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Sample records for antisense dna parameters

  1. The use of nano-sized acicular material, sliding friction, and antisense DNA oligonucleotides to silence bacterial genes.

    Science.gov (United States)

    Mitsudome, Yuya; Takahama, Mamiko; Hirose, Jun; Yoshida, Naoto

    2014-01-01

    Viable bacterial cells impaled with a single particle of a nano-sized acicular material formed when a mixture containing the cells and the material was exposed to a sliding friction field between polystyrene and agar gel; hereafter, we refer to these impaled cells as penetrons. We have used nano-sized acicular material to establish a novel method for bacterial transformation. Here, we generated penetrons that carried antisense DNA adsorbed on nano-sized acicular material (α-sepiolite) by providing sliding friction onto the surface of agar gel; we then investigated whether penetron formation was applicable to gene silencing techniques. Antisense DNA was artificially synthesized as 15 or 90mer DNA oligonucleotides based on the sequences around the translation start codon of target mRNAs. Mixtures of bacterial cells with antisense DNA adsorbed on α-sepiolite were stimulated by sliding friction on the surface of agar gel for 60 s. Upon formation of Escherichia coli penetrons, β-lactamase and β-galactosidase expression was evaluated by counting the numbers of colonies formed on LB agar containing ampicillin and by measuring β-galactosidase activity respectively. The numbers of ampicillin resistant colonies and the β-galactosidase activity derived from penetrons bearing antisense DNA (90mer) was repressed to 15% and 25%, respectively, of that of control penetrons which lacked antisense DNA. Biphenyl metabolite, ring cleavage yellow compound produced by Pseudomonas pseudoalcaligenes penetron treated with antisense oligonucleotide DNA targeted to bphD increased higher than that lacking antisense DNA. This result indicated that expression of bphD in P. pseudoalcaligenes penetrons was repressed by antisense DNA that targeted bphD mRNA. Sporulation rates of Bacillus subtilis penetrons treated with antisense DNA (15mer) targeted to spo0A decreased to 24.4% relative to penetrons lacking antisense DNA. This novel method of gene silencing has substantial promise for

  2. Identification of novel endogenous antisense transcripts by DNA microarray analysis targeting complementary strand of annotated genes

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    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

  3. Human Immunodeficiency Virus-Type 1 LTR DNA contains an intrinsic gene producing antisense RNA and protein products

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    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

  4. Os DNA sintéticos anti-sentido Antisense Synthtetic DNA

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    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.

  5. The use of nano-sized acicular material, sliding friction, and antisense DNA oligonucleotides to silence bacterial genes

    OpenAIRE

    2014-01-01

    Viable bacterial cells impaled with a single particle of a nano-sized acicular material formed when a mixture containing the cells and the material was exposed to a sliding friction field between polystyrene and agar gel; hereafter, we refer to these impaled cells as penetrons. We have used nano-sized acicular material to establish a novel method for bacterial transformation. Here, we generated penetrons that carried antisense DNA adsorbed on nano-sized acicular material (α-sepiolite) by prov...

  6. 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.

  7. 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.

  8. Effect of antisense TIMP-1 cDNA on the expression of TIMP-1 and MMP-2 in lung tissue with pulmonary fibrosis induced by bleomycin.

    Science.gov (United States)

    Tang, Haiying; Mao, Jingwei; Gao, Lili; Liu, Jia; Wu, Taihua

    2013-01-01

    The aim of this study was to observe the effect of antisense tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) cDNA on the concentration of hydroxyproline (HYP) and the expression of TIMP-1 and matrix metalloproteinase-2 (MMP-2) in the lung tissue of rats with bleomycin (BLM)‑induced pulmonary fibrosis. Sprague-Dawley rats were randomly divided into 5 groups: the control, pulmonary fibrosis model, sense TIMP-1 transfection, antisense TIMP-1 transfection and empty vector transfection groups. For the transfection groups, following the intratracheal injection of BLM on days 1, 3, 7, 14, 28 and 60, the rats were treated with retroviral vectors and sacrificed on day 28. The control and pulmonary fibrosis groups were treated with normal saline at the same time‑points. The concentration of HYP and the expression levels of TIMP-1 and MMP-2 in the lung tissue were detected. The HYP concentration and lung tissue TIMP-1 expression levels of the antisense TIMP-1 group decreased significantly on days 1 and 3 compared with those of the empty vector and pulmonary fibrosis groups at the same time-points (Ppulmonary fibrosis groups on days 7, 14, 28 and 60. The lung expression levels of MMP-2 in all groups, with the exception of the control group, had no significant differences at all time-points (P>0.05). Antisense TIMP-1 cDNA retroviral vectors are able to suppress the development of pulmonary fibrosis in the early stages.

  9. 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.

  10. 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.

  11. Delivery of a survivin promoter-driven antisense survivin-expressing plasmid DNA as a cancer therapeutic: a proof-of-concept study

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    Lin KY

    2016-05-01

    Full Text Available Kun-Yuan Lin,1 Siao Muk Cheng,2 Shing-Ling Tsai,2 Ju-Ya Tsai,1 Chun-Hui Lin,1 Chun Hei Antonio Cheung1,2 1Department of Pharmacology, College of Medicine, National Cheng Kung University, Tainan, Taiwan, ROC; 2Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan, Taiwan, ROC Abstract: Survivin is a member of the inhibitor-of-apoptosis proteins family. It is overexpressed in many different cancer types but not in the differentiated normal tissue. In addition, overexpression of survivin promotes cancer cell survival and induces chemotherapeutic drug resistance, making it an attractive target for new anticancer interventions. Despite survivin being a promising molecular target for anticancer treatment, it is widely accepted that survivin is only a “semi-druggable” target. Therefore, it is important to develop a new strategy to target survivin for anticancer treatment. In this study, we constructed a novel survivin promoter-driven full-length antisense survivin (pSur/AS-Sur expression plasmid DNA. Promoter activity assay revealed that the activity of the survivin promoter of pSur/AS-Sur correlated with the endogenous expression of survivin at the transcriptional level in the transfected A549, MDA-MB-231, and PANC-1 cancer cells. Western blot analysis showed that liposomal delivery of pSur/AS-Sur successfully downregulated the expression of survivin in A549, MBA-MB-231, and PANC-1 cells in vitro. In addition, delivery of pSur/AS-Sur induced autophagy, caspase-dependent apoptosis, and caspase-independent apoptosis as indicated by the increased LC3B-II conversion, autophagosome formation, caspase-9/-3 and poly(ADP-ribose polymerase-1 cleavage, and apoptosis-inducing factor nuclear translocation in A549, MBA-MB-231, and PANC-1 cells. Importantly, liposomal delivery of pSur/AS-Sur was also capable of decreasing the proliferation of the survivin/MDR1 coexpressing multidrug-resistant KB-TAX50 cancer cells and

  12. 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.

  13. Binding Parameters of Alkaloids Berberine and Sanguinarine with DNA

    CERN Document Server

    Gumenyuk, V G; Kutovyy, S Yu; Yashchuk, V M; Zaika, L A

    2012-01-01

    We study the interaction of berberine and sanguinarine (plant alkaloids) with DNA in aqueous solutions, by using optical spectroscopy methods (absorption and fluorescence). The dependencies of alkaloid spectral characteristics on the concentration ratio N/c between the DNA base pairs and alkaloid molecules in the solutions are considered, and the manifestations of the alkaloid-DNA binding are revealed. The character of binding is found to depend on N/c. The parameters of the binding of berberine and sanguinarine with DNA are determined, by using the modified Scatchard and McGhee-von Hippel equations

  14. Construction and characterization of replication-deficient adenoviral vector containing the cDNA for human VEGF165 in an antisense orientation%反义VEGF165腺病毒重组体的构建及鉴定

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    王家宁; 黄永章; 王俊峰; 王卫民; 李瑞明; 张群林

    2001-01-01

    objective To construct the recombinant adenovirus vectorcontaining the cDNA for hVEGF165 in an antisense orientation for future study of tumor treatment by antisense hVEGF165 RNA strategy.Methods The VEGF165 cDNA has been extracted from pUCCAGGS/hVEGF165 with EcoRI and then inserted in an antisense orientation into the E1-deleted expression plasmid pHCMVsp1A shuttle vector, called pAd-ahVEGF165. pAd-ahVEGF165 was cotransfected with the plasmid pJM17 into the transformed human embryonic kidney cell line 293 cells by liposome-mediated method. Homologous recombination of the pAd-ahVEGF165 and pJM17 in 293 cells replaced the E1 region with the expression cassette from pAd-ahVEGF165. Ad-ahVEGF165 was confirmed by PCR.Ad-ahVEGF165 was propagated in 293 cells and then underwent CsCl density purification. subsequently, the preparations were didalyzed in dialysis buffer. The titer of each viral stock was determined by measuring the absorbance at 260nm. Results VEGF165 cDNA was successfully inserted into the shuttle vector pHCMVSPIA. pAd-ahVEGF165 was confirmed by NcoI and XhoI digestion. Ad-ahVEGF165 was characterized by PCR coamplification. The virus titer was 5.6×1011pfu/ml. Conclusions Ad-ahVEGF165 containing the antisense VEGF165 sequence was successfully constructd.This investigation provides the basis for future study of tumor treatment based on antisense VEGF RNA strategy.%目的:构建含人反义血管内皮生长因子165(VEGF165)基因的重组腺病毒载体,为采用反义VEGF165RNA防治肿瘤的研究奠定基础。方法:将人VEGF165 cDNA反向插入到穿梭质粒pHCMVSP1A的CMV启动子之下,即pAd-ahVEGF165。后者与pJM17通过脂质体共转染293细胞,经同源重组获得含人反义VEGF165基因的重组腺病毒Ad-ahVEGF165。通过PCR共扩增法鉴别Ad-ahVEGF165的正确与否。根据260nm的紫外光吸收值计算病毒滴度。结果:VEGF165 cDNA成功地反向插入了pHCMVSP1A载体,以重组病毒基因组DNA

  15. ADVANCED IN VIVO USE OF CRISPR/CAS9 AND ANTI-SENSE DNA INHIBITION FOR GENE MANIPULATION IN THE BRAIN

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    Brandon J Walters

    2016-01-01

    Full Text Available Gene-editing tools are essential for uncovering how genes mediate normal brain-behaviour relationships and contribute to neurodegenerative and neuropsychiatric disorders. Recent progress in gene-editing technology is now allowing 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 mature nervous system, particularly CRISPR/Cas9 and anti-sense nucleotide-based techniques. CRISPR/Cas9 is a flexible gene-editing tool, allowing the genome to be manipulated in diverse ways. For instance, CRISPR/Cas9 has been successfully used to knock-out genes, knock-in mutations, overexpress or inhibit gene activity, and provide scaffolding for recruiting specific epigenetic regulators to individual genes and gene regions. Moreover, the CRISPR/Cas9 system may be modified to target multiple genes at one time, affording simultaneous inhibition and overexpression of distinct genetic targets. Although many of the more advanced applications of CRISPR/Cas9 have not been applied to the nervous system, the tool-box is widely-accessible, such that it is poised to help advance neuroscience. Anti-sense nucleotide-based technologies can be used to rapidly knock down genes in the brain and are simple to use. A main advantage of anti-sense based tools is their independence of viral packaging, allowing for rapid gene delivery with minimal technical expertise. Here, we describe the main applications and functions of each of these systems with an emphasis on their many potential applications in neuroscience laboratories.

  16. DNA Duplex Length and Salt Concentration Dependence of Enthalpy−Entropy Compensation Parameters for DNA Melting

    KAUST Repository

    Starikov, E. B.

    2009-08-20

    Systematical differential calorimetry experiments on DNA oligomers with different lengths and placed in water solutions with various added salt concentrations may, in principle, unravel important information about the structure and dynamics of the DNA and their water-counterion surrounding. With this in mind, to reinterpret the most recent results of calorimetric experiments on DNA oligomers of such a kind, the recent enthalpy-entropy compensation theory has been used. It is demonstrated that the application of the latter could enable direct estimation of thermodynamic parameters of the microphase transitions connected to the changes in DNA dynamical regimes versus the length of the biopolymers and the ionic strengths of their water solutions, and this calls for much more systematical experimental and theoretical studies in this field. © 2009 American Chemical Society.

  17. DNA duplex length and salt concentration dependence of enthalpy-entropy compensation parameters for DNA melting.

    Science.gov (United States)

    Starikov, E B; Nordén, Bengt

    2009-08-20

    Systematical differential calorimetry experiments on DNA oligomers with different lengths and placed in water solutions with various added salt concentrations may, in principle, unravel important information about the structure and dynamics of the DNA and their water-counterion surrounding. With this in mind, to reinterpret the most recent results of calorimetric experiments on DNA oligomers of such a kind, the recent enthalpy-entropy compensation theory has been used. It is demonstrated that the application of the latter could enable direct estimation of thermodynamic parameters of the microphase transitions connected to the changes in DNA dynamical regimes versus the length of the biopolymers and the ionic strengths of their water solutions, and this calls for much more systematical experimental and theoretical studies in this field.

  18. An antisense transcript in the human cytomegalovirus UL87 gene region

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    Ma Yanping

    2011-11-01

    Full Text Available Abstract Background Rapid advances in research on antisense transcripts are gradually changing our comprehension of genomic and gene expression aspects of the Herpesviridae. One such herpesvirus is the human cytomegalovirus (HCMV. Although transcription of the HCMV UL87 gene has not been specifically investigated, cDNA clones of UL87 antisense transcripts were found in HCMV cDNA libraries previously. In this study, the transcription of the UL87 antisense strand was investigated in three clinically isolated HCMV strains. Results First, an 800 nucleotides transcript having an antisense orientation to the UL87 gene was found in a late HCMV cDNA library. Then, the UL87 antisense transcript was confirmed by Rapid amplification of cDNA ends (RACE and Northern blot in three HCMV clinical strains. Two ORFs were predicted in the antisense transcript. The putative protein of ORF 1 showed a high degree of conservation among HCMV and other CMV strains. Conclusion An 800nt antisense transcript in the UL87 gene region exists in HCMV clinical strains.

  19. Inhibition of AAC(6′)-Ib-Mediated Resistance to Amikacin in Acinetobacter baumannii by an Antisense Peptide-Conjugated 2′,4′-Bridged Nucleic Acid-NC-DNA Hybrid Oligomer

    Science.gov (United States)

    Lopez, Christina; Arivett, Brock A.; Actis, Luis A.

    2015-01-01

    Multiresistant Acinetobacter baumannii, a common etiologic agent of severe nosocomial infections in compromised hosts, usually harbors aac(6′)-Ib. This gene specifies resistance to amikacin and other aminoglycosides, seriously limiting the effectiveness of these antibiotics. An antisense oligodeoxynucleotide (ODN4) that binds to a duplicated sequence on the aac(6′)-Ib mRNA, one of the copies overlapping the initiation codon, efficiently inhibited translation in vitro. An isosequential nuclease-resistant hybrid oligomer composed of 2′,4′-bridged nucleic acid-NC (BNANC) residues and deoxynucleotides (BNANC-DNA) conjugated to the permeabilizing peptide (RXR)4XB (“X” and “B” stand for 6-aminohexanoic acid and β-alanine, respectively) (CPPBD4) inhibited translation in vitro at the same levels observed in testing ODN4. Furthermore, CPPBD4 in combination with amikacin inhibited growth of a clinical A. baumannii strain harboring aac(6′)-Ib in liquid cultures, and when both compounds were used as combination therapy to treat infected Galleria mellonella organisms, survival was comparable to that seen with uninfected controls. PMID:26169414

  20. EFFECT OF BcL-2 ANTISENSE DRUG WITH DIFFERENT STRUCTURE ON THE BIOLOGICAL FUNCTION OF K562 CELLS

    Institute of Scientific and Technical Information of China (English)

    雷小勇; 张洹; 何冬梅

    2004-01-01

    Objective: To study the differences and similarities of the antisense drugs with different structures on the biological functions of K562 cells. Methods: Cytotoxic effects were measured by use of a cell viability assay. Flow cytometric analysis and agarose gel electrophoresis of DNA fragmentation were also performed. The expression level of protein was assayed by immunofluorescence using fluoresce isothiocyanate label. Results: PNA targeting the coding region of the Bcl-2 messenger RNA could effectively inhibit K562 cell viability, down-regulate the synthesis of the Bcl-2 protein and increase cell apoptosis. By 72 h after the Bcl-2 antisense PNA treatment, K562 cells showed more reduction in the level of Bcl-2 protein compared with cells treated with the antisense ODN. After treatment with 10μmol/L of Bcl-2 antisense PNA or antisense ODN for 72 h, apoptotic rates of K562 cells were 13.15±1.13 and 11.72±1.12, respectively. Furthermore, there was significant difference in the percentage of apoptotic cells between antisense PNA group and antisense ODN group. Conclusion: The results suggest that antisense PNA targeting the coding region of Bcl-2 mRNA has better antisense effects than the antisense oligonucleotides on inducing apoptosis of K562 cells.

  1. Targeting Cancer with Antisense Oligomers

    Energy Technology Data Exchange (ETDEWEB)

    Hnatowich, DJ

    2008-10-28

    With financial assistance from the Department of Energy, we have shown definitively that radiolabeled antisense DNAs and other oligomers will accumulate in target cancer cells in vitro and in vivo by an antisense mechanism. We have also shown that the number of mRNA targets for our antisense oligomers in the cancer cell types that we have investigated so far is sufficient to provide and antisense image and/or radiotherapy of cancer in mice. These studies have been reported in about 10 publications. However our observation over the past several years has shown that radiolabeled antisense oligomers administered intravenously in their native and naked form will accumulate and be retained in target xenografts by an antisense mechanism but will also accumulate at high levels in normal organs such as liver, spleen and kidneys. We have investigated unsuccessfully several commercially available vectors. Thus the use of radiolabeled antisense oligomers for the imaging of cancer must await novel approaches to delivery. This laboratory has therefore pursued two new paths, optical imaging of tumor and Auger radiotherapy. We are developing a novel method of optical imaging tumor using antisense oligomers with a fluorophore is administered while hybridized with a shorter complementary oligomer with an inhibitor. In culture and in tumored mice that the duplex remains intact and thus nonfluorescent until it encounters its target mRNA at which time it dissociates and the antisense oligomer binds along with its fluorophore to the target. Simultaneous with the above, we have also observed, as have others, that antisense oligomers migrate rapidly and quantitatively to the nucleus upon crossing cell membranes. The Auger electron radiotherapy path results from this observation since the nuclear migration properties could be used effectively to bring and to retain in the nucleus an Auger emitting radionuclide such as 111In or 125I bound to the antisense oligomer. Since the object becomes

  2. 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

  3. 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.

  4. Reduction of polygalacturonase activity in tomato fruit by antisense RNA.

    Science.gov (United States)

    Sheehy, R E; Kramer, M; Hiatt, W R

    1988-12-01

    Polygalacturonase [PG; poly(1,4-alpha-D-galacturonide) glycanhydrolase; EC 3.2.1.15] is expressed in tomato only during the ripening stage of fruit development. PG becomes abundant during ripening and has a major role in cell wall degradation and fruit softening. Tomato plants were transformed to produce antisense RNA from a gene construct containing the cauliflower mosaic virus 35S promoter and a full-length PG cDNA in reverse orientation. The construct was integrated into the tomato genome by Agrobacterium-mediated transformation. The constitutive synthesis of PG antisense RNA in transgenic plants resulted in a substantial reduction in the levels of PG mRNA and enzymatic activity in ripening fruit. The steady-state levels of PG antisense RNA in green fruit of transgenic plants were lower than the levels of PG mRNA normally attained during ripening. However, analysis of transcription in isolated nuclei demonstrated that the antisense RNA construct was transcribed at a higher rate than the tomato PG gene(s). Analysis of fruit from transgenic plants demonstrated a reduction in PG mRNA and enzymatic activity of 70-90%. The reduction in PG activity did not prevent the accumulation of the red pigment lycopene.

  5. Differential modification of flavonoid and isoflavonoid biosynthesis with an antisense chalcone synthase construct in transgenic Lotus corniculatus.

    Science.gov (United States)

    Colliver, S P; Morris, P; Robbins, M P

    1997-11-01

    Three clonal genotypes of Lotus corniculatus L. (bird's foot trefoil) were transformed with an antisense chalcone synthase (CHS) gene construct made using a stress induced CHS17 cDNA from Phaseolus vulgaris under the control of the constitutive CaMV 35S promoter and Nos terminator via Agrobacterium rhizogenes. After initial screening, ten antisense and five control co-transformation events from each recipient clonal genotype were analysed. After elicitation with glutathione, the level of tannin accumulation was found to be increased in a number of antisense root cultures derived from the low (S33) and moderate (S50) tannin recipient genotypes. Six antisense and four control transformed lines from genotype S50 were selected for more detailed study. The antisense CHS construct was found to be integrated into the genome, with a copy number ranging from 1 to 5 and antisense orientation was confirmed by PCR. In transformed root cultures, increased CHS transcript levels were noted in a number of antisense lines. Biochemical analyses of glutathione-elicited-root cultures indicated a significant increase in tannin accumulation in antisense CHS lines and mean vestitol levels were reduced. These results show that the introduction of a heterologous antisense chalcone synthase construct into L. corniculatus resulted in an unpredicted molecular and biochemical phenotype. Such findings are discussed in relation to manipulation of this complex multigene family.

  6. 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.

  7. Reduction of tumorigenicity of SMMC-7721 hepatoma cells by vascular endothelial growth factor antisense gene therapy

    Institute of Scientific and Technical Information of China (English)

    Yu Cheng Tang; Yu Li; Guan Xiang Qian

    2001-01-01

    AIM To test the hypothesis to block VEGFexpression of SMMC-7721 hepatoma cells mayinhibit tumor growth using the rat hepatomamodel.METHODS Amplifiy the 200 VEGF cDNAfragment and insert it into human U6 genecassette in the reverse orientation transcribingsmall antisense RNA which could specificallyinteract with VEGF165, and VEGF121 mRNA.Construct the retroviral vector containing thisantisense VEGF U6 cassette and package thereplication-deficient recombinant retrovirus.SMMC-7721 cells were transduced with thesevirus and positive clones were selected withG418. PCR and Southern blot analysis wereperformed to determine if U6 cassette integratedinto the genomic DNA of positive clone.Transfected tumor cells were evaluated for RNAexpression by ribonuclease protection assays.The VEGF protein in the supernatant of parentaltumor cells and genetically modified tumor cellswas determined with ELISA. In vitro and in vivogrowth properties of antisense VEGF cell clonein nude mice were analyzed.RESULTS Restriction enzyme digestion andPCR sequencing verified that the antisense VEGFRNA retroviral vector was successfullyconstructed. After G418 selection, resistantSMMC-7721 cell clone was picked up. PCR andSouthern blot analysis suggested that U6cassette was integrated into the cell genomicDNA. Stable SMMC-7721 cell clone transducedwith U6 antisense RNA cassette could express200bp small antisense VEGF RNA and secretereduced levels of VEGF in culture condition.Production of VEGF by antisense transgeneexpressing cells was 65 ± 10 ng / L per 106 cells,420 ± 45 ng/L per 106 cells in sense group and 485± 30 ng/L per 106 cells in the negative control group, (P<0.05). The antisense-VEGF cell clone appeared phenotypically indistinguishable from SMMC-7721 cells and SMMC-7721 cells transfected sense VEGF. The growth rate of the antisense-VEGF cell clone was the same as the control cells. When S. C. was implanted into nude mice, growth of antisense-VEGF cell lines was greatly inhibited

  8. 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

  9. ANTISENSE TECHNIQUE TO TREAT BREAST CANCER – A REVIEW

    Directory of Open Access Journals (Sweden)

    Vijayalakshmi S

    2011-09-01

    Full Text Available There are many genes which are responsible for developing breast cancer especially, BRCA2 (Breast Cancer 2 and HER2 are extensively involved in developing breast cancer and hence it is the centre of attractions for all the researchers. Nano-particles conjugated with the anti-HER2 monoclonal antibodies are called as “Trastazumab” which directly target the HER2 gene. The major advantage of this technology is that the cells can be prevented before they evolve in to mature stages i.e. metastases production. The BRCA2 gene belongs to the family of tumor suppressor genes and its protein product is responsible for the error free repair mechanisms of DNA. This BRCA2 gene interacts with RAD51 gene to fix the DNA breaks. Mutation in BRCA2 gene such as insertion and deletion leads to breast cancer. More than 800 mutations are found in this gene that lead to increased risk of the breast cancer. Furthermore, BRCA2 gene is also associated with various cancers like prostate, ovarian, fallopian, male breast cancer. Researchers believe that altered products produced due to defects in this gene are unable to interact with the gene RAD51 and cannot repair the DNA. Antisense RNA is the tool which can used to block any RNA or DNA to synthesize its product. In this review we focus in using Antisense RNA against the sense RNA of an altered BRCA2 gene to block the altered affectivity of that gene on the DNA repair mechanism. However, Antisense RNA technique may not help in treating breast cancer, it can better manage the breast cancer to occur.

  10. EXPERIMENTAL STUDY ON THE GENE THERAPY OF MALIGNANT GLIOMA WITH ANTISENSE VEGF RNA

    Institute of Scientific and Technical Information of China (English)

    浦佩玉; 王建桢; 黄强; 张敬; 张云亭

    2003-01-01

    Objective: To study the effect of antisense VEGF RNA on rat C6 gliomas in vivo and find out the feasibility of antiangiogenesis therapy with antisense VEGF RNA for malignant gliomas. Methods: Parental rat C6 glioma cells and C6 cells transfected with antisense VEGF cDNA were implanted intracerebrally and subcutaneously into SD rats as control and transfected group. Rats bearing cerebral and subcutaneous C6 gliomas were treated with antisense VEGF cDNA as treated group and sense VEGF cDNA and empty vector as control of treated group. The general manifestation, survival time, MRI and histopathological changes of all rats were observed. The volume of subcutaneously implanted tumors was determined regularly. In situ hybridization and immunohistochemical staining were used for detection of VEGF gene expression of gliomas while PCNA immunostaining and TUNEL method for examination of proliferation activity and apoptosis of gliomas, respectively. Results: The survival of the rats in transfected and treated group was prolonged. There were two rats surviving over 90 d in the treated group and their tumors disappeared. The VEGF gene expression, the number of microvessels and the proliferation activity were decreased and a large amount of apoptotic cells could be found in cerebral and subcutaneous gliomas in treated and transfected groups. Conclusion: VEGF is one of the candidate genes for gene therapy of malignant gliomas. Antisense VEGF RNA combined with other therapies should be studied further for enhancing the therapeutic effect of malignant gliomas.

  11. Genetic modification of condensed tannin biosynthesis in Lotus corniculatus. 1. Heterologous antisense dihydroflavonol reductase down-regulates tannin accumulation in "hairy root" cultures.

    Science.gov (United States)

    Carron, T R; Robbins, M P; Morris, P

    1994-03-01

    An antisense dihydroflavonol reductase (DFR) gene-construct made using the cDNA for DFR from Antirrhinum majus was introduced into the genome of a series of clonal genotypes of Lotus corniculatus via Agrobacterium rhizogenes. After initial screening, 17 antisense and 11 control transformation events were analysed and tannin levels found to be reduced in antisense root cultures. The effect of this antisense construct, (pMAJ2), which consisted of the 5' half of the DFR cDNA sequence, was compared in three different recipient Lotus genotypes. This construct effectively down-regulated tannin biosynthesis in two of the recepient genotypes (s33 and s50); however, this construct was relatively ineffective in a third genotype (s41) which accumulated high levels of condensed tannins in derived transgenic root cultures. Four pMAJ2 antisense and three control lines derived from clonal genotypes s33 and s50 were selected and studied in greater detail. The antisense DFR construct was found to be integrated into the genome of the antisense "hairy root" cultures, and the antisense RNA was shown to be expressed. Tannin levels were much lower in antisense roots compared to the controls and this reduction in tannin levels was accompanied by a change in condensed tannin subunit composition.

  12. 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.

  13. 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.

  14. Functionalization of an Antisense Small RNA.

    Science.gov (United States)

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

    2016-02-27

    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.

  15. Evaluation of sperm DNA damage in bulls by TUNEL assay as a parameter of semen quality

    OpenAIRE

    TAKEDA, Kumiko; UCHIYAMA, Kyoko; KINUKAWA, Masashi; Tagami, Takahiro; Kaneda, Masahiro; Watanabe, Shinya

    2015-01-01

    Sperm DNA damage affects the conception rate resulting from human assisted reproduction technology. The objective of this study was to adapt the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay to provide a quality parameter for bull semen based on the detection of sperm DNA damage. Fresh semen was collected from two Japanese Black bulls (A, B) several times over the course of a year, and the percentage of TUNEL-positive spermatozoa (sperm TUNEL index) was d...

  16. Reduction of polygalacturonase activity in tomato fruit by antisense RNA

    OpenAIRE

    Sheehy, Raymond E.; Kramer, Matthew; Hiatt, William R

    1988-01-01

    Polygalacturonase [PG; poly(1,4-α-D-galacturonide) glycanhydrolase; EC 3.2.1.15] is expressed in tomato only during the ripening stage of fruit development. PG becomes abundant during ripening and has a major role in cell wall degradation and fruit softening. Tomato plants were transformed to produce antisense RNA from a gene construct containing the cauliflower mosaic virus 35S promoter and a full-length PG cDNA in reverse orientation. The construct was integrated into the tomato genome by A...

  17. Coexistence of sense and anti-sense mRNAs of variant surface protein in Giardia lamblia trophozoites.

    Science.gov (United States)

    Guo, Junli; Zheng, Wenyu; Wang, Yuehua; Li, Yao; Lu, Siqi; Feng, Xianmin

    2014-02-14

    A strategy of the parasitic protozoan Giardia lamblia to evade attack from the host immune system is periodic changes of its surface antigen, a member of the variant surface protein (VSP) family. A post-transcriptional gene silencing mechanism has been proposed to explain the presence of only one among many possible VSPs at any time. To investigate this phenomenon further, we extracted total RNA from cultured trophozoites of the G. lamblia C2 isolate, and cDNA was reverse-transcribed from the RNA. Sense and anti-sense VSPs were amplified from the total cDNA using nested PCR with primers designed from the 3'-conserved region and the known 5' or 3' end of the cDNA library. Sequence analyses of the amplified products revealed more than 34 full-length antisense VSPs and a smear of sense VSPs. Sequence alignments and comparisons revealed that these VSPs contained variable N-termini and conserved C-termini, and could be classified into 5 clades based on the sizes and variations of the N-terminal sequence. All antisense VSPs existed in the sense forms, but no corresponding antisense VSP existed for sense RNA (snsRNA) 16. The coexistence of sense and antisense VSP mRNAs in cultured G. lamblia supports the post-transcriptional regulation of VSP expression. We propose that VSPs transcribed simultaneously in the sense and antisense forms form double-stranded RNAs (dsRNAs) which are degraded by the Dicer endonuclease, while a VSP without an antisense transcription (e.g., snsRNA16) will be expressed on the surface of Giardia. In addition, in the course of this investigation VSPs were identified that were previously not known. PCR-based amplification of specific sense and antisense VSP cDNAs can be used to identify the specific VSP on G. lamblia trophozoites, which is easier than using specific monoclonal antibody approaches.

  18. 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

  19. 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 a...... or phosphorothioate-DNA segment flanked by LNA gaps, rivals siRNA as the technology of choice for target validation and therapeutic applications....... 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...

  20. [Molecular combing method in the research of DNA replication parameters in isolated organs of Drosophyla melanogaster].

    Science.gov (United States)

    Ivankin, A V; Kolesnikova, T D; Demakov, S A; Andreenkov, O V; Bil'danova, E R; Andreenkova, N G; Zhimulev, I F

    2011-01-01

    Methods of physical DNA mapping and direct visualization of replication and transcription in specific regions of genome play crucial role in the researches of structural and functional organization of eukaryotic genomes. Since DNA strands in the cells are organized into high-fold structure and present as highly compacted chromosomes, the majority of these methods have lower resolution at chromosomal level. One of the approaches to enhance the resolution and mapping accuracy is the method of molecular combing. The method is based on the process of stretching and alignment of DNA molecules that are covalently attached with one of the ends to the cover glass surface. In this article we describe the major methodological steps of molecular combing and their adaptation for researches of DNA replication parameters in polyploidy and diploid tissues of Drosophyla larvae.

  1. SENSITIZATION OF ACNU KILLING EFFECTS ON HeLa S3 CELLS BY MGMT ANTISENSE RNA TRANSFECTION

    Institute of Scientific and Technical Information of China (English)

    季守平; 由英; 吴英; 陈建敏; 杨军; 章扬培

    1998-01-01

    O’-methylguanlne-DNA-msthybransferase(MGMT)plays a very important role in the ceUular resistsnce to uitrosoureas drugs. Inhibition of MGMT might be a useful approach in tumor chemotherapy. In this study, the depletlon vii MGMT activity hy retroviral-mediated antisense RNA transfectkm were reported. Three retroviral vectors expressing MGMT antisense RNA were constructed and transfected into HeLa S3 cells. The difference of MGMT mRNA, MGMT activity as well as cellular resistance to ACNU before and after transtecfion were ohserved. It was found that antisense RNA targeting 5''region and whole length of MGMT mRNA could partially deplete MGMT activity and enhance killing effects of ACNU.However, 3'' region antisense RNA had no effect on MGMT modulation.

  2. 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.

  3. 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.

  4. Antisense expression increases gene expression variability and locus interdependency

    OpenAIRE

    Xu, Zhenyu; Wei, Wu; Gagneur, Julien; Clauder-Münster, Sandra; Smolik, Miłosz; Huber, Wolfgang; Steinmetz, Lars M.

    2011-01-01

    Genome-wide transcription profiling has revealed extensive expression of non-coding RNAs antisense to genes, yet their functions, if any, remain to be understood. In this study, we perform a systematic analysis of sense–antisense expression in response to genetic and environmental changes in yeast. We find that antisense expression is associated with genes of larger expression variability. This is characterized by more ‘switching off' at low levels of expression for genes with antisense compa...

  5. Longitudinal follow-up of oxidative stress and DNA damage parameters in detergent workers

    Directory of Open Access Journals (Sweden)

    Boojar Masoud Mashhadi

    2009-01-01

    Full Text Available Background: The aim of this study was the follow-up of work place enzyme and detergent dust exposure effects and smoking habit on DNA damage parameters of workers and the evaluation of their antioxidant enzyme activities and lipid peroxidation with regard to bag-filter installation in the work place. Material and Methods: All investigated parameters were studied in a group of 153 workers of enzyme-free detergent production plant (E-free and a group of 138 workers of enzyme-plus detergent plant (E-plus and compared with 45 controls 7.2 years before and 3.1 years after filter system installation. The following methods were used: antioxidant enzymes by an ultraviolet-visibles spectrophotometer, malondialdehyde (MDA, 8-hydroxy-2′deoxyguanosine (8OH-2′dG by high-performance liquid chromatography, trace elements by atomic absorption spectroscopy, and comet assay by single cell gel electrophoresis. Results: Compared with controls, significant increases were observed in both detergent-exposed groups with respect to the levels of MDA, antioxidant enzyme activities, and DNA damage parameters, including 8OH-2′dG, endonuclease III-sensitive sites, and DNA strand breaks, with enhancement effect of smoking before filter system installation. After filter installation, besides significant decrease in the detergent and enzyme dust of airborne and oxidative stress indicators, there was improvement in all DNA damage investigated parameters at the end of this study. The levels of cumulative exposure index of detergent dusts decreased significantly after airborne improvement and showed positive correlation with internal biochemical parameters. Conclusions: We concluded that high levels of enzyme and detergent contents of work place dusts had a cumulative effect and smoking had a synergistic effect on the imbalance of antioxidant status and lipid peroxidation, suggesting that oxidation stress is important in the occurrence and progression of DNA damage over

  6. Identification of resting cells by dual-parameter flow cytometry of statin expression and DNA content

    Energy Technology Data Exchange (ETDEWEB)

    Pellicciari, C.; Mangiarotti, R.; Bottone, M.G.; Danova, M. [Univ. of Pavia (Italy); Wang, E. [Jewish General Hospital, Montreal, Quebec (Canada)

    1995-12-01

    Statin, a 57-kDa nuclear protein, has been recognized as a unique marker of quiescent (G{sub 0}) cells; specific monoclonal antibodies (MoAb) against statin have been produced and used to label resting cells in tissue sections and in cultured cells. We present an improved method for the identification of G{sub 0} cells by dual-parameter flow cytometry of statin expression and DNA content. The appropriate technical conditions were set up by using resting and cycling human fibroblasts as a model cell system. Several fixatives proved to be suitable for the immunocytochemical detection of statin; among them, 70% ethanol was selected because this fixation procedure is suitable for DNA staining with intercalating dyes and is routinely used for the immunolabeling of proliferation markers (such as proliferating cell nuclear antigen [PCNA] and Ki-67) and of bromodeoxyuridine (BrdUrd) incorporation. Following cell permeabilization with detergent, exposure to the antistatin antibody (S-44), and indirect fluorescein isothiocyanate immunolabeling, cells were counterstained for DNA with propidium iodide and analyzed by dual-parameter flow cytometry. In cells from several animal sources (rat thymocytes and C6 glioma cells, mouse 3T3 cells, and human MCF-7 cells), under different experimental conditions, the expression of statin was found to correlate inversely with that of PCNA and Ki-67, and with the BrdUrd labeling index. In dual-parameter flow scattergrams, G{sub 0} (statin positive) cells can be discriminated from the potentially cycling (statin negative) G{sub 1} cells, i.e., within a cell fraction having the same DNA content. This approach can be envisaged as a powerful tool both for monitoring changes in the resting cell fraction and for investigating the process of G{sub 0}-G{sub 1} transition in unperturbed and drug-treated cell populations. 48 refs., 5 figs., 1 tab.

  7. 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.

  8. 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.

  9. 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.

  10. Superior Silencing by 2′,4′-BNANC-Based Short Antisense Oligonucleotides Compared to 2′,4′-BNA/LNA-Based Apolipoprotein B Antisense Inhibitors

    Directory of Open Access Journals (Sweden)

    Tsuyoshi Yamamoto

    2012-01-01

    Full Text Available The duplex stability with target mRNA and the gene silencing potential of a novel bridged nucleic acid analogue are described. The analogue, 2′,4′-BNANC antisense oligonucleotides (AONs ranging from 10- to 20-nt-long, targeted apolipoprotein B. 2′,4′-BNANC was directly compared to its conventional bridged (or locked nucleic acid (2′,4′-BNA/LNA-based counterparts. Melting temperatures of duplexes formed between 2′,4′-BNANC-based antisense oligonucleotides and the target mRNA surpassed those of 2′,4′-BNA/LNA-based counterparts at all lengths. An in vitro transfection study revealed that when compared to the identical length 2′,4′-BNA/LNA-based counterpart, the corresponding 2′,4′-BNANC-based antisense oligonucleotide showed significantly stronger inhibitory activity. This inhibitory activity was more pronounced in shorter (13-, 14-, and 16-mer oligonucleotides. On the other hand, the 2′,4′-BNANC-based 20-mer AON exhibited the highest affinity but the worst IC50 value, indicating that very high affinity may undermine antisense potency. These results suggest that the potency of AONs requires a balance between reward term and penalty term. Balance of these two parameters would depend on affinity, length, and the specific chemistry of the AON, and fine-tuning of this balance could lead to improved potency. We demonstrate that 2′,4′-BNANC may be a better alternative to conventional 2′,4′-BNA/LNA, even for “short” antisense oligonucleotides, which are attractive in terms of drug-likeness and cost-effective bulk production.

  11. Effects of Sperm DNA Fragmentation on Semen Parameters and ICSI Outcome Determined by an Improved SCD Test,Halosperm

    Directory of Open Access Journals (Sweden)

    Asuman Demiroglu Zergeroğlu

    2010-01-01

    Full Text Available Background: Sperm DNA fragmentation is known as an important cause of male infertility.The influence of sperm DNA damage on reproductive potential has been subject of many studiesindicating various results and remaining the subject controversial. In this study, we investigateddifferences of the semen parameters and intracytoplasmic sperm injection (ICSI outcome accordingto sperm DNA fragmentation levels (DFLs of patients.Materials and Methods: The DFLs were determined by Halosperm, a new improved spermchromatin dispersion (SCD test. Patients were grouped as low DNA fragmentation group (LFG≤30% and high fragmentation group (HFG >30%.Results: Our analysis showed that semen parameters including concentration of untreated spermand motility of prepared semen were low in HGF, whereas other parameters were not different.Sperm DNA fragmentation levels decreased in both groups after semen preparation by densitygradient technique.Conclusion: No difference was detected on ICSI outcomes (fertilization, embryo development,embryo cleavage, embryo quality and pregnancy rates between two group.

  12. 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.

  13. Influence of routes and administration parameters on antibody response of pigs following DNA vaccination

    DEFF Research Database (Denmark)

    Barfoed, Annette Malene; Kirstensen, Birte; Dannemann-Jensen, Tove

    2004-01-01

    Using the nucleoprotein of porcine reproductive and respiratory syndrome virus as model antigen, we optimised parameters for gene gun vaccination of pigs, including firing pressure and vaccination site. As criteria for optimisation, we characterised particle penetration and local tissue damage...... by histology. For selected combinations, vaccination efficiency in terms of antibody response was studied. Gene gun vaccination on ear alone was as efficient as a multi-site (ear, thorax, inguinal area, tongue mucosa) gene gun approach, and more efficient than combined intramuscular (i.m.)/intradermal (i.......d.) injection of plasmid DNA. This indicates, that the ear is an attractive site for gene gun vaccination of pigs....

  14. Effects of Sperm DNA Fragmentation on Semen Parameters and ICSI Outcome Determined by an Improved SCD Test,Halosperm

    OpenAIRE

    Asuman Demiroglu Zergeroğlu; Seda Yılmaz; Pelin Kutlu; Nuri Delikara; Kenan Sofuoglu

    2010-01-01

    Background: Sperm DNA fragmentation is known as an important cause of male infertility.The influence of sperm DNA damage on reproductive potential has been subject of many studiesindicating various results and remaining the subject controversial. In this study, we investigateddifferences of the semen parameters and intracytoplasmic sperm injection (ICSI) outcome accordingto sperm DNA fragmentation levels (DFLs) of patients.Materials and Methods: The DFLs were determined by Halosperm, a new im...

  15. Dopamine-induced apoptosis in human neuronal cells: inhibition by nucleic acides antisense to the dopamine transporter

    Energy Technology Data Exchange (ETDEWEB)

    Porat, S.; Gabbay, M.; Tauber, M.; Ratovitski, T.; Blinder, E.; Simantov, R. [Department of Molecular Genetics, Weizmann Institute of Science Rehovot 76100 (Israel)

    1996-09-01

    Human neuroblastoma NMB cells take up [{sup 3}H]dopamine in a selective manner indicating that dopamine transporters are responsible for this uptake. These cells were therefore used as a model to study dopamine neurotoxicity, and to elucidate the role of dopamine transporters in controlling cell death. Treatment with 0.05-0.4 mM dopamine changed cells' morphology within 4 h, accompanied by retraction of processes, shrinkage, apoptosis-like atrophy, accumulation of apoptotic particles, DNA fragmentation and cell death. Cycloheximide inhibited dopamine's effect, suggesting that induction of apoptosis by dopamine was dependent upon protein synthesis. Dopamine cytotoxicity, monitored morphologically by flow cytometric analysis, and by lactate dehydrogenase released, was blocked by cocaine but not by the noradrenaline and serotonin uptake blockers desimipramine and imipramine, respectively. Attempting to inhibit dopamine transport and toxicity in a drug-free and highly selective way, three 18-mer dopamine transporter antisense phosphorothioate oligonucleotides (numbers 1, 2 and 3) and a new plasmid vector expressing the entire rat dopamine transporter complementary DNA in the antisense orientation were prepared and tested. Antisense phosphorothioate oligonucleotide 3 inhibited [{sup 3}H]dopamine uptake in a time- and dose-dependent manner. Likewise, transient transfection of NMB cells with the plasmid expressing dopamine transporter complementary DNA in the antisense orientation partially blocked [{sup 3}H]dopamine uptake. Antisense phosphorothioate oligonucleotide 3 also decreased, dose-dependently, the toxic effect of dopamine and 6-hydroxydopamine. Western blot analysis with newly prepared anti-human dopamine transporter antibodies showed that antisense phosphorothioate oligonucleotide 3 decreased the transporter protein level. These studies contribute to better understand the mechanism of dopamine-induced apoptosis and neurotoxicity. (Copyright (c) 1996

  16. Evaluation of sperm DNA damage in bulls by TUNEL assay as a parameter of semen quality.

    Science.gov (United States)

    Takeda, Kumiko; Uchiyama, Kyoko; Kinukawa, Masashi; Tagami, Takahiro; Kaneda, Masahiro; Watanabe, Shinya

    2015-01-01

    Sperm DNA damage affects the conception rate resulting from human assisted reproduction technology. The objective of this study was to adapt the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay to provide a quality parameter for bull semen based on the detection of sperm DNA damage. Fresh semen was collected from two Japanese Black bulls (A, B) several times over the course of a year, and the percentage of TUNEL-positive spermatozoa (sperm TUNEL index) was determined. Individual differences in semen were detected using the sperm TUNEL index in these bulls (P bulls with a conception rate lower than 10%) and Holstein (n = 34) bulls were analyzed. The average sperm TUNEL index and conception rate resulting from artificial insemination (AI) were 4.7% and 55.7% for Japanese Black, and 4.9% and 39.5% for Holstein, respectively. A weak negative correlation between sperm TUNEL index and conception rate was observed in Holstein bulls (P bulls with more than 10% sperm TUNEL index were studied, and these samples showed low sperm viability. However, semen resulting in a very low conception rate did not have a high sperm TUNEL index. Although it would be difficult to predict a low conception rate resulting from AI using the sperm TUNEL index alone, the index can be used as an additional parameter to provide a more comprehensive description of semen quality.

  17. 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

  18. Antisense oligonucleotides in therapy for neurodegenerative disorders.

    Science.gov (United States)

    Evers, Melvin M; Toonen, Lodewijk J A; van Roon-Mom, Willeke M C

    2015-06-29

    Antisense oligonucleotides are synthetic single stranded strings of nucleic acids that bind to RNA and thereby alter or reduce expression of the target RNA. They can not only reduce expression of mutant proteins by breakdown of the targeted transcript, but also restore protein expression or modify proteins through interference with pre-mRNA splicing. There has been a recent revival of interest in the use of antisense oligonucleotides to treat several neurodegenerative disorders using different approaches to prevent disease onset or halt disease progression and the first clinical trials for spinal muscular atrophy and amyotrophic lateral sclerosis showing promising results. For these trials, intrathecal delivery is being used but direct infusion into the brain ventricles and several methods of passing the blood brain barrier after peripheral administration are also under investigation.

  19. 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

  20. 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.

  1. A novel HBV antisense RNA gene delivery system targeting hepatocellular carcinoma

    Institute of Scientific and Technical Information of China (English)

    Chun-Hong Ma; Xiao-Hong Liang; Wen-Sheng Sun; Pei-Kun Tian; Li-Fen Gao; Su-Xia Liu; Xiao-Yan Wang; Li-Ning Zhang; Ying-Lin Cao; Li-Hui Han

    2003-01-01

    AIM: To construct a novel HBV antisense RNA delivery system targeting hapatocellular carcinoma and study its inhibitory effect in vitro and in vivo.METHODS: GE7,a 16-peptide specific to EGFR, and HA20,a homologue of N-terminus of haemagglutinin of influenza viral envelope protein, were synthesized and conjugated with polylysin. The above conjugates were organized into the pEBAF-as-preS2, a hepatocarcinoma specific HBV antisense expression vector, to construct a novel HBV antisense RNA delivery system, named AFP-enhancing 4-element complex. Hepatocelluar carcinoma HepG2.2.15 cells was used to assay the in vitro inhibition of the complex on HBV. Expression of HBV antigen was assayed by ELISA. BALB/c nude mice bearing HepG2.2.15 cells were injected with AFP-enhancing 4-element complex. The expression of HBV antisense RNA was examined by RT-PCR and the size of tumor in nude mice were measured.RESULTS: The AFP-enhancing 4-element complex was constructed and DNA was completely trapped at the slot with no DNA migration when the ratio of polypeptide to plasmid was 1:1.The expression of HBsAg and HBeAg of HepG2.2.15 cells was greatly decreased after being transfected by AFP-enhancing 4-element complex. The inhibitory rates were 33.4 % and 58.5 % respectively. RTPCR showed HBV antisense RNA expressed specifically in liver tumor cells of tumor-bearing nude mice. After 4injections of AFP-enhancing 4-element complex containing 0.2 μg DNA, the diameter of the tumor was 0.995 cm±0.35,which was significantly smaller than that of the control groups (2.215 cm±0.25, P<0.05).CONCLUSION: AFP-enhancing 4-element complex could deliver HBV antisense RNA targeting on hepatocarcinoma and inhibit both HBV and liver tumor cells in vitro and in vivo.

  2. 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.

  3. 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.

  4. 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.

  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. 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.

  7. 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.

  8. 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.

  9. DAF optimization using Taguchi methods and the effect of thermal cycling parameters on DNA amplification.

    Science.gov (United States)

    Caetano-Anollés, G

    1998-09-01

    Taguchi methods, which are widely applied in industrial process design, were used to optimize DNA amplification finger-printing (DAF). Quadratic loss functions that penalize deviations from prediction values and L9 (3(4)) and L18 (3(8)) orthogonal arrays revealed effects and interactions of amplification reaction components and thermal cycling parameters. Analysis of variance (ANOVA) decomposed the contribution of individual factors to the experimental response (amplification yield and product number), while verification experiments established that optimum conditions were predictable, verifiable and reproducible. While several amplification components (primer, magnesium and enzyme) conditioned the amplification reaction, annealing temperature and time were the only important thermal cycling contributing factors. The Taguchi strategy defined a robust and transportable amplification protocol based on high annealing temperatures (typically 48 degrees C) and primer concentrations (typically 8 microM), which can be applied to the fingerprinting of a wide range of DNA templates of plant and fungal origin. The general strategy of robust experimental design holds potential as an optimization tool for other methods in molecular biology.

  10. INTEGRATIVE COMPUTER ANALYSIS OF ANTISENSE TRANSCRIPTS AND miRNA TARGETS IN PLANT GENOMES

    Directory of Open Access Journals (Sweden)

    Orlov Y.L.

    2012-08-01

    Full Text Available Non-coding RNA, including small interfering RNAs (siRNAs, are important components of gene expression in eukaryotes, forming a regulatory network. miRNAs are expressed through nucleolytic maturation of hairpin precursors transcribed by RNA Polymerase II or III. Such transcripts are involved in post-transcriptional gene regulation in plants, fungi and animals. miRNAs bind to target RNA transcripts and guide their cleavage (mostly for plants or act to prevent translation. siRNAs act via a similar mechanism of cleavage of their target genes, but they also can direct genomic DNA methylation and chromatin remodeling. It is estimated that large fraction, up to 30% of all human genes also may be post-transcriptionally regulated by miRNAs. For plant genomes numbers could be higher depending on quality of sequencing and genome annotation. Due to availability of genome and mRNA sequences genome-wide searches for sense-antisense transcripts have been reported, but few plant sense-antisense transcript pairs have been studied. Integration of these data in specialized databases is challenging problem of computer genomics. We have developed set of computer programs to define antisense transcripts and miRNA genes based on available sequencing data. We have analyzed data from PlantNATsDB (Plant Natural Antisense Transcripts DataBase which is a platform for annotating and discovering Natural Antisense Transcripts (NAT by integrating various data sources [1]. NATs can be grouped into two categories, cis-NATs and trans-NATs. Cis-NAT pairs are transcribed from opposing DNA strands at the same genomic locus and have a variety of orientations and differing lengths of overlap between the perfect sequence complementary regions, whereas trans-NAT pairs are transcribed from different loci and form partial complementarily. The database contains at the moment 69 plant species. The database provides an integrative, interactive and information-rich web graphical interface to

  11. Synthesis of antisense oligonucleotides containing acyclic alkynyl nucleoside analogs and their biophysical and biological properties.

    Science.gov (United States)

    Ogata, Aya; Maeda, Yusuke; Ueno, Yoshihito

    2017-02-17

    The synthesis of oligonucleotide (ON) analogs, which can be used as antisense molecules, has recently gained much attention. Here, we report the synthesis and properties of an ON analog containing acyclic thymidine and cytidine analogs with a 4-pentyl-1,2-diol instead of the d-ribofuranose moiety. The incorporation of these analogs into the ON improved its nuclease resistance to 3'-exonucleases. Furthermore, it was found that the incorporation of the acyclic thymidine analog into a DNA/RNA duplex accelerates the RNA cleavage of a DNA/RNA duplex by Escherichia coli RNase H.

  12. RNA Interference-Guided Targeting of Hepatitis C Virus Replication with Antisense Locked Nucleic Acid-Based Oligonucleotides Containing 8-oxo-dG Modifications.

    Science.gov (United States)

    Mutso, Margit; Nikonov, Andrei; Pihlak, Arno; Žusinaite, Eva; Viru, Liane; Selyutina, Anastasia; Reintamm, Tõnu; Kelve, Merike; Saarma, Mart; Karelson, Mati; Merits, Andres

    2015-01-01

    The inhibitory potency of an antisense oligonucleotide depends critically on its design and the accessibility of its target site. Here, we used an RNA interference-guided approach to select antisense oligonucleotide target sites in the coding region of the highly structured hepatitis C virus (HCV) RNA genome. We modified the conventional design of an antisense oligonucleotide containing locked nucleic acid (LNA) residues at its termini (LNA/DNA gapmer) by inserting 8-oxo-2'-deoxyguanosine (8-oxo-dG) residues into the central DNA region. Obtained compounds, designed with the aim to analyze the effects of 8-oxo-dG modifications on the antisense oligonucleotides, displayed a unique set of properties. Compared to conventional LNA/DNA gapmers, the melting temperatures of the duplexes formed by modified LNA/DNA gapmers and DNA or RNA targets were reduced by approximately 1.6-3.3°C per modification. Comparative transfection studies showed that small interfering RNA was the most potent HCV RNA replication inhibitor (effective concentration 50 (EC50): 0.13 nM), whereas isosequential standard and modified LNA/DNA gapmers were approximately 50-fold less efficient (EC50: 5.5 and 7.1 nM, respectively). However, the presence of 8-oxo-dG residues led to a more complete suppression of HCV replication in transfected cells. These modifications did not affect the efficiency of RNase H cleavage of antisense oligonucleotide:RNA duplexes but did alter specificity, triggering the appearance of multiple cleavage products. Moreover, the incorporation of 8-oxo-dG residues increased the stability of antisense oligonucleotides of different configurations in human serum.

  13. 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.

  14. 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

    -signaling proteins in pollen tubes from the lilly Agapanthus umbellatus. For the uptake of DNA pollen tubes represent a unique system since the growing tip is surrounded by a loose matrix of hemicellulose and pectins, exposing the plasma membrane7 and the first uptake of ODNs by pollen tubes was reported as early as 1994. A breakthrough in the employment of antisense ODN inhibition as a powerful approach in plant biology was recently presented through our work on intact barley leaves. As was illustrated by confocal microscopy and fluorescently labeled ODNs, naked ODNs were taken up through the leaf petiole and efficiently imported into the plant cell and the nucleus. The work portrayed in that study demonstrate the applicability of antisense ODN inhibition in plant biology, e.g. as a rapid antecedent to time-consuming transgenic studies, and that it operates through RNase H degradation. We employed the antisense ODN strategy to demonstrate the importance of the SUSIBA2 transcription factor in regulation of starch synthesis, and to depict a possible mechanism for sugar signaling in plants and how it might confer endosperm-specific gene expression during seed development. We also described the employment of the antisense ODN strategy for studies on in vitro spike cultures of barley. Here we present further evidence as to the value of the antisense ODN approach in plant biology by following the effects on starch branching enzyme (SBE) accumulation in barley leaves after suppression of individual SBE genes. In agreement with transcript analyses of SBE expression in barley leaves, a zymogram assay (Fig. 1) revealed that sucrose treatment of barley leaves increased the number of SBE activity bands as compared to sorbitol treatment. In the presence of antisense SBEI or SBEIIA ODNs, zymograms of sucrose-treated leaves displayed only a subset of these activities with bands in the top portion of the zymogram gel missing or diminished. With antisense SBEIIB ODN, all activity bands in the top

  15. 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

    -signaling proteins in pollen tubes from the lilly Agapanthus umbellatus. For the uptake of DNA pollen tubes represent a unique system since the growing tip is surrounded by a loose matrix of hemicellulose and pectins, exposing the plasma membrane7 and the first uptake of ODNs by pollen tubes was reported as early as 1994. A breakthrough in the employment of antisense ODN inhibition as a powerful approach in plant biology was recently presented through our work on intact barley leaves. As was illustrated by confocal microscopy and fluorescently labeled ODNs, naked ODNs were taken up through the leaf petiole and efficiently imported into the plant cell and the nucleus. The work portrayed in that study demonstrate the applicability of antisense ODN inhibition in plant biology, e.g. as a rapid antecedent to time-consuming transgenic studies, and that it operates through RNase H degradation. We employed the antisense ODN strategy to demonstrate the importance of the SUSIBA2 transcription factor in regulation of starch synthesis, and to depict a possible mechanism for sugar signaling in plants and how it might confer endosperm-specific gene expression during seed development. We also described the employment of the antisense ODN strategy for studies on in vitro spike cultures of barley. Here we present further evidence as to the value of the antisense ODN approach in plant biology by following the effects on starch branching enzyme (SBE) accumulation in barley leaves after suppression of individual SBE genes. In agreement with transcript analyses of SBE expression in barley leaves, a zymogram assay (Fig. 1) revealed that sucrose treatment of barley leaves increased the number of SBE activity bands as compared to sorbitol treatment. In the presence of antisense SBEI or SBEIIA ODNs, zymograms of sucrose-treated leaves displayed only a subset of these activities with bands in the top portion of the zymogram gel missing or diminished. With antisense SBEIIB ODN, all activity bands in the top

  16. Cocaine and amphetamine elicit differential effects in rats with a unilateral injection of dopamine transporter antisense oligodeoxynucleotides.

    Science.gov (United States)

    Silvia, C P; Jaber, M; King, G R; Ellinwood, E H; Caron, M G

    1997-02-01

    We have developed an antisense oligodeoxynucleotide to the dopamine transporter and used it to discriminate the behavioral properties of amphetamine and cocaine. In SK-N-MC cells permanently transfected with the dopamine transporter complementary DNA, treatment with 5 mM antisense oligodeoxynucleotide reduced dopamine uptake by 25% when compared to sense control. Unilateral intranigral administration of dopamine transporter antisense (50 microM) twice daily in freely moving rats for 2.5 days was sufficient to reduce dopamine transporter messenger RNA by 70% as measured by in situ hybridization, but not protein levels as measured by [3H]mazindol binding. However, intranigral treatment via implanted osmotic minipump over a period of seven days produced reductions in both dopamine transporter messenger RNA and protein levels (32%) at a dose of 500 pmol/day. These results indicate a longer half-life for the dopamine transporter than expected. Potassium chloride depolarization of ipsilateral striatal slices showed a greater than 200% increase in dopamine overflow on the antisense-treated side compared to the control side. Since imbalance of dopamine tone is known to induce rotational activity, we tested this behavioral paradigm in rats treated with various oligodeoxynucleotides at different doses and time-points. We have found that antisense-treated animals did not rotate spontaneously under any experimental conditions. Using various psychostimulants that target the dopamine transporter and increase dopamine levels, we found that the antisense-treated animals consistently rotated contralaterally in response to amphetamine (2 mg/kg), but not to cocaine (10 mg/kg) or nomifensine (10 mg/kg). These results bring in vivo evidence for a different mode of action of amphetamine and cocaine on the dopamine transporter and lend direct support to the view that amphetamine acts as a dopamine releaser, whereas cocaine acts by blocking dopamine transport.

  17. 反义DNA甲基化酶3b基因片断真核表达载体的构建及鉴定%Construction and indentification of eukaryotic expression vector for antisense DNA methylferase 3b gene fragment

    Institute of Scientific and Technical Information of China (English)

    左石; 罗剑; 刘民锋; 徐立宁; 董泾青; 郭伟; 邹声泉

    2005-01-01

    目的构建反义DNA甲基化酶3b(DNMT3b)基因片断真核表达载体,为研究DNMT3b基因功能提供工具.方法根据DNMT3b基因cDNA序列中编码序列设计PCR引物,在上下游引物5′端分别添加XbaⅠ和KpnⅠ酶切位点,RT-PCR从胆管癌细胞QBC-939中获得485bp的DNA片断;将该片断反向插入真核表达载体pcDNA3.1(+)的多克隆位点,构建反义DNMT3b基因片断真核表达载体,并用PCR、酶切法和DNA测序鉴定.结果 PCR鉴定得到467bp特异条带,双酶切鉴定得到471bp片断和5.4kb载体片断,DNA测序说明插入片断序列正确.结论本研究构建的反义DNMT3b基因片断真核表达载体可为进一步研究DNMT3b基因功能提供实验工具.

  18. Antisense repression of sucrose phosphate synthase in transgenic muskmelon alters plant growth and fruit development

    Energy Technology Data Exchange (ETDEWEB)

    Tian, Hongmei; Ma, Leyuan; Zhao, Cong; Hao, Hui; Gong, Biao [College of Horticulture Science and Engineering, State Key Laboratory of Crop Biology, Shandong Agricultural University, Tai' an, Shandong 271018 (China); Yu, Xiyan, E-mail: yuxiyan@sdau.edu.cn [College of Horticulture Science and Engineering, State Key Laboratory of Crop Biology, Shandong Agricultural University, Tai' an, Shandong 271018 (China); Wang, Xiufeng, E-mail: xfwang@sdau.edu.cn [College of Horticulture Science and Engineering, State Key Laboratory of Crop Biology, Shandong Agricultural University, Tai' an, Shandong 271018 (China)

    2010-03-12

    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.

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. 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.

  4. Impact of xanthohumol (a prenylated flavonoid from hops) on DNA stability and other health-related biochemical parameters

    DEFF Research Database (Denmark)

    Ferk, Franziska; Mišík, Miroslav; Nersesyan, Armen

    2016-01-01

    SCOPE: Xanthohumol (XN) is a hop flavonoid found in beers and refreshment drinks. Results of in vitro and animal studies indicate that it causes beneficial health effects due to DNA protective, anti-inflammatory, antioxidant, and phytoestrogenic properties. Aim of the present study was to find out...... if XN causes alterations of health-related parameters in humans. METHODS AND RESULTS: The effects of the flavonoid were investigated in a randomized crossover intervention trial (n = 22) in which the participants consumed a XN drink (12 mg XN/P/day). We monitored alterations of the DNA stability......'-deoxyguanosine and 8-oxo-guanosine in urine was reduced. The assumption that the flavonoid causes DNA protection was confirmed in a randomized follow-up study with pure XN (n = 10) with a parallel design. Other biochemical parameters reflecting the redox- and hormonal status and lipid- and glucose metabolism...

  5. Chromosomally encoded small antisense RNA in Corynebacterium glutamicum.

    Science.gov (United States)

    Zemanová, Martina; Kaderábková, Pavla; Pátek, Miroslav; Knoppová, Monika; Silar, Radoslav; Nesvera, Jan

    2008-02-01

    The first observation of chromosomally encoded small antisense RNA in Corynebacterium glutamicum is reported. Transcription oriented in the reverse direction to the transcription of the genes cg1934 and cg1935 was demonstrated within the chromosomal cg1934-cg1935 intergenic region. The transcription was found to be increased after heat shock. The transcriptional start point of this RNA designated ArnA was localized 21 bp upstream of the cg1935 translational start point by primer extension analysis, when the total RNA was isolated from cells grown at 30 degrees C. After heat shock, the transcriptional start point of an additional species of ArnA RNA was detected 19 bp upstream of the cg1935 translational start point. The stress-response sigma factor SigH was found to be involved in the synthesis of ArnA RNAs. The 3' end of the ArnA RNAs was identified using the 3'-rapid amplification of cDNA ends technique. The length of the two ArnA RNA species was thus determined to be 129 and 131 nt, respectively. The ArnA RNAs were found to overlap the 5'-untranslated region of the transcript of the cg1935 gene coding for a transcriptional regulator of the GntR family. These results suggest that the noncoding ArnA RNAs have a regulatory function.

  6. Ribonucleases, antisense RNAs and the control of bacterial plasmids.

    Science.gov (United States)

    Saramago, Margarida; Bárria, Cátia; Arraiano, Cecília M; Domingues, Susana

    2015-03-01

    In the last decade regulatory RNAs have emerged as powerful tools to regulate the expression of genes both in prokaryotes and in eukaryotes. RNases, by degrading these RNA molecules, control the right amount of regulatory RNAs, which is fundamental for an accurate regulation of gene expression in the cell. Remarkably the first antisense RNAs identified were plasmid-encoded and their detailed study was crucial for the understanding of prokaryotic antisense RNAs. In this review we highlight the role of RNases in the precise modulation of antisense RNAs that control plasmid replication, maintenance and transfer.

  7. On primordial sense-antisense coding.

    Science.gov (United States)

    Rodin, Andrei S; Rodin, Sergei N; Carter, Charles W

    2009-11-01

    The genetic code is implemented by aminoacyl-tRNA synthetases (aaRS). These 20 enzymes are divided into two classes that, despite performing same functions, have nothing common in structure. The mystery of this striking partition of aaRSs might have been concealed in their sterically complementary modes of tRNA recognition that, as we have found recently, protect the tRNAs with complementary anticodons from confusion in translation. This finding implies that, in the beginning, life increased its coding repertoire by the pairs of complementary codons (rather than one-by-one) and used both complementary strands of genes as templates for translation. The class I and class II aaRSs may represent one of the most important examples of such primordial sense-antisense (SAS) coding (Rodin and Ohno, Orig Life Evol Biosph 25:565-589, 1995). In this report, we address the issue of SAS coding in a wider scope. We suggest a variety of advantages that such coding would have had in exploring a wider sequence space before translation became highly specific. In particular, we confirm that in Achlya klebsiana a single gene might have originally coded for an HSP70 chaperonin (class II aaRS homolog) and an NAD-specific GDH-like enzyme (class I aaRS homolog) via its sense and antisense strands. Thus, in contrast to the conclusions in Williams et al. (Mol Biol Evol 26:445-450, 2009), this could indeed be a "Rosetta stone" gene (Carter and Duax, Mol Cell 10:705-708, 2002) (eroded somewhat, though) for the SAS origin of the two aaRS classes.

  8. 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.

  9. 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.

  10. Early and late effects of Ibuprofen on mouse sperm parameters, chromatin condensation, and DNA integrity in mice

    Directory of Open Access Journals (Sweden)

    Fatemeh Roodbari

    2015-11-01

    Full Text Available Background: There are few studies indicating the detrimental effects of ibuprofen on sperm fertility potential and DNA integrity. Objective: To determine the effects of Ibuprofen on sperm parameters, chromatin condensation and DNA integrity of mice. Materials and Methods: In this experimental study, 36 adult male mice with average weight 37 gr were divided into three groups, including control (group I, n=12, normal dosage of ibuprofen (group II, n=12 and high dosage (group III, n=12. Ibuprofen with different doses was dissolved in daily water of animals. After 35, 70 and 105 days, the cauda epididymis of mice were cut and incubated in Ham’s F10 media. Sperm samples were analyzed for parameters (motility, morphology and count, DNA integrity (SCD test and chromatin condensation (chromomycin A3 and Aniline blue staining. Results: After 35 days, in addition to above mentioned sperm parameters, all of the treated mice showed statistically significant increase in spermatozoa with immature chromatin (P<0.05. However, after 70 days, the rate of sperm DNA fragmentation assessed by SCD was increased in group II (66.5±0.7 and the percentage of immature spermatozoa (AB+ and CMA3+ was higher in group III (77.5±0.7 and 49.5±6.3 respectively than other groups. After 105 days, the AB+ spermatozoa were increased in both normal dose and high dose groups. Conclusion: Ibuprofen may cause a significant reduction in sperm parameters and sperm chromatin/DNA integrity in mice. It should be noted that these deleterious effects are dose-dependent and can be seen in early and late stage of drug treatments.

  11. 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.

  12. Natural antisense RNAs are involved in the regulation of CD45 expression in autoimmune diseases.

    Science.gov (United States)

    Rong, J; Yin, J; Su, Z

    2015-03-01

    CD45 is a transmembrane protein tyrosine phosphatase that is specifically expressed in hematopoietic cells and can initiate signal transduction via the dephosphorylation of tyrosine. Alternatively spliced transcript variants of this gene encode distinct isoforms, which indicate different functional states of CD45. Among these variants, CD45RO, which contains neither exon 4, 5, or 6, is over-expressed in lymphocytes in autoimmune diseases, including systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, and type I diabetes. The CD45 RO serves as a marker of the immune response activity and lymphocyte development. Previous studies have indicated that exon splicing is generally correlated with local hypermethylated DNA and acetylated histone modification, while autoimmune diseases are commonly associated with global hypomethylation and histone deacetylation in lymphocytes. Thus, the question arises of how exons 4, 5, and 6 of CD45RO are excluded under the status of global DNA hypomethylation and histone deacetylation in these autoimmune diseases. On the basis of the analyses of the context sequence of CD45 and its natural antisense RNA in GenBank, we proposed that the long noncoding RNA encoded by the natural antisense gene of CD45 contributes to the expressional regulation of the CD45RO splicing variant via recruitment of DNA methyltransferase and histone modification modulators specific to the sense gene CD45; thus, it is associated with the over-expression of CD45RO and the functional regulation of lymphocytes in the pathogenic development of autoimmune diseases.

  13. Immobilization of denatured DNA to macroporous supports: II. Steric and kinetic parameters of heterogeneous hybridization reactions.

    Science.gov (United States)

    Bünemann, H

    1982-11-25

    The accessibility of immobilized DNA has been shown to depend more crucially on the method of immobilization than on the type of support used for fixation. When sonicated denatured DNA is coupled via diazotization or via cyanogen bromide reaction to solid Sephadex G-25 and Cellex 410 or to macroporous Sephacryl S-500 and Sepharose C1-6B its accessibility varies from 100 to 24 percent. Generally the loss of accessibility is linked to a depression of the melting temperature of DNA helices formed on the support. This correlation shows a characteristic course for a particular coupling method. DNA coupled under denaturing conditions may become totally inaccessible when only 3 percent of its bases are involved in the covalent linkage. Kinetic experiments with sonicated E.coli DNA have shown that the rate constants for renaturation or hybridization reactions are very similar for DNA immobilized by different methods to solid or macroporous supports. Generally the second order rate constant for a heterogeneous reaction (between mobile and immobilized DNA) is about one order of magnitude smaller than that of the analogous homogeneous reaction (in solution).

  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. 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.

  16. Immobilization of denatured DNA to macroporous supports: II. Steric and kinetic parameters of heterogeneous hybridization reactions.

    OpenAIRE

    Bünemann, H

    1982-01-01

    The accessibility of immobilized DNA has been shown to depend more crucially on the method of immobilization than on the type of support used for fixation. When sonicated denatured DNA is coupled via diazotization or via cyanogen bromide reaction to solid Sephadex G-25 and Cellex 410 or to macroporous Sephacryl S-500 and Sepharose C1-6B its accessibility varies from 100 to 24 percent. Generally the loss of accessibility is linked to a depression of the melting temperature of DNA helices forme...

  17. Joint interaction of ethidium bromide and methylene blue with DNA. The effect of ionic strength on binding thermodynamic parameters.

    Science.gov (United States)

    Vardevanyan, Poghos O; Antonyan, Ara P; Parsadanyan, Marine A; Torosyan, Margarita A; Karapetian, Armen T

    2016-07-01

    Large amount of data of experimental and theoretical studies have shown that ethidium bromide (EtBr) and methylene blue (MB) may bind to nucleic acids via three modes: intercalation between two adjacent base pairs, insertion into the plane between neighboring bases in the same strand (semi-intercalation), and outside binding with negatively charged backbone phosphate groups. The aim of the given research is to examine the behavior of these two ligands at both separate and joint DNA binding. The obtained experimental data show that the effect of simultaneous binding of EtBr and MB on double-stranded DNA has a non-additive effect of separate binding. The analyses of the melting thermodynamic parameters of DNA complexes with two bound ligands suggest competitive mechanism of interaction.

  18. Assessment of delivery parameters with the multi-electrode array for development of a DNA vaccine against Bacillus anthracis.

    Science.gov (United States)

    Donate, Amy; Heller, Richard

    2013-12-01

    Gene electrotransfer (GET) enhances delivery of DNA vaccines by increasing both gene expression and immune responses. Our lab has developed the multi-electrode array (MEA) for DNA delivery to skin. The MEA was used at constant pulse duration (150 ms) and frequency (6.67 Hz). In this study, delivery parameters including applied voltage (5-45 V), amount of plasmid (100-300 μg), and number of treatments (2-3) were evaluated for delivery of a DNA vaccine. Mice were intradermally injected with plasmid expressing Bacillus anthracis protective antigen with or without GET and αPA serum titers measured. Within this experiment no significant differences were noted in antibody levels from varying dose or treatment number. However, significant differences were measured from applied voltages of 25 and 35 V. These voltages generated antibody levels between 20,000 and 25,000. Serum from animals vaccinated with these conditions also resulted in toxin neutralization in 40-60% of animals. Visual damage was noted at MEA conditions of 40 V. No damage was noted either visually or histologically from conditions of 35 V or below. These results reflect the importance of establishing appropriate electrical parameters and the potential for the MEA in non-invasive DNA vaccination against B. anthracis.

  19. Polarized expression of the membrane ASP protein derived from HIV-1 antisense transcription in T cells

    Directory of Open Access Journals (Sweden)

    Gay Bernard

    2011-09-01

    Full Text Available Abstract Background Retroviral gene expression generally depends on a full-length transcript that initiates in the 5' LTR, which is either left unspliced or alternatively spliced. We and others have demonstrated the existence of antisense transcription initiating in the 3' LTR in human lymphotropic retroviruses, including HTLV-1, HTLV-2, and HIV-1. Such transcripts have been postulated to encode antisense proteins important for the establishment of viral infections. The antisense strand of the HIV-1 proviral DNA contains an ORF termed asp, coding for a highly hydrophobic protein. However, although anti-ASP antibodies have been described to be present in HIV-1-infected patients, its in vivo expression requires further support. The objective of this present study was to clearly demonstrate that ASP is effectively expressed in infected T cells and to provide a better characterization of its subcellular localization. Results We first investigated the subcellular localization of ASP by transfecting Jurkat T cells with vectors expressing ASP tagged with the Flag epitope to its N-terminus. Using immunofluorescence microscopy, we found that ASP localized to the plasma membrane in transfected Jurkat T cells, but with different staining patterns. In addition to an entire distribution to the plasma membrane, ASP showed an asymmetric localization and could also be detected in membrane connections between two cells. We then infected Jurkat T cells with NL4.3 virus coding for ASP tagged with the Flag epitope at its C-terminal end. By this approach, we were capable of showing that ASP is effectively expressed from the HIV-1 3' LTR in infected T cells, with an asymmetric localization of the viral protein at the plasma membrane. Conclusion These results demonstrate for the first time that ASP can be detected when expressed from full-length HIV-1 proviral DNA and that its localization is consistent with Jurkat T cells overexpressing ASP.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. Induction of a mutant phenotype in human repair proficient cells after overexpression of a mutated human DNA repair gene.

    NARCIS (Netherlands)

    P.B.G.M. Belt; M.F. van Oostenrijk; H. Odijk (Hanny); J.H.J. Hoeijmakers (Jan); C.M.P. Backendorf (Claude)

    1991-01-01

    textabstractAntisense and mutated cDNA of the human excision repair gene ERCC-1 were overexpressed in repair efficient HeLa cells by means of an Epstein-Barr-virus derived CDNA expression vector. Whereas antisense RNA did not influence the survival of the transfected cells, a mutated cDNA generating

  5. Use of neuropathological tissue for molecular genetic studies: parameters affecting DNA extraction and polymerase chain reaction.

    Science.gov (United States)

    Kösel, S; Graeber, M B

    1994-01-01

    Nuclear and mitochondrial DNA were extracted from gray matter of human cerebral cortex which had either been formalin-fixed and embedded into paraffin or stored in formalin for up to 26 years. Extraction conditions were optimized for proteinase K digestion, i.e., enzyme concentration, digestion temperature and incubation time. Using the polymerase chain reaction (PCR), DNA was successfully amplified from archival material and sequenced employing a direct nonradioactive cycle sequencing protocol. In general, tissue embedded into paraffin following brief fixation in formalin gave good quantitative results, i.e., up to 1 microgram DNA/mg tissue were extracted. This yield was at least one order of magnitude higher than that obtained with tissue stored in formalin. However, paraffin-embedded neuropathological material was found to contain an as-yet-unidentified PCR inhibitor, and a deleterious effect of long-term fixation in unbuffered low-grade formalin was clearly detectable. Importantly, both paraffin-embedded tissue blocks and human brain that had been stored in formalin for many years yielded DNA sufficient for qualitative analysis. The implications of these findings for the use of neuropathological material in molecular genetic studies are discussed.

  6. An Effective Security Management of Database through DNA Fingerprinting Recognition using Geometric Parameters

    Directory of Open Access Journals (Sweden)

    Radha Krishna Rambol

    2012-08-01

    Full Text Available History bears testimony to the fact that the march of any civilization has been on the shoulders of science and technology. Almost everything that distinguishes the modern world from earlier centuries is attributable to science specially information technology. With the rapid growth in computing technology and its application in all spheres of modern society, databases have become an integral component of our everyday life. Database management system manages all the information to our use. The database needs to ensure its security and make it personalized and secure. To make it personalized up to family or royalty, DNA fingerprinting is only the aspect that serve best. In this present article, the researcher has endeavored to make all possible attempts to clarify the DNA database as the tool for Database Security that maintain the hierarchy of family inheritance. In this work, we are going to recognize the DNA sample present in the database with a feature based approach. The approach here is mainly by using the histogram plot by this we are going to extract the characteristics of the image features. By comparing these image features with images present in the database, we can decide whether the test image is from among DNA sample already known or not. If the test image is find in the database then system is displaying the identity of the user as well as works as the password to access the particular database on the basis of biological inheritance.

  7. Calculation of the Electronic Parameters of an Al/DNA/p-Si Schottky Barrier Diode Influenced by Alpha Radiation

    Directory of Open Access Journals (Sweden)

    Hassan Maktuff Jaber Al-Ta'ii

    2015-02-01

    Full Text Available Many types of materials such as inorganic semiconductors have been employed as detectors for nuclear radiation, the importance of which has increased significantly due to recent nuclear catastrophes. Despite the many advantages of this type of materials, the ability to measure direct cellular or biological responses to radiation might improve detector sensitivity. In this context, semiconducting organic materials such as deoxyribonucleic acid or DNA have been studied in recent years. This was established by studying the varying electronic properties of DNA-metal or semiconductor junctions when exposed to radiation. In this work, we investigated the electronics of aluminium (Al/DNA/silicon (Si rectifying junctions using their current-voltage (I-V characteristics when exposed to alpha radiation. Diode parameters such as ideality factor, barrier height and series resistance were determined for different irradiation times. The observed results show significant changes with exposure time or total dosage received. An increased deviation from ideal diode conditions (7.2 to 18.0 was observed when they were bombarded with alpha particles for up to 40 min. Using the conventional technique, barrier height values were observed to generally increase after 2, 6, 10, 20 and 30 min of radiation. The same trend was seen in the values of the series resistance (0.5889–1.423 Ω for 2–8 min. These changes in the electronic properties of the DNA/Si junctions could therefore be utilized in the construction of sensitive alpha particle detectors.

  8. Correlation between the expression of DNA topoisomerases I and IIalpha and clinical parameters in kidney disease.

    Science.gov (United States)

    Ivanova, L V; Rudolph, P; Shilov, Y M; Gieseler, F; Alm, P; Tareeva, I E; Proppe, D

    2001-11-01

    Multiple factors interact during the evolution of renal diseases. In the present study, we examined the expression of DNA topoisomerases type I and IIalpha, which reflect gene transcription and DNA replication, respectively. Enzyme content was assessed by immunohistochemistry using two specific monoclonal antibodies, C21 and Ki-S4, on 81 archival punch-biopsy specimens from patients with renal diseases, including minimal change disease (MCD; n = 10), focal segmental glomerular sclerosis (FSGS; n = 6), mesangial proliferative glomerulonephritis (MPGN; n = 11), membranous glomerulonephritis (MGN; n = 10), mesangial capillary glomerulonephritis (MCGN; n = 7), rapidly progressive glomerulonephritis (RPGN; n = 12), lupus nephritis (LN; n = 15), and tubulointerstitial nephritis (TIN; n = 10). Both enzymes were strongly expressed in diseases tending to rapid progression, notably RPGN and LN, whereas MCD and MGN showed low protein levels in both the glomerular and tubular compartments. Moreover, topoisomerase expression was significantly associated with the density of monocytogenic infiltrates (monitored by means of the monoclonal antibody Ki-M1p), such pathogenesis-associated factors as antinuclear antibodies and paranuclear antineutrophilic antibodies, and serum immunoglobulin levels. There also was a positive correlation with serum creatinine levels and an inverse association with proteinuria and nephrotic syndrome. We conclude that the expression of DNA topoisomerases may be linked to pathogenetic mechanisms and may provide prognostic information. Because of their comparatively low nephrotoxicity, topoisomerase inhibitors might prove to be useful therapeutic agents in the treatment of renal diseases.

  9. Biomonitoring of Human Exposure to Prestige Oil: Effects on DNA and Endocrine Parameters

    Science.gov (United States)

    Pérez-Cadahía, Beatriz; Méndez, Josefina; Pásaro, Eduardo; Lafuente, Anunciación; Cabaleiro, Teresa; Laffon, Blanca

    2008-01-01

    Since 1960, about 400 tankers spilled more than 377765 tons of oil, with the Prestige accident (Galician coast, NW Spain, November 2002) the most recent. Taking into account the consistent large number of individuals exposed to oil that exists all over the world, it seems surprising the absence in the literature of studies focused on the chronic effects of this exposure on human health. In this work we evaluated the level of DNA damage by means of comet assay, and the potential endocrine alterations (prolactin and cortisol) caused by Prestige oil exposure in a population of 180 individuals, classified in 3 groups according to the tasks performed, and 60 controls. Heavy metals in blood were determined as exposure biomarkers, obtaining significant increases of aluminum, nickel and lead in the exposed groups as compared to controls. Higher levels of genetic damage and endocrine alterations were also observed in the exposed population. DNA damage levels were influenced by age, sex, and the use of protective clothes, and prolactin concentrations by the last two factors. Surprisingly, the use of mask did not seem to protect individuals from genetic or endocrine alterations. Moreover, polymorphisms in genes encoding for the main enzymes involved in the metabolism of oil components were analyzed as susceptibility biomarkers. CYP1A1-3′UTR and EPHX1 codons 113 and 139 variant alleles were related to higher damage levels, while lower DNA damage was observed in GSTM1 and GSTT1 null individuals. PMID:21572833

  10. Biomonitoring of Human Exposure to Prestige Oil: Effects on DNA and Endocrine Parameters

    Directory of Open Access Journals (Sweden)

    Beatriz Pérez-Cadahía

    2008-01-01

    Full Text Available Since 1960, about 400 tankers spilled more than 377765 tons of oil, with the Prestige accident (Galician coast, NW Spain, November 2002 the most recent. Taking into account the consistent large number of individuals exposed to oil that exists all over the world, it seems surprising the absence in the literature of studies focused on the chronic effects of this exposure on human health. In this work we evaluated the level of DNA damage by means of comet assay, and the potential endocrine alterations (prolactin and cortisol caused by Prestige oil exposure in a population of 180 individuals, classified in 3 groups according to the tasks performed, and 60 controls. Heavy metals in blood were determined as exposure biomarkers, obtaining significant increases of aluminum, nickel and lead in the exposed groups as compared to controls. Higher levels of genetic damage and endocrine alterations were also observed in the exposed population. DNA damage levels were influenced by age, sex, and the use of protective clothes, and prolactin concentrations by the last two factors. Surprisingly, the use of mask did not seem to protect individuals from genetic or endocrine alterations. Moreover, polymorphisms in genes encoding for the main enzymes involved in the metabolism of oil components were analyzed as susceptibility biomarkers. CYP1A1-3’UTR and EPHX1 codons 113 and 139 variant alleles were related to higher damage levels, while lower DNA damage was observed in GSTM1 and GSTT1 null individuals.

  11. Antisense downregulation of mutant huntingtin in a cell model

    DEFF Research Database (Denmark)

    Hasholt, L.; Abell, K.; Norremolle, A.;

    2003-01-01

    of specific neurons in the brains of HD patients correlate with the expression of mutant huntingtin. Therefore, we have studied whether mutant huntingtin expression can be downregulated by antisense technique. Methods NT2 precursor cells and differentiated postmitotic NT2-N neurons, respectively, were...... transfected with plasmid constructs containing exon 1 of the HD gene with expanded CAG repeats in frame with the reporter protein EGFP. The transfected cell cultures were treated with a phosphorothioated antisense oligonucleotide (PS-ASHD/20+) or a control oligonucleotide either by cotransfection...... or by addition to the culture medium. Results Expression of the fusion protein containing the mutant huntingtin fragment resulted in diffuse green fluorescence in the cytoplasm and formation of aggregates in some of the NT2 cells and NT2-N neurons. We obtained antisense sequence-specific inhibition of expression...

  12. Short locked nucleic acid antisense oligonucleotides potently reduce apolipoprotein B mRNA and serum cholesterol in mice and non-human primates

    DEFF Research Database (Denmark)

    Straarup, Ellen Marie; Fisker, Niels; Hedtjärn, Maj;

    2010-01-01

    the design of short antisense oligonucleotides (12- to 13-mers) that possessed high affinity and increased potency both in vitro and in vivo compared to longer oligonucleotides. The short LNA oligonucleotides were more target specific, and they exhibited the same biodistribution and tissue half......-high-density lipoprotein (non-HDL) cholesterol without increasing serum liver toxicity markers. The data presented here show that oligonucleotide length as a parameter needs to be considered in the design of antisense oligonucleotide and that potent short oligonucleotides with sufficient target affinity can be generated...

  13. 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...

  14. OPTIMIZATION OF ELECTROPORATION PARAMETERS FOR TRANSFECTION OF PLASMID DNA INTO MURINE BONE MARROW-DERIVED DENDRITIC CELL

    Institute of Scientific and Technical Information of China (English)

    KE Shan; CHEN Xue-hua; LI Hao; LI Jian-fang; GU Qin-long; ZHU Zheng-gang; LIU Bing-ya

    2006-01-01

    Objective To explore the optimal electroporation parameters for transfection of plasmid DNA into murine bone marrow-derived dendritic cells. Methods Murine bone marrow-derived dendritic cells (DCs) were electroporated with plasmid DNA in varied conditions, such as electrical voltage, pulse time,pre-electroporation cell condition and serum concentration in electrical buffer. Inverted fluorescence microscope and flow cytometer were used to determine the transfection efficiency. Some of the DCs genetically modified under different conditions were stained with trypan-blue and its viability was observed microscopically 48h after electroporation. Results Highest transfection efficiency (22.10%) could be reached when electrical voltage was 250V and pulse time was 20ms. Refreshing the culture medium pre-electroporation may help the cells recover more easily from gene transfer.Besides, electrical buffer containing serum may benefit the viability of DC after electroporation and temperature may has little influence on transfection efficiency. Conclusion Our observations demonstrated plasmid DNA could be efficiently transferred into murine bone marrow-derived DCs by electroporation. These data may helpful for cancer research related to murine DC transfection.

  15. The Relationship between Seminal Melatonin with Sperm Parameters, DNA Fragmentation and Nuclear Maturity in Intra-Cytoplasmic Sperm Injection Candidates

    Directory of Open Access Journals (Sweden)

    Mina Sharbatoghli

    2015-10-01

    Full Text Available Objective: Melatonin, the chief secretory product of the pineal gland, regulates dynamic physiological adaptations that occur in seasonally breeding mammals as a response to changes in daylight hours. Because of the presence of melatonin in semen and the membrane melatonin receptor in spermatozoa, the impact of melatonin on the regulation of male infertility is still questionable. The aim of this study was to determine the effects of endogenous melatonin on human semen parameters (sperm concentration, motility and normal morphology, DNA fragmentation (DF and nuclear maturity. Materials and Methods: In this clinical prospective study, semen samples from 75 infertile men were routinely analyzed and assessed for melatonin and total antioxidant capacity (TAC levels using the enzyme-linked immunosorbent assay (ELISA and colorimetric assay kits, respectively. DF was examined by the sperm chromatin dispersion (SCD test. Acidic aniline blue staining was used to detect chromatin defects in the sperm nuclei. Results: There was no significant correlation between seminal plasma melatonin and TAC with sperm parameters and nuclear maturity. However, we observed a positive significant correlation between DF and melatonin level (r=0.273, P<0.05. Conclusion: Melatonin in seminal plasma is positively correlated with damaged sperm DNA of infertile patients. The mechanism of this phenomenon needs further study.

  16. Early apoptotic changes in human spermatozoa and their relationships with conventional semen parameters and sperm DNA fragmentation

    Institute of Scientific and Technical Information of China (English)

    Hao-Bo Zhang; Shao-Ming Lu; Chun-Yan Ma; Li Wang; Xiao Li; Zi-Jiang Chen

    2008-01-01

    Aim: To investigate whether early apoptotic changes in spermatozoa can be significant markers for sperm quality.Methods: Two early apoptotic changes in the semen of 56 men were assessed using Annexin V (AN)/propidium iodide (PI) staining for phosphatidylserine externalization and JC-1 staining for mitochondrial membrane potential (MMP). The results were compared with conventional semen parameters and DNA fragmentation identified using the TUNEL assay. Results: The different labeling patterns in the bivariate Annexin V/PI analysis identified four distinc-tive spermatozoa populations. The percentage of AN-/PI- spermatozoa positively correlated with conventional semen parameters and MMP, but negatively correlated with TUNEL (+) spermatozoa. As for the AN-/PI+ fraction, we found an opposite result in comparison to AN-/PI- spermatozoa. The level of early apoptotic AN+/PI- spermatozoa negatively correlated with MMP and sperm motility. The level of late apoptotic AN+/PI+ spermatozoa negatively correlated with conventional semen parameters and MMP, and positively correlated with TUNEL (+) spermatozoa. MMP positively correlated with conventional semen parameters, but negatively correlated with TUNEL (+) spermatozoa. Conclusion:Although early apoptotic AN+/PI- spermatozoa only negatively correlates with sperm motility, the differences in proportion of each subpopulation of spermatozoa (especially, the percentage of AN-/PI-spermatozoa), and decreased MMP might be significant markers for diagnosing male infertility. They possibly bring additional information to predict the outcome of in vitro fertilization.

  17. 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.

  18. 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.

  19. 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.

  20. 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...

  1. Natural antisense transcripts associated with salinity response in alfalfa

    Science.gov (United States)

    Natural antisense transcripts (NATs) are long non-coding RNAs (lncRNAs) complimentary to the messenger (sense) RNA (Wang et al. 2014). Many of them are involved in regulation of their own sense transcripts thus playing pivotal biological roles in all processes of organismal development and responses...

  2. 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.

  3. Inhibitory effects of antisense RNA of HAb18G/CD147 on invasion of hepatocellular carcinoma cells in vitro

    Institute of Scientific and Technical Information of China (English)

    Yu Li; Peng Shang; Ai-Rong Qian; Li Wang; Yong Yang; Zhi-Nan Chen

    2003-01-01

    AIM: To study the inhibitory effects of antisense RNA of HAb18G/CD147 on invasion of hepatocellular carcinoma (HCC) cells in vitro.METHODS: Antisense RNA of HAb18G/CD147 vector PCIasHAb18G was constructed by reversely inserting HAb18G/CD147 cDNA to eukaryotic expression vector PCI-neo. The HCC cell line HHCC was transfected by PCI-asHAb18G via cation liposome. Expression of HAb18G/CD147 of transfected cells selected by G418 (geneticin) was observed by immunohistochemical SP staining and FACS (fluorescence activated cell sorting). Gelatin zymography was used to determine the effect of PCI-asHAb18G on reducing secretions of MMP2 and MMP-9 of the transfected cells. Boyden chamber was employed to test the invasion of HCC cells in vitro.RESULTS: The construction of antisense RNA vector PCIasHAb18G was verified correct by partial nucleotide sequencing and restricted endonuclease digestion. The expression of HAb18G/CD147 in transfected HHCC was inhibited by PCI-asHAb18G. Secretions of MMP-2 and MMP9 of transfected HHCC were reduced and the invasion of transfected HHCC was inhibited compared to HHCC,respectively.CONCLUSION: Invasion of HCC cells can be inhibited by antisense RNA of HAb18G/CD147. HAb18G/CD147 may be used as a potential target of drugs for anti-invasion and metastasis of HCC.

  4. Inhibitory effect of antisense vascular endothelial growth factor 165 eukaryotic expression vector on proliferation of hepatocellular carcinoma cells

    Institute of Scientific and Technical Information of China (English)

    Song Gu; Chang-Jian Liu; Tong Qiao; Xue-Mei Sun; Lei-Lei Chen; Le Zhang

    2004-01-01

    AIM: To construct antisense VEGF165 eukaryotic expression vector PCDNA3-as-VEGF165 and to study its expression and effect on the proliferation of hepatocarcinoma SMMC-7721 cells.METHODS: VEGF165 cDNA was inserted into polylinker sites of eukaryotic expression vector PCDNA3 to construct PCDNA3-as-VEGF165. Then the vector was transferred into human hepatocarcinoma cell strain SMMC-7721 with cation lipofectamine 2000 mediated methods to evaluate the expression of VEGF protein and the inhibitory effect on the proliferation of hepatocarcinoma SMMC-7721 cells.RESULTS: The detection indicated the presence of VEGF cDNA in normally cultured SMMC-7721 cells by PCR. VEGF mRNA expression was notably decreased in SMMC-7721 cells by RT-PCR after PCDNA3-as-VEGF165 transfection. The expression of VEGF protein was dramatically inhibited (142.01±7.95 vs 1 625.52±64.46 pg·ml-1, P<0.01) 2 days after transfection,which correlated with the dose of PCDNA3-as-VEGF165 gene.VEGF protein was most expressed in PCDNA3 transferred SMMC-7721 cells but few in PCDNA3-as-VEGF165 transferred cells by immunohistochemical staining. The apoptotic rate of hepatocarcinoma SMMC-7721 cells was significantly promoted (17.98±0.86% vs4.86±0.27%, P<0.01) and the survival rate was notably decreased (80.99±3.20% vs 93.52±3.93%, P<0.05) due to antisense VEGF165 by flow cytometry (FCM). The transfection of antisense VEGF165 gene resulted in the inhibitory effect on the proliferation of hepatocarcinoma cells by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and the death of all hepatocarcinoma cells on day 6 after transfection.CONCLUSION: It is confirmed that antisense VEGF165 can inhibit the expression of VEGF protein, interfere with the proliferation and induce the apoptosis of hepatocarcinoma cells in our study. Antisense VEGF165 gene therapy may play an important role in the treatment of human hepatocarcinoma.

  5. 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

  6. Physicochemical and biological properties of self-assembled antisense/poly(amidoamine dendrimer nanoparticles: the effect of dendrimer generation and charge ratio

    Directory of Open Access Journals (Sweden)

    Alireza Nomani

    2010-05-01

    Full Text Available Alireza Nomani1,6, Ismaeil Haririan1,5, Ramin Rahimnia2,4, Shamileh Fouladdel2, Tarane Gazori1, Rassoul Dinarvand1, Yadollah Omidi3, Ebrahim Azizi2,41Department of Pharmaceutics, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran; 2Molecular Research Lab, Department of Pharmacology and Toxicology, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran; 3Department of Pharmaceutics, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran; 4Department of Medical Biotechnology, School of Advanced Medical Technologies, Tehran University of Medical Sciences, Tehran, Iran; 5Biomaterials Research Center (BRC Tehran, Iran; 6Department of Pharmaceutics, Faculty of Pharmacy, Zanjan University of Medical Sciences, Zanjan, IranAbstract: To gain a deeper understanding of the physicochemical phenomenon of self-assembled nanoparticles of different generations and ratios of poly (amidoamine dendrimer (PAMAM dendrimer and a short-stranded DNA (antisense oligonucleotide, multiple methods were used to characterize these nanoparticles including photon correlation spectroscopy (PCS; zeta potential measurement; and atomic force microscopy (AFM. PCS and AFM results revealed that, in contrast to larger molecules of DNA, smaller molecules produce more heterodisperse and large nanoparticles when they are condensed with a cationic dendrimer. AFM images also showed that such nanoparticles were spherical. The stability of the antisense content of the nanoparticles was investigated over different charge ratios using polyacrylamide gel electrophoresis. It was clear from such analyses that much more than charge neutrality point was required to obtain stable nanoparticles. For cell uptake, self-assembled nanoparticles were prepared with PAMAM G5 and 5’-FITC labeled antisense and the uptake experiment was carried out in T47D cell culture. This investigation also shows that the cytotoxicity of the nanoparticles was

  7. Better estimation of protein-DNA interaction parameters improve prediction of functional sites

    Directory of Open Access Journals (Sweden)

    O'Flanagan Ruadhan A

    2008-12-01

    Full Text Available Abstract Background Characterizing transcription factor binding motifs is a common bioinformatics task. For transcription factors with variable binding sites, we need to get many suboptimal binding sites in our training dataset to get accurate estimates of free energy penalties for deviating from the consensus DNA sequence. One procedure to do that involves a modified SELEX (Systematic Evolution of Ligands by Exponential Enrichment method designed to produce many such sequences. Results We analyzed low stringency SELEX data for E. coli Catabolic Activator Protein (CAP, and we show here that appropriate quantitative analysis improves our ability to predict in vitro affinity. To obtain large number of sequences required for this analysis we used a SELEX SAGE protocol developed by Roulet et al. The sequences obtained from here were subjected to bioinformatic analysis. The resulting bioinformatic model characterizes the sequence specificity of the protein more accurately than those sequence specificities predicted from previous analysis just by using a few known binding sites available in the literature. The consequences of this increase in accuracy for prediction of in vivo binding sites (and especially functional ones in the E. coli genome are also discussed. We measured the dissociation constants of several putative CAP binding sites by EMSA (Electrophoretic Mobility Shift Assay and compared the affinities to the bioinformatics scores provided by methods like the weight matrix method and QPMEME (Quadratic Programming Method of Energy Matrix Estimation trained on known binding sites as well as on the new sites from SELEX SAGE data. We also checked predicted genome sites for conservation in the related species S. typhimurium. We found that bioinformatics scores based on SELEX SAGE data does better in terms of prediction of physical binding energies as well as in detecting functional sites. Conclusion We think that training binding site detection

  8. Simultaneous analysis of mitochondrial activity and DNA content in Ehrlich ascites tumor cells by dual parameter flow cytometry.

    Science.gov (United States)

    Hämmerle, T; Löffler, M

    1989-01-01

    Ehrlich ascites tumor cells were permeabilized using low concentrations of digitonin, 8 micrograms/10(6) cells. Permeabilization was monitored by the assay of lactate dehydrogenase released into the incubation medium and of hexokinase partially bound to mitochondria. Integrity of the cellular organelles was unaffected as determined by assay of the mitochondrial enzyme glutamate dehydrogenase. Cells were stained with rhodamine 123 as a mitochondrial specific dye and propidium iodide/mithramycin as DNA specific dyes. The green fluorescence of bound rhodamine 123 versus red fluorescence of DNA in individual cells was analysed by dual parameter flow cytometry. Incubation of cells with inhibitors of mitochondrial energy metabolism, such as, potassium cyanide and carbonyl cyanide m-chlorophenylhydrazone abolished binding of rhodamine 123. Flow cytometric data allowed a correlation between cell position in the mitotic cycle with total mitochondrial activity. In addition, comparison of the characteristics of propidium iodide and ethidium bromide staining further elucidated the molecular basis of the staining with the positively-charged fluorescent dye rhodamine 123.

  9. 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.

  10. 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.

  11. Cloning of NHE-1 gene fragment from human lung cancer cells and construction of its antisense expression vector

    Institute of Scientific and Technical Information of China (English)

    WU Guo-ming; HUANG Gui-jun; QIAN Gui-sheng

    2001-01-01

    To clone the partial sequence of Na+/H+ exchanger-1 (NHE-1) gene of human lung cancer cells and insert it reversely into the multiclone site of pLXSN in order to construct an antisense expression vector for tumor gene therapy in vivo. Methods: With use of the upstream and downstream primers containing Bam H I and EcoR I in their 5' ends respectively, a partial sequence of the first exon of NHE-1 gene was cloned in a length of 454 bp from genomic DNA of human lung cancer cell A549 with PCR method. The product was then directionally and reversely insert into the multiclone site of pLXSN. Finally, the constructed recombinant was identified with agarose gel electrophoresis and DNA sequencing. Results: The cloned fragment was 461 bp in length and successfully ligated to pLXSN with the identification by agarose gel electrophoresis. DNA sequencing confirmed that the fragment cloned and inserted into the vector was identical with the targeted one. Conclusion: The targeted fragment is successfully cloned and reversely inserted into pLXSN in our experiment. The antisense expression vector ofNHE-1, pNHE- 1, was constructed successfully.

  12. Lysine metabolism in antisense C-hordein barley grains

    DEFF Research Database (Denmark)

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

    2015-01-01

    ) and five antisense C-hordein transgenic barley lines. Considering the amounts of soluble and protein-bound aspartate-derived amino acids together with the analysis of key enzymes of aspartate metabolic pathway, we suggest that the C-hordein suppression did not only alter the metabolism of at least one......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 increased lysine, methionine and threonine contents. The objective of the study was to investigate the possible changes in the regulation of key enzymes of the aspartate metabolic pathway and the contents of aspartate-derived amino acids in the nontransgenic line (Hordeum vulgare L. cv. Golden Promise...

  13. Targeting several CAG expansion diseases by a single antisense oligonucleotide.

    Directory of Open Access Journals (Sweden)

    Melvin M Evers

    Full Text Available To date there are 9 known diseases caused by an expanded polyglutamine repeat, with the most prevalent being Huntington's disease. Huntington's disease is a progressive autosomal dominant neurodegenerative disorder for which currently no therapy is available. It is caused by a CAG repeat expansion in the HTT gene, which results in an expansion of a glutamine stretch at the N-terminal end of the huntingtin protein. This polyglutamine expansion plays a central role in the disease and results in the accumulation of cytoplasmic and nuclear aggregates. Here, we make use of modified 2'-O-methyl phosphorothioate (CUGn triplet-repeat antisense oligonucleotides to effectively reduce mutant huntingtin transcript and protein levels in patient-derived Huntington's disease fibroblasts and lymphoblasts. The most effective antisense oligonucleotide, (CUG(7, also reduced mutant ataxin-1 and ataxin-3 mRNA levels in spinocerebellar ataxia 1 and 3, respectively, and atrophin-1 in dentatorubral-pallidoluysian atrophy patient derived fibroblasts. This antisense oligonucleotide is not only a promising therapeutic tool to reduce mutant huntingtin levels in Huntington's disease but our results in spinocerebellar ataxia and dentatorubral-pallidoluysian atrophy cells suggest that this could also be applicable to other polyglutamine expansion disorders as well.

  14. The sense and antisense expression of gibberellin 20-oxidase gene (rga5) in rice and its effects on GA1 level and agronomic traits

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    A gibberellin 20-oxidase gene rga5 was isolated by PCR from genomic DNA of rice (Oryza sativa ssp indica) cultivars 'Aizizhan' and 'Nante'. Compared with the reported OsGA20ox, the rga5 was partial-frame-shifted with 11 different amino acids. Then the rga5 with CaMV 35S promotor and NOS terminator was inserted into the polylinker site of pCambia1301 to construct sense and antisense gene expressing vectors pSrga5 and pArga5. The transgenic plants were obtained by biolistic bombardment with pSrga5 or pArga5. The transgenic rice plants showed that the over- expression and antisense-expression of rga5 have remarkable effects on the biological characters of rice. The sense transgenic plants showed heightening with longer spike, more seed-bearing and unaffected flowering, whereas antisense transgenic plants showed dwarfing, early-flowering with slender stem, dark leaf color, shorter leaf and shorter spike. The PCR amplification and Southern blot hybridization showed that the rga5 has been integrated into the transgenic rice genome and the transcription of rga5 was identified by Northern blot hybridization. In the sense transgenic plants the GA1 content increased of about 50%, however, the antisense transgenic rice decreased of 90% approximately compared with control plant 'Zhonghua 8'. These results demonstrated that the rga5 is a functional gene encoding gibberellin 20-oxidase in rice. Over-expressing rga5 significantly increases the endogenous GA1 level and plant height of rice, whereas the expression of antisense rga5 decreases the GA1 level and plant height of rice dramatically.

  15. The effects on DNA migration of altering parameters in the comet assay protocol such as agarose density, electrophoresis conditions and durations of the enzyme or the alkaline treatments.

    Science.gov (United States)

    Ersson, Clara; Möller, Lennart

    2011-11-01

    The single cell gel electrophoresis (comet assay) is a popular method for measuring DNA migration as an estimate of DNA damage. No standardised comet assay protocol exists, which make comparisons between studies complicated. In a previous inter-laboratory validation study of the comet assay, we identified important parameters in the protocol that might affect DNA migration. The aim of this study was to assess how different comet assay protocols affect DNA migration. The results in this study suggest that (i) there is a significant linear dose-response relationship between the agarose gel's density and DNA migration and that damaged cells are more sensitive to the agarose gel's density; (ii) incubation with formamidopyrimidine DNA glycosylase for 10 min is inadequate, whereas 30 min is sufficient; (iii) the typically used 20 min of alkaline treatment might be to short when analysing samples that contain particular alkali-labile sites (ALS) and (iv) the duration of electrophoresis as well as the strength of the electric field applied affects the DNA migration. By using protocol-specific calibration curves, it is possible to reduce the variation in DNA migration caused by differences in comet assay protocols. This does, however, not completely remove the impact of the durations of alkaline treatment and electrophoresis when analysing cells containing ALS that are relatively resistant to high alkaline treatment.

  16. Nutrient-regulated antisense and intragenic RNAs modulate a signal transduction pathway in yeast.

    Directory of Open Access Journals (Sweden)

    Masafumi Nishizawa

    2008-12-01

    Full Text Available The budding yeast Saccharomyces cerevisiae alters its gene expression profile in response to a change in nutrient availability. The PHO system is a well-studied case in the transcriptional regulation responding to nutritional changes in which a set of genes (PHO genes is expressed to activate inorganic phosphate (Pi metabolism for adaptation to Pi starvation. Pi starvation triggers an inhibition of Pho85 kinase, leading to migration of unphosphorylated Pho4 transcriptional activator into the nucleus and enabling expression of PHO genes. When Pi is sufficient, the Pho85 kinase phosphorylates Pho4, thereby excluding it from the nucleus and resulting in repression (i.e., lack of transcription of PHO genes. The Pho85 kinase has a role in various cellular functions other than regulation of the PHO system in that Pho85 monitors whether environmental conditions are adequate for cell growth and represses inadequate (untimely responses in these cellular processes. In contrast, Pho4 appears to activate some genes involved in stress response and is required for G1 arrest caused by DNA damage. These facts suggest the antagonistic function of these two players on a more general scale when yeast cells must cope with stress conditions. To explore general involvement of Pho4 in stress response, we tried to identify Pho4-dependent genes by a genome-wide mapping of Pho4 and Rpo21 binding (Rpo21 being the largest subunit of RNA polymerase II using a yeast tiling array. In the course of this study, we found Pi- and Pho4-regulated intragenic and antisense RNAs that could modulate the Pi signal transduction pathway. Low-Pi signal is transmitted via certain inositol polyphosphate (IP species (IP7 that are synthesized by Vip1 IP6 kinase. We have shown that Pho4 activates the transcription of antisense and intragenic RNAs in the KCS1 locus to down-regulate the Kcs1 activity, another IP6 kinase, by producing truncated Kcs1 protein via hybrid formation with the KCS1 m

  17. Correlation between male age, WHO sperm parameters, DNA fragmentation, chromatin packaging and outcome in assisted reproduction technology.

    Science.gov (United States)

    Nijs, M; De Jonge, C; Cox, A; Janssen, M; Bosmans, E; Ombelet, W

    2011-06-01

    In the human, male ageing results in reproductive hormonal and cellular changes that can influence semen quality (volume, motility, concentration and morphology) and ultimately result in a reduced fertilising capacity and a longer 'time to pregnancy' for ageing men as well as an increased risk for miscarriage. This prospective cohort study of 278 patients undergoing a first in vitro fertilisation or intracytoplasmic sperm injection treatment was undertaken to examine whether patient's age was reflected in sperm motility, concentration, morphology as well as in DNA fragmentation (DFI) and immature chromatin (unprocessed nuclear proteins and/or poorly condensed chromatin) as measured by the sperm chromatin structure assay. This study also investigated the possible influence of male age (after correcting for female age) on their fertilising capacity, on obtaining a pregnancy and a healthy baby at home. Logistic regression analysis did not reveal any male age-related influences on sperm parameters like concentration, motility or morphology. No significant male age-related increase in DFI or immature chromatin was demonstrable for these patients. Elevated male age, after correcting for female age, was not related to lower fertilisation rates or significant decreases in the chance for a healthy baby at home.

  18. 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.

  19. 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.

  20. Measurement of X-ray-induced DNA double-strand breaks at various stages of the cell cycle using the total fluorescence as a comet assay parameter

    Energy Technology Data Exchange (ETDEWEB)

    Attia, Atef M.M. [Department of Biochemistry, Biophysical laboratory, National Research Center, Dokki, Cairo (Egypt); Nabil, Ghada M., E-mail: gmnabilnooh@hotmail.com [Department of Biochemistry, Biophysical laboratory, National Research Center, Dokki, Cairo (Egypt); Frankenberg, Dieter; Frankenberg-Schwager, M. [Abteilung Klinische Strahlenbiologie und Klinische Strahlenphysik, Zentrum, Radiologie, Georg-August-Universitaet Goettingen, Von-Siebold-Str.3 (Germany)

    2011-11-15

    The aim of the study was to develop a protocol for both estimating cell cycle position and the level of ionizing radiation-induced DNA dsb using the neutral comet assay. Using DNA histograms, cell cycle positions were determined for human dermal fibroblasts. The tail intensity was used to estimate the level of DNA damage induced by X-rays, at different positions of the cell cycle. The results of tail intensity versus DNA content bivariate analysis of exponentially growing cells showed a remarkable decrease in tail intensity with transition of cells from G1 to S-phase and increases slightly with transition to G2/M phase. This effect is observed at all doses including unirradiated cells, indicating that the effect is not caused by X-rays and the comet assay based on the current tail parameters is not relevant to measure DNA damage at various stages of the cell cycle. The results of dose response curves showed a linear decrease in the comet fluorescence with the X-ray dose. This observation provides a basis for estimating the fraction of damaged DNA, based on the fluorescence decrement induced by ionizing radiation. The results of this new approach showed a linear increase in DNA damage with dose, at various stages of the cell cycle, with rates, which vary in the following order G0>G2/M>S/G1 cells. These results suggest that G0 and G2/M cells are the most sensitive to X-rays among all phases of the cell cycle and suggest synchronization of cells at these phases to increase the cellular radiosensitivity during radiotherapy. - Display Omitted Highlights: > Increase in DNA damage with dose. > Introduction of a new technique for measuring DNA damage using a new approach of the neutral comet assay. > Estimation of DNA damage in mammalian cells.

  1. Lycopene and resveratrol improve post-thaw bull sperm parameters: sperm motility, mitochondrial activity and DNA integrity.

    Science.gov (United States)

    Bucak, M N; Ataman, M B; Başpınar, N; Uysal, O; Taşpınar, M; Bilgili, A; Öztürk, C; Güngör, Ş; İnanç, M E; Akal, E

    2015-06-01

    We focussed on evaluating the protective effect of lycopene and resveratrol on post-thaw bull sperm and oxidative stress parameters. Nine ejaculates for each bull were used in the study. Each ejaculate, splitted into three equal aliquots and diluted at 37 °C with base extenders containing lycopene (1 × 10(-3)  g ml(-1) ) and resveratrol (1 mm), and no antioxidant (control), was cooled to 5 °C and then frozen. Frozen straws were thawed in a water bath for evaluation. The supplementation of the semen extender with lycopene and resveratrol increased the percentages of post-thawed computer-assisted sperm analysis (CASA) motility (55.8 ± 3.8 and 61.9 ± 4.0%) and progressive motility (38 ± 2.4 and 37 ± 8.8), compared with the controls (50.7 ± 2.65 and 33.3 ± 3.74%, respectively, P < 0.05). Resveratrol provided a higher ALH (4.3 ± 0.1), in comparison with the control (3.9 ± 0.3, P < 0.05). The supplementation of the semen extender with lycopene and resveratrol produced a higher mitochondrial activity (24.6 ± 2.9 and 30.1 ± 6.5% respectively), compared with that of the control (11.8 ± 9.5%, P < 0.05). It was determined that both antioxidants resulted in a lower percentage of sperm with damaged DNA than that of the control (P < 0.05). Sperm motion characteristics except for ALH, acrosome integrity, sperm viability and oxidative stress parameters were not affected by the adding of lycopene and resveratrol.

  2. A riboswitch-regulated antisense RNA in Listeria monocytogenes.

    Science.gov (United States)

    Mellin, J R; Tiensuu, Teresa; Bécavin, Christophe; Gouin, Edith; Johansson, Jörgen; Cossart, Pascale

    2013-08-06

    Riboswitches are ligand-binding elements located in 5' untranslated regions of messenger RNAs, which regulate expression of downstream genes. In Listeria monocytogenes, a vitamin B12-binding (B12) riboswitch was identified, not upstream of a gene but downstream, and antisense to the adjacent gene, pocR, suggesting it might regulate pocR in a nonclassical manner. In Salmonella enterica, PocR is a transcription factor that is activated by 1,2-propanediol, and subsequently activates expression of the pdu genes. The pdu genes mediate propanediol catabolism and are implicated in pathogenesis. As enzymes involved in propanediol catabolism require B12 as a cofactor, we hypothesized that the Listeria B12 riboswitch might be involved in pocR regulation. Here we demonstrate that the B12 riboswitch is transcribed as part of a noncoding antisense RNA, herein named AspocR. In the presence of B12, the riboswitch induces transcriptional termination, causing aspocR to be transcribed as a short transcript. In contrast, in the absence of B12, aspocR is transcribed as a long antisense RNA, which inhibits pocR expression. Regulation by AspocR ensures that pocR, and consequently the pdu genes, are maximally expressed only when both propanediol and B12 are present. Strikingly, AspocR can inhibit pocR expression in trans, suggesting it acts through a direct interaction with pocR mRNA. Together, this study demonstrates how pocR and the pdu genes can be regulated by B12 in bacteria and extends the classical definition of riboswitches from elements governing solely the expression of mRNAs to a wider role in controlling transcription of noncoding RNAs.

  3. 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.

  4. Splice-switching antisense oligonucleotides as therapeutic drugs

    OpenAIRE

    Havens, Mallory A.; Hastings, Michelle L.

    2016-01-01

    Splice-switching oligonucleotides (SSOs) are short, synthetic, antisense, modified nucleic acids that base-pair with a pre-mRNA and disrupt the normal splicing repertoire of the transcript by blocking the RNA–RNA base-pairing or protein–RNA binding interactions that occur between components of the splicing machinery and the pre-mRNA. Splicing of pre-mRNA is required for the proper expression of the vast majority of protein-coding genes, and thus, targeting the process offers a means to manipu...

  5. The influence of physicochemical parameters on the efficacy of non-viral DNA transfection complexes : A comparative study

    NARCIS (Netherlands)

    Kneuer, Carsten; Ehrhardt, Carsten; Bakowsky, Heike; Kumar, M. N. V. Ravi; Oberle, Volker; Lehr, Claus M.; Hoekstra, Dick; Bakowsky, Udo

    2006-01-01

    Various polycationic vehicles have been developed to facilitate the transfer of foreign DNA into mammalian cells. Structure-activity studies suggested that biophysical properties, such as size, charge, and morphology of the resulting DNA complexes determine transfection efficiency within one class o

  6. Is it possible to study the kinetic parameters of interaction between PNA and parallel and antiparallel DNA by stopped-flow fluorescence?

    Science.gov (United States)

    Barbero, N; Cauteruccio, S; Thakare, P; Licandro, E; Viscardi, G; Visentin, S

    2016-10-01

    Peptide nucleic acids (PNAs) are among the most interesting and versatile artificial structural mimics of nucleic acids and exhibit peculiar and important properties (i.e. high chemical stability, and a high resistance to cellular enzymes and nucleases). Despite their unnatural structure, they are able to recognize and bind DNA and RNA in a very high, specific and selective manner. One of the most popular, easy and reliable method to measure the stability of PNA-DNA hybrid systems is the melting temperature but the thermodynamic data are obtained using a big quantity of materials failing to provide information on the kinetics of the interaction. In the present work, the PNA decamer 6, with the TCACTAGATG sequence of nucleobases, and the corresponding fluorescent PNA-FITU (fluorescein isothiourea) decamer 8 were synthesized with standard manual Boc-based chemistry. The interaction of the PNA-FITU with parallel and antiparallel DNA has been studied by stopped-flow fluorescence, which is proposed as an alternative technique to obtain the kinetic parameters of the binding. The great advantage of using the stopped-flow technique is the possibility of studying the kinetics of the PNA-DNA duplex formation in a physiological environment. In particular, fluorescence stopped-flow technique has been exploited to compare the affinity of two PNA-DNA duplexes since it can discriminate between parallel and antiparallel DNA binding.

  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. 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...

  9. Cellular Antisense Activity of PNA-Oligo(bicycloguanidinium) Conjugates forming Self-Assembled Nano-aggregates

    DEFF Research Database (Denmark)

    Valero, Julian; Shiraishi, Takehiko; de Mendoza, Javier;

    2015-01-01

    A series of peptide nucleic acid-oligo(bicycloguanidinium) (PNA-BGn) conjugates have been synthesized and characterized in terms of cellular antisense activity using the pLuc750HeLa cell splice correction assay. PNA-BG4 conjugates exhibit low micromolar antisense activity and the cellular activit...

  10. 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

  11. Extensive polycistronism and antisense transcription in the mammalian Hox clusters.

    Directory of Open Access Journals (Sweden)

    Gaëll Mainguy

    Full Text Available The Hox clusters play a crucial role in body patterning during animal development. They encode both Hox transcription factor and micro-RNA genes that are activated in a precise temporal and spatial sequence that follows their chromosomal order. These remarkable collinear properties confer functional unit status for Hox clusters. We developed the TranscriptView platform to establish high resolution transcriptional profiling and report here that transcription in the Hox clusters is far more complex than previously described in both human and mouse. Unannotated transcripts can represent up to 60% of the total transcriptional output of a cluster. In particular, we identified 14 non-coding Transcriptional Units antisense to Hox genes, 10 of which (70% have a detectable mouse homolog. Most of these Transcriptional Units in both human and mouse present conserved sizeable sequences (>40 bp overlapping Hox transcripts, suggesting that these Hox antisense transcripts are functional. Hox clusters also display at least seven polycistronic clusters, i.e., different genes being co-transcribed on long isoforms (up to 30 kb. This work provides a reevaluated framework for understanding Hox gene function and dys-function. Such extensive transcriptions may provide a structural explanation for Hox clustering.

  12. Antisense-mediated exon skipping to reframe transcripts.

    Science.gov (United States)

    Turczynski, Sandrina; Titeux, Matthias; Pironon, Nathalie; Hovnanian, Alain

    2012-01-01

    Numerous genetic disorders are caused by loss-of-function mutations that disrupt the open reading frame of the gene either by nonsense or by frameshift (insertion, deletion, indel, or splicing) mutations. Most of the time, the result is the absence of functional protein synthesis due to mRNA degradation by nonsense-mediated mRNA decay, or rapid degradation of a truncated protein. Antisense-based splicing modulation is a powerful tool that has the potential to treat genetic disorders by restoring the open reading frame through selective removal of the mutated exon, or by restoring correct splicing.We have developed this approach for a severe genetic skin disorder, recessive dystrophic epidermolysis bullosa, caused by mutations in the COL7A1 gene encoding type VII collagen. This gene is particularly suited for exon-skipping approaches due to its unique genomic structure. It is composed of 118 exons, 83 of which are in frame. Moreover, these exons encode a single repetitive collagenous domain.Using this gene as an example, we describe general methods that demonstrate the feasibility and efficacy of the antisense-mediated exon-skipping strategy to reframe transcripts.

  13. 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.

  14. Data in support of transcriptional regulation and function of Fas-antisense long noncoding RNA during human erythropoiesis

    Directory of Open Access Journals (Sweden)

    Olga Villamizar

    2016-06-01

    Full Text Available This paper describes data related to a research article titled, “Fas-antisense long noncoding RNA is differentially expressed during maturation of human erythrocytes and confers resistance to Fas-mediated cell death” [1]. Long noncoding RNAs (lncRNAs are increasingly appreciated for their capacity to regulate many steps of gene expression. While recent studies suggest that many lncRNAs are functional, the scope of their actions throughout human biology is largely undefined including human red blood cell development (erythropoiesis. Here we include expression data for 82 lncRNAs during early, intermediate and late stages of human erythropoiesis using a commercial qPCR Array. From these data, we identified lncRNA Fas-antisense 1 (Fas-AS1 or Saf described in the research article. Also included are 5′ untranslated sequences (UTR for lncRNA Saf with transcription factor target sequences identified. Quantitative RT-PCR data demonstrate relative levels of critical erythroid transcription factors, GATA-1 and KLF1, in K562 human erythroleukemia cells and maturing erythroblasts derived from human CD34+ cells. End point and quantitative RT-PCR data for cDNA prepared using random hexamers versus oligo(dT18 revealed that lncRNA Saf is not effectively polyadenylated. Finally, we include flow cytometry histograms demonstrating Fas levels on maturing erythroblasts derived from human CD34+ cells transduced using mock conditions or with lentivirus particles encoding for Saf.

  15. 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.

  16. Antisense expression of a gene encoding a calcium-binding protein in transgenic tobacco leads to altered morphology and enhanced chlorophyll

    Indian Academy of Sciences (India)

    Girdhar K Pandey; Amita Pandey; Vanga Siva Reddy; Renu Deswal; Alok Bhattacharya; Kailash C Upadhyaya; Sudhir K Sopory

    2007-03-01

    Entamoeba histolytica contains a novel calcium-binding protein like calmodulin, which was discovered earlier, and we have reported the presence of its homologue(s) and a dependent protein kinase in plants. To understand the functions of these in plants, a cDNA encoding a calcium-binding protein isolated from Entamoeba histolytica (EhCaBP) was cloned into vector pBI121 in antisense orientation and transgenic tobacco plants were raised. These plants showed variation in several phenotypic characters, of which two distinct features, more greenness and leaf thickness, were inherited in subsequent generations. The increase in the level of total chlorophyll in different plants ranged from 60% to 70%. There was no major change in chloroplast structure and in the protein level of D1, D2, LHCP and RuBP carboxylase. These morphological changes were not seen in antisense calmodulin transgenic tobacco plants, nor was the calmodulin level altered in EhCaBP antisense plants.

  17. Measurement of X-ray-induced DNA double-strand breaks at various stages of the cell cycle using the total fluorescence as a comet assay parameter

    Science.gov (United States)

    Attia, Atef M. M.; Nabil, Ghada M.; Frankenberg, Dieter; Frankenberg-Schwager, M.

    2011-11-01

    The aim of the study was to develop a protocol for both estimating cell cycle position and the level of ionizing radiation-induced DNA dsb using the neutral comet assay. Using DNA histograms, cell cycle positions were determined for human dermal fibroblasts. The tail intensity was used to estimate the level of DNA damage induced by X-rays, at different positions of the cell cycle. The results of tail intensity versus DNA content bivariate analysis of exponentially growing cells showed a remarkable decrease in tail intensity with transition of cells from G1 to S-phase and increases slightly with transition to G2/M phase. This effect is observed at all doses including unirradiated cells, indicating that the effect is not caused by X-rays and the comet assay based on the current tail parameters is not relevant to measure DNA damage at various stages of the cell cycle. The results of dose response curves showed a linear decrease in the comet fluorescence with the X-ray dose. This observation provides a basis for estimating the fraction of damaged DNA, based on the fluorescence decrement induced by ionizing radiation. The results of this new approach showed a linear increase in DNA damage with dose, at various stages of the cell cycle, with rates, which vary in the following order G0>G2/M>S/G1 cells.These results suggest that G0 and G2/M cells are the most sensitive to X-rays among all phases of the cell cycle and suggest synchronization of cells at these phases to increase the cellular radiosensitivity during radiotherapy.

  18. 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

  19. 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.

  20. 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.

  1. Silencing of the AV2 gene by antisense RNA protects transgenic plants against a bipartite begomovirus

    Directory of Open Access Journals (Sweden)

    Zafar Yusuf

    2007-01-01

    Full Text Available Abstract Whitefly-transmitted geminiviruses (genus Begomovirus are phytopathogens that cause heavy losses to crops worldwide. Efforts to engineer resistance against these viruses are focused mainly on silencing of complementary-sense virus genes involved in virus replication. Here we have targeted a virion-sense gene (AV2 to develop resistance against Tomato leaf curl New Delhi virus, a bipartite begomovirus prevalent throughout the Indian subcontinent. We show that tobacco plants transformed with an antisense construct targeting this gene are resistant to the virus. Following challenged with the virus, transgenic plants remained symptomless, although viral DNA could be detected in some plants by PCR. This is the first report of transgenic resistance against a bipartite begomovirus obtained by targeting a virion-sense gene. The relatively conserved nature of the gene suggests that the technology may be useful to develop broad-spectrum resistance which is required because of the fact that plants are often infected with multiple begomoviruses in the field.

  2. Ultra deep sequencing of Listeria monocytogenes sRNA transcriptome revealed new antisense RNAs.

    Directory of Open Access Journals (Sweden)

    Sebastian Behrens

    Full Text Available Listeria monocytogenes, a gram-positive pathogen, and causative agent of listeriosis, has become a widely used model organism for intracellular infections. Recent studies have identified small non-coding RNAs (sRNAs as important factors for regulating gene expression and pathogenicity of L. monocytogenes. Increased speed and reduced costs of high throughput sequencing (HTS techniques have made RNA sequencing (RNA-Seq the state-of-the-art method to study bacterial transcriptomes. We created a large transcriptome dataset of L. monocytogenes containing a total of 21 million reads, using the SOLiD sequencing technology. The dataset contained cDNA sequences generated from L. monocytogenes RNA collected under intracellular and extracellular condition and additionally was size fractioned into three different size ranges from 150 nt. We report here, the identification of nine new sRNAs candidates of L. monocytogenes and a reevaluation of known sRNAs of L. monocytogenes EGD-e. Automatic comparison to known sRNAs revealed a high recovery rate of 55%, which was increased to 90% by manual revision of the data. Moreover, thorough classification of known sRNAs shed further light on their possible biological functions. Interestingly among the newly identified sRNA candidates are antisense RNAs (asRNAs associated to the housekeeping genes purA, fumC and pgi and potentially their regulation, emphasizing the significance of sRNAs for metabolic adaptation in L. monocytogenes.

  3. Ultra Deep Sequencing of Listeria monocytogenes sRNA Transcriptome Revealed New Antisense RNAs

    Science.gov (United States)

    Behrens, Sebastian; Widder, Stefanie; Mannala, Gopala Krishna; Qing, Xiaoxing; Madhugiri, Ramakanth; Kefer, Nathalie; Mraheil, Mobarak Abu; Rattei, Thomas; Hain, Torsten

    2014-01-01

    Listeria monocytogenes, a gram-positive pathogen, and causative agent of listeriosis, has become a widely used model organism for intracellular infections. Recent studies have identified small non-coding RNAs (sRNAs) as important factors for regulating gene expression and pathogenicity of L. monocytogenes. Increased speed and reduced costs of high throughput sequencing (HTS) techniques have made RNA sequencing (RNA-Seq) the state-of-the-art method to study bacterial transcriptomes. We created a large transcriptome dataset of L. monocytogenes containing a total of 21 million reads, using the SOLiD sequencing technology. The dataset contained cDNA sequences generated from L. monocytogenes RNA collected under intracellular and extracellular condition and additionally was size fractioned into three different size ranges from 150 nt. We report here, the identification of nine new sRNAs candidates of L. monocytogenes and a reevaluation of known sRNAs of L. monocytogenes EGD-e. Automatic comparison to known sRNAs revealed a high recovery rate of 55%, which was increased to 90% by manual revision of the data. Moreover, thorough classification of known sRNAs shed further light on their possible biological functions. Interestingly among the newly identified sRNA candidates are antisense RNAs (asRNAs) associated to the housekeeping genes purA, fumC and pgi and potentially their regulation, emphasizing the significance of sRNAs for metabolic adaptation in L. monocytogenes. PMID:24498259

  4. 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.

  5. Antisense oligonucleotide targeting midkine suppresses in vivo angiogenesis

    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 antisense oligonucleotide targeting midkine (MK-AS) on angiogenesis in chick chorioallantoic membrane (CAM) andin situ human hepatocellular carcinoma (HCC).METHODS: An in situ human hepatocellular carcinoma (HCC) model and CAM assay were used in this experiment. The effect of MK-AS on angiogenesis was evaluated by cell proliferation assay and hematoxylineosin (HE) staining.RESULTS: MK-AS significantly inhibited human umbilical vein endothelial cells (HUVEC) and in situ human HCC growth. At the same time, MK-AS suppressed the angiogenesis both in human hepatocellular carcinoma cell line (HEPG2)-induced CAM and in situ human HCC tissues.CONCLUSION: MK-AS is an effective antiangiogenesis agent in vivo.

  6. Isolation of a Calmodulin cDNA from Salvia miltiorrhiza Bunge and Construction of its Antisense Expression Vector%丹参钙调蛋白cDNA的克隆及其反义表达载体的构建

    Institute of Scientific and Technical Information of China (English)

    张成; 王喆之

    2006-01-01

    丹参是著名的传统中药,对心血管疾病和癌症有广泛的疗效.钙调蛋白是细胞信号转导途径中的重要蛋白,在各种生理活动中起着重要的作用,其序列比较保守.国内外对中药丹参的分子学研究很少,本文利用分子生物学手段克隆了丹参钙调蛋白基因.从丹参成熟叶片中提取总RNA,cDNA第一链经反转录合成,利用设计的引物,PCR扩增,得到丹参钙调蛋白基因,测定其全序列.在GeneBank进行了注册.其次构建了丹参钙调蛋白基因反义表达载体,以后可进行基因敲除.通过Southern杂交,进行拷贝数推测,丹参钙调蛋白cDNA至少为2个拷贝.序列分析结果表明:得到的丹参钙调蛋白基因具有完整地读码框,由454个核苷酸组成,编码150个氨基酸.与别的植物钙调蛋白基因相比有很高的同源性,核苷酸序列同源性在80%以上,编码的氨基酸序列同源性在80%以上.以上结果能使我们更加深入地研究和了解钙调蛋白.%Salvia miltiorrhiza Bunge is a famous Chinese traditional medicinal herb to cure heart and cancer diseases. Calmodulin is a key regulatory protein involved in the signal transduction pathway and plays important roles in all aspects. There are few molecular researches on Salvia miltiorrhiza Bunge. The Calmodulin gene was cloned from Salvia miltiorrhiza Bunge. The total RNA was isolated from mature leaves of Salvia miltiorrhiza Bunge. The Calmodulin gene was amplified by PCR after reverse transcription with the conserved primer sequences. A band of 454bp in length of PCR product was subsequently cloned with T-easy vector. The gene was registered in GenBank.The antisense express vector was constructed. The copy number of the gene is estimated to be at least two copies per genome by using southern blot. The result showed that the PCR product had complete Open Reading Frame which had 454bp nucleotides. This gene shared more than 80% homologies with the calmodulian cDNAs of

  7. 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

  8. 正义-反义多肽的相互作用%The Interaction between Sense and Antisense Peptides

    Institute of Scientific and Technical Information of China (English)

    吕凤林; 朱锡华

    1999-01-01

    @@ 遗传学中心法则指出,DNA有义链(即正义链,sense RNA strand)可转录为单链mRNA,其核苷酸序列决定蛋白质的氨基酸序列.虽然DNA无义链(即反义DNA链,antisense DNA strand)能以框转录方式转录成反义mRNA,甚至能以框翻译方式翻译成蛋白质,但在正常情况下,反义DNA在细胞中不表达具有功能意义的蛋白质.反义RNA能与mRNA特异地互补结合,从而抑制该mRNA的加工与翻译.

  9. Isolation of Promoters and Fragments of Genes Controlling Endosperm Development Without Fertilization in Arabidopsis and Engineering of the Antisense Constructions

    Directory of Open Access Journals (Sweden)

    Grigory A. Gerashchenkov

    2015-06-01

    Full Text Available Apomixis is asexual seed reproduction without both meiosis and fertilization based on the complex developmental processes such as apomeiosis, parthenogenesis and specific endosperm development. This investigation is aimed at engineering of apomixis in Arabidopsis thaliana with sexual seed reproduction. The fragments of known genes of endosperm formation MEA, FIE, FIS2 and gene of apomeiosis DYAD (as control were isolated using Q5 high fidelity DNA polymerase. These gene fragments of interest at the antisense orientation were fused with isolated constitutive and meiosis specific promoters of Arabidopsis at NcoI sites. The fused promoter-gene fragment modules were cloned in pCambia1301 at SalI cites. The engineered constructions will be used for the floral dip transformation of Arabidopsis and down regulation of these genes at engineering of apomixis.

  10. Mechanism of adsorption of single and double stranded DNA on gold and silver nanoparticles: Investigating some important parameters in bio-sensing applications.

    Science.gov (United States)

    Farkhari, Nahid; Abbasian, Sara; Moshaii, Ahmad; Nikkhah, Maryam

    2016-12-01

    The mechanism of adsorption of single and double stranded DNAs on colloidal gold and silver nanoparticles has been studied by measuring the resistance of the nanoparticles, surrounded by various oligonucleotides, against salt induced aggregation. It is shown that both single and double stranded DNAs can be adsorbed on the metal nanoparticles and the adsorption strength is determined by the interaction between various bases of DNA and the nanoparticles. By changing the salt concentration, the difference between adsorption of various DNA strands on the nanoparticles can be specified. The results indicate that a key parameter in success of a sensing assay of DNA hybridization is the salt concentration which should be greater than a minimum threshold depending on the nanoparticles characteristics. We have also investigated the interaction mechanism between various DNA bases with the metal nanoparticles. For both gold and silver nanoparticles, adenine has the highest and thymine has the lowest attachment to the nanoparticles. From surface enhanced Raman spectroscopy (SERS) data of various bases in the presence of gold nanoparticles, the probable interaction points in the bases with the nanoparticles have been determined, which are mainly the nitrogen sites of these oligonucleotides.

  11. Selection of optimal antisense accessible sites of survivin and its application in treatment of gastric cancer

    Institute of Scientific and Technical Information of China (English)

    Qiang-Song Tong; Li-Duan Zheng; Fang-Min Chen; Fu-Qing Zeng; Liang Wang; Ji-Hua Dong; Gong-Cheng Lu

    2005-01-01

    AIM: To select the optimal antisense accessible sites of survivin, a highly expressed gene in tumor tissues, in order to explore a novel approach to improve biological therapy of gastric cancer.METHODS: The 20 mer random oligonucleotide library was synthesized, hybridized with in vitro transcribed total survivin cRNA, then digested by RNase H. After primer extension and autoradiography, the antisense accessible sites (AAS) of survivin were selected. Then RNADraw software was used to analyze and choose the AAS with obvious stem-loop structures, according to which the complementary antisense oligonucleotides (AS-ODNs) were synthesized and transferred into survivin highly- expressing gastric cancer cell line MKN-45. Survivin expression was detected by RT-PCR and Western Blotting. Cellular growth activities were assayed by tetrazolium bromide (MTT)colorimetry. Cellular ultrastructure was observed by electronic microscopy, while apoptosis was detected by annexin V-FITC and propidium iodide staining flow cytometry.RESULTS: Thirteen AAS of survivin were selected in vitro.Four AAS with stem-loop structures were chosen, locating at 207-226 bp, 187-206 bp, 126-145 bp and 44-63 bp of survivin cDNA respectively. When compared with nontranfection controls, their corresponding AS-ODNs (AS-ODN1,AS-ODN2, AS-ODN3 and AS-ODN4) could reduce Survivin mRNA levels in MKN-45 cells by 54.3±1.1% (t= 6.12, P<0.01),86.1±1.0% (t= 5.27, P<0.01), 32.2±1.3% (t= 7.34, P<0.01)and 56.2±0.9% (t= 6.45, P<0.01) respectively, while survivin protein levels were decreased by 42.2±2.5% (t = 6.26,P<0.01), 75.4±3.1% (t= 7.11, P<0.01), 28.3±2.0% (t= 6.04,P<0.01) and 45.8±1.2% (t = 6.38, P<0.01) respectively.After transfection with 600 nmol/L AS-ODN1~AS-ODN4 for24 h, cell growth was inhibited by 28.12±1.54% (t= 7.62,P<0.01), 38.42±3.12% (t = 7.75, P<0.01), 21.46±2.63%(t= 5.94, P<0.01) and 32.12±1.77% (t= 6.17, P<0.01)respectively. Partial cancer cells presented the

  12. Site specificity of the Arabidopsis METI DNA methyltransferase demonstrated through hypermethylation of the superman locus.

    Science.gov (United States)

    Kishimoto, N; Sakai, H; Jackson, J; Jacobsen, S E; Meyerowitz, E M; Dennis, E S; Finnegan, E J

    2001-05-01

    Plants with low levels of DNA methylation show a range of developmental abnormalities including homeotic transformation of floral organs. Two independent DNA METHYLTRANSFERASEI (METI) antisense transformants with low levels of DNA methylation had flowers with increased numbers of stamens which resembled flowers seen on the loss-of-function superman (sup) mutant plants and on transgenic plants that ectopically express APETALA3 (AP3). These METI antisense plants have both increased and decreased methylation in and around the sup gene, compared with untransformed controls. DNA from the antisense plants was demethylated at least 4 kb upstream of the sup gene, while there was dense methylation around the start of transcription and within the coding region of this gene; these regions were unmethylated in control DNA. Methylation within the sup gene was correlated with an absence of SUP transcripts. The pattern and density of methylation was heterogeneous among different DNA molecules from the same plant, with some molecules being completely unmethylated. Methylcytosine occurred in asymmetric sites and in symmetric CpA/TpG but rarely in CpG dinucleotides in the antisense plants. In contrast, segregants lacking the METI antisense construct and epimutants with a hypermethylated allele of sup (clark kent 3), both of which have active METI genes, showed a higher frequency of methylation of CpG dinucleotides and of asymmetric cytosines. We conclude that METI is the predominant CpG methyltransferase and directly or indirectly affects asymmetric methylation.

  13. 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.

  14. 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.

  15. 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...

  16. Analysis on correlation between sperm DNA fragments and semen parameters%精子 DNA 碎片与精液各参数的相关性分析

    Institute of Scientific and Technical Information of China (English)

    韩小克; 栾世巍; 苑辉; 关立军; 郑波; 邱喜豹; 王胜涛; 安先贵; 杨志伟; 胡一珍

    2016-01-01

    Objective To investigate the relationship between sperm DNA fragmentation and semen parameters in male infertile patients and to study the application value of sperm DNA integrity in the assessment of male fertility .Methods From January 2014 to November 2015, 265 cases of male infertility in the reproductive center of Xingtai Infertility Specialist Hospital were analyzed retrospectively .Collection of semen samples , routine semen analysis by computer assisted , the sperm morphology and sperm DNA fragmentation rate were detected by the Wright-Ji staining microscopic examination .Comparison and correlation analysis between the DNA fragment rate and age , and the parameters of semen ( sperm concentration、forward moving sperm、normal sperm morphology ) were compared.Results There were significant differences in the DNA fragments of different age groups ( P 0.05);There was a significant difference between the two groups in the pre-active sperm and the normal form sperm ( P<0.05);There was a positive correlation between age and sperm DNA fragmentation .Forward movement sperm rate , normal sperm morphology and sperm DNA fragments were negatively correlated .Conclusion Age is the factor that affects the DNA damage of sperm .Sperm DNA damage degree can lead to the decline of sperm motility and normal sperm morphology .%目的:探讨男性不育患者精子DNA碎片与精液各参数的相关性,研究精子DNA完整性在评估男性生育力方面的应用价值。方法回顾性分析2014年1月至2015年11月就诊于邢台不孕不育专科医院生殖中心男科的不育患者265例,采集精液标本,通过计算机辅助行常规精液分析,采用瑞-吉染色镜检观察精子形态及通过精子染色体扩散试验( SCD法)行精子DNA碎片率检测,对精子DNA碎片率与年龄、精液各参数(精子浓度、前向运动精子、正常精子形态)进行分组比较及相关分析。结果不同年龄段精子DNA碎片组间

  17. 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.

  18. 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

  19. On scaling laws at the phase transition of systems with divergent order parameter and/or internal length the example of DNA denaturation

    CERN Document Server

    Buyukdagli, S; Buyukdagli, Sahin; Joyeux, Marc

    2006-01-01

    We used the Transfer-Integral method to compute, with an uncertainty smaller than 5%, the six fundamental characteristic exponents of two dynamical models for DNA thermal denaturation and investigate the validity of the scaling laws. Doubts concerning this point arise because the investigated systems (i) have a divergent internal length, (ii) are described by a divergent order parameter, (iii) are of dimension 1. We found that the assumption that the free energy can be described by a single homogeneous function is robust, despite the divergence of the order parameter, so that Rushbrooke's and Widom's identities are valid relations. Josephson's identity is instead not satisfied. This is probably due to the divergence of the internal length, which invalidates the assumption that the correlation length is solely responsible for singular contributions to thermodynamic quantities. Fisher's identity is even wronger. We showed that this is due to the d=1 dimensionality and obtained an alternative law, which is well ...

  20. Factors that affect the efficiency of antisense oligodeoxyribonucleotide transfection by insonated gas-filled lipid microbubbles

    Science.gov (United States)

    Zhao, Ying-Zheng; Lu, Cui-Tao

    2008-03-01

    Objective: To investigate the factors that affect the efficiency of antisense oligodeoxyribonucleotide(AS-ODNs) transfection by insonated gas-filled lipid microbubbles. Methods: Lipid microbubbles filled with two types of gases-air and C3F8, were prepared respectively. An AS-ODNs sequence HA824 and a breast cancer cell line SK-BR-3 were used to define the various operating variables determining the transfection efficiency of insonated microbubbles. Two mixing methods, three levels of mixing speed, different mixing durations and various ultrasound initiation time after mixing were examined respectively. Transfection efficiency was detected by fluorescence microscopy. Results: C3F8 microbubbles gave higher levels of AS-ODNs transfection efficiency than air microbubbles in all test conditions. Transfection efficiency resulted from mixing method A (incubation of HA824 and microbubbles before mixing cells) did not show significant difference with that of mixing method B (without incubation of HA824 and microbubbles before mixing cells). Mixing speed, duration of mixing and ultrasound initiation time after mixing were central to determining HA824 transfection efficiency in vitro. The optimum parameters for SK-BR-3 cells were found at a mixing speed of 40-50 rpm for 30-60 s with less than 60 s delay before ultrasound. Conclusion: Ultrasound-mediated AS-ODNs transfection enhanced by C3F8-filled lipid microbubbles represents an effective avenue for AS-ODNs transfer.

  1. Factors that affect the efficiency of antisense oligodeoxyribonucleotide transfection by insonated gas-filled lipid microbubbles

    Energy Technology Data Exchange (ETDEWEB)

    Zhao Yingzheng [General Hospital of Beijing Military Command of PLA, Department of Clinical Pharmacology (China)], E-mail: lctuua@yahoo.com.cn; Lu Cuitao [Madam Medical Management Group (China)

    2008-03-15

    Objective: To investigate the factors that affect the efficiency of antisense oligodeoxyribonucleotide(AS-ODNs) transfection by insonated gas-filled lipid microbubbles. Methods: Lipid microbubbles filled with two types of gases-air and C{sub 3}F{sub 8}, were prepared respectively. An AS-ODNs sequence HA824 and a breast cancer cell line SK-BR-3 were used to define the various operating variables determining the transfection efficiency of insonated microbubbles. Two mixing methods, three levels of mixing speed, different mixing durations and various ultrasound initiation time after mixing were examined respectively. Transfection efficiency was detected by fluorescence microscopy. Results: C{sub 3}F{sub 8} microbubbles gave higher levels of AS-ODNs transfection efficiency than air microbubbles in all test conditions. Transfection efficiency resulted from mixing method A (incubation of HA824 and microbubbles before mixing cells) did not show significant difference with that of mixing method B (without incubation of HA824 and microbubbles before mixing cells). Mixing speed, duration of mixing and ultrasound initiation time after mixing were central to determining HA824 transfection efficiency in vitro. The optimum parameters for SK-BR-3 cells were found at a mixing speed of 40-50 rpm for 30-60 s with less than 60 s delay before ultrasound. Conclusion: Ultrasound-mediated AS-ODNs transfection enhanced by C{sub 3}F{sub 8}-filled lipid microbubbles represents an effective avenue for AS-ODNs transfer.

  2. BayesPI - a new model to study protein-DNA interactions: a case study of condition-specific protein binding parameters for Yeast transcription factors

    Directory of Open Access Journals (Sweden)

    Morigen

    2009-10-01

    Full Text Available Abstract Background We have incorporated Bayesian model regularization with biophysical modeling of protein-DNA interactions, and of genome-wide nucleosome positioning to study protein-DNA interactions, using a high-throughput dataset. The newly developed method (BayesPI includes the estimation of a transcription factor (TF binding energy matrices, the computation of binding affinity of a TF target site and the corresponding chemical potential. Results The method was successfully tested on synthetic ChIP-chip datasets, real yeast ChIP-chip experiments. Subsequently, it was used to estimate condition-specific and species-specific protein-DNA interaction for several yeast TFs. Conclusion The results revealed that the modification of the protein binding parameters and the variation of the individual nucleotide affinity in either recognition or flanking sequences occurred under different stresses and in different species. The findings suggest that such modifications may be adaptive and play roles in the formation of the environment-specific binding patterns of yeast TFs and in the divergence of TF binding sites across the related yeast species.

  3. Disruption of erythrocyte antioxidant defense system, hematological parameters, induction of pro-inflammatory cytokines and DNA damage in liver of co-exposed rats to aluminium and acrylamide.

    Science.gov (United States)

    Ghorbel, Imen; Maktouf, Sameh; Kallel, Choumous; Ellouze Chaabouni, Semia; Boudawara, Tahia; Zeghal, Najiba

    2015-07-05

    The individual toxic effects of aluminium and acrylamide are well known but there are no data on their combined effects. The present study was undertaken to determine (i) hematological parameters during individual and combined chronic exposure to aluminium and acrylamide (ii) correlation of oxidative stress in erythrocytes with pro-inflammatory cytokines expression, DNA damage and histopathological changes in the liver. Rats were exposed to aluminium (50 mg/kg body weight) in drinking water and acrylamide (20 mg/kg body weight) by gavage, either individually or in combination for 3 weeks. Exposure rats to AlCl3 or/and ACR provoked an increase in MDA, AOPP, H2O2 and a decrease in GSH and NPSH levels in erythrocytes. Activities of catalase, glutathione peroxidase and superoxide dismutase were decreased in all treated rats. Our results showed that all treatments induced an increase in WBC, erythrocyte osmotic fragility and a decrease in RBC, Hb and Ht. While MCV, MCH, MCHC remained unchanged. Hepatic pro-inflammatory cytokines expression including tumor necrosis factor-α, interleukin-6, interleukin-1β was increased suggesting leucocytes infiltration in the liver. A random DNA degradation was observed on agarose gel only in the liver of co-exposed rats to AlCl3 and ACR treatment. Interestingly, co-exposure to these toxicants exhibited synergism based on physical and biochemical variables in erythrocytes, pro-inflammatory cytokines and DNA damage in liver.

  4. Strand-specific RNA-seq reveals widespread occurrence of novel cis-natural antisense transcripts in rice

    Directory of Open Access Journals (Sweden)

    Lu Tingting

    2012-12-01

    Full Text Available Abstract Background Cis-natural antisense transcripts (cis-NATs are RNAs transcribed from the antisense strand of a gene locus, and are complementary to the RNA transcribed from the sense strand. Common techniques including microarray approach and analysis of transcriptome databases are the major ways to globally identify cis-NATs in various eukaryotic organisms. Genome-wide in silico analysis has identified a large number of cis-NATs that may generate endogenous short interfering RNAs (nat-siRNAs, which participate in important biogenesis mechanisms for transcriptional and post-transcriptional regulation in rice. However, the transcriptomes are yet to be deeply sequenced to comprehensively investigate cis-NATs. Results We applied high-throughput strand-specific complementary DNA sequencing technology (ssRNA-seq to deeply sequence mRNA for assessing sense and antisense transcripts that were derived under salt, drought and cold stresses, and normal conditions, in the model plant rice (Oryza sativa. Combined with RAP-DB genome annotation (the Rice Annotation Project Database build-5 data set, 76,013 transcripts corresponding to 45,844 unique gene loci were assembled, in which 4873 gene loci were newly identified. Of 3819 putative rice cis-NATs, 2292 were detected as expressed and giving rise to small RNAs from their overlapping regions through integrated analysis of ssRNA-seq data and small RNA data. Among them, 503 cis-NATs seemed to be associated with specific conditions. The deep sequence data from isolated epidermal cells of rice seedlings further showed that 54.0% of cis-NATs were expressed simultaneously in a population of homogenous cells. Nearly 9.7% of rice transcripts were involved in one-to-one or many-to-many cis-NATs formation. Furthermore, only 17.4-34.7% of 223 many-to-many cis-NAT groups were all expressed and generated nat-siRNAs, indicating that only some cis-NAT groups may be involved in complex regulatory networks. Conclusions

  5. Investigation of the Relationship of Some Antihypertensive Drugs with Oxidant/Antioxidant Parameters and DNA Damage on Rat Uterus Tissue

    Directory of Open Access Journals (Sweden)

    Mustafa Talip Sener

    2011-01-01

    Full Text Available Background: In this study, we investigated the effects of treatment with chronic antihypertensivedrugs (clonidine, methyldopa, amlodipine, ramipril and rilmenidine on oxidant-antioxidantparameters and toxic effects on DNA in rat uterus tissue. In addition, uterus tissues were examinedhistopathologically.Materials and Methods: A total of 36 albino Wistar rats were divided into the following six groups:0.075 mg/kg clonidine group; 100 mg/kg methyldopa group; 2 mg/kg amlodipine group; 2.5 mg/kgramipril group; 0.5 mg/kg rilmenidine group; and the healthy group. Rats underwent chronic drugadministration for 30 days and at the end, biochemical and histopathological examinations wereperformed. All data were subjected to one-way ANOVA test.Results: We divided these drugs into the following three groups according to their effects on ratuteri: (I mild negative effects (clonidine, (II moderate negative effects (rilmenidine, methyldopaand (III drugs which had severe negative effects (amlodipine, ramipril.Conclusion: These data may help with selection of antihypertensive drugs, in order to determinewhich drugs have the lowest toxicity in pregnant and non-pregnant (pre-pregnancy women.

  6. Antisense oligonucleotide inhibition of hepatitis C virus genotype 4 replication in HepG2 cells

    Directory of Open Access Journals (Sweden)

    Omran Moataza H

    2006-06-01

    Full Text Available Abstract Background Hepatitis C (HCV viral infection is a serious medical problem in Egypt and it has a devastating impact on the Egyptian economy. It is estimated that over 15% of Egyptians are infected by the virus and thus finding a cure for this disease is of utmost importance. Current therapies for hepatitis C virus (HCV genotype 4 with interferon/ribavirin have not been successful and thus the development of alternative therapy for this genotype is disparately needed. Results Although previous studies utilizing viral subgenomic or full cDNA fragments linked to reporter genes transfected into adhered cells or in a cell free system showed promise, demonstration of efficient viral replication was lacking. Thus, we utilized HepG2 cells infected with native HCV RNA genomes in a replication competent system and used antisense phosphorothioate Oligonucleotides (S-ODN against stem loop IIId and the AUG translation start site of the viral polyprotein precursor to monitor viral replication. We were able to show complete arrest of intracellular replication of HCV-4 at 1 uM S-ODN, thus providing a proof of concept for the potential antiviral activity of S-ODN on native genomic replication of HCV genotype 4. Conclusion We have successfully demonstrated that by using two S-ODNs [(S-ODN1 (nt 326–348 and S-ODN-2 (nt 264–282], we were able to completely inhibit viral replication in culture, thus confirming earlier reports on subgenomic constructs and suggesting a potential therapeutic value in HCV type 4.

  7. Antihepatoma effect of alpha-fetoprotein antisense phosphorothioate oligodeoxyribonucleotides in vitro and in mice

    Institute of Scientific and Technical Information of China (English)

    Xing Wang Wang; Jin Hui Yuan; Ru Gang Zhang; Li Xia Guo; Yong Xie; Hong Xie

    2001-01-01

    AIM To evaluate antihepatoma effect ofantisense phosphorothioate oligodeo-xyribonucleotides (S-ODNs) targeted to alpha-fetoprotein (AFP) genes in vitro and in nudemice.METHODS AFP gene expression was examinedby immunocytochemical method or enzyme-linked immunosorbent assay. Effect of S-ODNson SMMC-7721 human hepatoma cell growth invitro was determined using microculturetetrazolium assay. In vivo antitumor activitiesof S-ODNs were monitored by measuring tumorweight differences in treated and control micebearing SMMC-7721 xenografts. Induction of cellapoptosis was evaluated by fluorescence-activated cell sorter (FACS) analysis.RESULTS Antisense S-ODN treatment led toreduced AFP gene expression. Specificantisense S-ODNs, but not control S-ODNs,inhibited the growth of heaptoma cells in vitro.In vivo. only antisense S-ODNs exhibitedobvious antitumor activities. FACS analysisrevealed that the growth inhibition by antisenseS. ODNs was associated with their cell apoptosisinduction.CONCLUSION Antisense S-ODNs targeted toAFP genes inhibit the growth of human hepatomacells and solid hepatoma, which is related totheir cell apoptosis induction.

  8. Expression of XIST sense and antisense in bovine fetal organs and cell cultures.

    Science.gov (United States)

    Farazmand, Ali; Basrur, Parvathi K; Stranzinger, Gerald; Graphodatskaya, Daria; Reyes, Ed R; King, W Allan

    2004-01-01

    Untranslated RNAs transcribed from sense and antisense strands of a gene referred to as X-inactive specific transcript (XIST) play crucial roles in the genetic inactivation and condensation of one of the two X chromosomes in the somatic cells of female mammals. X inactivation is also thought to occur in mammalian male germ cells mainly based on the formation of a condensed structure referred to as a sex body or XY-body, during spermatogenesis. Molecular identity of the sex body, the roles of sense and antisense XIST RNAs in its formation, and the relevance of the sex body to spermatogenesis are not known. Here we report the results of our strand-specific RT-PCR approach to identify the amplicon detected in fetal bovine testes previously referred to as XIST and to test for sense/antisense expression in male and female organs and cell cultures of different sex chromosome constitution. Our results showed that the transcript detected consistently in male gonads and variably in somatic organs represents XIST antisense RNA and that XIST sense and antisense RNAs are co-expressed in female somatic tissues and cultured cells including cells of sex chromosome aneuploids (XXY and XXX). Our results, which differ from those of other investigators in this area, are discussed in the light of the recently reported differences in the expression pattern of murine Xist/Tsix loci and their structural and functional differences in different mammalian species.

  9. 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.

  10. 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.

  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. Profiled support vector machines for antisense oligonucleotide efficacy prediction

    Directory of Open Access Journals (Sweden)

    Martín-Guerrero José D

    2004-09-01

    Full Text Available Abstract Background This paper presents the use of Support Vector Machines (SVMs for prediction and analysis of antisense oligonucleotide (AO efficacy. The collected database comprises 315 AO molecules including 68 features each, inducing a problem well-suited to SVMs. The task of feature selection is crucial given the presence of noisy or redundant features, and the well-known problem of the curse of dimensionality. We propose a two-stage strategy to develop an optimal model: (1 feature selection using correlation analysis, mutual information, and SVM-based recursive feature elimination (SVM-RFE, and (2 AO prediction using standard and profiled SVM formulations. A profiled SVM gives different weights to different parts of the training data to focus the training on the most important regions. Results In the first stage, the SVM-RFE technique was most efficient and robust in the presence of low number of samples and high input space dimension. This method yielded an optimal subset of 14 representative features, which were all related to energy and sequence motifs. The second stage evaluated the performance of the predictors (overall correlation coefficient between observed and predicted efficacy, r; mean error, ME; and root-mean-square-error, RMSE using 8-fold and minus-one-RNA cross-validation methods. The profiled SVM produced the best results (r = 0.44, ME = 0.022, and RMSE= 0.278 and predicted high (>75% inhibition of gene expression and low efficacy (http://aosvm.cgb.ki.se/. Conclusions The SVM approach is well suited to the AO prediction problem, and yields a prediction accuracy superior to previous methods. The profiled SVM was found to perform better than the standard SVM, suggesting that it could lead to improvements in other prediction problems as well.

  13. Cloning of full-length cDNA encoding ACC synthase of Pyrus pyrifolia Nakai and construction of its antisense and sense expression vector%砂梨ACC合酶cDNA全长克隆及其反义与正义表达载体构建

    Institute of Scientific and Technical Information of China (English)

    乔玉山; 宋长年; 王新卫; 李志强; 熊爱生; 姚泉洪; 章镇

    2008-01-01

    为构建干扰砂梨(Pyrus pyrifolia Nakai)ACC合酶基因表达的遗传转化载体,利用RACE技术从'早生新水'成熟果实cDNA中克隆了ACC合酶的cDNA,该cDNA全长为1 939 bp,命名为Pyp-ACS,GenBank登录号为EF566865.Pyp-ACS核苷酸序列含有5′末端非翻译区109 bp、1 488 bp完整的开放阅读框和3′末端非翻译区342 bp(含25 bp的Ploy+(A)).Pyp-ACS编码495个氨基酸,与白梨(Pyrus×bretschneideri Rehd.)、西洋梨(Pyrus communis L.)、中国李(Prunus salicina Lindl.)、桃(Prunus persica(L.)Batsch)、梅(Prunus mume Seib.et Zucc.)、苹果(Malus×domestica Borkh.)和柿(Diospyros kaki Thunb.)有较高的同源性.该氨基酸序列具备ACC合酶7个保守区和组成该酶活性中心的12个氨基酸残基,即SLSKDMGFPGLR,进一步验证了克隆的正确性.以pYPX145载体为基础,分别将Pyp-ACS编码区序列反向和正向插入相应位点构建反义和正义表达载体,并分别命名为pPyp-ACS(-)和pPyp-ACS(+),目的基因由双35S启动子所控制.分别将这2个表达载体导入根癌农杆菌菌株(Agrobacterium tumefaciens)EHA105,为耐贮藏转基因梨的遗传转化提供载体.

  14. 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.

  15. Zinc fixation preserves flow cytometry scatter and fluorescence parameters and allows simultaneous analysis of DNA content and synthesis, and intracellular and surface epitopes

    DEFF Research Database (Denmark)

    Jensen, Uffe Birk; Owens, David; Pedersen, Søren;

    2010-01-01

    Zinc salt-based fixation (ZBF) has proved advantageous in histochemical analyses conducted on intact tissues but has not been exploited in flow cytometry procedures that focus on quantitative analysis of individual cells. Here, we show that ZBF performs equally well to paraformaldehyde in the pre......Zinc salt-based fixation (ZBF) has proved advantageous in histochemical analyses conducted on intact tissues but has not been exploited in flow cytometry procedures that focus on quantitative analysis of individual cells. Here, we show that ZBF performs equally well to paraformaldehyde...... allowing subsequent quantitative PCR analysis or labeling for incorporation of the thymidine analog EdU following surface and intracellular epitope staining. Finally, ZBF treatment allows for long-term storage of labeled cells with little change in these parameters. Thus, we present a protocol for zinc...... salt fixation of cells that allows for the simultaneous analysis of DNA and intracellular and cell surface proteins by flow cytometry....

  16. 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.

  17. The influence of low-level laser therapy on parameters of oxidative stress and DNA damage on muscle and plasma in rats with heart failure.

    Science.gov (United States)

    Biasibetti, Micheli; Rojas, Denise B; Hentschke, Vítor S; Moura, Dinara Jaqueline; Karsten, Marlus; Wannmacher, Clóvis M D; Saffi, Jenifer; Dal Lago, Pedro

    2014-11-01

    In heart failure (HF), there is an imbalance between the production of reactive oxygen species and the synthesis of antioxidant enzymes, causing damage to the cardiovascular function and increased susceptibility to DNA damage. The aim of this study was to evaluate the influence of low-level laser therapy (LLLT) on parameters of oxidative stress and DNA damage in skeletal muscle and plasma of rats with HF. Wistar rats were allocated into six groups: "placebo" HF rats (P-HF, n = 9), "placebo" Sham rats (P-sham, n = 8), HF rats at a dose 3 J/cm(2) of LLLT (3 J/cm(2)-HF, n = 8), sham rats at a dose 3 J/cm(2) of LLLT (3 J/cm(2)-sham, n = 8), HF rats at a dose 21 J/cm(2) of LLLT (21 J/cm(2)-HF, n = 8) and sham rats at a dose 21 J/cm(2) of LLLT (21 J/cm(2)-sham, n = 8). Animals were submitted to a LLLT protocol for 10 days at the right gastrocnemius muscle. Comparison between groups showed a significant reduction in superoxide dismutase (SOD) activity in the 3 J/cm(2)-HF group (p = 0.03) and the 21 J/cm(2)-HF group (p = 0.01) compared to the P-HF group. 2',7'-Dihydrodichlorofluorescein (DCFH) oxidation levels showed a decrease when comparing 3 J/cm(2)-sham to P-sham (p = 0.02). The DNA damage index had a significant increase either in 21 J/cm(2)-HF or 21 J/cm(2)-sham in comparison to P-HF (p = 0.004) and P-sham (p = 0.001) and to 3 J/cm(2)-HF (p = 0.007) and 3 J/cm(2)-sham (p = 0.037), respectively. Based on this, laser therapy appears to reduce SOD activity and DCFH oxidation levels, changing the oxidative balance in the skeletal muscle of HF rats. Otherwise, high doses of LLLT seem to increase DNA damage.

  18. 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.

  19. 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...

  20. The role of natural antisense transcripts in the pathogenesis of nervous system diseases

    Directory of Open Access Journals (Sweden)

    Lei XIANG

    2015-03-01

    Full Text Available Mammalian genomes encode numerous natural antisense transcripts (NATs. These antisense transcripts are now recognized as an important component of molecular mechanisms involved in the regulation of gene expression. NATs are particularly prevalent in the mammalian nervous system. The importance of NATs in the normal functioning of nervous system is becoming increasingly evident. They are not only involved in neuronal differentiation, myelination and ion channel regulation, but also in advanced cognitive processes, such as synapse plasticity and memory formation. This paper focuses on the potential involvement of NATs in various neurodegenerative disorders. DOI: 10.3969/j.issn.1672-6731.2015.03.014

  1. 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

    We have explored the merits of a novel delivery strategy for the antisense oligomers based on cell penetrating peptide (CPP) conjugated to a carrier PNA with sequence complementary to part of the antisense oligomer. The effect of these carrier CPP-PNAs was evaluated by using antisense PNA targeting...... 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...

  2. 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.

  3. 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.

  4. 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

  5. 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

    This dissertation focuses on the development and investigation of antisense imaging and therapeutic agents, combined with nanotechnology, to detect and suppress inducible nitric oxide synthase (iNOS) expression for the diagnosis and treatment of acute lung injury (ALI). To achieve this goal, several efforts were made. The first effort was the identification and characterization of high binding affinity antisense peptide nucleic acids (PNAs) and shell-crosslinked knedel-like nanoparticle (SCK)-PNA conjugates to the iNOS mRNA. Antisense binding sites on the iNOS mRNA were first mapped by a procedure for rapidly generating a library of antisense accessible sites on native mRNAs (MASL) which involves reverse transcription of whole cell mRNA extracts with a random oligodeoxynucleotide primer followed by mRNA-specific PCR. Antisense PNAs against the antisense accessible sites were accordingly synthesized and characterized. The second effort was the investigation of cationic shell crosslinked knedel-like nanoparticle (cSCK)-mediated siRNA delivery to suppress iNOS expression for the treatment of ALI. siRNA with its unique gene-specific properties could serve as a promising therapeutic agent, however success in this area has been challenged by a lack of efficient biocompatible transfection agents. cSCK with its nanometer size and positive charge previously showed efficient cellular delivery of phosphorothioate ODNs (oligodeoxynucleotides), plasmid DNA and PNA. Herein, cSCK showed good siRNA binding and facilitated efficient siRNA transfection in HeLa, a mouse macrophage cell line and other human cell lines. cSCK led to greater silencing efficiency than Lipofectamine 2000 in HeLa cells as determined by the viability following transfection with cytotoxic and non-cytotoxic siRNAs, as well in 293T and HEK cells, and was comparable in BEAS-2B and MCF10a cells. The third effort was the preparation of an iNOS imaging probe through electrostatic complexation between a radiolabeled

  6. 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

  7. Modulation of the cellulose content of tuber cell walls by antisense expression of different potato (Solanum tuberosum L.) CesA clones.

    Science.gov (United States)

    Oomen, Ronald J F J; Tzitzikas, Emmanouil N; Bakx, Edwin J; Straatman-Engelen, Irma; Bush, Maxwell S; McCann, Maureen C; Schols, Henk A; Visser, Richard G F; Vincken, Jean-Paul

    2004-03-01

    Four potato cellulose synthase (CesA) homologs (StCesA1, 2, 3 and 4) were isolated by screening a cDNA library made from developing tubers. Based on sequence comparisons and the fact that all four potato cDNAs were isolated from this single cDNA-library, all four StCesA clones are likely to play a role in primary cell wall biosynthesis. Several constructs were generated to modulate cellulose levels in potato plants in which the granule-bound starch synthase promoter was used to target the modification to the tubers. The StCesA3 was used for up- and down-regulation of the cellulose levels by sense (SE-StCesA3) and antisense (AS-StCesA3) expression of the complete cDNA. Additionally, the class-specific regions (CSR) of all four potato cellulose synthase genes were used for specific down-regulation (antisense) of the corresponding CesA genes (csr1, 2, 3 and 4). None of the transformants showed an overt developmental phenotype. Sections of tubers were screened for altered cell wall structure by Fourier Transform Infrared microspectroscopy (FTIR) and exploratory Principal Component Analysis (PCA), and those plants discriminating from WT plants were analysed for cellulose content and monosaccharide composition. Several transgenic lines were obtained with mainly decreased levels of cellulose. These results show that the cellulose content in potato tubers can be reduced down to 40% of the WT level without affecting normal plant development, and that constructs based on the CSR alone are specific and sufficient to down-regulate cellulose biosynthesis.

  8. The novel cis-encoded antisense RNA AsrC positively regulates the expression of rpoE-rseABC operon and thus enhances the motility of Salmonella enterica serovar Typhi.

    Directory of Open Access Journals (Sweden)

    Qi eZhang

    2015-09-01

    Full Text Available Bacterial noncoding RNAs are essential in many cellular processes, including response to environmental stress, and virulence. Deep sequencing analysis of the Salmonella enterica serovar Typhi (S. Typhi transcriptome revealed a novel antisense RNA transcribed in cis on the strand complementary to rseC, an activator gene of sigma factor RpoE. In this study, expression of this antisense RNA was confirmed in S. Typhi by Northern hybridization. Rapid amplification of cDNA ends and sequence analysis identified an 893 bp sequence from the antisense RNA coding region that covered all of the rseC coding region in the reverse direction of transcription. This sequence of RNA was named as AsrC. After overexpression of AsrC with recombinantant plasmid in S. Typhi, the bacterial motility was increased obviously. To explore the mechanism of AsrC function, regulation of rseC and rpoE expression by AsrC was investigated. We found that AsrC increased the levels of rseC mRNA and protein. The expression of rpoE was also increased in S. Typhi after overexpression of AsrC, which was dependent on rseC. Thus, we propose that AsrC increased RseC level and indirectly activating RpoE which can initiate fliA expression and promote the motility of S. Typhi.

  9. Thermodynamic and kinetic characterization of duplex formation between 2'-O, 4'-C-methylene-modified oligoribonucleotides, DNA and RNA

    DEFF Research Database (Denmark)

    Christensen, Ulla

    2007-01-01

    2'-O,4'-C-methylene-linked ribonucleotide derivatives, named LNA (locked nucleic acid) and BNA (bridged nucleic acid) are nucleic acid analogoues that have shown high-affinity recognition of DNA and RNA, and the employment of LNA oligomers for antisense activity, gene regulation and nucleic acid...... the strength of duplexes formed with the complementary DNA and RNA....

  10. 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...

  11. 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

    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...

  12. 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

  13. Tumor delivery of antisense oligomer using trastuzumab within a streptavidin nanoparticle

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yi [University of Massachusetts Medical School, Division of Nuclear Medicine, Department of Radiology, Worcester, MA (United States); Yale University, Yale PET Center, Department of Diagnostic Radiology, New Haven, CT (United States); Liu, Xinrong; Chen, Ling; Cheng, Dengfeng; Rusckowski, Mary [University of Massachusetts Medical School, Division of Nuclear Medicine, Department of Radiology, Worcester, MA (United States); Hnatowich, Donald J. [University of Massachusetts Medical School, Division of Nuclear Medicine, Department of Radiology, Worcester, MA (United States); Umass Medical School, Department of Radiology, Worcester, MA (United States)

    2009-12-15

    Trastuzumab (Herceptin trademark) is often internalized following binding to Her2+ tumor cells. The objective of this study was to investigate whether trastuzumab can be used as a specific carrier to deliver antisense oligomers into Her2+ tumor cells both in vitro and in vivo. A biotinylated MORF oligomer antisense to RhoC mRNA and its biotinylated sense control were labeled with either lissamine for fluorescence detection or {sup 99m}Tc for radioactivity detection and were linked to biotinylated trastuzumab via streptavidin. The nanoparticles were studied in SUM190 (RhoC+, Her2+) study and SUM149 (RhoC+, Her2-) control cells in culture and as xenografts in mice. As evidence of unimpaired Her2+ binding of trastuzumab within the nanoparticle, accumulations were clearly higher in SUM190 compared to SUM149 cells and, by whole-body imaging, targeting of SUM190 tumor was similar to that expected for a radiolabeled trastuzumab. As evidence of internalization, fluorescence microscopy images of cells grown in culture and obtained from xenografts showed uniform cytoplasm distribution of the lissamine-MORF. An invasion assay showed decreased RhoC expression in SUM190 cells when incubated with the antisense MORF nanoparticles at only 100 nM. Both in cell culture and in animals, the nanoparticle with trastuzumab as specific carrier greatly improved tumor delivery of the antisense oligomer against RhoC mRNA into tumor cells overexpressing Her2 and may be of general utility. (orig.)

  14. 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

  15. 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.

  16. Identification of novel non-coding RNAs as potential antisense regulators in the archaeon Sulfolobus solfataricus

    DEFF Research Database (Denmark)

    tang, T. H.; Polacek, N.; Zywicki, M.;

    2005-01-01

    to target the 3'-untranslated regions of certain mRNAs. Furthermore, one of the ncRNAs that does not show antisense elements is transcribed from a repeat unit of a cluster of small regularly spaced repeats in S. solfataricus which is potentially involved in replicon partitioning. In conclusion...

  17. Efficient hammerhead ribozyme and antisense RNA targeting in a slow ribosome Escherichia coli mutant.

    Science.gov (United States)

    Chen, H; Ferbeyre, G; Cedergren, R

    1997-05-01

    We have evaluated inhibition of the plasmid-born chloramphenicol acetyl transferase gene (CAT) by the hammerhead ribozyme and antisense RNA in Escherichia coli where the translation and transcription rates have been modified. Whereas neither antisense nor the hammerhead had an inhibitory effect on CAT activity in wild-type E. coli, both reduced the level of the messenger RNA and the activity of the CAT gene by almost 60% in a slow ribosome mutant. Streptomycin, which increases the speed of translation in this mutant strain, restored full CAT activity. The level of CAT activity expressed from a T7 RNA polymerase promoter was not affected by the presence of either antisense RNA or the hammerhead ribozyme. When the target gene was expressed from a chromosomal locus in wild-type E. coli, both antisense RNA and the hammerhead ribozyme showed some inhibitory activity, but the level of inhibition was significantly increased in the slow ribosome strain. This bacterial system offers a unique entry to the study of cellular factors which mediate the activity of ribozymes in vivo.

  18. Antisense long noncoding RNAs regulate var gene activation in the malaria parasite Plasmodium falciparum.

    Science.gov (United States)

    Amit-Avraham, Inbar; Pozner, Guy; Eshar, Shiri; Fastman, Yair; Kolevzon, Netanel; Yavin, Eylon; Dzikowski, Ron

    2015-03-03

    The virulence of Plasmodium falciparum, the causative agent of the deadliest form of human malaria, is attributed to its ability to evade human immunity through antigenic variation. These parasites alternate between expression of variable antigens, encoded by members of a multicopy gene family named var. Immune evasion through antigenic variation depends on tight regulation of var gene expression, ensuring that only a single var gene is expressed at a time while the rest of the family is maintained transcriptionally silent. Understanding how a single gene is chosen for activation is critical for understanding mutually exclusive expression but remains a mystery. Here, we show that antisense long noncoding RNAs (lncRNAs) initiating from var introns are associated with the single active var gene at the time in the cell cycle when the single var upstream promoter is active. We demonstrate that these antisense transcripts are incorporated into chromatin, and that expression of these antisense lncRNAs in trans triggers activation of a silent var gene in a sequence- and dose-dependent manner. On the other hand, interference with these lncRNAs using complement peptide nucleic acid molecules down-regulated the active var gene, erased the epigenetic memory, and induced expression switching. Altogether, our data provide evidence that these antisense lncRNAs play a key role in regulating var gene activation and mutually exclusive expression.

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. Revised genomic structure of the human ghrelin gene and identification of novel exons, alternative splice variants and natural antisense transcripts

    Directory of Open Access Journals (Sweden)

    Herington Adrian C

    2007-08-01

    Full Text Available Abstract Background Ghrelin is a multifunctional peptide hormone expressed in a range of normal tissues and pathologies. It has been reported that the human ghrelin gene consists of five exons which span 5 kb of genomic DNA on chromosome 3 and includes a 20 bp non-coding first exon (20 bp exon 0. The availability of bioinformatic tools enabling comparative analysis and the finalisation of the human genome prompted us to re-examine the genomic structure of the ghrelin locus. Results We have demonstrated the presence of an additional novel exon (exon -1 and 5' extensions to exon 0 and 1 using comparative in silico analysis and have demonstrated their existence experimentally using RT-PCR and 5' RACE. A revised exon-intron structure demonstrates that the human ghrelin gene spans 7.2 kb and consists of six rather than five exons. Several ghrelin gene-derived splice forms were detected in a range of human tissues and cell lines. We have demonstrated ghrelin gene-derived mRNA transcripts that do not code for ghrelin, but instead may encode the C-terminal region of full-length preproghrelin (C-ghrelin, which contains the coding region for obestatin and a transcript encoding obestatin-only. Splice variants that differed in their 5' untranslated regions were also found, suggesting a role of these regions in the post-transcriptional regulation of preproghrelin translation. Finally, several natural antisense transcripts, termed ghrelinOS (ghrelin opposite strand transcripts, were demonstrated via orientation-specific RT-PCR, 5' RACE and in silico analysis of ESTs and cloned amplicons. Conclusion The sense and antisense alternative transcripts demonstrated in this study may function as non-coding regulatory RNA, or code for novel protein isoforms. This is the first demonstration of putative obestatin and C-ghrelin specific transcripts and these findings suggest that these ghrelin gene-derived peptides may also be produced independently of preproghrelin

  6. Recombinant adeno-associated virus-mediated delivery of antisense angiotensin Ⅱ receptor 1 gene attenuates hypertension development

    Institute of Scientific and Technical Information of China (English)

    Xu-guang LI; Jiang-tao YAN; Xi-zheng XU; Jia-ning WANG; Li-ming CHENG; Tao WANG; Ping ZUO; Dao-wen WANG

    2007-01-01

    Aim:The renin-angiotensin system plays a crucial role in the development and establishment of hypertension,and the pharmacological blockade of the system results in a reduction in blood pressure. In the present study,we investigated whether the effects of a novel,double-stranded,recombinant adeno-associated virus vector (rAAV)-mediated antisense angiotensin Ⅱ receptor l (AT1R) gene efficiently prevents the development of hypertension induced by a high-salt diet in adult,male Sprague-Dawley (SD) rats. Methods:A rAAV was prepared with a cassette containing a cytomegalovirus promoter and partial cDNA (660 base pairs) for the AT1R inserted in the antisense direction (rAAV-AT1AS). A single tail vein injection of the rAAV-AT1-AS or rAAV-GFP (green fluorescent protein,a reporter gene) was performed in adult,male SD rats. Two weeks after injection,the animals were fed a diet containing 8% NaCI,and the systolic blood pressure was measured weekly using the tail-cuff method for 12 weeks. Results:The high-salt diet induced a significant rise in systolic blood pressure in the rAAV-GFP-treated animals;however,the rAAV-AT:AS treatment attenuated the rise in blood pressure (142.7±4.5 mmHg vs 117±3.8 mmHg,P<0.01),and the hypotensive effect was maintained until the experiments ended at 12 weeks. In the rAAV-GFP-treated animals AT1 was overexpressed in various tissues,especially in the aorta and kidney at mRNA levels;in contrast,rAAV-AT:AS treatment markedly attenuated AT1 expression. Furthermore,rAAV-AT:AS treatment prevented target organ damages from hypertension,including cardiac dysfunction and renal injury compared to the rAAV-GFP group. Conclusion:These results suggest that rAAVmediated anti-AT1 delivery attenuates the development of hypertension and protects against renal injury and cardiac remodeling.

  7. Variation of DNA Fragmentation Levels During Density Gradient Sperm Selection for Assisted Reproduction Techniques: A Possible New Male Predictive Parameter of Pregnancy?

    Science.gov (United States)

    Muratori, Monica; Tarozzi, Nicoletta; Cambi, Marta; Boni, Luca; Iorio, Anna Lisa; Passaro, Claudia; Luppino, Benedetta; Nadalini, Marco; Marchiani, Sara; Tamburrino, Lara; Forti, Gianni; Maggi, Mario; Baldi, Elisabetta; Borini, Andrea

    2016-05-01

    Predicting the outcome of in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) is one main goal of the present research on assisted reproduction. To understand whether density gradient centrifugation (DGC), used to select sperm, can affect sperm DNA integrity and impact pregnancy rate (PR), we prospectively evaluated sperm DNA fragmentation (sDF) by TUNEL/PI, before and after DGC. sDF was studied in a cohort of 90 infertile couples the same day of IVF/ICSI treatment. After DGC, sDF increased in 41 samples (Group A, median sDF value: 29.25% [interquartile range, IQR: 16.01-41.63] in pre- and 60.40% [IQR: 32.92-93.53] in post-DGC) and decreased in 49 (Group B, median sDF value: 18.84% [IQR: 13.70-35.47] in pre- and 8.98% [IQR: 6.24-15.58] in post-DGC). PR was 17.1% and 34.4% in Group A and B, respectively (odds ratio [OR]: 2.58, 95% confidence interval [CI]: 0.95-7.04, P = 0.056). After adjustment for female factor, female and male age and female BMI, the estimated OR increased to 3.12 (95% CI: 1.05-9.27, P = 0.041). According to the subgroup analysis for presence/absence of female factor, heterogeneity in the association between the Group A and B and PR emerged (OR: 4.22, 95% CI: 1.16-15.30 and OR: 1.53, 95% CI: 0.23-10.40, respectively, for couples without, n = 59, and with, n = 31, female factor).This study provides the first evidence that the DGC procedure produces an increase in sDF in about half of the subjects undergoing IVF/ICSI, who then show a much lower probability of pregnancy, raising concerns about the safety of this selection procedure. Evaluation of sDF before and after DGC configures as a possible new prognostic parameter of pregnancy outcome in IVF/ICSI. Alternative sperm selection strategies are recommended for those subjects who undergo the damage after DGC.

  8. Protein expression and methylation of MGMT, a DNA repair gene and their correlation with clinicopathological parameters in invasive ductal carcinoma of the breast.

    Science.gov (United States)

    Asiaf, Asia; Ahmad, Shiekh Tanveer; Malik, Ajaz Ahmad; Aziz, Shiekh Aejaz; Rasool, Zubaida; Masood, Akbar; Zargar, Mohammad Afzal

    2015-08-01

    Epigenetic mechanisms such as DNA methylation are being increasingly recognized to play an important role in cancer and may serve as a cancer biomarker. The aim of this study was to evaluate the promoter methylation status of MGMT (O6-methylguanine-DNA methyltransferase) and a possible correlation with the expression of MGMT and standard clinicopathological parameters in invasive ductal breast carcinoma patients (IDC) of Kashmir. Methylation-specific PCR was carried out to investigate the promoter methylation status of MGMT in breast tumors paired with the corresponding normal tissue samples from 128 breast cancer patients. The effect of promoter methylation on protein expression in the primary breast cancer and adjacent normal tissues was evaluated by immunohistochemistry (n = 128) and western blotting (n = 30). The frequency of tumor hypermethylation was 39.8 % and a significant difference in methylation frequency among breast tumors were found (p MGMT in 68/128 (53.1 %) tumors. MGMT promoter methylation mediated gene silencing was associated with loss of its protein expression (rs = -0.285, p = 0.001, OR = 3.38, 95 % CI = 1.59-7.17). A significant correlation was seen between loss of MGMT and lymph node involvement (p = 0.030), tumor grade (p MGMT methylation was found to be associated with tumor grade (p = 0.011), tumor stage (p = 0.009), and loss of ER (p = 0.003) and PR receptors (p = 0.009). To our knowledge, our findings, for the first time, in Kashmiri population, indicate that MGMT is aberrantly methylated in breast cancer and promoter hypermethylation could be attributed to silencing of MGMT gene expression in breast cancer. Our data suggests that MGMT promoter hypermethylation could have a potential function as molecular biomarker of breast oncogenesis. Also, based on their predictive value of response to therapy, the immunohistochemical evaluation and interpretation of MGMT may also help in future to

  9. 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

    AND AIM: Transitional cell carcinoma (TCC) of the bladder represents the fifth most prevalent malignancy in Western population, with peak incidence found in males of the 50-to 70- year-old age group. A major problem in the management of bladder cancer is the low sensitivity of a large proportion (approximately 40%) among bladder tumors to chemotherapy and the high risk for recurrence of bladder tumors after transurethral resection. So drug resistance, especially in its multiple type forms, remains a major and difficult problem to resolve in bladder cancer therapy. This phenomenon has often been ascribed to strictly pharmacolo-gic factors, such as the overexpression of multidrug transporters P-glycoprotein, multidrug resistance related protein (MRP), and other variables closely implicated DNA repair and induction/modulation of apoptosis, such as P53 and the Bcl-protein family. Furthermore, it has been recently shown that certain growth factors(IGFs etc) may be involved in the mechanism of drug resistance. Clearly, these findings suggest the design of new strategies that might improve bladder tumor response to chemotherapy. Results have previously shown that human bladder tumor cell lines may be adapted to grow in the complete absence of serum or any other growth supplement and that this can be explained on the basis of autocrine stimulation. The acquirement of autonomous growth capacity was likely to be an important element in the oncogenesis of bladder tumors. Furthermore, criss-cross experiments showed that supernatants stimulated not only proliferation of the autologous cell line of bladder cancer, but also growth of the other bladder cancer cell lines, suggesting the production of common autocrine factors in bladder tumor cells. Some factors or their receptors involved in autocrine loop mechanism of bladder tumor cells have been confirmed, such as IL-6, the epidermal growth factor receptor, IFN-beta, transferrins-like substance etc. But certain factors which may

  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. Inhibitory effects of PIN1 antisense gene on the proliferation of human osteosarcoma cells

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Objective: To evaluate the inhibitory effects of PIN1 antisense gene on the proliferation of human osteosarcoma cells. Methods: Different doses of antisense PIN1 gene (0,20,50,100,200,250μl) were transfected into osteosarcoma MG-63 cells. The cells and the culture supernatants before and after transfection were collected. The cell growth curve was made using MTT method. The cell growth cycle and apoptosis were detected by FCM. The expression of PIN1 was detected by Western blot. The expression of PIN1 mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR). Results: MTT and FCM assays indicated that the transfection of antisense PIN1 gene could inhibit proliferation of MG-63 cells and lead to cell apoptosis. Western-blot assays revealed the MG-63 cells transfected with antisense PIN1 gene had weaker expression than those without transfection with antisense PIN1 gene, and the band intensity was negatively related with doses. The cells transfected with different doses of gene (0,20,50,100,200,250 μl) 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 ), with significant difference assessed by F and q test ( P < 0.05). The absorbance rate of PINI mRNA 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 PINlmRNA in MG-63 cells could be inhibited by antisense PIN1 gene, and then the expression of PIN1 was reduced and depressed, and so the proliferation of human osteosarcoma cells MG-63 was inhibited.

  12. 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.

  13. 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.

  14. 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.

  15. The Cellular Processing Capacity Limits the Amounts of Chimeric U7 snRNA Available for Antisense Delivery

    OpenAIRE

    2012-01-01

    Many genetic diseases are induced by mutations disturbing the maturation of pre-mRNAs, often affecting splicing. Antisense oligoribonucleotides (AONs) have been used to modulate splicing thereby circumventing the deleterious effects of mutations. Stable delivery of antisense sequences is achieved by linking them to small nuclear RNA (snRNAs) delivered by viral vectors, as illustrated by studies where therapeutic exon skipping was obtained in animal models of Duchenne muscular dystrophy (DMD)....

  16. Expression of Vascular Endothelial Growth Factor (VEGF) in Human Osteosarcoma Cells Transfected with Adeno-associated Virus-antisense VEGF

    Institute of Scientific and Technical Information of China (English)

    徐卫国; 陈安民; 张衣北; 易成腊

    2004-01-01

    Summary: The expression of protein vascular endothelial growth factor (VEGF) in osteosarcoma cells transfected with adeno-associated virus (rAAV)-antisense VEGF was studied to provide the foundation of osteosarcoma treatment through antivascularization. The rAAV-antisense VEGF at different doses (0, 20, 50, 100, 200, 240 μl) was transfected into osteosarcoma MG-63 cell. The cells and culture supernatants were collected before and after tansfection. The expression of VEGF protein was detected by using immunohistochemical staining (SP) and Western blot. SP and Western-blot tests revealed that the MG-63 Cells transfected with rAAV-antisense VEGF had less staining than those without transfection with rAAV-antisense VEGF, and the staining intensity was negatively correlated with the doses of genes. The corresponding A values of transfected genes with different doses of rAAV-antisense VEGF (0, 20, 50, 100, 200, 240 μA) were 86 614±13 776, 73 245±15 414, 61 078±12 124, 54 657±10 953, 39 802±11 308, 32 014±15 057 respectively,w ith the difference being significant (P<0.05). It was concluded that the expression of VEGF protein in MG-63 cells could be inhibited by rAAV-antisense VEGF.

  17. Cross-protective effect of antisense oligonucleotide developed against the common 3' NCR of influenza A virus genome.

    Science.gov (United States)

    Kumar, Prashant; Kumar, Binod; Rajput, Roopali; Saxena, Latika; Banerjea, Akhil C; Khanna, Madhu

    2013-11-01

    The influenza A virus (IAV) has eight segmented single-stranded RNA genome containing a common and evolutionarily conserved non-coding region (NCRs) at 5' and 3' ends that are important for the virus replication. In this study, we designed an antisense oligonucleotide against the 3' NCR of vital segments of the IAV genome to inhibit its replication. The results demonstrated that the co-transfection of Madine Darby Canine Kidney (MDCK) cells with the antisense oligonucleotide and the plasmids encoding the viral genes led to the down-regulation of the viral gene expression. The designed antisense molecules reduced the cytopathic effect caused by A/PR/8/34 (H1N1), A/Udorn/307/72 (H3N2), and A/New Caledonia/20/99 (H1N1) strains of IAV for almost 48 h. Furthermore, the intra-venous delivery of this oligonucleotide significantly reduced the viral titers in the lungs of infected mice and protected the mice from lethal effects of all the strains of influenza virus. The study demonstrated that the antisense oligonucleotide designed against the NCR region inhibits the expression of the viral genome. The decrease of the cytopathic effect in the MDCK cells and increase in survival of mice confirmed the reduction of virus multiplication and pathogenesis in the presence of antisense oligonucleotide. Thus, we demonstrate that a single antisense oligonucleotide is capable of providing protection against more than one strains of the IAV.

  18. Regulation of chromatin structure by long noncoding RNAs: focus on natural antisense transcripts.

    Science.gov (United States)

    Magistri, Marco; Faghihi, Mohammad Ali; St Laurent, Georges; Wahlestedt, Claes

    2012-08-01

    In the decade following the publication of the Human Genome, noncoding RNAs (ncRNAs) have reshaped our understanding of the broad landscape of genome regulation. During this period, natural antisense transcripts (NATs), which are transcribed from the opposite strand of either protein or non-protein coding genes, have vaulted to prominence. Recent findings have shown that NATs can exert their regulatory functions by acting as epigenetic regulators of gene expression and chromatin remodeling. Here, we review recent work on the mechanisms of epigenetic modifications by NATs and their emerging role as master regulators of chromatin states. Unlike other long ncRNAs, antisense RNAs usually regulate their counterpart sense mRNA in cis by bridging epigenetic effectors and regulatory complexes at specific genomic loci. Understanding the broad range of effects of NATs will shed light on the complex mechanisms that regulate chromatin remodeling and gene expression in development and disease.

  19. Chemical Modifications of Antisense Morpholino Oligomers Enhance Their Efficacy against Ebola Virus Infection

    Science.gov (United States)

    2009-05-01

    specific PMOs in infected cells and mice during lethal Ebola virus challenge. Members of the Filoviridae family of viruses , Ebola virus (EBOV) and Marburg ...American Society for Microbiology. All Rights Reserved. Chemical Modifications of Antisense Morpholino Oligomers Enhance Their Efficacy against Ebola Virus ...sequence is complementary to a region spanning the start codon of VP24 mRNA were protected against lethal Ebola virus challenge. In the present study, we

  20. Propionic and Methylmalonic Acidemia: Antisense Therapeutics for Intronic Variations Causing Aberrantly Spliced Messenger RNA

    OpenAIRE

    Rincón, A. ; Aguado, C. ; Desviat, L. R. ; Sánchez-Alcudia, R. ; Ugarte, M. ; Pérez, B. 

    2007-01-01

    We describe the use of antisense morpholino oligonucleotides (AMOs) to restore normal splicing caused by intronic molecular defects identified in methylmalonic acidemia (MMA) and propionic acidemia (PA). The three new point mutations described in deep intronic regions increase the splicing scores of pseudoexons or generate consensus binding motifs for splicing factors, such as SRp40, which favor the intronic inclusions in MUT (r.1957ins76), PCCA (r.1284ins84), or PCCB (r.654ins72) messenger R...

  1. Effects of multidrug resistance, antisense RNA on the chemosensitivity of hepatocellular carcinoma cells

    Institute of Scientific and Technical Information of China (English)

    Bo Li; Jian-Ping Gong; Tian Ye; Lei Zhao; De-Hua Li; Xing-Hua Gou; Lan-Ying Zhao; Lei Han; Lin Chen; Lu-Nan Yan

    2006-01-01

    BACKGROUND: Multidrug resistance is a major obstacle in cancer chemotherapy. We examined whether the antisense RNA of multidrug resistance gene 1 (mdr1) could reverse multidrug resistance in the human hepatocellular carcinoma (HCC) cell line SMMC7721/ADM. METHODS: The recombinant adenoviruses pAdEasy-GFP-ASmdr1 product was produced by the adenoviral vector AdEasy system, which can express antisense RNA against the mdr1 gene. Following that, the recombinant adenovirus was transfected into the P-glycoprotein-producing multidrug resistance cell line, SMMC7721/ADM human HCC cells resistant to adriamycin (ADM) and daunorubicin (DNR). In order to investigate the reversal of multidrug resistance phenotype, we measured the expression of mdr1 mRNA by RT-PCR and the production of P-glycoprotein by lfow cytometry. The sensitivities for ADM and DNR SMMC7721/ADM cells were examined by [3-(4, 5-dimethylthi-azol-2-yl)-2,5 diphenyl-terazolium bromide] (MTT) analysis. RESULTS: The low-level expression of mdr1 mRNA and P-glycoprotein production were observed in parental sensitive cells SMMC/7721 in addition to the overexpression of mdr1 mRNA and P-glycoprotein in SMMC7721/ADM cells. The transfection of antisense-RNA into SMMC7721/ADM cells resulted in decreases of mdr1 mRNA and P-glycoprotein, but increase of drug sensitivities. The sensitivities of transfected SMMC7721/ADM cells to ADM and DNR in IC50 reduced by 31.25% and 62.96%respectively. CONCLUSIONS: Mdr1 antisense RNA can increase the sensitivities of SMMC7721/ADM cells to anticancer drug by decreasing the expression of the mdr1 gene and inhibiting P-glycoprotein expression. This strategy may be applicable to cancer patients with P-glycoportein mediated multidrug resistance.

  2. 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.

  3. 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.

  4. New methods as alternative or corrective measures for the pitfalls and artifacts of reverse transcription and polymerase chain reactions (RT-PCR) in cloning chimeric or antisense-accompanied RNA.

    Science.gov (United States)

    Yuan, Chengfu; Liu, Yongming; Yang, Min; Liao, D Joshua

    2013-06-01

    We established new methods for cloning cDNA ends that start with reverse transcription (RT) and soon proceed with the synthesis of the second cDNA strand, avoiding manipulations of fragile RNA. Our 3'-end cloning method does not involve poly-dT primers and polymerase chain reactions (PCR), is low in efficiency but high in fidelity and can clone those RNAs without a poly-A tail. We also established a cDNA protection assay to supersede RNA protection assay. The protected cDNA can be amplified, cloned and sequenced, enhancing sensitivity and fidelity. We report that RT product using gene-specific primer (GSP) cannot be gene- or strand-specific because RNA sample contains endogenous random primers (ERP). The gene-specificity may be improved by adding a linker sequence at the 5'-end of the GSP to prime RT and using the linker as a primer in the ensuing PCR. The strand-specificity may be improved by using strand-specific DNA oligos in our protection assay. The CDK4 mRNA and TSPAN31 mRNA are transcribed from the opposite DNA strands and overlap at their 3' ends. Using this relationship as a model, we found that the overlapped sequence might serve as a primer with its antisense as the template to create a wrong-template extension in RT or PCR. We infer that two unrelated RNAs or cDNAs overlapping at the 5'- or 3'-end might create a spurious chimera in this way, and many chimeras with a homologous sequence may be such artifacts. The ERP and overlapping antisense together set complex pitfalls, which one should be aware of.

  5. Presymptomatic Treatment with Acetylcholinesterase Antisense Oligonucleotides Prolongs Survival in ALS (G93A-SOD1 Mice

    Directory of Open Access Journals (Sweden)

    Gotkine Marc

    2013-01-01

    Full Text Available Objective. Previous research suggests that acetylcholinesterase (AChE may be involved in ALS pathogenesis. AChE enzyme inhibitors can upregulate AChE transcription which in certain contexts can have deleterious (noncatalytic effects, making them theoretically harmful in ALS, whilst AChE antisense-oligonucleotides (mEN101, which downregulate AChE may be beneficial. Our aim was to investigate whether downregulation of AChE using mEN101 is beneficial in an ALS mouse model. Methods. ALS (G93A-SOD1 mice received saline, mEN101, inverse-EN101, or neostigmine. Treatments were administered from 5 weeks. Disease-onset and survival were recorded. Additional mice were sacrificed for pathological analysis at 15 weeks of age. In a follow-up experiment treatment was started at the symptomatic stage at a higher dose. Results. mEN101 given at the presymptomatic (but not symptomatic stage prolonged survival and attenuated motor-neuron loss in ALS mice. In contrast, neostigmine exacerbated the clinical parameters. Conclusions. These results suggest that AChE may be involved in ALS pathogenesis. The accelerated disease course with neostigmine suggests that any beneficial effects of mEN101 occur through a non-catalytic rather than cholinergic mechanism.

  6. 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.

  7. 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.

  8. 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

    2012-09-07

    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.

  9. Antisense oligonucleotide therapy for the treatment of C9ORF72 ALS/FTD diseases.

    Science.gov (United States)

    Riboldi, Giulietta; Zanetta, Chiara; Ranieri, Michela; Nizzardo, Monica; Simone, Chiara; Magri, Francesca; Bresolin, Nereo; Comi, Giacomo P; Corti, Stefania

    2014-12-01

    Motor neuron disorders, and particularly amyotrophic lateral sclerosis (ALS), are fatal diseases that are due to the loss of motor neurons in the brain and spinal cord, with progressive paralysis and premature death. It has been recently shown that the most frequent genetic cause of ALS, frontotemporal dementia (FTD), and other neurological diseases is the expansion of a hexanucleotide repeat (GGGGCC) in the non-coding region of the C9ORF72 gene. The pathogenic mechanisms that produce cell death in the presence of this expansion are still unclear. One of the most likely hypotheses seems to be the gain-of-function that is achieved through the production of toxic RNA (able to sequester RNA-binding protein) and/or toxic proteins. In recent works, different authors have reported that antisense oligonucleotides complementary to the C9ORF72 RNA transcript sequence were able to significantly reduce RNA foci generated by the expanded RNA, in affected cells. Here, we summarize the recent findings that support the idea that the buildup of "toxic" RNA containing the GGGGCC repeat contributes to the death of motor neurons in ALS and also suggest that the use of antisense oligonucleotides targeting this transcript is a promising strategy for treating ALS/frontotemporal lobe dementia (FTLD) patients with the C9ORF72 repeat expansion. These data are particularly important, given the state of the art antisense technology, and they allow researchers to believe that a clinical application of these discoveries will be possible soon.

  10. ASBEL, an ANA/BTG3 antisense transcript required for tumorigenicity of ovarian carcinoma.

    Science.gov (United States)

    Yanagida, Satoshi; Taniue, Kenzui; Sugimasa, Hironobu; Nasu, Emiko; Takeda, Yasuko; Kobayashi, Mana; Yamamoto, Tadashi; Okamoto, Aikou; Akiyama, Tetsu

    2013-01-01

    Mammalian genomes encode numerous antisense non-coding RNAs, which are assumed to be involved in the regulation of the sense gene expression. However, the mechanisms of their action and involvement in the development of diseases have not been well elucidated. The ANA/BTG3 protein is an antiproliferative protein whose expression is downregulated in prostate and lung cancers. Here we show that an antisense transcript of the ANA/BTG3 gene, termed ASBEL, negatively regulates the levels of ANA/BTG3 protein, but not of ANA/BTG3 mRNA and is required for proliferation and tumorigenicity of ovarian clear cell carcinoma. We further show that knockdown of ANA/BTG3 rescues growth inhibition caused by ASBEL knockdown. Moreover, we demonstrate that ASBEL forms duplexes with ANA/BTG3 mRNA in the nucleus and suppresses its cytoplasmic transportation. Our findings illustrate a novel function for an antisense transcript that critically promotes tumorigenesis by suppressing translation of the sense gene by inhibiting its cytoplasmic transportation.

  11. Regulation of apoptosis by fau revealed by functional expression cloning and antisense expression.

    Science.gov (United States)

    Mourtada-Maarabouni, Mirna; Kirkham, Lucy; Farzaneh, Farzin; Williams, Gwyn T

    2004-12-16

    Functional expression cloning is a powerful strategy for identifying critical steps in biological pathways independently of prior assumptions. It is particularly suitable for the identification of molecules crucial to the control of apoptosis. Our screen for sequences suppressing T-cell apoptosis isolated a sequence antisense to fau (Finkel-Biskis-Reilly murine sarcoma virus (FBR-MuSV)-associated ubiquitously expressed gene). The fox gene in FBR murine osteosarcoma virus is also antisense to fau and several reports have indicated that fau displays tumour suppressor and oncogenic properties in different contexts. Our observations indicate that the fau antisense sequence suppresses expression of endogenous fau mRNA and produces resistance to apoptosis induced both by the glucocorticoid analogue dexamethasone' by ultraviolet radiation, and by the anticancer drug cisplatin. In all cases, colony-forming ability is protected, indicating that fau affects the critical events prior to commitment to cell death. Overexpression of fau in the sense orientation induces cell death, which is inhibited both by Bcl-2 and by inhibition of caspases, in line with its proposed role in apoptosis.

  12. 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.

  13. Effect of antisense RNA targeting polo-like kinase 1 on cell cycle and proliferation in A549 cells

    Institute of Scientific and Technical Information of China (English)

    周琼; 白明; 苏远

    2004-01-01

    Background Expression of polo-like kinase 1 (Plk1) is elevated in lung cancer and has been proposed as having prognostic value and related to resistance to chemotherapy and radiation. In addition, Plk1 has several functions in mitotic progression. In this study, the authors investigated the effect of Plk1 depletion on cell cycle progression and proliferation in A549 cells, a lung cancer cell line.Methods A recombinant plasmid containing antisense RNA targeting Plk1 (pcDNA3-Plk1) was transfected into A549 cells. Reverse transcription-polymerase chain reaction and Western blot were used to examine Plk1 gene expression. Cell proliferation was evaluated by direct cell counting and bromodeoxyuridine (BrdU) labelling. Cell cycle and apoptosis were examined by flow cytometry. Expression of α-tubulin was detected by immunofluorescence, and the inhibition rate (IR) by chemotherapeutic agents was determined by 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl-tetrazolium bromide assay.Results After transfection into A549 cells, pcDNA3-Plk1 reduced Plk1 mRNA by 46.75% for 24 hours and by 61.84% for 48 hours. Plk1 protein was significantly decreased simultaneously (P<0.05). Abnormal morphological changes of cells and growth inhibition were observed in pcDNA3-Plk1 transfected groups. The BrdU labelling index was 25.59% 48 hours after transfection, which was significantly lower than that of the control groups (P<0.05). Forty-eight hours after transfection, there showed absence of microtubule polymerization and spindle abnormalities in staining for α-tubulin. A549 cells showed a strong G2/M arrest and apoptosis 72 hours post transfection. IR of vinorelbine in pcDNA3-Plk1 transfected groups was significantly higher than that of the other groups (P<0.05, respectively).Conclusions Plk1 depletion interferes with spindle formation, induces cell cycle arrest and apoptosis, and consequently inhibits cell proliferation in A549 cells. Moreover, it sensitizes lung cancer cells to chemotherapy.

  14. [Connection of magnetic antisense probe with SK-Br-3 oncocyte mRNA nucleotide detected by high resolution atomic force microscope].

    Science.gov (United States)

    Tan, Shude; Ouyang, Yu; Li, Xinyou; Wen, Ming; Li, Shaolin

    2011-06-01

    The present paper is aimed to detect superparamagnetic iron oxide labeled c-erbB2 oncogene antisense oligonucleotide probe (magnetic antisense probe) connected with SK-Br-3 oncocyte mRNA nucleotide by high resolution atomic force microscope (AFM). We transfected SK-Br-3 oncocyte with magnetic antisense probe, then observed the cells by AFM with high resolution and detected protein expression and magnetic resonance imagine (MRI). The high resolution AFM clearly showed the connection of the oligonucleotide remote end of magnetic antisense probe with the mRNA nucleotide of oncocyte. The expression of e-erbB2 protein in SK-Br3 cells were highly inhibited by using magnetic antisense probe. We then obtained the lowest signal to noise ratio (SNR) of SK-Br-3 oncocyte transfected with magnetic antisense probe by MRI (PSK-Br-3 mRNA of tumor cell nuclear.

  15. Electrophoretic mobility of semi-flexible double-stranded DNA in defect-controlled polymer networks: Mechanism investigation and role of structural parameters.

    Science.gov (United States)

    Khairulina, Kateryna; Li, Xiang; Nishi, Kengo; Shibayama, Mitsuhiro; Chung, Ung-il; Sakai, Takamasa

    2015-06-21

    Our previous studies have reported an empirical model, which explains the electrophoretic mobility (μ) of double-stranded DNA (dsDNA) as a combination of a basic migration term (Rouse-like or reptation) and entropy loss term in polymer gels with ideal network structure. However, this case is of exception, considering a large amount of heterogeneity in the conventional polymer gels. In this study, we systematically tune the heterogeneity in the polymer gels and study the migration of dsDNA in these gels. Our experimental data well agree with the model found for ideal networks. The basic migration mechanism (Rouse-like or reptation) persists perfectly in the conventional heterogeneous polymer gel system, while the entropy loss term continuously changes with increase in the heterogeneity. Furthermore, we found that in the limit where dsDNA is shorter than dsDNA persistence length, the entropy loss term may be related to the collisional motions between DNA fragments and the cross-links.

  16. 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

  17. Involvement of Bcl-2 and Bax in photodynamic therapy-mediated apoptosis. Antisense Bcl-2 oligonucleotide sensitizes RIF 1 cells to photodynamic therapy apoptosis.

    Science.gov (United States)

    Srivastava, M; Ahmad, N; Gupta, S; Mukhtar, H

    2001-05-04

    Photodynamic therapy (PDT), a promising treatment modality, is an oxidative stress that induces apoptosis in many cancer cells in vitro and tumors in vivo. Understanding the mechanism(s) involved in PDT-mediated apoptosis may improve its therapeutic efficacy. Although studies suggest the involvement of multiple pathways, the triggering event(s) responsible for PDT-mediated apoptotic response is(are) not clear. To investigate the role of Bcl-2 in PDT-mediated apoptosis, we employed Bcl-2-antisense and -overexpression approaches in two cell types differing in their responses toward PDT apoptosis. In the first approach, we treated radiation-induced fibrosarcoma (RIF 1) cells, which are resistant to silicon phthalocyanine (Pc 4)-PDT apoptosis, with Bcl-2-antisense oligonucleotide. This treatment resulted in sensitization of RIF 1 cells to PDT-mediated apoptosis as demonstrated by i) cleavage of poly(ADP-ribose) polymerase, ii) DNA ladder formation, iii) terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells, and iv) DEVDase activity. This treatment also resulted in oligonucleotide concentration-dependent decrease in cell viability and down-regulation of Bcl-2 protein with a concomitant increase in apoptosis. However, the level of Bax, a pro-apoptotic member of Bcl-2 family, remained unaltered. In the second approach, an overexpression of Bcl-2 in PDT apoptosis-sensitive human epidermoid carcinoma (A431) cells resulted in enhanced apoptosis and up-regulation of Bax following PDT. In both the approaches, the increased Bax/Bcl-2 ratio was associated with an increased apoptotic response of PDT. Our data also demonstrated that PDT results in modulation of other Bcl-2 family members in a way that the overall ratio of pro-apoptotic and anti-apoptotic member proteins favors apoptosis.

  18. 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.

  19. Antisense PMO found in dystrophic dog model was effective in cells from exon 7-deleted DMD patient.

    Directory of Open Access Journals (Sweden)

    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.

  20. Bacterial resistance to antisense peptide phosphorodiamidate morpholino oligomers.

    Science.gov (United States)

    Puckett, Susan E; Reese, Kaleb A; Mitev, Georgi M; Mullen, Valerie; Johnson, Rudd C; Pomraning, Kyle R; Mellbye, Brett L; Tilley, Lucas D; Iversen, Patrick L; Freitag, Michael; Geller, Bruce L

    2012-12-01

    Peptide phosphorodiamidate morpholino oligomers (PPMOs) are synthetic DNA mimics that bind cRNA and inhibit bacterial gene expression. The PPMO (RFF)(3)RXB-AcpP (where R is arginine, F, phenylalanine, X is 6-aminohexanoic acid, B is β-alanine, and AcpP is acyl carrier protein) is complementary to 11 bases of the essential gene acpP (which encodes acyl carrier protein). The MIC of (RFF)(3)RXB-AcpP was 2.5 μM (14 μg/ml) in Escherichia coli W3110. The rate of spontaneous resistance of E. coli to (RFF)(3)RXB-AcpP was 4 × 10(-7) mutations/cell division. A spontaneous (RFF)(3)RXB-AcpP-resistant mutant (PR200.1) was isolated. The MIC of (RFF)(3)RXB-AcpP was 40 μM (224 μg/ml) for PR200.1. The MICs of standard antibiotics for PR200.1 and W3110 were identical. The sequence of acpP was identical in PR200.1 and W3110. PR200.1 was also resistant to other PPMOs conjugated to (RFF)(3)RXB or peptides with a similar composition or pattern of cationic and nonpolar residues. Genomic sequencing of PR200.1 identified a mutation in sbmA, which encodes an active transport protein. In separate experiments, a (RFF)(3)RXB-AcpP-resistant isolate (RR3) was selected from a transposome library, and the insertion was mapped to sbmA. Genetic complementation of PR200.1 or RR3 with sbmA restored susceptibility to (RFF)(3)RXB-AcpP. Deletion of sbmA caused resistance to (RFF)(3)RXB-AcpP. We conclude that resistance to (RFF)(3)RXB-AcpP was linked to the peptide and not the phosphorodiamidate morpholino oligomer, dependent on the composition or repeating pattern of amino acids, and caused by mutations in sbmA. The data further suggest that (RFF)(3)R-XB PPMOs may be transported across the plasma membrane by SbmA.

  1. DNA damage response inhibition at dysfunctional telomeres by modulation of telomeric DNA damage response RNAs.

    Science.gov (United States)

    Rossiello, Francesca; Aguado, Julio; Sepe, Sara; Iannelli, Fabio; Nguyen, Quan; Pitchiaya, Sethuramasundaram; Carninci, Piero; d'Adda di Fagagna, Fabrizio

    2017-02-27

    The DNA damage response (DDR) is a set of cellular events that follows the generation of DNA damage. Recently, site-specific small non-coding RNAs, also termed DNA damage response RNAs (DDRNAs), have been shown to play a role in DDR signalling and DNA repair. Dysfunctional telomeres activate DDR in ageing, cancer and an increasing number of identified pathological conditions. Here we show that, in mammals, telomere dysfunction induces the transcription of telomeric DDRNAs (tDDRNAs) and their longer precursors from both DNA strands. DDR activation and maintenance at telomeres depend on the biogenesis and functions of tDDRNAs. Their functional inhibition by sequence-specific antisense oligonucleotides allows the unprecedented telomere-specific DDR inactivation in cultured cells and in vivo in mouse tissues. In summary, these results demonstrate that tDDRNAs are induced at dysfunctional telomeres and are necessary for DDR activation and they validate the viability of locus-specific DDR inhibition by targeting DDRNAs.

  2. The Cellular Processing Capacity Limits the Amounts of Chimeric U7 snRNA Available for Antisense Delivery.

    Science.gov (United States)

    Eckenfelder, Agathe; Tordo, Julie; Babbs, Arran; Davies, Kay E; Goyenvalle, Aurélie; Danos, Olivier

    2012-06-26

    Many genetic diseases are induced by mutations disturbing the maturation of pre-mRNAs, often affecting splicing. Antisense oligoribonucleotides (AONs) have been used to modulate splicing thereby circumventing the deleterious effects of mutations. Stable delivery of antisense sequences is achieved by linking them to small nuclear RNA (snRNAs) delivered by viral vectors, as illustrated by studies where therapeutic exon skipping was obtained in animal models of Duchenne muscular dystrophy (DMD). Yet, clinical translation of these approaches is limited by the amounts of vector to be administered. In this respect, maximizing the amount of snRNA antisense shuttle delivered by the vector is essential. Here, we have used a muscle- and heart-specific enhancer (MHCK) to drive the expression of U7 snRNA shuttles carrying antisense sequences against the human or murine DMD pre-mRNAs. Although antisense delivery and subsequent exon skipping were improved both in tissue culture and in vivo, we observed the formation of additional U7 snRNA by-products following gene transfer. These included aberrantly 3' processed as well as unprocessed species that may arise because of the saturation of the cellular processing capacity. Future efforts to increase the amounts of functional U7 shuttles delivered into a cell will have to take this limitation into account.

  3. Antisense bcl-2 retrovirus vector increases the sensitivity of a human gastric adenocarcinoma cell line to photodynamic therapy.

    Science.gov (United States)

    Zhang, W G; Ma, L P; Wang, S W; Zhang, Z Y; Cao, G D

    1999-05-01

    The bcl-2 oncoprotein directly prolongs cellular survival by blocking apoptosis and its overexpression is associated with cellular resistance to killing by chemotherapeutic drugs and gamma-irradiation. Meanwhile, it has been shown that bcl-2 antisense oligonucleotide can induce apoptosis or increase toxicity of the treatment in tumors in vivo and in vitro. However, it is difficult to obtain stable transfection by this approach and there are no reports about the effect of an antisense bcl-2 on the sensitivity to oxidative stress induced by photodynamic therapy (PDT). Here we investigated the effect of an antisense bcl-2 RNA retrovirus vector transfer on the sensitivity of 2-butylamino-2-demethoxy-hypocrellin A (2-BA-2-DMHA) photosensitization in a human gastric adenocarcinoma MGC803 cell line. The results indicate that antisense bcl-2-infected MGC803 cells expressed exogenous antisense bcl-2 mRNA measured by reverse transcription polymerase chain reaction and significantly reduced bcl-2 protein determined by western blotting analysis. The decreased expression of bcl-2 protein was accompanied by increased phototoxicity and susceptibility to apoptosis induced by 2-BA-2-DMHA PDT. Our finding suggests that reduction of bcl-2 protein in gastric cancers, and possibly also in a variety of other tumors, may be a novel and rational approach to improve photosensitivity and the treatment outcome.

  4. 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

  5. Antisense-mediated RNA targeting: versatile and expedient genetic manipulation in the brain

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    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.

  6. Sense and Antisense DMPK RNA Foci Accumulate in DM1 Tissues during Development.

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    Lise Michel

    Full Text Available Myotonic dystrophy type 1 (DM1 is caused by an unstable expanded CTG repeat located within the DMPK gene 3'UTR. The nature, severity and age at onset of DM1 symptoms are very variable in patients. Different forms of the disease are described, among which the congenital form (CDM is the most severe. Molecular mechanisms of DM1 are well characterized for the adult form and involve accumulation of mutant DMPK RNA forming foci in the nucleus. These RNA foci sequester proteins from the MBNL family and deregulate CELF proteins. These proteins are involved in many cellular mechanisms such as alternative splicing, transcriptional, translational and post-translational regulation miRNA regulation as well as mRNA polyadenylation and localization. All these mechanisms can be impaired in DM1 because of the deregulation of CELF and MBNL functions. The mechanisms involved in CDM are not clearly described. In order to get insight into the mechanisms underlying CDM, we investigated if expanded RNA nuclear foci, one of the molecular hallmarks of DM1, could be detected in human DM1 fetal tissues, as well as in embryonic and neonatal tissues from transgenic mice carrying the human DMPK gene with an expanded CTG repeat. We observed very abundant RNA foci formed by sense DMPK RNA and, to a lesser extent, antisense DMPK RNA foci. Sense DMPK RNA foci clearly co-localized with MBNL1 and MBNL2 proteins. In addition, we studied DMPK sense and antisense expression during development in the transgenic mice. We found that DMPK sense and antisense transcripts are expressed from embryonic and fetal stages in heart, muscle and brain and are regulated during development. These results suggest that mechanisms underlying DM1 and CDM involved common players including toxic expanded RNA forming numerous nuclear foci at early stages during development.

  7. Morpholino antisense oligonucleotides targeting intronic repressor Element1 improve phenotype in SMA mouse models

    OpenAIRE

    Osman, Erkan Y.; Miller, Madeline R.; Robbins, Kate L.; Lombardi, Abby M.; Atkinson, Arleigh K.; Brehm, Amanda J.; Lorson, Christian L.

    2014-01-01

    Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by the loss of Survival Motor Neuron-1 (SMN1). In all SMA patients, a nearly identical copy gene called SMN2 is present, which produces low levels of functional protein owing to an alternative splicing event. To prevent exon-skipping, we have targeted an intronic repressor, Element1 (E1), located upstream of SMN2 exon 7 using Morpholino-based antisense oligonucleotides (E1MO-ASOs). A single intracerebroventricular injection i...

  8. 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...... essential bacterial gene involved in fatty acid synthesis) of P. aeruginosa (PA01) and characterized these compounds according to their antimicrobial activity and mode of action. Four antisense PNA oligomers conjugated to the H-(R-Ahx-R)(4)-Ahx-ßala or the H-(R-Ahx)(6)-ßala peptide exhibited complete growth...

  9. 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

    Prolamins are the predominant storage proteins in barley and wheat grains, accounting for 50 to 80% of total seed protein. However, the prolamins are not optimal feed for monogastric animals as they have a low content of certain essential amino acids such as lysine, threonine and tryptophan...... 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...

  10. 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.

  11. 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

  12. Search for antisense copies of beta-globin mRNA in anemic mouse spleen

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    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.

  13. Suppression of Glioma-Cell Survival by Antisense and Dominant-Negative AKT2 RNA

    Institute of Scientific and Technical Information of China (English)

    Peiyu Pu; Chunsheng Kang; Jie Li; Guangxiu Wang

    2005-01-01

    OBJECTIVE Overexpression of growth factors and their receptors such as PDGF, FGF, VEGF, IGF, EGF, TGFα etc. Play a critical role in the development and progression of malignant gliomas. AKT, one of the most potent downstream signaling effectors of these growth factor receptors is usually overactivated in malignant gliomas. The present study was undertaken to investigate the effects of antisense and dominant negative AKT2 RNA on the survival of glioma cells with overexpression of AKT2.METHODS Antisense and dominant negative AKT2 constructs (AS-AKT2,DN-AKT2) were transfected into human glioblastoma cell line TJ905 with overexpression of AKT2. Using Western blotting, MTT assay, Ki67 labeling index (Ki67 LI), flow cytometry and the TUNEL method, the expression of AKT2 and GFAP, the proliferation rate and apoptosis of glioma cells transfected with AS-AKT2 or DN-AKT2 were compared to those characteristics of parental and glioma cells transfected with an empty vector.RESULTS Cell proliferation was inhibited in glioma cells transfected with ASAKT2 and DN-AKT2 RNA, while GFAP expression and apoptosis were markedly increased in those cells.CONCLUSION AKT is an important mediator in the growth signaling pathway of malignant gliomas and is a potential promising therapeutic target for malignant gliomas.

  14. 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.

  15. 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.

  16. Translation efficiency of mRNAs is increased by antisense oligonucleotides targeting upstream open reading frames.

    Science.gov (United States)

    Liang, Xue-Hai; Shen, Wen; Sun, Hong; Migawa, Michael T; Vickers, Timothy A; Crooke, Stanley T

    2016-08-01

    Increasing the levels of therapeutic proteins in vivo remains challenging. Antisense oligonucleotides (ASOs) are often used to downregulate gene expression or to modify RNA splicing, but antisense technology has not previously been used to directly increase the production of selected proteins. Here we used a class of modified ASOs that bind to mRNA sequences in upstream open reading frames (uORFs) to specifically increase the amounts of protein translated from a downstream primary ORF (pORF). Using ASO treatment, we increased the amount of proteins expressed from four genes by 30-150% in a dose-dependent manner in both human and mouse cells. Notably, systemic treatment of mice with ASO resulted in an ∼80% protein increase of LRPPRC. The ASO-mediated increase in protein expression was sequence-specific, occurred at the level of translation and was dependent on helicase activity. We also found that the type of RNA modification and the position of modified nucleotides in ASOs affected translation of a pORF. ASOs are a useful class of therapeutic agents with broad utility.

  17. 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.

  18. Antisense RNA regulation and application in the development of novel antibiotics to combat multidrug resistant bacteria.

    Science.gov (United States)

    Ji, Yinduo; Lei, Ting

    2013-01-01

    Despite the availability of antibiotics and vaccines, infectious diseases remain one of most dangerous threats to humans and animals. The overuse and misuse of antibacterial agents have led to the emergence of multidrug resistant bacterial pathogens. Bacterial cells are often resilient enough to survive in even the most extreme environments. To do so, the organisms have evolved different mechanisms, including a variety of two-component signal transduction systems, which allow the bacteria to sense the surrounding environment and regulate gene expression in order to adapt and respond to environmental stimuli. In addition, some bacteria evolve resistance to antibacterial agents while many bacterial cells are able to acquire resistance genes from other bacterial species to enable them to survive in the presence of toxic antimicrobial agents. The crisis of antimicrobial resistance is an unremitting menace to human health and a burden on public health. The rapid increase in antimicrobial resistant organisms and limited options for development of new classes of antibiotics heighten the urgent need to develop novel potent antibacterial therapeutics in order to combat multidrug resistant infections. In this review, we introduce the regulatory mechanisms of antisense RNA and significant applications of regulated antisense RNA interference technology in early drug discovery. This includes the identification and evaluation of drug targets in vitro and in vivo, the determination of mode of action for antibiotics and new antibacterial agents, as well as the development of peptide-nucleic acid conjugates as novel antibacterials.

  19. 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.

  20. RNA sequencing uncovers antisense RNAs and novel small RNAs in Streptococcus pyogenes.

    Science.gov (United States)

    Le Rhun, Anaïs; Beer, Yan Yan; Reimegård, Johan; Chylinski, Krzysztof; Charpentier, Emmanuelle

    2016-01-01

    Streptococcus pyogenes is a human pathogen responsible for a wide spectrum of diseases ranging from mild to life-threatening infections. During the infectious process, the temporal and spatial expression of pathogenicity factors is tightly controlled by a complex network of protein and RNA regulators acting in response to various environmental signals. Here, we focus on the class of small RNA regulators (sRNAs) and present the first complete analysis of sRNA sequencing data in S. pyogenes. In the SF370 clinical isolate (M1 serotype), we identified 197 and 428 putative regulatory RNAs by visual inspection and bioinformatics screening of the sequencing data, respectively. Only 35 from the 197 candidates identified by visual screening were assigned a predicted function (T-boxes, ribosomal protein leaders, characterized riboswitches or sRNAs), indicating how little is known about sRNA regulation in S. pyogenes. By comparing our list of predicted sRNAs with previous S. pyogenes sRNA screens using bioinformatics or microarrays, 92 novel sRNAs were revealed, including antisense RNAs that are for the first time shown to be expressed in this pathogen. We experimentally validated the expression of 30 novel sRNAs and antisense RNAs. We show that the expression profile of 9 sRNAs including 2 predicted regulatory elements is affected by the endoribonucleases RNase III and/or RNase Y, highlighting the critical role of these enzymes in sRNA regulation.

  1. NrrA directly regulates expression of the fraF gene and antisense RNAs for fraE in the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120.

    Science.gov (United States)

    Ehira, Shigeki; Ohmori, Masayuki

    2014-05-01

    The heterocystous cyanobacterium Anabaena sp. strain PCC 7120 grows as linear multicellular filaments that can contain hundreds of cells. Heterocysts, which are specialized cells for nitrogen fixation, are regularly intercalated among photosynthetic vegetative cells, and these cells are metabolically dependent on each other. Thus, multicellularity is essential for diazotrophic growth of heterocystous cyanobacteria. In Anabaena sp. strain PCC 7120, the fraF gene, which is required to limit filament length, is induced by nitrogen deprivation. The fraF transcripts extend to the fraE gene, which lies on the opposite DNA strand and could possess dual functionality, mRNAs for fraF and antisense RNAs for fraE. In the present study, we found that NrrA, a nitrogen-regulated response regulator, directly regulated expression of fraF. Induction of fraF by nitrogen deprivation was abolished by the nrrA disruption. NrrA specifically bound to the promoter region of fraF, and recognized an inverted repeat sequence. Thus, it is concluded that NrrA controls expression of mRNAs for fraF and antisense RNAs for fraE in response to nitrogen deprivation.

  2. Charge-Transfer Matrix Elements by FMO-LCMO Approach: Hole Transfer in DNA with Parameter Tuned Range-Separated DFT

    CERN Document Server

    Kitoh-Nishioka, Hirotaka

    2016-01-01

    A scheme for computing charge-transfer matrix elements with the linear combination of fragment molecular orbitals and the 'nonempirically tuned range-separated' density functional is presented. It takes account of the self-consistent orbital relaxation induced by environmental Coulomb field and the exchange interaction in fragment pairs at low computational scaling along the system size. The accuracy was confirmed numerically on benchmark systems of imidazole and furane homo-dimer cations. Applications to hole transfers in DNA nucleobase pairs and in a $\\pi$-stack adenine octomer highlight the effects of orbital relaxation.

  3. The Cloning of the Human Tumor Supressor Gene INGI: DNA Cloning into Plasmid Vector and DNA Analysis by Restriction Enzymes

    Directory of Open Access Journals (Sweden)

    Elza Ibrahim Auerkari

    2015-11-01

    Full Text Available DNA cloning is one of the most important techniques In the field of molecular biology, with a critical role in analyzing the structure and function of genes and their adjacent regulatory regions. DNA cloning is helpful in learning fundamental molecular biological techniques, since DNA cloning involves a series of them, such as polymerase chain reaction (PCR, DNA ligation, bacterial transformation, bacterial culture, plasmid DNA extraction, DNA digestion with restriction enzymes and agarose gel electrophoresis. In this paper the cloning of the human tumor suppressor gene INGI has been used to illustrate the methodology. The gene was amplified by PCR, cloned into a TA-cloning vectore, and restriction enzyme mapping was used to distinguish the sense INGI construct from the antisense INGI construct.

  4. Protective effects of curcumin against oxidative stress parameters and DNA damage in the livers and kidneys of rats with biliary obstruction.

    Science.gov (United States)

    Tokaç, Mehmet; Taner, Gökçe; Aydın, Sevtap; Ozkardeş, Alper Bilal; Dündar, Halit Ziya; Taşlıpınar, Mine Yavuz; Arıkök, Ata Türker; Kılıç, Mehmet; Başaran, Arif Ahmet; Basaran, Nursen

    2013-11-01

    Curcumin, a most active antioxidant compound, has been suggested to have potential beneficial effects against most metabolic and psychological disorders, including cholestasis. In the present study, the effects of curcumin against oxidative stress and DNA damage induced by bile duct ligation (BDL) in Wistar albino rats for 14 days were investigated. The rats were divided into three following groups: Sham group, the BDL group and the BDL+curcumin group. A daily dose of 50mg/kg curcumin was given to the BDL+curcumin group intragastrically for 14 days. The biomarkers of hepatocellular damage were decreased in the BDL+curcumin group compared to the BDL group, indicating that curcumin recovered the liver functions. DNA damage as assessed by the alkaline comet assay was also found to be low in the BDL+curcumin group. Curcumin significantly reduced malondialdehyde and nitric oxide levels, and enchanced reduced glutathione levels and catalase, superoxide dismutase, and glutathione S-transferase enzymes activities in the livers and kidneys of BDL group. Curcumin treatment in BDL group was found to decrease tumor necrosis factor-alpha levels in the livers of rats. These results suggest that curcumin might have protective effects on the cholestasis-induced injuries in the liver and kidney tissues of rats.

  5. Aberrant expression of proteins involved in signal transduction and DNA repair pathways in lung cancer and their association with clinical parameters.

    Directory of Open Access Journals (Sweden)

    Yong He

    Full Text Available BACKGROUND: Because cell signaling and cell metabolic pathways are executed through proteins, protein signatures in primary tumors are useful for identifying key nodes in signaling networks whose alteration is associated with malignancy and/or clinical outcomes. This study aimed to determine protein signatures in primary lung cancer tissues. METHODOLOGY/ PRINCIPAL FINDINGS: We analyzed 126 proteins and/or protein phosphorylation sites in case-matched normal and tumor samples from 101 lung cancer patients with reverse-phase protein array (RPPA assay. The results showed that 18 molecules were significantly different (p<0.05 by at least 30% between normal and tumor tissues. Most of those molecules play roles in cell proliferation, DNA repair, signal transduction and lipid metabolism, or function as cell surface/matrix proteins. We also validated RPPA results by Western blot and/or immunohistochemical analyses for some of those molecules. Statistical analyses showed that Ku80 levels were significantly higher in tumors of nonsmokers than in those of smokers. Cyclin B1 levels were significantly overexpressed in poorly differentiated tumors while Cox2 levels were significantly overexpressed in neuroendocrinal tumors. A high level of Stat5 is associated with favorable survival outcome for patients treated with surgery. CONCLUSIONS/ SIGNIFICANCE: Our results revealed that some molecules involved in DNA damage/repair, signal transductions, lipid metabolism, and cell proliferation were drastically aberrant in lung cancer tissues, and Stat5 may serve a molecular marker for prognosis of lung cancers.

  6. Ancient DNA

    DEFF Research Database (Denmark)

    Willerslev, Eske; Cooper, Alan

    2004-01-01

    ancient DNA, palaeontology, palaeoecology, archaeology, population genetics, DNA damage and repair......ancient DNA, palaeontology, palaeoecology, archaeology, population genetics, DNA damage and repair...

  7. 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.

  8. 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

    oligomer. This modification of the PNA does not interfere with the nucleic acid target binding affinity based on thermal stability of the PNA/RNA duplexes. When delivered to cultured HeLa pLuc705 cells by Lipofectamine, the PNAs showed dose-dependent nuclear antisense activity in the nanomolar range...

  9. Loop structures in the 5' untranslated region and antisense RNA mediate pilE gene expression in Neisseria gonorrhoeae.

    Science.gov (United States)

    Masters, Thao L; Wachter, Jenny; Hill, Stuart A

    2016-11-01

    Regulation of the Neisseria gonorrhoeae pilE gene is ill-defined. In this study, post-transcriptional effects on expression were assessed. In silico analysis predicts the formation of three putative stable stem-loop structures with favourable free energies within the 5' untranslated region of the pilE message. Using quantitative reverse transcriptase PCR analyses, we show that each loop structure forms, with introduced destabilizing stem-loop mutations diminishing loop stability. Utilizing a series of pilE translational fusions, deletion of either loop 1 or loop 2 caused a significant reduction of pilE mRNA resulting in reduced expression of the reporter gene. Consequently, the formation of the loops apparently protects the pilE transcript from degradation. Putative loop 3 contains the pilE ribosomal binding site. Consequently, its formation may influence translation. Analysis of a small RNA transcriptome revealed an antisense RNA being produced upstream of the pilE promoter that is predicted to hybridize across the 5' untranslated region loops. Insertional mutants were created where the antisense RNA is not transcribed. In these mutants, pilE transcript levels are greatly diminished, with any residual message apparently not being translated. Complementation of these insertion mutants in trans with the antisense RNA gene facilitates pilE translation yielding a pilus + phenotype. Overall, this study demonstrates a complex relationship between loop-dependent transcript protection and antisense RNA in modulating pilE expression levels.

  10. 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.

  11. 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...

  12. Regulation of Nav1.7 : A Conserved SCN9A Natural Antisense Transcript Expressed in Dorsal Root Ganglia

    NARCIS (Netherlands)

    Koenig, Jennifer; Werdehausen, Robert; Linley, John E; Habib, Abdella M; Vernon, Jeffrey; Lolignier, Stephane; Eijkelkamp, Niels; Zhao, Jing; Okorokov, Andrei L; Woods, C Geoffrey; Wood, John N; Cox, James J

    2015-01-01

    The Nav1.7 voltage-gated sodium channel, encoded by SCN9A, is critical for human pain perception yet the transcriptional and post-transcriptional mechanisms that regulate this gene are still incompletely understood. Here, we describe a novel natural antisense transcript (NAT) for SCN9A that is conse

  13. 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.

  14. The Effect of Telomerase Antisense RNA on Telomerase Activity and Cell Proliferation of HL-60%端粒酶反义RNA 对HL-60细胞端粒酶活性及细胞增殖的影响

    Institute of Scientific and Technical Information of China (English)

    刘利; 孙秉中; 梁英民; 张传山; 张惠中; 阎严; 张方信

    2001-01-01

    Objective To study the effect of telomerase antisense RNA ontelomerase activity and cell proliferation of HL-60 cells.Methods By using lipofectin mediated DNA transfection,telomerase antisense RNA was successfully introduced into HL-60 cells.By means of TRAP、dynamics of cell growth、flucytometry the changes of HL-60 cells in aspect of telomerase activity,proliferation were detected.Results Telomerase antisense RNA can significantly inhibit the telomerase activity and the proliferation of HL-60 cells and induce the apoptosis of HL-60 cells.Conclusion It seems to be a new methods to treat leukemia by using telomerase antisense to inhibit telomerase activity and the proliferation of HL-60 cells.telomerase is a new latent target to treat leukemia.%目的 探讨端粒酶反义RNA对HL-60细胞端粒酶活性及细胞增殖的影响。方法 用脂质体介导法将pBBS212-hTR(反义端粒酶RNA真核表达载体)导入人髓性白血病细胞系HL-60中,采用TRAP法测定端粒酶活性,细胞生长动力学检测,FCM分析等方法观察其对HL-60细胞的端粒酶活性及细胞增殖影响。结果 端粒酶反义RNA能显著抑制HL-60细胞的端粒酶活性,抑制HL-60细胞的增殖并诱导其凋亡。结论 以端粒酶反义RNA抑制端粒酶活性是治疗白血病的潜在途径。

  15. 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.

  16. 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.

  17. Lipid-based delivery of combinations of antisense oligodeoxynucleotides for the in vitro inhibition of HIV-1 replication.

    Science.gov (United States)

    Lavigne, C; Yelle, J; Sauvé, G; Thierry, A G

    2001-01-01

    We evaluated a new approach to AIDS therapy by using combinations of oligodeoxynucleotides (ODNs), delivered with a lipid-based carrier system, that target different HIV viral genome sites. We identified some of the factors that seem to influence the effectiveness of a combination strategy in cell cultures including ODN concentrations, type of infection (acute vs chronic), backbone modification of the ODN, and the number of sequences. When delivered by the DLS carrier system, some advantages of using a combination of ODNs over treatment with only one ODN could be observed in acute infection assays but not in the chronic infection model. These results suggest that in the acute infection model, the 3 different antisense ODNs in the "cocktail" might block an early step of virus replication by combined inhibitory effects. Various combinations of phosphorothioate-modified (PS) and unmodified oligonucleotides delivered by the DLS system were compared for their antiviral activity in a long-term acute assay using HIV-1 (IIIB strain)-infected MOLT-3 cells. The most effective combination had 3 phosphorothioate antisense ODNs: Srev, SDIS, and SPac (>99% inhibition at 100 pM). However, the additive effect determined when using ODN combinations was rather low, revealing the high level of nonsequence specificity in HIV-1 cell culture models. Data illustrated the high sequence nonspecific activity of ODNs, especially when comparing activity of antisense ODNs with activity of random control sequence ODNs. The latter exhibited an inhibitory effect similar to that of antisense ODNs under our experimental conditions. Nevertheless, we demonstrated that it is possible to achieve high anti-HIV activity by using, in combination, picomolar range concentrations of antisense oligonucleotides complexed to a lipid-based carrier system such as the DLS system, without increasing cell toxicity.

  18. Antisense RNA modulation of alkyl hydroperoxide reductase levels in Helicobacter pylori correlates with organic peroxide toxicity but not infectivity.

    Science.gov (United States)

    Croxen, Matthew A; Ernst, Peter B; Hoffman, Paul S

    2007-05-01

    Much of the gene content of the human gastric pathogen Helicobacter pylori ( approximately 1.7-Mb genome) is considered essential. This view is based on the completeness of metabolic pathways, infrequency of nutritional auxotrophies, and paucity of pathway redundancies typically found in bacteria with larger genomes. Thus, genetic analysis of gene function is often hampered by lethality. In the absence of controllable promoters, often used to titrate gene function, we investigated the feasibility of an antisense RNA interference strategy. To test the antisense approach, we targeted alkyl hydroperoxide reductase (AhpC), one of the most abundant proteins expressed by H. pylori and one whose function is essential for both in vitro growth and gastric colonization. Here, we show that antisense ahpC (as-ahpC) RNA expression from shuttle vector pDH37::as-ahpC achieved an approximately 72% knockdown of AhpC protein levels, which correlated with increased susceptibilities to hydrogen peroxide, cumene, and tert-butyl hydroperoxides but not with growth efficiency. Compensatory increases in catalase levels were not observed in the knockdowns. Expression of single-copy antisense constructs (expressed under the urease promoter and containing an fd phage terminator) from the rdxA locus of mouse-colonizing strain X47 achieved a 32% knockdown of AhpC protein levels (relative to wild-type X47 levels), which correlated with increased susceptibility to organic peroxides but not with mouse colonization efficiency. Our studies indicate that high levels of AhpC are not required for in vitro growth or for primary gastric colonization. Perhaps AhpC, like catalase, assumes a greater role in combating exogenous peroxides arising from lifelong chronic inflammation. These studies also demonstrate the utility of antisense RNA interference in the evaluation of gene function in H. pylori.

  19. 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.

  20. 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.

  1. Engineering Resistance Against Mungbean yellow mosaic India virus Using Antisense RNA.

    Science.gov (United States)

    Haq, Q M I; Ali, Arif; Malathi, V G

    2010-06-01

    Yellow mosaic disease of cultivated legumes in South-East Asia, is caused by Mungbean yellow mosaic India virus (MYMIV) and Mungbean yellow mosaic virus (MYMV) belonging to the genus Begomovirus of the family Geminiviridae. Efforts to engineer resistance against the genus Begomovirus are focused mainly on silencing of complementary-sense virus genes involved in virus replication. Here we have targeted a complementary-sense gene (ACI) encoding Replication initiation Protein (Rep) to develop resistance against soybean isolate of Mungbean yellow mosaic India virus-[India:New Delhi:Soybean 2:1999], a bipartite begomovirus prevalent throughout the Indian subcontinent. We show that the legume host plants co-agroinoculated with infectious constructs of soybean isolate of Mungbean yellow mosaic India virus [India:New Delhi:Soybean 2:1999] along with this antisense Rep gene construct show resistance to the virus.

  2. 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.

  3. 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.

  4. Characteristics of photosynthesis in rice plants transformed with an antisense Rubisco activase gene.

    Science.gov (United States)

    Jin, Song-Heng; Jiang, De-An; Li, Xue-Qin; Sun, Jun-Wei

    2004-08-01

    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 in Rubsico activase and/or the reduction in carbamylation.

  5. 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.

  6. 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.

  7. 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.

  8. Study on Apoptosis-Inducing Effect of XIAP Antisense Oligonucleotides on Glioblastoma Cells in Vitro

    Institute of Scientific and Technical Information of China (English)

    Zhongwei Zhao; Zhengchun Sun; Yunhan Zhang; Ming Zhang; Xudong Ma

    2009-01-01

    OBJECTIVE To investigate the apoptosis-inducing effect of XIAP antisense oligonucleotides on glioblastoma cells in vitro.METHODS There were 4 groups in our experiment. Group A,as a cell control group, had normal cell culture and no treatment applied. Group B, as a blank control group, had normal cell culture and no liposome control of ASODN. Group C was N-ODN.Group D was the ASODN group. RT-PCR and Western blot assay were conducted to detect the expression of XIAP in all A-172cell groups after treatment with XIAP antisense oligonucleotides (ASODN). MTT assay and flow-cytometry (FCM) detection were used to detect the ability of cell anchoring growth and apoptotic rates of all groups. The processing time was 72 h.RESULTS The expression of XIAP in the A-172 cells was greatly down-regulated, after treated with XIAP-ASODN. Among different concentrations of ASODN, the 300nM was the most optimal one. The down-regulation of XIAP obviously inhibited the succinate dehydrogenase (SDH) activity of the A-172 cells and the increased apoptotic rate of A-172 cells (87.45%) was significantly higher than that of the A-172 in the control groups. There was a statistically significant difference between the treatment and control groups (P < 0.01).CONCLUSION The XIAP-ASODN can effectively regulate the expression of the XIAP down, as a result, inhibit the growth of the glioblastoma cells (A-172) and obviously increase the apoptotic rate of the A-172 cells. The results of the study manifest an overt killing role of XIAP-ASODN to the glioblastoma cells.

  9. Biodegradable polymer nanocarriers for therapeutic antisense microRNA delivery in living animals

    Science.gov (United States)

    Paulmurugan, Ramasamy; Sekar, Narayana M.; Sekar, Thillai V.

    2012-03-01

    MicroRNAs are endogenous regulators of gene expression, deregulated in several cellular diseases including cancer. Altering the cellular microenvironment by modulating the microRNAs functions can regulate different genes involved in major cellular processes, and this approach is now being investigated as a promising new generation of molecularly targeted anti-cancer therapies. AntagomiRs (Antisense-miRNAs) are a novel class of chemically modified stable oligonucleotides used for blocking the functions of endogenous microRNAs, which are overexpressed. A key challenge in achieving effective microRNAbased therapeutics lies in the development of an efficient delivery system capable of specifically delivering antisense oligonucleotides and target cancer cells in living animals. We are now developing an effective delivery system designed to selectively deliver antagomiR- 21 and antagomiR-10b to triple negative breast cancer cells, and to revert tumor cell metastasis and invasiveness. The FDA-approved biodegradable PLGA-nanoparticles were selected as a carrier for antagomiRs delivery. Chemically modified antagomiRs (antagomiR-21 and antagomiR-10b) were co-encapsulated in PEGylated-PLGA-nanoparticles by using the double-emulsification (W/O/W) solvent evaporation method, and the resulting average particle size of 150-200nm was used for different in vitro and in vivo experiments. The antagomiR encapsulated PLGA-nanoparticles were evaluated for their in vitro antagomiRs delivery, intracellular release profile, and antagomiRs functional effects, by measuring the endogenous cellular targets, and the cell growth and metastasis. The xenografts of tumor cells in living mice were used for evaluating the anti-metastatic and anti-invasive properties of cells. The results showed that the use of PLGA for antagomiR delivery is not only efficient in crossing cell membrane, but can also maintain functional intracellular antagomiRs level for a extended period of time and achieve

  10. 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.

  11. Antisense-Based Progerin Downregulation in HGPS-Like Patients’ Cells

    Directory of Open Access Journals (Sweden)

    Karim Harhouri

    2016-07-01

    Full Text Available Progeroid laminopathies, including Hutchinson-Gilford Progeria Syndrome (HGPS, OMIM #176670, are premature and accelerated aging diseases caused by defects in nuclear A-type Lamins. Most HGPS patients carry a de novo point mutation within exon 11 of the LMNA gene encoding A-type Lamins. This mutation activates a cryptic splice site leading to the deletion of 50 amino acids at its carboxy-terminal domain, resulting in a truncated and permanently farnesylated Prelamin A called Prelamin A Δ50 or Progerin. Some patients carry other LMNA mutations affecting exon 11 splicing and are named “HGPS-like” patients. They also produce Progerin and/or other truncated Prelamin A isoforms (Δ35 and Δ90 at the transcriptional and/or protein level. The results we present show that morpholino antisense oligonucleotides (AON prevent pathogenic LMNA splicing, markedly reducing the accumulation of Progerin and/or other truncated Prelamin A isoforms (Prelamin A Δ35, Prelamin A Δ90 in HGPS-like patients’ cells. Finally, a patient affected with Mandibuloacral Dysplasia type B (MAD-B, carrying a homozygous mutation in ZMPSTE24, encoding an enzyme involved in Prelamin A maturation, leading to accumulation of wild type farnesylated Prelamin A, was also included in this study. These results provide preclinical proof of principle for the use of a personalized antisense approach in HGPS-like and MAD-B patients, who may therefore be eligible for inclusion in a therapeutic trial based on this approach, together with classical HGPS patients.

  12. 微流控芯片精子分选法对精子常规参数及DNA完整性的影响%Effects of a microfluidic sperm sorter on sperm routine parameters and DNA integrity

    Institute of Scientific and Technical Information of China (English)

    王维; 梁广铁; 彭娅娅; 刘大渔; 周小棉

    2011-01-01

    Objective: To investigate the effects of a microfluidic sperm sorter on the routine parameters and DNA integrity of human sperm. Methods: We divided 40 semen samples into two aliquets and performed sperm sorting using a self-made pelydimethylsiloxane microfluidic sperm sorter and the swim-up method, respectively. Then we evaluated and compared the effects of these two methods on the sperm routine parameters and DNA integrity by computer-assisted sperm analysis and sperm chromatin dispersion test. Results: After processing, sperm motility, normal morphology and tail hypoosmotic swelling rate were significantiy improved,while sperm DNA damage remarkably decreased (P <0.01 ). The microfluidic sperm sorter achieved a significantly lower rate of sperm DNA damage than the swim-up method ( [ 8.4 ± 5.8 ] % vs [ 16.4 ± 9.2 ] %, P < 0.01 ), but no statistically significant differences were found in all other parameters between the two methods. Conclusion: High-quality sperm with less DNA integrity damage could be obtained in sperm sorting with the microfluidic sperm sorter.%目的:研究微流控芯片精子优选技术对精子常规参数及DNA完整性的影响.方法:自行设计制作微流控芯片,利用芯片处理技术和上游法对40例精液标本进行精子优选,通过计算机辅助精液分析系统及染色质扩散试验从精子常规参数及DNA完整性两个方面评价精液体外处理对精子的影响.结果:精液经微流控芯片法和上游法处理后,精子活力、精子正常形态率以及精子尾部肿胀率均有显著提高(P<0.01),精子的DNA损伤率明显降低(P<0.01).微流控芯片法与上游法相比,优选后前者精子DNA损伤率明显低于后者[(8.4±5.8)%vs(16.4±9.2)%,P<0.01],而其他参数差异无显著性.结论:微流控芯片技术在精子优选中能获得精子DNA损伤程度小的高质量精子.

  13. Rational genomics I: antisense open reading frames and codon bias in short-chain oxido reductase enzymes and the evolution of the genetic code.

    Science.gov (United States)

    Duax, William L; Huether, Robert; Pletnev, Vladimir Z; Langs, David; Addlagatta, Anthony; Connare, Sonjay; Habegger, Lukas; Gill, Jay

    2005-12-01

    The short-chain oxidoreductase (SCOR) family of enzymes includes over 6000 members, extending from bacteria and archaea to humans. Nucleic acid sequence analysis reveals that significant numbers of these genes are remarkably free of stopcodons in reading frames other than the coding frame, including those on the antisense strand. The genes from this subset also use almost entirely the GC-rich half of the 64 codons. Analysis of a million hypothetical genes having random nucleotide composition shows that the percentage of SCOR genes having multiple open reading frames exceeds random by a factor of as much as 1 x 10(6). Nevertheless, screening the content of the SWISS-PROT TrEMBL database reveals that 15% of all genes contain multiple open reading frames. The SCOR genes having multiple open reading frames and a GC-rich coding bias exhibit a similar GC bias in the nucleotide triple composition of their DNA. This bias is not correlated with the GC content of the species in which the SCOR genes are found. One possible explanation for the conservation of multiple open reading frames and extreme bias in nucleic acid composition in the family of Rossman folds is that the primordial member of this family was encoded early using only very stable GC-rich DNA and that evolution proceeded with extremely limited introduction of any codons having two or more adenine or thymine nucleotides. These and other data suggest that the SCOR family of enzymes may even have diverged from a common ancestor before most of the AT-rich half of the genetic code was fully defined.

  14. Digital Droplet PCR for the Absolute Quantification of Exon Skipping Induced by Antisense Oligonucleotides in (Pre-)Clinical Development for Duchenne Muscular Dystrophy

    Science.gov (United States)

    Verheul, Ruurd C.; van Deutekom, Judith C. T.; Datson, Nicole A.

    2016-01-01

    Antisense oligonucleotides (AONs) in clinical development for Duchenne muscular dystrophy (DMD) aim to induce skipping of a specific exon of the dystrophin transcript during pre-mRNA splicing. This results in restoration of the open reading frame and consequently synthesis of a dystrophin protein with a shorter yet functional central rod domain. To monitor the molecular therapeutic effect of exon skip-inducing AONs in clinical studies, accurate quantification of pre- and post-treatment exon skip levels is required. With the recent introduction of 3rd generation digital droplet PCR (ddPCR), a state-of-the-art technology became available which allows absolute quantification of transcript copy numbers with and without specific exon skip with high precision, sensitivity and reproducibility. Using Taqman assays with probes targeting specific exon-exon junctions, we here demonstrate that ddPCR reproducibly quantified cDNA fragments with and without exon 51 of the DMD gene over a 4-log dynamic range. In a comparison of conventional nested PCR, qPCR and ddPCR using cDNA constructs with and without exon 51 mixed in different molar ratios using, ddPCR quantification came closest to the expected outcome over the full range of ratios (0–100%), while qPCR and in particular nested PCR overestimated the relative percentage of the construct lacking exon 51. Highest accuracy was similarly obtained with ddPCR in DMD patient-derived muscle cells treated with an AON inducing exon 51 skipping. We therefore recommend implementation of ddPCR for quantification of exon skip efficiencies of AONs in (pre)clinical development for DMD. PMID:27612288

  15. 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.

  16. 细菌染色体编码的反义RNA调节系统%The Regulation System of Antisense RNAs Encoded by Bacterial Chromosomes

    Institute of Scientific and Technical Information of China (English)

    龚阳敏; 高宏

    2011-01-01

    The research progress on regulation systems of antisense RNAs in bacteria was reviewed from several aspects, including the attributes of antisense RNAs Regulation systems of antisense RNAs encoded by the chromosomes of Escherichia coli ,cyanobacteria and gram-positive bacteri-a,and ribonucleases III involved in antisense RNA regulation.%从反义RNA的特征、大肠杆菌、蓝细菌和革兰氏阳性菌染色体编码的反义RNA及其调控系统,以及与反义RNA调控相关的核糖核酸酶Ⅲ等方面介绍了细菌染色体编码的反义RNA调控系统.

  17. Initiation of adenovirus DNA replication.

    OpenAIRE

    Reiter, T; Fütterer, J; Weingärtner, B; Winnacker, E L

    1980-01-01

    In an attempt to study the mechanism of initiation of adenovirus DNA replication, an assay was developed to investigate the pattern of DNA synthesis in early replicative intermediates of adenovirus DNA. By using wild-type virus-infected cells, it was possible to place the origin of adenovirus type 2 DNA replication within the terminal 350 to 500 base pairs from either of the two molecular termini. In addition, a variety of parameters characteristic of adenovirus DNA replication were compared ...

  18. 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.

  19. 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.

  20. 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.

  1. Targeting antisense mitochondrial ncRNAs inhibits murine melanoma tumor growth and metastasis through reduction in survival and invasion factors

    Science.gov (United States)

    Lobos-González, Lorena; Silva, Verónica; Araya, Mariela; Restovic, Franko; Echenique, Javiera; Oliveira-Cruz, Luciana; Fitzpatrick, Christopher; Briones, Macarena; Villegas, Jaime; Villota, Claudio; Vidaurre, Soledad; Borgna, Vincenzo; Socias, Miguel; Valenzuela, Sebastián; Lopez, Constanza; Socias, Teresa; Varas, Manuel; Díaz, Jorge; Burzio, Luis O.; Burzio, Verónica A.

    2016-01-01

    We reported that knockdown of the antisense noncoding mitochondrial RNAs (ASncmtRNAs) induces apoptotic death of several human tumor cell lines, but not normal cells, suggesting this approach for selective therapy against different types of cancer. In order to translate these results to a preclinical scenario, we characterized the murine noncoding mitochondrial RNAs (ncmtRNAs) and performed in vivo knockdown in syngeneic murine melanoma models. Mouse ncmtRNAs display structures similar to the human counterparts, including long double-stranded regions arising from the presence of inverted repeats. Knockdown of ASncmtRNAs with specific antisense oligonucleotides (ASO) reduces murine melanoma B16F10 cell proliferation and induces apoptosis in vitro through downregulation of pro-survival and metastasis markers, particularly survivin. For in vivo studies, subcutaneous B16F10 melanoma tumors in C57BL/6 mice were treated systemically with specific and control antisense oligonucleotides (ASO). For metastasis studies, tumors were resected, followed by systemic administration of ASOs and the presence of metastatic nodules in lungs and liver was assessed. Treatment with specific ASO inhibited tumor growth and metastasis after primary tumor resection. In a metastasis-only assay, mice inoculated intravenously with cells and treated with the same ASO displayed reduced number and size of melanoma nodules in the lungs, compared to controls. Our results suggest that ASncmtRNAs could be potent targets for melanoma therapy. To our knowledge, the ASncmtRNAs are the first potential non-nuclear targets for melanoma therapy. PMID:27507060

  2. Antisense downregulation of the barley limit dextrinase inhibitor modulates starch granule size distribution, starch composition and amylopectin structure.

    Science.gov (United States)

    Stahl, Yvonne; Coates, Steve; Bryce, James H; Morris, Peter C

    2004-08-01

    The barley protein limit dextrinase inhibitor (LDI), structurally related to the alpha-amylase/trypsin inhibitor family, is an inhibitor of the starch debranching enzyme limit dextrinase (LD). In order to investigate the function of LDI, and the consequences for starch metabolism of reduced LDI activity, transgenic barley plants designed to downregulate LDI by antisense were generated. Homozygous antisense lines with reduced LDI protein level and activity were analysed and found to have enhanced free LD activity in both developing and germinating grains. In addition the antisense lines showed unpredicted pleiotropic effects on numerous enzyme activities, for example, alpha- and beta-amylases and starch synthases. Analysis of the starch showed much reduced numbers of the small B-type starch granules, as well as reduced amylose relative to amylopectin levels and reduced total starch. The chain length distribution of the amylopectin was modified with less of the longer chains (>25 units) and enhanced number of medium chains (10-15 units). These results suggest an important role for LDI and LD during starch synthesis as well as during starch breakdown.

  3. 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.

  4. Study on the Transformation of Antisense Soybean Fat Acid Desaturase to Agrobacterium tumefacien%大豆脂肪酸脱饱和酶反义基因转化根癌农杆菌研究

    Institute of Scientific and Technical Information of China (English)

    周延清; 田苗苗; 王芳; 陈艳梅; 张永华; 苑保军

    2011-01-01

    根据NCBI中大豆fad2-1序列设计引物,用PCR方法从大豆的基因组DNA中扩增大豆脂肪酸脱饱和酶基因,克隆到pMD18-T vector中,转化大肠杆菌JM109菌株,进行测序与比对.然后,将其反向克隆到表达载体pBt,转化农杆菌菌株LBA4404,经双酶切鉴定和PCR扩增检测,获得具有该基因的农杆菌工程菌.结果表明,克隆的fad2-1基因为1 196 bp,基因序列与NCBI中已发表的fad2-1序列只有4%的差异,相似性大于95%,说明克隆的基因是大豆fad2-1基因;构建了该基因的反向表达载体,转入农杆菌内.这为利用农杆菌介导法把该反义基因转入大豆,改良脂肪酸成分奠定了基础.%In this study,the primers were designed based on the sequences of soybean fat acid desaturase (fad21) in NCBI database to amplify fad2-1 gene from the genomic DNA of soybean leaves. The amplicon was cloned into pMD18-T vector to be introduced to E. coli JM109 ,and then was sequenced and aligned with the sequence of soybean fad2-1 in NCBI database, and reversely inserted in pBt expression vector to construct plant antisense expression veetor,which was mobilized into Agrobacterium tumefacien strains LBA4404 by freeze-thawing method to get genetically modified strain LBA4404 confirmed by double enzyme digestion and PCR detection. The results indicated that the size of the isolated gene was 1 196 bp,bearing 95% identity with that in NCBI database showing that it was soybean fad21, and that antisense fad2-1 expression vector was successfully constructed and transferred into Agrobacterium tumefacien strains LBA4404. This will build up the foundation for the improvement in soybean faty acids by antisense technique.

  5. Analysis of antisense expression by whole genome tiling microarrays and siRNAs suggests mis-annotation of Arabidopsis orphan protein-coding genes.

    Directory of Open Access Journals (Sweden)

    Casey R Richardson

    Full Text Available BACKGROUND: MicroRNAs (miRNAs and trans-acting small-interfering RNAs (tasi-RNAs are small (20-22 nt long RNAs (smRNAs generated from hairpin secondary structures or antisense transcripts, respectively, that regulate gene expression by Watson-Crick pairing to a target mRNA and altering expression by mechanisms related to RNA interference. The high sequence homology of plant miRNAs to their targets has been the mainstay of miRNA prediction algorithms, which are limited in their predictive power for other kingdoms because miRNA complementarity is less conserved yet transitive processes (production of antisense smRNAs are active in eukaryotes. We hypothesize that antisense transcription and associated smRNAs are biomarkers which can be computationally modeled for gene discovery. PRINCIPAL FINDINGS: We explored rice (Oryza sativa sense and antisense gene expression in publicly available whole genome tiling array transcriptome data and sequenced smRNA libraries (as well as C. elegans and found evidence of transitivity of MIRNA genes similar to that found in Arabidopsis. Statistical analysis of antisense transcript abundances, presence of antisense ESTs, and association with smRNAs suggests several hundred Arabidopsis 'orphan' hypothetical genes are non-coding RNAs. Consistent with this hypothesis, we found novel Arabidopsis homologues of some MIRNA genes on the antisense strand of previously annotated protein-coding genes. A Support Vector Machine (SVM was applied using thermodynamic energy of binding plus novel expression features of sense/antisense transcription topology and siRNA abundances to build a prediction model of miRNA targets. The SVM when trained on targets could predict the "ancient" (deeply conserved class of validated Arabidopsis MIRNA genes with an accuracy of 84%, and 76% for "new" rapidly-evolving MIRNA genes. CONCLUSIONS: Antisense and smRNA expression features and computational methods may identify novel MIRNA genes and other non

  6. Msx1 expression regulation by its own antisense RNA: consequence on tooth development and bone regeneration.

    Science.gov (United States)

    Babajko, Sylvie; Petit, Stéphane; Fernandes, Isabelle; Méary, Fleur; LeBihan, Johanne; Pibouin, Laurence; Berdal, Ariane

    2009-01-01

    Msx homeogenes play an important role in epithelial-mesenchymal interactions leading development. Msx1 is relevant for dental and craniofacial morphogenesis, as suggested by phenotypes of Msx1 mutations in human and Msx1 KO mice. During adulthood, Msx1 is still expressed in the skeleton where its role is largely unknown. Our group showed that the Msx1 gene is submitted to bidirectional transcription generating a long noncoding antisense (AS) RNA. During tooth development, Msx1 sense (S) and AS RNAs showed specific patterns of expression. Thus, the aim of the present study was to analyze the relation between Msx1 S and AS RNAs. In vivo mapping in adult mice showed that both Msx1 RNAs were detected in tested tissues such as bone. In vitro, Msx1 AS RNA decreased endogenous Msx1 S expression and modified Msx1 protein cell distribution. Regulations of Dlx5 and Bmp4 expression involving Msx1 S and AS RNAs showed that Msx1 AS RNA could modulate Msx1 function. The study of Msx1 S and AS RNA status is interesting in the case of tooth agenesis and bone loss to see if a disturbance of this balance could be associated with a disturbance of bone homeostasis. In that sense, our current results suggest a clear involvement of Msx1 in alveolar bone.

  7. In vivo correction of a Menkes disease model using antisense oligonucleotides.

    Science.gov (United States)

    Madsen, Erik C; Morcos, Paul A; Mendelsohn, Bryce A; Gitlin, Jonathan D

    2008-03-11

    Although the molecular basis of many inherited metabolic diseases has been defined, the availability of effective therapies in such disorders remains problematic. Menkes disease is a fatal neurodegenerative disorder due to loss-of-function mutations in the ATP7A gene encoding a copper-transporting P-type Atpase. To develop therapeutic approaches in affected patients, we have identified a zebrafish model of Menkes disease termed calamity that results from splicing defects in the zebrafish orthologue of the ATP7A gene. Embryonic-recessive lethal mutants have impaired copper homeostasis that results in absent melanin pigmentation, impaired notochord formation, and hindbrain neurodegeneration. In this current study, we have attempted to rescue these striking phenotypic alterations by using a series of antisense morpholino oligonucleotides directed against the splice-site junctions of two mutant calamity alleles. Our findings reveal a robust and complete correction of the copper-deficient defects of calamity in association with the generation of the WT Menkes protein in all rescued mutants. Interestingly, a quantitative analysis of atp7a-specific transcripts suggests that competitive translational regulation may account for the synthesis of WT protein in these embryos. This in vivo correction of Menkes disease through the rescue of aberrant splicing may provide therapeutic options in this fatal disease and illustrates the potential for zebrafish models of human genetic disease in the development of treatments based on the principles of interactions of synthetic oligonucleotide analogues with mRNA.

  8. Antisense oligonucleotide-mediated exon skipping as a strategy to reduce proteolytic cleavage of ataxin-3.

    Science.gov (United States)

    Toonen, Lodewijk J A; Schmidt, Iris; Luijsterburg, Martijn S; van Attikum, Haico; van Roon-Mom, Willeke M C

    2016-10-12

    Spinocerebellar ataxia type-3 (SCA3) is a neurodegenerative disorder caused by a polyglutamine repeat expansion in the ataxin-3 protein. Cleavage of mutant ataxin-3 by proteolytic enzymes yields ataxin-3 fragments containing the polyglutamine stretch. These shorter ataxin-3 fragments are thought to be involved in SCA3 pathogenesis due to their increased cellular toxicity and their involvement in formation of the characteristic neuronal aggregates. As a strategy to prevent formation of toxic cleavage fragments, we investigated an antisense oligonucleotide-mediated modification of the ataxin-3 pre-mRNA through exon skipping of exon 8 and 9, resulting in the removal of a central 88 amino acid region of the ataxin-3 protein. This removed protein region contains several predicted cleavage sites and two ubiquitin-interacting motifs. In contrast to unmodified mutant ataxin-3, the internally truncated ataxin-3 protein did not give rise to potentially toxic cleavage fragments when incubated with caspases. In vitro experiments did not show cellular toxicity of the modified ataxin-3 protein. However, the modified protein was incapable of binding poly-ubiquitin chains, which may interfere with its normal deubiquitinating function. Low exon skipping efficiencies combined with reduction in important ataxin-3 protein functions suggest that skipping of exon 8 and 9 is not a viable therapeutic option for SCA3.

  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. 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.

  11. 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.

  12. Short antisense-locked nucleic acids (all-LNAs) correct alternative splicing abnormalities in myotonic dystrophy.

    Science.gov (United States)

    Wojtkowiak-Szlachcic, Agnieszka; Taylor, Katarzyna; Stepniak-Konieczna, Ewa; Sznajder, Lukasz J; Mykowska, Agnieszka; Sroka, Joanna; Thornton, Charles A; Sobczak, Krzysztof

    2015-03-31

    Myotonic dystrophy type 1 (DM1) is an autosomal dominant multisystemic disorder caused by expansion of CTG triplet repeats in 3'-untranslated region of DMPK gene. The pathomechanism of DM1 is driven by accumulation of toxic transcripts containing expanded CUG repeats (CUG(exp)) in nuclear foci which sequester several factors regulating RNA metabolism, such as Muscleblind-like proteins (MBNLs). In this work, we utilized very short chemically modified antisense oligonucleotides composed exclusively of locked nucleic acids (all-LNAs) complementary to CUG repeats, as potential therapeutic agents against DM1. Our in vitro data demonstrated that very short, 8- or 10-unit all-LNAs effectively bound the CUG repeat RNA and prevented the formation of CUG(exp)/MBNL complexes. In proliferating DM1 cells as well as in skeletal muscles of DM1 mouse model the all-LNAs induced the reduction of the number and size of CUG(exp) foci and corrected MBNL-sensitive alternative splicing defects with high efficacy and specificity. The all-LNAs had low impact on the cellular level of CUG(exp)-containing transcripts and did not affect the expression of other transcripts with short CUG repeats. Our data strongly indicate that short all-LNAs complementary to CUG repeats are a promising therapeutic tool against DM1.

  13. Cymbidium mosaic virus coat protein gene in antisense confers resistance to transgenic Nicotiana occidentalis.

    Science.gov (United States)

    Lim, S H; Ko, M K; Lee, S J; La, Y J; Kim, B D

    1999-12-31

    The nucleotide sequence of the 3'-terminal region of the Korean isolate of cymbidium mosaic virus (CyMV-Ca) from a naturally infected cattleya was determined. The sequence contains an open reading frame (ORF) coding for the viral coat protein (CP) at the 3'-end and three other ORFs (triple gene block or movement protein) of CyMV. The CP gene encodes a polypeptide chain of 220 amino acids with a molecular mass of 23,760 Da. The deduced CP sequence showed a strong homology with those of two CyMVs reported. A construct of the CyMV-Ca CP gene in the antisense orientation in the plant expression vector pMBP1 was transferred via Agrobacterium tumefaciens-mediated transformation into Nicotiana occidentalis which is a propagation host of CyMV. The T1 progeny of the transgenic plants were inoculated with CyMV and found to be highly resistant to CyMV infection.

  14. 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.

  15. 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.

  16. Correlation between sperm DNA damage and sperm routine parameters in infertile males%不育患者精子DNA损伤和精液常规参数关系分析

    Institute of Scientific and Technical Information of China (English)

    麦选诚; 董云华; 陈斌; 孙杨; 欧绍峰

    2016-01-01

    Objective To investigate the application value of sperm DNA fragmentation index(DFI) in male patients, and explore the correlation between DFI and semen parameters including motility and concentration. Methods Semen samples were obtained from subfertile men. All semen samples were divided into the normal group and the abnormal groups including asthenozoospermia group,oligozoospermia group and oligoasthenozoospermia group according to World Health Organization guideline. Sperm DNA damage frequency was evaluated by the sperm chromatin dispersion (SCD) test. Results Significant differences were found in sperm DFI between the normal group and the abnormal groups (P0.05) was determined. Area under ROC curve of sperm DFI and sperm parameters reached 0.693, and the cut-off value was 19.5%(sensitivity:41.8%, specificity:89.3%). Conclusion Sperm DFI increased significantly in parallel with the decrease in sperm quality, indicating that sperm DNA damage might be an important cause of male infertility. Sperm DFI evaluates semen quality, with the cut-off level of 19.5%.The test of sperm DNA fragmentation was helpful to evaluate male reproductive function and the outcome of assisted reproductive technology.%目的:探讨男性不育患者的精子DNA碎片率(DFI)与精液参数间相关性及精子DFI评价男性生育力方面的应用价值。方法精液标本均取自不育患者,以WHO精液分析标准行精液检测,根据结果分为精液参数正常组、弱精子症组、少精子症组、少弱精子症组。用染色质扩散实验法(SCD)检测精子DFI。结果精液参数正常组与参数异常组(包括弱精子症组、少精子症组和少弱精子症组)间的DFI有明显的差异(P<0.05),不育组间DFI也存在差异(P<0.01)。DFI与精子活动率、前向运动精子率存在明显的负相关性(r=-0.575,r=-0.547, P<0.01),与精子浓度之间无相关性(r=-0.049,P>0.05);精子DFI评价精液参

  17. Evolution of the Antisense Overlap between Genes for Thyroid Hormone Receptor and Rev-erbα and Characterization of an Exonic G-Rich Element That Regulates Splicing of TRα2 mRNA.

    Directory of Open Access Journals (Sweden)

    Stephen H Munroe

    Full Text Available The α-thyroid hormone receptor gene (TRα codes for two functionally distinct proteins: TRα1, the α-thyroid hormone receptor; and TRα2, a non-hormone-binding variant. The final exon of TRα2 mRNA overlaps the 3' end of Rev-erbα mRNA, which encodes another nuclear receptor on the opposite strand of DNA. To understand the evolution of this antisense overlap, we sequenced these genes and mRNAs in the platypus Orthorhynchus anatinus. Despite its strong homology with other mammals, the platypus TRα/Rev-erbα locus lacks elements essential for expression of TRα2. Comparative analysis suggests that alternative splicing of TRα2 mRNA expression evolved in a stepwise fashion before the divergence of eutherian and marsupial mammals. A short G-rich element (G30 located downstream of the alternative 3'splice site of TRα2 mRNA and antisense to the 3'UTR of Rev-erbα plays an important role in regulating TRα2 splicing. G30 is tightly conserved in eutherian mammals, but is absent in marsupials and monotremes. Systematic deletions and substitutions within G30 have dramatically different effects on TRα2 splicing, leading to either its inhibition or its enhancement. Mutations that disrupt one or more clusters of G residues enhance splicing two- to three-fold. These results suggest the G30 sequence can adopt a highly structured conformation, possibly a G-quadruplex, and that it is part of a complex splicing regulatory element which exerts both positive and negative effects on TRα2 expression. Since mutations that strongly enhance splicing in vivo have no effect on splicing in vitro, it is likely that the regulatory role of G30 is mediated through linkage of transcription and splicing.

  18. 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.

  19. Influence of seminal oxidative stress on sperm DNA integrity and other parameters in males with infertility%不育患者精液的氧化应激对精子DNA完整性等参数的影响

    Institute of Scientific and Technical Information of China (English)

    焦瑞宝; 冯恒孝; 唐吉斌; 方军; 凤俊蓉; 詹三华; 周萍; 陈然

    2013-01-01

    cases,Group Ⅲ) and leucocyte sperm disease group (referred as white essence disease group,sperm white blood cell count > 1 × 106/mL,9 cases).A total of 21 healthy subjects were as healthy controls.The semen,sperm concentration and motility rate,PR,sperm membrane function,sperm shape normality,malonaldehyde(MDA),total antioxidant capacity(TAC) and sperm DNA integrity parameters were determined.Results In comparison with healthy control group,modified MDA concentration of each group was significantly higher than that in healthy control group (P < 0.05 or P < 0.01),and with the decline of sperm motility,sperm MDA increased.The MDA concentrations in Group Ⅰ,Ⅱ and Ⅲ had statistical significance with those in oligospermia group and leucoyte sperm disease group (P < 0.01).Semen indices of oxidative stress with sperm concentration,total number of sperm,MDA content and sperm DNA fragmentation index(DFI) had positive correlation,and those with sperm motility rate,PR,hypoosmotic swelling rate (HOS),fast linear motion concentration,fast linear motion number,path velocity (VAP),linear velocity (VSL),TAC and sperm shape normality had negative correlation.Conclusions Seminal oxidative stress can cause the changes of sperm membrane function,dynamic parameter,shape parameter and sperm nuclear DNA damage,and may be one of the mechanisms causing male infertility.

  20. 神经肽Y-Y5受体反义基因治疗诱导大鼠脂肪细胞凋亡研究%Apoptosis and Lipolysis of White Adipocytes Induced by Neuropeptide Y-Y5 Receptor Antisense Oligodeoxynucleotides

    Institute of Scientific and Technical Information of China (English)

    龚海霞; 郭锡熔; 陈荣华; 费莉; 郭梅; 刘倩琦; 倪毓辉

    2003-01-01

    目的:探讨神经肽Y-Y5受体反义基因治疗后正常SD大鼠减肥减重效应与外周白色脂肪细胞体积、数目变化的关系.方法:侧脑室插管,注射针对Y5受体编码起始区的反义、正义、错义寡聚脱氧核糖核酸及生理盐水,采用MPLAS-500多媒体彩色病理图文分析系统计算平均脂肪细胞面积,基因组DNA提取物凝胶电泳检测脂肪细胞凋亡,RT-PCR分析凋亡相关基因Bcl-2、Bax表达的改变.结果:①Y5受体反义基因治疗后大鼠进食量与体重显著降低,外周白色脂肪组织湿重与平均脂肪细胞面积明显减少;②脂肪组织基因组DNA提取物凝胶电泳出现凋亡特征性梯状条带;③凋亡相关基因Bcl-2表达下调、Bax表达上调.结论:平均脂肪细胞面积减小、脂肪细胞凋亡增加可能是Y5受体反义基因治疗减肥减重的重要原因.%Objective:To investigate the influence of central administration of neuropeptideY-Y5 receptor antisense oligodeoxynucleotides (ODNs) on body weight, fat pads of SD rats, and the ef-fects of white adipocytes lipolysis and apoptosis. Methods:Y 5 receptor antisense, sense, mismatcbedODNs or vehicle was intracerebroventricularly ( i. c. v. ) injected. Average adipocyte area was calculat-ed. DNA ladders were measured to evaluate adipocyte apoptosis, and RT-PCR was used to analyse theexpresswn of Bcl-2 and Box gene. Results:Central administration of Y5 antisense ODNs significant-ly decreased body weight, and average adipocyte area. DNA fragmentation was present after electrophore-sis at epididymal adipose tissue. The expression of Bcl-2 gene was downregulated, while the expressionof Box upregulated. Conclusion:Lipolysis and adipocyte apoptosis may be important mechanisms forY5 antisense therapy.

  1. 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.

  2. Overexpression of a natural chloroplast-encoded antisense RNA in tobacco destabilizes 5S rRNA and retards plant growth

    Directory of Open Access Journals (Sweden)

    Stern David B

    2010-09-01

    Full Text Available Abstract Background The roles of non-coding RNAs in regulating gene expression have been extensively studied in both prokaryotes and eukaryotes, however few reports exist as to their roles in organellar gene regulation. Evidence for accumulation of natural antisense RNAs (asRNAs in chloroplasts comes from the expressed sequence tag database and cDNA libraries, while functional data have been largely obtained from artificial asRNAs. In this study, we used Nicotiana tabacum to investigate the effect on sense strand transcripts of overexpressing a natural chloroplast asRNA, AS5, which is complementary to the region which encodes the 5S rRNA and tRNAArg. Results AS5-overexpressing (AS5ox plants obtained by chloroplast transformation exhibited slower growth and slightly pale green leaves. Analysis of AS5 transcripts revealed four distinct species in wild-type (WT and AS5ox plants, and additional AS5ox-specific products. Of the corresponding sense strand transcripts, tRNAArg overaccumulated several-fold in transgenic plants whereas 5S rRNA was unaffected. However, run-on transcription showed that the 5S-trnR region was transcribed four-fold more in the AS5ox plants compared to WT, indicating that overexpression of AS5 was associated with decreased stability of 5S rRNA. In addition, polysome analysis of the transformants showed less 5S rRNA and rbcL mRNA associated with ribosomes. Conclusions Our results suggest that AS5 can modulate 5S rRNA levels, giving it the potential to affect Chloroplast translation and plant growth. More globally, overexpression of asRNAs via chloroplast transformation may be a useful strategy for defining their functions.

  3. 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.

  4. An analysis of the use of genomic DNA as a universal reference in two channel DNA microarrays

    Directory of Open Access Journals (Sweden)

    Kapur Vivek

    2005-05-01

    Full Text Available Abstract Background DNA microarray is an invaluable tool for gene expression explorations. In the two-dye microarray, fluorescence intensities of two samples, each labeled with a different dye, are compared after hybridization. To compare a large number of samples, the 'reference design' is widely used, in which all RNA samples are hybridized to a common reference. Genomic DNA is an attractive candidate for use as a universal reference, especially for bacterial systems with a low percentage of non-coding sequences. However, genomic DNA, comprising of both the sense and anti-sense strands, is unlike the single stranded cDNA usually used in microarray hybridizations. The presence of the antisense strand in the 'reference' leads to reactions between complementary labeled strands in solution and may cause the assay result to deviate from true values. Results We have developed a mathematical model to predict the validity of using genomic DNA as a reference in the microarray assay. The model predicts that the assay can accurately estimate relative concentrations for a wide range of initial cDNA concentrations. Experimental results of DNA microarray assay using genomic DNA as a reference correlated well to those obtained by a direct hybridization between two cDNA samples. The model predicts that the initial concentrations of labeled genomic DNA strands and immobilized strands, and the hybridization time do not significantly affect the assay performance. At low values of the rate constant for hybridization between immobilized and mobile strands, the assay performance varies with the hybridization time and initial cDNA concentrations. For the case where a microarray with immobilized single strands is used, results from hybridizations using genomic DNA as a reference will correspond to true ratios under all conditions. Conclusion Simulation using the mathematical model, and the experimental study presented here show the potential utility of microarray

  5. Correlation of sperm DNA fragmentation index and semen parameters%精子DN A碎片指数与精液参数的相关性研究

    Institute of Scientific and Technical Information of China (English)

    秦文松; 刘英; 段金良

    2014-01-01

    目的:探讨男性精子DNA碎片与精液常规参数及精子顶体酶活性之间的关系。方法:收集生殖中心门诊553例男性患者精液标本,采用改良精子染色质扩散实验(SCD)检测精子 DNA 碎片指数(DFI),按精子DFI结果分为DFI<15%(I组)、15%≤DFI<25%(II组)、DFI≥25%(III组),同时检测精子密度、总数、(a+b)级精子百分率、畸形率及顶体酶活性,对DFI与精液各项参数的相关性进行分析。结果:精子密度及精子总数与DFI无显著相关性;前向运动精子百分率随DFI升高逐渐降低,III组与I组间存在显著性差异;精子正常形态率随DFI的增加有下降的趋势,但是差异不显著;精子顶体酶活性随DFI升高而降低,组间均有显著性差异。结论:精子DNA碎片指数与前向精子百分率及精子顶体酶活性呈负相关。因此DFI可作为评估精子功能的一个较好的参考指标。%Objectives:To explore the relationship between the male sperm DNA fragmentation,semen pa-rameters and acrosome activity.Method:Semen samples of 553 male patients were collected from our center. Sperm DNA fragmentation index(DFI)was detected by Sperm Chromatin Dispersion(SCD)methods.According to the results of sperm DFI,all the values were divided into group I (DFI<15%),group II (15%≤DFI <25%) and group III (DFI≥25%),and the sperm density,count,percentage of progressive motility sperm,morphology and acrosome activity were detected at the same time.The correction of DFI and semen parameters were analyzed. Results:Sperm density and total sperm count had no significant correlation with the DFI.The percentage of progres-sive motility sperm gradually decreased with the augment of DFI,and there were significant differences between group I and group III.Normal sperm morphology rate also decreased with the augment of DFI,and the difference was not significant.Acrosome activity gradually decreased

  6. An antisense oligodeoxynucleotide that depletes RI alpha subunit of cyclic AMP-dependent protein kinase induces growth inhibition in human cancer cells.

    Science.gov (United States)

    Yokozaki, H; Budillon, A; Tortora, G; Meissner, S; Beaucage, S L; Miki, K; Cho-Chung, Y S

    1993-02-15

    Enhanced expression of the RI alpha subunit of cyclic AMP-dependent protein kinase type I has been correlated with cancer cell growth. We provide evidence that RI alpha is a growth-inducing protein that may be essential for neoplastic cell growth. Human colon, breast, and gastric carcinoma and neuroblastoma cell lines exposed to a 21-mer human RI alpha antisense phosphorothioate oligodeoxynucleotide (S-oligodeoxynucleotide) exhibited growth inhibition with no sign of cytotoxicity. Mismatched sequence (random) S-oligodeoxynucleotides of the same length exhibited no effect. The growth inhibitory effect of RI alpha antisense oligomer correlated with a decrease in the RI alpha mRNA and protein levels and with an increase in RII beta (the regulatory subunit of protein kinase type II) expression. The growth inhibition was abolished, however, when cells were exposed simultaneously to both RI alpha and RII beta antisense S-oligodeoxynucleotides. The RII beta antisense S-oligodeoxynucleotide alone, exhibiting suppression of RII beta along with enhancement of RI alpha expression, led to slight stimulation of cell growth. These results demonstrate that two isoforms of cyclic AMP receptor proteins, RI alpha and RII beta, are reciprocally related in the growth control of cancer cells and that the RI alpha antisense oligodeoxynucleotide, which efficiently depletes the growth stimulatory RI alpha, is a powerful biological tool toward suppression of malignancy.

  7. Inhibitory effect of 2 '-o-methoxyethyl-modified antisense oligonucleotides targeting vascular endothelial growth factor A on SKOV3 human ovarian cancer cells

    Institute of Scientific and Technical Information of China (English)

    FU Yi-bing; WEN Ze-qing; ZHAO Xing-bo; YAN Lei; ZHANG Chun-hua; WANG Fei

    2011-01-01

    Background Ovarian cancers are often at an advanced stage at diagnosis because early detection is difficult. The poor prognosis of ovarian cancers highlights the crucial need to develop better therapeutic agents and strategies. The objective of this study was to investigate the inhibitory effects of a new modified antisense oligonucleotides targeting vascular endothelial growth factor A (VEGF-A) in SKOV3 ovarian cancer cells.Methods Antisense oligonucleotides targeting VEGF-A was designed, synthesized and transfected into SKOV3ovarian cancer cells. Western blotting and real-time RT-PCR were used to analyze the inhibitory effects of antisense oligonucleotides on VEGF-A protein and mRNA expression. Transwell matrix assay was used to detect cell migration inhibition.Results The antisense oligonucleotides targeting VEGF-A significantly decreased VEGF-A protein and mRNA expression and inhibited cell migration in SKOV3 ovarian cancer cells.Conclusions This new modified antisense oligonucleotides targeting VEGF-A can decrease VEGF-A expression and inhibit cell migration in SKOV3 ovarian cancer cells. This new oligonucleotides may be a promising therapeutic agent for ovarian cancers.

  8. A single administration of morpholino antisense oligomer rescues spinal muscular atrophy in mouse.

    Science.gov (United States)

    Porensky, Paul N; Mitrpant, Chalermchai; McGovern, Vicki L; Bevan, Adam K; Foust, Kevin D; Kaspar, Brain K; Wilton, Stephen D; Burghes, Arthur H M

    2012-04-01

    Spinal muscular atrophy (SMA) is an autosomal-recessive disorder characterized by α-motor neuron loss in the spinal cord anterior horn. SMA results from deletion or mutation of the Survival Motor Neuron 1 gene (SMN1) and retention of SMN2. A single nucleotide difference between SMN1 and SMN2 results in exclusion of exon 7 from the majority of SMN2 transcripts, leading to decreased SMN protein levels and development of SMA. A series of splice enhancers and silencers regulate incorporation of SMN2 exon 7; these splice motifs can be blocked with antisense oligomers (ASOs) to alter SMN2 transcript splicing. We have evaluated a morpholino (MO) oligomer against ISS-N1 [HSMN2Ex7D(-10,-29)], and delivered this MO to postnatal day 0 (P0) SMA pups (Smn-/-, SMN2+/+, SMNΔ7+/+) by intracerebroventricular (ICV) injection. Survival was increased markedly from 15 days to >100 days. Delayed CNS MO injection has moderate efficacy, and delayed peripheral injection has mild survival advantage, suggesting that early CNS ASO administration is essential for SMA therapy consideration. ICV treatment increased full-length SMN2 transcript as well as SMN protein in neural tissue, but only minimally in peripheral tissue. Interval analysis shows a decrease in alternative splice modification over time. We suggest that CNS increases of SMN will have a major impact on SMA, and an early increase of the SMN level results in correction of motor phenotypes. Finally, the early introduction by intrathecal delivery of MO oligomers is a potential treatment for SMA patients.

  9. Peripheral reduction of FGFR4 with antisense oligonucleotides increases metabolic rate and lowers adiposity in diet-induced obese mice.

    Directory of Open Access Journals (Sweden)

    Xing Xian Yu

    Full Text Available Obesity is a primary risk factor for multiple metabolic disorders. Many drugs for the treatment of obesity, which mainly act through CNS as appetite suppressants, have failed during development or been removed from the market due to unacceptable adverse effects. Thus, there are very few efficacious drugs available and remains a great unmet medical need for anti-obesity drugs that increase energy expenditure by acting on peripheral tissues without severe side effects. Here, we report a novel approach involving antisense inhibition of fibroblast growth factor receptor 4 (FGFR4 in peripheral tissues. Treatment of diet-induce obese (DIO mice with FGFR4 antisense oligonucleotides (ASO specifically reduced liver FGFR4 expression that not only resulted in decrease in body weight (BW and adiposity in free-feeding conditions, but also lowered BW and adiposity under caloric restriction. In addition, combination treatment with FGFR4 ASO and rimonabant showed additive reduction in BW and adiposity. FGFR4 ASO treatment increased basal metabolic rate during free-feeding conditions and, more importantly, prevented adaptive decreases of metabolic rate induced by caloric restriction. The treatment increased fatty acid oxidation while decreased lipogenesis in both liver and fat. Mechanistic studies indicated that anti-obesity effect of FGFR4 ASO was mediated at least in part through an induction of plasma FGF15 level resulted from reduction of hepatic FGFR4 expression. The anti-obesity effect was accompanied by improvement in plasma glycemia, whole body insulin sensitivity, plasma lipid levels and liver steatosis. Therefore, FGFR4 could be a potential novel target and antisense reduction of hepatic FGFR4 expression could be an efficacious therapy as an adjunct to diet restriction or to an appetite suppressant for the treatment of obesity and related metabolic disorders.

  10. Hfq restructures RNA-IN and RNA-OUT and facilitates antisense pairing in the Tn10/IS10 system.

    Science.gov (United States)

    Ross, Joseph A; Ellis, Michael J; Hossain, Shahan; Haniford, David B

    2013-05-01

    Hfq functions in post-transcriptional gene regulation in a wide range of bacteria, usually by promoting base-pairing of mRNAs and trans-encoded sRNAs that share partial sequence complementarity. It is less clear if Hfq is required for pairing of cis-encoded RNAs (i.e., antisense RNAs) with their target mRNAs. In the current work, we have characterized the interactions between Escherichia coli Hfq and the components of the Tn10/IS10 antisense system, RNA-IN and RNA-OUT. We show that Hfq interacts with RNA-OUT through its proximal RNA-binding surface, as is typical for Hfq and trans-encoded sRNAs. In contrast, RNA-IN binds both proximal and distal RNA-binding surfaces in Hfq with a higher affinity for the latter, as is typical for mRNA interactions in canonical sRNA-mRNA pairs. Importantly, an amino acid substitution in Hfq that interferes with RNA binding to the proximal site negatively impacts RNA-IN:OUT pairing in vitro and suppresses the ability of Hfq to negatively regulate IS10 transposition in vivo. We also show that Hfq binding to RNA-IN and RNA-OUT alters secondary structure elements in both of these RNAs and speculate that this could be important in how Hfq facilitates RNA-IN:OUT pairing. Based on the results presented here, we suggest that Hfq could be involved in regulating RNA pairing in other antisense systems, including systems encoded by other transposable elements.

  11. Evaluation of cystine transport in cultured human kidney cells and establishment of cystinuria type I phenotype by antisense technology.

    Science.gov (United States)

    Wendt-Nordahl, Gunnar; Sagi, Sreedhar; Bolenz, Christian; Alken, Peter; Michel, Maurice Stephan; Knoll, Thomas

    2008-02-01

    Cystinuria is a rare hereditary disease resulting in recurrent stone formation and the need for repeated invasive interventions. So far, two responsible genes have been identified which encode the two transporters, rBAT and b(0,+)AT forming a heterodimer to transport cystine in proximal tubular cells (PTC) and whose defect results in increased excretion of cystine. A human cell line mimicing the phenotype of cystinuria in vitro is yet to be developed. Human kidney (HK)-2 is a PTC line derived from normal HK. After determining the presence of rBAT gene by RT-PCR and Western blot analysis, radioactively labeled cystine (S(35)) was used to evaluate the functional presence of the amino acid transport in HK-2 cells when cultured in vitro. To achieve a cystinuria type I phenotype in HK-2 cells, the rBAT gene was silenced using antisense oligonucleotides complimentary to human rBAT mRNA. The reduced transport activity of cystine was then determined by radiolabeled cystine uptake measurements. RT-PCR and Western blot confirmed the expression of the rBAT gene in HK-2 cells. Considerable transport of the radio labeled cystine was observed in HK-2 cells and was linearly dependent on the incubation time with the amino acid. The cystine transport in rBAT knockdown cells after incubation with antisense oligonucleotides was significantly lower compared to control (0.76 vs. 0.98%; P=0.0008), proving a transient knock-down of the rBAT gene. This study demonstrates the presence of the b(0,+) amino acid transport system in human proximal tubular HK-2 cells when cultured in vitro. Inhibition of this transport system is possible by using antisense technology. A permanent inhibition of the cystine transport, based on our model, would be useful for the development and evaluation gene therapeutic approaches.

  12. Peripheral reduction of FGFR4 with antisense oligonucleotides increases metabolic rate and lowers adiposity in diet-induced obese mice.

    Science.gov (United States)

    Yu, Xing Xian; Watts, Lynnetta M; Manchem, Vara Prasad; Chakravarty, Kaushik; Monia, Brett P; McCaleb, Michael L; Bhanot, Sanjay

    2013-01-01

    Obesity is a primary risk factor for multiple metabolic disorders. Many drugs for the treatment of obesity, which mainly act through CNS as appetite suppressants, have failed during development or been removed from the market due to unacceptable adverse effects. Thus, there are very few efficacious drugs available and remains a great unmet medical need for anti-obesity drugs that increase energy expenditure by acting on peripheral tissues without severe side effects. Here, we report a novel approach involving antisense inhibition of fibroblast growth factor receptor 4 (FGFR4) in peripheral tissues. Treatment of diet-induce obese (DIO) mice with FGFR4 antisense oligonucleotides (ASO) specifically reduced liver FGFR4 expression that not only resulted in decrease in body weight (BW) and adiposity in free-feeding conditions, but also lowered BW and adiposity under caloric restriction. In addition, combination treatment with FGFR4 ASO and rimonabant showed additive reduction in BW and adiposity. FGFR4 ASO treatment increased basal metabolic rate during free-feeding conditions and, more importantly, prevented adaptive decreases of metabolic rate induced by caloric restriction. The treatment increased fatty acid oxidation while decreased lipogenesis in both liver and fat. Mechanistic studies indicated that anti-obesity effect of FGFR4 ASO was mediated at least in part through an induction of plasma FGF15 level resulted from reduction of hepatic FGFR4 expression. The anti-obesity effect was accompanied by improvement in plasma glycemia, whole body insulin sensitivity, plasma lipid levels and liver steatosis. Therefore, FGFR4 could be a potential novel target and antisense reduction of hepatic FGFR4 expression could be an efficacious therapy as an adjunct to diet restriction or to an appetite suppressant for the treatment of obesity and related metabolic disorders.

  13. Long noncoding RNA FGFR3-AS1 promotes osteosarcoma growth through regulating its natural antisense transcript FGFR3.

    Science.gov (United States)

    Sun, Jiabing; Wang, Xuming; Fu, Chunjiang; Wang, Xiaoyu; Zou, Jilong; Hua, Hanbing; Bi, Zhenggang

    2016-05-01

    Long noncoding RNAs (lncRNAs), a new class of RNAs with no protein-coding potential, have been reported to have crucial roles in the regulation of a variety of tumors. However, the functions and molecular mechanisms of lncRNAs to osteosarcoma are still largely unknown. The purpose of this study is to examine the expression, functions and molecular mechanisms of a new lncRNA FGFR3 antisense transcript 1 (FGFR3-AS1) in osteosarcoma. The expression of FGFR3-AS1 was examined by real-time quantitative PCR. The regulation of FGFR3 by FGFR3-AS1 was examined by RNase protection assay, real-time quantitative PCR, western blotting, and luciferase reporter assay. The effects of FGFR3-AS1 on osteosarcoma cell proliferation and cell cycle were determined by Cell Counting Kit-8, Ethynyl deoxyuridine incorporation assay and flow cytometry. FGFR3-AS1 was upregulated in osteosarcoma. Increased FGFR3-AS1 expression correlates with large tumor size, advanced Enneking stage, metastasis and poor survival. Through antisense pairing with FGFR3 3'UTR, FGFR3-AS1 increases FGFR3 mRNA stability and upregulates FGFR3 expression. The expression of FGFR3-AS1 and FGFR3 is positively correlated in osteosarcoma tissues. Knockdown of FGFR3-AS1 inhibits the proliferation and cell cycle progression of osteosarcoma cells in vitro. Moreover, knockdown of FGFR3-AS1 inhibits xenograft tumor growth of osteosarcoma cells in vivo. These data demonstrate the mechanisms of how antisense noncoding RNA regulate the expression of sense genes, and show the pivotal functions of FGFR3-AS1 in osteosarcoma.

  14. Modulation of γ2-MSH hepatoprotection by antisense peptides and melanocortin subtype 3 and 4 receptor antagonists.

    Science.gov (United States)

    Turcic, Petra; Stambuk, Nikola; Konjevoda, Pasko; Kelava, Tomislav; Gabricevic, Mario; Stojkovic, Ranko; Aralica, Gorana

    2015-01-01

    Melanocortins, i.e., melanocyte stimulating hormones (MSH) are peptides with strong antiinflammatory effects. The most investigated aspects of γ2-MSH are related to cardiovascular effects and natriuresis, with limited research available about its anti-inflammatory and cytoprotective effects. The aims of this study were: 1) to examine the effects of γ2-MSH and its derivative [D-Trp(8)]-γ2-MSH on the acetaminophen model of liver damage in CBA mice; 2) to evaluate the modulation of γ2-MSH hepatoprotection by melanocortin subtypes 3 and 4 receptor antagonists SHU 9119 and HS 024; 3) to define the importance of central MSH pharmacophore region (HFRW) by using antisense peptides LVKAT and VKAT. In this study, specific antagonists and antisense peptides were used to target central pharmacophore region of γ2-MSH and [D-Trp(8)]-γ2-MSH, enabling the evaluation of hepatoprotection from the standpoint of the receptor and pharmacophore blockade. The criteria for monitoring the effects of the hormones on the liver damage were alanine transaminase, aspartate transaminase activities (U/L), and pathohistological scoring of liver necrosis (scale 0-5). γ2-MSH (0.24 mg/kg) indicated hepatoprotective effects in comparison to control (p < 0.001). In contrast, [D-Trp(8)]-γ2-MSH did not show any hepatoprotective effects. Application of antagonists SHU 9119 and HS 024, and antisense peptides LVKAT and VKAT, also did not show any hepatoprotective effects. In fact, when combined with γ2-MSH, it annulled its hepatoprotective effect. The results provide evidence for hepatoprotective and antiinflammatory effects of the γ2-MSH in the liver.

  15. 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.

  16. 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...

  17. Evidence for the antisense transcription in the proviral R29-127 strain of bovine immunodeficiency virus

    Institute of Scientific and Technical Information of China (English)

    Bin; Liu; Xuechao; Zhao; Wenyuan; Shen; Xiaohong; Kong

    2015-01-01

    <正>Dear Editor,In the late stages of the retroviral life cycle,the transcription of viral pre-m RNA is initiated at the 5′long terminal repeat(5′LTR)and terminated at the 3′LTR;the full-length and spliced viral RNAs are transported out of the nucleus and serve as templates for the translation of viral proteins or alternatively as the full-length viral genome to be packaged into virus particles.Antisense

  18. Parameter Estimation

    DEFF Research Database (Denmark)

    Sales-Cruz, Mauricio; Heitzig, Martina; Cameron, Ian;

    2011-01-01

    of optimisation techniques coupled with dynamic solution of the underlying model. Linear and nonlinear approaches to parameter estimation are investigated. There is also the application of maximum likelihood principles in the estimation of parameters, as well as the use of orthogonal collocation to generate a set...

  19. Articulatory Parameters.

    Science.gov (United States)

    Ladefoged, Peter

    1980-01-01

    Summarizes the 16 parameters hypothesized to be necessary and sufficient for linguistic phonetic specifications. Suggests seven parameters affecting tongue shapes, three determining the positions of the lips, one controlling the position of the velum, four varying laryngeal actions, and one controlling respiratory activity. (RL)

  20. Antisense Down-Regulation of Metallothionein: A Therapeutic Approach for Prostate Cancer

    Science.gov (United States)

    1999-08-01

    buffer containing dATP and 3 U of T4 DNA ligase in 50 i.l final volume at 160C overnight. The reaction was stopped by heating at 75°C for 10 min. The...for 10 min. Blunt end ligation was carried out as shown above in presence of 6 U of T4 DNA ligase overnight. Reaction was stopped and samples were...initially ligated to the shuttle plasmid pSV-ICEU I using 1X ligation buffer, 3 U of T4 DNA ligase , 0.25 .tg of shuttle plasmid with three-fold molar excess

  1. Hybridization of different antisense oligonucleotides on the surface of gold nanoparticles to silence zinc metalloproteinase gene after uptake by Leishmania major.

    Science.gov (United States)

    Jebali, Ali; Anvari-Tafti, Mohammad Hosssein

    2015-05-01

    The use of antisense oligonucleotides is a novel strategy to treat infectious diseases. In this approach, vital mRNAs are targeted by antisense oligonucleotides. The aim of this study was to evaluate the effects of gold nanoparticles hybridized with different antisense oligonucleotides on Leishmania (L) major. In this project, gold nanoparticles were first synthesized, and then conjugated with primary oligonucleotides, 3'-AAA-5'. Next, conjugated gold nanoparticles (NP1) were separately hybridized with three types of antisense oligonucleotide from coding reign of GP63 gene (NP2), non-coding reign of GP63 gene (NP3), and both coding and non-coding reigns of GP63 (NP4). Then, 1mL of L. major suspension was separately added to 1mL of different hybridized gold nanoparticles at serial concentrations (1-200μg/mL), and incubated for 24, 48, and 72h at 37°C. Next, the uptake of each nanoparticle was separately measured by atomic absorption spectroscopy. After incubation, the cell viability was separately evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay. Also, the expression of GP63 gene was read out by quantitative-real-time PCR. This study showed that NP2 and NP3 had higher (5-fold) uptake than NP1 and NP4. Moreover, NP2 and NP3 led to less cell viability and gene expression, compared with NP1 and NP4. It could be concluded that both sequence and size of antisense oligonucleotide were important for transfection of L. major. Importantly, these antisense oligonucleotides can be obtained from both coding and non-coding reign of GP63 gene. Moreover, hybridized gold nanoparticles not only could silence GP63 gene, but also could kill L. major.

  2. 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

  3. 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

  4. Evaluation of the effects of chemically different linkers on hepatic accumulations, cell tropism and gene silencing ability of cholesterol-conjugated antisense oligonucleotides.

    Science.gov (United States)

    Wada, Shunsuke; Yasuhara, Hidenori; Wada, Fumito; Sawamura, Motoki; Waki, Reiko; Yamamoto, Tsuyoshi; Harada-Shiba, Mariko; Obika, Satoshi

    2016-03-28

    Cholesterol conjugation of oligonucleotides is an attractive way to deliver the oligonucleotides specifically to the liver. However cholesterol-conjugated antisense oligonucleotides (ASOs) mainly accumulate in non-parenchymal cells (NPCs) such as Kupffer cells. In this study, to increase the hepatic accumulation of cholesterol-conjugated ASOs, we prepared a variety of linkers for cholesterol conjugation to anti-Pcsk9 ASOs and examined their effects on pharmacological parameters. Hepatic accumulation of ASO was dramatically increased with cholesterol conjugation. The increase in hepatic accumulation depended largely on the linker chemistry of each cholesterol-conjugated ASO. In addition to hepatic accumulation, the cell tropism of each cholesterol-conjugated ASO tended to depend on their linker. Although a linker bearing a disulfide bond accumulated mainly in NPCs, hexamethylene succinimide linker accumulated mainly in hepatocytes. To estimate the benefits of releasing ASO from the conjugated cholesterol in hepatocyte, we designed another linker based on hexamethylene succinimide, which has a phosphodiester bond between the linker and the ASO. The cholesterol-conjugated ASO bearing such a phosphodiester bond showed a significantly improved Pcsk9 mRNA inhibitory effect compared to its counterpart, cholesterol-conjugated ASO with a phosphorothioate bond, while the hepatic accumulation of both cholesterol-conjugated ASOs was comparable, indicating the effectiveness of removing the conjugated cholesterol for ASO activity. In toxicity analysis, some of the linkers induced lethal toxicities when they were injected at high concentrations (>600μM). These toxicities were attributed to decreased platelet levels in the blood, suggesting an interaction between cholesterol-conjugated ASO and platelets. Our findings may provide a guideline for the design of molecule-conjugated ASOs.

  5. The mycobacterial DNA-binding protein 1 (MDP1 from Mycobacterium bovis BCG influences various growth characteristics

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    Maurischat Sven

    2008-06-01

    Full Text Available Abstract Background Pathogenic mycobacteria such as M. tuberculosis, M. bovis or M. leprae are characterised by their extremely slow growth rate which plays an important role in mycobacterial virulence and eradication of the bacteria. Various limiting factors influence the generation time of mycobacteria, and the mycobacterial DNA-binding protein 1 (MDP1 has also been implicated in growth regulation. Our strategy to investigate the role of MDP1 in mycobacterial growth consisted in the generation and characterisation of a M. bovis BCG derivative expressing a MDP1-antisense gene. Results The expression rate of the MDP1 protein in the recombinant M. bovis BCG containing the MDP1-antisense plasmid was reduced by about 50% compared to the reference strain M. bovis BCG containing the empty vector. In comparison to this reference strain, the recombinant M. bovis BCG grew faster in broth culture and reached higher cell masses in stationary phase. Likewise its intracellular growth in mouse and human macrophages was ameliorated. Bacterial clumping in broth culture was reduced by the antisense plasmid. The antisense plasmid increased the susceptibility of the bacteria towards Ampicillin. 2-D protein gels of bacteria maintained under oxygen-poor conditions demonstrated a reduction in the number and the intensity of many protein spots in the antisense strain compared to the reference strain. Conclusion The MDP1 protein has a major impact on various growth characteristics of M. bovis BCG. It plays an important role in virulence-related traits such as aggregate formation and intracellular multiplication. Its impact on the protein expression in a low-oxygen atmosphere indicates a role in the adaptation to the hypoxic conditions present in the granuloma.

  6. 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.

  7. Antisense Oligonucleotide Targeting TGF-β1 Abrogates Tumorigenicity of Rhabdomyosarcoma in vivo

    Institute of Scientific and Technical Information of China (English)

    Shouli Wang; Huihua Yao; Lingling Guo; Liang Dong; Shigang Li; Haizhen Deng; Maomin Sun

    2008-01-01

    OBJECTIVE Over-expression of transforming growth factor β1 (TGF-β1) has been observed in many advanced cancers.The present study was aimed at developing potential antisense oligonucleotides (ASONs) to repress TGF-β1 expression in rhabdomyosarcoma (RMS) RD cells, and to examine their effect on tumorigenicity of RD cells in vivo.METHODS ASONs targeting the region surrounding the start codon of TGF-β1 were synthesized and transferred into cells in the form of complexes with Lipofectamine 2000. The TGF-β1 protein was determined by immunofluorescence and ELISA.The cell viability and cell cycle were examined by MTT and flow cytometry. The RD cells, with or without TGF-β1ASON, in 50 μl of serum-free EMDM medium were injected subcutaneously into the right flank of nude mice. The tumors were then measured and weighed.RESULTS The ASON sequence targeting the first start site at bases 841-855 of the human TGF-β1 gene had the greatest effect on attenuating the expression of TGF-β1 (P<0.05). The ASONs induced a decrease in OD values after 6 d (P<0.05). Analysis of the cell cycle revealed that the ASON induced a significant decrease in cells in the S phase and an increase in cells in the G1 phase (P<0.05). In the nude mice model, the mean tumor volume, after 2 weeks of treatment with Lipofectamine or ASON,decreased to 88.5% or 55% respectively, compared to the control tumor size, resulting in a significant difference (P<0.01).CONCLUSION The sequence of the ASON, which targeted the start condon at the bases 841-855 of the human TGF-β1 gene, was demonstrated to be a useful agent for studying the regulation of TGF-β1 over-expression in RD cells, and has important therapeutic potential for suppressing the tumorigenicity of human RMS in vivo.

  8. Dual Toxic-Peptide-Coding Staphylococcus aureus RNA under Antisense Regulation Targets Host Cells and Bacterial Rivals Unequally

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    Marie-Laure Pinel-Marie

    2014-04-01

    Full Text Available Produced from the pathogenicity islands of Staphylococcus aureus clinical isolates, stable SprG1 RNA encodes two peptides from a single internal reading frame. These two peptides accumulate at the membrane, and inducing their expression triggers S. aureus death. Replacement of the two initiation codons by termination signals reverses this toxicity. During growth, cis-antisense RNA SprF1 is expressed, preventing mortality by reducing SprG1 RNA and peptide levels. The peptides are secreted extracellularly, where they lyse human host erythrocytes, a process performed more efficiently by the longer peptide. The two peptides also inactivate Gram-negative and -positive bacteria, with the shorter peptide more effective against S. aureus rivals. Two peptides are secreted from an individual RNA containing two functional initiation codons. Thus, we present an unconventional type I toxin-antitoxin system expressed from a human pathogen producing two hemolytic and antibacterial peptides from a dual-coding RNA, negatively regulated by a dual-acting antisense RNA.

  9. 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.

  10. Delayed Time-to-Treatment of an Antisense Morpholino Oligomer Is Effective against Lethal Marburg Virus Infection in Cynomolgus Macaques.

    Science.gov (United States)

    Warren, Travis K; Whitehouse, Chris A; Wells, Jay; Welch, Lisa; Charleston, Jay S; Heald, Alison; Nichols, Donald K; Mattix, Marc E; Palacios, Gustavo; Kugleman, Jeffrey R; Iversen, Patrick L; Bavari, Sina

    2016-02-01

    Marburg virus (MARV) is an Ebola-like virus in the family Filovirdae that causes sporadic outbreaks of severe hemorrhagic fever with a case fatality rate as high as 90%. AVI-7288, a positively charged antisense phosphorodiamidate morpholino oligomer (PMOplus) targeting the viral nucleoprotein gene, was evaluated as a potential therapeutic intervention for MARV infection following delayed treatment of 1, 24, 48, and 96 h post-infection (PI) in a nonhuman primate lethal challenge model. A total of 30 cynomolgus macaques were divided into 5 groups of 6 and infected with 1,830 plaque forming units of MARV subcutaneously. AVI-7288 was administered by bolus infusion daily for 14 days at 15 mg/kg body weight. Survival was the primary endpoint of the study. While none (0 of 6) of the saline group survived, 83-100% of infected monkeys survived when treatment was initiated 1, 24, 48, or 96 h post-infection (PI). The antisense treatment also reduced serum viremia and inflammatory cytokines in all treatment groups compared to vehicle controls. The antibody immune response to virus was preserved and tissue viral antigen was cleared in AVI-7288 treated animals. These data show that AVI-7288 protects NHPs against an otherwise lethal MARV infection when treatment is initiated up to 96 h PI.

  11. Delayed Time-to-Treatment of an Antisense Morpholino Oligomer Is Effective against Lethal Marburg Virus Infection in Cynomolgus Macaques.

    Directory of Open Access Journals (Sweden)

    Travis K Warren

    2016-02-01

    Full Text Available Marburg virus (MARV is an Ebola-like virus in the family Filovirdae that causes sporadic outbreaks of severe hemorrhagic fever with a case fatality rate as high as 90%. AVI-7288, a positively charged antisense phosphorodiamidate morpholino oligomer (PMOplus targeting the viral nucleoprotein gene, was evaluated as a potential therapeutic intervention for MARV infection following delayed treatment of 1, 24, 48, and 96 h post-infection (PI in a nonhuman primate lethal challenge model. A total of 30 cynomolgus macaques were divided into 5 groups of 6 and infected with 1,830 plaque forming units of MARV subcutaneously. AVI-7288 was administered by bolus infusion daily for 14 days at 15 mg/kg body weight. Survival was the primary endpoint of the study. While none (0 of 6 of the saline group survived, 83-100% of infected monkeys survived when treatment was initiated 1, 24, 48, or 96 h post-infection (PI. The antisense treatment also reduced serum viremia and inflammatory cytokines in all treatment groups compared to vehicle controls. The antibody immune response to virus was preserved and tissue viral antigen was cleared in AVI-7288 treated animals. These data show that AVI-7288 protects NHPs against an otherwise lethal MARV infection when treatment is initiated up to 96 h PI.

  12. Improved antisense oligonucleotide design to suppress aberrant SMN2 gene transcript processing: towards a treatment for spinal muscular atrophy.

    Directory of Open Access Journals (Sweden)

    Chalermchai Mitrpant

    Full Text Available Spinal muscular atrophy (SMA is caused by loss of the Survival Motor Neuron 1 (SMN1 gene, resulting in reduced SMN protein. Humans possess the additional SMN2 gene (or genes that does produce low level of full length SMN, but cannot adequately compensate for loss of SMN1 due to aberrant splicing. The majority of SMN2 gene transcripts lack exon 7 and the resultant SMNΔ7 mRNA is translated into an unstable and non-functional protein. Splice intervention therapies to promote exon 7 retention and increase amounts of full-length SMN2 transcript offer great potential as a treatment for SMA patients. Several splice silencing motifs in SMN2 have been identified as potential targets for antisense oligonucleotide mediated splice modification. A strong splice silencer is located downstream of exon 7 in SMN2 intron 7. Antisense oligonucleotides targeting this motif promoted SMN2 exon 7 retention in the mature SMN2 transcripts, with increased SMN expression detected in SMA fibroblasts. We report here systematic optimisation of phosphorodiamidate morpholino oligonucleotides (PMO that promote exon 7 retention to levels that rescued the phenotype in a severe mouse model of SMA after intracerebroventricular delivery. Furthermore, the PMO gives the longest survival reported to date after a single dosing by ICV.

  13. Regulation of Nav1.7: A Conserved SCN9A Natural Antisense Transcript Expressed in Dorsal Root Ganglia.

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    Jennifer Koenig

    Full Text Available The Nav1.7 voltage-gated sodium channel, encoded by SCN9A, is critical for human pain perception yet the transcriptional and post-transcriptional mechanisms that regulate this gene are still incompletely understood. Here, we describe a novel natural antisense transcript (NAT for SCN9A that is conserved in humans and mice. The NAT has a similar tissue expression pattern to the sense gene and is alternatively spliced within dorsal root ganglia. The human and mouse NATs exist in cis with the sense gene in a tail-to-tail orientation and both share sequences that are complementary to the terminal exon of SCN9A/Scn9a. Overexpression analyses of the human NAT in human embryonic kidney (HEK293A and human neuroblastoma (SH-SY5Y cell lines show that it can function to downregulate Nav1.7 mRNA, protein levels and currents. The NAT may play an important role in regulating human pain thresholds and is a potential candidate gene for individuals with chronic pain disorders that map to the SCN9A locus, such as Inherited Primary Erythromelalgia, Paroxysmal Extreme Pain Disorder and Painful Small Fibre Neuropathy, but who do not contain mutations in the sense gene. Our results strongly suggest the SCN9A NAT as a prime candidate for new therapies based upon augmentation of existing antisense RNAs in the treatment of chronic pain conditions in man.

  14. Regulation of Nav1.7: A Conserved SCN9A Natural Antisense Transcript Expressed in Dorsal Root Ganglia.

    Science.gov (United States)

    Koenig, Jennifer; Werdehausen, Robert; Linley, John E; Habib, Abdella M; Vernon, Jeffrey; Lolignier, Stephane; Eijkelkamp, Niels; Zhao, Jing; Okorokov, Andrei L; Woods, C Geoffrey; Wood, John N; Cox, James J

    2015-01-01

    The Nav1.7 voltage-gated sodium channel, encoded by SCN9A, is critical for human pain perception yet the transcriptional and post-transcriptional mechanisms that regulate this gene are still incompletely understood. Here, we describe a novel natural antisense transcript (NAT) for SCN9A that is conserved in humans and mice. The NAT has a similar tissue expression pattern to the sense gene and is alternatively spliced within dorsal root ganglia. The human and mouse NATs exist in cis with the sense gene in a tail-to-tail orientation and both share sequences that are complementary to the terminal exon of SCN9A/Scn9a. Overexpression analyses of the human NAT in human embryonic kidney (HEK293A) and human neuroblastoma (SH-SY5Y) cell lines show that it can function to downregulate Nav1.7 mRNA, protein levels and currents. The NAT may play an important role in regulating human pain thresholds and is a potential candidate gene for individuals with chronic pain disorders that map to the SCN9A locus, such as Inherited Primary Erythromelalgia, Paroxysmal Extreme Pain Disorder and Painful Small Fibre Neuropathy, but who do not contain mutations in the sense gene. Our results strongly suggest the SCN9A NAT as a prime candidate for new therapies based upon augmentation of existing antisense RNAs in the treatment of chronic pain conditions in man.

  15. Tyrosine phosphorylation of RNA polymerase II CTD is associated with antisense promoter transcription and active enhancers in mammalian cells

    Science.gov (United States)

    Descostes, Nicolas; Heidemann, Martin; Spinelli, Lionel; Schüller, Roland; Maqbool, Muhammad Ahmad; Fenouil, Romain; Koch, Frederic; Innocenti, Charlène; Gut, Marta; Gut, Ivo; Eick, Dirk; Andrau, Jean-Christophe

    2014-01-01

    In mammals, the carboxy-terminal domain (CTD) of RNA polymerase (Pol) II consists of 52 conserved heptapeptide repeats containing the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. Post-translational modifications of the CTD coordinate the transcription cycle and various steps of mRNA maturation. Here we describe Tyr1 phosphorylation (Tyr1P) as a hallmark of promoter (5′ associated) Pol II in mammalian cells, in contrast to what was described in yeast. Tyr1P is predominantly found in antisense orientation at promoters but is also specifically enriched at active enhancers. Mutation of Tyr1 to phenylalanine (Y1F) prevents the formation of the hyper-phosphorylated Pol IIO form, induces degradation of Pol II to the truncated Pol IIB form, and results in a lethal phenotype. Our results suggest that Tyr1P has evolved specialized and essential functions in higher eukaryotes associated with antisense promoter and enhancer transcription, and Pol II stability. DOI: http://dx.doi.org/10.7554/eLife.02105.001 PMID:24842994

  16. Antisense Oligonucleotides Targeting Parasite Inositol 1,4,5-Trisphosphate Receptor Inhibits Mammalian Host Cell Invasion by Trypanosoma cruzi

    Science.gov (United States)

    Hashimoto, Muneaki; Nara, Takeshi; Hirawake, Hiroko; Morales, Jorge; Enomoto, Masahiro; Mikoshiba, Katsuhiko

    2014-02-01

    Chagas disease is caused by an intracellular parasitic protist, Trypanosoma cruzi. As there are no highly effective drugs against this agent that also demonstrate low toxicity, there is an urgent need for development of new drugs to treat Chagas disease. We have previously demonstrated that the parasite inositol 1,4,5-trisphosphate receptor (TcIP3R) is crucial for invasion of the mammalian host cell by T. cruzi. Here, we report that TcIP3R is a short-lived protein and that its expression is significantly suppressed in trypomastigotes. Treatment of trypomastigotes, an infective stage of T. cruzi, with antisense oligonucleotides specific to TcIP3R deceased TcIP3R protein levels and impaired trypomastigote invasion of host cells. Due to the resulting instability and very low expression level of TcIP3R in trypomastigotes indicates that TcIP3R is a promising target for antisense therapy in Chagas disease.

  17. RNA toxicity from the ALS/FTD C9ORF72 expansion is mitigated by antisense intervention.

    Science.gov (United States)

    Donnelly, Christopher J; Zhang, Ping-Wu; Pham, Jacqueline T; Haeusler, Aaron R; Heusler, Aaron R; Mistry, Nipun A; Vidensky, Svetlana; Daley, Elizabeth L; Poth, Erin M; Hoover, Benjamin; Fines, Daniel M; Maragakis, Nicholas; Tienari, Pentti J; Petrucelli, Leonard; Traynor, Bryan J; Wang, Jiou; Rigo, Frank; Bennett, C Frank; Blackshaw, Seth; Sattler, Rita; Rothstein, Jeffrey D

    2013-10-16

    A hexanucleotide GGGGCC repeat expansion in the noncoding region of the C9ORF72 gene is the most common genetic abnormality in familial and sporadic amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The function of the C9ORF72 protein is unknown, as is the mechanism by which the repeat expansion could cause disease. Induced pluripotent stem cell (iPSC)-differentiated neurons from C9ORF72 ALS patients revealed disease-specific (1) intranuclear GGGGCCexp RNA foci, (2) dysregulated gene expression, (3) sequestration of GGGGCCexp RNA binding protein ADARB2, and (4) susceptibility to excitotoxicity. These pathological and pathogenic characteristics were confirmed in ALS brain and were mitigated with antisense oligonucleotide (ASO) therapeutics to the C9ORF72 transcript or repeat expansion despite the presence of repeat-associated non-ATG translation (RAN) products. These data indicate a toxic RNA gain-of-function mechanism as a cause of C9ORF72 ALS and provide candidate antisense therapeutics and candidate human pharmacodynamic markers for therapy.

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

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    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.

  19. Antisense RNA Controls LRP1 Sense Transcript Expression through Interaction with a Chromatin-Associated Protein, HMGB2

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    Yasunari Yamanaka

    2015-05-01

    Full Text Available Long non-coding RNAs (lncRNAs, including natural antisense transcripts (NATs, are expressed more extensively than previously anticipated and have widespread roles in regulating gene expression. Nevertheless, the molecular mechanisms of action of the majority of NATs remain largely unknown. Here, we identify a NAT of low-density lipoprotein receptor-related protein 1 (Lrp1, referred to as Lrp1-AS, that negatively regulates Lrp1 expression. We show that Lrp1-AS directly binds to high-mobility group box 2 (Hmgb2 and inhibits the activity of Hmgb2 to enhance Srebp1a-dependent transcription of Lrp1. Short oligonucleotides targeting Lrp1-AS inhibit the interaction of antisense transcript and Hmgb2 protein and increase Lrp1 expression by enhancing Hmgb2 activity. Quantitative RT-PCR analysis of brain tissue samples from Alzheimer’s disease patients and aged-matched controls revealed upregulation of LRP1-AS and downregulation of LRP1. Our data suggest a regulatory mechanism whereby a NAT interacts with a ubiquitous chromatin-associated protein to modulate its activity in a locus-specific fashion.

  20. Antisense RNA controls LRP1 Sense transcript expression through interaction with a chromatin-associated protein, HMGB2.

    Science.gov (United States)

    Yamanaka, Yasunari; Faghihi, Mohammad Ali; Magistri, Marco; Alvarez-Garcia, Oscar; Lotz, Martin; Wahlestedt, Claes

    2015-05-12

    Long non-coding RNAs (lncRNAs), including natural antisense transcripts (NATs), are expressed more extensively than previously anticipated and have widespread roles in regulating gene expression. Nevertheless, the molecular mechanisms of action of the majority of NATs remain largely unknown. Here, we identify a NAT of low-density lipoprotein receptor-related protein 1 (Lrp1), referred to as Lrp1-AS, that negatively regulates Lrp1 expression. We show that Lrp1-AS directly binds to high-mobility group box 2 (Hmgb2) and inhibits the activity of Hmgb2 to enhance Srebp1a-dependent transcription of Lrp1. Short oligonucleotides targeting Lrp1-AS inhibit the interaction of antisense transcript and Hmgb2 protein and increase Lrp1 expression by enhancing Hmgb2 activity. Quantitative RT-PCR analysis of brain tissue samples from Alzheimer's disease patients and aged-matched controls revealed upregulation of LRP1-AS and downregulation of LRP1. Our data suggest a regulatory mechanism whereby a NAT interacts with a ubiquitous chromatin-associated protein to modulate its activity in a locus-specific fashion.

  1. In depth analysis of the Sox4 gene locus that consists of sense and natural antisense transcripts.

    Science.gov (United States)

    Ling, King-Hwa; Brautigan, Peter J; Moore, Sarah; Fraser, Rachel; Leong, Melody Pui-Yee; Leong, Jia-Wen; Zainal Abidin, Shahidee; Lee, Han-Chung; Cheah, Pike-See; Raison, Joy M; Babic, Milena; Lee, Young Kyung; Daish, Tasman; Mattiske, Deidre M; Mann, Jeffrey R; Adelson, David L; Thomas, Paul Q; Hahn, Christopher N; Scott, Hamish S

    2016-06-01

    SRY (Sex Determining Region Y)-Box 4 or Sox4 is an important regulator of the pan-neuronal gene expression during post-mitotic cell differentiation within the mammalian brain. Sox4 gene locus has been previously characterized with multiple sense and overlapping natural antisense transcripts [1], [2]. Here we provide accompanying data on various analyses performed and described in Ling et al. [2]. The data include a detail description of various features found at Sox4 gene locus, additional experimental data derived from RNA-Fluorescence in situ Hybridization (RNA-FISH), Western blotting, strand-specific reverse-transcription quantitative polymerase chain reaction (RT-qPCR), gain-of-function and in situ hybridization (ISH) experiments. All the additional data provided here support the existence of an endogenous small interfering- or PIWI interacting-like small RNA known as Sox4_sir3, which origin was found within the overlapping region consisting of a sense and a natural antisense transcript known as Sox4ot1.

  2. Asymmetric localization of natural antisense RNA of neuropeptide sensorin in Aplysia sensory neurons during aging and activity

    Directory of Open Access Journals (Sweden)

    Beena eKadakkuzha

    2014-04-01

    Full Text Available Despite the advances in our understanding of transcriptome, regulation and function of its noncoding components continue to be poorly understood. Here we searched for natural antisense transcript for sensorin (NAT-SRN, a neuropeptide expressed in the presynaptic sensory neurons of gill-withdrawal reflex of the marine snail Aplysia californica. Sensorin (SRN has a key role in learning and long-term memory storage in Aplysia. We have identified NAT-SRN in the central nervous system (CNS and have confirmed its expression by northern blotting and fluorescent RNA in situ hybridization. Quantitative analysis of NAT-SRN in micro dissected cell bodies and processes of sensory neurons suggest that NAT-SRN is present in the distal neuronal processes along with sense transcripts. Importantly, aging is associated with reduced levels of NAT-SRN in sensory neuron processes. Furthermore, we find that forskolin, an activator of CREB signaling, differentially alters the distribution of SRN and NAT-SRN. These studies reveal novel insights into physiological regulation of natural antisense RNAs.

  3. In depth analysis of the Sox4 gene locus that consists of sense and natural antisense transcripts

    Directory of Open Access Journals (Sweden)

    King-Hwa Ling

    2016-06-01

    Full Text Available SRY (Sex Determining Region Y-Box 4 or Sox4 is an important regulator of the pan-neuronal gene expression during post-mitotic cell differentiation within the mammalian brain. Sox4 gene locus has been previously characterized with multiple sense and overlapping natural antisense transcripts [1,2]. Here we provide accompanying data on various analyses performed and described in Ling et al. [2]. The data include a detail description of various features found at Sox4 gene locus, additional experimental data derived from RNA-Fluorescence in situ Hybridization (RNA-FISH, Western blotting, strand-specific reverse-transcription quantitative polymerase chain reaction (RT-qPCR, gain-of-function and in situ hybridization (ISH experiments. All the additional data provided here support the existence of an endogenous small interfering- or PIWI interacting-like small RNA known as Sox4_sir3, which origin was found within the overlapping region consisting of a sense and a natural antisense transcript known as Sox4ot1.

  4. 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

  5. Synthesis and evaluation of a fluorine-18 labeled antisense oligonucleotide as a potential PET tracer for NOS mRNA expression

    NARCIS (Netherlands)

    de Vries, EFJ; Vroegh, J; Dijkstra, G; Moshage, H; Elsinga, PH; Jansen, PLM; Vaalburg, W

    2004-01-01

    Inducible NO synthase (iNOS) is overexpressed in inflammatory bowel diseases. An antisense oligonucleotide with good hybridization properties for iNOS mRNA was selected using RT-PCR. The oligonucleotide was reliably labeled with fluorine-18 using N-(4-[F-18]fluorobenzyl)-2-bromoacetamide. Cellular u

  6. 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.

  7. Data in support of a functional analysis of splicing mutations in the IDS gene and the use of antisense oligonucleotides to exploit an alternative therapy for MPS II

    Directory of Open Access Journals (Sweden)

    Liliana Matos

    2015-12-01

    The interpretation of these data and further extensive experiments into the analysis of these three mutations and also into the methodology applied to correct one of them can be found in “Functional analysis of splicing mutations in the IDS gene and the use of antisense oligonucleotides to exploit an alternative therapy for MPS II” Matos et al. (2015 [1].

  8. Thiolated carboxymethyl dextran as a nanocarrier for colon delivery of hSET1 antisense: In vitro stability and efficiency study

    Energy Technology Data Exchange (ETDEWEB)

    Kiani, Melika, E-mail: Melika.kiani@gmail.com [Department of Pharmaceutical Nanotechnology, Faculty of Pharmacy, Tehran University of Medical Sciences, P.O. Box 14155-6451, Tehran (Iran, Islamic Republic of); Mirzazadeh Tekie, Farnaz Sadat, E-mail: mirzazadehf@yahoo.com [Department of Pharmaceutical Nanotechnology, Faculty of Pharmacy, Tehran University of Medical Sciences, P.O. Box 14155-6451, Tehran (Iran, Islamic Republic of); Dinarvand, Meshkat, E-mail: mdinarvand@hotmail.com [Department of Pharmaceutical Nanotechnology, Faculty of Pharmacy, Tehran University of Medical Sciences, P.O. Box 14155-6451, Tehran (Iran, Islamic Republic of); Soleimani, Masoud, E-mail: soleim_m@modares.ac.ir [Stem Cell Technology Research Centre, P.O. Box 14155-3174, Tehran (Iran, Islamic Republic of); Department of Hematology, School of Medical Sciences, Tarbiat Modares University, P.O. Box: 14115-111, Tehran (Iran, Islamic Republic of); Dinarvand, Rassoul, E-mail: dinarvand@tums.ac.ir [Department of Pharmaceutical Nanotechnology, Faculty of Pharmacy, Tehran University of Medical Sciences, P.O. Box 14155-6451, Tehran (Iran, Islamic Republic of); Nanotechnology Research Centre, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of); Atyabi, Fatemeh, E-mail: atyabifa@tums.ac.ir [Department of Pharmaceutical Nanotechnology, Faculty of Pharmacy, Tehran University of Medical Sciences, P.O. Box 14155-6451, Tehran (Iran, Islamic Republic of); Nanotechnology Research Centre, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of)

    2016-05-01

    Gene therapy is an optimistic approach in cancer treatment. However, for efficient delivery of gene materials, designing an appropriate vector is necessary. Polyelectrolyte complexes (PECs) of chitosan and dextran could be considered a proper nanoparticulate carrier for sensitive biomaterials. In this study, PECs of chitosan and thiolated dextran were used as either an injectable or oral gene delivery system. hSET1 antisense was loaded into the PECs to suppress proliferation of colon cancer cell line. The prepared nanoparticles have ~ 115 nm diameter size and positive zeta potential with high mucoadhesion properties. They are able to protect antisense from degradation in serum and biorelevant fluids (FaSSIF and FaSSGF). Furthermore, prepared nanoparticles demonstrated superior cellular penetration and inhibitory effect on SW480 colon cancer cell proliferation. All nanoparticles significantly down regulated hSET1 in comparison with naked antisense. It can be concluded that thiolated PECs have potential use for injectable or oral delivery of nucleic acids such as antisense. - Highlights: • Formation of stable nanoparticle with dextran and chitosan derivatives for oral and intravenous gene delivery. • Satifactory cellular uptake of nanoparticles and approximately complete suppression of hSET1 expression in SW480 cell lines • Prolonged stability of nanoparticles against biorelevent media with desirable release rate.

  9. Antisense-mediated suppression of C-hordein biosynthesis in the barley grain results in correlated changes in the transcriptome, protein profile, and amino acid composition

    DEFF Research Database (Denmark)

    Hansen, Mette; Lange, Marianne; Friis, Carsten;

    2007-01-01

    Antisense- or RNAi-mediated suppression of the biosynthesis of nutritionally inferior storage proteins is a promising strategy for improving the amino acid profile of seeds. However, the potential pleiotropic effects of this on interconnected pathways and the agronomic quality traits need...

  10. SINEUPs are modular antisense long-non coding RNAs that increase synthesis of target proteins in cells

    Directory of Open Access Journals (Sweden)

    Silvia eZucchelli

    2015-05-01

    Full Text Available Despite recent efforts in discovering novel long non-coding RNAs (lncRNAs and unveiling their functions in a wide range of biological processes their applications as biotechnological or therapeutic tools are still at their infancy. We have recently shown that AS Uchl1, a natural lncRNA antisense to the Parkinson’s disease-associated gene Ubiquitin carboxyl-terminal esterase L1 (Uchl1, is able to increase UchL1 protein synthesis at post-transcriptional level. Its activity requires two RNA elements: an embedded inverted SINEB2 sequence to increase translation and the overlapping region to target its sense mRNA. This functional organization is shared with several mouse lncRNAs antisense to protein coding genes. The potential use of AS Uchl1-derived lncRNAs as enhancers of target mRNA translation remains unexplored. Here we define AS Uchl1 as the representative member of a new functional class of natural and synthetic antisense lncRNAs that activate translation. We named this class of RNAs SINEUPs for their requirement of the inverted SINEB2 sequence to UP-regulate translation in a gene-specific manner. The overlapping region is indicated as the Binding Doman (BD while the embedded inverted SINEB2 element is the Effector Domain (ED. By swapping BD, synthetic SINEUPs are designed targeting mRNAs of interest. SINEUPs function in an array of cell lines and can be efficiently directed towards N-terminally tagged proteins. Their biological activity is retained in a miniaturized version within the range of small RNAs length. Its modular structure was exploited to successfully design synthetic SINEUPs targeting endogenous Parkinson’s disease-associated DJ-1 and proved to be active in different neuronal cell lines.In summary, SINEUPs represent the first scalable tool to increase synthesis of proteins of interest. We propose SINEUPs as reagents for molecular biology experiments, in protein manufacturing as well as in therapy of haploinsufficiencies.

  11. Study of HIV-2 primer-template initiation complex using antisense oligonucleotides

    DEFF Research Database (Denmark)

    Boulmé, F; Freund, F; Gryaznov, S;

    2000-01-01

    HIV-2 reverse transcription is initiated by the retroviral DNA polymerase (reverse transcriptase) from a cellular tRNALys3 partially annealed to the primer binding site in the 5'-region of viral RNA. The HIV-2 genome has two A-rich regions upstream of the primer binding site. In contrast to HIV-1...

  12. 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.

  13. DNA Nanotechnology

    Science.gov (United States)

    Taniguchi, Masateru; Kawai, Tomoji

    2002-11-01

    DNA is one candidate of promising molecules for molecular electronic devices, since it has the double helix structure with pi-electron bases for electron transport, the address at 0.4 nm intervals, and the self-assembly. Electrical conductivity and nanostructure of DNA and modified DNA molecules are investigated in order to research the application of DNA in nanoelectronic devices. It has been revealed that DNA is a wide-gap semiconductor in the absence of doping. The conductivity of DNA has been controlled by chemical doping, electric field doping, and photo-doping. It has found that Poly(dG)[middle dot]Poly(dC) has the best conductivity and can function as a conducting nanowire. The pattern of DNA network is controlled by changing the concentration of the DNA solution.

  14. DNA Methylation

    OpenAIRE

    Alokail, Majed S.; Alenad, Amal M.

    2015-01-01

    The DNA of E. coli contains 19,120 6-methyladenines and 12,045 5-methylcytosines in addition to the four regular bases and these are formed by the postreplicative action of three DNA methyltransferases. The majority of the methylated bases are formed by the Dam and Dcm methyltransferases encoded by the dam (DNA adenine methyltransferase) and dcm (DNA cytosine methyltransferase) genes. Although not essential, Dam methylation is important for strand discrimination during repair of replication e...

  15. Context dependent DNA evolutionary models

    DEFF Research Database (Denmark)

    Jensen, Jens Ledet

    This paper is about stochastic models for the evolution of DNA. For a set of aligned DNA sequences, connected in a phylogenetic tree, the models should be able to explain - in probabilistic terms - the differences seen in the sequences. From the estimates of the parameters in the model one can...

  16. Generation of stable 'low phytic acid' transgenic rice through antisense repression of the 1D-myo-inositol 3-phosphate synthase gene (RINO1) using the 18-kDa oleosin promoter.

    Science.gov (United States)

    Kuwano, Mio; Mimura, Tetsuro; Takaiwa, Fumio; Yoshida, Kaoru T

    2009-01-01

    Phytic acid acts as the major storage form of phosphorus in plant seeds and is poorly digested by monogastric animals. The degradation of phytic acid in animal diets is necessary to overcome both environmental and nutritional issues. The enzyme 1D-myo-inositol 3-phosphate [Ins(3)P(1)] synthase (EC 5.5.1.4) catalyses the first step of myo-inositol biosynthesis and directs phytic acid biosynthesis in seeds. The rice Ins(3)P(1) synthase gene (RINO1) is highly expressed in developing seed embryos and in the aleurone layer, where phytic acid is synthesized and stored. In rice seeds, 18-kDa oleosin (Ole18) is expressed in a seed-specific manner, and its transcripts are restricted to the embryo and the aleurone layer. Therefore, to effectively suppress phytic acid biosynthesis, antisense RINO1 cDNA was expressed under the control of the Ole18 promoter, directing the same spatial pattern in seeds as RINO1 in transgenic rice plants. The generated transgenic rice plants showed strong 'low phytic acid' (lpa) phenotypes, in which seed phytic acid was reduced by 68% and free available phosphate was concomitantly increased. No negative effects on seed weight, germination or plant growth were observed. The available phosphate levels of the stable transgenic plants surpassed those of currently available rice lpa mutants.

  17. 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.

  18. The antisense expression of AhPEPC1 increases seed oil production in peanuts (Arachis hypogaea L.

    Directory of Open Access Journals (Sweden)

    L. Pan

    2016-12-01

    Full Text Available Although phosphoenolpyruvate carboxylases (PEPCs are reported to be involved in fatty acid accumulation, nitrogen assimilation, and salt and drought stresses, knowledge regarding PEPC gene functions is still limited, particularly in peanuts (Arachis hypogaea L.. In this study, the antisense expression of the peanut PEPC isoform 1 (AhPEPC1 gene increased the lipid content by 5.7%–10.3%. This indicated that AhPEPC1 might be related to plant lipid accumulation. The transgenic plants underwent more root elongation than the wild-type under salinity stress. Additionally, the specific down regulation of the AhPEPC1 gene improved the salt tolerance in peanuts. This is the first report on the role of PEPC in lipid accumulation and salt tolerance in peanuts.

  19. 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.

  20. Kink solitons in DNA

    CERN Document Server

    Zdravković, S; Daniel, M

    2012-01-01

    We here examine the nonlinear dynamics of artificial homogeneous DNA chain relying on the plain-base rotator model. It is shown that such dynamics can exhibit kink and antikink solitons of sine-Gordon type. In that respect we propose possible experimental assays based on single molecule micromanipulation techniques. The aim of these experiments is to excite the rotational waves and to determine their speeds along excited DNA. We propose that these experiments should be conducted either for the case of double stranded (DS) or single stranded (SS) DNA. A key question is to compare the corresponding velocities of the rotational waves indicating which one is bigger. The ratio of these velocities appears to be related with the sign of the model parameter representing ratio of the hydrogen-bonding and the covalent-bonding interaction within the considered DNA chain.

  1. Detection of very long antisense transcripts by whole transcriptome RNA-Seq analysis of Listeria monocytogenes by semiconductor sequencing technology.

    Science.gov (United States)

    Wehner, Stefanie; Mannala, Gopala K; Qing, Xiaoxing; Madhugiri, Ramakanth; Chakraborty, Trinad; Mraheil, Mobarak A; Hain, Torsten; Marz, Manja

    2014-01-01

    The Gram-positive bacterium Listeria monocytogenes is the causative agent of listeriosis, a severe food-borne infection characterised by abortion, septicaemia, or meningoencephalitis. L. monocytogenes causes outbreaks of febrile gastroenteritis and accounts for community-acquired bacterial meningitis in humans. Listeriosis has one of the highest mortality rates (up to 30%) of all food-borne infections. This human pathogenic bacterium is an important model organism for biomedical research to investigate cell-mediated immunity. L. monocytogenes is also one of the best characterised bacterial systems for the molecular analysis of intracellular parasitism. Recently several transcriptomic studies have also made the ubiquitous distributed bacterium as a model to understand mechanisms of gene regulation from the environment to the infected host on the level of mRNA and non-coding RNAs (ncRNAs). We have used semiconductor sequencing technology for RNA-seq to investigate the repertoire of listerial ncRNAs under extra- and intracellular growth conditions. Furthermore, we applied a new bioinformatic analysis pipeline for detection, comparative genomics and structural conservation to identify ncRNAs. With this work, in total, 741 ncRNA locations of potential ncRNA candidates are now known for L. monocytogenes, of which 611 ncRNA candidates were identified by RNA-seq. 441 transcribed ncRNAs have never been described before. Among these, we identified novel long non-coding antisense RNAs with a length of up to 5,400 nt e.g. opposite to genes coding for internalins, methylases or a high-affinity potassium uptake system, namely the kdpABC operon, which were confirmed by qRT-PCR analysis. RNA-seq, comparative genomics and structural conservation of L. monocytogenes ncRNAs illustrate that this human pathogen uses a large number and repertoire of ncRNA including novel long antisense RNAs, which could be important for intracellular survival within the infected eukaryotic host.

  2. Thiolated chitosan nanoparticles as a delivery system for antisense therapy: evaluation against EGFR in T47D breast cancer cells

    Directory of Open Access Journals (Sweden)

    Talaei F

    2011-09-01

    Full Text Available Fatemeh Talaei1, Ebrahim Azizi2, Rassoul Dinarvand3, Fatemeh Atyabi31Novel Drug Delivery Systems Lab, 2Molecular Research Lab, Department of Pharmacology and Toxicology, 3Nanotechnology Research Centre, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, IranAbstract: Thiolated chitosan has high transfection and mucoadhesive properties. We investigated the potential of two recently synthesized polymers: NAC-C (N-acetyl cysteine-chitosan and NAP-C (N-acetyl penicillamine-chitosan in anticancer drug delivery targeting epidermal growth factor receptor (EGFR. Doxorubicin (DOX and antisense oligonucleotide (ASOND-loaded polymer nanoparticles were prepared in water by a gelation process. Particle characterization, drug loading, and drug release were evaluated. To verify drug delivery efficiency in vitro experiments on a breast cancer cell line (T47D were performed. EGFR gene and protein expression was analyzed by real time quantitative polymerase chain reaction and Western blotting, respectively. A loading percentage of 63% ± 5% for ASOND and 70% ± 5% for DOX was achieved. Drug release data after 15 hours showed that ASOND and DOX were completely released from chitosan-based particles while a lower and more sustained release of only 22% ± 8% was measured for thiolated particles. In a cytosol simulated release medium/reducing environment, such as found intracellularly, polymer-based nanoparticles dissociated, liberating approximately 50% of both active substances within 7 hours. ASOND-loaded polymer nanoparticles had higher stability and high mucoadhesive properties. The ASOND-loaded thiolated particles significantly suppressed EGFR gene expression in T47D cells compared with ASOND-loaded chitosan particles and downregulated EGFR protein expression in cells. This study could facilitate future investigations into the functionality of NAP-C and NAC-C polymers as an efficient ASOND delivery system in vitro and in vivo

  3. An antisense RNA in a lytic cyanophage links psbA to a gene encoding a homing endonuclease.

    Science.gov (United States)

    Millard, Andrew D; Gierga, Gregor; Clokie, Martha R J; Evans, David J; Hess, Wolfgang R; Scanlan, David J

    2010-09-01

    Cyanophage genomes frequently possess the psbA gene, encoding the D1 polypeptide of photosystem II. This protein is believed to maintain host photosynthetic capacity during infection and enhance phage fitness under high-light conditions. Although the first documented cyanophage-encoded psbA gene contained a group I intron, this feature has not been widely reported since, despite a plethora of new sequences becoming available. In this study, we show that in cyanophage S-PM2, this intron is spliced during the entire infection cycle. Furthermore, we report the widespread occurrence of psbA introns in marine metagenomic libraries, and with psbA often adjacent to a homing endonuclease (HE). Bioinformatic analysis of the intergenic region between psbA and the adjacent HE gene F-CphI in S-PM2 showed the presence of an antisense RNA (asRNA) connecting these two separate genetic elements. The asRNA is co-regulated with psbA and F-CphI, suggesting its involvement with their expression. Analysis of scaffolds from global ocean survey datasets shows this asRNA to be commonly associated with the 3' end of cyanophage psbA genes, implying that this potential mechanism of regulating marine 'viral' photosynthesis is evolutionarily conserved. Although antisense transcription is commonly found in eukaryotic and increasingly also in prokaryotic organisms, there has been no indication for asRNAs in lytic phages so far. We propose that this asRNA also provides a means of preventing the formation of mobile group I introns within cyanophage psbA genes.

  4. PU.1 antisense lncRNA against its mRNA translation promotes adipogenesis in porcine preadipocytes.

    Science.gov (United States)

    Wei, N; Wang, Y; Xu, R-X; Wang, G-Q; Xiong, Y; Yu, T-Y; Yang, G-S; Pang, W-J

    2015-04-01

    Antisense long non-coding RNAs (AS lncRNAs) play important roles in refined regulation of animal gene expression. However, their functions and molecular mechanisms for domestic animal adipogenesis are largely unknown. Here, we found a novel AS lncRNA transcribed from the porcine PU.1 gene (also known as SPI1) by strand-specific RT-PCR. Results showed that PU.1 AS lncRNA was expressed and generally lower than the level of PU.1 mRNA in porcine subcutaneous adipose, heart, liver, spleen, lympha, skeletal muscle and kidney tissues. We further found that the levels of PU.1 mRNA and PU.1 protein were significantly lower in subcutaneous and intermuscular adipose than in mesenteric and greater omentum adipose, whereas the levels of PU.1 AS lncRNA showed no difference in porcine adipose tissues from four different parts of the body. During porcine adipogenesis, levels of PU.1 mRNA increased at day 2 and then gradually decreased. Meanwhile, PU.1 AS lncRNA exhibited an expression trend similar to PU.1 mRNA but sharply decreased after day 2. Interestingly, PU.1 protein level rose during differentiation. In addition, at day 6 after differentiation, knockdown of endogenous PU.1 promoted adipogenesis, whereas knockdown of endogenous PU.1 AS lncRNA had the opposite effect. Moreover, peroxisome proliferator-activated receptor gamma (PPARG) and fatty acid synthase (FASN) were significantly upregulated in the PU.1 shRNA treatment group (P PU.1 AS shRNA treatment group (P PU.1 mRNA/PU.1 AS lncRNA duplex was detected by an endogenous ribonuclease protection assay combined with RT-PCR. Based on the above results, we suggest that PU.1 AS lncRNA (vs. its mRNA translation) promotes adipogenesis through the formation of a sense-antisense RNA duplex with PU.1 mRNA.

  5. Detection of very long antisense transcripts by whole transcriptome RNA-Seq analysis of Listeria monocytogenes by semiconductor sequencing technology.

    Directory of Open Access Journals (Sweden)

    Stefanie Wehner

    Full Text Available The Gram-positive bacterium Listeria monocytogenes is the causative agent of listeriosis, a severe food-borne infection characterised by abortion, septicaemia, or meningoencephalitis. L. monocytogenes causes outbreaks of febrile gastroenteritis and accounts for community-acquired bacterial meningitis in humans. Listeriosis has one of the highest mortality rates (up to 30% of all food-borne infections. This human pathogenic bacterium is an important model organism for biomedical research to investigate cell-mediated immunity. L. monocytogenes is also one of the best characterised bacterial systems for the molecular analysis of intracellular parasitism. Recently several transcriptomic studies have also made the ubiquitous distributed bacterium as a model to understand mechanisms of gene regulation from the environment to the infected host on the level of mRNA and non-coding RNAs (ncRNAs. We have used semiconductor sequencing technology for RNA-seq to investigate the repertoire of listerial ncRNAs under extra- and intracellular growth conditions. Furthermore, we applied a new bioinformatic analysis pipeline for detection, comparative genomics and structural conservation to identify ncRNAs. With this work, in total, 741 ncRNA locations of potential ncRNA candidates are now known for L. monocytogenes, of which 611 ncRNA candidates were identified by RNA-seq. 441 transcribed ncRNAs have never been described before. Among these, we identified novel long non-coding antisense RNAs with a length of up to 5,400 nt e.g. opposite to genes coding for internalins, methylases or a high-affinity potassium uptake system, namely the kdpABC operon, which were confirmed by qRT-PCR analysis. RNA-seq, comparative genomics and structural conservation of L. monocytogenes ncRNAs illustrate that this human pathogen uses a large number and repertoire of ncRNA including novel long antisense RNAs, which could be important for intracellular survival within the infected

  6. Detection of Very Long Antisense Transcripts by Whole Transcriptome RNA-Seq Analysis of Listeria monocytogenes by Semiconductor Sequencing Technology

    Science.gov (United States)

    Wehner, Stefanie; Mannala, Gopala K.; Qing, Xiaoxing; Madhugiri, Ramakanth; Chakraborty, Trinad; Mraheil, Mobarak A.; Hain, Torsten; Marz, Manja

    2014-01-01

    The Gram-positive bacterium Listeria monocytogenes is the causative agent of listeriosis, a severe food-borne infection characterised by abortion, septicaemia, or meningoencephalitis. L. monocytogenes causes outbreaks of febrile gastroenteritis and accounts for community-acquired bacterial meningitis in humans. Listeriosis has one of the highest mortality rates (up to 30%) of all food-borne infections. This human pathogenic bacterium is an important model organism for biomedical research to investigate cell-mediated immunity. L. monocytogenes is also one of the best characterised bacterial systems for the molecular analysis of intracellular parasitism. Recently several transcriptomic studies have also made the ubiquitous distributed bacterium as a model to understand mechanisms of gene regulation from the environment to the infected host on the level of mRNA and non-coding RNAs (ncRNAs). We have used semiconductor sequencing technology for RNA-seq to investigate the repertoire of listerial ncRNAs under extra- and intracellular growth conditions. Furthermore, we applied a new bioinformatic analysis pipeline for detection, comparative genomics and structural conservation to identify ncRNAs. With this work, in total, 741 ncRNA locations of potential ncRNA candidates are now known for L. monocytogenes, of which 611 ncRNA candidates were identified by RNA-seq. 441 transcribed ncRNAs have never been described before. Among these, we identified novel long non-coding antisense RNAs with a length of up to 5,400 nt e.g. opposite to genes coding for internalins, methylases or a high-affinity potassium uptake system, namely the kdpABC operon, which were confirmed by qRT-PCR analysis. RNA-seq, comparative genomics and structural conservation of L. monocytogenes ncRNAs illustrate that this human pathogen uses a large number and repertoire of ncRNA including novel long antisense RNAs, which could be important for intracellular survival within the infected eukaryotic host. PMID

  7. Dna Sequencing

    Science.gov (United States)

    Tabor, Stanley; Richardson, Charles C.

    1995-04-25

    A method for sequencing a strand of DNA, including the steps off: providing the strand of DNA; annealing the strand with a primer able to hybridize to the strand to give an annealed mixture; incubating the mixture with four deoxyribonucleoside triphosphates, a DNA polymerase, and at least three deoxyribonucleoside triphosphates in different amounts, under conditions in favoring primer extension to form nucleic acid fragments complementory to the DNA to be sequenced; labelling the nucleic and fragments; separating them and determining the position of the deoxyribonucleoside triphosphates by differences in the intensity of the labels, thereby to determine the DNA sequence.

  8. The role of DNA methylation in cancer development.

    Directory of Open Access Journals (Sweden)

    Michał W Luczak

    2006-09-01

    Full Text Available Epigenetic modifications include DNA methylation and covalent modification of histones. These alterations are reversible but very stable and exert a significant impact on the regulation of gene expression. Changes in methylation of promoter or first exon may mimic the effect of mutations of various tumor suppressor genes (TSGs or protooncogenes. Carcinogenesis can also result from aberrations in genomic DNA methylation that include hypermethylation and hypomethylation of promoter or first exon of cancer-related genes. Hypermethylation of promoter of various TSGs causes their transcriptional silencing. However, hypomethylation of regulatory DNA sequences activates transcription of protooncogenes, retrotransposons, as well as genes encoding proteins involved in genomic instability and malignant cell metastasis. The methylation of genomic DNA in malignant cells is catalyzed by DNA methyltransferases DNMT1 and DNMT3B, revealing significantly elevated expression in different types of cancers. The reversibility of hypermethylation can be used as target of therapeutic treatment in cancer. DNMT 1 and DNMT3B inhibitors including 5-Aza-2'-deoxycytidine and antisense oligonucleotides have been applied in clinical trials of such treatment. Identification of aberrations of DNA methylation in cancer cells is a new field of investigation in carcinogenesis. We believe that epigenetic cancer diagnostic and therapy will be achieved in the next decades.

  9. [Integration of different T-DNA structures of ACC oxidase gene into carnation genome extended cut flower vase-life differently].

    Science.gov (United States)

    Yu, Yi-Xun; Bao, Man-Zhu

    2004-09-01

    The cultivar 'Master' of carnation (Dianthus caryophyllus L.) was transformed with four T-DNA structures containing sense, antisense, sense direct repeat and antisense direct repeat gene of ACC oxidase mediated by Agrobacterium tumefaciens. Southern blotting detection showed that foreign gene was integrated into the carnation genome and 14 transgenic lines were obtained. The transgenic plants were transplanted to soil and grew normally in greenhouse. Of the 12 transgenic lines screened, the cut flower vase life of 8 transgenic lines is up to 11 days and the longest one is 12.8 days while the vase life of the control is 5.8 days under 25 degrees C. The vase life of 2 lines out of 3 with single sense ACO gene is same as that of the control, while the vase life of 3 lines out of 4 with single antisense ACO gene is prolonged. The vase life of cut flowers of 5 lines with direct repeat ACO genes is all prolonged by about 6 days, while the vase life of 3 out of 7 lines with single ACO gene is same as that of the control. During the senescence of cut flowers, the ethylene production of the most of the transgenic lines decreased significantly, and the production of ethylene is not detectable in lines T456, T556 and T575. The results of the research demonstrate that antisense foreign gene inhibits expression of endogenesis gene more significantly than sense one. Both sense direct repeat and antisense direct repeat foreign genes can suppress endogenous gene expression more significantly comparing to single foreign genes. The transgenic lines obtained from this research are useful to minimize carnation cut flower transportation and storage expenses.

  10. Supergranular Parameters

    Science.gov (United States)

    Udayashankar, Paniveni

    2016-07-01

    I study the complexity of supergranular cells using intensity patterns from Kodaikanal solar observatory. The chaotic and turbulent aspect of the solar supergranulation can be studied by examining the interrelationships amongst the parameters characterizing supergranular cells namely size, horizontal flow field, lifetime and physical dimensions of the cells and the fractal dimension deduced from the size data. The findings are supportive of Kolmogorov's theory of turbulence. The Data consists of visually identified supergranular cells, from which a fractal dimension 'D' for supergranulation is obtained according to the relation P α AD/2 where 'A' is the area and 'P' is the perimeter of the supergranular cells. I find a fractal dimension close to about 1.3 which is consistent with that for isobars and suggests a possible turbulent origin. The cell circularity shows a dependence on the perimeter with a peak around (1.1-1.2) x 105 m. The findings are supportive of Kolmogorov's theory of turbulence.

  11. Single and repeated moderate consumption of native or dealcoholized red wine show different effects on antioxidant parameters in blood and DNA strand breaks in peripheral leukocytes in healthy volunteers: a randomized controlled trial [ISRCTN68505294

    Directory of Open Access Journals (Sweden)

    Spengler Ulrich

    2005-11-01

    Full Text Available Abstract Background Red wine (RW is rich in antioxidant polyphenols that might protect from oxidative stress related diseases, such as cardiovascular disease and cancer. Antioxidant effects after single ingestion of RW or dealcoholized RW (DRW have been observed in several studies, but results after regular consumption are contradictory. Thus, we examined if single or repeated consumption of moderate amounts of RW or DRW exert antioxidant activity in vivo. Methods Total phenolic content and concentration of other antioxidants in plasma/serum, total antioxidant capacity (TEAC in plasma as well as DNA strand breaks in peripheral leukocytes were measured in healthy non-smokers A before, 90 and 360 min after ingestion of one glass of RW, DRW or water; B before and after consumption of one glass of RW or DRW daily for 6 weeks. DNA strand breaks (SB were determined by single cell gel electrophoresis (Comet Assay in untreated cells and after induction of oxidative stress ex vivo with H2O2 (300 μM, 20 min. Results Both RW and DRW transiently increased total phenolic content in plasma after single consumption, but only RW lead to a sustained increase if consumed regularly. Plasma antioxidant capacity was not affected by single or regular consumption of RW or DRW. Effects of RW and DRW on DNA SB were conflicting. DNA strand breaks in untreated cells increased after a single dose of RW and DRW, whereas H2O2 induced SB were reduced after DRW. In contrast, regular RW consumption reduced SB in untreated cells but did not affect H2O2 induced SB. Conclusion The results suggest that consumption of both RW and DRW leads to an accumulation of phenolic compounds in plasma without increasing plasma antioxidant capacity. Red wine and DRW seem to affect the occurrence of DNA strand breaks, but this cannot be referred to antioxidant effects.

  12. Automated DNA extraction from pollen in honey.

    Science.gov (United States)

    Guertler, Patrick; Eicheldinger, Adelina; Muschler, Paul; Goerlich, Ottmar; Busch, Ulrich

    2014-04-15

    In recent years, honey has become subject of DNA analysis due to potential risks evoked by microorganisms, allergens or genetically modified organisms. However, so far, only a few DNA extraction procedures are available, mostly time-consuming and laborious. Therefore, we developed an automated DNA extraction method from pollen in honey based on a CTAB buffer-based DNA extraction using the Maxwell 16 instrument and the Maxwell 16 FFS Nucleic Acid Extraction System, Custom-Kit. We altered several components and extraction parameters and compared the optimised method with a manual CTAB buffer-based DNA isolation method. The automated DNA extraction was faster and resulted in higher DNA yield and sufficient DNA purity. Real-time PCR results obtained after automated DNA extraction are comparable to results after manual DNA extraction. No PCR inhibition was observed. The applicability of this method was further successfully confirmed by analysis of different routine honey samples.

  13. The detection and analysis of sperm motion parameters and DNA fragmentation index in asthenozoospermia patients%弱精子症患者精子运动参数及DNA 碎片化指数的检测与分析

    Institute of Scientific and Technical Information of China (English)

    焦瑞宝; 姚余有; 唐吉斌; 冯恒孝; 凤俊蓉

    2015-01-01

    Objective To study the changes of sperm motion parameters and DNA fragmentation index (DFI) in patients with asthenozoospermia.Methods The routine analysis, semen analyzer activity detection and fluorescence microzooscopy of 41 cases of asthenozoospermia patients ( asthenozoospermia group) and 48 cases of normal vitality patients(normal vitality group) were performed.Semen routine parameters(sperm concentration, total number of sperm and sperm activity rate), sperm motion parameters [ progressive motility ( PR), average path velocity ( VAP), curvilinear velocity ( VCL), straight line velocity ( VSL), linearity ( LIN), wobble ( WOB), straightness ( STR), amplitude of lateral head displacement(ALH), beat-cross frequency(BCF) and mean angular displacement(MAD)] and DFI in patients with asthenozoospermia and normal vitality group were compared. Results The sperm concentration, total number of sperm, sperm activity rate, PR, VAP, VCL, VSL, LIN, WOB, STR, ALH, BCF and MAD of asthenozoospermia group were (69.0 ±49.1) ×10 6 /mL, (203 ±159) ×10 6 , 60.8% ±15.7%, 21.1% ± 8.0%,(10.7 ±2.8)μm/s, (20.6 ±4.9)μm/s, (6.3 ±2.2)μm/s, 29.9% ±8.1%,51.4% ±5.8%, 57.2% ± 10.4%, (0.53 ±0.12)μm, (3.20 ±0.85)Hz and (14.2 ±3.2)°, respectively.These parameters were significantly lower than those of normal vitality group[(98.8 ±38.2) ×10 6 /mL(P <0.05),(281 ±171) ×10 6 (P <0.05), 82.0%±7.9%(P <0.01), 45.1% ±8.5%(P <0.01), (18.5 ±3.0)μm/s(P <0.01), (30.7 ±5.3)μm/s(P <0.01), (12.0 ±2.3)μm/s (P <0.01), 39.6% ±7.0%(P <0.01), 60.5% ±5.0%(P <0.01), 65.1% ±7.2%(P <0.01), (0.72 ±0.13)μm (P <0.01), (5.07 ±0.81)Hz(P <0.01) and (17.8 ±2.5)°(P <0.01)].The DFI in asthenozoospermia group(17.9% ±12.5%) was significantly higher than that in normal vitality group(8.5 ±6.4%, P <0.01).Conclusions Besides the performance of low sperm motility, asthenozoospermia is still accompanied by decreasing sperm concentration, total number of sperm and sperm motion parameters and

  14. 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.

  15. 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

    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...

  16. The human PINK1 locus is regulated in vivo by a non-coding natural antisense RNA during modulation of mitochondrial function

    DEFF Research Database (Denmark)

    Scheele, Camilla; Petrovic, Natasa; Faghihi, Mohammad A

    2007-01-01

    BACKGROUND: Mutations in the PTEN induced putative kinase 1 (PINK1) are implicated in early-onset Parkinson's disease. PINK1 is expressed abundantly in mitochondria rich tissues, such as skeletal muscle, where it plays a critical role determining mitochondrial structural integrity in Drosophila....... RESULTS: Herein we characterize a novel splice variant of PINK1 (svPINK1) that is homologous to the C-terminus regulatory domain of the protein kinase. Naturally occurring non-coding antisense provides sophisticated mechanisms for diversifying genomes and we describe a human specific non-coding antisense...... expressed at the PINK1 locus (naPINK1). We further demonstrate that PINK1 varies in vivo when human skeletal muscle mitochondrial content is enhanced, supporting the idea that PINK1 has a physiological role in mitochondrion. The observation of concordant regulation of svPINK1 and naPINK1 during in vivo...

  17. GPR39 splice variants versus antisense gene LYPD1: expression and regulation in gastrointestinal tract, endocrine pancreas, liver, and white adipose tissue

    DEFF Research Database (Denmark)

    Egerod, Kristoffer L; Holst, Birgitte; Petersen, Pia S;

    2007-01-01

    five-transmembrane form, GPR39-1b. The 3' exon of the GPR39 gene overlaps with an antisense gene called LYPD1 (Ly-6/PLAUR domain containing 1). Quantitative RT-PCR analysis demonstrated that GPR39-1a is expressed selectively throughout the gastrointestinal tract, including the liver and pancreas...... as well as in the kidney and adipose tissue, whereas the truncated GPR39-1b form has a more broad expression pattern, including the central nervous system but with highest expression in the stomach and small intestine. In contrast, the LYPD1 antisense gene is highly expressed throughout the central...... nervous system as characterized with both quantitative RT-PCR and in situ hybridization analysis. A functional analysis of the GPR39 promoter region identified sites for the hepatocyte nuclear factors 1alpha and 4alpha (HNF-1alpha and -4alpha) and specificity protein 1 (SP1) transcription factors as being...

  18. 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.

  19. Role of Leishmania (Leishmania chagasi amastigote cysteine protease in intracellular parasite survival: studies by gene disruption and antisense mRNA inhibition

    Directory of Open Access Journals (Sweden)

    Kucknoor Ashwini S

    2005-02-01

    Full Text Available Abstract Background The parasitic protozoa belonging to Leishmania (L. donovani complex possess abundant, developmentally regulated cathepsin L-like cysteine proteases. Previously, we have reported the isolation of cysteine protease gene, Ldccys2 from Leishmania (L. chagasi. Here, we have further characterized this cysteine protease gene and demonstrated its role during infection and survival of Leishmania (L. chagasi within the U937 macrophage cells. Results The amastigote specific Ldccys2 genes of L. (L. chagasi and L. (L. donovani have identical gene organization, as determined by southern blots. In vivo expression analyses by Northern blots showed that Ldccys2 is amastigote specific. Western blot using anti-Ldccys2 antibody confirmed the amastigote specific protein expression. Recombinant expression of Ldccys2, a 30 kDA protein, was functionally active in a gelatin assay. Results from Ldccys2 heterozygous knockout mutants showed its role during macrophage infection and in intra-macrophage survival of the parasites. Since attempts to generate null mutants failed, we used antisense RNA inhibition to regulate Ldcccys2 gene expression. Not surprisingly, the results from antisense studies further confirmed the results from heterozygous knockout mutants, reiterating the importance of amastigote specific cysteine proteases in Leishmania infection and pathogenesis. Conclusions The study shows that Ldccys2 is a developmentally regulated gene and that Ldccys2 is expressed only in infectious amastigote stages of the parasite. The collective results from both the heterozygous knockout mutants and antisense mRNA inhibition studies shows that Ldccys2 helps in infection and survival of L. (L. chagasi amastigotes within the macrophage cells. Finally, antisense RNA technique can be used as an alternate approach to gene knockout, for silencing gene expression in L. (L. chagasi, especially in cases such as this, where a null mutant cannot be achieved by

  20. Arabidopsis RRP6L1 and RRP6L2 function in FLOWERING LOCUS C silencing via regulation of antisense RNA synthesis.

    Directory of Open Access Journals (Sweden)

    Jun-Hye Shin

    2014-09-01

    Full Text Available The exosome complex functions in RNA metabolism and transcriptional gene silencing. Here, we report that mutations of two Arabidopsis genes encoding nuclear exosome components AtRRP6L1 and AtRRP6L2, cause de-repression of the main flowering repressor FLOWERING LOCUS C (FLC and thus delay flowering in early-flowering Arabidopsis ecotypes. AtRRP6L mutations affect the expression of known FLC regulatory antisense (AS RNAs AS I and II, and cause an increase in Histone3 K4 trimethylation (H3K4me3 at FLC. AtRRP6L1 and AtRRP6L2 function redundantly in regulation of FLC and also act independently of the exosome core complex. Moreover, we discovered a novel, long non-coding, non-polyadenylated antisense transcript (ASL, for Antisense Long originating from the FLC locus in wild type plants. The AtRRP6L proteins function as the main regulators of ASL synthesis, as these mutants show little or no ASL transcript. Unlike ASI/II, ASL associates with H3K27me3 regions of FLC, suggesting that it could function in the maintenance of H3K27 trimethylation during vegetative growth. AtRRP6L mutations also affect H3K27me3 levels and nucleosome density at the FLC locus. Furthermore, AtRRP6L1 physically associates with the ASL transcript and directly interacts with the FLC locus. We propose that AtRRP6L proteins participate in the maintenance of H3K27me3 at FLC via regulating ASL. Furthermore, AtRRP6Ls might participate in multiple FLC silencing pathways by regulating diverse antisense RNAs derived from the FLC locus.

  1. 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...

  2. Effects of Intrathecally Administerd NaV1.8 Antisense Oligonucleotide on the Expression of Sodium Channel mRNA in Dorsal Root Ganglion

    Institute of Scientific and Technical Information of China (English)

    LIU Yongmin; YAO Shanglong; SONG Wenge; WANG Yuelan; LIU Dong; ZEN Lian

    2005-01-01

    Neuropathic pain has been hypothesized to be the result of aberrant expression and function of sodium channels at the site of injury. To investigate the effects of NaV1.8 antisense oligonucleotide on the expression of sodium channel mRNA in dorsal root ganglion (DRG) neurons in chronic neuropathic pain. 24 Sprague-Dawley rats weighing 200-260 g were anesthetized with the in of sciatic nerve trunk by 4-0 chromic gut. The mechanical and thermal pain threshold were measured before operation and 1, 3, 5, 7, 9, 11, 13 days after operation. A PE-10 catheter was implanted in subarachnoid space at lumbar region. On the 7th postoperative day the animals were randomly divided into 4 groups. The drugs were injected intrathecally twice a day for 5 consecutive days in group 2-4. The animals were decapitated 14 days after the surgery. The L4-L6 DRG of the operated side was removed and crushed, and total RNA was extracted with Trizol reagent. The contralateral side was used as control. The change of NaV1.8 sodium channel transcripts was determined by RT-PCR. Pain threshold was significantly lowered after CCI as compared with that in control group and was elevated 3 days after antisense oligonucleotide injection. Sensory neuron specific TTX-R sodium channel NaV1.8 transcript was down-regulated after antisense oligonucleotide injection at the dosage of 45 μg as compared with that in CCI group (P<0.01), and it was even greater at the dosage of 90 μg. The intrathecally injected NaV1.8 antisense oligonucleotide can reduce the mechanical allodynia and thermal hyperalgesia partially by downregulating the SNS transcript expression.

  3. DNA glue

    DEFF Research Database (Denmark)

    Filichev, Vyacheslav V; Astakhova, Irina V.; Malakhov, Andrei D.

    2008-01-01

    Significant alterations in thermal stability of parallel DNA triplexes and antiparallel duplexes were observed upon changing the attachment of ethynylpyrenes from para to ortho in the structure of phenylmethylglycerol inserted as a bulge into DNA (TINA). Insertions of two ortho-TINAs as a pseudo...

  4. 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

  5. Antisense targeting of 3' end elements involved in DUX4 mRNA processing is an efficient therapeutic strategy for facioscapulohumeral dystrophy: a new gene-silencing approach.

    Science.gov (United States)

    Marsollier, Anne-Charlotte; Ciszewski, Lukasz; Mariot, Virginie; Popplewell, Linda; Voit, Thomas; Dickson, George; Dumonceaux, Julie

    2016-04-15

    Defects in mRNA 3'end formation have been described to alter transcription termination, transport of the mRNA from the nucleus to the cytoplasm, stability of the mRNA and translation efficiency. Therefore, inhibition of polyadenylation may lead to gene silencing. Here, we choose facioscapulohumeral dystrophy (FSHD) as a model to determine whether or not targeting key 3' end elements involved in mRNA processing using antisense oligonucleotide drugs can be used as a strategy for gene silencing within a potentially therapeutic context. FSHD is a gain-of-function disease characterized by the aberrant expression of the Double homeobox 4 (DUX4) transcription factor leading to altered pathogenic deregulation of multiple genes in muscles. Here, we demonstrate that targeting either the mRNA polyadenylation signal and/or cleavage site is an efficient strategy to down-regulate DUX4 expression and to decrease the abnormally high-pathological expression of genes downstream of DUX4. We conclude that targeting key functional 3' end elements involved in pre-mRNA to mRNA maturation with antisense drugs can lead to efficient gene silencing and is thus a potentially effective therapeutic strategy for at least FSHD. Moreover, polyadenylation is a crucial step in the maturation of almost all eukaryotic mRNAs, and thus all mRNAs are virtually eligible for this antisense-mediated knockdown strategy.

  6. Construction of Bcl-2 Antisense RNA Expressed by Defect Retroviral Vector%反义bcl-2基因逆转录病毒表达载体的构建

    Institute of Scientific and Technical Information of China (English)

    林祥华; 陈志哲; 杨婷; 吕联煌

    2001-01-01

    目的克隆bcl-2基因,构建其不同反义核酸的逆转录病毒表达载体。方法设计一对两端带有EcoRⅠ酶切位点的引物,RT-PCR克隆含bcl-2全部编码区的848 bp片段,并连接到T载体,测序正确后正反向亚克隆到pLXSN中EcoRⅠ位点;从pBluescriptⅡSK(-)bcl-2中用BamHⅠ切下含有bcl-2阅读框起始部位的部分编码区亚克隆到pLXSN BamHⅠ位点。结果构建带有正反向含bcl-2阅读框的全部编码区和部分编码区的逆转录病毒表达载体。结论在已知基因序列克隆中,联合RT-PCR和T载体是一个简单、快速、有效的方法,为基因转移和表达研究提供基础。%Objective To clone bcl-2 gene and develop two different bcl-2 antisense RNA expressed by defect retroviral vector. Methods Both upper and down primers with the end of EcoRⅠ site were designed to amplify 848 bp cDNA fragement including the whole bcl-2 exon by RT-PCR. After confirmation of the sequences of cloned bcl-2 exon by T vector, both forward and backward of bcl-2 cDNA were subcloned into the EcoRⅠ site of retroviral vector pLXSN. In the other way, 600 bp cDNA fragement with the beginning of coding bcl-2 exon was obtained by digestion of pBluescriptⅡ SK(-) bcl-2 with BamHⅠ restriction enzyme and also subcloned into the BamHⅠ site of retroviral vector pLXSN. Results Retroviral vectors with both forward and backward, whole and part of bcl-2 exon were successfully constructed. Conclusion Under the knowledge of targeting cDNA sequences, the approach for clonning of targeting gene by combination of RT-PCR with T vector is simple, rapid, effective, and offering the underlying bases of gene transfer and expression.

  7. Direct current hopping conductance along DNA chain

    Institute of Scientific and Technical Information of China (English)

    Ma Song-Shan; Xu Hui; Liu Xiao-Liang; Li Ming-Jun

    2007-01-01

    This paper proposes a model of direct current(DC) electron hopping transport in DNA,in which DNA is considered as a binary one-dimensional disordered system.To quantitatively study the DC conductivity in DNA,it numerically calculates the DC conductivity of DNA chains with difierent parameter values.The result shows that the DC conductivity of DNA chain increases with the increase of temperature.And the conductivity of DNA chain is depended on the probability P.which represents the degree of compositional disorder in a DNA sequence to some extent.For P<0.5,the conductivity of DNA chain decreases with the increase of P,while for P≥0.5,the conductivity increases with the increase of p.The DC conductivity in DNA chain also varies with the change of the electric field,it presents non-Ohm's law conductivity characteristics.

  8. Biomarkers of genotoxicity of air pollution (the AULIS project): bulky DNA adducts in subjects with moderate to low exposures to airborne polycyclic aromatic hydrocarbons and their relationship to environmental tobacco smoke and other parameters

    DEFF Research Database (Denmark)

    Georgiadis, P.; Topinka, J.; Stoikidou, M.

    2001-01-01

    tobacco smoke (ETS), namely (i) declared times of exposure to ETS during the 24 h prior to blood donation, (ii) plasma cotinine levels and (iii) chrysene/benzo[g,h,i]perylene ratios in the profile of personal PAH exposure. Furthermore, among the Halkida campus area subjects (but not the remaining subjects......, exposure to ETS was a significant determinant of the observed DNA damage. Gender had a consistent and significant effect on adduct levels (males having higher levels), which remained significant even after multiple regression analysis. Habitual consumption of roasted meat was significantly associated...

  9. [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 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 expression of apollon mRNA, and inhibit the proliferation, induce apoptosis, arrest cell cycle at S phase of colorectal cancer Lovo cells in vitro and enhance the chemo-sensitivity to 5-FU, DDP and EPI.

  10. Lack of clinical pharmacodynamic and pharmacokinetic drug-drug interactions between warfarin and the antisense oligonucleotide mipomersen.

    Science.gov (United States)

    Li, Zhaoyang; Hard, Marjie L; Grundy, John S; Singh, Tejdip; von Moltke, Lisa L; Boltje, Ingrid

    2014-08-01

    Mipomersen is a second-generation antisense oligonucleotide indicated as an adjunct therapy for homozygous familial hypercholesterolemia (HoFH). Warfarin is commonly prescribed for a variety of cardiac disorders in homozygous familial hypercholesterolemia population, and concurrent use of warfarin and mipomersen is likely. This open-label, single-sequence 2-period phase 1 study in healthy subjects evaluated the potential drug-drug interactions between mipomersen and warfarin. The subjects received a single oral 25 mg dose of warfarin alone on day 1, and after a 7-day washout period, received 200 mg mipomersen alone subcutaneously every other day on days 8-12, and received both concurrently on day 14. Coadministration of mipomersen did not change the pharmacodynamics (international normalized ratio, prothrombin time, and activated partial thromboplastin time) and pharmacokinetics (PK) of warfarin. There were no clinically significant changes in the PK of mipomersen with concurrent administration of warfarin. There were no events indicative of an increase in bleeding tendency when warfarin was coadministered with mipomersen, and the adverse event profile of mipomersen did not appear to be altered in combination with warfarin, as compared with that of the respective reference treatment. The combination of these 2 medications appeared to be safe and well tolerated. These results suggest that the dosage adjustment of warfarin or mipomersen is not expected to be necessary with coadministration.

  11. Allele-specific suppression of mutant huntingtin using antisense oligonucleotides: providing a therapeutic option for all Huntington disease patients.

    Directory of Open Access Journals (Sweden)

    Niels H Skotte

    Full Text Available Huntington disease (HD is an inherited, fatal neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. The mutant protein causes neuronal dysfunction and degeneration resulting in motor dysfunction, cognitive decline, and psychiatric disturbances. Currently, there is no disease altering treatment, and symptomatic therapy has limited benefit. The pathogenesis of HD is complicated and multiple pathways are compromised. Addressing the problem at its genetic root by suppressing mutant huntingtin expression is a promising therapeutic strategy for HD. We have developed and evaluated antisense oligonucleotides (ASOs targeting single nucleotide polymorphisms that are significantly enriched on HD alleles (HD-SNPs. We describe our structure-activity relationship studies for ASO design and find that adjusting the SNP position within the gap, chemical modifications of the wings, and shortening the unmodified gap are critical for potent, specific, and well tolerated silencing of mutant huntingtin. Finally, we show that using two distinct ASO drugs targeting the two allelic variants of an HD-SNP could provide a therapeutic option for all persons with HD; allele-specifically for roughly half, and non-specifically for the remainder.

  12. Effective exon skipping and dystrophin restoration by 2'-o-methoxyethyl antisense oligonucleotide in dystrophin-deficient mice.

    Directory of Open Access Journals (Sweden)

    Lu Yang

    Full Text Available Antisense oligonucleotide (AO-mediated exon-skipping therapy is one of the most promising therapeutic strategies for Duchenne Muscular Dystrophy (DMD and several AO chemistries have been rigorously investigated. In this report, we focused on the effect of 2'-O-methoxyethyl oligonucleotides (MOE on exon skipping in cultured mdx myoblasts and mice. Efficient dose-dependent skipping of targeted exon 23 was achieved in myoblasts with MOE AOs of different lengths and backbone chemistries. Furthermore, we established that 25-mer MOE phosphorothioate (PS AOs provided the greatest exon-skipping efficacy. When compared with 2'O methyl phosphorothioate (2'OmePS AOs, 25-mer MOE (PS AOs also showed higher exon-skipping activity in vitro and in mdx mice after intramuscular injections. Characterization of uptake in vitro corroborated with exon-skipping results, suggesting that increased uptake of 25-mer MOE PS AOs might partly contribute to the difference in exon-skipping activity observed in vitro and in mdx mice. Our findings demonstrate the substantial potential for MOE PS AOs as an alternative option for the treatment of DMD.

  13. 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.

  14. Inhibition of adenovirus replication by the E1A antisense transcript initiated from hsp70 and VA-1 promoters.

    Science.gov (United States)

    Miroshnichenko, O I; Borisenko, A S; Ponomareva, T I; Tikhonenko, T I

    1990-03-01

    The E1A region of the adenoviral genome, important for initiation of virus infection and activation of other viral genes, was chosen as a target for engineering antisense RNA (asRNA) to inhibit adenovirus 5 (Ad5) replication in COS-1 cell culture in vitro. The hsp70 promoter, taken from the appropriate heat-shock-protein gene of Drosophila melanogaster, and the VA-1 RNA promoter, derived from the Ad5 gene coding for low-molecular-mass VA-1 RNA and recognized by RNA polymerase III were used as regulatory elements of transcription. The two types of recombinant constructs contained E1A fragments of 710 bp (hsp70 constructs) or 380 or 740 bp (VA-1 RNA constructs) in reverse orientation relative to the promoter position, as well as a transcription termination signal, the SV40 ori, and the gene controlling Geneticin (antibiotic G418) resistance (G418R). After selection of transfected COS-1 cells in the presence of G418, a number of stable G418R cell lines were raised which expressed engineered asRNAs. Plating of Ad5 suspensions of known titre on monolayers of transfected COS-1 cells clearly showed strong inhibition of adenovirus replication by asRNAs: 75% with the hsp70 promoter and 90% with the VA-1 RNA promoter.

  15. Wild-type mouse models to screen antisense oligonucleotides for exon-skipping efficacy in Duchenne muscular dystrophy.

    Directory of Open Access Journals (Sweden)

    Limin Cao

    Full Text Available A readily available animal model is essential for rapidly identifying effective treatments for Duchenne muscular dystrophy (DMD, a devastating neuromuscular disorder caused by the lack of dystrophin protein, which results from frame-disrupting mutations in the DMD gene. Currently, the mdx mouse is the most commonly used model for antisense oligonucleotide (AO-mediated exon skipping pre-clinical studies, with a mild phenotype. However, the accessibility of mdx mouse colonies particularly in developing countries can constrain research. Therefore in this study we explore the feasibility of using wild-type mice as models to establish exon-skipping efficiency of various DMD AO chemistries and their conjugates. Four different strains of wild-type mice and six different AO chemistries were investigated intramuscularly and the results indicated that the same exon-skipping efficiency was achieved for all tested AOs as that from mdx mice. Notably, levels of exon-skipping obtained in C57BL6 and C3H and mdx mice were most closely matched, followed by ICR and BALB/C mice. Systemic validation revealed that wild-type mice are less responsive to AO-mediated exon skipping than mdx mice. Our study provides evidence for the first time that wild-type mice can be appropriate models for assessing DMD AO exon-skipping efficiency with similar sensitivity to that of mdx mice and this finding can further accelerate the development of effective DMD AOs.

  16. Regulated expression of PTPRJ/CD148 and an antisense long noncoding RNA in macrophages by proinflammatory stimuli.

    Directory of Open Access Journals (Sweden)

    Richa K Dave

    Full Text Available PTPRJ/CD148 is a tyrosine phosphatase that has tumour suppressor-like activity. Quantitative PCR of various cells and tissues revealed that it is preferentially expressed in macrophage-enriched tissues. Within lymphoid tissues immunohistochemistry revealed that PTPRJ/CD148 co-localised with F4/80, indicating that macrophages most strongly express the protein. Macrophages express the highest basal level of ptprj, and this is elevated further by treatment with LPS and other Toll-like receptor ligands. In contrast, CSF-1 treatment reduced basal and stimulated Ptprj expression in human and mouse cells, and interferon also repressed Ptprj expression. We identified a 1006 nucleotide long noncoding RNA species, Ptprj-as1 that is transcribed antisense to Ptprj. Ptprj-as1 was highly expressed in macrophage-enriched tissue and was transiently induced by Toll-like receptor ligands with a similar time course to Ptprj. Finally, putative transcription factor binding sites in the promoter region of Ptprj were identified.

  17. In vitro and in vivo rescue of aberrant splicing in CEP290-associated LCA by antisense oligonucleotide delivery.

    Science.gov (United States)

    Garanto, Alejandro; Chung, Daniel C; Duijkers, Lonneke; Corral-Serrano, Julio C; Messchaert, Muriël; Xiao, Ru; Bennett, Jean; Vandenberghe, Luk H; Collin, Rob W J

    2016-06-15

    Leber congenital amaurosis (LCA) is a severe disorder resulting in visual impairment usually starting in the first year of life. The most frequent genetic cause of LCA is an intronic mutation in CEP290 (c.2991 + 1655A > G) that creates a cryptic splice donor site resulting in the insertion of a pseudoexon (exon X) into CEP290 mRNA. Previously, we showed that naked antisense oligonucleotides (AONs) effectively restored normal CEP290 splicing in patient-derived lymphoblastoid cells. We here explore the therapeutic potential of naked and adeno-associated virus (AAV)-packaged AONs in vitro and in vivo In both cases, AON delivery fully restored CEP290 pre-mRNA splicing, significantly increased CEP290 protein levels and rescued a ciliary phenotype present in patient-derived fibroblast cells. Moreover, administration of naked and AAV-packaged AONs to the retina of a humanized mutant Cep290 mouse model, carrying the intronic mutation, showed a statistically significant reduction of exon X-containing Cep290 transcripts, without compromising the retinal structure. Together, our data highlight the tremendous therapeutic prospective of AONs for the treatment of not only CEP290-associated LCA but potentially many other subtypes of retinal dystrophy caused by splicing mutations.

  18. Regulated Expression of PTPRJ/CD148 and an Antisense Long Noncoding RNA in Macrophages by Proinflammatory Stimuli

    Science.gov (United States)

    Dave, Richa K.; Dinger, Marcel E.; Andrew, Megan; Askarian-Amiri, Marjan; Hume, David A.; Kellie, Stuart

    2013-01-01

    PTPRJ/CD148 is a tyrosine phosphatase that has tumour suppressor-like activity. Quantitative PCR of various cells and tissues revealed that it is preferentially expressed in macrophage-enriched tissues. Within lymphoid tissues immunohistochemistry revealed that PTPRJ/CD148 co-localised with F4/80, indicating that macrophages most strongly express the protein. Macrophages express the highest basal level of ptprj, and this is elevated further by treatment with LPS and other Toll-like receptor ligands. In contrast, CSF-1 treatment reduced basal and stimulated Ptprj expression in human and mouse cells, and interferon also repressed Ptprj expression. We identified a 1006 nucleotide long noncoding RNA species, Ptprj-as1 that is transcribed antisense to Ptprj. Ptprj-as1 was highly expressed in macrophage-enriched tissue and was transiently induced by Toll-like receptor ligands with a similar time course to Ptprj. Finally, putative transcription factor binding sites in the promoter region of Ptprj were identified. PMID:23840844

  19. Antisense suppression of cucumber (Cucumis sativus L.) sucrose synthase 3 (CsSUS3) reduces hypoxic stress tolerance.

    Science.gov (United States)

    Wang, Hongyun; Sui, Xiaolei; Guo, Jinju; Wang, Zhenyu; Cheng, Jintao; Ma, Si; Li, Xiang; Zhang, Zhenxian

    2014-03-01

    Sucrose synthase (SUS; EC 2.4.1.13) plays important roles in sugar metabolism and abiotic stress response. But the genes encoding SUS in cucumber (Cucumis sativus L.) have not been well studied. Here, we isolated four cucumber sucrose synthase genes (CsSUS). Among them, CsSUS3, which highly expressed in the roots, was chosen for further study. Immunolocalization and subcellular localization analysis indicated that CsSUS3 localized in the cytosol and the plasma membrane, and mainly existed in the companion cells of phloem in the roots. When suffering hypoxia stress from flooding, CsSUS3 expression and SUS activity in roots increased, especially in the lateral roots; moreover, the soluble SUS activity increased clearly, but the membrane fraction hardly changed. Compared with the wild-type cucumbers, the transgenic lines with antisense expression of CsSUS3 were more sensitive to flooding. After 6 d of flooding, the SUS activity, soluble sugar and uridine 5'-diphosphate glucose (UDPG) content and the ratio of ATP/ADP in the roots of transgenic plants were significantly lower than that in wild-type plants. Moreover, the transgenic lines grew more slowly with more yellow necrosis in the leaves. These findings suggested CsSUS3 participated in resisting hypoxic stress. Furthermore, the mechanism of CsSUS3 in resisting hypoxic stress was also discussed.

  20. 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.

  1. 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.

  2. 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.

  3. Apoptosis of drug-resistant human ovarian carcinoma cell line COC1/DDP induced by survivin antisense oligonucleotides

    Institute of Scientific and Technical Information of China (English)

    ZHENG Fei; RUAN Fei; XIE Xian-kuan; LIU Shao-yang

    2006-01-01

    @@ Currently, surgery-oriented treatment plays a major role in the treatment of ovarian cancer patients. But 5-year survival rate of patients is still around 30%. One of the main reasons for the Iow survival rate is the drug resistance of tumor cells against chemotherapy.1,2 The function of antiapoptosis in the course of initiation and progress of cancer has a close relationship with drug resistance of tumor cells. Survivin is a new discovered anti-apoptosis gene, its expression levels correlating with more aggressive disease and poor clinical outcome in many of these tumors. It has been reported that survivin is expressed during fetal development and in cancer tissues.3 Furthermore,survivin overexpression, by disrupting the balance between cell proliferation/differentiation and apoptosis, may relate with the resistance to a variety of apoptotic stimuli, including chemotherapy.4,5 We designed antisense oligonucleotides of survivin to treat the drug-resistant human ovarian carcinoma cell line COC1/DDP, and studied its effects on inducing COC1/DDP apoptosis. The purpose of this study was to find a novel approach to improve the sensitivity of ovarian carcinoma chemotherapy.

  4. Antisense-mediated suppression of tomato thylakoidal ascorbate peroxidase influences anti-oxidant network during chilling stress.

    Science.gov (United States)

    Duan, Ming; Ma, Na-Na; Li, Dong; Deng, Yong-Sheng; Kong, Fan-Ying; Lv, Wei; Meng, Qing-Wei

    2012-09-01

    Photosynthesis is a well-established source of reactive oxygen species (ROS) in plants particularly under chilling stress. Ascorbate peroxidase (APXs) plays an important role in the anti-oxidant system by utilizing AsA as specific electron donor to reduce H(2)O(2) to water. In order to investigate the possible mechanisms of ascorbate peroxidsae (APX) in photoprotection under chilling stress, a tomato (Lycopersicon esculentum Mill.) thylakoidal ascorbate peroxidase gene (LetAPX) was isolated and antisense transgenic tomato plants were produced. Under chilling stress, transgenic plants accumulated more H(2)O(2), and showed higher levels of ion leakage and malondialdehyde (MDA), lower net photosynthetic rate (Pn), lower maximal photochemical efficiency of PSII (Fv/Fm) and less content of D1 protein compared with wild type (WT) plants. On the other hand, after chilling stress, transgenic plants showed higher reduced ascorbate (AsA) and activities of catalase (CAT) and superoxide dismutase (SOD) than those in WT plants, and the expression of several known stress-responsive and antioxidative genes was also higher at the end of chilling treatment. These results suggested that the suppression of LetAPX gene induced compensatory anti-oxidant mechanisms in tomato, and inactivation of tAPX may have a regulatory role in facilitating redox signaling pathways under chilling stress. Furthermore, transient increases in ROS levels also have a vital role in stress signaling and thereby in the survival of plants under chilling conditions.

  5. PLGA-PEG-PLGA microspheres as a delivery vehicle for antisense oligonucleotides to CTGF: Implications on post-surgical peritoneal adhesion prevention

    Science.gov (United States)

    Azeke, John Imuetinyan-Jesu, Jr.

    Abdominal adhesions are the aberrant result of peritoneal wound healing commonly associated with surgery and inflammation. A subject of a large number of studies since the first half of the last century, peritoneal adhesion prevention has, for the most part, evaded the scientific community and continues to cost Americans an estimated $2-4 billion annually. It is known that transforming growth factor-beta (TGF-beta) plays a key role in the wound healing cascade; however, suppression of this multifunctional growth factor's activity may have more harmful consequences than can be tolerated. As a result, much attention has fallen on connective tissue growth factor (CTGF), a downstream mediator of TGF-beta's fibrotic action. It has been demonstrated in several in vitro models, that the suppression of CTGF hinders fibroblast proliferation, a necessary condition for fibrosis. Furthermore, antisense oligonucleotides (antisense oligos, AO) to CTGF have been shown to knock down CTGF mRNA levels by specifically hindering the translation of CTGF protein. Antisense technologies have met with a great deal of excitement as a viable means of preventing diseases such as adhesions by hindering protein translation at the mRNA level. However, the great challenge associated with the use of these drugs lies in the short circulation time when administered "naked". Viral delivery systems, although excellent platforms in metabolic studies, are not ideal for diagnostic use because of the inherent danger associated with viral vectors. Microparticles made of biodegradable polymers have therefore presented themselves as a viable means of delivering these drugs to target cells over extended periods. Herein, we present two in vivo studies confirming the up-regulation of TGF-beta protein and CTGF mRNA following injury to the uterine tissues of female rats. We were able to selectively knockdown post-operative CTGF protein levels following surgery, however, our observations led us to conclude that

  6. 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.

  7. Light-Triggered Release of DNA from Plasmon-Resonant Nanoparticles

    Science.gov (United States)

    Huschka, Ryan

    Plasmon-resonant nanoparticle complexes show promising potential for lighttriggered, controllable delivery of deoxyribonucleic acids (DNA) for research and therapeutic purposes. For example, the approach of RNA interference (RNAi) . using antisense DNA or RNA oligonucleotides to silence activity of a specific pathogenic gene transcript and reduce expression of the encoded protein . is very useful in dissecting genetic function and holds promise as a molecular therapeutic. Herein, we investigate the mechanism and probe the in vitro therapeutic potential of DNA light-triggered release from plasmonic nanoparticles. First, we investigate the mechanism of light-triggered release by dehybridizing double-stranded (dsDNA) via laser illumination from two types of nanoparticle substrates: gold (Au) nanoshells and Au nanorods. Both light-triggered and thermally induced releases are distinctly observable from nanoshell-based complexes. Surprisingly, no analogous measurable light-triggered release was observable from nanorod-based complexes below the DNA melting temperature. These results suggest that a nonthermal mechanism may play a role in light-triggered DNA release. Second, we demonstrate the in vitro light-triggered release of molecules noncovalently attached within dsDNA bound to the Au nanoshell surface. DAPI (4',6- diamidino-2-phenylindole), a bright blue fluorescent molecule that binds reversibly to double-stranded DNA, was chosen to visualize this intracellular light-induced release process. Illumination through the cell membrane of the nanoshell-dsDNA-DAPI complexes dehybridizes the DNA and releases the DAPI molecules within living cells. The DAPI molecules diffuse to the nucleus and associate with the cell's endogenous DNA. This work could have future applications towards drug delivery of molecules that associate with dsDNA. Finally, we demonstrate an engineered Au nanoshell (AuNS)-based therapeutic oligonucleotide delivery vehicle, designed to release its cargo on

  8. A-DNA and B-DNA: Comparing Their Historical X-Ray Fiber Diffraction Images

    Science.gov (United States)

    Lucas, Amand A.

    2008-01-01

    A-DNA and B-DNA are two secondary molecular conformations (among other allomorphs) that double-stranded DNA drawn into a fiber can assume, depending on the relative water content and other chemical parameters of the fiber. They were the first two forms to be observed by X-ray fiber diffraction in the early 1950s, respectively by Wilkins and…

  9. DNA methylation

    DEFF Research Database (Denmark)

    Williams, Kristine; Christensen, Jesper; Helin, Kristian

    2012-01-01

    DNA methylation is involved in key cellular processes, including X-chromosome inactivation, imprinting and transcriptional silencing of specific genes and repetitive elements. DNA methylation patterns are frequently perturbed in human diseases such as imprinting disorders and cancer. The recent...... discovery that the three members of the TET protein family can convert 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) has provided a potential mechanism leading to DNA demethylation. Moreover, the demonstration that TET2 is frequently mutated in haematopoietic tumours suggests that the TET...... proteins are important regulators of cellular identity. Here, we review the current knowledge regarding the function of the TET proteins, and discuss various mechanisms by which they contribute to transcriptional control. We propose that the TET proteins have an important role in regulating DNA methylation...

  10. DNA data

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Raw DNA chromatogram data produced by the ABI 373, 377, 3130 and 3730 automated sequencing machines in ABI format. These are from fish (primarily Sebastes spp.,...

  11. DNA adductomics.

    Science.gov (United States)

    Balbo, Silvia; Turesky, Robert J; Villalta, Peter W

    2014-03-17

    Systems toxicology is a broad-based approach to describe many of the toxicological features that occur within a living system under stress or subjected to exogenous or endogenous exposures. The ultimate goal is to capture an overview of all exposures and the ensuing biological responses of the body. The term exposome has been employed to refer to the totality of all exposures, and systems toxicology investigates how the exposome influences health effects and consequences of exposures over a lifetime. The tools to advance systems toxicology include high-throughput transcriptomics, proteomics, metabolomics, and adductomics, which is still in its infancy. A well-established methodology for the comprehensive measurement of DNA damage resulting from every day exposures is not fully developed. During the past several decades, the (32)P-postlabeling technique has been employed to screen the damage to DNA induced by multiple classes of genotoxicants; however, more robust, specific, and quantitative methods have been sought to identify and quantify DNA adducts. Although triple quadrupole and ion trap mass spectrometry, particularly when using multistage scanning (LC-MS(n)), have shown promise in the field of DNA adductomics, it is anticipated that high-resolution and accurate-mass LC-MS(n) instrumentation will play a major role in assessing global DNA damage. Targeted adductomics should also benefit greatly from improved triple quadrupole technology. Once the analytical MS methods are fully mature, DNA adductomics along with other -omics tools will contribute greatly to the field of systems toxicology.

  12. DNA rendering of polyhedral meshes at the nanoscale

    Science.gov (United States)

    Benson, Erik; Mohammed, Abdulmelik; Gardell, Johan; Masich, Sergej; Czeizler, Eugen; Orponen, Pekka; Högberg, Björn

    2015-07-01

    It was suggested more than thirty years ago that Watson-Crick base pairing might be used for the rational design of nanometre-scale structures from nucleic acids. Since then, and especially since the introduction of the origami technique, DNA nanotechnology has enabled increasingly more complex structures. But although general approaches for creating DNA origami polygonal meshes and design software are available, there are still important constraints arising from DNA geometry and sense/antisense pairing, necessitating some manual adjustment during the design process. Here we present a general method of folding arbitrary polygonal digital meshes in DNA that readily produces structures that would be very difficult to realize using previous approaches. The design process is highly automated, using a routeing algorithm based on graph theory and a relaxation simulation that traces scaffold strands through the target structures. Moreover, unlike conventional origami designs built from close-packed helices, our structures have a more open conformation with one helix per edge and are therefore stable under the ionic conditions usually used in biological assays.

  13. DNA expressions - A formal notation for DNA

    NARCIS (Netherlands)

    Vliet, Rudy van

    2015-01-01

    We describe a formal notation for DNA molecules that may contain nicks and gaps. The resulting DNA expressions denote formal DNA molecules. Different DNA expressions may denote the same molecule. Such DNA expressions are called equivalent. We examine which DNA expressions are minimal, which

  14. Influence of Morse Potential on DNA Dynamics

    Institute of Scientific and Technical Information of China (English)

    ZDRAVKOVI(C) S.; SATARI(C) M.V.

    2006-01-01

    Based on the Peyrard-Bishop-Dauxois (i.e.the extended Peyrard-Bishop) model of DNA dynamics,the transversal hydrogen interaction is modelled by Morse potential and the impact of the Morse parameters on the DNA dynamics is investigated.In particular,we show how modulation of the signal,moving through the DNA chain, depends on those parameters,It is also shown that the DNA dynamics represents the interplay between dispersion and nonlinearity.Finally,we discuss the values of coupling constants κ and K.

  15. Mechanism of p53 downstream effectors p21 and Gadd45 in DNA damage surveillance

    Institute of Scientific and Technical Information of China (English)

    孟祥兵; 董燕; 孙志贤

    1999-01-01

    Both p21 (WAF1/CIP1) and Gadd45 were activated in a p53-dependent manner in MCF-7 cells after being exposed to ionizing radiation. In order to investigate their roles in DNA damage surveillance, p21as/MCF-7 cells stably transfected by p21 antisense expression plasmid pC-WAF1-AS and Gadd45as/MCF-7 stably transfected by Gadd45 antisense expression plasmid pCMVas45 were established. It was observed that G1 arrest induced by radiation was significantly reduced in Gadd45as/MCF-7 cells as well as in p21as/MCF-7 cells. Repair of radiation damaged report gene greatly reduced in Gadd45as/MCF-7 and p21as/MCF-7 cells. Apoptosis significantly increased in p21as/MCF-7 after exposure to radiation. These results suggest that both p21 and Gadd45 support cellular survival by taking roles in G1 arrest and DNA repair, furthermore, p21 protects cells from death by inhibiting apoptosis after exposure to ionizing radiation.

  16. In vitro transcription and translation inhibition via DNA functionalized gold nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Conde, J; Baptista, P V [Centro de Investigacao em Genetica Molecular Humana (CIGMH), Departamento de Ciencias da Vida, Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica (Portugal); De la Fuente, J M, E-mail: pmvb@fct.unl.pt [Instituto de Nanociencia de Aragon, Universidad de Zaragoza, Pedro Cerbuna 12, 50009 Zaragoza (Spain)

    2010-12-17

    The use of gold nanoparticles (AuNPs) has been gaining momentum as vectors for gene silencing strategies, combining the AuNPs' ease of functionalization with DNA and/or siRNA, high loading capacity and fast uptake by target cells. Here, we used AuNP functionalized with thiolated oligonucleotides to specifically inhibit transcription in vitro, demonstrating the synergetic effect between AuNPs and a specific antisense sequence that blocks the T7 promoter region. Also, AuNPs efficiently protect the antisense oligonucleotide against nuclease degradation, which can thus retain its inhibitory potential. In addition, we demonstrate that AuNPs functionalized with a thiolated oligonucleotide complementary to the ribosome binding site and the start codon, effectively shut down in vitro translation. Together, these two approaches can provide for a simple yet robust experimental set up to test for efficient gene silencing of AuNP-DNA conjugates. What is more, these results show that appropriate functionalization of AuNPs can be used as a dual targeting approach to an enhanced control of gene expression-inhibition of both transcription and translation.

  17. DNA and RNA sensor

    Institute of Scientific and Technical Information of China (English)

    LIU; Tao; LIN; Lin; ZHAO; Hong; JIANG; Long

    2005-01-01

    This review summarizes recent advances in DNA sensor. Major areas of DNA sensor covered in this review include immobilization methods of DNA, general techniques of DNA detection and application of nanoparticles in DNA sensor.

  18. What Is Mitochondrial DNA?

    Science.gov (United States)

    ... DNA What is mitochondrial DNA? What is mitochondrial DNA? Although most DNA is packaged in chromosomes within ... proteins. For more information about mitochondria and mitochondrial DNA: Molecular Expressions, a web site from the Florida ...

  19. Molecular cloning and identification of naturally occurring human antisense angiopoietin-1: Gna-1

    Institute of Scientific and Technical Information of China (English)

    ZHANG; Keman

    2001-01-01

    [1]Li, Z. Y., Xia, G. M., Chen, H. M., Somatic embryogenesis and plant regeneration from protoplasts isolated from embryo-genic cell suspensions of wheat (Triticum aestivum L.), Plant Cell, Tissue and Organ Culture, 1992, 98: 79-85.[2]Zhou, A. F., Xia, G. M., Chen, H. M., Asymmetric somatic hybridization between Triticum aestivum and Haynaldia vil-losa, Science in China, Ser. C, 1996, 39(6): 617-626.[3]Zhou, A. F., Xia, G. M., Chen, H. M. et al., Effect of 60Co-g radiation on calli of Haynaldia villosa, Chinese J. of Bio-technology, 1996, 12(Supplement): 127-130.[4]Xia, G. M., Chen, H. M., Wang, H., Somatic hybridization and regeneration capacity complementation between common wheat (T. aestivum) and Agropyron elongatum. J. Shandong University, 1995, 30(3): 325-330.[5]Xia, G. M., Chen, H. M., Plant regeneration from intergeneric somatic hybridization between Triticum aestivum L. and Leymus chinensis (Trin.)Tzvel., Plant Science, 1996, 120: 197-203.[6]Doyle, J. J., Doyle, J. L., Isolation of plant DNA from fresh tissue, Focus, 1990, 12: 13-15.[7]Zhou, A. F., Xu, C. H., Xiang, F. N. et al., Study on the identification of somatic hybrids by PCR with 5S rDNA spacer se-quence primers, Chinese J. of Biotechnology, 1999, 15(4): 529-532.[8]Ma, J. X., Zhou, R. H., Jia, J. Z. et al., Identification of wheat-Haynaldia villosa substitution lines conferring resistance to powdery mildew using genomic in situ hybridization (GISH) and RFLP markers, Acta Genetica Sinica, 1997, 24(5): 447-452.[9]Zhou, R. H., Jia, J. Z., Dong, Y. C. et al., Characterization of progenies of Triticum aestivum-Psathyrostachys juncea de-rivatives by using genomic in-situ hybridization, Science in China, Ser. C, 1997, 40(6): 657-664.[10]Parokonny, A. S., Kenton, A. Y., Gleba, Y. Y. et al., Genomic reorganization in Nicotiana asymmetric somatic hybrids analysed by in situ hybridization. Plant Journal, 1992, 2: 863-874.[11]Piastuch, W. C., Bates, G. W

  20. Enhanced therapeutic effects for human pancreatic cancer by application K-ras and IGF-IR antisense oligodeoxynucleotides

    Institute of Scientific and Technical Information of China (English)

    Yong-Mei Shen; Xiao-Chun Yang; Chen Yang; Jun-Kang Shen

    2008-01-01

    AIM:To investigate the combined effects of K-ras antisense oligodeoxynucleotide(K-ras ASODN)specific to GTT point mutation at codon 12 and type I insulin-like growth factor receptor(IGF-IR)antisense oligodeoxynucleotide(IGF-IR ASODN)on proliferation and apoptosis of human pancreatic cancer Patu8988 cells in vitro and in vivo.METHODS:K-ras gene point mutation and its style at codon 12 of human pancreatic cancer cell line Patu8988 were detected by using polymerase chain reaction with special sequence primers(PCR-SSP)and sequence analysis.According to the mutation style,K-ras mutation ASODN specific to K-ras point mutation at codon 12 was designed and composed.After K-ras ASODN and IGF-IR ASODN treated on Patu8988 cells respectively or cooperatively,the proliferation and morphological change of Patu8988 cells were analyzed by 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide(MTT)assay,colony forming assay and transmission electron microscopy;the expression of K-ras and IGF-IR mRNA and protein in the treated cells was measured by reverse-transcript polymerase chain reaction(RT-PCR)and flow cytometry respectively;apoptosis was determined by flow cytometry.The combined antitumor activity of K-ras ASODN and IGFIR ASODN was evaluated in BALB/C nude mice bearing human pancreatic cancer inoculated with Patu8988 cells.RESULTS:The results of PCR-SSP and sequence analysis showed that the human Dancreatic cancer cell line Patu8988 had point mutation at coclon 12,and the mutation style was GGT→GTT.2-32 μg/mL K-ras ASODN and 2-32 μg/mL IGF-IR ASODN could inhibit Patu8988 cells' growth,induce apoptosis and decrease the expression of K-ras and IGF-IR mRNA and protein alone.However,there was much more effective inhibition of growth and induction of apoptosis by their combination than by each one alone.In tumor bearing mice,the combination of K-ras ASODN and IGF-IR ASODN showed a significant inhibitory effect on the growth of transplanted pancreatic cancer,resulting in

  1. DNA vaccines

    Science.gov (United States)

    Gregersen, Jens-Peter

    2001-12-01

    Immunization by genes encoding immunogens, rather than with the immunogen itself, has opened up new possibilities for vaccine research and development and offers chances for new applications and indications for future vaccines. The underlying mechanisms of antigen processing, immune presentation and regulation of immune responses raise high expectations for new and more effective prophylactic or therapeutic vaccines, particularly for vaccines against chronic or persistent infectious diseases and tumors. Our current knowledge and experience of DNA vaccination is summarized and critically reviewed with particular attention to basic immunological mechanisms, the construction of plasmids, screening for protective immunogens to be encoded by these plasmids, modes of application, pharmacokinetics, safety and immunotoxicological aspects. DNA vaccines have the potential to accelerate the research phase of new vaccines and to improve the chances of success, since finding new immunogens with the desired properties is at least technically less demanding than for conventional vaccines. However, on the way to innovative vaccine products, several hurdles have to be overcome. The efficacy of DNA vaccines in humans appears to be much less than indicated by early studies in mice. Open questions remain concerning the persistence and distribution of inoculated plasmid DNA in vivo, its potential to express antigens inappropriately, or the potentially deleterious ability to insert genes into the host cell's genome. Furthermore, the possibility of inducing immunotolerance or autoimmune diseases also needs to be investigated more thoroughly, in order to arrive at a well-founded consensus, which justifies the widespread application of DNA vaccines in a healthy population.

  2. DNA nanotechnology

    Directory of Open Access Journals (Sweden)

    Nadrian C Seeman

    2003-01-01

    We are all aware that the DNA found in cells is a double helix consisting of two antiparallel strands held together by specific hydrogen-bonded base pairs; adenine (A always pairs with thymine (T, and guanine (G always pairs with cytosine (C. The specificity of this base pairing and the ability to ensure that it occurs in this fashion (and not some other1 is key to the use of DNA in materials applications. The double helical arrangement of the two molecules leads to a linear helix axis, linear not in the geometrical sense of being a straight line, but in the topological sense of being unbranched. Genetic engineers discovered in the 1970s how to splice together pieces of DNA to add new genes to DNA molecules2, and synthetic chemists worked out convenient syntheses for short pieces of DNA (up to ∼100–150 units in the 1980s3. Regardless of the impact of these technologies on biological systems, hooking together linear molecules leads only to longer linear molecules, with circles, knots, and catenanes perhaps resulting from time to time.

  3. 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.

  4. Antisense oligonucleotide-induced alternative splicing of the APOB mRNA generates a novel isoform of APOB

    Directory of Open Access Journals (Sweden)

    Chew Shern L

    2007-01-01

    Full Text Available Abstract Background Apolipoprotein B (APOB is an integral part of the LDL, VLDL, IDL, Lp(a and chylomicron lipoprotein particles. The APOB pre-mRNA consists of 29 constitutively-spliced exons. APOB exists as two natural isoforms: the full-length APOB100 isoform, assembled into LDL, VLDL, IDL and Lp(a and secreted by the liver in humans; and the C-terminally truncated APOB48, assembled into chylomicrons and secreted by the intestine in humans. Down-regulation of APOB100 is a potential therapy to lower circulating LDL and cholesterol levels. Results We investigated the ability of 2'O-methyl RNA antisense oligonucleotides (ASOs to induce the skipping of exon 27 in endogenous APOB mRNA in HepG2 cells. These ASOs are directed towards the 5' and 3' splice-sites of exon 27, the branch-point sequence (BPS of intron 26–27 and several predicted exonic splicing enhancers within exon 27. ASOs targeting either the 5' or 3' splice-site, in combination with the BPS, are the most effective. The splicing of other alternatively spliced genes are not influenced by these ASOs, suggesting that the effects seen are not due to non-specific changes in alternative splicing. The skip 27 mRNA is translated into a truncated isoform, APOB87SKIP27. Conclusion The induction of APOB87SKIP27 expression in vivo should lead to decreased LDL and cholesterol levels, by analogy to patients with hypobetalipoproteinemia. As intestinal APOB mRNA editing and APOB48 expression rely on sequences within exon 26, exon 27 skipping should not affect APOB48 expression unlike other methods of down-regulating APOB100 expression which also down-regulate APOB48.

  5. Design and screening of antisense oligodeoxynucleotides against PAI-1 mRNA in endothelial cells in vitro

    Institute of Scientific and Technical Information of China (English)

    Qi-sheng JIANG; Sheng-qi WANG

    2006-01-01

    Aim: To design and screen antisense oligodeoxynucleotides (ASODNs), which inhibit type-1 plasminogen activator inhibitor (PAI-1) expression in human umbilical vein endothelial cells (HUVEC) in vitro. Methods: Twenty seven ASODNs against different sites of PAI-1 mRNA were designed and transfected to HUVEC by lipofectin in vitro. The effects of ASODNs on PAI-1 antigen, PAI-1 activity and PAI-1 mRNA expression were detected by ELISA, amidolytical assay and RT-PCR, respectively. Results: Transforming growth factor β1 (TGF-β1)-treated HUVEC increased the expression of PAI-1 compared with the normal HUVEC. Five among twenty seven designed ASODNs were effective in inhibiting the increase in PAI-1 antigen and PAI-1 activity in a dose-dependent manner after 48-h transfection. In particular, ASODN 14 (AO14) exhibited the best inhibitory effect. The control sequences of AO14, including sense, scramble, and mismatch sequences, did not significantly inhibit PAI-1 activity. It was revealed that the inhibitory efficacy of AO14 was in a sequence-specific manner. RT-PCR showed that ASODN 1, 7, 8, 14, and 15 decreased PAI-1 mRNA expression induced by TGF-β1 and AO14 showed the best inhibitory effect. Conclusion: ASODN 1,7,8, 14, and 15, among twenty seven designed ASODNs against PAI-1 mRNA, significantly decreased PAI-1 antigen and PAI-1 activity induced by TGF-β1 in a dosedependent manner in HUVEC in vitro. AO14 showed the best inhibitory effect on PAI-1 expression in a sequence-specific manner. The results of RT-PCR indicated that inhibitory effects of ASODNs on PAI-1 biosynthesis occurred at the mRNA level. Four among five effective target sites of ASODNs located at the translation initiation site or within the translation area of PAI-1 mRNA, suggesting that these sites may be promising sites for the design of effective ASODNs.

  6. 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.

  7. Antisense sequencing improves the accuracy and precision of A-to-I editing measurements using the peak height ratio method

    Directory of Open Access Journals (Sweden)

    Rinkevich Frank D

    2012-01-01

    Full Text Available Abstract Background A-to-I RNA editing is found in all phyla of animals and contributes to transcript diversity that may have profound impacts on behavior and physiology. Many transcripts of genes involved in axonal conductance, synaptic transmission and modulation are the targets of A-to-I RNA editing. There are a number of methods to measure the extent of A-to-I RNA editing, but they are generally costly and time consuming. One way to determine the frequency of A-to-I RNA editing is the peak height ratio method, which compares the size of peaks on electropherograms that represent unedited and edited sites. Findings Sequencing of 4 editing sites of the Dα6 nicotinic acetylcholine receptor subunit with an antisense primer (which uses T/C peaks to measure unedited and edited sites, respectively showed very accurate and precise measurements of A-to-I RNA editing. The accuracy and precision were excellent for all editing sites, including those edited with high or low frequencies. The frequency of A-to-I RNA editing was comparable to the editing frequency as measured by clone counting from the same sample. Sequencing these same sites with the sense primer (which uses A/G peaks yielded inaccurate and imprecise measurements. Conclusions We have validated and improved the accuracy and precision of the peak height ratio method to measure the frequency of A-to-I RNA editing, and shown that results are primer specific. Thus, the correct sequencing primer must be utilized for the most dependable data. When compared to other methods used to measure the frequency of A-to-I RNA editing, the major benefits of the peak height ratio are that this method is inexpensive, fast, non-labor intensive and easily adaptable to many laboratory and field settings.

  8. Dynamics of co-transcriptional pre-mRNA folding influences the induction of dystrophin exon skipping by antisense oligonucleotides.

    Directory of Open Access Journals (Sweden)

    Keng Boon Wee

    Full Text Available Antisense oligonucleotides (AONs mediated exon skipping offers potential therapy for Duchenne muscular dystrophy. However, the identification of effective AON target sites remains unsatisfactory for lack of a precise method to predict their binding accessibility. This study demonstrates the importance of co-transcriptional pre-mRNA folding in determining the accessibility of AON target sites for AON induction of selective exon skipping in DMD. Because transcription and splicing occur in tandem, AONs must bind to their target sites before splicing factors. Furthermore, co-transcriptional pre-mRNA folding forms transient secondary structures, which redistributes accessible binding sites. In our analysis, to approximate transcription elongation, a "window of analysis" that included the entire targeted exon was shifted one nucleotide at a time along the pre-mRNA. Possible co-transcriptional secondary structures were predicted using the sequence in each step of transcriptional analysis. A nucleotide was considered "engaged" if it formed a complementary base pairing in all predicted secondary structures of a particular step. Correlation of frequency and localisation of engaged nucleotides in AON target sites accounted for the performance (efficacy and efficiency of 94% of 176 previously reported AONs. Four novel insights are inferred: (1 the lowest frequencies of engaged nucleotides are associated with the most efficient AONs; (2 engaged nucleotides at 3' or 5' ends of the target site attenuate AON performance more than at other sites; (3 the performance of longer AONs is less attenuated by engaged nucleotides at 3' or 5' ends of the target site compared to shorter AONs; (4 engaged nucleotides at 3' end of a short target site attenuates AON efficiency more than at 5' end.

  9. XRN2 is required for the degradation of target RNAs by RNase H1-dependent antisense oligonucleotides

    Energy Technology Data Exchange (ETDEWEB)

    Hori, Shin-Ichiro; Yamamoto, Tsuyoshi; Obika, Satoshi, E-mail: obika@phs.osaka-u.ac.jp

    2015-08-21

    Antisense oligonucleotides (ASOs) can suppress the expression of a target gene by cleaving pre-mRNA and/or mature mRNA via RNase H1. Following the initial endonucleolytic cleavage by RNase H1, the target RNAs are degraded by a mechanism that is poorly understood. To better understand this degradation pathway, we depleted the expression of two major 5′ to 3′ exoribonucleases (XRNs), named XRN1 and XRN2, and analyzed the levels of 3′ fragments of the target RNAs in vitro. We found that the 3′ fragments of target pre-mRNA generated by ASO were almost completely degraded from their 5′ ends by nuclear XRN2 after RNase H1-mediated cleavage, whereas the 3′ fragments of mature mRNA were partially degraded by XRN2. In contrast to ASO, small interference RNA (siRNA) could reduce the expression level of only mature mRNA, and the 3′ fragment was degraded by cytoplasmic XRN1. Our findings indicate that the RNAs targeted by RNase H1-dependent ASO are rapidly degraded in the nucleus, contrary to the cytoplasmic degradation pathway mediated by siRNA. - Highlights: • We compared the degradation mechanism of the transcript targeted by ASO and siRNA. • We focused on two 5′ to 3′ exoribonucleases, cytoplasmic XRN1, and nuclear XRN2. • The 3′ fragment of target pre-mRNA generated by ASO was degraded by XRN2. • The 3′ fragment of target mRNA generated by ASO was partially degraded by XRN2. • XRN1 depletion promoted accumulation of the 3′ fragment of mRNA generated by siRNA.

  10. Decreased Pollen Viability and Thicken Pollen Intine in Antisense Silenced Brassica campestris Mutant of BcMF19

    Institute of Scientific and Technical Information of China (English)

    LIU Jin-long; GAO Ming-hui; LIU Ying; CAO Jia-shu

    2014-01-01

    Brassica campestris male fertility 19 (BcMF19;GenBank accession number GQ902048.1), a gene that is specially expressed in tapetum cells and microspores during anther development in B. campestris ssp. chinensis, which is learned from the previous in situ hybridization study. In the present study, we constructed antisense-silenced plants of BcMF19 using B. campestris ssp. chinensis to validate this prediction. The morphology of the pistils, long anthers, and short anthers was signiifcantly affected in 35sbcmf19 compared with the control samples. 4´-6-Diamidino-2-phenylindole staining revealed that two generative nuclei and one large vegetative nucleus were not affected in the mutant compared with control. Statistical analysis of Alexander’s staining results showed that 96% of the control pollen grains had vitality, whereas only 86% of the mutant pollen grains did. Under scanning electron microscopy, the mutant demonstrated numerous abnormal pollen grains and resembled dried persimmon. The frequency of normal pollen grains was approximately 18%. Under transmission electron microscopy, the pollen intine during the binucleate and mature pollen stages in 35sbcmf19 exhibited abnormal thickening, especially at the germinal furrows, compared with control. In vitro pollen germination test showed that the tips of the mutant pollen tubes transformed into globular alveoli and stopped growing compared with control. On the other hand, in vivo pollen germination test suggested that BcMF19 affected the pollen tube extension in the pistil. These ifndings indicate that BcMF19 is essential to the pollen development and pollen tube extension of B. campestris ssp. chinensis.

  11. Influence of connective tissue growth factor antisense oligonucleotide on angiotensin Ⅱ-induced epithelial mesenchymal transition in HK2 cells

    Institute of Scientific and Technical Information of China (English)

    Long CHEN; Bi-cheng LIU; Xiao-liang ZHANG; Jian-dong ZHANG; Hong LIU; Min-xia LI

    2006-01-01

    Aim: The present study was designed to further investigate the effect of connective tissue growth factor antisense oligonucleotide (CTGF-AS) on angiotensin Ⅱ (Ang Ⅱ)-induced tubular cell epithelial mesenchymal transition (EMT) in vitro. Methods: The human proximal tubular cell line (HK2) was grown in Dulbecco's modified Eagle's medium containing 10% heat inactivated fetal calf serum. After being rested in serum-free medium for 24 h, the influence of CTGF-AS (20 μg/mL) on Ang Ⅱ-induced (1×10-7 mol/L) CTGF mRNA and the protein expression were examined by using reverse transcription-polymerase chain reaction and indirectimmunofluorescence. The effect of CTGF-AS on Ang Ⅱ-induced cellular ultrastructure was observed using a transmissive electronic microscope. The expression of α-smooth action (α-SMA) was assayed by immunocytochemistry. In all experiments, the control group was treated with scrambled oligonucleotide. Results: It was shown that Ang Ⅱ significantly induced the increasing expression of CTGF mRNA and protein (P<0.01, respectively), which were significantly abolished by treatment with CTGF-AS. After stimulating cells with Ang Ⅱ, the cellular ultrastructure showed mesenchymal features. These effects were partially inhibited by CTGF-AS. Ang Ⅱ significantly resulted in the expression of α-SMA in time dependent manner, which was markedly attenuated by the treatment with CTGF-AS (P<0.01, respectively). In contrast, no similar effects were observed in the control group treated with scrambled oligonucleotide. Conclusion: Ang Ⅱ-induced EMT in human proximal tubular epithelial cells (PTC) can be attenuated by treatment with CTGF-AS. Our data provides further evidence that CTGF might be involved in Ang Ⅱ-induced EMT in PTC.

  12. Fusions of Dendritic Cells and C6 Cells Transfected with TGF-β1 Antisense in Treatment of Intracranial Gliomas

    Institute of Scientific and Technical Information of China (English)

    Jin Gui-shan; Liu Fu-sheng; Chai Qi; Wang Jian-jao; Li Jun-hua

    2007-01-01

    Objective: To investigate the immunotherapy efficacy of fusion cells (dendritic-C6anti-TGF-β1 cells) in the treatment of intracranial gliomas. Methods: Dendritic cells were isolated from rat bone-marrow precursors stimulated in vitro with granulocyte-macrophage colony-stimulating factor (GM-CSF) and Interleukin-4 (IL-4). C6anti-TGF-β1 cells originally from C6 cell line of a rat glioblastoma were tr