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Sample records for antisense elements genetics

  1. 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

  2. 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...

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

    Directory of Open Access Journals (Sweden)

    Ioannis eZalachoras

    2011-07-01

    Full Text Available A limiting factor in brain research still is the difficulty to evaluate in vivo the role of the increasing number of proteins implicated in neuronal processes. We discuss here the potential of antisense-mediated RNA targeting approaches. We mainly focus on those that manipulate splicing (exon skipping and exon inclusion, but will also briefly discuss mRNA targeting. Classic knockdown of expression by mRNA targeting is only one possible application of antisense oligonucleotides (AON in the control of gene function. Exon skipping and inclusion are based on the interference of AONs with splicing of pre-mRNAs. These are powerful, specific and particularly versatile techniques, which can be used to circumvent pathogenic mutations, shift splice variant expression, knock down proteins, or to create molecular models using in-frame deletions. Pre-mRNA targeting is currently used both as a research tool, e.g. in models for motor neuron disease, and in clinical trials for Duchenne muscular dystrophy and amyotrophic lateral sclerosis.AONs are particularly promising in relation to brain research, as the modified AONs are taken up extremely fast in neurons and glial cells with a long residence, and without the need for viral vectors or other delivery tools, once inside the blood brain barrier. In this review we cover 1. The principles of antisense-mediated techniques, chemistry and efficacy.2. The pros and cons of AON approaches in the brain compared to other techniques of interfering with gene function, such as transgenesis and short hairpin RNAs, in terms of specificity of the manipulation, spatial and temporal control over gene expression, toxicity and delivery issues.3. The potential applications for Neuroscience. We conclude that there is good evidence from animal studies that the CNS can be successfully targeted, but the potential of the diverse AON-based approaches appears to be under-recognized.

  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.

    1992-01-01

    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.

  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.

    1992-12-31

    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.

  6. Mobile genetic elements in Methanobacterium thermoformicicum.

    NARCIS (Netherlands)

    Nölling, J.

    1993-01-01

    The identification of the Archaea as a third primary lineage of life and their adaptation to extreme environmental conditions have generated considerable interest in the molecular biology of these organisms. Most progress in the investigation of archaeal mobile genetic elements, i.e. viruses, plasmi

  7. Mobile genetic elements in protozoan parasites

    Indian Academy of Sciences (India)

    Sudha Bhattacharya; Abhijeet Bakre; Alok Bhattacharya

    2002-08-01

    Mobile genetic elements, by virtue of their ability to move to new chromosomal locations, are considered important in shaping the evolutionary course of the genome. They are widespread in the biological kingdom. Among the protozoan parasites several types of transposable elements are encountered. The largest variety is seen in the trypanosomatids—Trypanosoma brucei, Trypanosoma cruzi and Crithidia fasciculata. They contain elements that insert site-specifically in the spliced-leader RNA genes, and others that are dispersed in a variety of genomic locations. Giardia lamblia contains three families of transposable elements. Two of these are subtelomeric in location while one is chromosome-internal. Entamoeba histolytica has an abundant retrotransposon dispersed in the genome. Nucleotide sequence analysis of all the elements shows that they are all retrotransposons, and, with the exception of one class of elements in T. cruzi, all of them are non-long-terminal-repeat retrotransposons. Although most copies have accumulated mutations, they can potentially encode reverse transcriptase, endonuclease and nucleic-acid-binding activities. Functionally and phylogenetically they do not belong to a single lineage, showing that retrotransposons were acquired early in the evolution of protozoan parasites. Many of the potentially autonomous elements that encode their own transposition functions have nonautonomous counterparts that probably utilize the functions in trans. In this respect these elements are similar to the mammalian LINEs and SINEs (long and short interspersed DNA elements), showing a common theme in the evolution of retrotransposons. So far there is no report of a DNA transposon in any protozoan parasite. The genome projects that are under way for most of these organisms will help understand the evolution and possible function of these genetic elements.

  8. Stable propagation of `selfish' genetic elements

    Indian Academy of Sciences (India)

    Soundarapandian Velmurugan; Shwetal Mehta; Dina Uzri; Makkuni Jayaram

    2003-09-01

    Extrachromosomal or chromosomally integrated genetic elements are common among prokaryotic and eukaryotic cells. These elements exhibit a variety of `selfish’ strategies to ensure their replication and propagation during the growth of their host cells. To establish long-term persistence, they have to moderate the degree of selfishness so as not to imperil the fitness of their hosts. Earlier genetic and biochemical studies together with more recent cell biological investigations have revealed details of the partitioning mechanisms employed by low copy bacterial plasmids. At least some bacterial chromosomes also appear to rely on similar mechanisms for their own segregation. The 2 m plasmid of Saccharomyces cerevisiae and related yeast plasmids provide models for optimized eukaryotic selfish DNA elements. Selfish DNA elements exploit the genetic endowments of their hosts without imposing an undue metabolic burden on them. The partitioning systems of these plasmids appear to make use of a molecular trick by which the plasmids feed into the segregation pathway established for the host chromosomes.

  9. Molecular genetics and epigenetics of CACTA elements

    KAUST Repository

    Fedoroff, Nina V.

    2013-08-21

    The CACTA transposons, so named for a highly conserved motif at element ends, comprise one of the most abundant superfamilies of Class 2 (cut-and-paste) plant transposons. CACTA transposons characteristically include subterminal sequences of several hundred nucleotides containing closely spaced direct and inverted repeats of a short, conserved sequence of 14-15 bp. The Supressor-mutator (Spm) transposon, identified and subjected to detailed genetic analysis by Barbara McClintock, remains the paradigmatic element of the CACTA family. The Spm transposon encodes two proteins required for transposition, the transposase (TnpD) and a regulatory protein (TnpA) that binds to the subterminal repeats. Spm expression is subject to both genetic and epigenetic regulation. The Spm-encoded TnpA serves as an activator of the epigenetically inactivated, methylated Spm, stimulating both transient and heritable activation of the transposon. TnpA also serves as a negative regulator of the demethylated active element promoter and is required, in addition to the TnpD, for transposition. © Springer Science+Business Media, New York 2013.

  10. 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.

  11. Progress toward therapy with antisense-mediated splicing modulation

    OpenAIRE

    Du, Liutao; Gatti, Richard A.

    2009-01-01

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

  12. Antisense Therapy in Neurology

    OpenAIRE

    Lee, Joshua J.A.; Toshifumi Yokota

    2013-01-01

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

  13. Antisense 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. 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.

  15. The mobile genetic element Alu in the human genome

    Energy Technology Data Exchange (ETDEWEB)

    Novick, G.E. [Florida International Univ., Miami, FL (United States); Batzer, M.A.; Deininger, P.L. [Louisiana State Univ. Medical Center, New Orleans, LA (United States)] [and others

    1996-01-01

    Genetic material has been traditionally envisioned as relatively static with the exception of occasional, often deleterious mutations. The sequence DNA-to-RNA-to-protein represented for many years the central dogma relating gene structure and function. Recently, the field of molecular genetics has provided revolutionary information on the dynamic role of repetitive elements in the function of the genetic material and the evolution of humans and other organisms. Alu sequences represent the largest family of short interspersed repetitive elements (SINEs) in humans, being present in an excess of 500,000 copies per haploid genome. Alu elements, as well as the other repetitive elements, were once considered to be useless. Today, the biology of Alu transposable elements is being widely examined in order to determine the molecular basis of a growing number of identified diseases and to provide new directions in genome mapping and biomedical research. 66 refs., 5 figs.

  16. Enterprise Projects Set Risk Element Transmission Chaotic Genetic Model

    OpenAIRE

    Cunbin Li; Peng Li; Gongshu Lu

    2012-01-01

    In order to research projects set risk transfer process and improve risk management efficiency in projects management, combining chaos theory and genetic algorithm, put forward enterprise projects set risk element transmission chaos genetic model. Using logistic chaos mapping and chebyshev chaos mapping mixture, constructed a hybrid chaotic mapping system. The steps of adopting hybrid chaos mapping for genetic operation include projects set initialization, calculation of fitness, selection, c...

  17. Modularization of genetic elements promotes synthetic metabolic engineering.

    Science.gov (United States)

    Qi, Hao; Li, Bing-Zhi; Zhang, Wen-Qian; Liu, Duo; Yuan, Ying-Jin

    2015-11-15

    In the context of emerging synthetic biology, metabolic engineering is moving to the next stage powered by new technologies. Systematical modularization of genetic elements makes it more convenient to engineer biological systems for chemical production or other desired purposes. In the past few years, progresses were made in engineering metabolic pathway using synthetic biology tools. Here, we spotlighted the topic of implementation of modularized genetic elements in metabolic engineering. First, we overviewed the principle developed for modularizing genetic elements and then discussed how the genetic modules advanced metabolic engineering studies. Next, we picked up some milestones of engineered metabolic pathway achieved in the past few years. Last, we discussed the rapid raised synthetic biology field of "building a genome" and the potential in metabolic engineering.

  18. Transposable elements and genetic instabilities in crop plants

    Energy Technology Data Exchange (ETDEWEB)

    Burr, B.; Burr, F.

    1981-04-10

    Transposable elements have long been associated with certain unstable loci in maize and have been intensively studied by McClintock and others. It is known that a transposable element can control the expression of the structural genes at the locus where it resides. These controlling elements in maize are now beginning to be studied at the molecular level. Using recombinant molecular probes we have been able to describe the changes induced by the controlling element Ds at the shrunken locus. Ds elements appear to be large and dissimilar insertions into the wild-type locus - two elements actually map within the transcribed region of the gene. Genetic instabilities have been described in other economically important plants but the bases for these phenomena have not been understood. We believe that it is likely that some of these instabilities are the result of transposable element activity much as in the case of maize.

  19. Enterprise Projects Set Risk Element Transmission Chaotic Genetic Model

    Directory of Open Access Journals (Sweden)

    Cunbin Li

    2012-08-01

    Full Text Available In order to research projects set risk transfer process and improve risk management efficiency in projects management, combining chaos theory and genetic algorithm, put forward enterprise projects set risk element transmission chaos genetic model. Using logistic chaos mapping and chebyshev chaos mapping mixture, constructed a hybrid chaotic mapping system. The steps of adopting hybrid chaos mapping for genetic operation include projects set initialization, calculation of fitness, selection, crossover and mutation operators, fitness adjustment and condition judgment. The results showed that the model can simulate enterprise projects set risk transmission process very well and it also provides the basis for the enterprise managers to make decisions.

  20. The ecology of transfer of mobile genetic elements

    NARCIS (Netherlands)

    Elsas, van J.D.; Bailey, M.J.

    2002-01-01

    The ecological aspects of the transfer and spread of mobile genetic elements (MGE) are reviewed in the context of the emerging evidence for the dominant role that horizontal gene transfer (HGT) has played in the evolutionary shaping of bacterial communities. Novel tools are described that allow a re

  1. 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...

  2. Sense antisense DNA strand?

    Science.gov (United States)

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

    1994-01-01

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

  3. Mammalian small nucleolar RNAs are mobile genetic elements.

    Directory of Open Access Journals (Sweden)

    Michel J Weber

    2006-12-01

    Full Text Available Small nucleolar RNAs (snoRNAs of the H/ACA box and C/D box categories guide the pseudouridylation and the 2'-O-ribose methylation of ribosomal RNAs by forming short duplexes with their target. Similarly, small Cajal body-specific RNAs (scaRNAs guide modifications of spliceosomal RNAs. The vast majority of vertebrate sno/scaRNAs are located in introns of genes transcribed by RNA polymerase II and processed by exonucleolytic trimming after splicing. A bioinformatic search for orthologues of human sno/scaRNAs in sequenced mammalian genomes reveals the presence of species- or lineage-specific sno/scaRNA retroposons (sno/scaRTs characterized by an A-rich tail and an approximately 14-bp target site duplication that corresponds to their insertion site, as determined by interspecific genomic alignments. Three classes of snoRTs are defined based on the extent of intron and exon sequences from the snoRNA parental host gene they contain. SnoRTs frequently insert in gene introns in the sense orientation at genomic hot spots shared with other genetic mobile elements. Previously characterized human snoRNAs are encoded in retroposons whose parental copies can be identified by phylogenic analysis, showing that snoRTs can be faithfully processed. These results identify snoRNAs as a new family of mobile genetic elements. The insertion of new snoRNA copies might constitute a safeguard mechanism by which the biological activity of snoRNAs is maintained in spite of the risk of mutations in the parental copy. I furthermore propose that retroposition followed by genetic drift is a mechanism that increased snoRNA diversity during vertebrate evolution to eventually acquire new RNA-modification functions.

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

    OpenAIRE

    Guzowski, John F.; McGaugh, James L.

    1997-01-01

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

  5. Identification of genetic elements associated with EPSPs gene amplification.

    Directory of Open Access Journals (Sweden)

    Todd A Gaines

    Full Text Available Weed populations can have high genetic plasticity and rapid responses to environmental selection pressures. For example, 100-fold amplification of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS gene evolved in the weed species Amaranthus palmeri to confer resistance to glyphosate, the world's most important herbicide. However, the gene amplification mechanism is unknown. We sequenced the EPSPS gene and genomic regions flanking EPSPS loci in A. palmeri, and searched for mobile genetic elements or repetitive sequences. The EPSPS gene was 10,229 bp, containing 8 exons and 7 introns. The gene amplification likely proceeded through a DNA-mediated mechanism, as introns exist in the amplified gene copies and the entire amplified sequence is at least 30 kb in length. Our data support the presence of two EPSPS loci in susceptible (S A. palmeri, and that only one of these was amplified in glyphosate-resistant (R A. palmeri. The EPSPS gene amplification event likely occurred recently, as no sequence polymorphisms were found within introns of amplified EPSPS copies from R individuals. Sequences with homology to miniature inverted-repeat transposable elements (MITEs were identified next to EPSPS gene copies only in R individuals. Additionally, a putative Activator (Ac transposase and a repetitive sequence region were associated with amplified EPSPS genes. The mechanism controlling this DNA-mediated amplification remains unknown. Further investigation is necessary to determine if the gene amplification may have proceeded via DNA transposon-mediated replication, and/or unequal recombination between different genomic regions resulting in replication of the EPSPS gene.

  6. Genetic optimization of reliability design of machine element

    Institute of Scientific and Technical Information of China (English)

    郭观七; 喻寿益

    2001-01-01

    Canonical genetic algorithms have the defects of prematurity and stagnation when applied in optimization problems. The causes resulting in such phenomena were analyzed and a class of improved genetic algorithm with niche implemented by crossover of similar individuals and (μ+λ) selection was proposed. According to the reliability design theory of machine components, the genetic optimization model of jack clutch was obtained. An optimization instance and some results calculated by improved genetic algorithm were presented. The results of emulations and application show that the improved genetic algorithm with the niche technique can achieve the reliable global convergence and stable convergent velocity almost without any additional calculation expense.

  7. Scheme optimization of AT shifting element based on genetic algorithm

    Institute of Scientific and Technical Information of China (English)

    岳会军; 刘艳芳; 马明月; 徐向阳; 王书翰

    2015-01-01

    In order to realize the computer aided design of AT shifting element schemes, a mathematical model of shifting element schemes which can be easily identified by computers was built. Taking the transmission ratio sequence as an optimization objective and simple shifting logic between adjacent gears through operating only one shifting element as a constraint condition, a fitness function of shifting element schemes was proposed. ZF-8AT shifting element schemes were optimized based on GA work-box of MATLAB, and the feasibility of the optimization algorithm was verified.

  8. Antisense approaches in prostate cancer.

    Science.gov (United States)

    Chi, Kim N; Gleave, Martin E

    2004-06-01

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

  9. Antisense Oligonucleotide Therapy for Inherited Retinal Dystrophies.

    Science.gov (United States)

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

    2016-01-01

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

  10. Transfer of Clostridium difficile Genetic Elements Conferring Resistance to Macrolide-Lincosamide-Streptogramin B (MLSB) Antibiotics.

    Science.gov (United States)

    Barbanti, Fabrizio; Wasels, François; Spigaglia, Patrizia

    2016-01-01

    Molecular analysis is an important tool to investigate Clostridium difficile resistance to macrolide-lincosamide-streptogramin B (MLSB). In particular, the protocols described in this chapter have been designed to investigate the genetic organization of erm(B)-containing elements and to evaluate the capability of these elements to transfer in C. difficile recipient strains using filter mating assay. PMID:27507342

  11. Interaction of α-Melanocortin and Its Pentapeptide Antisense LVKAT: Effects on Hepatoprotection in Male CBA Mice

    Directory of Open Access Journals (Sweden)

    Paško Konjevoda

    2011-08-01

    Full Text Available The genetic code defines nucleotide patterns that code for individual amino acids and their complementary, i.e., antisense, pairs. Peptides specified by the complementary mRNAs often bind to each other with a higher specificity and efficacy. Applications of this genetic code property in biomedicine are related to the modulation of peptide and hormone biological function, selective immunomodulation, modeling of discontinuous and linear epitopes, modeling of mimotopes, paratopes and antibody mimetics, peptide vaccine development, peptidomimetic and drug design. We have investigated sense-antisense peptide interactions and related modulation of the peptide function by modulating the effects of a-MSH on hepatoprotection with its antisense peptide LVKAT. First, transcription of complementary mRNA sequence of a-MSH in 3’→5’ direction was used to design antisense peptide to the central motif that serves as a-MSH pharmacophore for melanocortin receptors. Second, tryptophan spectrofluorometric titration was applied to evaluate the binding of a-MSH and its central pharmacophore motif to the antisense peptide, and it was concluded that this procedure represents a simple and efficient method to evaluate sense-antisense peptide interaction in vitro. Third, we showed that antisense peptide LVKAT abolished potent hepatoprotective effects of a-MSH in vivo.

  12. Mobile Genetic Elements Provide Evidence for a Bovine Origin of Clonal Complex 17 of Streptococcus agalactiae▿

    OpenAIRE

    Héry-Arnaud, Geneviève; Bruant, Guillaume; Lanotte, Philippe; Brun, Stella; Picard, Bertrand; Rosenau, Agnès; Van Ver Mee-Marquet, Nathalie; Rainard, Pascal; Quentin, Roland; Mereghetti, Laurent

    2007-01-01

    International audience We sought an explanation for epidemiological changes in Streptococcus agalactiae infections by investigating the link between ecological niches of the bacterium by determining the prevalence of 11 mobile genetic elements. The prevalence of nine of these elements differed significantly according to the human or bovine origin of the isolate. Correlating this distribution with the phylogeny obtained by multilocus sequence analysis, we observed that human isolates harbor...

  13. Circadian rhythms of sense and antisense transcription in sugarcane, a highly polyploid crop.

    Directory of Open Access Journals (Sweden)

    Carlos Takeshi Hotta

    Full Text Available Commercial sugarcane (Saccharum hybrid is a highly polyploid and aneuploid grass that stores large amounts of sucrose in its stem. We have measured circadian rhythms of sense and antisense transcription in a commercial cultivar (RB855453 using a custom oligoarray with 14,521 probes that hybridize to sense transcripts (SS and 7,380 probes that hybridize to antisense transcripts (AS.We estimated that 32% of SS probes and 22% AS probes were rhythmic. This is a higher proportion of rhythmic probes than the usually found in similar experiments in other plant species. Orthologs and inparalogs of Arabidopsis thaliana, sugarcane, rice, maize and sorghum were grouped in ortholog clusters. When ortholog clusters were used to compare probes among different datasets, sugarcane also showed a higher proportion of rhythmic elements than the other species. Thus, it is possible that a higher proportion of transcripts are regulated by the sugarcane circadian clock. Thirty-six percent of the identified AS/SS pairs had significant correlated time courses and 64% had uncorrelated expression patterns. The clustering of transcripts with similar function, the anticipation of daily environmental changes and the temporal compartmentation of metabolic processes were some properties identified in the circadian sugarcane transcriptome. During the day, there was a dominance of transcripts associated with photosynthesis and carbohydrate metabolism, including sucrose and starch synthesis. During the night, there was dominance of transcripts associated with genetic processing, such as histone regulation and RNA polymerase, ribosome and protein synthesis. Finally, the circadian clock also regulated hormone signalling pathways: a large proportion of auxin and ABA signalling components were regulated by the circadian clock in an unusual biphasic distribution.

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

    Science.gov (United States)

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

    1996-11-01

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

  15. Genetic organisation, mobility and predicted functions of genes on integrated, mobile genetic elements in sequenced strains of Clostridium difficile.

    Directory of Open Access Journals (Sweden)

    Michael S M Brouwer

    Full Text Available BACKGROUND: Clostridium difficile is the leading cause of hospital-associated diarrhoea in the US and Europe. Recently the incidence of C. difficile-associated disease has risen dramatically and concomitantly with the emergence of 'hypervirulent' strains associated with more severe disease and increased mortality. C. difficile contains numerous mobile genetic elements, resulting in the potential for a highly plastic genome. In the first sequenced strain, 630, there is one proven conjugative transposon (CTn, Tn5397, and six putative CTns (CTn1, CTn2 and CTn4-7, of which, CTn4 and CTn5 were capable of excision. In the second sequenced strain, R20291, two further CTns were described. RESULTS: CTn1, CTn2 CTn4, CTn5 and CTn7 were shown to excise from the genome of strain 630 and transfer to strain CD37. A putative CTn from R20291, misleadingly termed a phage island previously, was shown to excise and to contain three putative mobilisable transposons, one of which was capable of excision. In silico probing of C. difficile genome sequences with recombinase gene fragments identified new putative conjugative and mobilisable transposons related to the elements in strains 630 and R20291. CTn5-like elements were described occupying different insertion sites in different strains, CTn1-like elements that have lost the ability to excise in some ribotype 027 strains were described and one strain was shown to contain CTn5-like and CTn7-like elements arranged in tandem. Additionally, using bioinformatics, we updated previous gene annotations and predicted novel functions for the accessory gene products on these new elements. CONCLUSIONS: The genomes of the C. difficile strains examined contain highly related CTns suggesting recent horizontal gene transfer. Several elements were capable of excision and conjugative transfer. The presence of antibiotic resistance genes and genes predicted to promote adaptation to the intestinal environment suggests that CTns play a

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

    Institute of Scientific and Technical Information of China (English)

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

    2006-01-01

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

  17. Systems genetics reveals key genetic elements of drought induced gene regulation in diploid potato.

    Science.gov (United States)

    van Muijen, Dennis; Anithakumari, A M; Maliepaard, Chris; Visser, Richard G F; van der Linden, C Gerard

    2016-09-01

    In plants, tolerance to drought stress is a result of numerous minor effect loci in which transcriptional regulation contributes significantly to the observed phenotypes. Under severe drought conditions, a major expression quantitative trait loci hotspot was identified on chromosome five in potato. A putative Nuclear factor y subunit C4 was identified as key candidate in the regulatory cascade in response to drought. Further investigation of the eQTL hotspots suggests a role for a putative Homeobox leucine zipper protein 12 in relation to drought in potato. Genes strongly co-expressed with Homeobox leucine zipper protein 12 were plant growth regulators responsive to water deficit stress in Arabidopsis thaliana, implying a possible conserved mechanism. Integrative analysis of genetic, genomic, phenotypic and transcriptomic data provided insights in the downstream functional components of the drought response. The abscisic acid- and environmental stress-inducible protein TAS14 was highly induced by severe drought in potato and acts as a reliable biomarker for the level of stress perceived by the plant. The systems genetics approach supported a role for multiple genes responsive to severe drought stress of Solanum tuberosum. The combination of gene regulatory networks, expression quantitative trait loci mapping and phenotypic analysis proved useful for candidate gene selection. PMID:27353051

  18. Finding an optimization of the plate element of Egyptian research reactor using genetic algorithm

    Institute of Scientific and Technical Information of China (English)

    WAHED Mohamed; IBRAHIM Wesam; EFFAT Ahmed

    2008-01-01

    The second Egyptian research reactor ET-RR-2 went critical on the 27th of November 1997. The National Center of Nuclear Safety and Radiation Control (NCNSRC) has the responsibility of the evaluation and assessment of the safety of this reactor. The purpose of this paper is to present an approach to optimization of the fuel element plate.For an efficient search through the solution space we use a multi objective genetic algorithm which allows us to identify a set of Pareto optimal solutions providing the decision maker with the complete spectrum of optimal solutions with respect to the various targets. The aim of this paper is to propose a new approach for optimizing the fuel element plate in the reactor. The fuel element plate is designed with a view to improve reliability and lifetime and it is one of the most important elements during the shut down. In this present paper, we present a conceptual design approach for fuel element plate, in conjunction with a genetic algorithm to obtain a fuel plate that maximizes a fitness value to optimize the safety design of the fuel plate.

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

    Directory of Open Access Journals (Sweden)

    Hsiao Chiu-Bin

    2006-11-01

    Full Text Available Abstract Background While viruses have long been shown to capitalize on their limited genomic size by utilizing both strands of DNA or complementary DNA/RNA intermediates to code for viral proteins, it has been assumed that human retroviruses have all their major proteins translated only from the plus or sense strand of RNA, despite their requirement for a dsDNA proviral intermediate. Several studies, however, have suggested the presence of antisense transcription for both HIV-1 and HTLV-1. More recently an antisense transcript responsible for the HTLV-1 bZIP factor (HBZ protein has been described. In this study we investigated the possibility of an antisense gene contained within the human immunodeficiency virus type 1 (HIV-1 long terminal repeat (LTR. Results Inspection of published sequences revealed a potential transcription initiator element (INR situated downstream of, and in reverse orientation to, the usual HIV-1 promoter and transcription start site. This antisense initiator (HIVaINR suggested the possibility of an antisense gene responsible for RNA and protein production. We show that antisense transcripts are generated, in vitro and in vivo, originating from the TAR DNA of the HIV-1 LTR. To test the possibility that protein(s could be translated from this novel HIV-1 antisense RNA, recombinant HIV antisense gene-FLAG vectors were designed. Recombinant protein(s were produced and isolated utilizing carboxy-terminal FLAG epitope (DYKDDDDK sequences. In addition, affinity-purified antisera to an internal peptide derived from the HIV antisense protein (HAP sequences identified HAPs from HIV+ human peripheral blood lymphocytes. Conclusion HIV-1 contains an antisense gene in the U3-R regions of the LTR responsible for both an antisense RNA transcript and proteins. This antisense transcript has tremendous potential for intrinsic RNA regulation because of its overlap with the beginning of all HIV-1 sense RNA transcripts by 25 nucleotides. The

  20. [Mobile genetic element MDG4 (gypsy) in Drosophila melanogaster. Features of structure and regulation of transposition].

    Science.gov (United States)

    Kusulidu, L K; Karpova, N N; Razorenova, O V; Glukhov, I A; Kim, A I; Liubomirskaia, N V; Il'in, Iu V

    2001-12-01

    Distribution of two structural functional variants of the MDG4 (gypsy) mobile genetic element was examined in 44 strains of Drosophila melanogaster. The results obtained suggest that less transpositionally active MDG4 variant is more ancient component of the Drosophila genome. Using Southern blotting, five strains characterized by increased copy number of MDG4 with significant prevalence of the active variant over the less active one were selected for further analysis. Genetic analysis of these strains led to the suggestion that some of them carry factors that mobilize MDG4 independently from the cellular flamenco gene known to be responsible for transposition of this element. Other strains probably contained a suppressor of the flam- mutant allele causing active transpositions of the MDG4. Thus, the material for studying poorly examined relationships between the retrovirus and the host cell genome was obtained. PMID:11785284

  1. Smoothed Finite Element and Genetic Algorithm based optimization for Shape Adaptive Composite Marine Propellers

    OpenAIRE

    Herath, Manudha T; Natarajan, Sundararajan; Prusty, B Gangadhara; John, Nigel St

    2013-01-01

    An optimization scheme using the Cell-based Smoothed Finite Element Method (CS-FEM) combined with a Genetic Algorithm (GA) framework is proposed in this paper to design shape adaptive laminated composite marine propellers. The proposed scheme utilise the bend-twist coupling characteristics of the composites to achieve the required performance. An iterative procedure to evaluate the unloaded shape of the propeller blade is proposed, confirming the manufacturing requirements at the initial stag...

  2. Design of a Four-Element, Hollow-Cube Corner Retroreflector for Satellites by use of a Genetic Algorithm.

    Science.gov (United States)

    Minato, A; Sugimoto, N

    1998-01-20

    A four-element retroreflector was designed for satellite laser ranging and Earth-satellite-Earth laser long-path absorption measurement of the atmosphere. The retroreflector consists of four symmetrically located corner retroreflectors. Each retroreflector element has curved mirrors and tuned dihedral angles to correct velocity aberrations. A genetic algorithm was employed to optimize dihedral angles of each element and the directions of the four elements. The optimized four-element retroreflector has high reflectance with a reasonably broad angular coverage. It is also shown that the genetic algorithm is effective for optimizing optics with many parameters.

  3. Functionalization of an Antisense Small RNA

    Science.gov (United States)

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

    2016-01-01

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

  4. Circulating nucleic acids: a new class of physiological mobile genetic elements [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Indraneel Mittra

    2015-09-01

    Full Text Available Mobile genetic elements play a major role in shaping biotic genomes and bringing about evolutionary transformations. Herein, a new class of mobile genetic elements is proposed in the form of circulating nucleic acids (CNAs derived from the billions of cells that die in the body every day due to normal physiology and that act intra-corporeally. A recent study shows that CNAs can freely enter into healthy cells, integrate into their genomes by a unique mechanism and cause damage to their DNA. Being ubiquitous and continuously arising, CNA-induced DNA damage may be the underlying cause of ageing, ageing-related disabilities and the ultimate demise of the organism. Thus, DNA seems to act in the paradoxical roles of both preserver and destroyer of life. This new class of mobile genetic element may be relevant not only to multi-cellular organisms with established circulatory systems, but also to other multi-cellular organisms in which intra-corporeal mobility of nucleic acids may be mediated via the medium of extra-cellular fluid.

  5. Key genetic elements and regulation systems in methicillin-resistant Staphylococcus aureus.

    Science.gov (United States)

    Hao, Haihong; Dai, Menghong; Wang, Yulian; Huang, Lingli; Yuan, Zonghui

    2012-11-01

    Methicillin-resistant Staphylococcus aureus (MRSA), popularly known as a type of superbug, has been a serious challenge for animal and human health. S. aureus has developed methicillin resistance mainly by expression of β-lactamase and PBP2a, which is regulated by the blaZ-blaI-blaR1 and mecA-mecI-mecRI systems. Other genetic elements, including murE and femA, also participate in expression of methicillin resistance, but the mechanism remains unclear. The evolution of the staphylococcal cassette chromosome mec determines the epidemiological risk of MRSA. The plasmid-located gene cfr might contribute to multiresistance and transmission of MRSA. Some virulence factors, including Panton-Valentine leukocidin, phenol-soluble modulin, arginine catabolic mobile element and other toxin elements enhance the pathogenesis and fitness of MRSA. Two-component regulation systems (agr, saeRS and vraRS) are closely associated with pathogenesis and drug resistance of MRSA. The systematic exploration of key genetic elements and regulation systems involved in multidrug resistance/pathogenesis/transmission of MRSA is conclusively integrated into this review, providing fundamental information for the development of new antimicrobial agents and the establishment of reasonable antibiotic stewardship to reduce the risk of this superbug. PMID:23075449

  6. sp3-hybridized framework structure of group-14 elements discovered by genetic algorithm

    Energy Technology Data Exchange (ETDEWEB)

    Nguyen, Manh Cuong [Ames Laboratory; Zhao, Xin [Ames Laboratory; Wang, Cai-Zhuang [Ames Laboratory; Ho, Kai-Ming [Ames Laboratory

    2014-05-01

    Group-14 elements, including C, Si, Ge, and Sn, can form various stable and metastable structures. Finding new metastable structures of group-14 elements with desirable physical properties for new technological applications has attracted a lot of interest. Using a genetic algorithm, we discovered a new low-energy metastable distorted sp3-hybridized framework structure of the group-14 elements. It has P42/mnm symmetry with 12 atoms per unit cell. The void volume of this structure is as large as 139.7Å3 for Si P42/mnm, and it can be used for gas or metal-atom encapsulation. Band-structure calculations show that P42/mnm structures of Si and Ge are semiconducting with energy band gaps close to the optimal values for optoelectronic or photovoltaic applications. With metal-atom encapsulation, the P42/mnm structure would also be a candidate for rattling-mediated superconducting or used as thermoelectric materials.

  7. 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.

  8. 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.

  9. Tye7 regulates yeast Ty1 retrotransposon sense and antisense transcription in response to adenylic nucleotides stress.

    Science.gov (United States)

    Servant, Géraldine; Pinson, Benoit; Tchalikian-Cosson, Aurélie; Coulpier, Fanny; Lemoine, Sophie; Pennetier, Carole; Bridier-Nahmias, Antoine; Todeschini, Anne Laure; Fayol, Hélène; Daignan-Fornier, Bertrand; Lesage, Pascale

    2012-07-01

    Transposable elements play a fundamental role in genome evolution. It is proposed that their mobility, activated under stress, induces mutations that could confer advantages to the host organism. Transcription of the Ty1 LTR-retrotransposon of Saccharomyces cerevisiae is activated in response to a severe deficiency in adenylic nucleotides. Here, we show that Ty2 and Ty3 are also stimulated under these stress conditions, revealing the simultaneous activation of three active Ty retrotransposon families. We demonstrate that Ty1 activation in response to adenylic nucleotide depletion requires the DNA-binding transcription factor Tye7. Ty1 is transcribed in both sense and antisense directions. We identify three Tye7 potential binding sites in the region of Ty1 DNA sequence where antisense transcription starts. We show that Tye7 binds to Ty1 DNA and regulates Ty1 antisense transcription. Altogether, our data suggest that, in response to adenylic nucleotide reduction, TYE7 is induced and activates Ty1 mRNA transcription, possibly by controlling Ty1 antisense transcription. We also provide the first evidence that Ty1 antisense transcription can be regulated by environmental stress conditions, pointing to a new level of control of Ty1 activity by stress, as Ty1 antisense RNAs play an important role in regulating Ty1 mobility at both the transcriptional and post-transcriptional stages. PMID:22379133

  10. Tye7 regulates yeast Ty1 retrotransposon sense and antisense transcription in response to adenylic nucleotides stress

    Science.gov (United States)

    Servant, Géraldine; Pinson, Benoit; Tchalikian-Cosson, Aurélie; Coulpier, Fanny; Lemoine, Sophie; Pennetier, Carole; Bridier-Nahmias, Antoine; Todeschini, Anne Laure; Fayol, Hélène; Daignan-Fornier, Bertrand; Lesage, Pascale

    2012-01-01

    Transposable elements play a fundamental role in genome evolution. It is proposed that their mobility, activated under stress, induces mutations that could confer advantages to the host organism. Transcription of the Ty1 LTR-retrotransposon of Saccharomyces cerevisiae is activated in response to a severe deficiency in adenylic nucleotides. Here, we show that Ty2 and Ty3 are also stimulated under these stress conditions, revealing the simultaneous activation of three active Ty retrotransposon families. We demonstrate that Ty1 activation in response to adenylic nucleotide depletion requires the DNA-binding transcription factor Tye7. Ty1 is transcribed in both sense and antisense directions. We identify three Tye7 potential binding sites in the region of Ty1 DNA sequence where antisense transcription starts. We show that Tye7 binds to Ty1 DNA and regulates Ty1 antisense transcription. Altogether, our data suggest that, in response to adenylic nucleotide reduction, TYE7 is induced and activates Ty1 mRNA transcription, possibly by controlling Ty1 antisense transcription. We also provide the first evidence that Ty1 antisense transcription can be regulated by environmental stress conditions, pointing to a new level of control of Ty1 activity by stress, as Ty1 antisense RNAs play an important role in regulating Ty1 mobility at both the transcriptional and post-transcriptional stages. PMID:22379133

  11. Optimization of Zoom Lens with Discrete State of Liquid Lens Elements by Using Genetic Algorithm

    Directory of Open Access Journals (Sweden)

    Cheng-Mu Tsai

    2015-01-01

    Full Text Available This paper is to employ liquid lens elements to design a lens with zoom function by using the genetic algorithm (GA optimization. The liquid lens elements used in the proposal can apply voltage adjustment to generate the electrical field that induces the liquid with electric conductivity to vary the surface curvature between two different kinds of liquids. According to the voltage level, the liquid lens element makes the discrete variation of the curvature and thickness realize the zoom function without moving the lens groups so that the overall length can be reduced. However, it is difficult to design the zoom lens under the discrete variation of the curvature and thickness in the liquid lens elements and the mechanical space that is constantly limited. The GA offers a flexible way for lens optimization. We regarded the spot size as the fitness function to look for the optimum curvatures, thickness, and the corresponding statuses of liquid lens elements for the zoom lens. As a result, the zoom lens with constant space can be realized by running the selection, crossover, and mutation operation in the GA optimization.

  12. Functional correction by antisense therapy of a splicing mutation in the GALT gene.

    Science.gov (United States)

    Coelho, Ana I; Lourenço, Sílvia; Trabuco, Matilde; Silva, Maria João; Oliveira, Anabela; Gaspar, Ana; Diogo, Luísa; Tavares de Almeida, Isabel; Vicente, João B; Rivera, Isabel

    2015-04-01

    In recent years, antisense therapy has emerged as an increasingly important therapeutic approach to tackle several genetic disorders, including inborn errors of metabolism. Intronic mutations activating cryptic splice sites are particularly amenable to antisense therapy, as the canonical splice sites remain intact, thus retaining the potential for restoring constitutive splicing. Mutational analysis of Portuguese galactosemic patients revealed the intronic variation c.820+13A>G as the second most prevalent mutation, strongly suggesting its pathogenicity. The aim of this study was to functionally characterize this intronic variation, to elucidate its pathogenic molecular mechanism(s) and, ultimately, to correct it by antisense therapy. Minigene splicing assays in two distinct cell lines and patients' transcript analyses showed that the mutation activates a cryptic donor splice site, inducing an aberrant splicing of the GALT pre-mRNA, which in turn leads to a frameshift with inclusion of a premature stop codon (p.D274Gfs*17). Functional-structural studies of the recombinant wild-type and truncated GALT showed that the latter is devoid of enzymatic activity and prone to aggregation. Finally, two locked nucleic acid oligonucleotides, designed to specifically recognize the mutation, successfully restored the constitutive splicing, thus establishing a proof of concept for the application of antisense therapy as an alternative strategy for the clearly insufficient dietary treatment in classic galactosemia. PMID:25052314

  13. The Gypsy Database (GyDB) of mobile genetic elements: release 2.0.

    Science.gov (United States)

    Llorens, Carlos; Futami, Ricardo; Covelli, Laura; Domínguez-Escribá, Laura; Viu, Jose M; Tamarit, Daniel; Aguilar-Rodríguez, Jose; Vicente-Ripolles, Miguel; Fuster, Gonzalo; Bernet, Guillermo P; Maumus, Florian; Munoz-Pomer, Alfonso; Sempere, Jose M; Latorre, Amparo; Moya, Andres

    2011-01-01

    This article introduces the second release of the Gypsy Database of Mobile Genetic Elements (GyDB 2.0): a research project devoted to the evolutionary dynamics of viruses and transposable elements based on their phylogenetic classification (per lineage and protein domain). The Gypsy Database (GyDB) is a long-term project that is continuously progressing, and that owing to the high molecular diversity of mobile elements requires to be completed in several stages. GyDB 2.0 has been powered with a wiki to allow other researchers participate in the project. The current database stage and scope are long terminal repeats (LTR) retroelements and relatives. GyDB 2.0 is an update based on the analysis of Ty3/Gypsy, Retroviridae, Ty1/Copia and Bel/Pao LTR retroelements and the Caulimoviridae pararetroviruses of plants. Among other features, in terms of the aforementioned topics, this update adds: (i) a variety of descriptions and reviews distributed in multiple web pages; (ii) protein-based phylogenies, where phylogenetic levels are assigned to distinct classified elements; (iii) a collection of multiple alignments, lineage-specific hidden Markov models and consensus sequences, called GyDB collection; (iv) updated RefSeq databases and BLAST and HMM servers to facilitate sequence characterization of new LTR retroelement and caulimovirus queries; and (v) a bibliographic server. GyDB 2.0 is available at http://gydb.org. PMID:21036865

  14. Viruses-to-mobile genetic elements skew in the deep Atlantis II brine pool sediments

    Science.gov (United States)

    Adel, Mustafa; Elbehery, Ali H. A.; Aziz, Sherry K.; Aziz, Ramy K.; Grossart, Hans-Peter; Siam, Rania

    2016-01-01

    The central rift of the Red Sea has 25 brine pools with different physical and geochemical characteristics. Atlantis II (ATIID), Discovery Deeps (DD) and Chain Deep (CD) are characterized by high salinity, temperature and metal content. Several studies reported microbial communities in these brine pools, but few studies addressed the brine pool sediments. Therefore, sediment cores were collected from ATIID, DD, CD brine pools and an adjacent brine-influenced site. Sixteen different lithologic sediment sections were subjected to shotgun DNA pyrosequencing to generate 1.47 billion base pairs (1.47 × 109 bp). We generated sediment-specific reads and attempted to annotate all reads. We report the phylogenetic and biochemical uniqueness of the deepest ATIID sulfur-rich brine pool sediments. In contrary to all other sediment sections, bacteria dominate the deepest ATIID sulfur-rich brine pool sediments. This decrease in virus-to-bacteria ratio in selected sections and depth coincided with an overrepresentation of mobile genetic elements. Skewing in the composition of viruses-to-mobile genetic elements may uniquely contribute to the distinct microbial consortium in sediments in proximity to hydrothermally active vents of the Red Sea and possibly in their surroundings, through differential horizontal gene transfer. PMID:27596223

  15. Viruses-to-mobile genetic elements skew in the deep Atlantis II brine pool sediments

    Science.gov (United States)

    Adel, Mustafa; Elbehery, Ali H. A.; Aziz, Sherry K.; Aziz, Ramy K.; Grossart, Hans-Peter; Siam, Rania

    2016-09-01

    The central rift of the Red Sea has 25 brine pools with different physical and geochemical characteristics. Atlantis II (ATIID), Discovery Deeps (DD) and Chain Deep (CD) are characterized by high salinity, temperature and metal content. Several studies reported microbial communities in these brine pools, but few studies addressed the brine pool sediments. Therefore, sediment cores were collected from ATIID, DD, CD brine pools and an adjacent brine-influenced site. Sixteen different lithologic sediment sections were subjected to shotgun DNA pyrosequencing to generate 1.47 billion base pairs (1.47 × 109 bp). We generated sediment-specific reads and attempted to annotate all reads. We report the phylogenetic and biochemical uniqueness of the deepest ATIID sulfur-rich brine pool sediments. In contrary to all other sediment sections, bacteria dominate the deepest ATIID sulfur-rich brine pool sediments. This decrease in virus-to-bacteria ratio in selected sections and depth coincided with an overrepresentation of mobile genetic elements. Skewing in the composition of viruses-to-mobile genetic elements may uniquely contribute to the distinct microbial consortium in sediments in proximity to hydrothermally active vents of the Red Sea and possibly in their surroundings, through differential horizontal gene transfer.

  16. Inteins, introns, and homing endonucleases: recent revelations about the life cycle of parasitic genetic elements

    Directory of Open Access Journals (Sweden)

    Hilario Elena

    2006-11-01

    Full Text Available Abstract Self splicing introns and inteins that rely on a homing endonuclease for propagation are parasitic genetic elements. Their life-cycle and evolutionary fate has been described through the homing cycle. According to this model the homing endonuclease is selected for function only during the spreading phase of the parasite. This phase ends when the parasitic element is fixed in the population. Upon fixation the homing endonuclease is no longer under selection, and its activity is lost through random processes. Recent analyses of these parasitic elements with functional homing endonucleases suggest that this model in its most simple form is not always applicable. Apparently, functioning homing endonuclease can persist over long evolutionary times in populations and species that are thought to be asexual or nearly asexual. Here we review these recent findings and discuss their implications. Reasons for the long-term persistence of a functional homing endonuclease include: More recombination (sexual and as a result of gene transfer than previously assumed for these organisms; complex population structures that prevent the element from being fixed; a balance between active spreading of the homing endonuclease and a decrease in fitness caused by the parasite in the host organism; or a function of the homing endonuclease that increases the fitness of the host organism and results in purifying selection for the homing endonuclease activity, even after fixation in a local population. In the future, more detailed studies of the population dynamics of the activity and regulation of homing endonucleases are needed to decide between these possibilities, and to determine their relative contributions to the long term survival of parasitic genes within a population. Two outstanding publications on the amoeba Naegleria group I intron (Wikmark et al. BMC Evol Biol 2006, 6:39 and the PRP8 inteins in ascomycetes (Butler et al.BMC Evol Biol 2006, 6:42 provide

  17. Evaluation on the nutritional value of protein in genetically modified rice with double antisense starch-branching enzyme gene%转双反义淀粉分支酶基因大米中蛋白质的营养价值评价

    Institute of Scientific and Technical Information of China (English)

    李敏; 胡贻椿; 朴建华; 杨晓光

    2011-01-01

    目的 对转双反义淀粉分支酶基因大米和亲本大米蛋白质的营养价值进行评价.方法 采用通用的非生物学评价方法,即化学评分、氨基酸评分、蛋白质消化率校正后的氨基酸评分、必需氨基酸指数、营养指数、氨基酸比值系数和氨基酸比值系数分,评价大米蛋白质的营养价值.结果 转基因大米和亲本大米的化学评分分别为62,59;氨基酸评分分别为75,62;蛋白质消化率校正后的氨基酸评分分别为65,56;必需氨基酸指数分别为79,80;营养指数分别为11,8;氨基酸比值系数分分别为76.7,67.0;氨基酸比值系数都是l.1.结论 转双反义淀粉分支氨基因大来中抗性淀粉含童显著提高,但并未影响到其自身蛋白质的营养价值.该转基因大米蛋白质的营养价值与亲本大米具有"实质等同性".%Objective To assess the nutritional value of protein in the genetically modified rice with double antisense starch-branching enzyme (SBE) gene and its parental rice.Methods The adopted nutritional value assessment methods, including chemical score (CS), amino acid score (AAS), protein digestibility-corrected amino acid score (PDCAAS), essential amino acid index (EAAI), nutritional index (NI), ratio coefficient of amino acid (RCAA) and score of RCAA ( SRCAA ) was applied to assess the protein nutritional value of the rice.Results The CS value of genetically modified rice and parental rice were 62 and 59, AAS value were 75 and 62, PDCAAS value were 65 and 56,EAAI were 79 and 80, NI were 11 and 8, SRCAA were 76.7 and 67.0, respectively.RCAA were the same.Conclusion The nutritional value of protein in the genetically modified rice with double antisense SBE gene were not greatly changed by the increase of resistant starch content in the rice.So, the nutritional value of protein in the genetically modified rice and its parental rice was substantial equivalent.

  18. 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.

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

    Directory of Open Access Journals (Sweden)

    Ting Ni

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

  20. 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.

  1. Antisense Oligonucleotide-Mediated Exon Skipping for Duchenne Muscular Dystrophy: Progress and Challenges.

    OpenAIRE

    Arechavala-Gomeza, V.; Anthony, K.; Morgan, J; Muntoni, F.

    2012-01-01

    Duchenne muscular dystrophy (DMD) is the most common childhood neuromuscular disorder. It is caused by mutations in the DMD gene that disrupt the open reading frame (ORF) preventing the production of functional dystrophin protein. The loss of dystrophin ultimately leads to the degeneration of muscle fibres, progressive weakness and premature death. Antisense oligonucleotides (AOs) targeted to splicing elements within DMD pre-mRNA can induce the skipping of targeted exons, restoring the ORF an...

  2. Genetic Innovation in Vertebrates: Gypsy Integrase Genes and Other Genes Derived from Transposable Elements

    Directory of Open Access Journals (Sweden)

    Domitille Chalopin

    2012-01-01

    Full Text Available Due to their ability to drive DNA rearrangements and to serve as a source of new coding and regulatory sequences, transposable elements (TEs are considered as powerful evolutionary agents within genomes. In this paper, we review the mechanism of molecular domestication, which corresponds to the formation of new genes derived from TE sequences. Many genes derived from retroelements and DNA transposons have been identified in mammals and other vertebrates, some of them fulfilling essential functions for the development and survival of their host organisms. We will particularly focus on the evolution and expression of Gypsy integrase (GIN genes, which have been formed from ancient event(s of molecular domestication and have evolved differentially in some vertebrate sublineages. What we describe here is probably only the tip of the evolutionary iceberg, and future genome analyses will certainly uncover new TE-derived genes and biological functions driving genetic innovation in vertebrates and other organisms.

  3. Mobile genetic elements of Pseudomonas aeruginosa isolates from hydrotherapy facility and respiratory infections.

    Science.gov (United States)

    Pereira, S G; Cardoso, O

    2014-03-01

    The content of mobile genetic elements in Pseudomonas aeruginosa isolates of a pristine natural mineral water system associated with healthcare was compared with clinical isolates from respiratory infections. One isolate, from the therapy pool circuit, presented a class 1 integron, with 100% similarity to a class 1 integron contained in plasmid p4800 of the Klebsiella pneumoniae Kp4800 strain, which is the first time it has been reported in P. aeruginosa. Class 1 integrons were found in 25.6% of the clinical isolates. PAGI1 orf3 was more prevalent in environmental isolates, while PAGI2 c105 and PAGI3 sg100 were more prevalent in clinical isolates. Plasmids were not observed in either population.

  4. Genetic evidence for conserved non-coding element function across species--the ears have it

    Directory of Open Access Journals (Sweden)

    Eric E Turner

    2014-01-01

    Full Text Available Comparison of genomic sequences from diverse vertebrate species has revealed numerous highly conserved regions that do not appear to encode proteins or functional RNAs. Often these conserved non-coding elements, or CNEs, direct gene expression to specific tissues in transgenic models, demonstrating they have regulatory function. CNEs are frequently found near ‘developmental’ genes, particularly transcription factors, implying that these elements have essential regulatory roles in development. However, actual examples demonstrating CNE regulatory functions across species have been few, and recent loss-of-function studies of several CNEs in mice have shown relatively minor effects. In this Perspectives article, we discuss new findings in fancy rats and Highland cattle demonstrating that function of a CNE near the Hmx1 gene is crucial for normal external ear development and resembles loss-of function Hmx1 coding mutations in mice and humans. These findings provide important support for similar developmental roles of CNEs in divergent species, and reinforce the concept that CNEs should be examined systematically in the ongoing search for genetic causes of human developmental disorders in the era of genome-scale sequencing.

  5. Voltage-gated calcium channel and antisense oligonucleotides thereto

    Science.gov (United States)

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

    1998-01-01

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

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

    Science.gov (United States)

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

    2015-07-01

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

  7. Integration of Experiments across Diverse Environments Identifies the Genetic Determinants of Variation in Sorghum bicolor Seed Element Composition.

    Science.gov (United States)

    Shakoor, Nadia; Ziegler, Greg; Dilkes, Brian P; Brenton, Zachary; Boyles, Richard; Connolly, Erin L; Kresovich, Stephen; Baxter, Ivan

    2016-04-01

    Seedling establishment and seed nutritional quality require the sequestration of sufficient element nutrients. The identification of genes and alleles that modify element content in the grains of cereals, including sorghum (Sorghum bicolor), is fundamental to developing breeding and selection methods aimed at increasing bioavailable element content and improving crop growth. We have developed a high-throughput work flow for the simultaneous measurement of multiple elements in sorghum seeds. We measured seed element levels in the genotyped Sorghum Association Panel, representing all major cultivated sorghum races from diverse geographic and climatic regions, and mapped alleles contributing to seed element variation across three environments by genome-wide association. We observed significant phenotypic and genetic correlation between several elements across multiple years and diverse environments. The power of combining high-precision measurements with genome-wide association was demonstrated by implementing rank transformation and a multilocus mixed model to map alleles controlling 20 element traits, identifying 255 loci affecting the sorghum seed ionome. Sequence similarity to genes characterized in previous studies identified likely causative genes for the accumulation of zinc, manganese, nickel, calcium, and cadmium in sorghum seeds. In addition to strong candidates for these five elements, we provide a list of candidate loci for several other elements. Our approach enabled the identification of single-nucleotide polymorphisms in strong linkage disequilibrium with causative polymorphisms that can be evaluated in targeted selection strategies for plant breeding and improvement. PMID:26896393

  8. Cotton transgenics with Antisense AC1 gene for resistance against cotton leaf curl virus

    Directory of Open Access Journals (Sweden)

    J.Amudha, G.Balasubramani, V.G.Malathi, D.Monga, K.C.Bansal and K.R.Kranthi

    2010-07-01

    Full Text Available Cotton leaf curl virus is a devastating pest in the North India and in small pockets of Southern states. Cotton leaf curldisease (CLCuD is caused by a Geminivirus, transmitted by whitefly Bemisia tabaci vector. This is a serious problem inthe northern region and leads to yield losses up to 58% and 69% (ICAC recorder, 1999. Genetic engineering for cottontransgenics resistant to leaf curl disease (CLCuD through antisense RNA approach is potential to tackle the disease incotton. Cotton transgenics resistant to leaf curl disease (CLCuD using Antisense (rep (Replicase protein gene wasdeveloped via Agrobacterium mediated transformation. A binary vector carrying the Antisense rep gene along with thenpt II (neomycin phospho transferase gene driven by CaMV-35S promoter and NOS (nopaline synthase terminator wasused for transformation. The confirmation of the rep and npt II genes in the transgenic plants were verified by PCR andintegration of T-DNA into the plant genome was confirmed by Southern analysis. The individual transgenics were raisedin the green house and screened for the virus resistance. T2 progeny analysis showed classical Mendelian pattern ofinheritance.

  9. Resistance determinants and mobile genetic elements of an NDM-1-encoding Klebsiella pneumoniae strain.

    Directory of Open Access Journals (Sweden)

    Corey M Hudson

    Full Text Available Multidrug-resistant Enterobacteriaceae are emerging as a serious infectious disease challenge. These strains can accumulate many antibiotic resistance genes though horizontal transfer of genetic elements, those for β-lactamases being of particular concern. Some β-lactamases are active on a broad spectrum of β-lactams including the last-resort carbapenems. The gene for the broad-spectrum and carbapenem-active metallo-β-lactamase NDM-1 is rapidly spreading. We present the complete genome of Klebsiella pneumoniae ATCC BAA-2146, the first U.S. isolate found to encode NDM-1, and describe its repertoire of antibiotic-resistance genes and mutations, including genes for eight β-lactamases and 15 additional antibiotic-resistance enzymes. To elucidate the evolution of this rich repertoire, the mobile elements of the genome were characterized, including four plasmids with varying degrees of conservation and mosaicism and eleven chromosomal genomic islands. One island was identified by a novel phylogenomic approach, that further indicated the cps-lps polysaccharide synthesis locus, where operon translocation and fusion was noted. Unique plasmid segments and mosaic junctions were identified. Plasmid-borne blaCTX-M-15 was transposed recently to the chromosome by ISEcp1. None of the eleven full copies of IS26, the most frequent IS element in the genome, had the expected 8-bp direct repeat of the integration target sequence, suggesting that each copy underwent homologous recombination subsequent to its last transposition event. Comparative analysis likewise indicates IS26 as a frequent recombinational junction between plasmid ancestors, and also indicates a resolvase site. In one novel use of high-throughput sequencing, homologously recombinant subpopulations of the bacterial culture were detected. In a second novel use, circular transposition intermediates were detected for the novel insertion sequence ISKpn21 of the ISNCY family, suggesting that it uses

  10. Metagenomic profiling of antibiotic resistance genes and mobile genetic elements in a tannery wastewater treatment plant.

    Directory of Open Access Journals (Sweden)

    Zhu Wang

    Full Text Available Antibiotics are often used to prevent sickness and improve production in animal agriculture, and the residues in animal bodies may enter tannery wastewater during leather production. This study aimed to use Illumina high-throughput sequencing to investigate the occurrence, diversity and abundance of antibiotic resistance genes (ARGs and mobile genetic elements (MGEs in aerobic and anaerobic sludge of a full-scale tannery wastewater treatment plant (WWTP. Metagenomic analysis showed that Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria dominated in the WWTP, but the relative abundance of archaea in anaerobic sludge was higher than in aerobic sludge. Sequencing reads from aerobic and anaerobic sludge revealed differences in the abundance of functional genes between both microbial communities. Genes coding for antibiotic resistance were identified in both communities. BLAST analysis against Antibiotic Resistance Genes Database (ARDB further revealed that aerobic and anaerobic sludge contained various ARGs with high abundance, among which sulfonamide resistance gene sul1 had the highest abundance, occupying over 20% of the total ARGs reads. Tetracycline resistance genes (tet were highly rich in the anaerobic sludge, among which tet33 had the highest abundance, but was absent in aerobic sludge. Over 70 types of insertion sequences were detected in each sludge sample, and class 1 integrase genes were prevalent in the WWTP. The results highlighted prevalence of ARGs and MGEs in tannery WWTPs, which may deserve more public health concerns.

  11. Resistance Genes and Genetic Elements Associated with Antibiotic Resistance in Clinical and Commensal Isolates of Streptococcus salivarius.

    Science.gov (United States)

    Chaffanel, Fanny; Charron-Bourgoin, Florence; Libante, Virginie; Leblond-Bourget, Nathalie; Payot, Sophie

    2015-06-15

    The diversity of clinical (n = 92) and oral and digestive commensal (n = 120) isolates of Streptococcus salivarius was analyzed by multilocus sequence typing (MLST). No clustering of clinical or commensal strains can be observed in the phylogenetic tree. Selected strains (92 clinical and 46 commensal strains) were then examined for their susceptibilities to tetracyclines, macrolides, lincosamides, aminoglycosides, and phenicol antibiotics. The presence of resistance genes tet(M), tet(O), erm(A), erm(B), mef(A/E), and catQ and associated genetic elements was investigated by PCR, as was the genetic linkage of resistance genes. High rates of erythromycin and tetracycline resistance were observed among the strains. Clinical strains displayed either the erm(B) (macrolide-lincosamide-streptogramin B [MLSB] phenotype) or mef(A/E) (M phenotype) resistance determinant, whereas almost all the commensal strains harbored the mef(A/E) resistance gene, carried by a macrolide efflux genetic assembly (MEGA) element. A genetic linkage between a macrolide resistance gene and genes of Tn916 was detected in 23 clinical strains and 5 commensal strains, with a predominance of Tn3872 elements (n = 13), followed by Tn6002 (n = 11) and Tn2009 (n = 4) elements. Four strains harboring a mef(A/E) gene were also resistant to chloramphenicol and carried a catQ gene. Sequencing of the genome of one of these strains revealed that these genes colocalized on an IQ-like element, as already described for other viridans group streptococci. ICESt3-related elements were also detected in half of the isolates. This work highlights the potential role of S. salivarius in the spread of antibiotic resistance genes both in the oral sphere and in the gut. PMID:25862227

  12. System-Level Genetic Codes Using a Transposable Element-Like Mechanism with Applications to Cancer

    OpenAIRE

    McGowan, John F.

    2000-01-01

    A system-level genetic code is a hypothetical genetic code that exclusively or preferentially codes systems of interacting coadapted parts. System-level genetic codes differ from part-level genetic codes in which each discrete part is coded independently. In general, a system-level genetic code requires coding discrete interacting parts such as organs or proteins in an interdependent way. Changing a single symbol or "gene" in a system-level genetic code affects two or more parts in a coordina...

  13. Undetected antisense tRNAs in mitochondrial genomes?

    Directory of Open Access Journals (Sweden)

    Seligmann Hervé

    2010-06-01

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

  14. Virulence determinants, drug resistance and mobile genetic elements of Laribacter hongkongensis: a genome-wide analysis

    Directory of Open Access Journals (Sweden)

    Lau Susanna KP

    2011-04-01

    Full Text Available Abstract Background Laribacter hongkongensis is associated with community-acquired gastroenteritis and traveler's diarrhea. In this study, we performed an in-depth annotation of the genes in its genome related to the various steps in the infective process, drug resistance and mobile genetic elements. Results For acid and bile resistance, L. hongkongensis possessed a urease gene cassette, two arc gene clusters and bile salt efflux systems. For intestinal colonization, it possessed a putative adhesin of the autotransporter family homologous to those of diffusely adherent Escherichia coli (E. coli and enterotoxigenic E. coli. To evade from host defense, it possessed superoxide dismutase and catalases. For lipopolysaccharide biosynthesis, it possessed the same set of genes that encode enzymes for synthesizing lipid A, two Kdo units and heptose units as E. coli, but different genes for its symmetrical acylation pattern, and nine genes for polysaccharide side chains biosynthesis. It contained a number of CDSs that encode putative cell surface acting (RTX toxin and hemolysins and intracellular cytotoxins (patatin-like proteins and enzymes for invasion (outer membrane phospholipase A. It contained a broad variety of antibiotic resistance-related genes, including genes related to β-lactam (n = 10 and multidrug efflux (n = 54. It also contained eight prophages, 17 other phage-related CDSs and 26 CDSs for transposases. Conclusions The L. hongkongensis genome possessed genes for acid and bile resistance, intestinal mucosa colonization, evasion of host defense and cytotoxicity and invasion. A broad variety of antibiotic resistance or multidrug resistance genes, a high number of prophages, other phage-related CDSs and CDSs for transposases, were also identified.

  15. Gene invasion in distant eukaryotic lineages: discovery of mutually exclusive genetic elements reveals marine biodiversity.

    Science.gov (United States)

    Monier, Adam; Sudek, Sebastian; Fast, Naomi M; Worden, Alexandra Z

    2013-09-01

    Inteins are rare, translated genetic parasites mainly found in bacteria and archaea, while spliceosomal introns are distinctly eukaryotic features abundant in most nuclear genomes. Using targeted metagenomics, we discovered an intein in an Atlantic population of the photosynthetic eukaryote, Bathycoccus, harbored by the essential spliceosomal protein PRP8 (processing factor 8 protein). Although previously thought exclusive to fungi, we also identified PRP8 inteins in parasitic (Capsaspora) and predatory (Salpingoeca) protists. Most new PRP8 inteins were at novel insertion sites that, surprisingly, were not in the most conserved regions of the gene. Evolutionarily, Dikarya fungal inteins at PRP8 insertion site a appeared more related to the Bathycoccus intein at a unique insertion site, than to other fungal and opisthokont inteins. Strikingly, independent analyses of Pacific and Atlantic samples revealed an intron at the same codon as the Bathycoccus PRP8 intein. The two elements are mutually exclusive and neither was found in cultured Bathycoccus or other picoprasinophyte genomes. Thus, wild Bathycoccus contain one of few non-fungal eukaryotic inteins known and a rare polymorphic intron. Our data indicate at least two Bathycoccus ecotypes exist, associated respectively with oceanic or mesotrophic environments. We hypothesize that intein propagation is facilitated by marine viruses; and, while intron gain is still poorly understood, presence of a spliceosomal intron where a locus lacks an intein raises the possibility of new, intein-primed mechanisms for intron gain. The discovery of nucleus-encoded inteins and associated sequence polymorphisms in uncultivated marine eukaryotes highlights their diversity and reveals potential sexual boundaries between populations indistinguishable by common marker genes. PMID:23635865

  16. Detection and linkage to mobile genetic elements of tetracycline resistance gene tet(M) in Escherichia coli isolates from pigs

    DEFF Research Database (Denmark)

    Jurado-Rabadán, Sonia; de la Fuente, Ricardo; A Ruiz-Santa-Quiteria, José;

    2014-01-01

    (M) has been identified in E. coli, to our knowledge, there are no previous reports studying the linkage of the tet(M) gene in E. coli to different mobile genetic elements. The aim of this study was to determine the occurrence of tet(A), tet(B), and tet(M) genes in doxycycline-resistant E. coli isolates...... from pigs, as well as the detection of mobile genetic elements linked to tet(M) in E. coli and its possible transfer from enterococci. RESULTS: tet(A) was the most frequently detected gene (87.9%) in doxycycline-resistant isolates. tet(M) was found in 13.1% E. coli isolates. The tet(M) gene...

  17. Trace Ratio Criterion-Based Kernel Discriminant Analysis for Fault Diagnosis of Rolling Element Bearings Using Binary Immune Genetic Algorithm

    Directory of Open Access Journals (Sweden)

    Wen-An Yang

    2016-01-01

    Full Text Available The rolling element bearing is a core component of many systems such as aircraft, train, steamboat, and machine tool, and their failure can lead to reduced capability, downtime, and even catastrophic breakdowns. Due to misoperation, manufacturing deficiencies, or the lack of monitoring and maintenance, it is often found to be the most unreliable component within these systems. Therefore, effective and efficient fault diagnosis of rolling element bearings has an important role in ensuring the continued safe and reliable operation of their host systems. This study presents a trace ratio criterion-based kernel discriminant analysis (TR-KDA for fault diagnosis of rolling element bearings. The binary immune genetic algorithm (BIGA is employed to solve the trace ratio problem in TR-KDA. The numerical results obtained using extensive simulation indicate that the proposed TR-KDA using BIGA (called TR-KDA-BIGA can effectively and efficiently classify different classes of rolling element bearing data, while also providing the capability of real-time visualization that is very useful for the practitioners to monitor the health status of rolling element bearings. Empirical comparisons show that the proposed TR-KDA-BIGA performs better than existing methods in classifying different classes of rolling element bearing data. The proposed TR-KDA-BIGA may be a promising tool for fault diagnosis of rolling element bearings.

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

    DEFF Research Database (Denmark)

    Good, L; Nielsen, P E

    1999-01-01

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

  19. Structural optimisation of vertical-axis wind turbine composite blades based on finite element analysis and genetic algorithm

    OpenAIRE

    Wang, Lin; Kolios, Athanasios; Nishino, Takafumi; DELAFIN, Pierre-Luc; Bird, Theodore

    2016-01-01

    A wind turbine blade generally has complex structures including several layers of composite materials with shear webs, making its structure design very challenging. In this paper, a structural optimisation model for wind turbine composite blades has been developed based on a parametric FEA (finite element analysis) model and a GA (genetic algorithm) model. The optimisation model minimises the mass of composite blades with multi-criteria constraints. The number of unidirectional plies, the loc...

  20. The landscape of antisense gene expression in human cancers

    OpenAIRE

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

    2015-01-01

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

  1. Extended spectrum β-lactamases, carbapenemases and mobile genetic elements responsible for antibiotics resistance in Gram-negative bacteria.

    Science.gov (United States)

    El Salabi, Allaaeddin; Walsh, Timothey R; Chouchani, Chedly

    2013-05-01

    Infectious diseases due to Gram-negative bacteria are a leading cause of morbidity and mortality worldwide. Antimicrobial agents represent one major therapeutic tools implicated to treat these infections. The misuse of antimicrobial agents has resulted in the emergence of resistant strains of Gram-negatives in particular Enterobacteriaceae and non-fermenters; they have an effect not only on a human but on the public health when bacteria use the resistance mechanisms to spread in the hospital environment and to the community outside the hospitals by means of mobile genetic elements. Gram-negative bacteria have become increasingly resistant to antimicrobial agents. They have developed several mechanisms by which they can withstand to antimicrobials, these mechanisms include the production of Extended-spectrum β-lactamases (ESBLs) and carbapenemases, furthermore, Gram-negative bacteria are now capable of spreading such resistance between members of the family Enterobacteriaceae and non-fermenters using mobile genetic elements as vehicles for such resistance mechanisms rendering antibiotics useless. Therefore, addressing the issue of mechanisms of antimicrobial resistance is considered one of most urgent priorities. This review will help to illustrate different resistance mechanisms; ESBLs, carbapenemases encoded by genes carried by mobile genetic elements, which are used by Gram-negative bacteria to escape antimicrobial effect.

  2. Suicidal genetic elements and their use in biological containment of bacteria

    DEFF Research Database (Denmark)

    Molin, Søren; Boe, Lars; Jensen, Lars Bogø;

    1993-01-01

    . The possibilities of reducing such potential risks and increasing the predictability of the organisms are discussed for genetically engineered bacteria. Different approaches towards designing disabled strains without seriously reducing their beneficial effects are presented. Principally two types of strain design...... are discussed: actively contained bacteria based on the introduction of controlled suicide systems, and passively contained strains based on genetic interference with their survival under environmental-stress conditions.......The potential risks of unintentional releases of genetically modified organisms, and the lack of predictable behavior of these in the environment, are the subject of considerable concern. This concern is accentuated in connection with the next phase of gene technology comprising deliberate releases...

  3. Optimizing antisense oligonucleotides using phosphorodiamidate morpholino oligomers.

    Science.gov (United States)

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

    2012-01-01

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

  4. Genetics

    Science.gov (United States)

    ... Inheritance; Heterozygous; Inheritance patterns; Heredity and disease; Heritable; Genetic markers ... The chromosomes are made up of strands of genetic information called DNA. Each chromosome contains sections of ...

  5. 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.

  6. Multi-exon Skipping Using Cocktail Antisense Oligonucleotides in the Canine X-linked Muscular Dystrophy.

    Science.gov (United States)

    Miskew Nichols, Bailey; Aoki, Yoshitsugu; Kuraoka, Mutsuki; Lee, Joshua J A; Takeda, Shin'ichi; Yokota, Toshifumi

    2016-01-01

    Duchenne muscular dystrophy (DMD) is one of the most common lethal genetic diseases worldwide, caused by mutations in the dystrophin (DMD) gene. Exon skipping employs short DNA/RNA-like molecules called antisense oligonucleotides (AONs) that restore the reading frame and produce shorter but functional proteins. However, exon skipping therapy faces two major hurdles: limited applicability (up to only 13% of patients can be treated with a single AON drug), and uncertain function of truncated proteins. These issues were addressed with a cocktail AON approach. While approximately 70% of DMD patients can be treated by single exon skipping (all exons combined), one could potentially treat more than 90% of DMD patients if multiple exon skipping using cocktail antisense drugs can be realized. The canine X-linked muscular dystrophy (CXMD) dog model, whose phenotype is more similar to human DMD patients, was used to test the systemic efficacy and safety of multi-exon skipping of exons 6 and 8. The CXMD dog model harbors a splice site mutation in intron 6, leading to a lack of exon 7 in dystrophin mRNA. To restore the reading frame in CXMD requires multi-exon skipping of exons 6 and 8; therefore, CXMD is a good middle-sized animal model for testing the efficacy and safety of multi-exon skipping. In the current study, a cocktail of antisense morpholinos targeting exon 6 and exon 8 was designed and it restored dystrophin expression in body-wide skeletal muscles. Methods for transfection/injection of cocktail oligos and evaluation of the efficacy and safety of multi-exon skipping in the CXMD dog model are presented. PMID:27285612

  7. Structural optimisation of a high speed Organic Rankine Cycle generator using a genetic algorithm and a finite element method

    Energy Technology Data Exchange (ETDEWEB)

    Palko, S. [Machines Division, ABB industry Oy, Helsinki (Finland)

    1997-12-31

    The aim in this work is to design a 250 kW high speed asynchronous generator using a genetic algorithm and a finite element method for Organic Rankine Cycle. The characteristics of the induction motors are evaluated using two-dimensional finite element method (FEM) The movement of the rotor and the non-linearity of the iron is included. In numerical field problems it is possible to find several local extreme for an optimisation problem, and therefore the algorithm has to be capable of determining relevant changes, and to avoid trapping to a local minimum. In this work the electromagnetic (EM) losses at the rated point are minimised. The optimisation includes the air gap region. Parallel computing is applied to speed up optimisation. (orig.) 2 refs.

  8. cAMP response element binding protein (CREB activates transcription via two distinct genetic elements of the human glucose-6-phosphatase gene

    Directory of Open Access Journals (Sweden)

    Stefano Luisa

    2005-01-01

    Full Text Available Abstract Background The enzyme glucose-6-phosphatase catalyzes the dephosphorylation of glucose-6-phosphatase to glucose, the final step in the gluconeogenic and glycogenolytic pathways. Expression of the glucose-6-phosphatase gene is induced by glucocorticoids and elevated levels of intracellular cAMP. The effect of cAMP in regulating glucose-6-phosphatase gene transcription was corroborated by the identification of two genetic motifs CRE1 and CRE2 in the human and murine glucose-6-phosphatase gene promoter that resemble cAMP response elements (CRE. Results The cAMP response element is a point of convergence for many extracellular and intracellular signals, including cAMP, calcium, and neurotrophins. The major CRE binding protein CREB, a member of the basic region leucine zipper (bZIP family of transcription factors, requires phosphorylation to become a biologically active transcriptional activator. Since unphosphorylated CREB is transcriptionally silent simple overexpression studies cannot be performed to test the biological role of CRE-like sequences of the glucose-6-phosphatase gene. The use of a constitutively active CREB2/CREB fusion protein allowed us to uncouple the investigation of target genes of CREB from the variety of signaling pathways that lead to an activation of CREB. Here, we show that this constitutively active CREB2/CREB fusion protein strikingly enhanced reporter gene transcription mediated by either CRE1 or CRE2 derived from the glucose-6-phosphatase gene. Likewise, reporter gene transcription was enhanced following expression of the catalytic subunit of cAMP-dependent protein kinase (PKA in the nucleus of transfected cells. In contrast, activating transcription factor 2 (ATF2, known to compete with CREB for binding to the canonical CRE sequence 5'-TGACGTCA-3', did not transactivate reporter genes containing CRE1, CRE2, or both CREs derived from the glucose-6-phosphatase gene. Conclusions Using a constitutively active CREB2

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

    OpenAIRE

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

    1986-01-01

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

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

    Science.gov (United States)

    Brolin, Camilla; Shiraishi, Takehiko

    2011-01-01

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

  11. 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...

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2016-08-01

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

  14. Extinction probabilities and stationary distributions of mobile genetic elements in prokaryotes: The birth-death-diversification model.

    Science.gov (United States)

    Drakos, Nicole E; Wahl, Lindi M

    2015-12-01

    Theoretical approaches are essential to our understanding of the complex dynamics of mobile genetic elements (MGEs) within genomes. Recently, the birth-death-diversification model was developed to describe the dynamics of mobile promoters (MPs), a particular class of MGEs in prokaryotes. A unique feature of this model is that genetic diversification of elements was included. To explore the implications of diversification on the longterm fate of MGE lineages, in this contribution we analyze the extinction probabilities, extinction times and equilibrium solutions of the birth-death-diversification model. We find that diversification increases both the survival and growth rate of MGE families, but the strength of this effect depends on the rate of horizontal gene transfer (HGT). We also find that the distribution of MGE families per genome is not necessarily monotonically decreasing, as observed for MPs, but may have a peak in the distribution that is related to the HGT rate. For MPs specifically, we find that new families have a high extinction probability, and predict that the number of MPs is increasing, albeit at a very slow rate. Additionally, we develop an extension of the birth-death-diversification model which allows MGEs in different regions of the genome, for example coding and non-coding, to be described by different rates. This extension may offer a potential explanation as to why the majority of MPs are located in non-promoter regions of the genome.

  15. Micro-scale spatial expansion of microbial cells and mobile genetic elements

    DEFF Research Database (Denmark)

    Smets, Barth F.; Kreft, Jan-Ulrich; Or, Dani;

    Microbes can actively explore their local spatial environment when sufficiently hydrated pathways are present - mobile gene elements can also travel in local environments when cellular density is sufficient. In this presentation, I will present our efforts at predicting the dynamics of these two ...

  16. 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.

  17. 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.

  18. Lysine metabolism in antisense C-hordein barley grains

    DEFF Research Database (Denmark)

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

    2015-01-01

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

  19. Antisense bcl-2 treatment increases programmed cell death in non-small cell lung cancer cell lines.

    Science.gov (United States)

    Koty, P P; Zhang, H; Levitt, M L

    1999-02-01

    Programmed cell death (PCD) is a genetically regulated pathway that is altered in many cancers. This process is, in part, regulated by the ratio of PCD inducers (Bax) or inhibitors (Bcl-2). An abnormally high ratio of Bcl-2 to Bax prevents PCD, thus contributing to resistance to chemotherapeutic agents, many of which are capable of inducing PCD. Non-small cell lung cancer (NSCLC) cells demonstrate resistance to these PCD-inducing agents. If Bcl-2 prevents NSCLC cells from entering the PCD pathway, then reducing the amount of endogenous Bcl-2 product may allow these cells to spontaneously enter the PCD pathway. Our purpose was to determine the effects of bcl-2 antisense treatment on the levels of programmed cell death in NSCLC cells. First, we determined whether bcl-2 and bax mRNA were expressed in three morphologically distinct NSCLC cell lines: NCI-H226 (squamous), NCI-H358 (adenocarcinoma), and NCI-H596 (adenosquamous). Cells were then exposed to synthetic antisense bcl-2 oligonucleotide treatment, after which programmed cell death was determined, as evidenced by DNA fragmentation. Bcl-2 protein expression was detected immunohistochemically. All three NSCLC cell lines expressed both bcl-2 and bax mRNA and had functional PCD pathways. Synthetic antisense bcl-2 oligonucleotide treatment resulted in decreased Bcl-2 levels, reduced cell proliferation, decreased cell viability, and increased levels of spontaneous PCD. This represents the first evidence that decreasing Bcl-2 in three morphologically distinct NSCLC cell lines allows the cells to spontaneously enter a PCD pathway. It also indicates the potential therapeutic use of antisense bcl-2 in the treatment of NSCLC. PMID:10217615

  20. Genetic and phenotypic diversity in Burkholderia: contributions by prophage and phage-like elements

    Directory of Open Access Journals (Sweden)

    Ulrich Ricky L

    2010-07-01

    Full Text Available Abstract Background Burkholderia species exhibit enormous phenotypic diversity, ranging from the nonpathogenic, soil- and water-inhabiting Burkholderia thailandensis to the virulent, host-adapted mammalian pathogen B. mallei. Genomic diversity is evident within Burkholderia species as well. Individual isolates of Burkholderia pseudomallei and B. thailandensis, for example, carry a variety of strain-specific genomic islands (GIs, including putative pathogenicity and metabolic islands, prophage-like islands, and prophages. These GIs may provide some strains with a competitive advantage in the environment and/or in the host relative to other strains. Results Here we present the results of analysis of 37 prophages, putative prophages, and prophage-like elements from six different Burkholderia species. Five of these were spontaneously induced to form bacteriophage particles from B. pseudomallei and B. thailandensis strains and were isolated and fully sequenced; 24 were computationally predicted in sequenced Burkholderia genomes; and eight are previously characterized prophages or prophage-like elements. The results reveal numerous differences in both genome structure and gene content among elements derived from different species as well as from strains within species, due in part to the incorporation of additional DNA, or 'morons' into the prophage genomes. Implications for pathogenicity are also discussed. Lastly, RNAseq analysis of gene expression showed that many of the genes in ϕ1026b that appear to contribute to phage and lysogen fitness were expressed independently of the phage structural and replication genes. Conclusions This study provides the first estimate of the relative contribution of prophages to the vast phenotypic diversity found among the Burkholderiae.

  1. Identification and assembly of genomes and genetic elements in complex metagenomic samples without using reference genomes

    DEFF Research Database (Denmark)

    Nielsen, Henrik Bjørn; Almeida, Mathieu; Juncker, Agnieszka;

    2014-01-01

    Most current approaches for analyzing metagenomic data rely on comparisons to reference genomes, but the microbial diversity of many environments extends far beyond what is covered by reference databases. De novo segregation of complex metagenomic data into specific biological entities......, such as particular bacterial strains or viruses, remains a largely unsolved problem. Here we present a method, based on binning co-abundant genes across a series of metagenomic samples, that enables comprehensive discovery of new microbial organisms, viruses and co-inherited genetic entities and aids assembly...... of microbial genomes without the need for reference sequences. We demonstrate the method on data from 396 human gut microbiome samples and identify 7,381 co-abundance gene groups (CAGs), including 741 metagenomic species (MGS). We use these to assemble 238 high-quality microbial genomes and identify...

  2. The dissemination of C10 cysteine protease genes in Bacteroides fragilis by mobile genetic elements

    LENUS (Irish Health Repository)

    Thornton, Roibeard F

    2010-04-23

    Abstract Background The C10 family of cysteine proteases includes enzymes that contribute to the virulence of bacterial pathogens, such as SpeB in Streptococcus pyogenes. The presence of homologues of cysteine protease genes in human commensal organisms has not been examined. Bacteroides fragilis is a member of the dominant Bacteroidetes phylum of the human intestinal microbiota, and is a significant opportunistic pathogen. Results Four homologues of the streptococcal virulence factor SpeB were identified in the B. fragilis genome. These four protease genes, two were directly contiguous to open reading frames predicted to encode staphostatin-like inhibitors, with which the protease genes were co-transcribed. Two of these protease genes are unique to B. fragilis 638R and are associated with two large genomic insertions. Gene annotation indicated that one of these insertions was a conjugative Tn-like element and the other was a prophage-like element, which was shown to be capable of excision. Homologues of the B. fragilis C10 protease genes were present in a panel of clinical isolates, and in DNA extracted from normal human faecal microbiota. Conclusions This study suggests a mechanism for the evolution and dissemination of an important class of protease in major members of the normal human microbiota.

  3. The dissemination of C10 cysteine protease genes in Bacteroides fragilis by mobile genetic elements

    Directory of Open Access Journals (Sweden)

    Kagawa Todd F

    2010-04-01

    Full Text Available Abstract Background The C10 family of cysteine proteases includes enzymes that contribute to the virulence of bacterial pathogens, such as SpeB in Streptococcus pyogenes. The presence of homologues of cysteine protease genes in human commensal organisms has not been examined. Bacteroides fragilis is a member of the dominant Bacteroidetes phylum of the human intestinal microbiota, and is a significant opportunistic pathogen. Results Four homologues of the streptococcal virulence factor SpeB were identified in the B. fragilis genome. These four protease genes, two were directly contiguous to open reading frames predicted to encode staphostatin-like inhibitors, with which the protease genes were co-transcribed. Two of these protease genes are unique to B. fragilis 638R and are associated with two large genomic insertions. Gene annotation indicated that one of these insertions was a conjugative Tn-like element and the other was a prophage-like element, which was shown to be capable of excision. Homologues of the B. fragilis C10 protease genes were present in a panel of clinical isolates, and in DNA extracted from normal human faecal microbiota. Conclusions This study suggests a mechanism for the evolution and dissemination of an important class of protease in major members of the normal human microbiota.

  4. Os DNA sintéticos anti-sentido Antisense Synthtetic DNA

    Directory of Open Access Journals (Sweden)

    Alfredo Cravador

    1998-07-01

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

  5. Antisense treatment of caliciviridae: an emerging disease agent of animals and humans.

    Science.gov (United States)

    Smith, Alvin W; Matson, David O; Stein, David A; Skilling, Douglas E; Kroeker, Andrew D; Berke, Tamas; Iversen, Patrick L

    2002-04-01

    The Earth's oceans are the primary reservoir for an emerging family of RNA viruses, the Caliciviridae, which can cause a spectrum of diseases in marine animals, wildlife, farm animals, pets and humans. Certain members of this family have unusually broad host ranges, and some are zoonotic (transmissible from animals to humans). The RNA virus replicative processes lack effective genetic repair mechanisms, and, therefore, virtually every calicivirus replicate is a mutant. Hence, traditional therapeutics dependent on specific nucleic acid sequences or protein epitopes lack the required diversity of sequence or conformational specificity that would be required to reliably detect, prevent or treat infections from these mutant clusters (quasi-species) of RNA viruses, including the Caliciviridae. Antisense technology using phosphorodiamidate morpholino oligomers shows promise in overcoming these current diagnostic and therapeutic problems inherent with newly emerging viral diseases. PMID:12044040

  6. Metabolite profiling of Arabidopsis thaliana (L.) plants transformed with an antisense chalcone synthase gene

    DEFF Research Database (Denmark)

    Le Gall, G.; Metzdorff, Stine Broeng; Pedersen, Jan W.;

    2005-01-01

    A metabolite profiling study has been carried out on Arabidopsis thaliana (L.) Heynh. ecotype Wassilewskija and a series of transgenic lines of the ecotype transformed with a CHS (chalcone synthase) antisense construct. Compound identifications by LC/MS and H-1 NMR are discussed. The glucosinolate...... be expected in the transgenic lines relative to the wild type. In practice the reductions achieved were highly variable both between lines and within a given line on different occasions when the plants were grown. Possible reasons for this variability are discussed with reference to current models of gene...... silencing. The metabolite profiles of the transgenic lines were examined for unintended effects of the modification. An apparently major effect on the glucosinolate composition was shown to result from an unusual genetic variation in the ecotype and not from the modification. The modification did produce...

  7. Detection and linkage to mobile genetic elements of tetracycline resistance gene tet(M) in Escherichia coli isolates from pigs

    DEFF Research Database (Denmark)

    Jurado-Rabadan, Sonia; de la Fuente, Ricardo; Ruiz-Santa-Quiteria, Jose A.;

    2014-01-01

    from pigs, as well as the detection of mobile genetic elements linked to tet(M) in E. coli and its possible transfer from enterococci. Results: tet(A) was the most frequently detected gene (87.9%) in doxycycline-resistant isolates. tet(M) was found in 13.1% E. coli isolates. The tet(M) gene...... was detected in relation with conjugative transposons in 10 out of 36 enterococci isolates analyzed but not in any of E. coli isolates positive for tet(M). Southern blot showed that in E. coli and in most of the enterococci isolates the tet(M) gene was carried on a plasmid. According to the phylogenetic...... analysis, E. coli contained a new tet(M) allele grouping separately. Mating experiments revealed that tet(M) was carried on a mobile element successfully transferred between enterococci and between enterococci and E. coli. Conclusions: The detection of tet(M) in E. coli isolates from pigs was higher than...

  8. Aquaculture changes the profile of antibiotic resistance and mobile genetic element associated genes in Baltic Sea sediments.

    Science.gov (United States)

    Muziasari, Windi I; Pärnänen, Katariina; Johnson, Timothy A; Lyra, Christina; Karkman, Antti; Stedtfeld, Robert D; Tamminen, Manu; Tiedje, James M; Virta, Marko

    2016-04-01

    Antibiotics are commonly used in aquaculture and they can change the environmental resistome by increasing antibiotic resistance genes (ARGs). Sediment samples were collected from two fish farms located in the Northern Baltic Sea, Finland, and from a site outside the farms (control). The sediment resistome was assessed by using a highly parallel qPCR array containing 295 primer sets to detect ARGs, mobile genetic elements and the 16S rRNA gene. The fish farm resistomes were enriched in transposon and integron associated genes and in ARGs encoding resistance to antibiotics which had been used to treat fish at the farms. Aminoglycoside resistance genes were also enriched in the farm sediments despite the farms not having used aminoglycosides. In contrast, the total relative abundance values of ARGs were higher in the control sediment resistome and they were mainly genes encoding efflux pumps followed by beta-lactam resistance genes, which are found intrinsically in many bacteria. This suggests that there is a natural Baltic sediment resistome. The resistome associated with fish farms can be from native ARGs enriched by antibiotic use at the farms and/or from ARGs and mobile elements that have been introduced by fish farming. PMID:26976842

  9. RNA aptamers as genetic control devices: the potential of riboswitches as synthetic elements for regulating gene expression.

    Science.gov (United States)

    Berens, Christian; Groher, Florian; Suess, Beatrix

    2015-02-01

    RNA utilizes many different mechanisms to control gene expression. Among the regulatory elements that respond to external stimuli, riboswitches are a prominent and elegant example. They consist solely of RNA and couple binding of a small molecule ligand to the so-called "aptamer domain" with a conformational change in the downstream "expression platform" which then determines system output. The modular organization of riboswitches and the relative ease with which ligand-binding RNA aptamers can be selected in vitro against almost any molecule have led to the rapid and widespread adoption of engineered riboswitches as artificial genetic control devices in biotechnology and synthetic biology over the past decade. This review highlights proof-of-principle applications to demonstrate the versatility and robustness of engineered riboswitches in regulating gene expression in pro- and eukaryotes. It then focuses on strategies and parameters to identify aptamers that can be integrated into synthetic riboswitches that are functional in vivo, before finishing with a reflection on how to improve the regulatory properties of engineered riboswitches, so that we can not only further expand riboswitch applicability, but also finally fully exploit their potential as control elements in regulating gene expression. PMID:25676052

  10. Isolation and Characterization of Mobile Genetic Elements from Microbial Assemblages Obtained from the Field Research Center Site

    Energy Technology Data Exchange (ETDEWEB)

    Patricia Sobecky; Cassie Hodges; Kerri Lafferty; Mike Humphreys; Melanie Raimondo; Kristin Tuttle; Tamar Barkay

    2004-03-17

    Considerable knowledge has been gained from the intensive study of a relatively limited group of bacterial plasmids. Recent efforts have begun to focus on the characterization of, at the molecular level, plasmid populations and associated mobile genetic elements (e.g., transposons, integrons) occurring in a wider range of aquatic and terrestrial habitats. Surprisingly, however, little information is available regarding the incidence and distribution of mobile genetic elements extant in contaminated subsurface environments. Such studies will provide greater knowledge on the ecology of plasmids and their contributions to the genetic plasticity (and adaptation) of naturally occurring subsurface microbial communities. We requested soil cores from the DOE NABIR Field Research Center (FRC) located on the Oak Ridge Reservation. The cores, received in February 2003, were sampled from four areas on the Oak Ridge Site: Area 1, Area 2, Area 3 (representing contaminated subsurface locales) and the background reference sites. The average core length (24 in) was subdivided into three profiles and soil pH and moisture content were determined. Uranium concentration was also determined in bulk samples. Replicate aliquots were fixed for total cell counts and for bacterial isolation. Four different isolation media were used to culture aerobic and facultative microbes from these four study areas. Colony forming units ranged from a minimum of 100 per gram soil to a maximum of 10,000 irrespective of media composition used. The vast majority of cultured subsurface isolates were gram-positive isolates and plasmid characterization was conducted per methods routinely used in the Sobecky laboratory. The percentage of plasmid incidence ranged from 10% to 60% of all isolates tested. This frequency appears to be somewhat higher than the incidence of plasmids we have observed in other habitats and we are increasing the number of isolates screened to confirm this observation. We are also

  11. Detection of small RNAs in Bordetella pertussis and identification of a novel repeated genetic element

    Directory of Open Access Journals (Sweden)

    Wulbrecht Bérénice

    2011-04-01

    Full Text Available Abstract Background Small bacterial RNAs (sRNAs have been shown to participate in the regulation of gene expression and have been identified in numerous prokaryotic species. Some of them are involved in the regulation of virulence in pathogenic bacteria. So far, little is known about sRNAs in Bordetella, and only very few sRNAs have been identified in the genome of Bordetella pertussis, the causative agent of whooping cough. Results An in silico approach was used to predict sRNAs genes in intergenic regions of the B. pertussis genome. The genome sequences of B. pertussis, Bordetella parapertussis, Bordetella bronchiseptica and Bordetella avium were compared using a Blast, and significant hits were analyzed using RNAz. Twenty-three candidate regions were obtained, including regions encoding the already documented 6S RNA, and the GCVT and FMN riboswitches. The existence of sRNAs was verified by Northern blot analyses, and transcripts were detected for 13 out of the 20 additional candidates. These new sRNAs were named Bordetella pertussis RNAs, bpr. The expression of 4 of them differed between the early, exponential and late growth phases, and one of them, bprJ2, was found to be under the control of BvgA/BvgS two-component regulatory system of Bordetella virulence. A phylogenetic study of the bprJ sequence revealed a novel, so far undocumented repeat of ~90 bp, found in numerous copies in the Bordetella genomes and in that of other Betaproteobacteria. This repeat exhibits certain features of mobile elements. Conclusion We shown here that B. pertussis, like other pathogens, expresses sRNAs, and that the expression of one of them is controlled by the BvgA/BvgS system, similarly to most virulence genes, suggesting that it is involved in virulence of B. pertussis.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    OpenAIRE

    DeVos, Sarah L.; Miller, Timothy M.

    2013-01-01

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

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

    OpenAIRE

    Brolin, Camilla; Shiraishi, Takehiko

    2011-01-01

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

  15. Progress in therapeutic antisense applications for neuromuscular disorders

    OpenAIRE

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

    2009-01-01

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

  16. A selfish genetic element influencing longevity correlates with reactive behavioural traits in female house mice (Mus domesticus.

    Directory of Open Access Journals (Sweden)

    Yannick Auclair

    Full Text Available According to theory in life-history and animal personality, individuals with high fitness expectations should be risk-averse, while individuals with low fitness expectations should be more bold. In female house mice, a selfish genetic element, the t haplotype, is associated with increased longevity under natural conditions, representing an appropriate case study to investigate this recent theory empirically. Following theory, females heterozygous for the t haplotype (+/t are hypothesised to express more reactive personality traits and be more shy, less explorative and less active compared to the shorter-lived homozygous wildtype females (+/+. As males of different haplotype do not differ in survival, no similar pattern is expected. We tested these predictions by quantifying boldness, exploration, activity, and energetic intake in both +/t and +/+ mice. +/t females, unlike +/+ ones, expressed some reactive-like personality traits: +/t females were less active, less prone to form an exploratory routine and tended to ingest less food. Taken together these results suggest that differences in animal personality may contribute to the survival advantage observed in +/t females but fail to provide full empirical support for recent theory.

  17. A selfish genetic element influencing longevity correlates with reactive behavioural traits in female house mice (Mus domesticus).

    Science.gov (United States)

    Auclair, Yannick; König, Barbara; Lindholm, Anna K

    2013-01-01

    According to theory in life-history and animal personality, individuals with high fitness expectations should be risk-averse, while individuals with low fitness expectations should be more bold. In female house mice, a selfish genetic element, the t haplotype, is associated with increased longevity under natural conditions, representing an appropriate case study to investigate this recent theory empirically. Following theory, females heterozygous for the t haplotype (+/t) are hypothesised to express more reactive personality traits and be more shy, less explorative and less active compared to the shorter-lived homozygous wildtype females (+/+). As males of different haplotype do not differ in survival, no similar pattern is expected. We tested these predictions by quantifying boldness, exploration, activity, and energetic intake in both +/t and +/+ mice. +/t females, unlike +/+ ones, expressed some reactive-like personality traits: +/t females were less active, less prone to form an exploratory routine and tended to ingest less food. Taken together these results suggest that differences in animal personality may contribute to the survival advantage observed in +/t females but fail to provide full empirical support for recent theory.

  18. 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.

  19. Archaeal extrachromosomal genetic elements

    DEFF Research Database (Denmark)

    Wang, Haina; Peng, Nan; Shah, Shiraz Ali;

    2015-01-01

    viruses and plasmids. In particular, it has been suggested that ECE-host interactions have shaped the coevolution of ECEs and their archaeal hosts. Furthermore, archaeal hosts have developed defense systems, including the innate restriction-modification (R-M) system and the adaptive CRISPR (clustered...

  20. A screen for genetic suppressor elements of hepatitis C virus identifies a supercharged protein inhibitor of viral replication.

    Science.gov (United States)

    Simeon, Rudo L; Chen, Zhilei

    2013-01-01

    Genetic suppressor elements (GSEs) are biomolecules derived from a gene or genome of interest that act as transdominant inhibitors of biological functions presumably by disruption of critical biological interfaces. We exploited a cell death reporter cell line for hepatitis C virus (HCV) infection, n4mBid, to develop an iterative selection/enrichment strategy for the identification of anti-HCV GSEs. Using this approach, a library of fragments of an HCV genome was screened for sequences that suppress HCV infection. A 244 amino acid gene fragment, B1, was strongly enriched after 5 rounds of selection. B1 derives from a single-base frameshift of the enhanced green fluorescent protein (eGFP) which was used as a filler during fragment cloning. B1 has a very high net positive charge of 43 at neutral pH and a high charge-to-mass (kDa) ratio of 1.5. We show that B1 expression specifically inhibits HCV replication. In addition, five highly positively charged B1 fragments produced from progressive truncation at the C-terminus all retain the ability to inhibit HCV, suggesting that a high positive charge, rather than a particular motif in B1, likely accounts for B1's anti-HCV activity. Another supercharged protein, +36GFP, was also found to strongly inhibit HCV replication when added to cells at the time of infection. This study reports a new methodology for HCV inhibitor screening and points to the anti-HCV potential of positively charged proteins/peptides.

  1. A screen for genetic suppressor elements of hepatitis C virus identifies a supercharged protein inhibitor of viral replication.

    Directory of Open Access Journals (Sweden)

    Rudo L Simeon

    Full Text Available Genetic suppressor elements (GSEs are biomolecules derived from a gene or genome of interest that act as transdominant inhibitors of biological functions presumably by disruption of critical biological interfaces. We exploited a cell death reporter cell line for hepatitis C virus (HCV infection, n4mBid, to develop an iterative selection/enrichment strategy for the identification of anti-HCV GSEs. Using this approach, a library of fragments of an HCV genome was screened for sequences that suppress HCV infection. A 244 amino acid gene fragment, B1, was strongly enriched after 5 rounds of selection. B1 derives from a single-base frameshift of the enhanced green fluorescent protein (eGFP which was used as a filler during fragment cloning. B1 has a very high net positive charge of 43 at neutral pH and a high charge-to-mass (kDa ratio of 1.5. We show that B1 expression specifically inhibits HCV replication. In addition, five highly positively charged B1 fragments produced from progressive truncation at the C-terminus all retain the ability to inhibit HCV, suggesting that a high positive charge, rather than a particular motif in B1, likely accounts for B1's anti-HCV activity. Another supercharged protein, +36GFP, was also found to strongly inhibit HCV replication when added to cells at the time of infection. This study reports a new methodology for HCV inhibitor screening and points to the anti-HCV potential of positively charged proteins/peptides.

  2. Phylogenomics of the reproductive parasite Wolbachia pipientis wMel: a streamlined genome overrun by mobile genetic elements.

    Directory of Open Access Journals (Sweden)

    Martin Wu

    2004-03-01

    Full Text Available The complete sequence of the 1,267,782 bp genome of Wolbachia pipientis wMel, an obligate intracellular bacteria of Drosophila melanogaster, has been determined. Wolbachia, which are found in a variety of invertebrate species, are of great interest due to their diverse interactions with different hosts, which range from many forms of reproductive parasitism to mutualistic symbioses. Analysis of the wMel genome, in particular phylogenomic comparisons with other intracellular bacteria, has revealed many insights into the biology and evolution of wMel and Wolbachia in general. For example, the wMel genome is unique among sequenced obligate intracellular species in both being highly streamlined and containing very high levels of repetitive DNA and mobile DNA elements. This observation, coupled with multiple evolutionary reconstructions, suggests that natural selection is somewhat inefficient in wMel, most likely owing to the occurrence of repeated population bottlenecks. Genome analysis predicts many metabolic differences with the closely related Rickettsia species, including the presence of intact glycolysis and purine synthesis, which may compensate for an inability to obtain ATP directly from its host, as Rickettsia can. Other discoveries include the apparent inability of wMel to synthesize lipopolysaccharide and the presence of the most genes encoding proteins with ankyrin repeat domains of any prokaryotic genome yet sequenced. Despite the ability of wMel to infect the germline of its host, we find no evidence for either recent lateral gene transfer between wMel and D. melanogaster or older transfers between Wolbachia and any host. Evolutionary analysis further supports the hypothesis that mitochondria share a common ancestor with the alpha-Proteobacteria, but shows little support for the grouping of mitochondria with species in the order Rickettsiales. With the availability of the complete genomes of both species and excellent genetic tools for

  3. High prevalence of multidrug resistance and random distribution of mobile genetic elements among uropathogenic Escherichia coli (UPEC) of the four major phylogenetic groups.

    Science.gov (United States)

    Rijavec, Matija; Starcic Erjavec, Marjanca; Ambrozic Avgustin, Jerneja; Reissbrodt, Rolf; Fruth, Angelika; Krizan-Hergouth, Veronika; Zgur-Bertok, Darja

    2006-08-01

    One hundred and ten UTI Escherichia coli strains, from Ljubljana, Slovenia, were analyzed for antibiotic resistances, mobile DNA elements, serotype, and phylogenetic origin. A high prevalence of drug resistance and multidrug resistance was found. Twenty-six percent of the isolates harbored a class 1 integron, while a majority of the strains (56%) harbored rep sequences characteristic of F-like plasmids. int as well as rep sequences were found to be distributed in a random manner among strains of the four major phylogenetic groups indicating that all groups have a similar tendency to acquire and maintain mobile genetic elements frequently associated with resistance determinants.

  4. 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…

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

    Institute of Scientific and Technical Information of China (English)

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

    2005-01-01

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

  6. Isolation of genetic suppressor elements, inducing resistance to topoisomerase II-interactive cytotoxic drugs, from human topoisomerase II cDNA.

    OpenAIRE

    Gudkov, A V; Zelnick, C R; Kazarov, A R; Thimmapaya, R; Suttle, D P; Beck, W T; Roninson, I B

    1993-01-01

    Many cytotoxic anticancer drugs act at topoisomerase II (topo II) by stabilizing cleavable complexes with DNA formed by this enzyme. Several cell lines, selected for resistance to topo II-interactive drugs, show decreased expression or activity of topo II, suggesting that such a decrease may be responsible for drug resistance. In the present study, etoposide resistance was used as the selection strategy to isolate genetic suppressor elements (GSEs) from a retroviral library expressing random ...

  7. Screening of Exist Genetically Modified Elements in Local and Commercial Rice Available in Baghdad Markets Using PCR and Real-time PCR

    Directory of Open Access Journals (Sweden)

    Hayba Q. Younan

    2013-11-01

    Full Text Available Rice is one of the staple foods of the Iraqi population; therefore large amounts of rice cultivated and imported in Iraq. Because of increasing the production of GM crops especially rice crop, it was necessary to investigate if there is any genetically modified rice (GM rice in Baghdad markets. Conventional PCR and Real-Time PCR used to create this investigation. Genomic DNA extracted from 7 samples of rice seeds that cultivated in Iraq; 31 samples of commercial rice seeds and 4 samples of kids' food, where rice is one of their ingredients. The primers RM 171 selected to amplify the Rice Microsatellite Region (RM for the DNA integrity inspection and rice species detection especially samples of kids' food, as well as, the primers P35S selected to amplify the CaMV35S promoter region (P35S for Genetically Modified elements (GM elements detection. There was no existence of GM elements in all samples except one, which was one of kids' food samples. These results indicate that the ability to produce GM crops and monitoring of the GM food entry are limitable in Iraq. Moreover, no commitment in the labeling regulations of genetically modified food.

  8. 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.

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

    Science.gov (United States)

    Seetharaman, Mahadevan; Musier-Forsyth, Karin

    2003-12-01

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

  10. Induction of endogenous telomerase (hTERT) by c-Myc in WI-38 fibroblasts transformed with specific genetic elements.

    Science.gov (United States)

    Casillas, Mark A; Brotherton, Scott L; Andrews, Lucy G; Ruppert, J Michael; Tollefsbol, Trygve O

    2003-10-16

    Elucidation of the mechanisms governing expression of the human telomerase reverse transcriptase (hTERT) is important for understanding cancer pathogenesis. Approximately 90% of tumors express hTERT, the major catalytic component of telomerase. Activation of telomerase is an early event, and high levels of this activity correlate with poor prognosis. Recent studies have shown that the transcription factors c-Myc and Mad1 activate and repress hTERT, respectively. It is not clear how these transcription factors compete for the same recognition sequence in the hTERT core promoter region. Studies have shown that the combined expression of SV40 large T antigen (T-Ag), hTERT, and H-Ras is able to transform human cells. In this study, we used a distinct human cell type, WI-38 fetal lung fibroblasts used extensively for senescence studies. We transduced cells with amphotropic retroviral constructs containing SV40 T antigen, hTERT, and activated H-ras. Transduced cells exhibited anchorage independence in soft agar and expressed increased levels of c-Myc and endogenous hTERT. These effects were observed by 25 population doublings (PDs) following the establishment of the neoplastic cell line. During the process of transformation, we observed a switch from Mad1/Max to c-Myc/Max binding to oligonucleotide sequences containing the hTERT promoter distal and proximal E-boxes. c-Myc can bind specifically to the hTERT promoter in vitro, indicating that c-Myc expression in tumors may account for the increased expression of hTERT observed in vivo. These findings indicate that the widely used model system of WI-38 fibroblasts can be employed for transformation studies using defined genetic elements and that the endogenous hTERT and c-Myc are induced in these cells during early tumorigenesis. Such studies should have important implications in the mechanisms of hTERT and c-Myc induction in the beginning stages of tumorigenesis and facilitate extension of these studies to novel models of

  11. From Cryptic Toward Canonical Pre-mRNA Splicing in Pompe Disease: a Pipeline for the Development of Antisense Oligonucleotides.

    Science.gov (United States)

    Bergsma, Atze J; In 't Groen, Stijn Lm; Verheijen, Frans W; van der Ploeg, Ans T; Pijnappel, Wwm Pim

    2016-01-01

    While 9% of human pathogenic variants have an established effect on pre-mRNA splicing, it is suspected that an additional 20% of otherwise classified variants also affect splicing. Aberrant splicing includes disruption of splice sites or regulatory elements, or creation or strengthening of cryptic splice sites. For the majority of variants, it is poorly understood to what extent and how these may affect splicing. We have identified cryptic splicing in an unbiased manner. Three types of cryptic splicing were analyzed in the context of pathogenic variants in the acid α-glucosidase gene causing Pompe disease. These involved newly formed deep intronic or exonic cryptic splice sites, and a natural cryptic splice that was utilized due to weakening of a canonical splice site. Antisense oligonucleotides that targeted the identified cryptic splice sites repressed cryptic splicing at the expense of canonical splicing in all three cases, as shown by reverse-transcriptase-quantitative polymerase chain reaction analysis and by enhancement of acid α-glucosidase enzymatic activity. This argues for a competition model for available splice sites, including intact or weakened canonical sites and natural or newly formed cryptic sites. The pipeline described here can detect cryptic splicing and correct canonical splicing using antisense oligonucleotides to restore the gene defect. PMID:27623443

  12. 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

  13. Optimization of amplitude-frequency characteristic three elements band-pass filter with dielectric resonator by genetic algorithm

    Directory of Open Access Journals (Sweden)

    A. A. Trubin

    2010-01-01

    Full Text Available It’s proposed the program that is searching the coupling coefficients of dielectric resonators answering to the necessary gain-frequency characteristic of band pass microwave filter. The program is based on a genetic algorithm.

  14. Behavior of a modified Dissociation element in barley: a tool for genetic studies and for breeding transgenic barley

    Science.gov (United States)

    Maize-derived sequences from the transposable elements Activator (Ac) and Dissociation (Ds) have enabled studies of gene function via transposon tagging. The characteristics of synthetic, transgene-containing Ds elements constructed for some of these studies has demonstrated their ability to resolve...

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

    Institute of Scientific and Technical Information of China (English)

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

    2004-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2000-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    LEI Xiao-yong; ZHANG Huan

    2005-01-01

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

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

    OpenAIRE

    Lee, Eun-Jin; Groisman, Eduardo A.

    2010-01-01

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

  19. 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.

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

    Science.gov (United States)

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

    2002-09-27

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

  1. LNA-antisense rivals siRNA for gene silencing

    DEFF Research Database (Denmark)

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

    2004-01-01

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

  2. Tuning growth cycles of Brassica crops via natural antisense transcripts of BrFLC.

    Science.gov (United States)

    Li, Xiaorong; Zhang, Shaofeng; Bai, Jinjuan; He, Yuke

    2016-03-01

    Several oilseed and vegetable crops of Brassica are biennials that require a prolonged winter cold for flowering, a process called vernalization. FLOWERING LOCUS C (FLC) is a central repressor of flowering. Here, we report that the overexpression of natural antisense transcripts (NATs) of Brassica rapa FLC (BrFLC) greatly shortens plant growth cycles. In rapid-, medium- and slow-cycling crop types, there are four copies of the BrFLC genes, which show extensive variation in sequences and expression levels. In Bre, a biennial crop type that requires vernalization, five NATs derived from the BrFLC2 locus are rapidly induced under cold conditions, while all four BrFLC genes are gradually down-regulated. The transgenic Bre lines overexpressing a long NAT of BrFLC2 do not require vernalization, resulting in a gradient of shortened growth cycles. Among them, a subset of lines both flower and set seeds as early as Yellow sarson, an annual crop type in which all four BrFLC genes have non-sense mutations and are nonfunctional in flowering repression. Our results demonstrate that the growth cycles of biennial crops of Brassica can be altered by changing the expression levels of BrFLC2 NATs. Thus, BrFLC2 NATs and their transgenic lines are useful for the genetic manipulation of crop growth cycles. PMID:26250982

  3. 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

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

    Institute of Scientific and Technical Information of China (English)

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

    2006-01-01

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

  5. Insight on trace element detoxification in the Black-tailed Godwit (Limosa limosa) through genetic, enzymatic and metallothionein analyses

    Energy Technology Data Exchange (ETDEWEB)

    Lucia, Magali, E-mail: m.lucia33@laposte.net [Littoral, Environnement et Societes (LIENSs), UMR 7266 CNRS-Universite de La Rochelle, 2 rue Olympe de Gouges, 17000 La Rochelle (France); Bocher, Pierrick [Littoral, Environnement et Societes (LIENSs), UMR 7266 CNRS-Universite de La Rochelle, 2 rue Olympe de Gouges, 17000 La Rochelle (France); Cosson, Richard P. [Mer Molecules Sante (MMS), Universite de Nantes, EA 2663, 2 rue de la Houssiniere, BP 92208, 44322 Nantes Cedex 3 (France); Churlaud, Carine; Robin, Frederic; Bustamante, Paco [Littoral, Environnement et Societes (LIENSs), UMR 7266 CNRS-Universite de La Rochelle, 2 rue Olympe de Gouges, 17000 La Rochelle (France)

    2012-04-15

    Trace element concentrations (Ag, As, Cd, Co, Cr, Cu, Fe, Hg, Mn, Ni, Pb, Se, Zn) were investigated in the liver, kidneys, muscle and feathers of 31 black-tailed godwits (Limosa limosa) accidentally killed during catches by mist net in the Pertuis Charentais, Atlantic coast of France. Analyses of carbon and nitrogen stable isotope ratios were carried out in liver, muscle and feathers in order to elucidate dietary patterns and to determine whether differences in diet explained the variation in elemental uptake. This study also aimed to have a preliminary assessment of sub-lethal effects triggered by trace elements through the investigation of gene expressions by quantitative real-time PCR, antioxidant enzyme activities (catalase, superoxide dismutase, glutathione peroxidase), and metallothionein (MT) levels. The results showed that Cr and Ni concentrations in tissues of adults were lower than in juveniles in part because adults may have eliminated these trace elements through moulting. Except for Cd and Ni, trace element concentrations were negatively correlated to the body mass of godwits. Ag, As, Hg and Se concentrations were positively linked with the trophic position of birds. The diet could be considered as a fundamental route of exposure for these elements demonstrating therefore the qualitative linkage between dietary habits of godwits and their contaminant concentrations. Our results strongly suggest that even though trace element concentrations were mostly below toxicity threshold level, the elevated concentrations of As, Ag, Cd, Cu, Fe and Se may however trigger sub-lethal effects. Trace elements appear to enhance expression of genes involved in oxidative stress defence, which indicates the production of reactive oxygen species. Moreover, birds with the highest concentrations appeared to have an increased mitochondrial metabolism suggesting that the fight against trace element toxicity requires additional energetic needs notably to produce detoxification

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

    Directory of Open Access Journals (Sweden)

    Kyu Hong eCho

    2015-03-01

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

  7. 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.

  8. 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.

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

    Science.gov (United States)

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

    2016-08-12

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

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

    Institute of Scientific and Technical Information of China (English)

    Maolin He; Anmin Chen

    2005-01-01

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

  11. 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

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

    OpenAIRE

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

    2011-01-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

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

    OpenAIRE

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

    1989-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Jung Yong-chul

    2008-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2012-01-01

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

  18. 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.

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

    Science.gov (United States)

    Takeda, Shin'ichi

    2009-11-01

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

  20. Mobile genetic elements related to the diffusion of plasmid-mediated AmpC β-lactamases or carbapenemases from Enterobacteriaceae: findings from a multicenter study in Spain.

    Science.gov (United States)

    Zamorano, L; Miró, E; Juan, C; Gómez, L; Bou, G; González-López, J J; Martínez-Martínez, L; Aracil, B; Conejo, M C; Oliver, A; Navarro, F

    2015-09-01

    We examined the genetic context of 74 acquired ampC genes and 17 carbapenemase genes from 85 of 640 Enterobacteriaceae isolates collected in 2009. Using S1 pulsed-field gel electrophoresis and Southern hybridization, 37 of 74 bla AmpC genes were located on large plasmids of different sizes belonging to six incompatibility groups. We used sequencing and PCR mapping to investigate the regions flanking the acquired ampC genes. The bla CMY-2-like genes were associated with ISEcp1; the surrounding bla DHA genes were similar to Klebsiella pneumoniae plasmid pTN60013 associated with IS26 and the psp and sap operons; and the bla ACC-1 genes were associated with IS26 elements inserted into ISEcp1. All of the carbapenemase genes (bla VIM-1, bla IMP-22, and bla IMP-28) were located in class 1 integrons. Therefore, although plasmids are the main cause of the rapid dissemination of ampC genes among Enterobacteriaceae, we need to be aware that other mobile genetic elements, such as insertion sequences, transposons, or integrons, can be involved in the mobilization of these genes of chromosomal origin. Additionally, three new integrons (In846 to In848) are described in this study. PMID:26077249

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

    OpenAIRE

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

    2014-01-01

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

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

    Directory of Open Access Journals (Sweden)

    López-Barragán María J

    2011-11-01

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

  3. Platinum-group Elements Geochemistry of the Yangliuping Magmatic Ni-Cu-PGE Sulfide Deposit:Implications of Its Genetic Link with the Extrusive Basalts

    Institute of Scientific and Technical Information of China (English)

    ZHENG Jianbin; CAO Zhimin; SONG Xieyan; AN Wei; LIU Ji

    2004-01-01

    Primitive mantle-normalized Platinum-group elements (PGE) concentration patterns for the Zhengziyanwo intrusion and Dashibao Formation basalts are of positive slope, similar to most of the world-class magmatic Ni-Cu-PGE sulfide deposits. Characters of this intrusion and its related ores and Dashibao Formation basalts are their negative Pt-anomaly and high concentration of Rh relative to Pt and Pd, facts being interpreted to be the results of crystallization and fractionation of Pt-alloys and spinel phase-free crystallization history for the magma, respectively. PGE parameters of the Dashibao Formation basalts are consistent with the general trend of those found for the Zhengziyanwo intrusion, and this might infer a genetic link between them.

  4. Mycobacterium avium restriction fragment lenght polymorphism-IS IS1245 and the simple double repetitive element polymerase chain reaction typing method to screen genetic diversity in Brazilian strains

    Directory of Open Access Journals (Sweden)

    Patrícia Carvalho de Sequeira

    2005-11-01

    Full Text Available Simple double repetitive element polymerase chain reaction (MaDRE-PCR and Pvu II-IS1245 restriction fragment length polymorphism (RFLP typing methods were used to type 41 Mycobacterium avium isolates obtained from 14 Aids inpatients and 10 environment and animals specimens identified among 53 mycobacteria isolated from 237 food, chicken, and pig. All environmental and animals strains showed orphan patterns by both methods. By MaDRE-PCR four patients, with multiple isolates, showed different patterns, suggesting polyclonal infection that was confirmed by RFLP in two of them. This first evaluation of MaDRE-PCR on Brazilian M. avium strains demonstrated that the method seems to be useful as simple and less expensive typing method for screening genetic diversity in M. avium strains on selected epidemiological studies, although with limitation on analysis identical patterns except for one band.

  5. Similarity in the Structure of tetD-Carrying Mobile Genetic Elements in Bacterial Strains of Different Genera Isolated from Cultured Yellowtail.

    Science.gov (United States)

    Furushita, Manabu; Akagi, Hiroshi; Kaneoka, Azusa; Maeda, Toshimichi; Fukuda, Tsubasa; Tatsuno, Ryohei; Shiba, Tsuneo

    2016-01-01

    Structure analysis was performed on the antibiotic-resistance-gene region of conjugative plasmids of four fish farm bacteria.The kanamycin resistance gene, IS26, and tetracycline resistance gene (tetA(D)) were flanked by two IS26s in opposite orientation in Citrobacter sp. TA3 and TA6, and Alteromonas sp. TA55 from fish farm A. IS26-Inner was disrupted with ISRSB101. The chloramphenicol resistance gene, IS26 and tetA (D) were flanked by two IS26s in direct orientation in Salmonella sp. TC67 from farm C. Structures of tetA (D) and IS26 were identical among the four bacteria, but there was no insertion within the IS26-Inner of Salmonella sp. TC67. Horizontal gene transfer between the strains of two different genera in fish farm A was suggested by the structure homologies of mobile genetic elements and antibiotic resistance genes. PMID:27667524

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

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

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

  7. 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.

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

    Science.gov (United States)

    Ma; Itharat; Fluharty; Sakai

    1997-10-01

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

  9. 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.

  10. Depletion of Bcl-2 by an antisense oligonucleotide induces apoptosis accompanied by oxidation and externalization of phosphatidylserine in NCI-H226 lung carcinoma cells.

    Science.gov (United States)

    Koty, Patrick P; Tyurina, Yulia Y; Tyurin, Vladimir A; Li, Shang-Xi; Kagan, Valerian E

    2002-01-01

    Oxidant-induced apoptosis involves oxidation of many different and essential molecules including phospholipids. As a result of this non-specific oxidation, any signaling role of a particular phospholipid-class of molecules is difficult to elucidate. To determine whether preferential oxidation of phosphatidylserine (PS) is an early event in apoptotic signaling related to PS externalization and is independent of direct oxidant exposure, we chose a genetic-based induction of apoptosis. Apoptosis was induced in the lung cancer cell line NCI-H226 by decreasing the amount of Bcl-2 protein expression by preventing the translation of bcl-2 mRNA using an antisense bcl-2 oligonucleotide. Peroxidation of phospholipids was assayed using a fluorescent technique based on metabolic integration of an oxidation-sensitive and fluorescent fatty acid, cis-parinaric acid (PnA), into cellular phospholipids and subsequent HPLC separation of cis-PnA-labeled phospholipids. We found a decrease in Bcl-2 was associated with a selective oxidation of PS in a sub-population of the cells with externalized PS. No significant difference in oxidation of cis-PnA-labeled phospholipids was observed in cells treated with medium alone or a nonsense oligonucleotide. Treatment with either nonsensc or antisense bcl-2 oligonucleotides was not associated with changes in the pattern of individual phospholipid classes as determined by HPTLC. These metabolic and topographical changes in PS arrangement in plasma membrane appear to be early responses to antisense bcl-2 exposure that trigger a PS-dependent apoptotic signaling pathway. This observed externalization of PS may facilitate the 'labeling' of apoptotic cells for recognition by macrophage scavenger receptors and subsequent phagocytic clearance. PMID:12162425

  11. Transcription and translation products of the cytolysin gene psm-mec on the mobile genetic element SCCmec regulate Staphylococcus aureus virulence.

    Directory of Open Access Journals (Sweden)

    Chikara Kaito

    Full Text Available The F region downstream of the mecI gene in the SCCmec element in hospital-associated methicillin-resistant Staphylococcus aureus (HA-MRSA contains two bidirectionally overlapping open reading frames (ORFs, the fudoh ORF and the psm-mec ORF. The psm-mec ORF encodes a cytolysin, phenol-soluble modulin (PSM-mec. Transformation of the F region into the Newman strain, which is a methicillin-sensitive S. aureus (MSSA strain, or into the MW2 (USA400 and FRP3757 (USA300 strains, which are community-acquired MRSA (CA-MRSA strains that lack the F region, attenuated their virulence in a mouse systemic infection model. Introducing the F region to these strains suppressed colony-spreading activity and PSMα production, and promoted biofilm formation. By producing mutations into the psm-mec ORF, we revealed that (i both the transcription and translation products of the psm-mec ORF suppressed colony-spreading activity and promoted biofilm formation; and (ii the transcription product of the psm-mec ORF, but not its translation product, decreased PSMα production. These findings suggest that both the psm-mec transcript, acting as a regulatory RNA, and the PSM-mec protein encoded by the gene on the mobile genetic element SCCmec regulate the virulence of Staphylococcus aureus.

  12. 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.

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

    DEFF Research Database (Denmark)

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

    2008-01-01

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

  14. 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.

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

    Directory of Open Access Journals (Sweden)

    Benoit eBarbeau

    2013-08-01

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

  16. 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

  17. Functional analysis of splicing mutations in the IDS gene and the use of antisense oligonucleotides to exploit an alternative therapy for MPS II.

    Science.gov (United States)

    Matos, Liliana; Gonçalves, Vânia; Pinto, Eugénia; Laranjeira, Francisco; Prata, Maria João; Jordan, Peter; Desviat, Lourdes R; Pérez, Belén; Alves, Sandra

    2015-12-01

    Mucopolysaccharidosis II is a lysosomal storage disorder caused by mutations in the IDS gene, including exonic alterations associated with aberrant splicing. In the present work, cell-based splicing assays were performed to study the effects of two splicing mutations in exon 3 of IDS, i.e., c.241C>T and c.257C>T, whose presence activates a cryptic splice site in exon 3 and one in exon 8, i.e., c.1122C>T that despite being a synonymous mutation is responsible for the creation of a new splice site in exon 8 leading to a transcript shorter than usual. Mutant minigene analysis and overexpression assays revealed that SRSF2 and hnRNP E1 might be involved in the use and repression of the constitutive 3' splice site of exon 3 respectively. For the c.1122C>T the use of antisense therapy to correct the splicing defect was explored, but transfection of patient fibroblasts with antisense morpholino oligonucleotides (n=3) and a locked nucleic acid failed to abolish the abnormal transcript; indeed, it resulted in the appearance of yet another aberrant splicing product. Interestingly, the oligonucleotides transfection in control fibroblasts led to the appearance of the aberrant transcript observed in patients' cells after treatment, which shows that the oligonucleotides are masking an important cis-acting element for 5' splice site regulation of exon 8. These results highlight the importance of functional studies for understanding the pathogenic consequences of mis-splicing and highlight the difficulty in developing antisense therapies involving gene regions under complex splicing regulation. PMID:26407519

  18. Bacteriophages of Staphylococcus aureus efficiently package various bacterial genes and mobile genetic elements including SCCmec with different frequencies.

    Science.gov (United States)

    Mašlaňová, Ivana; Doškař, Jiří; Varga, Marian; Kuntová, Lucie; Mužík, Jan; Malúšková, Denisa; Růžičková, Vladislava; Pantůček, Roman

    2013-02-01

    Staphylococcus aureus is a serious human and veterinary pathogen in which new strains with increasing virulence and antimicrobial resistance occur due to acquiring new genes by horizontal transfer. It is generally accepted that temperate bacteriophages play a major role in gene transfer. In this study, we proved the presence of various bacterial genes of the S. aureus COL strain directly within the phage particles via qPCR and quantified their packaging frequency. Non-parametric statistical analysis showed that transducing bacteriophages φ11, φ80 and φ80α of serogroup B, in contrast to serogroup A bacteriophage φ81, efficiently package selected chromosomal genes localized in 4 various loci of the chromosome and 8 genes carried on variable elements, such as staphylococcal cassette chromosome SCCmec, staphylococcal pathogenicity island SaPI1, genomic islands vSaα and vSaβ, and plasmids with various frequency. Bacterial gene copy number per ng of DNA isolated from phage particles ranged between 1.05 × 10(2) for the tetK plasmid gene and 3.86 × 10(5) for the SaPI1 integrase gene. The new and crucial finding that serogroup B bacteriophages can package concurrently ccrA1 (1.16 × 10(4)) and mecA (1.26 × 10(4)) located at SCCmec type I into their capsids indicates that generalized transduction plays an important role in the evolution and emergence of new methicillin-resistant clones.

  19. Isolation and antisense suppression of flavonoid 3', 5'-hydroxylase modifies flower pigments and colour in cyclamen

    Directory of Open Access Journals (Sweden)

    Patel Deepa

    2010-06-01

    Full Text Available Abstract Background Cyclamen is a popular and economically significant pot plant crop in several countries. Molecular breeding technologies provide opportunities to metabolically engineer the well-characterized flavonoid biosynthetic pathway for altered anthocyanin profile and hence the colour of the flower. Previously we reported on a genetic transformation system for cyclamen. Our aim in this study was to change pigment profiles and flower colours in cyclamen through the suppression of flavonoid 3', 5'-hydroxylase, an enzyme in the flavonoid pathway that plays a determining role in the colour of anthocyanin pigments. Results A full-length cDNA putatively identified as a F3'5'H (CpF3'5'H was isolated from cyclamen flower tissue. Amino acid and phylogeny analyses indicated the CpF3'5'H encodes a F3'5'H enzyme. Two cultivars of minicyclamen were transformed via Agrobacterium tumefaciens with an antisense CpF3'5'H construct. Flowers of the transgenic lines showed modified colour and this correlated positively with the loss of endogenous F3'5'H transcript. Changes in observed colour were confirmed by colorimeter measurements, with an overall loss in intensity of colour (C in the transgenic lines and a shift in hue from purple to red/pink in one cultivar. HPLC analysis showed that delphinidin-derived pigment levels were reduced in transgenic lines relative to control lines while the percentage of cyanidin-derived pigments increased. Total anthocyanin concentration was reduced up to 80% in some transgenic lines and a smaller increase in flavonol concentration was recorded. Differences were also seen in the ratio of flavonol types that accumulated. Conclusion To our knowledge this is the first report of genetic modification of the anthocyanin pathway in the commercially important species cyclamen. The effects of suppressing a key enzyme, F3'5'H, were wide ranging, extending from anthocyanins to other branches of the flavonoid pathway. The results

  20. 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.

  1. 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.

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

    OpenAIRE

    Nepveu, A; Marcu, K B

    1986-01-01

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

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

    OpenAIRE

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

    1991-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2005-01-01

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

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

    OpenAIRE

    Jacques, J P; Susskind, M M

    1991-01-01

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

  6. 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...

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

    Institute of Scientific and Technical Information of China (English)

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

    2010-01-01

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

  8. 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.

  9. 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

  10. 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

  11. 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

  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. 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

  14. Evolution and Diversity of the Antimicrobial Resistance Associated Mobilome in Streptococcus suis: A Probable Mobile Genetic Elements Reservoir for Other Streptococci

    Science.gov (United States)

    Huang, Jinhu; Ma, Jiale; Shang, Kexin; Hu, Xiao; Liang, Yuan; Li, Daiwei; Wu, Zuowei; Dai, Lei; Chen, Li; Wang, Liping

    2016-01-01

    Streptococcus suis is a previously neglected, newly emerging multidrug-resistant zoonotic pathogen. Mobile genetic elements (MGEs) play a key role in intra- and interspecies horizontal transfer of antimicrobial resistance (AMR) determinants. Although, previous studies showed the presence of several MGEs, a comprehensive analysis of AMR-associated mobilome as well as their interaction and evolution has not been performed. In this study, we presented the AMR-associated mobilome and their insertion hotspots in S. suis. Integrative conjugative elements (ICEs), prophages and tandem MGEs were located at different insertion sites, while 86% of the AMR-associated MGEs were inserted at rplL and rum loci. Comprehensive analysis of insertions at rplL and rum loci among four pathogenic Streptococcus species (Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, and S. suis) revealed the existence of different groups of MGEs, including Tn5252, ICESp1108, and TnGBS2 groups ICEs, Φm46.1 group prophage, ICE_ICE and ICE_prophage tandem MGEs. Comparative ICE genomics of ICESa2603 family revealed that module exchange and acquisition/deletion were the main mechanisms in MGEs' expansion and evolution. Furthermore, the observation of tandem MGEs reflected a novel mechanism for MGE diversity. Moreover, an in vitro competition assay showed no visible fitness cost was observed between different MGE-carrying isolates and a conjugation assay revealed the transferability of ICESa2603 family of ICEs. Our statistics further indicated that the prevalence and diversity of MGEs in S. suis is much greater than in other three species which prompted our hypothesis that S. suis is probably a MGEs reservoir for other streptococci. In conclusion, our results showed that acquisition of MGEs confers S. suis not only its capability as a multidrug resistance pathogen, but also represents a paradigm to study the modular evolution and matryoshkas of MGEs. PMID:27774436

  15. 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.

  16. 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

  17. Improving the nutritional quality of the barley and wheat grain storage proteins by antisense technology

    DEFF Research Database (Denmark)

    Sikdar, Md. Shafiqul Islam; Lange, Mette; Aaslo, Per;

    2011-01-01

    gliadins) are also available from Germany and UK. We have grown them under different N regimes (high, medium and low N) in semi-field conditions. Previously five different antisense C-hordein lines of barley have been characterized in our laboratory. The analyses revealed that the lysine, threonine...

  18. Potent Antibacterial Antisense Peptide-Peptide Nucleic Acid Conjugates Against Pseudomonas aeruginosa

    DEFF Research Database (Denmark)

    Ghosal, Anubrata; Nielsen, Peter E

    2012-01-01

    Pseudomonas aeruginosa is an opportunistic pathogen causing severe infections in hospital settings, especially with immune compromised patients, and the increasing prevalence of multidrug resistant strains urges search for new drugs with novel mechanisms of action. In this study we introduce...... significantly reduced bacterial survival. These results open the possibility of development of antisense antibacterials for treatment of Pseudomonas infections....

  19. 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.

  20. Molecular characterization of a stable antisense chalcone synthase phenotype in strawberry (Fragaria ananassa)

    NARCIS (Netherlands)

    Lunkenbein, S.; Coiner, H.; Vos, de C.H.; Schaart, J.G.; Boone, M.J.; Krens, F.A.; Schwab, W.

    2006-01-01

    An octaploid (Fragaria × ananassa cv. Calypso) genotype of strawberry was transformed with an antisense chalcone synthase (CHS) gene construct using a ripening related CHS cDNA from Fragaria × ananassa cv. Elsanta under the control of the constitutive CaMV 35S promoter via Agrobacterium tumefaciens.

  1. TSUNAMI: an antisense method to phenocopy splicing-associated diseases in animals

    OpenAIRE

    Sahashi, Kentaro; Hua, Yimin; Ling, Karen K Y; Hung, Gene; Rigo, Frank; Horev, Guy; Katsuno, Masahisa; Sobue, Gen; Ko, Chien-Ping; Bennett, C. Frank; Krainer, Adrian R.

    2012-01-01

    This study presents an antisense oligonucleotide methodology to phenocopy a disease—in this case, the motor neuron disease spinal muscular atrophy in mice. Sahashi et al. show that it is possible to fine-tune disease severity through dose-dependent effects on RNA splicing, making this a novel animal model for monitoring disease onset and progression as well as testing candidate therapeutics.

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

    NARCIS (Netherlands)

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

    1995-01-01

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

  3. 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.

  4. 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

  5. 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.

  6. Effects of antisense oligonucleotides on the expression of macrophage migration inhibitory factor on macrophages

    Institute of Scientific and Technical Information of China (English)

    WEIYINGCHEN; GUANGRANLI; XUEQINGYU; XIAOYANLI; XIAOYANG

    2005-01-01

    To investigate the effects of antisense oligonucleotides on the expression of macrophage migration inhibitory factor (MIF) on macrophages, the mouse phosphorothioate oligonucleotides were designed and synthesized with the sequences of antisense, 5'-TACGGATACAAGTAGCAC-3';Sense, 5'-ATGC-CTATGTTCATCGTG-3';Missense, 5'-CTCTCAGACTCGATCTGT-3'. These phosphorothioate oligonucleotides were then transfected into cultured macrophages (RAW264.7) by luciferase vector, and the transfected macrophages were incubated with Lipopolysaccharide (LPS) (1 ng/ml) for various periods of times and collected afterwards. The content of MIF protein in the cultural supernatants was determined by ELISA, cellular RNA extracted and the expression of MIF mRNA was examined by RT-PCR analysis.The experimental results showed that LPS could induce a time-dependent specific expression of MIF on macrophages, in which the MIF mRNA in cells and the MIF protein in cultural supernatants appeared after 3 h and reached their highest concentration at 9-12 h after LPS stimulation. The levels of mRNA and proteins in the macrophages treated with antisense olignucleotides were decreased significantly after stimulation with LPS in comparison with that of stimulation with LPS alone or with that with LPS plus sense or missense oligonucleotides.There were no differences among those without LPS stimulation. It is concluded that macrophages stimulated with LPS express MIF, and the antisense olignucleotides of MIF inhibitthe expression of MIF mRNA as well as the secretion of MIF proteins in macrophages.

  7. Regulation of Polyphosphate Kinase Production by Antisense RNA in Pseudomonas fluorescens Pf0-1

    OpenAIRE

    Silby, Mark W.; Julie S Nicoll; Levy, Stuart B.

    2012-01-01

    Pseudomonas spp. adapt rapidly to environmental fluctuations. Loss or overproduction of polyphosphate reduces the fitness of Pseudomonas fluorescens Pf0-1, indicating the importance of the fine-tuning of polyphosphate production. An antisense RNA was investigated and shown to regulate the polyphosphate kinase gene (ppk) by a posttranscriptional mechanism reducing ppk transcript abundance.

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

    International Nuclear Information System (INIS)

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

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

    Science.gov (United States)

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

    2016-07-01

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

  10. 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.

  11. 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

  12. 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

  13. 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.

  14. 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.

  15. Intricate interactions between the bloom-forming cyanobacterium Microcystis aeruginosa and foreign genetic elements, revealed by diversified clustered regularly interspaced short palindromic repeat (CRISPR) signatures.

    Science.gov (United States)

    Kuno, Sotaro; Yoshida, Takashi; Kaneko, Takakazu; Sako, Yoshihiko

    2012-08-01

    Clustered regularly interspaced short palindromic repeats (CRISPR) confer sequence-dependent, adaptive resistance in prokaryotes against viruses and plasmids via incorporation of short sequences, called spacers, derived from foreign genetic elements. CRISPR loci are thus considered to provide records of past infections. To describe the host-parasite (i.e., cyanophages and plasmids) interactions involving the bloom-forming freshwater cyanobacterium Microcystis aeruginosa, we investigated CRISPR in four M. aeruginosa strains and in two previously sequenced genomes. The number of spacers in each locus was larger than the average among prokaryotes. All spacers were strain specific, except for a string of 11 spacers shared in two closely related strains, suggesting diversification of the loci. Using CRISPR repeat-based PCR, 24 CRISPR genotypes were identified in a natural cyanobacterial community. Among 995 unique spacers obtained, only 10 sequences showed similarity to M. aeruginosa phage Ma-LMM01. Of these, six spacers showed only silent or conservative nucleotide mutations compared to Ma-LMM01 sequences, suggesting a strategy by the cyanophage to avert CRISPR immunity dependent on nucleotide identity. These results imply that host-phage interactions can be divided into M. aeruginosa-cyanophage combinations rather than pandemics of population-wide infectious cyanophages. Spacer similarity also showed frequent exposure of M. aeruginosa to small cryptic plasmids that were observed only in a few strains. Thus, the diversification of CRISPR implies that M. aeruginosa has been challenged by diverse communities (almost entirely uncharacterized) of cyanophages and plasmids.

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

    Institute of Scientific and Technical Information of China (English)

    李维方; 张光霁; 朱诚; 金由辛; 卢亦成

    2003-01-01

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

  17. Effects of a plasmid expressing antisense tissue inhibitor of metalloproteinase-1 on liver fibrosis in rats

    Institute of Scientific and Technical Information of China (English)

    JIANG Wei; WANG Ji-yao; YANG Chang-qing; LIU Wen-bin; WANG Yi-qing; HE Bo-ming

    2005-01-01

    Background No efficient therapy for liver fibrosis has been available. This study was aimed to provide evidence that the introduction of a plasmid expressing antisense tissue inhibitor of metalloproteinase-1 (TIMP-1) into a rat model of immunologically induced liver fibrosis can result in the increased activity of interstitial collagenase, thus enhancing the degradation of collagen.Methods Real-time nested polymerase chain reaction (RT-Nested-PCR) and gene recombination techniques were used to construct a rat antisense TIMP-1 recombinant plasmid that can be expressed in eukaryotic cells. Both the recombinant plasmid and an empty vector (pcDNA3) were encapsulated with glycosyl-poly-L-lysine and injected into rats suffering from pig serum-induced liver fibrosis. The expression of exogenous transfected plasmid was assessed by Northern blot, RT-PCR, and Western blot. Hepatic interstitial collagenase activity was detected using fluorescinisothiocyanate (FITC)-labeled type Ⅰ collagen. In addition to hepatic hydroxyproline content, hepatic collagen types Ⅰ and Ⅲ were detected by immunohistochemical staining, and the stages of liver fibrosis by Van Gieson staining.Results Exogenous antisense TIMP-1 was successfully expressed in vivo and could block the gene and protein expression of TIMP-1. Active and latent hepatic interstitial collagenase activities were elevated (P<0.01), hepatic hydroxyproline content and the accumulation of collagen types Ⅰ and Ⅲ were lowered, and liver fibrosis was alleviated in the antisense TIMP-1 group (P<0.01) as compared with the model group. Conclusion The results demonstrate that antisense TIMP-1 recombinant plasmids have some inhibitory effect on liver fibrosis.

  18. Antisense EGFR sequence enhances apoptosis in a human hepatoma cell line BEL—7404

    Institute of Scientific and Technical Information of China (English)

    FUTAO; HELIU; 等

    1996-01-01

    Effects of antisense epidermal growth factor receptor (EGFR) sequence on apoptotic cell death were examined in a human hepatoma cell line BEL-7404 cells.In the cells of JX-1,a sub clone of BEL-7404 stably transfected with antisense EGFR vector (Cell Research,3:75,1993),an enhanced rate(9.5%) of spontaneous apoptosis was detected by flow cytometry,whereas the rates of spontaneous apoptosis in JX-0 cells,a sub-clone of BEL-7404 transfected by control vector,and the parent BEL-7404 transfected by control vector,and the parent BEL-7404 transfected by control vector,and the parent BEL-7404 cells were almost equal and about 1.7%.Serum-starvation for 72h increased the rate of apoptosis of JX-lcells up to 33.7%,while JX-0 and BEL-7404 cells,under the same condition,produced less than 5% of apoptotic cells.Observation with electron microscope demonstrated that condensation and fragmentation of chromatin and formation of apoptotic bodies often occurred in JX-1 cells,especially during serumstarvation.These results,combined with the data of DNA fragmentation Elisa test,suggested that antisense EGFR sequence enhances apoptosis in the human hepatoma cells.Comparison of intracellular Ca2+ level and the responsiveness of JX-1 cells to the induced action of EGF and tharpsigargin (TG) treatment with that of control JX-0 cells indicated that antisense egfr might interrupt the EGF/EGFR sigaling pathway resulting in the decreass of intracellular Ca2+ pool content as well as the responsiveness of these cells to the extracellular signals.These findings suggest that antisense EGFR either directly or indirectly regulates Ca2+ storage in endoplasmic reticulum,thereby enhances apoptosis in the human hepatoma cells.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2008-01-15

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

  20. 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.

  1. 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

  2. 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.

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

    Institute of Scientific and Technical Information of China (English)

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

    2003-01-01

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

  4. 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

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

    OpenAIRE

    Hemenway, Cynthia; Fang, Rong-Xiang; Kaniewski, Wojciech K.; Chua, Nam-Hai; Tumer, Nilgun E.

    1988-01-01

    Transgenic tobacco plants engineered to express either the potato virus X (PVX) coat protein (CP+) or the antisense coat protein transcript (CP-antisense) were protected from infection by PVX, as indicated by reduced lesion numbers on inoculated leaves, delay or absence of systemic symptom development and reduction in virus accumulation in both inoculated and systemic leaves. The extent of protection observed in CP+ plants primarily depended upon the level of expression of the coat protein. P...

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

    Institute of Scientific and Technical Information of China (English)

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

    2006-01-01

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

  7. Cell number and transfection volume dependent peptide nucleic acid antisense activity by cationic delivery methods

    DEFF Research Database (Denmark)

    Llovera Nadal, Laia; Berthold, Peter; Nielsen, Peter E;

    2012-01-01

    Efficient intracellular delivery is essential for high activity of nucleic acids based therapeutics, including antisense agents. Several strategies have been developed and practically all rely on auxiliary transfection reagents such as cationic lipids, cationic polymers and cell penetrating...... peptides as complexing agents and carriers of the nucleic acids. However, uptake mechanisms remain rather poorly understood, and protocols always require optimization of transfection parameters. Considering that cationic transfection complexes bind to and thus may up-concentrate on the cell surface, we...... have now quantitatively compared the cellular activity (in the pLuc705 HeLa cell splice correction system) of PNA antisense oligomers using lipoplex delivery of cholesterol- and bisphosphonate-PNA conjugates, polyplex delivery via a PNA-polyethyleneimine conjugate and CPP delivery via a PNA...

  8. Lignin reduction in transgenic poplars by expressing antisense CCoAOMT gene

    Institute of Scientific and Technical Information of China (English)

    LU Jing; ZHAO Huayan; WEI Jianhua; HE Yikun; SHI Chao; WANG Hongzhi; SONG Yanru

    2004-01-01

    The antisense Caffeoyl CoA O-methyltransferase (CCoAOMT) cDNA was transformed into Chinese white poplar (Populus tomentosa) mediated by Agrobacterium tumefaciens. Many factors affecting the transformation efficiency were studied and a stable transformation system was established. PCR-Southern blot analysis indicated that antisense CCoAOMT cDNA had been integrated into the genome of the transgenic poplars. RT-PCR and Western blot analyses demonstrated that the endogenous CCoAOMT gene was suppressed at both transcriptional and translational levels. Klason lignin content assay exhibited the lignin reduction to different degrees in transgenic poplars. The stems of partial transgenic poplars with the remarkable lignin reduction turned red, and the color distribution was stripped or spotted. Taken together, these results suggested that CCoAOMT gene would be a potential useful gene in altering lignin biosynthesis by biotechnology for improving wood properties.

  9. Cellular delivery and antisense effects of peptide nucleic acid conjugated to polyethyleneimine via disulfide linkers

    DEFF Research Database (Denmark)

    Berthold, Peter R; Shiraishi, Takehiko; Nielsen, Peter E

    2010-01-01

    Peptide nucleic acid (PNA) is potentially an attractive antisense and antigene agent for which more efficient cellular delivery systems are still warranted. The cationic polymer polyethylenimine (PEI) is commonly used for cellular transfection of DNA and RNA complexes, but is not readily applicable...... for PNA due to the (inherent) charge neutrality of PNA. However, PEI could function as an efficient scaffold for PNA via chemical conjugation. Accordingly, we modified PEI with the amine-reactive heterobifunctional linker agent N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) (with and without a PEG...... moiety) and further reacted this with a cysteine PNA. The level of modification was determined spectrophotometrically with high accuracy, and the PNA transfection efficiency of the conjugates was evaluated in an antisense luciferase splice-correction assay using HeLa pLuc705 cells. We find that PEI...

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

    OpenAIRE

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

    2009-01-01

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

  11. Purification of noncoding RNA and bound proteins using FLAG peptide-conjugated antisense-oligonucleotides.

    Science.gov (United States)

    Adachi, Shungo; Natsume, Tohru

    2015-01-01

    To understand the function of certain RNAs, including noncoding RNAs, it is important to identify the proteins that interact with the RNAs. Here we describe the method for purification of ribonucleoprotein (RNP) complexes composed of specific cellular RNAs by pull-down with FLAG peptide-conjugated antisense oligonucleotide (ASO). Using this method, we identified a novel protein component of U7 snRNP complex.

  12. Over 20% of human transcripts might form sense–antisense pairs

    OpenAIRE

    Chen, Jianjun; Sun, Miao; Kent, W. James; Huang, Xiaoqiu; Xie, Hanqing; Wang, Wenquan; Zhou, Guolin; Shi, Run Zhang; Rowley, Janet D.

    2004-01-01

    The major challenge to identifying natural sense– antisense (SA) transcripts from public databases is how to determine the correct orientation for an expressed sequence, especially an expressed sequence tag sequence. In this study, we established a set of very stringent criteria to identify the correct orientation of each human transcript. We used these orientation-reliable transcripts to create 26 741 transcription clusters in the human genome. Our analysis shows that 22% (5880) of the human...

  13. Antisense Mediated Splicing Modulation For Inherited Metabolic Diseases: Challenges for Delivery

    OpenAIRE

    Pérez, Belen; Vilageliu, Lluisa; Grinberg, Daniel; Desviat, Lourdes R.

    2014-01-01

    In the past few years, research in targeted mutation therapies has experienced significant advances, especially in the field of rare diseases. In particular, the efficacy of antisense therapy for suppression of normal, pathogenic, or cryptic splice sites has been demonstrated in cellular and animal models and has already reached the clinical trials phase for Duchenne muscular dystrophy. In different inherited metabolic diseases, splice switching oligonucleotides (SSOs) have been used with suc...

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

    OpenAIRE

    López-Barragán María J; Lemieux Jacob; Quiñones Mariam; Williamson Kim C; Molina-Cruz Alvaro; Cui Kairong; Barillas-Mury Carolina; Zhao Keji; Su Xin-zhuan

    2011-01-01

    Abstract Background It has been shown that nearly a quarter of the initial predicted gene models in the Plasmodium falciparum genome contain errors. Although there have been efforts to obtain complete cDNA sequences to correct the errors, the coverage of cDNA sequences on the predicted genes is still incomplete, and many gene models for those expressed in sexual or mosquito stages have not been validated. Antisense transcripts have widely been reported in P. falciparum; however, the extent an...

  15. Antiviral Effects of Antisense Morpholino Oligomers in Murine Coronavirus Infection Models▿

    OpenAIRE

    Burrer, Renaud; Neuman, Benjamin W.; Ting, Joey P.C.; Stein, David A.; Moulton, Hong M.; Iversen, Patrick L.; Kuhn, Peter; Michael J Buchmeier

    2007-01-01

    The recent emergence of novel pathogenic human and animal coronaviruses has highlighted the need for antiviral therapies that are effective against a spectrum of these viruses. We have used several strains of murine hepatitis virus (MHV) in cell culture and in vivo in mouse models to investigate the antiviral characteristics of peptide-conjugated antisense phosphorodiamidate morpholino oligomers (P-PMOs). Ten P-PMOs directed against various target sites in the viral genome were tested in cell...

  16. 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.

  17. Delivering Antisense Morpholino Oligonucleotides to Target Telomerase Splice Variants in Human Embryonic Stem Cells.

    Science.gov (United States)

    Radan, Lida; Hughes, Chris S; Teichroeb, Jonathan H; Postovit, Lynne-Marie; Betts, Dean H

    2016-01-01

    Morpholino oligonucleotides (MO) are an innovative tool that provides a means for examining and modifying gene expression outcomes by antisense interaction with targeted RNA transcripts. The site-specific nature of their binding facilitates focused modulation to alter splice variant expression patterns. Here we describe the steric-blocking of human telomerase reverse transcriptase (hTERT) Δα and Δβ splice variants using MO to examine cellular outcomes related to pluripotency and differentiation in human embryonic stem cells.

  18. Behaviour of genetically modified amylose free potato clones as progenitors in a breeding program.

    NARCIS (Netherlands)

    Heeres, P.; Jacobsen, E.; Visser, R.G.F.

    1997-01-01

    Three amylose-free genetically modified potato clones were used both as male and female parents in a breeding program with non-GMO potato clones. Segregation data on the expression of the inserted antisense gene construct in tubers of progeny plants were in agreement with previous molecular analysis

  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. 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.

  2. 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.

  3. Targeted skipping of human dystrophin exons in transgenic mouse model systemically for antisense drug development.

    Directory of Open Access Journals (Sweden)

    Bo Wu

    Full Text Available Antisense therapy has recently been demonstrated with great potential for targeted exon skipping and restoration of dystrophin production in cultured muscle cells and in muscles of Duchenne Muscular Dystrophy (DMD patients. Therapeutic values of exon skipping critically depend on efficacy of the drugs, antisense oligomers (AOs. However, no animal model has been established to test AO targeting human dystrophin exon in vivo systemically. In this study, we applied Vivo-Morpholino to the hDMD mouse, a transgenic model carrying the full-length human dystrophin gene, and achieved for the first time more than 70% efficiency of targeted human dystrophin exon skipping in vivo systemically. We also established a GFP-reporter myoblast culture to screen AOs targeting human dystrophin exon 50. Antisense efficiency for most AOs is consistent between the reporter cells, human myoblasts and in the hDMD mice in vivo. However, variation in efficiency was also clearly observed. A combination of in vitro cell culture and a Vivo-Morpholino based evaluation in vivo systemically in the hDMD mice therefore may represent a prudent approach for selecting AO drug and to meet the regulatory requirement.

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

    Science.gov (United States)

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

    2012-09-01

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

  5. 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.

  6. 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.

  7. 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.

  8. 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.

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

    International Nuclear Information System (INIS)

    To unravel the roles of sucrose phosphate synthase (SPS) in muskmelon (Cucumis melo L.), we reduced its activity in transgenic muskmelon plants by an antisense approach. For this purpose, an 830 bp cDNA fragment of muskmelon sucrose phosphate synthase was expressed in antisense orientation behind the 35S promoter of the cauliflower mosaic virus. The phenotype of the antisense plants clearly differed from that of control plants. The transgenic plant leaves were markedly smaller, and the plant height and stem diameter were obviously shorter and thinner. Transmission electron microscope observation revealed that the membrane degradation of chloroplast happened in transgenic leaves and the numbers of grana and grana lamella in the chloroplast were significantly less, suggesting that the slow growth and weaker phenotype of transgenic plants may be due to the damage of the chloroplast ultrastructure, which in turn results in the decrease of the net photosynthetic rate. The sucrose concentration and levels of sucrose phosphate synthase decreased in transgenic mature fruit, and the fruit size was smaller than the control fruit. Together, our results suggest that sucrose phosphate synthase may play an important role in regulating the muskmelon plant growth and fruit development.

  10. Reassessment of the Listeria monocytogenes pan-genome reveals dynamic integration hotspots and mobile genetic elements as major components of the accessory genome

    Directory of Open Access Journals (Sweden)

    Kuenne Carsten

    2013-01-01

    Full Text Available Abstract Background Listeria monocytogenes is an important food-borne pathogen and model organism for host-pathogen interaction, thus representing an invaluable target considering research on the forces governing the evolution of such microbes. The diversity of this species has not been exhaustively explored yet, as previous efforts have focused on analyses of serotypes primarily implicated in human listeriosis. We conducted complete genome sequencing of 11 strains employing 454 GS FLX technology, thereby achieving full coverage of all serotypes including the first complete strains of serotypes 1/2b, 3c, 3b, 4c, 4d, and 4e. These were comparatively analyzed in conjunction with publicly available data and assessed for pathogenicity in the Galleria mellonella insect model. Results The species pan-genome of L. monocytogenes is highly stable but open, suggesting an ability to adapt to new niches by generating or including new genetic information. The majority of gene-scale differences represented by the accessory genome resulted from nine hyper variable hotspots, a similar number of different prophages, three transposons (Tn916, Tn554, IS3-like, and two mobilizable islands. Only a subset of strains showed CRISPR/Cas bacteriophage resistance systems of different subtypes, suggesting a supplementary function in maintenance of chromosomal stability. Multiple phylogenetic branches of the genus Listeria imply long common histories of strains of each lineage as revealed by a SNP-based core genome tree highlighting the impact of small mutations for the evolution of species L. monocytogenes. Frequent loss or truncation of genes described to be vital for virulence or pathogenicity was confirmed as a recurring pattern, especially for strains belonging to lineages III and II. New candidate genes implicated in virulence function were predicted based on functional domains and phylogenetic distribution. A comparative analysis of small regulatory RNA candidates

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

    Directory of Open Access Journals (Sweden)

    Kohama Chihiro

    2009-08-01

    Full Text Available Abstract Background Recent transcriptomic analyses in mammals have uncovered the widespread occurrence of endogenous antisense transcripts, termed natural antisense transcripts (NATs. NATs are transcribed from the opposite strand of the gene locus and are thought to control sense gene expression, but the mechanism of such regulation is as yet unknown. Although several thousand potential sense-antisense pairs have been identified in mammals, examples of functionally characterized NATs remain limited. To identify NAT candidates suitable for further functional analyses, we performed DNA microarray-based NAT screening using mouse adult normal tissues and mammary tumors to target not only the sense orientation but also the complementary strand of the annotated genes. Results First, we designed microarray probes to target the complementary strand of genes for which an antisense counterpart had been identified only in human public cDNA sources, but not in the mouse. We observed a prominent expression signal from 66.1% of 635 target genes, and 58 genes of these showed tissue-specific expression. Expression analyses of selected examples (Acaa1b and Aard confirmed their dynamic transcription in vivo. Although interspecies conservation of NAT expression was previously investigated by the presence of cDNA sources in both species, our results suggest that there are more examples of human-mouse conserved NATs that could not be identified by cDNA sources. We also designed probes to target the complementary strand of well-characterized genes, including oncogenes, and compared the expression of these genes between mammary cancerous tissues and non-pathological tissues. We found that antisense expression of 95 genes of 404 well-annotated genes was markedly altered in tumor tissue compared with that in normal tissue and that 19 of these genes also exhibited changes in sense gene expression. These results highlight the importance of NAT expression in the regulation

  12. Antisense expression of peach mildew resistance locus O (PpMlo1) gene confers cross-species resistance to powdery mildew in Fragaria x ananassa.

    Science.gov (United States)

    Jiwan, Derick; Roalson, Eric H; Main, Dorrie; Dhingra, Amit

    2013-12-01

    Powdery mildew (PM) is one of the major plant pathogens. The conventional method of PM control includes frequent use of sulfur-based fungicides adding to production costs and potential harm to the environment. PM remains a major scourge for Rosaceae crops where breeding approaches mainly resort to gene-for-gene resistance. We have tested an alternate source of PM resistance in Rosaceae. Mildew resistance locus O (MLO) has been well studied in barley due to its role in imparting broad spectrum resistance to PM. We identified PpMlo1 (Prunus persica Mlo) in peach and characterized it further to test if a similar mechanism of resistance is conserved in Rosaceae. Due to its recalcitrance in tissue culture, reverse genetic studies involving PpMloI were not feasible in peach. Therefore, Fragaria x ananassa LF9 line, a taxonomic surrogate, was used for functional analysis of PpMlo1. Agrobacterium-mediated transformation yielded transgenic strawberry plants expressing PpMlo1 in sense and antisense orientation. Antisense expression of PpMlo1 in transgenic strawberry plants conferred resistance to Fragaria-specific powdery mildew, Podosphaera macularis. Phylogenetic analysis of 208 putative Mlo gene copies from 35 plant species suggests a large number of duplications of this gene family prior to the divergence of monocots and eudicots, early in eudicot diversification. Our results indicate that the Mlo-based resistance mechanism is functional in Rosaceae, and that Fragaria can be used as a host to test mechanistic function of genes derived from related tree species. To the best of our knowledge, this work is one of the first attempts at testing the potential of using a Mlo-based resistance strategy to combat powdery mildew in Rosaceae.

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

    Science.gov (United States)

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

    1998-09-01

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

  14. 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.

  15. Splicing modulation therapy in the treatment of genetic diseases

    Directory of Open Access Journals (Sweden)

    Arechavala-Gomeza V

    2014-12-01

    Full Text Available Virginia Arechavala-Gomeza,1 Bernard Khoo,2 Annemieke Aartsma-Rus3 1Neuromuscular Disorders Group, BioCruces Health Research Institute, Barakaldo, Bizkaia, Spain; 2Endocrinology, Division of Medicine, University College London, London, UK; 3Department of Human Genetics, Leiden University Medical Center, Leiden, the Netherlands All authors contributed equally to this manuscript Abstract: Antisense-mediated splicing modulation is a tool that can be exploited in several ways to provide a potential therapy for rare genetic diseases. This approach is currently being tested in clinical trials for Duchenne muscular dystrophy and spinal muscular atrophy. The present review outlines the versatility of the approach to correct cryptic splicing, modulate alternative splicing, restore the open reading frame, and induce protein knockdown, providing examples of each. Finally, we outline a possible path forward toward the clinical application of this approach for a wide variety of inherited rare diseases. Keywords: splicing, therapy, antisense oligonucleotides, cryptic splicing, alternative splicing

  16. Cell type-specific termination of transcription by transposable element sequences

    OpenAIRE

    Conley Andrew B; Jordan I

    2012-01-01

    Abstract Background Transposable elements (TEs) encode sequences necessary for their own transposition, including signals required for the termination of transcription. TE sequences within the introns of human genes show an antisense orientation bias, which has been proposed to reflect selection against TE sequences in the sense orientation owing to their ability to terminate the transcription of host gene transcripts. While there is evidence in support of this model for some elements, the ex...

  17. 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.

  18. 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.

  19. 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

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

    Institute of Scientific and Technical Information of China (English)

    江军; 王洛夫; 方玉华; 靳风烁; 靳文生

    2004-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    杨雪; 贺海斌; 杨威; 宋涛; 郭成; 郑鑫; 刘青光

    2010-01-01

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

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

    OpenAIRE

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

    2003-01-01

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

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

    OpenAIRE

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

    2005-01-01

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

  5. Identification of an antisense transcript to ZIM2 in the primate lineage

    OpenAIRE

    Huang, Jennifer M.; Yu, Sungryul; Kim, Joomyeong

    2009-01-01

    In this study, we identified an antisense transcript to ZIM2 (zinc finger imprinted gene 2) in the human, called ZIM2as. Sequence analysis of the 110 kb region spanned by this transcript revealed a cluster of tandemly repeated sequence in the human, orangutan, and chimpanzee as well as a loss of approximately 70 kb from the corresponding region in the rhesus. The homologous region in most mammals contains a cluster of olfactory receptor (OLFR) genes, but this gene cluster has been lost from t...

  6. 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.

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

    Institute of Scientific and Technical Information of China (English)

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

    2004-01-01

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

  8. 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.

  9. 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.

  10. Strategic down-regulation of DNA polymerase beta by antisense RNA sensitizes mammalian cells to specific DNA damaging agents.

    OpenAIRE

    Horton, J K; Srivastava, D K; Zmudzka, B Z; Wilson, S H

    1995-01-01

    Previously, mouse NIH 3T3 cells were stably transfected with human DNA polymerase beta (beta-pol) cDNA in the antisense orientation and under the control of a metallothionein promoter [Zmudzka, B.Z. and Wilson, S.H. (1990) Som. Cell Mol. Gen., 16, 311-320]. To assess the feasibility of enhancing the efficacy of chemotherapy by an antisense approach and to confirm a role for beta-pol in cellular DNA repair, we looked for increased sensitivity to DNA damaging agents under conditions where beta-...

  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. 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

  13. Peptide nucleic acid (PNA) cell penetrating peptide (CPP) conjugates as carriers for cellular delivery of antisense oligomers

    DEFF Research Database (Denmark)

    Shiraishi, Takehiko; Nielsen, Peter E

    2012-01-01

    splicing correction of the mutated luciferase gene in the HeLa pLuc705 cell line, reporting cellular (nuclear) uptake of the antisense PNA via luciferase activity measurement. Carrier CPP-PNA constructs were studied in terms of construct modification (with octaarginine and/or decanoic acid) and carrier PNA...... that the carrier might facilitate endosomal escape. Furthermore, 50% downregulation of luciferase expression at 60 nM siRNA was obtained using this carrier CPP-PNA delivery strategy (with CQ co-treatment) for a single stranded antisense RNA targeting normal luciferase mRNA. These results indicated that CPP...

  14. Antisense suppression of LOX3 gene expression in rice endosperm enhances seed longevity.

    Science.gov (United States)

    Xu, Huibin; Wei, Yidong; Zhu, Yongsheng; Lian, Ling; Xie, Hongguang; Cai, Qiuhua; Chen, Qiushi; Lin, Zhongping; Wang, Zonghua; Xie, Huaan; Zhang, Jianfu

    2015-05-01

    Lipid peroxidation plays a major role in seed longevity and viability. In rice grains, lipid peroxidation is catalyzed by the enzyme lipoxygenase 3 (LOX3). Previous reports showed that grain from the rice variety DawDam in which the LOX3 gene was deleted had less stale flavour after grain storage than normal rice. The molecular mechanism by which LOX3 expression is regulated during endosperm development remains unclear. In this study, we expressed a LOX3 antisense construct in transgenic rice (Oryza sativa L.) plants to down-regulate LOX3 expression in rice endosperm. The transgenic plants exhibited a marked decrease in LOX mRNA levels, normal phenotypes and a normal life cycle. We showed that LOX3 activity and its ability to produce 9-hydroperoxyoctadecadienoic acid (9-HPOD) from linoleic acid were significantly lower in transgenic seeds than in wild-type seeds by measuring the ultraviolet absorption of 9-HPOD at 234 nm and by high-performance liquid chromatography. The suppression of LOX3 expression in rice endosperm increased grain storability. The germination rate of TS-91 (antisense LOX3 transgenic line) was much higher than the WT (29% higher after artificial ageing for 21 days, and 40% higher after natural ageing for 12 months). To our knowledge, this is the first report to demonstrate that decreased LOX3 expression can preserve rice grain quality during storage with no impact on grain yield, suggesting potential applications in agricultural production. PMID:25545811

  15. 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.

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

    Science.gov (United States)

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

    2011-01-01

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

  17. Antisense mediated splicing modulation for inherited metabolic diseases: challenges for delivery.

    Science.gov (United States)

    Pérez, Belen; Vilageliu, Lluisa; Grinberg, Daniel; Desviat, Lourdes R

    2014-02-01

    In the past few years, research in targeted mutation therapies has experienced significant advances, especially in the field of rare diseases. In particular, the efficacy of antisense therapy for suppression of normal, pathogenic, or cryptic splice sites has been demonstrated in cellular and animal models and has already reached the clinical trials phase for Duchenne muscular dystrophy. In different inherited metabolic diseases, splice switching oligonucleotides (SSOs) have been used with success in patients' cells to force pseudoexon skipping or to block cryptic splice sites, in both cases recovering normal transcript and protein and correcting the enzyme deficiency. However, future in vivo studies require individual approaches for delivery depending on the gene defect involved, given the different patterns of tissue and organ expression. Herein we review the state of the art of antisense therapy targeting RNA splicing in metabolic diseases, grouped according to their expression patterns-multisystemic, hepatic, or in central nervous system (CNS)-and summarize the recent progress achieved in the field of in vivo delivery of oligonucleotides to each organ or system. Successful body-wide distribution of SSOs and preferential distribution in the liver after systemic administration have been reported in murine models for different diseases, while for CNS limited data are available, although promising results with intratechal injections have been achieved. PMID:24506780

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

    Directory of Open Access Journals (Sweden)

    Taylor John M

    2001-03-01

    Full Text Available Abstract Background Previous studies by Volloch and coworkers have reported that during the expression of high levels of β-globin mRNA in the spleen of anemic mice, they could also detect small but significant levels of an antisense (AS globin RNA species, which they postulated might have somehow arisen by RNA-directed RNA synthesis. For two reasons we undertook to confirm and possibly extend these studies. First, previous studies in our lab have focussed on what is an unequivocal example of host RNA-directed RNA polymerase activity on the RNA genome of human hepatitis delta virus. Second, if AS globin species do exist they could in turn form double-stranded RNA species which might induce post-transcriptional gene silencing, a phenomenon somehow provoked in eukaryotic cells by AS RNA sequences. Results We reexamined critical aspects of the previous globin studies. We used intraperitoneal injections of phenylhydrazine to induce anemia in mice, as demonstrated by the appearance and ultimate disappearance of splenomegaly. While a 30-fold increase in globin mRNA was detected in the spleen, the relative amount of putative AS RNA could be no more than 0.004%. Conclusions Contrary to earlier reports, induction of a major increase in globin transcripts in the mouse spleen was not associated with a detectable level of antisense RNA to globin mRNA.

  19. 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.

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

    Science.gov (United States)

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

    2013-01-01

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

  1. 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.

  2. Identification of novel sense and antisense transcription at the TRPM2 locus in cancer

    Institute of Scientific and Technical Information of China (English)

    Ugo Orfanelli; Ann-Kathrin Wenke; Claudio Doglioni; Vincenzo Russo; Anja Katrin Bosserhoff; Giovanni Lavorgna

    2008-01-01

    It has been proposed that in cancer, where the bulk of the genome becomes hypomethylated, there is an increase in transcriptional noise that might lead to the generation of antisense transcripts that could affect the function of key oncosuppressor genes, ultimately leading to malignant transformation. Here, we describe the computational identifi-cation of a melanoma-enriched antisense transcript, TRPM2-AS, mapped within the locus of TRPM2, an ion channel capable of mediating susceptibility to cell death. Analysis of the TRPM2-AS genomic region indicated the presence in the same region of another tumor-enriched TRPM2 transcript, TRPM2-TE, located across a CpG island shared with TRPM2-AS. Quantitative PCR experiments confirmed that TRPM2-AS and TRPM2-TE transcripts were up-regu-lated in melanoma, and their activation was consistent with the methylation status of the shared CpG island. Func-tional knock-out of TRPM2-TE, as well as over-expression of wild-type TRPM2, increased melanoma susceptibility to apoptosis and necrosis. Finally, expression analysis in other cancer types indicated that TRPM2-AS and TRPM2-TE over-expression might have an even wider role than anticipated, reinforcing the relevance of our computational approach in identifying new potential therapeutic targets.

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

    Directory of Open Access Journals (Sweden)

    Daniel W Thomson

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2006-01-01

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

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

    International Nuclear Information System (INIS)

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

  6. 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.

  7. 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.

  8. 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.

  9. Antisense oligonucleotides targeting midkine induced apoptosis and increased chemosensitivity in hepatocellular carcinoma cells

    Institute of Scientific and Technical Information of China (English)

    Li-cheng DAI; Xiang WANG; Xing YAO; Yong-liang LU; Jin-liang PING; Jian-fang HE

    2006-01-01

    Aim: Overexpression of midkine (MK) has been observed in many malignancies. This aim of this study is to screen for suitable antisense oligonucleotides (ASODN) targeting MK in hepatocellular carcinoma (HCC) cells and evaluate its antitumor activity. Methods: Ten ASODN targeting MK were designed and synthesized. After transfection with ASODN, cell proliferation was analyzed with MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2//-tetrazolium, inner salt] assay. In addition, MK mRNA, protein levels, as well as apoptosis and caspase-3 activity were also examined in HepG2 cells. Cell proliferation was then analyzed after treatment with both ASODN and chemotherapeu-tic drugs. Results: In this experiment, the ASODN5 among the 10 ASODN showed higher inhibitory activity against proliferation of hepatocellular carcinoma cells in a dose-dependent manner. In HepG2 cells, ASODN5 could significantly reduce the MK mRNA level and protein content. After transfection with ASODN5 for 48 h, accompanied with a decline of survivin and Bcl-2 protein content, a remarkable increase of apoptosis and caspase-3 activity was observed in HepG2 cells. Furthermore, ASODN5 transfer can significantly increase chemosensitivity in HepG2 cells. Conclusion: Antisense oligonucleotides targeting MK shows therapeutic effects on HCC; ASODN5 has the possibility to be developed as an effective antitumor agent.

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

    Institute of Scientific and Technical Information of China (English)

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

    2005-01-01

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

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

    International Nuclear Information System (INIS)

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

  12. Streptococcus gallolyticus subsp. gallolyticus from human and animal origins: genetic diversity, antimicrobial susceptibility, and characterization of a vancomycin-resistant calf isolate carrying a vanA-Tn1546-like element.

    Science.gov (United States)

    Romero-Hernández, Beatriz; Tedim, Ana P; Sánchez-Herrero, José Francisco; Librado, Pablo; Rozas, Julio; Muñoz, Gloria; Baquero, Fernando; Cantón, Rafael; Del Campo, Rosa

    2015-04-01

    The aim of this work was to characterize the antibiotic susceptibility and genetic diversity of 41 Streptococcus gallolyticus subsp. gallolyticus isolates: 18 isolates obtained from animals and 23 human clinical isolates. Antibiotic susceptibility was determined by the semiautomatic Wider system and genetic diversity by pulsed-field gel electrophoresis (PFGE) with SmaI. Animal isolates grouped separately in the PFGE analysis, but no statistical differences in antimicrobial resistance were found between the two groups. The LMG 17956 sequence type 28 (ST28) strain recovered from the feces of a calf exhibited high levels of resistance to vancomycin and teicoplanin (MIC, ≥256 mg/liter). Its glycopeptide resistance mechanism was characterized by Southern blot hybridization and a primer-walking strategy, and finally its genome, determined by whole-genome sequencing, was compared with four closely related S. gallolyticus subsp. gallolyticus genomes. Hybridization experiments demonstrated that a Tn1546-like element was integrated into the bacterial chromosome. In agreement with this finding, whole-genome sequencing confirmed a partial deletion of the vanY-vanZ region and partial duplication of the vanH gene. The comparative genomic analyses revealed that the LMG 17956 ST28 strain had acquired an unusually high number of transposable elements and had experienced extensive chromosomal rearrangements, as well as gene gain and loss events. In conclusion, S. gallolyticus subsp. gallolyticus isolates from animals seem to belong to lineages separate from those infecting humans. In addition, we report a glycopeptide-resistant isolate from a calf carrying a Tn1546-like element integrated into its chromosome.

  13. Improved cellular activity of antisense peptide nucleic acids by conjugation to a cationic peptide-lipid (CatLip) domain

    DEFF Research Database (Denmark)

    Koppelhus, Uffe; Shiraishi, Takehiko; Zachar, Vladimir;

    2008-01-01

    for future in vivo applications. We find that simply conjugating a lipid domain (fatty acid) to the cationic peptide (a CatLip conjugate) increases the biological effect of the corresponding PNA (CatLip) conjugates in a luciferase cellular antisense assay up to 2 orders of magnitude. The effect increases...

  14. 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.

  15. 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.

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

    Institute of Scientific and Technical Information of China (English)

    Lin Ma; Voahangy Randrianarison; Fabien Calvo

    2005-01-01

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

  17. Design and synthesis of dendritic molecular transporter that achieves efficient in vivo delivery of morpholino antisense oligo.

    Science.gov (United States)

    Li, Yong-Fu; Morcos, Paul A

    2008-07-01

    Safe and efficient in vivo delivery of Morpholino antisense oligos was probably the last and most difficult challenge for the broad application of antisense in animal research and therapeutics. Several arginine-rich peptides effective for in vivo delivery of Morpholino antisense oligos require rather complex and expensive procedures for synthesis and conjugation. This work describes the design and synthesis of a dendritic transporter in a most concise manner where the selection of the core scaffold, functional group multiplication, orthogonal protecting group manipulation, solid phase conjugation, and off-resin perguanidinylation of the transporter structure are all orchestrated for efficient assembly. We utilized triazine as a core to provide a site for on-column conjugation to the Morpholino oligo and to anchor functional side arms which, after extension, multiplication, and deprotection, are subsequently converted from primary amines to the eight guanidinium headgroups that serve for transport across cell membranes. Intravenous administration of the delivery-enabled Morpholino into a splice-reporter strain of transgenic living mice results in de novo expression of splice-corrected green fluorescent protein in a broad range of tissues and organs in those treated mice. This rigorously demonstrates that this new dendritic transporter achieves effective delivery of a Morpholino oligo into the cytosol/nuclear compartment of cells systemically in vivo. The practical conjugation process may overcome any availability limitation for routine use by the scientific community, and the efficient delivery ability of this transporter may advance the application of Morpholino antisense technology in animals.

  18. Improved cellular uptake of antisense Peptide nucleic acids by conjugation to a cell-penetrating Peptide and a lipid domain

    DEFF Research Database (Denmark)

    Shiraishi, Takehiko; Nielsen, Peter E

    2011-01-01

    Unaided cellular uptake of RNA interference agents such as antisense oligonucleotides and siRNA is extremely poor, and in vivo bioavailability is also limited. Thus, effective delivery strategies for such potential drugs are in high demand. Recently, a novel approach using a class of short cationic...

  19. 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.

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

    Institute of Scientific and Technical Information of China (English)

    童强松; 曾甫清; 林晨; 赵军; 鲁功成

    2003-01-01

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

  1. 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.

  2. An in silico approach reveals associations between genetic and epigenetic factors within regulatory elements in B cells from primary Sjögren’s syndrome patients

    Directory of Open Access Journals (Sweden)

    Orsia D. Konsta

    2015-08-01

    Full Text Available Recent advances in genetics have highlighted several regions and candidate genes associated with primary Sjögren's syndrome (SS, a systemic autoimmune epithelitis that combines exocrine gland dysfunctions, and focal lymphocytic infiltrations. In addition to genetic factors, it is now clear that epigenetic deregulations are present during SS and restricted to specific cell type subsets such as lymphocytes and salivary gland epithelial cells. In this study, 72 single nucleotide polymorphisms (SNPs associated with 43 SS gene risk factors were selected from publicly available and peer reviewed literature for further in silico analysis. SS risk variant location was tested revealing a broad distribution in coding sequences (5.6%, intronic sequences (55.6%, upstream/downstream genic regions (30.5%, and intergenic regions (8.3%. Moreover, a significant enrichment of regulatory motifs (promoter, enhancer, insulator, DNAse peak and eQTL characterizes SS risk variants (94.4%. Next, screening SNPs in high linkage disequilibrium (r2 ≥ 0.8 in Caucasians revealed 645 new variants including 5 SNPs with missense mutations, and indicated an enrichment of transcriptionally active motifs according to the cell type (B cells > monocytes > T cells >> A549. Finally, we looked at SS risk variants for histone markers in B cells (GM12878, monocytes (CD14+ and epithelial cells (A548. Active histone markers were associated with SS risk variants at both promoters and enhancers in B cells, and within enhancers in monocytes. In conclusion and based on the obtained in silico results, that need further confirmation, associations were observed between SS genetic risk factors and epigenetic factors and these associations predominate in B cells such as those observed at the FAM167A-BLK locus.

  3. Screening of Exist Genetically Modified Elements in Local and Commercial Rice Available in Baghdad Markets Using PCR and Real-time PCR

    OpenAIRE

    Hayba Q. Younan

    2013-01-01

    Rice is one of the staple foods of the Iraqi population; therefore large amounts of rice cultivated and imported in Iraq. Because of increasing the production of GM crops especially rice crop, it was necessary to investigate if there is any genetically modified rice (GM rice) in Baghdad markets. Conventional PCR and Real-Time PCR used to create this investigation. Genomic DNA extracted from 7 samples of rice seeds that cultivated in Iraq; 31 samples of commercial rice seeds and 4 samples of k...

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

    International Nuclear Information System (INIS)

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

  5. The genomes of closely related Pantoea ananatis maize seed endophytes having different effects on the host plant differ in secretion system genes and mobile genetic elements

    Directory of Open Access Journals (Sweden)

    Raheleh eSheibani-Tezerji

    2015-05-01

    Full Text Available The seed as a habitat for microorganisms is as yet under-explored and has quite distinct characteristics as compared to other vegetative plant tissues. In this study, we investigated three closely related P. ananatis strains (named S6, S7 and S8, which were isolated from maize seeds of healthy plants. Plant inoculation experiments revealed that each of these strains exhibited a different phenotype ranging from weak pathogenic (S7, commensal (S8, to a beneficial, growth-promoting effect (S6 in maize. We performed a comparative genomics analysis in order to find genetic determinants responsible for the differences observed. Recent studies provided exciting insight into the genetic drivers of niche adaption and functional diversification of the genus Pantoea. However, we report here for the first time on the analysis of P. ananatis strains colonizing the same ecological niche but showing distinct interaction strategies with the host plant. Our comparative analysis revealed that genomes of these three strains are highly similar. However, genomic differences in genes encoding protein secretion systems and putative effectors, and transposase/integrases/phage related genes could be observed.

  6. The genomes of closely related Pantoea ananatis maize seed endophytes having different effects on the host plant differ in secretion system genes and mobile genetic elements.

    Science.gov (United States)

    Sheibani-Tezerji, Raheleh; Naveed, Muhammad; Jehl, Marc-André; Sessitsch, Angela; Rattei, Thomas; Mitter, Birgit

    2015-01-01

    The seed as a habitat for microorganisms is as yet under-explored and has quite distinct characteristics as compared to other vegetative plant tissues. In this study, we investigated three closely related P. ananatis strains (named S6, S7, and S8), which were isolated from maize seeds of healthy plants. Plant inoculation experiments revealed that each of these strains exhibited a different phenotype ranging from weak pathogenic (S7), commensal (S8), to a beneficial, growth-promoting effect (S6) in maize. We performed a comparative genomics analysis in order to find genetic determinants responsible for the differences observed. Recent studies provided exciting insight into the genetic drivers of niche adaption and functional diversification of the genus Pantoea. However, we report here for the first time on the analysis of P. ananatis strains colonizing the same ecological niche but showing distinct interaction strategies with the host plant. Our comparative analysis revealed that genomes of these three strains are highly similar. However, genomic differences in genes encoding protein secretion systems and putative effectors, and transposase/integrases/phage related genes could be observed.

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

    Institute of Scientific and Technical Information of China (English)

    LI Shuang; MA Ding; ZHU Changhong

    2007-01-01

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

  8. Transposable element origins of epigenetic gene regulation.

    Science.gov (United States)

    Lisch, Damon; Bennetzen, Jeffrey L

    2011-04-01

    Transposable elements (TEs) are massively abundant and unstable in all plant genomes, but are mostly silent because of epigenetic suppression. Because all known epigenetic pathways act on all TEs, it is likely that the specialized epigenetic regulation of regular host genes (RHGs) was co-opted from this ubiquitous need for the silencing of TEs and viruses. With their internally repetitive and rearranging structures, and the acquisition of fragments of RHGs, the expression of TEs commonly makes antisense RNAs for both TE genes and RHGs. These antisense RNAs, particularly from heterochromatic reservoirs of 'zombie' TEs that are rearranged to form variously internally repetitive structures, may be advantageous because their induction will help rapidly suppress active TEs of the same family. RHG fragments within rapidly rearranging TEs may also provide the raw material for the ongoing generation of miRNA genes. TE gene expression is regulated by both environmental and developmental signals, and insertions can place nearby RHGs under the regulation (both standard and epigenetic) of the TE. The ubiquity of TEs, their frequent preferential association with RHGs, and their ability to be programmed by epigenetic signals all indicate that RGHs have nearly unlimited access to novel regulatory cassettes to assist plant adaptation. PMID:21444239

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

    OpenAIRE

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

    1996-01-01

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

  10. Expression of antisense small RNAs in response to stress in Pseudomonas aeruginosa

    DEFF Research Database (Denmark)

    Gómez Lozano, María; Marvig, Rasmus Lykke; Tulstrup, Monica Vera-Lise;

    2014-01-01

    . We found a significant overrepresentation of asRNAs that are transcribed opposite to genes involved in cell division and in cell wall, lipopolysaccharide (LPS), and capsule biosynthesis, most likely reflecting the conditions used in this study. A substantial number of asRNAs significantly changed......Background: RNA sequencing technologies reveal that bacteria express RNA molecules other than mRNA, rRNA or tRNA. During the last years genome-wide bacterial transcriptomes have been shown to comprise intergenic RNA, antisense RNA, and untranslated regions, all capable of performing diverse...... that the extent of overlap between the studies is very limited. Conclusions: RNA-seq experiments are revealing hundreds of novel transcripts in all bacterial genomes investigated. The comparison between independent studies that used RNA-seq to detect novel asRNAs in P. aeruginosa shows that the overlap between...

  11. 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.

  12. A cytoplasmic pathway for gapmer antisense oligonucleotide-mediated gene silencing in mammalian cells

    Science.gov (United States)

    Castanotto, Daniela; Lin, Min; Kowolik, Claudia; Wang, LiAnn; Ren, Xiao-Qin; Soifer, Harris S.; Koch, Troels; Hansen, Bo Rode; Oerum, Henrik; Armstrong, Brian; Wang, Zhigang; Bauer, Paul; Rossi, John; Stein, C.A.

    2015-01-01

    Antisense oligonucleotides (ASOs) are known to trigger mRNA degradation in the nucleus via an RNase H-dependent mechanism. We have now identified a putative cytoplasmic mechanism through which ASO gapmers silence their targets when transfected or delivered gymnotically (i.e. in the absence of any transfection reagent). We have shown that the ASO gapmers can interact with the Ago-2 PAZ domain and can localize into GW-182 mRNA-degradation bodies (GW-bodies). The degradation products of the targeted mRNA, however, are not generated by Ago-2-directed cleavage. The apparent identification of a cytoplasmic pathway complements the previously known nuclear activity of ASOs and concurrently suggests that nuclear localization is not an absolute requirement for gene silencing. PMID:26433227

  13. 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.

  14. 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.

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

    Institute of Scientific and Technical Information of China (English)

    ZHANGYuan; LIWenyu

    2004-01-01

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

  16. 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.

  17. 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.

  18. 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.

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

    Institute of Scientific and Technical Information of China (English)

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

    2006-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2009-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2005-01-01

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

  2. 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.

  3. 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.

  4. 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.

  5. 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.

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

    Science.gov (United States)

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

    2016-12-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2004-01-01

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

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

    Science.gov (United States)

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

    2016-02-01

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

  9. Transuranium elements

    International Nuclear Information System (INIS)

    In this paper, the authors introduce this volume of historical and research papers on the transuranium elements by reviewing the period of discovery of elements 93 and beyond. Then the authors highlight the research achievements that focus on the unique properties of the 5f elements. Finally, the authors outline the technology and applications of transuranium elements and their radiobiology

  10. Elements for the Design of a Decision-making Information System for activities related to genetically modified organisms: Contributions from a case study

    International Nuclear Information System (INIS)

    In Colombia, decisions related to genetically modified organisms (GMOs) must be supported by assessment of the risk to biodiversity, human health and agricultural production. Based on this assessment, authorities can make decisions involving authorization or denial of the requested activities. The rationality of the decision-making process is very well established with respect to human health, particularly toxicity and allergenicity, but that is not the case for biodiversity issues. One of the biggest problems in this area is the lack of definition of a decision-making methodology, which leads to decisions made in an intuitive and non-systematic manner. Authorities in the field have recognized the need for a decision-making information system to help solve this situation. A proposal for the basic structure of a decision-making information system oriented to authorities involved in the process is presented. The proposal was developed based on a review of the main existing methodologies for GMO risk assessment and on a case study of the gene flow from GMOs to wild relatives. The structure is presented as a broad entity-relationship model from which the detailed design of the system can be developed. The proposal emphasizes the documentation of the decision protocols and the rationality of use of the information inputs.

  11. 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.

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

    Science.gov (United States)

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

    2014-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Yuanyuan Hong

    2014-01-01

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

  14. Scavenger Receptor-Mediated Delivery of Antisense Mini-Exon Phosphorothioate Oligonucleotide to Leishmania-Infected Macrophages: SELECTIVE AND EFFICIENT ELIMINATION OF THE PARASITE

    OpenAIRE

    Chaudhuri, Gautam

    1997-01-01

    Targeted delivery of a 17-mer antisense phosphorothioate oligodeoxyribonucleotide, complementary to the common 5′-end of every mRNA of the parasite cells, to the phagolysosomes of cultured murine macrophages infected with Leishmania mexicana amazonensis selectively and efficiently eliminated the parasite cells without causing any detectable harm to the host cells. The antisense mini-exon oligonucleotide (ASM) was encapsulated into liposomes coated with maleylated bovine serum albumin (MBSA), ...

  15. High genetic variation and recombination events in the vicinity of non-autonomous transposable elements from ‘Candidatus Liberibacter asiaticus’

    Institute of Scientific and Technical Information of China (English)

    WANG Xue-feng; CHEN Jiao-yue; TAN Jin; DUAN Suo; DENG Xiao-ling; CHEN Jian-chi; ZHOU Chang-yong

    2015-01-01

    Two miniature inverted-repeat transposable elements (MITEs), MCLas-A and MCLas-B, were recently identiifed from ‘Candidatus Liberibacter asiaticus’ known to be associated with citrus Huanglongbing (HLB, yelow shoot disease). MCLas-A was suggested as an active MITE because of its mobility. The immediate upstream gene of the two MITEs was predicted to be a putative transposase. The goal of this study is to analyze the sequence variation in the upstream putative transposase of MITEs and explore the possible correlation between sequence variation of transposase gene and MITE activity. PCR and sequence analysis showed that 12 sequence types were found in six major amplicon types from 43 representative ‘Ca. L. asiaticus’ isolates from China, the United States and Brazil. Out of the 12 sequence types, three (T4, T5-2, T6) were reported for the ifrst time. Recombination events were found in the two unique sequence types (T5-2 and T6) which were detected in al Brazilian isolates. Notably, no sequence variation or recombination events were detected in the upstream putative transposase gene of MCLas-A, suggesting the conservation of the transposase gene might be closely related with the MITE activity. Phylogenetic analysis demonstrated two wel supported clades including ifve subclades were identiifed, clearly relfecting the geographical origins of isolates, especialy that of Ruili isolates, São Paulo isolates and a few Florida isolates.

  16. Splicing Regulatory Elements and mRNA-abundance of dlg1 and capt, Genetically Interacting with dFMRP in Drosophila Brain

    Directory of Open Access Journals (Sweden)

    Maria Petrova

    2014-09-01

    Full Text Available To further understand the molecular and cellular mechanisms underlying the disease, we used the Drososphila FraX model and investigated a not well studied role of Drosophila Fragile X Mental Retardation Protein (dFMRP in alternative splicing of neuronal mRNAs to which it binds via a G-quartet sequence. By means of qRT-PCR we established the relative abundance of some isoforms of the gene dlg1, resulting from alternative exon skipping nearby a G-quartet and an exonic ESE-sequence, both acting as exonic splicing enhancers. We also investigated the relative mRNA-abundance of all capt-isoforms and the pre-mRNAs of both genes. We proposed a possible involvement of dFMRP in alternative splicing of genes, interacting with dfmr1. In the absence of dFMRP in larval and pupal brains, we found a change in the mRNA-level of one of the studied isoforms of dlg1 and of its pre-mRNA.We also established previously reported splicing regulatory elements and predicted computationally novel hexamere sequences in the exonic/intronic ends of both genes with p upative regulatory roles in alternative splicing.

  17. 基于遗传算法多段翼型外形优化设计%Optimization Design of Multi-element Airfoil Shape Based on Genetic Algorithm

    Institute of Scientific and Technical Information of China (English)

    魏闯; 张铁军; 廖应文

    2014-01-01

    为提高多段翼型增升效能,开展襟翼外形和缝道参数同时优化设计研究。优化算法采用遗传算法,以求解RANS方程为气动特性分析方法,通过椭圆方程控制生成多段翼型外形,同时优化缝宽、搭接量、襟翼偏角等位置参数和襟翼外形控制参数,实现多段翼型优化设计。设计实践表明,与只优化位置参数相比,同时优化襟翼外形和位置参数得到的多段翼型有更大的升力系数,方法是可行的,具有一定的工程应用前景。%The purpose is to enhance the aerodynamic performance of multi-element airfoil by searching for optimal shape and setting parameters .The procedure is driven by a genetic algorithm coupled with a RANS equations solver .Elliptic equations are used to automatically generate the multi-element airfoil shape,some design variables such as gap ,overlap,flap deflection and control parameters of flap shape are optimized to get excellent aerodynamic characteristic .Compared with the result that position parameters are only optimized ,multi-element airfoil optimized by the present method has higher lift coefficient .

  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. Down-regulation of protein kinase Ceta by antisense oligonucleotides sensitises A549 lung cancer cells to vincristine and paclitaxel.

    Science.gov (United States)

    Sonnemann, Jürgen; Gekeler, Volker; Ahlbrecht, Katrin; Brischwein, Klaus; Liu, Chao; Bader, Peter; Müller, Cornelia; Niethammer, Dietrich; Beck, James F

    2004-06-25

    Previous studies point to protein kinase C (PKC) isozyme eta as a resistance factor in cancer cells. Therefore, we investigated whether down-regulation of PKCeta with second generation antisense oligonucleotides (ODNs) would sensitise A549 human lung carcinoma cells to cytostatics. The effects were compared to the outcome of Bcl-xL down-regulation. Upon treatment with antisense ODNs, PKCeta and Bcl-xL were both significantly reduced on mRNA and protein level. Down-regulation of either PKCeta or Bcl-xL in combination with vincristine or paclitaxel resulted in a significant increase in caspase-3 activity compared to that in the control oligonucleotide treated cells. In addition, PKCeta down-regulation augmented vincristine-induced dissipation of mitochondrial transmembrane potential. In conclusion, these results confirm that PKCeta might represent a considerable resistance factor and an interesting target to improve anticancer chemotherapy. PMID:15159020

  20. 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.

  1. Prokaryotic homologs of Argonaute proteins are predicted to function as key components of a novel system of defense against mobile genetic elements

    Directory of Open Access Journals (Sweden)

    van der Oost John

    2009-08-01

    Full Text Available Abstract Background In eukaryotes, RNA interference (RNAi is a major mechanism of defense against viruses and transposable elements as well of regulating translation of endogenous mRNAs. The RNAi systems recognize the target RNA molecules via small guide RNAs that are completely or partially complementary to a region of the target. Key components of the RNAi systems are proteins of the Argonaute-PIWI family some of which function as slicers, the nucleases that cleave the target RNA that is base-paired to a guide RNA. Numerous prokaryotes possess the CRISPR-associated system (CASS of defense against phages and plasmids that is, in part, mechanistically analogous but not homologous to eukaryotic RNAi systems. Many prokaryotes also encode homologs of Argonaute-PIWI proteins but their functions remain unknown. Results We present a detailed analysis of Argonaute-PIWI protein sequences and the genomic neighborhoods of the respective genes in prokaryotes. Whereas eukaryotic Ago/PIWI proteins always contain PAZ (oligonucleotide binding and PIWI (active or inactivated nuclease domains, the prokaryotic Argonaute homologs (pAgos fall into two major groups in which the PAZ domain is either present or absent. The monophyly of each group is supported by a phylogenetic analysis of the conserved PIWI-domains. Almost all pAgos that lack a PAZ domain appear to be inactivated, and the respective genes are associated with a variety of predicted nucleases in putative operons. An additional, uncharacterized domain that is fused to various nucleases appears to be a unique signature of operons encoding the short (lacking PAZ pAgo form. By contrast, almost all PAZ-domain containing pAgos are predicted to be active nucleases. Some proteins of this group (e.g., that from Aquifex aeolicus have been experimentally shown to possess nuclease activity, and are not typically associated with genes for other (putative nucleases. Given these observations, the apparent extensive

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

    OpenAIRE

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

    2011-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    帖君

    2006-01-01

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

  4. Optimization of Peptide Nucleic Acid Antisense Oligonucleotides for Local and Systemic Dystrophin Splice Correction in the mdx Mouse

    OpenAIRE

    Yin, Haifang; Betts, Corinne; Saleh, Amer F; Ivanova, Gabriela D; Lee, Hyunil; Seow, Yiqi; Kim, Dalsoo; Gait, Michael J.; Wood, Matthew JA

    2010-01-01

    Antisense oligonucleotides (AOs) have the capacity to alter the processing of pre-mRNA transcripts in order to correct the function of aberrant disease-related genes. Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle degenerative disease that arises from mutations in the DMD gene leading to an absence of dystrophin protein. AOs have been shown to restore the expression of functional dystrophin via splice correction by intramuscular and systemic delivery in animal models of DMD and ...

  5. Cationic polyelectrolyte-mediated delivery of antisense morpholino oligonucleotides for exon-skipping in vitro and in mdx mice

    OpenAIRE

    Wang, Mingxing; Wu, Bo; Tucker, Jason D; Lu, Peijuan; Lu, Qilong

    2015-01-01

    In this study, we investigated a series of cationic polyelectrolytes (PEs) with different size and composition for their potential to improve delivery of an antisense phosphorodiamidate morpholino oligomer (PMO) both in vitro and in vivo. The results showed that the poly(diallyldimethylammonium chloride) (PDDAC) polymer series, especially PE-3 and PE-4, improves the delivery efficiency of PMO, comparable with Endoporter-mediated PMO delivery in vitro. The enhanced PMO delivery and targeting t...

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

    OpenAIRE

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

    1992-01-01

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

  7. 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.

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

    Science.gov (United States)

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

    1988-05-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2007-01-01

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

  10. 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.

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

    Science.gov (United States)

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

    1999-11-01

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

  12. 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.

  13. Knockdown of gene expression by antisense morpholino oligos in preimplantation mouse embryos cultured in vitro.

    Science.gov (United States)

    Sato, Yuki; Sato, Shiori; Kikuchi, Takahiro; Nonaka, Asumi; Kumagai, Yuki; Sasaki, Akira; Kobayashi, Masayuki

    2016-09-15

    Knockdown of gene expression by antisense morpholino oligos (MOs) is a simple and effective method for analyzing the roles of genes in mammalian cells. Here, we demonstrate the efficient delivery of MOs by Endo-Porter (EP), a special transfection reagent for MOs, into preimplantation mouse embryos cultured in vitro. A fluorescein-labeled control MO was applied for monitoring the incorporation of MOs into developing 2-cell embryos in the presence of varying amounts of EP and bovine serum albumin. In optimized conditions, fluorescence was detected in 2-cell embryos within a 3-h incubation period. In order to analyze the validity of the optimized conditions, an antisense Oct4 MO was applied for knockdown of the synthesis of OCT4 protein in developing embryos from the 2-cell stage. In blastocysts, the antisense Oct4 MO induced a decrease in the amount in OCT4 protein to less than half. An almost complete absence of OCT4-positive cells and nearly complete disappearance of the inner cell mass in the outgrowths of blastocysts were also noted. These phenotypes corresponded with those of Oct4-deficient mouse embryos. Overall, we suggest that the delivery of MOs using EP is useful for the knockdown of gene expression in preimplantation mouse embryos cultured in vitro. PMID:27381842

  14. 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.

  15. 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.

  16. 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.

  17. An element

    Energy Technology Data Exchange (ETDEWEB)

    Nakamura, K.; Iidzima, K.

    1983-03-30

    An anode of a light metal is used in the element, along with an electrolyte which consists of an ether solvent and an ionogenic additive in the form of a salt of dithiocarbamic acid. The element has good discharge characteristics.

  18. [Genetics and genetic counseling].

    Science.gov (United States)

    Izzi, Claudia; Liut, Francesca; Dallera, Nadia; Mazza, Cinzia; Magistroni, Riccardo; Savoldi, Gianfranco; Scolari, Francesco

    2016-01-01

    Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the most frequent genetic disease, characterized by progressive development of bilateral renal cysts. Two causative genes have been identified: PKD1 and PKD2. ADPKD phenotype is highly variable. Typically, ADPKD is an adult onset disease. However, occasionally, ADPKD manifests as very early onset disease. The phenotypic variability of ADPKD can be explained at three genetic levels: genic, allelic and gene modifier effects. Recent advances in molecular screening for PKD gene mutations and the introduction of the new next generation sequencing (NGS)- based genotyping approach have generated considerable improvement regarding the knowledge of genetic basis of ADPKD. The purpose of this article is to provide a comprehensive review of the genetics of ADPKD, focusing on new insights in genotype-phenotype correlation and exploring novel clinical approach to genetic testing. Evaluation of these new genetic information requires a multidisciplinary approach involving a nephrologist and a clinical geneticist. PMID:27067213

  19. Genetic Transformation of Citrus Paradisi with Antisense and untranslatable RNA-dependent RNA Polymerase Genes of Citrus Tristeza Closterovirus

    Science.gov (United States)

    Expression of the RNA-dependent RNA polymerase (RdRp) of Citrus tristeza virus (CTV) was studied in vivo and in vitro using a polyclonal antiserum raised against the recombinant CTV-RdRp protein. Although 56 kDa CTV-RdRp is thought to be expressed by a +1 translational frameshift at the carboxyl te...

  20. Characterization of extended-spectrum beta-lactamase, carbapenemase, and plasmid quinolone determinants in Klebsiella pneumoniae isolates carrying distinct types of 16S rRNA methylase genes, and their association with mobile genetic elements.

    Science.gov (United States)

    Wei, Dan-Dan; Wan, La-Gen; Yu, Yang; Xu, Qun-Fei; Deng, Qiong; Cao, Xian-Wei; Liu, Yang

    2015-04-01

    Eighty-four multidrug-resistant Klebsiella pneumoniae (MDR-KP) isolates from a Chinese hospital from January to October 2012 were evaluated to characterize the coexistence of 16S rRNA methylase, extended-spectrum β-lactamase, carbapenemase, and plasmid-mediated quinolone resistance determinants and their association with mobile genetic elements. Among the 84 MDR-KP isolates studied, 19 isolates exhibited high-level resistance to amikacin mediated by the production of the 16S rRNA methylase. They carried 19 armA genes (22.9%) and three rmtB genes (3.6%). CTX-M genes were found in all of the isolates. Among these armA- or rmtB/CTX-M-producing K. pneumoniae isolates, 31.6% carried the carbapenemase genes (blaKPC-2 [26.3%], blaIMP-4 [10.5%], and blaNDM-1 [5.3%]), which made them resistant to imipenem (minimum inhibitory concentration [MIC] ≥16 mg/L). All positive strains possessed qnr-like genes (16 qnrA1, 10 qnrS1, and 7 qnrB4 genes) and 18 harbored an aac(6')-Ib-cr gene. Mobile elements ISEcp1, IS26, ISCR1, ISAba125, and sul-1 integrons were detected in 19/19 (100%), 16/19 (84.2%), 18/19 (94.7%), 9/19 (47.4%), and 18/19 (94.7%) isolates, respectively. The mobilizing elements occurred in different combinations in the study isolates. Majority of armA and qnr genes were in MDR-KP strains carrying integrons containing the ISCR1. Close to 80% of blaTEM-1 and blaSHV-12 were linked to IS26 while ≥90% of blaCTX-Ms and blaCMYs were linked to ISEcp1. ISAba125 was located upstream of blaNDM-1 and some blaCMY-2 genes. In addition, seven transconjugants were available for further analysis, and armA, qnrS1, acc(6')-Ib-cr, blaCTX-M-15, blaTEM-1, and blaNDM-1 were cotransferred. This study points to the dissemination of 16S rRNA methylase genes and the prevalence of selected elements implicated in evolution of resistance determinants in collection of clinical K. pneumoniae in China.

  1. Ribonuclease H1-dependent hepatotoxicity caused by locked nucleic acid-modified gapmer antisense oligonucleotides.

    Science.gov (United States)

    Kasuya, Takeshi; Hori, Shin-Ichiro; Watanabe, Ayahisa; Nakajima, Mado; Gahara, Yoshinari; Rokushima, Masatomo; Yanagimoto, Toru; Kugimiya, Akira

    2016-01-01

    Gapmer antisense oligonucleotides cleave target RNA effectively in vivo, and is considered as promising therapeutics. Especially, gapmers modified with locked nucleic acid (LNA) shows potent knockdown activity; however, they also cause hepatotoxic side effects. For developing safe and effective gapmer drugs, a deeper understanding of the mechanisms of hepatotoxicity is required. Here, we investigated the cause of hepatotoxicity derived from LNA-modified gapmers. Chemical modification of gapmer's gap region completely suppressed both knockdown activity and hepatotoxicity, indicating that the root cause of hepatotoxicity is related to intracellular gapmer activity. Gene silencing of hepatic ribonuclease H1 (RNaseH1), which catalyses gapmer-mediated RNA knockdown, strongly supressed hepatotoxic effects. Small interfering RNA (siRNA)-mediated knockdown of a target mRNA did not result in any hepatotoxic effects, while the gapmer targeting the same position on mRNA as does the siRNA showed acute toxicity. Microarray analysis revealed that several pre-mRNAs containing a sequence similar to the gapmer target were also knocked down. These results suggest that hepatotoxicity of LNA gapmer is caused by RNAseH1 activity, presumably because of off-target cleavage of RNAs inside nuclei. PMID:27461380

  2. Global transcriptional start site mapping using differential RNA sequencing reveals novel antisense RNAs in Escherichia coli.

    Science.gov (United States)

    Thomason, Maureen K; Bischler, Thorsten; Eisenbart, Sara K; Förstner, Konrad U; Zhang, Aixia; Herbig, Alexander; Nieselt, Kay; Sharma, Cynthia M; Storz, Gisela

    2015-01-01

    While the model organism Escherichia coli has been the subject of intense study for decades, the full complement of its RNAs is only now being examined. Here we describe a survey of the E. coli transcriptome carried out using a differential RNA sequencing (dRNA-seq) approach, which can distinguish between primary and processed transcripts, and an automated prediction algorithm for transcriptional start sites (TSS). With the criterion of expression under at least one of three growth conditions examined, we predicted 14,868 TSS candidates, including 5,574 internal to annotated genes (iTSS) and 5,495 TSS corresponding to potential antisense RNAs (asRNAs). We examined expression of 14 candidate asRNAs by Northern analysis using RNA from wild-type E. coli and from strains defective for RNases III and E, two RNases reported to be involved in asRNA processing. Interestingly, nine asRNAs detected as distinct bands by Northern analysis were differentially affected by the rnc and rne mutations. We also compared our asRNA candidates with previously published asRNA annotations from RNA-seq data and discuss the challenges associated with these cross-comparisons. Our global transcriptional start site map represents a valuable resource for identification of transcription start sites, promoters, and novel transcripts in E. coli and is easily accessible, together with the cDNA coverage plots, in an online genome browser.

  3. A lignin-specific peroxidase in tobacco whose antisense suppression leads to vascular tissue modification

    Science.gov (United States)

    Blee, Kristopher A.; Choi, Joon W.; O'Connell, Ann P.; Schuch, Wolfgang; Lewis, Norman G.; Bolwell, G. Paul

    2003-01-01

    A tobacco peroxidase isoenzyme (TP60) was down-regulated in tobacco using an antisense strategy, this affording transformants with lignin reductions of up to 40-50% of wild type (control) plants. Significantly, both guaiacyl and syringyl levels decreased in essentially a linear manner with the reductions in lignin amounts, as determined by both thioacidolysis and nitrobenzene oxidative analyses. These data provisionally suggest that a feedback mechanism is operative in lignifying cells, which prevents build-up of monolignols should oxidative capacity for their subsequent metabolism be reduced. Prior to this study, the only known rate-limiting processes in the monolignol/lignin pathways involved that of Phe supply and the relative activities of cinnamate-4-hydroxylase/p-coumarate-3-hydroxylase, respectively. These transformants thus provide an additional experimental means in which to further dissect and delineate the factors involved in monolignol targeting to precise regions in the cell wall, and of subsequent lignin assembly. Interestingly, the lignin down-regulated tobacco phenotypes displayed no readily observable differences in overall growth and development profiles, although the vascular apparatus was modified.

  4. Serial incorporation of a monovalent GalNAc phosphoramidite unit into hepatocyte-targeting antisense oligonucleotides.

    Science.gov (United States)

    Yamamoto, Tsuyoshi; Sawamura, Motoki; Wada, Fumito; Harada-Shiba, Mariko; Obika, Satoshi

    2016-01-01

    The targeting of abundant hepatic asialoglycoprotein receptors (ASGPR) with trivalent N-acetylgalactosamine (GalNAc) is a reliable strategy for efficiently delivering antisense oligonucleotides (ASOs) to the liver. We here experimentally demonstrate the high systemic potential of the synthetically-accessible, phosphodiester-linked monovalent GalNAc unit when tethered to the 5'-terminus of well-characterised 2',4'-bridged nucleic acid (also known as locked nucleic acid)-modified apolipoprotein B-targeting ASO via a bio-labile linker. Quantitative analysis of the hepatic disposition of the ASOs revealed that phosphodiester is preferable to phosphorothioate as an interunit linkage in terms of ASGPR binding of the GalNAc moiety, as well as the subcellular behavior of the ASO. The flexibility of this monomeric unit was demonstrated by attaching up to 5 GalNAc units in a serial manner and showing that knockdown activity improves as the number of GalNAc units increases. Our study suggests the structural requirements for efficient hepatocellular targeting using monovalent GalNAc and could contribute to a new molecular design for suitably modifying ASO.

  5. Evaluation of 2'-Deoxy-2'-fluoro Antisense Oligonucleotides for Exon Skipping in Duchenne Muscular Dystrophy

    Science.gov (United States)

    Jirka, Silvana M G; Tanganyika-de Winter, Christa L; Boertje-van der Meulen, Joke W; van Putten, Maaike; Hiller, Monika; Vermue, Rick; de Visser, Peter C; Aartsma-Rus, Annemieke

    2015-01-01

    Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder typically caused by frame-shifting mutations in the DMD gene. Restoration of the reading frame would allow the production of a shorter but partly functional dystrophin protein as seen in Becker muscular dystrophy patients. This can be achieved with antisense oligonucleotides (AONs) that induce skipping of specific exons during pre-mRNA splicing. Different chemical modifications have been developed to improve AON properties. The 2'-deoxy-2'-fluoro (2F) RNA modification is attractive for exon skipping due to its ability to recruit ILF2/3 proteins to the 2F/pre-mRNA duplex, which resulted in enhanced exon skipping in spinal muscular atrophy models. In this study, we examined the effect of two different 2'-substituted AONs (2'-F phosphorothioate (2FPS) and 2'-O-Me phosphorothioate (2OMePS)) on exon skipping in DMD cell and animal models. In human cell cultures, 2FPS AONs showed higher exon skipping levels than their isosequential 2OMePS counterparts. Interestingly, in the mdx mouse model, 2FPS was less efficient than 2OMePS and suggested safety issues as evidenced by increased spleen size and weight loss. Our results do not support a clinical application for 2FPS AON. PMID:26623937

  6. Optimizing RNA/ENA chimeric antisense oligonucleotides using in vitro splicing.

    Science.gov (United States)

    Takeshima, Yasuhiro; Yagi, Mariko; Matsuo, Masafumi

    2012-01-01

    A molecular therapy for Duchenne muscular dystrophy (DMD) that converts dystrophin mRNA from out-of-frame to in-frame transcripts by inducing exon skipping with antisense oligonucleotides (AOs) is now approaching clinical application. To exploit the broad therapeutic applicability of exon skipping therapy, it is necessary to identify AOs that are able to induce efficient and specific exon skipping. To optimize AOs, we have established an in vitro splicing system using cultured DMD myocytes. Here, we describe the process of identifying the best AO.Cultured DMD myocytes are established from a biopsy sample and the target exon is chosen. A series of AOs are designed to cover the whole target exon sequence. As AOs, we use 15-20-mer chimeric oligonucleotides consisting of 2'-O-methyl RNA and modified nucleic acid (2'-O, 4'-C-ethylene-bridged nucleic acid). Each AO is transfected individually into cultured DMD myocytes, and the resulting mRNA is analyzed by reverse transcription-PCR. The ability of each AO to induce exon skipping is examined by comparing the amount of cDNA with and without exon skipping. If necessary, having roughly localized the target region, another set of AOs are designed and the exon skipping abilities of the new AOs are examined. Finally, one AO is determined as the best for the molecular therapy.Our simple and reliable methods using an in vitro splicing system have enabled us to identify optimized AOs against many exons of the DMD gene.

  7. RNA synthetic biology inspired from bacteria: construction of transcription attenuators under antisense regulation

    International Nuclear Information System (INIS)

    Among all biopolymers, ribonucleic acids or RNA have unique functional versatility, which led to the early suggestion that RNA alone (or a closely related biopolymer) might have once sustained a primitive form of life based on a single type of biopolymer. This has been supported by the demonstration of processive RNA-based replication and the discovery of 'riboswitches' or RNA switches, which directly sense their metabolic environment. In this paper, we further explore the plausibility of this 'RNA world' scenario and show, through synthetic molecular design guided by advanced RNA simulations, that RNA can also perform elementary regulation tasks on its own. We demonstrate that RNA synthetic regulatory modules directly inspired from bacterial transcription attenuators can efficiently activate or repress the expression of other RNA by merely controlling their folding paths 'on the fly' during transcription through simple RNA–RNA antisense interaction. Factors, such as NTP concentration and RNA synthesis rate, affecting the efficiency of this kinetic regulation mechanism are also studied and discussed in the light of evolutionary constraints. Overall, this suggests that direct coupling among synthesis, folding and regulation of RNAs may have enabled the early emergence of autonomous RNA-based regulation networks in absence of both DNA and protein partners

  8. Thiolated polycarbophil as an adjuvant for permeation enhancement in nasal delivery of antisense oligonucleotides.

    Science.gov (United States)

    Vetter, A; Martien, R; Bernkop-Schnürch, A

    2010-03-01

    The purpose of this study was to investigate the effect of thiolated polycarbophil as an adjuvant to enhance the permeation and improve the stability of a phosphorothioate antisense oligonucleotide (PTO-ODN) on the nasal mucosa. Polycarbophil-cysteine (PCP-Cys) was synthesized by the covalent attachment of L-cysteine to the polymeric backbone. Cytotoxicity tests were examined on human nasal epithelial cells from surgery of nasal polyps confirmed by histological studies. Deoxyribonuclease I activity in respiratory region of the porcine nasal cavity was analyzed by an enzymatic assay. The enzymatic degradation of PTO-ODNs on freshly excised porcine nasal mucosa was analyzed and protection of PCP-cysteine toward DNase I degradation was evaluated. Permeation studies were performed in Ussing-type diffusion chambers. PCP-Cys/GSH did not arise a remarkable mortal effect. Porcine respiratory mucosa was shown to possess nuclease activity corresponding to 0.69 Kunitz units/mL. PTO-ODNs were degraded by incubation with nasal mucosa. In the presence of 0.45% thiolated polycarbophil and 0.5% glutathione (GSH), this degradation process could be lowered. In the presence of thiolated polycarbophil and GSH the uptake of PTO-ODNs from the nasal mucosa was 1.7-fold improved. According to these results thiolated polycarbophil/GSH might be a promising excipient for nasal administration of PTO-ODNs.

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

    Directory of Open Access Journals (Sweden)

    Zhao Yongsheng

    2011-04-01

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

  10. 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.

  11. 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.

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

    Institute of Scientific and Technical Information of China (English)

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

    2003-01-01

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

  13. Development and Substance Accumulation of Caryopsis in Transgenic Rice with Antisense Wx Gene

    Institute of Scientific and Technical Information of China (English)

    CHEN Gang; WANG Zhong; LIU Qiao-quan; KIONG Fei; GU Yun-jie; GU Guo-jun

    2006-01-01

    The development and substance accumulation of rice caryopsis were studied by using the transgenic japonica and indica rice with antisense Wx gene. The weight of caryopses in transgenic rice was lower than that in non-transgenic one, and the reduction in weight was significantly correlated to the reduction in amylose content. In caryopsis of transgenic rice, the number of endosperm cells was less than that in caryopsis of non-transgenic one, but the proliferation speed was considerably higher during the first six days after flowering (DAF). During the first nine DAF, the soluble sugar content of transgenic rice caryopsis was less than that of the non-transgenic one, but the situation was reverse after nine DAF. Moreover, the total starch content also declined with the decrease in amylose content of transgenic rice caryopsis, while the amylopectin content increased accordingly. Therefore, the composition of starch in caryopsis also changed, but it did not affect the accumulation of protein in transgenic rice caryopsis.

  14. New Genetics

    Science.gov (United States)

    ... Home > Science Education > The New Genetics The New Genetics Living Laboratories Classroom Poster Order a Free Copy ... Piece to a Century-Old Evolutionary Puzzle Computing Genetics Model Organisms RNA Interference The New Genetics is ...

  15. 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.

  16. Transuranium elements

    International Nuclear Information System (INIS)

    This paper reports that neutron irradiation of uranium, beginning in 1934, led to the reported discovery of such transuranium elements as eka-rhenium. After a few years of investigation these were correctly identified, in December 1938, as fission products. The first real transuranium elements, with atomic numbers 93 and 94 (neptumiun and plutionium), were discovered in 1940-1941. Plutomiun-239 was shown to be fission-able with slow neutrons, and chemical methods for its separation in quantity were developed. Not until it was recognized that transuranium elements should be part of a 14-member actinide series could elements 95 and 96 (americium and curium) be chemically identified after their nuclear synthesis. This new view was the key to the synthesis and identified after their nuclear synthesis. This new view was the key to the synthesis and identification of the next seven transuranium elements, resulting in the completion of the actinide series at element 103 in 1961. The transactinide elements could, according to the actinide concept, also be correctly placed in the periodic table, and the chemical properties of the first transactinide elements, elements 104 and 105 (rutherfordium and hafnium), confirm this point of view. Transuranium elements through number 109 are now known

  17. 基因工程食品%Genetic engineering food

    Institute of Scientific and Technical Information of China (English)

    汪秋安

    2003-01-01

    @@ 1 概述 近年来,生物技术在食品行业的应用迅速发展,食品生物技术包括基因工程(genetic engineering)、蛋白质工程(protein enginering)、酶工程(enzyme engineering)、发酵技术(fermentation technology)、组织与细胞培养(tissue and cell culture)、反义RNA(antisense RNA)技术等.

  18. 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.

  19. 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.

  20. Osteoporosis and trace elements

    DEFF Research Database (Denmark)

    Aaseth, J.; Boivin, G.; Andersen, Ole

    2012-01-01

    More than 200 million people are affected by osteoporosis worldwide, as estimated by 2 million annual hip fractures and other debilitating bone fractures (vertebrae compression and Colles' fractures). Osteoporosis is a multi-factorial disease with potential contributions from genetic, endocrine...... functional, exercise related and nutritional factors. Of particular considerations are calcium (Ca) status, vitamin D, fluoride, magnesium and other trace elements. Several trace elements such as zinc and copper are essential for normal development of the skeleton in humans and animals. Fluoride accumulates...... in new bone and results in a net gain in bone mass, but may be associated with a tissue of poor quality. Aluminum induces impairment of bone formation. Gallium and cadmium suppresses bone turnover. However, exact involvements of the trace elements in osteoporosis have not yet been fully clarified...

  1. Hsp90 protein interacts with phosphorothioate oligonucleotides containing hydrophobic 2'-modifications and enhances antisense activity.

    Science.gov (United States)

    Liang, Xue-Hai; Shen, Wen; Sun, Hong; Kinberger, Garth A; Prakash, Thazha P; Nichols, Joshua G; Crooke, Stanley T

    2016-05-01

    RNase H1-dependent antisense oligonucleotides (ASOs) are chemically modified to enhance pharmacological properties. Major modifications include phosphorothioate (PS) backbone and different 2'-modifications in 2-5 nucleotides at each end (wing) of an ASO. Chemical modifications can affect protein binding and understanding ASO-protein interactions is important for better drug design. Recently we identified many intracellular ASO-binding proteins and found that protein binding could affect ASO potency. Here, we analyzed the structure-activity-relationships of ASO-protein interactions and found 2'-modifications significantly affected protein binding, including La, P54nrb and NPM. PS-ASOs containing more hydrophobic 2'-modifications exhibit higher affinity for proteins in general, although certain proteins, e.g. Ku70/Ku80 and TCP1, are less affected by 2'-modifications. We found that Hsp90 protein binds PS-ASOs containing locked-nucleic-acid (LNA) or constrained-ethyl-bicyclic-nucleic-acid ((S)-cEt) modifications much more avidly than 2'-O-methoxyethyl (MOE). ASOs bind the mid-domain of Hsp90 protein. Hsp90 interacts with more hydrophobic 2' modifications, e.g. (S)-cEt or LNA, in the 5'-wing of the ASO. Reduction of Hsp90 protein decreased activity of PS-ASOs with 5'-LNA or 5'-cEt wings, but not with 5'-MOE wing. Together, our results indicate Hsp90 protein enhances the activity of PS/LNA or PS/(S)-cEt ASOs, and imply that altering protein binding of ASOs using different chemical modifications can improve therapeutic performance of PS-ASOs. PMID:26945041

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

    Institute of Scientific and Technical Information of China (English)

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

    2004-01-01

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

  3. 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

  4. The effect of in vitro exposure to antisense oligonucleotides on macrophage morphology and function

    Directory of Open Access Journals (Sweden)

    Ann Brasey

    2011-11-01

    Full Text Available Antisense oligonucleotides (AON delivered via inhalation are in drug development for respiratory diseases. In rodents and monkeys, repeated exposure to high doses of inhaled phosphorothioate (PS AON can lead to microscopic changes in the lungs, including accumulation of alveolar macrophages in the lower airway that have a foamy appearance. The functional consequences that result from this morphological change are unclear as there is controversy whether the vacuoles/inclusion bodies reflect normal clearance of the inhaled AON or are early indicators of lung toxicity. The morphological and functional responses of macrophage to PS AON were characterized in vitro using the comparator drug amiodarone, as a known inducer of foamy macrophages. Morphological changes of increased vacuolization with the presence of lamellated structures were observed in macrophages in response to both amiodarone and AON treatment. Functional responses to the drugs clearly differed with amiodarone treatment leading to apoptosis of cells and cell death, release of proinflammatory mediators IL-1RA, MIP-1α and TNFα, decrease in IP-10, a cytokine shown to be involved in protection against pulmonary fibrosis and altered phagocytosis capacity of the cells. In contrast, AON in concentrations up to 30 μM, had no effect on cell viability or apoptosis, had minimal effects on pro-inflammatory cytokines, increased IP-10 levels and did not alter the phagocytic capacity of the cells. Exposure of macrophages to AON in vitro, led to morphological changes of increased vacuolization, but did not lead to functional consequences which were observed with another vacuolization-inducing drug, suggesting that the in vivo phenotypic changes observed following inhalation of AON may be consistent with a clearance mechanism and not an activation or impairment of macrophages.

  5. Affinity capture of (Arg sup 8 )vasopressin-receptor complex using immobilized antisense peptide

    Energy Technology Data Exchange (ETDEWEB)

    Feng Xian Lu; Aiyar, N.; Chaiken, I. (SmithKline Beecham, King of Prussia, PA (United States))

    1991-05-01

    Solubilized noncovalent complexes of (Arg{sup 8})-vasopressin (AVP) with receptor proteins from rat liver membranes were isolated by selective binding to silica-immobilized antisense (AS) peptide. The affinity chromatographic support was prepared with a chemically synthesized AS peptide whose sequence is encoded by the AS DNA corresponding to the 20 amino-terminal residues of the AVP bovine neurophysin II biosynthetic precursor (pro-AVP/BNPII-(20-1)), region that includes the AVP sequence at residues 1-9. The AS peptide-AVP interaction mechanism hypothesized, contact by hydropathic complementarity at multiple sites along the peptide chains, led to the prediction that AVP bound to its receptor would still have enough free surface to interact with immobilized AS peptide. To test this prediction of a three-way interaction, ({sup 3}H)AVP-receptor was obtained as a solubilized, partially purified fraction from rat liver membrane. Covalently crosslinked ({sup 3}H)AVP complex also was bound to the AS peptide column; binding was blocked by competition with unlabeled AVP in the elution buffer. Since the AVP-linked 31- and 38-kDa proteins have the same apparent molecular mass on SDS/PAGE as found previously by photoaffinity labeling, the authors conclude that the AS peptide column has affinity-captured AVP-receptor complexes. The 15-kDa protein appears to be an active AVP-receptor fragment of one or both of the larger proteins. It is generally concluded that immobilized AS peptides may be useful to isolate peptide and protein receptor complexes in other systems as well.

  6. Sense-antisense gene-pairs in breast cancer and associated pathological pathways

    Science.gov (United States)

    Grinchuk, Oleg V.; Motakis, Efthymios; Yenamandra, Surya Pavan; Ow, Ghim Siong; Jenjaroenpun, Piroon; Tang, Zhiqun; Yarmishyn, Aliaksandr A.; Ivshina, Anna V.; Kuznetsov, Vladimir A.

    2015-01-01

    More than 30% of human protein-coding genes form hereditary complex genome architectures composed of sense-antisense (SA) gene pairs (SAGPs) transcribing their RNAs from both strands of a given locus. Such architectures represent important novel components of genome complexity contributing to gene expression deregulation in cancer cells. Therefore, the architectures might be involved in cancer pathways and, in turn, be used for novel drug targets discovery. However, the global roles of SAGPs in cancer pathways has not been studied. Here we investigated SAGPs associated with breast cancer (BC)-related pathways using systems biology, prognostic survival and experimental methods. Gene expression analysis identified 73 BC-relevant SAGPs that are highly correlated in BC. Survival modelling and metadata analysis of the 1161 BC patients allowed us to develop a novel patient prognostic grouping method selecting the 12 survival-significant SAGPs. The qRT-PCR-validated 12-SAGP prognostic signature reproducibly stratified BC patients into low- and high-risk prognostic subgroups. The 1381 SAGP-defined differentially expressed genes common across three studied cohorts were identified. The functional enrichment analysis of these genes revealed the GABPA gene network, including BC-relevant SAGPs, specific gene sets involved in cell cycle, spliceosomal and proteasomal pathways. The co-regulatory function of GABPA in BC cells was supported using siRNA knockdown studies. Thus, we demonstrated SAGPs as the synergistically functional genome architectures interconnected with cancer-related pathways and associated with BC patient clinical outcomes. Taken together, SAGPs represent an important component of genome complexity which can be used to identify novel aspects of coordinated pathological gene networks in cancers. PMID:26517092

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

    Institute of Scientific and Technical Information of China (English)

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

    2007-01-01

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

  8. Cellular Selenoprotein mRNA Tethering via Antisense Interactions with Ebola and HIV-1 mRNAs May Impact Host Selenium Biochemistry.

    Science.gov (United States)

    Taylor, Ethan Will; Ruzicka, Jan A; Premadasa, Lakmini; Zhao, Lijun

    2016-01-01

    Regulation of protein expression by non-coding RNAs typically involves effects on mRNA degradation and/or ribosomal translation. The possibility of virus-host mRNA-mRNA antisense tethering interactions (ATI) as a gain-of-function strategy, via the capture of functional RNA motifs, has not been hitherto considered. We present evidence that ATIs may be exploited by certain RNA viruses in order to tether the mRNAs of host selenoproteins, potentially exploiting the proximity of a captured host selenocysteine insertion sequence (SECIS) element to enable the expression of virally-encoded selenoprotein modules, via translation of in-frame UGA stop codons as selenocysteine. Computational analysis predicts thermodynamically stable ATIs between several widely expressed mammalian selenoprotein mRNAs (e.g., isoforms of thioredoxin reductase) and specific Ebola virus mRNAs, and HIV-1 mRNA, which we demonstrate via DNA gel shift assays. The probable functional significance of these ATIs is further supported by the observation that, in both viruses, they are located in close proximity to highly conserved in-frame UGA stop codons at the 3' end of open reading frames that encode essential viral proteins (the HIV-1 nef protein and the Ebola nucleoprotein). Significantly, in HIV/AIDS patients, an inverse correlation between serum selenium and mortality has been repeatedly documented, and clinical benefits of selenium in the context of multi-micronutrient supplementation have been demonstrated in several well-controlled clinical trials. Hence, in the light of our findings, the possibility of a similar role for selenium in Ebola pathogenesis and treatment merits serious investigation. PMID:26369818

  9. Cellular Selenoprotein mRNA Tethering via Antisense Interactions with Ebola and HIV-1 mRNAs May Impact Host Selenium Biochemistry

    Science.gov (United States)

    Taylor, Ethan Will; Ruzicka, Jan A.; Premadasa, Lakmini; Zhao, Lijun

    2016-01-01

    Regulation of protein expression by non-coding RNAs typically involves effects on mRNA degradation and/or ribosomal translation. The possibility of virus-host mRNA-mRNA antisense tethering interactions (ATI) as a gain-of-function strategy, via the capture of functional RNA motifs, has not been hitherto considered. We present evidence that ATIs may be exploited by certain RNA viruses in order to tether the mRNAs of host selenoproteins, potentially exploiting the proximity of a captured host selenocysteine insertion sequence (SECIS) element to enable the expression of virally-encoded selenoprotein modules, via translation of in-frame UGA stop codons as selenocysteine. Computational analysis predicts thermodynamically stable ATIs between several widely expressed mammalian selenoprotein mRNAs (e.g., isoforms of thioredoxin reductase) and specific Ebola virus mRNAs, and HIV-1 mRNA, which we demonstrate via DNA gel shift assays. The probable functional significance of these ATIs is further supported by the observation that, in both viruses, they are located in close proximity to highly conserved in-frame UGA stop codons at the 3′ end of open reading frames that encode essential viral proteins (the HIV-1 nef protein and the Ebola nucleoprotein). Significantly, in HIV/AIDS patients, an inverse correlation between serum selenium and mortality has been repeatedly documented, and clinical benefits of selenium in the context of multi-micronutrient supplementation have been demonstrated in several well-controlled clinical trials. Hence, in the light of our findings, the possibility of a similar role for selenium in Ebola pathogenesis and treatment merits serious investigation. PMID:26369818

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

    Directory of Open Access Journals (Sweden)

    Baldi Alfonso

    2011-07-01

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

  11. Clinical and genetic basis of familial amyotrophic lateral sclerosis

    Directory of Open Access Journals (Sweden)

    Paulo Victor Sgobbi de Souza

    2015-01-01

    Full Text Available Amyotrophic lateral sclerosis represents the most common neurodegenerative disease leading to upper and lower motor neuron compromise. Although the vast majority of cases are sporadic, substantial gain has been observed in the knowledge of the genetic forms of the disease, especially of familial forms. There is a direct correlation between the profile of the mutated genes in sporadic and familial forms, highlighting the main role ofC9orf72 gene in the clinical forms associated with frontotemporal dementia spectrum. The different genes related to familial and sporadic forms represent an important advance on the pathophysiology of the disease and genetic therapeutic perspectives, such as antisense therapy. The objective of this review is to signal and summarize clinical and genetic data related to familial forms of amyotrophic lateral sclerosis.

  12. Global analysis of cis-natural antisense transcripts and their heat-responsive nat-siRNAs in Brassica rapa

    OpenAIRE

    Yu, Xiang; Yang, Jun; Li, Xiaorong; Liu, Xuxin; Sun, Chuanbao; Wu, Feijie; He, Yuke

    2013-01-01

    Background Brassica rapa includes several important leaf vegetable crops whose production is often damaged by high temperature. Cis-natural antisense transcripts (cis-NATs) and cis-NATs-derived small interfering RNAs (nat-siRNAs) play important roles in plant development and stress responses. However, genome-wide cis-NATs in B. rapa are not known. The NATs and nat-siRNAs that respond to heat stress have never been well studied in B. rapa. Here, we took advantage of RNA-seq and small RNA (sRNA...

  13. Therapeutic Liabilities of in Vivo Viral Vector Tropism: Adeno-Associated Virus Vectors, NMDAR1 Antisense, and Focal Seizure Sensitivity

    OpenAIRE

    Haberman, Rebecca P.; Criswell, Hugh E.; Snowdy, Stephen; Ming, Zhen; Breese, George R.; Samulski, R. Jude; McCown, Thomas J.

    2002-01-01

    The N-methyl-d-aspartic acid (NMDA) receptor provides a potential target for gene therapy of focal seizure disorders. To test this approach, we cloned a 729-bp NMDA receptor (NMDAR1) cDNA fragment in the antisense orientation into adeno-associated virus (AAV) vectors, where expression was driven by either a tetracycline-off regulatable promoter (AAV-tTAK-NR1A) or a cytomegalovirus (CMV) promoter (AAV-CMV-NR1A). After infection of primary cultured cortical neurons with recombinant AAV-tTAK-NR1...

  14. 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

  15. Defining a role for Hfq in Gram-positive bacteria: evidence for Hfq-dependent antisense regulation in Listeria monocytogenes

    OpenAIRE

    Nielsen, Jesper Sejrup; Lei, Lisbeth Kristensen; Ebersbach, Tine; Olsen, Anders Steno; Klitgaard, Janne Kudsk; Valentin-Hansen, Poul; Kallipolitis, Birgitte Haahr

    2009-01-01

    Small trans-encoded RNAs (sRNAs) modulate the translation and decay of mRNAs in bacteria. In Gram-negative species, antisense regulation by trans-encoded sRNAs relies on the Sm-like protein Hfq. In contrast to this, Hfq is dispensable for sRNA-mediated riboregulation in the Gram-positive species studied thus far. Here, we provide evidence for Hfq-dependent translational repression in the Gram-positive human pathogen Listeria monocytogenes, which is known to encode at least 50 sRNAs. We show t...

  16. 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.

  17. 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.

  18. 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

  19. Preparation of 99Tcm labeled survivin mRNA antisense PNA and gene imaging in nude mice bearing lung carcinoma A549 xenografts

    International Nuclear Information System (INIS)

    Objective: To prepare the 99Tcm-survivin mRNA antisense peptide nucleic acid (PNA)and investigate its value as a gene imaging agent in tumor bearing mice and early diagnosis in tumor. Methods: Survivin mRNA antisense PNA and mismatch PNA were synthesized. Four amino acids (Gly- (D)Ala-Gly-Gly) and Aba (4-aminobutyric acid) were linked to the 5' end of PNA. Gly- (D)Ala-Gly-Gly served as a chelating moiety for strong chelation of 99Tcm and Aba acted as a spacer to minimize the steric hindrance. PNAs were labeled with 99Tcm by the ligand-exchange method. The labeling efficiency and radiochemical purity were measured by HPLC and ITLC methods. There were five BALB/c nude mice bearing human lung carcinoma (A549) in each of antisense PNA and mismatch PNA groups. Gene imaging of 99Tcm-survivin mRNA antisense and mismatch PNAs were performed at 1, 2 and 4 h post the injection, respectively, and the T/NT ratio was measured by the method of ROI. The statistical comparisons of average values were performed with the two-group t-test for independent sample by SPSS 13.0. Results: The product kept stable in vitro. The labeling efficiency of 99Tcm-survivin mRNA antisense PNA was (95.48 ±1.92)% and more than 85% after the incubation for 24 h in serum. The radiochemical purity was >95%. The labeling efficiency of mismatch PNA was similar to the antisense PNA. 99Tcm-survivin mRNA antisense PNA was especially uptaken by tumor lesion, and its accumulation reached the top at 4 h post the injection. T/NT ratios at 1, 2, and 4 h were 2.70 ± 0.28, 3.44 ± 0.35,4.21 ± 0.63, respectively. In the comparison, the T/NT ratio of 99Tcm-survivin mRNA mismatch PNA at 4 h (3.12 ±0.50) was significantly lower (t=2.918, P=0.019). Conclusions: 99Tcm-survivin mRNA antisense PNA has high labeling efficiency,good stability and no need of purification. Its characteristic of especial uptake by tumor lesion provides the potential value in early diagnosis of tumor. (authors)

  20. 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

  1. Genetic Counseling

    Science.gov (United States)

    Genetic counseling provides information and support to people who have, or may be at risk for, genetic disorders. A ... meets with you to discuss genetic risks. The counseling may be for yourself or a family member. ...

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

    Institute of Scientific and Technical Information of China (English)

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

    2004-01-01

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

  3. Sterilization of sterlet Acipenser ruthenus by using knockdown agent, antisense morpholino oligonucleotide, against dead end gene.

    Science.gov (United States)

    Linhartová, Zuzana; Saito, Taiju; Kašpar, Vojtěch; Rodina, Marek; Prášková, Eva; Hagihara, Seishi; Pšenička, Martin

    2015-10-15

    Sturgeons (chondrostean, acipenseridae) are ancient fish species, widely known for their caviar. Nowadays, most of them are critically endangered. The sterlet (Acipenser ruthenus) is a common Eurasian sturgeon species with a small body size and the fastest reproductive cycle among sturgeons. Such species can be used as a host for surrogate production; application is of value for recovery of critically endangered and huge sturgeon species with an extremely long reproductive cycle. One prerequisite for production of the donor's gametes only is to have a sterile host. Commonly used sterilization techniques in fishes such as triploidization or hybridization do not guarantee sterility in sturgeon. Alternatively, sterilization can be achieved by using a temporary germ cell exclusion-specific gene by a knockdown agent, the antisense morpholino oligonucleotide (MO). The targeted gene for the MO is the dead end gene (dnd) which is a vertebrate-specific gene encoding a RNA-binding protein which is crucial for migration and survival of primordial germ cells (PGCs). For this purpose, a dnd homologue of Russian sturgeon (Agdnd), resulting in the same sequence in the start codon region with isolated fragments of sterlet dnd (Ardnd), was used. Reverse transcription polymerase chain reaction confirmed tissue-specific expression of Ardnd only in the gonads of both sexes. Dnd-MO for depletion of PGCs together with fluorescein isothiocyanate (FITC)-biotin-dextran for PGCs labeling was injected into the vegetal region of one- to four-cell-stage sterlet embryos. In the control groups, only FITC was injected to validate the injection method and labeling of PGCs. After optimization of MO concentration together with volume injection, 250-μM MO was applied for sterilization of sturgeon embryos. Primordial germ cells were detected under a fluorescent stereomicroscope in the genital ridge of the FITC-labeled control group only, whereas no PGCs were present in the body cavities of morphants

  4. 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.

  5. 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.

  6. 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.

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

    Institute of Scientific and Technical Information of China (English)

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

    2005-01-01

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

  8. 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.

  9. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  11. 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.

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

    Science.gov (United States)

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

    2006-10-01

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

  13. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1999-01-01

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

  15. Defining a role for Hfq in Gram-positive bacteria: evidence for Hfq-dependent antisense regulation in Listeria monocytogenes

    Science.gov (United States)

    Nielsen, Jesper Sejrup; Lei, Lisbeth Kristensen; Ebersbach, Tine; Olsen, Anders Steno; Klitgaard, Janne Kudsk; Valentin-Hansen, Poul; Kallipolitis, Birgitte Haahr

    2010-01-01

    Small trans-encoded RNAs (sRNAs) modulate the translation and decay of mRNAs in bacteria. In Gram-negative species, antisense regulation by trans-encoded sRNAs relies on the Sm-like protein Hfq. In contrast to this, Hfq is dispensable for sRNA-mediated riboregulation in the Gram-positive species studied thus far. Here, we provide evidence for Hfq-dependent translational repression in the Gram-positive human pathogen Listeria monocytogenes, which is known to encode at least 50 sRNAs. We show that the Hfq-binding sRNA LhrA controls the translation and degradation of its target mRNA by an antisense mechanism, and that Hfq facilitates the binding of LhrA to its target. The work presented here provides the first experimental evidence for Hfq-dependent riboregulation in a Gram-positive bacterium. Our findings indicate that modulation of translation by trans-encoded sRNAs may occur by both Hfq-dependent and -independent mechanisms, thus adding another layer of complexity to sRNA-mediated riboregulation in Gram-positive species. PMID:19942685

  16. The expression of ELK transcription factors in adult DRG: Novel isoforms, antisense transcripts and upregulation by nerve damage.

    Science.gov (United States)

    Kerr, Niall; Pintzas, Alexander; Holmes, Fiona; Hobson, Sally-Ann; Pope, Robert; Wallace, Mark; Wasylyk, Christine; Wasylyk, Bohdan; Wynick, David

    2010-06-01

    ELK transcription factors are known to be expressed in a number of regions in the nervous system. We show by RT-PCR that the previously described Elk1, Elk3/Elk3b/Elk3c and Elk4 mRNAs are expressed in adult dorsal root ganglia (DRG), together with the novel alternatively spliced isoforms Elk1b, Elk3d and Elk4c/Elk4d/Elk4e. These isoforms are also expressed in brain, heart, kidney and testis. In contrast to Elk3 protein, the novel Elk3d isoform is cytoplasmic, fails to bind ETS binding sites and yet can activate transcription by an indirect mechanism. The Elk3 and Elk4 genes are overlapped by co-expressed Pctk2 (Cdk17) and Mfsd4 genes, respectively, with the potential formation of Elk3/Pctaire2 and Elk4/Mfsd4 sense-antisense mRNA heteroduplexes. After peripheral nerve injury the Elk3 mRNA isoforms are each upregulated approximately 2.3-fold in DRG (P<0.005), whereas the natural antisense Pctaire2 isoforms show only a small increase (21%, P<0.01) and Elk1 and Elk4 mRNAs are unchanged. PMID:20304071

  17. 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.

  18. 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.

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

    Science.gov (United States)

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

    2015-08-01

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

  20. Graphical models for genetic analyses

    DEFF Research Database (Denmark)

    Lauritzen, Steffen Lilholt; Sheehan, Nuala A.

    2003-01-01

    This paper introduces graphical models as a natural environment in which to formulate and solve problems in genetics and related areas. Particular emphasis is given to the relationships among various local computation algorithms which have been developed within the hitherto mostly separate areas...... of graphical models and genetics. The potential of graphical models is explored and illustrated through a number of example applications where the genetic element is substantial or dominating....

  1. Transposable elements: The enemies within.

    Science.gov (United States)

    Scarfò, Irene; Pellegrino, Elisa; Mereu, Elisabetta; Inghirami, Giorgio; Piva, Roberto

    2016-10-01

    Understanding transformation mechanisms other than genetic aberrations has recently captured the attention of cancer researchers. To date, the role of transposable elements (TEs) in tumor development remains largely undefined. However, an increasing number of studies have reported that loss of epigenetic control causes TE reactivation and consequent oncogenic transcription. Here, we discuss principal examples of TEs-driven oncogenesis. Available data suggest that long terminal repeats and long interspersed nuclear elements play a pivotal role as alternative promoters. These findings provide definitive experimental evidence that repetitive elements are a powerful underestimated force toward oncogenesis and open the possibility to new therapeutic treatments.

  2. PELTIER ELEMENTS

    CERN Document Server

    Tani, Laurits

    2015-01-01

    To control Peltier elements, temperature controller was used. I used TEC-1091 that was manufactured my Meerstetter Engineering. To gain control with the temperature controller, software had to be intalled on a controlling PC. There were different modes to control the Peltier: Tempererature controller to control temperature, Static current/voltage to control voltage and current and LIVE ON/OFF to auto-tune the controller respectively to the system. Also, since near the collision pipe there is much radiation, radiation-proof Peltier elements have to be used. To gain the best results, I had to find the most efficient Peltier elements and try to get their cold side to -40 degrees Celsius.

  3. Radiographic element

    International Nuclear Information System (INIS)

    Radiographic elements are disclosed having first and second silver halide emulsion layers comprised of a dispersing medium and radiation-sensitive silver halide grains, and a support interposed between said silver halide emulsion layers capable of transmitting radiation to which said second silver halide emulsion layer is responsive. These elements are characterized in that at least said first silver halide emulsion layer contains tabular silver halide grains and spectral sensitizing dye adsorbed to the surface of the grains. Crossover can be improved in relation to the imaging characteristics. (author)

  4. 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 to be a...

  5. 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.

  6. Antisense down-regulation of strawberry endo-beta (1,4)-glucanase genes does not prevent fruit softening during ripening

    NARCIS (Netherlands)

    Palomer, X.; Llop-Tous, I.; Vendrell, M.; Krens, F.A.; Schaart, J.G.; Boone, M.J.; Valk, van der H.C.P.M.; Salentijn, E.M.J.

    2006-01-01

    Strawberry (Fragaria × ananassa Duch.) fruit softening during ripening is associated with the overlapping presence of two divergent endo-ß-(1,4)-glucanases (EC 3.2.1.4; EGases), Cel1 and Cel2. Antisense down-regulation of both genes was performed to assess the precise role of these endo-ß-(1,4)-gluc

  7. 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

  8. 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

  9. 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.

  10. 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.

  11. Comparative study on imaging of 99Tcm-survivin mRNA antisense peptide nucleic acid in tumor and inflammation animal models

    International Nuclear Information System (INIS)

    Objective: To investigate the value of 99Tcm labeled survivin mRNA antisense peptide nucleic acid (PNA) as an imaging agent in the specific diagnosis for carcinoma. Methods: Survivin mRNA antisense PNA was labeled directly with 99Tcm by the ligand-exchange method. Twenty nude mice with lung carcinoma A549 xenografts were randomly divided into 4 groups. Three groups were used for biodistribution study and one group was used for imaging study. Other twenty mice infected by staphylococcus aureus underwent the same procedure. The biodistribution and imaging of 99Tcm-survivin mRNA antisense PNA was studied at 1, 2 and 4 h respectively after the intravenous injection in nude mice bearing lung carcinoma A549 xenografts or inflammation models. SPSS 13.0 was used in the study and all data were analyzed by t test. Results: Biodistribution results showed that the highest radioactivity was found in the liver,and then in the kidney. Four hours after the administration of the imaging agent, the radioactivity ratios of target-to-non target (T/NT, tumor or inflammatory lesions to the contralateral regions) in tumor model group were significantly higher than those in inflammation model group (3.69 ± 1.13 vs 2.03 ± 0.47, t=3.01, P=0.02). Tumors were clearly visible in the tumor model groups at 0.5 h and still clearly seen at 4 h after the injection of antisense PNA. On the contrary,inflammatory lesions could not be seen clearly. Conclusion: 99Tcm labeled survivin mRNA antisense PNA can be used to distinguish tumor from inflammation and it may provide a new feasible method for specific tumor diagnosis. (authors)

  12. On the structure of AP-4 responsive element in the LTR of Jembrana disease virus

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Previous studies with deletion and sequence analysis of JDV LTR showed that there is a putative AP-4 responsive element in LTR. By antisense transient assay and gel shifting assay, we for the first time demonstrated that AP-4 modulated JDV gene expression by binding DNA directly to bovine cells. The results, derived from site-directed mutagenesis experiments, suggest that the six base pairs of AP-4 binding site (CAGCTG) have different effects on JDV gene expression. When the first two base pairs changed to GC, JDV gene expression is severely decreased.

  13. Novel Cationic Carotenoid Lipids as Delivery Vectors of Antisense Oligonucleotides for Exon Skipping in Duchenne Muscular Dystrophy

    Directory of Open Access Journals (Sweden)

    Vassilia Partali

    2012-01-01

    Full Text Available Duchenne Muscular Dystrophy (DMD is a common, inherited, incurable, fatal muscle wasting disease caused by deletions that disrupt the reading frame of the DMD gene such that no functional dystrophin protein is produced. Antisense oligonucleotide (AO-directed exon skipping restores the reading frame of the DMD gene, and truncated, yet functional dystrophin protein is expressed. The aim of this study was to assess the efficiency of two novel rigid, cationic carotenoid lipids, C30-20 and C20-20, in the delivery of a phosphorodiamidate morpholino (PMO AO, specifically designed for the targeted skipping of exon 45 of DMD mRNA in normal human skeletal muscle primary cells (hSkMCs. The cationic carotenoid lipid/PMO-AO lipoplexes yielded significant exon 45 skipping relative to a known commercial lipid, 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (EPC.

  14. Formulation and drug-content assay of microencapsulated antisense oligonucleotide to NF-κB using ATR-FTIR

    International Nuclear Information System (INIS)

    Antisense oligonucleotide to NF-κB sequence: 5′-GGA AAC ACA TCC TCC ATG-3′, was microencapsulated in an albumin matrix by the method of spray dryingTM. Spectral analysis was performed on varying drug loading formulations of both drugs by mid-IR attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR). An out of plane O–H bending vibration at 948 cm−1, unique to both the native and microencapsulated drugs was identified. The calculated peak areas corresponded to the drug loadings in the microsphere formulations. A standard curve could then be used to determine the drug content of an unknown microsphere formulation. Accuracy and precision were determined to be comparable to other analytical techniques such as HPLC. (paper)

  15. 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.

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

    Institute of Scientific and Technical Information of China (English)

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

    2004-01-01

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

  17. 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.

  18. 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.

  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. Formulation and drug-content assay of microencapsulated antisense oligonucleotide to NF-κB using ATR-FTIR

    Science.gov (United States)

    Siwale, Rodney; Meadows, Fred; Mody, Vicky V.; Shah, Samit

    2013-09-01

    Antisense oligonucleotide to NF-κB sequence: 5‧-GGA AAC ACA TCC TCC ATG-3‧, was microencapsulated in an albumin matrix by the method of spray dryingTM. Spectral analysis was performed on varying drug loading formulations of both drugs by mid-IR attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR). An out of plane O-H bending vibration at 948 cm-1, unique to both the native and microencapsulated drugs was identified. The calculated peak areas corresponded to the drug loadings in the microsphere formulations. A standard curve could then be used to determine the drug content of an unknown microsphere formulation. Accuracy and precision were determined to be comparable to other analytical techniques such as HPLC.

  1. 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

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

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

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

  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. Genetic Disorders

    Science.gov (United States)

    ... This can cause a medical condition called a genetic disorder. You can inherit a gene mutation from ... during your lifetime. There are three types of genetic disorders: Single-gene disorders, where a mutation affects ...

  6. Radiographic element

    International Nuclear Information System (INIS)

    Radiographic elements are disclosed comprised of first and second silver halide emulsion layers separated by an interposed support capable of transmitting radiation to which the second image portion is responsive. At least the first imaging portion contains a silver halide emulsion in which thin tubular silver halide grains of intermediate aspect ratios (from 5:1 to 8:1) are present. Spectral sensitizing dye is adsorbed to the surface of the tubular grains. Increased photographic speeds can be realized at comparable levels of crossover. (author)

  7. Alu element-containing RNAs maintain nucleolar structure and function.

    Science.gov (United States)

    Caudron-Herger, Maïwen; Pankert, Teresa; Seiler, Jeanette; Németh, Attila; Voit, Renate; Grummt, Ingrid; Rippe, Karsten

    2015-11-12

    Non-coding RNAs play a key role in organizing the nucleus into functional subcompartments. By combining fluorescence microscopy and RNA deep-sequencing-based analysis, we found that RNA polymerase II transcripts originating from intronic Alu elements (aluRNAs) were enriched in the nucleolus. Antisense-oligo-mediated depletion of aluRNAs or drug-induced inhibition of RNA polymerase II activity disrupted nucleolar structure and impaired RNA polymerase I-dependent transcription of rRNA genes. In contrast, overexpression of a prototypic aluRNA sequence increased both nucleolus size and levels of pre-rRNA, suggesting a functional link between aluRNA, nucleolus integrity and pre-rRNA synthesis. Furthermore, we show that aluRNAs interact with nucleolin and target ectopic genomic loci to the nucleolus. Our study suggests an aluRNA-based mechanism that links RNA polymerase I and II activities and modulates nucleolar structure and rRNA production.

  8. Genetic Counseling

    Science.gov (United States)

    ... go for genetic counseling, such as: A family history of a genetic condition To learn about genetic screening for diseases that are more common in certain ethnic groups (e.g., sickle cell disease in African Americans and Tay-Sachs disease in Ashkenazi Jews) To discuss abnormal results ...

  9. Genetic principles.

    Science.gov (United States)

    Abuelo, D

    1987-01-01

    The author discusses the basic principles of genetics, including the classification of genetic disorders and a consideration of the rules and mechanisms of inheritance. The most common pitfalls in clinical genetic diagnosis are described, with emphasis on the problem of the negative or misleading family history.

  10. Imaging Genetics

    Science.gov (United States)

    Munoz, Karen E.; Hyde, Luke W.; Hariri, Ahmad R.

    2009-01-01

    Imaging genetics is an experimental strategy that integrates molecular genetics and neuroimaging technology to examine biological mechanisms that mediate differences in behavior and the risks for psychiatric disorder. The basic principles in imaging genetics and the development of the field are discussed.

  11. Superheavy elements

    CERN Document Server

    Hofmann, S

    1999-01-01

    The outstanding aim of experimental investigations of heavy nuclei is the exploration of spherical 'SuperHeavy Elements' (SHEs). On the basis of the nuclear shell model, the next double magic shell-closure beyond sup 2 sup 0 sup 8 Pb is predicted at proton numbers between Z=114 and 126 and at neutron number N=184. All experimental efforts aiming at identifying SHEs (Z>=114) were negative so far. A highly sensitive search experiment was performed in November-December 1995 at SHIP. The isotope sup 2 sup 9 sup 0 116 produced by 'radiative capture' was searched for in the course of a 33 days irradiation of a sup 2 sup 0 sup 8 Pb target with sup 8 sup 2 Se projectiles, however, only cross-section limits were measured. Positive results were obtained in experiments searching for elements from 110 to 112 using cold fusion and the 1n evaporation channel. The produced isotopes were unambiguously identified by means of alpha-alpha correlations. Not fission, but alpha emission is the dominant decay mode. The measurement ...

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

    Science.gov (United States)

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

    1997-12-01

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

  13. Genetics Home Reference: Genetic Conditions

    Science.gov (United States)

    Skip to main content Your Guide to Understanding Genetic Conditions Enable Javascript for addthis links to activate. ... Conditions Genes Chromosomes & mtDNA Resources Help Me Understand Genetics Home Health Conditions Health Conditions Explore the signs ...

  14. Darwin's contributions to genetics.

    Science.gov (United States)

    Liu, Y-S; Zhou, X-M; Zhi, M-X; Li, X-J; Wang, Q-L

    2009-01-01

    Darwin's contributions to evolutionary biology are well known, but his contributions to genetics are much less known. His main contribution was the collection of a tremendous amount of genetic data, and an attempt to provide a theoretical framework for its interpretation. Darwin clearly described almost all genetic phenomena of fundamental importance, such as prepotency (Mendelian inheritance), bud variation (mutation), heterosis, reversion (atavism), graft hybridization (Michurinian inheritance), sex-limited inheritance, the direct action of the male element on the female (xenia and telegony), the effect of use and disuse, the inheritance of acquired characters (Lamarckian inheritance), and many other observations pertaining to variation, heredity and development. To explain all these observations, Darwin formulated a developmental theory of heredity - Pangenesis - which not only greatly influenced many subsequent theories, but also is supported by recent evidence. PMID:19638672

  15. 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.

  16. 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

    -life as longer oligonucleotides. Pharmacology studies in both mice and non-human primates were conducted with a 13-mer LNA oligonucleotide against apoB, and the data showed that repeated dosing of the 13-mer at 1-2 mg/kg/week was sufficient to provide a significant and long lasting lowering of non-high-density......The potency and specificity of locked nucleic acid (LNA) antisense oligonucleotides was investigated as a function of length and affinity. The oligonucleotides were designed to target apolipoprotein B (apoB) and were investigated both in vitro and in vivo. The high affinity of LNA enabled...... 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...

  17. 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.

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

    OpenAIRE

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

    2016-01-01

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

  19. 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...

  20. Cationic polyelectrolyte-mediated delivery of antisense morpholino oligonucleotides for exon-skipping in vitro and in mdx mice

    OpenAIRE

    Wang, Mingxing

    2015-01-01

    Mingxing Wang, Bo Wu, Jason D Tucker, Peijuan Lu, Qilong Lu Department of Neurology, McColl-Lockwood Laboratory for Muscular Dystrophy Research, Cannon Research Center, Carolinas Medical Center, Charlotte, NC, USA Abstract: In this study, we investigated a series of cationic polyelectrolytes (PEs) with different size and composition for their potential to improve delivery of an antisense phosphorodiamidate morpholino oligomer (PMO) both in vitro and in vivo. The results showed that...

  1. Cationic polyelectrolyte-mediated delivery of antisense morpholino oligonucleotides for exon-skipping in vitro and in mdx mice

    OpenAIRE

    Wang M; Wu B; Tucker JD; Lu P; Lu Q

    2015-01-01

    Mingxing Wang, Bo Wu, Jason D Tucker, Peijuan Lu, Qilong Lu Department of Neurology, McColl-Lockwood Laboratory for Muscular Dystrophy Research, Cannon Research Center, Carolinas Medical Center, Charlotte, NC, USA Abstract: In this study, we investigated a series of cationic polyelectrolytes (PEs) with different size and composition for their potential to improve delivery of an antisense phosphorodiamidate morpholino oligomer (PMO) both in vitro and in vivo. The results showed that the...

  2. Gene-Specific Effects of Antisense Phosphorodiamidate Morpholino Oligomer-Peptide Conjugates on Escherichia coli and Salmonella enterica Serovar Typhimurium in Pure Culture and in Tissue Culture

    OpenAIRE

    Lucas D Tilley; Hine, Orion S.; Kellogg, Jill A.; Hassinger, Jed N.; Weller, Dwight D.; Iversen, Patrick L.; Geller, Bruce L.

    2006-01-01

    The objective was to improve efficacy of antisense phosphorodiamidate morpholino oligomers (PMOs) by improving their uptake into bacterial cells. Four different bacterium-permeating peptides, RFFRFFRFFXB, RTRTRFLRRTXB, RXXRXXRXXB, and KFFKFFKFFKXB (X is 6-aminohexanoic acid and B is β-alanine), were separately coupled to two different PMOs that are complementary to regions near the start codons of a luciferase reporter gene (luc) and a gene required for viability (acpP). Luc peptide-PMOs targ...

  3. 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.

  4. 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.

  5. Genetic barcodes

    Science.gov (United States)

    Weier, Heinz -Ulrich G

    2015-08-04

    Herein are described multicolor FISH probe sets termed "genetic barcodes" targeting several cancer or disease-related loci to assess gene rearrangements and copy number changes in tumor cells. Two, three or more different fluorophores are used to detect the genetic barcode sections thus permitting unique labeling and multilocus analysis in individual cell nuclei. Gene specific barcodes can be generated and combined to provide both numerical and structural genetic information for these and other pertinent disease associated genes.

  6. 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

  7. 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.

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

    Institute of Scientific and Technical Information of China (English)

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

    2001-01-01

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

  9. 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.

  10. 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

  11. 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.

  12. Cationic polyelectrolyte-mediated delivery of antisense morpholino oligonucleotides for exon-skipping in vitro and in mdx mice

    Directory of Open Access Journals (Sweden)

    Wang M

    2015-09-01

    Full Text Available Mingxing Wang, Bo Wu, Jason D Tucker, Peijuan Lu, Qilong Lu Department of Neurology, McColl-Lockwood Laboratory for Muscular Dystrophy Research, Cannon Research Center, Carolinas Medical Center, Charlotte, NC, USA Abstract: In this study, we investigated a series of cationic polyelectrolytes (PEs with different size and composition for their potential to improve delivery of an antisense phosphorodiamidate morpholino oligomer (PMO both in vitro and in vivo. The results showed that the poly(diallyldimethylammonium chloride (PDDAC polymer series, especially PE-3 and PE-4, improves the delivery efficiency of PMO, comparable with Endoporter-mediated PMO delivery in vitro. The enhanced PMO delivery and targeting to dystrophin exon 23 was further observed in mdx mice, up to fourfold with the PE-4, compared with PMO alone. The cytotoxicity of the PEs was lower than that of Endoporter and polyethylenimine 25,000 Da in vitro, and was not clearly detected in muscle in vivo under the tested concentrations. Together, these results demonstrate that optimization of PE molecular size, composition, and distribution of cationic charge are key factors to achieve enhanced PMO exon-skipping efficiency. The increased efficiency and lower toxicity show this PDDAC series to be capable gene/antisense oligonucleotide delivery-enhancing agents for treating muscular dystrophy and other diseases. Keywords: cationic polyelectrolytes, antisense delivery, exon-skipping, PMO, muscular dystrophy

  13. 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.

  14. Moving into a new era of periodontal genetic studies: relevance of large case-control samples using severe phenotypes for genome-wide association studies.

    Science.gov (United States)

    Vaithilingam, R D; Safii, S H; Baharuddin, N A; Ng, C C; Cheong, S C; Bartold, P M; Schaefer, A S; Loos, B G

    2014-12-01

    Studies to elucidate the role of genetics as a risk factor for periodontal disease have gone through various phases. In the majority of cases, the initial 'hypothesis-dependent' candidate-gene polymorphism studies did not report valid genetic risk loci. Following a large-scale replication study, these initially positive results are believed to be caused by type 1 errors. However, susceptibility genes, such as CDKN2BAS (Cyclin Dependend KiNase 2B AntiSense RNA; alias ANRIL [ANtisense Rna In the Ink locus]), glycosyltransferase 6 domain containing 1 (GLT6D1) and cyclooxygenase 2 (COX2), have been reported as conclusive risk loci of periodontitis. The search for genetic risk factors accelerated with the advent of 'hypothesis-free' genome-wide association studies (GWAS). However, despite many different GWAS being performed for almost all human diseases, only three GWAS on periodontitis have been published - one reported genome-wide association of GLT6D1 with aggressive periodontitis (a severe phenotype of periodontitis), whereas the remaining two, which were performed on patients with chronic periodontitis, were not able to find significant associations. This review discusses the problems faced and the lessons learned from the search for genetic risk variants of periodontitis. Current and future strategies for identifying genetic variance in periodontitis, and the importance of planning a well-designed genetic study with large and sufficiently powered case-control samples of severe phenotypes, are also discussed. PMID:24528298

  15. Genetic modification and genetic determinism

    Directory of Open Access Journals (Sweden)

    Vorhaus Daniel B

    2006-06-01

    Full Text Available Abstract In this article we examine four objections to the genetic modification of human beings: the freedom argument, the giftedness argument, the authenticity argument, and the uniqueness argument. We then demonstrate that each of these arguments against genetic modification assumes a strong version of genetic determinism. Since these strong deterministic assumptions are false, the arguments against genetic modification, which assume and depend upon these assumptions, are therefore unsound. Serious discussion of the morality of genetic modification, and the development of sound science policy, should be driven by arguments that address the actual consequences of genetic modification for individuals and society, not by ones propped up by false or misleading biological assumptions.

  16. Alu elements in ANRIL non-coding RNA at chromosome 9p21 modulate atherogenic cell functions through trans-regulation of gene networks.

    Directory of Open Access Journals (Sweden)

    Lesca M Holdt

    Full Text Available The chromosome 9p21 (Chr9p21 locus of coronary artery disease has been identified in the first surge of genome-wide association and is the strongest genetic factor of atherosclerosis known today. Chr9p21 encodes the long non-coding RNA (ncRNA antisense non-coding RNA in the INK4 locus (ANRIL. ANRIL expression is associated with the Chr9p21 genotype and correlated with atherosclerosis severity. Here, we report on the molecular mechanisms through which ANRIL regulates target-genes in trans, leading to increased cell proliferation, increased cell adhesion and decreased apoptosis, which are all essential mechanisms of atherogenesis. Importantly, trans-regulation was dependent on Alu motifs, which marked the promoters of ANRIL target genes and were mirrored in ANRIL RNA transcripts. ANRIL bound Polycomb group proteins that were highly enriched in the proximity of Alu motifs across the genome and were recruited to promoters of target genes upon ANRIL over-expression. The functional relevance of Alu motifs in ANRIL was confirmed by deletion and mutagenesis, reversing trans-regulation and atherogenic cell functions. ANRIL-regulated networks were confirmed in 2280 individuals with and without coronary artery disease and functionally validated in primary cells from patients carrying the Chr9p21 risk allele. Our study provides a molecular mechanism for pro-atherogenic effects of ANRIL at Chr9p21 and suggests a novel role for Alu elements in epigenetic gene regulation by long ncRNAs.

  17. 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

  18. 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.

  19. Genetic Engineering

    Science.gov (United States)

    Phillips, John

    1973-01-01

    Presents a review of genetic engineering, in which the genotypes of plants and animals (including human genotypes) may be manipulated for the benefit of the human species. Discusses associated problems and solutions and provides an extensive bibliography of literature relating to genetic engineering. (JR)

  20. 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.

  1. A Polyethylenimine-Containing and Transferrin-Conjugated Lipid Nanoparticle System for Antisense Oligonucleotide Delivery to AML

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    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.

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

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    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.

  3. 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.

  4. 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.

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

    Institute of Scientific and Technical Information of China (English)

    LIU Ge-xiu

    2005-01-01

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

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

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    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.

  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. Staphylococcus aureus RNAIII coordinately represses the synthesis of virulence factors and the transcription regulator Rot by an antisense mechanism

    Science.gov (United States)

    Boisset, Sandrine; Geissmann, Thomas; Huntzinger, Eric; Fechter, Pierre; Bendridi, Nadia; Possedko, Maria; Chevalier, Clément; Helfer, Anne Catherine; Benito, Yvonne; Jacquier, Alain; Gaspin, Christine; Vandenesch, François; Romby, Pascale

    2007-01-01

    RNAIII is the intracellular effector of the quorum-sensing system in Staphylococcus aureus. It is one of the largest regulatory RNAs (514 nucleotides long) that are known to control the expression of a large number of virulence genes. Here, we show that the 3′ domain of RNAIII coordinately represses at the post-transcriptional level, the expression of mRNAs that encode a class of virulence factors that act early in the infection process. We demonstrate that the 3′ domain acts primarily as an antisense RNA and rapidly anneals to these mRNAs, forming long RNA duplexes. The interaction between RNAIII and the mRNAs results in repression of translation initiation and triggers endoribonuclease III hydrolysis. These processes are followed by rapid depletion of the mRNA pool. In addition, we show that RNAIII and its 3′ domain mediate translational repression of rot mRNA through a limited number of base pairings involving two loop–loop interactions. Since Rot is a transcriptional regulatory protein, we proposed that RNAIII indirectly acts on many downstream genes, resulting in the activation of the synthesis of several exoproteins. These data emphasize the multitude of regulatory steps affected by RNAIII and its 3′ domain in establishing a network of S. aureus virulence factors. PMID:17545468

  9. 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.

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

    Institute of Scientific and Technical Information of China (English)

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

    2003-01-01

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

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

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    Zheng Xuelian

    2008-10-01

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

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

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    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. In Vitro and In Vivo Enhancement of Antitumoral Activity of Liposomal Antisense Oligonucleotides by Cineole as a Chemical Penetration Enhancer

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    Hamid Reza Moghimi

    2015-01-01

    Full Text Available Cellular uptake and cytoplasmic release of liposomal antisense oligonucleotides (AsODNs, which can act as rate-limiting steps, are still remained to be completely optimized. Here, the possibility of enhancing such processes at cellular and animal levels by cineole, as a penetration enhancer, was investigated. A cationic nanoliposome containing an AsODN against PKC-α and a cineole-containing nanoliposome were prepared and characterized. The effect of nanoliposomal cineole on sequence-specific cytotoxicity of nanoliposomal AsODN against A549, was studied in vitro (MTT, flow cytometry, fluorescence microscopy, and real time PCR and in vivo (xenograft lung tumor in nude mice using different concentrations and treatment times. Results showed specific cytotoxicity of nanoliposomal AsODN was increased significantly from 11% to 25% when A549 cells were exposed to 10 µg/mL cineole for 1 or 4 hours. This inhibitory effect was further increased to about 40% when the concentration was increased to 40 µg/mL for 1 hour. In animal studies, cineole significantly decreased the tumor volume (about 75% and increased its doubling time from 13 days to 31 days. A linear relationship exists between cineole concentration and its enhancement effects. Finally it was concluded that cineole, and possibly other membrane fluidizers, can improve nanoliposomal gene therapy at cellular and animal levels.

  14. Histopathological Defects in Intestine in Severe Spinal Muscular Atrophy Mice Are Improved by Systemic Antisense Oligonucleotide Treatment.

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    Palittiya Sintusek

    Full Text Available Gastrointestinal (GI defects, including gastroesophageal reflux, constipation and delayed gastric emptying, are common in patients with spinal muscular atrophy (SMA. Similar GI dysmotility has been identified in mouse models with survival of motor neuron (SMN protein deficiency. We previously described vascular defects in skeletal muscle and spinal cord of SMA mice and we hypothesized that similar defects could be involved in the GI pathology observed in these mice. We therefore investigated the gross anatomical structure, enteric vasculature and neurons in the small intestine in a severe mouse model of SMA. We also assessed the therapeutic response of GI histopathology to systemic administration of morpholino antisense oligonucleotide (AON designed to increase SMN protein expression. Significant anatomical and histopathological abnormalities, with striking reduction of vascular density, overabundance of enteric neurons and increased macrophage infiltration, were detected in the small intestine in SMA mice. After systemic AON treatment in neonatal mice, all the abnormalities observed were significantly restored to near-normal levels. We conclude that the observed GI histopathological phenotypes and functional defects observed in these SMA mice are strongly linked to SMN deficiency which can be rescued by systemic administration of AON. This study on the histopathological changes in the gastrointestinal system in severe SMA mice provides further indication of the complex role that SMN plays in multiple tissues and suggests that at least in SMA mice restoration of SMN production in peripheral tissues is essential for optimal outcome.

  15. 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.

  16. 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

    ., Chromosomal analysis of Nicotiana asymmetric somatic hybrids by dot blotting and in situ hybridization, Mol .Gen. Genet., 1990, 222: 97-103.[12]Buiteveld, J., Suo, Y., van Lookeren Campagne, M. M. et al., Production and characterization of somatic hybrid plants between leek (Allium ampeloprasum L.) and onion (Allium cepa L.), Theor. Appl. Genet., 1998, 96: 765-775.[13]Wolters, A. M. A., Schoenmakers, H. C. H., Kamstra, S. et al., Mitotic and meiotic irregularities in somatic hybrids of Ly-copersicon esculentum and Solanum tuberosum, Genome, 1994, 37: 726-735.[14]Shieder, O., Somatic hybrids or Datura irnoxia Mill + Datura discolor Bernn and of Datiura irnoxia Mill + Datura stra-monium L. var tatula L (I)-- Selection and characterization, Mol. Gen. Genet., 1978, 162: 113-119.[15]Handley, L. W., Nickels, R. L., Cameron, M. W. et al., Somatic hybrid plants between Lycopersicon esculentum and Sola-num lycopersicoides, Theor. Appl. Genet., 1986, 71: 691-697.[16]Preiszner, J., Feher, A., Veisz, O. et al., Characterization of morphological variation and cold resistance in interspecific somatic hybrids between potato (Solanum tuberosum L.) and S. brevidens Phil., Euphytica, 1991, 57: 37-49.[17]Polgar, Z. S., Preiszner, J., Dudits, D. et al., Vigorous growth of fusion products allows highly efficient selection of inter-specific potato somatic hybrids: molecular proofs, Plant Cell Reports, 1993, 12: 399-402.[18]Xia, G. M., Wang, H., Chen, H. M., Plant regeneration from intergeneric asymmetric somatic hybridization between wheat (Triticum aestivum L.) and Russian wildrye (Psathyrostichs juncea (Fisch) Neveski) and couch grass (Agropyron elonga-tum Host Neviski), Chin. Sci. Bull., 1996, 41(15): 1423-1426.

  17. Genetic therapy for the nervous system.

    Science.gov (United States)

    Bowers, William J; Breakefield, Xandra O; Sena-Esteves, Miguel

    2011-04-15

    Genetic therapy is undergoing a renaissance with expansion of viral and synthetic vectors, use of oligonucleotides (RNA and DNA) and sequence-targeted regulatory molecules, as well as genetically modified cells, including induced pluripotent stem cells from the patients themselves. Several clinical trials for neurologic syndromes appear quite promising. This review covers genetic strategies to ameliorate neurologic syndromes of different etiologies, including lysosomal storage diseases, Alzheimer's disease and other amyloidopathies, Parkinson's disease, spinal muscular atrophy, amyotrophic lateral sclerosis and brain tumors. This field has been propelled by genetic technologies, including identifying disease genes and disruptive mutations, design of genomic interacting elements to regulate transcription and splicing of specific precursor mRNAs and use of novel non-coding regulatory RNAs. These versatile new tools for manipulation of genetic elements provide the ability to tailor the mode of genetic intervention to specific aspects of a disease state.

  18. An improved optimal elemental method for updating finite element models

    Institute of Scientific and Technical Information of China (English)

    Duan Zhongdong(段忠东); Spencer B.F.; Yan Guirong(闫桂荣); Ou Jinping(欧进萍)

    2004-01-01

    The optimal matrix method and optimal elemental method used to update finite element models may not provide accurate results. This situation occurs when the test modal model is incomplete, as is often the case in practice. An improved optimal elemental method is presented that defines a new objective function, and as a byproduct, circumvents the need for mass normalized modal shapes, which are also not readily available in practice. To solve the group of nonlinear equations created by the improved optimal method, the Lagrange multiplier method and Matlab function fmincon are employed. To deal with actual complex structures,the float-encoding genetic algorithm (FGA) is introduced to enhance the capability of the improved method. Two examples, a 7-degree of freedom (DOF) mass-spring system and a 53-DOF planar frame, respectively, are updated using the improved method.Thc example results demonstrate the advantages of the improved method over existing optimal methods, and show that the genetic algorithm is an effective way to update the models used for actual complex structures.

  19. 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

  20. Genetic Breakthrough

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    A new calf breeding technique shows promise for treating malignant tumors Chinese scientists have successfully bred a genetically altered cow capable of producing cancer-curing proteins for human beings.