射增敏作用

    Institute of Scientific and Technical Information of China (English)

    Lina Xing; Li Qi

    2009-01-01

    Objective: To determine the impact of antisense oligonucleotides targeting vascular endothelial growth factor (VEGF) on radiosensitivity of uterine cervix cancer Hela cells. Methods: VEGF antisense oligodeoxynucleotides (ASODN) was transfected into Hela cells by liposome-mediated method. Cells transfected with the oligodeoxynuclecotide and saline were used as control groups. Cells were irradiated by 6 MV X ray at the dose of 0 Gy, 2 Gy, 4 Gy and 6 Gy respectively. The expression of VEGF mRNA was determined by RT-PCR. Apoptosis were evaluated using FCM. Cloning efficiency was deter-mined by colony formation assay. Results: The expression of VEGF mRNA was inhibited by ASODN (P < 0.01) in Hela cells. The inhibited activation which was influenced by radiation resulted in increasing apoptosis (P < 0.01) and inhibiting plating efficiency (P < 0.01). Conclusion: The expression of VEGF induced by Ⅹ irradiation in Hela cells can be blocked by VEGF ASODN. Treatment with VEGF might increase apoptosis in HeLa cells and enhance radiosensitivity.

  5. Chemosensitization by antisense oligonucleotides targeting MDM2.

    Science.gov (United States)

    Bianco, Roberto; Ciardiello, Fortunato; Tortora, Giampaolo

    2005-02-01

    The MDM2 oncogene is overexpressed in many human cancers, including sarcomas, certain hematologic malignancies, and breast, colon and prostate cancers. The p53-MDM2 interaction pathway has been suggested as a novel target for cancer therapy. To that end, several strategies have been explored, including the use of small polypeptides targeted to the MDM2-p53 binding domain, anti-MDM2 antisense oligonucleotides, and natural agents. Different generations of anti-human-MDM2 oligonucleotides have been tested in in vitro and in vivo human cancer models, revealing specific inhibition of MDM2 expression and significant antitumor activity. Use of antisense oligos potentiated the effects of growth inhibition, p53 activation and p21 induction by several chemotherapeutic agents. Increased therapeutic effectiveness of chemotherapeutic drugs in human cancer cell lines carrying p53 mutations or deletions have shown the ability of MDM2 inhibitors to act as chemosensitizers in various types of tumors through both p53-dependent and p53-independent mechanisms. Inhibiting MDM2 appears to also have a role in radiation therapy for human cancer, regardless of p53 status, providing a rationale for the development of a new class of radiosensitizers. Moreover, MDM2 antisense oligonucleotides potentiate the effect of epidermal growth factor receptor (EGFR) inhibitors by affecting in vitro and in vivo proliferation, apoptosis and protein expression in hormone-refractory and hormone-dependent human prostate cancer cells. These data support the development, among other MDM2 inhibitors, of anti-MDM2 antisense oligonucleotides as a novel class of anticancer agents, and suggest a potentially relevant role for the oligonucleotides when integrated with conventional treatments and/or other signaling inhibitors in novel therapeutic strategies.

  6. The Use of Antisense-Mediated Inhibition to Delineate The Role of Inflammatory Agents in The Pathophysiology of Spinal Cord Injury

    Directory of Open Access Journals (Sweden)

    Damien D. Pearse

    2002-01-01

    Full Text Available Injuries to the central nervous system (CNS usually lead to a potent and acute inflammatory response[1]. During this period, glia and immune cells respond to chemical cues associated with the debris of lysed neurons, disrupted axons, and a broken blood-brain-barrier by releasing a battery of cytokines including tumor necrosis factor-α (TNF-α and, interleukin-β (IL-1β as well as reactive oxygen species such as nitric oxide (NO-[2]. The secretion of these factors may be primarily responsible for secondary damage to surrounding uninjured tissue that potentiates the initial injury[3]. Antisense oligonucleotides (ASOs are designed to hybridize to specific regions of specific mRNAs. Hybridization of the oligonucleotide to the mRNA then interferes with the normal processing of that mRNA at the ribosome or targets the RNA duplex for cleavage by the RNA digestive enzyme, ribonuclease H, resulting in greatly reduced expression of the coded protein. This effectively reduces the amount of corresponding translated protein product and experiments can be designed to examine the requirement of particular inflammatory agents in eliciting specific deleterious responses after injury, e.g., cell death.

  7. Modulation of tumor eIF4E by antisense inhibition: A phase I/II translational clinical trial of ISIS 183750-an antisense oligonucleotide against eIF4E-in combination with irinotecan in solid tumors and irinotecan-refractory colorectal cancer.

    Science.gov (United States)

    Duffy, A G; Makarova-Rusher, O V; Ulahannan, S V; Rahma, O E; Fioravanti, S; Walker, M; Abdullah, S; Raffeld, M; Anderson, V; Abi-Jaoudeh, N; Levy, E; Wood, B J; Lee, S; Tomita, Y; Trepel, J B; Steinberg, S M; Revenko, A S; MacLeod, A R; Peer, C J; Figg, W D; Greten, T F

    2016-10-01

    The eukaryotic translation initiation factor 4E (eIF4E) is a potent oncogene that is found to be dysregulated in 30% of human cancer, including colorectal carcinogenesis (CRC). ISIS 183750 is a second-generation antisense oligonucleotide (ASO) designed to inhibit the production of the eIF4E protein. In preclinical studies we found that EIF4e ASOs reduced expression of EIF4e mRNA and inhibited proliferation of colorectal carcinoma cells. An additive antiproliferative effect was observed in combination with irinotecan. We then performed a clinical trial evaluating this combination in patients with refractory cancer. No dose-limiting toxicities were seen but based on pharmacokinetic data and tolerability the dose of irinotecan was reduced to 160 mg/m(2) biweekly. Efficacy was evaluated in 15 patients with irinotecan-refractory colorectal cancer. The median time of disease control was 22.1 weeks. After ISIS 183750 treatment, peripheral blood levels of eIF4E mRNA were decreased in 13 of 19 patients. Matched pre- and posttreatment tumor biopsies showed decreased eIF4E mRNA levels in five of nine patients. In tumor tissue, the intracellular and stromal presence of ISIS 183750 was detected by IHC in all biopsied patients. Although there were no objective responses stable disease was seen in seven of 15 (47%) patients who were progressing before study entry, six of whom were stable at the time of the week 16 CT scan. We were also able to confirm through mandatory pre- and posttherapy tumor biopsies penetration of the ASO into the site of metastasis. PMID:27194579

  8. Overexpression of D-Xylose Reductase (xyl1) Gene and Antisense Inhibition of D-Xylulokinase (xyiH) Gene Increase Xylitol Production in Trichoderma reesei

    Science.gov (United States)

    Hong, Yuanyuan; Dashtban, Mehdi; Kepka, Greg; Chen, Sanfeng; Qin, Wensheng

    2014-01-01

    T. reesei is an efficient cellulase producer and biomass degrader. To improve xylitol production in Trichoderma reesei strains by genetic engineering, two approaches were used in this study. First, the presumptive D-xylulokinase gene in T. reesei (xyiH), which has high homology to known fungi D-xylulokinase genes, was silenced by transformation of T. reesei QM9414 strain with an antisense construct to create strain S6-2-2. The expression of the xyiH gene in the transformed strain S6-2-2 decreased at the mRNA level, and D-xylulokinase activity decreased after 48 h of incubation. This led to an increase in xylitol production from undetectable levels in wild-type T. reesei QM9414 to 8.6 mM in S6-2-2. The T. reesei Δxdh is a xylose dehydrogenase knockout strain with increased xylitol production compared to the wild-type T. reesei QM9414 (22.8 mM versus undetectable). The copy number of the xylose reductase gene (xyl1) in T. reesei Δxdh strain was increased by genetic engineering to create a new strain Δ9-5-1. The Δ9-5-1 strain showed a higher xyl1 expression and a higher yield of xylose reductase, and xylitol production was increased from 22.8 mM to 24.8 mM. Two novel strains S6-2-2 and Δ9-5-1 are capable of producing higher yields of xylitol. T. reesei has great potential in the industrial production of xylitol. PMID:25013760

  9. Overexpression of D-Xylose Reductase (xyl1 Gene and Antisense Inhibition of D-Xylulokinase (xyiH Gene Increase Xylitol Production in Trichoderma reesei

    Directory of Open Access Journals (Sweden)

    Yuanyuan Hong

    2014-01-01

    Full Text Available T. reesei is an efficient cellulase producer and biomass degrader. To improve xylitol production in Trichoderma reesei strains by genetic engineering, two approaches were used in this study. First, the presumptive D-xylulokinase gene in T. reesei (xyiH, which has high homology to known fungi D-xylulokinase genes, was silenced by transformation of T. reesei QM9414 strain with an antisense construct to create strain S6-2-2. The expression of the xyiH gene in the transformed strain S6-2-2 decreased at the mRNA level, and D-xylulokinase activity decreased after 48 h of incubation. This led to an increase in xylitol production from undetectable levels in wild-type T. reesei QM9414 to 8.6 mM in S6-2-2. The T. reesei Δxdh is a xylose dehydrogenase knockout strain with increased xylitol production compared to the wild-type T. reesei QM9414 (22.8 mM versus undetectable. The copy number of the xylose reductase gene (xyl1 in T. reesei Δxdh strain was increased by genetic engineering to create a new strain Δ9-5-1. The Δ9-5-1 strain showed a higher xyl1 expression and a higher yield of xylose reductase, and xylitol production was increased from 22.8 mM to 24.8 mM. Two novel strains S6-2-2 and Δ9-5-1 are capable of producing higher yields of xylitol. T. reesei has great potential in the industrial production of xylitol.

  10. 125I decay in synthetic oligodeoxynucleotides

    International Nuclear Information System (INIS)

    The availability of synthetic oligodeoxynucleotides provides the opportunity to study the effects of 125I in DNA with added precision and detail, compared to an earlier study (Martin and Haseltine, Science 214, 296, 1981). The authors have designed a template/primer system which enables incorporation of 125IdC into a defined location in a 31mer. The main advantage of this approach is that relatively large amounts (a few μCi) of DNA with both 32P and 125I can be produced quite easily. Consequently, when 125I-induced DNA damage is analyzed on DNA sequencing gels, the distribution of 32P-labeled cleavage products can be determined more accurately, following accumulation of decay events under various conditions (e.g. temperature, buffer components, free radical scavengers). The results of preliminary experiments suggest that the yield of DNA strand breaks per decay approaches one, at least for the 125I labeled strand. Extension of this experimental approach will also enable investigation of the nature of the terminii (e.g. phophoryl-versus phophorylglycollate) at either side of 125I decay-induced DNA strand breaks. The question of energy migration is also being investigated in the oligodeoxynucleotide system, by studying the effects of inclusion of BrUdR nucleotides at various distances from the 125IdC

  11. Gene therapeutic approaches to inhibit hepatitis B virusreplication

    Institute of Scientific and Technical Information of China (English)

    Maren Gebbing; Thorsten Bergmann; Eric Schulz; Anja Ehrhardt

    2015-01-01

    Acute and chronic hepatitis B virus (HBV) infectionsremain to present a major global health problem. Theinfection can be associated with acute symptomaticor asymptomatic hepatitis which can cause chronicinflammation of the liver and over years this can leadto cirrhosis and the development of hepatocellularcarcinomas. Currently available therapeutics forchronically infected individuals aim at reducing viralreplication and to slow down or stop the progressionof the disease. Therefore, novel treatment options areneeded to efficiently combat and eradicate this disease.Here we provide a state of the art overview of genetherapeutic approaches to inhibit HBV replication. Wediscuss non-viral and viral approaches which wereexplored to deliver therapeutic nucleic acids aiming atreducing HBV replication. Types of delivered therapeuticnucleic acids which were studied since many yearsinclude antisense oligodeoxynucleotides and antisenseRNA, ribozymes and DNAzymes, RNA interference,and external guide sequences. More recently designernucleases gained increased attention and wereexploited to destroy the HBV genome. In addition wemention other strategies to reduce HBV replicationbased on delivery of DNA encoding dominant negativemutants and DNA vaccination. In combination withavailable cell culture and animal models for HBVinfection, in vitro and in vivo studies can be performedto test efficacy of gene therapeutic approaches. Recentprogress but also challenges will be specified andfuture perspectives will be discussed. This is an excitingtime to explore such approaches because recentsuccesses of gene therapeutic strategies in the clinicto treat genetic diseases raise hope to find alternativetreatment options for patients chronically infected withHBV.

  12. Overexpression of members of the AP-1 transcriptional factor family from an early stage of renal carcinogenesis and inhibition of cell growth by AP-1 gene antisense oligonucleotides in the Tsc2 gene mutant (Eker) rat model.

    Science.gov (United States)

    Urakami, S; Tsuchiya, H; Orimoto, K; Kobayashi, T; Igawa, M; Hino, O

    1997-12-01

    We previously isolated subtracted cDNA clones for genes having increased expression in Tsc2 gene mutant (Eker) rat renal carcinomas (RCs). Among them, fra-1 encoding a transcriptional factor activator protein 1 (AP-1) was identified. We have therefore investigated whether other members of the AP-1 transcription factor family might also be involved in renal carcinogenesis in the Eker rat model. In the present study, overexpression of fra-1, fra-2, c-jun, junB, and junD mRNAs was demonstrated in RCs by Northern blot analysis. Interestingly, AP-1 proteins were highly expressed even in the earliest preneoplastic lesions (e.g., phenotypically altered tubules) as suggested by immunohistochemistry. Moreover, 12-O-tetradecanoylphorbol-13-acetate-responsive element (TRE)-binding activity of AP-1 proteins was observed in RC cell extracts by electrophoretic mobility shift assay. As a next step, we transfected antisense oligonucleotides targeting AP-1 genes into RC cells and demonstrated that their growth was strongly inhibited. Thus, the data suggest that overexpression of AP-1 genes might play a crucial role in renal carcinogenesis in the Eker rat model. PMID:9405228

  13. Sense antisense DNA strand?

    Science.gov (United States)

    Boldogkói, Z; Kaliman, A V; Murvai, J; Fodor, I

    1994-01-01

    Recent evidence indicates that alphaherpesviruses express latency associated transcripts (LATs) from the antisense strand of immediate-early (IE) genes of the viral genome. It has been suggested that LATs containing extended open reading frames (ORFs), might be translated into (a) protein product(s). We found that a salient feature of some herpesvirus DNAs is a high GC preference at the third codon positions. The consequence of this feature is that the probability of a stop-codon appearing at two of the six reading frames of the DNA strand is very low. Therefore, the presence of an extended ORF does not necessarily mean that it is relevant to real translation. PMID:7810418

  14. NF-κB诱骗剂——环状哑铃寡核苷酸抑制膀胱肿瘤细胞侵袭能力的研究%Experimental study on the invasiveness inhibition of bladder cancer cells by nuclear factor-κB decoy——circular dumbbell oligodeoxynucleotides

    Institute of Scientific and Technical Information of China (English)

    文博; 周四维; 杨为民

    2006-01-01

    Objective: To determine whether NF-κB is constitutively activated in human bladder cancer cell and, if so, to determine the invasiveness inhibition of bladder cancer cells by nuclear factor-κB decoy-circular dumbbell oligodeoxynucleotides (CD-ODN). Methods: NF-κBp65 activation was determined by immunohistochemical analysis of formalin-fixed, paraffin-embedded specimens from 38 cases of bladder transitional cell carcinoma patients. We quantified nuclear staining of RelA as a marker of NF-κBp65 activation. CD-ODN were transfected into human bladder cancer cell line BIU87 by lipofectamine. Luciferase reporter were applied to detecting NF-κB DNA binding activity. The expression levels of uPA were detected by RT-PCR and the cells' invasion ability by transwell cell culture chamber. Results: P65 excessive activation existed in tumor cell (P<0.01), the activation degree correlated significantly with the expression of uPA (r=0.89, P<0.01), as well as related to tumor invasion-related clinicopathological features such as lymphatic metastasis (P<0.01) and pathological ranking (P<0.05); After transfection with CD-ODN, the activation of NF-κB in BIU87 cell line was suppressed remarkably, the expression level of uPA was decreased and the cells' invasiveness was weakened as well. Conclusion: Excessively activated NF-κB is related to tumor progression possibly due to its transcriptional regulation of invasion-related factors such as uPA. CD-ODN can efficiently suppress DNA binding activity of NF-κB to reduce the invasive potency of tumor.

  15. Effect of decoy-oligodeoxynucleotides on expression of inflammation mediators in pMΦ cells from rats

    Institute of Scientific and Technical Information of China (English)

    国华; 梁华平; 吕凤林; 徐祥; 安兵; 郝天智; 王付龙; 杨文军

    2003-01-01

    Objective: To study the effect of decoy-oligodeoxynucleotides (decoy-ODNs) in dumbbell shape with the oligodeoxynucleotide sequence similar to nuclear factor kappa B (NF-κB) cis-elements on expression of inflammation mediators in pMΦ cells from rats.Methods: With carriers of cationic liposomes, decoy-ODNs were transfected into pMΦ cells of rats. Then the inhibiting effects of the decoy-ODNs on tumor necrosis factor α (TNFα), interleukin-6 (IL-6) and IL-10 were analyzed.Results: Decoy-ODNs could decrease the expression of TNFα and IL-6 in dose-dependent fashion but had weaker inhibiting effect on IL-10. Conclusions: Decoy-ODNs targeting NF-κB can decrease the expression of inflammatory mediators in pMΦ cells from rats.

  16. Antisense CTGF inhibits the expression of CTGF in hypertrophic scar of rabbit ears%药物抑制兔耳病理性瘢痕中结缔组织生长因子的实验

    Institute of Scientific and Technical Information of China (English)

    王少华; 李健; 王燕华; 吕建平; 李记森; 王勇

    2011-01-01

    Objective To investigate a medicine which can inhibit the expression of connective tissue growth factor (CTGF) in hypertrophic scar of rabbit ears. Methods 24 bigears white rabbits were used to establish a model of hypertrophic scar of rabbit ears, which was randomly divided into four groups. The hypertrophic scar was injected intralesionally with antisense CTGF (group A), betamethason (group B), triamcinolone acetonide (group C) and physiological saline (group D). Some scar tissue samples were sectioned in every group when the scar was treated after 7, 14, 30, and 60 days, respectively. The expression of CTGF mRNA in the scar was assessed by in situ hybridization and hematoxylin and eosin stain (HE stain) in every samples. Results The expression of CTGF mRNA and the counting of fibroblasts decreased in group A, which showed statistical difference as compared with groups B, C and D. Conclusions The results suggesz that antisense CTGF is able to inhibit the proliferation process of hypertrophic scar in rabbit ears and to remarkably decrease the degree of fiborsis in the scar.%目的 探讨能有效抑制结缔组织生长因子(CTGF)的药物,以便为治疗病理性瘢痕提供依据.方法 选择24只大耳白兔建立兔耳病理性瘢痕模型,随机分为4组:A组注射CTGF反义寡核苷酸,B组注射复方倍他米松,C组注射醋酸曲安奈得,D组为对照组,仅注射生理盐水.通过原位杂交法分别检测瘢痕组织中不同治疗组不同时段的CTGF表达,并通过苏木精-伊红(HE)染色法检测瘢痕组织中不同治疗组不同时段的成纤维细胞数.结果 A、B、C 3组在同一时段其CTGF表达均比D组低,差异有统计学意义(P<0.05),A组较B、C两组CTGF表达低,差异也具有统计学意义(P<0.05).B、C两组之间CTGF表达差异无统计学意义(P>0.05).成纤维细胞计数结果与以上结果基本一致.结论 CTGF反义寡核苷酸、复方倍他米松、醋酸曲安奈得均可抑制病理性瘢痕

  17. Construction of antisense Bmi-1 expression plasmid and its inhibitory effect on K562 cells proliferation

    Institute of Scientific and Technical Information of China (English)

    MENG Xiu-xiang; LIU Wei-hong; LIU Dan-dan; ZHAO Xin-yu; SU Ben-li

    2005-01-01

    Background Bmi-1 gene determines the proliferative capacity of normal and leukemia stem cells. Expression of Bmi-1 has been found in all types of myeloid leukemia cells in both humans and mice. This study aimed at assessing the effect of antisense Bmi-1 expression on K562 cells proliferation and p16 protein (p16) expression.Results K562 cells transfected with antisense Bmi-1 plasmid grew significantly slower than that of controls (the parental K562 and cells transfected with empty plasmid). The colony forming ability of antisense Bmi-1 plasmid transfected cells decreased significantly (P<0.01) compared with controls. The p16 expression of cells transfected with antisense Bmi-1 was upgraded more apparently than that of controls.Conclusion The antisense Bmi-1 gene can inhibit the growth of K562 cell and upgrade expression of p16 in K562 cells.

  18. Combination of telomerase antisense oligonucleotides simultaneously targeting hTR and hTERT produces synergism of inhibition of telomerase activity and growth in human colon cancer cell line

    Institute of Scientific and Technical Information of China (English)

    Xiao-Hua Fu; Jian-Song Zhang; Na Zhang; Yang-De Zhang

    2005-01-01

    AIM: To investigate synergism of inhibition of telomerase activity and proliferation of human colon cancer cells by combination of telomerase antisense oligonucleotides (ASODNs) simultaneously targeting human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT)in vitro.METHODS: ASODN of hTR and ASODN of hTERT were transfected into human colon cancer SW480 cells by liposomal transfection reagents. Telomerase activity of SW480 cells was examined using telomeric repeat amplification protocol (TRAP)-enzyme-linked immunosorbent assay (PCR-ELISA). Proliferation activity of SW480 cells was tested by methyl thiazolyl tetrazolium assay. Apoptosis and cell cycle were analyzed by flow cytometry.RESULTS: The telomerase activity and cell survival rate in SW480 cells transfected with 0.2 μmol/L of ASODN of hTR or ASODN of hTERT for 24-72 h were significantly decreased in a time-dependent manner compared with those after treatment with sense oligonucleotides and untreated (telomerase activity: 24 h, 73%, 74% vs 99%,98%; 48 h, 61%, 55% vs 98%, 99%; 72 h, 41%, 37% vs 99%, 97%; P<0.01; cell survival rate: 24 h, 88%, 86%vs94%, 98%; 48 h, 49%, 47% vs94%, 97%; 72 h, 44%,42% vs 92%, 96%; P<0.01). Moreover, the telomerase activity and the cell survival rate in SW480 cells treated by the combination of telomerase anti-hTR and anti-hTERT were more significantly suppressed than single anti-hTR or anti-hTERT (telomerase activity: 24 h, 59% vs 73%,74%; 48 h, 43% vs61%, 55%; 72 h, 18% vs41%, 37%;P<0.01; cell survival rate: 24 h, 64% vs88%, 86%; 48 h,37% vs49%, 47%; 72 h, 25% vs44%, 42%; P<0.01).Meanwhile, the apoptosis rates in the combination group were markedly increased compared with those in the single group (24 h, 18.0% vs 7.2%, 7.4%; 48 h, 23.0%vs 13.0%, 14.0%; 72 h, 28.6% vs13.2%, 13.75; P<0.01).Cells in combination group were arrested at G0/G1 phase.CONCLUSION: Telomerase anti-hRT and anti-hTERT suppress telomerase activity, and inhibit growth of human

  19. Detection and quantitation of PS20,a phosphorothioate oligodeoxynucleotides in monkey plasma

    Institute of Scientific and Technical Information of China (English)

    Ming-mei SHANG; Hai-feng SONG; Xiu-wen LIU; Zhong-ming TANG

    2004-01-01

    AIM: To establish the method for quantitation of the phosphorothioate oligodeoxynucleotides (S-ODNs) in plasma.METHODS: Two solid-phase extraction columns combined with a strong anion-exchange column were utilized to remove proteins and lipids in plasma, and the salts were removed by a reverse-phase column followed by dialysis with a 2500 Da-cutoff membrane. The concentration of the tested S-ODNs, PS20, and its metabolites extracted from the plasma were determined by the method of non-gel sieving capillary electrophoresis (NGCE) with diode array detection in the presence of internal standard (IS). RESULTS: The method was with good base number specificity. Relative standard deviation % of both intra and inter assay were all less than 10 %, and the total mean recovery was about 91%. The methodology was successfully used to determine the PK behavior of an anti-tumor antisense S-ODNs in monkeys and identify the metabolites with single base difference. CONCLUSION: The combined method of solid-phase extraction and NGCE could be used to study the pharmacokinetics of S-ODNs,and the main parameters of the methodology met the requirement of PK study.

  20. Advancements of antisense oligonucleotides in treatment of breast cancer

    Institute of Scientific and Technical Information of China (English)

    YANGShuan-Ping; SONGSan-Tai; 等

    2003-01-01

    Breast cancer is one kind of multi-gene related malignancy.Overexpression of some oncogenes such as HER-2(c-erbB-2,Neu),bcl-2/bcl-xL,protein kinase A(PKA),and transferrin receptor gene(TfR gene),etc significantly affect the prognosis of breast cancer.It was shown that specific suppression of the overexpressed genes above resulted in the improvement of the therapy of breast cancer.Antisense interference.one of useful tools for inhibiting the overexpression of specific oncogenes,was involved in the therapy of breast cancer in recent years. Data indicated that antisense oligonucleotides(ON)could inhibit specially the expression of the target genes on mRNA or protein levels in most of cases;some ON candidates showed encouraging therapeutic effects in vitro and in vivo on breast cancer cell lines or xenografts.Furthermore,the combination use of the antisense ON and normal chemotherapeutic agents indicated synergistic antitumor effects,which was probably the best utilization of antisense ON in the treatment of breast cancer.

  1. EFFECTS OF CYCLOOXYGENASE-2 ANTISENSE VECTOR ON PROLIFERATION OF HUMAN CHOLANGIOCARCINOMA CELLS

    Institute of Scientific and Technical Information of China (English)

    Gao-song Wu; Sheng-quan Zou; Xiao-yong Wu; Fa-zu Qiu

    2004-01-01

    Objective To transfect antisense vector of human cyclooxygenase-2 (COX-2) gene into COX-2 highly expressing cholangiocarcinoma cell line QBC939 and explore its biological activities and role in carcinogenesis.Methods QBC939 cells were transfected with antisense vector of human COX-2 gene using LipoVecTM transfecting technique. Transfected cells were selected with G418; COX-2 mRNA was examined using reverse transcription polymerase chain reaction (RT-PCR) and COX-2 protein expression was detected by immunocytochemistry using isozyme selective antibodies. The proliferative status of transfected cells was measured by using methabenzthiazuron (MTT) assay; Cell cycle and apoptosis were analyzed by using flow cytometry.Results RT-PCR showed a lower COX-2 mRNA level in antisense vector transfected cells and immunocytochemistry showed a weaker COX-2 protein expression in antisense vector transfected cells. The antisense vector transfected cells proliferative index decreased significantly (P< 0.01), the percentage of S phase decreased remarkably (P< 0.05) in antisense vector transfected cells (9.27% ± 1.91%) compared with that in QBC939 cells without transfection(16.35% ± 2.87%), and the percentage of G0/G 1 phase increased remarkably (P < 0.05) in antisense vector transfected cells (75.16%±4.13%) compared with that in QBC939 cells without transfection (57.31%± 10.16%). Transfection with antisense vector of human COX-2 gene had no significant influence on the apoptosis in QBC939 cells (P > 0.05).Conclusion Transfection with antisense vector of human COX-2 gene could inhibit the proliferation of human cholangiocarcinoma QBC939 cells.

  2. Construction and transfection of sense/antisense eukaryotic expression vectors for human cathepsin L gene

    Institute of Scientific and Technical Information of China (English)

    Maolin He; Anmin Chen

    2005-01-01

    Objective: To obtain sense/antisense eukaryotic expression vectors for human cathepsin L gene, and study the biological effects on human osteosarcoma cell line MG-63 after transfection. Methods: Cathepsin L gene sense/antisense eukaryotic expression vectors were constructed with recombinant technology and transfected into the human osteosarcoma cell line MG-63. The expression of cathepsin L gene mRNA was examined with RT-PCR and the expression of cathepsin L was examined with Western blot. Results: The sense/antisense recombinant eukaryotic expression vectors for cathepsin L were successfully constructed and transfected into MG-63 cell.Conclusion: Antisense cathepsin L gene can significantly inhibit the expression of cathepsin L mRNA and protein.

  3. Bolaamphiphile-based nanocomplex delivery of phosphorothioate gapmer antisense oligonucleotides as a treatment for Clostridium difficile

    Science.gov (United States)

    Hegarty, John P; Krzeminski, Jacek; Sharma, Arun K; Guzman-Villanueva, Diana; Weissig, Volkmar; Stewart, David B

    2016-01-01

    Despite being a conceptually appealing alternative to conventional antibiotics, a major challenge toward the successful implementation of antisense treatments for bacterial infections is the development of efficient oligonucleotide delivery systems. Cationic vesicles (bolasomes) composed of dequalinium chloride (“DQAsomes”) have been used to deliver plasmid DNA across the cardiolipin-rich inner membrane of mitochondria. As cardiolipin is also a component of many bacterial membranes, we investigated the application of cationic bolasomes to bacteria as an oligonucleotide delivery system. Antisense sequences designed in silico to target the expression of essential genes of the bacterial pathogen, Clostridium difficile, were synthesized as 2′-O-methyl phosphorothioate gapmer antisense oligonucleotides (ASO). These antisense gapmers were quantitatively assessed for their ability to block mRNA translation using luciferase reporter and C. difficile protein expression plasmid constructs in a coupled transcription–translation system. Cationic bolaamphiphile compounds (dequalinium derivatives) of varying alkyl chain length were synthesized and bolasomes were prepared via probe sonication of an aqueous suspension. Bolasomes were characterized by particle size distribution, zeta potential, and binding capacities for anionic oligonucleotide. Bolasomes and antisense gapmers were combined to form antisense nanocomplexes. Anaerobic C. difficile log phase cultures were treated with serial doses of gapmer nanocomplexes or equivalent amounts of empty bolasomes for 24 hours. Antisense gapmers for four gene targets achieved nanomolar minimum inhibitory concentrations for C. difficile, with the lowest values observed for oligonucleotides targeting polymerase genes rpoB and dnaE. No inhibition of bacterial growth was observed from treatments at matched dosages of scrambled gapmer nanocomplexes or plain, oligonucleotide-free bolasomes compared to untreated control cultures. We

  4. Preparation of oligodeoxynucleotide encapsulated cationic liposomes and release study with models of cellular membranes

    Directory of Open Access Journals (Sweden)

    Tamaddon AM.

    2007-05-01

    Full Text Available Cationic liposomes are used for cellular delivery of antisense oligodeoxynucleotide (AsODN, where release of encapsulated AsODN is mainly controlled by endocytosis and fusion mechanisms. In this investigation, it was tried to model such a release process that is difficult to evaluate in cell culture. For this purpose, an AsODN model (against protein kinase C-α was encapsulated in a DODAP-containing cationic liposome and evaluated for size, zeta-potential, encapsulation and ODN stability. Vesicular models of outer layer and total plasma membranes and early and late endosomal membranes were developed, based on lipid content and pH, using ether injection method. ODN release was determined by the fluorescence dequenching of encapsulated FITC-ODN. Zeta potential, size and ODN encapsulation efficiency of the prepared liposomes were -2.49 ± 7.15 mV, 108.4 nm and 73% respectively. ODN protection was 3-4 times more than that of conventional liposome/ODN complexation method. There was a correlation between model concentration and percent of ODN release. At 7.5 µM, the percent of released ODN was 76% for the cholesterol-free model of the late endosome and 16% for the early endosomal membrane; while the release was less than 11% for the models of plasma membrane. ODN release increased with temperature in the range of 4-37◦C for the late endosomal model, but not for others, possibly due to their high cholesterol contents or acidic pH. The interaction was fast and completed within 5 minutes and didn’t change in the range of 5-60 minutes. Our data are in agreement with published cell culture studies and reveal that cell-liposomes interaction can be modeled by lamellar membranes.

  5. Bifunctional oligodeoxynucleotide/antagomiR constructs: evaluation of a new tool for microRNA silencing.

    Science.gov (United States)

    Ziegler, Saskia; Eberle, Mariel Esther; Wölfle, Sabine J; Heeg, Klaus; Bekeredjian-Ding, Isabelle

    2013-12-01

    MicroRNAs (miRNAs) are fine-tuners in cellular processes, including those of the immune response. To study their functions and effects in immune cells, it is necessary to achieve specific silencing of individual miRNAs. To date, introduction of antisense microRNAs (antagomiRs) into primary cells is based on electroporation, lipofection, and viral vectors. However, these techniques often compromise viability, proliferative capacity, and differentiation. Furthermore, efficiency varies depending on the cell type and some are not suitable for in vivo approaches. To overcome these limitations we exploited the property of phosphorothioate (PTO)-modified DNA oligodeoxynucleotides (ODN) to enter cells with high efficacy: we developed and evaluated ODN/antagomiR constructs that consist of a PTO-ODN carrier covalently linked to a fully methylated antagomiR RNA sequence. Using these constructs, we achieved transfection efficiency of approximately 99% in leukocytes-in particular, in B lymphocytes that are hard to transfect with other methods. Our data demonstrate that miRNA silencing by the antagomiR portion of the constructs was specific and efficient, which could be further confirmed by an increase in target protein under silencing conditions. The constructs were successfully tested in human B cells, plasmacytoid dendritic cells, monocytes, and monocyte-derived dendritic cells, thus demonstrating their versatility. Moreover, introduction of stimulatory CpG sequences into the ODN portion conveys immune stimulatory quality when intended. Thus, bifunctional ODN/antagomiR constructs represent a highly efficient, versatile, and easy-to-handle tool to manipulate cellular miRNA expression levels and to allow the subsequent investigation of specific miRNA functions. PMID:24236889

  6. THE EFFECT OF ANTISENSE OLIGONUCLEOTIDE ON THE INTERLEUKIN-5 IN THE SUPERNATANTS OF SPLEEN CELL CULTURES OF ASTHMATIC MICE

    Institute of Scientific and Technical Information of China (English)

    王美琴; 白春学; 钮善福; 方晓惠; 陈常庆; 陈波

    2001-01-01

    To explore the effect of antisense oligonucleotide on the production of IL-5 by mouse spleen T lymphocytes.Methods Based on the IL-5 cDNA sequence of mouse, a segment of antisense oligonucleotide was designed and synthesized. 5’-labeling of antisense oligonucleotide was signed by T4 PNK in order that the efficiency of stearylamine liposome in transfecting antisense oligonucleotide can be evaluated. Asthma model was duplicated with ovalbumin(OVA) absorbed to aluminum hydroxide. T lymphocytes of mice were separated by nylon fiber method, then T lymphocytes transfected with different concentration of antisense oligonucleotide with cation stearylamine liposme were incubated respectively in order to observe the effect of antisense oligonucleotide on Il-5 production by T lymphocytes. IL-5 levels in the supernatants of T lymphocyte cultures were determined by ELISA.Results Stearylamine liposome could markedly increase the efficiency of antisense oligonucleotide transfection. The transfection efficiency of antisense oligouncleotide increased approximately 12 times at a ratio of 1: 15m/m (antisense oligonucleotide to SA liposome). In healthy and asthma Balb/c mice, IL-5 was not detectable in the supernatants of T lymphocyte cultures without stimulated with OVA; however, IL-5 was increased markedly in the supernatants of T lymphocyte cultures stimulated with OVA. After transfection with different concentrations of antisense oligonucleotide, IL-5 levels in the supernatants of T lymphocyte cultures were significantly lower than those in control cultured without antisense oligonucleotide transfection. IL-5 levels decreased from 44.60±6.23 pg/ml to 30.70±7.362 pg/ml, 17.20±6.181 pg/ml and 8.16±2.34 pg/ml respectively. And IL-5 synthesis was inhibited by 31.17%, 61.43% and 81.7% respectively.Conclusion IL-5 synthesis could be obviously inhibited by antisense oligonucleotide and showed a markedly correlation between dose and effectiveness. It suggests the production

  7. Antisense oligonucleotide to insulin—like growth factor Ⅱ induces apotosis in human ovarian cancer AO cell line

    Institute of Scientific and Technical Information of China (English)

    YINDELING; LUPU; 等

    1998-01-01

    The effects of antisense oligonucleotide to insulin0like growth factor -Ⅱ(IGFⅡ)to induce apotosis in human ovarian cancer cells were evaluated.Antiproliferation effects of antisense to IGFⅡin ovarian cancer AO cells were determined by 3H-thymidine incorporation.Apoptosis of the IGFⅡ antisense-treated cells was quantitated by both nuclear condensation and flow cytometry after cells were stained with propidium iodide,IGFⅡ antisense(4.5μM) treatment of 48h maximally inhibited proliferation of AO cells,More than 25% of IGFⅡantisense-treated cells(4.5μM for 24h) had undergone apoptosis,whereas less than 3% of the cells were apoptotic in either IGFⅡ sense-treated cells or untreated cells.Antisense oligonucleotide to IGFⅡ significantly inhibited cell proliferation and induced apoptosis in human ovarian cancer AO cell.These data suggest that IGFII may be a potential target in treatment of ovarian cancer and antisense oligonucleotide to IGFⅡ may serve as a therapeutic approach.

  8. The Inhibitive Effects of Combining an Antisense Oligodeoxynucleotides Targeted to ASGPR with Anti-HBV Drugs against Hepatitis B Virus in vitro%靶向ASGPR的反义核酸与抗HBV药物联合用药的体外抗HBV作用

    Institute of Scientific and Technical Information of China (English)

    丁晓然; 杨静; 伯晓晨; 张敏丽; 陈苏红; 王升启

    2006-01-01

    研究以宿主基因ASGPR为靶的反义核酸与抗乙肝病毒(hepatitis B virus,HBV)药物联合应用的抗-HBV作用,为HBV感染的联合治疗和用药提供新的思路.以HepG2.2.15细胞为靶细胞,脂质体为载体将靶向ASGPR的硫代反义寡核苷酸(ASODN)转染至HepG2.2.15细胞中,6h后加入抗病毒药拉米夫定(3TC)或阿地福韦(ADV),72h后收集细胞培养液,采用酶联免疫法及荧光定量PCR法测定ASODN与抗-HBV药物联合应用后对细胞培养液中HBsAg、HBeAg及细胞分泌HBV DNA的抑制作用.采用金正均Q值法对数据进行分析.拉米夫定(3TC)和阿地福韦(ADV)分别与ASODN联用对HBsAg的抑制呈相加或协同作用.随着ADV浓度的增加,两药的协同作用下降.3TC与ASODN联用对HBeAg的抑制呈拮抗、相加和协同作用,其拮抗作用随着3TC浓度的增加逐渐减弱.ADV与ASODN联用对HBeAg的抑制只呈相加作用.3TC和ADV分别与ASODN联用对HBV DNA的抑制均表现为相加作用或协同作用,取ADV0.5μmol/L与取ASODN0.2μmol/L联用时对HBV DNA的抑制率可提高到72.6%.ASODN在一定条件下分别与3TC及ADV联用有相加或协同抗HBsAg、HBeAg及HBV DNA的表达,尤以抗HBsAg作用显著增强.

  9. Inhibition of ICAM-1 expression in renal tubular epithelial cells with ICAM-1 antisense oligodeoxynucleotide%ICAM-1反义寡核苷酸对小鼠肾小管上皮细胞ICAM-1表达的影响

    Institute of Scientific and Technical Information of China (English)

    程庆砾; 陈香美; 傅博; 叶一舟

    2000-01-01

    目的:探讨ICAM-1反义寡核苷酸对小鼠肾小管上皮细胞表达ICAM-1的影响,为利用ICAM-1反义寡核苷酸类药物治疗肾小管间质病变奠定基础.方法:小鼠ICAM-1反义寡核苷酸及其对照物参照文献合成并行全硫代磷酸化修饰.部分ICAM-1反义寡核苷酸在其5'端作FITC荧光标记.将ICAM-1反义寡核苷酸单独或脂质体转染试剂DOTAP混合后转染至培养的肾小管上皮细胞之中,转染成功后加入IL-1β诱导细胞产生ICAM-1.采用对照寡核苷酸及不含寡核苷酸的细胞转染液作为实验对照及空白对照.利用免疫组化染色的方法检测细胞ICAM-1表达变化并提取细胞总RNA行逆转录PCR及Northern杂交观察ICAM-1 mRNA的表达的变化.结果:两个不同浓度的ICAM-1反义寡核苷酸均可明显阻断IL-1β诱导的肾小管上皮细胞ICAM-1及ICAM-1 mRNA的表达;对照的寡核苷酸不能减少IL-1β诱导的肾小管上皮细胞ICAM-1及ICAM-1 mRNA的表达.结论:小鼠ICAM-1反义寡核苷酸可明显抑制IL-1β诱导的小鼠肾小管上皮细胞ICAM-1及ICAM-1 mRNA 的表达,提示ICAM-1的反义寡核苷酸有可能应用于肾小管间质病变的实验治疗之中.

  10. Double-stranded RNA transcribed from vector-based oligodeoxynucleotide acts as transcription factor decoy

    Energy Technology Data Exchange (ETDEWEB)

    Xiao, Xiao [State Key Laboratory of Cancer Biology and Xijing Hospital of Digestive Diseases, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, Shaanxi Province (China); Gang, Yi [State Key Laboratory of Cancer Biology and Xijing Hospital of Digestive Diseases, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, Shaanxi Province (China); Department of Infectious Diseases, Tangdu Hospital, Fourth Military Medical University, Xi’an 710038, Shaanxi Province (China); Wang, Honghong [No. 518 Hospital of Chinese People’s Liberation Army, Xi’an 710043, Shaanxi Province (China); Wang, Jiayin [The Genome Institute, Washington University in St. Louis, St. Louis, MO 63108 (United States); Zhao, Lina [Department of Radiation Oncology, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, Shaanxi Province (China); Xu, Li, E-mail: lxuhelen@163.com [State Key Laboratory of Cancer Biology and Xijing Hospital of Digestive Diseases, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, Shaanxi Province (China); Liu, Zhiguo, E-mail: liuzhiguo@fmmu.edu.cn [State Key Laboratory of Cancer Biology and Xijing Hospital of Digestive Diseases, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, Shaanxi Province (China)

    2015-02-06

    Highlights: • A shRNA vector based transcription factor decoy, VB-ODN, was designed. • VB-ODN for NF-κB inhibited cell viability in HEK293 cells. • VB-ODN inhibited expression of downstream genes of target transcription factors. • VB-ODN may enhance nuclear entry ratio for its feasibility of virus production. - Abstract: In this study, we designed a short hairpin RNA vector-based oligodeoxynucleotide (VB-ODN) carrying transcription factor (TF) consensus sequence which could function as a decoy to block TF activity. Specifically, VB-ODN for Nuclear factor-κB (NF-κB) could inhibit cell viability and decrease downstream gene expression in HEK293 cells without affecting expression of NF-κB itself. The specific binding between VB-ODN produced double-stranded RNA and NF-κB was evidenced by electrophoretic mobility shift assay. Moreover, similar VB-ODNs designed for three other TFs also inhibit their downstream gene expression but not that of themselves. Our study provides a new design of decoy for blocking TF activity.

  11. An oligodeoxynucleotide with CCT repeats restrains CpG ODN-induced TLR9 trafficking.

    Science.gov (United States)

    Zhang, Xiaoling; Sun, Wei; Wu, Xiuli; Wang, Hua; Yan, Youyou; Guo, Sheng; Song, Dandan; Li, Hainan; Gao, Shuang; Wang, Luowei; Yu, Yongli; Wang, Liying

    2014-01-01

    Toll-like receptor 9 (TLR9) can sense pathogen DNA and CpG ODN or even self-DNA by trafficking assisted by Unc93B1, an endoplasmic reticulum (ER) transmembrane protein, from ER to endolysosomes or cell surface. In previous study, we found that an oligodeoxynucleotide with CCT repeats (SAT05f) could selectively inhibit TLR7/9 activation. However, the mechanism for the inhibitory activity of SAT05f is still unknown. In present research, it was found that SAT05f could inhibit CpG ODN-induced the intracellular trafficking of TLR9 and Unc93B1 with feedback the responses of decreased surface TLR9 and enhanced TLR9 mRNA expression but not influence TLR9 protein level by using human plasmacytoid dendritic cell line CAL-1 cells, suggesting that SAT05f inhibits TLR9 activation by restraining TLR9 trafficking. Since the mitochondrial DNA released from injured tissue can cause systemic inflammatory response syndrome (SIRS), this study may provide valuable data for prevention and treatment of SIRS and rescue severe trauma patients. PMID:25374030

  12. Antisense expression of the fasciclin-like arabinogalactan protein FLA6 gene in Populus inhibits expression of its homologous genes and alters stem biomechanics and cell wall composition in transgenic trees.

    Science.gov (United States)

    Wang, Haihai; Jiang, Chunmei; Wang, Cuiting; Yang, Yang; Yang, Lei; Gao, Xiaoyan; Zhang, Hongxia

    2015-03-01

    Fasciclin-like arabinogalactan proteins (FLAs) play important roles in the growth and development of roots, stems, and seeds in Arabidopsis. However, their biological functions in woody plants are largely unknown. In this work, we investigated the possible function of PtFLA6 in poplar. Quantitative real-time PCR, PtFLA6-yellow fluorescent protein (YFP) fusion protein subcellular localization, Western blotting, and immunohistochemical analyses demonstrated that the PtFLA6 gene was expressed specifically in the xylem of mature stem, and PtFLA6 protein was distributed ubiquitous in plant cells and accumulated predominantly in stem xylem fibres. Antisense expression of PtFLA6 in the aspen hybrid clone Poplar davidiana×Poplar bolleana reduced the transcripts of PtFLA6 and its homologous genes. Transgenic plants that showed a significant reduction in the transcripts of PtFLAs accumulated fewer PtFLA6 and arabinogalactan proteins than did the non-transgenic plants, leading to reduced stem flexural strength and stiffness. Further studies revealed that the altered stem biomechanics of transgenic plants could be attributed to the decreased cellulose and lignin composition in the xylem. In addition expression of some xylem-specific genes involved in cell wall biosynthesis was downregulated in these transgenic plants. All these results suggest that engineering the expression of PtFLA6 and its homologues could modulate stem mechanical properties by affecting cell wall composition in trees.

  13. Effects of CIITA antisense RNA on the expression of HLA class Ⅱ molecules

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    To study the effect of the major histocompatibility complex class Ⅱ (MHCⅡ) transactivator (CIITA) antisense RNA on the expression of the human leukemia (HLA) class Ⅱ molecules, 5′ end cDNA sequence of CIITA gene was cloned, and antisense RNA expression vector pcDNA-Ⅱ was constructed. HeLa cells transfected with pcDNA-Ⅱ and pcDNA3 were induced by IFN-g for 3 d. The expression of HLA class Ⅱ molecules on HeLa/pcDNA-Ⅱ cells was significantly decreased, while it has no effect on the expression of HLA class Ⅰ molecules. This result suggests that the CIITA antisense RNA can inhibit the expression of HLA class Ⅱ molecules in HeLa cells. It also implies a promising approach to generate immune tolerance in graft transplantation.

  14. Intracerebroventricular Administration of Mineralocorticoid Receptor Antisense Oligonucleotides Attenuates Salt Appetite in the Rat.

    Science.gov (United States)

    Ma; Itharat; Fluharty; Sakai

    1997-10-01

    The anterior ventral third ventricle (AV3V) region of the brain contains high concentrations of mineralocorticoid receptors (MR) and glucocorticoid receptors (GR) that are important in the maintenance of body fluid and electrolyte balance as well as other physiological processes. Daily intracerebroventricular pulse injections of MR antisense oligonucleotides significantly suppressed deoxycorticosterone acetate (DOCA) induced salt appetite in a dose-related manner. Similar administration of GR antisense or scrambled/sense oligonucleotide into the third ventricle failed to inhibit salt appetite. Salt appetite aroused after adrenalectomy was not suppressed by MR antisense oligonucleotide treatments but was suppressed by an antisense oligonucleotide directed against the angiotensin II AT1 receptor subtype. Receptor binding analysis demonstrated that MR and GR oligonucleotide treatments each reduced their respective receptor subtypes. Finally, although GR antisense oligonucleotide treatment was ineffective in suppressing DOCA-induced salt appetite, this treatment did increase stress induced corticosterone release as well as delayed the recovery of corticosterone to basal levels after stress. PMID:9787254

  15. Obstructive Effects of Ultrasonic Microbubble Intensifier on CHG-5 Cell with Survivin Antisense Oligonucleotides Transfection

    Institute of Scientific and Technical Information of China (English)

    CAO Hong-ying; CAO You-de; WANG Zhi-gang; LI Pan

    2008-01-01

    Objective:To study the effects on human glioma cell line CHG-5 by ultrasonic microbubble intensifier with survivin antisense oligonucleotides (ASODN)transfection. Methods: Antisense oligonucleotides targeting survivin mRNA was designed and synthesized.Four regimen groups were designed,group A:survivin antisense oligonucleotides transfected with ultrasonic microbubble intensifier combined with ultrasound irradiation,group B: survivin antisense oligonucleotides transfected with lipofectamine combined with ultrasound irradiation,group C:survivin antisense oligonucelotides with lipofectamine transfection.group D:blank control.The expression changes of surviving protein were measured by immunohistochemical staining and Western blotting,and MTr assay was used to measure the changes of proliferation.Results:Survivin protein expression in group A was decreased significantly in human glioma cell line CHG-5 than other groups(P<0.05),and the proliferating rate of CHG-5 in group A was also significantly inhibited(P<0.05).Conclusion:Ultrasonic microbubble intensifier transfection combined with ultrasound irradiation is a promising method in gene transfection effectively and noninvasively.

  16. Structure-dependent immunostimulatory effect of CpG oligodeoxynucleotides and their delivery system

    Directory of Open Access Journals (Sweden)

    Hanagata N

    2012-04-01

    Full Text Available Nobutaka HanagataNanotechnology Innovation Station, National Institute for Materials Science, Tsukuba, Ibaraki, and Graduate School of Life Science, Hokkaido University, Kita-ku, Sapporo, JapanAbstract: Unmethylated cytosine-phosphate-guanosine (CpG oligodeoxynucleotides (ODNs are recognized by Toll-like receptor 9 (TLR9 found in antigen-presenting cells and B cells and can activate the immune system. Using CpG ODNs as an adjuvant has been found to be effective for treating infectious diseases, cancers, and allergies. Because natural ODNs with only a phosphodiester backbone are easily degraded by nuclease (deoxyribonuclease [DNase] in serum, CpG ODNs with a phosphorothioate backbone have been studied for clinical application. CpG ODNs with a phosphorothioate backbone have raised concern regarding undesirable side effects; however, several CpG ODNs with only a phosphodiester backbone have been reported to be stable in serum and to show an immunostimulatory effect. In recent years, research has been conducted on delivery systems for CpG ODNs using nanoparticles (NPs. The advantages of NP-based delivery of CpG ODN include (1 it can protect CpG ODN from DNase, (2 it can retain CpG ODN inside the body for a long period of time, (3 it can improve the cellular uptake efficiency of CpG ODN, and (4 it can deliver CpG ODN to the target tissues. Because the target cells of CpG ODN are cells of the immune system and TLR9, the receptor of CpG ODN is localized in endolysosomes, CpG ODN delivery systems are required to have qualities different from other nucleic acid drugs such as antisense DNA and small interfering RNA. Studies until now have reported various NPs as carriers for CpG ODN delivery. This review presents DNase-resistant CpG ODNs with various structures and their immunostimulatory effects and also focuses on delivery systems of CpG ODNs that utilize NPs. Because CpG ODNs interact with TLR9 and activate both the innate and the adaptive immune

  17. Inhibitory effect of human telomerase antisense oligodeoxyribonucleotides on the growth of gastric cancer cell lines in variant tumor pathological subtype

    Institute of Scientific and Technical Information of China (English)

    Jing Ye; Yun-Lin Wu; Shu Zhang; Zi Chen; Li-Xia Guo; Ruo-Yu Zhou; Hong Xie

    2005-01-01

    AIM: To investigate the inhibitory effect of specialized human telomerase antisense oligodeoxyribonucleotides on the growth of well (MKN-28), moderately (SGC-7901)and poorly (MKN-45) differentiated gastric cancer cell lines under specific conditions and its inhibition mechanism,and to observe the correlation between the growth inhibition ratio and the tumor pathologic subtype of gastric cancer cells.METHODS: Telomerase activity in three gastric cancer cell lines of variant tumor pathologic subtype was determined by modified TRAP assay before and after the specialized human telomerase antisense oligodeoxyribonucleotides were dealt with under specific conditions. Effect of antisense oligomer under specific conditions of the growth and viability of gastric cancer cell lines was explored by using trypan blue dye exclusion assay, and cell apoptosis was detected by cell morphology observation, flow cytometry and TUNEL assay.RESULTS: Telomerase activity was detected in well,moderately and poorly differentiated gastric cancer cell lines (the quantification expression of telomerase activity was 43.7TPG, 56.5TPG, 76.7TPG, respectively).Telomerase activity was controlled to 30.2TPG, 36.3TPG and 35.2TPG for MKN-28, SGC-7901 and MKN-45 cell lines respectively after treatment with human telomerase antisense oligomers at the concentration of 5 μmol/L, and was entirely inhibited at 10 μmol/L, against the template region of telomerase RNA component, whereas no inhibition effect was detected in missense oligomers (P<0.05). After treatment with antisense oligomers at different concentrations under specific conditions for 96 h, significant growth inhibition effects were found in MKN-45 and SGC-7901gastric cancer cell lines (the inhibition ratio was 40.89%and 71.28%), but not in MKN-28 cell lines (15.86%). The ratio of inactive SGC-7901 cells increased according to the prolongation of treatment from 48 to 96 h. Missense oligomers could not lead to the same effect (P<0

  18. Variable coordination of cotranscribed genes in Escherichia coli following antisense repression

    Directory of Open Access Journals (Sweden)

    Kulyté Agne

    2006-11-01

    Full Text Available Abstract Background A majority of bacterial genes belong to tight clusters and operons, which complicates gene functional studies using conventional knock-out methods. Antisense agents can down-regulate the expression of genes without disrupting the genome because they bind mRNA and block its expression. However, it is unclear how antisense inhibition affects expression from genes that are cotranscribed with the target. Results To examine the effects of antisense inhibition on cotranscribed genes, we constructed a plasmid expressing the two reporter genes gfp and DsRed as one transcriptional unit. Incubation with antisense peptide nucleic acid (PNA targeted to the mRNA start codon region of either the upstream gfp or the downstream DsRed gene resulted in a complete expression discoordination from this artificial construct. The same approach was applied to the three cotranscribed genes in the endogenously expressed lac-operon (lacZ, Y and A and partial downstream expression coordination was seen when the lacZ start codon was targeted with antisense PNA. Targeting the lacY mRNA start codon region showed no effect on the upstream lacZ gene expression whereas expression from the downstream lacA gene was affected as strongly as the lacY gene. Determination of lacZ and lacY mRNA levels revealed a pattern of reduction that was similar to the Lac-proteins, indicating a relation between translation inhibition and mRNA degradation as a response to antisense PNA treatment. Conclusion The results show that antisense mediated repression of genes within operons affect cotranscribed genes to a variable degree. Target transcript stability appears to be closely related to inhibition of translation and presumably depends on translating ribosomes protecting the mRNA from intrinsic decay mechanisms. Therefore, for genes within operons and clusters it is likely that the nature of the target transcript will determine the inhibitory effects on cotranscribed genes

  19. Functionalization of an Antisense Small RNA

    Science.gov (United States)

    Rodrigo, Guillermo; Prakash, Satya; Cordero, Teresa; Kushwaha, Manish; Jaramillo, Alfonso

    2016-01-01

    In order to explore the possibility of adding new functions to preexisting genes, we considered a framework of riboregulation. We created a new riboregulator consisting of the reverse complement of a known riboregulator. Using computational design, we engineered a cis-repressing 5′ untranslated region that can be activated by this new riboregulator. As a result, both RNAs can orthogonally trans-activate translation of their cognate, independent targets. The two riboregulators can also repress each other by antisense interaction, although not symmetrically. Our work highlights that antisense small RNAs can work as regulatory agents beyond the antisense paradigm and that, hence, they could be interfaced with other circuits used in synthetic biology. PMID:26756967

  20. Functional comparison of antisense proteins of HTLV-1 and HTLV-2 in viral pathogenesis

    Directory of Open Access Journals (Sweden)

    Benoit eBarbeau

    2013-08-01

    Full Text Available The production of antisense transcripts from the 3’ long terminal repeat (LTR in human T-lymphotropic retroviruses has now been clearly demonstrated. After the identification of the antisense strand-encoded HTLV-1 bZIP (HBZ factor, we reported that HBZ could interact with CREB transcription factors and consequently turn off the important activating potential of the viral Tax protein on HTLV-1 5’ LTR promoter activity. We have recently accumulated new results demonstrating that antisense transcripts also exist in HTLV-2, -3 and -4. Furthermore, our data have confirmed the existence of encoded proteins from these antisense transcripts (termed antisense proteins of HTLVs or APHs. APHs are also involved in the down-regulation of Tax-dependent viral transcription. In this review, we will focus on the different molecular mechanisms used by HBZ and APH-2 to control viral expression. While HBZ interacts with CREB through its basic zipper domain, APH-2 binds to this cellular factor through a five amino acid motif localized in its carboxyl terminus. Moreover, unlike APH-2, HBZ possesses an N-terminal activation domain that also contributes to the inhibition of the viral transcription by interacting with the KIX domain of p300/CBP. On the other hand, HBZ was found to induce T-cell proliferation while APH-2 was unable to promote such proliferation. Interestingly, HTLV-2 has not been causally linked to human T-cell leukemia, while HTLV-1 is responsible for the development of the Adult T-cell Leukemia/Lymphoma (ATLL. We will further discuss the possible role played by antisense proteins in the establishment of pathologies induced by viral infection.

  1. Antisense oligonucleotides-induced local blockade of T-bet expression leads to airway inflammation in rats

    Institute of Scientific and Technical Information of China (English)

    Gang WANG; Chun-tao LIU; Zeng-li WANG; Li-li JIANG; Cuniang YAN; Feng-min LUO

    2006-01-01

    Aim: To explore whether local blockade of T-box expressed in T cells (T-bet) expression in the 1ungs could lead to airway inflammation. Methods: Twenty-four rats were randomly divided into 4 groups: saline group, ovalbumin (OVA)-sensitized group, nonsense group, and the antisense group. The OVA-sensitized rats were sensitized and challenged with OVA, and the rats in the nonsense and antisense groups were subjected to an aerosol delivery of the nonsense and antisense oligonucleotides (AS-ODN)of T-bet(0.1%, w/v). The levels of interferon-γ(IFN-γ), interleukin-4(IL-4), and IL-5 in the bronchoalveolar lavage fluid (BALF) were detected by ELISA, and the mRNA and the protein expression of T-bet and GATA-3 genes were examined by in situ hybridization and Western blot analysis, respectively. Results: The expression of T-bet mRNA and protein in the lungs of the rats in the antisense group were inhibited effectively. The lungs of the rats in the antisense and OVA-sensitized groups showed eosinophil and lymphocyte inflammatory infiltration, and eosinophilia located predominantly around the airways. The number of GATA-3 mRNA-positive cells and the level of GAllA-3 protein in the 1ungs of the rats in the antisense and the OVA-sensitized groups significantly increased. The level of IL-4 and IL-5 in the BALF in the antisense and OVA-sensitized groups were elevated, but the level of IFN-γ decreased markedly. Conclusion: Antisense ODN-induced local blockade of T-bet expression leads to airway inflammation with a selective alteration in patterns of cytokine expression and recruitment of eosinophil cells similar to that in the OVA-sensitized

  2. Bloqueo del receptor del factor de crecimiento semejante a la Insulina Tipo I utilizando oligodeoxinucleótidos antisentido en cáncer de mama experimental Type I insulin-like growth factor receptor antisense strategies in experimental breast cancer

    Directory of Open Access Journals (Sweden)

    Mariana Salatino

    2004-04-01

    Full Text Available Evaluamos el efecto del bloqueo de la expresión del receptor del factor de crecimiento semejante a la insulina tipo I (IGF-IR sobre el crecimiento in vivo de cáncer de mama empleando una estrategia "antisentido". Utilizamos el adenocarcinoma mamario murino progestágeno-dependiente C4HD. La administración intratumoral o sistémica de oligodeoxinucleótidos antisentido fosfotiolados al ARNm del IGF-IR (AS[S]ODN inhibió el crecimiento tumoral. El efecto antitumoral fue específico debido a su dosis-dependencia y a la falta de efecto en ratones tratados con el S[S]ODN "sentido". Los tumores obtenidos de ratones tratados con AS[S]ODN mostraron: disminución en la expresión de IGF-IR y en la fosforilación del sustrato del receptor de insulina-1, inhibición de la activación de PI-3K/Akt, p42/p44MAPK y ErbB-2, mientras que la expresión y activación del receptor de progesterona no se afectó. Es la primera demostración que el crecimiento de cáncer de mama puede ser inhibido por la administración in vivo de AS[S]ODN al IGF-IR.We addressed the effect of targeting type I insulin-like growth factor receptor (IGF-IR, with antisense strategies in in vivo growth of breast cancer cells. We used C4HD tumors from an experimental model of hormonal carcinogenesis in which medroxyprogesterone acetate induced mammary adenocarcinomas in Balb/c mice. Intratumor or systemic administration of phosphorothiolated antisense oligodeoxynucleotides (AS[S]ODN to IGF-IR mRNA resulted in a significant inhibition of C4HD tumor growth. The antitumor effect was specific since inhibition of tumor growth was dose-dependent and no effect was observed in mice treated with sense S[S]ODN. Tumors from AS[S]ODN-treated mice showed a decrease in IGF-IR expression and in insulin receptor substrate-1 tyrosine phosphorylation. Activation of PI-3K/Akt, p42/p44 MAPK and ErbB-2 was abolished in tumors treated with AS[S]ODN. Progesterone receptor expression or activity remained

  3. Identification and characterization of high affinity antisense PNAs for the human unr (upstream of N-ras) mRNA which is uniquely overexpressed in MCF-7 breast cancer cells.

    Science.gov (United States)

    Fang, Huafeng; Yue, Xuan; Li, Xiaoxu; Taylor, John-Stephen

    2005-01-01

    We have recently shown that an MCF-7 tumor can be imaged in a mouse by PET with 64Cu-labeled Peptide nucleic acids (PNAs) tethered to the permeation peptide Lys4 that recognize the uniquely overexpressed and very abundant upstream of N-ras or N-ras related gene (unr mRNA) expressed in these cells. Herein we describe how the high affinity antisense PNAs to the unr mRNA were identified and characterized. First, antisense binding sites on the unr mRNA were mapped by an reverse transcriptase random oligonucleotide library (RT-ROL) method that we have improved, and by a serial analysis of antisense binding sites (SAABS) method that we have developed which is similar to another recently described method. The relative binding affinities of oligodeoxynucleotides (ODNs) complementary to the antisense binding sites were then qualitatively ranked by a new Dynabead-based dot blot assay. Dissociation constants for a subset of the ODNs were determined by a new Dynabead-based solution assay and were found to be 300 pM for the best binders in 1 M salt. PNAs corresponding to the ODNs with the highest affinities were synthesized with an N-terminal CysTyr and C-terminal Lys4 sequence. Dissociation constants of these hybrid PNAs were determined by the Dynabead-based solution assay to be about 10 pM for the highest affinity binders. PMID:16314303

  4. Integrinβ1 asODN-inhibition of Cell Attachment and Invasion of the Human Gastric Cancer Cell Line SGC7901

    Institute of Scientific and Technical Information of China (English)

    Wu Dong; Yuehong Cui; Huimian Xu

    2005-01-01

    OBJECTIVE To study the inhibition of adhesion and invasion of SGC7901cells into the ECM by integrinβ1 antisense oligodeoxynucleotide asODN.METHODS asODN and control ODN were transfected into SGC7901 cells using liposomes as vectors. The distribution of the ODN was followed by immunochemistry and changes in the expression of integrinβ1 mRNA and protein were determined by RT-PCR and FCM, respectively. The adhesion and invasion into the ECM were measured by the MTT and Boyden chamber methods, respectively.RESULTS Integrinβ1 asODN which was transfected into SGC7901 cells distributed evenly in the cytoplasm and nucleus. PCR and FCM revealed a weakened band at 489bp and a left-shift curve, respectively. Adhesion and invasion assays showed decreased activity with an inhibition rate of 54% and 76%. The extent of decrease induced by integrinβ1 asODN was larger than that caused by random control ODN (P<0.001).CONCLUSION Transfection of integrinβ1 asODN into SGC7901 cells induced a decrease in the expression of integrinβ1 mRNA and protein,resulting in a decrease in adhesion and invasion into the ECM, with a greater effect than random control ODN.

  5. Antisense RNA: a genetic approach to cell resistance against Parvovirus. RNA antisentido: una aproximacion de resistencia genetica a Parvovirus

    Energy Technology Data Exchange (ETDEWEB)

    Ramirez Martinez, J.C.

    1992-01-01

    The Minute Virus of Mice (MVMp), an autonomous Parvovirus that replicates cytolytically in the A9 mouse fibroblast cell line, was interfered by constitutive expression of an antisense RNA targeted against the major non-structural NS-1 protein. Permanently transfected A9 clones expressing NS-1 antisense, showed increased proliferative capacity upon virus infection, and likewise cultures infected at low multiplicity by MVMp reached confluence overcoming virus growth. Correspondingly, an inhibition in virus multiplication was demonstrated by a significant lower virus production and plaque forming ability in clones expressing antisense RNa. At the molecular level, several fold reduction in viral DNA, RNA and proteins was quantitated by respective analysis of Southern, RNase protection and bidimensional gels. Remarkably, the accumulation of all three viral messengers(R1,R2,R3) was decreased both in the cytoplasm and in the nucleus, suggesting that antisense-mediated inhibition is primarily exerted at the level of viral transcription or nuclear post-transcriptional events. Thus, this system illustrates the possibility to create an antisense-mediated protective stage to highly cytotoxic viruses in permissive cells, by down-modulation the expression of a transactivator of virus genes. (author)180 refs., 25 figs.

  6. Antisense RNA: a genetic approach to cell resistance against Parvovirus; RNA antisentido: una aproximacion de resistencia genetica a Parvovirus

    Energy Technology Data Exchange (ETDEWEB)

    Ramirez Martinez, J.C.

    1992-12-31

    The Minute Virus of Mice (MVMp), an autonomous Parvovirus that replicates cytolytically in the A9 mouse fibroblast cell line, was interfered by constitutive expression of an antisense RNA targeted against the major non-structural NS-1 protein. Permanently transfected A9 clones expressing NS-1 antisense, showed increased proliferative capacity upon virus infection, and likewise cultures infected at low multiplicity by MVMp reached confluence overcoming virus growth. Correspondingly, an inhibition in virus multiplication was demonstrated by a significant lower virus production and plaque forming ability in clones expressing antisense RNa. At the molecular level, several fold reduction in viral DNA, RNA and proteins was quantitated by respective analysis of Southern, RNase protection and bidimensional gels. Remarkably, the accumulation of all three viral messengers(R1,R2,R3) was decreased both in the cytoplasm and in the nucleus, suggesting that antisense-mediated inhibition is primarily exerted at the level of viral transcription or nuclear post-transcriptional events. Thus, this system illustrates the possibility to create an antisense-mediated protective stage to highly cytotoxic viruses in permissive cells, by down-modulation the expression of a transactivator of virus genes. (author)180 refs., 25 figs.

  7. Effects of recombinant epidermal growth factor receptor antisense adenovirus combined with irradiation on breast cancer cells

    International Nuclear Information System (INIS)

    Objective: To investigate the effects of a recombinant antisense adenovirus for epidermal growth factor receptor (EGFR) combined with irradiation on breast cancer cells. Methods: Human EGFR cDNA fragment was subcloned in the opposite orientation to the cytomegaloviral promoter and inserted into a E1/E3-deleted type 5 adenoviral vector to obtain AdE5 construct which expresses EGFR antisense RNA. Combined with γ-ray irradiation, its effects on clonogenicity and cell cycle phase distribution were studied in a human breast cancer line MDA-MB-23. Results: EGFR protein expression was dramatically inhibited in MDA-MB-231 cells after AdE5 infection. The post-irradiation clonogenicity was reduced by AdE5 in a viral and irradiation dose-dependent manner. Further cytometric analysis showed that AdE5 infection at a MOI of 300 pfu/cell induced a cell cycle progression from radio-resistant G0 + G1 phases to radiosensitive G2 + M phases, resulting in a synergistic effect after combination of these two treatments. Conclusions: The transduction of EGFR antisense RNA by adenoviral vector is effective for antisense strategy targeting EGFR, and increases the cell-killing effect of ionizing radiation on breast cancer cells.(authors)

  8. CpG Oligodeoxynucleotide1826 combined with radioresistant cancer cell vaccine confers significant antitumor effects.

    Science.gov (United States)

    Zhuang, X B; Xing, N; Zhang, Q; Yuan, S J; Chen, W; Qiao, T K

    2015-01-01

    Immunotherapy is a hot issue in cancer research over the years and tumor cell vaccine is one of the increasing number of studies. Although the whole tumor cell vaccine can provide the best source of immunizing antigens, there is still a limitation that most tumors are not naturally immunogenic. CpG Oligodeoxynucleotides (CpG ODNs), synthetic oligonucleotides containing a cytosine-phosphate-guanine(CpG) motif, was shown to enhance immune responses to a wide variety of antigens. In this study, we generated the radioresistant Lewis lung cancer cell by repeated X-ray radiation and inactivated it as a whole tumor cell vaccine to enhance the immunogenicity of tumor cell vaccine. Mice were subcutaneously immunized with this inactivated vaccine combined with CpG ODN1826 and then inoculated with autologous Lewis lung cancer (LLC) to estimate the antitumor efficacy. The results showed that the radioresistant tumor cell vaccine combined with CpG ODN1826 could significantly inhibit tumor growth, increased survival of the mice and with 20% of the mice surviving tumor free in vivo compared with the unimmunized mice bearing LLC tumor. A significant increase of apoptosis was also observed in the tumor prophylactically immunized with vaccine of inactivated radioresistant tumor cell plus CpG ODN1826. The potent antitumor effect correlated with higher secretion levels of tumor necrosis factor-alpha(TNF-α) and lower levels of interleukin-10(IL-10) concentration in serum. Furthermore, the results suggested that the antitumor mechanism was probably depended on the decreased level of programmed death ligand-1(PD-L1) which plays an important role in the negative regulation of immune response by the inhibition of tumor antigen-specific T cell activation. These findings clearly demonstrated that the radioresistant tumor cell vaccine combined with CpG ODN1826 as an appropriate adjuvant could induce effective antitumor immunity in vivo. PMID:26458317

  9. The Inhibitory Effects of an Antisense u-PAR Vector on Invasion of Highly Invasive Human Prostate Carcinoma PC-3M Cell Subclones

    Institute of Scientific and Technical Information of China (English)

    廖国宁; 李清芬; 冯友梅; 邓耀祖; 李卓娅; 龚非力; 马丁

    2003-01-01

    Summary: To observe the inhibitory effects of an antisense u-PAR vector on invasion of highly inva-sive PC-3M cell subclones, the effects of the antisense u-PAR on activity of MMP-9 in those highlyinvasive cell subclones were detected by a quantitative RT-PCR and zymography. The monolayer in-vasion assay and colony formation assay in soft agar were used. And tumorigenesis rate and invasionsby the cell subclones with or without the antisense u-PAR were observed in nude mice. It was foundthat in vitro growth of highly invasive PC-3M cell subclones transfected with the antisense u-PARwas declined, and the ability of anchorage-independent growth of those cell subclones was found de-creased sharply, with the inhibiting rate becoming 79 % and 60 %, respectively. Although the anti-sense u-PAR didn't change MMP-9 gene transcription, they could inhibit the activation of MMP-9 ofhighly invasive PC-3M cell subclones. Moreover, the tumorigenesis rate of the cell subclones with theantisense u-PAR decreased and the growth of a neoplasm also slowed down. Thet tests showed thedifference between experimental and control groups was statistically significant (P<0. 01). The anti-sense u-PAR vector could not only inhibit the invasion ability of highly invasive PC-3M cell subclonesin vitro but also restrain the growth of those cell subclones in vivo.

  10. Effect of antisense human telomerase RNA on malignant behaviors of gastric carcinoma cell line SGC-7901

    Institute of Scientific and Technical Information of China (English)

    YANG Jin-liang; FANG Dian-chun; YANG Shi-ming; LUO Yuan-hui; LUO Kun-lun; LU Rong; LIU Wei-wen

    2001-01-01

    Objective: To study the effects of antisense human telomerase RNA (ahTR) transfection on the malignant behaviors of gastric carcinoma cell line SGC-7901 and its potential role in gene therapy for tumor. Methods: An antisense hTR eukaryotic expression vector containing the sequence of template region of telomere repeats was transfected into gastric carcinoma cell line SGC-7901 with liposome DOTAP. The expressions of hTR RNA and antisense hTR RNA were observed with RT-PCR, telomerase activity with PCR-ELISA. Telomere length was measured with Southern blot. Cell morphology and cellular proliferation capacity were studied with MTT assay. Cell cycle distribution and apoptotic state were observed with flow cytometry. Efficiency of clone formation in soft agar and tumorigencity in nude mice were examined and evaluated in ahTR-transfected 7901 cells, and plasmid pCL-neo transfected 7901 cells and parental 7901 cells served as control. Results: An antisense hTR eukaryotic expression vector was transfected into 7901 cells successfully. The telomerase activity in ahTR-transfected 7901 cells was decreased from 100% to about 25%, and telomere length in the cells shortened from 4.08 kb to 3.35 kb at 60 population doublings (PDs). Compared with parental 7901 and pCL-neo transfected 7901 cells, ahTR-transfected 7901 cells displayed some morphological changes, including decreased cell atypia and nucleus/cytoplasm ratio under light microscope. Furthermore, ahTR-transfected 7901 cells displayed growth inhibition, decreased invasive capacity in Borden's chamber invasive model, increased G0/G1 phase rate and apoptotic rate, and restored contact inhibition and density inhibition. Surprisingly, ahTR-transfected 7901 cells lost their capacity of clone formation in soft agar and carcinogensis in nude mice. Conclusion: Antisense hTR transfection can induce 7901 cell differentiation and reverse its malignant phenotype. This study provides an exciting approach for cancer therapy through the

  11. Effect of NHE1 antisense gene transfection on the biological behavior of SGC-7901 human gastric carcinoma cells

    Institute of Scientific and Technical Information of China (English)

    Hai-Feng Liu; Xiao-Chun Teng; Jing-Chen Zheng; Gang Chen; Xing-Wei Wang

    2008-01-01

    AIM: To study the effect of type 1 Na+/H+ exchanger (NHE1) antisense human gene transfection on the biological behavior of gastric carcinoma cell line SGC-7901.METHODS: Antisense NHE1 eukaryotic expression on vector pcDNA3.1 was constructed by recombinant DNA technique and transfected into gastric carcinoma cell line SGC-7901 with DOTAP liposome transfection method.Morphological changes of cells were observed with optic and electron microscopes. Changes in cell proliferative capacity, apoptosis, intracellular pH (pH1), cell cycle,clone formation in two-layer soft agar, and tumorigenicity in nude mice were examined.RESULTS: Antisense eukaryotic expressing vectors were successfully constructed and transfected into 5GC-7901.The transfectant obtained named 7901-antisense (7901-,45) stablely produced antisense NHE1. There was a significant difference between the pH1 of 7901-AS cells (6.77 ± 0.05) and that of 7901-zeo cells and SGC-7901 cells (7.24 ± 0.03 and 7.26 ± 0.03, P < 0.01). Compared with SGC-7901 and 7901-zeo cells, 7901-AS cells mostly showed cell proliferation inhibition, G1/Go phase arrest, increased cell apoptotic rate, recovery of contact inhibition, and density contact. The tumorigenicity in nude mice and cloning efficiency in the two-layer soft agar were dearly inhibited.CONCLUSION: NHE1 antisense gene significantly restrains the malignant behavior of human gastric carcinoma cells, suppresses cell growth and induces cell apoptosis, and partially reverses the malignant phenotypes of SGC-7901. These results suggest a potential role for human tumor gene therapy.

  12. The prevalence and regulation of antisense transcripts in Schizosaccharomyces pombe.

    Directory of Open Access Journals (Sweden)

    Ting Ni

    Full Text Available A strand-specific transcriptome sequencing strategy, directional ligation sequencing or DeLi-seq, was employed to profile antisense transcriptome of Schizosaccharomyces pombe. Under both normal and heat shock conditions, we found that polyadenylated antisense transcripts are broadly expressed while distinct expression patterns were observed for protein-coding and non-coding loci. Dominant antisense expression is enriched in protein-coding genes involved in meiosis or stress response pathways. Detailed analyses further suggest that antisense transcripts are independently regulated with respect to their sense transcripts, and diverse mechanisms might be potentially involved in the biogenesis and degradation of antisense RNAs. Taken together, antisense transcription may have profound impacts on global gene regulation in S. pombe.

  13. Using both strands: The fundamental nature of antisense transcription.

    Science.gov (United States)

    Murray, Struan C; Mellor, Jane

    2016-01-01

    Non-coding transcription across the antisense strands of genes is an abundant, pervasive process in eukaryotes from yeast to humans, however its biological function remains elusive. Here, we provide commentary on a recent study of ours, which demonstrates a genome-wide role for antisense transcription: establishing a unique, dynamic chromatin architecture over genes. Antisense transcription increases the level of nucleosome occupancy and histone acetylation at the promoter and body of genes, without necessarily modulating the level of protein-coding sense transcription. It is also associated with high levels of histone turnover. By allowing genes to sample a wider range of chromatin configurations, antisense transcription could serve to make genes more sensitive to changing signals, priming them for responses to developmental programs or stressful cellular environments. Given the abundance of antisense transcription and the breadth of these chromatin changes, we propose that antisense transcription represents a fundamental, canonical feature of eukaryotic genes.

  14. CpG-Oligodeoxynucleotide Treatment Protects against Ionizing Radiation-Induced Intestine Injury.

    Directory of Open Access Journals (Sweden)

    Chao Zhang

    Full Text Available the bone marrow and the intestine are the major sites of ionizing radiation (IR-induced injury. Our previous study demonstrated that CpG-oligodeoxynucleotide (ODN treatment mitigated IR-induced bone marrow injury, but its effect on the intestine is not known. In this study, we sought to determine if CpG-ODN have protective effect on IR-induced intestine injury, and if so, to determine the mechanism of its effect.Mice were treated with CpG-ODN after IR. The body weight and survival were daily monitored for 30 days consecutively after exposure. The number of surviving intestinal crypt was assessed by the microcolony survival assay. The number and the distribution of proliferating cell in crypt were evaluated by TUNEL assay and BrdU assay. The expression of Bcl-2, Bax and caspase-3 in crypt were analyzed by Immunohistochemistry assay. The findings showed that the treatment for irradiated mice with CpG-ODN diminished body weight loss, improved 30 days survival, enhanced intestinal crypts survival and maintained proliferating cell population and regeneration in crypt. The reason might involve that CpG-ODN up-regulated the expression of Bcl-2 protein and down-regulated the expression of Bax protein and caspase-3 protein.CpG-ODN was effective in protection of IR-induced intestine injury by enhancing intestinal crypts survival and maintaining proliferating cell population and regeneration in crypt. The mechanism might be that CpG-ODN inhibits proliferating cell apoptosis through regulating the expression of apoptosis-related protein, such as Bax, Bcl-2 and caspase-3.

  15. Immunostimulatory oligodeoxynucleotide from Bifidobacterium longum suppresses Th2 immune responses in a murine model.

    Science.gov (United States)

    Takahashi, N; Kitazawa, H; Iwabuchi, N; Xiao, J Z; Miyaji, K; Iwatsuki, K; Saito, T

    2006-07-01

    We have reported previously that novel immunostimulatory sequence (ISS) oligodeoxynucleotide (ODN) BL07S from a probiotic strain of Bifidobacterium longum inhibited immunoglobulin (Ig) E production in vitro. However, whether ISS-ODNs from probiotics regulate T helper type 2 (Th2)-polarized immune reactions in vivo remains unclear. To evaluate the inhibitory effects of ODN BL07S on type I allergic response, BALB/c mice were injected with or without ODN BL07S in the presence of ovalbumin (OVA) on days 0 and 14. Serum Ig levels (IgE, IgG1 and IgG2a) and cytokine levels (interferon (IFN)-gamma, interleukin (IL)-12, IL-4, IL-5, IL-10 and IL-13) were investigated in splenocyte cultures from days 14-28. Production of OVA-specific and total IgE were significantly suppressed by administration of ODN BL07S, but not by ODN BL06S, a non-ISS-ODN. Compared to controls, ODN BL07S induced significantly lower levels of Th2 cytokines (IL-4 and IL-5) in splenocyte cultures, and significantly higher levels of serum OVA-specific IgG2a. These effects of ODN BL07S on modulation of Th2 immune response were dose-dependent. The present results demonstrate that ODN BL07S from genomic DNA of B. longum BB536 prevents antigen-induced Th2 immune responses in vivo, suggesting that ISS-ODNs from probiotics might be useful in preventing allergic disease.

  16. Amphiregulin Antisense RNA Delivered by Adnovirus Suppresses Growth of Breast Cancer Cells In Vitro and In Vivo

    Institute of Scientific and Technical Information of China (English)

    Lin Ma; Voahangy Randrianarison; Fabien Calvo

    2005-01-01

    OBJECTIVE To investigate the therapeutic potential of amphiregulin antisense RNA delivered by adenoviral vector in a human breast cancer model.METHODS Human amphiregulin cDNA was subcloned in the opposite orientation to the cytomegaloviral promoter and inserted into an E1/E3-deleted type 5 adenoviral vector to obtain an AdA4 construct which expresses amphiregulin antisense mRNA. Both in vitro and in vivo antiproliferative effects of the antisense RNA were studied by infecting transformed human breast epithelial NS2T2A1 cells and tumors.RESULTS Amphiregulin protein expression was inhibited dramatically in the NS2T2A1 cells after infection with AdA4. The in vitro cell growth was inhibited significantly at day 4 post-AdA4 infection compared with control empty virus AdC1 at a MOl of 200 and 400 pfu/cell to 69.3% and 49.8%, respectively (P<0.02, P<0.005). After 3 intra-tumoral injections of 109 pfu AdA4, tumor volumes were reduced to 40.6% of that of the control group at day 35 (P<0.005).CONCLUSION The transfer of amphiregulin RNA antisense by adenoviral vector is effective for amphiregulin targeting strategy, leading to an inhibition of in vitro cell proliferation and in vivo tumor growth in this breast cancer model.

  17. Evaluation of Morpholino Antisense Oligos’ Role on BCR-ABL Gene Silencing in the K562 Cell Line

    Directory of Open Access Journals (Sweden)

    Bahman Delalat

    2010-01-01

    Full Text Available Objective: Chronic myeloid leukemia (CML develops when a hematopoietic stem cellacquires the BCR/ABL fusion gene. This causes these transformed hematopoietic cellsto have a greater than normal proliferation rate. Scientists attempt to improve the CMLtreatment process by silencing the BCR/ABL oncogene. In this work, we used morpholinoantisense oligos to silence the BCR/ABL oncogene.Materials and Methods: In this study, the K562 was used as a BCR/ABL fusion-genepositive cell line and the Jurkat cell line as a control. We explored the inhibiting capacityof morpholino antisense oligos in the the expression of the BCR/ABL oncogene andstudied their p210 BCR/ABL suppression, inhibition of cell proliferation and stimulation ofapoptosis in the K562 cells after 24 and 48 hours. Endo-Porter was used for delivery ofmorpholino antisense oligos into cell cytosols. Meanwhile, flow cytometric analysis wasperformed in order to determine the appropriate concentration of morpholino antisenseoligos.Results: Prolonged exposure of the K562 cell line to the morpholino antisense oligostargeted against the BCR-ABL gene showed proliferation inhibition as its main feature.After western blotting, we found that complete silencing of BCR/ABL was achieved, butflow cytometric analysis showed no broad apoptosis.Conclusion: The results indicate that the Morpholino antisense oligo is able to inhibitp210 BCR/ABL; however, it cannot induce broad apoptosis due to co-silencing of BCR.

  18. Diversification of antisense research and development: review of the Ringberg meeting, April 1994. Mechanisms of antisense-mediated gene silencing.

    Science.gov (United States)

    Hawkins, J W; Nellen, W

    1994-01-01

    Antisense technology has established itself as a new and vibrant entrant into the discipline of molecular biology. As such, it has contributed to basic research by providing tools for the molecular dissection of diverse experimental systems. In applied research, antisense approaches have contributed to development of agricultural products (D. Grierson) now coming to market and to the design of a number of oligonucleotide drugs, now in clinical trials. However, few activities to date have focused on the study of antisense per se. Further, few conceptual perspectives have regarded antisense as an integral part of cellular function and genetic regulation. The Ringberg conference showcased a number of systems that would seem unrelated if we regard antisense as a superficial tool to be imposed on nature. On the other hand, if we want to begin to regard antisense as a field of its own with deeper biological and genetic rationales, the Ringberg meeting provided much tantalizing evidence to do so.

  19. Response of porcine peripheral blood mononuclear cells to CpG-containing oligodeoxynucleotide

    DEFF Research Database (Denmark)

    Kamstrup, Søren; Verthelyi, D.; Klinman, D.M.

    2001-01-01

    in bacterial but not vertebrate DNA. Different motifs are optimally stimulatory in different species. This work examines whether oligodeoxynucleotides (ODNs) containing CpG motifs stimulate peripheral blood mononuclear cells from pigs. Results show that pigs respond to CpG ODN by proliferating and secreting IL...

  20. 78 FR 26794 - Prospective Grant of Exclusive License: Use of Oligodeoxynucleotide as Neuroprotectants in...

    Science.gov (United States)

    2013-05-08

    ... Oligodeoxynucleotide as Neuroprotectants in Cerebral and Other Ischemic Injury AGENCY: National Institutes of Health... neuroprotectants in cerebral ischemic injury. The field of use may be limited to pharmacological preconditioning against excitotoxic injury, ischemia and/or hypoxia. Properly filed competing applications for a...

  1. Voltage-gated calcium channel and antisense oligonucleotides thereto

    Science.gov (United States)

    Hruska, Keith A. (Inventor); Friedman, Peter A. (Inventor); Barry, Elizabeth L. R. (Inventor); Duncan, Randall L. (Inventor)

    1998-01-01

    An antisense oligonucleotide of 10 to 35 nucleotides in length that can hybridize with a region of the .alpha..sub.1 subunit of the SA-Cat channel gene DNA or mRNA is provided, together with pharmaceutical compositions containing and methods utilizing such antisense oligonucleotide.

  2. The landscape of antisense gene expression in human cancers.

    Science.gov (United States)

    Balbin, O Alejandro; Malik, Rohit; Dhanasekaran, Saravana M; Prensner, John R; Cao, Xuhong; Wu, Yi-Mi; Robinson, Dan; Wang, Rui; Chen, Guoan; Beer, David G; Nesvizhskii, Alexey I; Chinnaiyan, Arul M

    2015-07-01

    High-throughput RNA sequencing has revealed more pervasive transcription of the human genome than previously anticipated. However, the extent of natural antisense transcripts' (NATs) expression, their regulation of cognate sense genes, and the role of NATs in cancer remain poorly understood. Here, we use strand-specific paired-end RNA sequencing (ssRNA-seq) data from 376 cancer samples covering nine tissue types to comprehensively characterize the landscape of antisense expression. We found consistent antisense expression in at least 38% of annotated transcripts, which in general is positively correlated with sense gene expression. Investigation of sense/antisense pair expressions across tissue types revealed lineage-specific, ubiquitous and cancer-specific antisense loci transcription. Comparisons between tumor and normal samples identified both concordant (same direction) and discordant (opposite direction) sense/antisense expression patterns. Finally, we provide OncoNAT, a catalog of cancer-related genes with significant antisense transcription, which will enable future investigations of sense/antisense regulation in cancer. Using OncoNAT we identified several functional NATs, including NKX2-1-AS1 that regulates the NKX2-1 oncogene and cell proliferation in lung cancer cells. Overall, this study provides a comprehensive account of NATs and supports a role for NATs' regulation of tumor suppressors and oncogenes in cancer biology. PMID:26063736

  3. Antisense transcription as a tool to tune gene expression.

    Science.gov (United States)

    Brophy, Jennifer A N; Voigt, Christopher A

    2016-01-14

    A surprise that has emerged from transcriptomics is the prevalence of genomic antisense transcription, which occurs counter to gene orientation. While frequent, the roles of antisense transcription in regulation are poorly understood. We built a synthetic system in Escherichia coli to study how antisense transcription can change the expression of a gene and tune the response characteristics of a regulatory circuit. We developed a new genetic part that consists of a unidirectional terminator followed by a constitutive antisense promoter and demonstrate that this part represses gene expression proportionally to the antisense promoter strength. Chip-based oligo synthesis was applied to build a large library of 5,668 terminator-promoter combinations that was used to control the expression of three repressors (PhlF, SrpR, and TarA) in a simple genetic circuit (NOT gate). Using the library, we demonstrate that antisense promoters can be used to tune the threshold of a regulatory circuit without impacting other properties of its response function. Finally, we determined the relative contributions of antisense RNA and transcriptional interference to repressing gene expression and introduce a biophysical model to capture the impact of RNA polymerase collisions on gene repression. This work quantifies the role of antisense transcription in regulatory networks and introduces a new mode to control gene expression that has been previously overlooked in genetic engineering.

  4. Neonate intestinal immune response to CpG oligodeoxynucleotide stimulation.

    Directory of Open Access Journals (Sweden)

    Sonia Lacroix-Lamandé

    Full Text Available BACKGROUND: The development of mucosal vaccines is crucial to efficiently control infectious agents for which mucosae are the primary site of entry. Major drawbacks of these protective strategies are the lack of effective mucosal adjuvant. Synthetic oligodeoxynucleotides that contain several unmethylated cytosine-guanine dinucleotide (CpG-ODN motifs are now recognized as promising adjuvants displaying mucosal adjuvant activity through direct activation of TLR9-expressing cells. However, little is known about the efficacy of these molecules in stimulating the intestinal immune system in neonates. METHODOLOGY/PRINCIPAL FINDINGS: First, newborn mice received CpG-ODN orally, and the intestinal cytokine and chemokine response was measured. We observed that oral administration of CpG-ODN induces CXC and CC chemokine responses and a cellular infiltration in the intestine of neonates as detected by immunohistochemistry. We next compared the efficiency of the oral route to intraperitoneal administration in stimulating the intestinal immune responses of both adults and neonates. Neonates were more responsive to TLR9-stimulation than adults whatever the CpG-ODN administration route. Their intestinal epithelial cells (IECs indirectly responded to TLR9 stimulation and contributed to the CXC chemokine response, whereas other TLR9-bearing cells of the lamina-propria produced CC chemokines and Th1-type cytokines. Moreover, we showed that the intestine of adult exhibited a significantly higher level of IL10 at homeostasis than neonates, which might be responsible for the unresponsiveness to TLR9-stimulation, as confirmed by our findings in IL10-deficient mice. CONCLUSIONS/SIGNIFICANCE: This is the first report that deciphers the role played by CpG-ODN in the intestine of neonates. This work clearly demonstrates that an intraperitoneal administration of CpG-ODN is more efficient in neonates than in adults to stimulate an intestinal chemokine response due to their

  5. Immunostimulatory and anti-neoplasm effects of a novel palindrome CpG oligodeoxynucleotide in mice

    Institute of Scientific and Technical Information of China (English)

    Hai-yan DU; Zhong-ming TANG; Hai-feng SONG; Li-hou DONG; Bi-jun ZHAO; Jie FU; Qing-qing WANG; FangCHEN; Lun OU; Na LI; Xiao SUN

    2012-01-01

    Aim:DNAs containing unmethylated CpG motifs can stimulate innate and adaptive immunity.The aim of this study was to investigate the immunostimulatory and anti-neoplasm effects of a novel CpG oligodeoxynucleotide,ODN10,in tumor-bearing mice.Methods:B16 melanoma-bearing C57BL/6 mice were administered ip or sc with ODN10 or conventional CpG ODN1826 on the indicated days post inoculation.The animal survival rate and the inhibitory effect on tumor growth were observed in vivo.B and T lymphocyte proliferation,natural killing cell cytotoxicity and the phagocytic ability of peritoneal macrophages from the animals were determined using [3H]-thymidine incorporation assay,4-h 51Cr release assay and neutral red chromometry method,respectively.The serum levels of IL-12,IL-4,and IgE were quantified using ELISA assays.Histological examination of tumor tissues was performed after HE staining,and the expression of PCNA,CD63,and CD80 in tumor tissues was analyzed with immunohistochemistry.Results:ODN10 (1,5,and 25 mg/kg) significantly inhibited the growth and metastasis of the tumor,and significantly prolonged the survival of tumor-bearing mice,as compared with ODN1826,The immune status was suppressed in tumor-bearing mice.Both ODN10 and ODN1826 significantly reversed the suppressed immunoactivities in tumor-bearing mice,which included promoting B and T lymphocyte proliferation,enhancing NK cell and peritoneal macrophage activities,inducing IL-12 secretion and inhibiting IL-4 and IgE secretion.Further,CpG ODNs decreased PCNA and CD63 expression while induced expression of CD80.ODN10 presented more potent activity,and displayed the most prominent immunostimulatory potential.Conclusion:ODN10 produces prominent immunomodulatory effects on cellular immunity in tumor-bearing mice,which might help reverse the established Th2-type responses to the Th1-type responses,thus may be used as a potent anti-tumor immunotherapy agent or adjuvant.

  6. EFFECTS OF CALM /AF10 ANTISENSES ON PRIMARY LEUKEMIC CELLS WITH CALM /AF10 FUSION TRANSCRIPTS IN VITRO

    Institute of Scientific and Technical Information of China (English)

    LIU Ge-xiu

    2005-01-01

    Objectives: To define the involvement of CALM and AF10 fusion transcripts in primary leukaemias with t(10; 11). Methods: The AF10 and CALM fusion in five t(10; 11) leukemia samples were checked by reverse transcriptase-polymerase chain reaction (RT-PCR), and effects of CALM/AF10 antisense phosphorothioate oligodeoxynucleotides (AS PS-ODNs) on chemotherapy sensitivity and apoptosis of leukemia cells in vitro were observed. Results: Five different-sized AF10-CALM products and four different-sized CALM/AF10 products were detected by RT-PCR. The chemotherapy sensitivity of leukemic cells with t(10; 11) to drugs in vitro was lower than that of leukemic cells without t(10; 11). AS PS-ODNs increased the chemotherapy sensitivity and apoptotic rate. There were 4 cases positive at 5 (mol/L concentration, a11 cases positive at 10 (mol/L and 20 (mol/L concentration, P0.05). Conclusion: The CALM and AF10 fusion transcripts are involved in the pathogenesis of haematological malignancies with t(10, 11), and is associated with a poor prognosis. AS-PS-ODNs might be useful in therapy of t(10, 11) leukemia.

  7. Antisense-Mediated Depletion of Tomato Chloroplast Omega-3 Fatty Acid Desaturase Enhances Thermal Tolerance

    Institute of Scientific and Technical Information of China (English)

    Xun-Yan Liu; Jing-Hua Yang; Bin Li; Xiu-Mei Yang; Qing-Wei Meng

    2006-01-01

    A chloroplast-localized tomato (Lycopersicon esculentum Mill.) ω-3 fatty acid desaturase gene (LeFAD7) was isolated and characterized with regard to its sequence, response to various temperatures, and function in antisense transgenic tomato plants. The deduced amino acid sequence had four histidine-rich regions, of which three regions were highly conserved throughout the whole ω-3 fatty acid desaturase gene family.Southern blotting analysis showed that LeFAD7was encoded by a single copy gene and had two homologous genes in the tomato genome. Northern blot showed that LeFAD7was expressed in all organs and was especially abundant in leaf tissue. Meanwhile, expression of LeFAD7was induced by chilling stress (4 ℃),but was inhibited by high temperature (45 ℃), in leaves. Transgenic tomato plants were produced by integration of the antisense LeFAD7 DNA under the control of a CaMV35S promoter into the genome. Antisense transgenic plants with lower 18: 3 content could maintain a higher maximal photochemical efficiency (Fv/Fm)and O2 evolution rate than wild-type plants. These results suggested that silence of the LeFAD7 gene alleviated high-temperature stress. There was also a correlation between the low content of 18: 3 resulting from silence of the LeFAD7 gene and tolerance to high-temperature stress.

  8. Murine neurofibroma reversion by antisense RNA for HTLV-I tax

    Institute of Scientific and Technical Information of China (English)

    李昌本; Mark; C.Horowitz; Nancy; H.Ruddle

    1999-01-01

    Neurofibroma cell lines derived from mice transgenic for HTLV-I LTR tax express high levels of HTLV-I tax mRNA and protein and exhibit a transformed phenotype. A retrovirus vector carrying HTLV-I tax cDNA in reversed transcriptional orientation was stably transfected into the neurofibroma cells. Antisense RNA inhibited expression of the tax gene with a decrease of more than 40 % in both tax mRNA and protein. Tax antisense RNA reversed the transformed phenotype as exhibited by dramatic changes in cell morphology and growth characteristics. Expression of several cellular genes which are activated by Tax protein including GM-CSF, IL-6, LT/TNF, c-myc and LIF was down-regulated, while M-CSF and c-src proto-oncogene expressions were up-regulated. Accumulation of β-actin mRNA was not affected. The changes that occurred in the tax antisense expressing neurofibroma cells could be the consequence of the decreased concentration of Tax protein. These results also indicate that HTLV-I Tax protein is crucial for main

  9. HTLV-I antisense transcripts initiating in the 3'LTR are alternatively spliced and polyadenylated

    Directory of Open Access Journals (Sweden)

    Marriott Susan J

    2006-03-01

    Full Text Available Abstract Background Antisense transcription in retroviruses has been suggested for both HIV-1 and HTLV-I, although the existence and coding potential of these transcripts remain controversial. Thorough characterization is required to demonstrate the existence of these transcripts and gain insight into their role in retrovirus biology. Results This report provides the first complete characterization of an antisense retroviral transcript that encodes the previously described HTLV-I HBZ protein. In this study, we show that HBZ-encoding transcripts initiate in the 3' long terminal repeat (LTR at several positions and consist of two alternatively spliced variants (SP1 and SP2. Expression of the most abundant HBZ spliced variant (SP1 could be detected in different HTLV-I-infected cell lines and importantly in cellular clones isolated from HTLV-I-infected patients. Polyadenylation of HBZ RNA occurred at a distance of 1450 nucleotides downstream of the HBZ stop codon in close proximity of a typical polyA signal. We have also determined that translation mostly initiates from the first exon located in the 3' LTR and that the HBZ isoform produced from the SP1 spliced variant demonstrated inhibition of Tax and c-Jun-dependent transcriptional activation. Conclusion These results conclusively demonstrate the existence of antisense transcription in retroviruses, which likely plays a role in HTLV-I-associated pathogenesis through HBZ protein synthesis.

  10. Inhibitory effect of antisense vascular endothelial growth factor RNA on the profile of hepatocellular carcinoma cell line in vitro and in vivo

    Institute of Scientific and Technical Information of China (English)

    Ji-Hui Hao; Ming Yu; Hui-Kai Li; Yu-Rong Shi; Qiang Li; Xi-Shan Hao

    2006-01-01

    AIM: To evaluate the effect of antisense vascular endothelial growth factor (VEGF) RNA (PCMV-FGEV)transfection on the profile of hepatocellular carcinoma (HCC) SMMC-7721 cells in vitro and in vivo.METHODS: SMMC-7721 cells were transfected with PCMV-FGEV antisense, PCMV-VEGF sense and empty vector plasmid encapsulated by lipofectamine as antisense group, sense group and control group respectively. The positive cell clones were selected with G418. The stable transfection and expression of VEGF in the cells were determined by RT-PCR and immunohistochemistry. Cell proliferation was observed by MTT assay. FACS analysis was used to determine the effect of PCMV-FGEV transfection on cell apoptosis. The growth of transfected cells in vivo was also observed in nude mice.RESULTS: VEGFexpression was reduced in SMMC-7721transfected with PCMV-FGEV, which was confirmed by RT-PCR and immunohistochemistry. No effect of PCMVFGEV transfection was found on cell proliferation and cell apoptosis of SMMC-7721 in vitro. The growth of cells transfected with PCMV-FGEV was slow in nude mice and accompanied with obvious apoptosis. The latent time of tumors in the antisense group was 25.0± 1.8d, which was longer than that in sense and control groups (F=19.455, P<0.01). The average tumor weight in antisense group (0.96g±0.28 g) was the smallest among the three groups (F=21.501, P<0.01).CONCLUSION: The expression of VEGF can be inhibited by antisense PCMV-FGEV. Antisense PCMV-FGEV has no effect on cell proliferation and apoptosis of SMMC-7721in vitro but can inhibit tumor growth and induce cell apoptosis in vivo.

  11. c-FLIP antisense oligonucleotide-loaded nanoparticles inhibit growth of human orbital rhabdomyosarcoma xenograft in nude mice%c-FLIP反义寡核苷酸纳米粒抑制人眼眶横纹肌肉瘤裸鼠移植瘤的生长

    Institute of Scientific and Technical Information of China (English)

    梁莉; 魏锐利

    2013-01-01

    Objective To investigate the effect of cellular Fas-associated death domain-like interleukin-1β-converting enzyme-inhibitory protein (c-FLIP) antisense oligonucleotide (ASODN)-loaded nanoparticles (NP) on the human orbital rhabdomyosarcoma xenograft in nude mice,so as to assess the feasibility of nanoparticles as a gene vector.Methods The model of human orbital rhabdomyosarcoma xenograft was established in nude mice,and the tumors were injected with c-FLIP ASODN NP,c-FLIP ASODN or normal saline (NS).The tumor volume and histopathological changes of tumor were observed.Western blotting analysis and immunohistochemical analysis were used to examine the expression of c-FLIP in tumor tissues of each group.Apoptosis of tumor cells was detected using TUNEL method.Results The growth of human orbital rhabdomyosarcoma in nude mice was significantly inhibited in ASODN NP group compared with the other two groups.Western blotting analysis showed that c-FLIP protein expression in ASODN NP and ASODN groups was significantly decreased compared with NS group (P<0.05).Immunohistochemical study showed that c-FLIP expression was found in the endochylema,and the c-FLIP positive cells in ASODN NP group was significantly less than those in the other two groups (P<0.05).Tumor cell apoptosis was observed in both ASODN NP and ASODN groups,with more found in the former,and only a few apoptotic cells were found in the NS group.Conclusion c-FLIP ASODN NP can effectively inhibit the growth of human orbital rhabdomyosarcoma xenograft in nude mice,indicating that nanoparticles may serve as a safe and effective vector for ASODN.%目的 探讨c-FLIP反义寡核苷酸(c-FLIP ASODN)纳米粒(NP)对裸鼠体内人眼眶横纹肌肉瘤移植瘤生长的影响,评估纳米粒作为基因载体的可行性.方法 皮下种植法建立裸鼠人眼眶横纹肌肉瘤动物模型,瘤体内分别注射c-FLIP反义寡核苷酸纳米粒(ASODN NP组)、未包裹的c-FLIP反义寡核苷酸(ASODN组)及生

  12. Undetected antisense tRNAs in mitochondrial genomes?

    Directory of Open Access Journals (Sweden)

    Seligmann Hervé

    2010-06-01

    Full Text Available Abstract Background The hypothesis that both mitochondrial (mt complementary DNA strands of tRNA genes code for tRNAs (sense-antisense coding is explored. This could explain why mt tRNA mutations are 6.5 times more frequently pathogenic than in other mt sequences. Antisense tRNA expression is plausible because tRNA punctuation signals mt sense RNA maturation: both sense and antisense tRNAs form secondary structures potentially signalling processing. Sense RNA maturation processes by default 11 antisense tRNAs neighbouring sense genes. If antisense tRNAs are expressed, processed antisense tRNAs should have adapted more for translational activity than unprocessed ones. Four tRNA properties are examined: antisense tRNA 5' and 3' end processing by sense RNA maturation and its accuracy, cloverleaf stability and misacylation potential. Results Processed antisense tRNAs align better with standard tRNA sequences with the same cognate than unprocessed antisense tRNAs, suggesting less misacylations. Misacylation increases with cloverleaf fragility and processing inaccuracy. Cloverleaf fragility, misacylation and processing accuracy of antisense tRNAs decrease with genome-wide usage of their predicted cognate amino acid. Conclusions These properties correlate as if they adaptively coevolved for translational activity by some antisense tRNAs, and to avoid such activity by other antisense tRNAs. Analyses also suggest previously unsuspected particularities of aminoacylation specificity in mt tRNAs: combinations of competition between tRNAs on tRNA synthetases with competition between tRNA synthetases on tRNAs determine specificities of tRNA amino acylations. The latter analyses show that alignment methods used to detect tRNA cognates yield relatively robust results, even when they apparently fail to detect the tRNA's cognate amino acid and indicate high misacylation potential. Reviewers This article was reviewed by Dr Juergen Brosius, Dr Anthony M Poole and

  13. Effects of sense and antisense centromere/kinetochore complex protein-B (CENP-B) in cell cycle regulation

    Institute of Scientific and Technical Information of China (English)

    LUO Song; LIN Haiyan; QI Jianguo; WANG Yongchao

    2005-01-01

    This paper investigates the effects of sense and antisense centromere/kinetochore complex protein-B (CENP-B) in cell cycle regulation. Full-length cenpb cDNA was subcloned into pBI-EGFP eukaryotic expression vector in both sense and antisense orientation. HeLa-Tet-Off cells were transfected with sense or antisense cenpb vectors. Sense transfection of HeLa-Tet-Off cells resulted in the formation of a large centromere/kinetochore complex, and apoptosis of cells following several times of cell division. A stable antisense cenpb transfected cell line, named HACPB, was obtained. The centromere/kinetochore complex of HACPB cells became smaller than control HeLa-Tet-Off cells and scattered, and the expression of CENP-B was down-regulated. In addition, delayed cell cycle progression, inhibited malignant phenotype, restrained ability of tumor formation in nude mice, and delayed entry from G2/M phase into next G1 phase were observed in HACPB cells. Furthermore, the expression of cyclin-dependent kinases (CDKs), cyclins, and CDK inhibitors (CKIs) were modulated during different phases of the cell cycle. CENP-B is an essential protein for the maintenance of the structure and function of centromere/kinetochore complex, and plays important roles in cell cycle regulation.

  14. Effects of HSP70 Antisense Oligonucleotide on the Proliferation and Apoptosis of Human Hepatocellular Carcinoma Cells

    Institute of Scientific and Technical Information of China (English)

    杨雪; 贺海斌; 杨威; 宋涛; 郭成; 郑鑫; 刘青光

    2010-01-01

    The study investigated the effects of heat shock protein 70(HSP70) antisense oligonucleotide(ASODN) on the proliferation and apoptosis of a human hepatocellular carcinoma cell line(SMMC-7721 cells) in vitro.HSP70 oligonucleotide was transfected into SMMC-7721 cells by the mediation of SofastTM transfection reagent.Inhibition rate of SMMC-7721 cells was determined by using MTT method.Apoptosis rate and cell cycle distribution were measured by flow cytometry.Immunocytochemistry staining was used to observe th...

  15. The landscape of antisense gene expression in human cancers

    OpenAIRE

    Balbin, O. Alejandro; Malik, Rohit; Dhanasekaran, Saravana M.; Prensner, John R.; Cao, Xuhong; Wu, Yi-Mi; Robinson, Dan; Wang, Rui; Chen, Guoan; Beer, David G.; NesvizhskiI, Alexey I.; Arul M Chinnaiyan

    2015-01-01

    High-throughput RNA sequencing has revealed more pervasive transcription of the human genome than previously anticipated. However, the extent of natural antisense transcripts’ (NATs) expression, their regulation of cognate sense genes, and the role of NATs in cancer remain poorly understood. Here, we use strand-specific paired-end RNA sequencing (ssRNA-seq) data from 376 cancer samples covering nine tissue types to comprehensively characterize the landscape of antisense expression. We found c...

  16. Advances in Antisense Oligonucleotide Development for Target Identification, Validation, and as Novel Therapeutics

    Directory of Open Access Journals (Sweden)

    Moizza Mansoor

    2008-01-01

    Full Text Available Antisense oligonucleotides (As-ODNs are single stranded, synthetically prepared strands of deoxynucleotide sequences, usually 18–21 nucleotides in length, complementary to the mRNA sequence of the target gene. As-ODNs are able to selectively bind cognate mRNA sequences by sequence-specific hybridization. This results in cleavage or disablement of the mRNA and, thus, inhibits the expression of the target gene. The specificity of the As approach is based on the probability that, in the human genome, any sequence longer than a minimal number of nucleotides (nt, 13 for RNA and 17 for DNA, normally occurs only once. The potential applications of As-ODNs are numerous because mRNA is ubiquitous and is more accessible to manipulation than DNA. With the publication of the human genome sequence, it has become theoretically possible to inhibit mRNA of almost any gene by As-ODNs, in order to get a better understanding of gene function, investigate its role in disease pathology and to study novel therapeutic targets for the diseases caused by dysregulated gene expression. The conceptual simplicity, the availability of gene sequence information from the human genome, the inexpensive availability of synthetic oligonucleotides and the possibility of rational drug design makes As-ODNs powerful tools for target identification, validation and therapeutic intervention. In this review we discuss the latest developments in antisense oligonucleotide design, delivery, pharmacokinetics and potential side effects, as well as its uses in target identification and validation, and finally focus on the current developments of antisense oligonucleotides in therapeutic intervention in various diseases.

  17. Antisense oligodeoxynucleotide-mediated disruption of hippocampal cAMP response element binding protein levels impairs consolidation of memory for water maze training

    OpenAIRE

    Guzowski, John F.; McGaugh, James L.

    1997-01-01

    Extensive evidence suggests that long term memory (LTM) formation is dependent on the activation of neuronal second messenger systems and requires protein synthesis. The cAMP response element binding protein (CREB) is a constitutively expressed regulatory transcription factor that couples changes in second messenger levels to changes in cellular transcription. Several recent studies suggest that CREB and related transcription factors regulate gene expression necessary for neuronal plasticity ...

  18. CREB Antisense Oligodeoxynucleotide Administration into the Dorsal Hippocampal CA3 Region Impairs Long- but Not Short-Term Spatial Memory in Mice

    Science.gov (United States)

    Florian, Cedrick; Mons, Nicole; Roullet, Pascal

    2006-01-01

    The transcription factor cAMP response-element binding protein (CREB) has a pivotal role in hippocampal synaptic plasticity and hippocampus-dependent long-term memory. We recently demonstrated that the dorsal hippocampal CA3 region is involved in memory consolidation of spatial information tested on a Morris water maze in mice. To test whether…

  19. Effect of the Antisense BcMF12 Driven by the BcA9 Promoter on Gene Silencing in Brassica campestris L. ssp. chinensis

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The study analyzed the silencing of BcMF12 gene regulated by BcA9 promoter in the transgenic pakchoi and confirmed the effect of antisense BcMF12 gene on the pollen development. A conserved BcMF12 gene fragment was amplified from the cDNA of flower buds in pakchoi (Brassica campestris L. ssp. chinensis, syn. B. rapa L. ssp. chinensis) and was fused to the anther specific BcA9 promoter. The plant antisense expression vector was constructed and then introduced into pakchoi via Agrobacterium-mediated transformation. The transgenic plants were screened by antibiotics and molecular analysis. PCR and Southern blot revealed that the antisense BcMF12-GUS fusion gene regulated by BcA9 promoter was integrated into transgenic plants. Northern blot suggested that the expression of BcMF12 gene was down-regulated significantly. The pollen germination rate of transgenic plants with antisense BcMF12 gene decreased as compared with that of the control plants. The expression of the gene BcMF12 related to the pollen development was inhibited by the antisense BcMF12 driven by BcA9 promoter, which consequently affected the pollen development in pakchoi.

  20. Antisense Oligonucleotide Therapy for Inherited Retinal Dystrophies.

    Science.gov (United States)

    Gerard, Xavier; Garanto, Alejandro; Rozet, Jean-Michel; Collin, Rob W J

    2016-01-01

    Inherited retinal dystrophies (IRDs) are an extremely heterogeneous group of genetic diseases for which currently no effective treatment strategies exist. Over the last decade, significant progress has been made utilizing gene augmentation therapy for a few genetic subtypes of IRD, although several technical challenges so far prevent a broad clinical application of this approach for other forms of IRD. Many of the mutations leading to these retinal diseases affect pre-mRNA splicing of the mutated genes . Antisense oligonucleotide (AON)-mediated splice modulation appears to be a powerful approach to correct the consequences of such mutations at the pre-mRNA level , as demonstrated by promising results in clinical trials for several inherited disorders like Duchenne muscular dystrophy, hypercholesterolemia and various types of cancer. In this mini-review, we summarize ongoing pre-clinical research on AON-based therapy for a few genetic subtypes of IRD , speculate on other potential therapeutic targets, and discuss the opportunities and challenges that lie ahead to translate splice modulation therapy for retinal disorders to the clinic. PMID:26427454

  1. Optimizing antisense oligonucleotides using phosphorodiamidate morpholino oligomers.

    Science.gov (United States)

    Popplewell, Linda J; Malerba, Alberto; Dickson, George

    2012-01-01

    Duchenne muscular dystrophy (DMD) is caused by mutations that disrupt the reading frame of the human DMD gene. Selective removal of exons flanking an out-of-frame DMD mutation can result in an in-frame mRNA transcript that may be translated into an internally deleted Becker muscular dystrophy-like functionally active dystrophin protein with therapeutic activity. Antisense oligonucleotides (AOs) can be designed to bind to complementary sequences in the targeted mRNA and modify pre-mRNA splicing to correct the reading frame of a mutated transcript. AO-induced exon skipping resulting in functional truncated dystrophin has been demonstrated in animal models of DMD both in vitro and in vivo, in DMD patient cells in vitro in culture, and in DMD muscle explants. The recent advances made in this field suggest that it is likely that AO-induced exon skipping will be the first gene therapy for DMD to reach the clinic. However, it should be noted that personalized molecular medicine may be necessary, since the various reading frame-disrupting mutations are spread across the DMD gene. The different deletions that cause DMD would require skipping of different exons, which would require the optimization and clinical trial workup of many specific AOs. This chapter describes the methodologies available for the optimization of AOs, in particular phosphorodiamidate morpholino oligomers, for the targeted skipping of specific exons on the DMD gene. PMID:22454060

  2. On Measuring miRNAs after Transient Transfection of Mimics or Antisense Inhibitors

    Science.gov (United States)

    Thomson, Daniel W.; Bracken, Cameron P.; Szubert, Jan M.; Goodall, Gregory J.

    2013-01-01

    The ability to alter microRNA (miRNA) abundance is crucial for studying miRNA function. To achieve this there is widespread use of both exogenous double-stranded miRNA mimics for transient over-expression, and single stranded antisense RNAs (antimiRs) for miRNA inhibition. The success of these manipulations is often assessed using qPCR, but this does not accurately report the level of functional miRNA. Here, we draw attention to this discrepancy, which is overlooked in many published reports. We measured the functionality of exogenous miRNA by comparing the total level of transfected miRNA in whole cell extracts to the level of miRNA bound to Argonaute following transfection and show that the supraphysiological levels of transfected miRNA frequently seen using qPCR do not represent the functional levels, because the majority of transfected RNA that is detected is vesicular and not accessible for loading into Argonaute as functionally active miRNAs. In the case of microRNA inhibition by transient transfection with antisense inhibitors, there is also the potential for discrepancy, because following cell lysis the abundant inhibitor levels from cellular vesicles can directly interfere with the PCR reaction used to measure miRNA level. PMID:23358900

  3. On measuring miRNAs after transient transfection of mimics or antisense inhibitors.

    Directory of Open Access Journals (Sweden)

    Daniel W Thomson

    Full Text Available The ability to alter microRNA (miRNA abundance is crucial for studying miRNA function. To achieve this there is widespread use of both exogenous double-stranded miRNA mimics for transient over-expression, and single stranded antisense RNAs (antimiRs for miRNA inhibition. The success of these manipulations is often assessed using qPCR, but this does not accurately report the level of functional miRNA. Here, we draw attention to this discrepancy, which is overlooked in many published reports. We measured the functionality of exogenous miRNA by comparing the total level of transfected miRNA in whole cell extracts to the level of miRNA bound to Argonaute following transfection and show that the supraphysiological levels of transfected miRNA frequently seen using qPCR do not represent the functional levels, because the majority of transfected RNA that is detected is vesicular and not accessible for loading into Argonaute as functionally active miRNAs. In the case of microRNA inhibition by transient transfection with antisense inhibitors, there is also the potential for discrepancy, because following cell lysis the abundant inhibitor levels from cellular vesicles can directly interfere with the PCR reaction used to measure miRNA level.

  4. Antisense oligonucleotide targeting p53 increased apoptosis of MCF-7 cells induced by ionizing radiation

    Institute of Scientific and Technical Information of China (English)

    Li-cheng DAI; Xiang WANG; Xing YAO; Li-shan MIN; Fu-chu QIAN; Jian-fang HE

    2006-01-01

    Aim: To investigate the effect of antisense compounds (AS) targeting human p53 mRNA on radiosensitivity of MCF-7 cells. Methods: Western blotting and RT-PCR were used to analyze the protein content and mRNA level. Additionally, cell proliferation, cell cycle and cell apoptosis were all analyzed in irradiated or sham-irradiated cells. Results: Among the five antisense compounds (AS), AS3 was identified to efficiently inhibit p53 mRNA level and protein content. Interestingly, ASS transfer has little effect on cell proliferation in DU-145 cells (mutant p53) after ionizing radiation (IR). In contrast, a marked increase of cell apoptosis and growth inhibition were observed in MCF-7 cells (wild-type p53), suggesting that AS3 can increase radiosensitivity of MCF-7 cells. Additionally, it was also observed that the transfection of AS3 decreased the fraction of G1 phase cells, and increased the proportion of S phase cells compared to untreated cells 24 h after IR in MCF-7 cell lines. Conclusion: AS3 transfection increases MCF-7 cell apoptosis induced by 5 Gy-radiation, and this mechanism may be closely associated with abrogation of G1 phase arrest.

  5. Effect of antisense transfecting of monocarboxylate transporter gene on biological characteristics of lung adenocarcinoma A549 cells

    Institute of Scientific and Technical Information of China (English)

    ZHANG Gui-zhi; HUANG Gui-jun; GUO Xian-jian; QIAN Gui-sheng

    2002-01-01

    Objective: To study the influence of transfecting antisense expression vector of the first subtype of the monocarboxylate transporter (MCT1) gene into lung cancer cells on pHi regulation, lactate transportation and cell growth, Methods: MCT1 antisense gene recombinant vector was introduced into human lung cancer cell line A549 by electroporation. The transfected A549 cells resistant to G418 were selected. Positive clones were examined by using PCR. The changes of intracellular pH and lactate were examined with spectrophotometric method. Cell growth was studied with cell growth curve. Results: Intracellular pH and lactate were remarkably decreased in the cells transfected pLXSN-MCT1 in comparison with A549 cells without transfection (P<0. 001). The growth of A549 cells transfected pLXSN-MCT1 was also inhibited remarkably. Conclusion: MCT1 gene may play an important role in pHi regulation, lactate transportation and cell growth in tumor cells.

  6. A specific CpG oligodeoxynucleotide induces protective antiviral responses against grass carp reovirus in grass carp Ctenopharyngodon idella.

    Science.gov (United States)

    Su, Hang; Yuan, Gailing; Su, Jianguo

    2016-07-01

    CpG oligodeoxynucleotides (ODNs) show strong immune stimulatory activity in vertebrate, however, they possess specific sequence feature among species. In this study, we screened out an optimal CpG ODN sequence for grass carp (Ctenopharyngodon idella), 1670A 5'-TCGAACGTTTTAACGTTTTAACGTT-3', from six published sequences and three sequences designed by authors based on grass carp head kidney mononuclear cells and CIK (C. idella kidney) cells proliferation. VP4 mRNA expression was strongly inhibited by CpG ODN 1670A in CIK cells with GCRV infection, showing its strong antiviral activity. The mechanism via toll-like receptor 9 (TLR9)-mediated signaling pathway was measured by real-time quantitative RT-PCR, and TLR21 did not play a role in the immune response to CpG ODN. The late up-regulation of CiRIG-I mRNA expression indicated that RIG-I-like receptors (RLRs) signaling pathway participated in the immune response to CpG ODN which is the first report on the interaction between CpG and RLRs. We also found that the efficient CpG ODN can activates interferon system. Infected with GCRV, type I interferon expression was reduced and type II interferon was induced by the efficient CpG ODN in CIK cells, especially IFNγ2, suggesting that IFNγ2 played an important role in response to the efficient CpG ODN. These results provide a theoretical basis and new development trend for further research on CpG and the application of CpG vaccine adjuvant in grass carp disease control. PMID:26972738

  7. Interferon-α/β receptor-mediated selective induction of a gene cluster by CpG oligodeoxynucleotide 2006

    Directory of Open Access Journals (Sweden)

    Wakiguchi Hiroshi

    2003-07-01

    Full Text Available Abstract Background Oligodeoxynucleotides containing unmethylated CpG motifs (CpG ODN are known to exert a strong adjuvant effect on Th1 immune responses. Although several genes have been reported, no comprehensive study of the gene expression profiles in human cells after stimulation with CpG ODN has been reported. Results This study was designed to identify a CpG-inducible gene cluster that potentially predicts for the molecular mechanisms of clinical efficacy of CpG ODN, by determining mRNA expression in human PBMC after stimulation with CpG ODN. PBMCs were obtained from the peripheral blood of healthy volunteers and cultured in the presence or absence of CpG ODN 2006 for up to 24 hours. The mRNA expression profile was evaluated using a high-density oligonucleotide probe array, GeneChip®. Using hierarchical clustering-analysis, out of a total of 10,000 genes we identified a cluster containing 77 genes as having been up-regulated by CpG ODN. This cluster was further divided into two sub-clusters by means of time-kinetics. (1 Inflammatory cytokines such as IL-6 and GM-CSF were up-regulated predominantly 3 to 6 hours after stimulation with CpG ODN, presumably through activation of a transcription factor, NF-κB. (2 Interferon (IFN-inducible anti-viral proteins, including IFIT1, OAS1 and Mx1, and Th1 chemoattractant IP-10, were up-regulated predominantly 6 to 24 hours after stimulation. Blocking with mAb against IFN-α/β receptor strongly inhibited the induction of these IFN-inducible genes by CpG ODN. Conclusion This study provides new information regarding the possible immunomodulatory effects of CpG ODN in vivo via an IFN-α/β receptor-mediated paracrine pathway.

  8. Antisense mediated exon skipping therapy for duchenne muscular dystrophy (DMD).

    Science.gov (United States)

    Brolin, Camilla; Shiraishi, Takehiko

    2011-01-01

    Duchenne Muscular Dystrophy (DMD) is a lethal disease caused by mutations in the dystrophin gene (DMD) that result in the absence of essential muscle protein dystrophin. Among many different approaches for DMD treatment, exon skipping, mediated by antisense oligonucleotides, is one of the most promising methods for restoration of dystrophin expression. This approach has been tested extensively targeting different exons in numerous models both in vitro and in vivo. During the past 10 years, there has been a considerable progress by using DMD animal models involving three types of antisense oligonucleotides (2'-O-methyl phosphorothioate (2OME-PS), phosphorodiamidate morpholino oligomer (PMO)) and peptide nucleic acid (PNA). PMID:21686247

  9. Antisense mediated exon skipping therapy for duchenne muscular dystrophy (DMD)

    DEFF Research Database (Denmark)

    Brolin, Camilla; Shiraishi, Takehiko

    2011-01-01

    Duchenne Muscular Dystrophy (DMD) is a lethal disease caused by mutations in the dystrophin gene (DMD) that result in the absence of essential muscle protein dystrophin. Among many different approaches for DMD treatment, exon skipping, mediated by antisense oligonucleotides, is one of the most...... promising methods for restoration of dystrophin expression. This approach has been tested extensively targeting different exons in numerous models both in vitro and in vivo. During the past 10 years, there has been a considerable progress by using DMD animal models involving three types of antisense...

  10. Downstream Antisense Transcription Predicts Genomic Features That Define the Specific Chromatin Environment at Mammalian Promoters

    Science.gov (United States)

    Lavender, Christopher A.; Hoffman, Jackson A.; Trotter, Kevin W.; Gilchrist, Daniel A.; Bennett, Brian D.; Burkholder, Adam B.; Fargo, David C.; Archer, Trevor K.

    2016-01-01

    Antisense transcription is a prevalent feature at mammalian promoters. Previous studies have primarily focused on antisense transcription initiating upstream of genes. Here, we characterize promoter-proximal antisense transcription downstream of gene transcription starts sites in human breast cancer cells, investigating the genomic context of downstream antisense transcription. We find extensive correlations between antisense transcription and features associated with the chromatin environment at gene promoters. Antisense transcription downstream of promoters is widespread, with antisense transcription initiation observed within 2 kb of 28% of gene transcription start sites. Antisense transcription initiates between nucleosomes regularly positioned downstream of these promoters. The nucleosomes between gene and downstream antisense transcription start sites carry histone modifications associated with active promoters, such as H3K4me3 and H3K27ac. This region is bound by chromatin remodeling and histone modifying complexes including SWI/SNF subunits and HDACs, suggesting that antisense transcription or resulting RNA transcripts contribute to the creation and maintenance of a promoter-associated chromatin environment. Downstream antisense transcription overlays additional regulatory features, such as transcription factor binding, DNA accessibility, and the downstream edge of promoter-associated CpG islands. These features suggest an important role for antisense transcription in the regulation of gene expression and the maintenance of a promoter-associated chromatin environment. PMID:27487356

  11. Downstream Antisense Transcription Predicts Genomic Features That Define the Specific Chromatin Environment at Mammalian Promoters.

    Science.gov (United States)

    Lavender, Christopher A; Cannady, Kimberly R; Hoffman, Jackson A; Trotter, Kevin W; Gilchrist, Daniel A; Bennett, Brian D; Burkholder, Adam B; Burd, Craig J; Fargo, David C; Archer, Trevor K

    2016-08-01

    Antisense transcription is a prevalent feature at mammalian promoters. Previous studies have primarily focused on antisense transcription initiating upstream of genes. Here, we characterize promoter-proximal antisense transcription downstream of gene transcription starts sites in human breast cancer cells, investigating the genomic context of downstream antisense transcription. We find extensive correlations between antisense transcription and features associated with the chromatin environment at gene promoters. Antisense transcription downstream of promoters is widespread, with antisense transcription initiation observed within 2 kb of 28% of gene transcription start sites. Antisense transcription initiates between nucleosomes regularly positioned downstream of these promoters. The nucleosomes between gene and downstream antisense transcription start sites carry histone modifications associated with active promoters, such as H3K4me3 and H3K27ac. This region is bound by chromatin remodeling and histone modifying complexes including SWI/SNF subunits and HDACs, suggesting that antisense transcription or resulting RNA transcripts contribute to the creation and maintenance of a promoter-associated chromatin environment. Downstream antisense transcription overlays additional regulatory features, such as transcription factor binding, DNA accessibility, and the downstream edge of promoter-associated CpG islands. These features suggest an important role for antisense transcription in the regulation of gene expression and the maintenance of a promoter-associated chromatin environment. PMID:27487356

  12. Vector insert-targeted integrative antisense expression system for plasmid stabilization.

    Science.gov (United States)

    Luke, Jeremy M; Carnes, Aaron E; Hodgson, Clague P; Williams, James A

    2011-01-01

    Some DNA vaccine and gene therapy vector-encoded transgenes are toxic to the E. coli plasmid production host resulting in poor production yields. For plasmid products undergoing clinical evaluation, sequence modification to eliminate toxicity is undesirable because an altered vector is a new chemical entity. We hypothesized that: (1) insert-encoded toxicity is mediated by unintended expression of a toxic insert-encoded protein from spurious bacterial promoters; and (2) that toxicity could be eliminated with antisense RNA-mediated translation inhibition. We developed the pINT PR PL vector, a chromosomally integrable RNA expression vector, and utilized it to express insert-complementary (anti-insert) RNA from a single defined site in the bacterial chromosome. Anti-insert RNA eliminated leaky fluorescent protein expression from a target plasmid. A toxic retroviral gag pol helper plasmid produced in a gag pol anti-insert strain had fourfold improved plasmid fermentation yields. Plasmid fermentation yields were also fourfold improved when a DNA vaccine plasmid containing a toxic Influenza serotype H1 hemagglutinin transgene was grown in an H1 sense strand anti-insert production strain, suggesting that in this case toxicity was mediated by an antisense alternative reading frame-encoded peptide. This anti-insert chromosomal RNA expression technology is a general approach to improve production yields with plasmid-based vectors that encode toxic transgenes, or toxic alternative frame peptides. PMID:20607625

  13. Antitumor Effect of Antisense Ornithine Decarboxylase Adenovirus on Human Lung Cancer Cells

    Institute of Scientific and Technical Information of China (English)

    Hui TIAN; Lin LI; Xian-Xi LIU; Yan ZHANG

    2006-01-01

    Ornithine decarboxylase (ODC), the first enzyme of polyamine biosynthesis, was found to increase in cancer cells, especially lung cancer cells. Some chemotherapeutic agents aimed at decreasing ODC gene expression showed inhibitory effects on cancer cells. In this study, we examined the effects of adenoviral transduced antisense ODC on lung cancer cells. An adenovirus carrying antisense ODC (rAd-ODC/Ex3as) was used to infect lung cancer cell line A-549. The 3-(4,5-me thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to analyze the effect on cell growth. Expression of ODC and concentration of polyamines in cells were determined by Western blot analysis and high performance liquid chromatography. Terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick-end labeling was used to analyze cell apoptosis. The expression of ODC in A-549 cells was reduced to 54%, and that of three polyamines was also decreased through the rAd-ODC/Ex3as treatment. Consequently, cell growth was substantially inhibited and terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick-end labeling showed that rAd-ODC/Ex3as could lead to cell apoptosis, with apoptosis index of 46%. This study suggests that rAd-ODC/Ex3as has an antitumor effect on the human lung cancer cells.

  14. Highly expressed genes are associated with inverse antisense transcription in mouse

    Indian Academy of Sciences (India)

    Andras Györffy; Pawel Surowiak; Zsolt Tulassay; Balazs Györffy

    2007-08-01

    There is a growing evidence, that antisense transcription might have a key role in a range of human diseases. Although predefined sense–antisense pairs were extensively studied, the antisense expression of the known sense genes is rarely investigated. We retrieved and correlated the expression of sense and antisense sequences of 1182 mouse transcripts to assess the prevalence and to find the characteristic pattern of antisense transcription. We contrasted three Affymetrix MGU74A version 1 mouse genome chips to six MGU74A version 2 chips. For these 1182 transcripts, the version 1 chips contain the antisense sequences of the transcripts presented on the version 2 chips. The original data was taken from the GEO database (GDS431 and GDS432). As the Affymetrix data are semiquantitative, the relative expression levels of antisense partners were analysed. We detected antisense transcription, although the average antisense expression is shifted towards smaller expression values (MGU74A version 1, 516; version 2, 1688). An inverse direct correlation between sense and antisense expression values could be observed at high expression values. At a very high relative expression—above 40,000—the Pearson correlation coefficient is getting closer to −1. Transcripts with high inverse expression ratio may be correlated to the investigated gene (major histocompatibility complex class II trans activator). The ratio of sense to antisense transcripts varied among different chromosomes; on chromosomes 14 and 1 the level of antisense expression was higher than that of sense. We conclude that antisense transcription is a common phenomenon in the mouse genome. The hypothesis of regulatory role of antisense transcripts is supported by the inverse antisense gene expression of highly expressed genes.

  15. Analysis of the mechanism of protection in transgenic plants expressing the potato virus X coat protein or its antisense RNA.

    Science.gov (United States)

    Hemenway, C; Fang, R X; Kaniewski, W K; Chua, N H; Tumer, N E

    1988-05-01

    Transgenic tobacco plants engineered to express either the potato virus X (PVX) coat protein (CP+) or the antisense coat protein transcript (CP-antisense) were protected from infection by PVX, as indicated by reduced lesion numbers on inoculated leaves, delay or absence of systemic symptom development and reduction in virus accumulation in both inoculated and systemic leaves. The extent of protection observed in CP+ plants primarily depended upon the level of expression of the coat protein. Plants expressing antisense RNA were protected only at low inoculum concentrations. The extent of this protection was even lower than that observed in plants expressing low levels of CP. In contrast to previous reports for plants expressing tobacco mosaic virus or alfalfa mosaic virus CP, inoculation of plants expressing high levels of PVX CP with PVX RNA did not overcome the protection. Specifically, lesion numbers on inoculated leaves and PVX levels on inoculated and systemtic leaves of the CP+ plants were reduced to a similar extent in both virus and RNA inoculated plants. Although these results do not rule out that CP-mediated protection involves inhibition of uncoating of the challenge virus, they suggest that PVX CP (or its RNA) can moderate early events in RNA infection by a different mechanism. PMID:16453840

  16. CpG-A and B oligodeoxynucleotides enhance the efficacy of antibody therapy by activating different effector cell populations

    NARCIS (Netherlands)

    H.H. van Ojik; L. Bevaart; C.E. Dahle; A. Bakker (Annie); M.J.H. Jansen (Marco); M.J. van Vugt; J.G. van de Winkel; G.J. Weiner

    2003-01-01

    textabstractImmunostimulatory CpG oligodeoxynucleotides (ODNs) can enhance the therapeutic effect of monoclonal antibodies (mAbs) by enhancing antibody-dependent cell-mediated cytotoxicity (ADCC). Distinct classes of CpG ODNs have been found recently to stimulate different effector

  17. Lysine metabolism in antisense C-hordein barley grains

    DEFF Research Database (Denmark)

    Schmidt, Daiana; Rizzi, Vanessa; Gaziola, Salete A;

    2015-01-01

    The grain proteins of barley are deficient in lysine and threonine due to their low concentrations in the major storage protein class, the hordeins, especially in the C-hordein subgroup. Previously produced antisense C-hordein transgenic barley lines have an improved amino acid composition, with ...

  18. Os DNA sintéticos anti-sentido Antisense Synthtetic DNA

    Directory of Open Access Journals (Sweden)

    Alfredo Cravador

    1998-07-01

    Full Text Available One old dream of the chemist in the field of the drug research is to create molecules capable of reaching their target with the precision of a missile. To accomplish it these molecules must have the propriety of distinguishing qualitative differences between healthy and diseased cells. A therapy based on this principle, able of eradicating specifically defective cells, or cells affected by a pathogen has an enormous advantage with the regard to the classical approach in which the cytotoxic drugs merely exploit quantitative biochemical and kinetic differences between abnormal and normal cells. We present in this article a review on the chemical synthesis of analogues of desoxyribonucleotides and on results obtained on the specific and irreversible inhibition of undesired genetic expression using the antisense principle.

  19. Dynamics of human telomerase RNA structure revealed by antisense oligonucleotide technique.

    Science.gov (United States)

    Vasilkova, Daria V; Azhibek, Dulat M; Zatsepin, Timofei S; Naraikina, Yulia V; Prassolov, Vladimir S; Prokofjeva, Maria M; Zvereva, Maria I; Rubtsova, Maria P

    2013-12-01

    Telomeres are the nucleoprotein complexes that cap the linear chromosome ends. Telomerase is a ribonucleoprotein that maintains telomere length in stem, embryonic and cancer cells. Somatic cells don't contain active telomerase and telomere function as mitotic clock and telomere length determines the number of cell divisions. Telomerase RNA (TER) contains the template for telomere synthesis and serves as a structural scaffold for holoenzyme assembly. We compared different oligonucleotide based methods for telomerase RNA inhibition, such as antisense oligonucleotides, knockdown by transient siRNA transfection and silencing by miRNA derived from short expressed RNA hairpin in HEK293 cells. All of these methods were applied to different TER regions. Our results revealed that CR2/CR3 domain of TER is accessible in vitro and in vivo and could serve as an optimal site for oligonucleotide-based telomerase silencing.

  20. Antisense inhibition of hyaluronan synthase-2 in human osteosarcoma cells inhibits hyaluronan retention and tumorigenicity

    OpenAIRE

    Nishida, Yoshihiro; Knudson, Warren; Knudson, Cheryl B.; Ishiguro, Naoki

    2005-01-01

    Osteosarcoma is a common malignant bone tumor associated with childhood and adolescence. The results of numerous studies have suggested that hyaluronan plays an important role in regulating the aggressive behavior of various types of cancer cells. However, no studies have addressed hyaluronan with respect to osteosarcomas. In this investigation, the mRNA expression copy number of three mammalian hyaluronan synthases (HAS) was determined using competitive RT-PCR in the osteoblastic osteosarcom...

  1. Incorporation of cyclic azobenzene into oligodeoxynucleotides for the photo-regulation of DNA hybridization.

    Science.gov (United States)

    Eljabu, Fatma; Dhruval, Joshi; Yan, Hongbin

    2015-12-01

    Cyclic azobenzene carboxylic acid was synthesized using a shortened route. After reaction with D-threolinol, the resulting cyclic azobenzene-D-threolinol (cAB-Thr) building block was transformed into the corresponding DMTr-protected phosphoramidite, and incorporated into oligodeoxynucleotides at various positions and frequencies by solid phase synthesis. The melting temperatures of these modified oligonucleotides were determined by UV spectrometry. Photo-regulation of cAB-Thr-modified oligonucleotides with their complementary sequence was evaluated by Fluorescence Resonance Energy Transfer experiments using a fluorescein-Black Hole Quencher pair. Results suggest that while cis-cAB destabilizes DNA duplexes, trans-cAB can be accommodated in double stranded DNA.

  2. Bacterial antisense RNAs are mainly the product of transcriptional noise

    Science.gov (United States)

    Lloréns-Rico, Verónica; Cano, Jaime; Kamminga, Tjerko; Gil, Rosario; Latorre, Amparo; Chen, Wei-Hua; Bork, Peer; Glass, John I.; Serrano, Luis; Lluch-Senar, Maria

    2016-01-01

    cis-Encoded antisense RNAs (asRNAs) are widespread along bacterial transcriptomes. However, the role of most of these RNAs remains unknown, and there is an ongoing discussion as to what extent these transcripts are the result of transcriptional noise. We show, by comparative transcriptomics of 20 bacterial species and one chloroplast, that the number of asRNAs is exponentially dependent on the genomic AT content and that expression of asRNA at low levels exerts little impact in terms of energy consumption. A transcription model simulating mRNA and asRNA production indicates that the asRNA regulatory effect is only observed above certain expression thresholds, substantially higher than physiological transcript levels. These predictions were verified experimentally by overexpressing nine different asRNAs in Mycoplasma pneumoniae. Our results suggest that most of the antisense transcripts found in bacteria are the consequence of transcriptional noise, arising at spurious promoters throughout the genome. PMID:26973873

  3. In vitro and in vivo suppression of hepatocellular carcinoma growth by midkine-antisense oligonucleotide-loaded nanoparticles

    Institute of Scientific and Technical Information of China (English)

    Li-Cheng Dai; Xing Yao; Xiang Wang; Shu-Qiong Niu; Lin-Fu Zhou; Fang-Fang Fu; Shui-Xin Yang; Jin-Liang Ping

    2009-01-01

    AIM: To synthesize antisense oligonucleotides (ASODNs) of midkine (MK), package the ASODNs with nanoparticles, and to inhibit hepatocellular carcinoma (HCC) growth using these nanoparticles.METHODS: HepG2 cell proliferation was analyzed in vitro using the 3-(4,5-dimethythiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)- 2Htetrazolium, inner salt assay. The in vivo activity of nanoparticles delivering the MK-ASODNs was analyzed by histopathological and immunohistochemical staining and quantitative real time polymerase chain reaction (PCR).RESULTS: The in vitro proliferation of HepG2 cells was significantly inhibited by the nanoparticles packaged with MK-ASODNs (NANO-ASODNs). Furthermore, the NANOASODNs significantly inhibited the growth of HCC in the mouse model.

  4. Antisense Oligonucleotides: Treating Neurodegeneration at the Level of RNA

    OpenAIRE

    DeVos, Sarah L.; Miller, Timothy M.

    2013-01-01

    Adequate therapies are lacking for Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis, and other neurodegenerative diseases. The ability to use antisense oligonucleotides (ASOs) to target disease-associated genes by means of RNA may offer a potent approach for the treatment of these, and other, neurodegenerative disorders. In modifying the basic backbone chemistry, chemical groups, and target sequence, ASOs can act through numerous mechanisms to decr...

  5. Antisense mediated exon skipping therapy for duchenne muscular dystrophy (DMD)

    OpenAIRE

    Brolin, Camilla; Shiraishi, Takehiko

    2011-01-01

    Duchenne Muscular Dystrophy (DMD) is a lethal disease caused by mutations in the dystrophin gene (DMD) that result in the absence of essential muscle protein dystrophin. Among many different approaches for DMD treatment, exon skipping, mediated by antisense oligonucleotides, is one of the most promising methods for restoration of dystrophin expression. This approach has been tested extensively targeting different exons in numerous models both in vitro and in vivo. During the past 10 years, th...

  6. Progress in therapeutic antisense applications for neuromuscular disorders

    OpenAIRE

    Aartsma-Rus, Annemieke; van Ommen, Gert-Jan B.

    2009-01-01

    Neuromuscular disorders are a frequent cause of chronic disability in man. They often result from mutations in single genes and are thus, in principle, well suited for gene therapy. However, the tissues involved (muscle and the central nervous system) are post-mitotic, which poses a challenge for most viral vectors. In some cases, alternative approaches may use small molecules, for example, antisense oligonucleotides (AONs). These do not deliver a new gene, but rather modulate existing gene p...

  7. Modulation of p53 expression using antisense oligonucleotides complementary to the 5'-terminal region of p53 mRNA in vitro and in the living cells.

    Directory of Open Access Journals (Sweden)

    Agnieszka Gorska

    Full Text Available The p53 protein is a key player in cell response to stress events and cancer prevention. However, up-regulation of p53 that occurs during radiotherapy of some tumours results in radio-resistance of targeted cells. Recently, antisense oligonucleotides have been used to reduce the p53 level in tumour cells which facilitates their radiation-induced apoptosis. Here we describe the rational design of antisense oligomers directed against the 5'-terminal region of p53 mRNA aimed to inhibit the synthesis of p53 protein and its ΔNp53 isoform. A comprehensive analysis of the sites accessible to oligomer hybridization in this mRNA region was performed. Subsequently, translation efficiency from the initiation codons for both proteins in the presence of selected oligomers was determined in rabbit reticulocyte lysate and in MCF-7 cells. The antisense oligomers with 2'-OMe and LNA modifications were used to study the mechanism of their impact on translation. It turned out that the remaining RNase H activity of the lysate contributed to modulation of protein synthesis efficiency which was observed in the presence of antisense oligomers. A possibility of changing the ratio of the newly synthetized p53 and ΔNp53 in a controlled manner was revealed which is potentially very attractive considering the relationship between the functioning of these two proteins. Selected antisense oligonucleotides which were designed based on accessibility mapping of the 5'-terminal region of p53 mRNA were able to significantly reduce the level of p53 protein in MCF-7 cells. One of these oligomers might be used in the future as a support treatment in anticancer therapy.

  8. Effect of antisense oligonucleotide targeting bFGF on apoptosis of hepatoma cells%多层螺旋CT同层动态扫描结合MPR技术诊断肝外胆管癌的研究

    Institute of Scientific and Technical Information of China (English)

    Jielin Qi; Ning Wu; Li Li; Bing Bu; Dengfeng Zhou; Xiqin Zhang

    2009-01-01

    Objective:To investigate the cell cycle changes of hepatoma cells and the rote of antisense oligonucleotide targeting bFGF.Methods:Inhibition of bFGF protein expression was investigated by conical microscopy analysis and Western blot in the best condition of transfecting antisense oligonucleotide targeting bFGF.Cell cycle and apoptosis were detected with flow cytometry analysis.Results:Treatmenl with antisense oligonucleotide of bFGF not only reduced the expression of bFGF by conical microscopy and Western blot analysises,but also increased the apoptosis of HepG2 cells(P<0.01).Conclusion:bFGF may take part in apoptosis regulation of hepatoma cells and be used as a target of hepatocel-lular carcinoma therapy.

  9. A new usage of functionalized oligodeoxynucleotide probe for site-specific modification of a guanine base within RNA

    OpenAIRE

    Onizuka, Kazumitsu; Taniguchi, Yosuke; Sasaki, Shigeki

    2010-01-01

    Site-specific modification of RNA is of great significance to investigate RNA structure, function and dynamics. Recently, we reported a new method for sequence- and cytosine-selective chemical modification of RNA based on the functional group transfer reaction of the 1-phenyl-2-methylydene-1,3-diketone unit of the 6-thioguanosine base incorporated in the oligodeoxynucleotide probe. In this study, we describe that the functionality transfer rate is greatly enhanced and the selectivity is shift...

  10. Detection and kinetic studies of triplex formation by oligodeoxynucleotides using real-time biomolecular interaction analysis (BIA).

    OpenAIRE

    Bates, P. J.; Dosanjh, H. S.; S. Kumar; Jenkins, T. C.; Laughton, C A; Neidle, S

    1995-01-01

    Real-time biomolecular interaction analysis (BIA) has been applied to triplex formation between oligodeoxynucleotides. 5'-Biotinylated oligonucleotides were immobilised on the streptavidin-coated surface of a biosensor chip and subsequently hybridised to their complementary strand. Sequence-specific triplex formation was observed when a suitable third-strand oligopyrimidine was injected over the surface-bound duplex. In addition, a single-stranded oligonucleotide immobilised on the chip surfa...

  11. Investigation of the Reactivity of Oligodeoxynucleotides with Glyoxal and KMnO4 Chemical Probes by Electrospray Ionization Mass Spectrometry

    OpenAIRE

    Parr, Carol; Pierce, Sarah E.; Smith, Suncerae I.; Brodbelt, Jennifer S.

    2011-01-01

    The reactions of two well-known chemical probes, glyoxal and potassium permanganate (KMnO4), with oligodeoxynucleotides were monitored by electrospray ionization (ESI) mass spectrometry to evaluate the influence of the sequence of DNA, its secondary structure, and interactions with associated ligands on the reactivity of the two probes. Glyoxal, a guanine-reactive probe, incorporated a mass shift of 58 Da, and potassium permanganate (KMnO4) is a thymine-reactive probe that resulted in a mass ...

  12. Sense and antisense transcription are associated with distinct chromatin architectures across genes.

    Science.gov (United States)

    Murray, Struan C; Haenni, Simon; Howe, Françoise S; Fischl, Harry; Chocian, Karolina; Nair, Anitha; Mellor, Jane

    2015-09-18

    Genes from yeast to mammals are frequently subject to non-coding transcription of their antisense strand; however the genome-wide role for antisense transcription remains elusive. As transcription influences chromatin structure, we took a genome-wide approach to assess which chromatin features are associated with nascent antisense transcription, and contrast these with features associated with nascent sense transcription. We describe a distinct chromatin architecture at the promoter and gene body specifically associated with antisense transcription, marked by reduced H2B ubiquitination, H3K36 and H3K79 trimethylation and increased levels of H3 acetylation, chromatin remodelling enzymes, histone chaperones and histone turnover. The difference in sense transcription between genes with high or low levels of antisense transcription is slight; thus the antisense transcription-associated chromatin state is not simply analogous to a repressed state. Using mutants in which the level of antisense transcription is reduced at GAL1, or altered genome-wide, we show that non-coding transcription is associated with high H3 acetylation and H3 levels across the gene, while reducing H3K36me3. Set1 is required for these antisense transcription-associated chromatin changes in the gene body. We propose that nascent antisense and sense transcription have fundamentally distinct relationships with chromatin, and that both should be considered canonical features of eukaryotic genes.

  13. Construction of Antisense MT1-MMP Vector and Its Inhibitory Effects on Invasion of Human Ovarian Cancer Cells

    Institute of Scientific and Technical Information of China (English)

    WU Mingfu; SHI Yanyan; XI Lin; LI Qiong; LIAO Guo-Nin; HAN Zhi-Qiang; LU Yun-Ping; MA Ding

    2005-01-01

    Membrane-type 1 matrix metalloproteinase (MT1 MMP/MMP 14) plays crucial roles in tumor cell growth, invasion, and angiogenesis. To clarify whether the endogenously expressed MT1-MMP in metastatic human ovarian carcinoma cell lines SKOV3 plays a critical role in tumor cell invasiveness, antisense MT1-MMP cloned in eukaryotic expression vector pMMP14as was transferred into SKOV3 cells. 48h after transfection, decreased expression of endogenous MT1-MMP protein was detected in pMMP14as transfected SKOV3 cells and the activation of pro MMP2was inhibited markedly. The mean percentage of invasive cells was (62. 50 ±5. 30) % in pMMP14as-transfected cells, which was obviously less than that (97.20±6.90) % in the control.Thus, antisense MT1 MMP effectively inhibited the endogenous MT1 MMP expression and the invasiveness in SKOV3 cells, suggesting that MT1-MMP may be a therapeutic target molecule for human invasive ovarian cancers.

  14. Antisense expression of PKCα improved sensitivity of SGC7901/VCR cells to doxorubicin

    Institute of Scientific and Technical Information of China (English)

    Da-Long Wu; Feng-Ying Sui; Cheng Du; Cheng-Wen Zhang; Bin Hui; Shui-Ling Xu; Huan-Zhang Lu; Guo-Jie Song

    2009-01-01

    AIM: To explore whether antisense blocking of protein kinase C alpha (PKCα) would reverse multi-drug resistance (MDR) in the vincristine (VCR)-resistant human gastric cancer cell line SGC7901/VCR.METHODS: SGC7901/VCR cells expressing antisense PKCα, SGC7901/VCR/aPKC, were established by transfection with a recombinant plasmid reversely inserted with PKCα cDNA. Empty vector (PCI-neo)-transfected cell clones, SGC7901/VCR/neo, served as the control. Western blot method was used to detect PKCα content in SGC7901, SGC7901/VCR, SGC7901/VCR/neo and SGC7901/VCR/aPKC cells, using PKCα-specific antibody. The sensitivity of SGC7901, SGC7901/VCR, SGC7901/VCR/neo and SGC7901/VCR/aPKC cells to doxorubicin (DOX) in vitro was determined by MTT assay. The uptake of DOX in these cells was detected with fluorescence spectrophotometer.RESULTS: Western blot analysis showed that the PKCα protein level was about 8.7-fold higher in SGC7901/VCR cells than that in SGC7901 cells, whereas the protein expression of PKCα was reduced by 78% in SGC7901/VCR/aPKC cells when compared with the SGC7901/VCR cells. SGC7901/VCR/aPKC cells had a 4.2-fold increase in DOX cytotoxicity, accompanied by a 1.7-fold increase of DOX accumulation in comparison with SGC7901/VCR cells.CONCLUSION: PKCα positively regulates MDR in SGC7901 cells, and inhibition of PKCα can partially attenuate MDR in human gastric cancer cells.

  15. Effects of adenovirus-mediated human cyclooxygenase-2 antisense RNA on the growth of hepatocellular carcinoma

    Institute of Scientific and Technical Information of China (English)

    Xiao-Hu Wang; Sheng-Bao Li; Qiang Tong; Guo-Jian Xie; Qing-Ming Wu

    2005-01-01

    AIM: To investigate the relation between the expression of cyclooxygenase-2 (COX-2) and liver cancer, to construct the recombinant adenovirus encoding human COX-2antisense RNA, and to explore its effects on liver cancer cell proliferation.METHODS: We studied the expression of COX-2 in 34cases of hepatocellular carcinoma (HCC) and SMMC7402and SMMC7721 by immunohistochemical technique.Recombinant adenovirus Ad-AShcox-2 was constructed and transfected into human HCC cell lines SMMC7402and SMMC7721, and its effects on COX-2 expression, cell apoptosis and cell cycle were analyzed by flow cytometry.Cell proliferation was determined by colony-forming efficiency.RESULTS: We observed COX-2 expression in 82.4% of HCC and SMMC7402 cells, but no COX-2 expression in SMMC7721 cells. In addition, recombinant adenovirus encoding antisense COX-2 fragment Ad-AShcox-2 was obtained with the titer of 1.06× 1012 PFU/mL. Ad-AShcox-2 could reduce the expression of COX-2 and enhance the percentage of cells in G1/G0 phase in SMMC7402 cell line.The difference of apoptotic index between the Ad-AShcox2 group and control group was statistically significant(tcontrol group= 32.62 and tAd-LacZ= 10.93, P<0.001) in SMMC7402 but not in SMMC7721. Similarly, colony-forming rates of SMMC7402 and SMMC7721 cell lines, after the transfer of Ad-AShcox-2, were (2.7±0.94)% and(33.6±4.24)%, respectively.CONCLUSION: Reduction in the expression of COX-2 can inhibit COX-2 expressing HCC cells.

  16. Effect of Terminal Groups of Dendrimers in the Complexation with Antisense Oligonucleotides and Cell Uptake.

    Science.gov (United States)

    Márquez-Miranda, Valeria; Peñaloza, Juan Pablo; Araya-Durán, Ingrid; Reyes, Rodrigo; Vidaurre, Soledad; Romero, Valentina; Fuentes, Juan; Céric, Francisco; Velásquez, Luis; González-Nilo, Fernando D; Otero, Carolina

    2016-12-01

    Poly(amidoamine) dendrimers are the most recognized class of dendrimer. Amino-terminated (PAMAM-NH2) and hydroxyl-terminated (PAMAM-OH) dendrimers of generation 4 are widely used, since they are commercially available. Both have different properties, mainly based on their different overall charges at physiological pH. Currently, an important function of dendrimers as carriers of short single-stranded DNA has been applied. These molecules, known as antisense oligonucleotides (asODNs), are able to inhibit the expression of a target mRNA. Whereas PAMAM-NH2 dendrimers have shown to be able to transfect plasmid DNA, PAMAM-OH dendrimers have not shown the same successful results. However, little is known about their interaction with shorter and more flexible molecules such as asODNs. Due to several initiatives, the use of these neutral dendrimers as a scaffold to introduce other functional groups has been proposed. Because of its low cytotoxicity, it is relevant to understand the molecular phenomena involving these types of dendrimers. In this work, we studied the behavior of an antisense oligonucleotide in presence of both types of dendrimers using molecular dynamics simulations, in order to elucidate if they are able to form stable complexes. In this manner, we demonstrated at atomic level that PAMAM-NH2, unlike PAMAM-OH, could form a well-compacted complex with asODN, albeit PAMAM-OH can also establish stable interactions with the oligonucleotide. The biological activity of asODN in complex with PAMAM-NH2 dendrimer was also shown. Finally, we revealed that in contact with PAMAM-OH, asODN remains outside the cells as TIRF microscopy results showed, due to its poor interaction with this dendrimer and cell membranes.

  17. Effect of Terminal Groups of Dendrimers in the Complexation with Antisense Oligonucleotides and Cell Uptake.

    Science.gov (United States)

    Márquez-Miranda, Valeria; Peñaloza, Juan Pablo; Araya-Durán, Ingrid; Reyes, Rodrigo; Vidaurre, Soledad; Romero, Valentina; Fuentes, Juan; Céric, Francisco; Velásquez, Luis; González-Nilo, Fernando D; Otero, Carolina

    2016-12-01

    Poly(amidoamine) dendrimers are the most recognized class of dendrimer. Amino-terminated (PAMAM-NH2) and hydroxyl-terminated (PAMAM-OH) dendrimers of generation 4 are widely used, since they are commercially available. Both have different properties, mainly based on their different overall charges at physiological pH. Currently, an important function of dendrimers as carriers of short single-stranded DNA has been applied. These molecules, known as antisense oligonucleotides (asODNs), are able to inhibit the expression of a target mRNA. Whereas PAMAM-NH2 dendrimers have shown to be able to transfect plasmid DNA, PAMAM-OH dendrimers have not shown the same successful results. However, little is known about their interaction with shorter and more flexible molecules such as asODNs. Due to several initiatives, the use of these neutral dendrimers as a scaffold to introduce other functional groups has been proposed. Because of its low cytotoxicity, it is relevant to understand the molecular phenomena involving these types of dendrimers. In this work, we studied the behavior of an antisense oligonucleotide in presence of both types of dendrimers using molecular dynamics simulations, in order to elucidate if they are able to form stable complexes. In this manner, we demonstrated at atomic level that PAMAM-NH2, unlike PAMAM-OH, could form a well-compacted complex with asODN, albeit PAMAM-OH can also establish stable interactions with the oligonucleotide. The biological activity of asODN in complex with PAMAM-NH2 dendrimer was also shown. Finally, we revealed that in contact with PAMAM-OH, asODN remains outside the cells as TIRF microscopy results showed, due to its poor interaction with this dendrimer and cell membranes. PMID:26847692

  18. Effect of Terminal Groups of Dendrimers in the Complexation with Antisense Oligonucleotides and Cell Uptake

    Science.gov (United States)

    Márquez-Miranda, Valeria; Peñaloza, Juan Pablo; Araya-Durán, Ingrid; Reyes, Rodrigo; Vidaurre, Soledad; Romero, Valentina; Fuentes, Juan; Céric, Francisco; Velásquez, Luis; González-Nilo, Fernando D.; Otero, Carolina

    2016-02-01

    Poly(amidoamine) dendrimers are the most recognized class of dendrimer. Amino-terminated (PAMAM-NH2) and hydroxyl-terminated (PAMAM-OH) dendrimers of generation 4 are widely used, since they are commercially available. Both have different properties, mainly based on their different overall charges at physiological pH. Currently, an important function of dendrimers as carriers of short single-stranded DNA has been applied. These molecules, known as antisense oligonucleotides (asODNs), are able to inhibit the expression of a target mRNA. Whereas PAMAM-NH2 dendrimers have shown to be able to transfect plasmid DNA, PAMAM-OH dendrimers have not shown the same successful results. However, little is known about their interaction with shorter and more flexible molecules such as asODNs. Due to several initiatives, the use of these neutral dendrimers as a scaffold to introduce other functional groups has been proposed. Because of its low cytotoxicity, it is relevant to understand the molecular phenomena involving these types of dendrimers. In this work, we studied the behavior of an antisense oligonucleotide in presence of both types of dendrimers using molecular dynamics simulations, in order to elucidate if they are able to form stable complexes. In this manner, we demonstrated at atomic level that PAMAM-NH2, unlike PAMAM-OH, could form a well-compacted complex with asODN, albeit PAMAM-OH can also establish stable interactions with the oligonucleotide. The biological activity of asODN in complex with PAMAM-NH2 dendrimer was also shown. Finally, we revealed that in contact with PAMAM-OH, asODN remains outside the cells as TIRF microscopy results showed, due to its poor interaction with this dendrimer and cell membranes.

  19. 联合VEGF反义寡核苷酸和PDGF三链形成寡核苷酸抑制大鼠脑胶质瘤生长%Triplex forming oligonucleotide of PDGF-B chain combined with antisense oligonucleotide of VEGF inhibits glioma growth in rats

    Institute of Scientific and Technical Information of China (English)

    李维方; 周定标; 余新光; 金由辛

    2006-01-01

    目的:观察血小板源生长因子(PDGF) B链基因(PDGF-B)的三链形成寡核苷酸(triplex forming oligonucleotide,TFO)PDGF-TFO和血管内皮生长因子(VEGF)反义寡核苷酸(antisense oligonucleotide,AON)VEGF-AON对大鼠脑胶质瘤生长的抑制作用.方法:36只雄性SD大鼠,分为4组,所有大鼠均在立体定向导引下行右尾状核区微量灌注含1×106 C6胶质瘤细胞的生理盐水20 μl.在细胞接种后第8天,实验Ⅰ组6只大鼠原位注射含PDGF-TFO 1.5 mg的 20 μl生理盐水,实验Ⅱ组12只和实验Ⅲ组12只大鼠则分别原位注射含PDGF-TFO 1.5 mg+VEGF-AON 0.125 mg和PDGF-TFO 1.5 mg+VEGF-AON 0.25 mg的20 μl生理盐水.以后每隔72 h原位注射相同剂量的药物1次,共注射3次.对照组6只大鼠仅在相同时间原位注射20 μl生理盐水.实验3周时处死所有大鼠,观察肿瘤的生长情况,定性和定量观察肿瘤PDGF-B、VEGF和肿瘤核增殖抗原(PCNA)表达.结果:实验Ⅰ组的成瘤抑制率为53.1 %,实验Ⅱ组为81.4 %,实验Ⅲ组为93.1 %,3组比较有明显差异(P<0.01).PDGF-TFO对C6胶质瘤细胞PDGF-B、VEGF、PCNA表达有明显的抑制作用;联合应用PDGF-TFO和VEGF-AON能更好地抑制PDGF-B、VEGF、PCNA表达.结论:联合应用PDGF-TFO和VEGF-AON 比单用PFGF-TFO能更有效地抑制肿瘤生长.

  20. Dengue-1 envelope protein domain III along with PELC and CpG oligodeoxynucleotides synergistically enhances immune responses.

    Directory of Open Access Journals (Sweden)

    Chen-Yi Chiang

    Full Text Available The major weaknesses of subunit vaccines are their low immunogenicity and poor efficacy. Adjuvants can help to overcome some of these inherent defects with subunit vaccines. Here, we evaluated the efficacy of the newly developed water-in-oil-in-water multiphase emulsion system, termed PELC, in potentiating the protective capacity of dengue-1 envelope protein domain III. Unlike aluminum phosphate, dengue-1 envelope protein domain III formulated with PELC plus CpG oligodeoxynucleotides induced neutralizing antibodies against dengue-1 virus and increased the splenocyte secretion of IFN-γ after in vitro re-stimulation. The induced antibodies contained both the IgG1 and IgG2a subclasses. A rapid anamnestic neutralizing antibody response against a live dengue virus challenge was elicited at week 26 after the first immunization. These results demonstrate that PELC plus CpG oligodeoxynucleotides broaden the dengue-1 envelope protein domain III-specific immune responses. PELC plus CpG oligodeoxynucleotides is a promising adjuvant for recombinant protein based vaccination against dengue virus.

  1. Construction of the Antisense Eukaryotic Vector for Proliferating Cell Nuclear Antigen Gene and Its Expression in Bladder Cancer EJ Cell Line

    Institute of Scientific and Technical Information of China (English)

    童强松; 曾甫清; 齐义鹏; 朱朝晖; 鲁功成

    2002-01-01

    Summary: To explore a novel strategy for antisense gene therapy of cancer, the coding sequence ofhuman proliferating cell nuclear antigen (PCNA) cDNA was reversely inserted into the eukaryoticvector pLXSN by molecular cloning techniques and transferred into bladder cancer EJ cells with li-posome. The PCNA expression in transferred cells was dynamically detected by immunofluo-rescence and RT-PCR techniques. Changes of proliferation activities of cancer cells were assayedby MTT colorimetric and cloning formation methods. In the experiment, the antisense eukaryoticvector was successfully constructed and named as pLAPSN. After transfection with it for 1-7days, PCNA protein and mRNA levels in cancer cells were blocked by 16. 74 % - 84.21% (P<0. 05) and 23.27 % - 86.15 % (P<0. 05) respectively. The proliferation activities of transferredcells were inhibited by 27.91% - 62.07 % (P<0. 01), with cloning formation abilities being de-creased by 50. 81% (P<0. 01). It was concluded that the in vitro proliferation activities of cancercells could be effectively inhibited by blocking PCNA expression with antisense technique, whichcould serve as an ideal strategy for gene therapy of bladder cancer.

  2. Construction and packaging of pseudotype retrovirus containing human N—ras cDNA antisense sequence and its biological effects on human hepatoma cells

    Institute of Scientific and Technical Information of China (English)

    JIALIBIN; WANGXIANG; 等

    1990-01-01

    N-ras is one of the transforming genes in human hepatic cancer cells.It has been found that N-ras was overexpressed at the mRNA and protein level in hepatoma cells.In order to explore the biological roles of N-ras in human hepatic carcinogenesis and the potential application in control of cancer cell growth,a preudotype retrovirus containing antisense sequence of human N-ras was constructed and packaged.A recombinant retrovirus vector containing antisense or sense sequences of N-ras cDNA was constructed by pZIP-NeoSV(X)1.The pseudotype virus was packaged ang rescued by transfection and infection in PA317 and ψ 2 helper cells.It has been demonstrated that the pseudotype retrovirus containing antisense N-ras sequence did inhibit the growth of human PLC/PRF/5 hepatoma cells accompanied with inhibition of p21 expression,while the retrovirus containing sense sequence had none.The pseudotype virus had no effect on human diploid fibroblasts.

  3. Construction of Antisense Transforming Growth Factorβ1 Gene and Its Effect on the Proliferation by Expression in Osteosarcoma Cells

    Institute of Scientific and Technical Information of China (English)

    刘勇; 郑启新; 杜靖远; 杨述华; 邵增务; 肖宝钧

    2003-01-01

    Summary: To construct the antisensc transforming growth factorβl (TGFβ1) gene and investigatethe effect of TGFβ1 autocrine loop blockage on the proliferation of osteosarcoma cells. TGFβ1 cDNAwas cloned by RT-PCR from human osteosarcoma cells (MG-63) and inserted into pcDNA3 to con-struct an antisense expression vector, which was dubbed pcDNA3-TGFβ1(- ). MTT was used to de-tect the proliferation of osteosarcoma cells transfected by antisense TGFβ1 gene. Our results showedthat the proliferation of the transfected osteosarcoma cells was suppressed markedly. It is concludedthat TGFβ1 autocrine loop blockage in osteosarcoma cells could inhibit cell proliferation, which mightbe helpful for gene therapy of osteosarcoma.

  4. CpG oligodeoxynucleotide stimulates production of anti-neutrophil cytoplasmic antibodies in ANCA associated vasculitis

    Directory of Open Access Journals (Sweden)

    Sarvestani Ghafar

    2008-07-01

    Full Text Available Abstract Background Wegener's Granulomatosis and Microscopic Polyangiitis are life-threatening systemic necrotizing vasculitides of unknown aetiology. The appearance of circulating antibodies to neutrophil cytoplasmic antigens (ANCA is strongly associated with the development of the disease. A link between infection and disease has long been suspected, and the appearance of ANCA antibodies has been reported following bacterial and viral infections. The depletion of circulating B cells with monoclonal antibody therapy can induce remission, and this observation suggests a pathogenic role for B cells in this disease. As bacterial DNA is known to induce B cell proliferation and antibody production via TLR-9 stimulation, we have explored the possibility that unmethylated CpG oligodeoxynucleotide, as found in bacterial and viral DNA, may play a role in stimulating circulating autoreactive B cells to produce ANCA in patients with vasculitis. Results We have confirmed that unmethylated CpG oligonucleotide is a potent stimulator of antibody production by PBMC in vitro. The stimulation of PBMC with CpG oligonucleutides resulted in the production of similar amounts of IgG in both ANCA+ patients and normal controls. In spite of this, PR3 ANCA+ patients synthesised significantly higher amount of IgG ANCA than normal controls. In MPO ANCA+ patients, there was a tendency for patients to produce higher amount of ANCA than controls, however, the difference did not reach significance. Furthermore, we were able to detect circulating MPO-reactive B cells by ELISpot assay from the peripheral blood of 2 MPO+ ANCA vasculitis patients. Together, this indicates that circulating anti-neutrophil autoreactive B cells are present in ANCA+ vasculitis patients, and they are capable of producing antibodies in response to CpG stimulation. Of note, CpG also induced the production of the relevant autoantibodies in patients with other types of autoimmune diseases. Conclusion

  5. Effect of c-fos antisense probe on prostaglandin E2-induced upregulation of vascular endothelial growth factor mRNA in human liver cancer cells

    Institute of Scientific and Technical Information of China (English)

    Yong-Qi Li; Kai-Shan Tao; Ning Ren; Yi-Hu Wang

    2005-01-01

    AIM: To examine the effect of prostaglandin E2 (PGE2) on the expression of vascular endothelial growth factor (VEGF) mRNA in the human hepatocellular carcinoma (HCC) HepG2 cells and the possible involvement of c-fos protein in this process.METHODS: Human HCC HepG2 cells were divided into three groups treated respectively with PGE2, a combination of PGE2 and c-fos antisense oligodeoxynucleotide (ASO),and PGE2 plus c-fos sense oligodeoxynudeotide (SO). The expression of VEGF mRNA in HepG2 cells after different treatments was detected by reverse transcriptase-polymerase chain reaction (RT-PCR). The relative expression level of VEGF mRNA in HepG2 cells in each group was measured.RESULTS: Administration of PGE2 resulted in an increased expression of c-fosand VEGF mRNA in HepG2 cells. The relative expression level of c-fos mRNA reached the peak at 3 h (68.4±4.7%) after PGE2 treatment, which was significantly higher than that at 0 h (20.6±1.7%, P<0.01).Whereas, the highest expression level of VEGF mRNA was observed at 6 h (100.5±6.1%) after PGE2 treatment, which was significantly higher than that at 0 h (33.2±2.4%,P<0.01). C-fos ASO significantly reduced PGE2-induced VEGF mRNA expression in HepG2 cells.CONCLUSION: PGE2 increases the expression and secretion of VEGF in HCC cells by activating the transcription factor c-fos, promotes the angiogenesis of HCC and plays an important role in the pathogenesis of liver cancer.

  6. Identification of antisense long noncoding RNAs that function as SINEUPs in human cells

    DEFF Research Database (Denmark)

    Schein, Aleks; Zucchelli, Silvia; Kauppinen, Sakari;

    2016-01-01

    , increasing PPP1R12A protein translation in human cells. The SINEUP activity depends on the aforementioned sense-antisense interaction and a free right Alu monomer repeat element at the 3' end of R12A-AS1. In addition, we identify another human antisense lncRNA with SINEUP activity. Our results demonstrate...

  7. Intra-Amygdala Injections of CREB Antisense Impair Inhibitory Avoidance Memory: Role of Norepinephrine and Acetylcholine

    Science.gov (United States)

    Canal, Clinton E.; Chang, Qing; Gold, Paul E.

    2008-01-01

    Infusions of CREB antisense into the amygdala prior to training impair memory for aversive tasks, suggesting that the antisense may interfere with CRE-mediated gene transcription and protein synthesis important for the formation of new memories within the amygdala. However, the amygdala also appears to modulate memory formation in distributed…

  8. Development of CpG-oligodeoxynucleotides for effective activation of rabbit TLR9 mediated immune responses.

    Directory of Open Access Journals (Sweden)

    Tsung-Hsien Chuang

    Full Text Available CpG-oligodeoxynucleotides (CpG-ODN are potent immune stimuli being developed for use as adjuvants in different species. Toll-like receptor 9 (TLR9 is the cellular receptor for CpG-ODN in mammalian cells. The CpG-ODN with 18-24 deoxynucleotides that are in current use for human and mouse cells, however, have low activity with rabbit TLR9. Using a cell-based activation assay, we developed a type of CpG-ODN containing a GACGTT or AACGTT motif in 12 phosphorothioate-modified deoxynucleotides with potent stimulatory activity for rabbit TLR9. The developed CpG-ODN have higher activities than other developed CpG-ODN in eliciting antigen-nonspecific immune responses in rabbit splenocytes. When mixed with an NJ85 peptide derived from rabbit hemorrhagic disease virus, they had potent activities to boost an antigen-specific T cell activation and antibody production in rabbits. Compared to Freund's adjuvant, the developed CpG-ODN are capable of boosting a potent and less toxic antibody response. The results of this study suggest that both the choice of CpG-motif and its length are important factors for CpG-ODN to effectively activate rabbit TLR9 mediated immune responses.

  9. Use of oligodeoxynucleotide signature probes for identification of physiological groups of methylotrophic bacteria

    International Nuclear Information System (INIS)

    Oligodeoxynucleotide sequences that uniquely complemented 16S rRNAs of each group of methylotrophs were synthesized and used as hybridization probes for the identification of methylotrophic bacteria possessing the serine and ribulose monophosphate (RuMP) pathways for formaldehyde fixation. The specificity of the probes was determined by hybridizing radiolabeled probes with slot-blotted RNAs of methylotrophs and other eubacteria followed by autoradiography. The washing temperature was determined experimentally to be 50 and 52 degrees C for 9-α (serine pathway) and 10-γ (RuMP pathway) probes, respectively. RNAs isolated from serine pathway methylotrophs bound to probe 9-α, and RNAs from RuMP pathway methylotrophs bound to probe 10-γ. Nonmethylotrophic eubacterial RNAs did not bind to either probe. The probes were also labeled with fluorescent dyes. Cells fixed to microscope slides were hybridized with these probes, washed, and examined in a fluorescence microscope equipped with appropriate filter sets. Cells of methylotrophic bacteria possessing the serine or RuMP pathway specifically bind probes designed for each group. Samples with a mixture of cells of type I and II methanotrophs were detected and differentiated with single probes or mixed probes labeled with different fluorescent dyes, which enabled the detection of both types of cells in the same microscopic field

  10. Cooperative stimulation of dendritic cells by Cryptococcus neoformans mannoproteins and CpG oligodeoxynucleotides.

    Directory of Open Access Journals (Sweden)

    Jennifer M Dan

    Full Text Available While mannosylation targets antigens to mannose receptors on dendritic cells (DC, the resultant immune response is suboptimal. We hypothesized that the addition of toll-like receptor (TLR ligands would enhance the DC response to mannosylated antigens. Cryptococcus neoformans mannoproteins (MP synergized with CpG-containing oligodeoxynucleotides to stimulate enhanced production of proinflammatory cytokines and chemokines from murine conventional and plasmacytoid DC. Synergistic stimulation required the interaction of mannose residues on MP with the macrophage mannose receptor (MR, CD206. Moreover, synergy with MP was observed with other TLR ligands, including tripalmitoylated lipopeptide (Pam3CSK4, polyinosine-polycytidylic acid (pI:C, and imiquimod. Finally, CpG enhanced MP-specific MHC II-restricted CD4(+ T-cell responses by a mechanism dependent upon DC expression of CD206 and TLR9. These data suggest a rationale for vaccination strategies that combine mannosylated antigens with TLR ligands and imply that immune responses to naturally mannosylated antigens on pathogens may be greatly augmented if TLR and MR are cooperatively stimulated.

  11. Employment of Oligodeoxynucleotide plus Interleukin-2 Improves Cytogenetic Analysis in Splenic Marginal Zone Lymphoma

    Directory of Open Access Journals (Sweden)

    Antonella Bardi

    2011-01-01

    Full Text Available To compare the efficiency of novel mitogenic agents and traditional mitosis inductors, 18 patients with splenic marginal zone lymphoma (SMZL were studied. Three cultures using oligodeoxynucleotide (ODN plus interleukin-2 (IL-2, or TPA, or LPS were setup in each patient. Seventeen/18 cases with ODN+IL2 had moderate/good proliferation (94,4% as compared with 10/18 cases with TPA and LPS (55% (P=.015; 14/18 (77,7% cases with ODN+IL2 had sufficient good quality of banding as compared with 8/18 cases (44,4% with TPA and LPS. The karyotype could be defined from ODN+IL2-stimulated cultures in all 18 patients, 14 of whom (77,7% had a cytogenetic aberration, whereas clonal aberrations could be documented in 9 and in 3 cases by stimulation with LPS and TPA, respectively. Recurrent chromosome aberrations in our series were represented by aberrations of chromosome 14q in 5 patients, by trisomy 12 and 7q deletion in 4 cases each, and by abnormalities involving 11q and 13q in two cases each. These findings show that stimulation with ODN+IL2 offers more mitotic figures of better quality and results in an increased rate of clonal aberrations in SMZL, making this method ideal for prospective studies aiming at the definition of the prognostic impact of cytogenetic aberrations in this disorder.

  12. CpG oligodeoxynucleotides as TLR9 agonists: therapeutic applications in cancer.

    Science.gov (United States)

    Murad, Yanal M; Clay, Timothy M

    2009-01-01

    Toll-like receptors (TLRs) are part of the innate immune system, and they belong to the pattern recognition receptors (PRR) family. The PRR family is designed to recognize and bind conserved pathogen-associated molecular patterns, which are not generated by the host and are restricted and essential to micro-organisms. TLR9, which recognizes unmethylated CpG (cytosine guanosine dinucleotide), is a very promising target for therapeutic activation. Stimulation of TLR9 activates human plasmacytoid dendritic cells and B cells, and results in potent T helper-1 (T(h)1)-type immune responses and antitumor responses in mouse tumor models and in patients. Several pharmaceutical companies, such as Pfizer, Idera, and Dynavax, are developing CpG oligodeoxynucleotides (ODNs) for the treatment of cancer, along with other conditions, such as infections and allergy. CpG ODNs have shown promising results as vaccine adjuvants and in combination with cancer immunotherapy. Several TLR9 agonists are being developed and have entered clinical trials to evaluate their safety and efficacy for the treatment of several hematopoietic and solid tumors. In this review, we discuss the use of CpG ODNs in several phase I and II clinical trials for the treatment of NHL, renal cell carcinoma, melanoma, and non-small cell lung cancer, either alone or in combination with other agents. PMID:19894778

  13. VaccineDA: Prediction, design and genome-wide screening of oligodeoxynucleotide-based vaccine adjuvants.

    Science.gov (United States)

    Nagpal, Gandharva; Gupta, Sudheer; Chaudhary, Kumardeep; Dhanda, Sandeep Kumar; Prakash, Satya; Raghava, Gajendra P S

    2015-01-01

    Immunomodulatory oligodeoxynucleotides (IMODNs) are the short DNA sequences that activate the innate immune system via toll-like receptor 9. These sequences predominantly contain unmethylated CpG motifs. In this work, we describe VaccineDA (Vaccine DNA adjuvants), a web-based resource developed to design IMODN-based vaccine adjuvants. We collected and analyzed 2193 experimentally validated IMODNs obtained from the literature. Certain types of nucleotides (e.g., T, GT, TC, TT, CGT, TCG, TTT) are dominant in IMODNs. Based on these observations, we developed support vector machine-based models to predict IMODNs using various compositions. The developed models achieved the maximum Matthews Correlation Coefficient (MCC) of 0.75 with an accuracy of 87.57% using the pentanucleotide composition. The integration of motif information further improved the performance of our model from the MCC of 0.75 to 0.77. Similarly, models were developed to predict palindromic IMODNs and attained a maximum MCC of 0.84 with the accuracy of 91.94%. These models were evaluated using a five-fold cross-validation technique as well as validated on an independent dataset. The models developed in this study were integrated into VaccineDA to provide a wide range of services that facilitate the design of DNA-based vaccine adjuvants (http://crdd.osdd.net/raghava/vaccineda/). PMID:26212482

  14. 以腺病毒为载体表达猪α(1,3)半乳糖基转移酶 反义RNA抑制Galα(1,3)Gal抗原表位的表达%Adenovirus-mediated expression of antisense RNA transcripts complementary to pig a(1,3) galactosyltransferase mRNA inhibits expression of Gal α(1,3) Gal epitope

    Institute of Scientific and Technical Information of China (English)

    邢力; 夏国宏; 白旭芳; 费俭; 郭礼和

    2000-01-01

    目的:尝试以反义RNA的方法抑制Galα(1,3)Gal抗原表位(gal抗原)的表达.方法:以人腺病毒载体表达猪α(1,3)半乳糖基转移酶基因的反义RNA.流式细胞术比较H血型抗原和gal抗原的表达水平.结果:构建了表达反义RNA的重组腺病毒载体Ad5anti-sGT600和Adanti-sGTll00.反义RNA的表达使NIH3T3细胞表面的gal抗原表位下降约30%.另外,反义RNA与人分泌型α(1,2)岩藻糖基转移酶的共同作用可使gal抗原表位的水平进一步下降.结论:重组腺病毒Adanti-sGT600和Ad5anti-sGTll00可有效降低gal抗原表位的表达.%AIM: To examine the effects of the expression of antisense RNA transcripts complementary to the pig α(1,3) galactosyltransferase [α(1,3)GT]mRNA on the expression of Gal α(1,3) Gal structure (gal epitope) in cultured cell lines. METHODS: Human adenoviral vectors were used to mediate the expression of antisense RNA. The expression levels of H blood group antigens and gal epitopes were analyzed by flow cytometry using FITC-UEAI and FITC-GS-IB4 lectins, respectively. RESULTS: Recombinant adenoviruses, Ad5anti-sGT600 and Ad5anti-sGTll00, which express antisense RNA complementary to different regions of the pig α (1,3) GT mRNA, were constructed and used to infect cell line of NIH3T3. The results showed about 30% reduction in the expression level of gal epitopes on the surface of NIH3T3 cells. In addition, co-expression of human secretor type α(1,2) fucosyltransferase [α(1,2)Fr]cDNA and antisense RNA complementary to the pig α(1 ,3) GT mRNA led to a further reduction in the gal epitope level. CONCLUSION: Recombinant adenoviruses, Ad5anti-sGT600 and Ad5anti-sGTll00, are effective to down-regulate the gal epitope expression.

  15. Cis-Antisense Transcription Gives Rise to Tunable Genetic Switch Behavior: A Mathematical Modeling Approach.

    Science.gov (United States)

    Bordoy, Antoni E; Chatterjee, Anushree

    2015-01-01

    Antisense transcription has been extensively recognized as a regulatory mechanism for gene expression across all kingdoms of life. Despite the broad importance and extensive experimental determination of cis-antisense transcription, relatively little is known about its role in controlling cellular switching responses. Growing evidence suggests the presence of non-coding cis-antisense RNAs that regulate gene expression via antisense interaction. Recent studies also indicate the role of transcriptional interference in regulating expression of neighboring genes due to traffic of RNA polymerases from adjacent promoter regions. Previous models investigate these mechanisms independently, however, little is understood about how cells utilize coupling of these mechanisms in advantageous ways that could also be used to design novel synthetic genetic devices. Here, we present a mathematical modeling framework for antisense transcription that combines the effects of both transcriptional interference and cis-antisense regulation. We demonstrate the tunability of transcriptional interference through various parameters, and that coupling of transcriptional interference with cis-antisense RNA interaction gives rise to hypersensitive switches in expression of both antisense genes. When implementing additional positive and negative feed-back loops from proteins encoded by these genes, the system response acquires a bistable behavior. Our model shows that combining these multiple-levels of regulation allows fine-tuning of system parameters to give rise to a highly tunable output, ranging from a simple-first order response to biologically complex higher-order response such as tunable bistable switch. We identify important parameters affecting the cellular switch response in order to provide the design principles for tunable gene expression using antisense transcription. This presents an important insight into functional role of antisense transcription and its importance towards

  16. Respirable antisense oligonucleotides: a new drug class for respiratory disease

    Directory of Open Access Journals (Sweden)

    Tanaka Makoto

    2000-12-01

    Full Text Available Abstract Respirable antisense oligonucleotides (RASONs, which attenuate specific disease-associated mRNAs, represent a new class of respiratory therapeutics with considerable potential. RASONs overcome previous obstacles that have impeded the development of antisense therapeutics targeting diseases in other organ systems. RASONs are delivered directly to the target tissue via inhalation; their uptake seems to be enhanced by cationic properties inherent in pulmonary surfactant, and, because of the markedly different target properties of mRNA and proteins, they can have very long durations of effect compared with traditional drugs targeting the protein of the same gene. RASONs contain chemical modifications that decrease their degradation by cellular nucleases. However, total insensitivity to nucleases is probably not an optimal design criterion for RASONs, because moderate nuclease sensitivity can prevent their systemic delivery, decreasing the potential for systemic toxicity. EPI-2010 is a 21-mer phosphorothioate RASON that attenuates bronchoconstriction, inflammation and surfactant depletion in preclinical models of human asthma, has a duration of effect of seven days, and seems to undergo minimal systemic delivery.

  17. Does Active Learning through an Antisense Jigsaw Make Sense?

    Science.gov (United States)

    Seetharaman, Mahadevan; Musier-Forsyth, Karin

    2003-12-01

    Three journal articles on nucleic acid antisense modification strategies were assigned to 12 students as part of an active learning "jigsaw" exercise for a graduate-level chemistry course on nucleic acids. Each student was required to read one of the three articles. This assignment was preceded by an hour-long lecture on the basic concepts in antisense antigene technology. On the day of the jigsaw, the students with the same article (three groups of four students) discussed their article briefly, and then formed four new groups where no one had read the same article. Each student spent about five minutes teaching his or her article to the other group members, using specific questions provided to guide the discussion. This exercise laid the foundation for bringing the discussion to the entire class, where most of the students actively participated. To test the students' comprehension of the reading materials, a problem set was designed that required not only an understanding of the three articles, but also application of the concepts learned. The effectiveness of this active learning strategy and its applicability to other topics are discussed in this article.

  18. Combination Antisense Treatment for Destructive Exon Skipping of Myostatin and Open Reading Frame Rescue of Dystrophin in Neonatal mdx Mice.

    Science.gov (United States)

    Lu-Nguyen, Ngoc B; Jarmin, Susan A; Saleh, Amer F; Popplewell, Linda; Gait, Michael J; Dickson, George

    2015-08-01

    The fatal X-linked Duchenne muscular dystrophy (DMD), characterized by progressive muscle wasting and muscle weakness, is caused by mutations within the DMD gene. The use of antisense oligonucleotides (AOs) modulating pre-mRNA splicing to restore the disrupted dystrophin reading frame, subsequently generating a shortened but functional protein has emerged as a potential strategy in DMD treatment. AO therapy has recently been applied to induce out-of-frame exon skipping of myostatin pre-mRNA, knocking-down expression of myostatin protein, and such an approach is suggested to enhance muscle hypertrophy/hyperplasia and to reduce muscle necrosis. Within this study, we investigated dual exon skipping of dystrophin and myostatin pre-mRNAs using phosphorodiamidate morpholino oligomers conjugated with an arginine-rich peptide (B-PMOs). Intraperitoneal administration of B-PMOs was performed in neonatal mdx males on the day of birth, and at weeks 3 and 6. At week 9, we observed in treated mice (as compared to age-matched, saline-injected controls) normalization of muscle mass, a recovery in dystrophin expression, and a decrease in muscle necrosis, particularly in the diaphragm. Our data provide a proof of concept for antisense therapy combining dystrophin restoration and myostatin inhibition for the treatment of DMD. PMID:25959011

  19. Abrupt reduction of c-myc expression by antisense RNA inducing terminal differentiation and apoptosis of a human esophageal cancer cell line

    Institute of Scientific and Technical Information of China (English)

    赵晓航; 王秀琴; 周传农; 彭仁玲; 阎水中; 吴旻

    1995-01-01

    A human esophageal cancer cell line (EC8712) expressing high-level Myc protein was infected with recombmant retroviral particles (pA-BD9) at a multiplicity of infection (MOI) 1:1. This viral particle contains a neomycin-resistant gene and a 1.53-kb antisense RNA spanning the 2nd exon and its flanking sequences of the human c-myc oncogene. The G418-resistant EC8712 clones showed an 86% inhibition of growth rate and morphological changes characteristic of terminal differentiation and apoptosis. A decrease of about 80% of Myc protein was also observed in these infected cells by ABC-ELISA assay. 12-24 h after the infection of ECS712 cells with pA-BD9 at a high viral particle concentration (MOI = 1:10), the integration of the extrinsic 1.53-kb antisense c-myc fragment into the cancer cell genome was evidenced by the Southern blot analysis. Northern blot analyses showed the expression of this antisense fragment and a decrease of the intrinsic c-myc expression by 74% in comparison with that of the parental EC8

  20. 中脑腹侧被盖区注射M5受体反义寡核苷酸抑制海洛因敏化大鼠伏隔核和海马中FosB表达%Microinjection of M5 muscarinic receptor antisense oligonucleotide into VTA inhibits FosB expression in the NAc and the hippocampns of heroin sensitized rats

    Institute of Scientific and Technical Information of China (English)

    刘惠芬; 周文华; 朱华强; 赖苗军; 陈为升

    2007-01-01

    Objective To investigate the effect of M5 muscarinic receptor subtype on the locomotor sensitization induced by heroin priming, and it's effect on the FosB expression in the nucleus accumbens (NAc) and the hippocampus in the heroin sensitized rats. Methods Locomotor activity was measured every 10 min for 1 h after subcutaneous injection of heroin. FosB expression was assayed by immunohistochemistry, and the antisense oligonucleotides (AS-ONs) targeting M5 muscarinic receptor was transferred with the lipofectin. Results Microinjection of AS-ONs targeting M5 muscarinic receptor in the ventral tegmental area (VTA) blocked the expression of behavioral sensitization induced by heroin priming in rats. Meanwhile, the expression of FosB-positive neurons in either the NAc or the dentate gyrus (DG) of the hippocampus increased in heroin-induced locomotor sensitized rats. The enhancement of FosB-positive neurons in the NAc or DG could be inhibited by microinjection of M5 muscarinic receptor AS-ONs into the VTA before the heroin-induced locomotor sensitization was performed. In contrast, microinjection of M5 muscarinic receptor sense oligonucleotide (S-ONs) into the VTA did not block the expression of behavioral sensitization or the expression of FosB in the NAc or DG in the heroin sensitized rats. Conclusion Blocking M5 muscarinic receptor in the VTA inhibits the expression of heroin-induced locomotor sensitization, which is associated with the regulation of FosB expression in the NAc and hippocampus neurons.M5 muscarinic receptor may be a useful pharmacological target for the treatment of heroin addiction.%目的 探讨M5毒蕈碱受体亚型对海洛因诱导的大鼠行为敏化以及敏化后大脑伏隔核(NAc)和海马中FosB蛋白表达的影响.方法 建立海洛因诱导的大鼠行为敏化模型,测定大鼠的自主活动量(locomotor activity,LA),观察M5毒蕈碱受体反义寡核苷酸(M5AS-ONs)对行为敏化表达的影响.用免疫组化法测定大鼠NAc

  1. Effect of the control proliferation of astrocyte on the formation of glial scars by antisense GFAP retrovirus

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Astrocytes play an important role in the formation of glial scars.In order to investigate the effect of inhibiting GFAP gene expression on normal,reactive astrocytes and on glial scar formation,the efficiency of the recombinant antisense GFAP retrovirus (PLBskG) on the growth,cell cycle,morphology and GFAP gene expression of astrocytes in vitro and on the formation of glial scars in vivo has been studied by cell growth curves,flow cytometry,immunocytochemistry,in situ hybridization,RT-PCR and Southern blot.The results confirm the recombinant retrovirus (PLBskG) produced growth suppression and G1 arrest of the normal and injured astrocytes.The infected cells become round or ellipoid.The cell processes become fine or retracted.The intensity of staining of GFAP is reduced.Expression of GFAP mRNA is down regulated.However,in the control experiment,no obvious effects on the morphology or synthesis of GFAP on cultured normal and scratched astrocytes infected by primary retrovirus vector (PLXSN) have been observed.The supernatant of PLBskG has been injected into an injured site by microinjection in vivo.The number and process lengths of GFAP positive cells are obviously reduced around the injured site.The formation of the glial scar is inhibited,showing that the recombinant antisense GFAP retrovirus can effectively inhibit the growth and GFAP expression of normal and injured astrocytes in vitro and the formation of glial scar in vivo.It is suggested that GFAP plays an important role in glial scar formation.

  2. CpG Oligodeoxynucleotide and Montanide ISA 51 Adjuvant Combination Enhanced the Protective Efficacy of a Subunit Malaria Vaccine

    OpenAIRE

    Kumar, Sanjai; Jones, Trevor R.; Oakley, Miranda S.; Zheng, Hong; Shanmuga P Kuppusamy; Taye, Alem; Krieg, Arthur M.; Stowers, Anthony W; Kaslow, David C.; Hoffman, Stephen L.

    2004-01-01

    Unmethylated CpG dinucleotide motifs present in bacterial genomes or synthetic oligodeoxynucleotides (ODNs) serve as strong immunostimulatory agents in mice, monkeys and humans. We determined the adjuvant effect of murine CpG ODN 1826 on the immunogenicity and protective efficacy of the Saccharomyces cerevisiae-expressed 19-kDa C-terminal region of merozoite surface protein 1 (yMSP119) of the murine malaria parasite Plasmodium yoelii. We found that in C57BL/6 mice, following sporozoite challe...

  3. Immunostimulatory oligodeoxynucleotides containing the CpG motif are effective as immune adjuvants in tumor antigen immunization

    OpenAIRE

    Weiner, George J; Liu, Hsin-Ming; Wooldridge, James E.; Dahle, Christopher E.; Krieg, Arthur M.

    1997-01-01

    Recent advances in our understanding of the immune response are allowing for the logical design of new approaches to cancer immunization. One area of interest is the development of new immune adjuvants. Immunostimulatory oligodeoxynucleotides containing the CpG motif (CpG ODN) can induce production of a wide variety of cytokines and activate B cells, monocytes, dendritic cells, and NK cells. Using the 38C13 B cell lymphoma model, we assessed whether CpG ODN can function as immune adjuvants in...

  4. Enhanced therapeutic effects of combined chemotherapeutic drugs and midkine antisense oligonucleotides for hepatocellular carcinoma

    Institute of Scientific and Technical Information of China (English)

    Li-Cheng Dai; Xiang Wang; Xing Yao; Yong-Liang Lu; Jin-Liang Ping; Jian-Fang He

    2007-01-01

    AIM: To evaluate the effect of combined antisense oligonucleotides targeting midkine (MK-AS) and chemotherapeutic drugs [cisplatin(DDP), 5-fluorouracil(5-FU) and adriamycin (ADM)] on inhibition of HepG2cell proliferation, and to analyze the efficacy of MK-AS used in combined ADM in in situ human hepatocellular carcinoma (HCC) model.METHODS: HepG2 cells were treated with MK-AS and/or chemotherapeutic drugs mediated by Lipofectin,and cell growth activity was determined by MTS assay.An in situ HCC model was used in this experiment. MKAS, ADM and MK-AS + ADM were given intravenously for 20 d, respectively. The animal body weight and their tumor weight were measured to assess the effect of the combined therapy in vivo.RESULTS: Combined treatment with MK-AS reduced the IC50 of DDP, 5-FU and ADM in HepG2 cells. MK-AS significantly increased the inhibition rate of DDP, 5-FU and ADM. Additionally, synergism (Q 1.15) occurred at a lower concentration of ADM, 5-FU and DDP with combined MK-AS. Combined treatment with MK-AS and ADM resulted in the more growth inhibition on in situ human HCC model compared with treatment with chemotherapeutic drugs alone.CONCLUSION: MK-AS increases the chemosensitivity in HepG2 cells and in situ human HCC model, and the combination of MK-AS and ADM has a much better in vitro and in vivo synergism.

  5. Functional Analysis of Polyphenol Oxidases by Antisense/Sense Technology

    Directory of Open Access Journals (Sweden)

    Jutharat Attajarusit

    2007-07-01

    Full Text Available Polyphenol oxidases (PPOs catalyze the oxidation of phenolics to quinones, the secondary reactions of which lead to oxidative browning and postharvest losses of many fruits and vegetables. PPOs are ubiquitous in angiosperms, are inducible by both biotic and abiotic stresses, and have been implicated in several physiological processes including plant defense against pathogens and insects, the Mehler reaction, photoreduction of molecular oxygen by PSI, regulation of plastidic oxygen levels, aurone biosynthesis and the phenylpropanoid pathway. Here we review experiments in which the roles of PPO in disease and insect resistance as well as in the Mehler reaction were investigated using transgenic tomato (Lycopersicon esculentum plants with modified PPO expression levels (suppressed PPO and overexpressing PPO. These transgenic plants showed normal growth, development and reproduction under laboratory, growth chamber and greenhouse conditions. Antisense PPO expression dramatically increased susceptibility while PPO overexpression increased resistance of tomato plants to Pseudomonas syringae. Similarly, PPO-overexpressing transgenic plants showed an increase in resistance to various insects, including common cutworm (Spodoptera litura (F., cotton bollworm (Helicoverpa armigera (Hübner and beet army worm (Spodoptera exigua (Hübner, whereas larvae feeding on plants with suppressed PPO activity had higher larval growth rates and consumed more foliage. Similar increases in weight gain, foliage consumption, and survival were also observed with Colorado potato beetles (Leptinotarsa decemlineata (Say feeding on antisense PPO transgenic tomatoes. The putative defensive mechanisms conferred by PPO and its interaction with other defense proteins are discussed. In addition, transgenic plants with suppressed PPO exhibited more favorable water relations and decreased photoinhibition compared to nontransformed controls and transgenic plants

  6. Topical CpG Oligodeoxynucleotide Adjuvant Enhances the Adaptive Immune Response against Influenza A Infections.

    Science.gov (United States)

    Cheng, Wing Ki; Plumb, Adam William; Lai, Jacqueline Cheuk-Yan; Abraham, Ninan; Dutz, Jan Peter

    2016-01-01

    Current influenza vaccines generate humoral immunity, targeting highly variable epitopes and thus fail to achieve long-term protection. T cells recognize and respond to several highly conserved epitopes across influenza serotypes. A strategy of raising strong cytotoxic T cell memory responses to epitopes conserved across serotypes would provide cross serotype protection, eliminating the need for annual vaccination. We explored the adjuvant potential of epicutaneous (ec) and subcutaneous (sc) delivery of CpG oligodeoxynucleotide in conjunction with sc protein immunization to improve protection against influenza A virus (IAV) infections using a mouse model. We found enhanced long-term protection with epicutaneous CpG ODN (ecCpG) compared to subcutaneous CpG ODN (scCpG) as demonstrated by reduced viral titers in the lungs. This correlated with increased antigen-specific CD8 T cells in the airways and the lungs. The memory T cell response after immunization with ecCpG adjuvant was comparable to memory response by priming with IAV infection in the lungs. In addition, ecCpG was more efficient than scCpG in inducing the generation of IFN-γ producing CD4 T cells. The adjuvant effect of ecCpG was accompanied with its ability to modulate tissue-homing molecules on T cells that may direct them to the site of infection. Together, this work provides evidence for using ecCpG to induce strong antibody and memory T cell responses to confer protection against IAV infection. PMID:27524984

  7. Therapeutic applications of synthetic CpG oligodeoxynucleotides as TLR9 agonists for immune modulation.

    Science.gov (United States)

    Jurk, Marion; Vollmer, Jörg

    2007-01-01

    Vertebrate toll-like receptors (TLRs) sense invading pathogens by recognizing bacterial and viral structures and, as a result, activate innate and adaptive immune responses. Ten human functional TLRs have been reported so far; three of these (TLR7, 8, and 9) are expressed in intracellular compartments and respond to single-stranded nucleic acids as natural ligands. The pathogen structure selectively recognized by TLR9 in bacterial or viral DNA was identified to be CpG dinucleotides in specific sequence contexts (CpG motifs). Short phosphorothioate-stabilized oligodeoxynucleotides (ODNs) containing such motifs are used as synthetic TLR9 agonists, and different classes of ODN TLR9 agonists have been identified with distinct immune modulatory profiles. The TLR9-mediated activation of the vertebrate immune system suggests using such TLR9 agonists as effective vaccine adjuvants for infectious disease, and for the treatment of cancer and asthma/allergy. Immune activation by CpG ODNs has been demonstrated to be beneficial in animal models as a vaccine adjuvant and for the treatment of a variety of viral, bacterial, and parasitic diseases. Antitumor activity of CpG ODNs has also been established in numerous mouse models. In clinical vaccine trials in healthy human volunteers or in immunocompromised HIV-infected patients, CpG ODNs strongly enhanced vaccination efficiency. Most encouraging results in the treatment of cancers have come from human phase I and II clinical trials using CpG ODNs as a tumor vaccine adjuvant, monotherapy, or in combination with chemotherapy. Therefore, CpG ODNs represent targeted immune modulatory drugs with a broad range of potential applications. PMID:18020622

  8. Synthesis of novel chitosan–silica/CpG oligodeoxynucleotide nanohybrids with enhanced delivery efficiency

    International Nuclear Information System (INIS)

    Chitosan–silica/CpG oligodeoxynucleotide (ODN) nanohybrids were synthesized to stimulate Toll-like receptor 9-mediated induction of interleukin-6 (IL-6). The chitosan–silica hybrid was first synthesized from a mixture of chitosan and 3-glycidoxypropyl trimethoxysilane under acidic conditions via a sol–gel process, and then used to condense CpG ODN2006x3-PD to yield chitosan–silica/CpG ODN nanohybrids. Scanning electron microscopy and atomic force microscopy showed that the chitosan–silica/CpG ODN nanohybrids had an elliptic shape with a diameter of 100–200 nm. After soaking in HAc–NaAc buffer solution (pH 5.5), the nanohybrids exhibited sustained release of CpG ODN. When the nanohybrids were separately exposed to 293XL-hTLR9 cells and peripheral blood mononuclear cells, no significant toxicity was observed. An immunochemical assay for cellular uptake revealed that the nanohybrids were taken up by the cells and located in endolysosomes. An enzyme-linked immunosorbent assay for cytokines indicated that the nanohybrids effectively stimulated the induction of IL-6. Chitosan–silica/CpG ODN nanohybrids underwent cellular uptake and enhanced induction of IL-6 to a greater degree than conventional chitosan/CpG ODN nanocomplexes, indicating that they have an enhanced delivery efficiency. - Highlights: • Chitosan-silica/CpG ODN nanohybrids (100 nm–200 nm) were synthesized. • Nanohybrids exhibited a sustained release behavior for CpG ODN. • Cellular uptake of nanohybrids was enhanced due to presence of silica. • The released CpG ODN stimulated production of cell cytokine

  9. Inhibitory effect of IGF-Ⅱ antisense RNA on malignant phenotype of hepatocellular carcinoma

    Institute of Scientific and Technical Information of China (English)

    Dong Hua Yang; Ming Qing Zhang; Han Rong Qin; Zi Rong Fan; Jiang Du; Chong Xu; Qiao Ming Liang; Ji Fang Mao

    2000-01-01

    @@INTRODUCTION According to the therapeutic effect and strategy of antisense RNA for hepatocellular carcinoma (HCC), we have specifically synthesized partial cDNA of human insulin-like growth factor Ⅱ (IGFⅡ ) and constructed IGF-Ⅱ cDNA antisense eukaryotic expression vector. The constructed vector was introduced into hepatoma cell line SMMC-7721 to block the intrinsic IGF- Ⅱexpression. The biological behavior changes of hepatoma cells were observed. All these would provide scientific basis for IGF- Ⅱ antisense RNA in the treatment of HCC.

  10. An antisense RNA that governs the expression kinetics of a multifunctional virulence gene

    OpenAIRE

    Lee, Eun-Jin; Groisman, Eduardo A.

    2010-01-01

    Genome-wide transcriptome analyses of several bacterial species have recently uncovered a hitherto unappreciated amount of antisense transcription. However, the physiological role, regulation and significance of such antisense transcripts are presently unclear. We now report the identification of a cis-encoded 1.2 kb long antisense RNA – termed AmgR – that is complementary to the mgtC portion of the mgtCBR polycistronic message from Salmonella enterica. The mgtCBR mRNA specifies the MgtC prot...

  11. LNA-antisense rivals siRNA for gene silencing

    DEFF Research Database (Denmark)

    Jepsen, Jan Stenvang; Wengel, Jesper; Stenvang, Jan

    2004-01-01

    Locked nucleic acid (LNA) is a class of nucleic acid analogs possessing unprecedented binding affinity toward complementary DNA and RNA while obeying the Watson-Crick base-pairing rules. For efficient gene silencing in vitro and in vivo, fully modified or chimeric LNA oligonucleotides have been...... applied. LNA oligonucleotides are commercially available, can be transfected using standard techniques, are non-toxic, lead to increased target accessibility, can be designed to activate RNase H, and function in steric block approaches. LNA-Antisense, including gapmer LNA containing a central DNA...... or phosphorothioate-DNA segment flanked by LNA gaps, rivals siRNA as the technology of choice for target validation and therapeutic applications....

  12. Antisense oligonucleotide induction of progerin in human myogenic cells.

    Directory of Open Access Journals (Sweden)

    Yue-Bei Luo

    Full Text Available We sought to use splice-switching antisense oligonucleotides to produce a model of accelerated ageing by enhancing expression of progerin, translated from a mis-spliced lamin A gene (LMNA transcript in human myogenic cells. The progerin transcript (LMNA Δ150 lacks the last 150 bases of exon 11, and is translated into a truncated protein associated with the severe premature ageing disease, Hutchinson-Gilford progeria syndrome (HGPS. HGPS arises from de novo mutations that activate a cryptic splice site in exon 11 of LMNA and result in progerin accumulation in tissues of mesodermal origin. Progerin has also been proposed to play a role in the 'natural' ageing process in tissues. We sought to test this hypothesis by producing a model of accelerated muscle ageing in human myogenic cells. A panel of splice-switching antisense oligonucleotides were designed to anneal across exon 11 of the LMNA pre-mRNA, and these compounds were transfected into primary human myogenic cells. RT-PCR showed that the majority of oligonucleotides were able to modify LMNA transcript processing. Oligonucleotides that annealed within the 150 base region of exon 11 that is missing in the progerin transcript, as well as those that targeted the normal exon 11 donor site induced the LMNA Δ150 transcript, but most oligonucleotides also generated variable levels of LMNA transcript missing the entire exon 11. Upon evaluation of different oligomer chemistries, the morpholino phosphorodiamidate oligonucleotides were found to be more efficient than the equivalent sequences prepared as oligonucleotides with 2'-O-methyl modified bases on a phosphorothioate backbone. The morpholino oligonucleotides induced nuclear localised progerin, demonstrated by immunostaining, and morphological nuclear changes typical of HGPS cells. We show that it is possible to induce progerin expression in myogenic cells using splice-switching oligonucleotides to redirect splicing of LMNA. This may offer a model

  13. The oligodeoxynucleotide sequences corresponding to never-expressed peptide motifs are mainly located in the non-coding strand

    Directory of Open Access Journals (Sweden)

    Bickis Mik

    2010-07-01

    Full Text Available Abstract Background We study the usage of specific peptide platforms in protein composition. Using the pentapeptide as a unit of length, we find that in the universal proteome many pentapeptides are heavily repeated (even thousands of times, whereas some are quite rare, and a small number do not appear at all. To understand the physico-chemical-biological basis underlying peptide usage at the proteomic level, in this study we analyse the energetic costs for the synthesis of rare and never-expressed versus frequent pentapeptides. In addition, we explore residue bulkiness, hydrophobicity, and codon number as factors able to modulate specific peptide frequencies. Then, the possible influence of amino acid composition is investigated in zero- and high-frequency pentapeptide sets by analysing the frequencies of the corresponding inverse-sequence pentapeptides. As a final step, we analyse the pentadecamer oligodeoxynucleotide sequences corresponding to the never-expressed pentapeptides. Results We find that only DNA context-dependent constraints (such as oligodeoxynucleotide sequence location in the minus strand, introns, pseudogenes, frameshifts, etc. provide a coherent mechanistic platform to explain the occurrence of never-expressed versus frequent pentapeptides in the protein world. Conclusions This study is of importance in cell biology. Indeed, the rarity (or lack of expression of specific 5-mer peptide modules implies the rarity (or lack of expression of the corresponding n-mer peptide sequences (with n

  14. Expression of heparanase mRNA in anti-sense oligonucleotide-transfected human esophageal cancer EC9706 cells

    Institute of Scientific and Technical Information of China (English)

    Kui-Sheng Chen; Lan Zhang; Lin Tang; Yun-Han Zhang; Dong-Ling Gao; Liang Yan; Lei Zhang

    2005-01-01

    AIM: To investigate the effects of anti-sense oligonucleotides (ASODNs) on mRNA expression of heparanase in human esophageal cancer EC9706 cells.METHODS: One non-sense oligonucleotide (N-ODN) and five ASODNs against different heparanase mRNA sites were transfected into EC9706 cells, then the expression of heparanase mRNA in EC9706 cells was studied byin situ hybridization.RESULTS: The expression of heparanase mRNA could be inhibited by ASODNs.There was no significant difference among five ASODNs (P>0.05), but there was a significant difference between ASODNs and N-ODN or non-transfected group (ASODN1: 2.25±0.25, ASODN2: 2.21±0.23, ASODN3:2.23±0.23, ASODN4:2.25±0.24 vs N-ODN: 3.47±2.80 or non- transfected group: 3.51±2.93 respectively, P<0.05).CONCLUSION: The expression of heparanase mRNA in EC9706 cells can be inhibited by ASODNs in vivo, and heparanase ASODNs can inhibit metastasis of esophageal squamous cell carcinoma or other tumors by inhibiting the expression of heparanase.

  15. Cis-encoded noncoding antisense RNAs in streptococci and other low GC Gram (+ bacterial pathogens.

    Directory of Open Access Journals (Sweden)

    Kyu Hong eCho

    2015-03-01

    Full Text Available Due to recent advances of bioinformatics and high throughput sequencing technology, discovery of regulatory noncoding RNAs in bacteria has been increased to a great extent. Based on this bandwagon, many studies searching for trans-acting small noncoding RNAs in streptococci have been performed intensively, especially in the important human pathogen, group A and B streptococci. However, studies for cis-encoded noncoding antisense RNAs in streptococci have been scarce. A recent study shows antisense RNAs are involved in virulence gene regulation in group B streptococcus, S. agalactiae. This suggests antisense RNAs could have important roles in the pathogenesis of streptococcal pathogens. In this review, we describe recent discoveries of chromosomal cis-encoded antisense RNAs in streptococcal pathogens and other low GC Gram (+ bacteria to provide a guide for future studies.

  16. The effects of antisense PTEN gene transfection on the growth and invasion of glioma cells

    Institute of Scientific and Technical Information of China (English)

    CHEN Hong-jie; ZHENG Zhao-cong; WANG Ru-mi; WANG Shou-sen; YANG Wei-zhong

    2006-01-01

    Objective:To study the effects of antisense PTEN gene on the growth and invasion of glioma cells. Methods:A pcDNA3. 1/Hygro (-) recombinant plasmid containing antisense PTEN gene fragment was constructed. Glioma cells of primary culture were transfected with antisense PTEN gene vector and stably transfected clones were selected. Then, the different growth and invasion abilities and the different MMP9 mRNA expressions of three kinds of cells were observed, including the transfected cells, untransfected cells and the cells transfected with empty vector. Results :The abilities of growth and invasion of the transfected cells and the expressions of MMP9 mRNA were obviously enhanced. Conclusion: Antisense PTEN gene could have a negative impact on the growth and invasion of primary culture glioma cells.

  17. Spt4 selectively regulates the expression of C9orf72 sense and antisense mutant transcripts.

    Science.gov (United States)

    Kramer, Nicholas J; Carlomagno, Yari; Zhang, Yong-Jie; Almeida, Sandra; Cook, Casey N; Gendron, Tania F; Prudencio, Mercedes; Van Blitterswijk, Marka; Belzil, Veronique; Couthouis, Julien; Paul, Joseph West; Goodman, Lindsey D; Daughrity, Lillian; Chew, Jeannie; Garrett, Aliesha; Pregent, Luc; Jansen-West, Karen; Tabassian, Lilia J; Rademakers, Rosa; Boylan, Kevin; Graff-Radford, Neill R; Josephs, Keith A; Parisi, Joseph E; Knopman, David S; Petersen, Ronald C; Boeve, Bradley F; Deng, Ning; Feng, Yanan; Cheng, Tzu-Hao; Dickson, Dennis W; Cohen, Stanley N; Bonini, Nancy M; Link, Christopher D; Gao, Fen-Biao; Petrucelli, Leonard; Gitler, Aaron D

    2016-08-12

    An expanded hexanucleotide repeat in C9orf72 causes amyotrophic lateral sclerosis and frontotemporal dementia (c9FTD/ALS). Therapeutics are being developed to target RNAs containing the expanded repeat sequence (GGGGCC); however, this approach is complicated by the presence of antisense strand transcription of expanded GGCCCC repeats. We found that targeting the transcription elongation factor Spt4 selectively decreased production of both sense and antisense expanded transcripts, as well as their translated dipeptide repeat (DPR) products, and also mitigated degeneration in animal models. Knockdown of SUPT4H1, the human Spt4 ortholog, similarly decreased production of sense and antisense RNA foci, as well as DPR proteins, in patient cells. Therapeutic targeting of a single factor to eliminate c9FTD/ALS pathological features offers advantages over approaches that require targeting sense and antisense repeats separately. PMID:27516603

  18. Antitumor activity of antisense oligonucleotide p45Skp2 in soft palate carcinoma cell squamous in vitro

    Directory of Open Access Journals (Sweden)

    Supriatno Supriatno

    2013-03-01

    Full Text Available Background: Human soft palate cancers are characterized by a high degree of local invasion and metastasis to the regional lymph nodes. Treatment options for this cancer are limited. However, a new strategy for refractory cancer, gene therapy is watched with keen interest. p45Skp2 gene as a tumor promoter gene is one of target of the oral cancer therapy. To inhibit the activity of p45Skp2 gene is carried-out the genetic engineering via antisense technique. Purpose: To examine the antitumor activity of p45Skp2 antisense (p45Skp2 AS gene therapy in human soft palate [Hamakawa-Inoue (HI] cancer cells. Methods: Pure laboratory experimental study with post test only control group design was conducted as a research design. To investigate the apoptosis induction of p45Skp2 AStransfected cell was evaluated by colorimetric caspase-3 assay and Flow cytometry. Furthermore, to detect the suppression of in vitro HI cell invasion and cell growth of p45Skp2 AS-treatment cell was examined by Boyden chamber kit and MTT assay, respectively. Results: The cell number of p45Skp2 AS-treated HI cell was significant decreased when compared with that of p45Skp2 sense (p45Skp2 S cells (p<0.05. p45Skp2 AS-treated cell induced apoptosis characterized by an increase in the early and late apoptosis, and activation of caspase-3 (p<0.05. Therefore, suppression of HI cell invasion and cell growth were markedly increased by p45Skp2 AS treatment (p<0.05. Conclusion: Antisense oligonucleotide p45Skp2 has a high antitumor activity in human soft palate cancer cell, targeting this molecule could represent a promising new therapeutics approach for this type of cancer.Latar belakang: Kanker palatum lunak mempunyai karakteristik invasi dan metastasis ke limfonodi regional yang tinggi. Pilihan perawatan kanker tersebut masih sangat terbatas. Walaupun demikian, strategi baru untuk penanganan kanker yaitu terapi gen menjadi pilihan utama. Gen p45Skp2 sebagai gen pemacu tumor merupakan salah

  19. Targeted Skipping of Human Dystrophin Exons in Transgenic Mouse Model Systemically for Antisense Drug Development

    OpenAIRE

    Bo Wu; Ehsan Benrashid; Peijuan Lu; Caryn Cloer; Allen Zillmer; Mona Shaban; Qi Long Lu

    2011-01-01

    Antisense therapy has recently been demonstrated with great potential for targeted exon skipping and restoration of dystrophin production in cultured muscle cells and in muscles of Duchenne Muscular Dystrophy (DMD) patients. Therapeutic values of exon skipping critically depend on efficacy of the drugs, antisense oligomers (AOs). However, no animal model has been established to test AO targeting human dystrophin exon in vivo systemically. In this study, we applied Vivo-Morpholino to the hDMD/...

  20. Microinjection of antisense c-mos oligonucleotides prevents meiosis II in the maturing mouse egg.

    OpenAIRE

    O'Keefe, S J; Wolfes, H; Kiessling, A A; Cooper, G M

    1989-01-01

    Injection of antisense oligonucleotides was used to investigate the function of c-mos in murine oocytes. Oocytes injected with antisense c-mos oligonucleotides completed the first meiotic division but failed to initiate meiosis II. Instead, loss of c-mos function led to chromosome decondensation, reformation of a nucleus after meiosis I, and cleavage to two cells. Therefore, c-mos is required for meiosis II during murine oocyte maturation.

  1. Genome-wide analysis of antisense transcription with Affymetrix exon array

    Directory of Open Access Journals (Sweden)

    Jung Yong-chul

    2008-01-01

    Full Text Available Abstract Background A large number of natural antisense transcripts have been identified in human and mouse genomes. Study of their potential functions clearly requires cost-efficient method for expression analysis. Results Here we show that Affymetrix Exon arrays, which were designed to detect conventional transcripts in the sense orientation, can be used to monitor antisense expression across all exonic loci in mammalian genomes. Through modification of the cDNA synthesis protocol, we labeled single-strand cDNA in the reverse orientation as in the standard protocol, thus enabling the detection of antisense transcripts using the same array. Applying this technique to human Jurkat cells, we identified antisense transcription at 2,088 exonic loci of 1,516 UniGene clusters. Many of these antisense transcripts were not observed previously and some were validated by orientation-specific RT-PCR. Conclusion Our results suggest that with a modified protocol Affymetrix human, mouse and rat Exon arrays can be used as a routine method for genome-wide analysis of antisense transcription in these genomes.

  2. Bidirectional regulation between WDR83 and its natural antisense transcript DHPS in gastric cancer

    Institute of Scientific and Technical Information of China (English)

    Wen-Yu Su; Xuan Kong; Qin-Yan Gao; Li-Ping Wei; Jing-Yuan Fang; Jiong-Tang Li; Yun Cui; Jie Hong; Wan Du; Ying-Chao Wang; Yan-Wei Lin; Hua Xiong; Ji-Lin Wang

    2012-01-01

    Natural antisense transcripts (NATs) exist ubiquitously in mammalian genomes and play roles in the regulation of gene expression.However,both the existence of bidirectional antisense RNA regulation and the possibility of proteincoding genes that function as antisense RNAs remain speculative.Here,we found that the protein-coding gene,deoxyhypusine synthase (DHPS),as the NAT of WDR83,concordantly regulated the expression of WDR83 mRNA and protein.Conversely,WDR83 also regulated DHPS by antisense pairing in a concordant manner.WDR83 and DHPS were capable of forming an RNA duplex at overlapping 3′ untranslated regions and this duplex increased their mutual stability,which was required for the bidirectional regulation.As a pair of protein-coding cis-sense/antisense transcripts,WDR83 and DHPS were upregulated simultaneously and correlated positively in gastric cancer (GC),driving GC pathophysiology by promoting cell proliferation.Furthermore,the positive relationship between WDR83 and DHPS was also observed in other cancers.The bidirectional regulatory relationship between WDR83 and DHPS not only enriches our understanding of antisense regulation,but also provides a more complete understanding of their functions in tumor development.

  3. [Exon skipping therapy for Duchenne muscular dystrophy by using antisense Morpholino].

    Science.gov (United States)

    Takeda, Shin'ichi

    2009-11-01

    Duchenne muscular dystrophy (DMD) is caused by the lack of dystrophin protein at the sarcolemma. Exon skipping by antisense oligonucleotides is a novel method to restore the reading frame of the mutated DMD gene, and rescue dystrophin production. We recently reported that systemic delivery of Morpholino antisense oligonucleotides targeting exon 6 and 8 of the canine DMD gene, efficiently recovered functional dystrophin proteins at the sarcolamma of dystrophic dogs, and improved performance of affected dogs without serious side effects (Yokota et al., Ann Neurol. 65 (6): 667-676, 2009). To optimize therapeutic antisense Morpholinos for more frequent mutations of the DMD gene, we designed antisense Morpholinos targeting exon 51 of the mouse DMD gene, and injected them separately or in combination into the muscles of mdx52 mice, in which exon 52 has been deleted by a gene targeting technique (Araki et al., 1997). We also tried systemic delivery of antisense Morpholino to skip exon 51 in mdx52 mice. It is important to verify the effectiveness and side effects of antisense Morpholino in experimental animal models such as dystrophic dogs or mdx52 mice, before clinical trials in DMD patients. PMID:20030230

  4. Nanoparticle Delivery of Antisense Oligonucleotides and Their Application in the Exon Skipping Strategy for Duchenne Muscular Dystrophy

    OpenAIRE

    Falzarano, Maria Sofia; Passarelli, Chiara; Ferlini, Alessandra

    2014-01-01

    Antisense therapy is a powerful tool for inducing post-transcriptional modifications and thereby regulating target genes associated with disease. There are several classes of antisense oligonucleotides (AONs) with therapeutic use, such as double-stranded RNAs (interfering RNAs, utilized for gene silencing, and single-stranded AONs with various chemistries, which are useful for antisense targeting of micro-RNAs and mRNAs. In particular, the use of AONs for exon skipping, by targeting pre-mRNA,...

  5. Directional gene expression and antisense transcripts in sexual and asexual stages of Plasmodium falciparum

    Directory of Open Access Journals (Sweden)

    López-Barragán María J

    2011-11-01

    Full Text Available Abstract Background It has been shown that nearly a quarter of the initial predicted gene models in the Plasmodium falciparum genome contain errors. Although there have been efforts to obtain complete cDNA sequences to correct the errors, the coverage of cDNA sequences on the predicted genes is still incomplete, and many gene models for those expressed in sexual or mosquito stages have not been validated. Antisense transcripts have widely been reported in P. falciparum; however, the extent and pattern of antisense transcripts in different developmental stages remain largely unknown. Results We have sequenced seven bidirectional libraries from ring, early and late trophozoite, schizont, gametocyte II, gametocyte V, and ookinete, and four strand-specific libraries from late trophozoite, schizont, gametocyte II, and gametocyte V of the 3D7 parasites. Alignment of the cDNA sequences to the 3D7 reference genome revealed stage-specific antisense transcripts and novel intron-exon splicing junctions. Sequencing of strand-specific cDNA libraries suggested that more genes are expressed in one direction in gametocyte than in schizont. Alternatively spliced genes, antisense transcripts, and stage-specific expressed genes were also characterized. Conclusions It is necessary to continue to sequence cDNA from different developmental stages, particularly those of non-erythrocytic stages. The presence of antisense transcripts in some gametocyte and ookinete genes suggests that these antisense RNA may play an important role in gene expression regulation and parasite development. Future gene expression studies should make use of directional cDNA libraries. Antisense transcripts may partly explain the observed discrepancy between levels of mRNA and protein expression.

  6. Expression of Toll-like receptor 9 and response to bacterial CpG oligodeoxynucleotides in human intestinal epithelium

    DEFF Research Database (Denmark)

    Pedersen, G; Andresen, Lars; Matthiessen, M W;

    2005-01-01

    were stimulated with a synthetic CpG-oligodeoxynucleotide (ODN), exhibiting strong immunostimulatory effects in B cells. Interleukin (IL)-8 secretion was determined by enzyme-linked immunosorbent assay, nuclear factor-kappaB (NF-kB) activity by electrophoretic mobility shift assay and Ik......B phosphorylation by Western blotting. TLR9 mRNA was equally expressed in colonic mucosa from controls (n = 6) and patients with ulcerative colitis or Crohn's disease disease (n = 13). HT-29 cells expressed TLR9 mRNA and protein and responded to CpG-ODN (P secreting IL...... in vitro despite spontaneous TLR9 gene expression. This suggests that the human epithelium is able to avoid inappropriate immune responses to luminal bacterial products through modulation of the TLR9 pathway....

  7. DNA strand breakage by 125I-decay in a synthetic oligodeoxynucleotide. Fragment distribution and evaluation of DMSO protection effect

    International Nuclear Information System (INIS)

    A double stranded oligodeoxynucleotide containing a single 125I-dC in a defined location was used to investigate DNA strand breakage resulting from 125I decay. Samples of a 41 bp oligodeoxynucleotide were incubated in 20 mM phosphate buffer (PB), or PB plus 2 M dimethylsulphoxide (DMSO), at 4 C during 18-20 days. The 32P-5'-end labelled DNA fragments produced by 125I decays were separated on denaturing polyacrylamide gels, and the 32P activity in each fragment was determined by scintillation counting after elution of fragments from gel. Most of the breaks, around 90%, occurred within 4-5 nucleotides of the 125I-dC, but DNA breaks were detected up to 16 nucleotides from the decay site. The 125I-dC was located at the 21st nucleotide from the 32P-5'-end label, and since 32P was not detected in fragments longer than 20 nucleotides, it was assumed that all 125I decay events produce at least one break in the 125I-labelled DNA strand. The results show a considerable protection effect of DMSO on DNA breaks at sites >5-6 nucleotides from the 125I location. The probability of breaks in this region was decreased with DMSO by a factor of 2 to 8-fold, suggesting significant role for radical-mediated DNA breaks at the more distant sites. However, the total protection effect of DMSO is rather small: 1.1, because of the small contribution of breakage at distant sites to the total yield. (orig.)

  8. Prolongation of liver allograft survival by dendritic cells modified with NF-κB decoy oligodeoxynucleotides

    Institute of Scientific and Technical Information of China (English)

    Ming-Qing Xu; Yu-Ping Suo; Jian-Ping Gong; Ming-Man Zhang; Lü-Nan Yan

    2004-01-01

    AIM: To induce the tolerance of rat liver allograft by dendritic cells (DCs) modified with NF-κB decoy oligodeoxynucleotides (ODNs).METHODS: Bone marrow (BM)-derived DCs from SD rats were propagated in the presence of GM-CSF or GM-CSF+IL-4to obtain immature DCs or mature DCs. GM-CSF+IL-4-propagated DCs were treated with double-strand NF-κB decoy ODNs containing two NF-κB binding sites or scrambled ODNs to ascertain whether NF-κB decoy ODNs might prevent DC maturation. GM-CSF-propagated DCs, GMCSF+NF-κB decoy ODNs or scrambled ODNs-propagated DCs were treated with LPS for 18 h to determine whether NF-κB decoy ODNs could prevent LPS-induced IL-12production in DCs. NF-κB binding activities, costimulatory molecule (CD40, CD80, CD86) surface expression, IL-12protein expression and allostimulatory capacity of DCs were measured with electrophoretic mobility shift assay (EMSA),flow cytometry, Western blotting, and mixed lymphocyte reaction (MLR), respectively. GM-CSF-propagated DCs, GMCSF+IL-4 -propagated DCs, and GM-CSF+NF-κB decoy ODNs or scrambled ODNs-propagated DCs were injected intravenously into recipient LEW rats 7 d prior to liver transplantation and immediately after liver transplantation.Histological grading of liver graft rejection was determined 7 d after liver transplantation. Expression of IL-2, IL-4 and IFN-γ mRNA in liver graft and in recipient spleen was analyzed by semiquantitative RT-PCR. Apoptosis of liver allograft-infiltrating cells was measured with TUNEL staining.RESULTS: GM-CSF-propagated DCs, GM-CSF+NF-κB decoy ODNs-propagated DCs and GM-CSF+ scrambled ODNspropagated DCs exhibited features of immature DCs, with similar low level of costimulatory molecule(CD40, CD80,CD86) surface expression, absence of NF-κB activation,and few allocostimulatory activities. GM-CSF+IL-4-propagated DCs displayed features of mature DCs, with high levels of costimulatory molecule (CD40, CD80, CD86) surface expression, marked NF-κB activation, and

  9. Effect of angiotensin Ⅱ receptor 1 antisense oligodoexynucleotides on physiological and pathophysiological growth of cardiomyocytes

    Institute of Scientific and Technical Information of China (English)

    Ying WANG; Jin-ming WANG; Shu-xun Yan; Ming-jiang LI; Jian-jun LI

    2004-01-01

    AIM: To evaluate the role of angiotensin Ⅱ receptor 1 antisense oligodexynucleotides (AT1R-AS-ODNs) on physiological and pathophysiological growth of cardiomyocytes from normotensive rats. METHODS: Cardiomyocytes were transfected with AT1R-AS-ODNs (200 nmol/L) followed by treatment with or without angiotensin Ⅱ (1 μmol/L).In situ hybridization and Western blot were used for AT1R mRNA and protein detection, respectively. c-Jun Nterminal protein kinase (JNK) activity was characterized by immune complex kinase assay. c-Jun protein expression was examined by immunocytochemistry. DNA content was detected by flow cytometric assay. Atrial natriuretic factor (ANF) expression was identified by radioimmunoassay. RESULTS: Treatment with AT1R-AS-ODNs for 24 h resulted in 51.2 % decrease in AT1R mRNA and 60.7 % in protein (P<0.05 vs control). However, the basal level of JNK activity, c-Jun protein expression, and DNA content were not altered by AT1R-AS treatment in absence of overactive hormonal system. After treatment with angiotensin Ⅱ for 30 min, both p46JNK and p54JNK were robustly activated. By 2 h, c-Jun protein expression was increased. By 24 h, angiotensin Ⅱ caused a marked increase both in G0/G1 and G2/M DNA content, and increased ANF expression by 1.8-fold. All these were inhibited by AT1R-AS-ODNs pretreatment. In contrast, sense sequence was ineffective. CONCLUSION: Decrease of AT1R expression by AS-ODNs did not interfere with normal growth, but protected cardiomyocytes from angiotensin Ⅱ-dependent pathophysiological growth.

  10. Expression of an Antisense BcMF3 Affects Microsporogenesis and Pollen Tube Growth in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    LIU Le-cheng; CAO Jia-shu; YU Xiao-lin; XIANG Xun; FEI Yong-jun

    2006-01-01

    In an effort to provide some information relevant to the molecular mechanism of genic male sterility in plants, BcMF3 gene that encodes a pectin methylesterase was isolated from the fertile B line of Chinese cabbage-pak-choi (Brassica rapa ssp.chinensis, syn. B. campestris ssp. chinensis). In the present paper, a 455-bp antisense cDNA fragment of BcMF3 was introduced to binary vector pBI121, and then was mobilized into Agrobacterium tumefaciens strain LBA4404. The A.tumefaciens harboring the BcMF3 antisense fragment was transformed to Arabidopsis thaliana by floral dip. Scanning electronic microscopy examination demonstrated that 47.8% of BcMF3 antisense pollen grains exhibited abnormal shape,which might lead to decreased germination of pollens, suggesting that the product of BcMF3 gene plays an important role during microsporogenesis. The evidence on burst of 45.7% of BcMF3 antisense pollen tubes in vitro and a majority of BcMF3 antisense pollens restricted within the stigmatic tissue revealed that BcMF3 is involved in aiding the growth of pollen tubes. The results suggest that BcMF3 acts at both stages of microsporogensis and pollen tube growth.

  11. Correction of a Cystic Fibrosis Splicing Mutation by Antisense Oligonucleotides.

    Science.gov (United States)

    Igreja, Susana; Clarke, Luka A; Botelho, Hugo M; Marques, Luís; Amaral, Margarida D

    2016-02-01

    Cystic fibrosis (CF), the most common life-threatening genetic disease in Caucasians, is caused by ∼2,000 different mutations in the CF transmembrane conductance regulator (CFTR) gene. A significant fraction of these (∼13%) affect pre-mRNA splicing for which novel therapies have been somewhat neglected. We have previously described the effect of the CFTR splicing mutation c.2657+5G>A in IVS16, showing that it originates transcripts lacking exon 16 as well as wild-type transcripts. Here, we tested an RNA-based antisense oligonucleotide (AON) strategy to correct the aberrant splicing caused by this mutation. Two AONs (AON1/2) complementary to the pre-mRNA IVS16 mutant region were designed and their effect on splicing was assessed at the RNA and protein levels, on intracellular protein localization and function. To this end, we used the 2657+5G>A mutant CFTR minigene stably expressed in HEK293 Flp-In cells that express a single copy of the transgene. RNA data from AON1-treated mutant cells show that exon 16 inclusion was almost completely restored (to 95%), also resulting in increased levels of correctly localized CFTR protein at the plasma membrane (PM) and with increased function. A novel two-color CFTR splicing reporter minigene developed here allowed the quantitative monitoring of splicing by automated microscopy localization of CFTR at the PM. The AON strategy is thus a promising therapeutic approach for the specific correction of alternative splicing.

  12. EGFR antisense RNA blocks expression of the epidermal growth factor receptor and partially reverse the malignant phenotype of human breast cancer MDA—MB—231 cells

    Institute of Scientific and Technical Information of China (English)

    FANWENHONG; YINGLINLU; 等

    1998-01-01

    The effects of human EGFR to the malignant phenotype of human breast cancer cell line MDA-MB-231 were investigated experimentally.A retroviral vector containing a 5'1350bp fragment of the human EGFR cDNA in the antisense orientation was transfected into targeted cells by lipofectamine.The effects on cell proliferation,cell cycle and adherent ability to extracellular matrix(ECM) components were studied after the expression of antisense transcripts to EGFR5'1350bp fragment in target cells,In vitro studies showed that the growth ability of the transfected cells was partialy inhibited in comparison to parental cells and to cells transfected with the plasmid containing the neomycin resistance gene only.It was found that EGF(10ng/ml) had an augmenation effect on the growth of transfected MDA-AS10 cells but not MDA-MB-231 cells.Flow cytometric analysis showed that the cell cycle of the transfected cells was abnormal with a decrease of cells in G2/M and S phases and an increase of cells in G1 phase,indicating a blockage in phase G1.Immunofluorescence of EGFR expression in transfectants stained with an antiEGFR antibody was decreased and their growth in soft agarose was also severely imparired.The transfected cells showed less adherence to laminin(LN) and fibronectin (FN).In short,EGFR antisense RNA decreases the expression of EGFR on MDA-MB-231 cells and partially reverses their malignant phenotype as well.

  13. Adrenalectomy promotes a permanent decrease of plasma corticoid levels and a transient increase of apoptosis and the expression of Transforming Growth Factor β1 (TGF-β1 in hippocampus: effect of a TGF-β1 oligo-antisense

    Directory of Open Access Journals (Sweden)

    Lara Hernán E

    2006-05-01

    Full Text Available Abstract Background Corticosterone reduction produced by adrenalectomy (ADX induces apoptosis in dentate gyrus (DG of the hippocampus, an effect related to an increase in the expression of the pro-apoptotic gene bax. However it has been reported that there is also an increase of the anti-apoptotic gene bcl-2, suggesting the promotion of a neuroprotective phenomenon, perhaps related to the expression of transforming growth factor β1 (TGF-β1. Thus, we have investigated whether TGF-β1 levels are induced by ADX, and whether apoptosis is increased by blocking the expression of TGF-β1 with an antisense oligonucleotide (ASO administered intracerebrally in corticosterone depleted rats. Results It was observed an increase of apoptosis in DG, 2 and 5 days after ADX, in agreement with a reduction of corticosterone levels. However, the effect of ADX on the number of apoptotic positive cells in DG was decreased 5 days after the lesion. In CA1–CA3 regions, the effect was only observed 2 days after ADX. TGF-β1 mRNA levels were increased 2 days after ADX. The sustained intracerebro-ventricular administration of a TGF-β1 ASO via an osmotic mini pump increased apoptosis levels in CA and DG regions 5 days after ADX as well as sham-operated control animals. No significant effect was observed following a scrambled-oligodeoxynucleotide treatment. Conclusion The changes in both the pattern and the magnitude of apoptotic-cell morphology observed 2 and 5 days after ADX suggest that, as a consequence of the reduction of corticosteroids, some trophic mechanisms restricting cell death to a particular time window are elicited. Sustained intracerebral administration of TGF-β1 ASO increased the apoptosis promoted by ADX, suggesting that TGF-β1 plays an anti-apoptotic role in vivo in hippocampus.

  14. Repair of Thalassemic Human β -globin mRNA in Mammalian Cells by Antisense Oligonucleotides

    Science.gov (United States)

    Sierakowska, Halina; Sambade, Maria J.; Agrawal, Sudhir; Kole, Ryszard

    1996-11-01

    In one form of β -thalassemia, a genetic blood disorder, a mutation in intron 2 of the β -globin gene (IVS2-654) causes aberrant splicing of β -globin pre-mRNA and, consequently, β -globin deficiency. Treatment of mammalian cells stably expressing the IVS2-654 human β -globin gene with antisense oligonucleotides targeted at the aberrant splice sites restored correct splicing in a dose-dependent fashion, generating correct human β -globin mRNA and polypeptide. Both products persisted for up to 72 hr posttreatment. The oligonucleotides modified splicing by a true antisense mechanism without overt unspecific effects on cell growth and splicing of other pre-mRNAs. This novel approach in which antisense oligonucleotides are used to restore rather than to down-regulate the activity of the target gene is applicable to other splicing mutants and is of potential clinical interest.

  15. CpG Oligodeoxynucleotides Adsorbed onto Polylactide-Co-Glycolide Microparticles Improve the Immunogenicity and Protective Activity of the Licensed Anthrax Vaccine

    OpenAIRE

    Xie, Hang; Gursel, Ihsan; Ivins, Bruce E.; Singh, Manmohan; O'Hagan, Derek T.; Ulmer, Jeffrey B.; Klinman, Dennis M.

    2005-01-01

    To reduce the biothreat posed by anthrax, efforts are under way to improve the protection afforded by vaccination. This work examines the ability of immunostimulatory CpG oligodeoxynucleotides (ODN) adsorbed onto cationic polylactide-co-glycolide (PLG) microparticles (CpG ODN-PLG) to accelerate and boost the protective immunity elicited by Anthrax Vaccine Adsorbed (AVA, the licensed human anthrax vaccine). The results indicate that coadministering CpG ODN-PLG with AVA induces a stronger and f...

  16. Subnanomolar antisense activity of phosphonate-peptide nucleic acid (PNA) conjugates delivered by cationic lipids to HeLa cells

    DEFF Research Database (Denmark)

    Shiraishi, Takehiko; Hamzavi, Ramin; Nielsen, Peter E

    2008-01-01

    as inferred from induced luciferase activity as a consequence of pre-mRNA splicing correction by the antisense-PNA. Antisense activity depended on the number of phosphonate moieties and the most potent hexa-bis-phosphonate-PNA showed at least 20-fold higher activity than that of an optimized PNA/DNA hetero...

  17. A Simple Three-Step Method for Design and Affinity Testing of New Antisense Peptides: An Example of Erythropoietin

    Directory of Open Access Journals (Sweden)

    Nikola Štambuk

    2014-05-01

    Full Text Available Antisense peptide technology is a valuable tool for deriving new biologically active molecules and performing peptide–receptor modulation. It is based on the fact that peptides specified by the complementary (antisense nucleotide sequences often bind to each other with a higher specificity and efficacy. We tested the validity of this concept on the example of human erythropoietin, a well-characterized and pharmacologically relevant hematopoietic growth factor. The purpose of the work was to present and test simple and efficient three-step procedure for the design of an antisense peptide targeting receptor-binding site of human erythropoietin. Firstly, we selected the carboxyl-terminal receptor binding region of the molecule (epitope as a template for the antisense peptide modeling; Secondly, we designed an antisense peptide using mRNA transcription of the epitope sequence in the 3'→5' direction and computational screening of potential paratope structures with BLAST; Thirdly, we evaluated sense–antisense (epitope–paratope peptide binding and affinity by means of fluorescence spectroscopy and microscale thermophoresis. Both methods showed similar Kd values of 850 and 816 µM, respectively. The advantages of the methods were: fast screening with a small quantity of the sample needed, and measurements done within the range of physicochemical parameters resembling physiological conditions. Antisense peptides targeting specific erythropoietin region(s could be used for the development of new immunochemical methods. Selected antisense peptides with optimal affinity are potential lead compounds for the development of novel diagnostic substances, biopharmaceuticals and vaccines.

  18. Large-scale analysis of antisense transcription in wheat using the Affymetrix GeneChip Wheat Genome Array

    Directory of Open Access Journals (Sweden)

    Settles Matthew L

    2009-05-01

    Full Text Available Abstract Background Natural antisense transcripts (NATs are transcripts of the opposite DNA strand to the sense-strand either at the same locus (cis-encoded or a different locus (trans-encoded. They can affect gene expression at multiple stages including transcription, RNA processing and transport, and translation. NATs give rise to sense-antisense transcript pairs and the number of these identified has escalated greatly with the availability of DNA sequencing resources and public databases. Traditionally, NATs were identified by the alignment of full-length cDNAs or expressed sequence tags to genome sequences, but an alternative method for large-scale detection of sense-antisense transcript pairs involves the use of microarrays. In this study we developed a novel protocol to assay sense- and antisense-strand transcription on the 55 K Affymetrix GeneChip Wheat Genome Array, which is a 3' in vitro transcription (3'IVT expression array. We selected five different tissue types for assay to enable maximum discovery, and used the 'Chinese Spring' wheat genotype because most of the wheat GeneChip probe sequences were based on its genomic sequence. This study is the first report of using a 3'IVT expression array to discover the expression of natural sense-antisense transcript pairs, and may be considered as proof-of-concept. Results By using alternative target preparation schemes, both the sense- and antisense-strand derived transcripts were labeled and hybridized to the Wheat GeneChip. Quality assurance verified that successful hybridization did occur in the antisense-strand assay. A stringent threshold for positive hybridization was applied, which resulted in the identification of 110 sense-antisense transcript pairs, as well as 80 potentially antisense-specific transcripts. Strand-specific RT-PCR validated the microarray observations, and showed that antisense transcription is likely to be tissue specific. For the annotated sense-antisense

  19. Antisense oligonucleotide targeting at the initiator of hTERT arrests growth of hepatoma cells

    Institute of Scientific and Technical Information of China (English)

    Su-Xia Liu; Wen-Sheng Sun; Ying-Lin Cao; Chun-Hong Ma; Li-Hui Han; Li-Ning Zhang; Zhen-Guang Wang; Fa-Liang Zhu

    2004-01-01

    AIM: To evaluate the inhibitory effect of antisense phosphorothioate oligonucleotide (asON) complementary to the initiator of human telomerase catalytic subunit (hTERT)on the growth of hepatoma cells.METHODS: The as-hTERT was synthesized by using a DNA synthesizer. HepG2.2.15 cells were treated with ashTERT at the concentration of 10 μmol/L. After 72 h, these cells were obtained for detecting growth inhibition,telomerase activity using the methods of MTT, TRAP-PCR-ELISA, respectively. BALB/c(nu/nu) mice were injected HepG2.2.15 cells and a human-nude mice model was obtained. There were three groups for anti-tumor activity study. Once tumors were established, these animals in the first group were administered as-hTERT and saline.Apoptosis of tumor cells was detected by FCM. In the 2nd group, the animals were injected HepG2.2.15 cells together with as-hTERT. In the third group, the animals were given as-hTERT 24 hours postinjection of HepG2.2.15 cells. The anti-HBV effects were assayed with ELISA ih vitro and in vivo.RESULTS: Growth inhibition was observed in cells treated with as-hTERT ih vitro. A significant different in the value of A570-A630 was found between cells treated with as-hTERT and control (P<0.01) by MTT method. The telomerase activity of tumor cells treated with as-hTERT was reduced,the value of A450 nm was 0.42 compared to control (1,49)with TRAP-PCR-ELISA. The peak of apoptosis in tumor cells given as-hTERT was 21. 12%, but not seen in saline-treated control. A prolonged period of carcinogenesis was observed in the second and third group animals. There was inhibitory effect on the expression of HBsAg and HBeAg ih vivo and in vitro.CONCLUSION: As-hTERT has an anti-tumor activity, which may be useful for gene therapy of tumors.

  20. Combination Adenovirus-Mediated HSV-tk/GCV and Antisense IGF-1 Gene Therapy for Rat Glioma

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective To investigate the effects of combination adenovirus-mediated HSV-tk/GCV system and antisense IGF-1 gene therapy for rat glioma and analyze the mechanism.Methods Using the recombinant adenovirus vector,GCV killing effeciency after combined gene transfer of HSV-tk and antisense IGF-1 was observed in vitro.Rat glioma was treated with HSV-tk/GCV and antisense IGF-1 and the survival rate of rats was observed.Results C6 cells transfected with tk and antisense IGF-1 gene were more sensitive to GCV than that transfected with tk gene alone.The survival of the combination gene therapy group was prolonged significantly and large amounts of CD+4,CD+8 lymphocytes were detected in the tumor tissues.Conclusion Antisense IGF-1 gene may enhance the tumor-killing effects of HSV-tk/GCV.

  1. Intragenic pausing and anti-sense transcription within the murine c-myc locus.

    OpenAIRE

    Nepveu, A; Marcu, K B

    1986-01-01

    We present a detailed analysis of strand-specific transcription in different regions of the murine c-myc locus. In normal and transformed cell lines, RNA polymerase II directed transcription occurs in the sense and anti-sense direction. Three noncontiguous regions show a high level of transcription in the anti-sense orientation: upstream of the first exon, within the first intron and in the 3' part of the gene (intron 2 and exon 3). In a cell line carrying a c-myc amplification (54c12), anti-...

  2. Use of electrophoretic mobility to determine the secondary structure of a small antisense RNA.

    OpenAIRE

    Jacques, J P; Susskind, M M

    1991-01-01

    Natural antisense RNAs have stem-loop (hairpin) secondary structures that are important for their function. The sar antisense RNA of phage P22 is unusual: the 3' half of the molecule forms an extensive stem-loop, but potential structures for the 5' half are not predicted to be thermodynamically stable. We devised a novel method to determine the secondary structure of sar RNA by examining the electrophoretic mobility on non-denaturing gels of numerous sar mutants. The results show that the wil...

  3. Antisense silencing of the creA gene in Aspergillus nidulans

    DEFF Research Database (Denmark)

    Bautista, L. F.; Aleksenko, Alexei Y.; Hentzer, Morten;

    2000-01-01

    Antisense expression of a portion of the gene encoding the major carbon catabolite repressor CREA in Aspergillus nidulans resulted in a substantial increase in the levels of glucose-repressible enzymes, both endogenous and heterologous, in the presence of glucose. The derepression effect was appr......Antisense expression of a portion of the gene encoding the major carbon catabolite repressor CREA in Aspergillus nidulans resulted in a substantial increase in the levels of glucose-repressible enzymes, both endogenous and heterologous, in the presence of glucose. The derepression effect...

  4. Construction of neuron specific vector of human antisense noggin gene expression

    Institute of Scientific and Technical Information of China (English)

    Shengnian Zhou; Chengshan Li; Xiansen Wei; Liqing Liu; Zhengda Zhang

    2010-01-01

    The noggin gene is present in the central nervous system in embryonic and postnatal mammals,and plays an important role in maintaining nervous system development and physiological function.A 0.76-kb sequence of human noggin gene was cloned by polymerase chain reaction with the digestion site of Hind Ⅲ and Xba l on the 5' end.The cloned fragment was reversely inserted into pCS2+[Tα1]-GFP plasmid,an neural cell-specific antisense eukaryotic expression vector.The plasmid expresses antisense for human noggin specifically in neurons,which may facilitate understanding of the physiological function of noggin.

  5. Effect of antisense oligonucleotides targeting telomerase catalytic subunit on tumor cell proliferationin vitro

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    To screen specific antitumor drugs targeting telomerase catalytic subunit (hEST2), 12 different hEST2 antisense oligonucleotides were designed based on hEST2 mRNA second structure and transfected into tumor cell lines by the lipofectin-mediated method. Cell growth activity was evaluated by MTT assay. hEST212 was picked out and its specificity, antitumor tree and continuous effect were analyzed. The results showed that hEST212 had promising antitumor activity in vitro, hEST2 can be used as a pratical target and an antisense drug candidate for cancer.

  6. The zebrafish progranulin gene family and antisense transcripts

    Directory of Open Access Journals (Sweden)

    Baranowski David

    2005-11-01

    Full Text Available Abstract Background Progranulin is an epithelial tissue growth factor (also known as proepithelin, acrogranin and PC-cell-derived growth factor that has been implicated in development, wound healing and in the progression of many cancers. The single mammalian progranulin gene encodes a glycoprotein precursor consisting of seven and one half tandemly repeated non-identical copies of the cystine-rich granulin motif. A genome-wide duplication event hypothesized to have occurred at the base of the teleost radiation predicts that mammalian progranulin may be represented by two co-orthologues in zebrafish. Results The cDNAs encoding two zebrafish granulin precursors, progranulins-A and -B, were characterized and found to contain 10 and 9 copies of the granulin motif respectively. The cDNAs and genes encoding the two forms of granulin, progranulins-1 and -2, were also cloned and sequenced. Both latter peptides were found to be encoded by precursors with a simplified architecture consisting of one and one half copies of the granulin motif. A cDNA encoding a chimeric progranulin which likely arises through the mechanism of trans-splicing between grn1 and grn2 was also characterized. A non-coding RNA gene with antisense complementarity to both grn1 and grn2 was identified which may have functional implications with respect to gene dosage, as well as in restricting the formation of the chimeric form of progranulin. Chromosomal localization of the four progranulin (grn genes reveals syntenic conservation for grna only, suggesting that it is the true orthologue of mammalian grn. RT-PCR and whole-mount in situ hybridization analysis of zebrafish grns during development reveals that combined expression of grna and grnb, but not grn1 and grn2, recapitulate many of the expression patterns observed for the murine counterpart. This includes maternal deposition, widespread central nervous system distribution and specific localization within the epithelial

  7. Delivery of antisense oligonucleotide to the cornea by iontophoresis.

    Science.gov (United States)

    Berdugo, M; Valamanesh, F; Andrieu, C; Klein, C; Benezra, D; Courtois, Y; Behar-Cohen, F

    2003-04-01

    We wished to evaluate the potential of iontophoresis to promote the delivery of antisense oligonucleotides (ODN) directed at the vascular endothelial growth factor (VEGF)-R2 receptor (KDR/Flk) to the cornea of the rat eye. Fluorescence (CY5)-labeled ODNs in phosphate-buffered saline (PBS) (20 microM) were locally administered to rat eyes, and their fate within the anterior segment was studied. Thirty-four male, 5-week-old Wistar rats were used for all experiments. The rats were divided in four groups. In group I (12 rats, 12 eyes), the ODNs (20 microM) were delivered by iontophoresis (300 microA for 5 minutes) using a specially designed corneal applicator. In group II (12 rats, 12 eyes), the ODNs (20 microM) were delivered using the same applicator, but no electrical current was applied. In group III (6 rats, 6 eyes), a corneal neovascular reaction was induced prior to the application of ODNs (20 microM), and iontophoresis electrical current was delivered as for group I rats. Group IV (4 rats, 4 eyes) received ODN (60 microM) iontophoresis application (300 microA for 5 minutes) and were used for ODN integrity studies. The animals were killed 5 minutes, 90 minutes, and 24 hours after a single ODN application and studied. Topically applied ODNs using the same iontophoresis applicator but without current do not penetrate the cornea and remain confined to the superficial epithelial layer. ODNs delivered with transcorneoscleral iontophoresis penetrate into all corneal layers and are also detected in the iris. In corneas with neovascularization, ODNs were particularly localized within the vascular endothelial cells of the stroma. ODNs extracted from eye tissues 24 hours after iontophoresis remained unaltered. The iontophoresis current did not cause any detectable ocular damage under these conditions. Iontophoresis promotes the delivery of ODNs to the anterior segment of the eye, including all corneal layers. Iontophoresis of ODNs directed at VEGF-R2 may be used for the

  8. The seeds of Lotus japonicus lines transformed with sense, antisense, and sense/antisense galactomannan galactosyltransferase constructs have structurally altered galactomannans in their endosperm cell walls.

    Science.gov (United States)

    Edwards, Mary E; Choo, Tze-Siang; Dickson, Cathryn A; Scott, Catherine; Gidley, Michael J; Reid, J S Grant

    2004-03-01

    Galactomannan biosynthesis in legume seed endosperms involves two Golgi membrane-bound glycosyltransferases, mannan synthase and galactomannan galactosyltransferase (GMGT). GMGT specificity is an important factor regulating the distribution and amount of (1-->6)-alpha-galactose (Gal) substitution of the (1-->4)-beta-linked mannan backbone. The model legume Lotus japonicus is shown now to have endospermic seeds with endosperm cell walls that contain a high-Gal galactomannan (mannose [Man]/Gal = 1.2-1.3). Galactomannan biosynthesis in developing L. japonicus endosperms has been mapped, and a cDNA encoding a functional GMGT has been obtained from L. japonicus endosperms during galactomannan deposition. L. japonicus has been transformed with sense, antisense, and sense/antisense ("hairpin loop") constructs of the GMGT cDNA. Some of the sense, antisense, and sense/antisense transgenic lines exhibited galactomannans with altered (higher) Man/Gal values in their (T(1) generation) seeds, at frequencies that were consistent with posttranscriptional silencing of GMGT. For T(1) generation individuals, transgene inheritance was correlated with galactomannan composition and amount in the endosperm. All the azygous individuals had unchanged galactomannans, whereas those that had inherited a GMGT transgene exhibited a range of Man/Gal values, up to about 6 in some lines. For Man/Gal values up to 4, the results were consistent with lowered Gal substitution of a constant amount of mannan backbone. Further lowering of Gal substitution was accompanied by a slight decrease in the amount of mannan backbone. Microsomal membranes prepared from the developing T(2) generation endosperms of transgenic lines showed reduced GMGT activity relative to mannan synthase. The results demonstrate structural modification of a plant cell wall polysaccharide by designed regulation of a Golgi-bound glycosyltransferase.

  9. Polyethyleneimine-functionalized boron nitride nanospheres as efficient carriers for enhancing the immunostimulatory effect of CpG oligodeoxynucleotides

    Directory of Open Access Journals (Sweden)

    Zhang HJ

    2015-08-01

    Full Text Available Huijie Zhang,1 Shini Feng,1 Ting Yan,1 Chunyi Zhi,2 Xiao-Dong Gao,1 Nobutaka Hanagata3,41The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, People’s Republic of China; 2Department of Physics and Materials Science, City University of Hong Kong, Kowlong, Hong Kong SAR, People’s Republic of China; 3Biomaterials Unit, International Center for Materials Nanoarchitectonics, National Institute for Materials Science, Ibaraki, Japan; 4Nanotechnology Innovation Station, National Institute for Materials Science, Ibaraki, JapanAbstract: CpG oligodeoxynucleotides (ODNs stimulate innate and adaptive immune responses. Thus, these molecules are promising therapeutic agents and vaccine adjuvants against various diseases. In this study, we developed a novel CpG ODNs delivery system based on polyethyleneimine (PEI-functionalized boron nitride nanospheres (BNNS. PEI was coated on the surface of BNNS via electrostatic interactions. The prepared BNNS–PEI complexes had positive zeta potential and exhibited enhanced dispersity and stability in aqueous solution. In vitro cytotoxicity assays revealed that the BNNS–PEI complexes with concentrations up to 100 µg/mL exhibited no obvious cytotoxicity. Furthermore, the positively charged surface of the BNNS–PEI complexes greatly improved the loading capacity and cellular uptake efficiency of CpG ODNs. Class B CpG ODNs loaded on the BNNS–PEI complexes enhanced the production of interleukin-6 and tumor necrosis factor-α from peripheral blood mononuclear cells compared with CpG ODNs directly loaded on BNNS. Contrary to the free CpG ODNs or CpG ODNs directly loaded on BNNS, class B CpG ODNs loaded on the BNNS–PEI complexes induced interferon-α simultaneously. PEI coating may have changed the physical form of class B CpG ODNs on BNNS, which further affected their interaction with Toll-like receptor 9 and induced interferon

  10. Toll-like receptor 9 ligand D-type oligodeoxynucleotide D35 as a broad inhibitor for influenza A virus replication that is associated with suppression of neuraminidase activity.

    Science.gov (United States)

    Yamada, Hiroshi; Nagase, Satoshi; Takahashi, Kazuo; Sakoda, Yoshihiro; Kida, Hiroshi; Okamoto, Shigefumi

    2016-05-01

    The most effective drugs available to treat influenza are neuraminidase (NA) inhibitors, which provide important additional measures for the control of influenza virus infections. However, since the emergence of NA inhibitor-resistant viruses may compromise the clinical utility of this class of anti-influenza agents, it is very important to develop new anti-influenza agents which target a different region in NA responsible for its sensitivity from that for NA inhibitors and could be used to treat NA inhibitors-resistant isolates. The oligodeoxynucleotide D35, multimerized and aggregated, suppressed replication of influenza A viruses except A/WSN/33 (WSN). The suppressive viral replication by D35 depended on G-terad and multimer formation. The range of the suppressive viral replication at the late stage, including virus assembly and release from infected cells, was much larger than that at the initial stage, viral attachment and entry. D35 suppressed NA activity of influenza A viruses. Furthermore, replacing the NA gene of A/Puerto Rico/8/34 (PR8), in which viral replication was inhibited by D35 at the late stage, with the NA gene from WSN, in which viral replication was not inhibited, eliminated the D35-dependent suppression. D35 showed an additive anti-influenza effect with oseltamivir. It was also effective in vivo. These results suggest that the influenza virus NA mainly contributes to the D35-suppressible virus release from infected cells at the late stage. In addition, because administration of D35 into the virus-infected mice suppressed viral replication and weight loss, clinical application of D35 could be considered. PMID:26923882

  11. Sequence-specific inhibition of duck hepatitis B virus reverse transcription by peptide nucleic acids (PNA)

    DEFF Research Database (Denmark)

    Robaczewska, Magdalena; Narayan, Ramamurthy; Seigneres, Beatrice;

    2005-01-01

    BACKGROUND/AIMS: Peptide nucleic acids (PNAs) appear as promising new antisense agents, that have not yet been examined as hepatitis B virus (HBV) inhibitors. Our aim was to study the ability of PNAs targeting the duck HBV (DHBV) encapsidation signal epsilon to inhibit reverse transcription (RT...

  12. An in vivo transcriptome data set of natural antisense transcripts from Plasmodium falciparum clinical isolates

    Directory of Open Access Journals (Sweden)

    Amit Kumar Subudhi

    2014-12-01

    Full Text Available Antisense transcription is pervasive among biological systems and one of the products of antisense transcription is natural antisense transcripts (NATs. Emerging evidences suggest that they are key regulators of gene expression. With the discovery of NATs in Plasmodium falciparum, it has been suggested that these might also be playing regulatory roles in this parasite. However, all the reports describing the diversity of NATs have come from parasites in culture condition except for a recent study published by us. In order to explore the in vivo diversity of NATs in P. falciparum clinical isolates, we performed a whole genome expression profiling using a strand-specific 244 K microarray that contains probes for both sense and antisense transcripts. In this report, we describe the experimental procedure and analysis thereof of the microarray data published recently in Gene Expression Omnibus (GEO under accession number GSE44921. This published data provide a wealth of information about the prevalence of NATs in P. falciparum clinical isolates from patients with diverse malaria related disease conditions. Supplementary information about the description and interpretation of the data can be found in a recent publication by Subudhi et al. in Experimental Parasitology (2014.

  13. Refinement of antisense oligonucleotide mediated exon skipping as therapy for Duchenne muscular dystrophy

    NARCIS (Netherlands)

    Heemskerk, Johannes Antonius

    2011-01-01

    In recent years, modulation of mRNA has emerged as a promising therapeutic tool. For instance, in the field of neuromuscular disorders therapeutic strategies are being developed for several diseases, including antisense oligonucleotide (AON) mediated exon skipping for Duchenne Muscular Dystrophy (DM

  14. Improving the nutritional quality of the barley and wheat grain storage proteins by antisense technology

    DEFF Research Database (Denmark)

    Sikdar, Md. Shafiqul Islam; Lange, Mette; Aaslo, Per;

    2011-01-01

    gliadins) are also available from Germany and UK. We have grown them under different N regimes (high, medium and low N) in semi-field conditions. Previously five different antisense C-hordein lines of barley have been characterized in our laboratory. The analyses revealed that the lysine, threonine...

  15. Potent Antibacterial Antisense Peptide-Peptide Nucleic Acid Conjugates Against Pseudomonas aeruginosa

    DEFF Research Database (Denmark)

    Ghosal, Anubrata; Nielsen, Peter E

    2012-01-01

    Pseudomonas aeruginosa is an opportunistic pathogen causing severe infections in hospital settings, especially with immune compromised patients, and the increasing prevalence of multidrug resistant strains urges search for new drugs with novel mechanisms of action. In this study we introduce...... significantly reduced bacterial survival. These results open the possibility of development of antisense antibacterials for treatment of Pseudomonas infections....

  16. Functional correction by antisense therapy of a splicing mutation in the GALT gene.

    Science.gov (United States)

    Coelho, Ana I; Lourenço, Sílvia; Trabuco, Matilde; Silva, Maria João; Oliveira, Anabela; Gaspar, Ana; Diogo, Luísa; Tavares de Almeida, Isabel; Vicente, João B; Rivera, Isabel

    2015-04-01

    In recent years, antisense therapy has emerged as an increasingly important therapeutic approach to tackle several genetic disorders, including inborn errors of metabolism. Intronic mutations activating cryptic splice sites are particularly amenable to antisense therapy, as the canonical splice sites remain intact, thus retaining the potential for restoring constitutive splicing. Mutational analysis of Portuguese galactosemic patients revealed the intronic variation c.820+13A>G as the second most prevalent mutation, strongly suggesting its pathogenicity. The aim of this study was to functionally characterize this intronic variation, to elucidate its pathogenic molecular mechanism(s) and, ultimately, to correct it by antisense therapy. Minigene splicing assays in two distinct cell lines and patients' transcript analyses showed that the mutation activates a cryptic donor splice site, inducing an aberrant splicing of the GALT pre-mRNA, which in turn leads to a frameshift with inclusion of a premature stop codon (p.D274Gfs*17). Functional-structural studies of the recombinant wild-type and truncated GALT showed that the latter is devoid of enzymatic activity and prone to aggregation. Finally, two locked nucleic acid oligonucleotides, designed to specifically recognize the mutation, successfully restored the constitutive splicing, thus establishing a proof of concept for the application of antisense therapy as an alternative strategy for the clearly insufficient dietary treatment in classic galactosemia. PMID:25052314

  17. Molecular characterization of a stable antisense chalcone synthase phenotype in strawberry (Fragaria ananassa)

    NARCIS (Netherlands)

    Lunkenbein, S.; Coiner, H.; Vos, de C.H.; Schaart, J.G.; Boone, M.J.; Krens, F.A.; Schwab, W.

    2006-01-01

    An octaploid (Fragaria × ananassa cv. Calypso) genotype of strawberry was transformed with an antisense chalcone synthase (CHS) gene construct using a ripening related CHS cDNA from Fragaria × ananassa cv. Elsanta under the control of the constitutive CaMV 35S promoter via Agrobacterium tumefaciens.

  18. TSUNAMI: an antisense method to phenocopy splicing-associated diseases in animals

    OpenAIRE

    Sahashi, Kentaro; Hua, Yimin; Ling, Karen K Y; Hung, Gene; Rigo, Frank; Horev, Guy; Katsuno, Masahisa; Sobue, Gen; Ko, Chien-Ping; Bennett, C. Frank; Krainer, Adrian R.

    2012-01-01

    This study presents an antisense oligonucleotide methodology to phenocopy a disease—in this case, the motor neuron disease spinal muscular atrophy in mice. Sahashi et al. show that it is possible to fine-tune disease severity through dose-dependent effects on RNA splicing, making this a novel animal model for monitoring disease onset and progression as well as testing candidate therapeutics.

  19. Mismatched single stranded antisense oligonucleotides can induce efficient dystrophin splice switching

    Directory of Open Access Journals (Sweden)

    Kole Ryszard

    2011-10-01

    Full Text Available Abstract Background Antisense oligomer induced exon skipping aims to reduce the severity of Duchenne muscular dystrophy by redirecting splicing during pre-RNA processing such that the causative mutation is by-passed and a shorter but partially functional Becker muscular dystrophy-like dystrophin isoform is produced. Normal exons are generally targeted to restore the dystrophin reading frame however, an appreciable subset of dystrophin mutations are intra-exonic and therefore have the potential to compromise oligomer efficiency, necessitating personalised oligomer design for some patients. Although antisense oligomers are easily personalised, it remains unclear whether all patient polymorphisms within antisense oligomer target sequences will require the costly process of producing and validating patient specific compounds. Methods Here we report preclinical testing of a panel of splice switching antisense oligomers, designed to excise exon 25 from the dystrophin transcript, in normal and dystrophic patient cells. These patient cells harbour a single base insertion in exon 25 that lies within the target sequence of an oligomer shown to be effective at removing exon 25. Results It was anticipated that such a mutation would compromise oligomer binding and efficiency. However, we show that, despite the mismatch an oligomer, designed and optimised to excise exon 25 from the normal dystrophin mRNA, removes the mutated exon 25 more efficiently than the mutation-specific oligomer. Conclusion This raises the possibility that mismatched AOs could still be therapeutically applicable in some cases, negating the necessity to produce patient-specific compounds.

  20. Antisense oligonucleotides as innovative therapeutic strategy in the treatment of high-grade gliomas.

    Science.gov (United States)

    Caruso, Gerardo; Caffo, Mariella; Raudino, Giuseppe; Alafaci, Concetta; Salpietro, Francesco M; Tomasello, Francesco

    2010-01-01

    Despite the intensive recent research in cancer therapy, the prognosis in patients affected by high-grade gliomas is still very unfavorable. The efficacy of classical anti-cancer strategies is seriously limited by lack of specific therapies against malignant cells. The extracellular matrix plays a pivotal role in processes such as differentiation, apoptosis, and migration in both the normal and the pathologic nervous system. Glial tumors seem to be able to create a favorable environment for the invasion of glioma cells in cerebral parenchyma when they combine with the extracellular matrix via cell surface receptors. Glioma cells synthesize matrix proteins, such as tenascin, laminin, fibronectin that facilitate the tumor cell's motility. New treatments have shown to hit the acting molecules in the tumor growth and to increase the efficacy and minimize the toxicity. Antisense oligonucleotides are synthetic stretches of DNA which hybridize with specific mRNA strands. The specificity of hybridization makes antisense method an interesting strategy to selectively modulate the expression of genes involved in tumorigenesis. In this review we will focus on the mechanisms of action of antisense oligonucleotides and report clinical and experimental studies on the treatment of high-grade gliomas. We will also report the patents of preclinical and/or clinical studies that adopt the antisense oligonucleotide therapy list in cerebral gliomas.

  1. Effects of antisense oligonucleotides on the expression of macrophage migration inhibitory factor on macrophages

    Institute of Scientific and Technical Information of China (English)

    WEIYINGCHEN; GUANGRANLI; XUEQINGYU; XIAOYANLI; XIAOYANG

    2005-01-01

    To investigate the effects of antisense oligonucleotides on the expression of macrophage migration inhibitory factor (MIF) on macrophages, the mouse phosphorothioate oligonucleotides were designed and synthesized with the sequences of antisense, 5'-TACGGATACAAGTAGCAC-3';Sense, 5'-ATGC-CTATGTTCATCGTG-3';Missense, 5'-CTCTCAGACTCGATCTGT-3'. These phosphorothioate oligonucleotides were then transfected into cultured macrophages (RAW264.7) by luciferase vector, and the transfected macrophages were incubated with Lipopolysaccharide (LPS) (1 ng/ml) for various periods of times and collected afterwards. The content of MIF protein in the cultural supernatants was determined by ELISA, cellular RNA extracted and the expression of MIF mRNA was examined by RT-PCR analysis.The experimental results showed that LPS could induce a time-dependent specific expression of MIF on macrophages, in which the MIF mRNA in cells and the MIF protein in cultural supernatants appeared after 3 h and reached their highest concentration at 9-12 h after LPS stimulation. The levels of mRNA and proteins in the macrophages treated with antisense olignucleotides were decreased significantly after stimulation with LPS in comparison with that of stimulation with LPS alone or with that with LPS plus sense or missense oligonucleotides.There were no differences among those without LPS stimulation. It is concluded that macrophages stimulated with LPS express MIF, and the antisense olignucleotides of MIF inhibitthe expression of MIF mRNA as well as the secretion of MIF proteins in macrophages.

  2. Regulation of Polyphosphate Kinase Production by Antisense RNA in Pseudomonas fluorescens Pf0-1

    OpenAIRE

    Silby, Mark W.; Julie S Nicoll; Levy, Stuart B.

    2012-01-01

    Pseudomonas spp. adapt rapidly to environmental fluctuations. Loss or overproduction of polyphosphate reduces the fitness of Pseudomonas fluorescens Pf0-1, indicating the importance of the fine-tuning of polyphosphate production. An antisense RNA was investigated and shown to regulate the polyphosphate kinase gene (ppk) by a posttranscriptional mechanism reducing ppk transcript abundance.

  3. Antisense precision polymer micelles require less poly(ethylenimine) for efficient gene knockdown

    Science.gov (United States)

    Fakhoury, Johans J.; Edwardson, Thomas G.; Conway, Justin W.; Trinh, Tuan; Khan, Farhad; Barłóg, Maciej; Bazzi, Hassan S.; Sleiman, Hanadi F.

    2015-12-01

    Therapeutic nucleic acids are powerful molecules for shutting down protein expression. However, their cellular uptake is poor and requires transport vectors, such as cationic polymers. Of these, poly(ethylenimine) (PEI) has been shown to be an efficient vehicle for nucleic acid transport into cells. However, cytotoxicity has been a major hurdle in the development of PEI-DNA complexes as clinically viable therapeutics. We have synthesized antisense-polymer conjugates, where the polymeric block is completely monodisperse and sequence-controlled. Depending on the polymer sequence, these can self-assemble to produce micelles of very low polydispersity. The introduction of linear poly(ethylenimine) to these micelles leads to aggregation into size-defined PEI-mediated superstructures. Subsequently, both cellular uptake and gene silencing are greatly enhanced over extended periods compared to antisense alone, while at the same time cellular cytotoxicity remains very low. In contrast, gene silencing is not enhanced with antisense polymer conjugates that are not able to self-assemble into micelles. Thus, using antisense precision micelles, we are able to achieve significant transfection and knockdown with minimal cytotoxicity at much lower concentrations of linear PEI then previously reported. Consequently, a conceptual solution to the problem of antisense or siRNA delivery is to self-assemble these molecules into `gene-like' micelles with high local charge and increased stability, thus reducing the amount of transfection agent needed for effective gene silencing.Therapeutic nucleic acids are powerful molecules for shutting down protein expression. However, their cellular uptake is poor and requires transport vectors, such as cationic polymers. Of these, poly(ethylenimine) (PEI) has been shown to be an efficient vehicle for nucleic acid transport into cells. However, cytotoxicity has been a major hurdle in the development of PEI-DNA complexes as clinically viable

  4. Effect of CD44 Suppression by Antisense Oligonucleotide on Attachment of Human Trabecular Meshwork Cells to HA

    Institute of Scientific and Technical Information of China (English)

    李中国; 张虹

    2004-01-01

    The effects of suppression of CD44 by CD44-specific antisense oligonucleotide on attachment of human trabecular meshwork cells to hyaluronic acid (HA) were observed and the possible relationship between CD44 and primary open-angle glaucoma (POAG) investigated. CD44-specific antisense oligonucleotide was delivered with cationic lipid to cultured human trabecular meshwork cells. The expression of CD44 suppressed by CD44-specific antisense oligonucleotide was detected by RT-PCR and Western blotting. The effect of CD44 suppression by specific antisense oligonucleotide on attachment of trabecular meshwork cells to HA was measured by MTT assay. Results showed that expression of CD44 was suppressed by CD4, specific antisense oligonucleotide. Antisense oligonucleotide also suppressed the adhesion of human trabecular meshwork cells to HA in a concentration dependent manner. It was concluded that attachment of human trabecular meshwork cells to HA was decreased when CD44 was suppressed by specific antisense oligonucleotide. CD44might play a role in pathogenesis of POAG by affecting the adhesion of trabecular meshwork cells to HA.

  5. Interaction of α-Melanocortin and Its Pentapeptide Antisense LVKAT: Effects on Hepatoprotection in Male CBA Mice

    Directory of Open Access Journals (Sweden)

    Paško Konjevoda

    2011-08-01

    Full Text Available The genetic code defines nucleotide patterns that code for individual amino acids and their complementary, i.e., antisense, pairs. Peptides specified by the complementary mRNAs often bind to each other with a higher specificity and efficacy. Applications of this genetic code property in biomedicine are related to the modulation of peptide and hormone biological function, selective immunomodulation, modeling of discontinuous and linear epitopes, modeling of mimotopes, paratopes and antibody mimetics, peptide vaccine development, peptidomimetic and drug design. We have investigated sense-antisense peptide interactions and related modulation of the peptide function by modulating the effects of a-MSH on hepatoprotection with its antisense peptide LVKAT. First, transcription of complementary mRNA sequence of a-MSH in 3’→5’ direction was used to design antisense peptide to the central motif that serves as a-MSH pharmacophore for melanocortin receptors. Second, tryptophan spectrofluorometric titration was applied to evaluate the binding of a-MSH and its central pharmacophore motif to the antisense peptide, and it was concluded that this procedure represents a simple and efficient method to evaluate sense-antisense peptide interaction in vitro. Third, we showed that antisense peptide LVKAT abolished potent hepatoprotective effects of a-MSH in vivo.

  6. Immunostimulatory Oligodeoxynucleotides Containing the CpG Motif are Effective as Immune Adjuvants in Tumor Antigen Immunization

    Science.gov (United States)

    Weiner, George J.; Liu, Hsin-Ming; Wooldridge, James E.; Dahle, Christopher E.; Krieg, Arthur M.

    1997-09-01

    Recent advances in our understanding of the immune response are allowing for the logical design of new approaches to cancer immunization. One area of interest is the development of new immune adjuvants. Immunostimulatory oligodeoxynucleotides containing the CpG motif (CpG ODN) can induce production of a wide variety of cytokines and activate B cells, monocytes, dendritic cells, and NK cells. Using the 38C13 B cell lymphoma model, we assessed whether CpG ODN can function as immune adjuvants in tumor antigen immunization. The idiotype served as the tumor antigen. Select CpG ODN were as effective as complete Freund's adjuvant at inducing an antigen-specific antibody response but were associated with less toxicity. These CpG ODN induced a higher titer of antigen-specific IgG2a than did complete Freund's adjuvant, suggesting an enhanced TH1 response. Mice immunized with CpG ODN as an adjuvant were protected from tumor challenge to a degree similar to that seen in mice immunized with complete Freund's adjuvant. We conclude that CpG ODN are effective as immune adjuvants and are attractive as part of a tumor immunization strategy.

  7. Systemic Administration of CpG Oligodeoxynucleotide and Levamisole as Adjuvants for Gene-Gun-Delivered Antitumor DNA Vaccines

    Directory of Open Access Journals (Sweden)

    Michal Šmahel

    2011-01-01

    Full Text Available DNA vaccines showed great promise in preclinical models of infectious and malignant diseases, but their potency was insufficient in clinical trials and is needed to be improved. In this study, we tested systemic administration of two conventional adjuvants, synthetic oligodeoxynucleotide carrying immunostimulatory CpG motifs (CpG-ODN and levamisole (LMS, and evaluated their effect on immune reactions induced by DNA vaccines delivered by a gene gun. DNA vaccination was directed either against the E7 oncoprotein of human papillomavirus type 16 or against the BCR-ABL1 oncoprotein characteristic for chronic myeloid leukemia. High doses of both adjuvants reduced activation of mouse splenic CD8+ T lymphocytes, but the overall antitumor effect was enhanced in both tumor models. High-dose CpG-ODN exhibited a superior adjuvant effect in comparison with any combination of CpG-ODN with LMS. In summary, our results demonstrate the benefit of combined therapy with gene-gun-delivered antitumor DNA vaccines and systemic administration of CpG-ODN or LMS.

  8. Immunogenicity and safety of liposome-vaccine encapsulating hepatitis B surface antigen and phosphodiester CpG oligodeoxynucleotides

    Institute of Scientific and Technical Information of China (English)

    CHUN YAN HE; QING LIANG LIU

    2006-01-01

    CpG oligodeoxynucleotides (CpG ODN) as adjuvant have been extensively studied in recent years. Phosphodiester CpG ODN (PO CpG ODN) can perfectly mimic bacterial DNA in enhancing immune response but are vulnerable to nucleases in vivo. This study aimed to evaluate the immunostimu latory potential and safety of phosphodiester CpG ODN encapsulated in nonphospholipid liposomes.BALB/c mice were immunized intramuscularly with different formulations of liposomes, CpG ODN and hepatitis B surface antigen (HBsAg). The results demonstrated that the encapsulated PO CpG ODN were protected against rapid degradation in vivo and retained their adjuvant activity. PO CpG ODN encapsulated with HBsAg in liposomes induced strong Th1-biased or Th1/Th2 mixed humoral immune response in mice with the magnitude similar to their phosphothioate equivalent in the same formulation.High IFN-gamma production induced by this formulation confirmed the generation of strong cellular immune response. Additionally, co-delivery of HBsAg and PO CpG ODN improved the immune response over that obtained with separate delivery. Safety experiment showed that liposome-encapsulaed PO CpG ODN and HBsAg caused mild systemic and moderate local adverse reaction. In conclusion, our data shows that PO CpG ODN encapsulated in liposomes fully exhibit their Th1-type adjuvant activity and act as a potential adjuvant for vaccines.

  9. The protein DIIIC-2, aggregated with a specific oligodeoxynucleotide and adjuvanted in alum, protects mice and monkeys against DENV-2.

    Science.gov (United States)

    Gil, Lázaro; Marcos, Ernesto; Izquierdo, Alienys; Lazo, Laura; Valdés, Iris; Ambala, Peris; Ochola, Lucy; Hitler, Rikoi; Suzarte, Edith; Álvarez, Mayling; Kimiti, Prisilla; Ndung'u, James; Kariuki, Thomas; Guzmán, María Guadalupe; Guillén, Gerardo; Hermida, Lisset

    2015-01-01

    Previously, we reported the ability of the chimeric protein DIIIC-2 (domain III of the dengue envelope protein fused to the capsid protein of dengue-2 virus), to induce immunity and protection in mice, when it is highly aggregated with a non-defined oligodeoxynucleotide (ODN) and adjuvanted in alum. In this work, three different defined ODNs were studied as aggregating agents. Our results suggest that the nature of the ODN influences the capacity of protein DIIIC-2 to activate cell-mediated immunity in mice. Consequently, the ODN 39M was selected to perform further experiments in mice and nonhuman primates. Mice receiving the preparation 39M-DIIIC-2 were solidly protected against dengue virus (DENV) challenge. Moreover, monkeys immunized with the same preparation developed neutralizing antibodies, as measured by four different neutralization tests varying the virus strains and the cell lines used. Two of the immunized monkeys were completely protected against challenge, whereas the third animal had a single day of low-titer viremia. This is the first work describing the induction of short-term protection in monkeys by a formulation that is suitable for human use combining a recombinant protein from DENV with alum.

  10. Effects of a plasmid expressing antisense tissue inhibitor of metalloproteinase-1 on liver fibrosis in rats

    Institute of Scientific and Technical Information of China (English)

    JIANG Wei; WANG Ji-yao; YANG Chang-qing; LIU Wen-bin; WANG Yi-qing; HE Bo-ming

    2005-01-01

    Background No efficient therapy for liver fibrosis has been available. This study was aimed to provide evidence that the introduction of a plasmid expressing antisense tissue inhibitor of metalloproteinase-1 (TIMP-1) into a rat model of immunologically induced liver fibrosis can result in the increased activity of interstitial collagenase, thus enhancing the degradation of collagen.Methods Real-time nested polymerase chain reaction (RT-Nested-PCR) and gene recombination techniques were used to construct a rat antisense TIMP-1 recombinant plasmid that can be expressed in eukaryotic cells. Both the recombinant plasmid and an empty vector (pcDNA3) were encapsulated with glycosyl-poly-L-lysine and injected into rats suffering from pig serum-induced liver fibrosis. The expression of exogenous transfected plasmid was assessed by Northern blot, RT-PCR, and Western blot. Hepatic interstitial collagenase activity was detected using fluorescinisothiocyanate (FITC)-labeled type Ⅰ collagen. In addition to hepatic hydroxyproline content, hepatic collagen types Ⅰ and Ⅲ were detected by immunohistochemical staining, and the stages of liver fibrosis by Van Gieson staining.Results Exogenous antisense TIMP-1 was successfully expressed in vivo and could block the gene and protein expression of TIMP-1. Active and latent hepatic interstitial collagenase activities were elevated (P<0.01), hepatic hydroxyproline content and the accumulation of collagen types Ⅰ and Ⅲ were lowered, and liver fibrosis was alleviated in the antisense TIMP-1 group (P<0.01) as compared with the model group. Conclusion The results demonstrate that antisense TIMP-1 recombinant plasmids have some inhibitory effect on liver fibrosis.

  11. Antisense EGFR sequence enhances apoptosis in a human hepatoma cell line BEL—7404

    Institute of Scientific and Technical Information of China (English)

    FUTAO; HELIU; 等

    1996-01-01

    Effects of antisense epidermal growth factor receptor (EGFR) sequence on apoptotic cell death were examined in a human hepatoma cell line BEL-7404 cells.In the cells of JX-1,a sub clone of BEL-7404 stably transfected with antisense EGFR vector (Cell Research,3:75,1993),an enhanced rate(9.5%) of spontaneous apoptosis was detected by flow cytometry,whereas the rates of spontaneous apoptosis in JX-0 cells,a sub-clone of BEL-7404 transfected by control vector,and the parent BEL-7404 transfected by control vector,and the parent BEL-7404 transfected by control vector,and the parent BEL-7404 cells were almost equal and about 1.7%.Serum-starvation for 72h increased the rate of apoptosis of JX-lcells up to 33.7%,while JX-0 and BEL-7404 cells,under the same condition,produced less than 5% of apoptotic cells.Observation with electron microscope demonstrated that condensation and fragmentation of chromatin and formation of apoptotic bodies often occurred in JX-1 cells,especially during serumstarvation.These results,combined with the data of DNA fragmentation Elisa test,suggested that antisense EGFR sequence enhances apoptosis in the human hepatoma cells.Comparison of intracellular Ca2+ level and the responsiveness of JX-1 cells to the induced action of EGF and tharpsigargin (TG) treatment with that of control JX-0 cells indicated that antisense egfr might interrupt the EGF/EGFR sigaling pathway resulting in the decreass of intracellular Ca2+ pool content as well as the responsiveness of these cells to the extracellular signals.These findings suggest that antisense EGFR either directly or indirectly regulates Ca2+ storage in endoplasmic reticulum,thereby enhances apoptosis in the human hepatoma cells.

  12. Cooperative inhibitory effects of antisense oligonucleotide of cell adhesion molecules and cimetidine on cancer cell adhesion

    Institute of Scientific and Technical Information of China (English)

    Nan-Hong Tang; Yan-Ling Chen; Xiao-Qian Wang; Xiu-Jin Li; Feng-Zhi Yin; Xiao-Zhong Wang

    2004-01-01

    AIM: To explore the cooperative effects of antisense oligonucleotide (ASON) of cell adhesion molecules and cimetidine on the expression of E-selectin and ICAM-1 in endothelial cells and their adhesion to tumor cells.METHODS: After treatment of endothelial cells with ASON and/or cimetidine and induction with TNF-α, the protein and mRNA changes of E-selectin and ICAM-1 in endothelial cells were examined by flow cytometry and RT-PCR,respectively. The adhesion rates of endothelial cells to tumor cells were measured by cell adhesion experiment.RESULTS: In comparison with TNF-α inducing group, lipoASON and lipo-ASON/cimetidine could significantly decrease the protein and mRNA levels of E-selectin and ICAM-1 in endothelial cells, and lipo-ASON/cimetidine had most significant inhibitory effect on E-selectin expression (from 36.37±1.56% to 14.23±1.07%, P<0.001). Meanwhile,cimetidine alone could inhibit the expression of E-selectin (36.37±1.56% vs 27.2±1.31%, P<0.001), but not ICAM-1 (69.34±2.50% vs68.07±2.10%,P>O.05)and the two kinds of mRNA, either. Compared with TNF-αα inducing group, the rate of adhesion was markedly decreased in lipo-E-selectin ASON and lipo-E-selectin ASON/cimetidine treated groups(P<0.05),and Jipo-E-selectin ASON/cimetidine worked better than lipo-E-selectin ASON alone except for HepG2/ECV304 group(P<0.05). However, the decrease of adhesion was not significant in lipo-ICAM-1 ASON and lipo-ICAM-1 ASON/cimetidine treated groups except for HepG2/ECV304 group (P >0.05).CONCLUSION: These data demonstrate that ASON in combination with cimetidine in vitro can significantly reduce the adhesion between endothelial cells and hepatic or colorectal cancer cells, which is stronger than ASON or cimetidine alone. This study provides some useful proofs for gene therapy of antiadhesion.

  13. Human Immunodeficiency Virus-Type 1 LTR DNA contains an intrinsic gene producing antisense RNA and protein products

    Directory of Open Access Journals (Sweden)

    Hsiao Chiu-Bin

    2006-11-01

    Full Text Available Abstract Background While viruses have long been shown to capitalize on their limited genomic size by utilizing both strands of DNA or complementary DNA/RNA intermediates to code for viral proteins, it has been assumed that human retroviruses have all their major proteins translated only from the plus or sense strand of RNA, despite their requirement for a dsDNA proviral intermediate. Several studies, however, have suggested the presence of antisense transcription for both HIV-1 and HTLV-1. More recently an antisense transcript responsible for the HTLV-1 bZIP factor (HBZ protein has been described. In this study we investigated the possibility of an antisense gene contained within the human immunodeficiency virus type 1 (HIV-1 long terminal repeat (LTR. Results Inspection of published sequences revealed a potential transcription initiator element (INR situated downstream of, and in reverse orientation to, the usual HIV-1 promoter and transcription start site. This antisense initiator (HIVaINR suggested the possibility of an antisense gene responsible for RNA and protein production. We show that antisense transcripts are generated, in vitro and in vivo, originating from the TAR DNA of the HIV-1 LTR. To test the possibility that protein(s could be translated from this novel HIV-1 antisense RNA, recombinant HIV antisense gene-FLAG vectors were designed. Recombinant protein(s were produced and isolated utilizing carboxy-terminal FLAG epitope (DYKDDDDK sequences. In addition, affinity-purified antisera to an internal peptide derived from the HIV antisense protein (HAP sequences identified HAPs from HIV+ human peripheral blood lymphocytes. Conclusion HIV-1 contains an antisense gene in the U3-R regions of the LTR responsible for both an antisense RNA transcript and proteins. This antisense transcript has tremendous potential for intrinsic RNA regulation because of its overlap with the beginning of all HIV-1 sense RNA transcripts by 25 nucleotides. The

  14. Depressive Effect of the Antisense Oligonucleotides of C-myc and PCNA on the Proliferation of VSMC

    Institute of Scientific and Technical Information of China (English)

    Qingxian Li; Yanfu Wang; Yuhua Liao; Huiling Zhang; Yanying Jiang

    2007-01-01

    To study the depressive effect of the antisense oligonuceotides (ASODN) of c-myc and proliferating cell nuclear antigen (PCNA) on the proliferation of VSMC.Methods Taking the VSMC obtained from rat aorta thoracalis cultured 4 ~ 8 generation as research object.The objects were divided into three groups to carry out control study:control group,PCNA ASODN group and c-myc ASODN group.The ASODNs' working concentration all were 1:50.The depressive effect of ASODN on VSMC proliferation was investigated by cell counting,MTT and 3H-TdR incorporation assay;PCNA and c-myc expression were detected by immunohistochemical method after transferring PCNA successfully;the corresponding gene was inhibited obviously;compared with control group ( P < 0.05 ).Conclusions PCNA and c-myc might play a considerable role in the VSMC proliferation process.The corresponding gene could be depressed successfully after transferring PCNA and c-myc ASODN into VSMC,and then the proliferation of VSMC was slowed down.This study presented a beneficial proposal and theoretical fundament for atherosclerotic treatment.

  15. A Polyethylenimine-Containing and Transferrin-Conjugated Lipid Nanoparticle System for Antisense Oligonucleotide Delivery to AML

    Directory of Open Access Journals (Sweden)

    Yiming Yuan

    2016-01-01

    Full Text Available Limited success of antisense oligonucleotides (ASO in clinical anticancer therapy calls for more effective delivery carriers. The goal of this study was to develop a nanoparticle system for delivery of ASO G3139, which targets mRNA of antiapoptotic protein Bcl-2, to acute myeloid leukemia (AML cells. The synthesized nanoparticle Tf-LPN-G3139 contained a small molecular weight polyethylenimine and two cationic lipids as condensing agents, with transferrin on its surface for selective binding and enhanced cellular uptake. The optimized nitrogen to phosphate (N/P ratio was 4 to achieve small particle size and high G3139 entrapment efficiency. The Tf-LPN-G3139 exhibited excellent colloidal stability during storage for at least 12 weeks and remained intact for 4 hours in nuclease-containing serum. The cellular uptake results showed extensive internalization of fluorescence-labelled G3139 in MV4-11 cells through Tf-LPN. Following transfection, Tf-LPN-G3139 at 1 µM ASO level induced 54% Bcl-2 downregulation and >20-fold apoptosis compared to no treatment. When evaluated in mice bearing human xenograft AML tumors, Tf-LPN-G3139 suppressed tumor growth by ~60% at the end of treatment period, accompanied by remarkable pharmacological effect of Bcl-2 inhibition in tumor. In conclusion, Tf-LPN-G3139 is a promising nanoparticle system for ASO G3139 delivery to AML and warrants further investigations.

  16. Effects of connective tissue growth factor antisense oligonucleotides on the proliferation and collagen synthesis of the cultured human keloid fibroblasts in vitro

    Institute of Scientific and Technical Information of China (English)

    刘剑毅; 李世荣; 纪淑兴

    2004-01-01

    Objective: To explore the effects of connective tissue growth factor (CTGF) on the pathogenesis of human keloid. Methods: CTGF antisense oligonucleotides (ASODN) conjugated with isothiocyananate fluorescence was encapsulated by liposome, and then added into the human keloid fibroblasts (HKFs) culture media. The intracellular distribution of CTGF ASODN was observed by fluorescence microscopy in the fixed HKFs. The proliferation of HKFs was measured by MTT test. The collagen synthesis of HKFs was measured by 3H-proline incorporation method. Results: Compared with control group, the CTGF ASODN can inhibit the proliferation and collagen synthesis of the HKFs (P < 0.01 ). Conclusion: CTGF ASODN has anti-fibrotic effects on keloid in vitro, and CTGF play an important role in promoting the fibrosis of keloid.

  17. Effect of molecular weight of polyethyleneimine on loading of CpG oligodeoxynucleotides onto flake-shell silica nanoparticles for enhanced TLR9-mediated induction of interferon-α

    Directory of Open Access Journals (Sweden)

    Manoharan Y

    2012-07-01

    Full Text Available Yuvaraj Manoharan,1,* Qingmin Ji,2,* Tomohiko Yamazaki,2,3 Shanmugavel Chinnathambi,1 Song Chen,2,4 Ganesan Singaravelu,1 Jonathan P Hill,2 Katsuhiko Ariga,2,5 Nobutaka Hanagata3,6 1Department of Medical Physics, Anna University, Chennai, India; 2Research Center for Materials Nanoarchitectonics, National Institute for Materials Science, Tsukuba, Ibarak, 3Graduate School of Life Science, Hokkaido University, Kita-ku, Sapporo, 4JSPS Research Fellow, Chiyoda-ku, Tokyo, 5JST and CREST, National Institute for Materials Science, Tsukuba, Ibaraki, Japan; 6Nanotechnology Innovation Station, National Institute for Materials Science, Tsukuba, Ibaraki, Japan*These authors contributed equally to this workBackground: Class B CpG oligodeoxynucleotides primarily interact with Toll-like receptor 9 (TLR9 in B cells and enhance the immune system through induction of various interleukins including interleukin-6 in these immune cells. Although free class B CpG oligodeoxynucleotides do not induce interferon (IFN-α production, CpG oligodeoxynucleotide molecules have been reported to induce IFN-α when loaded onto nanoparticles. Here, we investigated the in vitro induction of IFN-α by a nanocarrier delivery system for class B CpG oligodeoxynucleotide molecules.Methods: For improving the capacity to load CpG oligodeoxynucleotide molecules, flake-shell SiO2 nanoparticles with a specific surface area approximately 83-fold higher than that of smooth-surfaced SiO2 nanoparticles were prepared by coating SiO2 nanoparticles with polyethyleneimine (PEI of three different number-average molecular weights (Mns 600, 1800, and 10,000 Da.Results: The capacity of the flake-shell SiO2 nanoparticles to load CpG oligodeoxynucleotides was observed to be 5.8-fold to 6.7-fold higher than that of smooth-surfaced SiO2 nanoparticles and was found to increase with an increase in the Mn of the PEI because the Mn contributed to the positive surface charge density of the nanoparticles. Further

  18. Abrogation of heat-shock protein (HSP)70 expression induced cell growth inhibition and apoptosis in human androgen-independent prostate cancer cell line PC-3m

    Institute of Scientific and Technical Information of China (English)

    Zhi-GangZhao; Qing-ZhengMa; Chun-XiaoXu

    2004-01-01

    Aim: To investigate the effect of abrogating heat shock protein (HSP) 70 expression by antisense HSP70 oligonucleotides treatment on human androgen-independent prostate cancer cell line PC-3m growth. Methods: PC3m cells were treated with 0-16μmol/L antisense HSP70 oligomers for 0-100 hr. Cell growth inhibition was analyzed using a trypan blue dye exclusion test. Apoptotic cells were detected and confirmed by flow cytometric analysis and DNA fragmentation analysis. The protein expression of HSP70 and bcl-2 affected by antisense HSP70 oligomers were determined using Western blot. Results: Antisense HSP70 oligomer induced apoptosis and then inhibited proliferation of PC-3m cells in a dose- and time-dependent manner. Ladder-like patterns of DNA fragments were observed in PC-3m cells treated with 10μmol/L antisense HSP70 oligomer for 48 hr or 8μtmol/L for 72 hr on agarose gel electrophoresis. Antisense HSP70 oligomer pretreatment enhanced the subsequent induction of apoptosis by heat shock in PC-3m cells. In addition, undetectable HSP70 expression was observed at a concentration of 10μtmol/L antisense HSP70 oligomer treatment for 48 hr or 8μtmol/L for 72 hr in Western blot, which was paralleled by decreased expression levels of anti-apoptotic protein bcl-2. Conclusion: HSP70 antisense oligomer treatment abro-gates the expression of HSP70, which may disrupt HSP70-bcl-2-interactions and further down-regulate bcl-2 expression,in turn inducing apoptosis and inhibiting cell growth in PC-3m cells. (Asian JAndro12004 Dec;6:319-324)

  19. High-dose irradiation in combination with toll-like receptor 9 agonist CpG oligodeoxynucleotide 7909 downregulates PD-L1 expression via the NF-κB signaling pathway in non-small cell lung cancer cells

    Science.gov (United States)

    Chen, Xue; Zhang, Qi; Luo, Youjun; Gao, Caixia; Zhuang, Xibing; Xu, Guoxiong; Qiao, Tiankui

    2016-01-01

    Objectives Irradiation resistance appears as local recurrence and distant metastasis in advanced stages of non-small cell lung cancer (NSCLC). High-dose irradiation combined with immunotherapy improved overall survival and local control of NSCLC. This study explored the underlying molecular mechanism by which the effect of high-dose irradiation plus toll-like receptor 9 (TLR9) agonist CpG oligodeoxynucleotide (CpG ODN) 7909 on NSCLC. Materials and methods NSCLC H460 cells were exposed to constant high-dose irradiation (6.37 Gy) in irradiation (IR) group and the irradiation plus CpG group. Gene expression was assessed using quantitative reverse transcriptase-polymerase chain reaction and Western blot. Knockdown of nuclear factor kappa B (NF-κB) p65 expression was conducted using p65 siRNA. Results Expression of programmed death-ligand 1 (PD-L1) mRNA was significantly decreased in IR combined with CpG ODN 7909 group compared with the control or IR-alone groups (P<0.05). TLR9 expression was also obviously increased in the combination group compared with the control (P<0.05). Moreover, expression of NF-κB p65 was apparently reduced in the combination group compared with the control (P<0.05). However, expression of PD-L1 was significantly decreased after knockdown of p65 in IR group (P<0.05), but increased in the combination group (P<0.05) and slightly increased in CpG ODN-alone group (P<0.05), which was opposite to that without p65 knockdown group. Conclusion This study demonstrated that radiotherapy combined with CpG ODN 7909 was able to downregulate PD-L1 expression through inhibition via the NF-κB signaling pathway. PMID:27799798

  20. Antisense oligonucleotides and all-trans retinoic acid have a synergistic anti-tumor effect on oral squamous cell carcinoma

    Directory of Open Access Journals (Sweden)

    Chen Wantao

    2008-06-01

    Full Text Available Abstract Background Antisense oligonucleotides against hTR (As-ODN-hTR have shown promising results as treatment strategies for various human malignancies. All-trans retinoic acid (ATRA is a signalling molecule with important roles in differentiation and apoptosis. Biological responses to ATRA are currently used therapeutically in various human cancers. The aim of this study was to evaluate the anti-tumor effects of As-ODN-hTR combined with ATRA in vivo. Methods In situ human oral squamous cell carcinoma (OSCC models were established by subcutaneous injection of Tca8113 cells. Mice were treated with sense oligonucleotides against hTR(S-ODN-hTR alone, As-ODN-hTR alone, ATRA alone, As-ODN-hTR plus ATRA, or S-ODN-hTR plus ATRA. Tumor size and weight were assessed in the mice. Telomerase activity was detected by a TRAP assay, apoptotic cells were evaluated with a Tunel assay, the expression of apoptosis-related proteins (Bcl-2 and Bax was evaluated by immunohistochemistry and ultrastructural morphological changes in the tumor specimen were examined. Results Both As-ODN-hTR and ATRA can significantly inhibit tumor growth in this OSCC xenograft solid-tumor model, and the combination of the two agents had a synergistic anti-tumorogenic effect. We also demonstrated that this anti-tumor effect correlated with inhibition of telomerase activity. Furthermore, significant increases in the number of apoptotic cells, typical apoptotic morphology and a downregulation of the anti-apoptotic protein, bcl-2 were observed in the treated tissues. Conclusion The combination of As-ODN-hTR and ATRA has a synergistic anti-tumor effect. This anti-tumor effect can be mainly attributed to apoptosis induced by a decrease in telomerase activity. Bcl-2 plays an important role in this process. Therefore, combining As-ODN-hTR and ATRA may be an approach for the treatment of human oral squamous cell carcinoma.

  1. Analysis of the mechanism of protection in transgenic plants expressing the potato virus X coat protein or its antisense RNA

    OpenAIRE

    Hemenway, Cynthia; Fang, Rong-Xiang; Kaniewski, Wojciech K.; Chua, Nam-Hai; Tumer, Nilgun E.

    1988-01-01

    Transgenic tobacco plants engineered to express either the potato virus X (PVX) coat protein (CP+) or the antisense coat protein transcript (CP-antisense) were protected from infection by PVX, as indicated by reduced lesion numbers on inoculated leaves, delay or absence of systemic symptom development and reduction in virus accumulation in both inoculated and systemic leaves. The extent of protection observed in CP+ plants primarily depended upon the level of expression of the coat protein. P...

  2. The Effect of TLR9 Agonist CpG Oligodeoxynucleotides on the Intestinal Immune Response of Cobia (Rachycentron canadum

    Directory of Open Access Journals (Sweden)

    Omkar Byadgi

    2014-01-01

    Full Text Available Cytosine-guanine oligodeoxynucleotide (CpG ODN motifs of bacterial DNA are recognized through toll-like receptor 9 (TLR9 and are potent activators of innate immunity. However, the interaction between TLR9 and CpG ODN in aquatic species has not been well characterized. Hence, cobia TLR9 isoform B (RCTLR9B was cloned and its expression and induction in intestine were investigated. RCTLR9B cDNA consists of 3113bp encoding 1009 amino acids containing three regions, leucine rich repeats, transmembrane domain, and toll/interleukin-1 receptor (TIR domain. Intraperitoneal injection of CpG ODN 2395 upregulated RCTLR9 A and B and MyD88 and also induced the expressions of Mx, chemokine CC, and interleukin IL-1β. Cobia intraperitoneally injected with CpG ODN 1668 and 2395 had increased survival rates after challenge with Photobacterium damselae subsp. piscicida. In addition, formulation of CpG ODN with formalin-killed bacteria (FKB and aluminum hydroxide gel significantly increased expressions of RCTLR9 A (50 folds and B (30 folds isoforms at 10 dpi (CpG ODN 1668 and MyD88 (21 folds at 6 dpv (CpG ODN 2395. Subsequently, IL-1β increased at 6 dpv in 1668 group. No histopathological damage and inflammatory responses were observed in the injected cobia. Altogether, these results facilitate CpG ODNs as an adjuvant to increase bacterial disease resistance and efficacy of vaccines in cobia.

  3. Calculation of DNA strand breakage by neutralisation effect after 125I decays in a synthetic oligodeoxynucleotide using charge transfer theory

    International Nuclear Information System (INIS)

    The decay of the radioisotope 125I into 125Te is typically followed by the emission of two groups of approximately 10 electrons each via Auger processes. In deoxyribonucleic acid (DNA) with 125I incorporated, these electrons produce various types of damage to DNA, e.g. single strand breaks (SSBs) and double strand breaks (DSBs) through direct actions of physical tracks, or indirect actions of radicals produced in water. Among the direct actions one should consider not only the excitation and ionisation of DNA by Auger electrons, but also the neutralisation of highly charged 125mTe ions with electrons from neighbouring molecules. Comparison between experiment and simulation done recently revealed that without including neutralisation effect the simulated yield of SSBs was 50% less than the measured result. In the present work a calculation of DNA strand breakage by the neutralization effect in a 41-mer synthetic oligodeoxynucleotide (oligo-DNA) model was done using the charge transfer theory. Calculation based on transfer rate using the newly evaluated electronic coupling of DNA bases showed that the positive charge (hole) transfer rate is of the order of magnitudes of several 1013s-1, implying that a charge higher than 10 units might not build on a 125mTe atom. The potential energy accumulated on the decay base is transferred to bases along the DNA chain nearby and destroys those bases and ionises the sugar-phosphate group, leading a DNA SSB with a frequency of 0.2% per eV in average. (authors)

  4. Structure-efficacy relationships of immunostimulatory activity of CpG-containing oligodeoxynucleotides on mouse spleen cells

    Institute of Scientific and Technical Information of China (English)

    Hai-yan DU; Ai-guo JI; Zhong-ming TANG; Shao-you XIA; Hai-feng SONG; Na LI; Ming-mei SHANG; Jia ZOU; Qing-qing WANG; Lun OU; Xiao SUN

    2007-01-01

    Aim: To study the relationship between primary structures of oligodeoxynucleo-tides (ODN) containing unmethylated deoxycytidyl-deoxyguanosine (CpG)dinucleotide motifs and their immunostimulatory activities in mouse spleen cells.Methods: A series of CpG ODN with different primary structures were synthesized.Their capabilities to stimulate mouse spleen cell proliferation were determined by[3H]thymidine incorporation assay. Cytokine (interleukin JILl-6, IL-12, and IFN-α)secretion spectra induced by CpG ODN were assessed by ELISA. The ability ofCpG ODN to activate natural killer cells was evaluated by standard 4 h 51Cr-release assay. Flow cytometry was utilized to examine the expressions of various lymphocyte surface molecules on diverse immunocytes. An effective CpG ODN for routine,ODN1826, was set as the template of modification and the positive control.Results: The immunostimulatory activities of CpG ODN with different sequences and compositions varied markedly, both in character and in extent. It was useless for improving the immunostimulatory activity of ODN1826 by simply increasing the functional hexameric CpG motif number, modifying the site of CpG motifs, or changing the distance between multi-CpG motifs. However, an addition of a selfcomplementary palindrome structure at the 3'-end, but not the 5'-end of CpG ODN,aroused marked improvement in its activity. Several designed ODN had superior comprehensive immunostimulatory properties compared to ODN 1826. Conclusion:The immunostimulatory activity ofa CpG ODN was relevant to its primary structure.It was useless for promoting immunostimulatory activity to simply change CpG motif number, space, or distance. The 3'-end palindrome structure of CpG ODN is associated with enhanced immunostimulatory activity.

  5. Using Surface-Assisted Laser Desorption/Ionization Mass Spectrometry to Detect ss- and ds-Oligodeoxynucleotides

    Science.gov (United States)

    Chen, Wen-Tsen; Huang, Ming-Feng; Chang, Huan-Tsung

    2013-06-01

    We applied surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) with HgTe nanostructures as the matrix for the detection of single- and double-stranded oligodeoxynucleotides (ss-ODNs and ds-ODNs). The concentrations of surfactant and additives (metal ions, an amine) and the pH and ionic strength of the sample matrix played significantly different roles in the detection of ss- and ds-ODNs with various sequences. In the presence of Brij 76 (1.5 %), Hg2+ (7.5 μM), and cadaverine (10 μM) at pH 5.0, this SALDI-MS approach allowed the simultaneous detection of T15, T20, T33, and T40, with limits of detection at the femtomole-to-picomole level and sample-to-sample intensity variation <23 %. In the presence of Ag+ (1 μM) and cadaverine (10 μM) at pH 7.0, this technique allowed the detection of randomly sequenced ss- and ds-ODNs at concentrations down to the femtomole level. To the best of our knowledge, this paper is the first to report the detection of ss-ODNs (up to 50-mer) and ds-ODNs (up to 30 base pairs) through the combination of SALDI-MS with HgTe nanostructures as matrices. We demonstrated the practicality of this approach through analysis of a single nucleotide polymorphism that determines the fate of the valine residue in the β-globin of sickle cell megaloblasts.

  6. Tye7 regulates yeast Ty1 retrotransposon sense and antisense transcription in response to adenylic nucleotides stress.

    Science.gov (United States)

    Servant, Géraldine; Pinson, Benoit; Tchalikian-Cosson, Aurélie; Coulpier, Fanny; Lemoine, Sophie; Pennetier, Carole; Bridier-Nahmias, Antoine; Todeschini, Anne Laure; Fayol, Hélène; Daignan-Fornier, Bertrand; Lesage, Pascale

    2012-07-01

    Transposable elements play a fundamental role in genome evolution. It is proposed that their mobility, activated under stress, induces mutations that could confer advantages to the host organism. Transcription of the Ty1 LTR-retrotransposon of Saccharomyces cerevisiae is activated in response to a severe deficiency in adenylic nucleotides. Here, we show that Ty2 and Ty3 are also stimulated under these stress conditions, revealing the simultaneous activation of three active Ty retrotransposon families. We demonstrate that Ty1 activation in response to adenylic nucleotide depletion requires the DNA-binding transcription factor Tye7. Ty1 is transcribed in both sense and antisense directions. We identify three Tye7 potential binding sites in the region of Ty1 DNA sequence where antisense transcription starts. We show that Tye7 binds to Ty1 DNA and regulates Ty1 antisense transcription. Altogether, our data suggest that, in response to adenylic nucleotide reduction, TYE7 is induced and activates Ty1 mRNA transcription, possibly by controlling Ty1 antisense transcription. We also provide the first evidence that Ty1 antisense transcription can be regulated by environmental stress conditions, pointing to a new level of control of Ty1 activity by stress, as Ty1 antisense RNAs play an important role in regulating Ty1 mobility at both the transcriptional and post-transcriptional stages. PMID:22379133

  7. Tye7 regulates yeast Ty1 retrotransposon sense and antisense transcription in response to adenylic nucleotides stress

    Science.gov (United States)

    Servant, Géraldine; Pinson, Benoit; Tchalikian-Cosson, Aurélie; Coulpier, Fanny; Lemoine, Sophie; Pennetier, Carole; Bridier-Nahmias, Antoine; Todeschini, Anne Laure; Fayol, Hélène; Daignan-Fornier, Bertrand; Lesage, Pascale

    2012-01-01

    Transposable elements play a fundamental role in genome evolution. It is proposed that their mobility, activated under stress, induces mutations that could confer advantages to the host organism. Transcription of the Ty1 LTR-retrotransposon of Saccharomyces cerevisiae is activated in response to a severe deficiency in adenylic nucleotides. Here, we show that Ty2 and Ty3 are also stimulated under these stress conditions, revealing the simultaneous activation of three active Ty retrotransposon families. We demonstrate that Ty1 activation in response to adenylic nucleotide depletion requires the DNA-binding transcription factor Tye7. Ty1 is transcribed in both sense and antisense directions. We identify three Tye7 potential binding sites in the region of Ty1 DNA sequence where antisense transcription starts. We show that Tye7 binds to Ty1 DNA and regulates Ty1 antisense transcription. Altogether, our data suggest that, in response to adenylic nucleotide reduction, TYE7 is induced and activates Ty1 mRNA transcription, possibly by controlling Ty1 antisense transcription. We also provide the first evidence that Ty1 antisense transcription can be regulated by environmental stress conditions, pointing to a new level of control of Ty1 activity by stress, as Ty1 antisense RNAs play an important role in regulating Ty1 mobility at both the transcriptional and post-transcriptional stages. PMID:22379133

  8. Cell number and transfection volume dependent peptide nucleic acid antisense activity by cationic delivery methods

    DEFF Research Database (Denmark)

    Llovera Nadal, Laia; Berthold, Peter; Nielsen, Peter E;

    2012-01-01

    Efficient intracellular delivery is essential for high activity of nucleic acids based therapeutics, including antisense agents. Several strategies have been developed and practically all rely on auxiliary transfection reagents such as cationic lipids, cationic polymers and cell penetrating...... peptides as complexing agents and carriers of the nucleic acids. However, uptake mechanisms remain rather poorly understood, and protocols always require optimization of transfection parameters. Considering that cationic transfection complexes bind to and thus may up-concentrate on the cell surface, we...... have now quantitatively compared the cellular activity (in the pLuc705 HeLa cell splice correction system) of PNA antisense oligomers using lipoplex delivery of cholesterol- and bisphosphonate-PNA conjugates, polyplex delivery via a PNA-polyethyleneimine conjugate and CPP delivery via a PNA...

  9. Lignin reduction in transgenic poplars by expressing antisense CCoAOMT gene

    Institute of Scientific and Technical Information of China (English)

    LU Jing; ZHAO Huayan; WEI Jianhua; HE Yikun; SHI Chao; WANG Hongzhi; SONG Yanru

    2004-01-01

    The antisense Caffeoyl CoA O-methyltransferase (CCoAOMT) cDNA was transformed into Chinese white poplar (Populus tomentosa) mediated by Agrobacterium tumefaciens. Many factors affecting the transformation efficiency were studied and a stable transformation system was established. PCR-Southern blot analysis indicated that antisense CCoAOMT cDNA had been integrated into the genome of the transgenic poplars. RT-PCR and Western blot analyses demonstrated that the endogenous CCoAOMT gene was suppressed at both transcriptional and translational levels. Klason lignin content assay exhibited the lignin reduction to different degrees in transgenic poplars. The stems of partial transgenic poplars with the remarkable lignin reduction turned red, and the color distribution was stripped or spotted. Taken together, these results suggested that CCoAOMT gene would be a potential useful gene in altering lignin biosynthesis by biotechnology for improving wood properties.

  10. Cellular delivery and antisense effects of peptide nucleic acid conjugated to polyethyleneimine via disulfide linkers

    DEFF Research Database (Denmark)

    Berthold, Peter R; Shiraishi, Takehiko; Nielsen, Peter E

    2010-01-01

    Peptide nucleic acid (PNA) is potentially an attractive antisense and antigene agent for which more efficient cellular delivery systems are still warranted. The cationic polymer polyethylenimine (PEI) is commonly used for cellular transfection of DNA and RNA complexes, but is not readily applicable...... for PNA due to the (inherent) charge neutrality of PNA. However, PEI could function as an efficient scaffold for PNA via chemical conjugation. Accordingly, we modified PEI with the amine-reactive heterobifunctional linker agent N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) (with and without a PEG...... moiety) and further reacted this with a cysteine PNA. The level of modification was determined spectrophotometrically with high accuracy, and the PNA transfection efficiency of the conjugates was evaluated in an antisense luciferase splice-correction assay using HeLa pLuc705 cells. We find that PEI...

  11. Purification of noncoding RNA and bound proteins using FLAG peptide-conjugated antisense-oligonucleotides.

    Science.gov (United States)

    Adachi, Shungo; Natsume, Tohru

    2015-01-01

    To understand the function of certain RNAs, including noncoding RNAs, it is important to identify the proteins that interact with the RNAs. Here we describe the method for purification of ribonucleoprotein (RNP) complexes composed of specific cellular RNAs by pull-down with FLAG peptide-conjugated antisense oligonucleotide (ASO). Using this method, we identified a novel protein component of U7 snRNP complex.

  12. Over 20% of human transcripts might form sense–antisense pairs

    OpenAIRE

    Chen, Jianjun; Sun, Miao; Kent, W. James; Huang, Xiaoqiu; Xie, Hanqing; Wang, Wenquan; Zhou, Guolin; Shi, Run Zhang; Rowley, Janet D.

    2004-01-01

    The major challenge to identifying natural sense– antisense (SA) transcripts from public databases is how to determine the correct orientation for an expressed sequence, especially an expressed sequence tag sequence. In this study, we established a set of very stringent criteria to identify the correct orientation of each human transcript. We used these orientation-reliable transcripts to create 26 741 transcription clusters in the human genome. Our analysis shows that 22% (5880) of the human...

  13. Antisense Oligonucleotide-Mediated Exon Skipping for Duchenne Muscular Dystrophy: Progress and Challenges.

    OpenAIRE

    Arechavala-Gomeza, V.; Anthony, K.; Morgan, J; Muntoni, F.

    2012-01-01

    Duchenne muscular dystrophy (DMD) is the most common childhood neuromuscular disorder. It is caused by mutations in the DMD gene that disrupt the open reading frame (ORF) preventing the production of functional dystrophin protein. The loss of dystrophin ultimately leads to the degeneration of muscle fibres, progressive weakness and premature death. Antisense oligonucleotides (AOs) targeted to splicing elements within DMD pre-mRNA can induce the skipping of targeted exons, restoring the ORF an...

  14. Antisense Mediated Splicing Modulation For Inherited Metabolic Diseases: Challenges for Delivery

    OpenAIRE

    Pérez, Belen; Vilageliu, Lluisa; Grinberg, Daniel; Desviat, Lourdes R.

    2014-01-01

    In the past few years, research in targeted mutation therapies has experienced significant advances, especially in the field of rare diseases. In particular, the efficacy of antisense therapy for suppression of normal, pathogenic, or cryptic splice sites has been demonstrated in cellular and animal models and has already reached the clinical trials phase for Duchenne muscular dystrophy. In different inherited metabolic diseases, splice switching oligonucleotides (SSOs) have been used with suc...

  15. Directional gene expression and antisense transcripts in sexual and asexual stages of Plasmodium falciparum

    OpenAIRE

    López-Barragán María J; Lemieux Jacob; Quiñones Mariam; Williamson Kim C; Molina-Cruz Alvaro; Cui Kairong; Barillas-Mury Carolina; Zhao Keji; Su Xin-zhuan

    2011-01-01

    Abstract Background It has been shown that nearly a quarter of the initial predicted gene models in the Plasmodium falciparum genome contain errors. Although there have been efforts to obtain complete cDNA sequences to correct the errors, the coverage of cDNA sequences on the predicted genes is still incomplete, and many gene models for those expressed in sexual or mosquito stages have not been validated. Antisense transcripts have widely been reported in P. falciparum; however, the extent an...

  16. Antiviral Effects of Antisense Morpholino Oligomers in Murine Coronavirus Infection Models▿

    OpenAIRE

    Burrer, Renaud; Neuman, Benjamin W.; Ting, Joey P.C.; Stein, David A.; Moulton, Hong M.; Iversen, Patrick L.; Kuhn, Peter; Michael J Buchmeier

    2007-01-01

    The recent emergence of novel pathogenic human and animal coronaviruses has highlighted the need for antiviral therapies that are effective against a spectrum of these viruses. We have used several strains of murine hepatitis virus (MHV) in cell culture and in vivo in mouse models to investigate the antiviral characteristics of peptide-conjugated antisense phosphorodiamidate morpholino oligomers (P-PMOs). Ten P-PMOs directed against various target sites in the viral genome were tested in cell...

  17. Delivering Antisense Morpholino Oligonucleotides to Target Telomerase Splice Variants in Human Embryonic Stem Cells.

    Science.gov (United States)

    Radan, Lida; Hughes, Chris S; Teichroeb, Jonathan H; Postovit, Lynne-Marie; Betts, Dean H

    2016-01-01

    Morpholino oligonucleotides (MO) are an innovative tool that provides a means for examining and modifying gene expression outcomes by antisense interaction with targeted RNA transcripts. The site-specific nature of their binding facilitates focused modulation to alter splice variant expression patterns. Here we describe the steric-blocking of human telomerase reverse transcriptase (hTERT) Δα and Δβ splice variants using MO to examine cellular outcomes related to pluripotency and differentiation in human embryonic stem cells.

  18. Pseudomonas exotoxin antisense RNA selectively kills hepatitis B virus infected cells

    Institute of Scientific and Technical Information of China (English)

    Peter Hafkemeyer; Ulrich Brinkmann; Elizabeth Brinkmann; Ira Pastan; Hubert E Blum; Thomas F Baumert

    2008-01-01

    AIM: To present an approach for selectively killing retrovirus-infected cells that combines the toxicity of Pseudomonas exotoxin (PE) and the presence of reverse transcriptase (RT) in infected cells. METHODS: PE antisense toxin RNA has palindromic stem loops at its 5' and 3' ends enabling self-primed generation of cDNA in the presence of RT. The RT activity expressed in retrovirus-infected cells converts "antisense-toxin-RNA" into a lethal toxin gene exclusively in these cells. RESULTS: Using cotransfection studies with Peexpressing RNAs and β-gal expressing reporter plasmids, we show that, in HepG2 and HepG2. 2. 15 hepatomacells as well as in duck hepatitis B virus (DHBV) infected cells, HBV or DHBV-polymerase reverse transcribe a lethal cDNA copy of an antisense toxin RNA, which is composed of sequences complementary to a PE gene and eukaryotic transcription and translation signals. CONCLUSION: This finding may have important implications as a novel therapeutic strategy aimed at the elimination of HBV infection.

  19. Targeted skipping of human dystrophin exons in transgenic mouse model systemically for antisense drug development.

    Directory of Open Access Journals (Sweden)

    Bo Wu

    Full Text Available Antisense therapy has recently been demonstrated with great potential for targeted exon skipping and restoration of dystrophin production in cultured muscle cells and in muscles of Duchenne Muscular Dystrophy (DMD patients. Therapeutic values of exon skipping critically depend on efficacy of the drugs, antisense oligomers (AOs. However, no animal model has been established to test AO targeting human dystrophin exon in vivo systemically. In this study, we applied Vivo-Morpholino to the hDMD mouse, a transgenic model carrying the full-length human dystrophin gene, and achieved for the first time more than 70% efficiency of targeted human dystrophin exon skipping in vivo systemically. We also established a GFP-reporter myoblast culture to screen AOs targeting human dystrophin exon 50. Antisense efficiency for most AOs is consistent between the reporter cells, human myoblasts and in the hDMD mice in vivo. However, variation in efficiency was also clearly observed. A combination of in vitro cell culture and a Vivo-Morpholino based evaluation in vivo systemically in the hDMD mice therefore may represent a prudent approach for selecting AO drug and to meet the regulatory requirement.

  20. Cotton transgenics with Antisense AC1 gene for resistance against cotton leaf curl virus

    Directory of Open Access Journals (Sweden)

    J.Amudha, G.Balasubramani, V.G.Malathi, D.Monga, K.C.Bansal and K.R.Kranthi

    2010-07-01

    Full Text Available Cotton leaf curl virus is a devastating pest in the North India and in small pockets of Southern states. Cotton leaf curldisease (CLCuD is caused by a Geminivirus, transmitted by whitefly Bemisia tabaci vector. This is a serious problem inthe northern region and leads to yield losses up to 58% and 69% (ICAC recorder, 1999. Genetic engineering for cottontransgenics resistant to leaf curl disease (CLCuD through antisense RNA approach is potential to tackle the disease incotton. Cotton transgenics resistant to leaf curl disease (CLCuD using Antisense (rep (Replicase protein gene wasdeveloped via Agrobacterium mediated transformation. A binary vector carrying the Antisense rep gene along with thenpt II (neomycin phospho transferase gene driven by CaMV-35S promoter and NOS (nopaline synthase terminator wasused for transformation. The confirmation of the rep and npt II genes in the transgenic plants were verified by PCR andintegration of T-DNA into the plant genome was confirmed by Southern analysis. The individual transgenics were raisedin the green house and screened for the virus resistance. T2 progeny analysis showed classical Mendelian pattern ofinheritance.

  1. PCSK9 LNA antisense oligonucleotides induce sustained reduction of LDL cholesterol in nonhuman primates.

    Science.gov (United States)

    Lindholm, Marie W; Elmén, Joacim; Fisker, Niels; Hansen, Henrik F; Persson, Robert; Møller, Marianne R; Rosenbohm, Christoph; Ørum, Henrik; Straarup, Ellen M; Koch, Troels

    2012-02-01

    Proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged as a therapeutic target for the reduction of low-density lipoprotein cholesterol (LDL-C). PCSK9 increases the degradation of the LDL receptor, resulting in high LDL-C in individuals with high PCSK9 activity. Here, we show that two locked nucleic acid (LNA) antisense oligonucleotides targeting PCSK9 produce sustained reduction of LDL-C in nonhuman primates after a loading dose (20 mg/kg) and four weekly maintenance doses (5 mg/kg). PCSK9 messenger RNA (mRNA) and serum PCSK9 protein were reduced by 85% which resulted in a 50% reduction in circulating LDL-C. Serum total cholesterol (TC) levels were reduced to the same extent as LDL-C with no reduction in high-density lipoprotein levels, demonstrating a specific pharmacological effect on LDL-C. The reduction in hepatic PCSK9 mRNA correlated with liver LNA oligonucleotide content. This verified that anti-PCSK9 LNA oligonucleotides regulated LDL-C through an antisense mechanism. The compounds were well tolerated with no observed effects on toxicological parameters (liver and kidney histology, alanine aminotransferase, aspartate aminotransferase, urea, and creatinine). The pharmacologic evidence and initial safety profile of the compounds used in this study indicate that LNA antisense oligonucleotides targeting PCSK9 provide a viable therapeutic strategy and are potential complements to statins in managing high LDL-C.

  2. Polymerase-endonuclease amplification reaction (PEAR for large-scale enzymatic production of antisense oligonucleotides.

    Directory of Open Access Journals (Sweden)

    Xiaolong Wang

    Full Text Available Antisense oligonucleotides targeting microRNAs or their mRNA targets prove to be powerful tools for molecular biology research and may eventually emerge as new therapeutic agents. Synthetic oligonucleotides are often contaminated with highly homologous failure sequences. Synthesis of a certain oligonucleotide is difficult to scale up because it requires expensive equipment, hazardous chemicals and a tedious purification process. Here we report a novel thermocyclic reaction, polymerase-endonuclease amplification reaction (PEAR, for the amplification of oligonucleotides. A target oligonucleotide and a tandem repeated antisense probe are subjected to repeated cycles of denaturing, annealing, elongation and cleaving, in which thermostable DNA polymerase elongation and strand slipping generate duplex tandem repeats, and thermostable endonuclease (PspGI cleavage releases monomeric duplex oligonucleotides. Each round of PEAR achieves over 100-fold amplification. The product can be used in one more round of PEAR directly, and the process can be further repeated. In addition to avoiding dangerous materials and improved product purity, this reaction is easy to scale up and amenable to full automation. PEAR has the potential to be a useful tool for large-scale production of antisense oligonucleotide drugs.

  3. Re-sensitizing drug-resistant bacteria to antibiotics by designing Antisense Therapeutics

    Science.gov (United States)

    Courtney, Colleen; Chatterjee, Anushree

    2014-03-01

    ``Super-bugs'' or ``multi-drug resistant organisms'' are a serious international health problem, with devastating consequences to patient health care. The Center for Disease Control has identified antibiotic resistance as one of the world's most pressing public health problems as a significant fraction of bacterial infections contracted are drug resistant. Typically, antibiotic resistance is encoded by ``resistance-genes'' which express proteins that carryout the resistance causing functions inside the bacterium. We present a RNA based therapeutic strategy for designing antimicrobials capable of re-sensitizing resistant bacteria to antibiotics by targeting labile regions of messenger RNAs encoding for resistance-causing proteins. We perform in silico RNA secondary structure modeling to identify labile target regions in an mRNA of interest. A synthetic biology approach is then used to administer antisense nucleic acids to our model system of ampicillin resistant Escherichia coli. Our results show a prolonged lag phase and decrease in viability of drug-resistant E. colitreated with antisense molecules. The antisense strategy can be applied to alter expression of other genes in antibiotic resistance pathways or other pathways of interest.

  4. A vector library for silencing central carbon metabolism genes with antisense RNAs in Escherichia coli.

    Science.gov (United States)

    Nakashima, Nobutaka; Ohno, Satoshi; Yoshikawa, Katsunori; Shimizu, Hiroshi; Tamura, Tomohiro

    2014-01-01

    We describe here the construction of a series of 71 vectors to silence central carbon metabolism genes in Escherichia coli. The vectors inducibly express antisense RNAs called paired-terminus antisense RNAs, which have a higher silencing efficacy than ordinary antisense RNAs. By measuring mRNA amounts, measuring activities of target proteins, or observing specific phenotypes, it was confirmed that all the vectors were able to silence the expression of target genes efficiently. Using this vector set, each of the central carbon metabolism genes was silenced individually, and the accumulation of metabolites was investigated. We were able to obtain accurate information on ways to increase the production of pyruvate, an industrially valuable compound, from the silencing results. Furthermore, the experimental results of pyruvate accumulation were compared to in silico predictions, and both sets of results were consistent. Compared to the gene disruption approach, the silencing approach has an advantage in that any E. coli strain can be used and multiple gene silencing is easily possible in any combination.

  5. Antisense repression of sucrose phosphate synthase in transgenic muskmelon alters plant growth and fruit development

    International Nuclear Information System (INIS)

    To unravel the roles of sucrose phosphate synthase (SPS) in muskmelon (Cucumis melo L.), we reduced its activity in transgenic muskmelon plants by an antisense approach. For this purpose, an 830 bp cDNA fragment of muskmelon sucrose phosphate synthase was expressed in antisense orientation behind the 35S promoter of the cauliflower mosaic virus. The phenotype of the antisense plants clearly differed from that of control plants. The transgenic plant leaves were markedly smaller, and the plant height and stem diameter were obviously shorter and thinner. Transmission electron microscope observation revealed that the membrane degradation of chloroplast happened in transgenic leaves and the numbers of grana and grana lamella in the chloroplast were significantly less, suggesting that the slow growth and weaker phenotype of transgenic plants may be due to the damage of the chloroplast ultrastructure, which in turn results in the decrease of the net photosynthetic rate. The sucrose concentration and levels of sucrose phosphate synthase decreased in transgenic mature fruit, and the fruit size was smaller than the control fruit. Together, our results suggest that sucrose phosphate synthase may play an important role in regulating the muskmelon plant growth and fruit development.

  6. A multi-target antisense approach against PDE4 and PDE7 reduces smoke-induced lung inflammation in mice

    Directory of Open Access Journals (Sweden)

    Séguin Rosanne

    2009-05-01

    Full Text Available Abstract Background Recent development in the field of COPD has focused on strategies aimed at reducing the underlying inflammation through selective inhibition of the phosphodiesterase type IV (PDE4 isoform. Although the anti-inflammatory and bronchodilator activity of selective PDE4 inhibitors has been well documented, their low therapeutic ratio and dose-dependent systemic side effects have limited their clinical utility. This study examined the effect of 2'-deoxy-2'-Fluoro-β-D-Arabinonucleic Acid (FANA-containing antisense oligonucleotides (AON targeting the mRNA for the PDE4B/4D and 7A subtypes on lung inflammatory markers, both in vitro and in vivo. Methods Normal human bronchial epithelial (NHBE cells were transfected with FANA AON against PDE4B/4D and 7A alone or in combination. mRNA levels for target PDE subtypes, as well as secretion of pro-inflammatory chemokines were then measured following cell stimulation. Mice were treated with combined PDE4B/4D and 7A AON via endo-tracheal delivery, or with roflumilast via oral delivery, and exposed to cigarette smoke for one week. Target mRNA inhibition, as well as influx of inflammatory cells and mediators were measured in lung lavages. A two-week smoke exposure protocol was also used to test the longer term potency of PDE4B/4D and 7A AONs. Results In NHBE cells, PDE4B/4D and 7A AONs dose-dependently and specifically inhibited expression of their respective target mRNA. When used in combination, PDE4B/4D and 7A AONs significantly abrogated the cytokine-induced secretion of IL-8 and MCP-1 to near baseline levels. In mice treated with combined PDE4B/4D and 7A AONs and exposed to cigarette smoke, significant protection against the smoke-induced recruitment of neutrophils and production of KC and pro-MMP-9 was obtained, which was correlated with inhibition of target mRNA in cells from lung lavages. In this model, PDE AONs exerted more potent and broader anti-inflammatory effects against smoke

  7. 冬凌草甲素和survivin反义核苷酸对前列腺癌细胞作用的研究%Effects of survivin antisense oligodeoxynecleotides and Oridonin on PC-3 cells

    Institute of Scientific and Technical Information of China (English)

    李进; 杨罗艳; 吴洪涛

    2014-01-01

    Objective To explore the synergistic effects of survivin antisense oligonucleotides combined with Oridonin on growth, apoptosis, and the expression of survivin of PC-3 cells. Methods Human prostate carcinoma cells PC-3 on logarithmic growth phase were used in this study. The cell vitality was determined by MTT assay. The combination index (CI) was calculated using Pharmaconamics CalcuSynsoftware. The apoptotic rate was examined by flow cytometer (FCM). The expression of survivin was detected by Western Blot and Real-time Fluorescent Quantitation-PCR. Results After transfection with antisense Survivin RNAi, the proliferation of PC-3 cells was inhibited markedly. An obvious apoptosis was found in the transfected PC-3 cells. The inhibitory effect of combined administration of survivin antisense and Oridonin on cell proliferation was much stronger than that of the single way (P<0.01). It showed that there was a synergistic effect (Fa<0.80). Western Blot and RT-PCR assays demonstrated that survivin antisense and Oridonin all inhibited the expression of survivin(P <0.01). Conclusion Combined survivin antisense and Oridonin significantly inhibits cell proliferation, induces cell apoptosis and down-regulates survivin expression in PC-3 cells, indicating that survivin antisense and Oridonin have a synergistic effect on PC-3 cells.%目的:探讨冬凌草甲素联合survivin反义核苷酸(反义链)对前列腺癌PC-3细胞株增殖和凋亡以及survivin mRNA和蛋白的影响。方法常规培养PC-3细胞,用四甲基偶氮唑盐法(MTT法)检测survivin反义链联合冬凌草甲素对PC-3细胞增殖的影响;流式细胞仪(FCM)检测PC-3细胞凋亡率;以CalcuSyn药效学软件计算联合指数(CI)评价survivin反义链联合凌草甲素对PC-3细胞的联合效应,并通过荧光定量PCR和Western blot方法检测PC-3细胞survivin基因和蛋白表达变化。结果 survivin反义链转染PC-3细胞后,可以显著抑制PC-3

  8. Identification of novel endogenous antisense transcripts by DNA microarray analysis targeting complementary strand of annotated genes

    Directory of Open Access Journals (Sweden)

    Kohama Chihiro

    2009-08-01

    Full Text Available Abstract Background Recent transcriptomic analyses in mammals have uncovered the widespread occurrence of endogenous antisense transcripts, termed natural antisense transcripts (NATs. NATs are transcribed from the opposite strand of the gene locus and are thought to control sense gene expression, but the mechanism of such regulation is as yet unknown. Although several thousand potential sense-antisense pairs have been identified in mammals, examples of functionally characterized NATs remain limited. To identify NAT candidates suitable for further functional analyses, we performed DNA microarray-based NAT screening using mouse adult normal tissues and mammary tumors to target not only the sense orientation but also the complementary strand of the annotated genes. Results First, we designed microarray probes to target the complementary strand of genes for which an antisense counterpart had been identified only in human public cDNA sources, but not in the mouse. We observed a prominent expression signal from 66.1% of 635 target genes, and 58 genes of these showed tissue-specific expression. Expression analyses of selected examples (Acaa1b and Aard confirmed their dynamic transcription in vivo. Although interspecies conservation of NAT expression was previously investigated by the presence of cDNA sources in both species, our results suggest that there are more examples of human-mouse conserved NATs that could not be identified by cDNA sources. We also designed probes to target the complementary strand of well-characterized genes, including oncogenes, and compared the expression of these genes between mammary cancerous tissues and non-pathological tissues. We found that antisense expression of 95 genes of 404 well-annotated genes was markedly altered in tumor tissue compared with that in normal tissue and that 19 of these genes also exhibited changes in sense gene expression. These results highlight the importance of NAT expression in the regulation

  9. Defining global gene expression changes of the hypothalamic-pituitary-gonadal axis in female sGnRH-antisense transgenic common carp (Cyprinus carpio.

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    Jing Xu

    Full Text Available BACKGROUND: The hypothalamic-pituitary-gonadal (HPG axis is critical in the development and regulation of reproduction in fish. The inhibition of neuropeptide gonadotropin-releasing hormone (GnRH expression may diminish or severely hamper gonadal development due to it being the key regulator of the axis, and then provide a model for the comprehensive study of the expression patterns of genes with respect to the fish reproductive system. METHODOLOGY/PRINCIPAL FINDINGS: In a previous study we injected 342 fertilized eggs from the common carp (Cyprinus carpio with a gene construct that expressed antisense sGnRH. Four years later, we found a total of 38 transgenic fish with abnormal or missing gonads. From this group we selected the 12 sterile females with abnormal ovaries in which we combined suppression subtractive hybridization (SSH and cDNA microarray analysis to define changes in gene expression of the HPG axis in the present study. As a result, nine, 28, and 212 genes were separately identified as being differentially expressed in hypothalamus, pituitary, and ovary, of which 87 genes were novel. The number of down- and up-regulated genes was five and four (hypothalamus, 16 and 12 (pituitary, 119 and 93 (ovary, respectively. Functional analyses showed that these genes involved in several biological processes, such as biosynthesis, organogenesis, metabolism pathways, immune systems, transport links, and apoptosis. Within these categories, significant genes for neuropeptides, gonadotropins, metabolic, oogenesis and inflammatory factors were identified. CONCLUSIONS/SIGNIFICANCE: This study indicated the progressive scaling-up effect of hypothalamic sGnRH antisense on the pituitary and ovary receptors of female carp and provided comprehensive data with respect to global changes in gene expression throughout the HPG signaling pathway, contributing towards improving our understanding of the molecular mechanisms and regulative pathways in the

  10. Antisense PMO found in dystrophic dog model was effective in cells from exon 7-deleted DMD patient.

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    Takashi Saito

    Full Text Available BACKGROUND: Antisense oligonucleotide-induced exon skipping is a promising approach for treatment of Duchenne muscular dystrophy (DMD. We have systemically administered an antisense phosphorodiamidate morpholino oligomer (PMO targeting dystrophin exons 6 and 8 to a dog with canine X-linked muscular dystrophy in Japan (CXMD(J lacking exon 7 and achieved recovery of dystrophin in skeletal muscle. To date, however, antisense chemical compounds used in DMD animal models have not been directly applied to a DMD patient having the same type of exon deletion. We recently identified a DMD patient with an exon 7 deletion and tried direct translation of the antisense PMO used in dog models to the DMD patient's cells. METHODOLOGY/PRINCIPAL FINDINGS: We converted fibroblasts of CXMD(J and the DMD patient to myotubes by FACS-aided MyoD transduction. Antisense PMOs targeting identical regions of dog and human dystrophin exons 6 and 8 were designed. These antisense PMOs were mixed and administered as a cocktail to either dog or human cells in vitro. In the CXMD(J and human DMD cells, we observed a similar efficacy of skipping of exons 6 and 8 and a similar extent of dystrophin protein recovery. The accompanying skipping of exon 9, which did not alter the reading frame, was different between cells of these two species. CONCLUSION/SIGNIFICANCE: Antisense PMOs, the effectiveness of which has been demonstrated in a dog model, achieved multi-exon skipping of dystrophin gene on the FACS-aided MyoD-transduced fibroblasts from an exon 7-deleted DMD patient, suggesting the feasibility of systemic multi-exon skipping in humans.

  11. Experimental research for specific down-regulated expression of p53 gene by individual antisense RNA in vitro%个体性反义RNA特异性封闭突变p53基因的实验研究

    Institute of Scientific and Technical Information of China (English)

    Yahong Wang; Shaofeng Xu; Yuanyuan Zhang; Bin Zhang; Yumei Feng; Ruifang Niu; Li Fu

    2007-01-01

    Objective: To investigate the specific blockage effect of individual antisense RNA on mutant p53 gene in vitro.Methods: The single strand antisense transcription system containing mt-p53 exon 8 sequence (pGEM3zf(+/-)p53exon8)was constructed. The ligation of antisense RNA with mt-p53 gene was confirmed by in situ hybridization; MDA-MB-231 human breast cancer cells were transfected with ASp53exon8'RNA eationic liposome-mediated. Expression of mt-p53 protein was examined by immunocytochemical staining and Western blot. Cell proliferation was evaluated by MTT assay; Cell cycle distribution was determined by flow cytometry (FCM); Apoptosis was observed by TUNEL. Results: In transfected MDA-MB-231cells, hybridization signals were observed in cytoplasm. ASp53exon8'RNA transfection induced inhibition of cell proliferation,G2/M phase arrest and increasing apoptotic rates. In addition, expression of p53 protein was down-regulated. Conclusion:pGEM3zf(+/-)p53exon8 was well constructed and ASp53exon8'RNA can block mt-p53 gene expression specifically and then inhibit MDA-MB-231 cell proliferation in vitro, which may serve as therapeutic means for human malignancy.

  12. Emulsified nanoparticles containing inactivated influenza virus and CpG oligodeoxynucleotides critically influences the host immune responses in mice.

    Directory of Open Access Journals (Sweden)

    Ming-Hsi Huang

    Full Text Available BACKGROUND: Antigen sparing and cross-protective immunity are regarded as crucial in pandemic influenza vaccine development. Both targets can be achieved by adjuvantation strategy to elicit a robust and broadened immune response. We assessed the immunogenicity of an inactivated H5N1 whole-virion vaccine (A/Vietnam/1194/2004 NIBRG-14, clade 1 formulated with emulsified nanoparticles and investigated whether it can induce cross-clade protecting immunity. METHODOLOGY/PRINCIPAL FINDINGS: After formulation with PELC, a proprietary water-in-oil-in-water nanoemulsion comprising of bioresorbable polymer/Span(R85/squalene, inactivated virus was intramuscularly administered to mice in either one-dose or two-dose schedule. We found that the antigen-specific serum antibody responses elicited after two doses of non-adjuvanted vaccine were lower than those observed after a single dose of adjuvanted vaccine, PELC and the conventional alum adjuvant as well. Moreover, 5 microg HA of PELC-formulated inactivated virus were capable of inducing higher antibodies than those obtained from alum-adjuvanted vaccine. In single-dose study, we found that encapsulating inactivated virus into emulsified PELC nanoparticles could induce better antibody responses than those formulated with PELC-adsorbed vaccine. However, the potency was rather reduced when the inactivated virus and CpG (an immunostimulatory oligodeoxynucleotide containing unmethylated cytosine-guanosine motifs were co-encapsulated within the emulsion. Finally, the mice who received PELC/CpG(adsorption-vaccine could easily and quickly reach 100% of seroprotection against a homologous virus strain and effective cross-protection against a heterologous virus strain (A/Whooper swan/Mongolia/244/2005, clade 2.2. CONCLUSIONS/SIGNIFICANCE: Encapsulating inactivated H5N1 influenza virus and CpG into emulsified nanoparticles critically influences the humoral responses against pandemic influenza. These results demonstrated

  13. Identification and characterization of a cis-encoded antisense RNA associated with the replication process of Salmonella enterica serovar Typhi.

    Directory of Open Access Journals (Sweden)

    Isaac Dadzie

    Full Text Available Antisense RNAs that originate from the complementary strand of protein coding genes are involved in the regulation of gene expression in all domains of life. In bacteria, some of these antisense RNAs are transcriptional noise while others play a vital role to adapt the cell to changing environmental conditions. By deep sequencing analysis of transcriptome of Salmonella enterica serovar Typhi, a partial RNA sequence encoded in-cis to the dnaA gene was revealed. Northern blot and RACE analysis confirmed the transcription of this antisense RNA which was expressed mostly in the stationary phase of the bacterial growth and also under iron limitation and osmotic stress. Pulse expression analysis showed that overexpression of the antisense RNA resulted in a significant increase in the mRNA levels of dnaA, which will ultimately enhance their translation. Our findings have revealed that antisense RNA of dnaA is indeed transcribed not merely as a by-product of the cell's transcription machinery but plays a vital role as far as stability of dnaA mRNA is concerned.

  14. Preparation of liposomes/HIF-1α ASODN complexes and its inhibiting effect on HIF-1αexpression in bovine retinal endothelial cell%HIF-1α反义寡核苷酸脂质体的制备和对视网膜血管内皮细胞HIF-1α表达的影响

    Institute of Scientific and Technical Information of China (English)

    邓爱军; 姜德咏

    2005-01-01

    [Objective] To study inhibition of HIF-1α expression in bovine retinal endothelial cell(BREC) by HIF-1α fully phosporothioated antisense oligodeoxynucleotide (HIF-1α ASODN). [Methods] BRECs were incubated with HIF-1α ASODN and were cultured in COCl2-induced hypoxic condition. HIF-1α expression was detected with immunohistochemistry. [Results] Phosphorothioate ODNs labeled with FAM was transfected into BRECs by Lipofectin. Six hours later, many green fluorescence spots were observed in the cell with the fluromicroscope, and the transfective effect was (93.8 ± 3.4)%. In no-ASODN group, the expression of HIF-1α was very low at 0 h. But it began to increase significantly at 1 h after hypoxia, and reached the peak at 4 h, 16 h later, it declined. While in ASODN group, the expression of HIF-1α was always at very low level. The two groups had significant difference (P <0.01). [ Conclusions ] HIF-1α antisense oligonucleotides can suppress HIF-1α expression of bovine retinal endothelial cell effectively. It has good prospect in resisting retinal angiogenesis treatment.%目的应用HIF-1αASODN处理牛眼视网膜微血管内皮细胞,观察细胞摄取ASODN的情况和对缺氧时细胞HIF-1α表达的影响.方法对分离的牛视网膜血管内皮细胞进行HIF-1α反义寡核苷酸的转染,CoCl2模拟缺氧培养,采用免疫组化检测不同缺氧时间HIF-1α的表达.结果硫代化修饰的标有FAM的HIF-1α反义寡核苷酸能被Lipofectin转染进BREC细胞.转染6 h后,在荧光显微镜下可观察到细胞内的绿色荧光斑.计算细胞转染率为(93.8±3.4)%.未转染组开始时HIF-1α有极低表达,在缺氧1 h时表达量显著上升,4 h达到高峰,16h后下降.而ASODN转染组HIF-1α的表达始终在极低水平,两组间有显著性差异(P<0.01).结论以脂质体为载体能高效率地将HIF-1αASODN转染至视网膜血管内皮细胞内,明显抑制HIF-1α蛋白质的表达,这可能为HIF-1αASODN用于视网膜新生血管的治疗提供理论依据.

  15. Release profile and stability evaluation of optimized chitosan/alginate nanoparticles as EGFR antisense vector

    Directory of Open Access Journals (Sweden)

    Ebrahim Azizi

    2010-06-01

    Full Text Available Ebrahim Azizi1,4, Alireza Namazi1, Ismaeil Haririan2,5, Shamileh Fouladdel1, Mohammad R Khoshayand3, Parisa Y Shotorbani6, Alireza Nomani1,7, Taraneh Gazori1,21Molecular Research Lab, Department of Pharmacology and Toxicology, 2Department of Pharmaceutics, 3Department of Food and Drug Control, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran; 4Department of Medical Biotechnology, School of Advanced Medical Technologies, Tehran University of Medical Sciences, Tehran, Iran; 5Biomaterials Research Center (BRC Tehran, Iran; 6Pharmaceutical Sciences Branch, Azad University, Tehran, Iran; 7Department of Pharmaceutics, Faculty of Pharmacy, Zanjan University of Medical Sciences, Zanjan, IranAbstract: Chitosan/alginate nanoparticles which had been optimized in our previous study using two different N/P ratios were chosen and their ability to release epidermal growth factor receptor (EGFR antisense was investigated. In addition, the stability of these nanoparticles in aqueous medium and after freeze-drying was investigated. In the case of both N/P ratios (5, 25, nanoparticles started releasing EGFR antisense as soon as they were exposed to the medium and the release lasted for approximately 50 hours. Nanoparticle size, shape, zeta potential, and release profile did not show any significant change after the freeze-drying process (followed by reswelling. The nanoparticles were reswellable again after freeze-drying in phosphate buffer with a pH of 7.4 over a period of six hours. Agarose gel electrophoresis of the nanoparticles with the two different N/P ratios showed that these nanoparticles could protect EGFR antisense molecules for six hours.Keywords: chitosan/alginate nanoparticles, release profile, freeze-drying, agarose gel electrophoresis

  16. Antisense-mediated RNA targeting: versatile and expedient genetic manipulation in the brain

    Directory of Open Access Journals (Sweden)

    Ioannis eZalachoras

    2011-07-01

    Full Text Available A limiting factor in brain research still is the difficulty to evaluate in vivo the role of the increasing number of proteins implicated in neuronal processes. We discuss here the potential of antisense-mediated RNA targeting approaches. We mainly focus on those that manipulate splicing (exon skipping and exon inclusion, but will also briefly discuss mRNA targeting. Classic knockdown of expression by mRNA targeting is only one possible application of antisense oligonucleotides (AON in the control of gene function. Exon skipping and inclusion are based on the interference of AONs with splicing of pre-mRNAs. These are powerful, specific and particularly versatile techniques, which can be used to circumvent pathogenic mutations, shift splice variant expression, knock down proteins, or to create molecular models using in-frame deletions. Pre-mRNA targeting is currently used both as a research tool, e.g. in models for motor neuron disease, and in clinical trials for Duchenne muscular dystrophy and amyotrophic lateral sclerosis.AONs are particularly promising in relation to brain research, as the modified AONs are taken up extremely fast in neurons and glial cells with a long residence, and without the need for viral vectors or other delivery tools, once inside the blood brain barrier. In this review we cover 1. The principles of antisense-mediated techniques, chemistry and efficacy.2. The pros and cons of AON approaches in the brain compared to other techniques of interfering with gene function, such as transgenesis and short hairpin RNAs, in terms of specificity of the manipulation, spatial and temporal control over gene expression, toxicity and delivery issues.3. The potential applications for Neuroscience. We conclude that there is good evidence from animal studies that the CNS can be successfully targeted, but the potential of the diverse AON-based approaches appears to be under-recognized.

  17. Multi-exon Skipping Using Cocktail Antisense Oligonucleotides in the Canine X-linked Muscular Dystrophy.

    Science.gov (United States)

    Miskew Nichols, Bailey; Aoki, Yoshitsugu; Kuraoka, Mutsuki; Lee, Joshua J A; Takeda, Shin'ichi; Yokota, Toshifumi

    2016-01-01

    Duchenne muscular dystrophy (DMD) is one of the most common lethal genetic diseases worldwide, caused by mutations in the dystrophin (DMD) gene. Exon skipping employs short DNA/RNA-like molecules called antisense oligonucleotides (AONs) that restore the reading frame and produce shorter but functional proteins. However, exon skipping therapy faces two major hurdles: limited applicability (up to only 13% of patients can be treated with a single AON drug), and uncertain function of truncated proteins. These issues were addressed with a cocktail AON approach. While approximately 70% of DMD patients can be treated by single exon skipping (all exons combined), one could potentially treat more than 90% of DMD patients if multiple exon skipping using cocktail antisense drugs can be realized. The canine X-linked muscular dystrophy (CXMD) dog model, whose phenotype is more similar to human DMD patients, was used to test the systemic efficacy and safety of multi-exon skipping of exons 6 and 8. The CXMD dog model harbors a splice site mutation in intron 6, leading to a lack of exon 7 in dystrophin mRNA. To restore the reading frame in CXMD requires multi-exon skipping of exons 6 and 8; therefore, CXMD is a good middle-sized animal model for testing the efficacy and safety of multi-exon skipping. In the current study, a cocktail of antisense morpholinos targeting exon 6 and exon 8 was designed and it restored dystrophin expression in body-wide skeletal muscles. Methods for transfection/injection of cocktail oligos and evaluation of the efficacy and safety of multi-exon skipping in the CXMD dog model are presented. PMID:27285612

  18. Identification of an antisense transcript to ZIM2 in the primate lineage

    OpenAIRE

    Huang, Jennifer M.; Yu, Sungryul; Kim, Joomyeong

    2009-01-01

    In this study, we identified an antisense transcript to ZIM2 (zinc finger imprinted gene 2) in the human, called ZIM2as. Sequence analysis of the 110 kb region spanned by this transcript revealed a cluster of tandemly repeated sequence in the human, orangutan, and chimpanzee as well as a loss of approximately 70 kb from the corresponding region in the rhesus. The homologous region in most mammals contains a cluster of olfactory receptor (OLFR) genes, but this gene cluster has been lost from t...

  19. Effects of Cyclin D1 Antisense Oligodeoxyneucleotides on the Growth and Expression of G1 Phase Regulators in Gastric Carcinoma Cells

    Institute of Scientific and Technical Information of China (English)

    帅晓明; 韩高雄; 王国斌

    2003-01-01

    To investigate the effects of Cyclin D1 antisense oligodeoxyneucleotides (ASODN) on the growth, cell cycle progression and expression of G1 phase regulators in human gastric carcinoma cell lines SGC7901 and HS746T, phosphorothioate-modified Cyclin D1 ASODN were encapsulated by LipofectAMINE2000 and transfected into gastric carcinoma cells. Dose-dependent inhibitory effects were induced by Cyclin D1 ASODN in two gastric carcinoma cell lines. Treatment of gastric carcinoma cells with 0.2 μmol/L Cyclin D1 ASODN for 24 h could significantly inhibit their growth in vitro and in vivo, reduce expression of Cyclin DlmRNA to 26.3 % (SGC7901) and 17.3 %(HS746T) respectively. The percentage of cells in G0/G1 phase was increased as revealed by flow cytometry. Immunohistochemical staining showed that the expression of p21 was increased and the expression of Cyclin D1 and pRb was decreased in the two cell lines; the expression of p27 was increased in HS746T, but unchanged in SGC7901. Cyclin D1 ASODN could inhibit the growth and the expression of Cyclin D1 mRNA in gastric carcinoma cells, influence the cell cycle and expression of its regulators.

  20. Antisense EGFR sequence reverses the growth properties of human liver carcinoma cell line BEL-7404 in vitro

    Institute of Scientific and Technical Information of China (English)

    XUYONGHUA; WANLIJIANG; SUFENGPENG; YINGHUACHEN

    1993-01-01

    A recombinant plasmid containing a full length human epidermal growth factor receptor (EGFR) cDNA sequence in antisense orientation was transferred into cells of a human liver carcinoma cell line BEL-7404. Compared with the control cell clone JX-0 transferred with the vector plasmid and the parent BEL-7404 cells, the antisense EGFR transferred cell clone JX-1 showed a decreased EGFR gene expression and reduced significantly the growth potential either in anchorage-dependent or anchorage-independent growth. Furthermore. JX-1 cells appeared to be distinctly dependent on serum concentration for monolayer growth. The results suggested that antisense EGFR could partly block the EGFR gene ex-pression and reverse the malignant growth properties of human liver carcinoma cells in vitro.

  1. Characteristics of transgenic tomatoes antisensed for the ethylene receptor genes LeERT1 and LeERT2

    Institute of Scientific and Technical Information of China (English)

    WANG Zhong-feng; YING Tie-jin; ZHANG Ying; BAO Bi-li; HUANG Xiao-dan

    2006-01-01

    Two stable transformed lines containing antisense LeERT1 or LeERT2 sequences and their hybridized line were investigated to determine the effect of LeERT1 and LeERT2 specificity in the ethylene receptor family in tomato (Lycopersicon esculentum Mill.) on ethylene signaling. The transgenic line alel containing antisense LeERT1 displayed shorter length of seedling grown in the dark and adult plant in the light, severe epinastic petiole, and accelerated abscission of petiole explant and senescence of flower explant, compared with its wild type B1. The transgenic line ale2 containing antisense LeERT2 also exhibited shorter hypocotyls and slightly accelerated abscission. The phenotypes of cross line dale of LeERT1 and LeERT2 were close to alel in many aspects. These results suggested that LeERT1 probably plays a relatively important role in ethylene signaling of tomato growth and development.

  2. Lack of Interactions Between an Antisense Oligonucleotide with 2'-O-(2-Methoxyethyl) Modifications and Major Drug Transporters.

    Science.gov (United States)

    Yu, Rosie Z; Warren, Mark S; Watanabe, Tanya; Nichols, Brandon; Jahic, Mirza; Huang, Jane; Burkey, Jennifer; Geary, Richard S; Henry, Scott P; Wang, Yanfeng

    2016-04-01

    ISIS 141923 is a model compound of 2'-O-(2-methoxyethyl) (2'-MOE) modified antisense oligonucleotides (ASOs). The purpose of this study is to determine whether ISIS 141923 is a substrate or an inhibitor against a panel of nine major uptake or efflux drug transporters, namely breast cancer resistance protein (BCRP), P-glycoprotein (P-gp), organic anion transporter (OAT)1, OAT3, organic cation transporter (OCT)1, OCT2, organic anion transporting polypeptide 1B (OATP1B)1, OATP1B3, and bile salt export pump (BSEP), in vitro. The uptake test system for transporters in the solute carrier (SLC) family (OAT1, OAT3, OCT1, OCT2, OATP1B1, and OATP1B3) was studied in Madin-Darby canine kidney (MDCK)-II cells transfected to express the transporters of interest. BCRP was studied using carcinoma colon-2 (Caco-2) cells with endogenously expressed BCRP. P-gp transporter was studied in MDCK-multi-drug resistance 1 (MDR1) cells, while BSEP was studied using Spodoptera frugiperda 9 (Sf9) membrane vesicles containing human BSEP. The ISIS 141293 concentrations evaluated were 10 and 100 μM for the substrate and inhibition study, respectively. Cellular uptake of ISIS 141923 was analyzed using a high performance liquid chromatography-mass spectrometry method, while concentrations of known substrates (used as positive controls) of each transporters evaluated were determined by radiometric detection. At 10 μM ISIS 141923, there was no significant transporter-mediated uptake of ISIS 141923 (P > 0.05) in the SLC family, and the efflux ratios were not above 2.0 for either BCRP or P-gp. Therefore, no transporter-mediated uptake of ISIS 141923 was observed by any of the nine transporters studied. At 100 μM ISIS 141923, the % inhibition was in the range of -16.0% to 19.0% for the nine transporters evaluated. Therefore, ISIS 141923 is not considered as an inhibitor of the nine transporters studied. Overall, the results from this study suggest that it is unlikely that ISIS 141923 or similar 2

  3. Strategic down-regulation of DNA polymerase beta by antisense RNA sensitizes mammalian cells to specific DNA damaging agents.

    OpenAIRE

    Horton, J K; Srivastava, D K; Zmudzka, B Z; Wilson, S H

    1995-01-01

    Previously, mouse NIH 3T3 cells were stably transfected with human DNA polymerase beta (beta-pol) cDNA in the antisense orientation and under the control of a metallothionein promoter [Zmudzka, B.Z. and Wilson, S.H. (1990) Som. Cell Mol. Gen., 16, 311-320]. To assess the feasibility of enhancing the efficacy of chemotherapy by an antisense approach and to confirm a role for beta-pol in cellular DNA repair, we looked for increased sensitivity to DNA damaging agents under conditions where beta-...

  4. Peptide nucleic acid (PNA) cell penetrating peptide (CPP) conjugates as carriers for cellular delivery of antisense oligomers

    DEFF Research Database (Denmark)

    Shiraishi, Takehiko; Nielsen, Peter E

    2012-01-01

    splicing correction of the mutated luciferase gene in the HeLa pLuc705 cell line, reporting cellular (nuclear) uptake of the antisense PNA via luciferase activity measurement. Carrier CPP-PNA constructs were studied in terms of construct modification (with octaarginine and/or decanoic acid) and carrier PNA...... that the carrier might facilitate endosomal escape. Furthermore, 50% downregulation of luciferase expression at 60 nM siRNA was obtained using this carrier CPP-PNA delivery strategy (with CQ co-treatment) for a single stranded antisense RNA targeting normal luciferase mRNA. These results indicated that CPP...

  5. Antisense suppression of LOX3 gene expression in rice endosperm enhances seed longevity.

    Science.gov (United States)

    Xu, Huibin; Wei, Yidong; Zhu, Yongsheng; Lian, Ling; Xie, Hongguang; Cai, Qiuhua; Chen, Qiushi; Lin, Zhongping; Wang, Zonghua; Xie, Huaan; Zhang, Jianfu

    2015-05-01

    Lipid peroxidation plays a major role in seed longevity and viability. In rice grains, lipid peroxidation is catalyzed by the enzyme lipoxygenase 3 (LOX3). Previous reports showed that grain from the rice variety DawDam in which the LOX3 gene was deleted had less stale flavour after grain storage than normal rice. The molecular mechanism by which LOX3 expression is regulated during endosperm development remains unclear. In this study, we expressed a LOX3 antisense construct in transgenic rice (Oryza sativa L.) plants to down-regulate LOX3 expression in rice endosperm. The transgenic plants exhibited a marked decrease in LOX mRNA levels, normal phenotypes and a normal life cycle. We showed that LOX3 activity and its ability to produce 9-hydroperoxyoctadecadienoic acid (9-HPOD) from linoleic acid were significantly lower in transgenic seeds than in wild-type seeds by measuring the ultraviolet absorption of 9-HPOD at 234 nm and by high-performance liquid chromatography. The suppression of LOX3 expression in rice endosperm increased grain storability. The germination rate of TS-91 (antisense LOX3 transgenic line) was much higher than the WT (29% higher after artificial ageing for 21 days, and 40% higher after natural ageing for 12 months). To our knowledge, this is the first report to demonstrate that decreased LOX3 expression can preserve rice grain quality during storage with no impact on grain yield, suggesting potential applications in agricultural production. PMID:25545811

  6. Lipid-Albumin Nanoparticles (LAN) for Therapeutic Delivery of Antisense Oligonucleotide against HIF-1α.

    Science.gov (United States)

    Li, Hong; Quan, Jishan; Zhang, Mengzi; Yung, Bryant C; Cheng, Xinwei; Liu, Yang; Lee, Young B; Ahn, Chang-Ho; Kim, Deog Joong; Lee, Robert J

    2016-07-01

    Lipid-albumin nanoparticles (LAN) were synthesized for delivery of RX-0047, an antisense oligonucleotide (ASO) against the hypoxia inducible factor-1 alpha (HIF-1α) to solid tumor. These lipid nanoparticles (LNs) incorporated a human serum albumin-pentaethylenehexamine (HSA-PEHA) conjugate, which is cationic and can form electrostatic complexes with negatively charged oligonucleotides. The delivery efficiency of LAN-RX-0047 was investigated in KB cells and a KB murine xenograft model. When KB cells were treated with LAN-RX-0047, significant HIF-1α downregulation and enhanced cellular uptake were observed compared to LN-RX-0047. LN-RX-0047 and LAN-RX-0047 showed similar cytotoxicity against KB cells with IC50 values of 19.3 ± 3.8 and 20.1 ± 4.2 μM, respectively. LAN-RX-0047 was shown to be taken up by the cells via the macropinocytosis and caveolae-mediated endocytosis pathways while LN-RX-0047 was taken up by cells via caveolae-mediated endocytosis. In the KB xenograft tumor model, LAN-RX-0047 exhibited tumor suppressive activity and significantly reduced intratumoral HIF-1α expression compared to LN-RX-0047. Furthermore, LAN-RX-0047 greatly increased survival time of mice bearing KB-1 xenograft tumors at doses of either 3 mg/kg or 16 mg/kg. These results indicated that LAN-RX-0047 is a highly effective vehicle for therapeutic delivery of antisense agents to tumor.

  7. Antisense mediated splicing modulation for inherited metabolic diseases: challenges for delivery.

    Science.gov (United States)

    Pérez, Belen; Vilageliu, Lluisa; Grinberg, Daniel; Desviat, Lourdes R

    2014-02-01

    In the past few years, research in targeted mutation therapies has experienced significant advances, especially in the field of rare diseases. In particular, the efficacy of antisense therapy for suppression of normal, pathogenic, or cryptic splice sites has been demonstrated in cellular and animal models and has already reached the clinical trials phase for Duchenne muscular dystrophy. In different inherited metabolic diseases, splice switching oligonucleotides (SSOs) have been used with success in patients' cells to force pseudoexon skipping or to block cryptic splice sites, in both cases recovering normal transcript and protein and correcting the enzyme deficiency. However, future in vivo studies require individual approaches for delivery depending on the gene defect involved, given the different patterns of tissue and organ expression. Herein we review the state of the art of antisense therapy targeting RNA splicing in metabolic diseases, grouped according to their expression patterns-multisystemic, hepatic, or in central nervous system (CNS)-and summarize the recent progress achieved in the field of in vivo delivery of oligonucleotides to each organ or system. Successful body-wide distribution of SSOs and preferential distribution in the liver after systemic administration have been reported in murine models for different diseases, while for CNS limited data are available, although promising results with intratechal injections have been achieved. PMID:24506780

  8. Search for antisense copies of beta-globin mRNA in anemic mouse spleen

    Directory of Open Access Journals (Sweden)

    Taylor John M

    2001-03-01

    Full Text Available Abstract Background Previous studies by Volloch and coworkers have reported that during the expression of high levels of β-globin mRNA in the spleen of anemic mice, they could also detect small but significant levels of an antisense (AS globin RNA species, which they postulated might have somehow arisen by RNA-directed RNA synthesis. For two reasons we undertook to confirm and possibly extend these studies. First, previous studies in our lab have focussed on what is an unequivocal example of host RNA-directed RNA polymerase activity on the RNA genome of human hepatitis delta virus. Second, if AS globin species do exist they could in turn form double-stranded RNA species which might induce post-transcriptional gene silencing, a phenomenon somehow provoked in eukaryotic cells by AS RNA sequences. Results We reexamined critical aspects of the previous globin studies. We used intraperitoneal injections of phenylhydrazine to induce anemia in mice, as demonstrated by the appearance and ultimate disappearance of splenomegaly. While a 30-fold increase in globin mRNA was detected in the spleen, the relative amount of putative AS RNA could be no more than 0.004%. Conclusions Contrary to earlier reports, induction of a major increase in globin transcripts in the mouse spleen was not associated with a detectable level of antisense RNA to globin mRNA.

  9. Ustilago maydis natural antisense transcript expression alters mRNA stability and pathogenesis.

    Science.gov (United States)

    Donaldson, Michael E; Saville, Barry J

    2013-07-01

    Ustilago maydis infection of Zea mays leads to the production of thick-walled diploid teliospores that are the dispersal agent for this pathogen. Transcriptome analyses of this model biotrophic basidiomycete fungus identified natural antisense transcripts (NATs) complementary to 247 open reading frames. The U. maydis NAT cDNAs were fully sequenced and annotated. Strand-specific RT-PCR screens confirmed expression and identified NATs preferentially expressed in the teliospore. Targeted screens revealed four U. maydis NATs that are conserved in a related fungus. Expression of NATs in haploid cells, where they are not naturally occurring, resulted in increased steady-state levels of some complementary mRNAs. The expression of one NAT, as-um02151, in haploid cells resulted in a twofold increase in complementary mRNA levels, the formation of sense-antisense double-stranded RNAs, and unchanged Um02151 protein levels. This led to a model for NAT function in the maintenance and expression of stored teliospore mRNAs. In testing this model by deletion of the regulatory region, it was determined that alteration in NAT expression resulted in decreased pathogenesis in both cob and seedling infections. This annotation and functional analysis supports multiple roles for U. maydis NATs in controlling gene expression and influencing pathogenesis.

  10. Identification of novel sense and antisense transcription at the TRPM2 locus in cancer

    Institute of Scientific and Technical Information of China (English)

    Ugo Orfanelli; Ann-Kathrin Wenke; Claudio Doglioni; Vincenzo Russo; Anja Katrin Bosserhoff; Giovanni Lavorgna

    2008-01-01

    It has been proposed that in cancer, where the bulk of the genome becomes hypomethylated, there is an increase in transcriptional noise that might lead to the generation of antisense transcripts that could affect the function of key oncosuppressor genes, ultimately leading to malignant transformation. Here, we describe the computational identifi-cation of a melanoma-enriched antisense transcript, TRPM2-AS, mapped within the locus of TRPM2, an ion channel capable of mediating susceptibility to cell death. Analysis of the TRPM2-AS genomic region indicated the presence in the same region of another tumor-enriched TRPM2 transcript, TRPM2-TE, located across a CpG island shared with TRPM2-AS. Quantitative PCR experiments confirmed that TRPM2-AS and TRPM2-TE transcripts were up-regu-lated in melanoma, and their activation was consistent with the methylation status of the shared CpG island. Func-tional knock-out of TRPM2-TE, as well as over-expression of wild-type TRPM2, increased melanoma susceptibility to apoptosis and necrosis. Finally, expression analysis in other cancer types indicated that TRPM2-AS and TRPM2-TE over-expression might have an even wider role than anticipated, reinforcing the relevance of our computational approach in identifying new potential therapeutic targets.

  11. Small regulatory RNAs in lambdoid bacteriophages and phage-derived plasmids: Not only antisense.

    Science.gov (United States)

    Nejman-Faleńczyk, Bożena; Bloch, Sylwia; Licznerska, Katarzyna; Felczykowska, Agnieszka; Dydecka, Aleksandra; Węgrzyn, Alicja; Węgrzyn, Grzegorz

    2015-03-01

    Until recently, only two small regulatory RNAs encoded by lambdoid bacteriophages were known. These transcripts are derived from paQ and pO promoters. The former one is supposed to act as an antisense RNA for expression of the Q gene, encoding a transcription antitermination protein. The latter transcript, called oop RNA, was initially proposed to have a double role, in establishing expression of the cI gene and in providing a primer for DNA replication. Although the initially proposed mechanisms by which oop RNA could influence the choice between two alternative developmental pathways of the phage and the initiation of phage DNA replication were found not true, the pO promoter has been demonstrated to be important for both regulation of phage development and control of DNA replication. Namely, the pO-derived transcript is an antisense RNA for expression of the cII gene, and pO is a part of a dual promoter system responsible for regulation of initiation of DNA synthesis from the oriλ region. Very recent studies identified a battery of small RNAs encoded by lambdoid bacteriophages existing as prophages in chromosomes of enterohemorrhagic Escherichia coli strains. Some of them have very interesting functions, like anti-small RNAs.

  12. Antisense oligonucleotides targeting midkine induced apoptosis and increased chemosensitivity in hepatocellular carcinoma cells

    Institute of Scientific and Technical Information of China (English)

    Li-cheng DAI; Xiang WANG; Xing YAO; Yong-liang LU; Jin-liang PING; Jian-fang HE

    2006-01-01

    Aim: Overexpression of midkine (MK) has been observed in many malignancies. This aim of this study is to screen for suitable antisense oligonucleotides (ASODN) targeting MK in hepatocellular carcinoma (HCC) cells and evaluate its antitumor activity. Methods: Ten ASODN targeting MK were designed and synthesized. After transfection with ASODN, cell proliferation was analyzed with MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2//-tetrazolium, inner salt] assay. In addition, MK mRNA, protein levels, as well as apoptosis and caspase-3 activity were also examined in HepG2 cells. Cell proliferation was then analyzed after treatment with both ASODN and chemotherapeu-tic drugs. Results: In this experiment, the ASODN5 among the 10 ASODN showed higher inhibitory activity against proliferation of hepatocellular carcinoma cells in a dose-dependent manner. In HepG2 cells, ASODN5 could significantly reduce the MK mRNA level and protein content. After transfection with ASODN5 for 48 h, accompanied with a decline of survivin and Bcl-2 protein content, a remarkable increase of apoptosis and caspase-3 activity was observed in HepG2 cells. Furthermore, ASODN5 transfer can significantly increase chemosensitivity in HepG2 cells. Conclusion: Antisense oligonucleotides targeting MK shows therapeutic effects on HCC; ASODN5 has the possibility to be developed as an effective antitumor agent.

  13. Efficient gene silencing by delivery of locked nucleic acid antisense oligonucleotides, unassisted by transfection reagents.

    Science.gov (United States)

    Stein, C A; Hansen, J Bo; Lai, Johnathan; Wu, SiJian; Voskresenskiy, Anatoliy; Høg, Anja; Worm, Jesper; Hedtjärn, Maj; Souleimanian, Naira; Miller, Paul; Soifer, Harris S; Castanotto, Daniella; Benimetskaya, Luba; Ørum, Henrik; Koch, Troels

    2010-01-01

    For the past 15-20 years, the intracellular delivery and silencing activity of oligodeoxynucleotides have been essentially completely dependent on the use of a delivery technology (e.g. lipofection). We have developed a method (called 'gymnosis') that does not require the use of any transfection reagent or any additives to serum whatsoever, but rather takes advantage of the normal growth properties of cells in tissue culture in order to promote productive oligonucleotide uptake. This robust method permits the sequence-specific silencing of multiple targets in a large number of cell types in tissue culture, both at the protein and mRNA level, at concentrations in the low micromolar range. Optimum results were obtained with locked nucleic acid (LNA) phosphorothioate gap-mers. By appropriate manipulation of oligonucleotide dosing, this silencing can be continuously maintained with little or no toxicity for >240 days. High levels of oligonucleotide in the cell nucleus are not a requirement for gene silencing, contrary to long accepted dogma. In addition, gymnotic delivery can efficiently deliver oligonucleotides to suspension cells that are known to be very difficult to transfect. Finally, the pattern of gene silencing of in vitro gymnotically delivered oligonucleotides correlates particularly well with in vivo silencing. The establishment of this link is of particular significance to those in the academic research and drug discovery and development communities.

  14. Rapid generation of microRNA sponges for microRNA inhibition.

    Directory of Open Access Journals (Sweden)

    Joost Kluiver

    Full Text Available MicroRNA (miRNA sponges are transcripts with repeated miRNA antisense sequences that can sequester miRNAs from endogenous targets. MiRNA sponges are valuable tools for miRNA loss-of-function studies both in vitro and in vivo. We developed a fast and flexible method to generate miRNA sponges and tested their efficiency in various assays. Using a single directional ligation reaction we generated sponges with 10 or more miRNA binding sites. Luciferase and AGO2-immuno precipitation (IP assays confirmed effective binding of the miRNAs to the sponges. Using a GFP competition assay we showed that miR-19 sponges with central mismatches in the miRNA binding sites are efficient miRNA inhibitors while sponges with perfect antisense binding sites are not. Quantification of miRNA sponge levels suggests that this is at least in part due to degradation of the perfect antisense sponge transcripts. Finally, we provide evidence that combined inhibition of miRNAs of the miR-17∼92 cluster results in a more effective growth inhibition as compared to inhibition of individual miRNAs. In conclusion, we describe and validate a method to rapidly generate miRNA sponges for miRNA loss-of-function studies.

  15. Improved cellular activity of antisense peptide nucleic acids by conjugation to a cationic peptide-lipid (CatLip) domain

    DEFF Research Database (Denmark)

    Koppelhus, Uffe; Shiraishi, Takehiko; Zachar, Vladimir;

    2008-01-01

    for future in vivo applications. We find that simply conjugating a lipid domain (fatty acid) to the cationic peptide (a CatLip conjugate) increases the biological effect of the corresponding PNA (CatLip) conjugates in a luciferase cellular antisense assay up to 2 orders of magnitude. The effect increases...

  16. Reduction of methylviologen-mediated oxidative stress tolerance in antisense transgenic tobacco seedlings through restricted expression of StAPX.

    Science.gov (United States)

    Sun, Wei-Hong; Wang, Yong; He, Hua-Gang; Li, Xue; Song, Wan; Du, Bin; Meng, Qing-Wei

    2013-07-01

    Ascorbate peroxidases are directly involved in reactive oxygen species (ROS) scavenging by reducing hydrogen peroxide to water. The tomato thylakoid-bound ascorbate peroxidase gene (StAPX) was introduced into tobacco. RNA gel blot analysis confirmed that StAPX in tomato leaves was induced by methylviologen-mediated oxidative stress. The sense transgenic seedlings exhibited higher tAPX activity than that of the wild type (WT) plants under oxidative stress conditions, while the antisense seedlings exhibited lower tAPX activity. Lower APX activities of antisense transgenic seedlings caused higher malondialdehyde contents and relative electrical conductivity. The sense transgenic seedlings with higher tAPX activity maintained higher chlorophyll content and showed the importance of tAPX in maintaining the optimal chloroplast development under methylviologen stress conditions, whereas the antisense lines maintained lower chlorophyll content than WT seedlings. Results indicated that the over-expression of StAPX enhanced tolerance to methylviologen-mediated oxidative stress in sense transgenic tobacco early seedlings, whereas the suppression of StAPX in antisense transgenic seedlings showed high sensitivity to oxidative stress.

  17. Design and synthesis of dendritic molecular transporter that achieves efficient in vivo delivery of morpholino antisense oligo.

    Science.gov (United States)

    Li, Yong-Fu; Morcos, Paul A

    2008-07-01

    Safe and efficient in vivo delivery of Morpholino antisense oligos was probably the last and most difficult challenge for the broad application of antisense in animal research and therapeutics. Several arginine-rich peptides effective for in vivo delivery of Morpholino antisense oligos require rather complex and expensive procedures for synthesis and conjugation. This work describes the design and synthesis of a dendritic transporter in a most concise manner where the selection of the core scaffold, functional group multiplication, orthogonal protecting group manipulation, solid phase conjugation, and off-resin perguanidinylation of the transporter structure are all orchestrated for efficient assembly. We utilized triazine as a core to provide a site for on-column conjugation to the Morpholino oligo and to anchor functional side arms which, after extension, multiplication, and deprotection, are subsequently converted from primary amines to the eight guanidinium headgroups that serve for transport across cell membranes. Intravenous administration of the delivery-enabled Morpholino into a splice-reporter strain of transgenic living mice results in de novo expression of splice-corrected green fluorescent protein in a broad range of tissues and organs in those treated mice. This rigorously demonstrates that this new dendritic transporter achieves effective delivery of a Morpholino oligo into the cytosol/nuclear compartment of cells systemically in vivo. The practical conjugation process may overcome any availability limitation for routine use by the scientific community, and the efficient delivery ability of this transporter may advance the application of Morpholino antisense technology in animals.

  18. Improved cellular uptake of antisense Peptide nucleic acids by conjugation to a cell-penetrating Peptide and a lipid domain

    DEFF Research Database (Denmark)

    Shiraishi, Takehiko; Nielsen, Peter E

    2011-01-01

    Unaided cellular uptake of RNA interference agents such as antisense oligonucleotides and siRNA is extremely poor, and in vivo bioavailability is also limited. Thus, effective delivery strategies for such potential drugs are in high demand. Recently, a novel approach using a class of short cationic...

  19. Reduction of methylviologen-mediated oxidative stress tolerance in antisense transgenic tobacco seedlings through restricted expression of StAPX

    Institute of Scientific and Technical Information of China (English)

    Wei-hong SUN; Yong WANG; Hua-gang HE; Xue LI; Wan SONG; Bin DU; Qing-wei MENG

    2013-01-01

    Ascorbate peroxidases are directly involved in reactive oxygen species (ROS) scavenging by reducing hydrogen peroxide to water.The tomato thylakoid-bound ascorbate peroxidase gene (StAPX) was introduced into tobacco.RNA gel blot analysis confirmed that StAPX in tomato leaves was induced by methylviologen-mediated oxidative stress.The sense transgenic seedlings exhibited higher tAPX activity than that of the wild type (WT) plants under oxidative stress conditions,while the antisense seedlings exhibited lower tAPX activity.Lower APX activities of antisense transgenic seedlings caused higher malondialdehyde contents and relative electrical conductivity.The sense transgenic seedlings with higher tAPX activity maintained higher chlorophyll content and showed the importance of tAPX in maintaining the optimal chloroplast development under methylviologen stress conditions,whereas the antisense lines maintained lower chlorophyll content than WT seedlings.Results indicated that the over-expression of StAPX enhanced tolerance to methylviologen-mediated oxidative stress in sense transgenic tobacco early seedlings,whereas the suppression of StAPX in antisense transgenic seedlings showed high sensitivity to oxidative stress.

  20. Expression of RNA-interference/antisense transgenes by the cognate promoters of target genes is a better gene-silencing strategy to study gene functions in rice.

    Science.gov (United States)

    Li, Jing; Jiang, Dagang; Zhou, Hai; Li, Feng; Yang, Jiawei; Hong, Laifa; Fu, Xiao; Li, Zhibin; Liu, Zhenlan; Li, Jianming; Zhuang, Chuxiong

    2011-03-03

    Antisense and RNA interference (RNAi)-mediated gene silencing systems are powerful reverse genetic methods for studying gene function. Most RNAi and antisense experiments used constitutive promoters to drive the expression of RNAi/antisense transgenes; however, several reports showed that constitutive promoters were not expressed in all cell types in cereal plants, suggesting that the constitutive promoter systems are not effective for silencing gene expression in certain tissues/organs. To develop an alternative method that complements the constitutive promoter systems, we constructed RNAi and/or antisense transgenes for four rice genes using a constitutive promoter or a cognate promoter of a selected rice target gene and generated many independent transgenic lines. Genetic, molecular, and phenotypic analyses of these RNAi/antisense transgenic rice plants, in comparison to previously-reported transgenic lines that silenced similar genes, revealed that expression of the cognate promoter-driven RNAi/antisense transgenes resulted in novel growth/developmental defects that were not observed in transgenic lines expressing constitutive promoter-driven gene-silencing transgenes of the same target genes. Our results strongly suggested that expression of RNAi/antisense transgenes by cognate promoters of target genes is a better gene-silencing approach to discovery gene function in rice.

  1. Expression of RNA-interference/antisense transgenes by the cognate promoters of target genes is a better gene-silencing strategy to study gene functions in rice.

    Directory of Open Access Journals (Sweden)

    Jing Li

    Full Text Available Antisense and RNA interference (RNAi-mediated gene silencing systems are powerful reverse genetic methods for studying gene function. Most RNAi and antisense experiments used constitutive promoters to drive the expression of RNAi/antisense transgenes; however, several reports showed that constitutive promoters were not expressed in all cell types in cereal plants, suggesting that the constitutive promoter systems are not effective for silencing gene expression in certain tissues/organs. To develop an alternative method that complements the constitutive promoter systems, we constructed RNAi and/or antisense transgenes for four rice genes using a constitutive promoter or a cognate promoter of a selected rice target gene and generated many independent transgenic lines. Genetic, molecular, and phenotypic analyses of these RNAi/antisense transgenic rice plants, in comparison to previously-reported transgenic lines that silenced similar genes, revealed that expression of the cognate promoter-driven RNAi/antisense transgenes resulted in novel growth/developmental defects that were not observed in transgenic lines expressing constitutive promoter-driven gene-silencing transgenes of the same target genes. Our results strongly suggested that expression of RNAi/antisense transgenes by cognate promoters of target genes is a better gene-silencing approach to discovery gene function in rice.

  2. Increased cell survival by inhibition of BRCA1 using an antisense approach in an estrogen responsive ovarian carcinoma cell line

    OpenAIRE

    Annab, Lois A; Hawkins, Rebecca E; Solomon, Greg; Barrett, J Carl; Afshari, Cynthia A.

    2000-01-01

    Introduction: Germline mutations in the breast and ovarian cancer susceptibility gene BRCA1, which is located on chromosome 17q21, are associated with a predisposition to the development of cancer in these organs [1,2]. No mutations in the BRCA1 gene have been detected in sporadic breast cancer cases, but mutations have been detected in sporadic cases of ovarian cancer [3,4]. Although there is debate regarding the level of cancer risk associated with mutations in BRCA1 and the significance of...

  3. A locked nucleic acid antisense oligonucleotide (LNA silences PCSK9 and enhances LDLR expression in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Nidhi Gupta

    Full Text Available BACKGROUND: The proprotein convertase subtilisin/kexin type 9 (PCSK9 is an important factor in the etiology of familial hypercholesterolemia (FH and is also an attractive therapeutic target to reduce low density lipoprotein (LDL cholesterol. PCSK9 accelerates the degradation of hepatic low density lipoprotein receptor (LDLR and low levels of hepatic PCSK9 activity are associated with reduced levels of circulating LDL-cholesterol. METHODOLOGY/PRINCIPAL FINDINGS: The present study presents the first evidence for the efficacy of a locked nucleic acid (LNA antisense oligonucleotide (LNA ASO that targets both human and mouse PCSK9. We employed human hepatocytes derived cell lines HepG2 and HuH7 and a pancreatic mouse beta-TC3 cell line known to express high endogenous levels of PCSK9. LNA ASO efficiently reduced the mRNA and protein levels of PCSK9 with a concomitant increase in LDLR protein levels after transfection in these cells. In vivo efficacy of LNA ASO was further investigated in mice by tail vein intravenous administration of LNA ASO in saline solution. The level of PCSK9 mRNA was reduced by approximately 60%, an effect lasting more than 16 days. Hepatic LDLR protein levels were significantly up-regulated by 2.5-3 folds for at least 8 days and approximately 2 fold for 16 days. Finally, measurement of liver alanine aminotransferase (ALT levels revealed that long term LNA ASO treatment (7 weeks does not cause hepatotoxicity. CONCLUSION/SIGNIFICANCE: LNA-mediated PCSK9 mRNA inhibition displayed potent reduction of PCSK9 in cell lines and mouse liver. Our data clearly revealed the efficacy and safety of LNA ASO in reducing PCSK9 levels, an approach that is now ready for testing in primates. The major significance and take home message of this work is the development of a novel and promising approach for human therapeutic intervention of the PCSK9 pathway and hence for reducing some of the cardiovascular risk factors associated with the metabolic

  4. Effect of Dexamethasone and Aquaporin-1 Antisense Oligonucleotides on the Aquaporin-1 Expression in Cultured Human Trabecular Meshwork Cells

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    The changes in the expression of aquaporin-1 (AQP1) mRNA and protein in cultured human trabecular meshwork (HTM) cells treated with dexamethasone and transfected with antisense oligonucleotides (AS-ODN) were studied, and the implication of AQP1 regulation in corticosteroid-glaucoma and the possibility of AS-ODN inhibiting the AQP1 expression were evaluated.The cultured HTM cells in vitro were treated with different concentrations of dexamethasone and transfected with oligonucleotides for 5 days respectively. Then, total RNA and protein of HTM cells were extracted. The changes of AQP1 mRNA and protein were demonstrated qualitatively and quantitatively by RT-PCR and Western blot. Band intensities were detected by imaging analysis.There was a parallel relationship between the results of RT-PCR and those of Western blot. The expression levels of AQP1 mRNA and protein in dexamethasone-treated groups were increased initially and decreased later as dexamethasone concentration was stepped up. In the 0.04 μg/mL and 0.4 μg/mL groups, the levels of AQP1 were higher than in control group (0μg/mL). In the 4μg/mL and 40μg/mL groups, the AQP1 expression levels were lower than in control group. AS-ODN could down-regulate the expression of AQP1 mRNA and protein in a dose-dependent manner. At 5 μg/mL, down-regulation efficiency reached the maximum. There was no statistically significant difference in the expression of AQP1 mRNA and protein between all sense oligonucleotides groups and control group. It was suggested that dexamethasone may induce the changes of the AQP1 expression in HTM cells to be involved in the occurrence of corticosteroid-glaucoma. AS-ODN can down-regulate the AQP1 expression in HTM cells to some extent.

  5. STAT1 Antisense Oligonucleotides Attenuate the Proinflammatory Cytokine Release of Alveolar Macrophages in Bleomycin-Induced Fibrosis

    Institute of Scientific and Technical Information of China (English)

    Xianming Fan; Zengli Wang

    2005-01-01

    To investigate the effect of signal transducers and activators of transcription 1 (STAT1) antisense oligonucleotides (ASON) on concentrations of TNF-α, IL-8, NO secreted by alveolar macrophages (AMs) in bleomycin-induced rat pulmonary fibrosis, five adult female Wistar rats were intratracheally instilled with bleomycin. After 7 days, the rats were killed by right ventricle of heart exsanguinations under ketamine anaesthesia and bronchoalveolar lavage (BAL) was performed to obtain AMs. AMs were divided into four groups, treated with STAT1 ASON, STAT1 sense oligonucleotides (SON), dexamethasone (DEX) and medium alone (control), respectively. AMs and media were collected after culture for 36 h. The mRNA and protein expressions of STAT1 and ICAM-1 in AMs were detected by RT-PCR and ELISA, respectively. The concentrations of TNF-α, IL-8, NO in cultured medium were detected.The STAT1 mRNA expression by AMs in the STAT1 ASON group was lower than those of AMs in the STAT1 SON group, the DEX group and the control group (p < 0.05). Moreover, the STAT1 mRNA expression by AMs in the DEX group was also lower than those of AMs in the STAT1 SON group and the control group (p < 0.05), but the STAT1 mRNA expression by AMs in the STAT1 SON group was not different from that of the control group (p >0.05). The protein expressions of STAT1 and ICAM-1 and the mRNA expression of ICAM-1 showed similar changes to the STAT1 mRNA expression by AMs. The concentrations of TNF-α, IL-8, NO in cultured medium from STAT1 ASON group were lower than those from STAT1 SON, DEX and the control groups (p < 0.05). Moreover,the concentrations of TNF-α, IL-8, NO in cultured medium from DEX group were also lower than those from the control and STAT1 SON group (p < 0.05), but no difference between STAT1 SON group and the control (p > 0.05).The results suggest that STAT1 ASON could inhibit the secretion of TNF-α, IL-8, NO in AMs, and STAT1 could become a target of treating pulmonary fibrosis.

  6. Inhibition of clinical human immunodeficiency virus (HIV) type 1 isolates in primary CD4+ T lymphocytes by retroviral vectors expressing anti-HIV genes.

    OpenAIRE

    Vandendriessche, T.; Chuah, M. K.; L. Chiang; Chang, H K; Ensoli, B; Morgan, R A

    1995-01-01

    Gene therapy may be of benefit in human immunodeficiency virus type 1 (HIV-1)-infected individuals by virtue of its ability to inhibit virus replication and prevent viral gene expression. It is not known whether anti-HIV-1 gene therapy strategies based on antisense or transdominant HIV-1 mutant proteins can inhibit the replication and expression of clinical HIV-1 isolates in primary CD4+ T lymphocytes. We therefore transduced CD4+ T lymphocytes from uninfected individuals with retroviral vect...

  7. Expression of antisense small RNAs in response to stress in Pseudomonas aeruginosa

    DEFF Research Database (Denmark)

    Gómez Lozano, María; Marvig, Rasmus Lykke; Tulstrup, Monica Vera-Lise;

    2014-01-01

    . We found a significant overrepresentation of asRNAs that are transcribed opposite to genes involved in cell division and in cell wall, lipopolysaccharide (LPS), and capsule biosynthesis, most likely reflecting the conditions used in this study. A substantial number of asRNAs significantly changed......Background: RNA sequencing technologies reveal that bacteria express RNA molecules other than mRNA, rRNA or tRNA. During the last years genome-wide bacterial transcriptomes have been shown to comprise intergenic RNA, antisense RNA, and untranslated regions, all capable of performing diverse...... that the extent of overlap between the studies is very limited. Conclusions: RNA-seq experiments are revealing hundreds of novel transcripts in all bacterial genomes investigated. The comparison between independent studies that used RNA-seq to detect novel asRNAs in P. aeruginosa shows that the overlap between...

  8. Efficient Synthesis and Biological Evaluation of 5'-GalNAc Conjugated Antisense Oligonucleotides.

    Science.gov (United States)

    Østergaard, Michael E; Yu, Jinghua; Kinberger, Garth A; Wan, W Brad; Migawa, Michael T; Vasquez, Guillermo; Schmidt, Karsten; Gaus, Hans J; Murray, Heather M; Low, Audrey; Swayze, Eric E; Prakash, Thazha P; Seth, Punit P

    2015-08-19

    Conjugation of triantennary N-acetyl galactosamine (GalNAc) to oligonucleotide therapeutics results in marked improvement in potency for reducing gene targets expressed in hepatocytes. In this report we describe a robust and efficient solution-phase conjugation strategy to attach triantennary GalNAc clusters (mol. wt. ∼2000) activated as PFP (pentafluorophenyl) esters onto 5'-hexylamino modified antisense oligonucleotides (5'-HA ASOs, mol. wt. ∼8000 Da). The conjugation reaction is efficient and was used to prepare GalNAc conjugated ASOs from milligram to multigram scale. The solution phase method avoids loading of GalNAc clusters onto solid-support for automated synthesis and will facilitate evaluation of GalNAc clusters for structure activity relationship (SAR) studies. Furthermore, we show that transfer of the GalNAc cluster from the 3'-end of an ASO to the 5'-end results in improved potency in cells and animals.

  9. A cytoplasmic pathway for gapmer antisense oligonucleotide-mediated gene silencing in mammalian cells

    Science.gov (United States)

    Castanotto, Daniela; Lin, Min; Kowolik, Claudia; Wang, LiAnn; Ren, Xiao-Qin; Soifer, Harris S.; Koch, Troels; Hansen, Bo Rode; Oerum, Henrik; Armstrong, Brian; Wang, Zhigang; Bauer, Paul; Rossi, John; Stein, C.A.

    2015-01-01

    Antisense oligonucleotides (ASOs) are known to trigger mRNA degradation in the nucleus via an RNase H-dependent mechanism. We have now identified a putative cytoplasmic mechanism through which ASO gapmers silence their targets when transfected or delivered gymnotically (i.e. in the absence of any transfection reagent). We have shown that the ASO gapmers can interact with the Ago-2 PAZ domain and can localize into GW-182 mRNA-degradation bodies (GW-bodies). The degradation products of the targeted mRNA, however, are not generated by Ago-2-directed cleavage. The apparent identification of a cytoplasmic pathway complements the previously known nuclear activity of ASOs and concurrently suggests that nuclear localization is not an absolute requirement for gene silencing. PMID:26433227

  10. Gene-Specific Countermeasures against Ebola Virus Based on Antisense Phosphorodiamidate Morpholino Oligomers.

    Directory of Open Access Journals (Sweden)

    2006-01-01

    Full Text Available The filoviruses Marburg virus and Ebola virus (EBOV quickly outpace host immune responses and cause hemorrhagic fever, resulting in case fatality rates as high as 90% in humans and nearly 100% in nonhuman primates. The development of an effective therapeutic for EBOV is a daunting public health challenge and is hampered by a paucity of knowledge regarding filovirus pathogenesis. This report describes a successful strategy for interfering with EBOV infection using antisense phosphorodiamidate morpholino oligomers (PMOs. A combination of EBOV-specific PMOs targeting sequences of viral mRNAs for the viral proteins (VPs VP24, VP35, and RNA polymerase L protected rodents in both pre- and post-exposure therapeutic regimens. In a prophylactic proof-of-principal trial, the PMOs also protected 75% of rhesus macaques from lethal EBOV infection. The work described here may contribute to development of designer, "druggable" countermeasures for filoviruses and other microbial pathogens.

  11. Gene-specific countermeasures against Ebola virus based on antisense phosphorodiamidate morpholino oligomers.

    Directory of Open Access Journals (Sweden)

    Kelly L Warfield

    2006-01-01

    Full Text Available The filoviruses Marburg virus and Ebola virus (EBOV quickly outpace host immune responses and cause hemorrhagic fever, resulting in case fatality rates as high as 90% in humans and nearly 100% in nonhuman primates. The development of an effective therapeutic for EBOV is a daunting public health challenge and is hampered by a paucity of knowledge regarding filovirus pathogenesis. This report describes a successful strategy for interfering with EBOV infection using antisense phosphorodiamidate morpholino oligomers (PMOs. A combination of EBOV-specific PMOs targeting sequences of viral mRNAs for the viral proteins (VPs VP24, VP35, and RNA polymerase L protected rodents in both pre- and post-exposure therapeutic regimens. In a prophylactic proof-of-principal trial, the PMOs also protected 75% of rhesus macaques from lethal EBOV infection. The work described here may contribute to development of designer, "druggable" countermeasures for filoviruses and other microbial pathogens.

  12. Antisense treatment of caliciviridae: an emerging disease agent of animals and humans.

    Science.gov (United States)

    Smith, Alvin W; Matson, David O; Stein, David A; Skilling, Douglas E; Kroeker, Andrew D; Berke, Tamas; Iversen, Patrick L

    2002-04-01

    The Earth's oceans are the primary reservoir for an emerging family of RNA viruses, the Caliciviridae, which can cause a spectrum of diseases in marine animals, wildlife, farm animals, pets and humans. Certain members of this family have unusually broad host ranges, and some are zoonotic (transmissible from animals to humans). The RNA virus replicative processes lack effective genetic repair mechanisms, and, therefore, virtually every calicivirus replicate is a mutant. Hence, traditional therapeutics dependent on specific nucleic acid sequences or protein epitopes lack the required diversity of sequence or conformational specificity that would be required to reliably detect, prevent or treat infections from these mutant clusters (quasi-species) of RNA viruses, including the Caliciviridae. Antisense technology using phosphorodiamidate morpholino oligomers shows promise in overcoming these current diagnostic and therapeutic problems inherent with newly emerging viral diseases. PMID:12044040

  13. Metabolite profiling of Arabidopsis thaliana (L.) plants transformed with an antisense chalcone synthase gene

    DEFF Research Database (Denmark)

    Le Gall, G.; Metzdorff, Stine Broeng; Pedersen, Jan W.;

    2005-01-01

    A metabolite profiling study has been carried out on Arabidopsis thaliana (L.) Heynh. ecotype Wassilewskija and a series of transgenic lines of the ecotype transformed with a CHS (chalcone synthase) antisense construct. Compound identifications by LC/MS and H-1 NMR are discussed. The glucosinolate...... be expected in the transgenic lines relative to the wild type. In practice the reductions achieved were highly variable both between lines and within a given line on different occasions when the plants were grown. Possible reasons for this variability are discussed with reference to current models of gene...... silencing. The metabolite profiles of the transgenic lines were examined for unintended effects of the modification. An apparently major effect on the glucosinolate composition was shown to result from an unusual genetic variation in the ecotype and not from the modification. The modification did produce...

  14. Characteristics of photosynthesis in rice plants transformed with an antisense Rubisco activase gene

    Institute of Scientific and Technical Information of China (English)

    金松恒; 蒋德安; 李雪芹; 孙骏威

    2004-01-01

    Transgenic rice plants with an antisense gene inserted via Agrobacterium tumefaciens were used to explore the impact of the reduction of Rubisco activase (RCA) on Rubisco and photosynthesis. In this study, transformants containing 15% to 35% wild type Rubisco activase were selected, which could survive in ambient CO2 concentration but grew slowly compared with wild type controls. Gas exchange measurements indicated that the rate of photosynthesis decreased sig-nificantly, while stomatal conductance and transpiration rate did not change; and that the intercellular CO2 concentration even increased. Rubisco determination showed that these plants had approximately twice as much Rubisco as the wild types,although they showed 70% lower rate of photosynthesis, whichRubsico activase and/or the reduction in carbamylation.was likely an acclimation response to the reduction in Rubsico activase and/or the reduction in carbamylation.

  15. Characteristics of photosynthesis in rice plants transformed with an antisense Rubisco activase gene

    Institute of Scientific and Technical Information of China (English)

    金松恒; 蒋德安; 李雪芹; 孙骏威

    2004-01-01

    Transgenic rice plants with an antisense gene inserted via Agrobacterium tumefaciens were used to explore the impact of the reduction of Rubisco activase (RCA) on Rubisco and photosynthesis. In this study, transformants containing 15% to 35% wild type Rubisco activase were selected, which could survive in ambient CO2 concentration but grew slowly compared with wild type controls. Gas exchange measurements indicated that the rate of photosynthesis decreased significantly, while stomatal conductance and transpiration rate did not change; and that the intercellular CO2 concentration even increased. Rubisco determination showed that these plants had approximately twice as much Rubisco as the wild types,although they showed 70% lower rate of photosynthesis, which was likely an acclimation response to the reduction inRubsico activase and/or the reduction in carbamylation.

  16. Multiple Defects of Cell Cycle Checkpoints in U937-ASPI3K, an U937 Cell Mutant Stably Expressing Anti-Sense ATM Gene cDNA

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    (Ataxia-telangiectasia mutated gene (ATM) functions in control of cell cycle checkpoints in responding to DNA damage and protects cells from undergoing apoptosis. Knock-out within tumor cells of endogenous ATM will achieve therapeutic benefits and nable a better understanding of the decisive mechanisms of cell death or survival in response to DNA damaging agents. ) In present paper, we sought to characterize the cell cycle checkpoint profiles in U937-ASPI3K, a U937 cell mutant that was previously established with endogenous ATM knock-out phenotype. Synchronized U937-ASPI3K was exposed to 137Cs irradiation, G1, S, G2/M cell cycle checkpoint profiles were evaluated by determining cell cycle kinetics, p53/p21 protein, cyclin dependent kinase 2 (CDK2) and p34CDC2 kinase activity in response to irradiation. U937-ASPI3K exhibited multiple defects in cell cycle checkpoints as defined by failing to arrest cells upon irradiation. The accumulation of cellular p53/p21 protein and inhibition of CDK kinase was also abolished in U937-ASPI3K. It was concluded that the stable expression of anti-sense PI3K cDNA fragment completely abolished multiple cell cycle checkpoints in U937-ASPI3K, and hence U937-ASPI3K with an AT-like phenotype could serves as a valuable model system for investigating the signal transduction pathway in responding to DNA damaging-based cancer therapy.

  17. Unconventional genomic architecture in the budding yeast saccharomyces cerevisiae masks the nested antisense gene NAG1.

    Science.gov (United States)

    Ma, Jun; Dobry, Craig J; Krysan, Damian J; Kumar, Anuj

    2008-08-01

    The genomic architecture of the budding yeast Saccharomyces cerevisiae is typical of other eukaryotes in that genes are spatially organized into discrete and nonoverlapping units. Inherent in this organizational model is the assumption that protein-coding sequences do not overlap completely. Here, we present evidence to the contrary, defining a previously overlooked yeast gene, NAG1 (for nested antisense gene) nested entirely within the coding sequence of the YGR031W open reading frame in an antisense orientation on the opposite strand. NAG1 encodes a 19-kDa protein, detected by Western blotting of hemagglutinin (HA)-tagged Nag1p with anti-HA antibodies and by beta-galactosidase analysis of a NAG1-lacZ fusion. NAG1 is evolutionarily conserved as a unit with YGR031W in bacteria and fungi. Unlike the YGR031WP protein product, however, which localizes to the mitochondria, Nag1p localizes to the cell periphery, exhibiting properties consistent with those of a plasma membrane protein. Phenotypic analysis of a site-directed mutant (nag1-1) disruptive for NAG1 but silent with respect to YGR031W, defines a role for NAG1 in yeast cell wall biogenesis; microarray profiling of nag1-1 indicates decreased expression of genes contributing to cell wall organization, and the nag1-1 mutant is hypersensitive to the cell wall-perturbing agent calcofluor white. Furthermore, production of Nag1p is dependent upon the presence of the cell wall integrity pathway mitogen-activated protein kinase Slt2p and its downstream transcription factor Rlm1p. Thus, NAG1 is important for two reasons. First, it contributes to yeast cell wall biogenesis. Second, its genomic context is novel, raising the possibility that other nested protein-coding genes may exist in eukaryotic genomes.

  18. Antisense-induced exon skipping for duplications in Duchenne muscular dystrophy

    Directory of Open Access Journals (Sweden)

    van Ommen Gert-Jan B

    2007-07-01

    Full Text Available Abstract Background Antisense-mediated exon skipping is currently one of the most promising therapeutic approaches for Duchenne muscular dystrophy (DMD. Using antisense oligonucleotides (AONs targeting specific exons the DMD reading frame is restored and partially functional dystrophins are produced. Following proof of concept in cultured muscle cells from patients with various deletions and point mutations, we now focus on single and multiple exon duplications. These mutations are in principle ideal targets for this approach since the specific skipping of duplicated exons would generate original, full-length transcripts. Methods Cultured muscle cells from DMD patients carrying duplications were transfected with AONs targeting the duplicated exons, and the dystrophin RNA and protein were analyzed. Results For two brothers with an exon 44 duplication, skipping was, even at suboptimal transfection conditions, so efficient that both exons 44 were skipped, thus generating, once more, an out-of-frame transcript. In such cases, one may resort to multi-exon skipping to restore the reading frame, as is shown here by inducing skipping of exon 43 and both exons 44. By contrast, in cells from a patient with an exon 45 duplication we were able to induce single exon 45 skipping, which allowed restoration of wild type dystrophin. The correction of a larger duplication (involving exons 52 to 62, by combinations of AONs targeting the outer exons, appeared problematic due to inefficient skipping and mistargeting of original instead of duplicated exons. Conclusion The correction of DMD duplications by exon skipping depends on the specific exons targeted. Its options vary from the ideal one, restoring for the first time the true, wild type dystrophin, to requiring more 'classical' skipping strategies, while the correction of multi-exon deletions may need the design of tailored approaches.

  19. Circadian rhythms of sense and antisense transcription in sugarcane, a highly polyploid crop.

    Directory of Open Access Journals (Sweden)

    Carlos Takeshi Hotta

    Full Text Available Commercial sugarcane (Saccharum hybrid is a highly polyploid and aneuploid grass that stores large amounts of sucrose in its stem. We have measured circadian rhythms of sense and antisense transcription in a commercial cultivar (RB855453 using a custom oligoarray with 14,521 probes that hybridize to sense transcripts (SS and 7,380 probes that hybridize to antisense transcripts (AS.We estimated that 32% of SS probes and 22% AS probes were rhythmic. This is a higher proportion of rhythmic probes than the usually found in similar experiments in other plant species. Orthologs and inparalogs of Arabidopsis thaliana, sugarcane, rice, maize and sorghum were grouped in ortholog clusters. When ortholog clusters were used to compare probes among different datasets, sugarcane also showed a higher proportion of rhythmic elements than the other species. Thus, it is possible that a higher proportion of transcripts are regulated by the sugarcane circadian clock. Thirty-six percent of the identified AS/SS pairs had significant correlated time courses and 64% had uncorrelated expression patterns. The clustering of transcripts with similar function, the anticipation of daily environmental changes and the temporal compartmentation of metabolic processes were some properties identified in the circadian sugarcane transcriptome. During the day, there was a dominance of transcripts associated with photosynthesis and carbohydrate metabolism, including sucrose and starch synthesis. During the night, there was dominance of transcripts associated with genetic processing, such as histone regulation and RNA polymerase, ribosome and protein synthesis. Finally, the circadian clock also regulated hormone signalling pathways: a large proportion of auxin and ABA signalling components were regulated by the circadian clock in an unusual biphasic distribution.

  20. Relationship between telomerase activity and its subunit expression and inhibitory effect of antisense hTR on pancreatic carcinoma

    Institute of Scientific and Technical Information of China (English)

    Jia-Hua Zhou; Hong-Mei Zhang; Quan Chen; Dong-Dong Han; Fei Pei; Li-Shan Zhang; De-Tong Yang

    2003-01-01

    AIM: To directly investigate the relationship between telomerase activity and its subunit expression and the inhibitory effect of antisense hTR on pancreatic carcinogenesis.METHODS: We examined the telomerase activity and its subunit expression by cell culture, polymerase chain reaction (PCR), PCR-silver staining, PCR-ELISA, DNA sequencing, MTT and flow cytometry methods.RESULTS: PCR-silver staining and PCR-ELISA methods had the same specificity and sensitivity as the TRAP method.Telomerase activity was detected in the extract of the 10th,20th and 30th passages of P3 cells,while it was absent in fibroblasts. Furthermore, after the 30th generation, the proliferation period of fibroblast cells was significantly prolonged. Telomerase activity and hTERTmRNA were detected in two pancreatic carcinoma cell lines, but were found to be negative in human fibroblast cells. Telomerase activity and hTERTmRNA were tested in pancreatic carcinoma specimens of 24 cases. The telomerase activity was positive in 21 of the 24 cases (87.5 %), and the hTERTmRNA in 20 cases (83.3 %). In adjacent normal tissues positive rates were both 12.5 %. There was a significant difference between the two groups. This indicated a significant correlation between the expression level of telomerase activity and histologic differentiation,metastasis and advanced clinical stage of pancreatic carcinoma. Our findings showed that the expressions of hTR and TP1mRNA were not correlated with the activity of telomerase but the expression of hTERTmRNA was. After treatment with PS-ODNs, telomerase activity in P3 cells weakened and the inhibiting effect became stronger with an increase in PS-ODNs concentration. There was a significant difference between different PS-ODN groups (P<0.05). Inhibition of telomerase activity occurred most significant with PS-ODN1.The results of the FCM test of pancreatic cancer P3 cells showed an increase in the apoptotic rate with increasing PS-ODN1 and PS-ODN2concentrations

  1. Constitutive Expression of Sense & Antisense PtAP3, an AP3 Homologue Gene of Populus tomentosa, Affects Growth and Flowering Time in Transgenic Tobacco

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    To analyze the function of PtAP3, an APETALA3 (AP3) homologue gene isolated from Populus tomentosa Carr., the full length sequence (1 797 bp) and a fragment (870 bp) of PtAP3 were fused to a CaMV 35S promoter of pBI121 to generate the sense and antisense constructs of PtAP3. These constructs were transformed into tobacco by Agrobacterium infection of leaf disks and selection on kanamycin medium. Some sense and antisense transgenic tobacco plants were obtained by PCR and Southern blot analysis. Great phenotypic differences in transgenic tobacco plants were observed. Almost all of sense PtAP3 to transgenic tobaccos showed a higher growth rate than those of antisense transformants and a few developed pregnancy earlier than wild type seedlings and antisense transformants under the same conditions.

  2. Latent membrane protein 1 inhibits apoptosis induced by 60 irradiation via Survivin triggering signal-pathway

    International Nuclear Information System (INIS)

    Objective: To investigate the anti-apoptosis mechanism of EB virus encoden latent membrane protein 1 (LMP1) via the survivin signal transduction pathway after irradiation induction. Methods: Tet-on- LMP1 HNE2 cells, as a model, were detected with morphological assay, flowcytometry and Caspase 3 assay after 60Co irradiation with LMP1 induced by doxycycline. The apoptosis in the anti-sense survivin transfected cells was tested. Results: The results showed that, with LMP1 expression, the apoptosis rates from morphological assay and flowcytometry were 32.7%±2.1% and 6.3%, which showed that they were all lower than that without LMP1 expression (66.0%±3.0% and 29.6%). When anti-sense of survivin was induced, the apoptosis rates were 59.0%±3.2% and 3.0% respectively, and caspase 3 activity was 3.78 nmol/106 cells, which were higher than that of the control (26.0%±2.6%, 8.6% and 2.79 nmol/106). Survivin restrained the cell apoptosis induced by irradiation, but anti-sense of survivin could release this inhibition of cell apoptosis triggered by LMP1 expression. Conclusion: LMP1 inhibits the irradiation-induced cell apoptosis via triggering survivin expression. Survivin may be targeted in some certain therapy

  3. 转反义LeETR2基因番茄的表型%Phenotypes of Transgenic Tomato with Antisense LeETR2

    Institute of Scientific and Technical Information of China (English)

    应铁进; 杨虎清; 向庆宁; 杜荣茂

    2003-01-01

    转反义LeETR2基因番茄植株的表型与普通番茄有所不同.用乙烯25 μL/L处理,转基因番茄能够表现出正常的"三重反应",但根的伸长和根毛形成受到显著抑制.同时,转基因番茄植株对乙烯处理的偏上生长反应敏感度不及普通番茄,叶柄和花柄的脱落被延迟.这几方面的表型特点并不完全一致,我们推测LeETR2在番茄发育的不同阶段可能发挥不同的功能.%Stable transformation of tomato (Lycopersicon esculentum Mill) plants with a construct containing the antisense sequence for the 3′-untranslated portion of the tomato ethylene receptor (encoded by LeETR2) controlled by an enhanced CaMV35S promoter resulted in some expected and unexpected phenotypes. Seedlings (T1) of transgenic plants exposed to ethylene 25 μL/L displayed the normal triple response except that root elongation was strongly inhibited by ethylene and root hair formation was completely inhibited. Much less epinasty in transgenic plants (4 weeks old) is observed after 24 h of exposure to ethylene 25 μL/L in air at 25℃ than that of control. The most typical character of transgenic plants was delayed abscission of leaf petiole and flower. These phenotypes of transgenic plants were not completely uniform. We suppose that LeETR2 gene may display differential functions during the course of tomato development.

  4. Scavenger Receptor-Mediated Delivery of Antisense Mini-Exon Phosphorothioate Oligonucleotide to Leishmania-Infected Macrophages: SELECTIVE AND EFFICIENT ELIMINATION OF THE PARASITE

    OpenAIRE

    Chaudhuri, Gautam

    1997-01-01

    Targeted delivery of a 17-mer antisense phosphorothioate oligodeoxyribonucleotide, complementary to the common 5′-end of every mRNA of the parasite cells, to the phagolysosomes of cultured murine macrophages infected with Leishmania mexicana amazonensis selectively and efficiently eliminated the parasite cells without causing any detectable harm to the host cells. The antisense mini-exon oligonucleotide (ASM) was encapsulated into liposomes coated with maleylated bovine serum albumin (MBSA), ...

  5. An immunostimulatory DNA sequence from a probiotic strain of Bifidobacterium longum inhibits IgE production in vitro.

    Science.gov (United States)

    Takahashi, Noritoshi; Kitazawa, Haruki; Shimosato, Takeshi; Iwabuchi, Noriyuki; Xiao, Jin-Zhong; Iwatsuki, Keiji; Kokubo, Sadayuki; Saito, Tadao

    2006-04-01

    The immunostimulatory oligodeoxynucleotide (ODN) BL07 (5'-GCGTCGGTTTCGGTGCTCAC-3') was identified from the genomic DNA of the probiotic strain Bifidobacterium longum BB536. ODN BL07 stimulated B-lymphocyte proliferation and induced interleukin-12 (IL-12) production in macrophage-like J774.1 cells. ODNs BL07 and BL07S (modified with phosphorothioate backbone) significantly inhibited immunoglobulin E (IgE) production and stimulated interferon-gamma (IFN-gamma) and IL-12 production, but did not affect IL-4 secretion in murine splenic cells of ovalbumin-primed BALB/c mice. These ODNs also significantly inhibited production of IgE in purified murine B cells in the presence of IL-4 and anti-CD40. The results suggest the potential of ODNs BL07 and BL07S in preventing IgE-related immune responses and the possible involvement of ODN BL07 in the antiallergic efficacy of B. longum BB536.

  6. Inhibition of Ly-6A antigen expression prevents T cell activation

    OpenAIRE

    1990-01-01

    Antisense oligonucleotides complementary to the 5' end of the mRNA encoding the Ly-6A protein were used to block the expression of that protein. Using this approach we could inhibit the expression of Ly-6A by 60-80% in antigen-primed lymph node (LN) T cells as well as in the D10 T cell clone. Inhibition of Ly-6 expression resulted in the inability to restimulate in vitro, antigen-primed T cells. It also blocked the activation of normal spleen cells by Con A, monoclonal antibody (mAb) to CD3, ...

  7. Response of immune response genes to adjuvants poly [di(sodium carboxylatoethylphenoxy)phosphazene] (PCEP), CpG oligodeoxynucleotide and emulsigen at intradermal injection site in pigs.

    Science.gov (United States)

    Magiri, R B; Lai, K; Chaffey, A M; Wilson, H L; Berry, W E; Szafron, M L; Mutwiri, G K

    2016-07-01

    Understanding the mechanisms by which adjuvants mediate their effects provide critical information on how innate immunity influences the development of adaptive immunity. Despite being a critical vaccine component, the mechanisms by which adjuvants mediate their effects are not fully understood and this is especially true when they are used in large animals. This lack of understanding limits our ability to design effective vaccines. In the present study, we administered polyphosphazene (PCEP), CpG oligodeoxynucleotides (CpG), emulsigen or saline via an intradermal injection into pigs and assessed the impact on the expression of reported 'adjuvant response genes' over time. CpG induced a strong upregulation of the chemokine CXL10 several 'Interferon Response Genes', as well as TNFα, and IL-10, and a down-regulation of IL-17 genes. Emulsigen upregulated expression of chemokines CCL2 and CCL5, proinflammatory cytokines IL-6 and TNFα, as well as TLR9, and several IFN response genes. PCEP induced the expression of chemokine CCL2 and proinflammatory cytokine IL-6. These results suggest that emulsigen and CpG may promote recruitment of innate immune cells and Th1 type cytokine production but that PCEP may promote a Th-2 type immune response through the induction of IL-6, an inducer of B cell activity and differentiation. PMID:27269793

  8. Transcriptomic Insights into the Response of Placenta and Decidua Basalis to the CpG Oligodeoxynucleotide Stimulation in Non-Obese Diabetic Mice and Wild-Type Controls

    Directory of Open Access Journals (Sweden)

    Xiao-Rui Liu

    2016-08-01

    Full Text Available Intrauterine infection is one of the most frequent causes of miscarriage. CpG oligodeoxynucleotide (CpG ODN can mimic intrauterine infection. CpG ODN-induced embryo-resorption was observed consistently in the NK-cell deficient non-obese diabetic (NOD mice but not in the wild-type (WT mice. To elucidate the molecular mechanisms of differential pregnancy outcomes, differentially expressed genes (DEGs in the placenta and decidua basalis was revealed by RNA-Seq with CpG ODN or control ODN treatment. Common DEGs in the WT and NOD mice were enriched in antimicrobial/antibacterial humoral responses that may be activated as a primary response to bacterial infection. The susceptibility to CpG ODN-induced embryo-resorption in the NOD mice might mainly be attributed to M1 macrophage polarization and the immunodeficient status, such as the down-regulation in antigen processing and presentation, allograft rejection, and natural killer cell mediated cytotoxicity. In contrast, the WT mice with normal immune systems could activate multiple immune responses and be resistant to CpG ODN-induced embryo-resorption, such as M2 macrophage differentiation and activation regulated by complement component C1q and peroxisome proliferation-activated receptor (PPAR signaling pathways. Collectively, this study suggests that the immunodeficient status of NOD mice and the macrophage polarization regulated by C1q and PPAR signaling might be the basis for differential pregnancy outcomes between the NOD and WT mice.

  9. Combining Single Strand Oligodeoxynucleotides and CRISPR/Cas9 to Correct Gene Mutations in β-Thalassemia-induced Pluripotent Stem Cells.

    Science.gov (United States)

    Niu, Xiaohua; He, Wenyin; Song, Bing; Ou, Zhanhui; Fan, Di; Chen, Yuchang; Fan, Yong; Sun, Xiaofang

    2016-08-01

    β-Thalassemia (β-Thal) is one of the most common genetic diseases in the world. The generation of patient-specific β-Thal-induced pluripotent stem cells (iPSCs), correction of the disease-causing mutations in those cells, and then differentiation into hematopoietic stem cells offers a new therapeutic strategy for this disease. Here, we designed a CRISPR/Cas9 to specifically target the Homo sapiens hemoglobin β (HBB) gene CD41/42(-CTTT) mutation. We demonstrated that the combination of single strand oligodeoxynucleotides with CRISPR/Cas9 was capable of correcting the HBB gene CD41/42 mutation in β-Thal iPSCs. After applying a correction-specific PCR assay to purify the corrected clones followed by sequencing to confirm mutation correction, we verified that the purified clones retained full pluripotency and exhibited normal karyotyping. Additionally, whole-exome sequencing showed that the mutation load to the exomes was minimal after CRISPR/Cas9 targeting. Furthermore, the corrected iPSCs were selected for erythroblast differentiation and restored the expression of HBB protein compared with the parental iPSCs. This method provides an efficient and safe strategy to correct the HBB gene mutation in β-Thal iPSCs. PMID:27288406

  10. Down-regulation of protein kinase Ceta by antisense oligonucleotides sensitises A549 lung cancer cells to vincristine and paclitaxel.

    Science.gov (United States)

    Sonnemann, Jürgen; Gekeler, Volker; Ahlbrecht, Katrin; Brischwein, Klaus; Liu, Chao; Bader, Peter; Müller, Cornelia; Niethammer, Dietrich; Beck, James F

    2004-06-25

    Previous studies point to protein kinase C (PKC) isozyme eta as a resistance factor in cancer cells. Therefore, we investigated whether down-regulation of PKCeta with second generation antisense oligonucleotides (ODNs) would sensitise A549 human lung carcinoma cells to cytostatics. The effects were compared to the outcome of Bcl-xL down-regulation. Upon treatment with antisense ODNs, PKCeta and Bcl-xL were both significantly reduced on mRNA and protein level. Down-regulation of either PKCeta or Bcl-xL in combination with vincristine or paclitaxel resulted in a significant increase in caspase-3 activity compared to that in the control oligonucleotide treated cells. In addition, PKCeta down-regulation augmented vincristine-induced dissipation of mitochondrial transmembrane potential. In conclusion, these results confirm that PKCeta might represent a considerable resistance factor and an interesting target to improve anticancer chemotherapy. PMID:15159020

  11. Optimization of Peptide Nucleic Acid Antisense Oligonucleotides for Local and Systemic Dystrophin Splice Correction in the mdx Mouse

    OpenAIRE

    Yin, Haifang; Betts, Corinne; Saleh, Amer F; Ivanova, Gabriela D; Lee, Hyunil; Seow, Yiqi; Kim, Dalsoo; Gait, Michael J.; Wood, Matthew JA

    2010-01-01

    Antisense oligonucleotides (AOs) have the capacity to alter the processing of pre-mRNA transcripts in order to correct the function of aberrant disease-related genes. Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle degenerative disease that arises from mutations in the DMD gene leading to an absence of dystrophin protein. AOs have been shown to restore the expression of functional dystrophin via splice correction by intramuscular and systemic delivery in animal models of DMD and ...

  12. Antisense bcl-2 treatment increases programmed cell death in non-small cell lung cancer cell lines.

    Science.gov (United States)

    Koty, P P; Zhang, H; Levitt, M L

    1999-02-01

    Programmed cell death (PCD) is a genetically regulated pathway that is altered in many cancers. This process is, in part, regulated by the ratio of PCD inducers (Bax) or inhibitors (Bcl-2). An abnormally high ratio of Bcl-2 to Bax prevents PCD, thus contributing to resistance to chemotherapeutic agents, many of which are capable of inducing PCD. Non-small cell lung cancer (NSCLC) cells demonstrate resistance to these PCD-inducing agents. If Bcl-2 prevents NSCLC cells from entering the PCD pathway, then reducing the amount of endogenous Bcl-2 product may allow these cells to spontaneously enter the PCD pathway. Our purpose was to determine the effects of bcl-2 antisense treatment on the levels of programmed cell death in NSCLC cells. First, we determined whether bcl-2 and bax mRNA were expressed in three morphologically distinct NSCLC cell lines: NCI-H226 (squamous), NCI-H358 (adenocarcinoma), and NCI-H596 (adenosquamous). Cells were then exposed to synthetic antisense bcl-2 oligonucleotide treatment, after which programmed cell death was determined, as evidenced by DNA fragmentation. Bcl-2 protein expression was detected immunohistochemically. All three NSCLC cell lines expressed both bcl-2 and bax mRNA and had functional PCD pathways. Synthetic antisense bcl-2 oligonucleotide treatment resulted in decreased Bcl-2 levels, reduced cell proliferation, decreased cell viability, and increased levels of spontaneous PCD. This represents the first evidence that decreasing Bcl-2 in three morphologically distinct NSCLC cell lines allows the cells to spontaneously enter a PCD pathway. It also indicates the potential therapeutic use of antisense bcl-2 in the treatment of NSCLC. PMID:10217615

  13. Cationic polyelectrolyte-mediated delivery of antisense morpholino oligonucleotides for exon-skipping in vitro and in mdx mice

    OpenAIRE

    Wang, Mingxing; Wu, Bo; Tucker, Jason D; Lu, Peijuan; Lu, Qilong

    2015-01-01

    In this study, we investigated a series of cationic polyelectrolytes (PEs) with different size and composition for their potential to improve delivery of an antisense phosphorodiamidate morpholino oligomer (PMO) both in vitro and in vivo. The results showed that the poly(diallyldimethylammonium chloride) (PDDAC) polymer series, especially PE-3 and PE-4, improves the delivery efficiency of PMO, comparable with Endoporter-mediated PMO delivery in vitro. The enhanced PMO delivery and targeting t...

  14. Antisense expression of a rice cellular apoptosis susceptibility gene (OsCAS) alters the height of transgenic rice

    Institute of Scientific and Technical Information of China (English)

    XU Chunxiao; HE Chaozu

    2007-01-01

    Cellular apoptosis susceptibility (CAS) gene plays important roles in mitosis, development and export of importin αfrom the nucleus, but its function in plant is unknown. In this study, a rice CAS ortholog (OsCAS), which encodes a predicted protein of 983 amino acids with 62% similarity to human CAS, was identified. DNA gel blot analysis revealed a single copy of OsCAS in the rice genome. A 973 bp fragment at the 3' end of OsCAS cDNA was cloned from rice cDNA library and transferred into rice in the antisense direction under the control of CaMV 35S promoter via Agrobacterium-mediated transformation method, 105 transgenic lines were obtained. Expression of OsCAS was suppressed in the antisense transgenic lines as revealed by semi-quantitative RT-PCR. The antisense transgenic lines showed dwarf phenotypes. The results indicated that OsCAS was involved in culm development of rice.

  15. Antisense suppression of an acid invertase gene (MAI1) in muskmelon alters plant growth and fruit development.

    Science.gov (United States)

    Yu, Xiyan; Wang, Xiufeng; Zhang, Wenqian; Qian, Tingting; Tang, Guimin; Guo, Yankui; Zheng, Chengchao

    2008-01-01

    To unravel the roles of soluble acid invertase in muskmelon (Cucumis melo L.), its activity in transgenic muskmelon plants was reduced by an antisense approach. For this purpose, a 1038 bp cDNA fragment of muskmelon soluble acid invertase was expressed in antisense orientation behind the 35S promoter of the cauliflower mosaic virus. The phenotype of the antisense plants clearly differed from that of control plants. The transgenic plant leaves were markedly smaller, and the stems were obviously thinner. Transmission electron microscopy revealed that degradation of the chloroplast membrane occurred in transgenic leaves and the number of grana in the chloroplast was significantly reduced, suggesting that the slow growth and weaker phenotype of the transgenic plants may be due to damage to the chloroplast ultrastructure, which in turn resulted in a decrease in net photosynthetic rate. The sucrose concentration increased and levels of acid invertase decreased in transgenic fruit, and the fruit size was 60% smaller than that of the control. In addition, transgenic fruit reached full-slip at 25 d after pollination (DAP), approximately 5 d before the control fruit (full-slip at 30 DAP), and this accelerated maturity correlated with a dramatic elevation of ethylene production at the later stages of fruit development. Together, these results suggest that soluble acid invertase not only plays an important role during muskmelon plant and fruit development but also controls the sucrose content in muskmelon fruit.

  16. Knockdown of gene expression by antisense morpholino oligos in preimplantation mouse embryos cultured in vitro.

    Science.gov (United States)

    Sato, Yuki; Sato, Shiori; Kikuchi, Takahiro; Nonaka, Asumi; Kumagai, Yuki; Sasaki, Akira; Kobayashi, Masayuki

    2016-09-15

    Knockdown of gene expression by antisense morpholino oligos (MOs) is a simple and effective method for analyzing the roles of genes in mammalian cells. Here, we demonstrate the efficient delivery of MOs by Endo-Porter (EP), a special transfection reagent for MOs, into preimplantation mouse embryos cultured in vitro. A fluorescein-labeled control MO was applied for monitoring the incorporation of MOs into developing 2-cell embryos in the presence of varying amounts of EP and bovine serum albumin. In optimized conditions, fluorescence was detected in 2-cell embryos within a 3-h incubation period. In order to analyze the validity of the optimized conditions, an antisense Oct4 MO was applied for knockdown of the synthesis of OCT4 protein in developing embryos from the 2-cell stage. In blastocysts, the antisense Oct4 MO induced a decrease in the amount in OCT4 protein to less than half. An almost complete absence of OCT4-positive cells and nearly complete disappearance of the inner cell mass in the outgrowths of blastocysts were also noted. These phenotypes corresponded with those of Oct4-deficient mouse embryos. Overall, we suggest that the delivery of MOs using EP is useful for the knockdown of gene expression in preimplantation mouse embryos cultured in vitro. PMID:27381842

  17. Inhibitory Effects of Anti-sense PTTG on Malignant Phenotype of Human Ovarian Carcinoma Cell Line SK-OV-3

    Institute of Scientific and Technical Information of China (English)

    陈刚; 李静; 李辅军; 李箫; 周剑锋; 卢运萍; 马丁

    2004-01-01

    To construct eukaryotic expression vector expressing full length anti-sense pituitary tumor transforming gene (PTTG) mRNA and observe its blocking effect on the potential invasion of human ovarian carcinoma cell line SK-OV-3. PCR primers containing designed enzyme cut sites were used for cloning full-length PTTG gene fragment, and the resulting PCR product was inserted into the eukaryotic vector pcDNA3. 1 in the antisense direction. The recombinant vector was then transfected into SK-OV-3 by Lipofectamine. The positive cell clone was screened by G418, PTTG and bFGF at protein level expression were detected by Western blot. The biological behavior change of transfection positive cells was observed by colony formation in soft agar assay. Our results showed that SK-OV-3 clones stably expressing full-length recombinant pcDNA3. 1-PTTGas were obtained. The expressions of PTTG and bFGF protein in transfected cells were decreased by 61.5 % and 52.3%, respectively as compared with non-transfected ones. The number of colony formation was reduced significantly in transfected cells as compared with empty vector transfected and non-transfected cells. It is concluded that the recombinant vector pcDNA3. 1-PTTGas is a novel tool and provides an alternative anti-sense gene therapy targeted at PTTG in human carcinoma.

  18. Antisense mRNA for NPY-Y1 receptor in the medial preoptic area increases prolactin secretion

    Directory of Open Access Journals (Sweden)

    N.A. Silveira

    1999-09-01

    Full Text Available We investigated the participation of neuropeptide Y-Y1 receptors within the medial preoptic area in luteinizing hormone, follicle-stimulating hormone and prolactin release. Four bilateral microinjections of sense (control or antisense 18-base oligonucleotides of messenger ribonucleic acid (mRNA (250 ng corresponding to the NH2-terminus of the neuropeptide Y1 receptor were performed at 12-h intervals for two days into the medial preoptic area of ovariectomized Wistar rats (N = 16, weighing 180 to 200 g, treated with estrogen (50 µg and progesterone (25 mg two days before the experiments between 8.00 and 10:00 a.m. Blockade of Y1 receptor synthesis in the medial preoptic area by the antisense mRNA did not change plasma luteinizing hormone or follicle-stimulating hormone but did increase prolactin from 19.6 ± 5.9 ng/ml in the sense group to 52.9 ± 9.6 ng/ml in the antisense group. The plasma hormones were measured by radioimmunoassay and the values are reported as mean ± SEM. These data suggest that endogenous neuropeptide Y in the medial preoptic area has an inhibitory action on prolactin secretion through Y1 receptors.

  19. Inhibition of DNA glycosylases via small molecule purine analogs.

    Directory of Open Access Journals (Sweden)

    Aaron C Jacobs

    Full Text Available Following the formation of oxidatively-induced DNA damage, several DNA glycosylases are required to initiate repair of the base lesions that are formed. Recently, NEIL1 and other DNA glycosylases, including OGG1 and NTH1 were identified as potential targets in combination chemotherapeutic strategies. The potential therapeutic benefit for the inhibition of DNA glycosylases was validated by demonstrating synthetic lethality with drugs that are commonly used to limit DNA replication through dNTP pool depletion via inhibition of thymidylate synthetase and dihydrofolate reductase. Additionally, NEIL1-associated synthetic lethality has been achieved in combination with Fanconi anemia, group G. As a prelude to the development of strategies to exploit the potential benefits of DNA glycosylase inhibition, it was necessary to develop a reliable high-throughput screening protocol for this class of enzymes. Using NEIL1 as the proof-of-principle glycosylase, a fluorescence-based assay was developed that utilizes incision of site-specifically modified oligodeoxynucleotides to detect enzymatic activity. This assay was miniaturized to a 1536-well format and used to screen small molecule libraries for inhibitors of the combined glycosylase/AP lyase activities. Among the top hits of these screens were several purine analogs, whose postulated presence in the active site of NEIL1 was consistent with the paradigm of NEIL1 recognition and excision of damaged purines. Although a subset of these small molecules could inhibit other DNA glycosylases that excise oxidatively-induced DNA adducts, they could not inhibit a pyrimidine dimer-specific glycosylase.

  20. Tuning growth cycles of Brassica crops via natural antisense transcripts of BrFLC.

    Science.gov (United States)

    Li, Xiaorong; Zhang, Shaofeng; Bai, Jinjuan; He, Yuke

    2016-03-01

    Several oilseed and vegetable crops of Brassica are biennials that require a prolonged winter cold for flowering, a process called vernalization. FLOWERING LOCUS C (FLC) is a central repressor of flowering. Here, we report that the overexpression of natural antisense transcripts (NATs) of Brassica rapa FLC (BrFLC) greatly shortens plant growth cycles. In rapid-, medium- and slow-cycling crop types, there are four copies of the BrFLC genes, which show extensive variation in sequences and expression levels. In Bre, a biennial crop type that requires vernalization, five NATs derived from the BrFLC2 locus are rapidly induced under cold conditions, while all four BrFLC genes are gradually down-regulated. The transgenic Bre lines overexpressing a long NAT of BrFLC2 do not require vernalization, resulting in a gradient of shortened growth cycles. Among them, a subset of lines both flower and set seeds as early as Yellow sarson, an annual crop type in which all four BrFLC genes have non-sense mutations and are nonfunctional in flowering repression. Our results demonstrate that the growth cycles of biennial crops of Brassica can be altered by changing the expression levels of BrFLC2 NATs. Thus, BrFLC2 NATs and their transgenic lines are useful for the genetic manipulation of crop growth cycles. PMID:26250982

  1. Ribonuclease H1-dependent hepatotoxicity caused by locked nucleic acid-modified gapmer antisense oligonucleotides.

    Science.gov (United States)

    Kasuya, Takeshi; Hori, Shin-Ichiro; Watanabe, Ayahisa; Nakajima, Mado; Gahara, Yoshinari; Rokushima, Masatomo; Yanagimoto, Toru; Kugimiya, Akira

    2016-01-01

    Gapmer antisense oligonucleotides cleave target RNA effectively in vivo, and is considered as promising therapeutics. Especially, gapmers modified with locked nucleic acid (LNA) shows potent knockdown activity; however, they also cause hepatotoxic side effects. For developing safe and effective gapmer drugs, a deeper understanding of the mechanisms of hepatotoxicity is required. Here, we investigated the cause of hepatotoxicity derived from LNA-modified gapmers. Chemical modification of gapmer's gap region completely suppressed both knockdown activity and hepatotoxicity, indicating that the root cause of hepatotoxicity is related to intracellular gapmer activity. Gene silencing of hepatic ribonuclease H1 (RNaseH1), which catalyses gapmer-mediated RNA knockdown, strongly supressed hepatotoxic effects. Small interfering RNA (siRNA)-mediated knockdown of a target mRNA did not result in any hepatotoxic effects, while the gapmer targeting the same position on mRNA as does the siRNA showed acute toxicity. Microarray analysis revealed that several pre-mRNAs containing a sequence similar to the gapmer target were also knocked down. These results suggest that hepatotoxicity of LNA gapmer is caused by RNAseH1 activity, presumably because of off-target cleavage of RNAs inside nuclei. PMID:27461380

  2. Global transcriptional start site mapping using differential RNA sequencing reveals novel antisense RNAs in Escherichia coli.

    Science.gov (United States)

    Thomason, Maureen K; Bischler, Thorsten; Eisenbart, Sara K; Förstner, Konrad U; Zhang, Aixia; Herbig, Alexander; Nieselt, Kay; Sharma, Cynthia M; Storz, Gisela

    2015-01-01

    While the model organism Escherichia coli has been the subject of intense study for decades, the full complement of its RNAs is only now being examined. Here we describe a survey of the E. coli transcriptome carried out using a differential RNA sequencing (dRNA-seq) approach, which can distinguish between primary and processed transcripts, and an automated prediction algorithm for transcriptional start sites (TSS). With the criterion of expression under at least one of three growth conditions examined, we predicted 14,868 TSS candidates, including 5,574 internal to annotated genes (iTSS) and 5,495 TSS corresponding to potential antisense RNAs (asRNAs). We examined expression of 14 candidate asRNAs by Northern analysis using RNA from wild-type E. coli and from strains defective for RNases III and E, two RNases reported to be involved in asRNA processing. Interestingly, nine asRNAs detected as distinct bands by Northern analysis were differentially affected by the rnc and rne mutations. We also compared our asRNA candidates with previously published asRNA annotations from RNA-seq data and discuss the challenges associated with these cross-comparisons. Our global transcriptional start site map represents a valuable resource for identification of transcription start sites, promoters, and novel transcripts in E. coli and is easily accessible, together with the cDNA coverage plots, in an online genome browser.

  3. A lignin-specific peroxidase in tobacco whose antisense suppression leads to vascular tissue modification

    Science.gov (United States)

    Blee, Kristopher A.; Choi, Joon W.; O'Connell, Ann P.; Schuch, Wolfgang; Lewis, Norman G.; Bolwell, G. Paul

    2003-01-01

    A tobacco peroxidase isoenzyme (TP60) was down-regulated in tobacco using an antisense strategy, this affording transformants with lignin reductions of up to 40-50% of wild type (control) plants. Significantly, both guaiacyl and syringyl levels decreased in essentially a linear manner with the reductions in lignin amounts, as determined by both thioacidolysis and nitrobenzene oxidative analyses. These data provisionally suggest that a feedback mechanism is operative in lignifying cells, which prevents build-up of monolignols should oxidative capacity for their subsequent metabolism be reduced. Prior to this study, the only known rate-limiting processes in the monolignol/lignin pathways involved that of Phe supply and the relative activities of cinnamate-4-hydroxylase/p-coumarate-3-hydroxylase, respectively. These transformants thus provide an additional experimental means in which to further dissect and delineate the factors involved in monolignol targeting to precise regions in the cell wall, and of subsequent lignin assembly. Interestingly, the lignin down-regulated tobacco phenotypes displayed no readily observable differences in overall growth and development profiles, although the vascular apparatus was modified.

  4. Serial incorporation of a monovalent GalNAc phosphoramidite unit into hepatocyte-targeting antisense oligonucleotides.

    Science.gov (United States)

    Yamamoto, Tsuyoshi; Sawamura, Motoki; Wada, Fumito; Harada-Shiba, Mariko; Obika, Satoshi

    2016-01-01

    The targeting of abundant hepatic asialoglycoprotein receptors (ASGPR) with trivalent N-acetylgalactosamine (GalNAc) is a reliable strategy for efficiently delivering antisense oligonucleotides (ASOs) to the liver. We here experimentally demonstrate the high systemic potential of the synthetically-accessible, phosphodiester-linked monovalent GalNAc unit when tethered to the 5'-terminus of well-characterised 2',4'-bridged nucleic acid (also known as locked nucleic acid)-modified apolipoprotein B-targeting ASO via a bio-labile linker. Quantitative analysis of the hepatic disposition of the ASOs revealed that phosphodiester is preferable to phosphorothioate as an interunit linkage in terms of ASGPR binding of the GalNAc moiety, as well as the subcellular behavior of the ASO. The flexibility of this monomeric unit was demonstrated by attaching up to 5 GalNAc units in a serial manner and showing that knockdown activity improves as the number of GalNAc units increases. Our study suggests the structural requirements for efficient hepatocellular targeting using monovalent GalNAc and could contribute to a new molecular design for suitably modifying ASO.

  5. Evaluation of 2'-Deoxy-2'-fluoro Antisense Oligonucleotides for Exon Skipping in Duchenne Muscular Dystrophy

    Science.gov (United States)

    Jirka, Silvana M G; Tanganyika-de Winter, Christa L; Boertje-van der Meulen, Joke W; van Putten, Maaike; Hiller, Monika; Vermue, Rick; de Visser, Peter C; Aartsma-Rus, Annemieke

    2015-01-01

    Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder typically caused by frame-shifting mutations in the DMD gene. Restoration of the reading frame would allow the production of a shorter but partly functional dystrophin protein as seen in Becker muscular dystrophy patients. This can be achieved with antisense oligonucleotides (AONs) that induce skipping of specific exons during pre-mRNA splicing. Different chemical modifications have been developed to improve AON properties. The 2'-deoxy-2'-fluoro (2F) RNA modification is attractive for exon skipping due to its ability to recruit ILF2/3 proteins to the 2F/pre-mRNA duplex, which resulted in enhanced exon skipping in spinal muscular atrophy models. In this study, we examined the effect of two different 2'-substituted AONs (2'-F phosphorothioate (2FPS) and 2'-O-Me phosphorothioate (2OMePS)) on exon skipping in DMD cell and animal models. In human cell cultures, 2FPS AONs showed higher exon skipping levels than their isosequential 2OMePS counterparts. Interestingly, in the mdx mouse model, 2FPS was less efficient than 2OMePS and suggested safety issues as evidenced by increased spleen size and weight loss. Our results do not support a clinical application for 2FPS AON. PMID:26623937

  6. Optimizing RNA/ENA chimeric antisense oligonucleotides using in vitro splicing.

    Science.gov (United States)

    Takeshima, Yasuhiro; Yagi, Mariko; Matsuo, Masafumi

    2012-01-01

    A molecular therapy for Duchenne muscular dystrophy (DMD) that converts dystrophin mRNA from out-of-frame to in-frame transcripts by inducing exon skipping with antisense oligonucleotides (AOs) is now approaching clinical application. To exploit the broad therapeutic applicability of exon skipping therapy, it is necessary to identify AOs that are able to induce efficient and specific exon skipping. To optimize AOs, we have established an in vitro splicing system using cultured DMD myocytes. Here, we describe the process of identifying the best AO.Cultured DMD myocytes are established from a biopsy sample and the target exon is chosen. A series of AOs are designed to cover the whole target exon sequence. As AOs, we use 15-20-mer chimeric oligonucleotides consisting of 2'-O-methyl RNA and modified nucleic acid (2'-O, 4'-C-ethylene-bridged nucleic acid). Each AO is transfected individually into cultured DMD myocytes, and the resulting mRNA is analyzed by reverse transcription-PCR. The ability of each AO to induce exon skipping is examined by comparing the amount of cDNA with and without exon skipping. If necessary, having roughly localized the target region, another set of AOs are designed and the exon skipping abilities of the new AOs are examined. Finally, one AO is determined as the best for the molecular therapy.Our simple and reliable methods using an in vitro splicing system have enabled us to identify optimized AOs against many exons of the DMD gene.

  7. RNA synthetic biology inspired from bacteria: construction of transcription attenuators under antisense regulation

    International Nuclear Information System (INIS)

    Among all biopolymers, ribonucleic acids or RNA have unique functional versatility, which led to the early suggestion that RNA alone (or a closely related biopolymer) might have once sustained a primitive form of life based on a single type of biopolymer. This has been supported by the demonstration of processive RNA-based replication and the discovery of 'riboswitches' or RNA switches, which directly sense their metabolic environment. In this paper, we further explore the plausibility of this 'RNA world' scenario and show, through synthetic molecular design guided by advanced RNA simulations, that RNA can also perform elementary regulation tasks on its own. We demonstrate that RNA synthetic regulatory modules directly inspired from bacterial transcription attenuators can efficiently activate or repress the expression of other RNA by merely controlling their folding paths 'on the fly' during transcription through simple RNA–RNA antisense interaction. Factors, such as NTP concentration and RNA synthesis rate, affecting the efficiency of this kinetic regulation mechanism are also studied and discussed in the light of evolutionary constraints. Overall, this suggests that direct coupling among synthesis, folding and regulation of RNAs may have enabled the early emergence of autonomous RNA-based regulation networks in absence of both DNA and protein partners

  8. Thiolated polycarbophil as an adjuvant for permeation enhancement in nasal delivery of antisense oligonucleotides.

    Science.gov (United States)

    Vetter, A; Martien, R; Bernkop-Schnürch, A

    2010-03-01

    The purpose of this study was to investigate the effect of thiolated polycarbophil as an adjuvant to enhance the permeation and improve the stability of a phosphorothioate antisense oligonucleotide (PTO-ODN) on the nasal mucosa. Polycarbophil-cysteine (PCP-Cys) was synthesized by the covalent attachment of L-cysteine to the polymeric backbone. Cytotoxicity tests were examined on human nasal epithelial cells from surgery of nasal polyps confirmed by histological studies. Deoxyribonuclease I activity in respiratory region of the porcine nasal cavity was analyzed by an enzymatic assay. The enzymatic degradation of PTO-ODNs on freshly excised porcine nasal mucosa was analyzed and protection of PCP-cysteine toward DNase I degradation was evaluated. Permeation studies were performed in Ussing-type diffusion chambers. PCP-Cys/GSH did not arise a remarkable mortal effect. Porcine respiratory mucosa was shown to possess nuclease activity corresponding to 0.69 Kunitz units/mL. PTO-ODNs were degraded by incubation with nasal mucosa. In the presence of 0.45% thiolated polycarbophil and 0.5% glutathione (GSH), this degradation process could be lowered. In the presence of thiolated polycarbophil and GSH the uptake of PTO-ODNs from the nasal mucosa was 1.7-fold improved. According to these results thiolated polycarbophil/GSH might be a promising excipient for nasal administration of PTO-ODNs.

  9. Programmable control of bacterial gene expression with the combined CRISPR and antisense RNA system.

    Science.gov (United States)

    Lee, Young Je; Hoynes-O'Connor, Allison; Leong, Matthew C; Moon, Tae Seok

    2016-03-18

    A central goal of synthetic biology is to implement diverse cellular functions by predictably controlling gene expression. Though research has focused more on protein regulators than RNA regulators, recent advances in our understanding of RNA folding and functions have motivated the use of RNA regulators. RNA regulators provide an advantage because they are easier to design and engineer than protein regulators, potentially have a lower burden on the cell and are highly orthogonal. Here, we combine the CRISPR system from Streptococcus pyogenes and synthetic antisense RNAs (asRNAs) in Escherichia coli strains to repress or derepress a target gene in a programmable manner. Specifically, we demonstrate for the first time that the gene target repressed by the CRISPR system can be derepressed by expressing an asRNA that sequesters a small guide RNA (sgRNA). Furthermore, we demonstrate that tunable levels of derepression can be achieved (up to 95%) by designing asRNAs that target different regions of a sgRNA and by altering the hybridization free energy of the sgRNA-asRNA complex. This new system, which we call the combined CRISPR and asRNA system, can be used to reversibly repress or derepress multiple target genes simultaneously, allowing for rational reprogramming of cellular functions.

  10. Development and Substance Accumulation of Caryopsis in Transgenic Rice with Antisense Wx Gene

    Institute of Scientific and Technical Information of China (English)

    CHEN Gang; WANG Zhong; LIU Qiao-quan; KIONG Fei; GU Yun-jie; GU Guo-jun

    2006-01-01

    The development and substance accumulation of rice caryopsis were studied by using the transgenic japonica and indica rice with antisense Wx gene. The weight of caryopses in transgenic rice was lower than that in non-transgenic one, and the reduction in weight was significantly correlated to the reduction in amylose content. In caryopsis of transgenic rice, the number of endosperm cells was less than that in caryopsis of non-transgenic one, but the proliferation speed was considerably higher during the first six days after flowering (DAF). During the first nine DAF, the soluble sugar content of transgenic rice caryopsis was less than that of the non-transgenic one, but the situation was reverse after nine DAF. Moreover, the total starch content also declined with the decrease in amylose content of transgenic rice caryopsis, while the amylopectin content increased accordingly. Therefore, the composition of starch in caryopsis also changed, but it did not affect the accumulation of protein in transgenic rice caryopsis.

  11. A palindromic CpG-containing phosphodiester oligodeoxynucleotide as a mucosal adjuvant stimulates plasmacytoid dendritic cell-mediated T(H1 immunity.

    Directory of Open Access Journals (Sweden)

    Jun-ichi Maeyama

    Full Text Available BACKGROUND: CpG oligodeoxynucleotides (ODNs, resembling bacterial DNA, are currently tested in clinical trials as vaccine adjuvants. They have the nuclease-resistant phosphorothioate bond; the immune responses elicited differ according to the CpG ODN sequence and vaccination method. To develop a CpG ODN that can induce plasmacytoid dendritic cell (pDC-mediated T(H1 immunity through the mucosa, we constructed phosphodiester G9.1 comprising one palindromic CpG motif with unique polyguanosine-runs that allows degradation similar to naturally occurring bacterial DNA. METHODS: T(H1 and T(H2 immunity activation was evaluated by cytokine production pattern and T-bet/GATA-3 ratio in human peripheral blood mononuclear cells and mouse bone marrow cells. Adjuvanticity was evaluated in mice administered G9.1 with diphtheria toxoid (DT through nasal vaccination. RESULTS: G9.1 exhibited stronger IFN-α-inducing activity than A-class CpG ODN2216 and increased T-bet/GATA-3 ratio by enhancing T-bet expression. Nasally administered G9.1 plus DT induced DT-specific mucosal IgA and serum IgG, but not IgE, responses with antitoxin activity in C57BL/6 and BALB/c mice, possibly due to IFN/BAFF production. Induction of T(H1, but not T(H2-type Abs depended completely on pDCs, the first in vivo demonstration by CpG ODNs. CONCLUSIONS: G9.1 is a promising mucosal adjuvant for induction of pDC-mediated T(H1 immunity.

  12. Modulatory effect of CpG oligodeoxynucleotide on a DNA vaccine against nervous necrosis virus in orange-spotted grouper (Epinephelus coioides).

    Science.gov (United States)

    Chen, Shiang-Peng; Peng, Ran-Hong; Chiou, Pinwen P

    2015-08-01

    We report the development of a DNA vaccine pcMGNNV2 against nervous necrosis virus (NNV), a leading cause of mass mortality in grouper larvae. In addition, the modulatory effect of CpG oligodeoxynucleotide (ODN), a Toll-like receptor 9 agonist, on the DNA vaccine was evaluated. The DNA vaccine alone elicited the production of NNV-specific antibodies, indicating that the vaccine was capable of triggering adaptive humoral response. Furthermore, significant induction of TLR9, Mx and IL-1β was observed in the spleen on day 7 post-vaccination, supporting that the vaccine could trigger TLR9 signaling. The incorporation of CpG ODN at high dose did not significantly affect the level of NNV-specific antibodies, but was able to moderately enhance the expression of Mx and IL-1β on day 7, indicating its ability in modulating innate response. After challenge with NNV, the vaccine alone enhanced the survival rate in infected larvae at both 1 and 2 weeks post-vaccination. The combination of CpG ODN further increased the survival rate at week 1 but not week 2. Interestingly, at week 2 the ODN appeared to induce a Th1-like response, as indicated by upregulation of T-bet (a Th1 marker) and downregulation of GATA-3 (a Th2 marker). Thus, the results suggest that the boosted Th1 response by CpG ODN does not augment the protection efficacy of pcMGNNV2 vaccine. To our best knowledge, this is the first report of a successful DNA vaccine against NNV in grouper.

  13. Gibberellic Acid, Synthetic Auxins, and Ethylene Differentially Modulate α-l-Arabinofuranosidase Activities in Antisense 1-Aminocyclopropane-1-Carboxylic Acid Synthase Tomato Pericarp Discs1

    Science.gov (United States)

    Sozzi, Gabriel O.; Greve, L. Carl; Prody, Gerry A.; Labavitch, John M.

    2002-01-01

    α-l-Arabinofuranosidases (α-Afs) are plant enzymes capable of releasing terminal arabinofuranosyl residues from cell wall matrix polymers, as well as from different glycoconjugates. Three different α-Af isoforms were distinguished by size exclusion chromatography of protein extracts from control tomatoes (Lycopersicon esculentum) and an ethylene synthesis-suppressed (ESS) line expressing an antisense 1-aminocyclopropane-1-carboxylic synthase transgene. α-Af I and II are active throughout fruit ontogeny. α-Af I is the first Zn-dependent cell wall enzyme isolated from tomato pericarp tissues, thus suggesting the involvement of zinc in fruit cell wall metabolism. This isoform is inhibited by 1,10-phenanthroline, but remains stable in the presence of NaCl and sucrose. α-Af II activity accounts for over 80% of the total α-Af activity in 10-d-old fruit, but activity drops during ripening. In contrast, α-Af III is ethylene dependent and specifically active during ripening. α-Af I released monosaccharide arabinose from KOH-soluble polysaccharides from tomato cell walls, whereas α-Af II and III acted on Na2CO3-soluble pectins. Different α-Af isoform responses to gibberellic acid, synthetic auxins, and ethylene were followed by using a novel ESS mature-green tomato pericarp disc system. α-Af I and II activity increased when gibberellic acid or 2,4-dichlorophenoxyacetic acid was applied, whereas ethylene treatment enhanced only α-Af III activity. Results suggest that tomato α-Afs are encoded by a gene family under differential hormonal controls, and probably have different in vivo functions. The ESS pericarp explant system allows comprehensive studies involving effects of physiological levels of different growth regulators on gene expression and enzyme activity with negligible wound-induced ethylene production. PMID:12114586

  14. Comparison of hepatic transcription profiles of locked ribonucleic acid antisense oligonucleotides: evidence of distinct pathways contributing to non-target mediated toxicity in mice.

    Science.gov (United States)

    Kakiuchi-Kiyota, Satoko; Koza-Taylor, Petra H; Mantena, Srinivasa R; Nelms, Linda F; Enayetallah, Ahmed E; Hollingshead, Brett D; Burdick, Andrew D; Reed, Lori A; Warneke, James A; Whiteley, Lawrence O; Ryan, Anne M; Mathialagan, Nagappan

    2014-03-01

    Development of LNA gapmers, antisense oligonucleotides used for efficient inhibition of target RNA expression, is limited by non-target mediated hepatotoxicity issues. In the present study, we investigated hepatic transcription profiles of mice administered non-toxic and toxic LNA gapmers. After repeated administration, a toxic LNA gapmer (TS-2), but not a non-toxic LNA gapmer (NTS-1), caused hepatocyte necrosis and increased serum alanine aminotransferase levels. Microarray data revealed that, in addition to gene expression patterns consistent with hepatotoxicity, 17 genes in the clathrin-mediated endocytosis (CME) pathway were altered in the TS-2 group. TS-2 significantly down-regulated myosin 1E (Myo1E), which is involved in release of clathrin-coated pits from plasma membranes. To map the earliest transcription changes associated with LNA gapmer-induced hepatotoxicity, a second microarray analysis was performed using NTS-1, TS-2, and a severely toxic LNA gapmer (HTS-3) at 8, 16, and 72 h following a single administration in mice. The only histopathological change observed was minor hepatic hypertrophy in all LNA groups across time points. NTS-1, but not 2 toxic LNA gapmers, increased immune response genes at 8 and 16 h but not at 72 h. TS-2 significantly perturbed the CME pathway only at 72 h, while Myo1E levels were decreased at all time points. In contrast, HTS-3 modulated DNA damage pathway genes at 8 and 16 h and also modulated the CME pathway genes (but not Myo1E) at 16 h. Our results may suggest that different LNAs modulate distinct transcriptional genes and pathways contributing to non-target mediated hepatotoxicity in mice.

  15. HIF-1α反义寡核苷酸对骨肉瘤细胞系MG-63 HIF-1α和VEGF表达的影响%Effects of HIF-1α Antisense Oligonucleotides on the Expression of HIF-1α and VEGF in the Osteosarcoma Cell Line MG-63

    Institute of Scientific and Technical Information of China (English)

    蔡文涛; 陈安民; 郭风劲; 朱波

    2006-01-01

    目的:观察缺氧诱导因子HIF-1α反义寡核苷酸(antisense oligonucleotides,ASODN)在缺氧环境下对骨肉瘤细胞系MG-63 HIF-1α和血管内皮生长因子VEGF表达的影响.方法:设计针对HIF-1αRNA亚基模板序列的反义、正义寡核苷酸(sense oligodeoxynucleotides,SODN),并建立骨肉瘤细胞体外缺氧培养模型,观察缺氧培养不同时相HIF-1α反义寡核苷酸对骨肉瘤细胞系MG-63 HIF-1α和VEGF的表达的影响.RT-PCR方法检测HIF-1α和VEGF mRNA水平,免疫组化和免疫印迹方法检测HIF-1α和VEGF蛋白表达情况.结果:与常氧组比较,单纯缺氧组及SODN缺氧组的HIF-1α转录水平未见明显改变,蛋白表达水平却随缺氧时间延长明显升高;而VEGF mRNA以及蛋白的表达水平较常氧组均显著增强.然而在ASODN缺氧组,HIF-1α、VEGF mRNA活性以及蛋白的表达水平明显减弱,且HIF-1α蛋白水平与VEGF转录活性有明显的相关性.结论:在缺氧环境下,转染HIF-1α反义寡核苷酸能够抑制HIF-1α活性,从而使VEGF mRNA和蛋白的表达水平明显下降.

  16. THE EFFECT OF ANTISENSE EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR) RNA ON THE PROLIFERATION OF HUMAN GLIOMA CELLS AND INDUCTION OF CELL APOPTOSIS

    Institute of Scientific and Technical Information of China (English)

    PU Pei-yu; LIU Xu-wen; LIU Ai-xue; WANG Chun-yan; WANG Guang-xiu

    1999-01-01

    Objective: To study the effect of antisense EGFR RNA on the growth of human glioma cells in vitro and evaluate the feasibility of targeting EGFR gene for gene therapy of gliomas. Methods: Southern and Northern blot analysis,in situ hybridization and immunohistochemical staining were used to detect the integration and expression of antisense EGFR constructs. MTT assay and the average number of AgNOR for evaluation of cell proliferation, and the TUNEL method and ultrastructural change for observation of cell apoptosis. Results: Exogenous antisense EGFR cDNA was integrated into the genome of glioma cells and highly expressed, which resulted in a dramatic decrease of endogenous EGFR mRNA and GEPR protein levels.Clones with high expression of the antisense construct showed a lower proliferation activity and the induction of apoptosis in vitro. Conclusion: This study suggests that EGFR plays an important role in the genesis of gliomas; it may be used as a target for antisense gene therapy of gliomas.

  17. Hsp90 protein interacts with phosphorothioate oligonucleotides containing hydrophobic 2'-modifications and enhances antisense activity.

    Science.gov (United States)

    Liang, Xue-Hai; Shen, Wen; Sun, Hong; Kinberger, Garth A; Prakash, Thazha P; Nichols, Joshua G; Crooke, Stanley T

    2016-05-01

    RNase H1-dependent antisense oligonucleotides (ASOs) are chemically modified to enhance pharmacological properties. Major modifications include phosphorothioate (PS) backbone and different 2'-modifications in 2-5 nucleotides at each end (wing) of an ASO. Chemical modifications can affect protein binding and understanding ASO-protein interactions is important for better drug design. Recently we identified many intracellular ASO-binding proteins and found that protein binding could affect ASO potency. Here, we analyzed the structure-activity-relationships of ASO-protein interactions and found 2'-modifications significantly affected protein binding, including La, P54nrb and NPM. PS-ASOs containing more hydrophobic 2'-modifications exhibit higher affinity for proteins in general, although certain proteins, e.g. Ku70/Ku80 and TCP1, are less affected by 2'-modifications. We found that Hsp90 protein binds PS-ASOs containing locked-nucleic-acid (LNA) or constrained-ethyl-bicyclic-nucleic-acid ((S)-cEt) modifications much more avidly than 2'-O-methoxyethyl (MOE). ASOs bind the mid-domain of Hsp90 protein. Hsp90 interacts with more hydrophobic 2' modifications, e.g. (S)-cEt or LNA, in the 5'-wing of the ASO. Reduction of Hsp90 protein decreased activity of PS-ASOs with 5'-LNA or 5'-cEt wings, but not with 5'-MOE wing. Together, our results indicate Hsp90 protein enhances the activity of PS/LNA or PS/(S)-cEt ASOs, and imply that altering protein binding of ASOs using different chemical modifications can improve therapeutic performance of PS-ASOs. PMID:26945041

  18. Antisense oligonucleotide induced exon skipping and the dystrophin gene transcript: cocktails and chemistries

    Directory of Open Access Journals (Sweden)

    Fletcher Sue

    2007-07-01

    Full Text Available Abstract Background Antisense oligonucleotides (AOs can interfere with exon recognition and intron removal during pre-mRNA processing, and induce excision of a targeted exon from the mature gene transcript. AOs have been used in vitro and in vivo to redirect dystrophin pre-mRNA processing in human and animal cells. Targeted exon skipping of selected exons in the dystrophin gene transcript can remove nonsense or frame-shifting mutations that would otherwise have lead to Duchenne Muscular Dystrophy, the most common childhood form of muscle wasting. Results Although many dystrophin exons can be excised using a single AO, several exons require two motifs to be masked for efficient or specific exon skipping. Some AOs were inactive when applied individually, yet pronounced exon excision was induced in transfected cells when the AOs were used in select combinations, clearly indicating synergistic rather than cumulative effects on splicing. The necessity for AO cocktails to induce efficient exon removal was observed with 2 different chemistries, 2'-O-methyl modified bases on a phosphorothioate backbone and phosphorodiamidate morpholino oligomers. Similarly, other trends in exon skipping, as a consequence of 2'-O-methyl AO action, such as removal of additional flanking exons or variations in exon skipping efficiency with overlapping AOs, were also seen when the corresponding sequences were prepared as phosphorodiamidate morpholino oligomers. Conclusion The combination of 2 AOs, directed at appropriate motifs in target exons was found to induce very efficient targeted exon skipping during processing of the dystrophin pre-mRNA. This combinatorial effect is clearly synergistic and is not influenced by the chemistry of the AOs used to induce exon excision. A hierarchy in exon skipping efficiency, observed with overlapping AOs composed of 2'-O-methyl modified bases, was also observed when these same sequences were evaluated as phosphorodiamidate morpholino

  19. The effect of in vitro exposure to antisense oligonucleotides on macrophage morphology and function

    Directory of Open Access Journals (Sweden)

    Ann Brasey

    2011-11-01

    Full Text Available Antisense oligonucleotides (AON delivered via inhalation are in drug development for respiratory diseases. In rodents and monkeys, repeated exposure to high doses of inhaled phosphorothioate (PS AON can lead to microscopic changes in the lungs, including accumulation of alveolar macrophages in the lower airway that have a foamy appearance. The functional consequences that result from this morphological change are unclear as there is controversy whether the vacuoles/inclusion bodies reflect normal clearance of the inhaled AON or are early indicators of lung toxicity. The morphological and functional responses of macrophage to PS AON were characterized in vitro using the comparator drug amiodarone, as a known inducer of foamy macrophages. Morphological changes of increased vacuolization with the presence of lamellated structures were observed in macrophages in response to both amiodarone and AON treatment. Functional responses to the drugs clearly differed with amiodarone treatment leading to apoptosis of cells and cell death, release of proinflammatory mediators IL-1RA, MIP-1α and TNFα, decrease in IP-10, a cytokine shown to be involved in protection against pulmonary fibrosis and altered phagocytosis capacity of the cells. In contrast, AON in concentrations up to 30 μM, had no effect on cell viability or apoptosis, had minimal effects on pro-inflammatory cytokines, increased IP-10 levels and did not alter the phagocytic capacity of the cells. Exposure of macrophages to AON in vitro, led to morphological changes of increased vacuolization, but did not lead to functional consequences which were observed with another vacuolization-inducing drug, suggesting that the in vivo phenotypic changes observed following inhalation of AON may be consistent with a clearance mechanism and not an activation or impairment of macrophages.

  20. Affinity capture of (Arg sup 8 )vasopressin-receptor complex using immobilized antisense peptide

    Energy Technology Data Exchange (ETDEWEB)

    Feng Xian Lu; Aiyar, N.; Chaiken, I. (SmithKline Beecham, King of Prussia, PA (United States))

    1991-05-01

    Solubilized noncovalent complexes of (Arg{sup 8})-vasopressin (AVP) with receptor proteins from rat liver membranes were isolated by selective binding to silica-immobilized antisense (AS) peptide. The affinity chromatographic support was prepared with a chemically synthesized AS peptide whose sequence is encoded by the AS DNA corresponding to the 20 amino-terminal residues of the AVP bovine neurophysin II biosynthetic precursor (pro-AVP/BNPII-(20-1)), region that includes the AVP sequence at residues 1-9. The AS peptide-AVP interaction mechanism hypothesized, contact by hydropathic complementarity at multiple sites along the peptide chains, led to the prediction that AVP bound to its receptor would still have enough free surface to interact with immobilized AS peptide. To test this prediction of a three-way interaction, ({sup 3}H)AVP-receptor was obtained as a solubilized, partially purified fraction from rat liver membrane. Covalently crosslinked ({sup 3}H)AVP complex also was bound to the AS peptide column; binding was blocked by competition with unlabeled AVP in the elution buffer. Since the AVP-linked 31- and 38-kDa proteins have the same apparent molecular mass on SDS/PAGE as found previously by photoaffinity labeling, the authors conclude that the AS peptide column has affinity-captured AVP-receptor complexes. The 15-kDa protein appears to be an active AVP-receptor fragment of one or both of the larger proteins. It is generally concluded that immobilized AS peptides may be useful to isolate peptide and protein receptor complexes in other systems as well.

  1. Sense-antisense gene-pairs in breast cancer and associated pathological pathways

    Science.gov (United States)

    Grinchuk, Oleg V.; Motakis, Efthymios; Yenamandra, Surya Pavan; Ow, Ghim Siong; Jenjaroenpun, Piroon; Tang, Zhiqun; Yarmishyn, Aliaksandr A.; Ivshina, Anna V.; Kuznetsov, Vladimir A.

    2015-01-01

    More than 30% of human protein-coding genes form hereditary complex genome architectures composed of sense-antisense (SA) gene pairs (SAGPs) transcribing their RNAs from both strands of a given locus. Such architectures represent important novel components of genome complexity contributing to gene expression deregulation in cancer cells. Therefore, the architectures might be involved in cancer pathways and, in turn, be used for novel drug targets discovery. However, the global roles of SAGPs in cancer pathways has not been studied. Here we investigated SAGPs associated with breast cancer (BC)-related pathways using systems biology, prognostic survival and experimental methods. Gene expression analysis identified 73 BC-relevant SAGPs that are highly correlated in BC. Survival modelling and metadata analysis of the 1161 BC patients allowed us to develop a novel patient prognostic grouping method selecting the 12 survival-significant SAGPs. The qRT-PCR-validated 12-SAGP prognostic signature reproducibly stratified BC patients into low- and high-risk prognostic subgroups. The 1381 SAGP-defined differentially expressed genes common across three studied cohorts were identified. The functional enrichment analysis of these genes revealed the GABPA gene network, including BC-relevant SAGPs, specific gene sets involved in cell cycle, spliceosomal and proteasomal pathways. The co-regulatory function of GABPA in BC cells was supported using siRNA knockdown studies. Thus, we demonstrated SAGPs as the synergistically functional genome architectures interconnected with cancer-related pathways and associated with BC patient clinical outcomes. Taken together, SAGPs represent an important component of genome complexity which can be used to identify novel aspects of coordinated pathological gene networks in cancers. PMID:26517092

  2. Different effects of antisense RelA p65 and NF-κB1 p50 oligonucleotides on the nuclear factor-κB mediated expression of ICAM-1 in human coronary endothelial and smooth muscle cells

    Directory of Open Access Journals (Sweden)

    Both Anton

    2001-08-01

    Full Text Available Abstract Background Activation of nuclear factor-κB (NF-κB is one of the key events in early atherosclerosis and restenosis. We hypothesized that tumor necrosis factor-α (TNF-α induced and NF-κB mediated expression of intercellular adhesion molecule-1 (ICAM-1 can be inhibited by antisense RelA p65 and NF-κB1 p50 oligonucleotides (RelA p65 and NF-κB1 p50. Results Smooth muscle cells (SMC from human coronary plaque material (HCPSMC, plaque material of 52 patients, SMC from the human coronary media (HCMSMC, human endothelial cells (EC from umbilical veins (HUVEC, and human coronary EC (HCAEC were successfully isolated (HCPSMC, HUVEC, identified and cultured (HCPSMC, HCMSMC, HUVEC, HCAEC. 12 hrs prior to TNF-α stimulus (20 ng/mL, 6 hrs RelA p65 and NF-κB1 p50 (1, 2, 4, 10, 20, and 30 μM and controls were added for a period of 18 hrs. In HUVEC and HCAEC there was a dose dependent inhibition of ICAM-1 expression after adding of both RelA p65 and NF-κB1 p50. No inhibitory effect was seen after incubation of HCMSMC with RelA p65 and NF-κB1 p50. A moderate inhibition of ICAM-1 expression was found after simultaneous addition of RelA p65 and NF-κB1 p50 to HCPSMC, no inhibitory effect was detected after individual addition of RelA p65 and NF-κB1 p50. Conclusions The data point out that differences exist in the NF-κB mediated expression of ICAM-1 between EC and SMC. Experimental antisense strategies directed against RelA p65 and NF-κB1 p50 in early atherosclerosis and restenosis are promising in HCAEC but will be confronted with redundant pathways in HCMSMC and HCPSMC.

  3. Restoration of contact inhibition in human glioblastoma cell lines after MIF knockdown

    International Nuclear Information System (INIS)

    Studies of the role of the cytokine macrophage-migration-inhibitory-factor (MIF) in malignant tumors have revealed its stimulating influence on cell-cycle progression, angiogenesis and anti-apoptosis. Here we show that in vitro targeting MIF in cultures of human malignant glioblastoma cells by either antisense plasmid introduction or anti-MIF antibody treatment reduced the growth rates of tumor cells. Of note is the marked decrease of proliferation under confluent and over-confluent conditions, implying a role of MIF in overcoming contact inhibition. Several proteins involved in contact inhibition including p27, p21, p53 and CEBPalpha are upregulated in the MIF antisense clones indicating a restoration of contact inhibition in the tumor cells. Correspondingly, we observed a marked increase in MIF mRNA and protein content under higher cell densities in LN18 cells. Furthermore, we showed the relevance of the enzymatic active site of MIF for the proliferation of glioblastoma cells by using the MIF-tautomerase inhibitor ISO-1. Our study adds another puzzle stone to the role of MIF in tumor growth and progression by showing the importance of MIF for overcoming contact inhibition

  4. Restoration of contact inhibition in human glioblastoma cell lines after MIF knockdown

    Directory of Open Access Journals (Sweden)

    Meyer Bernhard

    2009-12-01

    Full Text Available Abstract Background Studies of the role of the cytokine macrophage-migration-inhibitory-factor (MIF in malignant tumors have revealed its stimulating influence on cell-cycle progression, angiogenesis and anti-apoptosis. Results Here we show that in vitro targeting MIF in cultures of human malignant glioblastoma cells by either antisense plasmid introduction or anti-MIF antibody treatment reduced the growth rates of tumor cells. Of note is the marked decrease of proliferation under confluent and over-confluent conditions, implying a role of MIF in overcoming contact inhibition. Several proteins involved in contact inhibition including p27, p21, p53 and CEBPalpha are upregulated in the MIF antisense clones indicating a restoration of contact inhibition in the tumor cells. Correspondingly, we observed a marked increase in MIF mRNA and protein content under higher cell densities in LN18 cells. Furthermore, we showed the relevance of the enzymatic active site of MIF for the proliferation of glioblastoma cells by using the MIF-tautomerase inhibitor ISO-1. Conclusion Our study adds another puzzle stone to the role of MIF in tumor growth and progression by showing the importance of MIF for overcoming contact inhibition.

  5. Global analysis of cis-natural antisense transcripts and their heat-responsive nat-siRNAs in Brassica rapa

    OpenAIRE

    Yu, Xiang; Yang, Jun; Li, Xiaorong; Liu, Xuxin; Sun, Chuanbao; Wu, Feijie; He, Yuke

    2013-01-01

    Background Brassica rapa includes several important leaf vegetable crops whose production is often damaged by high temperature. Cis-natural antisense transcripts (cis-NATs) and cis-NATs-derived small interfering RNAs (nat-siRNAs) play important roles in plant development and stress responses. However, genome-wide cis-NATs in B. rapa are not known. The NATs and nat-siRNAs that respond to heat stress have never been well studied in B. rapa. Here, we took advantage of RNA-seq and small RNA (sRNA...

  6. Natural genetic variation impacts expression levels of coding, non-coding, and antisense transcripts in fission yeast

    DEFF Research Database (Denmark)

    Clément-Ziza, Mathieu; Marsellach, Francesc X.; Codlin, Sandra;

    2014-01-01

    Our current understanding of how natural genetic variation affects gene expression beyond well-annotated coding genes is still limited. The use of deep sequencing technologies for the study of expression quantitative trait loci (eQTLs) has the potential to close this gap. Here, we generated...... the first recombinant strain library for fission yeast and conducted an RNA-seq-based QTL study of the coding, non-coding, and antisense transcriptomes. We show that the frequency of distal effects (trans-eQTLs) greatly exceeds the number of local effects (cis-eQTLs) and that non-coding RNAs are as likely...

  7. Therapeutic Liabilities of in Vivo Viral Vector Tropism: Adeno-Associated Virus Vectors, NMDAR1 Antisense, and Focal Seizure Sensitivity

    OpenAIRE

    Haberman, Rebecca P.; Criswell, Hugh E.; Snowdy, Stephen; Ming, Zhen; Breese, George R.; Samulski, R. Jude; McCown, Thomas J.

    2002-01-01

    The N-methyl-d-aspartic acid (NMDA) receptor provides a potential target for gene therapy of focal seizure disorders. To test this approach, we cloned a 729-bp NMDA receptor (NMDAR1) cDNA fragment in the antisense orientation into adeno-associated virus (AAV) vectors, where expression was driven by either a tetracycline-off regulatable promoter (AAV-tTAK-NR1A) or a cytomegalovirus (CMV) promoter (AAV-CMV-NR1A). After infection of primary cultured cortical neurons with recombinant AAV-tTAK-NR1...

  8. Defining a role for Hfq in Gram-positive bacteria: evidence for Hfq-dependent antisense regulation in Listeria monocytogenes

    OpenAIRE

    Nielsen, Jesper Sejrup; Lei, Lisbeth Kristensen; Ebersbach, Tine; Olsen, Anders Steno; Klitgaard, Janne Kudsk; Valentin-Hansen, Poul; Kallipolitis, Birgitte Haahr

    2009-01-01

    Small trans-encoded RNAs (sRNAs) modulate the translation and decay of mRNAs in bacteria. In Gram-negative species, antisense regulation by trans-encoded sRNAs relies on the Sm-like protein Hfq. In contrast to this, Hfq is dispensable for sRNA-mediated riboregulation in the Gram-positive species studied thus far. Here, we provide evidence for Hfq-dependent translational repression in the Gram-positive human pathogen Listeria monocytogenes, which is known to encode at least 50 sRNAs. We show t...

  9. A Novel Vector for Abundant Expression of Antisense RNA, Triplex-forming RNA and Ribozyme in vivo

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    For abundant expression of antisense RNA, triplex-forming RNA and Ribozyme in vivo, a novel vector pBSKneorU6' was constructed by PCR cloning. This vector contains the intact human snRNA U6 gene expression unit, yet replacing the 61-nt-sequence in the middle of U6 snRNA coding region with three restriction enzyme sites. Hela nuclear extract in vitro transcription experiments demonstrated that this vector can effectively express U6 mutant RNA. Containing neor at the same time, stably transfected pBSKneorU6' can be selected easily.

  10. Abrupt decrease of c—myc expression by antisense transcripts induses terminal differentiation and apoptosis in human promyelocytic leukemia HL—60 cells

    Institute of Scientific and Technical Information of China (English)

    HAOXIUJUAN; PEIHSIENTANG; 等

    1996-01-01

    This study was designed using c-myc antisense transcripts to evaluate how alteration of c-myc expression in human myeloid leukemic HL-60 cells could influence the myelomonocytic differentiation and induction of apoptosis.The recombinant plasmid pDACx expressing antisense transcripts to c-myc fragment containing a part of intron 1 and 137 nt exon 2 was constructed.pDACx was transfected into HL-60 cell line by lipofectin reagent.Cytochemical stainings including NBT reduction,peroxidase and α-NAE as well as detection of CD13 and CD33 antigens by flow cytometric analysis indicated occurrence of myelomonocytic differentiation in cells expressing antisense transcripts to c-myc.DNA degradation measured by DNA gel electrophoresis and typical morphological changes observed under electron microscope proved the swith-on of apoptosis in terminally differentiating HL-60 cells.

  11. Preparation of 99Tcm labeled survivin mRNA antisense PNA and gene imaging in nude mice bearing lung carcinoma A549 xenografts

    International Nuclear Information System (INIS)

    Objective: To prepare the 99Tcm-survivin mRNA antisense peptide nucleic acid (PNA)and investigate its value as a gene imaging agent in tumor bearing mice and early diagnosis in tumor. Methods: Survivin mRNA antisense PNA and mismatch PNA were synthesized. Four amino acids (Gly- (D)Ala-Gly-Gly) and Aba (4-aminobutyric acid) were linked to the 5' end of PNA. Gly- (D)Ala-Gly-Gly served as a chelating moiety for strong chelation of 99Tcm and Aba acted as a spacer to minimize the steric hindrance. PNAs were labeled with 99Tcm by the ligand-exchange method. The labeling efficiency and radiochemical purity were measured by HPLC and ITLC methods. There were five BALB/c nude mice bearing human lung carcinoma (A549) in each of antisense PNA and mismatch PNA groups. Gene imaging of 99Tcm-survivin mRNA antisense and mismatch PNAs were performed at 1, 2 and 4 h post the injection, respectively, and the T/NT ratio was measured by the method of ROI. The statistical comparisons of average values were performed with the two-group t-test for independent sample by SPSS 13.0. Results: The product kept stable in vitro. The labeling efficiency of 99Tcm-survivin mRNA antisense PNA was (95.48 ±1.92)% and more than 85% after the incubation for 24 h in serum. The radiochemical purity was >95%. The labeling efficiency of mismatch PNA was similar to the antisense PNA. 99Tcm-survivin mRNA antisense PNA was especially uptaken by tumor lesion, and its accumulation reached the top at 4 h post the injection. T/NT ratios at 1, 2, and 4 h were 2.70 ± 0.28, 3.44 ± 0.35,4.21 ± 0.63, respectively. In the comparison, the T/NT ratio of 99Tcm-survivin mRNA mismatch PNA at 4 h (3.12 ±0.50) was significantly lower (t=2.918, P=0.019). Conclusions: 99Tcm-survivin mRNA antisense PNA has high labeling efficiency,good stability and no need of purification. Its characteristic of especial uptake by tumor lesion provides the potential value in early diagnosis of tumor. (authors)

  12. The Application of Cytidyl Guanosyl Oligodeoxynucleotide Can Affect the Antitumor Immune Response Induced by a Combined Protocol of Cryoablation and Dendritic Cells in Lewis Lung Cancer Model.

    Science.gov (United States)

    Zhang, Mi; Yin, Tianquan; Lu, Yuan; Feng, Huasong

    2016-01-01

    BACKGROUND Recently, several combined therapeutic strategies and targeted agents have been under investigation for their potential role in lung cancer. The combined administration of dendritic cells (DCs) and immune-adjuvant cytidyl guanosyl oligodeoxynucleotide (CpG-ODN) after cryosurgery has proven to be an effective strategy for treating lung cancer. However, whether the application of CpG-ODN could affect the therapeutic results remained to be further explored. MATERIAL AND METHODS The Lewis lung cancer (LLC)-bearing mice received cryoablation and injection of ex vivo-cultured DCs into the peritumoral zone. Subsequently, CpG-ODN was administered to experimental animals 6 hours, 12 hours, and 24 hours after DC injection. The mice in the control group received coadministration of DCs and CpG-ODN simultaneously. Therapeutic effects were evaluated by survival rates. The resistance to rechallenge of LLC cell was assessed by lung metastasis and in vitro cytotoxicity of splenocytes. Furthermore, T-cell subsets and multiple cytokines (interleukin [IL]-4, -10, and-12; interferon [IFN]-γ; tumor necrosis factor [TNF]-α) in the blood were assessed to elucidate the underlying mechanisms. RESULTS Higher ratios of CD4+ and CD8+ T cells and higher levels of IL-12, IFN-γ, and TNF-α were found in the blood of the mice that received CpG-ODN therapy 12 h after DC injection. The cytotoxicity potency of the splenocytes of these mice was significantly higher compared with the mice in other groups. Moreover, the mice receiving CpG-ODN therapy 12 h after DC injection showed significantly better resistance to rechallenge. Compared with the mice in other groups, the mice receiving CpG-ODN therapy 12 h after DC injection were superior in survival rates and antimetastatic effects. CONCLUSIONS Our study suggested that the therapeutic efficacy was closely associated wi