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Sample records for inducible antisense expression

  1. Antisense imaging of epidermal growth factor-induced p21{sup WAF-1/CIP-1} gene expression in MDA-MB-468 human breast cancer xenografts

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Judy; Chen, Paul; Mrkobrada, Marko [Leslie Dan Faculty of Pharmacy, University of Toronto, 19 Russell Street, M5S 2S2, Toronto, Ontario (Canada); Hu, Meiduo [Leslie Dan Faculty of Pharmacy, University of Toronto, 19 Russell Street, M5S 2S2, Toronto, Ontario (Canada); Department of Pharmaceutical Sciences, University of Toronto, Toronto, Ontario (Canada); Vallis, Katherine A. [Department of Radiation Oncology, Princess Margaret Hospital, University Health Network, 610 University Avenue, Toronto, Ontario (Canada); Department of Radiation Oncology, University of Toronto, Toronto, Ontario (Canada); Department of Medical Biophysics, University of Toronto, Toronto, Ontario (Canada); Reilly, Raymond M. [Department of Medical Imaging, University of Toronto, Toronto, Ontario (Canada)

    2003-09-01

    Molecular imaging of the expression of key genes which determine the response to DNA damage following cancer treatment may predict the effectiveness of a particular treatment strategy. A prominent early response gene for DNA damage is the gene encoding p21{sup WAF-1/CIP-1}, a cyclin-dependent kinase inhibitor that regulates progression through the cell cycle. In this study, we explored the feasibility of imaging p21{sup WAF-1/CIP-1} gene expression at the mRNA level using an 18-mer phosphorothioated antisense oligodeoxynucleotide (ODN) labeled with {sup 111}In. The known induction of the p21{sup WAF-1/CIP-1} gene in MDA-MB-468 human breast cancer cells following exposure to epidermal growth factor (EGF) was used as an experimental tool. Treatment of MDA-MB-468 cells in vitro with EGF (20 nM) increased the ratio of p21{sup WAF-1/CIP-1} mRNA/{beta}-actin mRNA threefold within 2 h as measured by the reverse transcription polymerase chain reaction (RT-PCR). A concentration-dependent inhibition of EGF-induced p21{sup WAF-1/CIP-1} protein expression was achieved in MDA-MB-468 cells by treatment with antisense ODNs with up to a tenfold decrease observed at 1 {mu}M. There was a fourfold lower inhibition of p21{sup WAF-1/CIP-1} protein expression by control sense or random sequence ODNs. Intratumoral injections of EGF (15 {mu}g/day x 3 days) were employed to induce p21{sup WAF-1/CIP-1} gene expression in MDA-MB-468 xenografts implanted subcutaneously into athymic mice. RT-PCR of explanted tumors showed a threefold increased level of p21{sup WAF-1/CIP-1} mRNA compared with normal saline-treated tumors. Successful imaging of EGF-induced p21{sup WAF-1/CIP-1} gene expression in MDA-MB-468 xenografts was achieved at 48 h post injection of {sup 111}In-labeled antisense ODNs (3.7 MBq; 2 {mu}g). Tumors displaying basal levels of p21{sup WAF-1/CIP-1} gene expression in the absence of EGF treatment could not be visualized. Biodistribution studies showed a significantly higher tumor

  2. Alteration of rice growth and development via antisense expression ...

    African Journals Online (AJOL)

    user

    OsGA20ox2 in regulating plant growth and development, we used reverse genomic approach to ... pathways. Similarly, Carmen et al. (2007) suggested that. Carrizo citrange plants have produced antisense ... universal SP6 and T7 primers to conform their reality (Sangon, ..... Optimising the tissue culture conditions for.

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

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    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.

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

    International Nuclear Information System (INIS)

    Porat, S.; Gabbay, M.; Tauber, M.; Ratovitski, T.; Blinder, E.; Simantov, R.

    1996-01-01

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

  5. Simultaneous Expression from Both the Sense and Antisense Strand of the Erythropoietin Receptor Gene Mitigates Acute Lung Injury

    Science.gov (United States)

    2017-09-01

    concept efficacy that increasing EpoR or RopE expression by cDNA delivery to lung cells in vitro enhances cytoprotection against hyperoxia-induced injury...oxidative damage, cell culture, rodent model, inhalation cDNA delivery, sense and antisense erythropoietin receptor transcripts 16. SECURITY...prevention of acute lung injury. 1-6 50% Subtask 1: Prepare plasmid cDNA of EpoR and RopE in nanoparticle formulation. 1 Completed 06.2017 Subtask 2

  6. G3139, a Bcl-2 antisense oligodeoxynucleotide, induces clinical responses in VAD refractory myeloma

    NARCIS (Netherlands)

    van de Donk, N. W. C. J.; de Weerdt, O.; Veth, G.; Eurelings, M.; van Stralen, E.; Frankel, S. R.; Hagenbeek, A.; Bloem, A. C.; Lokhorst, H. M.

    2004-01-01

    Expression of Bcl-2 in multiple myeloma is associated with resistance to chemotherapeutic drugs. Conversely, suppression of Bcl-2 enhanced the chemosensitivity of myeloma cells in vitro. G3139 is an antisense oligodeoxynucleotide targeted to the first six codons of the Bcl-2 mRNA open reading frame.

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

    Science.gov (United States)

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

    2013-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Xing Xian Yu

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

  9. 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.

  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

    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. Antisense oligonucleotides suppress cell-volume-induced activation of chloride channels.

    Science.gov (United States)

    Gschwentner, M; Nagl, U O; Wöll, E; Schmarda, A; Ritter, M; Paulmichl, M

    1995-08-01

    Cell volume regulation is an essential feature of most cells. After swelling in hypotonic media, the simultaneous activation of potassium and chloride channels is believed to be the initial, time-determining step in cell volume regulation. The activation of both pathways is functionally linked and enables the cells to lose ions and water, subsequently leading to cell shrinkage and readjustment of the initial volume. NIH 3T3 fibroblasts efficiently regulate their volume after swelling and bear chloride channels that are activated by decreasing extracellular osmolarity. The chloride current elicited in these cells after swelling is reminiscent of the current found in oocytes expressing an outwardly rectifying chloride current termed ICln. Introduction of antisense oligodeoxynucleotides complementary to the first 30 nucleotides of the coding region of the ICln channel into NIH 3T3 fibroblasts suppresses the activation of the swelling-induced chloride current. The experiments directly demonstrate an unambiguous link between a volume-activated chloride current and a cloned protein involved in chloride transport.

  12. Antisense-induced suppression of taxoid 14β- hydroxylase gene ...

    African Journals Online (AJOL)

    Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that the 14OH mRNA level in transgenic cells dropped dramatically, suggesting that the expression of endogenous14OH gene was significantly suppressed by the exogenous as14OH gene. Correspondingly, the total yield of three major C-14 ...

  13. Multilevel Regulation of Bacterial Gene Expression with the Combined STAR and Antisense RNA System.

    Science.gov (United States)

    Lee, Young Je; Kim, Soo-Jung; Moon, Tae Seok

    2018-03-16

    Synthetic small RNA regulators have emerged as a versatile tool to predictably control bacterial gene expression. Owing to their simple design principles, small size, and highly orthogonal behavior, these engineered genetic parts have been incorporated into genetic circuits. However, efforts to achieve more sophisticated cellular functions using RNA regulators have been hindered by our limited ability to integrate different RNA regulators into complex circuits. Here, we present a combined RNA regulatory system in Escherichia coli that uses small transcription activating RNA (STAR) and antisense RNA (asRNA) to activate or deactivate target gene expression in a programmable manner. Specifically, we demonstrated that the activated target output by the STAR system can be deactivated by expressing two different types of asRNAs: one binds to and sequesters the STAR regulator, affecting the transcription process, while the other binds to the target mRNA, affecting the translation process. We improved deactivation efficiencies (up to 96%) by optimizing each type of asRNA and then integrating the two optimized asRNAs into a single circuit. Furthermore, we demonstrated that the combined STAR and asRNA system can control gene expression in a reversible way and can regulate expression of a gene in the genome. Lastly, we constructed and simultaneously tested two A AND NOT B logic gates in the same cell to show sophisticated multigene regulation by the combined system. Our approach establishes a methodology for integrating multiple RNA regulators to rationally control multiple genes.

  14. The endogenous retroviral insertion in the human complement C4 gene modulates the expression of homologous genes by antisense inhibition.

    Science.gov (United States)

    Schneider, P M; Witzel-Schlömp, K; Rittner, C; Zhang, L

    2001-02-01

    Intron 9 contains the complete endogenous retrovirus HERV-K(C4) as a 6.4-kb insertion in 60% of human C4 genes. The retroviral insertion is in reverse orientation to the C4 coding sequence. Therefore, expression of C4 could lead to the transcription of an antisense RNA, which might protect against exogenous retroviral infections. To test this hypothesis, open reading frames from the HERV sequence were subcloned in sense orientiation into a vector allowing expression of a beta-galactosidase fusion protein. Mouse L cells which had been stably transfected with either the human C4A or C4B gene both carrying the HERV insertion (LC4 cells), and L(Tk-) cells without the C4 gene were transiently transfected either with a retroviral construct or with the wild-type vector. Expression was monitored using an enzymatic assay. We demonstrated that (1) HERV-K(C4) antisense mRNA transcripts are present in cells constitutively expressing C4, (2) expression of retroviral-like constructs is significantly downregulated in cells expressing C4, and (3) this downregulation is further modulated in a dose-dependent fashion following interferon-gamma stimulation of C4 expression. These results support the hypothesis of a genomic antisense strategy mediated by the HERV-K(C4) insertion as a possible defense mechanism against exogenous retroviral infections.

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

    International Nuclear Information System (INIS)

    Lu, Zheng; Ping, Liang; JianBo, Zhou; XiaoBing, Huang; Yu, Wen; Zheng, Wang; Jing, Li

    2012-01-01

    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 HepG 2 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 HepG 2 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 HepG 2 cells and inhibited their proliferation in vitro (P < 0.01). More cells were arrested by the antisense vector at the G 1 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

  16. Antisense-induced exon skipping for duplications in Duchenne muscular dystrophy

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    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.

  17. Sense and antisense transcripts of the developmentally regulated murine hsp70.2 gene are expressed in distinct and only partially overlapping areas in the adult brain

    Science.gov (United States)

    Murashov, A. K.; Wolgemuth, D. J.

    1996-01-01

    We have examined the spatial pattern of expression of a member of the hsp70 gene family, hsp70.2, in the mouse central nervous system. Surprisingly, RNA blot analysis and in situ hybridization revealed abundant expression of an 'antisense' hsp70.2 transcript in several areas of adult mouse brain. Two different transcripts recognized by sense and antisense riboprobes for the hsp70.2 gene were expressed in distinct and only partially overlapping neuronal populations. RNA blot analysis revealed low levels of the 2.7 kb transcript of hsp70.2 in several areas of the brain, with highest signal in the hippocampus. Abundant expression of a slightly larger (approximately 2.8 kb) 'antisense' transcript was detected in several brain regions, notably in the brainstem, cerebellum, mesencephalic tectum, thalamus, cortex, and hippocampus. In situ hybridization revealed that the sense and antisense transcripts were both predominantly neuronal and localized to the same cell types in the granular layer of the cerebellum, trapezoid nucleus of the superior olivary complex, locus coeruleus and hippocampus. The hsp70.2 antisense transcripts were particularly abundant in the frontal cortex, dentate gyrus, subthalamic nucleus, zona incerta, superior and inferior colliculi, central gray, brainstem, and cerebellar Purkinje cells. Our findings have revealed a distinct cellular and spatial localization of both sense and antisense transcripts, demonstrating a new level of complexity in the function of the heat shock genes.

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

    Directory of Open Access Journals (Sweden)

    Lin KY

    2016-05-01

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

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

    Directory of Open Access Journals (Sweden)

    Yasunari Yamanaka

    2015-05-01

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

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

    Directory of Open Access Journals (Sweden)

    Casey R Richardson

    2010-05-01

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

  1. Anti-sense suppression of epidermal growth factor receptor expression alters cellular proliferation, cell-adhesion and tumorigenicity in ovarian cancer cells.

    Science.gov (United States)

    Alper, O; De Santis, M L; Stromberg, K; Hacker, N F; Cho-Chung, Y S; Salomon, D S

    2000-11-15

    Over-expression of epidermal growth factor receptor (EGFR) in ovarian cancer has been well documented. Human NIH:OVCAR-8 ovarian carcinoma cells were transfected with an expression vector containing the anti-sense orientation of truncated human EGFR cDNA. EGFR anti-sense over-expression resulted in decreased EGFR protein and mRNA expression, cell proliferation and tumor formation in nude mice. In accordance with the reduced levels of EGFR in EGFR anti-sense-expressing cells, tyrosine phosphorylation of EGFR was decreased compared to untransfected parental cells treated with EGF. In EGFR anti-sense-transfected cells, expression of erbB-3, but not erbB-2, was increased. In addition, basal and heregulin-beta 1-stimulated tyrosine phosphorylation of erbB-3 was higher in EGFR anti-sense vector-transfected cells. A morphological alteration in EGFR anti-sense gene-expressing cells was correlated with a decrease in the expression of E-cadherin, alpha-catenin and, to a lesser extent, beta-catenin. Changes in the expression of these proteins were associated with a reduction in complex formation among E-cadherin, beta-catenin and alpha-catenin and between beta-catenin and EGFR in EGFR anti-sense-expressing cells compared to sense-transfected control cells. These results demonstrate that EGFR expression in ovarian carcinoma cells regulates expression of cell adhesion proteins that may enhance cell growth and invasiveness. Copyright 2000 Wiley-Liss, Inc.

  2. Antisense expression of a gene encoding a calcium-binding protein ...

    Indian Academy of Sciences (India)

    PRAKASH

    using the transgenic approach. The transformation of ... methods using EhCaBP or AtCaM3 gene-specific primers in ... acetone) was added, mixed and incubated for 15–18 h in the dark at .... as expected from the design of the AtCaM3 antisense construct .... Thus, there seems to be a positive qualitative correlation between ...

  3. Expression of c-Fos and c-Jun in the cornea, lens, and retina after ultraviolet irradiation of the rat eye and effect of topical antisense oligodeoxynucleotides

    International Nuclear Information System (INIS)

    Gillardon, F.; Zimmermann, M.

    1995-01-01

    Aims - Immunohistochemical techniques were used to investigate c-Fos and c-Jun proto-oncogene expression in the cornea, lens, and retina after ultraviolet irradiation of the rat eye. Methods -Eyes of anaesthetised rats were exposed to 1.5 J/cm 2 of ultraviolet radiation (280-380 nm). Animals were perfused 1, 6, or 24 hours after irradiation and tissue sections were incubated with specific antiserum to c-Fos and c-Jun, respectively. Non-irradiated contralateral eyes displayed no c-Fos and c-Jun immunoreactivity. One and 6 hours after ultraviolet exposure numerous c-Fos and c-Jun immunopositive nuclei were observed mainly in the epithelial cell layers of the cornea and the lens epithelium. Scattered labelled nuclei were detectable in the retinal ganglion cell layer and the inner nuclear layer. Twenty four hours after irradiation c-Fos and c-Jun protein expression returned to near control levels. Histological signs of ultraviolet damage (for example, chromatin condensation, nuclear fragmentation) were first recognisable in the corneal epithelium 6 hours after irradiation and became more apparent at later times. The rapid and sustained activation of c-Fos and c-Jun expression in the eye after single ultraviolet exposure may represent the molecular mechanism underlying ultraviolet induced photodamage and initiation of cell death. Furthermore, topical application of a c-fos antisense oligode-oxynucleotide to the ultraviolet exposed rat eye inhibited the increase in c-Fos expression in the cornea, suggesting therapeutic activity of antisense drugs in corneal malignant and infectious diseases. (author)

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

    Energy Technology Data Exchange (ETDEWEB)

    Pan, L.; Zhang, J.; Chi, X.; Chen, N.; Chen, M.; Wang, M.; Wang, T.; Yang, Z.; Zhang, Z.; Wan, Y.; Yu, S.; Liu, F.

    2016-07-01

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

  5. Anti-sense expression of a metallopeptidase gene enhances nuclear entry of HBV-DNA

    International Nuclear Information System (INIS)

    Yeh, C.-T.; Lai, H.-Y.; Chu, S.-P.; Tseng, I-Chu

    2004-01-01

    Although several putative hepatitis B virus (HBV) receptors have been identified, none of them is capable of initiating HBV replication in a non-permissive human cell line. Using an Epstein-Barr virus-based extrachromosomal replication system, we have screened through a human liver cDNA library and successfully identified a clone capable of facilitating nuclear transport of HBV-DNA during the early phase of HBV infection. This clone contained a cDNA encoding a metallopeptidase-like protein in anti-sense orientation. Pretreatment of naive HepG2 cells with 1,10-phenanthroline, an inhibitor for liver metallopeptidases, led to nuclear entry of HBV-DNA after HBV infection. However, cccDNA was still undetectable in the nuclei, indicating other cellular factors required to complete the replication cycle were still missing. Our present data suggest that in the initial stage of HBV infection, liver metallopeptidase constitutes a barrier for effective nuclear entry of HBV genomic DNA. Attenuation of metallopeptidase activity may facilitate HBV infection

  6. In vitro and in vivo inhibition of proangiogenic retinal phenotype by an antisense oligonucleotide downregulating uPAR expression.

    Science.gov (United States)

    Lulli, Matteo; Cammalleri, Maurizio; Granucci, Irene; Witort, Ewa; Bono, Silvia; Di Gesualdo, Federico; Lupia, Antonella; Loffredo, Rosa; Casini, Giovanni; Dal Monte, Massimo; Capaccioli, Sergio

    2017-08-26

    Neoangiogenesis is the main pathogenic event involved in a variety of retinal diseases. It has been recently demonstrated that inhibiting the urokinase-type plasminogen activator receptor (uPAR) results in reduced angiogenesis in a mouse model of oxygen-induced retinopathy (OIR), establishing uPAR as a therapeutic target in proliferative retinopathies. Here, we evaluated in cultured human retinal endothelial cells (HRECs) and in OIR mice the potential of a specific antisense oligodeoxyribonucleotide (ASO) in blocking the synthesis of uPAR and in providing antiangiogenic effects. uPAR expression in HRECs was inhibited by lipofection with the phosphorotioated 5'-CGGCGGGTGACCCATGTG-3' ASO-uPAR, complementary to the initial translation site of uPAR mRNA. Inhibition of uPAR expression via ASO-uPAR was evaluated in HRECs by analyzing VEGF-induced tube formation and migration. In addition, the well-established and reproducible murine OIR model was used to induce retinal neovascularization in vivo. OIR mice were injected intraperitoneally with ASO-uPAR and retinopathy was evaluated considering the extent of the avascular area in the central retina and neovascular tuft formation. The ASO-uPAR specifically decreased uPAR mRNA and protein levels in HRECs and mitigated VEGF-induced tube formation and cell migration. Noteworthy, in OIR mice ASO-uPAR administration reduced both the avascular area and the formation of neovascular tufts. In conclusion, although the extrapolation of these experimental findings to the clinic is not straightforward, ASO-uPAR may be considered a potential therapeutic tool for treatment of proliferative retinal diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Knockdown of long noncoding antisense RNA brain-derived neurotrophic factor attenuates hypoxia/reoxygenation-induced nerve cell apoptosis through the BDNF-TrkB-PI3K/Akt signaling pathway.

    Science.gov (United States)

    Zhong, Jian-Bin; Li, Xie; Zhong, Si-Ming; Liu, Jiu-Di; Chen, Chi-Bang; Wu, Xiao-Yan

    2017-09-27

    Brain-derived neurotrophic factor (BDNF) plays an important role in neuronal cell apoptosis. The antisense RNA of brain-derived neurotrophic factor (BDNF-AS) is a natural antisense transcript that is transcribed opposite the gene that encodes BDNF. The aim of this study was to determine whether knockdown of BDNF-AS can suppress hypoxia/reoxygenation (H/R)-induced neuronal cell apoptosis and whether this is mediated by the BDNF-TrkB-PI3K/Akt pathway. We detected the expression of BDNF and BDNF-AS in brain tissue from 20 patients with cerebral infarction and five patients with other diseases (but no cerebral ischemia). We found that BDNF expression was significantly downregulated in patients with cerebral infarction, whereas the expression of BDNF-AS was significantly upregulated. In both human cortical neurons (HCN2) and human astrocytes, H/R significantly induced the expression of BDNF-AS, but significantly decreased BDNF expression. H/R also significantly induced apoptosis and reduced the mitochondrial membrane potential in these cells. Following downregulation of BDNF-AS by siRNA in human cortical neurons and human astrocyte cells, BDNF expression was significantly upregulated and the H/R-induced upregulation of BDNF-AS was significantly attenuated. BDNF-AS siRNA inhibited H/R-induced cell apoptosis and ameliorated the H/R-induced suppression of mitochondrial membrane potential. H/R inhibited the expression of BDNF, p-AKT/AKT, and TrKB, and this inhibition was recovered by BDNF-AS siRNA. In summary, this study indicates that BDNF-AS siRNA induces activation of the BDNF-TrkB-PI3K/Akt pathway following H/R-induced neurotoxicity. These findings will be useful toward the application of BDNF-AS siRNA for the treatment of neurodegenerative diseases.

  8. Repression of meiotic genes by antisense transcription and by Fkh2 transcription factor in Schizosaccharomyces pombe.

    Science.gov (United States)

    Chen, Huei-Mei; Rosebrock, Adam P; Khan, Sohail R; Futcher, Bruce; Leatherwood, Janet K

    2012-01-01

    In S. pombe, about 5% of genes are meiosis-specific and accumulate little or no mRNA during vegetative growth. Here we use Affymetrix tiling arrays to characterize transcripts in vegetative and meiotic cells. In vegetative cells, many meiotic genes, especially those induced in mid-meiosis, have abundant antisense transcripts. Disruption of the antisense transcription of three of these mid-meiotic genes allowed vegetative sense transcription. These results suggest that antisense transcription represses sense transcription of meiotic genes in vegetative cells. Although the mechanism(s) of antisense mediated transcription repression need to be further explored, our data indicates that RNAi machinery is not required for repression. Previously, we and others used non-strand specific methods to study splicing regulation of meiotic genes and concluded that 28 mid-meiotic genes are spliced only in meiosis. We now demonstrate that the "unspliced" signal in vegetative cells comes from the antisense RNA, not from unspliced sense RNA, and we argue against the idea that splicing regulates these mid-meiotic genes. Most of these mid-meiotic genes are induced in mid-meiosis by the forkhead transcription factor Mei4. Interestingly, deletion of a different forkhead transcription factor, Fkh2, allows low levels of sense expression of some mid-meiotic genes in vegetative cells. We propose that vegetative expression of mid-meiotic genes is repressed at least two independent ways: antisense transcription and Fkh2 repression.

  9. Repression of Meiotic Genes by Antisense Transcription and by Fkh2 Transcription Factor in Schizosaccharomyces pombe

    Science.gov (United States)

    Chen, Huei-Mei; Rosebrock, Adam P.; Khan, Sohail R.; Futcher, Bruce; Leatherwood, Janet K.

    2012-01-01

    In S. pombe, about 5% of genes are meiosis-specific and accumulate little or no mRNA during vegetative growth. Here we use Affymetrix tiling arrays to characterize transcripts in vegetative and meiotic cells. In vegetative cells, many meiotic genes, especially those induced in mid-meiosis, have abundant antisense transcripts. Disruption of the antisense transcription of three of these mid-meiotic genes allowed vegetative sense transcription. These results suggest that antisense transcription represses sense transcription of meiotic genes in vegetative cells. Although the mechanism(s) of antisense mediated transcription repression need to be further explored, our data indicates that RNAi machinery is not required for repression. Previously, we and others used non-strand specific methods to study splicing regulation of meiotic genes and concluded that 28 mid-meiotic genes are spliced only in meiosis. We now demonstrate that the “unspliced” signal in vegetative cells comes from the antisense RNA, not from unspliced sense RNA, and we argue against the idea that splicing regulates these mid-meiotic genes. Most of these mid-meiotic genes are induced in mid-meiosis by the forkhead transcription factor Mei4. Interestingly, deletion of a different forkhead transcription factor, Fkh2, allows low levels of sense expression of some mid-meiotic genes in vegetative cells. We propose that vegetative expression of mid-meiotic genes is repressed at least two independent ways: antisense transcription and Fkh2 repression. PMID:22238674

  10. Repression of meiotic genes by antisense transcription and by Fkh2 transcription factor in Schizosaccharomyces pombe.

    Directory of Open Access Journals (Sweden)

    Huei-Mei Chen

    Full Text Available In S. pombe, about 5% of genes are meiosis-specific and accumulate little or no mRNA during vegetative growth. Here we use Affymetrix tiling arrays to characterize transcripts in vegetative and meiotic cells. In vegetative cells, many meiotic genes, especially those induced in mid-meiosis, have abundant antisense transcripts. Disruption of the antisense transcription of three of these mid-meiotic genes allowed vegetative sense transcription. These results suggest that antisense transcription represses sense transcription of meiotic genes in vegetative cells. Although the mechanism(s of antisense mediated transcription repression need to be further explored, our data indicates that RNAi machinery is not required for repression. Previously, we and others used non-strand specific methods to study splicing regulation of meiotic genes and concluded that 28 mid-meiotic genes are spliced only in meiosis. We now demonstrate that the "unspliced" signal in vegetative cells comes from the antisense RNA, not from unspliced sense RNA, and we argue against the idea that splicing regulates these mid-meiotic genes. Most of these mid-meiotic genes are induced in mid-meiosis by the forkhead transcription factor Mei4. Interestingly, deletion of a different forkhead transcription factor, Fkh2, allows low levels of sense expression of some mid-meiotic genes in vegetative cells. We propose that vegetative expression of mid-meiotic genes is repressed at least two independent ways: antisense transcription and Fkh2 repression.

  11. Nucleobase-modified antisense oligonucleotides containing 5-(phenyltriazol)-2′-deoxyuridine nucleotides induce exon-skipping

    DEFF Research Database (Denmark)

    Le, Bao T.; Hornum, Mick; Sharma, Pawan K.

    2017-01-01

    Chemically-modified antisense oligonucleotide-mediated exon-skipping has been validated as a therapeutic strategy for tackling several disease pathologies, particularly duchenne muscular dystrophy. To date, only sugar-modified and internucleotide linkage-modified oligonucleotide chemistries have...

  12. Identification of a novel antisense long non-coding RNA PLA2G16-AS that regulates the expression of PLA2G16 in pigs.

    Science.gov (United States)

    Liu, Pengliang; Jin, Long; Zhao, Lirui; Long, Keren; Song, Yang; Tang, Qianzi; Ma, Jideng; Wang, Xun; Tang, Guoqing; Jiang, Yanzhi; Zhu, Li; Li, Xuewei; Li, Mingzhou

    2018-05-31

    Natural antisense transcripts (NATs) are widely present in mammalian genomes and act as pivotal regulator molecules to control gene expression. However, studies on the NATs of pigs are relatively rare. Here, we identified a novel antisense transcript, designated PLA2G16-AS, transcribed from the phospholipase A2 group XVI locus (PLA2G16) in the porcine genome, which is a well-known regulatory molecule of fat deposition. PLA2G16-AS and PLA2G16 were dominantly expressed in porcine adipose tissue, and were differentially expressed between Tibetan pigs and Rongchang pigs. In addition, PLA2G16-AS has a weak sequence conservation among different vertebrates. PLA2G16-AS was also shown to form an RNA-RNA duplex with PLA2G16, and to regulate PLA2G16 expression at the mRNA level. Moreover, the overexpression of PLA2G16-AS increased the stability of PLA2G16 mRNA in porcine cells. We envision that our findings of a NAT for a regulatory gene associated with lipolysis might further our understanding of the molecular regulation of fat deposition. Copyright © 2017. Published by Elsevier B.V.

  13. A locked nucleic acid antisense oligonucleotide (LNA silences PCSK9 and enhances LDLR expression in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Nidhi Gupta

    2010-05-01

    Full Text Available The proprotein convertase subtilisin/kexin type 9 (PCSK9 is an important factor in the etiology of familial hypercholesterolemia (FH and is also an attractive therapeutic target to reduce low density lipoprotein (LDL cholesterol. PCSK9 accelerates the degradation of hepatic low density lipoprotein receptor (LDLR and low levels of hepatic PCSK9 activity are associated with reduced levels of circulating LDL-cholesterol.The present study presents the first evidence for the efficacy of a locked nucleic acid (LNA antisense oligonucleotide (LNA ASO that targets both human and mouse PCSK9. We employed human hepatocytes derived cell lines HepG2 and HuH7 and a pancreatic mouse beta-TC3 cell line known to express high endogenous levels of PCSK9. LNA ASO efficiently reduced the mRNA and protein levels of PCSK9 with a concomitant increase in LDLR protein levels after transfection in these cells. In vivo efficacy of LNA ASO was further investigated in mice by tail vein intravenous administration of LNA ASO in saline solution. The level of PCSK9 mRNA was reduced by approximately 60%, an effect lasting more than 16 days. Hepatic LDLR protein levels were significantly up-regulated by 2.5-3 folds for at least 8 days and approximately 2 fold for 16 days. Finally, measurement of liver alanine aminotransferase (ALT levels revealed that long term LNA ASO treatment (7 weeks does not cause hepatotoxicity.LNA-mediated PCSK9 mRNA inhibition displayed potent reduction of PCSK9 in cell lines and mouse liver. Our data clearly revealed the efficacy and safety of LNA ASO in reducing PCSK9 levels, an approach that is now ready for testing in primates. The major significance and take home message of this work is the development of a novel and promising approach for human therapeutic intervention of the PCSK9 pathway and hence for reducing some of the cardiovascular risk factors associated with the metabolic syndrome.

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

    International Nuclear Information System (INIS)

    Kinuya, S.; Yokoyama, K; Fukuoka, M.; Michigishi, T.; Tonami, N.; Shiba, K.; Mori, H.; Watanabe, N.; Shuke, N.

    2006-01-01

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

  15. Defining global gene expression changes of the hypothalamic-pituitary-gonadal axis in female sGnRH-antisense transgenic common carp (Cyprinus carpio.

    Directory of Open Access Journals (Sweden)

    Jing Xu

    Full Text Available BACKGROUND: The hypothalamic-pituitary-gonadal (HPG axis is critical in the development and regulation of reproduction in fish. The inhibition of neuropeptide gonadotropin-releasing hormone (GnRH expression may diminish or severely hamper gonadal development due to it being the key regulator of the axis, and then provide a model for the comprehensive study of the expression patterns of genes with respect to the fish reproductive system. METHODOLOGY/PRINCIPAL FINDINGS: In a previous study we injected 342 fertilized eggs from the common carp (Cyprinus carpio with a gene construct that expressed antisense sGnRH. Four years later, we found a total of 38 transgenic fish with abnormal or missing gonads. From this group we selected the 12 sterile females with abnormal ovaries in which we combined suppression subtractive hybridization (SSH and cDNA microarray analysis to define changes in gene expression of the HPG axis in the present study. As a result, nine, 28, and 212 genes were separately identified as being differentially expressed in hypothalamus, pituitary, and ovary, of which 87 genes were novel. The number of down- and up-regulated genes was five and four (hypothalamus, 16 and 12 (pituitary, 119 and 93 (ovary, respectively. Functional analyses showed that these genes involved in several biological processes, such as biosynthesis, organogenesis, metabolism pathways, immune systems, transport links, and apoptosis. Within these categories, significant genes for neuropeptides, gonadotropins, metabolic, oogenesis and inflammatory factors were identified. CONCLUSIONS/SIGNIFICANCE: This study indicated the progressive scaling-up effect of hypothalamic sGnRH antisense on the pituitary and ovary receptors of female carp and provided comprehensive data with respect to global changes in gene expression throughout the HPG signaling pathway, contributing towards improving our understanding of the molecular mechanisms and regulative pathways in the

  16. Use of versican variant V3 and versican antisense expression to engineer cultured human skin containing increased content of insoluble elastin.

    Science.gov (United States)

    Merrilees, Mervyn J; Falk, Ben A; Zuo, Ning; Dickinson, Michelle E; May, Barnaby C H; Wight, Thomas N

    2017-01-01

    Skin substitutes for repair of dermal wounds are deficient in functional elastic fibres. We report that the content of insoluble elastin in the dermis of cultured human skin can be increased though the use of two approaches that enhance elastogenesis by dermal fibroblasts, forced expression of versican variant V3, which lacks glycosaminoglycan (GAG) chains, and forced expression of versican antisense to decrease levels of versican variant V1 with GAG chains. Human dermal fibroblasts transduced with V3 or anti-versican were cultured under standard conditions over a period of 4 weeks to produce dermal sheets, with growth enhanced though multiple seedings for the first 3 weeks. Human keratinocytes, cultured in supplemented media, were added to the 4-week dermal sheets and the skin layer cultured for a further week. At 5 weeks, keratinocytes were multilayered and differentiated, with desmosome junctions thoughout and keratin deposits in the upper squamous layers. The dermal layer was composed of layered fibroblasts surrounded by extracellular matrix of collagen bundles and, in control cultures, small scattered elastin deposits. Forced expression of V3 and versican antisense slowed growth, decreased versican V1 expression, increased tropoelastin expression and/or the deposition of large aggregates of insoluble elastin in the dermal layer, and increased tissue stiffness, as measured by nano-indentation. Skin sheets were also cultured on Endoform Dermal Template™, the biodegradable wound dressing made from the lamina propria of sheep foregut. Skin structure and the enhanced deposition of elastin by forced expression of V3 and anti-versican were preserved on this supportive substrate. Copyright © 2014 John Wiley & Sons, Ltd. Copyright © 2014 John Wiley & Sons, Ltd.

  17. Defining Global Gene Expression Changes of the Hypothalamic-Pituitary-Gonadal Axis in Female sGnRH-Antisense Transgenic Common Carp (Cyprinus carpio)

    Science.gov (United States)

    Xu, Jing; Huang, Wei; Zhong, Chengrong; Luo, Daji; Li, Shuangfei; Zhu, Zuoyan; Hu, Wei

    2011-01-01

    Background The hypothalamic-pituitary-gonadal (HPG) axis is critical in the development and regulation of reproduction in fish. The inhibition of neuropeptide gonadotropin-releasing hormone (GnRH) expression may diminish or severely hamper gonadal development due to it being the key regulator of the axis, and then provide a model for the comprehensive study of the expression patterns of genes with respect to the fish reproductive system. Methodology/Principal Findings In a previous study we injected 342 fertilized eggs from the common carp (Cyprinus carpio) with a gene construct that expressed antisense sGnRH. Four years later, we found a total of 38 transgenic fish with abnormal or missing gonads. From this group we selected the 12 sterile females with abnormal ovaries in which we combined suppression subtractive hybridization (SSH) and cDNA microarray analysis to define changes in gene expression of the HPG axis in the present study. As a result, nine, 28, and 212 genes were separately identified as being differentially expressed in hypothalamus, pituitary, and ovary, of which 87 genes were novel. The number of down- and up-regulated genes was five and four (hypothalamus), 16 and 12 (pituitary), 119 and 93 (ovary), respectively. Functional analyses showed that these genes involved in several biological processes, such as biosynthesis, organogenesis, metabolism pathways, immune systems, transport links, and apoptosis. Within these categories, significant genes for neuropeptides, gonadotropins, metabolic, oogenesis and inflammatory factors were identified. Conclusions/Significance This study indicated the progressive scaling-up effect of hypothalamic sGnRH antisense on the pituitary and ovary receptors of female carp and provided comprehensive data with respect to global changes in gene expression throughout the HPG signaling pathway, contributing towards improving our understanding of the molecular mechanisms and regulative pathways in the reproductive system of

  18. Attenuation of alpha2A-adrenergic receptor expression in neonatal rat brain by RNA interference or antisense oligonucleotide reduced anxiety in adulthood.

    Science.gov (United States)

    Shishkina, G T; Kalinina, T S; Dygalo, N N

    2004-01-01

    Brain alpha2-adrenergic receptors (alpha2-ARs) have been implicated in the regulation of anxiety, which is associated with stress. Environmental treatments during neonatal development could modulate the level of brain alpha2-AR expression and alter anxiety in adults, suggesting possible involvement of these receptors in early-life programming of anxiety state. The present study was undertaken to determine whether the reduction of the expression of A subtype of these receptors most abundant in the neonatal brain affects anxiety-related behavior in adulthood. We attenuated the expression of alpha2A-ARs during neonatal life by two different sequence specific approaches, antisense technology and RNA interference. Treatment of rats with the antisense oligodeoxynucleotide or short interfering RNA (siRNA) against alpha2A-ARs on the days 2-4 of their life, produced a marked acute decrease in the levels of both alpha2A-AR mRNA and [3H]RX821002 binding sites in the brainstem into which drugs were injected. The decrease of alpha2A-AR expression in the neonatal brainstem influenced the development of this receptor system in the brain regions as evidenced by the increased number of [3H]RX821002 binding sites in the hypothalamus of adult animals with both neonatal alpha2A-AR knockdown treatments; also in the frontal cortex of antisense-treated, and in the hippocampus of siRNA-treated adult rats. These adult animals also demonstrated a decreased anxiety in the elevated plus-maze as evidenced by an increased number of the open arm entries, greater proportion of time spent in the open arms, and more than a two-fold increase in the number of exploratory head dips. The results provide the first evidence that the reduction in the brain expression of a gene encoding for alpha2A-AR during neonatal life led to the long-term neurochemical and behavioral alterations. The data suggests that alterations in the expression of the receptor-specific gene during critical periods of brain

  19. Reversible antisense inhibition of Shaker-like Kv1.1 potassium channel expression impairs associative memory in mouse and rat

    Science.gov (United States)

    Meiri, Noam; Ghelardini, Carla; Tesco, Giuseppina; Galeotti, Nicoletta; Dahl, Dennis; Tomsic, Daniel; Cavallaro, Sebastiano; Quattrone, Alessandro; Capaccioli, Sergio; Bartolini, Alessandro; Alkon, Daniel L.

    1997-01-01

    Long-term memory is thought to be subserved by functional remodeling of neuronal circuits. Changes in the weights of existing synapses in networks might depend on voltage-gated potassium currents. We therefore studied the physiological role of potassium channels in memory, concentrating on the Shaker-like Kv1.1, a late rectifying potassium channel that is highly localized within dendrites of hippocampal CA3 pyramidal and dentate gyrus granular cells. Repeated intracerebroventricular injection of antisense oligodeoxyribonucleotide to Kv1.1 reduces expression of its particular intracellular mRNA target, decreases late rectifying K+ current(s) in dentate granule cells, and impairs memory but not other motor or sensory behaviors, in two different learning paradigms, mouse passive avoidance and rat spatial memory. The latter, hippocampal-dependent memory loss occurred in the absence of long-term potentiation changes recorded both from the dentate gyrus or CA1. The specificity of the reversible antisense targeting of mRNA in adult animal brains may avoid irreversible developmental and genetic background effects that accompany transgenic “knockouts”. PMID:9114006

  20. Reversible antisense inhibition of Shaker-like Kv1.1 potassium channel expression impairs associative memory in mouse and rat.

    Science.gov (United States)

    Meiri, N; Ghelardini, C; Tesco, G; Galeotti, N; Dahl, D; Tomsic, D; Cavallaro, S; Quattrone, A; Capaccioli, S; Bartolini, A; Alkon, D L

    1997-04-29

    Long-term memory is thought to be subserved by functional remodeling of neuronal circuits. Changes in the weights of existing synapses in networks might depend on voltage-gated potassium currents. We therefore studied the physiological role of potassium channels in memory, concentrating on the Shaker-like Kv1.1, a late rectifying potassium channel that is highly localized within dendrites of hippocampal CA3 pyramidal and dentate gyrus granular cells. Repeated intracerebroventricular injection of antisense oligodeoxyribonucleotide to Kv1.1 reduces expression of its particular intracellular mRNA target, decreases late rectifying K+ current(s) in dentate granule cells, and impairs memory but not other motor or sensory behaviors, in two different learning paradigms, mouse passive avoidance and rat spatial memory. The latter, hippocampal-dependent memory loss occurred in the absence of long-term potentiation changes recorded both from the dentate gyrus or CA1. The specificity of the reversible antisense targeting of mRNA in adult animal brains may avoid irreversible developmental and genetic background effects that accompany transgenic "knockouts".

  1. Quantitative Analysis of Survivin Protein Expression and Its Therapeutic Depletion by an Antisense Oligonucleotide in Human Lung Tumors

    Directory of Open Access Journals (Sweden)

    Anna L Olsen

    2012-01-01

    Full Text Available RNA-directed antisense and interference therapeutics are a promising treatment option for cancer. The demonstration of depletion of target proteins within human tumors in vivo using validated methodology will be a key to the application of this technology. Here, we present a flow cytometric-based approach to quantitatively determine protein levels in solid tumor material derived by fiber optic brushing (FOB of non-small cell lung cancer (NSCLC patients. Focusing upon the survivin protein, and its depletion by an antisense oligonucleotide (ASO (LY2181308, we show that we can robustly identify a subpopulation of survivin positive tumor cells in FOB samples, and, moreover, detect survivin depletion in tumor samples from a patient treated with LY2181308. Survivin depletion appears to be a result of treatment with this ASO, because a tumor treated with conventional cytotoxic chemotherapy did not exhibit a decreased percentage of survivin positive cells. Our approach is likely to be broadly applicable to, and useful for, the quantification of protein levels in tumor samples obtained as part of clinical trials and studies, facilitating the proof-of-principle testing of novel targeted therapies.

  2. Antisense myb inhibition of purified erythroid progenitors in development and differentiation is linked to cycling activity and expression of DNA polymerase alpha

    International Nuclear Information System (INIS)

    Valtieri, M.; Venturelli, D.; Care, A.; Fossati, C.; Pelosi, E.; Labbaye, C.; Mattia, G.; Gewirtz, A.M.; Calabretta, B.; Peschle, C.

    1991-01-01

    These studies aimed to determine the expression and functional role of c-myb in erythroid progenitors with different cycling activities. In the first series of experiments the erythroid burst-forming unit (BFU-E) and colony-forming unit (CFU-E) populations from adult peripheral blood (PB), bone marrow (BM), and embryonic-fetal liver (FL) were treated with either c-myb antisense oligomers or 3H-thymidine (3H-TdR). A direct correlation was always observed between the inhibitory effect of anti-myb oligomers and the level of cycling activity. Thus, the inhibitory effect of antisense c-myb on the number of BFU-E colonies was 28.3% +/- 15.8% in PB, 53.4% +/- 9.3% in BM, and 68.2% +/- 24.5% in FL. Both adult and embryonic CFU-E were markedly inhibited. Using purified PB progenitors, we observed a similar pattern, although with slightly lower inhibitory effects. In the 3H-TdR suicide assay the killing index of BFU-E was 8.9% +/- 4.2% in PB, 29.4% +/- 6.5% in BM, and 40.1% +/- 9.6% in FL. The values for adult and embryonic CFU-E were 55.7% +/- 7.9% and 60.98% +/- 6.6%, respectively. We then investigated the kinetics of c-myb mRNA level during the erythroid differentiation of purified adult PB and FL BFU-E, as evaluated in liquid-phase culture by reverse transcription-polymerase chain reaction. Adult erythroid precursors showed a gradual increase of c-myb mRNA from day 4 through day 8 of culture and a sharp decrease at later times, whereas the expression of c-myb mRNA and protein in differentiation embryonic precursors peaked 2 days earlier. In both cases, c-myb mRNA level peaked at the CFU-E stage of differentiation. Finally, highly purified adult PB BFU-E were stimulated into cycling by a 3-day treatment with interleukin-3 in liquid phase: both the sensitivity to c-myb antisense oligomers and the 3H-TdR suicide index showed a gradual, strictly parallel increase

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

    Science.gov (United States)

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

    2015-03-03

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

  4. Different effects of antisense RelA p65 and NF-κB1 p50 oligonucleotides on the nuclear factor-κB mediated expression of ICAM-1 in human coronary endothelial and smooth muscle cells

    Directory of Open Access Journals (Sweden)

    Both Anton

    2001-08-01

    Full Text Available Abstract Background Activation of nuclear factor-κB (NF-κB is one of the key events in early atherosclerosis and restenosis. We hypothesized that tumor necrosis factor-α (TNF-α induced and NF-κB mediated expression of intercellular adhesion molecule-1 (ICAM-1 can be inhibited by antisense RelA p65 and NF-κB1 p50 oligonucleotides (RelA p65 and NF-κB1 p50. Results Smooth muscle cells (SMC from human coronary plaque material (HCPSMC, plaque material of 52 patients, SMC from the human coronary media (HCMSMC, human endothelial cells (EC from umbilical veins (HUVEC, and human coronary EC (HCAEC were successfully isolated (HCPSMC, HUVEC, identified and cultured (HCPSMC, HCMSMC, HUVEC, HCAEC. 12 hrs prior to TNF-α stimulus (20 ng/mL, 6 hrs RelA p65 and NF-κB1 p50 (1, 2, 4, 10, 20, and 30 μM and controls were added for a period of 18 hrs. In HUVEC and HCAEC there was a dose dependent inhibition of ICAM-1 expression after adding of both RelA p65 and NF-κB1 p50. No inhibitory effect was seen after incubation of HCMSMC with RelA p65 and NF-κB1 p50. A moderate inhibition of ICAM-1 expression was found after simultaneous addition of RelA p65 and NF-κB1 p50 to HCPSMC, no inhibitory effect was detected after individual addition of RelA p65 and NF-κB1 p50. Conclusions The data point out that differences exist in the NF-κB mediated expression of ICAM-1 between EC and SMC. Experimental antisense strategies directed against RelA p65 and NF-κB1 p50 in early atherosclerosis and restenosis are promising in HCAEC but will be confronted with redundant pathways in HCMSMC and HCPSMC.

  5. Beneficial metabolic effects of CB1R anti-sense oligonucleotide treatment in diet-induced obese AKR/J mice.

    Directory of Open Access Journals (Sweden)

    Yuting Tang

    Full Text Available An increasing amount of evidence supports pleiotropic metabolic roles of the cannibinoid-1 receptor (CB1R in peripheral tissues such as adipose, liver, skeletal muscle and pancreas. To further understand the metabolic consequences of specific blockade of CB1R function in peripheral tissues, we performed a 10-week-study with an anti-sense oligonucleotide directed against the CB1R in diet-induced obese (DIO AKR/J mice. DIO AKR/J mice were treated with CB1R ASO Isis-414930 (6.25, 12.5 and 25 mg/kg/week or control ASO Isis-141923 (25 mg/kg/week via intraperitoneal injection for 10 weeks. At the end of the treatment, CB1R mRNA from the 25 mg/kg/week CB1R ASO group in the epididymal fat and kidney was decreased by 81% and 63%, respectively. Body weight gain was decreased in a dose-dependent fashion, significantly different in the 25 mg/kg/week CB1R ASO group (46.1±1.0 g vs veh, 51.2±0.9 g, p<0.05. Body fat mass was reduced in parallel with attenuated body weight gain. CB1R ASO treatment led to decreased fed glucose level (at week 8, 25 mg/kg/week group, 145±4 mg/dL vs veh, 195±10 mg/dL, p<0.05. Moreover, CB1R ASO treatment dose-dependently improved glucose excursion during an oral glucose tolerance test, whereas control ASO exerted no effect. Liver steatosis was also decreased upon CB1R ASO treatment. At the end of the study, plasma insulin and leptin levels were significantly reduced by 25 mg/kg/week CB1R ASO treatment. SREBP1 mRNA expression was decreased in both epididymal fat and liver. G6PC and fatty acid translocase/CD36 mRNA levels were also reduced in the liver. In summary, CB1R ASO treatment in DIO AKR/J mice led to improved insulin sensitivity and glucose homeostasis. The beneficial effects of CB1R ASO treatment strongly support the notion that selective inhibition of the peripheral CB1R, without blockade of central CB1R, may serve as an effective approach for treating type II diabetes, obesity and the metabolic syndrome.

  6. Antiviral effects of herpes simplex virus specific anti-sense nucleic acids.

    Science.gov (United States)

    Cantin, E M; Podsakoff, G; Willey, D E; Openshaw, H

    1992-01-01

    We have targeted mRNA sequences encompassing the translation initiation codon of the essential herpes simplex virus type 1 (HSV-1) IE3 gene with three kinds of anti-sense molecule. Addition of a 15mer oligodeoxyribonucleoside methylphosphonate to tissue culture cells resulted in suppression of viral replication. HSV-1 replication was also inhibited in cultured cells containing anti-sense vectors expressing transcripts complementary to the IE3 mRNA. We have also constructed a ribozyme which upon base pairing with the target IE3 mRNA induces cleavage at the predicted GUC site. A major obstacle to anti-sense studies in animals is drug delivery of preformed antisense molecules to ganglionic neurons, the site of HSV latency and reactivation. We speculate as to how this may be accomplished through carrier compounds which are taken up by nerve terminals and transported by retrograde axoplasmic flow. By the same route, HSV itself may be used as an anti-sense vector.

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

    DEFF Research Database (Denmark)

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

    2007-01-01

    nervous system as characterized with both quantitative RT-PCR and in situ hybridization analysis. A functional analysis of the GPR39 promoter region identified sites for the hepatocyte nuclear factors 1alpha and 4alpha (HNF-1alpha and -4alpha) and specificity protein 1 (SP1) transcription factors as being......G protein-coupled receptor 39 (GPR39) is a constitutively active, orphan member of the ghrelin receptor family that is activated by zinc ions. GPR39 is here described to be expressed in a full-length, biologically active seven-transmembrane form, GPR39-1a, as well as in a truncated splice variant...... five-transmembrane form, GPR39-1b. The 3' exon of the GPR39 gene overlaps with an antisense gene called LYPD1 (Ly-6/PLAUR domain containing 1). Quantitative RT-PCR analysis demonstrated that GPR39-1a is expressed selectively throughout the gastrointestinal tract, including the liver and pancreas...

  8. Cis-Natural Antisense Transcripts Are Mainly Co-expressed with Their Sense Transcripts and Primarily Related to Energy Metabolic Pathways during Muscle Development.

    Science.gov (United States)

    Zhao, Yunxia; Hou, Ye; Zhao, Changzhi; Liu, Fei; Luan, Yu; Jing, Lu; Li, Xinyun; Zhu, Mengjin; Zhao, Shuhong

    2016-01-01

    Cis-natural antisense transcripts (cis-NATs) are a new class of RNAs identified in various species. However, the biological functions of cis-NATs are largely unknown. In this study, we investigated the transcriptional characteristics and functions of cis-NATs in the muscle tissue of lean Landrace and indigenous fatty Lantang pigs. In total, 3,306 cis-NATs of 2,469 annotated genes were identified in the muscle tissue of pigs. More than 1,300 cis-NATs correlated with their sense genes at the transcriptional level, and approximately 80% of them were co-expressed in the two breeds. Furthermore, over 1,200 differentially expressed cis-NATs were identified during muscle development. Function annotation showed that the cis-NATs participated in muscle development mainly by co-expressing with genes involved in energy metabolic pathways, including citrate cycle (TCA cycle), glycolysis or gluconeogenesis, mitochondrial activation and so on. Moreover, these cis-NATs and their sense genes abruptly increased at the transition from the late fetal stages to the early postnatal stages and then decreased along with muscle development. In conclusion, the cis-NATs in the muscle tissue of pigs were identified and determined to be mainly co-expressed with their sense genes. The co-expressed cis-NATs and their sense gene were primarily related to energy metabolic pathways during muscle development in pigs. Our results offered novel evidence on the roles of cis-NATs during the muscle development of pigs.

  9. Inhibition of B cell proliferation by antisense DNA to both alpha and beta forms of Fc epsilon R II.

    Science.gov (United States)

    Bhatti, L; Behle, K; Stevens, R H

    1992-10-01

    Epstein-Barr Virus (EBV) infection activates B lymphocyte proliferation through partially understood mechanisms, resulting in phenotypic changes, including the appearance of new antigens. One such antigen is Fc epsilon R II/CD-23 which may be relevant for B cell proliferation. We have used anti-sense oligonucleotides to study the importance of the two forms of this molecule for proliferation in the EBV-transformed, Fc epsilon R II +ve lymphoblastoid B cell line, RPMI 8866. Anti-sense oligodeoxynucleotides were generated to the two forms of Fc epsilon R II; Fc epsilon R IIa (alpha) and IIb (beta) which differ only in their intracytoplasmic domains. Addition of increasing concentrations of anti-sense oligonucleotides, ranging from 1 to 30 microM, significantly decreased cellular proliferation as measured by the incorporation of [3H]thymidine (inhibition range 8-88%). Optimum inhibition of cellular proliferation was apparent at 15 microM concentration of both anti-sense Fc epsilon R IIa and IIb (Fc epsilon R IIa, mean +/- SE = 75 +/- 7% inhibition, p less than 0.001; Fc epsilon R IIb, mean +/- SE = 71 +/- 7% inhibition, p less than 0.001). Anti-sense oligonucleotides complementary to the common part of Fc epsilon R II resulted in a similar inhibition of proliferation. Sense oligonucleotides did not induce significant inhibition. Preincubation of sense and anti-sense oligonucleotides resulted in an abrogation of proliferation inhibition. Moreover, none of these oligonucleotides had any effect on a Fc epsilon R II -ve cell line. Incubation with both anti-sense IIa and IIb resulted in additive, but not synergistic inhibition of proliferation. Addition of soluble Fc epsilon R II did not reverse inhibition of proliferation, suggesting that membrane-bound or intracellular rather than soluble Fc epsilon R II was important for the induced proliferation. Analysis of cell surface expression for Fc epsilon II indicated that while there was a pronounced effect on cell number

  10. 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.

  11. Effects of CD49d-targeted antisense-oligonucleotide on α4 integrin expression and function of acute lymphoblastic leukemia cells: Results of in vitro and in vivo studies.

    Directory of Open Access Journals (Sweden)

    Yann Duchartre

    Full Text Available We recently demonstrated the effectiveness of blocking CD49d with anti-functional antibodies or small molecule inhibitors as a rational targeted approach to the treatment of acute leukemia in combination with chemotherapy. Antisense oligonucleotide promises to be no less specific than antibodies and inhibitors, but more interesting for pharmacokinetics and pharmacodynamics. We addressed this using the published CD49d antisense drug ATL1102. In vitro, we incubated/nucleofected the ALL cell line Kasumi-2 with ATL1102. In vivo, immunodeficient hosts were engrafted with primary ALL cells and treated with ATL1102. Changes in expression of CD49d mRNA and CD49d protein, and of cooperating gene products, including ß1 integrin and CXCR4, as well as survival in the mouse experiments were quantified. We observed dose-dependent down-regulation of CD49d mRNA and protein levels and its partner integrin ß1 cell surface protein level and, up-regulation of CXCR4 surface expression. The suppression was more pronounced after nucleofection than after incubation, where down-regulation was significant only at the higher doses. In vivo effects of ATL1102 were not sufficient to translate into "clinical" benefit in the leukemia model. In summary, antisense oligonucleotides are successful tools for specifically modulating gene expression but sufficient delivery to down-regulate CD49d in vivo may be difficult to achieve.

  12. The antisense RNA As1_flv4 in the Cyanobacterium Synechocystis sp. PCC 6803 prevents premature expression of the flv4-2 operon upon shift in inorganic carbon supply.

    Science.gov (United States)

    Eisenhut, Marion; Georg, Jens; Klähn, Stephan; Sakurai, Isamu; Mustila, Henna; Zhang, Pengpeng; Hess, Wolfgang R; Aro, Eva-Mari

    2012-09-28

    The functional relevance of natural cis-antisense transcripts is mostly unknown. Here we have characterized the association of three antisense RNAs and one intergenically encoded noncoding RNA with an operon that plays a crucial role in photoprotection of photosystem II under low carbon conditions in the cyanobacterium Synechocystis sp. PCC 6803. Cyanobacteria show strong gene expression dynamics in response to a shift of cells from high carbon to low levels of inorganic carbon (C(i)), but the regulatory mechanisms are poorly understood. Among the most up-regulated genes in Synechocystis are flv4, sll0218, and flv2, which are organized in the flv4-2 operon. The flavodiiron proteins encoded by this operon open up an alternative electron transfer route, likely starting from the Q(B) site in photosystem II, under photooxidative stress conditions. Our expression analysis of cells shifted from high carbon to low carbon demonstrated an inversely correlated transcript accumulation of the flv4-2 operon mRNA and one antisense RNA to flv4, designated as As1_flv4. Overexpression of As1_flv4 led to a decrease in flv4-2 mRNA. The promoter activity of as1_flv4 was transiently stimulated by C(i) limitation and negatively regulated by the AbrB-like transcription regulator Sll0822, whereas the flv4-2 operon was positively regulated by the transcription factor NdhR. The results indicate that the tightly regulated antisense RNA As1_flv4 establishes a transient threshold for flv4-2 expression in the early phase after a change in C(i) conditions. Thus, it prevents unfavorable synthesis of the proteins from the flv4-2 operon.

  13. The Antisense RNA As1_flv4 in the Cyanobacterium Synechocystis sp. PCC 6803 Prevents Premature Expression of the flv4-2 Operon upon Shift in Inorganic Carbon Supply*

    Science.gov (United States)

    Eisenhut, Marion; Georg, Jens; Klähn, Stephan; Sakurai, Isamu; Mustila, Henna; Zhang, Pengpeng; Hess, Wolfgang R.; Aro, Eva-Mari

    2012-01-01

    The functional relevance of natural cis-antisense transcripts is mostly unknown. Here we have characterized the association of three antisense RNAs and one intergenically encoded noncoding RNA with an operon that plays a crucial role in photoprotection of photosystem II under low carbon conditions in the cyanobacterium Synechocystis sp. PCC 6803. Cyanobacteria show strong gene expression dynamics in response to a shift of cells from high carbon to low levels of inorganic carbon (Ci), but the regulatory mechanisms are poorly understood. Among the most up-regulated genes in Synechocystis are flv4, sll0218, and flv2, which are organized in the flv4-2 operon. The flavodiiron proteins encoded by this operon open up an alternative electron transfer route, likely starting from the QB site in photosystem II, under photooxidative stress conditions. Our expression analysis of cells shifted from high carbon to low carbon demonstrated an inversely correlated transcript accumulation of the flv4-2 operon mRNA and one antisense RNA to flv4, designated as As1_flv4. Overexpression of As1_flv4 led to a decrease in flv4-2 mRNA. The promoter activity of as1_flv4 was transiently stimulated by Ci limitation and negatively regulated by the AbrB-like transcription regulator Sll0822, whereas the flv4-2 operon was positively regulated by the transcription factor NdhR. The results indicate that the tightly regulated antisense RNA As1_flv4 establishes a transient threshold for flv4-2 expression in the early phase after a change in Ci conditions. Thus, it prevents unfavorable synthesis of the proteins from the flv4-2 operon. PMID:22854963

  14. Antisense targeting of TGF-β1 augments BMP-induced upregulation of osteopontin, type I collagen and Cbfa1 in human Saos-2 cells

    International Nuclear Information System (INIS)

    Shen, Zhong-Jian; Kook Kim, Sang; Youn Jun, Do; Park, Wan; Ho Kim, Young; Malter, James S.; Jo Moon, Byung

    2007-01-01

    Despite commonalities in signal transduction in osteoblasts from different species, the role of TGF-β1 on bone formation remains elusive. In particular, the role of autocrine TGF-β1 on human osteoblasts is largely unknown. Here we show the effect of TGF-β1 knock-down on the proliferation and differentiation of osteoblasts induced by BMP2. Treatment with antisense TGF-β1 moderately increased the rate of cell proliferation, which was completely reversed by the exogenous addition of TGF-β1. Notably, TGF-β1 blockade significantly enhanced BMP2-induced upregulation of mRNAs encoding osteopontin, type I collagen and Cbfa1, which was suppressed by exogenous TGF-β1. Moreover, TGF-β1 knock-down increased BMP2-induced phosphorylation of Smad1/5 as well as their nuclear import, which paralleled a reduction of inhibitory Smad6. These data suggest autocrine TGF-β1 antagonizes BMP signaling through modulation of inducible Smad6 and the activity of BMP specific Smad1/5

  15. Cocaine alters Homer1 natural antisense transcript in the nucleus accumbens.

    Science.gov (United States)

    Sartor, Gregory C; Powell, Samuel K; Velmeshev, Dmitry; Lin, David Y; Magistri, Marco; Wiedner, Hannah J; Malvezzi, Andrea M; Andrade, Nadja S; Faghihi, Mohammad A; Wahlestedt, Claes

    2017-12-01

    Natural antisense transcripts (NATs) are an abundant class of long noncoding RNAs that have recently been shown to be key regulators of chromatin dynamics and gene expression in nervous system development and neurological disorders. However, it is currently unclear if NAT-based mechanisms also play a role in drug-induced neuroadaptations. Aberrant regulation of gene expression is one critical factor underlying the long-lasting behavioral abnormalities that characterize substance use disorder, and it is possible that some drug-induced transcriptional responses are mediated, in part, by perturbations in NAT activity. To test this hypothesis, we used an automated algorithm that mines the NCBI AceView transcriptomics database to identify NAT overlapping genes linked to addiction. We found that 22% of the genes examined contain NATs and that expression of Homer1 natural antisense transcript (Homer1-AS) was altered in the nucleus accumbens (NAc) of mice 2h and 10days following repeated cocaine administration. In in vitro studies, depletion of Homer1-AS lead to an increase in the corresponding sense gene expression, indicating a potential regulatory mechanisms of Homer1 expression by its corresponding antisense transcript. Future in vivo studies are needed to definitely determine a role for Homer1-AS in cocaine-induced behavioral and molecular adaptations. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. An antisense oligodeoxynucleotide targeted against the type IIβ regulatory subunit mRNA of protein kinase inhibits cAMP-induced differentiation in HL-60 leukemia cells without affecting phorbol ester effects

    International Nuclear Information System (INIS)

    Tortora, G.; Clair, T.; Cho-Chung, Y.S.

    1990-01-01

    The type II β regulatory subunit of cAMP-dependent protein kinase (RII β ) has been hypothesized to play an important role in the growth inhibition and differentiation induced by site-selective cAMP analogs in human cancer cells, but direct proof of this function has been lacking. To address this tissue, HL-60 human promyelocytic leukemia cells were exposed to RII β antisense synthetic oligodeoxynucleotide, and the effects on cAMP-induced growth regulation were examined. Exposure of these cells to RII β antisense oligodeoxynucleotide resulted in a decrease in cAMP analog-induced growth inhibition and differentiation without apparent effect on differentiation induced by phorbol esters. This loss in cAMP growth regulatory function correlated with a decrease in basal and induced levels of RII β protein. Exposure to RII β sense, RI α and RII α antisense, or irrelevant oligodeoxynucleotides had no such effect. These results show that the RII β regulatory subunit of protein kinase plays a critical role in the cAMP-induced growth regulation of HL-60 leukemia cells

  17. Anti-sense expression of putrescine N-methyltransferase confirms defensive role of nicotine in Nicotiana sylvestris against Manduca sexta

    NARCIS (Netherlands)

    Voelckel, C.; Krugel, T.; Gase, K.; Heidrich, N.; Van Dam, N.M.; Winz, R.; Baldwin, I.T.

    2001-01-01

    Several lines of evidence support the defensive function of nicotine production in the Nicotiana genus against a range of herbivores, but the evidence is largely correlative. To suppress nicotine production in planta and to test its defensive function, we expressed DNA of putrescine N-methyl

  18. Inhibition of 3T3-L1 adipocyte differentiation by expression of acyl-CoA-binding protein antisense RNA

    DEFF Research Database (Denmark)

    Mandrup, S; Sorensen, R V; Helledie, T

    1998-01-01

    Several lines of evidence have recently underscored the significance of fatty acids or fatty acid-derived metabolites as signaling molecules in adipocyte differentiation. The acyl-CoA-binding protein (ACBP), which functions as an intracellular acyl-CoA pool former and transporter, is induced duri...

  19. Scientific and Regulatory Policy Committee Points-to-consider Paper*: Drug-induced Vascular Injury Associated with Nonsmall Molecule Therapeutics in Preclinical Development: Part 2. Antisense Oligonucleotides.

    Science.gov (United States)

    Engelhardt, Jeffery A; Fant, Pierluigi; Guionaud, Silvia; Henry, Scott P; Leach, Michael W; Louden, Calvert; Scicchitano, Marshall S; Weaver, James L; Zabka, Tanja S; Frazier, Kendall S

    2015-10-01

    Drug-induced vascular injury (DIVI) is a recurrent challenge in the development of novel pharmaceutical agents. In recent years, DIVI has been occasionally observed in nonhuman primates given RNA-targeting therapeutics such as antisense oligonucleotide therapies (ASOs) during chronic toxicity studies. While DIVI in laboratory animal species has been well characterized for vasoactive small molecules, and immune-mediated responses against large molecule biotherapeutics have been well described, there is little published information regarding DIVI induced by ASOs to date. Preclinical DIVI findings in monkeys have caused considerable delays in development of promising new ASO therapies, because of the uncertainty about whether DIVI in preclinical studies is predictive of effects in humans, and the lack of robust biomarkers of DIVI. This review of DIVI discusses clinical and microscopic features of vasculitis in monkeys, their pathogenic mechanisms, and points to consider for the toxicologist and pathologist when confronted with ASO-related DIVI. Relevant examples of regulatory feedback are included to provide insight into risk assessment of ASO therapies. © 2015 by The Author(s).

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

    Directory of Open Access Journals (Sweden)

    Christopher A Lavender

    2016-08-01

    Full Text Available 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.

  1. Plant 7SL RNA and tRNA(Tyr) genes with inserted antisense sequences are efficiently expressed in an in vitro transcription system from Nicotiana tabacum cells

    Czech Academy of Sciences Publication Activity Database

    Yukawa, Y.; Matoušek, Jaroslav; Grimm, M.; Vrba, Lukáš; Steger, G.; Sugiura, M.; Beier, H.

    2002-01-01

    Roč. 50, - (2002), s. 713-723 ISSN 0167-4412 R&D Projects: GA ČR GA521/99/1591; GA MŠk ME 463 Keywords : antisense RNA Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.529, year: 2002

  2. Identification of sequence motifs significantly associated with antisense activity

    Directory of Open Access Journals (Sweden)

    Peek Andrew S

    2007-06-01

    mediators to speed the process along like the RNA Induced Silencing Complex (RISC in RNAi. The independence of motif position and antisense activity also allows us to bypass consideration of this feature in the modelling process, promoting model efficiency and reducing the chance of overfitting when predicting antisense activity. The increase in SVR correlation with significant features compared to nearest-neighbour features indicates that thermodynamics alone is likely not the only factor in determining antisense efficiency.

  3. Structure and expression of two nuclear receptor genes in marsupials: insights into the evolution of the antisense overlap between the α-thyroid hormone receptor and Rev-erbα

    Directory of Open Access Journals (Sweden)

    Brown M Scott

    2010-12-01

    Full Text Available Abstract Background Alternative processing of α-thyroid hormone receptor (TRα, NR1A1 mRNAs gives rise to two functionally antagonistic nuclear receptors: TRα1, the α-type receptor, and TRα2, a non-hormone binding variant that is found only in mammals. TRα2 shares an unusual antisense coding overlap with mRNA for Rev-erbα (NR1D1, another nuclear receptor protein. In this study we examine the structure and expression of these genes in the gray short-tailed opossum, Monodelphis domestica, in comparison with that of eutherian mammals and three other marsupial species, Didelphis virginiana, Potorous tridactylus and Macropus eugenii, in order to understand the evolution and regulatory role of this antisense overlap. Results The sequence, expression and genomic organization of mRNAs encoding TRα1 and Rev-erbα are very similar in the opossum and eutherian mammals. However, the sequence corresponding to the TRα2 coding region appears truncated by almost 100 amino acids. While expression of TRα1 and Rev-erbα was readily detected in all tissues of M. domestica ages 0 days to 18 weeks, TRα2 mRNA was not detected in any tissue or stage examined. These results contrast with the widespread and abundant expression of TRα2 in rodents and other eutherian mammals. To examine requirements for alternative splicing of TRα mRNAs, a series of chimeric minigenes was constructed. Results show that the opossum TRα2-specific 5' splice site sequence is fully competent for splicing but the sequence homologous to the TRα2 3' splice site is not, even though the marsupial sequences are remarkably similar to core splice site elements in rat. Conclusions Our results strongly suggest that the variant nuclear receptor isoform, TRα2, is not expressed in marsupials and that the antisense overlap between TRα and Rev-erbα thus is unique to eutherian mammals. Further investigation of the TRα and Rev-erbα genes in marsupial and eutherian species promises to yield

  4. Structure and expression of two nuclear receptor genes in marsupials: insights into the evolution of the antisense overlap between the α-thyroid hormone receptor and Rev-erbα

    Science.gov (United States)

    2010-01-01

    Background Alternative processing of α-thyroid hormone receptor (TRα, NR1A1) mRNAs gives rise to two functionally antagonistic nuclear receptors: TRα1, the α-type receptor, and TRα2, a non-hormone binding variant that is found only in mammals. TRα2 shares an unusual antisense coding overlap with mRNA for Rev-erbα (NR1D1), another nuclear receptor protein. In this study we examine the structure and expression of these genes in the gray short-tailed opossum, Monodelphis domestica, in comparison with that of eutherian mammals and three other marsupial species, Didelphis virginiana, Potorous tridactylus and Macropus eugenii, in order to understand the evolution and regulatory role of this antisense overlap. Results The sequence, expression and genomic organization of mRNAs encoding TRα1 and Rev-erbα are very similar in the opossum and eutherian mammals. However, the sequence corresponding to the TRα2 coding region appears truncated by almost 100 amino acids. While expression of TRα1 and Rev-erbα was readily detected in all tissues of M. domestica ages 0 days to 18 weeks, TRα2 mRNA was not detected in any tissue or stage examined. These results contrast with the widespread and abundant expression of TRα2 in rodents and other eutherian mammals. To examine requirements for alternative splicing of TRα mRNAs, a series of chimeric minigenes was constructed. Results show that the opossum TRα2-specific 5' splice site sequence is fully competent for splicing but the sequence homologous to the TRα2 3' splice site is not, even though the marsupial sequences are remarkably similar to core splice site elements in rat. Conclusions Our results strongly suggest that the variant nuclear receptor isoform, TRα2, is not expressed in marsupials and that the antisense overlap between TRα and Rev-erbα thus is unique to eutherian mammals. Further investigation of the TRα and Rev-erbα genes in marsupial and eutherian species promises to yield additional insight into the

  5. Antisense downregulation of mutant huntingtin in a cell model

    DEFF Research Database (Denmark)

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

    2003-01-01

    or by addition to the culture medium. Results Expression of the fusion protein containing the mutant huntingtin fragment resulted in diffuse green fluorescence in the cytoplasm and formation of aggregates in some of the NT2 cells and NT2-N neurons. We obtained antisense sequence-specific inhibition of expression...... of the fusion protein and/or suppression of the aggregate formation in both cell types. In the NT2 cells the antisense effect was dependent on the way of administration of the oligo. Conclusions The PS-antisense oligo is effective in downregulation of mutant huntingtin, and the reduction of aggregate formation...... is a sensitive biological marker. The findings suggest that antisense knockdown of huntingtin could be a useful strategy for treatment of HD, and could also be suitable for studies of the normal and pathological function of huntingtin in different cellular model systems....

  6. 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...

  7. The design and partial analysis of RNAseIII anti-PVS antisense complex system to induce plant resistance

    Czech Academy of Sciences Publication Activity Database

    Matoušek, Jaroslav; Schubert, J.; Kuchař, M.; Dědič, P.; Ptáček, J.; Vrba, Lukáš; Lichtenstein, C. P.

    2001-01-01

    Roč. 33, - (2001), s. 381-394 ISSN 0323-5408 R&D Projects: GA ČR GA521/96/1308; GA MZe(CZ) EP9111; GA MŠk ME 463 Grant - others:NAZV(CZ) EP9111 Program:EP Keywords : Plant genetic * induced resistance Subject RIV: EB - Genetics ; Molecular Biology

  8. Repression of Meiotic Genes by Antisense Transcription and by Fkh2 Transcription Factor in Schizosaccharomyces pombe

    OpenAIRE

    Chen, Huei-Mei; Rosebrock, Adam P.; Khan, Sohail R.; Futcher, Bruce; Leatherwood, Janet K.

    2012-01-01

    In S. pombe, about 5% of genes are meiosis-specific and accumulate little or no mRNA during vegetative growth. Here we use Affymetrix tiling arrays to characterize transcripts in vegetative and meiotic cells. In vegetative cells, many meiotic genes, especially those induced in mid-meiosis, have abundant antisense transcripts. Disruption of the antisense transcription of three of these mid-meiotic genes allowed vegetative sense transcription. These results suggest that antisense transcription ...

  9. A regulatory effect of INMAP on centromere proteins: antisense INMAP induces CENP-B variation and centromeric halo.

    Directory of Open Access Journals (Sweden)

    Tan Tan

    Full Text Available CENP-B is a highly conserved protein that facilitates the assembly of specific centromere structures both in interphase nuclei and on mitotic chromosomes. INMAP is a conserved protein that localizes at nucleus in interphase cells and at mitotic apparatus in mitotic cells. Our previous results showed that INMAP over-expression leads to spindle defects, mitotic arrest and formation of polycentrosomal and multinuclear cells, indicating that INMAP may modulate the function of (a key protein(s in mitotic apparatus. In this study, we demonstrate that INMAP interacts with CENP-B and promotes cleavage of the N-terminal DNA binding domain from CENP-B. The cleaved CENP-B cannot associate with centromeres and thus lose its centromere-related functions. Consistent with these results, CENP-B in INMAP knockdown cells becomes more diffused around kinetochores. Although INMAP knockdown cells do not exhibit gross defects in mitotic spindle formation, these cells go through mitosis, especially prophase and metaphase, with different relative timing, indicating subtle abnormality. These results identify INMAP as a model regulator of CENP-B and support the notion that INMAP regulates mitosis through modulating CENP-B-mediated centromere organization.

  10. SiRNAs conjugated with aromatic compounds induce RISC-mediated antisense strand selection and strong gene-silencing activity

    Energy Technology Data Exchange (ETDEWEB)

    Kubo, Takanori, E-mail: kubo-t@yasuda-u.ac.jp [Faculty of Pharmacy, Yasuda Women' s University, 6-13-1 Yasuhigashi, Asaminami-ku, Hiroshima 731-0153 (Japan); Yanagihara, Kazuyoshi [Faculty of Pharmacy, Yasuda Women' s University, 6-13-1 Yasuhigashi, Asaminami-ku, Hiroshima 731-0153 (Japan); Division of Genetics, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); Takei, Yoshifumi [Department of Biochemistry, Nagoya University Graduate School of Medicine, 65 Tsurumi-cho, Showa-ku, Nagoya 466-8550 (Japan); Mihara, Keichiro [Department of Hematology and Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553 (Japan); Sato, Yuichiro; Seyama, Toshio [Faculty of Pharmacy, Yasuda Women' s University, 6-13-1 Yasuhigashi, Asaminami-ku, Hiroshima 731-0153 (Japan)

    2012-10-05

    Highlights: Black-Right-Pointing-Pointer SiRNAs conjugated with aromatic compounds (Ar-siRNAs) at 5 Prime -sense strand were synthesized. Black-Right-Pointing-Pointer Ar-siRNAs increased resistance against nuclease degradation. Black-Right-Pointing-Pointer Ar-siRNAs were thermodynamically stable compared with the unmodified siRNA. Black-Right-Pointing-Pointer High levels of cellular uptake and cytoplasmic localization were found. Black-Right-Pointing-Pointer Strong gene-silencing efficacy was exhibited in the Ar-siRNAs. -- Abstract: Short interference RNA (siRNA) is a powerful tool for suppressing gene expression in mammalian cells. In this study, we focused on the development of siRNAs conjugated with aromatic compounds in order to improve the potency of RNAi and thus to overcome several problems with siRNAs, such as cellular delivery and nuclease stability. The siRNAs conjugated with phenyl, hydroxyphenyl, naphthyl, and pyrenyl derivatives showed strong resistance to nuclease degradation, and were thermodynamically stable compared with unmodified siRNA. A high level of membrane permeability in HeLa cells was also observed. Moreover, these siRNAs exhibited enhanced RNAi efficacy, which exceeded that of locked nucleic acid (LNA)-modified siRNAs, against exogenous Renilla luciferase in HeLa cells. In particular, abundant cytoplasmic localization and strong gene-silencing efficacy were found in the siRNAs conjugated with phenyl and hydroxyphenyl derivatives. The novel siRNAs conjugated with aromatic compounds are promising candidates for a new generation of modified siRNAs that can solve many of the problems associated with RNAi technology.

  11. Nanoparticle for delivery of antisense γPNA oligomers targeting CCR5.

    Science.gov (United States)

    Bahal, Raman; McNeer, Nicole Ali; Ly, Danith H; Saltzman, W Mark; Glazer, Peter M

    2013-01-01

    The development of a new class of peptide nucleic acids (PNAs), i.e., gamma PNAs (γPNAs), creates the need for a general and effective method for its delivery into cells for regulating gene expression in mammalian cells. Here we report the antisense activity of a recently developed hydrophilic and biocompatible diethylene glycol (miniPEG)-based gamma peptide nucleic acid called MPγPNAs via its delivery by poly(lactide-co-glycolide) (PLGA)-based nanoparticle system. We show that MPγPNA oligomers designed to bind to the selective region of chemokine receptor 5 (CC R5) transcript, induce potent and sequence-specific antisense effects as compared with regular PNA oligomers. In addition, PLGA nanoparticle delivery of MPγPNAs is not toxic to the cells. The findings reported in this study provide a combination of γPNA technology and PLGA-based nanoparticle delivery method for regulating gene expression in live cells via the antisense mechanism.

  12. Antisense Treatments for Biothreat Agents

    National Research Council Canada - National Science Library

    Warfield, Kelly L; Panchal, Rekha G; Aman, M J; Bavari, Sina

    2006-01-01

    ... a variety of pathogens in cell culture studies and nonhuman primate models of infection. For these reasons, antisense technologies are being pursued as treatments against biothreat agents such as Ebola virus, dengue virus and Bacillus anthracis...

  13. Comparison of three techniques for generation of tolerogenic dendritic cells: siRNA, oligonucleotide antisense, and antibody blocking.

    Science.gov (United States)

    Karimi, Mohammad Hossein; Ebadi, Padideh; Pourfathollah, Ali Akbar; Moazzeni, Mohammad; Soheili, Zahra Soheila; Samiee, Shahram

    2010-12-01

    In recent years, a new view of dendritic cells (DCs) as a main regulator of immunity to induce and maintain tolerance has been established. In vitro manipulation of their development and maturation is a topic of DC therapeutic application, which utilizes their inherent tolerogenicity. In this field, the therapeutic potential of antisense, siRNA, and blocking antibody are an interesting goal. In the present study, the efficiency of these three methods--siRNA, antisense, and blocking antibody--against CD40 molecule and its function in DCs and BCL1 cell line are compared. DCs were separated from mouse spleen and then cultured in vitro using Lipofectamine 2000 to deliver both silencers; the efficacy of transfection was estimated by flow cytometry. mRNA expression and protein synthesis were assessed by real time-PCR and flow cytometry, respectively. By Annexin V and propidium iodine staining, we could evaluate the viability of transfected cells. Knocking down the CD40 gene into separate groups of DCs by siRNA, antisense, and blocking antibody treated DCs can cause an increase in IL-4, decrease in IL-12, IFN-γ production, and allostimulation activity. Our results indicated that, in comparison to antisense and blocking antibody, siRNAs appear to be quantitatively more efficient in CD40 downregulation and their differences are significant.

  14. Small RNAs and the regulation of cis-natural antisense transcripts in Arabidopsis

    Directory of Open Access Journals (Sweden)

    Lonardi Stefano

    2008-01-01

    Full Text Available Abstract Background In spite of large intergenic spaces in plant and animal genomes, 7% to 30% of genes in the genomes encode overlapping cis-natural antisense transcripts (cis-NATs. The widespread occurrence of cis-NATs suggests an evolutionary advantage for this type of genomic arrangement. Experimental evidence for the regulation of two cis-NAT gene pairs by natural antisense transcripts-generated small interfering RNAs (nat-siRNAs via the RNA interference (RNAi pathway has been reported in Arabidopsis. However, the extent of siRNA-mediated regulation of cis-NAT genes is still unclear in any genome. Results The hallmarks of RNAi regulation of NATs are 1 inverse regulation of two genes in a cis-NAT pair by environmental and developmental cues and 2 generation of siRNAs by cis-NAT genes. We examined Arabidopsis transcript profiling data from public microarray databases to identify cis-NAT pairs whose sense and antisense transcripts show opposite expression changes. A subset of the cis-NAT genes displayed negatively correlated expression profiles as well as inverse differential expression changes under at least one of the examined developmental stages or treatment conditions. By searching the Arabidopsis Small RNA Project (ASRP and Massively Parallel Signature Sequencing (MPSS small RNA databases as well as our stress-treated small RNA dataset, we found small RNAs that matched at least one gene in 646 pairs out of 1008 (64% protein-coding cis-NAT pairs, which suggests that siRNAs may regulate the expression of many cis-NAT genes. 209 putative siRNAs have the potential to target more than one gene and half of these small RNAs could target multiple members of a gene family. Furthermore, the majority of the putative siRNAs within the overlapping regions tend to target only one transcript of a given NAT pair, which is consistent with our previous finding on salt- and bacteria-induced nat-siRNAs. In addition, we found that genes encoding plastid- or

  15. Effective intracellular delivery of oligonucleotides in order to make sense of antisense

    NARCIS (Netherlands)

    Shi, FX; Hoekstra, D

    2004-01-01

    For more than two decades, antisense oligonucleotides (ODNs) have been used to modulate gene expression for the purpose of applications in cell biology and for development of novel sophisticated medical therapeutics. Conceptually, the antisense approach represents an elegant strategy, involving the

  16. 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

  17. Blocking CHK1 Expression Induces Apoptosis and Abrogates the G2 Checkpoint Mechanism

    Directory of Open Access Journals (Sweden)

    Yan Luo

    2001-01-01

    Full Text Available Checkpoint kinase 1 (Chki is a checkpoint gene that is activated after DNA damage. It phosphorylates and inactivates the Cdc2 activating phosphatase Cdc25C. This in turn inactivates Cdc2, which leads to G2/M arrest. We report that blocking Chki expression by antisense or ribozymes in mammalian cells induces apoptosis and interferes with the G2/M arrest induced by adriamycin. The Chki inhibitor UCN-01 also blocks the G2 arrest after DNA damage and renders cells more susceptible to adriamycin. These results indicate that Chki is an essential gene for the checkpoint mechanism during normal cell proliferation as well as in the DNA damage response.

  18. Hydrogel-Assisted Antisense LNA Gapmer Delivery for In Situ Gene Silencing in Spinal Cord Injury

    DEFF Research Database (Denmark)

    Moreno, Pedro M.D.; Ferreira, Ana R.; Salvador, Daniela

    2018-01-01

    )-modified AON gapmers in combination with a fibrin hydrogel bridging material to induce gene silencing in situ at a SCI lesion site. LNA gapmers were effectively developed against two promising gene targets aiming at enhancing axonal regeneration—RhoA and GSK3β. The fibrin-matrix-assisted AON delivery system......After spinal cord injury (SCI), nerve regeneration is severely hampered due to the establishment of a highly inhibitory microenvironment at the injury site, through the contribution of multiple factors. The potential of antisense oligonucleotides (AONs) to modify gene expression at different levels...

  19. Inhibitory PAS domain protein is a negative regulator of hypoxia-inducible gene expression

    Science.gov (United States)

    Makino, Yuichi; Cao, Renhai; Svensson, Kristian; Bertilsson, Göran; Asman, Mikael; Tanaka, Hirotoshi; Cao, Yihai; Berkenstam, Anders; Poellinger, Lorenz

    2001-11-01

    Alteration of gene expression is a crucial component of adaptive responses to hypoxia. These responses are mediated by hypoxia-inducible transcription factors (HIFs). Here we describe an inhibitory PAS (Per/Arnt/Sim) domain protein, IPAS, which is a basic helix-loop-helix (bHLH)/PAS protein structurally related to HIFs. IPAS contains no endogenous transactivation function but demonstrates dominant negative regulation of HIF-mediated control of gene expression. Ectopic expression of IPAS in hepatoma cells selectively impairs induction of genes involved in adaptation to a hypoxic environment, notably the vascular endothelial growth factor (VEGF) gene, and results in retarded tumour growth and tumour vascular density in vivo. In mice, IPAS was predominantly expressed in Purkinje cells of the cerebellum and in corneal epithelium of the eye. Expression of IPAS in the cornea correlates with low levels of expression of the VEGF gene under hypoxic conditions. Application of an IPAS antisense oligonucleotide to the mouse cornea induced angiogenesis under normal oxygen conditions, and demonstrated hypoxia-dependent induction of VEGF gene expression in hypoxic corneal cells. These results indicate a previously unknown mechanism for negative regulation of angiogenesis and maintenance of an avascular phenotype.

  20. Expression of a natural antisense transcript of Cg-Foxl2 during the gonadic differentiation of the oyster Crassostrea gigas: first demonstration in the gonads of a lophotrochozoa species.

    Science.gov (United States)

    Santerre, C; Sourdaine, P; Martinez, A-S

    2012-01-01

    In the oyster Crassostrea gigas, a successive hermaphrodite, Cg-Foxl2, an ortholog of Foxl2, is suspected to be involved in vitellogenesis or female sex determination. The existence of a natural antisense transcript (NAT) of this factor has been suspected in gonads but needs to be confirmed to better understand the early events of the gonadic differentiation. The occurrence of this NAT was studied by orientation-specific RT-PCR. The NAT and its mRNA expressions were investigated during the development and in adults by real-time PCR and in situ hybridization. The presence of stable in vivo RNA-RNA duplexes was also explored by RNase protection-based approach. This work is the first evidence of characterization of a NAT in the gonads of mollusks and Lophotrochozoa. This NAT named Cg-Foxl2os is supposed to be polyadenylated and forms RNA-RNA duplexes with its mRNA. Cg-Foxl2os is significantly more expressed than Cg-Foxl2 in 2-month-old spats and in mature males. It is co-localized with the mRNA in the cytoplasm of spermatogonia and spermatocytes. The results of this study demonstrate the existence of a NAT of Cg-Foxl2 in the gonads of C. gigas. It may regulate its mRNA expression through formation of cytoplasmic RNA-RNA duplexes during the oyster gonadic differentiation. Copyright © 2012 S. Karger AG, Basel.

  1. Combination of vascular endothelial growth factor antisense oligonucleotide therapy and radiotherapy increases the curative effects against maxillofacial VX2 tumors in rabbits

    Energy Technology Data Exchange (ETDEWEB)

    Zheng Linfeng, E-mail: zhenglinfeng04@yahoo.com.cn [Department of Radiology, Shanghai First People' s Hospital, Medical College, Shanghai Jiaotong University, Hanning Road, 100, 200080 Shanghai (China); Li Yujie, E-mail: yujieli01@yahoo.com.cn [Department of Radiology, Shanghai First People' s Hospital, Medical College, Shanghai Jiaotong University, Hanning Road, 100, 200080 Shanghai (China); Wang Han, E-mail: bingowh@hotmail.com [Department of Radiology, Shanghai First People' s Hospital, Medical College, Shanghai Jiaotong University, Hanning Road, 100, 200080 Shanghai (China); Zhao Jinglong, E-mail: jinglongz@yahoo.com [Department of Radiology, Shanghai First People' s Hospital, Medical College, Shanghai Jiaotong University, Hanning Road, 100, 200080 Shanghai (China); Wang Xifu, E-mail: wangxiechen001@163.com [Department of Radiology, Shanghai First People' s Hospital, Medical College, Shanghai Jiaotong University, Hanning Road, 100, 200080 Shanghai (China); Hu Yunsheng, E-mail: springmorninghu@163.com [Department of Radiology, Shanghai First People' s Hospital, Medical College, Shanghai Jiaotong University, Hanning Road, 100, 200080 Shanghai (China); Zhang Guixiang, E-mail: guixiangzhang@sina.com [Department of Radiology, Shanghai First People' s Hospital, Medical College, Shanghai Jiaotong University, Hanning Road, 100, 200080 Shanghai (China)

    2011-05-15

    Purpose: To study the effects of combination of vascular endothelial growth factor (VEGF) antisense oligonucleotide therapy and radiotherapy on maxillofacial VX2 tumors in rabbits. Methods: We used 24 New Zealand white rabbits as a model to induce maxillofacial VX2 tumor. The rabbits were randomly divided into the following 4 groups: radiotherapy group (group A), treated with 16 Gy of radiotherapy; VEGF antisense oligonucleotide treatment group (group B), treated with an injection of 150 {mu}g of VEGF antisense oligonucleotide into the local tumor; VEGF antisense oligonucleotide combined with radiotherapy group (group C), treated with an injection of 150 {mu}g of VEGF antisense oligonucleotide into the local tumor immediately after 16 Gy of radiotherapy; and control group (group D), treated with an injection of 300 {mu}l 5% aqueous glucose solution into the local tumor. On days 3 and 14 after treatment, dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) was performed to calculate maximal enhancement ratio (MER), slope of enhancement (SLE), and tumor volume change. Rabbits were killed on day 14 to obtain samples for pathological examination and immunohistochemical staining for VEGF. Results: In group C, tumor volume was significantly reduced on day 14 after treatment, and the difference was statistically different as compared to that before treatment, on day 3 after treatment and other groups (P < 0.01). Values of both MER and SLE after treatment were significantly lower than the values before treatment (P < 0.05). Pathological specimen revealed tumor cell edema, bleeding, necrosis, vascular wall thickening and occlusion, and decreased VEGF expression. The immunohistochemical score (IHS) of group C was significantly different from groups A and D respectively (P < 0.05). Conclusion: Injecting the tumor with VEGF antisense oligonucleotide immediately after radiotherapy can enhance the curative effect on rabbit maxillofacial VX2 tumor, and DCE-MRI can serve

  2. Developmental transitions in Arabidopsis are regulated by antisense RNAs resulting from bidirectionally transcribed genes.

    Science.gov (United States)

    Krzyczmonik, Katarzyna; Wroblewska-Swiniarska, Agata; Swiezewski, Szymon

    2017-07-03

    Transcription terminators are DNA elements located at the 3' end of genes that ensure efficient cleavage of nascent RNA generating the 3' end of mRNA, as well as facilitating disengagement of elongating DNA-dependent RNA polymerase II. Surprisingly, terminators are also a potent source of antisense transcription. We have recently described an Arabidopsis antisense transcript originating from the 3' end of a master regulator of Arabidopsis thaliana seed dormancy DOG1. In this review, we discuss the broader implications of our discovery in light of recent developments in yeast and Arabidopsis. We show that, surprisingly, the key features of terminators that give rise to antisense transcription are preserved between Arabidopsis and yeast, suggesting a conserved mechanism. We also compare our discovery to known antisense-based regulatory mechanisms, highlighting the link between antisense-based gene expression regulation and major developmental transitions in plants.

  3. Dyslipidemia, sense, antisense or nonsense?

    NARCIS (Netherlands)

    Visser, M.E.

    2011-01-01

    Maartje Visser onderzocht het remmen van de synthese van apoB met behulp van antisense - een nieuwe farmacologische techniek. Dit blijkt het slechte LDL-cholesterol op een effectieve manier te verlagen. Bij sommige proefpersonen resulteerde dit in leververvetting. Of dit op de lange termijn

  4. Antisense gene silencing

    DEFF Research Database (Denmark)

    Nielsen, Troels T; Nielsen, Jørgen E

    2013-01-01

    Since the first reports that double-stranded RNAs can efficiently silence gene expression in C. elegans, the technology of RNA interference (RNAi) has been intensively exploited as an experimental tool to study gene function. With the subsequent discovery that RNAi could also be applied...

  5. Development of Novel Antisense Oligonucleotides for the Functional Regulation of RNA-Induced Silencing Complex (RISC) by Promoting the Release of microRNA from RISC.

    Science.gov (United States)

    Ariyoshi, Jumpei; Momokawa, Daiki; Eimori, Nao; Kobori, Akio; Murakami, Akira; Yamayoshi, Asako

    2015-12-16

    MicroRNAs (miRNAs) are known to be important post-transcription regulators of gene expression. Aberrant miRNA expression is associated with pathological disease processes, including carcinogenesis. Therefore, miRNAs are considered significant therapeutic targets for cancer therapy. MiRNAs do not act alone, but exhibit their functions by forming RNA-induced silencing complex (RISC). Thus, the regulation of RISC activity is a promising approach for cancer therapy. MiRNA is a core component of RISC and is an essential to RISC for recognizing target mRNA. Thereby, it is expected that development of the method to promote the release of miRNA from RISC would be an effective approach for inhibition of RISC activity. In this study, we synthesized novel peptide-conjugated oligonucleotides (RINDA-as) to promote the release of miRNA from RISC. RINDA-as showed a high rate of miRNA release from RISC and high level of inhibitory effect on RISC activity.

  6. 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.

  7. Antisense sequences and antagomiR 155 in therapy for B lymphomas over expressing miR-155: preclinical models and identification of target mRNAs

    International Nuclear Information System (INIS)

    Marziali, G.; Peschle, C.

    2009-01-01

    Micro RNAs (miRNAs) are a conserved class of small noncoding RNAs (22-25 nucleotides), which modulate gene expression at post-transcriptional level by base pairing to the 3'UTR of the target mRNAs, thus causing messenger degradation or inhibiting its translation. Experimental evidence indicate that several miRNAs are deregulated in human tumors. MIRN155 has been shown to be highly expressed in a variety of human B cell lymphomas, especially diffuse large B cells, Hodgkin, and a subset of Burkitt lymphomas. Its expression is physiologically increased in activated B and T cells and it plays a key role in regulating the homeostasis and function of the immune system

  8. Quantitative Antisense Screening and Optimization for Exon 51 Skipping in Duchenne Muscular Dystrophy.

    Science.gov (United States)

    Echigoya, Yusuke; Lim, Kenji Rowel Q; Trieu, Nhu; Bao, Bo; Miskew Nichols, Bailey; Vila, Maria Candida; Novak, James S; Hara, Yuko; Lee, Joshua; Touznik, Aleksander; Mamchaoui, Kamel; Aoki, Yoshitsugu; Takeda, Shin'ichi; Nagaraju, Kanneboyina; Mouly, Vincent; Maruyama, Rika; Duddy, William; Yokota, Toshifumi

    2017-11-01

    Duchenne muscular dystrophy (DMD), the most common lethal genetic disorder, is caused by mutations in the dystrophin (DMD) gene. Exon skipping is a therapeutic approach that uses antisense oligonucleotides (AOs) to modulate splicing and restore the reading frame, leading to truncated, yet functional protein expression. In 2016, the US Food and Drug Administration (FDA) conditionally approved the first phosphorodiamidate morpholino oligomer (morpholino)-based AO drug, eteplirsen, developed for DMD exon 51 skipping. Eteplirsen remains controversial with insufficient evidence of its therapeutic effect in patients. We recently developed an in silico tool to design antisense morpholino sequences for exon skipping. Here, we designed morpholino AOs targeting DMD exon 51 using the in silico tool and quantitatively evaluated the effects in immortalized DMD muscle cells in vitro. To our surprise, most of the newly designed morpholinos induced exon 51 skipping more efficiently compared with the eteplirsen sequence. The efficacy of exon 51 skipping and rescue of dystrophin protein expression were increased by up to more than 12-fold and 7-fold, respectively, compared with the eteplirsen sequence. Significant in vivo efficacy of the most effective morpholino, determined in vitro, was confirmed in mice carrying the human DMD gene. These findings underscore the importance of AO sequence optimization for exon skipping. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

  9. Inhaled ENaC antisense oligonucleotide ameliorates cystic fibrosis-like lung disease in mice.

    Science.gov (United States)

    Crosby, Jeff R; Zhao, Chenguang; Jiang, Chong; Bai, Dong; Katz, Melanie; Greenlee, Sarah; Kawabe, Hiroshi; McCaleb, Michael; Rotin, Daniela; Guo, Shuling; Monia, Brett P

    2017-11-01

    Epithelial sodium channel (ENaC, Scnn1) hyperactivity in the lung leads to airway surface dehydration and mucus accumulation in cystic fibrosis (CF) patients and in mice with CF-like lung disease. We identified several potent ENaC specific antisense oligonucleotides (ASOs) and tested them by inhalation in mouse models of CF-like lung disease. The inhaled ASOs distributed into lung airway epithelial cells and decreased ENaC expression by inducing RNase H1-dependent degradation of the targeted Scnn1a mRNA. Aerosol delivered ENaC ASO down-regulated mucus marker expression and ameliorated goblet cell metaplasia, inflammation, and airway hyper-responsiveness. Lack of systemic activity of ASOs delivered via the aerosol route ensures the safety of this approach. Our results demonstrate that antisense inhibition of ENaC in airway epithelial cells could be an effective and safe approach for the prevention and reversal of lung symptoms in CF and potentially other inflammatory diseases of the lung. Copyright © 2017 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

  10. 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.

  11. 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

  12. Upregulation of PEDF expression by PARP inhibition contributes to the decrease in hyperglycemia-induced apoptosis in HUVECs

    International Nuclear Information System (INIS)

    Chen Haibing; Jia Weiping; Xu Xun; Fan Ying; Zhu Dongqing; Wu Haixiang; Xie Zhenggao; Zheng Zhi

    2008-01-01

    Poly(ADP-ribose)polymerase (PARP) inhibitors decrease angiogenesis through reducing vascular endothelium growth factor (VEGF) induced proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs). In contrast to VEGF, pigment epithelium-derived factor (PEDF) has been demonstrated to act as a strong endogenous inhibitor of angiogenesis. Here, we show that PARP inhibition with a specific inhibitor PJ-34 or specific PARP antisense oligonucleotide upregulates hyperglycemia-induced PEDF expression in HUVECs in a dose-dependent manner. This results in the retard of activation of p38 MAP kinase and the concomitant decrease in cell apoptosis. These results give the first direct demonstration that PEDF might represent a target for PARP inhibition treatment and the effects of PEDF on endothelial cells growth are context dependent

  13. Regulation of Peripheral Myelination through Transcriptional Buffering of Egr2 by an Antisense Long Non-coding RNA

    Directory of Open Access Journals (Sweden)

    Margot Martinez-Moreno

    2017-08-01

    Full Text Available Precise regulation of Egr2 transcription is fundamentally important to the control of peripheral myelination. Here, we describe a long non-coding RNA antisense to the promoter of Egr2 (Egr2-AS-RNA. During peripheral nerve injury, the expression of Egr2-AS-RNA is increased and correlates with decreased Egr2 transcript and protein levels. Ectopic expression of Egr2-AS-RNA in dorsal root ganglion (DRG cultures inhibits the expression of Egr2 mRNA and induces demyelination. In vivo inhibition of Egr2-AS-RNA using oligonucleotide GapMers released from a biodegradable hydrogel following sciatic nerve injury reverts the EGR2-mediated gene expression profile and significantly delays demyelination. Egr2-AS-RNA gradually recruits H3K27ME3, AGO1, AGO2, and EZH2 on the Egr2 promoter following sciatic nerve injury. Furthermore, expression of Egr2-AS-RNA is regulated through ERK1/2 signaling to YY1, while loss of Ser184 of YY1 regulates binding to Egr2-AS-RNA. In conclusion, we describe functional exploration of an antisense long non-coding RNA in peripheral nervous system (PNS biology.

  14. Antisense Suppression of 2-Cysteine Peroxiredoxin in Arabidopsis Specifically Enhances the Activities and Expression of Enzymes Associated with Ascorbate Metabolism But Not Glutathione Metabolism1

    Science.gov (United States)

    Baier, Margarete; Noctor, Graham; Foyer, Christine H.; Dietz, Karl-Josef

    2000-01-01

    The aim of this study was to characterize the effect of decreased 2-cysteine peroxiredoxin (2-CP) on the leaf anti-oxidative system in Arabidopsis. At three stages of leaf development, two lines of transgenic Arabidopsis mutants with decreased contents of chloroplast 2-CP were compared with wild type and a control line transformed with an empty vector. Glutathione contents and redox state were similar in all plants, and no changes in transcript levels for enzymes involved in glutathione metabolism were observed. Transcript levels for chloroplastic glutathione peroxidase were much lower than those for 2-CP, and both cytosolic and chloroplastic glutathione peroxidase were not increased in the mutants. In contrast, the foliar ascorbate pool was more oxidized in the mutants, although the difference decreased with plant age. The activities of thylakoid and stromal ascorbate peroxidase and particularly monodehydroascorbate reductase were increased as were transcripts for these enzymes. No change in dehydroascorbate reductase activity was observed, and effects on transcript abundance for glutathione reductase, catalase, and superoxide dismutase were slight or absent. The results demonstrate that 2-CP forms an integral part of the anti-oxidant network of chloroplasts and is functionally interconnected with other defense systems. Suppression of 2-CP leads to increased expression of other anti-oxidative genes possibly mediated by increased oxidation state of the leaf ascorbate pool. PMID:11027730

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

    Science.gov (United States)

    Schein, Aleks; Zucchelli, Silvia; Kauppinen, Sakari; Gustincich, Stefano; Carninci, Piero

    2016-09-20

    Mammalian genomes encode numerous natural antisense long noncoding RNAs (lncRNAs) that regulate gene expression. Recently, an antisense lncRNA to mouse Ubiquitin carboxyl-terminal hydrolase L1 (Uchl1) was reported to increase UCHL1 protein synthesis, representing a new functional class of lncRNAs, designated as SINEUPs, for SINE element-containing translation UP-regulators. Here, we show that an antisense lncRNA to the human protein phosphatase 1 regulatory subunit 12A (PPP1R12A), named as R12A-AS1, which overlaps with the 5' UTR and first coding exon of the PPP1R12A mRNA, functions as a SINEUP, 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 for the first time that human natural antisense lncRNAs can up-regulate protein translation, suggesting that endogenous SINEUPs may be widespread and present in many mammalian species.

  16. Drug-induced regulation of target expression

    DEFF Research Database (Denmark)

    Iskar, Murat; Campillos, Monica; Kuhn, Michael

    2010-01-01

    Drug perturbations of human cells lead to complex responses upon target binding. One of the known mechanisms is a (positive or negative) feedback loop that adjusts the expression level of the respective target protein. To quantify this mechanism systems-wide in an unbiased way, drug......-induced differential expression of drug target mRNA was examined in three cell lines using the Connectivity Map. To overcome various biases in this valuable resource, we have developed a computational normalization and scoring procedure that is applicable to gene expression recording upon heterogeneous drug treatments....... In 1290 drug-target relations, corresponding to 466 drugs acting on 167 drug targets studied, 8% of the targets are subject to regulation at the mRNA level. We confirmed systematically that in particular G-protein coupled receptors, when serving as known targets, are regulated upon drug treatment. We...

  17. A dystrophic Duchenne mouse model for testing human antisense oligonucleotides.

    Directory of Open Access Journals (Sweden)

    Marcel Veltrop

    Full Text Available Duchenne muscular dystrophy (DMD is a severe muscle-wasting disease generally caused by reading frame disrupting mutations in the DMD gene resulting in loss of functional dystrophin protein. The reading frame can be restored by antisense oligonucleotide (AON-mediated exon skipping, allowing production of internally deleted, but partially functional dystrophin proteins as found in the less severe Becker muscular dystrophy. Due to genetic variation between species, mouse models with mutations in the murine genes are of limited use to test and further optimize human specific AONs in vivo. To address this we have generated the del52hDMD/mdx mouse. This model carries both murine and human DMD genes. However, mouse dystrophin expression is abolished due to a stop mutation in exon 23, while the expression of human dystrophin is abolished due to a deletion of exon 52. The del52hDMD/mdx model, like mdx, shows signs of muscle dystrophy on a histological level and phenotypically mild functional impairment. Local administration of human specific vivo morpholinos induces exon skipping and dystrophin restoration in these mice. Depending on the number of mismatches, occasional skipping of the murine Dmd gene, albeit at low levels, could be observed. Unlike previous models, the del52hDMD/mdx model enables the in vivo analysis of human specific AONs targeting exon 51 or exon 53 on RNA and protein level and muscle quality and function. Therefore, it will be a valuable tool for optimizing human specific AONs and genome editing approaches for DMD.

  18. Citrus nobiletin suppresses inducible nitric oxide synthase gene expression in interleukin-1β-treated hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Yoshigai, Emi [Department of Biomedical Sciences, College of Life Sciences, Kusatsu, Shiga (Japan); Ritsumeikan Global Innovation Research Organization (R-GIRO), Kusatsu, Shiga (Japan); Machida, Toru [Department of Biomedical Sciences, College of Life Sciences, Kusatsu, Shiga (Japan); Okuyama, Tetsuya [Ritsumeikan Global Innovation Research Organization (R-GIRO), Kusatsu, Shiga (Japan); Mori, Masatoshi; Murase, Hiromitsu; Yamanishi, Ryota [Department of Biomedical Sciences, College of Life Sciences, Kusatsu, Shiga (Japan); Okumura, Tadayoshi [Research Organization of Science and Technology, Ritsumeikan University, Kusatsu, Shiga (Japan); Department of Surgery, Kansai Medical University, Hirakata, Osaka (Japan); Ikeya, Yukinobu [Department of Pharmacy, College of Pharmaceutical Sciences, Ritsumeikan University, Kusatsu, Shiga (Japan); Nishino, Hoyoku [Ritsumeikan Global Innovation Research Organization (R-GIRO), Kusatsu, Shiga (Japan); Department of Biochemistry, Kyoto Prefectural University of Medicine, Kyoto (Japan); Nishizawa, Mikio, E-mail: nishizaw@sk.ritsumei.ac.jp [Department of Biomedical Sciences, College of Life Sciences, Kusatsu, Shiga (Japan)

    2013-09-13

    Highlights: •Nobiletin is a polymethoxylated flavone that is abundant in citrus peels. •Nobiletin is a major constituent of the Citrus unshiu peel extract. •Nobiletin suppresses induction of NO and reduces iNOS expression in hepatocytes. •Nobiletin reduces the iNOS promoter activity and the DNA-binding activity of NF-κB. -- Abstract: Background: Nobiletin is a polymethoxylated flavone that is abundant in the peels of citrus fruits, such as Citrus unshiu (Satsuma mandarin) and Citrus sinensis. The dried peels of C. unshiu (chinpi) have been included in several formulae of Japanese Kampo medicines. Nobiletin may suppress the induction of inducible nitric oxide synthase (iNOS), which synthesizes the inflammatory mediator nitric oxide (NO) in hepatocytes. Methods: A C. unshiu peel (CUP) extract was prepared. Primary cultured rat hepatocytes were treated with the CUP extract or nobiletin in the presence of interleukin 1β (IL-1β), which induces iNOS expression. NO production and iNOS gene expression were analyzed. Results: High-performance liquid chromatography analyses revealed that the nobiletin content in the CUP extract was 0.14%. Nobiletin dose-dependently reduced the NO levels and decreased iNOS expression at the protein, mRNA and antisense transcript levels. Flavone, which does not contain any methoxy groups, also suppressed iNOS induction. Nobiletin reduced the transcriptional activity of iNOS promoter-luciferase constructs and the DNA-binding activity of nuclear factor κB (NF-κB) in the nuclei. Conclusions: The suppression of iNOS induction by nobiletin suggests that nobiletin may be responsible for the anti-inflammatory effects of citrus peels and have a therapeutic potential for liver diseases.

  19. Heme oxygenase-1 gene expression modulates angiotensin II-induced increase in blood pressure.

    Science.gov (United States)

    Yang, Liming; Quan, Shuo; Nasjletti, Alberto; Laniado-Schwartzman, Michal; Abraham, Nader G

    2004-06-01

    The heme-heme oxygenase (HO) system has been implicated in the regulation of vascular reactivity and blood pressure. This study examines the notion that overexpression of HO decreases pressor responsiveness to angiotensin II (Ang II). Five-day-old Sprague-Dawley rats received an intraleft ventricular injection of approximately 5x10(9) cfu/mL of retroviruses containing human HO-1 sense (LSN-HHO-1), rat HO-1 antisense (LSN-RHO-1-AS), or control retrovirus (LXSN). Three months later, rats were instrumented with femoral arterial and venous catheters for mean arterial pressure (MAP) determination and Ang II administration, respectively. Rats injected with LSN-HHO-1, but not with LXSN, expressed human HO-1 mRNA and protein in several tissues. BP increased with administration of Ang II in rats expressing and not expressing human HO-1. However, the Ang II-induced pressor response (mm Hg) in LSN-HHO-1 rats (16+/-3, 27+/-3, and 38+/-3 at 0.5, 2, and 10 ng) was surpassed (PHHO-1 rats with the HO inhibitor tin mesoporphyrin (SnMP) enhanced (P<0.05) the Ang II-induced pressor response to a level not different from that observed in LXSN rats. Rats injected with LSN-RHO-1-AS showed a decrease in renal HO-1 protein expression and HO activity relative to control LXSN rats. Administration of Ang II (0.1 to 2 ng) caused small (4 to 5 mm Hg) but significant increases in MAP in rats injected with LSN-RHO-1-AS (P<0.05) compared with rats injected with LXSN. These data demonstrate that overexpression of HO-1 brings about a reduction in pressor responsiveness to Ang II, which is most likely due to increased generation of an HO-1 product, presumably CO, with the ability to inhibit vascular reactivity to constrictor stimuli.

  20. An antisense peptide nucleic acid against Pseudomonas aeruginosa inhibiting bacterial-induced inflammatory responses in the cystic fibrosis IB3-1 cellular model system

    DEFF Research Database (Denmark)

    Montagner, Giulia; Bezzerri, Valentino; Cabrini, Giulio

    2017-01-01

    of the essential acpP gene of P. aeruginosa, and previously shown to inhibit bacterial growth, concomitantly also strongly inhibits induced up-regulation of the pro-inflammatory markers IL-8, IL-6, G-CSF, IFN-γ, IP-10, MCP-1 and TNF-α in IB3-1 cystic fibrosis cells infected by P. aeruginosa PAO1. Remarkably...... are significant considering the key role of this protein in the cystic fibrosis inflammatory process exacerbated by P. aeruginosa infection....

  1. p8 inhibits the growth of human pancreatic cancer cells and its expression is induced through pathways involved in growth inhibition and repressed by factors promoting cell growth

    Directory of Open Access Journals (Sweden)

    Vasseur Sophie

    2003-11-01

    Full Text Available Abstract Background p8 is a stress-induced protein with multiple functions and biochemically related to the architectural factor HMG-I/Y. We analyzed the expression and function of p8 in pancreatic cancer-derived cells. Methods Expression of p8 was silenced in the human pancreatic cancer cell lines Panc-1 and BxPc-3 by infection with a retrovirus expressing p8 RNA in the antisense orientation. Cell growth was measured in control and p8-silenced cells. Influence on p8 expression of the induction of intracellular pathways promoting cellular growth or growth arrest was monitored. Results p8-silenced cells grew more rapidly than control cells transfected with the empty retrovirus. Activation of the Ras→Raf→MEK→ERK and JNK intracellular pathways down-regulated p8 expression. In addition, the MEK1/2 inhibitor U0126 and the JNK inhibitor SP600125 up-regulates expression of p8. Conversely, p38 or TGFβ-1 induced p8 expression whereas the specific p38 inhibitor SB203580 down-regulated p8 expression. Finally, TGFβ-1 induction was in part mediated through p38. Conclusions p8 inhibits the growth of human pancreatic cancer cells. p8 expression is induced through pathways involved in growth inhibition and repressed by factors that promote cell growth. These results suggest that p8 belongs to a pathway regulating the growth of pancreatic cancer cells.

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

    DEFF Research Database (Denmark)

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

    2005-01-01

    By generating a specialized cDNA library from the archaeon Sulfolobus solfataricus, we have identified 57 novel small non-coding RNA (ncRNA) candidates and confirmed their expression by Northern blot analysis. The majority was found to belong to one of two classes, either antisense or antisense...... elements by inhibiting expression of the transposase mRNA. Surprisingly, the class of antisense RNAs also contained RNAs complementary to tRNAs or sRNAs (small-nucleolar-like RNAs). For the antisense-box ncRNAs, the majority could be assigned to the class of C/D sRNAs, which specify 2'-O-methylation sites...... on rRNAs or tRNAs. Five C/D sRNAs of this group are predicted to target methylation at six sites in 13 different tRNAs, thus pointing to the widespread role of these sRNA species in tRNA modification in Archaea. Another group of antisense-box RNAs, lacking typical C/D sRNA motifs, was predicted...

  3. Transfection of mouse cytotoxic T lymphocyte with an antisense granzyme A vector reduces lytic activity.

    Science.gov (United States)

    Talento, A; Nguyen, M; Law, S; Wu, J K; Poe, M; Blake, J T; Patel, M; Wu, T J; Manyak, C L; Silberklang, M

    1992-12-15

    Murine CTL have seven serine proteases, known as granzymes, in their lytic granules. Despite considerable effort, convincing evidence that these enzymes play an obligatory role in the lytic process has not been presented. To investigate the function of one of these proteases, granzyme A (GA), we utilized an antisense expression vector to lower the level of the enzyme in the cells. An expression vector containing antisense cDNA for GA and the gene for hygromycin B resistance was constructed and electroporated into the murine CTL line, AR1. Transfectants were selected based on resistance to hygromycin B, and a number of stable lines were developed. One of the antisense lines had greatly reduced levels of GA mRNA, when compared to the parental cells or to control lines transfected with the vector lacking the antisense DNA. The message levels for two other CTL granule proteins, granzyme B and perforin, were unaffected by the antisense vector. The amount of GA, as measured by enzymatic activity, was 3- to 10-fold lower in the transfectant. Most significantly, this line also consistently showed 50 to 70% lower ability to lyse nucleated target cells and to degrade their DNA. Furthermore, it exhibited 90 to 95% lower lytic activity to anti-CD3-coated SRBC. Conjugate formation with target cells, however, was normal. These data provide strong evidence that GA plays an important role in the cytolytic cycle, and that the quantity of enzyme is a limiting factor in these cytolytic cells.

  4. Profiled support vector machines for antisense oligonucleotide efficacy prediction

    Directory of Open Access Journals (Sweden)

    Martín-Guerrero José D

    2004-09-01

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

  5. TGF-beta induces connexin43 gene expression in normal murine mammary gland epithelial cells via activation of p38 and PI3K/AKT signaling pathways.

    Science.gov (United States)

    Tacheau, Charlotte; Fontaine, Juliette; Loy, Jennifer; Mauviel, Alain; Verrecchia, Franck

    2008-12-01

    One of the shared physiological roles between TGF-beta and connexin family members is to inhibit epithelial cell cycle progression and consequently, to provide protection against malignant transformation. Herein, we demonstrated that TGF-beta1 induces the expression of connexin43 (Cx43) in normal murine mammary gland (NMuMG) cell lines at the protein and mRNA levels, and transcriptionally. Using overexpression of a truncated dominant-negative form of Cx43, we determined that the modulation of gap junctional communication by TGF-beta1 plays a key role in the control of NMuMG cells proliferation by TGF-beta1. In addition, using overexpression of truncated dominant-negative forms of either Smad2 or Smad3, and MDA-MB-468 human breast carcinoma cells deficient for Smad4, we determined that the Smad cascade is not implicated in TGF-beta1 effect on Cx43 expression. Using specific pharmacologic inhibitors for JNK, ERK, p38, and PI3K/AKT signaling pathways, we demonstrated the cooperative role of p38 and PI3K/AKT signaling in TGF-beta1-induced Cx43 expression and gap junctional communication. Furthermore, transfection of a c-jun antisense expression vector significantly prevented TGF-beta1-induced Cx43 gene expression demonstrating the involvement of c-Jun/AP-1 pathway together with p38 and PI3K/AKT pathways in mediating TGF-beta1-induced Cx43 gene expression.

  6. Role of natural antisense transcripts pertaining to tumor suppressor genes in human carcinomas

    International Nuclear Information System (INIS)

    Pelicci, G.; Pierotti, M.

    2009-01-01

    Overlapping transcripts in opposite orientations can potentially form perfect sense-antisense duplex RNA. Recently, several studies have revealed the extent of natural antisense transcripts (NATs) and their role in important biological phenomena also in higher organisms. In order to test the hypothesis that the function of NATs in man might represent an essential element in the regulation of gene expression, especially at transcriptional level, in this study we planned to look for, systematically examine, and characterize NATs belonging in the human genome to the tumour suppressor class of genes, so to identify physiological (and potentially pathological) modulators in this gene class

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

    DEFF Research Database (Denmark)

    Shiraishi, Takehiko; Nielsen, Peter E

    2012-01-01

    We have explored the merits of a novel delivery strategy for the antisense oligomers based on cell penetrating peptide (CPP) conjugated to a carrier PNA with sequence complementary to part of the antisense oligomer. The effect of these carrier CPP-PNAs was evaluated by using antisense PNA targeting......-PNA (cPNA1(7)-(D-Arg)8) and hexamer carrier decanoyl-CPP-PNA (Deca-cPNA1(6)-(D-Arg)8), respectively, without showing significant additional cellular toxicity. Most interestingly, the activity reached the same level obtained by enhancement with endosomolytic chloroquine (CQ) treatment, suggesting...... 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...

  8. 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...

  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.

  10. Growth inhibition of human pancreatic cancer cells by lipofection mediated IGF-1R antisense oligodeoxynucletides in combination with ionizing radiation

    International Nuclear Information System (INIS)

    Pan Yaozhen; Sun Chengyi; Wang Yuzhi

    2004-01-01

    Objective: To study the growth inhibition of human pancreatic cancer cells (PC-3) by lipofection-mediated and ionizing radiation improving transfection of IGF-1R antisense oligodeoxynucletides (ASON) in vitro. Methods: Colonigenicity of PC-3 cells in vitro after 60 Co γ-radiation was observed for ascertaining their radiosensitivity and optimal radiation dose was selected according to the radiation sensitivity. PC-3 cells were transfected by two ways: 1) by lipofection-mediated IGF-1R ASON combined with ionizing radiation. 2) by lipo-ASON alone without ionizing radiation. Cell growth was assessed by MTT method. The expression of IGF-1R at mRNA level was examined by RT-PCR. Flow cytometry was used to demonstrate apoptotic changes in lipo-ASON-treated cells. Results: The inhibitory efficiency of lipo-ASON combined with ionizing radiation was higher than that without ionizing radiation (P < 0.05). The apoptotic efficiency and the decreased level of IGF-1R at mRNA were significantly improved (P < 0.05). Conclusion: Lipofection-mediated and ionizing radiation-promoted transfection of IGF-1R antisense oligodeoxynucletides (ASON) significantly decreases IGF-1R at mRNA level and induces apoptosis of human pancreatic cancer cells in vitro

  11. Scintigraphic imaging of oncogenes with antisense probes: does it make sense?

    International Nuclear Information System (INIS)

    Urbain, J.L.C.; Shore, S.K.; Vekemans, M.C.; Cosenza, S.C.; DeRiel, K.; Patel, G.V.; Charkes, N.D.; Malmud, L.S.; Reddy, E.P.

    1995-01-01

    The aim of this study was to demonstrate that cells which are expressing a particular mRNA transcript do preferentially and specifically retain the antisense probe targeting that mRNA. Using a mouse plasmacytoma cell line (MOPC315) which produces high levels of IgA heavy chain mRNA, a control mouse pre B cell line (7OZ/3B), a human mammary cell line (MCF7) which expresses the erbB2 or neu oncogene, MOPC315 cells as neu-negative controls, and antisense DNA oligonucleotides complementary to the 5' region of the mRNAs and the sense sequence, we have shown that there is a preferential, specific retention of the IgA and neu antisense sequence in MOPC315 and MCF7 cells, respectively. We have further demonstrated that this retention is time and concentration dependent with a maximum at 24 h. We conclude that cancer cells which express a particular oncogene are suitable targets for radiolabeled antisense deoxyoligonucleotides directed toward the oncogene transcript. (orig.)

  12. Hypoxia-inducible factor-2α-dependent hypoxic induction of Wnt10b expression in adipogenic cells.

    Science.gov (United States)

    Park, Young-Kwon; Park, Bongju; Lee, Seongyeol; Choi, Kang; Moon, Yunwon; Park, Hyunsung

    2013-09-06

    Adipocyte hyperplasia and hypertrophy in obesity can lead to many changes in adipose tissue, such as hypoxia, metabolic dysregulation, and enhanced secretion of cytokines. In this study, hypoxia increased the expression of Wnt10b in both human and mouse adipogenic cells, but not in hypoxia-inducible factor (HIF)-2α-deficient adipogenic cells. Chromatin immunoprecipitation analysis revealed that HIF-2α, but not HIF-1α, bound to the Wnt10b enhancer region as well as upstream of the Wnt1 gene, which is encoded by an antisense strand of the Wnt10b gene. Hypoxia-conditioned medium (H-CM) induced phosphorylation of lipoprotein-receptor-related protein 6 as well as β-catenin-dependent gene expression in normoxic cells, which suggests that H-CM contains canonical Wnt signals. Furthermore, adipogenesis of both human mesenchymal stem cells and mouse preadipocytes was inhibited by H-CM even under normoxic conditions. These results suggest that O2 concentration gradients influence the formation of Wnt ligand gradients, which are involved in the regulation of pluripotency, cell proliferation, and cell differentiation.

  13. Ets-2 and p53 mediate cAMP-induced MMP-2 expression, activity and trophoblast invasion

    Directory of Open Access Journals (Sweden)

    Goldman Shlomit

    2009-11-01

    Full Text Available Abstract Background We have previously shown that Matrix metalloproteinase (MMP -2 is a key-enzyme in early trophoblast invasion and that Protein Kinase A (PKA increases MMP-2 expression and trophoblast invasion. The aim of this study was to examine MMP -2 regulation by PKA in invasive trophoblasts: JAR choriocarcinoma cell-line and 6-8 w first trimester trophoblasts. Methods The effect of Forskolin (PKA on MMP-2 expression was assessed by Northern Blot and RT-PCR. Possible transcription factors binding to consensus MMP-2 promoter sequences in response to Forskolin, were detected by EMSA binding assay and their expression assessed by western blot analysis. Antisense transfection of relevant transcription factors was performed and the inhibitory effect assessed on MMP-2 expression (RT-PCR, secretion (zymography and trophoblast invasiveness (transwell migration assay. Results We found that Forskolin increased MMP-2 mRNA in JAR cells within 24 hours, and induced binding to p53, Ets, C/EBP and AP-2. Transcription factors Ets-2, phospho- p53, C/EBP epsilon, C/EBP lambda and AP-2 alpha bound to their respective binding sequences in response to Forskolin and the expressions of these transcription factors were all elevated in Forskolin- treated cells. Inhibition of Ets-2 and p53 reduced MMP-2 expression, secretion and invasiveness of Forskolin treated cells. Conclusion MMP-2 is regulated by PKA through several binding sites and transcription factors including Ets-2, p53, C/EBP, C/EBP lambda and AP-2 alpha. Ets-2 and p53 mediate cAMP- induced trophoblast invasiveness, through regulation of MMP-2.

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

    Directory of Open Access Journals (Sweden)

    Wahlestedt Claes

    2007-03-01

    Full Text Available Abstract Background Mutations in the PTEN induced putative kinase 1 (PINK1 are implicated in early-onset Parkinson's disease. PINK1 is expressed abundantly in mitochondria rich tissues, such as skeletal muscle, where it plays a critical role determining mitochondrial structural integrity in Drosophila. Results Herein we characterize a novel splice variant of PINK1 (svPINK1 that is homologous to the C-terminus regulatory domain of the protein kinase. Naturally occurring non-coding antisense provides sophisticated mechanisms for diversifying genomes and we describe a human specific non-coding antisense expressed at the PINK1 locus (naPINK1. We further demonstrate that PINK1 varies in vivo when human skeletal muscle mitochondrial content is enhanced, supporting the idea that PINK1 has a physiological role in mitochondrion. The observation of concordant regulation of svPINK1 and naPINK1 during in vivo mitochondrial biogenesis was confirmed using RNAi, where selective targeting of naPINK1 results in loss of the PINK1 splice variant in neuronal cell lines. Conclusion Our data presents the first direct observation that a mammalian non-coding antisense molecule can positively influence the abundance of a cis-transcribed mRNA under physiological abundance conditions. While our analysis implies a possible human specific and dsRNA-mediated mechanism for stabilizing the expression of svPINK1, it also points to a broader genomic strategy for regulating a human disease locus and increases the complexity through which alterations in the regulation of the PINK1 locus could occur.

  15. Protective effect of c-fos antisense oligonucleotides on brain damage induced by glutamate%c-fos反义寡核苷酸对谷氨酸神经毒性鼠脑损伤的防护

    Institute of Scientific and Technical Information of China (English)

    岳少杰; 陶永光; 罗自强; 冯德云; 伍赶球

    2001-01-01

    Objective To investigate the relation between glutamate neurotoxicity and c-fos gene expression. Methods c-fos antisense oligonucleotides (AS ODN) was injected into the right lateral ventricles of 9 SD rats to block the c-fos gene expression in brain tissue. c-fos sense oligonucleotides (S ODN)was used a control. The numbers and morphology of neurons in both cerebral cortex and hippocampal CA1 were detected by MIAS-300 image analysing instrument. c-fos gene expression in brain was observed by immunohistochemical method. The content of water and electrolytes in the brain tissue and Ca2+ in the synapse were measured. Results The c-fos AS ODN blocked the c-fos gene expression and reduced the content of both water and sodium in brain tissue and Ca2+ in symptosome, thus alleviating the morphological damage in neuron. S ODN did not have such effect. Conclusion c-fos gene expression plays an important role in mediating the effect of glutamate neurotoxicity. Blocking the c-fos gene expression could antagonize glutamate neurotoxicity.%目的 探讨c-fos基因的表达在谷氨酸神经毒性中的作用。方法 在9只SD大鼠侧脑室注射c-fos反义寡核苷酸以阻断脑组织c-fos基因的表达,并用c-fos正义寡核苷酸为对照。观察脑组织中水、电解质含量和突触体内Ca2+浓度的变化,并采用细胞形态计量分析及免疫组织化学方法,观察大脑皮质、海马CA1区神经细胞数目、形态的变化及c-fos基因的表达。结果 c-fos反义寡核苷酸可有效地阻断脑组织c-fos基因的表达,降低脑组织c-fos阳性细胞率(9.4%±2.8%和74%±3%,P<0.01),抑制谷氨酸神经毒性所致的脑组织含水量(79.9%±0.4%和82.3%±0.8%,P<0.01)、钠(5.05 mg/g干重±0.39 mg/g干重和5.98 mg/g干重±0.50 mg/g干重,P<0.01)及细胞内Ca2+(176 nmol/L±35 nmol/L和344.12±50.13,P<0.01)含量的增加,抑制谷氨酸所致大脑皮质(157±10和145±7,P<0

  16. 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.

  17. 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.

  18. A Transcriptional Regulatory Network Containing Nuclear Receptors and Long Noncoding RNAs Controls Basal and Drug-Induced Expression of Cytochrome P450s in HepaRG Cells.

    Science.gov (United States)

    Chen, Liming; Bao, Yifan; Piekos, Stephanie C; Zhu, Kexin; Zhang, Lirong; Zhong, Xiao-Bo

    2018-07-01

    Cytochrome P450 (P450) enzymes are responsible for metabolizing drugs. Expression of P450s can directly affect drug metabolism, resulting in various outcomes in therapeutic efficacy and adverse effects. Several nuclear receptors are transcription factors that can regulate expression of P450s at both basal and drug-induced levels. Some long noncoding RNAs (lncRNAs) near a transcription factor are found to participate in the regulatory functions of the transcription factors. The aim of this study is to determine whether there is a transcriptional regulatory network containing nuclear receptors and lncRNAs controlling both basal and drug-induced expression of P450s in HepaRG cells. Small interfering RNAs or small hairpin RNAs were applied to knock down four nuclear receptors [hepatocyte nuclear factor 1 α (HNF1 α ), hepatocyte nuclear factor 4 α (HNF4 α ), pregnane X receptor (PXR), and constitutive androstane receptor (CAR)] as well as two lncRNAs [HNF1 α antisense RNA 1 (HNF1 α -AS1) and HNF4 α antisense RNA 1 (HNF4 α -AS1)] in HepaRG cells with or without treatment of phenobarbital or rifampicin. Expression of eight P450 enzymes was examined in both basal and drug-induced levels. CAR and PXR mainly regulated expression of specific P450s. HNF1 α and HNF4 α affected expression of a wide range of P450s as well as other transcription factors. HNF1 α and HNF4 α controlled the expression of their neighborhood lncRNAs, HNF1 α -AS1 and HNF4 α -AS1, respectively. HNF1 α -AS1 and HNF4 α -AS1 was also involved in the regulation of P450s and transcription factors in diverse manners. Altogether, our study concludes that a transcription regulatory network containing the nuclear receptors and lncRNAs controls both basal and drug-induced expression of P450s in HepaRG cells. Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.

  19. Suppression of cell division by pKi-67 antisense-RNA and recombinant protein.

    Science.gov (United States)

    Duchrow, M; Schmidt, M H; Zingler, M; Anemüller, S; Bruch, H P; Broll, R

    2001-01-01

    The human antigen defined by the monoclonal antibody Ki-67 (pKi-67) is a human nuclear protein strongly associated with cell proliferation and found in all tissues studied. It is widely used as a marker of proliferating cells, yet its function is unknown. To investigate its function we suppressed pKi-67 expression by antisense RNA and overexpressed a partial structure of pKi-67 in HeLa cells. A BrdU-incorporation assay showed a significant decrease in DNA synthesis after antisense inhibition. Cell cycle analysis indicated a higher proportion of cells in G1 phase and a lower proportion of cells in S phase while the number of G(2)/M phase cells remained constant. Overexpression of a recombinant protein encoding three of the repetitive elements from exon 13 of pKi-67 had a similar effect to that obtained by antisense inhibition. The similarity of the effect of expressing 'Ki-67 repeats' and pKi-67 antisense RNA could be explained by a negative effect on the folding of the endogenous protein in the endoplasmatic reticulum. Furthermore excessive self-association of pKi-67 via the repeat structure could inhibit its nuclear transport, preventing it from getting to its presumptive site of action. We conclude that the Ki-67 protein has an important role in the regulation of the cell cycle, which is mediated in part by its repetitive elements. Copyright 2001 S. Karger AG, Basel

  20. Down-regulation of the antisense mitochondrial non-coding RNAs (ncRNAs) is a unique vulnerability of cancer cells and a potential target for cancer therapy.

    Science.gov (United States)

    Vidaurre, Soledad; Fitzpatrick, Christopher; Burzio, Verónica A; Briones, Macarena; Villota, Claudio; Villegas, Jaime; Echenique, Javiera; Oliveira-Cruz, Luciana; Araya, Mariela; Borgna, Vincenzo; Socías, Teresa; Lopez, Constanza; Avila, Rodolfo; Burzio, Luis O

    2014-09-26

    Hallmarks of cancer are fundamental principles involved in cancer progression. We propose an additional generalized hallmark of malignant transformation corresponding to the differential expression of a family of mitochondrial ncRNAs (ncmtRNAs) that comprises sense and antisense members, all of which contain stem-loop structures. Normal proliferating cells express sense (SncmtRNA) and antisense (ASncmtRNA) transcripts. In contrast, the ASncmtRNAs are down-regulated in tumor cells regardless of tissue of origin. Here we show that knockdown of the low copy number of the ASncmtRNAs in several tumor cell lines induces cell death by apoptosis without affecting the viability of normal cells. In addition, knockdown of ASncmtRNAs potentiates apoptotic cell death by inhibiting survivin expression, a member of the inhibitor of apoptosis (IAP) family. Down-regulation of survivin is at the translational level and is probably mediated by microRNAs generated by dicing of the double-stranded stem of the ASncmtRNAs, as suggested by evidence presented here, in which the ASncmtRNAs are bound to Dicer and knockdown of the ASncmtRNAs reduces reporter luciferase activity in a vector carrying the 3'-UTR of survivin mRNA. Taken together, down-regulation of the ASncmtRNAs constitutes a vulnerability or Achilles' heel of cancer cells, suggesting that the ASncmtRNAs are promising targets for cancer therapy. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Down-regulation of the Antisense Mitochondrial Non-coding RNAs (ncRNAs) Is a Unique Vulnerability of Cancer Cells and a Potential Target for Cancer Therapy*

    Science.gov (United States)

    Vidaurre, Soledad; Fitzpatrick, Christopher; Burzio, Verónica A.; Briones, Macarena; Villota, Claudio; Villegas, Jaime; Echenique, Javiera; Oliveira-Cruz, Luciana; Araya, Mariela; Borgna, Vincenzo; Socías, Teresa; Lopez, Constanza; Avila, Rodolfo; Burzio, Luis O.

    2014-01-01

    Hallmarks of cancer are fundamental principles involved in cancer progression. We propose an additional generalized hallmark of malignant transformation corresponding to the differential expression of a family of mitochondrial ncRNAs (ncmtRNAs) that comprises sense and antisense members, all of which contain stem-loop structures. Normal proliferating cells express sense (SncmtRNA) and antisense (ASncmtRNA) transcripts. In contrast, the ASncmtRNAs are down-regulated in tumor cells regardless of tissue of origin. Here we show that knockdown of the low copy number of the ASncmtRNAs in several tumor cell lines induces cell death by apoptosis without affecting the viability of normal cells. In addition, knockdown of ASncmtRNAs potentiates apoptotic cell death by inhibiting survivin expression, a member of the inhibitor of apoptosis (IAP) family. Down-regulation of survivin is at the translational level and is probably mediated by microRNAs generated by dicing of the double-stranded stem of the ASncmtRNAs, as suggested by evidence presented here, in which the ASncmtRNAs are bound to Dicer and knockdown of the ASncmtRNAs reduces reporter luciferase activity in a vector carrying the 3′-UTR of survivin mRNA. Taken together, down-regulation of the ASncmtRNAs constitutes a vulnerability or Achilles' heel of cancer cells, suggesting that the ASncmtRNAs are promising targets for cancer therapy. PMID:25100722

  2. Molecular characterization of long direct repeat (LDR) sequences expressing a stable mRNA encoding for a 35-amino-acid cell-killing peptide and a cis-encoded small antisense RNA in Escherichia coli.

    Science.gov (United States)

    Kawano, Mitsuoki; Oshima, Taku; Kasai, Hiroaki; Mori, Hirotada

    2002-07-01

    Genome sequence analyses of Escherichia coli K-12 revealed four copies of long repetitive elements. These sequences are designated as long direct repeat (LDR) sequences. Three of the repeats (LDR-A, -B, -C), each approximately 500 bp in length, are located as tandem repeats at 27.4 min on the genetic map. Another copy (LDR-D), 450 bp in length and nearly identical to LDR-A, -B and -C, is located at 79.7 min, a position that is directly opposite the position of LDR-A, -B and -C. In this study, we demonstrate that LDR-D encodes a 35-amino-acid peptide, LdrD, the overexpression of which causes rapid cell killing and nucleoid condensation of the host cell. Northern blot and primer extension analysis showed constitutive transcription of a stable mRNA (approximately 370 nucleotides) encoding LdrD and an unstable cis-encoded antisense RNA (approximately 60 nucleotides), which functions as a trans-acting regulator of ldrD translation. We propose that LDR encodes a toxin-antitoxin module. LDR-homologous sequences are not pre-sent on any known plasmids but are conserved in Salmonella and other enterobacterial species.

  3. 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. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2009-12-15

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

  5. Peripheral administration of antisense oligonucleotides targeting the amyloid-β protein precursor reverses AβPP and LRP-1 overexpression in the aged SAMP8 mouse brain.

    Science.gov (United States)

    Erickson, Michelle A; Niehoff, Michael L; Farr, Susan A; Morley, John E; Dillman, Lucy A; Lynch, Kristin M; Banks, William A

    2012-01-01

    The senescence accelerated mouse-prone 8 (SAMP8) mouse model of Alzheimer's disease has a natural mutation leading to age-related increases in the amyloid-β protein precursor (AβPP) and amyloid-β (Aβ) in the brain, memory impairment, and deficits in Aβ removal from the brain. Previous studies show that centrally administered antisense oligonucleotide directed against AβPP can decrease AβPP expression and Aβ production in the brains of aged SAMP8 mice, and improve memory. The same antisense crosses the blood-brain barrier and reverses memory deficits when injected intravenously. Here, we give 6 μg of AβPP or control antisense 3 times over 2 week intervals to 12 month old SAMP8 mice. Object recognition test was done 48 hours later, followed by removal of whole brains for immunoblot analysis of AβPP, low-density lipoprotein-related protein-1 (LRP-1), p-glycoprotein (Pgp), receptor for advanced glycation endproducts (RAGE), or ELISA of soluble Aβ(40). Our results show that AβPP antisense completely reverses a 30% age-associated increase in AβPP signal (p < 0.05 versus untreated 4 month old SAMP8). Soluble Aβ(40) increased with age, but was not reversed by antisense. LRP-1 large and small subunits increased significantly with age (147.7%, p < 0.01 and 123.7%, p < 0.05 respectively), and AβPP antisense completely reversed these increases (p < 0.05). Pgp and RAGE were not significantly altered with age or antisense. Antisense also caused improvements in memory (p < 0.001). Together, these data support the therapeutic potential of AβPP antisense and show a unique association between AβPP and LRP-1 expression in the SAMP8 mouse.

  6. Antisense and sense expression of cDNA coding for CYP73A15, a class II cinnamate 4-hydroxylase, leads to a delayed and reduced production of lignin in tobacco

    Science.gov (United States)

    Blee, K.; Choi, J. W.; O'Connell, A. P.; Jupe, S. C.; Schuch, W.; Lewis, N. G.; Bolwell, G. P.

    2001-01-01

    A number of plant species contain the class II of genes encoding the cytochrome P450, CYP73, the cognate protein of which cinnamic acid 4-hydroxylase, is the second enzyme of the phenylpropanoid pathway. In order to begin to determine possible functionality, tobacco has been transformed with a truncated French bean class II cinnamate hydroxylase (CYP73A15) in the sense and antisense orientations. Signals for C4H protein could be detected in vascular tissue from wild-type plants using heterologous probes. The transformed plants showed a normal phenotype, even though detectable C4H protein was much reduced in tissue prints. Young propagated transformants displayed a range of reduced C4H activities, as well as either reduced or no phloroglucinol-stainable lignin. However, all mature tobacco plants showed the accumulation of lignin, even though its deposition was apparently delayed. This was not due to induction of tyrosine ammonia-lyase activity, which was not detected, but instead it is presumed due to sufficient C4H residual activity. Analysis of the lignin content of the plants showed reductions of up to 30% with a slightly reduced syringyl to guaiacyl ratio as compared to wild type. This reduction level was favourable in comparison with some other targets in the lignification pathway that have been manipulated including that of class I cinnamate 4-hydroxylase. It is proposed that the class II cinnamate 4-hydroxylase might also function in lignification in a number of species including French bean and tobacco, based on these data.

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

    International Nuclear Information System (INIS)

    Annab, Lois A; Hawkins, Rebecca E; Solomon, Greg; Barrett, J Carl; Afshari, Cynthia A

    2000-01-01

    antisense clone that had significantly low levels of BRCA1 protein was able to form twice as many tumors in ovariectomized nude mice with a decreased latency compared with a control clone. In multicellular mammalian organisms, a balance between cell proliferation and cell death is extremely important for the maintenance of normal healthy tissues. In support of this hypothesis, it has been shown that p53 and BRCA1 can form stable complexes, and can coactivate p21 and bax genes, which may lead to the activation of the apoptosis pathway [15]. The present data, which show that cells with a reduction of BRCA1 have a survival advantage in conditions where control cells fail to thrive, also supports this hypothesis. BRCA1 levels appear to affect the ability of cells to arrest growth or die in the absence of estrogenic growth-inducing conditions. Although mutations in this gene are uncommon in sporadic breast and ovarian tumors, BRCA1 expression levels and protein levels have been found to be reduced in sporadic human breast carcinomas [16,17,18,19]. In addition it has been demonstrated [20] that hormone-dependent tumors such as breast and ovarian cancers have a decreased ability to undergo apoptosis. Other mechanisms involving gene regulation may allow for decreased expression of BRCA1 in sporadic tumors. The response of BRCA1 mRNA and protein levels to mitogens and hormones in vitro suggests that BRCA1 may play a role in regulation of cell growth or maintenance [21]. The BRCA1 gene product may be involved in the regulation of hormone response pathways, and the present results demonstrate that loss of BRCA1 may result in loss of inhibitory control of these mitogenic pathways. These studies show that reduction in BRCA1 mRNA and protein can result in increased proliferation of BG-1 ovarian cancer cells in both in vitro and in vivo conditions, suggesting that BRCA1 may normally be acting as a growth inhibitor. Low BRCA1 levels found in sporadic cancers may be an important factor in

  8. 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.

  9. Wound-induced expression of horseradish peroxidase.

    Science.gov (United States)

    Kawaoka, A; Kawamoto, T; Ohta, H; Sekine, M; Takano, M; Shinmyo, A

    1994-01-01

    Peroxidases have been implicated in the responses of plants to physiological stress and to pathogens. Wound-induced peroxidase of horseradish (Armoracia rusticana) was studied. Total peroxidase activity was increased by wounding in cell wall fractions extracted from roots, stems and leaves of horseradish. On the other hand, wounding decreased the peroxidase activity in the soluble fraction from roots. The enzyme activities of the basic isozymes were induced by wounding in horseradish leaves based on data obtained by fractionation of crude enzyme in isoelectric focusing gel electrophoresis followed by activity staining. We have previously isolated genomic clones for four peroxidase genes, namely, prxC1a, prxC1b, prxC2 and prxC3. Northern blot analysis using gene-specific probes showed that mRNA of prxC2, which encodes a basic isozyme, accumulated by wounding, while the mRNAs for other peroxidase genes were not induced. Tobacco (Nicotiana tabacum) plants were transformed with four chimeric gene constructs, each consisting of a promoter from one of the peroxidase genes and the β-glucuronidase (GUS) structural gene. High level GUS activity induced in response to wounding was observed in tobacco plants containing the prxC2-GUS construct.

  10. Ligand Binding Domain Protein in Tetracycline-Inducible Expression

    African Journals Online (AJOL)

    Purpose: To investigate tetracycline-inducible expression system for producing clinically usable, highquality liver X receptor ligand-binding domain recombinant protein. Methods: In this study, we have expressed and purified the recombinant liver X receptor β-ligand binding domain proteins in E. coli using a tetracycline ...

  11. 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

  12. Recent advances in antisense oligonucleotide therapy in genetic neuromuscular diseases

    Directory of Open Access Journals (Sweden)

    Ashok Verma

    2018-01-01

    Full Text Available Genetic neuromuscular diseases are caused by defective expression of nuclear or mitochondrial genes. Mutant genes may reduce expression of wild-type proteins, and strategies to activate expression of the wild-type proteins might provide therapeutic benefits. Also, a toxic mutant protein may cause cell death, and strategies that reduce mutant gene expression may provide therapeutic benefit. Synthetic antisense oligonucleotide (ASO can recognize cellular RNA and control gene expression. In recent years, advances in ASO chemistry, creation of designer ASO molecules to enhance their safety and target delivery, and scientific controlled clinical trials to ascertain their therapeutic safety and efficacy have led to an era of plausible application of ASO technology to treat currently incurable neuromuscular diseases. Over the past 1 year, for the first time, the United States Food and Drug Administration has approved two ASO therapies in genetic neuromuscular diseases. This overview summarizes the recent advances in ASO technology, evolution and use of synthetic ASOs as a therapeutic platform, and the mechanism of ASO action by exon-skipping in Duchenne muscular dystrophy and exon-inclusion in spinal muscular atrophy, with comments on their advantages and limitations.

  13. Differential expression of ozone-induced gene during exposures to ...

    African Journals Online (AJOL)

    Differential expression of ozone-induced gene during exposures to salt stress in Polygonum sibiricum Laxm leaves, stem and underground stem. ... PcOZI-1 mRNA in untreated plants was detected at low levels in underground stem, leaves and at higher levels in stem. PcOZI-1 mRNA accumulation was transiently induced ...

  14. Central and peripheral administration of antisense oligonucleotide targeting amyloid-β protein precursor improves learning and memory and reduces neuroinflammatory cytokines in Tg2576 (AβPPswe) mice.

    Science.gov (United States)

    Farr, Susan A; Erickson, Michelle A; Niehoff, Michael L; Banks, William A; Morley, John E

    2014-01-01

    Alzheimer's disease (AD) is a progressive neurodegenerative disease. Currently, there are no therapies to stop or reverse the symptoms of AD. We have developed an antisense oligonucleotide (OL-1) against the amyloid-β protein precursor (AβPP) that can decrease AβPP expression and amyloid-β protein (Aβ) production. This antisense rapidly crosses the blood-brain barrier, reverses learning and memory impairments, reduces oxidative stress, and restores brain-to-blood efflux of Aβ in SAMP8 mice. Here, we examined the effects of this AβPP antisense in the Tg2576 mouse model of AD. We administered the OL-1 antisense into the lateral ventricle 3 times at 2week intervals. Seventy-two hours after the third injection, we tested learning and memory in T-maze foot shock avoidance. In the second study, we injected the mice with OL-1 antisense 3 times at 2-week intervals via the tail vein. Seventy-two hours later, we tested learning and memory T-maze, novel object recognition, and elevated plus maze. At the end of behavioral testing, brain tissue was collected. OL-1 antisense administered centrally improved acquisition and retention of T-maze foot shock avoidance. OL-1 antisense administered via tail vein improved learning and memory in both T-maze foot shock avoidance and novel object-place recognition. In the elevated plus maze, the mice which received OL-1 antisense spent less time in the open arms and had fewer entries into the open arms indicating reduced disinhibitation. Biochemical analyses reveal significant reduction of AβPP signal and a reduction of measures of neuroinflammation. The current findings support the therapeutic potential of OL-1 AβPP antisense.

  15. Targeting TGF-β Signaling by Antisense Oligonucleotide-mediated Knockdown of TGF-β Type I Receptor

    Directory of Open Access Journals (Sweden)

    Dwi U Kemaladewi

    2014-01-01

    Full Text Available Duchenne muscular dystrophy (DMD is caused by lack of functional dystrophin and results in progressive myofiber damage and degeneration. In addition, impaired muscle regeneration and fibrosis contribute to the progressive pathology of DMD. Importantly, transforming growth factor-β (TGF-β is implicated in DMD pathology and is known to stimulate fibrosis and inhibit muscle regeneration. In this study, we present a new strategy to target TGF-β signaling cascades by specifically inhibiting the expression of TGF-β type I receptor TGFBR1 (ALK5. Antisense oligonucleotides (AONs were designed to specifically induce exon skipping of mouse ALK5 transcripts. AON-induced exon skipping of ALK5 resulted in specific downregulation of full-length receptor transcripts in vitro in different cell types, repression of TGF-β activity, and enhanced C2C12 myoblast differentiation. To determine the effect of these AONs in dystrophic muscles, we performed intramuscular injections of ALK5 AONs in mdx mice, which resulted in a decrease in expression of fibrosis-related genes and upregulation of Myog expression compared to control AON-injected muscles. In summary, our study presents a novel method to target TGF-β signaling cascades with potential beneficial effects for DMD.

  16. Antisense pre-treatment increases gene therapy efficacy in dystrophic muscles.

    Science.gov (United States)

    Peccate, Cécile; Mollard, Amédée; Le Hir, Maëva; Julien, Laura; McClorey, Graham; Jarmin, Susan; Le Heron, Anita; Dickson, George; Benkhelifa-Ziyyat, Sofia; Piétri-Rouxel, France; Wood, Matthew J; Voit, Thomas; Lorain, Stéphanie

    2016-08-15

    In preclinical models for Duchenne muscular dystrophy, dystrophin restoration during adeno-associated virus (AAV)-U7-mediated exon-skipping therapy was shown to decrease drastically after six months in treated muscles. This decline in efficacy is strongly correlated with the loss of the therapeutic AAV genomes, probably due to alterations of the dystrophic myofiber membranes. To improve the membrane integrity of the dystrophic myofibers at the time of AAV-U7 injection, mdx muscles were pre-treated with a single dose of the peptide-phosphorodiamidate morpholino (PPMO) antisense oligonucleotides that induced temporary dystrophin expression at the sarcolemma. The PPMO pre-treatment allowed efficient maintenance of AAV genomes in mdx muscles and enhanced the AAV-U7 therapy effect with a ten-fold increase of the protein level after 6 months. PPMO pre-treatment was also beneficial to AAV-mediated gene therapy with transfer of micro-dystrophin cDNA into muscles. Therefore, avoiding vector genome loss after AAV injection by PPMO pre-treatment would allow efficient long-term restoration of dystrophin and the use of lower and thus safer vector doses for Duchenne patients. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  17. Antisense Oligonucleotide-mediated Exon Skipping as a Systemic Therapeutic Approach for Recessive Dystrophic Epidermolysis Bullosa.

    Science.gov (United States)

    Bremer, Jeroen; Bornert, Olivier; Nyström, Alexander; Gostynski, Antoni; Jonkman, Marcel F; Aartsma-Rus, Annemieke; van den Akker, Peter C; Pasmooij, Anna Mg

    2016-10-18

    The "generalized severe" form of recessive dystrophic epidermolysis bullosa (RDEB-gen sev) is caused by bi-allelic null mutations in COL7A1, encoding type VII collagen. The absence of type VII collagen leads to blistering of the skin and mucous membranes upon the slightest trauma. Because most patients carry exonic point mutations or small insertions/deletions, most exons of COL7A1 are in-frame, and low levels of type VII collagen already drastically improve the disease phenotype, this gene seems a perfect candidate for antisense oligonucleotide (AON)-mediated exon skipping. In this study, we examined the feasibility of AON-mediated exon skipping in vitro in primary cultured keratinocytes and fibroblasts, and systemically in vivo using a human skin-graft mouse model. We show that treatment with AONs designed against exon 105 leads to in-frame exon 105 skipping at the RNA level and restores type VII collagen protein production in vitro. Moreover, we demonstrate that systemic delivery in vivo induces de novo expression of type VII collagen in skin grafts generated from patient cells. Our data demonstrate strong proof-of-concept for AON-mediated exon skipping as a systemic therapeutic strategy for RDEB.

  18. Encapsulation of c-myc antisense oligodeoxynucleotides in lipid particles improves antitumoral efficacy in vivo in a human melanoma line.

    Science.gov (United States)

    Leonetti, C; Biroccio, A; Benassi, B; Stringaro, A; Stoppacciaro, A; Semple, S C; Zupi, G

    2001-06-01

    Phosphorothioate c-myc antisense oligodeoxynucleotides [S]ODNs (free INX-6295) were encapsulated in a new liposome formulation and the antitumor activity was compared to the unencapsulated antisense in a human melanoma xenograft. The systemic administration of INX-6295 encapsulated in stabilized antisense lipid particles (SALP INX-6295) improved plasma AUC (area under the plasma concentration-time curve) and initial half-life of free INX-6295, resulting in a significant enhancement in tumor accumulation and improvement in tumor distribution of antisense oligodeoxynucleotides. Animals treated with SALP INX-6295 exhibited a prolonged reduction of c-myc expression, reduced tumor growth and increased mice survival. When administered in combination with cisplatin (DDP), SALP INX-6295 produced a complete tumor regression in approximately 30% of treated mice, which persisted for at least 60 days following the first cycle of treatment. Finally, the median survival of mice treated with DDP/SALP INX-6295 increased by 105% compared to 84% for animals treated with the combination DDP/free INX-6295. These data indicate that the biological activity and the therapeutic efficacy of c-myc antisense therapy may be improved when these agents are administered in lipid-based delivery systems.

  19. Hippocampal Arc (Arg3.1) expression is induced by memory recall and required for memory reconsolidation in trace fear conditioning.

    Science.gov (United States)

    Chia, Chester; Otto, Tim

    2013-11-01

    Mounting evidence suggests that long-lasting, protein synthesis-dependent changes in synaptic strength accompany both the initial acquisition and subsequent recall of specific memories. Within brain areas thought to be important for learning and memory, including the hippocampus, learning-related plasticity is likely mediated in part by NMDA receptor activation and experience-dependent changes in gene expression. In the present study, we examined the role of activity-regulated cytoskeletal-associated protein (Arc/Arg3.1) expression in the acquisition, recall, and reconsolidation of memory in a trace fear conditioning paradigm. First, we show that the expression of Arc protein in ventral hippocampus (VH) is dramatically enhanced by memory recall 24h after the acquisition of trace fear conditioning, and that both memory recall and the associated recall-induced enhancement of Arc expression are blocked by pre-training administration of 2-amino-5-phosphonovaleric acid (APV). Next, we show that while infusion of Arc antisense oligodeoxynucleotides (ODNs) into VH prior to testing had little effect on memory recall, it significantly reduced both Arc protein expression and freezing behavior during subsequent testing sessions. Collectively, these results suggest that Arc/Arg3.1 protein plays an important functional role in both the initial acquisition of hippocampal-dependent memory and the reconsolidation of these memories after recall. Copyright © 2013 Elsevier Inc. All rights reserved.

  20. Neuroinflammation induces glial aromatase expression in the uninjured songbird brain

    Directory of Open Access Journals (Sweden)

    Saldanha Colin J

    2011-07-01

    Full Text Available Abstract Background Estrogens from peripheral sources as well as central aromatization are neuroprotective in the vertebrate brain. Under normal conditions, aromatase is only expressed in neurons, however following anoxic/ischemic or mechanical brain injury; aromatase is also found in astroglia. This increased glial aromatization and the consequent estrogen synthesis is neuroprotective and may promote neuronal survival and repair. While the effects of estradiol on neuroprotection are well studied, what induces glial aromatase expression remains unknown. Methods Adult male zebra finches (Taeniopygia guttata were given a penetrating injury to the entopallium. At several timepoints later, expression of aromatase, IL-1β-like, and IL-6-like were examined using immunohisotchemistry. A second set of zebra birds were exposed to phytohemagglutinin (PHA, an inflammatory agent, directly on the dorsal surface of the telencephalon without creating a penetrating injury. Expression of aromatase, IL-1β-like, and IL-6-like were examined using both quantitative real-time polymerase chain reaction to examine mRNA expression and immunohistochemistry to determine cellular expression. Statistical significance was determined using t-test or one-way analysis of variance followed by the Tukey Kramers post hoc test. Results Following injury in the zebra finch brain, cytokine expression occurs prior to aromatase expression. This temporal pattern suggests that cytokines may induce aromatase expression in the damaged zebra finch brain. Furthermore, evoking a neuroinflammatory response characterized by an increase in cytokine expression in the uninjured brain is sufficient to induce glial aromatase expression. Conclusions These studies are among the first to examine a neuroinflammatory response in the songbird brain following mechanical brain injury and to describe a novel neuroimmune signal to initiate aromatase expression in glia.

  1. Expression of angiopoietin-1 in hypoxic pericytes: Regulation by hypoxia-inducible factor-2α and participation in endothelial cell migration and tube formation.

    Science.gov (United States)

    Park, Yoon Shin; Kim, Gyungah; Jin, Yoon Mi; Lee, Jee Young; Shin, Jong Wook; Jo, Inho

    2016-01-08

    We previously reported that hypoxia increases angiopoietin-1 (Ang1), but not Ang2, mRNA expression in bovine retinal pericytes (BRP). However, the mechanism underlying Ang1 expression is unknown. Here, we report that Ang1 protein expression increased in hypoxic BRP in a dose- and time-dependent manner. This increase was accompanied by an increase in hypoxia-inducible factor-2α (HIF2α) expression. Transfection with an antisense oligonucleotide for HIF2α partially inhibited the hypoxia-induced increase in Ang1 expression. HIF2α overexpression further potentiated hypoxia-stimulated Ang1 expression, suggesting that HIF2α plays an important role in Ang1 regulation in BRP. When fused the Ang1 promoter (-3040 to +199) with the luciferase reporter gene, we found that hypoxia significantly increased promoter activity by 4.02 ± 1.68 fold. However, progressive 5'-deletions from -3040 to -1799, which deleted two putative hypoxia response elements (HRE), abolished the hypoxia-induced increase in promoter activity. An electrophoretic mobility shift assay revealed that HIF2α was predominantly bound to a HRE site, located specifically at nucleotides -2715 to -2712. Finally, treatment with conditioned medium obtained from hypoxic pericytes stimulated endothelial cell migration and tube formation, which was completely blocked by co-treatment with anti-Ang1 antibody. This study is the first to demonstrate that hypoxia upregulates Ang1 expression via HIF2α-mediated transcriptional activation in pericytes, which plays a key role in angiogenesis. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Lithium ions induce prestalk-associated gene expression and inhibit prespore gene expression in Dictyostelium discoideum

    NARCIS (Netherlands)

    Peters, Dorien J.M.; Lookeren Campagne, Michiel M. van; Haastert, Peter J.M. van; Spek, Wouter; Schaap, Pauline

    1989-01-01

    We investigated the effect of Li+ on two types of cyclic AMP-regulated gene expression and on basal and cyclic AMP-stimulated inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) levels. Li+ effectively inhibits cyclic AMP-induced prespore gene expression, half-maximal inhibition occurring at about 2mM-LiCl.

  3. HTLV Deregulation of the NF-κB Pathway: An Update on Tax and Antisense Proteins Role.

    Science.gov (United States)

    Fochi, Stefania; Mutascio, Simona; Bertazzoni, Umberto; Zipeto, Donato; Romanelli, Maria G

    2018-01-01

    Human T-cell lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia (ATL), an aggressive CD4 + /CD25 + T-cell malignancy and of a severe neurodegenerative disease, HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). The chronic activation or deregulation of the canonical and non-canonical nuclear factor kappa B (NF-κB) pathways play a crucial role in tumorigenesis. The HTLV-1 Tax-1 oncoprotein is a potent activator of the NF-κB transcription factors and the NF-κB response is required for promoting the development of HTLV-1 transformed cell lines. The homologous retrovirus HTLV-2, which also expresses a Tax-2 transforming protein, is not associated with ATL. In this review, we provide an updated synopsis of the role of Tax-1 in the deregulation of the NF-κB pathway, highlighting the differences with the homologous Tax-2. Special emphasis is directed toward the understanding of the molecular mechanisms involved in NF-κB activation resulting from Tax interaction with host factors affecting several cellular processes, such as cell cycle, apoptosis, senescence, cell proliferation, autophagy, and post-translational modifications. We also discuss the current knowledge on the role of the antisense viral protein HBZ in down-regulating the NF-κB activation induced by Tax, and its implication in cellular senescence. In addition, we review the recent studies on the mechanism of HBZ-mediated inhibition of NF-κB activity as compared to that exerted by the HTLV-2 antisense protein, APH-2. Finally, we discuss recent advances aimed at understanding the role exerted in the development of ATL by the perturbation of NF-κB pathway by viral regulatory proteins.

  4. HTLV Deregulation of the NF-κB Pathway: An Update on Tax and Antisense Proteins Role

    Science.gov (United States)

    Fochi, Stefania; Mutascio, Simona; Bertazzoni, Umberto; Zipeto, Donato; Romanelli, Maria G.

    2018-01-01

    Human T-cell lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia (ATL), an aggressive CD4+/CD25+ T-cell malignancy and of a severe neurodegenerative disease, HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). The chronic activation or deregulation of the canonical and non-canonical nuclear factor kappa B (NF-κB) pathways play a crucial role in tumorigenesis. The HTLV-1 Tax-1 oncoprotein is a potent activator of the NF-κB transcription factors and the NF-κB response is required for promoting the development of HTLV-1 transformed cell lines. The homologous retrovirus HTLV-2, which also expresses a Tax-2 transforming protein, is not associated with ATL. In this review, we provide an updated synopsis of the role of Tax-1 in the deregulation of the NF-κB pathway, highlighting the differences with the homologous Tax-2. Special emphasis is directed toward the understanding of the molecular mechanisms involved in NF-κB activation resulting from Tax interaction with host factors affecting several cellular processes, such as cell cycle, apoptosis, senescence, cell proliferation, autophagy, and post-translational modifications. We also discuss the current knowledge on the role of the antisense viral protein HBZ in down-regulating the NF-κB activation induced by Tax, and its implication in cellular senescence. In addition, we review the recent studies on the mechanism of HBZ-mediated inhibition of NF-κB activity as compared to that exerted by the HTLV-2 antisense protein, APH-2. Finally, we discuss recent advances aimed at understanding the role exerted in the development of ATL by the perturbation of NF-κB pathway by viral regulatory proteins. PMID:29515558

  5. Transforming growth factor-β1 induces expression of human coagulation factor XII via Smad3 and JNK signaling pathways in human lung fibroblasts.

    Science.gov (United States)

    Jablonska, Ewa; Markart, Philipp; Zakrzewicz, Dariusz; Preissner, Klaus T; Wygrecka, Malgorzata

    2010-04-09

    Coagulation factor XII (FXII) is a liver-derived serine protease involved in fibrinolysis, coagulation, and inflammation. The regulation of FXII expression is largely unknown. Transforming growth factor-beta1 (TGF-beta1) is a multifunctional cytokine that has been linked to several pathological processes, including tissue fibrosis by modulating procoagulant and fibrinolytic activities. This study investigated whether TGF-beta1 may regulate FXII expression in human lung fibroblasts. Treatment of human lung fibroblasts with TGF-beta1 resulted in a time-dependent increase in FXII production, activation of p44/42, p38, JNK, and Akt, and phosphorylation and translocation into the nucleus of Smad3. However, TGF-beta1-induced FXII expression was repressed only by the JNK inhibitor and JNK and Smad3 antisense oligonucleotides but not by MEK, p38, or phosphoinositide 3-kinase blockers. JNK inhibition had no effect on TGF-beta1-induced Smad3 phosphorylation, association with Smad4, and its translocation into the nucleus but strongly suppressed Smad3-DNA complex formation. FXII promoter analysis revealed that the -299/+1 region was sufficient for TGF-beta1 to induce FXII expression. Sequence analysis of this region detected a potential Smad-binding element at position -272/-269 (SBE-(-272/-269)). Chromatin immunoprecipitation and streptavidin pulldown assays demonstrated TGF-beta1-dependent Smad3 binding to SBE-(-272/-269). Mutation or deletion of SBE-(-272/-269) substantially reduced TGF-beta1-mediated activation of the FXII promoter. Clinical relevance was demonstrated by elevated FXII levels and its co-localization with fibroblasts in the lungs of patients with acute respiratory distress syndrome. Our results show that JNK/Smad3 pathway plays a critical role in TGF-beta1-induced FXII expression in human lung fibroblasts and implicate its possible involvement in pathological conditions characterized by elevated TGF-beta1 levels.

  6. Photodynamic antisense regulation of mRNA having a point mutation with psoralen-conjugated oligonucleotide.

    Science.gov (United States)

    Higuchi, Maiko; Yamayoshi, Asako; Kobori, Akio; Murakami, Akira

    2008-01-01

    Nucleic acid-based drugs, such as antisense oligonucleotide, ribozyme, and small interfering RNA, are specific compounds that inhibit gene expression at the post-transcriptional level. To develop more effective nucleic acid-based drugs, we focused on photo-reactive antisense oligonucleotides. We have optimized the structure of psoralen-conjugated oligonucleotide to improve their sequence selectivity and photo-crosslinking efficiency. Previously, we reported that photo reactive oligonucleotides containing 2'-O-psoralenyl-methoxyethyl adenosine (2'-Ps-eom) showed drastic photo-reactivity with a strictly sequence specific manner in vitro. In this report, we evaluated the binding ability toward intracellular target mRNA. The 2'-Ps-eom selectively photo-cross-linked to the target mRNA extracted from cells. The 2'-Ps-eom also cross-linked to target mRNA in cells. Furthermore, 2'-Ps-eom did not cross-link to mRNA having a mismatch base. These results suggest that 2'-Ps-eom is a powerful antisense molecule to inhibit the expression of mRNA having a point mutation.

  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. Preparation and quality test of superparamagnetic iron oxide labeled antisense oligodeoxynucleotide probe: a preliminary study.

    Science.gov (United States)

    Wen, Ming; Li, Bibo; Ouyang, Yu; Luo, Yi; Li, Shaolin

    2009-06-01

    transfected SK-Br-3 oncocytes by antisense probe had the lowest signal of all. The SPIO-labeled ASODN probe shows unique features including well-distributed spherical morphology, high conjugating rate and loading efficiency, and the signal intensity of SPIO-labeled ASODN-transfected SK-Br-3 oncocytes is reduced in MR imaging. These results indicate that the SPIO-labeled ASODN probe is potentially useful as a MR targeting contrast enhancing agent to specifically diagnose tumors which had over-expression of the c-erbB2 oncogene at an early stage.

  9. Tetracycline-inducible gene expression system in Leishmania mexicana

    Czech Academy of Sciences Publication Activity Database

    Kraeva, N.; Ishemgulova, A.; Lukeš, Julius; Yurchenko, Vyacheslav

    2014-01-01

    Roč. 198, č. 1 (2014), s. 11-13 ISSN 0166-6851 R&D Projects: GA MŠk(CZ) EE2.3.30.0032 Institutional support: RVO:60077344 Keywords : Leishmania mexicana * Gene expression * Tet-inducible system Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.787, year: 2014

  10. Identification of salt-stress induced differentially expressed genes in ...

    African Journals Online (AJOL)

    Identification of salt-stress induced differentially expressed genes in barley leaves using the annealingcontrol- primer-based GeneFishing technique. S Lee, K Lee, K Kim, GJ Choi, SH Yoon, HC Ji, S Seo, YC Lim, N Ahsan ...

  11. Downregulation of miR-210 expression inhibits proliferation, induces apoptosis and enhances radiosensitivity in hypoxic human hepatoma cells in vitro

    International Nuclear Information System (INIS)

    Yang, Wei; Sun, Ting; Cao, Jianping; Liu, Fenju; Tian, Ye; Zhu, Wei

    2012-01-01

    Hypoxia is a common feature of solid tumors and an important contributor to tumor radioresistance. miR-210 is the most consistently and robustly induced microRNA under hypoxia in different types of tumor cells and normal cells. In the present study, to explore the feasibility of miR-210 as an effective therapeutic target, lentiviral-mediated anti-sense miR-210 gene transfer technique was employed to downregulate miR-210 expression in hypoxic human hepatoma SMMC-7721, HepG2 and HuH7 cells, and phenotypic changes of which were analyzed. Hypoxia led to an increased hypoxia inducible factor-1α (HIF-1α) and miR-210 expression and cell arrest in the G 0 /G 1 phase in all cell lines. miR-210 downregulation significantly suppressed cell viability, induced cell arrest in the G 0 /G 1 phase, increased apoptotic rate and enhanced radiosensitivity in hypoxic human hepatoma cells. Moreover, apoptosis-inducing factor, mitochondrion-associated, 3 (AIFM3) was identified as a direct target gene of miR-210. AIFM3 downregulation by siRNA attenuated radiation induced apoptosis in miR-210 downregulated hypoxic human hepatoma cells. Taken together, these data suggest that miR-210 might be a potential therapeutic target and specific inhibition of miR-210 expression in combination with radiotherapy might be expected to exert strong anti-tumor effect on hypoxic human hepatoma cells. -- Highlights: ► miR-210 downregulation radiosensitized hypoxic hepatoma. ► AIFM3 was identified as a direct target gene of miR-210. ► miR-210 might be a therapeutic target to hypoxic hepatoma.

  12. Downregulation of miR-210 expression inhibits proliferation, induces apoptosis and enhances radiosensitivity in hypoxic human hepatoma cells in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Wei, E-mail: detachedy@yahoo.com.cn [Department of Radiobiology, School of Radiological Medicine and Protection, Soochow University, Suzhou (China); Sun, Ting [Brain and Nerve Research Laboratory, The First Affiliated Hospital, Soochow University, Suzhou (China); Cao, Jianping; Liu, Fenju [Department of Radiobiology, School of Radiological Medicine and Protection, Soochow University, Suzhou (China); Tian, Ye [Department of Radiotherapy and Oncology, The Second Affiliated Hospital, Soochow University, Suzhou (China); Zhu, Wei [Department of Radiobiology, School of Radiological Medicine and Protection, Soochow University, Suzhou (China)

    2012-05-01

    Hypoxia is a common feature of solid tumors and an important contributor to tumor radioresistance. miR-210 is the most consistently and robustly induced microRNA under hypoxia in different types of tumor cells and normal cells. In the present study, to explore the feasibility of miR-210 as an effective therapeutic target, lentiviral-mediated anti-sense miR-210 gene transfer technique was employed to downregulate miR-210 expression in hypoxic human hepatoma SMMC-7721, HepG2 and HuH7 cells, and phenotypic changes of which were analyzed. Hypoxia led to an increased hypoxia inducible factor-1{alpha} (HIF-1{alpha}) and miR-210 expression and cell arrest in the G{sub 0}/G{sub 1} phase in all cell lines. miR-210 downregulation significantly suppressed cell viability, induced cell arrest in the G{sub 0}/G{sub 1} phase, increased apoptotic rate and enhanced radiosensitivity in hypoxic human hepatoma cells. Moreover, apoptosis-inducing factor, mitochondrion-associated, 3 (AIFM3) was identified as a direct target gene of miR-210. AIFM3 downregulation by siRNA attenuated radiation induced apoptosis in miR-210 downregulated hypoxic human hepatoma cells. Taken together, these data suggest that miR-210 might be a potential therapeutic target and specific inhibition of miR-210 expression in combination with radiotherapy might be expected to exert strong anti-tumor effect on hypoxic human hepatoma cells. -- Highlights: Black-Right-Pointing-Pointer miR-210 downregulation radiosensitized hypoxic hepatoma. Black-Right-Pointing-Pointer AIFM3 was identified as a direct target gene of miR-210. Black-Right-Pointing-Pointer miR-210 might be a therapeutic target to hypoxic hepatoma.

  13. Angiotensin II (AngII) induces the expression of suppressor of cytokine signaling (SOCS)-3 in rat hypothalamus - a mechanism for desensitization of AngII signaling.

    Science.gov (United States)

    Torsoni, Márcio A; Carvalheira, José B; Calegari, Vivian C; Bezerra, Rosangela M N; Saad, Mário J A; Gontijo, José A; Velloso, Lício A

    2004-04-01

    Angiotensin II exerts a potent dypsogenic stimulus on the hypothalamus, which contributes to its centrally mediated participation in the control of water balance and blood pressure. Repetitive intracerebroventricular (i.c.v.) injections of angiotensin II lead to a loss of effect characterized as physiological desensitization to the peptide's action. In the present study, we demonstrate that angiotensin II induces the expression of suppressor of cytokine signaling (SOCS)-3 via angiotensin receptor 1 (AT1) and JAK-2, mostly located at the median preoptic lateral and anterodorsal preoptic nuclei. SOCS-3 produces an inhibitory effect upon the signal transduction pathways of several cytokines and hormones that employ members of the JAK/STAT families as intermediaries. The partial inhibition of SOCS-3 translation by antisense oligonucleotide was sufficient to significantly reduce the refractoriness of repetitive i.c.v. angiotensin II injections, as evaluated by water ingestion. Thus, by acting through AT1 on the hypothalamus, angiotensin II induces the expression of SOCS-3 which, in turn, blocks further activation of the pathway and consequently leads to desensitization to angiotensin II stimuli concerning its dypsogenic effect.

  14. Inflammatory Cytokines Induce Podoplanin Expression at the Tumor Invasive Front.

    Science.gov (United States)

    Kunita, Akiko; Baeriswyl, Vanessa; Meda, Claudia; Cabuy, Erik; Takeshita, Kimiko; Giraudo, Enrico; Wicki, Andreas; Fukayama, Masashi; Christofori, Gerhard

    2018-05-01

    Tumor invasion is a critical first step in the organismic dissemination of cancer cells and the formation of metastasis in distant organs, the most important prognostic factor and the actual cause of death in most of the cancer patients. We report herein that the cell surface protein podoplanin (PDPN), a potent inducer of cancer cell invasion, is conspicuously expressed by the invasive front of squamous cell carcinomas (SCCs) of the cervix in patients and in the transgenic human papillomavirus/estrogen mouse model of cervical cancer. Laser capture microscopy combined with gene expression profiling reveals that the expression of interferon-responsive genes is up-regulated in PDPN-expressing cells at the tumor invasive front, which are exposed to CD45-positive inflammatory cells. Indeed, PDPN expression can be induced in cultured SCC cell lines by single or combined treatments with interferon-γ, transforming growth factor-β, and/or tumor necrosis factor-α. Notably, shRNA-mediated ablation of either PDPN or STAT1 in A431 SCC cells repressed cancer cell invasion on s.c. transplantation into immunodeficient mice. The results highlight the induction of tumor cell invasion by the inflammatory cytokine-stimulated expression of PDPN in the outermost cell layers of cervical SCC. Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  15. Development of inducer-free expression plasmids based on IPTG-inducible promoters for Bacillus subtilis.

    Science.gov (United States)

    Tran, Dinh Thi Minh; Phan, Trang Thi Phuong; Huynh, Thanh Kieu; Dang, Ngan Thi Kim; Huynh, Phuong Thi Kim; Nguyen, Tri Minh; Truong, Tuom Thi Tinh; Tran, Thuoc Linh; Schumann, Wolfgang; Nguyen, Hoang Duc

    2017-07-25

    Besides Escherichia coli, Bacillus subtilis is an important bacterial species for the production of recombinant proteins. Recombinant genes are inserted into shuttle expression vectors which replicate in both E. coli and in B. subtilis. The ligation products are first transformed into E. coli cells, analyzed for correct insertions, and the correct recombinant plasmids are then transformed into B. subtilis. A major problem using E. coli cells can be the strong basal level of expression of the recombinant protein which may interfere with the stability of the cells. To minimize this problem, we developed strong expression vectors being repressed in E. coli and inducer-free in B. subtilis. In general, induction of IPTG-inducible expression vectors is determined by the regulatory lacI gene encoding the LacI repressor in combination with the lacO operator on the promoter. To investigate the inducer-free properties of the vectors, we constructed inducer-free expression plasmids by removing the lacI gene and characterized their properties. First, we examined the ability to repress a reporter gene in E. coli, which is a prominent property facilitating the construction of the expression vectors carrying a target gene. The β-galactosidase (bgaB gene) basal levels expressed from Pgrac01-bgaB could be repressed at least twice in the E. coli cloning strain. Second, the inducer-free production of BgaB from four different plasmids with the Pgrac01 promoter in B. subtilis was investigated. As expected, BgaB expression levels of inducer-free constructs are at least 37 times higher than that of the inducible constructs in the absence of IPTG, and comparable to those in the presence of the inducer. Third, using efficient IPTG-inducible expression vectors containing the strong promoter Pgrac100, we could convert them into inducer-free expression plasmids. The BgaB production levels from the inducer-free plasmid in the absence of the inducer were at least 4.5 times higher than that of

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

    International Nuclear Information System (INIS)

    Tian, Hongmei; Ma, Leyuan; Zhao, Cong; Hao, Hui; Gong, Biao; Yu, Xiyan; Wang, Xiufeng

    2010-01-01

    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.

  17. Review on investigations of antisense oligonucleotides with the use of mass spectrometry.

    Science.gov (United States)

    Studzińska, Sylwia

    2018-01-01

    Antisense oligonucleotides have been investigated as potential drugs for years. They inhibit target gene or protein expression. The present review summarizes their modifications, modes of action, and applications of liquid chromatography coupled with mass spectrometry for qualitative and quantitative analysis of these compounds. The most recent reports on a given topic were given prominence, while some early studies were reviewed in order to provide a theoretical background. The present review covers the issues of using ion-exchange chromatography, ion-pair reversed-phase high performance liquid chromatography and hydrophilic interaction chromatography for the separation of antisense oligonucleotides. The application of mass spectrometry was described with regard to the ionization type used for the determination of these potential therapeutics. Moreover, the current approaches and applications of mass spectrometry for quantitative analysis of antisense oligonucleotides and their metabolites as well as their impurities during in vitro and in vivo studies were discussed. Finally, certain conclusions and perspectives on the determination of therapeutic oligonucleotides in various samples were briefly described. Copyright © 2017 Elsevier B.V. All rights reserved.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-03-12

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

  19. Sense-antisense (complementary) peptide interactions and the proteomic code; potential opportunities in biology and pharmaceutical science.

    Science.gov (United States)

    Miller, Andrew D

    2015-02-01

    A sense peptide can be defined as a peptide whose sequence is coded by the nucleotide sequence (read 5' → 3') of the sense (positive) strand of DNA. Conversely, an antisense (complementary) peptide is coded by the corresponding nucleotide sequence (read 5' → 3') of the antisense (negative) strand of DNA. Research has been accumulating steadily to suggest that sense peptides are capable of specific interactions with their corresponding antisense peptides. Unfortunately, although more and more examples of specific sense-antisense peptide interactions are emerging, the very idea of such interactions does not conform to standard biology dogma and so there remains a sizeable challenge to lift this concept from being perceived as a peripheral phenomenon if not worse, into becoming part of the scientific mainstream. Specific interactions have now been exploited for the inhibition of number of widely different protein-protein and protein-receptor interactions in vitro and in vivo. Further, antisense peptides have also been used to induce the production of antibodies targeted to specific receptors or else the production of anti-idiotypic antibodies targeted against auto-antibodies. Such illustrations of utility would seem to suggest that observed sense-antisense peptide interactions are not just the consequence of a sequence of coincidental 'lucky-hits'. Indeed, at the very least, one might conclude that sense-antisense peptide interactions represent a potentially new and different source of leads for drug discovery. But could there be more to come from studies in this area? Studies on the potential mechanism of sense-antisense peptide interactions suggest that interactions may be driven by amino acid residue interactions specified from the genetic code. If so, such specified amino acid residue interactions could form the basis for an even wider amino acid residue interaction code (proteomic code) that links gene sequences to actual protein structure and function, even

  20. The equine herpesvirus-1 IR3 gene that lies antisense to the sole immediate-early (IE) gene is trans-activated by the IE protein, and is poorly expressed to a protein

    International Nuclear Information System (INIS)

    Ahn, Byung Chul; Breitenbach, Jonathan E.; Kim, Seong K.; O'Callaghan, Dennis J.

    2007-01-01

    The unique IR3 gene of equine herpesvirus 1 (EHV-1) is expressed as a late 1.0-kb transcript. Previous studies confirmed the IR3 transcription initiation site and tentatively identified other cis-acting elements specific to IR3 such as a TATA box, a 443 base pair 5'untranslated region (UTR), a 285 base pair open reading frame (ORF), and a poly adenylation (A) signal [Holden, V.R., Harty, R.N., Yalamanchili, R.R., O'Callaghan, D.J., 1992. The IR3 gene of equine herpesvirus type 1: a unique gene regulated by sequences within the intron of the immediate-early gene. DNA Seq. 3, 143-152]. Transient transfection assays revealed that the IR3 promoter is strongly trans-activated by the IE protein (IEP) and that coexpression of the IEP with the early EICP0 and IR4 regulatory proteins results in maximal trans-activation of the IR3 promoter. Gel shift assays revealed that the IEP directly binds to the IR3 promoter region. Western blot analysis showed that the IR3 protein produced in E. coli was detected by antibodies to IR3 synthetic peptides; however, the IR3 protein was not detected in EHV-1 infected cell extracts by these same anti-IR3 antibodies, even though the IR3 transcript was detected by northern blot. These findings suggest that the IR3 may not be expressed to a protein. Expression of an IR3/GFP fusion gene was not observed, but expression of a GFP/IR3 fusion gene was detected by fluorescent microscopy. In further attempts to detect the IR3/GFP fusion protein using anti-GFP antibody, western blot analysis showed that the IR3/GFP fusion protein was not detected in vivo. Interestingly, a truncated form of the GFP/IR3 protein was synthesized from the GFP/IR3 fusion gene. However, GFP/IR3 and IR3/GFP fusion proteins of the predicted sizes were synthesized by in vitro coupled transcription and translation of the fusion genes, suggesting poor expression of the IR3 protein in vivo. The possible role of the IR3 transcript in EHV-1 infection is discussed

  1. Plasticity-Related Gene Expression During Eszopiclone-Induced Sleep.

    Science.gov (United States)

    Gerashchenko, Dmitry; Pasumarthi, Ravi K; Kilduff, Thomas S

    2017-07-01

    Experimental evidence suggests that restorative processes depend on synaptic plasticity changes in the brain during sleep. We used the expression of plasticity-related genes to assess synaptic plasticity changes during drug-induced sleep. We first characterized sleep induced by eszopiclone in mice during baseline conditions and during the recovery from sleep deprivation. We then compared the expression of 18 genes and two miRNAs critically involved in synaptic plasticity in these mice. Gene expression was assessed in the cerebral cortex and hippocampus by the TaqMan reverse transcription polymerase chain reaction and correlated with sleep parameters. Eszopiclone reduced the latency to nonrapid eye movement (NREM) sleep and increased NREM sleep amounts. Eszopiclone had no effect on slow wave activity (SWA) during baseline conditions but reduced the SWA increase during recovery sleep (RS) after sleep deprivation. Gene expression analyses revealed three distinct patterns: (1) four genes had higher expression either in the cortex or hippocampus in the group of mice with increased amounts of wakefulness; (2) a large proportion of plasticity-related genes (7 out of 18 genes) had higher expression during RS in the cortex but not in the hippocampus; and (3) six genes and the two miRNAs showed no significant changes across conditions. Even at a relatively high dose (20 mg/kg), eszopiclone did not reduce the expression of plasticity-related genes during RS period in the cortex. These results indicate that gene expression associated with synaptic plasticity occurs in the cortex in the presence of a hypnotic medication. © Sleep Research Society 2017. Published by Oxford University Press on behalf of the Sleep Research Society. All rights reserved. For permissions, please e-mail journals.permissions@oup.com.

  2. Expression of extracellular matrix metalloproteinase inducer in odontogenic cysts.

    Science.gov (United States)

    Ali, Mohammad Abdulhadi Abbas

    2008-08-01

    Extracellular matrix metalloproteinase inducer (EMMPRIN) is known to induce matrix metalloproteinase (MMP) production. The expression of EMMPRIN in odontogenic cysts has not been previously studied. This study was done to determine the presence and the variability of EMMPRIN expression in various types of odontogenic cysts. An immunohistochemical study using a polyclonal anti-EMMPRIN antibody was done using 48 odontogenic cyst cases: 13 odontogenic keratocysts (OKCs), 18 dentigerous cysts (DCs), and 17 periapical cysts (PAs). Twelve cases of normal dental follicles (DFs) were also included in this study for comparison. EMMPRIN immunoreactivity was detected in all of the cysts and DFs studied. In odontogenic cysts, EMMPRIN immunoreactivity was generally higher in basal cells than in suprabasal cells. The overall EMMPRIN expression in the epithelial lining of the 3 different types of odontogenic cyst was significantly higher than in the DFs. Overall EMMPRIN expression was also found to be significantly higher in the epithelial lining of OKCs than in the other types of cysts. This study confirmed that EMMPRIN is present in odontogenic cysts and DFs. The higher EMMPRIN expression in OKCs suggests that it may be involved in the aggressive behavior of this type of cyst.

  3. Specific RNP capture with antisense LNA/DNA mixmers.

    Science.gov (United States)

    Rogell, Birgit; Fischer, Bernd; Rettel, Mandy; Krijgsveld, Jeroen; Castello, Alfredo; Hentze, Matthias W

    2017-08-01

    RNA-binding proteins (RBPs) play essential roles in RNA biology, responding to cellular and environmental stimuli to regulate gene expression. Important advances have helped to determine the (near) complete repertoires of cellular RBPs. However, identification of RBPs associated with specific transcripts remains a challenge. Here, we describe "specific ribonucleoprotein (RNP) capture," a versatile method for the determination of the proteins bound to specific transcripts in vitro and in cellular systems. Specific RNP capture uses UV irradiation to covalently stabilize protein-RNA interactions taking place at "zero distance." Proteins bound to the target RNA are captured by hybridization with antisense locked nucleic acid (LNA)/DNA oligonucleotides covalently coupled to a magnetic resin. After stringent washing, interacting proteins are identified by quantitative mass spectrometry. Applied to in vitro extracts, specific RNP capture identifies the RBPs bound to a reporter mRNA containing the Sex-lethal (Sxl) binding motifs, revealing that the Sxl homolog sister of Sex lethal (Ssx) displays similar binding preferences. This method also revealed the repertoire of RBPs binding to 18S or 28S rRNAs in HeLa cells, including previously unknown rRNA-binding proteins. © 2017 Rogell et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  4. Exercise-induced metallothionein expression in human skeletal muscle fibres

    DEFF Research Database (Denmark)

    Penkowa, Milena; Keller, Pernille; Keller, Charlotte

    2005-01-01

    in both type I and II muscle fibres. This is the first report demonstrating that MT-I + II are significantly induced in human skeletal muscle fibres following exercise. As MT-I + II are antioxidant factors that protect various tissues during pathological conditions, the MT-I + II increases post exercise......Exercise induces free oxygen radicals that cause oxidative stress, and metallothioneins (MTs) are increased in states of oxidative stress and possess anti-apoptotic effects. We therefore studied expression of the antioxidant factors metallothionein I and II (MT-I + II) in muscle biopsies obtained...... in response to 3 h of bicycle exercise performed by healthy men and in resting controls. Both MT-I + II proteins and MT-II mRNA expression increased significantly in both type I and II muscle fibres after exercise. Moreover, 24 h after exercise the levels of MT-II mRNA and MT-I + II proteins were still highly...

  5. Src Induces Podoplanin Expression to Promote Cell Migration*

    Science.gov (United States)

    Shen, Yongquan; Chen, Chen-Shan; Ichikawa, Hitoshi; Goldberg, Gary S.

    2010-01-01

    Nontransformed cells can force tumor cells to assume a normal morphology and phenotype by the process of contact normalization. Transformed cells must escape this process to become invasive and malignant. However, mechanisms underlying contact normalization have not been elucidated. Here, we have identified genes that are affected by contact normalization of Src-transformed cells. Tumor cells must migrate to become invasive and malignant. Src must phosphorylate the adaptor protein Cas (Crk-associated substrate) to promote tumor cell motility. We report here that Src utilizes Cas to induce podoplanin (Pdpn) expression to promote tumor cell migration. Pdpn is a membrane-bound extracellular glycoprotein that associates with endogenous ligands to promote tumor cell migration leading to cancer invasion and metastasis. In fact, Pdpn expression accounted for a major part of the increased migration seen in Src-transformed cells. Moreover, nontransformed cells suppressed Pdpn expression in adjacent Src-transformed cells. Of >39,000 genes, Pdpn was one of only 23 genes found to be induced by transforming Src activity and suppressed by contact normalization of Src-transformed cells. In addition, we found 16 genes suppressed by Src and induced by contact normalization. These genes encode growth factor receptors, adaptor proteins, and products that have not yet been annotated and may play important roles in tumor cell growth and migration. PMID:20123990

  6. Hypoxic-induced stress protein expression in rat cardiac myocytes

    International Nuclear Information System (INIS)

    Howard, G.; Geoghegan, T.E.

    1986-01-01

    Mammalian stress proteins can be induced in cells and tissues exposed to a variety of conditions including hyperthermia and diminished O 2 supply. The authors have previously shown that the expression of three stress proteins (71, 85, and 95 kDa) was induced in cardiac tissue from mice exposed to hypoxic conditions. The expression of mRNAs coding for the 85 and 95 kDa proteins increase with time of exposure to hypoxia, while the mRNA coding for the 71 kDa protein is transiently induced. The authors extended these studies to investigate the expression of stress proteins in isolated rat cardiac myocytes. Freshly prepared myocytes were exposed to control, hypoxic, anoxic, or heat-shock environments for up to 16 h. The proteins were then labeled for 6 hours with [ 35 S]methionine. Analysis of the solubilized proteins by SDS-PAGE and autoradiography showed that there was a 6-fold increase in synthesis of the 85 kDa protein upon exposure to hypoxia but not heat-shock conditions. The 71 kDa protein was present at high levels in both control and treated myocyte protein preparations, and presumably had been induced during the isolation procedure. Total RNA isolated from intact rat heart and isolated myocytes was compared by cell-free translation analysis and showed induction of RNAs coding for several stress proteins in the myocyte preparation. The induced proteins at 85 and 95 kDa have molecular weights similar to reported cell stress and/or glucose-regulated proteins

  7. Fetuin-A induces cytokine expression and suppresses adiponectin production.

    Directory of Open Access Journals (Sweden)

    Anita M Hennige

    Full Text Available BACKGROUND: The secreted liver protein fetuin-A (AHSG is up-regulated in hepatic steatosis and the metabolic syndrome. These states are strongly associated with low-grade inflammation and hypoadiponectinemia. We, therefore, hypothesized that fetuin-A may play a role in the regulation of cytokine expression, the modulation of adipose tissue expression and plasma concentration of the insulin-sensitizing and atheroprotective adipokine adiponectin. METHODOLOGY AND PRINCIPAL FINDINGS: Human monocytic THP1 cells and human in vitro differenttiated adipocytes as well as C57BL/6 mice were treated with fetuin-A. mRNA expression of the genes encoding inflammatory cytokines and the adipokine adiponectin (ADIPOQ was assessed by real-time RT-PCR. In 122 subjects, plasma levels of fetuin-A, adiponectin and, in a subgroup, the multimeric forms of adiponectin were determined. Fetuin-A treatment induced TNF and IL1B mRNA expression in THP1 cells (p<0.05. Treatment of mice with fetuin-A, analogously, resulted in a marked increase in adipose tissue Tnf mRNA as well as Il6 expression (27- and 174-fold, respectively. These effects were accompanied by a decrease in adipose tissue Adipoq mRNA expression and lower circulating adiponectin levels (p<0.05, both. Furthermore, fetuin-A repressed ADIPOQ mRNA expression of human in vitro differentiated adipocytes (p<0.02 and induced inflammatory cytokine expression. In humans in plasma, fetuin-A correlated positively with high-sensitivity C-reactive protein, a marker of subclinical inflammation (r = 0.26, p = 0.01, and negatively with total- (r = -0.28, p = 0.02 and, particularly, high molecular weight adiponectin (r = -0.36, p = 0.01. CONCLUSIONS AND SIGNIFICANCE: We provide novel evidence that the secreted liver protein fetuin-A induces low-grade inflammation and represses adiponectin production in animals and in humans. These data suggest an important role of fatty liver in the pathophysiology of insulin resistance and

  8. Inducible expression of trehalose synthase in Bacillus licheniformis.

    Science.gov (United States)

    Li, Youran; Gu, Zhenghua; Zhang, Liang; Ding, Zhongyang; Shi, Guiyang

    2017-02-01

    Trehalose synthase (TreS) could transform maltose into trehalose via isomerization. It is a crucial enzyme in the process of trehalose enzymatical transformation. In this study, plasmid-based inducible expression systems were constructed to produce Thermomonospora curvata TreS in B. licheniformis. Xylose operons from B. subtilis, B. licheniformis and B. megaterium were introduced to regulate the expression of the gene encoding TreS. It was functionally expressed, and the BlsTs construct yielded the highest enzyme activity (12.1 U/mL). Furthermore, the effect of different cultural conditions on the inducible expression of BlsTs was investigated, and the optimal condition was as follows: 4% maltodextrin, 0.4% soybean powder, 1% xylose added after 10 h of growth and an induction time of 12 h at 37 °C. As a result, the maximal yield reached 24.7 U/mL. This study contributes to the industrial application of B. licheniformis, a GRAS workhorse for enzyme production. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Differentially expressed genes in iron-induced prion protein conversion

    International Nuclear Information System (INIS)

    Kim, Minsun; Kim, Eun-hee; Choi, Bo-Ran; Woo, Hee-Jong

    2016-01-01

    The conversion of the cellular prion protein (PrP C ) to the protease-resistant isoform is the key event in chronic neurodegenerative diseases, including transmissible spongiform encephalopathies (TSEs). Increased iron in prion-related disease has been observed due to the prion protein-ferritin complex. Additionally, the accumulation and conversion of recombinant PrP (rPrP) is specifically derived from Fe(III) but not Fe(II). Fe(III)-mediated PK-resistant PrP (PrP res ) conversion occurs within a complex cellular environment rather than via direct contact between rPrP and Fe(III). In this study, differentially expressed genes correlated with prion degeneration by Fe(III) were identified using Affymetrix microarrays. Following Fe(III) treatment, 97 genes were differentially expressed, including 85 upregulated genes and 12 downregulated genes (≥1.5-fold change in expression). However, Fe(II) treatment produced moderate alterations in gene expression without inducing dramatic alterations in gene expression profiles. Moreover, functional grouping of identified genes indicated that the differentially regulated genes were highly associated with cell growth, cell maintenance, and intra- and extracellular transport. These findings showed that Fe(III) may influence the expression of genes involved in PrP folding by redox mechanisms. The identification of genes with altered expression patterns in neural cells may provide insights into PrP conversion mechanisms during the development and progression of prion-related diseases. - Highlights: • Differential genes correlated with prion degeneration by Fe(III) were identified. • Genes were identified in cell proliferation and intra- and extracellular transport. • In PrP degeneration, redox related genes were suggested. • Cbr2, Rsad2, Slc40a1, Amph and Mvd were expressed significantly.

  10. Gene expression profiling reveals multiple toxicity endpoints induced by hepatotoxicants

    Energy Technology Data Exchange (ETDEWEB)

    Huang Qihong; Jin Xidong; Gaillard, Elias T.; Knight, Brian L.; Pack, Franklin D.; Stoltz, James H.; Jayadev, Supriya; Blanchard, Kerry T

    2004-05-18

    Microarray technology continues to gain increased acceptance in the drug development process, particularly at the stage of toxicology and safety assessment. In the current study, microarrays were used to investigate gene expression changes associated with hepatotoxicity, the most commonly reported clinical liability with pharmaceutical agents. Acetaminophen, methotrexate, methapyrilene, furan and phenytoin were used as benchmark compounds capable of inducing specific but different types of hepatotoxicity. The goal of the work was to define gene expression profiles capable of distinguishing the different subtypes of hepatotoxicity. Sprague-Dawley rats were orally dosed with acetaminophen (single dose, 4500 mg/kg for 6, 24 and 72 h), methotrexate (1 mg/kg per day for 1, 7 and 14 days), methapyrilene (100 mg/kg per day for 3 and 7 days), furan (40 mg/kg per day for 1, 3, 7 and 14 days) or phenytoin (300 mg/kg per day for 14 days). Hepatic gene expression was assessed using toxicology-specific gene arrays containing 684 target genes or expressed sequence tags (ESTs). Principal component analysis (PCA) of gene expression data was able to provide a clear distinction of each compound, suggesting that gene expression data can be used to discern different hepatotoxic agents and toxicity endpoints. Gene expression data were applied to the multiplicity-adjusted permutation test and significantly changed genes were categorized and correlated to hepatotoxic endpoints. Repression of enzymes involved in lipid oxidation (acyl-CoA dehydrogenase, medium chain, enoyl CoA hydratase, very long-chain acyl-CoA synthetase) were associated with microvesicular lipidosis. Likewise, subsets of genes associated with hepatotocellular necrosis, inflammation, hepatitis, bile duct hyperplasia and fibrosis have been identified. The current study illustrates that expression profiling can be used to: (1) distinguish different hepatotoxic endpoints; (2) predict the development of toxic endpoints; and

  11. Long Noncoding RNA HOXC-AS1 Suppresses Ox-LDL-Induced Cholesterol Accumulation Through Promoting HOXC6 Expression in THP-1 Macrophages.

    Science.gov (United States)

    Huang, Chuan; Hu, Yan-Wei; Zhao, Jing-Jing; Ma, Xin; Zhang, Yuan; Guo, Feng-Xia; Kang, Chun-Min; Lu, Jing-Bo; Xiu, Jian-Cheng; Sha, Yan-Hua; Gao, Ji-Juan; Wang, Yan-Chao; Li, Pan; Xu, Bang-Ming; Zheng, Lei; Wang, Qian

    2016-11-01

    Atherosclerosis is a common pathological basis of cardiovascular disease, which remains the leading cause of mortality. Long noncoding RNAs (lncRNAs) are newly studied non-protein-coding RNAs involved in gene regulation, but how lncRNAs exert regulatory effect on atherosclerosis remains unclear. In this study, we found that lncRNA HOXC cluster antisense RNA 1 (HOXC-AS1) and homeobox C6 (HOXC6) were downregulated in carotid atherosclerosis by performing microarray analysis. The results were verified in atherosclerotic plaques and normal arterial intima tissues by quantitative reverse transcription PCR and western blot analysis. Lentivirus-mediated overexpression of HOXC-AS1 induced HOXC6 expression at mRNA and protein levels in THP-1 macrophages. Besides, oxidized low-density lipoprotein (Ox-LDL) decreased expression of HOXC-AS1 and HOXC6 in a time-dependent manner. Induction of cholesterol accumulation by Ox-LDL could be partly suppressed by overexpression of HOXC-AS1.

  12. Gemcitabine-induced CXCL8 expression counteracts its actions by inducing tumor neovascularization

    International Nuclear Information System (INIS)

    Song, Yao; Baba, Tomohisa; Li, Ying-Yi; Furukawa, Kaoru; Tanabe, Yamato; Matsugo, Seiichi; Sasaki, Soichiro; Mukaida, Naofumi

    2015-01-01

    Patients with pancreatic ductal adenocarcinoma (PDAC) are frequently complicated with metastatic disease or locally advanced tumors, and consequently need chemotherapy. Gemcitabine is commonly used for PDAC treatment, but with limited efficacy. The capacity of gemcitabine to generate reactive oxygen species (ROS) in human pancreatic cancer cells, prompted us to examine its effects on the expression of pro-inflammatory cytokines and chemokines. We observed that gemcitabine enhanced selectively the expression of CXCL8 in human pancreatic cancer cells through ROS generation and NF-κB activation. In vitro blocking of CXCL8 failed to modulate gemcitabine-mediated inhibition of cell proliferation in human pancreatic cancer cells. Gemcitabine also enhanced CXCL8 expression in pancreatic cancer cells in xenografted tumor tissues. Moreover, anti-CXCL8 antibody treatment in vivo attenuated tumor formation as well as intra-tumoral vascularity in nude mice, which were transplanted with Miapaca-2 cells and treated with gemcitabine. Thus, gemcitabine-induced CXCL8 may counteract the drug through inducing neovascularization. - Highlights: • Gemcitabine induced CXCL8 expression in human pancreatic cancer cells. • CXCL8 expression required ROS generation and NF-κB activation. • CXCL8 did not affect in vitro proliferation of human pancreatic cancer cells. • CXCL8 in vivo counteracted gemcitabine by inducing neovascularization

  13. Gemcitabine-induced CXCL8 expression counteracts its actions by inducing tumor neovascularization

    Energy Technology Data Exchange (ETDEWEB)

    Song, Yao; Baba, Tomohisa [Division of Molecular Bioregulation, Cancer Research Institute, Kanazawa University, Kanazawa, Ishikawa 920-1192 (Japan); Li, Ying-Yi [Cancer Research Institute, Fudan University Shanghai Cancer Center, Department of Oncology, Shanghai Medical College, Fudan University, Shanghai (China); Furukawa, Kaoru; Tanabe, Yamato [Division of Molecular Bioregulation, Cancer Research Institute, Kanazawa University, Kanazawa, Ishikawa 920-1192 (Japan); School of Natural System Bioengineering Course, College of Science and Engineering, Kanazawa University, Kanazawa, Ishikawa (Japan); Matsugo, Seiichi [School of Natural System Bioengineering Course, College of Science and Engineering, Kanazawa University, Kanazawa, Ishikawa (Japan); Sasaki, Soichiro [Division of Molecular Bioregulation, Cancer Research Institute, Kanazawa University, Kanazawa, Ishikawa 920-1192 (Japan); Mukaida, Naofumi, E-mail: mukaida@staff.kanazawa-u.ac.jp [Division of Molecular Bioregulation, Cancer Research Institute, Kanazawa University, Kanazawa, Ishikawa 920-1192 (Japan)

    2015-03-06

    Patients with pancreatic ductal adenocarcinoma (PDAC) are frequently complicated with metastatic disease or locally advanced tumors, and consequently need chemotherapy. Gemcitabine is commonly used for PDAC treatment, but with limited efficacy. The capacity of gemcitabine to generate reactive oxygen species (ROS) in human pancreatic cancer cells, prompted us to examine its effects on the expression of pro-inflammatory cytokines and chemokines. We observed that gemcitabine enhanced selectively the expression of CXCL8 in human pancreatic cancer cells through ROS generation and NF-κB activation. In vitro blocking of CXCL8 failed to modulate gemcitabine-mediated inhibition of cell proliferation in human pancreatic cancer cells. Gemcitabine also enhanced CXCL8 expression in pancreatic cancer cells in xenografted tumor tissues. Moreover, anti-CXCL8 antibody treatment in vivo attenuated tumor formation as well as intra-tumoral vascularity in nude mice, which were transplanted with Miapaca-2 cells and treated with gemcitabine. Thus, gemcitabine-induced CXCL8 may counteract the drug through inducing neovascularization. - Highlights: • Gemcitabine induced CXCL8 expression in human pancreatic cancer cells. • CXCL8 expression required ROS generation and NF-κB activation. • CXCL8 did not affect in vitro proliferation of human pancreatic cancer cells. • CXCL8 in vivo counteracted gemcitabine by inducing neovascularization.

  14. Low doses of neutrons induce changes in gene expression

    International Nuclear Information System (INIS)

    Woloschak, G.E.; Chang-Liu, C.M.; Panozzo, J.; Libertin, C.R.

    1993-01-01

    Studies were designed to identify genes induced following low-dose neutron but not following γ-ray exposure in fibroblasts. Our past work had shown differences in the expression of β-protein kinase C and c-fos genes, both being induced following γ-ray but not neutron exposure. We have identified two genes that are induced following neutron, but not γ-ray, exposure: Rp-8 (a gene induced by apoptosis) and the long terminal repeat (LTR) of the human immunodeficiency (HIV). Rp-8 mRNA induction was demonstrated in Syrian hamster embryo fibroblasts and was found to be induced in cells exposed to neutrons administered at low (0.5 cGy/min) and at high dose rate (12 cGy/min). The induction of transcription from the LTR of HIV was demonstrated in HeLa cells bearing a transfected construct of the chloramphenicol acetyl transferase (CAT) gene driven by the HIV-LTR promoter. Measures of CAT activity and CAT transcripts following irradiation demonstrated an unresponsiveness to γ rays over a broad range of doses. Twofold induction of the HIV-LTR was detected following neutron exposure (48 cGy) administered at low (0.5 cGy/min) but not high (12 cGy/min) dose rates. Ultraviolet-mediated HIV-LTR induction was inhibited by low-dose-rate neutron exposure

  15. HIV-1 induces DCIR expression in CD4+ T cells.

    Directory of Open Access Journals (Sweden)

    Alexandra A Lambert

    2010-11-01

    Full Text Available The C-type lectin receptor DCIR, which has been shown very recently to act as an attachment factor for HIV-1 in dendritic cells, is expressed predominantly on antigen-presenting cells. However, this concept was recently challenged by the discovery that DCIR can also be detected in CD4(+ T cells found in the synovial tissue from rheumatoid arthritis (RA patients. Given that RA and HIV-1 infections share common features such as a chronic inflammatory condition and polyclonal immune hyperactivation status, we hypothesized that HIV-1 could promote DCIR expression in CD4(+ T cells. We report here that HIV-1 drives DCIR expression in human primary CD4(+ T cells isolated from patients (from both aviremic/treated and viremic/treatment naive persons and cells acutely infected in vitro (seen in both virus-infected and uninfected cells. Soluble factors produced by virus-infected cells are responsible for the noticed DCIR up-regulation on uninfected cells. Infection studies with Vpr- or Nef-deleted viruses revealed that these two viral genes are not contributing to the mechanism of DCIR induction that is seen following acute infection of CD4(+ T cells with HIV-1. Moreover, we report that DCIR is linked to caspase-dependent (induced by a mitochondria-mediated generation of free radicals and -independent intrinsic apoptotic pathways (involving the death effector AIF. Finally, we demonstrate that the higher surface expression of DCIR in CD4(+ T cells is accompanied by an enhancement of virus attachment/entry, replication and transfer. This study shows for the first time that HIV-1 induces DCIR membrane expression in CD4(+ T cells, a process that might promote virus dissemination throughout the infected organism.

  16. Nanoparticle for delivery of antisense γPNA oligomers targeting CCR5

    OpenAIRE

    Bahal, Raman; McNeer, Nicole Ali; Ly, Danith H.; Saltzman, W. Mark; Glazer, Peter M.

    2013-01-01

    The development of a new class of peptide nucleic acids (PNAs), i.e., gamma PNAs (γPNAs), creates the need for a general and effective method for its delivery into cells for regulating gene expression in mammalian cells. Here we report the antisense activity of a recently developed hydrophilic and biocompatible diethylene glycol (miniPEG)-based gamma peptide nucleic acid called MPγPNAs via its delivery by poly(lactide-co-glycolide) (PLGA)-based nanoparticle system. We show that MPγPNA oligome...

  17. EGR-1 regulates Ho-1 expression induced by cigarette smoke

    International Nuclear Information System (INIS)

    Chen, Huaqun; Wang, Lijuan; Gong, Tao; Yu, Yang; Zhu, Chunhua; Li, Fen; Wang, Li; Li, Chaojun

    2010-01-01

    As an anti-oxidant molecule, heme oxygenase-1 (HO-1) has been implicated in the protection of lung injury by cigarette smoke (CS). The mechanisms regulating its expression have not been defined. In this report, the role of early growth response 1 (EGR-1) in the regulation of Ho-1 expression was investigated. In C57BL/6 mice with CS exposure, HO-1 was greatly increased in bronchial epithelial cells and alveolar inflammatory cells. In primary cultured mouse lung fibroblasts and RAW264.7 cells exposed to cigarette smoke water extract (CSE), an increase in HO-1 protein level was detected. In addition, CSE induced HO-1 expression was decreased in Egr-1 deficient mouse embryo fibroblasts (Egr-1 -/- MEFs). Nuclear localization of EGR-1 was examined in mouse lung fibroblasts after exposure to CSE. Luciferase reporter activity assays showed that the enhancer region of the Ho-1 gene containing a proposed EGR-1 binding site was responsible for the induction of HO-1. A higher increase of alveolar mean linear intercept (Lm) was observed in lung tissues, and a larger increase in the number of total cells and monocytes/macrophages from bronchial alveolar lavage fluid was found in CS-exposed mice by loss of function of EGR-1 treatment. In summary, the present data demonstrate that EGR-1 plays a critical role in HO-1 production induced by CS.

  18. Mechanoactive scaffold induces tendon remodeling and expression of fibrocartilage markers.

    Science.gov (United States)

    Spalazzi, Jeffrey P; Vyner, Moira C; Jacobs, Matthew T; Moffat, Kristen L; Lu, Helen H

    2008-08-01

    Biological fixation of soft tissue-based grafts for anterior cruciate ligament (ACL) reconstruction poses a major clinical challenge. The ACL integrates with subchondral bone through a fibrocartilage enthesis, which serves to minimize stress concentrations and enables load transfer between two distinct tissue types. Functional integration thus requires the reestablishment of this fibrocartilage interface on reconstructed ACL grafts. We designed and characterized a novel mechanoactive scaffold based on a composite of poly-alpha-hydroxyester nanofibers and sintered microspheres; we then used the scaffold to test the hypothesis that scaffold-induced compression of tendon grafts would result in matrix remodeling and the expression of fibrocartilage interface-related markers. Histology coupled with confocal microscopy and biochemical assays were used to evaluate the effects of scaffold-induced compression on tendon matrix collagen distribution, cellularity, proteoglycan content, and gene expression over a 2-week period. Scaffold contraction resulted in over 15% compression of the patellar tendon graft and upregulated the expression of fibrocartilage-related markers such as Type II collagen, aggrecan, and transforming growth factor-beta3 (TGF-beta3). Additionally, proteoglycan content was higher in the compressed tendon group after 1 day. The data suggest the potential of a mechanoactive scaffold to promote the formation of an anatomic fibrocartilage enthesis on tendon-based ACL reconstruction grafts.

  19. Lipopolysaccharide induces autotaxin expression in human monocytic THP-1 cells

    International Nuclear Information System (INIS)

    Li Song; Zhang Junjie

    2009-01-01

    Autotaxin (ATX) is a secreted enzyme with lysophospholipase D (lysoPLD) activity, which converts lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA), a bioactive phospholipid involved in numerous biological activities, including cell proliferation, differentiation, and migration. In the present study, we found that bacterial lipopolysaccharide (LPS), a well-known initiator of the inflammatory response, induced ATX expression in monocytic THP-1 cells. The activation of PKR, JNK, and p38 MAPK was required for the ATX induction. The LPS-induced ATX in THP-1 cells was characterized as the β isoform. In the presence of LPC, ATX could promote the migrations of THP-1 and Jurkat cells, which was inhibited by pertussis toxin (PTX), an inhibitor of Gi-mediated LPA receptor signaling. In summary, LPS induces ATX expression in THP-1 cells via a PKR, JNK and p38 MAPK-mediated mechanism, and the ATX induction is likely to enhance immune cell migration in proinflammatory response by regulating LPA levels in the microenvironment.

  20. The Dynamics of Compound, Transcript, and Protein Effects After Treatment With 2OMePS Antisense Oligonucleotides in mdx Mice

    Directory of Open Access Journals (Sweden)

    Ingrid E C Verhaart

    2014-01-01

    Full Text Available Antisense-mediated exon skipping is currently in clinical development for Duchenne muscular dystrophy (DMD to amend the consequences of the underlying genetic defect and restore dystrophin expression. Due to turnover of compound, transcript, and protein, chronic treatment with effector molecules (antisense oligonucleotides will be required. To investigate the dynamics and persistence of antisense 2′-O-methyl phosphorothioate oligonucleotides, exon skipping, and dystrophin expression after dosing was concluded, mdx mice were treated subcutaneously for 8 weeks with 100 mg/kg oligonucleotides twice weekly. Thereafter, mice were sacrificed at different time points after the final injection (36 hours–24 weeks. Oligonucleotide half-life was longer in heart (~65 days compared with that in skeletal muscle, liver, and kidney (~35 days. Exon skipping half-lives varied between 33 and 53 days, whereas dystrophin protein showed a long half-life (>100 days. Oligonucleotide and exon-skipping levels peaked in the first week and declined thereafter. By contrast, dystrophin expression peaked after 3–8 weeks and then slowly declined, remaining detectable after 24 weeks. Concordance between levels of oligonucleotides, exon skipping, and proteins was observed, except in heart, wherein high oligonucleotide levels but low exon skipping and dystrophin expression were seen. Overall, these results enhance our understanding of the pharmacokinetics and pharmacodynamics of 2′-O-methyl phosphorothioate oligos used for the treatment of DMD.

  1. Involvement of over-expressed BMP4 in pentylenetetrazol kindling-induced cell proliferation in the dentate gyrus of adult rats

    International Nuclear Information System (INIS)

    Yin Jinbo; Ma Yuxin; Yin Qing; Xu Haiwei; An Ning; Liu Shiyong; Fan Xiaotang; Yang Hui

    2007-01-01

    The dentate gyrus (DG) of the hippocampus is one of a few regions in the adult mammalian brain characterized by ongoing neurogenesis. Proliferation of neural precursors in the granule cell layer of the DG has been identified in pentylenetetrazol (PTZ) kindling epilepsy model, however, little is known about the molecular mechanism. We previously reported that the expression pattern of bone morphogenetic proteins-4 (BMP4) mRNA in the hippocampus was developmentally regulated and mainly localized in the DG of the adult. To explore the role of BMP4 in epileptic activity, we detected BMP4 expression in the DG during PTZ kindling process and explore its correlation with cell proliferation combined with bromodeoxyuridine (BrdU) labeling technique. We found that dynamic changes in BMP4 level and BrdU labeled cells dependent on the kindling stage of PTZ induced seizure-prone state. The number of BMP4 mRNA-positive cells and BrdU labeled cells reached the top level 1 day after PTZ kindled, then declined to base level 2 months later. Furthermore, there was a significant correlation between increased BMP4 mRNA expression and increased number of BrdU labeled cells. After effectively blocked expression of BMP4 with antisense oligodeoxynucleotides(ASODN), the BrdU labeled cells in the dentate gyrus subgranular zone(DG-SGZ) and hilus were significantly decreased 16d after First PTZ injection and 1, 3, 7, 14d after kindled respectively. These findings suggest that increased proliferation in the DG of the hippocampus resulted from kindling epilepsy elicited by PTZ maybe be modulated by BMP4 over-expression

  2. Ecstasy-Induced Caspase Expression Alters Following Ginger Treatment

    Directory of Open Access Journals (Sweden)

    Sara Soleimani Asl

    2013-11-01

    Full Text Available Introduction: Exposure to 3-4, methylenedioxymethamphetamine (MDMA leads to cell death. Herein, we studied the protective effects of ginger on MDMA- induced apoptosis. Methods: 15 Sprague dawley male rats were administrated with 0, 10 mg/kg MDMA, or MDMA along with 100mg/kg ginger, IP for 7 days. Brains were removed to study the caspase 3, 8, and 9 expressions in the hippocampus by RT-PCR. Data was analyzed by SPSS 16 software using the one-way ANOVA test. Results: MDMA treatment resulted in a significant increase in caspase 3, 8, and 9 as compared to the sham group (p<0.001. Ginger administration however, appeared to significantly decrease the same (p<0.001. Discussion: Our findings suggest that ginger consumption may lead to the improvement of MDMA-induced neurotoxicity.

  3. Glutamine deprivation induces interleukin-8 expression in ataxia telangiectasia fibroblasts.

    Science.gov (United States)

    Kim, Min-Hyun; Kim, Aryung; Yu, Ji Hoon; Lim, Joo Weon; Kim, Hyeyoung

    2014-05-01

    To investigate whether glutamine deprivation induces expression of inflammatory cytokine interleukin-8 (IL-8) by determining NF-κB activity and levels of oxidative indices (ROS, reactive oxygen species; hydrogen peroxide; GSH, glutathione) in fibroblasts isolated from patients with ataxia telangiectasia (A-T). We used A-T fibroblasts stably transfected with empty vector (Mock) or with human full-length ataxia telangiectasia mutated (ATM) cDNA (YZ5) and mouse embryonic fibroblasts (MEFs) transiently transfected with ATM small interfering RNA (siRNA) or with non-specific control siRNA. The cells were cultured with or without glutamine or GSH. ROS levels were determined using a fluorescence reader and confocal microscopy. IL-8 or murine IL-8 homolog, keratinocyte chemoattractant (KC), and hydrogen peroxide levels in the medium were determined by enzyme-linked immunosorbent assay and colorimetric assay. GSH level was assessed by enzymatic assay, while IL-8 (KC) mRNA level was measured by reverse transcription-polymerase chain reaction (RT-PCR) and/or quantitative real-time PCR. NF-κB DNA-binding activity was determined by electrophoretic mobility shift assay. Catalase activity and ATM protein levels were determined by O2 generation and Western blotting. While glutamine deprivation induced IL-8 expression and increased NF-κB DNA-binding activity in Mock cells, both processes were decreased by treatment of cells with glutamine or GSH or both glutamine and GSH. Glutamine deprivation had no effect on IL-8 expression or NF-κB DNA-binding activity in YZ5 cells. Glutamine-deprived Mock cells had higher oxidative stress indices (increases in ROS and hydrogen peroxide, reduction in GSH) than glutamine-deprived YZ5 cells. In Mock cells, glutamine deprivation-induced oxidative stress indices were suppressed by treatment with glutamine or GSH or both glutamine and GSH. GSH levels and catalase activity were lower in Mock cells than YZ5 cells. MEFs transfected with ATM siRNA and

  4. Decreased inducibility of TNF expression in lipid-loaded macrophages

    Directory of Open Access Journals (Sweden)

    Kallin Bengt

    2002-10-01

    Full Text Available Abstract Background Inflammation and immune responses are considered to be very important in the pathogenesis of atherosclerosis. Lipid accumulation in macrophages of the arterial intima is a characteristic feature of atherosclerosis which can influence the inflammatory potential of macrophages. We studied the effects of lipid loading on the regulation of TNF expression in human monocyte-derived macrophages. Results In macrophages incubated with acetylated low density lipoprotein (ac-LDL for 2 days, mRNA expression of TNF in cells stimulated with TNF decreased by 75%. In cell cultures stimulated over night with IL-1β, lipid loading decreased secretion of TNF into culture medium by 48%. These results suggest that lipid accumulation in macrophages makes them less responsive to inflammatory stimuli. Decreased basal activity and inducibility of transcription factor AP-1 was observed in lipid-loaded cells, suggesting a mechanism for the suppression of cytokine expression. NF-κB binding activity and inducibility were only marginally affected by ac-LDL. LDL and ac-LDL did not activate PPARγ. In contrast, oxidized LDL stimulated AP-1 and PPARγ but inhibited NF-κB, indicating that the effects of lipid loading with ac-LDL were not due to oxidation of lipids. Conclusions Accumulation of lipid, mainly cholesterol, results in down-regulation of TNF expression in macrophages. Since monocytes are known to be activated by cell adhesion, these results suggest that foam cells in atherosclerotic plaques may contribute less potently to an inflammatory reaction than newly arrived monocytes/macrophages.

  5. 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.

  6. In vivo knockdown of antisense non-coding mitochondrial RNAs by a lentiviral-encoded shRNA inhibits melanoma tumor growth and lung colonization.

    Science.gov (United States)

    Varas-Godoy, Manuel; Lladser, Alvaro; Farfan, Nicole; Villota, Claudio; Villegas, Jaime; Tapia, Julio C; Burzio, Luis O; Burzio, Veronica A; Valenzuela, Pablo D T

    2018-01-01

    The family of non-coding mitochondrial RNAs (ncmtRNA) is differentially expressed according to proliferative status. Normal proliferating cells express sense (SncmtRNA) and antisense ncmtRNAs (ASncmtRNAs), whereas tumor cells express SncmtRNA and downregulate ASncmtRNAs. Knockdown of ASncmtRNAs with oligonucleotides induces apoptotic cell death of tumor cells, leaving normal cells unaffected, suggesting a potential application for developing a novel cancer therapy. In this study, we knocked down the ASncmtRNAs in melanoma cell lines with a lentiviral-encoded shRNA approach. Transduction with lentiviral constructs targeted to the ASncmtRNAs induced apoptosis in murine B16F10 and human A375 melanoma cells in vitro and significantly retarded B16F10 primary tumor growth in vivo. Moreover, the treatment drastically reduced the number of lung metastatic foci in a tail vein injection assay, compared to controls. These results provide additional proof of concept to the knockdown of ncmtRNAs for cancer therapy and validate lentiviral-shRNA vectors for gene therapy. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  7. Radiation-induced gene expression in human subcutaneous fibroblasts is predictive of radiation-induced fibrosis

    DEFF Research Database (Denmark)

    Rødningen, Olaug Kristin; Børresen-Dale, Anne-Lise; Alsner, Jan

    2008-01-01

    BACKGROUND AND PURPOSE: Breast cancer patients show a large variation in normal tissue reactions after ionizing radiation (IR) therapy. One of the most common long-term adverse effects of ionizing radiotherapy is radiation-induced fibrosis (RIF), and several attempts have been made over the last...... years to develop predictive assays for RIF. Our aim was to identify basal and radiation-induced transcriptional profiles in fibroblasts from breast cancer patients that might be related to the individual risk of RIF in these patients. MATERIALS AND METHODS: Fibroblast cell lines from 31 individuals......-treated fibroblasts. Transcriptional differences in basal and radiation-induced gene expression profiles were investigated using 15K cDNA microarrays, and results analyzed by both SAM and PAM. RESULTS: Sixty differentially expressed genes were identified by applying SAM on 10 patients with the highest risk of RIF...

  8. One week's holiday sun exposure induces expression of photoaging biomarkers.

    Science.gov (United States)

    Lesiak, Aleksandra; Rogowski-Tylman, Michal; Danilewicz, Marian; Wozniacka, Anna; Narbutt, Joanna

    2016-01-01

    Skin aging is accompanied by the upregulation of the expression of various matrix metalloproteinases (MMPs). It was shown that exposure to ultraviolet radiation (UVR) may induce skin expression of MMPs and dysregulation of the transforming growth factor beta (TGF-β)/Smad pathway. The aim of our study was to compare the effects of short holiday UVR exposure and lifetime UVR exposure, on the expression of MMP-8, TGF-β1, and Smad2 in human skin biopsies. Skin biopsies were taken from the outer upper arm of 15 elderly people with significant photoaging (mean age 64.1 years) (Group 1) and from 15 healthy young adult volunteers (mean age 24.1 y) who participated in a six-day sun holiday. Biopsies were taken twice: 24 hours before leaving for holiday (Group 2a) and 24 hours after returning (Group 2b). The expression of TGF-β1, Smad2, and MMP-8 was examined by immunochemistry and measured semiquantitatively by two independent pathologists. The mean expression of TGF-β1 in dermal fibroblasts and keratinocytes in Group 1 and Group 2b was significantly lower than in Group 2a (0.54% ± 0.44% and 0.48% ± 0.51% vs. 1.48% ± 0.72%, respectively). The percentage of Smad2 (+) cells in Group 1 and Group 2b was lower than in Group 2a (2.13% ± 1.39% and 1.81% ± 1.16% vs. 4.13% ± 1.58%, respectively). The MMP-8 expression in Group 2b was 1.36% ± 0.68% and was significantly higher than in Group 1 (0.34% ± 0.42%) and Group 2a in which the protein was not detected (p < 0.001). We conclude that the decrease in the expression of TGF-β1 and Smad2 is a persistent biomarker of skin photoaging, while the increased expression of MMP-8 in keratinocytes can be regarded as a marker of acute sun exposure.

  9. Antitumor effects of radioiodinated antisense oligonucleotide mediated by VIP receptor

    International Nuclear Information System (INIS)

    Ou Xiaohong; Tan Tianzhi; Li Yunchun; Kuang Anren

    2004-01-01

    Purpose: we had constructed a targeting delivery system based on intestinal peptide (VIP) for antisense oligonucleotide (ASON) transfer into VIP receptor-positive cells in previous study. The aims of present studies are to observe the antitumor effect of VIP-131I-ASON in HT29 human colon adenocarcinoma xenografts. Methods: A 15-met phosphorothioate ASON, which was complementary to the translation start region of the C-myc oncogene mRNA, was labeled with 131I and the labelled compound was linked to the VIP bound covalently 'to a polylysine chain so as to deliver oligonucleotide into tumor cells. Distribution experiments for evaluating the radiolabeled antisense complexe uptake in tumor tissue were performed in BALB/c nude mice bearing with HT29 tumor xenografts. Nude mice beating HT29 tumor xenografts were adminstered VIP-131I-ASON (3.7,7.4 MBq) or 131I-ASON (3.7 MBq), 131I labeled control sense and nosense DNA (3.7 MBq), or saline. Antitumor effects were assessed using endpoints of tumor growth delay. C-myc-encoded protein expression of tumor was measured by immunocytohistochemical staining. Results: Distribution experiment performed with athymic mice bearing human colon tumor xenografts revealed maximal accumulation of conjugated ASON in the tumor tissue 2 h after administration and significantly higher than that in nude mice injected unconjngated ASON [(5.89±1.03)%ID/g and(1.56±0.31)%ID/g, respectively; t=7.7954 P<0.001]. The radioratio of tumor to muscle was peaked 4h after administration. VIP-131I-ASON exhibited strong antitumor effects against HT29 xenografts, decreasing their growth rate 7-fold compare with that in saline-treated mice(tumor growth delay, 25.4±0.89 day). The antitumor effects of unconjugated 131I-ASON were much less profound than VIP-131I-ASON (tumor growth delay, 3.2±1.3 and 25.4±0.89 day, respectively; q=51.4126 P<0.01). Sense, nosense control ON with VIP carder caused no therapeutic effect. There was no progressive weight loss or

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

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    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.

  12. Alpha particles induce expression of immunogenic markers on tumour cells

    International Nuclear Information System (INIS)

    Gorin, J.B.; Gouard, S.; Cherel, M.; Davodeau, F.; Gaschet, J.; Morgenstern, A.; Bruchertseifer, F.

    2013-01-01

    The full text of the publication follows. Radioimmunotherapy (RIT) is an approach aiming at targeting the radioelements to tumours, usually through the use of antibodies specific for tumour antigens. The radiations emitted by the radioelements then induce direct killing of the targeted cells as well as indirect killing through bystander effect. Interestingly, it has been shown that ionizing radiations, in some settings of external radiotherapy, can foster an immune response directed against tumour cells. Our research team is dedicated to the development of alpha RIT, i.e RIT using alpha particle emitters, we therefore decided to study the effects of such particles on tumour cells in regards to their immunogenicity. First, we studied the effects of bismuth 213, an alpha emitter, on cellular death and autophagy in six different tumour cell lines. Then, we measured the expression of 'danger' signals and MHC molecules at the cell surface to determine whether irradiation with 213 Bi could cause the tumour cells to be recognized by the immune system. Finally a co-culture of dendritic cells with irradiated tumour cells was performed to test whether it would induce dendritic cells to mature. No apoptosis was detected within 48 hours after irradiation in any cell line, however half of them exhibited signs of autophagy. No increase in membrane expression of 'danger' signals was observed after treatment with 213 Bi, but we showed an increase in expression of MHC class I and II for some cell lines. Moreover, the co-culture experiment indicated that the immunogenicity of a human adenocarcinoma cell line (LS 174T) was enhanced in vitro after irradiation with alpha rays. These preliminary data suggest that alpha particles could be of interest in raising an immune response associated to RIT. (authors)

  13. Genistein-induced alterations of radiation-responsive gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Grace, M.B. [Armed Forces Radiobiology Research Institute, 8901 Wisconsin Avenue, Bethesda, MD 20889-5603 (United States)], E-mail: grace@afrri.usuhs.mil; Blakely, W.F.; Landauer, M.R. [Armed Forces Radiobiology Research Institute, 8901 Wisconsin Avenue, Bethesda, MD 20889-5603 (United States)

    2007-07-15

    In order to clarify the molecular mechanism of radioprotection and understand biological dosimetry in the presence of medical countermeasure-radioprotectants, their effects on ionizing radiation (IR)-responsive molecular biomarkers must be examined. We used genistein in a radiation model system and measured gene expression by multiplex QRT-PCR assay in drug-treated healthy human blood cultures. Genistein has been demonstrated to be a radiosensitizer of malignant cells and a radioprotector against IR-induced lethality in a mouse model. Whole-blood cultures were supplemented with 50, 100, and 200{mu}M concentrations of genistein, 16 h prior to receiving a 2-Gy ({sup 60}Co-{gamma} rays, 10 cGy/min) dose of IR. Total RNA was isolated from whole blood 24 h postirradiation for assessments. Combination treatments of genistein and IR resulted in no significant genistein effects on ddb2 and bax downstream transcripts to p53, or proliferating cell-nuclear antigen, pcna, necessary for DNA synthesis and cell-cycle progression. Use of these radiation-responsive targets would be recommended for dose-assessment applications. We also observed decreased expression of pro-survival transcript, bcl-2. Genistein and IR-increased expression of cdkn1a and gadd45a, showing that genistein also stimulates p53 transcriptional activity. These results confirm published molecular signatures for genistein in numerous in vitro models. Evaluation of gene biomarkers may be further exploited for devising novel radiation countermeasure and/or therapeutic strategies.

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

    Institute of Scientific and Technical Information of China (English)

    张新霞; 崔长琮; 李江; 崔翰斌; 徐仓宝; 朱参战

    2002-01-01

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

  15. Chemical modifications of antisense morpholino oligomers enhance their efficacy against Ebola virus infection.

    Science.gov (United States)

    Swenson, Dana L; Warfield, Kelly L; Warren, Travis K; Lovejoy, Candace; Hassinger, Jed N; Ruthel, Gordon; Blouch, Robert E; Moulton, Hong M; Weller, Dwight D; Iversen, Patrick L; Bavari, Sina

    2009-05-01

    Phosphorodiamidate morpholino oligomers (PMOs) are uncharged nucleic acid-like molecules designed to inactivate the expression of specific genes via the antisense-based steric hindrance of mRNA translation. PMOs have been successful at knocking out viral gene expression and replication in the case of acute viral infections in animal models and have been well tolerated in human clinical trials. We propose that antisense PMOs represent a promising class of therapeutic agents that may be useful for combating filoviral infections. We have previously shown that mice treated with a PMO whose sequence is complementary to a region spanning the start codon of VP24 mRNA were protected against lethal Ebola virus challenge. In the present study, we report on the abilities of two additional VP24-specific PMOs to reduce the cell-free translation of a VP24 reporter, to inhibit the in vitro replication of Ebola virus, and to protect mice against lethal challenge when the PMOs are delivered prior to infection. Additionally, structure-activity relationship evaluations were conducted to assess the enhancement of antiviral efficacy associated with PMO chemical modifications that included conjugation with peptides of various lengths and compositions, positioning of conjugated peptides to either the 5' or the 3' terminus, and the conferring of charge modifications by the addition of piperazine moieties. Conjugation with arginine-rich peptides greatly enhanced the antiviral efficacy of VP24-specific PMOs in infected cells and mice during lethal Ebola virus challenge.

  16. Variation in Protein Intake Induces Variation in Spider Silk Expression

    Science.gov (United States)

    Blamires, Sean J.; Wu, Chun-Lin; Tso, I-Min

    2012-01-01

    Background It is energetically expensive to synthesize certain amino acids. The proteins (spidroins) of spider major ampullate (MA) silk, MaSp1 and MaSp2, differ in amino acid composition. Glutamine and proline are prevalent in MaSp2 and are expensive to synthesize. Since most orb web spiders express high proline silk they might preferentially attain the amino acids needed for silk from food and shift toward expressing more MaSp1 in their MA silk when starved. Methodology/Principal Findings We fed three spiders; Argiope aetherea, Cyrtophora moluccensis and Leucauge blanda, high protein, low protein or no protein solutions. A. aetherea and L. blanda MA silks are high in proline, while C. moluccesnsis MA silks are low in proline. After 10 days of feeding we determined the amino acid compositions and mechanical properties of each species' MA silk and compared them between species and treatments with pre-treatment samples, accounting for ancestry. We found that the proline and glutamine of A. aetherea and L. blanda silks were affected by protein intake; significantly decreasing under the low and no protein intake treatments. Glutmaine composition in C. moluccensis silk was likewise affected by protein intake. However, the composition of proline in their MA silk was not significantly affected by protein intake. Conclusions Our results suggest that protein limitation induces a shift toward different silk proteins with lower glutamine and/or proline content. Contradictions to the MaSp model lie in the findings that C. moluccensis MA silks did not experience a significant reduction in proline and A. aetherea did not experience a significant reduction in serine on low/no protein. The mechanical properties of the silks could not be explained by a MaSp1 expressional shift. Factors other than MaSp expression, such as the expression of spidroin-like orthologues, may impact on silk amino acid composition and spinning and glandular processes may impact mechanics. PMID:22363691

  17. Factor XI Antisense Oligonucleotide for Prevention of Venous Thrombosis

    NARCIS (Netherlands)

    Büller, Harry R.; Bethune, Claudette; Bhanot, Sanjay; Gailani, David; Monia, Brett P.; Raskob, Gary E.; Segers, Annelise; Verhamme, Peter; Weitz, Jeffrey I.; Weitz, Jeffrey; Prins, Martin; Beenen, Ludo; Otten, Hans-Martin; Roos, Yvo; Slagboom, Ton; Vandenbriele, Christophe; Vanassche, Thomas; Dani, Vidhi; Schulz, Dan; Shapiro, Cara; Kwoh, Katherine; Jung, Bill; Gawinek-Samelczak, Agata; Kaemmer, Christina; Angelov, S.; Stavrev, V.; Kinov, P.; Dessouki, E.; Abuzgaya, F.; Baurovskis, A.; Peredistijs, A.; Petronis, S.; Danilyak, V.; Driagin, V.; Kuropatkin, G.; Parfeev, S.; Safronov, A.; Ankin, M.; Korzh, M.; Olinichenko, G.; Polivoda, A.; Shevchenko, V.; Sulyma, V.

    2015-01-01

    Background Experimental data indicate that reducing factor XI levels attenuates thrombosis without causing bleeding, but the role of factor XI in the prevention of postoperative venous thrombosis in humans is unknown. FXI-ASO (ISIS 416858) is a second-generation antisense oligonucleotide that

  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 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...

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

    Science.gov (United States)

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

    2016-09-06

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

  1. Chemical memory reactions induced bursting dynamics in gene expression.

    Science.gov (United States)

    Tian, Tianhai

    2013-01-01

    Memory is a ubiquitous phenomenon in biological systems in which the present system state is not entirely determined by the current conditions but also depends on the time evolutionary path of the system. Specifically, many memorial phenomena are characterized by chemical memory reactions that may fire under particular system conditions. These conditional chemical reactions contradict to the extant stochastic approaches for modeling chemical kinetics and have increasingly posed significant challenges to mathematical modeling and computer simulation. To tackle the challenge, I proposed a novel theory consisting of the memory chemical master equations and memory stochastic simulation algorithm. A stochastic model for single-gene expression was proposed to illustrate the key function of memory reactions in inducing bursting dynamics of gene expression that has been observed in experiments recently. The importance of memory reactions has been further validated by the stochastic model of the p53-MDM2 core module. Simulations showed that memory reactions is a major mechanism for realizing both sustained oscillations of p53 protein numbers in single cells and damped oscillations over a population of cells. These successful applications of the memory modeling framework suggested that this innovative theory is an effective and powerful tool to study memory process and conditional chemical reactions in a wide range of complex biological systems.

  2. Epithelial Cell Gene Expression Induced by Intracellular Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Xianglu Li

    2009-01-01

    Full Text Available HEp-2 cell monolayers were cocultured with intracellular Staphylococcus aureus, and changes in gene expression were profiled using DNA microarrays. Intracellular S. aureus affected genes involved in cellular stress responses, signal transduction, inflammation, apoptosis, fibrosis, and cholesterol biosynthesis. Transcription of stress response and signal transduction-related genes including atf3, sgk, map2k1, map2k3, arhb, and arhe was increased. In addition, elevated transcription of proinflammatory genes was observed for tnfa, il1b, il6, il8, cxcl1, ccl20, cox2, and pai1. Genes involved in proapoptosis and fibrosis were also affected at transcriptional level by intracellular S. aureus. Notably, intracellular S. aureus induced strong transcriptional down-regulation of several cholesterol biosynthesis genes. These results suggest that epithelial cells respond to intracellular S. aureus by inducing genes affecting immunity and in repairing damage caused by the organism, and are consistent with the possibility that the organism exploits an intracellular environment to subvert host immunity and promote colonization.

  3. Follicular thyroglobulin induces cathepsin H expression and activity in thyrocytes

    International Nuclear Information System (INIS)

    Oda, Kenzaburo; Luo, Yuqian; Yoshihara, Aya; Ishido, Yuko; Sekihata, Kengo

    2017-01-01

    Thyroglobulin (Tg) stored in thyroid follicles exerts a potent negative-feedback effect on each step of pre-hormone biosynthesis, including Tg gene transcription and iodine uptake and organification, by suppressing the expression of specific transcription factors that regulate these steps. Pre-hormones are stored in the follicular colloid before being reabsorbed. Following lysosomal proteolysis of its precursor, thyroid hormone (TH) is released from thyroid follicles. Although the suppressive effects of follicular Tg on each step of pre-hormone biosynthesis have been extensively characterized, whether follicular Tg accumulation also affects hormone reabsorption, proteolysis, and secretion is unclear. In this study we explored whether follicular Tg can regulate the expression and function of the lysosomal endopeptidases cathepsins. We found that in the rat thyroid cell line FRTL-5 follicular Tg induced cathepsin H mRNA and protein expression, as well as cathepsin H enzyme activity. Double immunofluorescence staining showed that Tg endocytosis promoted cathepsin H translocalization into lysosomes where it co-localized with internalized Tg. These results suggest that cathepsin H is an active participant in lysosome-mediated pre-hormone degradation, and that follicular Tg stimulates mobilization of pre-hormones by activating cathepsin H-associated proteolysis pathways. - Highlights: • Follicular Tg increases cathepsin H mRNA and protein levels in rat thyroid cells. • Follicular Tg increases cathepsin H enzyme activity in rat thyroid cells. • After Tg stimulation cathepsin H co-localizes to lysosomes with follicular Tg. • Cathepsin H promotes hormone secretion by lysosome-mediated mechanisms.

  4. High Frequency Electromagnetic Field Induces Lipocalin 2 Expression in

    Directory of Open Access Journals (Sweden)

    Amaneh Mohammadi Roushandeh

    2010-06-01

    Full Text Available Objective(sNeutrophil gelatinase-associated lipocalin (NGAL/Lcn2, comprise a group of small extracellular proteins with a common β-sheet-dominated 3-dimensional structure. In the past, it was assumed that the predominant role of lipocalin was acting as transport proteins. Recently it has been found that oxidative stress induces Lcn2 expression. It has been also proved that electromagnetic field (EMF produces reactive oxygen species (ROS in different tissues. Expression of Lcn2 following exposure to electromagnetic field has been investigated in this study. Materials and MethodsBalb/c mice (8 weeks old were exposed to 3 mT, 50 HZ EMF for 2 months, 4 hr/day. Afterwards, the mice were sacrificed by cervical dislocation and livers were removed. The liver specimens were stained with Haematoxylin- Eosin (H&E and analyzed under an optical microscope. Total RNA was extracted from liver and reverse transcription was performed by SuperScript III reverse transcriptase with 1 µg of total RNA. Assessment of Lcn2 expression was performed by semiquantitative and real time- PCR.ResultsThe light microscopic studies revealed that the number of lymphocyte cells was increased compared to control and dilation of sinosoids was observed in the liver. Lcn2 was up-regulated in the mice exposed to EMF both in mRNA and protein levels.ConclusionTo the extent of our knowledge, this is the first report dealing with up-regulation of Lcn2 in liver after exposure to EMF. The up-regulation might be a compensatory response that involves cell defense pathways and protective effects against ROS. However, further and complementary studies are required in this regards.

  5. Resveratrol reverses morphine-induced neuroinflammation in morphine-tolerant rats by reversal HDAC1 expression

    Directory of Open Access Journals (Sweden)

    Ru-Yin Tsai

    2016-06-01

    Conclusion: Resveratrol restores the antinociceptive effect of morphine by reversing morphine infusion-induced spinal cord neuroinflammation and increase in TNFR1 expression. The reversal of the morphine-induced increase in TNFR1 expression by resveratrol is partially due to reversal of the morphine infusion-induced increase in HDAC1 expression. Resveratrol pretreatment can be used as an adjuvant in clinical pain management for patients who need long-term morphine treatment or with neuropathic pain.

  6. Preliminary studies on gene therapy with TGF β1 antisense gene/liposome complexes and adenovirus transfer vector in RPF rats

    International Nuclear Information System (INIS)

    Liu Chunjie; Wang Dewen; Zhang Zhaoshan; Gao Yabing; Xiong Chengqi; Long Jianyin; Wang Huixin; Peng Ruiyun; Cui Xuemei

    2001-01-01

    Objective: To observed the efficiency of gene therapy with TGF β1 antisense gene/liposome complexes and adenovirus transfer vector in RPF rats. Methods: TGFβ1 sense and antisense gene expression vectors and adenovirus transfer vector were introduced into rat bronchus by way of intratracheal instillation. Results: At day 1.5 after TGFβ1 sense and antisense gene transfer, PCR amplification using neo gene-specific primer from lung tissue DNA was all positive. After day 5.5, 67% (2/3) of lung tissue DNA was positive. RNA dot blot hybridization indicated that TGFβ1 mRNA content of lung tissue transfected with pMAMneo-antiTGFβ1 gene decreased. Detection of lung hydroxyproline (Hyp) content after day 35 of gene transfer showed that even in lung of rats received pMAMneo-AntiTGFβ1 lipid complexes it raised remarkably (P 9 pfu/ml were instilled into bronchus at 0.5 ml per rat. After day 2 day 6, the lung tissues of all six rats (three per each group )expressed the transfected luciferase gene by luminometer. Conclusion: Cationic lipid-mediated TGFβ1 antisense gene therapy was a simple and easy method. It can slow down the course of pathogenesis of lung fibrosis. Replication-deficient recombinant adenovirus-mediated gene therapy of lung diseases is a good and efficient method

  7. Oncogenic tyrosine kinase NPM-ALK induces expression of the growth-promoting receptor ICOS

    DEFF Research Database (Denmark)

    Zhang, Qian; Wang, HongYi; Kantekure, Kanchan

    2011-01-01

    Here we report that T-cell lymphoma cells carrying the NPM-ALK fusion protein (ALK(+) TCL) frequently express the cell-stimulatory receptor ICOS. ICOS expression in ALK(+) TCL is moderate and strictly dependent on the expression and enzymatic activity of NPM-ALK. NPM-ALK induces ICOS expression v...

  8. FORMALDEHYDE-INDUCED GENE EXPRESSION IN F344 RAT NASAL RESPIRATORY EPITHELIUM.

    Science.gov (United States)

    Formaldehyde-induced gene expression in F344 rat nasal respiratory epithelium ABSTRACTFormaldehyde, an occupational and environmental toxicant used extensively in the manufacturing of many household and personal use products, is known to induce squamous cell carci...

  9. An inducible expression system for high-level expression of recombinant proteins in slow growing mycobacteria.

    Science.gov (United States)

    Leotta, Lisa; Spratt, Joanne M; Kong, Carlyn U; Triccas, James A

    2015-09-01

    A novel protein expression vector utilising the inducible hspX promoter of Mycobacterium tuberculosis was constructed and evaluated in this study. High-level induction of three mycobacterial antigens, comprising up to 9% of bacterial sonicate, was demonstrated in recombinant Mycobacterium bovis BCG when grown under low-oxygen tension, which serves to enhance hspX promoter activity. Recombinant proteins were efficiently purified from bacterial lysates in a soluble form by virtue of a C-terminal 6-histidine tag. Purification of the immunodominant M. tuberculosis Ag85B antigen using this system resulted in a recombinant protein that stimulated significant IFN-γ release from Ag85B-reactive T cells generated after vaccination of mice with an Ag85B-expressing vaccine. Further, the M. tuberculosis L-alanine dehydrogenase (Ald) protein purified from recombinant BCG displayed strong enzymatic activity in recombinant form. This study demonstrated that high levels of native-like recombinant mycobacterial proteins can be produced in mycobacterial hosts, and this may aid the analysis of mycobacterial protein function and the development of new treatments. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Knockdown of long non-coding RNA MAP3K20 antisense RNA 1 inhibits gastric cancer growth through epigenetically regulating miR-375.

    Science.gov (United States)

    Quan, Yongsheng; Zhang, Yan; Lin, Wei; Shen, Zhaohua; Wu, Shuai; Zhu, Changxin; Wang, Xiaoyan

    2018-03-04

    Emerging evidence has demonstrated that long noncoding RNAs (lncRNAs) play a critical role in tumorigenesis of gastric cancer. LncRNA MAP3K20 antisense RNA 1 (MLK7-AS1) has been identified as one of gastric cancer-specific lncRNAs. However, its precise role in gastric cancer remains unknown. In this study, we found that lncRNA MLK7-AS1 was significantly increased in gastric cancer tissues compared with in adjacent tissues. Gastric cancer patients with high MLK7-AS1 expression had a shorter survival and poorer prognosis. By loss-function assay, we demonstrated that knockdown of MLK7-AS1 inhibited cell proliferation and induced apoptosis in HGC27and MKN-45 cells. Furthermore, we identified miR-375 as a target of MLK7-AS1. MLK7-AS1 interacted with Dnmt1 and recruited it to miR-375 promotor, hyper-methylating miR-375 promotor and repressing miR-375 expression. Taken together, our findings demonstrate that knockdown of MLK7-AS1 by siRNA inhibits gastric cancer growth by epigenetically regulating miR-375. Thus, MLK7-AS1 may be a useful prognostic marker and therapeutic target for gastric cancer patients. Copyright © 2018 Elsevier Inc. All rights reserved.

  11. Bcl-2 antisense therapy in B-cell malignancies.

    Science.gov (United States)

    Chanan-Khan, Asher

    2005-07-01

    Bcl-2 is an apoptosis regulating protein, overexpression of which is associated with chemotherapy resistant disease, aggressive clinical course, and poor survival in patients with B-cell lymphoproliferative disorders. Overexpression of Bcl-2 protein results in an aberrant intrinsic apoptotic pathway that confers a protective effect on malignant cells against a death signal (e.g., chemotherapy or radiotherapy). Downregulation of this oncoprotein, thus, represents a possible new way to target clinically aggressive disease. Preclinical studies have shown that this oncoprotein can be effectively decreased by Bcl-2 antisense in malignant lymphoid cells and can reverse chemotherapy resistance, as well as enhance the anti-apoptotic potential of both chemotherapeutic and biologic agents. Ongoing clinical trials are exploring the role of Bcl-2 downregulation with oblimersen (Bcl-2 antisense) in patients with non-Hodgkin's lymphoma, chronic lymphocytic leukemia and multiple myeloma. Early results from these studies are promising and support the proof of the principle. As these studies are completed and mature data emerges, the role of Bcl-2 antisense therapy in the treatment of B-cell malignancies will become clearer.

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

    International Nuclear Information System (INIS)

    Kiani, Melika; Mirzazadeh Tekie, Farnaz Sadat; Dinarvand, Meshkat; Soleimani, Masoud; Dinarvand, Rassoul; Atyabi, Fatemeh

    2016-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-05-01

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

  14. Oxacillin sensitization of methicillin-resistant Staphylococcus aureus and methicillin-resistant Staphylococcus pseudintermedius by antisense peptide nucleic acids in vitro.

    Science.gov (United States)

    Goh, Shan; Loeffler, Anette; Lloyd, David H; Nair, Sean P; Good, Liam

    2015-11-11

    Antibiotic resistance genes can be targeted by antisense agents, which can reduce their expression and thus restore cellular susceptibility to existing antibiotics. Antisense inhibitors can be gene and pathogen specific, or designed to inhibit a group of bacteria having conserved sequences within resistance genes. Here, we aimed to develop antisense peptide nucleic acids (PNAs) that could be used to effectively restore susceptibility to β-lactams in methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus pseudintermedius (MRSP). Antisense PNAs specific for conserved regions of the mobilisable gene mecA, and the growth essential gene, ftsZ, were designed. Clinical MRSA and MRSP strains of high oxacillin resistance were treated with PNAs and assayed for reduction in colony forming units on oxacillin plates, reduction in target gene mRNA levels, and cell size. Anti-mecA PNA at 7.5 and 2.5 μM reduced mecA mRNA in MRSA and MRSP (p resistance in staphylococci. Further studies are warranted as clinical treatment alternatives are needed.

  15. 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.

  16. Antisense oligonucleotides and all-trans retinoic acid have a synergistic anti-tumor effect on oral squamous cell carcinoma

    International Nuclear Information System (INIS)

    Xu, Qin; Zhang, Zhiyuan; Zhang, Ping; Chen, Wantao

    2008-01-01

    Antisense oligonucleotides against hTR (As-ODN-hTR) have shown promising results as treatment strategies for various human malignancies. All-trans retinoic acid (ATRA) is a signalling molecule with important roles in differentiation and apoptosis. Biological responses to ATRA are currently used therapeutically in various human cancers. The aim of this study was to evaluate the anti-tumor effects of As-ODN-hTR combined with ATRA in vivo. In situ human oral squamous cell carcinoma (OSCC) models were established by subcutaneous injection of Tca8113 cells. Mice were treated with sense oligonucleotides against hTR(S-ODN-hTR) alone, As-ODN-hTR alone, ATRA alone, As-ODN-hTR plus ATRA, or S-ODN-hTR plus ATRA. Tumor size and weight were assessed in the mice. Telomerase activity was detected by a TRAP assay, apoptotic cells were evaluated with a Tunel assay, the expression of apoptosis-related proteins (Bcl-2 and Bax) was evaluated by immunohistochemistry and ultrastructural morphological changes in the tumor specimen were examined. Both As-ODN-hTR and ATRA can significantly inhibit tumor growth in this OSCC xenograft solid-tumor model, and the combination of the two agents had a synergistic anti-tumorogenic effect. We also demonstrated that this anti-tumor effect correlated with inhibition of telomerase activity. Furthermore, significant increases in the number of apoptotic cells, typical apoptotic morphology and a downregulation of the anti-apoptotic protein, bcl-2 were observed in the treated tissues. The combination of As-ODN-hTR and ATRA has a synergistic anti-tumor effect. This anti-tumor effect can be mainly attributed to apoptosis induced by a decrease in telomerase activity. Bcl-2 plays an important role in this process. Therefore, combining As-ODN-hTR and ATRA may be an approach for the treatment of human oral squamous cell carcinoma

  17. Nutrient Induced Type 2 and Chemical Induced Type 1 Experimental Diabetes Differently Modulate Gastric GLP-1 Receptor Expression

    Directory of Open Access Journals (Sweden)

    Olga Bloch

    2015-01-01

    Full Text Available T2DM patients demonstrate reduced GLP-1 receptor (GLP-1R expression in their gastric glands. Whether induced T2DM and T1DM differently affect the gastric GLP-1R expression is not known. This study assessed extrapancreatic GLP-1R system in glandular stomach of rodents with different types of experimental diabetes. T2DM and T1DM were induced in Psammomys obesus (PO by high-energy (HE diet and by streptozotocin (STZ in Sprague Dawly (SD rats, respectively. GLP-1R expression was determined in glandular stomach by RT PCR and immunohistomorphological analysis. The mRNA expression and cellular association of the GLP-1R in principal glands were similar in control PO and SD rats. However, nutrient and chemical induced diabetes resulted in opposite alterations of glandular GLP-1R expression. Diabetic PO demonstrated increased GLP-1R mRNA expression, intensity of cellular GLP-1R immunostaining, and frequency of GLP-1R positive cells in the neck area of principal glands compared with controls. In contrast, SD diabetic rats demonstrated decreased GLP-1 mRNA, cellular GLP-1R immunoreactivity, and frequency of GLP-1R immunoreactive cells in the neck area compared with controls. In conclusion, nutrient and chemical induced experimental diabetes result in distinct opposite alterations of GLP-1R expression in glandular stomach. These results suggest that induced T1DM and T2DM may differently modulate GLP-1R system in enteropancreatic axis.

  18. 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.

  19. Retroviral gene transfer of an antisense construct against membrane type 1 matrix metalloproteinase reduces the invasiveness of rheumatoid arthritis synovial fibroblasts.

    Science.gov (United States)

    Rutkauskaite, Edita; Volkmer, Dagmar; Shigeyama, Yukio; Schedel, Jörg; Pap, Geza; Müller-Ladner, Ulf; Meinecke, Ingmar; Alexander, Dorothea; Gay, Renate E; Drynda, Susanne; Neumann, Wolfram; Michel, Beat A; Aicher, Wilhelm K; Gay, Steffen; Pap, Thomas

    2005-07-01

    Membrane type 1 matrix metalloproteinase (MT1-MMP) is expressed prominently in rheumatoid arthritis synovial fibroblasts (RASFs), but the specific contribution of MT1-MMP to fibroblast-mediated destruction of articular cartilage is incompletely understood. This study used gene transfer of an antisense expression construct to assess the effects of MT1-MMP inhibition on the invasiveness of RASFs. Retroviral gene transfer of a pLXIN vector-based antisense RNA expression construct (MT1-MMPalphaS) to MT1-MMP was used to stably transduce RASFs. Levels of MT1-MMP RNA and protein were determined by quantitative polymerase chain reaction, Western blotting, and immunocytochemistry in MT1-MMPalphaS-transduced RASFs as well as in control cells, with monitoring for 60 days. The effects of MT1-MMPalphaS on the invasiveness of RASFs were analyzed in the SCID mouse co-implantation model of RA. MT1-MMPalphaS-transduced RASFs produced high levels of antisense RNA that exceeded endogenous levels of MT1-MMP messenger RNA by 15-fold and resulted in a down-regulation of MT1-MMP at the protein level. Inhibition of MT1-MMP production was maintained for 60 days and significantly reduced the invasiveness of RASFs in the SCID mouse model. Whereas prominent invasion into cartilage by non-transduced and mock-transduced RASFs was observed (mean invasion scores 3.0 and 3.1, respectively), MT1-MMPalphaS-transduced cells showed only moderate invasiveness (mean invasion score 1.8; P < 0.05). The data demonstrate that an antisense RNA expression construct against MT1-MMP can be generated and expressed in RASFs for at least 60 days. Inhibition of MT1-MMP significantly reduces the cartilage degradation by RASFs.

  20. Endogenous Msx1 antisense transcript: In vivo and in vitro evidences, structure, and potential involvement in skeleton development in mammals

    OpenAIRE

    Blin-Wakkach, C.; Lezot, F.; Ghoul-Mazgar, S.; Hotton, D.; Monteiro, S.; Teillaud, C.; Pibouin, L.; Orestes-Cardoso, S.; Papagerakis, P.; Macdougall, M.; Robert, B.; Berdal, A.

    2001-01-01

    Msx1 is a key factor for the development of tooth and craniofacial skeleton and has been proposed to play a pivotal role in terminal cell differentiation. In this paper, we demonstrated the presence of an endogenous Msx1 antisense RNA (Msx1-AS RNA) in mice, rats, and humans. In situ analysis revealed that this RNA is expressed only in differentiated dental and bone cells with an inverse correlation with Msx1 protein. These in vivo data and overexpression of Msx1 sense and AS RNA in an odontob...

  1. Widespread anti-sense transcription in apple is correlated with siRNA production and indicates a large potential for transcriptional and/or post-transcriptional control.

    Science.gov (United States)

    Celton, Jean-Marc; Gaillard, Sylvain; Bruneau, Maryline; Pelletier, Sandra; Aubourg, Sébastien; Martin-Magniette, Marie-Laure; Navarro, Lionel; Laurens, François; Renou, Jean-Pierre

    2014-07-01

    Characterizing the transcriptome of eukaryotic organisms is essential for studying gene regulation and its impact on phenotype. The realization that anti-sense (AS) and noncoding RNA transcription is pervasive in many genomes has emphasized our limited understanding of gene transcription and post-transcriptional regulation. Numerous mechanisms including convergent transcription, anti-correlated expression of sense and AS transcripts, and RNAi remain ill-defined. Here, we have combined microarray analysis and high-throughput sequencing of small RNAs (sRNAs) to unravel the complexity of transcriptional and potential post-transcriptional regulation in eight organs of apple (Malus × domestica). The percentage of AS transcript expression is higher than that identified in annual plants such as rice and Arabidopsis thaliana. Furthermore, we show that a majority of AS transcripts are transcribed beyond 3'UTR regions, and may cover a significant portion of the predicted sense transcripts. Finally we demonstrate at a genome-wide scale that anti-sense transcript expression is correlated with the presence of both short (21-23 nt) and long (> 30 nt) siRNAs, and that the sRNA coverage depth varies with the level of AS transcript expression. Our study provides a new insight on the functional role of anti-sense transcripts at the genome-wide level, and a new basis for the understanding of sRNA biogenesis in plants. © 2014 INRA. New Phytologist © 2014 New Phytologist Trust.

  2. Thymoquinone inhibits phorbol ester-induced activation of NF-κB and expression of COX-2, and induces expression of cytoprotective enzymes in mouse skin in vivo

    International Nuclear Information System (INIS)

    Kundu, Joydeb Kumar; Liu, Lijia; Shin, Jun-Wan; Surh, Young-Joon

    2013-01-01

    Highlights: •Thymoquinone inhibits phorbol ester-induced COX-2 expression in mouse skin. •Thymoquinone attenuates phosphorylation of IκBα and DNA binding of NF-κB in mouse skin. •Thymoquinone inhibits phosphorylation of p38 MAP kinase, JNK and Akt in mouse skin. •Thymoquinone induces the expression of cytoprotective proteins in mouse skin. -- Abstract: Thymoquinone (TQ), the active ingredient of Nigella sativa, has been reported to possess anti-inflammatory and chemopreventive properties. The present study was aimed at elucidating the molecular mechanisms of anti-inflammatory and antioxidative activities of thymoquinone in mouse skin. Pretreatment of female HR-1 hairless mouse skin with TQ attenuated 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced expression of cyclooxygenase-2 (COX-2). TQ diminished nuclear translocation and the DNA binding of nuclear factor-kappaB (NF-κB) via the blockade of phosphorylation and subsequent degradation of IκBα in TPA-treated mouse skin. Pretreatment with TQ attenuated the phosphorylation of Akt, c-Jun-N-terminal kinase and p38 mitogen-activated protein kinase, but not that of extracellular signal-regulated kinase-1/2. Moreover, topical application of TQ induced the expression of heme oxygenase-1, NAD(P)H-quinoneoxidoreductase-1, glutathione-S-transferase and glutamate cysteine ligase in mouse skin. Taken together, the inhibitory effects of TQ on TPA-induced COX-2 expression and NF-κB activation, and its ability to induce the expression of cytoprotective proteins provide a mechanistic basis of anti-inflammatory and antioxidative effects of TQ in hairless mouse skin

  3. Evaluation of novel inducible promoter/repressor systems for recombinant protein expression in Lactobacillus plantarum.

    Science.gov (United States)

    Heiss, Silvia; Hörmann, Angelika; Tauer, Christopher; Sonnleitner, Margot; Egger, Esther; Grabherr, Reingard; Heinl, Stefan

    2016-03-10

    Engineering lactic acid bacteria (LAB) is of growing importance for food and feed industry as well as for in vivo vaccination or the production of recombinant proteins in food grade organisms. Often, expression of a transgene is only desired at a certain time point or period, e.g. to minimize the metabolic burden for the host cell or to control the expression time span. For this purpose, inducible expression systems are preferred, though cost and availability of the inducing agent must be feasible. We selected the plasmid free strain Lactobacillus plantarum 3NSH for testing and characterization of novel inducible promoters/repressor systems. Their feasibility in recombinant protein production was evaluated. Expression of the reporter protein mCherry was monitored with the BioLector(®) micro-fermentation system. Reporter gene mCherry expression was compared under the control of different promoter/repressor systems: PlacA (an endogenous promoter/repressor system derived from L. plantarum 3NSH), PxylA (a promoter/repressor system derived from Bacillus megaterium DSMZ 319) and PlacSynth (synthetic promoter and codon-optimized repressor gene based on the Escherichia coli lac operon). We observed that PlacA was inducible solely by lactose, but not by non-metabolizable allolactose analoga. PxylA was inducible by xylose, yet showed basal expression under non-induced conditions. Growth on galactose (as compared to exponential growth phase on glucose) reduced basal mCherry expression at non-induced conditions. PlacSynth was inducible with TMG (methyl β-D-thiogalactopyranoside) and IPTG (isopropyl β-D-1-thiogalactopyranoside), but also showed basal expression without inducer. The promoter PlacSynth was used for establishment of a dual plasmid expression system, based on T7 RNA polymerase driven expression in L. plantarum. Comparative Western blot supported BioLector(®) micro-fermentation measurements. Conclusively, overall expression levels were moderate (compared to a

  4. Gene expression profiling distinguishes between spontaneous and radiation-induced rat mammary carcinomas

    International Nuclear Information System (INIS)

    Imaoka, Tatsuhiko; Nishimura, Mayumi; Kakinuma, Shizuko; Shimada, Yoshiya; Yamashita, Satoshi; Ushijima, Toshikazu

    2008-01-01

    The ability to distinguish between spontaneous and radiation-induced cancers in humans is expected to improve the resolution of estimated risk from low dose radiation. Mammary carcinomas were obtained from Sprague-Dawley rats that were either untreated (n=45) or acutely γ-irradiated (1 Gy; n=20) at seven weeks of age. Gene expression profiles of three spontaneous and four radiation-induced carcinomas, as well as those of normal mammary glands, were analyzed by microarrays. Differential expression of identified genes of interest was then verified by quantitative polymerase chain reaction (qPCR). Cluster analysis of global gene expression suggested that spontaneous carcinomas were distinguished from a heterogeneous population of radiation-induced carcinomas, though most gene expressions were common. We identified 50 genes that had different expression levels between spontaneous and radiogenic carcinomas. We then selected 18 genes for confirmation of the microarray data by qPCR analysis and obtained the following results: high expression of Plg, Pgr and Wnt4 was characteristic to all spontaneous carcinomas; Tnfsf11, Fgf10, Agtr1a, S100A9 and Pou3f3 showed high expression in a subset of radiation-induced carcinomas; and increased Gp2, Areg and Igf2 expression, as well as decreased expression of Ca3 and noncoding RNA Mg1, were common to all carcinomas. Thus, gene expression analysis distinguished between spontaneous and radiogenic carcinomas, suggesting possible differences in their carcinogenic mechanism. (author)

  5. Behavioral experience induces zif268 expression in mature granule cells but suppresses its expression in immature granule cells

    Science.gov (United States)

    Huckleberry, Kylie A.; Kane, Gary A.; Mathis, Rita J.; Cook, Sarah G.; Clutton, Jonathan E.; Drew, Michael R.

    2015-01-01

    Thousands of neurons are born each day in the dentate gyrus (DG), but many of these cells die before reaching maturity. Both death and survival of adult-born neurons are regulated by neuronal activity in the DG. The immediate-early gene (IEG) zif268 appears to be an important mediator of these effects, as its expression can be induced by neural activity and knockout of zif268 impairs survival of adult-born neurons (Richardson et al., 1992; Veyrac et al., 2013). Despite the apparent importance of zif268 for adult neurogenesis, its behavior-induced expression has not been fully characterized in adult-born neurons. Here we characterize behavior-evoked expression of zif268 in mature and newborn dentate granule cells (DGCs). We first quantified zif268 expression in doublecortin-positive (DCX+) immature neurons and in the general granule cell population after brief exposure to a novel environment (NE). In the general granule cell population, zif268 expression peaked 1 h after NE exposure and returned to baseline by 8 h post-exposure. However, in the DCX+ cells, zif268 expression was suppressed relative to home cage for at least 8 h post-exposure. We next asked whether suppression of zif268 in DCX+ immature cells occurs in other behavioral paradigms that recruit the hippocampus. Exposure to Morris water maze (MWM) training, an enriched environment, or a NE caused approximately equal suppression of zif268 expression in DCX+ cells and approximately equal activation of zif268 expression among the general granule cell population. The same behavioral procedures activated zif268 expression in 6-week-old BrdU-labeled adult-born neurons, indicating that zif268 suppression is specific to immature neurons. Finally, we asked whether zif268 suppression varied as a function of age within the DCX+ population, which ranges in age from 0 to approximately 4 weeks. NE exposure had no significant effect on zif268 expression in 2- or 4-week-old BrdU-labeled neurons, but it significantly

  6. PTP1B antisense oligonucleotide lowers PTP1B protein, normalizes blood glucose, and improves insulin sensitivity in diabetic mice

    Science.gov (United States)

    Zinker, Bradley A.; Rondinone, Cristina M.; Trevillyan, James M.; Gum, Rebecca J.; Clampit, Jill E.; Waring, Jeffrey F.; Xie, Nancy; Wilcox, Denise; Jacobson, Peer; Frost, Leigh; Kroeger, Paul E.; Reilly, Regina M.; Koterski, Sandra; Opgenorth, Terry J.; Ulrich, Roger G.; Crosby, Seth; Butler, Madeline; Murray, Susan F.; McKay, Robert A.; Bhanot, Sanjay; Monia, Brett P.; Jirousek, Michael R.

    2002-01-01

    The role of protein-tyrosine phosphatase 1B (PTP1B) in diabetes was investigated using an antisense oligonucleotide in ob/ob and db/db mice. PTP1B antisense oligonucleotide treatment normalized plasma glucose levels, postprandial glucose excursion, and HbA1C. Hyperinsulinemia was also reduced with improved insulin sensitivity. PTP1B protein and mRNA were reduced in liver and fat with no effect in skeletal muscle. Insulin signaling proteins, insulin receptor substrate 2 and phosphatidylinositol 3 (PI3)-kinase regulatory subunit p50α, were increased and PI3-kinase p85α expression was decreased in liver and fat. These changes in protein expression correlated with increased insulin-stimulated protein kinase B phosphorylation. The expression of liver gluconeogenic enzymes, phosphoenolpyruvate carboxykinase, and fructose-1,6-bisphosphatase was also down-regulated. These findings suggest that PTP1B modulates insulin signaling in liver and fat, and that therapeutic modalities targeting PTP1B inhibition may have clinical benefit in type 2 diabetes. PMID:12169659

  7. 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.

  8. Mucosal Progranulin expression is induced by H. pylori, but independent of Secretory Leukocyte Protease Inhibitor (SLPI expression

    Directory of Open Access Journals (Sweden)

    Treiber Gerhard

    2011-05-01

    Full Text Available Abstract Background Mucosal levels of Secretory Leukocyte Protease Inhibitor (SLPI are specifically reduced in relation to H. pylori-induced gastritis. Progranulin is an epithelial growth factor that is proteolytically degraded into fragments by elastase (the main target of SLPI. Considering the role of SLPI for regulating the activity of elastase, we studied whether the H. pylori-induced reduction of SLPI and the resulting increase of elastase-derived activity would reduce the Progranulin protein levels both ex vivo and in vitro. Methods The expression of Progranulin was studied in biopsies of H. pylori-positive, -negative and -eradicated subjects as well as in the gastric tumor cell line AGS by ELISA, immunohistochemistry and real-time RT-PCR. Results H. pylori-infected subjects had about 2-fold increased antral Progranulin expression compared to H. pylori-negative and -eradicated subjects (P H. pylori infection; both epithelial and infiltrating immune cells contributed to the higher Progranulin expression levels. The H. pylori-induced upregulation of Progranulin was verified in AGS cells infected by H. pylori. The down-regulation of endogenous SLPI expression in AGS cells by siRNA methodology did not affect the Progranulin expression independent of the infection by H. pylori. Conclusions Taken together, Progranulin was identified as novel molecule that is upregulated in context to H. pylori infection. In contrast to other diseases, SLPI seems not to have a regulatory role for Progranulin in H. pylori-mediated gastritis.

  9. Induced resistance and gene expression in wheat against leaf rust ...

    African Journals Online (AJOL)

    uvp

    2013-05-15

    May 15, 2013 ... 2Department of Soil, Crop and Climate Sciences, University of the Free State, P.O Box ... Key words: Wheat leaf rust, induced resistance, priming, gene ..... transformation: susceptibility of transgenic Nicotiana sylvestris plants.

  10. Ligand Binding Domain Protein in Tetracycline-Inducible Expression ...

    African Journals Online (AJOL)

    binding domain proteins in E. coli using a tetracycline inducible system. To allow for ... development of molecular ligands with improved therapeutic windows. Keywords: Nuclear receptor ..... functional recombinant cannabinoid receptor CB2 in ...

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

    Science.gov (United States)

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

    2012-03-01

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

  12. Effective photo-enhancement of cellular activity of fluorophore-octaarginine antisense PNA conjugates correlates with singlet oxygen formation, endosomal escape and chromophore lipophilicity

    DEFF Research Database (Denmark)

    Yarani, Reza; Shiraishi, Takehiko; Nielsen, Peter E.

    2018-01-01

    Photochemical internalization (PCI) is a cellular drug delivery method based on the generation of light-induced reactive oxygen species (ROS) causing damage to the endosomal membrane and thereby resulting in drug release to the cytoplasm. In our study a series of antisense fluorophore octaarginin...... indicate that efficient photodynamic endosomal escape is strongly dependent on the quantum yield for photochemical singlet oxygen formation, photostability as well as the lipophilicity of the chromophore....

  13. Bupivacaine-induced apoptosis independently of WDR35 expression in mouse neuroblastoma Neuro2a cells

    Science.gov (United States)

    2012-01-01

    Background Bupivacaine-induced neurotoxicity has been shown to occur through apoptosis. Recently, bupivacaine was shown to elicit reactive oxygen species (ROS) production and induce apoptosis accompanied by activation of p38 mitogen-activated protein kinase (MAPK) in a human neuroblastoma cell line. We have reported that WDR35, a WD40-repeat protein, may mediate apoptosis through caspase-3 activation. The present study was undertaken to test whether bupivacaine induces apoptosis in mouse neuroblastoma Neuro2a cells and to determine whether ROS, p38 MAPK, and WDR35 are involved. Results Our results showed that bupivacaine induced ROS generation and p38 MAPK activation in Neuro2a cells, resulting in apoptosis. Bupivacaine also increased WDR35 expression in a dose- and time-dependent manner. Hydrogen peroxide (H2O2) also increased WDR35 expression in Neuro2a cells. Antioxidant (EUK-8) and p38 MAPK inhibitor (SB202190) treatment attenuated the increase in caspase-3 activity, cell death and WDR35 expression induced by bupivacaine or H2O2. Although transfection of Neuro2a cells with WDR35 siRNA attenuated the bupivacaine- or H2O2-induced increase in expression of WDR35 mRNA and protein, in contrast to our previous studies, it did not inhibit the increase in caspase-3 activity in bupivacaine- or H2O2-treated cells. Conclusions In summary, our results indicated that bupivacaine induced apoptosis in Neuro2a cells. Bupivacaine induced ROS generation and p38 MAPK activation, resulting in an increase in WDR35 expression, in these cells. However, the increase in WDR35 expression may not be essential for the bupivacaine-induced apoptosis in Neuro2a cells. These results may suggest the existence of another mechanism of bupivacaine-induced apoptosis independent from WDR35 expression in Neuro2a cells. PMID:23227925

  14. Ebola virus infection induces irregular dendritic cell gene expression.

    Science.gov (United States)

    Melanson, Vanessa R; Kalina, Warren V; Williams, Priscilla

    2015-02-01

    Filoviruses subvert the human immune system in part by infecting and replicating in dendritic cells (DCs). Using gene arrays, a phenotypic profile of filovirus infection in human monocyte-derived DCs was assessed. Monocytes from human donors were cultured in GM-CSF and IL-4 and were infected with Ebola virus Kikwit variant for up to 48 h. Extracted DC RNA was analyzed on SuperArray's Dendritic and Antigen Presenting Cell Oligo GEArray and compared to uninfected controls. Infected DCs exhibited increased expression of cytokine, chemokine, antiviral, and anti-apoptotic genes not seen in uninfected controls. Significant increases of intracellular antiviral and MHC I and II genes were also noted in EBOV-infected DCs. However, infected DCs failed to show any significant difference in co-stimulatory T-cell gene expression from uninfected DCs. Moreover, several chemokine genes were activated, but there was sparse expression of chemokine receptors that enabled activated DCs to home to lymph nodes. Overall, statistically significant expression of several intracellular antiviral genes was noted, which may limit viral load but fails to stop replication. EBOV gene expression profiling is of vital importance in understanding pathogenesis and devising novel therapeutic treatments such as small-molecule inhibitors.

  15. Mast cells infiltration and decreased E-cadherin expression in ketamine-induced cystitis

    Directory of Open Access Journals (Sweden)

    Mengqiang Li

    2015-01-01

    Conclusions: Increased mast cells in bladder wall and downregulated expression of E-cadherin junction protein in epithelial cells were probably associated with interstitial inflammation and fissures in mucosa. It implied that ketamine induced an interstitial cystitis.

  16. Insulin decreases atherosclerosis by inducing endothelin receptor B expression

    DEFF Research Database (Denmark)

    Park, Kyoungmin; Mima, Akira; Li, Qian

    2016-01-01

    Endothelial cell (EC) insulin resistance and dysfunction, caused by diabetes, accelerates atherosclerosis. It is unknown whether specifically enhancing EC-targeted insulin action can decrease atherosclerosis in diabetes. Accordingly, overexpressing insulin receptor substrate-1 (IRS1...... induction of NO action, which increases endothelin receptor B (EDNRB) expression and intracellular [Ca(2+)]. Using the mice with knockin mutation of eNOS, which had Ser1176 mutated to alanine (AKI), deleting the only known mechanism for insulin to activate eNOS/NO pathway, we observed that IRS1...... overexpression in the endothelia of Aki/ApoE(-/-) mice significantly decreased atherosclerosis. Interestingly, endothelial EDNRB expression was selectively reduced in intima of arteries from diabetic patients and rodents. However, endothelial EDNRB expression was upregulated by insulin via P13K/Akt pathway...

  17. A new maltose-inducible high-performance heterologous expression system in Bacillus subtilis.

    Science.gov (United States)

    Yue, Jie; Fu, Gang; Zhang, Dawei; Wen, Jianping

    2017-08-01

    To improve heterologous proteins production, we constructed a maltose-inducible expression system in Bacillus subtilis. An expression system based on the promoter for maltose utilization constructed in B. subtilis. Successively, to improve the performance of the P malA -derived system, mutagenesis was employed by gradually shortening the length of P malA promoter and altering the spacing between the predicted MalR binding site and the -35 region. Furthermore, deletion of the maltose utilization genes (malL and yvdK) improved the P malA promoter activity. Finally, using this efficient maltose-inducible expression system, we enhanced the production of luciferase and D-aminoacylase, compared with the P hpaII system. A maltose-inducible expression system was constructed and evaluated. It could be used for high level expression of heterologous proteins production.

  18. Inhibition of interleukin-6 decreases atrogene expression and ameliorates tail suspension-induced skeletal muscle atrophy

    Science.gov (United States)

    Yakabe, Mitsutaka; Ota, Hidetaka; Iijima, Katsuya; Eto, Masato; Ouchi, Yasuyoshi; Akishita, Masahiro

    2018-01-01

    Background Interleukin-6 (IL-6) is an inflammatory cytokine. Whether systemic IL-6 affects atrogene expression and disuse-induced skeletal muscle atrophy is unclear. Methods Tail-suspended mice were used as a disuse-induced muscle atrophy model. We administered anti-mouse IL-6 receptor antibody, beta-hydroxy-beta-methylbutyrate (HMB) and vitamin D to the mice and examined the effects on atrogene expression and muscle atrophy. Results Serum IL-6 levels were elevated in the mice. Inhibition of IL-6 receptor suppressed muscle RING finger 1 (MuRF1) expression and prevented muscle atrophy. HMB and vitamin D inhibited the serum IL-6 surge, downregulated the expression of MuRF1 and atrogin-1 in the soleus muscle, and ameliorated atrophy in the mice. Conclusion Systemic IL-6 affects MuRF1 expression and disuse-induced muscle atrophy. PMID:29351340

  19. Inhibition of interleukin-6 decreases atrogene expression and ameliorates tail suspension-induced skeletal muscle atrophy.

    Directory of Open Access Journals (Sweden)

    Mitsutaka Yakabe

    Full Text Available Interleukin-6 (IL-6 is an inflammatory cytokine. Whether systemic IL-6 affects atrogene expression and disuse-induced skeletal muscle atrophy is unclear.Tail-suspended mice were used as a disuse-induced muscle atrophy model. We administered anti-mouse IL-6 receptor antibody, beta-hydroxy-beta-methylbutyrate (HMB and vitamin D to the mice and examined the effects on atrogene expression and muscle atrophy.Serum IL-6 levels were elevated in the mice. Inhibition of IL-6 receptor suppressed muscle RING finger 1 (MuRF1 expression and prevented muscle atrophy. HMB and vitamin D inhibited the serum IL-6 surge, downregulated the expression of MuRF1 and atrogin-1 in the soleus muscle, and ameliorated atrophy in the mice.Systemic IL-6 affects MuRF1 expression and disuse-induced muscle atrophy.

  20. Estradiol-induced gene expression in largemouth bass (Micropterus salmoides)

    Science.gov (United States)

    Bowman, C.J.; Kroll, K.J.; Gross, T.G.; Denslow, N.D.

    2002-01-01

    Vitellogenin (Vtg) and estrogen receptor (ER) gene expression levels were measured in largemouth bass to evaluate the activation of the ER-mediated pathway by estradiol (E2). Single injections of E2 ranging from 0.0005 to 5 mg/kg up-regulated plasma Vtg in a dose-dependent manner. Vtg and ER mRNAs were measured using partial cDNA sequences corresponding to the C-terminal domain for Vtg and the ligand-binding domain of ER?? sequences. After acute E2-exposures (2 mg/kg), Vtg and ER mRNAs and plasma Vtg levels peaked after 2 days. The rate of ER mRNA accumulation peaked 36-42 h earlier than Vtg mRNA. The expression window for ER defines the primary response to E2 in largemouth bass and that for Vtg a delayed primary response. The specific effect of E2 on other estrogen-regulated genes was tested during these same time windows using differential display RT-PCR. Specific up-regulated genes that are expressed in the same time window as Vtg were ERp72 (a membrane-bound disulfide isomerase) and a gene with homology to an expressed gene identified in zebrafish. Genes that were expressed in a pattern that mimics the ER include the gene for zona radiata protein ZP2, and a gene with homology to an expressed gene found in winter flounder. One gene for fibrinogen ?? was down-regulated and an unidentified gene was transiently up-regulated after 12 h of exposure and returned to basal levels by 48 h. Taken together these studies indicate that the acute molecular response to E2 involves a complex network of responses over time. ?? 2002 Elsevier Science Ireland Ltd. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Gaëll Mainguy

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

  2. 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.

  3. Connexin43 gene and its irradiation-induced expression

    International Nuclear Information System (INIS)

    Long Xianhui; Zhou Pingkun

    2005-01-01

    Gap junctions, composed of connexin protein subunits, provide the important channel for the intercellular communication. Connexin43, the most popular component of the connexin protein family, is widely expressed in multiple tissues and cell lines and plays an important role in cell proliferation, differention and tissue homeostasis. Recently it was reported that the expression of connexin43 gene is remarkedly up-regulated by low dose ionizing radiation, the available data suggest connexin43 gene to be a poten-tial sensitive bio-marker in radiation damage. (authors)

  4. GPBAR1/TGR5 mediates bile acid-induced cytokine expression in murine Kupffer cells.

    Directory of Open Access Journals (Sweden)

    Guiyu Lou

    Full Text Available GPBAR1/TGR5 is a novel plasma membrane-bound G protein-coupled bile acid (BA receptor. BAs are known to induce the expression of inflammatory cytokines in the liver with unknown mechanism. Here we show that without other external stimuli, TGR5 activation alone induced the expression of interleukin 1β (IL-1β and tumor necrosis factor-α (TNF-α in murine macrophage cell line RAW264.7 or murine Kupffer cells. The TGR5-mediated increase of pro-inflammatory cytokine expression was suppressed by JNK inhibition. Moreover, the induced pro-inflammatory cytokine expression in mouse liver by 1% cholic acid (CA diet was blunted in JNK-/- mice. TGR5 activation by its ligands enhanced the phosphorylation levels, DNA-binding and trans-activities of c-Jun and ATF2 transcription factors. Finally, the induced pro-inflammatory cytokine expression in Kupffer cells by TGR5 activation correlated with the suppression of Cholesterol 7α-hydroxylase (Cyp7a1 expression in murine hepatocytes. These results suggest that TGR5 mediates the BA-induced pro-inflammatory cytokine production in murine Kupffer cells through JNK-dependent pathway. This novel role of TGR5 may correlate to the suppression of Cyp7a1 expression in hepatocytes and contribute to the delicate BA feedback regulation.

  5. Function and regulation of ATF 3 expression induced by ionizing radiation

    International Nuclear Information System (INIS)

    Fan, Feiyue; Wang, Yong; Du, Liqin; Zhan, Qimin

    2008-01-01

    Full text: Ionizing radiation results in a series of damages of mammalian cells as a genotoxic stress. There are some genes expressed after cells damaged, in which ATF 3, a member of ATF/CREB family of transcription factors, is one of them. In this report, we demonstrate that ATF 3 can be induced by ionizing radiation. The induction of ATF 3 protein requires normal status of p53 function in cells. There are some quantitative relationships between ATF 3 induction and dosages of radiation or time post-irradiation. In another word, ATF 3 expression induced by ionizing radiation present dose- and time-dependent. The regulation of ATF 3 expression refers to program of promoter and transcription. Radiation induces ATF 3 expression by activating the promoter and RNA transcription. In method of tetracycline-inducible system (tet-off), we have found that over-expression of ATF 3 protein brings caspase/PARP proteins into cleavage, which induces cell programmed death, and suppresses cell growth. Meanwhile, it was found that ATF 3 expression could slow down progression of cell from G 1 to S phase. It indicates ATF 3 protein might play a negative role in the control of cell cycle progression. It is very excited that expression of ATF 3 protein did not only suppress cell growth, but also demonstrated protecting effect of cell growth suppression resulting from ionizing radiation. It is suggested that ATF 3 protein might take part in the damage repair process of cells. (author)

  6. Regulatable Transgene Expression for Prevention of Chemotherapy-Induced Peripheral Neuropathy

    Directory of Open Access Journals (Sweden)

    Daisuke Kawata

    2017-09-01

    Full Text Available Chemotherapy-induced peripheral neuropathy (CIPN is a debilitating complication associated with drug treatment of cancer for which there are no effective strategies of prevention or treatment. In this study, we examined the effect of intermittent expression of neurotophin-3 (NT-3 or interleukin-10 (IL-10 from replication-defective herpes simplex virus (HSV-based regulatable vectors delivered by subcutaneous inoculation to the dorsal root ganglion (DRG on the development of paclitaxel-induced peripheral neuropathy. We constructed two different tetracycline (tet-on-based regulatable HSV vectors, one expressing NT-3 and the other expressing IL-10, in which the transactivator expression in the tet-on system was under the control of HSV latency-associated promoter 2 (LAP-2, and expression of the transgene was controlled by doxycycline (DOX. We examined the therapeutic effect of intermittent expression of the transgene in animals with paclitaxel-induced peripheral neuropathy modeled by intraperitoneal injection of paclitaxel (16 mg/kg once a week for 5 weeks. Intermittent expression of either NT-3 or IL-10 3 days before and 1 day after paclitaxel administration protected animals against paclitaxel-induced peripheral neuropathy over the course of 5 weeks. These results suggest the potential of regulatable vectors for prevention of chemotherapy-induced peripheral neuropathy.

  7. Regulatable Transgene Expression for Prevention of Chemotherapy-Induced Peripheral Neuropathy.

    Science.gov (United States)

    Kawata, Daisuke; Wu, Zetang

    2017-09-15

    Chemotherapy-induced peripheral neuropathy (CIPN) is a debilitating complication associated with drug treatment of cancer for which there are no effective strategies of prevention or treatment. In this study, we examined the effect of intermittent expression of neurotophin-3 (NT-3) or interleukin-10 (IL-10) from replication-defective herpes simplex virus (HSV)-based regulatable vectors delivered by subcutaneous inoculation to the dorsal root ganglion (DRG) on the development of paclitaxel-induced peripheral neuropathy. We constructed two different tetracycline (tet)-on-based regulatable HSV vectors, one expressing NT-3 and the other expressing IL-10, in which the transactivator expression in the tet-on system was under the control of HSV latency-associated promoter 2 (LAP-2), and expression of the transgene was controlled by doxycycline (DOX). We examined the therapeutic effect of intermittent expression of the transgene in animals with paclitaxel-induced peripheral neuropathy modeled by intraperitoneal injection of paclitaxel (16 mg/kg) once a week for 5 weeks. Intermittent expression of either NT-3 or IL-10 3 days before and 1 day after paclitaxel administration protected animals against paclitaxel-induced peripheral neuropathy over the course of 5 weeks. These results suggest the potential of regulatable vectors for prevention of chemotherapy-induced peripheral neuropathy.

  8. Decitabine induces delayed reactive oxygen species (ROS) accumulation in leukemia cells and induces the expression of ROS generating enzymes.

    Science.gov (United States)

    Fandy, Tamer E; Jiemjit, Anchalee; Thakar, Manjusha; Rhoden, Paulette; Suarez, Lauren; Gore, Steven D

    2014-03-01

    Azanucleoside DNA methyltransferase (DNMT) inhibitors are currently approved by the U.S. Food and Drug Administration for treatment of myelodysplastic syndrome. The relative contributions of DNMT inhibition and other off-target effects to their clinical efficacy remain unclear. Data correlating DNA methylation reversal and clinical response have been conflicting. Consequently, it is necessary to investigate so-called off-target effects and their impact on cell survival and differentiation. Flow cytometry was used for cell cycle, apoptosis, and reactive oxygen species (ROS) accumulation analysis. Gene expression analysis was performed using real-time PCR. DNA methylation was detected by methylation-specific PCR. Mitochondrial membrane potential was analyzed using JC-1 dye staining. Western blotting was used for quantitative protein expression analysis. 5-Aza-2'-deoxycytidine (DAC) induced cell-cycle arrest and apoptosis in leukemia cells. p53 expression was dispensable for DAC-induced apoptosis. DAC induced delayed ROS accumulation in leukemia cells but not in solid tumor cells and p53 expression was dispensable for ROS increase. ROS increase was deoxycytidine kinase dependent, indicating that incorporation of DAC into nuclear DNA is required for ROS generation. ROS accumulation by DAC was caspase-independent and mediated the dissipation of the mitochondrial membrane potential. Concordantly, ROS scavengers diminished DAC-induced apoptosis. DAC induced the expression of different NADPH oxidase isoforms and upregulated Nox4 protein expression in an ATM-dependent manner, indicating the involvement of DNA damage signaling in Nox4 upregulation. These data highlight the importance of mechanisms other than DNA cytosine demethylation in modulating gene expression and suggest investigating the relevance of ROS accumulation to the clinical activity of DAC. ©2014 AACR

  9. RNA interference prevents lipopolysaccharide-induced preprotachykinin gene expression

    International Nuclear Information System (INIS)

    Lai, Y.-L.; Yu, S.C.; Chen, M.-J.

    2003-01-01

    We showed previously that lipopolysaccharide (LPS) induces noncholinergic airway hyperreactivity to capsaicin via an upregulation of tachykinin synthesis. This study was designed to test whether double-stranded preprotachykinin (ds PPT) RNA, RNA interference (RNAi), prevents the LPS-induced alterations. First, cultured primary nodose ganglial cells of newborn Brown-Norway rats were divided into four groups: control; LPS; LPS+RNAi; and LPS+RNAi+liposome. Second, young Brown-Norway rats for the in vivo study were divided into three groups (control; LPS; and LPS+RNAi), and ds PPT RNA was microinjected bilaterally into the nodose ganglia in the LPS+RNAi group. Then, ganglial cells were collected from the culture whereas the nodose ganglia and lungs were sampled from the animals, and PPT mRNA and substance P (SP) levels were analyzed. Also, airway reactivity to capsaicin was performed in vivo. LPS induced significant increases in PPT mRNA and SP levels in vitro and in vivo and an increase in airway reactivity to capsaicin in vivo. However, ds PPT RNA, but not scrambled RNA, prevented all LPS-induced alterations. The effect of ds PPT RNA was not enhanced by liposome in vitro. Therefore, we demonstrated that the local application of RNAi prevents effectively the activation of the noncholinergic system modulating the lungs/airways

  10. STAT5 induces miR-21 expression in cutaneous T cell lymphoma

    DEFF Research Database (Denmark)

    Lindahl, Lise M; Fredholm, Simon; Joseph, Claudine

    2016-01-01

    was inhibited by Tofacitinib (CP-690550), a clinical-grade JAK3 inhibitor. Chromatin immunoprecipitation (ChIP) analysis showed direct binding of STAT5 to the miR-21 promoter. Cytokine starvation ex vivo triggered a decrease in miR-21 expression, whereas IL-2 induced an increased miR-21 expression in primary SS...

  11. Nitric oxide is involved in the down-regulation of sost expression induced by mechanical loading

    NARCIS (Netherlands)

    Delgado-Calle, J.; Riancho, J.A.; Klein-Nulend, J.

    2014-01-01

    Mechanical stimulation reduces sclerostin expression in rodents. However, few data are available about the effect of physical stimuli in human systems. Recently we observed that the demethylating agent AzadC induces SOST expression in bone cells. This allowed us in this study to explore the effect

  12. Behavioral experience induces zif268 expression in mature granule cells but suppresses its expression in immature granule cells

    Directory of Open Access Journals (Sweden)

    Kylie A. Huckleberry

    2015-08-01

    Full Text Available Thousands of neurons are born each day in the dentate gyrus (DG, but many of these cells die before reaching maturity. Both death and survival of adult-born neurons are regulated by neuronal activity in DG. The immediate-early gene (IEG zif268 is an important mediator of these effects, as its expression is induced by neural activity and knockout of zif268 impairs survival of adult-born neurons (Veyrac et al., 2013. Despite the apparent importance of zif268 for adult neurogenesis, its behavior-induced expression has not been fully characterized in adult-born neurons. Here we characterize behavior-evoked expression of zif268 in mature and newborn dentate granule cells (DGCs. In the general granule cell population, zif268 expression peaked 1 hour after novel environment exposure and returned to baseline by 8 hours post-exposure. However, in the doublecortin-positive (DCX+ immature neurons, zif268 expression was suppressed relative to home cage for at least 8 hours post-exposure. We next determined that exposure to water maze training, an enriched environment, or a novel environment caused approximately equal suppression of zif268 expression in DCX+ cells and approximately equal activation of zif268 in the general DGC population and in 6-week-old adult-born neurons. Finally, we asked whether zif268 suppression varied as a function of age within the DCX+ population, which ranges in age from 0 to approximately 4 weeks. Novel environment exposure had no significant effect on zif268 expression in 2- or 4-week-old BrdU-labeled neurons, but it significantly suppressed zif268 expression in 3-week-old neurons. In summary, behavioral experience transiently activated expression of zif268 in mature DGCs but caused a more long-lasting suppression of zif268 expression in immature, adult-born DGCs. We hypothesize that zif268 suppression inhibits memory-related synaptic plasticity in immature DGCs or mediates learning-induced apoptosis of immature adult

  13. Antisense-MDM2 Sensitizes LNCaP Prostate Cancer Cells to Androgen Deprivation, Radiation, and the Combination In Vivo

    International Nuclear Information System (INIS)

    Stoyanova, Radka; Hachem, Paul; Hensley, Harvey; Khor, L.-Y.; Mu Zhaomei; Hammond, M. Elizabeth H.; Agrawal, Sudhir; Pollack, Alan

    2007-01-01

    Purpose: To test the effects of antisense (AS)-MDM2 alone and with androgen deprivation (AD), radiotherapy (RT), and AD + RT on wild-type LNCaP cells in an orthotopic in vivo model. Methods: Androgen-sensitive LNCaP cells were grown in the prostates of nude mice. Magnetic resonance imaging-based tumor volume and serum prostate-specific antigen (PSA) measurements were used to assess effects on tumor response. Tumor response was measured by biochemical and tumor volume failure definitions and doubling time estimates from fitted PSA and tumor volume growth curves. Expression of MDM2, p53, p21, and Ki-67 was quantified using immunohistochemical staining and image analysis of formalin-fixed tissue, analogous to methods used clinically. Results: Antisense-MDM2 significantly inhibited the growth of LNCaP tumors over the mismatch controls. The most significant increase in tumor growth delay and tumor doubling time was from AS-MDM2 + AD + RT, although the effect of AS-MDM2 + AD was substantial. Expression of MDM2 was significantly reduced by AS-MDM2 in the setting of RT. Conclusions: This is the first in vivo investigation of the effects of AS-MDM2 in an orthotopic model and the first to demonstrate incremental sensitization when added to AD and AD + RT. The results with AD underscore the potential to affect micrometastatic disease, which is probably responsible for treatment failure in 30-40% of men with high-risk disease

  14. 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.

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

    Science.gov (United States)

    Kadakkuzha, Beena M; Liu, Xin-An; Narvaez, Maria; Kaye, Alexandra; Akhmedov, Komolitdin; Puthanveettil, Sathyanarayanan V

    2014-01-01

    Despite the advances in our understanding of transcriptome, regulation and function of its non-coding 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 now 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 reduction in 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.

  16. Effect of antisense c-raf-1 on tumorigenicity and radiation sensitivity of a human squamous carcinoma

    International Nuclear Information System (INIS)

    Kasid, U.; Pfeifer, A.; Brennan, T.; Beckett, M.; Weichselbaum, R.R.; Dritschilo, A.; Mark, G.E.

    1989-01-01

    Antisense RNA-mediated inhibition of gene expression was used to investigate the biological function of the c-raf-1 gene in a radiation-resistant human squamous carcinoma cell line, SQ-20B. S1 nuclease protection assays revealed that transfection of full-length raf complementary DNA in the antisense orientation (AS) leads to a specific reduction (greater than tenfold) of steady-state levels of the endogenous c-raf-1 sense (S) transcript in SQ-20B cells. In nude mice, the malignant potential of SQ-20B cells transfected with raf (S) was significantly increased relative to that of SQ-20B cells transfected with raf (AS). SQ-20B cells containing transfected raf (S) maintained a radiation-resistant phenotype as compared to those cells harboring the AS version, which appeared to have enhanced radiation sensitivity. These data indicate that the reduced expression of endogenous c-raf-1 is sufficient to modulate the tumorigenicity and the radiation-resistant phenotype of SQ-20B cells, thus implicating c-raf-1 in a pathway important to the genesis of this type of cancer

  17. Implementation of the agmatine-controlled expression system for inducible gene expression in Lactococcus lactis.

    Science.gov (United States)

    Linares, Daniel M; Alvarez-Sieiro, Patricia; del Rio, Beatriz; Ladero, Victor; Redruello, Begoña; Martin, Ma Cruz; Fernandez, Maria; Alvarez, Miguel A

    2015-12-30

    Lactococcus lactis has been safely consumed in fermented foods for millennia. This Gram-positive bacterium has now become of industrial importance as an expression host for the overproduction of lipopolysaccharide-free recombinant proteins used as food ingredients, therapeutic proteins and biotechnological enzymes. This paper reports an agmatine-controlled expression (ACE) system for L. lactis, comprising the lactococcal agmatine-sensor/transcriptional activator AguR and its target promoter P(aguB). The usefulness and efficiency of this system was checked via the reporter gene gfp and by producing PEP (Myxococcus xanthus prolyl-endopeptidase), an enzyme of biomedical interest able to degrade the immunotoxic peptides produced during the gastrointestinal breakdown of gluten. The ACE system developed in this work was suitable for the efficient expression of the functional recombinant proteins GFP and PEP. The expression system was tightly regulated by the agmatine concentration and allowed high protein production without leakiness.

  18. ADAMTS13 expression in human chondrosarcoma cells induced by insulin

    Directory of Open Access Journals (Sweden)

    Rıdvan Fırat

    2014-06-01

    Full Text Available Objectives: A Disintegrin-like Metalloproteinase with Thrombospondin Motifs (ADAMTS proteins is a proteinase enzyme group that primarily located in the extracellular matrix (ECM. Insulin has been known to stimulate proteoglycan biosynthesis in chondrosarcoma chondrocytes and thereby the levels of ADAMTS proteins. The aim of this study is to evaluate the time-dependent effects of insulin on the ADAMTS13 expression in OUMS-27 human chondrosarcoma cell line to test the hypothesis that insulin diminishes ADAMTS13 expression because of its anabolic effects. Methods: To test this hypothesis OUMS-27 cells were cultured in Dulbecco’s modified Eagle’ medium (DMEM containing 10μg/mL insulin. The medium containing insulin was changed every other day up to 11th day. Cells were harvested at 1, 3, 7, and 11th days and protein and RNA isolations were performed at the proper times. The levels of RNA expression of ADAMTS13 was quantified by qRT-PCR using appropriate primers while protein levels was detected by Western blot technique using anti-ADAMTS13 antibody. Results: Although there was a decrease in both RNA and protein levels in insulin-applied groups compared to the control cells, it was not statistically significant. Conclusion: Under the light of our findings, it is suggested that insulin does not participate in regulation of ADAMTS13 in OUMS-27 chondrosarcoma cells. J Clin Exp Invest 2014; 5 (2: 226-232

  19. Modulation of splicing of the preceding intron by antisense oligonucleotide complementary to intra-exon sequence deleted in dystrophin Kobe

    Energy Technology Data Exchange (ETDEWEB)

    Takeshima, Y.; Matuso, M.; Sakamoto, H.; Nishio, H. [Kobe Univ. School of Medicine and Science (Japan)

    1994-09-01

    Molecular analysis of dystrophin Kobe showed that exon 19 of the dystrophin gene bearing a 52 bp deletion was skipped during splicing, although the known consensus sequences at the 5{prime} and 3{prime} splice site of exon 19 were maintained. These data suggest that the deleted sequence of exon 19 may function as a cis-acting factor for exact splicing for the upstream intron. To investigate this potential role, an in vitro splicing system using dystrophin precursors was established. A two-exon precursor containing exon 18, truncated intron 18, and exon 19 was accurately spliced. However, splicing of intron 18 was dramatically inhibited when wild exon 19 was replaced with mutated exon 19. Even though the length of exon 19 was restored to normal by replacing the deleted sequence with other sequence, splicing of intron 18 was not fully reactivated. Characteristically, splicing of intron 18 was inactivated more markedly when the replaced sequence contained less polypurine stretches. These data suggested that modification of the exon sequence would result in a splicing abnormality. Antisense 31 mer 2`-O-methyl ribonucleotide was targeted against 5{prime} end of deleted region of exon 19 to modulate splicing of the mRNA precursor. Splicing of intron 18 was inhibited in a dose- and time-dependent manner. This is the first in vitro evidence to show splicing of dystrophin pre-mRNA can be managed by antisense oligonucleotides. These experiments represent an approach in which antisense oligonucleotides are used to restore the function of a defective dystrophin gene in Duchenne muscular dystrophy by inducing skipping of certain exons during splicing.

  20. CAR expression and inducibility of CYP2B genes in liver of rats treated with PB-like inducers

    International Nuclear Information System (INIS)

    Pustylnyak, Vladimir O.; Gulyaeva, Lyudmila F.; Lyakhovich, Vyacheslav V.

    2005-01-01

    The expression of the CAR gene and inducibility of CYP2B protein in the liver of male Wistar rats treated with phenobarbital (PB) and triphenyldioxane (TPD) were investigated. To clarify the role of phosphorylation/dephosphorylation in these processes, rats were treated with inhibitors of Ca 2+ /calmodulin-dependent kinase II (W 7 ) or protein phosphatases PP1 and PP2A (OA) before induction. Constitutive expression of the CAR gene in livers of untreated rats was detected by multiplex RT-PCR. Treatment with W 7 resulted in a 2.8-fold induction of CAR gene expression, whereas OA led to a 2.4-fold decrease of the mRNA level. The same results were obtained for CYP2B genes expression, which were increased by W 7 treatment (two-fold) and decreased by OA (2.3-fold). PB-induction did not lead to significant alteration in the level of CAR gene expression, although CYP2B genes expression was enhanced two-fold over control values. TPD caused a two-fold increase of both CAR and CYP2B mRNA levels. Both inducers reduced the effects of inhibitors on CAR gene expression. Results of EMSA showed that PB, TPD or W 7 alone induced formation of complexes of NR1 with nuclear proteins. Appearance of the complexes correlated with an increase in CYP2B expression, and their intensities were modulated by the protein kinase inhibitors. Thus, our results demonstrate that constitutive expressions of CAR as well as CYP2B during induction are regulated by phosphorylation/dephosphorylation processes

  1. UV-induced changes in cell cycle and gene expression within rabbit lens epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Sidjanin, D. [Northern Illinois Univ., De Kalb, IL (United States). Dept. of Biological Sciences; Grdina, D. [Argonne National Lab., IL (United States); Woloschak, G.E. [Northern Illinois Univ., De Kalb, IL (United States). Dept. of Biological Sciences

    1994-11-01

    Damage to lens epithelial cells is a probable initiation process in cataract formation induced by ultraviolet radiation. These experiments investigated the ability of 254 nm radiation on cell cycle progression and gene expression in rabbit lens epithelial cell line N/N1003A. No changes in expression of c-fos, c-jun, alpha- tubulin, or vimentin was observed following UV exposure. Using flow cytometry, an accumulation of cells in G1/S phase of the cell cycle 1 hr following exposure. The observed changes in gene expression, especially the decreased histone transcripts reported here may play a role in UV induced inhibition of cell cycle progression.

  2. Light-dependent expression of flg22-induced defense genes in Arabidopsis

    Directory of Open Access Journals (Sweden)

    Satoshi eSano

    2014-10-01

    Full Text Available Chloroplasts have been reported to generate retrograde immune signals that activate defense gene expression in the nucleus. However, the roles of light and photosynthesis in plant immunity remain largely elusive. In this study, we evaluated the effects of light on the expression of defense genes induced by flg22, a peptide derived from bacterial flagellins which acts as a potent elicitor in plants. Whole-transcriptome analysis of flg22-treated Arabidopsis thaliana seedlings under light and dark conditions for 30 min revealed that a number of (30% genes strongly induced by flg22 (>4.0 require light for their rapid expression, whereas flg22-repressed genes include a significant number of genes that are down-regulated by light. Furthermore, light is responsible for the flg22-induced accumulation of salicylic acid, indicating that light is indispensable for basal defense responses in plants. To elucidate the role of photosynthesis in defense, we further examined flg22-induced defense gene expression in the presence of specific inhibitors of photosynthetic electron transport: 3-(3,4-dichlorophenyl-1,1-dimethylurea (DCMU and 2,5-dibromo-3-methyl-6-isopropyl-benzoquinone (DBMIB. Light-dependent expression of defense genes was largely suppressed by DBMIB, but only partially suppressed by DCMU. These findings suggest that photosynthetic electron flow plays a role in controling the light-dependent expression of flg22-inducible defense genes.

  3. HIF1 Contributes to Hypoxia-Induced Pancreatic Cancer Cells Invasion via Promoting QSOX1 Expression

    Directory of Open Access Journals (Sweden)

    Chen-Ye Shi

    2013-08-01

    Full Text Available Background: Quiescin sulfhydryl oxidase 1 (QSOX1, which oxidizes sulfhydryl groups to form disulfide bonds in proteins, is found to be over-expressed in various pancreatic cancer cell lines and patients. QSOX1 promotes invasion of pancreatic cancer cells by activating MMP-2 and MMP-9. However, its regulatory mechanism remains largely undefined. Methods: Real-time PCR and Western blot were employed to detect the expression of QSOX1 in human pancreatic cancer cell lines under hypoxic condition. Luciferase reporter and ChIP assays were used to assess the regulation of QSOX1 by hypoxia-inducible factor 1 (HIF-1. Small interfering RNA (siRNA was applied to knock down endogenous expression of QSOX1. Matrigel-coated invasion chamber essays were conducted to detect the invasion capacity of QSOX1-depleted cells. Results: Both hypoxia and hypoxia mimicking reagent up-regulated the expression of QSOX1 in human pancreatic cancer cell lines. Knockdown of HIF-1α eliminated hypoxia induced QSOX1 expression. HIF-1α was found directly bound to two hypoxia-response elements (HRE of QSOX1 gene, both of which were required for HIF-1 induced QSOX1 expression. Moreover, QSOX1 silencing blocked hypoxia-induced pancreatic cancer cells invasion. Conclusion: QSOX1 is a direct target of HIF-1 and may contribute to hypoxia-induced pancreatic cancer cells invasion.

  4. Characterization of insulin-like-growth factor II (IGF II mRNA positive hepatic altered foci and IGF II expression in hepatocellular carcinoma during diethylnitrosamine-induced hepatocarcinogenesis in rats

    Directory of Open Access Journals (Sweden)

    Mukherjee Biswajit

    2005-08-01

    Full Text Available Abstract Background Insulin-like-growth factor II (IGF II has been implicated in the pathogenesis of neoplasm of different tissues, including liver of rats and men. This growth factor is believed to exert its effect during cellular proliferation. During the process of development of hepatocellular carcinoma (HCC, different hepatic altered foci appear. They are believed to be the putative precursors of HCC in rats and in men. Thus, to study the role of the gene in a defined model of hepatocarcinogenesis was the target to elucidate its role in various cancer phenotypes during the entire development stage of cancer, right from earlier preneoplastic lesions to HCC Methods Antisense in situ hybridization technique was used here to characterize the type(s of foci in which IGF II mRNA had expressed during the development of hepatocarcinogenesis-induced by diethylnitrosamine and promoted by phenobarbital in rats. Various focal lesions have been categorized depending on the stages and sizes along with IGF II expression patterns in them. Immunohistochemical detection for proliferating cell nuclear antigen (PCNA was made to detect the role of the gene in preneoplastic and neoplastic cellular proliferation. Results IGF II expression was located in the glycogen-storage acidophilic cell foci maximally followed by mixed cell lesions and the least in basophilic lesions. The expression of IGF II was found to be predominant in the HCC. The expression of gene was also located at the peripheral cells of spongiosis hepatis which are believed to be the precursor of ito cell carcinoma. It was noted that there is a direct correlation between IGF II expression and Immunohistochemical detection for PCNA. Conclusion It may be concluded that IGF II gene expression plays an important role during the development of neoplasia and the gene expresses in the sequence of events leading from glycogen-rich-acidophilic lesions to glycogen poor basophilic lesions to HCC with an

  5. Construction and Development of a Cardiac Tissue-Specific and Hypoxia-Inducible Expression Vector

    Directory of Open Access Journals (Sweden)

    Shahrooz Ghaderi

    2018-03-01

    Full Text Available Purpose: Cardiovascular gene therapy is a sophisticated approach, thanks to the safety of vectors, stable transgene expression, delivery method, and different layers of the heart. To date, numerous expression vectors have been introduced in biotechnology and biopharmacy industries in relation to genetic manipulation. Despite the rapid growth of these modalities, they must be intelligently designed, addressing the cardiac-specific transgene expression and less side effects. Herein, we conducted a pilot project aiming to design a cardiac-specific hypoxia-inducible expression cassette. Methods: We explored a new approach to design an expression cassette containing cardiac specific enhancer, hypoxia response elements (HRE, cardiac specific promoter, internal ribosome entry site (IRES, and beta globin poly A sequence to elicit specific and inducible expression of the gene of interest. Enhanced green fluorescent protein (eGFP was sub-cloned by BglII and NotI into the cassette. The specificity and inducible expression of the cassette was determined in both mouse myoblast C2C12 and mammary glandular tumor 4T1 as ‘twin’ cells. eGFP expression was evaluated by immunofluorescence microscope and flow cytometry at 520 nm emission peak. Results: Our data revealed that the designed expression cassette provided tissue specific and hypoxia inducible (O2<1% transgene expression. Conclusion: It is suggested that cardiac-specific enhancer combined with cardiac-specific promoter are efficient for myoblast specific gene expression. As well, this is for the first time that HRE are derived from three well known hypoxia-regulated promoters. Therefore, there is no longer need to overlap PCR process for one repeated sequence just in one promoter.

  6. Ectopic ERK Expression Induces Phenotypic Conversion of C10 Cells and Alters DNA Methyltransferase Expression

    Energy Technology Data Exchange (ETDEWEB)

    Sontag, Ryan L.; Weber, Thomas J.

    2012-05-04

    In some model systems constitutive extracellular signal regulated kinase (ERK) activation is sufficient to promote an oncogenic phenotype. Here we investigate whether constitutive ERK expression influences phenotypic conversion in murine C10 type II alveolar epithelial cells. C10 cells were stably transduced with an ERK1-green fluorescent protein (ERK1-GFP) chimera or empty vector and ectopic ERK expression was associated with the acquisition of soft agar focus-forming potential in late passage, but not early passage cells. Late passage ERK1-GFP cells exhibited a significant increase in the expression of DNA methyl transferases (DNMT1 and 3b) and a marked increase in sensitivity to 5-azacytidine (5-azaC)-mediated toxicity, relative to early passage ERK1-GFP cells and vector controls. The expression of xeroderma pigmentosum complementation group A (XPA) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) were significantly increased in late passage cells, suggesting enhanced DNA damage recognition and repair activity which we interpret as a reflection of genomic instability. Phospho-ERK levels were dramatically decreased in late passage ERK1-GFP cells, relative to early passage and vector controls, and phospho-ERK levels were restored by treatment with sodium orthovanadate, indicating a role for phosphatase activity in this response. Collectively these observations suggest that ectopic ERK expression promotes phenotypic conversion of C10 cells that is associated with latent effects on epigenetic programming and phosphatase activities.

  7. Glucose-induced serum- and glucocorticoid-regulated kinase activation in oncofetal fibronectin expression

    International Nuclear Information System (INIS)

    Khan, Zia A.; Barbin, Yousef P.; Farhangkhoee, Hana; Beier, Norbert; Scholz, Wolfgang; Chakrabarti, Subrata

    2005-01-01

    Preferential expression of oncofetal extra domain-B fibronectin (EDB + FN), a proposed angiogenic marker, has been shown in proliferative diabetic retinopathy. High levels of glucose also increase EDB + FN expression in endothelial cells (ECs) via transforming growth factor-β1 (TGF-β1) and endothelin-1 (ET-1). The present study was aimed at elucidating the role of serum- and glucocorticoid-regulated kinase (SGK-1) in glucose-induced EDB + FN expression. Using human macro- and microvascular ECs, we show that high levels of glucose, TGF-β1, and ET-1 increase the EDB + FN expression via SGK-1 alteration at the mRNA, protein, and activity levels. Inhibition of TGF-β1 and ET-1 prevented glucose-induced SGK-1 activation and the EDB + FN expression. Furthermore, using siRNA-mediated SGK-1 gene silencing, we show that glucose-induced EDB + FN expression can be completely prevented. These findings provide first evidence of glucose-induced SGK-1 activation in altered EDB + FN expression and provide novel avenues for therapeutic modalities

  8. Low dose radiation induced protein and its effect on expression of CD25 molecule in lymphocytes

    International Nuclear Information System (INIS)

    Lu Duicai; Su Liaoyuan

    2001-01-01

    Objective: To find the substantial basis for effects of low dose radiation, on development, extraction, and the biogical activity of the low-dose radiation-induced proteins, and the effects of LDR induced proteins on CD25 molecule expression of human lymphocytes. Methods: 1. Healthy Kumning male mice exposed to radiation of 226 Ra γ-rays at 5, 10 and 15 cGy respectively. The mice were killed 2 hours after exposure, the spleen cells were broken with ultrasonic energy and then ultra-centrifugalized at low temperature (4 degree C). The LDR-induced proteins were obtained in the supernatant solution. Then the changes of CD25 molecule was measured by flow cytometry (FCM) with immunofluorescence technique, which was used to reflect the effect of LDR induced proteins on CD25 molecule expression of human lymphocytes. Results: LDR induced proteins were obtained from spleen cells in mice exposed to 5-15 cGy whole body radiation. Conclusion: The expression of CD25 molecule of lymphocytes was increased significantly after use of LDR induced proteins. LDR induced proteins can enhance expression of CD25 molecule of lymphocytes slightly

  9. Mucosal Progranulin expression is induced by H. pylori, but independent of Secretory Leukocyte Protease Inhibitor (SLPI) expression.

    Science.gov (United States)

    Wex, Thomas; Kuester, Doerthe; Schönberg, Cornelius; Schindele, Daniel; Treiber, Gerhard; Malfertheiner, Peter

    2011-05-26

    Mucosal levels of Secretory Leukocyte Protease Inhibitor (SLPI) are specifically reduced in relation to H. pylori-induced gastritis. Progranulin is an epithelial growth factor that is proteolytically degraded into fragments by elastase (the main target of SLPI). Considering the role of SLPI for regulating the activity of elastase, we studied whether the H. pylori-induced reduction of SLPI and the resulting increase of elastase-derived activity would reduce the Progranulin protein levels both ex vivo and in vitro. The expression of Progranulin was studied in biopsies of H. pylori-positive, -negative and -eradicated subjects as well as in the gastric tumor cell line AGS by ELISA, immunohistochemistry and real-time RT-PCR. H. pylori-infected subjects had about 2-fold increased antral Progranulin expression compared to H. pylori-negative and -eradicated subjects (P Progranulin and SLPI levels were identified. Immunohistochemical analysis confirmed the upregulation of Progranulin in relation to H. pylori infection; both epithelial and infiltrating immune cells contributed to the higher Progranulin expression levels. The H. pylori-induced upregulation of Progranulin was verified in AGS cells infected by H. pylori. The down-regulation of endogenous SLPI expression in AGS cells by siRNA methodology did not affect the Progranulin expression independent of the infection by H. pylori. Taken together, Progranulin was identified as novel molecule that is upregulated in context to H. pylori infection. In contrast to other diseases, SLPI seems not to have a regulatory role for Progranulin in H. pylori-mediated gastritis.

  10. Exercise-induced liver chemokine CXCL-1 expression is linked to muscle-derived interleukin-6 expression

    DEFF Research Database (Denmark)

    Pedersen, Line; Pilegaard, Henriette; Hansen, Jakob

    2011-01-01

    interleukin-6 (IL-6) and muscle IL-6 mRNA. In contrast, exercise-induced regulation of liver CXCL-1 mRNA expression was completely blunted in IL-6 knockout mice. Based on these findings, we examined the possible existence of a muscle-to-liver axis by overexpressing IL-6 in muscles. This resulted in increases...... in serum CXCL-1 (5-fold) and liver CXCL-1 mRNA expression (24-fold) compared with control. Because IL-6 expression and release are known to be augmented during exercise in glycogen-depleted animals, CXCL-1 and IL-6 expression were examined after exercise in overnight-fasted mice.We found that fasting...... significantly augmented serum CXCL-1, and CXCL-1 expression in liver and muscle. Taken together, these data indicate that liver is the main source of serum CXCL-1 during exercise in mice, and that the CXCL-1 expression in the liver is regulated by muscle-derived IL-6....

  11. Transcriptome analysis of trichothecene-induced gene expression in barley.

    Science.gov (United States)

    Boddu, Jayanand; Cho, Seungho; Muehlbauer, Gary J

    2007-11-01

    Fusarium head blight, caused primarily by Fusarium graminearum, is a major disease problem on barley (Hordeum vulgare L.). Trichothecene mycotoxins produced by the fungus during infection increase the aggressiveness of the fungus and promote infection in wheat (Triticum aestivum L.). Loss-of-function mutations in the TRI5 gene in F. graminearum result in the inability to synthesize trichothecenes and in reduced virulence on wheat. We examined the impact of pathogen-derived trichothecenes on virulence and the transcriptional differences in barley spikes infected with a trichothecene-producing wild-type strain and a loss-of-function tri5 trichothecene nonproducing mutant. Disease severity, fungal biomass, and floret necrosis and bleaching were reduced in spikes inoculated with the tri5 mutant strain compared with the wild-type strain, indicating that the inability to synthesize trichothecenes results in reduced virulence in barley. We detected 63 transcripts that were induced during trichothecene accumulation, including genes encoding putative trichothecene detoxification and transport proteins, ubiquitination-related proteins, programmed cell death-related proteins, transcription factors, and cytochrome P450s. We also detected 414 gene transcripts that were designated as basal defense response genes largely independent of trichothecene accumulation. Our results show that barley exhibits a specific response to trichothecene accumulation that can be separated from the basal defense response. We propose that barley responds to trichothecene accumulation by inducing at least two general responses. One response is the induction of genes encoding trichothecene detoxification and transport activities that may reduce the impact of trichothecenes. The other response is to induce genes encoding proteins associated with ubiquitination and cell death which may promote successful establishment of the disease.

  12. Genetic expression of induced rice sterility under alien-cytoplasm

    International Nuclear Information System (INIS)

    Wang Naiyuan; Cai Zhijun; Liang Kangjing; Li Yu

    2005-01-01

    Rice restorer lines were treated with 60 Co γ-ray and 4 male sterile mutants obtained with the fertility of controlled by 4 non-allelic recessive genes, respectively. Sixty combinations were made by using male sterile plants/fertile plants as male parents, and 15 different cytoplasmic substitution lines of the same cell nucleus as female parents. The result showed that F 1 spikelets were normal and fertile, and different numbers of male sterile plants were segregated in F 2 . Complete fertility genotype was not found among interactions between induced male sterile genes and alien-cytoplasms. (authors)

  13. Bacterial feeding, Leishmania infection and distinct infection routes induce differential defensin expression in Lutzomyia longipalpis.

    Science.gov (United States)

    Telleria, Erich L; Sant'Anna, Maurício R Viana; Alkurbi, Mohammad O; Pitaluga, André N; Dillon, Rod J; Traub-Csekö, Yara M

    2013-01-11

    Phlebotomine insects harbor bacterial, viral and parasitic pathogens that can cause diseases of public health importance. Lutzomyia longipalpis is the main vector of visceral leishmaniasis in the New World. Insects can mount a powerful innate immune response to pathogens. Defensin peptides take part in this response and are known to be active against Gram-positive and Gram-negative bacteria, and some parasites. We studied the expression of a defensin gene from Lutzomyia longipalpis to understand its role in sand fly immune response. We identified, sequenced and evaluated the expression of a L. longipalpis defensin gene by semi-quantitative RT-PCR. The gene sequence was compared to other vectors defensins and expression was determined along developmental stages and after exposure of adult female L. longipalpis to bacteria and Leishmania. Phylogenetic analysis showed that the L. longipalpis defensin is closely related to a defensin from the Old World sand fly Phlebotomus duboscqi. Expression was high in late L4 larvae and pupae in comparison to early larval stages and newly emerged flies. Defensin expression was modulated by oral infection with bacteria. The Gram-positive Micrococcus luteus induced early high defensin expression, whilst the Gram-negative entomopathogenic Serratia marcescens induced a later response. Bacterial injection also induced defensin expression in adult insects. Female sand flies infected orally with Leishmania mexicana showed no significant difference in defensin expression compared to blood fed insects apart from a lower defensin expression 5 days post Leishmania infection. When Leishmania was introduced into the hemolymph by injection there was no induction of defensin expression until 72 h later. Our results suggest that L. longipalpis modulates defensin expression upon bacterial and Leishmania infection, with patterns of expression that are distinct among bacterial species and routes of infection.

  14. Radiation-induced gene expression in the nematode caenorhabditis elegans

    International Nuclear Information System (INIS)

    Nelson, G.A.; Jones, T.A.; Chesnut, A.; Smith, A.L.

    2002-01-01

    We used the nematode C. elegans to characterize the genotoxic and cytotoxic effects of ionizing radiation in a simple animal model emphasizing the unique effects of charged particle radiation. Here we demonstrate by reverse transcription polymerase chain reaction (RT-PCR) differential display and whole genome microarray hybridization experiments that gamma rays, accelerated protons and iron ions at the same physical dose lead to unique transcription profiles. 599 of 17871 genes analyzed (3.4%) showed differential expression 3 hrs after exposure to 3 Gy of radiation. 193 were up-regulated, 406 were down-regulated and 90% were affected only by a single species of radiation. A novel statistical clustering technique identified the regulatory relationships between the radiation-modulated genes and showed that genes affected by each radiation species were associated with unique regulatory clusters. This suggests that independent homeostatic mechanisms are activated in response to radiation exposure as a function of track structure or ionization density. (author)

  15. Protective effects of L-selenomethionine on space radiation induced changes in gene expression.

    Science.gov (United States)

    Stewart, J; Ko, Y-H; Kennedy, A R

    2007-06-01

    Ionizing radiation can produce adverse biological effects in astronauts during space travel. Of particular concern are the types of radiation from highly energetic, heavy, charged particles known as HZE particles. The aims of our studies are to characterize HZE particle radiation induced biological effects and evaluate the effects of L-selenomethionine (SeM) on these adverse biological effects. In this study, microarray technology was used to measure HZE radiation induced changes in gene expression, as well as to evaluate modulation of these changes by SeM. Human thyroid epithelial cells (HTori-3) were irradiated (1 GeV/n iron ions) in the presence or in the absence of 5 microM SeM. At 6 h post-irradiation, all cells were harvested for RNA isolation. Gene Chip U133Av2 from Affymetrix was used for the analysis of gene expression, and ANOVA and EASE were used for a determination of the genes and biological processes whose differential expression is statistically significant. Results of this microarray study indicate that exposure to small doses of radiation from HZE particles, 10 and 20 cGy from iron ions, induces statistically significant differential expression of 196 and 610 genes, respectively. In the presence of SeM, differential expression of 77 out of 196 genes (exposure to 10 cGy) and 336 out of 610 genes (exposure to 20 cGy) is abolished. In the presence or in the absence of SeM, radiation from HZE particles induces differential expression of genes whose products have roles in the induction of G1/S arrest during the mitotic cell cycle, as well as heat shock proteins. Some of the genes, whose expressions were affected by radiation from HZE particles and were unchanged in irradiated cells treated with SeM, have been shown to have altered expression levels in cancer cells. The conclusions of this report are that radiation from HZE particles can induce differential expression of many genes, some of which are known to play roles in the same processes that have

  16. In Vivo Imaging of Local Gene Expression Induced by Magnetic Hyperthermia

    Directory of Open Access Journals (Sweden)

    Olivier Sandre

    2017-02-01

    Full Text Available The present work aims to demonstrate that colloidal dispersions of magnetic iron oxide nanoparticles stabilized with dextran macromolecules placed in an alternating magnetic field can not only produce heat, but also that these particles could be used in vivo for local and noninvasive deposition of a thermal dose sufficient to trigger thermo-induced gene expression. Iron oxide nanoparticles were first characterized in vitro on a bio-inspired setup, and then they were assayed in vivo using a transgenic mouse strain expressing the luciferase reporter gene under transcriptional control of a thermosensitive promoter. Iron oxide nanoparticles dispersions were applied topically on the mouse skin or injected subcutaneously with Matrigel™ to generate so-called pseudotumors. Temperature was monitored continuously with a feedback loop to control the power of the magnetic field generator and to avoid overheating. Thermo-induced luciferase expression was followed by bioluminescence imaging 6 h after heating. We showed that dextran-coated magnetic iron oxide nanoparticle dispersions were able to induce in vivo mild hyperthermia compatible with thermo-induced gene expression in surrounding tissues and without impairing cell viability. These data open new therapeutic perspectives for using mild magnetic hyperthermia as noninvasive modulation of tumor microenvironment by local thermo-induced gene expression or drug release.

  17. Specific Increase of Protein Levels by Enhancing Translation Using Antisense Oligonucleotides Targeting Upstream Open Frames.

    Science.gov (United States)

    Liang, Xue-Hai; Shen, Wen; Crooke, Stanley T

    2017-01-01

    A number of diseases are caused by low levels of key proteins; therefore, increasing the amount of specific proteins in human bodies is of therapeutic interest. Protein expression is downregulated by some structural or sequence elements present in the 5' UTR of mRNAs, such as upstream open reading frames (uORF). Translation initiation from uORF(s) reduces translation from the downstream primary ORF encoding the main protein product in the same mRNA, leading to a less efficient protein expression. Therefore, it is possible to use antisense oligonucleotides (ASOs) to specifically inhibit translation of the uORF by base-pairing with the uAUG region of the mRNA, redirecting translation machinery to initiate from the primary AUG site. Here we review the recent findings that translation of specific mRNAs can be enhanced using ASOs targeting uORF regions. Appropriately designed and optimized ASOs are highly specific, and they act in a sequence- and position-dependent manner, with very minor off-target effects. Protein levels can be increased using this approach in different types of human and mouse cells, and, importantly, also in mice. Since uORFs are present in around half of human mRNAs, the uORF-targeting ASOs may thus have valuable potential as research tools and as therapeutics to increase the levels of proteins for a variety of genes.

  18. Changes in Oleic Acid Content of Transgenic Soybeans by Antisense RNA Mediated Posttranscriptional Gene Silencing

    Directory of Open Access Journals (Sweden)

    Ling Zhang

    2014-01-01

    Full Text Available The Delta-12 oleate desaturase gene (FAD2-1, which converts oleic acid into linoleic acid, is the key enzyme determining the fatty acid composition of seed oil. In this study, we inhibited the expression of endogenous Delta-12 oleate desaturase GmFad2-1b gene by using antisense RNA in soybean Williams 82. By employing the soybean cotyledonary-node method, a part of the cDNA of soybean GmFad2-1b 801 bp was cloned for the construction of a pCAMBIA3300 vector under the soybean seed promoter BCSP. Leaf painting, LibertyLink strip, PCR, Southern blot, qRT-PCR, and fatty acid analysis were used to detect the insertion and expression of GmFad2-1b in the transgenic soybean lines. The results indicate that the metabolically engineered plants exhibited a significant increase in oleic acid (up to 51.71% and a reduction in palmitic acid (to <3% in their seed oil content. No structural differences were observed between the fatty acids of the transgenic and the nontransgenic oil extracts.

  19. Coffee induces breast cancer resistance protein expression in Caco-2 cells.

    Science.gov (United States)

    Isshiki, Marina; Umezawa, Kazuo; Tamura, Hiroomi

    2011-01-01

    Coffee is a beverage that is consumed world-wide on a daily basis and is known to induce a series of metabolic and pharmacological effects, especially in the digestive tract. However, little is known concerning the effects of coffee on transporters in the gastrointestinal tract. To elucidate the effect of coffee on intestinal transporters, we investigated its effect on expression of the breast cancer resistance protein (BCRP/ABCG2) in a human colorectal cancer cell line, Caco-2. Coffee induced BCRP gene expression in Caco-2 cells in a coffee-dose dependent manner. Coffee treatment of Caco-2 cells also increased the level of BCRP protein, which corresponded to induction of gene expression, and also increased cellular efflux activity, as judged by Hoechst33342 accumulation. None of the major constituents of coffee tested could induce BCRP gene expression. The constituent of coffee that mediated this induction was extractable with ethyl acetate and was produced during the roasting process. Dehydromethylepoxyquinomicin (DHMEQ), an inhibitor of nuclear factor (NF)-κB, inhibited coffee-mediated induction of BCRP gene expression, suggesting involvement of NF-κB in this induction. Our data suggest that daily consumption of coffee might induce BCRP expression in the gastrointestinal tract and may affect the bioavailability of BCRP substrates.

  20. Differences in gene expression profiles and signaling pathways in rhabdomyolysis-induced acute kidney injury.

    Science.gov (United States)

    Geng, Xiaodong; Wang, Yuanda; Hong, Quan; Yang, Jurong; Zheng, Wei; Zhang, Gang; Cai, Guangyan; Chen, Xiangmei; Wu, Di

    2015-01-01

    Rhabdomyolysis is a threatening syndrome because it causes the breakdown of skeletal muscle. Muscle destruction leads to the release of myoglobin, intracellular proteins, and electrolytes into the circulation. The aim of this study was to investigate the differences in gene expression profiles and signaling pathways upon rhabdomyolysis-induced acute kidney injury (AKI). In this study, we used glycerol-induced renal injury as a model of rhabdomyolysis-induced AKI. We analyzed data and relevant information from the Gene Expression Omnibus database (No: GSE44925). The gene expression data for three untreated mice were compared to data for five mice with rhabdomyolysis-induced AKI. The expression profiling of the three untreated mice and the five rhabdomyolysis-induced AKI mice was performed using microarray analysis. We examined the levels of Cyp3a13, Rela, Aldh7a1, Jun, CD14. And Cdkn1a using RT-PCR to determine the accuracy of the microarray results. The microarray analysis showed that there were 1050 downregulated and 659 upregulated genes in the rhabdomyolysis-induced AKI mice compared to the control group. The interactions of all differentially expressed genes in the Signal-Net were analyzed. Cyp3a13 and Rela had the most interactions with other genes. The data showed that Rela and Aldh7a1 were the key nodes and had important positions in the Signal-Net. The genes Jun, CD14, and Cdkn1a were also significantly upregulated. The pathway analysis classified the differentially expressed genes into 71 downregulated and 48 upregulated pathways including the PI3K/Akt, MAPK, and NF-κB signaling pathways. The results of this study indicate that the NF-κB, MAPK, PI3K/Akt, and apoptotic pathways are regulated in rhabdomyolysis-induced AKI.

  1. RAE-1 expression is induced during experimental autoimmune encephalomyelitis and is correlated with microglia cell proliferation.

    Science.gov (United States)

    Djelloul, Mehdi; Popa, Natalia; Pelletier, Florence; Raguénez, Gilda; Boucraut, José

    2016-11-01

    Retinoic acid early induced transcript-1 (RAE-1) glycoproteins are ligands of the activating immune receptor NKG2D. They are known as stress molecules induced in pathological conditions. We previously reported that progenitor cells express RAE-1 in physiological conditions and we described a correlation between RAE-1 expression and cell proliferation. In addition, we showed that Raet1 transcripts are induced in the spinal cord of experimental autoimmune encephalomyelitis (EAE) mice. EAE is a model for multiple sclerosis which is accompanied by microglia proliferation and activation, recruitment of immune cells and neurogenesis. We herein studied the time course expression of the two members of the Raet1 gene family present in C57BL/6 mice, namely Raet1d and Raet1e, in the spinal cord during EAE. We report that Raet1d and Raet1e genes are induced early upon EAE onset and reach a maximal expression at the peak of the pathology. We show that myeloid cells, i.e. macrophages as well as microglia, are cellular sources of Raet1 transcripts. We also demonstrate that only Raet1d expression is induced in microglia, whereas macrophages expressed both Raet1d and Raet1e. Furthermore, we investigated the dynamics of RAE-1 expression in microglia cultures. RAE-1 induction correlated with cell proliferation but not with M1/M2 phenotypic orientation. We finally demonstrate that macrophage colony-stimulating factor (M-CSF) is a major factor controlling RAE-1 expression in microglia. Copyright © 2016 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

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

    2016-01-01

    SRY (Sex Determining Region Y)-Box 4 or Sox4 is an important regulator of the pan-neuronal gene expression during post-mitotic cell differentiation within the mammalian brain. Sox4 gene locus has been previously characterized with multiple sense and overlapping natural antisense transcripts [1], [2]. Here we provide accompanying data on various analyses performed and described in Ling et al. [2]. The data include a detail description of various features found at Sox4 gene locus, additional experimental data derived from RNA-Fluorescence in situ Hybridization (RNA-FISH), Western blotting, strand-specific reverse-transcription quantitative polymerase chain reaction (RT-qPCR), gain-of-function and in situ hybridization (ISH) experiments. All the additional data provided here support the existence of an endogenous small interfering- or PIWI interacting-like small RNA known as Sox4_sir3, which origin was found within the overlapping region consisting of a sense and a natural antisense transcript known as Sox4ot1. PMID:26958646

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

    Directory of Open Access Journals (Sweden)

    King-Hwa Ling

    2016-06-01

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

  4. hREV3 is essential for error-prone translesion synthesis past UV or benzo[a]pyrene diol epoxide-induced DNA lesions in human fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Li Ziqiang; Zhang Hong; McManus, Terrence P.; McCormick, J. Justin; Lawrence, Christopher W.; Maher, Veronica M

    2002-12-29

    In S. cerevisiae, the REV3 gene, encoding the catalytic subunit of polymerase zeta, is involved in translesion synthesis and required for the production of mutations induced by ultraviolet radiation (UV) photoproducts and other DNA fork-blocking lesions, and for the majority of spontaneous mutations. To determine whether hREV3, the human homolog of yeast REV3, is similarly involved in error-prone translesion synthesis past UV photoproducts and other lesions that block DNA replication, an hREV3 antisense construct under the control of the TetP promoter was transfected into an infinite life span human fibroblast cell strain that expresses a high level of tTAk, the activator of that promoter. Three transfectant strains expressing high levels of hREV3 antisense RNA were identified and compared with their parental cell strain for sensitivity to the cytotoxic and mutagenic effects of UV. The three hREV3 antisense-expressing cell strains were not more sensitive than the parental strain to the cytotoxic effect of UV, but the frequency of mutants induced by UV in their HPRT gene was significantly reduced, i.e. to 14% that of the parent. Two of these hREV3 antisense-expressing cell strains were compared with the parental strain for sensitivity to ({+-})-7{beta},8{alpha}-dihydroxy-9{alpha},10{alpha}-epoxy-7,8,9,10-tetrahydro= benzo[a]pyrene (BPDE). They were not more sensitive than the parent strain to the cytotoxic effect of BPDE, but the frequency of mutants induced was significantly reduced, i.e. in one strain, to 17% that of the parent, and in the other, to 24%. DNA sequencing showed that the kinds of mutations induced by BPDE in the parental and the derivative strains did not differ and were similar to those found previously with finite life span human fibroblasts. The data strongly support the hypothesis that hRev3 plays a critical role in the induction of mutations by UV or BPDE. Because the level of hRev3 protein in human fibroblasts is below the level of antibody

  5. hREV3 is essential for error-prone translesion synthesis past UV or benzo[a]pyrene diol epoxide-induced DNA lesions in human fibroblasts

    International Nuclear Information System (INIS)

    Li Ziqiang; Zhang Hong; McManus, Terrence P.; McCormick, J. Justin; Lawrence, Christopher W.; Maher, Veronica M.

    2002-01-01

    In S. cerevisiae, the REV3 gene, encoding the catalytic subunit of polymerase zeta, is involved in translesion synthesis and required for the production of mutations induced by ultraviolet radiation (UV) photoproducts and other DNA fork-blocking lesions, and for the majority of spontaneous mutations. To determine whether hREV3, the human homolog of yeast REV3, is similarly involved in error-prone translesion synthesis past UV photoproducts and other lesions that block DNA replication, an hREV3 antisense construct under the control of the TetP promoter was transfected into an infinite life span human fibroblast cell strain that expresses a high level of tTAk, the activator of that promoter. Three transfectant strains expressing high levels of hREV3 antisense RNA were identified and compared with their parental cell strain for sensitivity to the cytotoxic and mutagenic effects of UV. The three hREV3 antisense-expressing cell strains were not more sensitive than the parental strain to the cytotoxic effect of UV, but the frequency of mutants induced by UV in their HPRT gene was significantly reduced, i.e. to 14% that of the parent. Two of these hREV3 antisense-expressing cell strains were compared with the parental strain for sensitivity to (±)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE). They were not more sensitive than the parent strain to the cytotoxic effect of BPDE, but the frequency of mutants induced was significantly reduced, i.e. in one strain, to 17% that of the parent, and in the other, to 24%. DNA sequencing showed that the kinds of mutations induced by BPDE in the parental and the derivative strains did not differ and were similar to those found previously with finite life span human fibroblasts. The data strongly support the hypothesis that hRev3 plays a critical role in the induction of mutations by UV or BPDE. Because the level of hRev3 protein in human fibroblasts is below the level of antibody detection, it was not

  6. Endogenous α-crystallin inhibits expression of caspase-3 induced by hypoxia in retinal neurons.

    Science.gov (United States)

    Ying, Xi; Peng, Yanli; Zhang, Jiaping; Wang, Xingli; Wu, Nan; Zeng, Yuxiao; Wang, Yi

    2014-08-28

    To investigate the expression of endogenous, hypoxic stress-induced α-crystallin and caspase-3 in rat retinal neurons in vitro. Retinal neurons were cultured from Long-Evans rats. The expression of endogenous α-crystallin was analyzed by immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR). Furthermore, hypoxic exposure was performed in cultured cells, and the expression of endogenous α-crystallin and caspase-3 was assayed by Western blotting. Positive α-crystallin staining was observed in cultured retinal neurons, and expression of endogenous α-crystallin mRNA peaked 3-5d after inoculation (Pendogenous, hypoxic stress-induced α-crystallin expression increased gradually, peaking 6h after hypoxia. The expression was more abundant compared to the control (Pendogenous α-crystallin in retinal neurons, especially over-expression induced by hypoxic stress, results in the down regulation of caspase-3. The data suggest that endogenous α-crystallin may act as an endogenous neuroprotective factor in retinal neurons. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. Effect of enhanced expression of connexin 43 on sunitinib-induced cytotoxicity in mesothelioma cells

    Directory of Open Access Journals (Sweden)

    Miaki Uzu

    2015-05-01

    Full Text Available Connexin (Cx makes up a type of intercellular channel called gap junction (GJ. GJ plays a regulatory role in cellular physiology. The Cx expression level is often decreased in cancer cells compared to that in healthy ones, and the restoration of its expression has been shown to exert antiproliferative effects. This work aims to evaluate the effect of the restoration of connexin 43 (Cx43 (the most ubiquitous Cx subtype expression on sunitinib (SU-induced cytotoxicity in malignant mesothelioma (MM cells. Increased Cx43 expression in an MM cell line (H28 improved the ability of SU to inhibit receptor tyrosine kinase (RTK signaling. Moreover, higher Cx43 expression promoted SU-induced apoptosis. The cell viability test revealed that Cx43 enhanced the cytotoxic effect of SU in a GJ-independent manner. The effect of Cx43 on a proapoptotic factor, Bax, was then investigated. The interaction between Cx43 and Bax was confirmed by immunoprecipitation. Furthermore, higher Cx43 expression increased the production of a cleaved (active form of Bax during SU-induced apoptosis with no alteration in total Bax expression. These findings indicate that Cx43 most likely increases sensitivity to SU in H28 through direct interaction with Bax. In conclusion, we found that Cx43 overcame the chemoresistance of MM cells.

  8. 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.

  9. REM sleep enhancement and behavioral cataplexy following orexin (hypocretin)-II receptor antisense perfusion in the pontine reticular formation.

    Science.gov (United States)

    Thakkar, M M; Ramesh, V; Cape, E G; Winston, S; Strecker, R E; McCarley, R W

    1999-01-01

    Orexin (hypocretin)-containing neurons of the hypothalamus project to brainstem sites that are involved in the neural control of REM sleep, including the locus coeruleus, the dorsal raphe nucleus, the cholinergic zone of the mesopontine tegmentum, and the pontine reticular formation (PRF). Orexin knockout mice exhibit narcolepsy/cataplexy, and a mutant and defective gene for the orexin type II receptor is present in dogs with an inherited form of narcolepsy/cataplexy. However, the physiological systems mediating these effects have not been described. We reasoned that, since the effector neurons for the majority of REM sleep signs, including muscle atonia, were located in the PRF, this region was likely implicated in the production of these orexin-related abnormalities. To test this possibility, we used microdialysis perfusion of orexin type II receptor antisense in the PRF of rats. Ten to 24 hours after antisense perfusion, REM sleep increased two- to three-fold during both the light period (quiescent phase) and the dark period (active phase), and infrared video showed episodes of behavioral cataplexy. Moreover, preliminary data indicated no REM-related effects following perfusion with nonsense DNA, or when perfusion sites were outside the PRF. More work is needed to provide precise localization of the most effective site of orexin-induced inhibition of REM sleep phenomena.

  10. 2'-Hydroxycinnamaldehyde induces apoptosis through HSF1-mediated BAG3 expression.

    Science.gov (United States)

    Nguyen, Hai-Anh; Kim, Soo-A

    2017-01-01

    BAG3, a member of BAG co-chaperone family, is induced by stressful stimuli such as heat shock and heavy metals. Through interaction with various binding partners, BAG3 is thought to play a role in cellular adaptive responses against stressful conditions in normal and neoplastic cells. 2'-Hydroxycinnamaldehyde (HCA) is a natural derivative of cinnamaldehyde and has antitumor activity in various cancer cells. In the present study, for the first time, we identified that HCA induced BAG3 expression and BAG3-mediated apoptosis in cancer cells. The apoptotic cell death induced by HCA was demonstrated by caspase-7, -9 and PARP activation, and confirmed by Annexin V staining in both SW480 and SW620 colon cancer cells. Notably, both the mRNA and protein levels of BAG3 were largely induced by HCA in a dose- and time-dependent manner. By showing transcription factor HSF1 activation, we demonstrated that HCA induces the expression of BAG3 through HSF1 activation. More importantly, knockdown of BAG3 expression using siRNA largely inhibited HCA-induced apoptosis, suggesting that BAG3 is actively involved in HCA-induced cancer cell death. Considering the importance of the stress response mechanism in cancer progression, our results strongly suggest that BAG3 could be a potential target for anticancer therapy.

  11. Pregnancy-induced gene expression changes in vivo among women with rheumatoid arthritis: a pilot study.

    Science.gov (United States)

    Goin, Dana E; Smed, Mette Kiel; Pachter, Lior; Purdom, Elizabeth; Nelson, J Lee; Kjærgaard, Hanne; Olsen, Jørn; Hetland, Merete Lund; Zoffmann, Vibeke; Ottesen, Bent; Jawaheer, Damini

    2017-05-25

    Little is known about gene expression changes induced by pregnancy in women with rheumatoid arthritis (RA) and healthy women because the few studies previously conducted did not have pre-pregnancy samples available as baseline. We have established a cohort of women with RA and healthy women followed prospectively from a pre-pregnancy baseline. In this study, we tested the hypothesis that pregnancy-induced changes in gene expression among women with RA who improve during pregnancy (pregDAS improved ) overlap substantially with changes observed among healthy women and differ from changes observed among women with RA who worsen during pregnancy (pregDAS worse ). Global gene expression profiles were generated by RNA sequencing (RNA-seq) from 11 women with RA and 5 healthy women before pregnancy (T0) and at the third trimester (T3). Among the women with RA, eight showed an improvement in disease activity by T3, whereas three worsened. Differential expression analysis was used to identify genes demonstrating significant changes in expression within each of the RA and healthy groups (T3 vs T0), as well as between the groups at each time point. Gene set enrichment was assessed in terms of Gene Ontology processes and protein networks. A total of 1296 genes were differentially expressed between T3 and T0 among the 8 pregDAS improved women, with 161 genes showing at least two-fold change (FC) in expression by T3. The majority (108 of 161 genes) were also differentially expressed among healthy women (qexpression between the pregDAS improved and pregDAS worse groups, all of which were inducible by type I interferon (IFN). These IFN-inducible genes were over-expressed at T3 compared to the T0 baseline among the pregDAS improved women. In our pilot RNA-seq dataset, increased pregnancy-induced expression of type I IFN-inducible genes was observed among women with RA who improved during pregnancy, but not among women who worsened. These findings warrant further investigation into

  12. Cimetidine attenuates vinorelbine-induced phlebitis in mice by militating E-selectin expression.

    Science.gov (United States)

    Wang, Zhuo; Ma, Lijuan; Wang, Xuebin; Cai, Heping; Huang, Jin; Liu, Jiyong; Hu, Jinhong; Su, Dingfeng

    2014-08-01

    We investigated E-selectin expression in mice and rabbits with vinorelbine-induced phlebitis and the effect of cimetidine. To find the relationship between E-selectin expression and vinorelbine-induced phlebitis. Mouse and rabbit model of vinorelbine-induced phlebitis was established by intravenous infusion of vinorelbine. Pathological observation, molecular-biological determination of E-selectin and protein function of it was evaluated. Grossly, we observed swelling, edema and cord-like vessel changes in mice receiving vinorelbine but only mild edema in mice pretreated with cimetidine. Pathological scoring yielded a total score of 37 for vinorelbine-treated mice and 17 for mice pretreated with cimetidine (P phlebitis in mice probably by suppressing increased expression of E-selectin.

  13. Salicylic acid inhibits UV- and Cis-Pt-induced human immunodeficiency virus expression

    International Nuclear Information System (INIS)

    Woloschak, G.E.; Panozzo, J.; Libertin, C.R.; Schreck, S.; South Carolina Univ., Columbia, SC

    1994-01-01

    Previous studies have shown that exposure of HeLa cells stably transfected with a human immunodeficiency virus-long terminal repeat-chloramphenicol acetyl transferase (HIV-LTR-CAT) construct to UV light-induced expression from the HIV LTR. By culturing the cells with salicylic acid we demonstrated dose-dependent repression of this induced HIV expression. Repression was evident if salicylic acid was administered 2 h before, at the same time as, or up to 6 h after exposure to the DNA-damaging agent. The kinetics were similar for UV- and for cis-Pt-induced HIV expression, and induction was dependent on the UV dose or cis-Pt concentration added to the culture. These results suggest a role for the prostaglandins or the cyclooxygenase pathway or both in HIV induction mediated by DNA-damaging agents

  14. Expanding the molecular toolbox for Lactococcus lactis: construction of an inducible thioredoxin gene fusion expression system

    LENUS (Irish Health Repository)

    Douillard, Francois P

    2011-08-09

    Abstract Background The development of the Nisin Inducible Controlled Expression (NICE) system in the food-grade bacterium Lactococcus lactis subsp. cremoris represents a cornerstone in the use of Gram-positive bacterial expression systems for biotechnological purposes. However, proteins that are subjected to such over-expression in L. lactis may suffer from improper folding, inclusion body formation and\\/or protein degradation, thereby significantly reducing the yield of soluble target protein. Although such drawbacks are not specific to L. lactis, no molecular tools have been developed to prevent or circumvent these recurrent problems of protein expression in L. lactis. Results Mimicking thioredoxin gene fusion systems available for E. coli, two nisin-inducible expression vectors were constructed to over-produce various proteins in L. lactis as thioredoxin fusion proteins. In this study, we demonstrate that our novel L. lactis fusion partner expression vectors allow high-level expression of soluble heterologous proteins Tuc2009 ORF40, Bbr_0140 and Tuc2009 BppU\\/BppL that were previously insoluble or not expressed using existing L. lactis expression vectors. Over-expressed proteins were subsequently purified by Ni-TED affinity chromatography. Intact heterologous proteins were detected by immunoblotting analyses. We also show that the thioredoxin moiety of the purified fusion protein was specifically and efficiently cleaved off by enterokinase treatment. Conclusions This study is the first description of a thioredoxin gene fusion expression system, purposely developed to circumvent problems associated with protein over-expression in L. lactis. It was shown to prevent protein insolubility and degradation, allowing sufficient production of soluble proteins for further structural and functional characterization.

  15. Farnesoid-X-receptor expression in monocrotaline-induced pulmonary arterial hypertension and right heart failure

    International Nuclear Information System (INIS)

    Ye, Lusi; Jiang, Ying; Zuo, Xiaoxia

    2015-01-01

    Objective: The farnesoid-X-receptor (FXR) is a metabolic nuclear receptor superfamily member that is highly expressed in enterohepatic tissue and is also expressed in the cardiovascular system. Multiple nuclear receptors, including FXR, play a pivotal role in cardiovascular disease (CVD). Pulmonary arterial hypertension (PAH) is an untreatable cardiovascular system disease that leads to right heart failure (RHF). However, the potential physiological/pathological roles of FXR in PAH and RHF are unknown. We therefore compared FXR expression in the cardiovascular system in PAH, RHF and a control. Methods and results: Hemodynamic parameters and morphology were assessed in blank solution-exposed control, monocrotaline (MCT)-exposed PAH (4 weeks) and RHF (7 weeks) Sprague–Dawley rats. Real-time reverse transcription polymerase chain reaction (real-time RT-PCR), Western blot (WB), immunohistochemistry (IHC) analysis and immunofluorescence (IF) analysis were performed to assess FXR levels in the lung and heart tissues of MCT-induced PAH and RHF rats. In normal rats, low FXR levels were detected in the heart, and nearly no FXR was expressed in rat lungs. However, FXR expression was significantly elevated in PAH and RHF rat lungs but reduced in PAH and RHF rat right ventricular (RV) tissues. FXR expression was reduced only in RHF rat left ventricular (LV) tissues. Conclusions: The differential expression of FXR in MCT-induced PAH lungs and heart tissues in parallel with PAH pathophysiological processes suggests that FXR contributes to PAH. - Highlights: • FXR was expressed in rat lung and heart tissues. • FXR expression increased sharply in the lung tissues of PAH and RHF rats. • FXR expression was reduced in PAH and RHF rat RV tissue. • FXR expression was unaltered in PAH LV but reduced in RHF rat LV tissue. • FXR expression was prominent in the neovascularization region.

  16. Farnesoid-X-receptor expression in monocrotaline-induced pulmonary arterial hypertension and right heart failure

    Energy Technology Data Exchange (ETDEWEB)

    Ye, Lusi [Department of Rheumatology, Xiangya Hospital, Central South University, 87 Xiangya Road, Changsha, Hunan 410008 (China); Department of Rheumatology, The First Affiliated Hospital, Wenzhou Medical University, Wenzhou, Zhejiang 325015 (China); Jiang, Ying [Department of Rheumatology, Xiangya Hospital, Central South University, 87 Xiangya Road, Changsha, Hunan 410008 (China); Zuo, Xiaoxia, E-mail: susanzuo@hotmail.com [Department of Rheumatology, Xiangya Hospital, Central South University, 87 Xiangya Road, Changsha, Hunan 410008 (China)

    2015-11-06

    Objective: The farnesoid-X-receptor (FXR) is a metabolic nuclear receptor superfamily member that is highly expressed in enterohepatic tissue and is also expressed in the cardiovascular system. Multiple nuclear receptors, including FXR, play a pivotal role in cardiovascular disease (CVD). Pulmonary arterial hypertension (PAH) is an untreatable cardiovascular system disease that leads to right heart failure (RHF). However, the potential physiological/pathological roles of FXR in PAH and RHF are unknown. We therefore compared FXR expression in the cardiovascular system in PAH, RHF and a control. Methods and results: Hemodynamic parameters and morphology were assessed in blank solution-exposed control, monocrotaline (MCT)-exposed PAH (4 weeks) and RHF (7 weeks) Sprague–Dawley rats. Real-time reverse transcription polymerase chain reaction (real-time RT-PCR), Western blot (WB), immunohistochemistry (IHC) analysis and immunofluorescence (IF) analysis were performed to assess FXR levels in the lung and heart tissues of MCT-induced PAH and RHF rats. In normal rats, low FXR levels were detected in the heart, and nearly no FXR was expressed in rat lungs. However, FXR expression was significantly elevated in PAH and RHF rat lungs but reduced in PAH and RHF rat right ventricular (RV) tissues. FXR expression was reduced only in RHF rat left ventricular (LV) tissues. Conclusions: The differential expression of FXR in MCT-induced PAH lungs and heart tissues in parallel with PAH pathophysiological processes suggests that FXR contributes to PAH. - Highlights: • FXR was expressed in rat lung and heart tissues. • FXR expression increased sharply in the lung tissues of PAH and RHF rats. • FXR expression was reduced in PAH and RHF rat RV tissue. • FXR expression was unaltered in PAH LV but reduced in RHF rat LV tissue. • FXR expression was prominent in the neovascularization region.

  17. Expression of manganese superoxide dismutase in rat blood, heart and brain during induced systemic hypoxia

    Directory of Open Access Journals (Sweden)

    Septelia I. Wanandi

    2011-02-01

    Full Text Available Background: Hypoxia results in an increased generation of ROS. Until now, little is known about the role of MnSOD - a major endogenous antioxidant enzyme - on the cell adaptation response against hypoxia. The aim of this study was to  determine the MnSOD mRNA expression and levels of specific activity in blood, heart and brain of rats during induced systemic hypoxia.Methods: Twenty-five male Sprague Dawley rats were subjected to systemic hypoxia in an hypoxic chamber (at 8-10% O2 for 0, 1, 7, 14 and 21 days, respectively. The mRNA relative expression of MnSOD was analyzed using Real Time RT-PCR. MnSOD specific activity was determined using xanthine oxidase inhibition assay.Results: The MnSOD mRNA relative expression in rat blood and heart was decreased during early induced systemic hypoxia (day 1 and increased as hypoxia continued, whereas the mRNA expression in brain was increased since day 1 and reached its maximum level at day 7. The result of MnSOD specific activity during early systemic hypoxia was similar to the mRNA expression. Under very late hypoxic condition (day 21, MnSOD specific activity in blood, heart and brain was significantly decreased. We demonstrate a positive correlation between MnSOD mRNA expression and specific activity in these 3 tissues during day 0-14 of induced systemic hypoxia. Furthermore, mRNA expression and specific activity levels in heart strongly correlate with those in blood.Conclusion: The MnSOD expression at early and late phases of induced systemic hypoxia is distinctly regulated. The MnSOD expression in brain differs from that in blood and heart revealing that brain tissue can  possibly survive better from induced systemic hypoxia than heart and blood. The determination of MnSOD expression in blood can be used to describe its expression in heart under systemic hypoxic condition. (Med J Indones 2011; 20:27-33Keywords: MnSOD, mRNA expression, ROS, specific activity, systemic hypoxia

  18. SIRT-1 regulates TGF-β-induced dermal fibroblast migration via modulation of Cyr61 expression.

    Science.gov (United States)

    Kwon, Eun-Jeong; Park, Eun-Jung; Yu, Hyeran; Huh, Jung-Sik; Kim, Jinseok; Cho, Moonjae

    2018-05-01

    SIRT1 is a NAD-dependent protein deacetylase that participates in cellular regulation. The increased migration of fibroblasts is an important phenotype in fibroblast activation. The role of SIRT1 in cell migration remains controversial as to whether SIRT1 acts as an activator or suppressor of cell migration. Therefore, we have established the role of SIRT1 in the migration of human dermal fibroblasts and explored targets of SIRT1 during dermal fibroblast migration. SIRT1 and Cyr61 were expressed in human dermal fibroblasts and the stimulation with TGF-β further induced their expression. Treatment with resveratrol (RSV), a SIRT1 agonist, or overexpression of SIRT1 also promoted the expression Cyr61 in human dermal fibroblasts, whereas the inhibition of SIRT1 activity by nicotinamide or knockdown of SIRT1 decreased the level of Cyr61, as well as TGF-β or RSV-induced Cyr61 expression. Blocking of ERK signaling by PD98509 reduced the expression of Cyr61 induced by TGF-β or RSV. TGF-β, RSV, or SIRT1 overexpression enhanced β-catenin as well as Cyr61 expression. This stimulation was reduced by the Wnt inhibitor XAV939. RSV increased migration and nicotinamide attenuated RSV-induced migration of human dermal fibroblasts. Furthermore, SIRT1 overexpression promoted cell migration, whereas blocking Cyr61 attenuated SIRT1-stimulated migration of human dermal fibroblasts. SIRT1 increased cell migration by stimulating Cyr61 expression and the ERK and Wnt/β-catenin signaling. SIRT1-induced Cyr61 activity is very important for human dermal fibroblasts migration.

  19. Dual-Specificity Anti-HER-2/neu Antisense DNA Agents for Breast Cancer Therapy

    National Research Council Canada - National Science Library

    Stein, Stanley

    2001-01-01

    .... To achieve high avidity and specificity, we designed chimeric antisense molecules consisting of a short active DNA fused to a short "anchor" 2'-0-methyl RNA complementary to non-contiguous single...

  20. BMP-2 Induced Expression of Alx3 That Is a Positive Regulator of Osteoblast Differentiation.

    Directory of Open Access Journals (Sweden)

    Takashi Matsumoto

    Full Text Available Bone morphogenetic proteins (BMPs regulate many aspects of skeletal development, including osteoblast and chondrocyte differentiation, cartilage and bone formation, and cranial and limb development. Among them, BMP-2, one of the most potent osteogenic signaling molecules, stimulates osteoblast differentiation, while it inhibits myogenic differentiation in C2C12 cells. To evaluate genes involved in BMP-2-induced osteoblast differentiation, we performed cDNA microarray analyses to compare BMP-2-treated and -untreated C2C12 cells. We focused on Alx3 (aristaless-like homeobox 3 which was clearly induced during osteoblast differentiation. Alx3, a homeobox gene related to the Drosophilaaristaless gene, has been linked to developmental functions in craniofacial structures and limb development. However, little is known about its direct relationship with bone formation. In the present study, we focused on the mechanisms of Alx3 gene expression and function during osteoblast differentiation induced by BMP-2. In C2C12 cells, BMP-2 induced increase of Alx3 gene expression in both time- and dose-dependent manners through the BMP receptors-mediated SMAD signaling pathway. In addition, silencing of Alx3 by siRNA inhibited osteoblast differentiation induced by BMP-2, as showed by the expressions of alkaline phosphatase (Alp, Osteocalcin, and Osterix, while over-expression of Alx3 enhanced osteoblast differentiation induced by BMP-2. These results indicate that Alx3 expression is enhanced by BMP-2 via the BMP receptors mediated-Smad signaling and that Alx3 is a positive regulator of osteoblast differentiation induced by BMP-2.

  1. Novel interactions between the HTLV antisense proteins HBZ and APH-2 and the NFAR protein family: Implications for the HTLV lifecycles

    Energy Technology Data Exchange (ETDEWEB)

    Murphy, Jane; Hall, William W. [Centre for Research in Infectious Diseases, School of Medicine and Medical Science, University College Dublin, Belfield, Dublin 4 (Ireland); Ratner, Lee [Department of Medicine, Division of Molecular Oncology, Washington University School of Medicine, Saint Louis, Missouri, United States of America (United States); Sheehy, Noreen [Centre for Research in Infectious Diseases, School of Medicine and Medical Science, University College Dublin, Belfield, Dublin 4 (Ireland)

    2016-07-15

    The human T-cell leukaemia virus type 1 and type 2 (HTLV-1/HTLV-2) antisense proteins HBZ and APH-2 play key roles in the HTLV lifecycles and persistence in the host. Nuclear Factors Associated with double-stranded RNA (NFAR) proteins NF90/110 function in the lifecycles of several viruses and participate in host innate immunity against infection and oncogenesis. Using GST pulldown and co-immunoprecipitation assays we demonstrate specific novel interactions between HBZ/APH-2 and NF90/110 and characterised the protein domains involved. Moreover we show that NF90/110 significantly enhance Tax mediated LTR activation, an effect that was abolished by HBZ but enhanced by APH-2. Additionally we found that HBZ and APH-2 modulate the promoter activity of survivin and are capable of antagonising NF110-mediated survivin activation. Thus interactions between HTLV antisense proteins and the NFAR protein family have an overall positive impact on HTLV infection. Hence NFARs may represent potential therapeutic targets in HTLV infected cells. - Highlights: • This study demonstrates for the first time interactions between NF90/110 and the HTLV antisense proteins HBZ and APH-2. • We show that NF90/110 significantly enhance LTR activation by the HTLV Tax protein, an effect that is abolished by HBZ but enhanced by APH-2. • The study shows that even though the HTLV antisense proteins activate survivin expression they antagonize the ability of NF90/110 to do so. • Overall we found that NF90/110 positively regulate HTLV infection and as such might represent a therapeutic target in infected cells.

  2. Novel interactions between the HTLV antisense proteins HBZ and APH-2 and the NFAR protein family: Implications for the HTLV lifecycles

    International Nuclear Information System (INIS)

    Murphy, Jane; Hall, William W.; Ratner, Lee; Sheehy, Noreen

    2016-01-01

    The human T-cell leukaemia virus type 1 and type 2 (HTLV-1/HTLV-2) antisense proteins HBZ and APH-2 play key roles in the HTLV lifecycles and persistence in the host. Nuclear Factors Associated with double-stranded RNA (NFAR) proteins NF90/110 function in the lifecycles of several viruses and participate in host innate immunity against infection and oncogenesis. Using GST pulldown and co-immunoprecipitation assays we demonstrate specific novel interactions between HBZ/APH-2 and NF90/110 and characterised the protein domains involved. Moreover we show that NF90/110 significantly enhance Tax mediated LTR activation, an effect that was abolished by HBZ but enhanced by APH-2. Additionally we found that HBZ and APH-2 modulate the promoter activity of survivin and are capable of antagonising NF110-mediated survivin activation. Thus interactions between HTLV antisense proteins and the NFAR protein family have an overall positive impact on HTLV infection. Hence NFARs may represent potential therapeutic targets in HTLV infected cells. - Highlights: • This study demonstrates for the first time interactions between NF90/110 and the HTLV antisense proteins HBZ and APH-2. • We show that NF90/110 significantly enhance LTR activation by the HTLV Tax protein, an effect that is abolished by HBZ but enhanced by APH-2. • The study shows that even though the HTLV antisense proteins activate survivin expression they antagonize the ability of NF90/110 to do so. • Overall we found that NF90/110 positively regulate HTLV infection and as such might represent a therapeutic target in infected cells.

  3. Endogenous brain IL-1 mediates LPS-induced anorexia and hypothalamic cytokine expression.

    Science.gov (United States)

    Layé, S; Gheusi, G; Cremona, S; Combe, C; Kelley, K; Dantzer, R; Parnet, P

    2000-07-01

    The present study was designed to determine the role of endogenous brain interleukin (IL)-1 in the anorexic response to lipopolysaccharide (LPS). Intraperitoneal administration of LPS (5-10 microgram/mouse) induced a dramatic, but transient, decrease in food intake, associated with an enhanced expression of proinflammatory cytokine mRNA (IL-1beta, IL-6, and tumor necrosis factor-alpha) in the hypothalamus. This dose of LPS also increased plasma levels of IL-1beta. Intracerebroventricular pretreatment with IL-1 receptor antagonist (4 microgram/mouse) attenuated LPS-induced depression of food intake and totally blocked the LPS-induced enhanced expression of proinflammatory cytokine mRNA measured in the hypothalamus 1 h after treatment. In contrast, LPS-induced increases in plasma levels of IL-1beta were not altered. These findings indicate that endogenous brain IL-1 plays a pivotal role in the development of the hypothalamic cytokine response to a systemic inflammatory stimulus.

  4. Precise integration of inducible transcriptional elements (PrIITE) enables absolute control of gene expression

    DEFF Research Database (Denmark)

    Pinto, Rita; Hansen, Lars; Hintze, John

    2017-01-01

    to be a limitation. Here, we report that the combined use of genome editing tools and last generation Tet-On systems can resolve these issues. Our principle is based on precise integration of inducible transcriptional elements (coined PrIITE) targeted to: (i) exons of an endogenous gene of interest (GOI) and (ii......Tetracycline-based inducible systems provide powerful methods for functional studies where gene expression can be controlled. However, the lack of tight control of the inducible system, leading to leakiness and adverse effects caused by undesirable tetracycline dosage requirements, has proven......) a safe harbor locus. Using PrIITE cells harboring a GFP reporter or CDX2 transcription factor, we demonstrate discrete inducibility of gene expression with complete abrogation of leakiness. CDX2 PrIITE cells generated by this approach uncovered novel CDX2 downstream effector genes. Our results provide...

  5. Inhibition of connective tissue growth factor (CTGF/CCN2) expression decreases the survival and myogenic differentiation of human rhabdomyosarcoma cells.

    Science.gov (United States)

    Croci, Stefania; Landuzzi, Lorena; Astolfi, Annalisa; Nicoletti, Giordano; Rosolen, Angelo; Sartori, Francesca; Follo, Matilde Y; Oliver, Noelynn; De Giovanni, Carla; Nanni, Patrizia; Lollini, Pier-Luigi

    2004-03-01

    Connective tissue growth factor (CTGF/CCN2), a cysteine-rich protein of the CCN (Cyr61, CTGF, Nov) family of genes, emerged from a microarray screen of genes expressed by human rhabdomyosarcoma cells. Rhabdomyosarcoma is a soft tissue sarcoma of childhood deriving from skeletal muscle cells. In this study, we investigated the role of CTGF in rhabdomyosarcoma. Human rhabdomyosarcoma cells of the embryonal (RD/12, RD/18, CCA) and the alveolar histotype (RMZ-RC2, SJ-RH4, SJ-RH30), rhabdomyosarcoma tumor specimens, and normal skeletal muscle cells expressed CTGF. To determine the function of CTGF, we treated rhabdomyosarcoma cells with a CTGF antisense oligonucleotide or with a CTGF small interfering RNA (siRNA). Both treatments inhibited rhabdomyosarcoma cell growth, suggesting the existence of a new autocrine loop based on CTGF. CTGF antisense oligonucleotide-mediated growth inhibition was specifically due to a significant increase in apoptosis, whereas cell proliferation was unchanged. CTGF antisense oligonucleotide induced a strong decrease in the level of myogenic differentiation of rhabdomyosarcoma cells, whereas the addition of recombinant CTGF significantly increased the proportion of myosin-positive cells. CTGF emerges as a survival and differentiation factor and could be a new therapeutic target in human rhabdomyosarcoma.

  6. Growth hormone-releasing factor induces c-fos expression in cultured primary pituitary cells

    DEFF Research Database (Denmark)

    Billestrup, Nils; Mitchell, R L; Vale, W

    1987-01-01

    GH-releasing factor (GRF) and somatostatin regulates the secretion and biosynthesis of GH as well as the proliferation of GH-producing cells. In order to further characterize the mitogenic effect of GRF, we studied the expression of the proto-oncogene c-fos in primary pituitary cells. Maximal...... induction of c-fos mRNA was observed 20-60 min after stimulation with 5 nM GRF, returning to basal levels after 2 h. Somatostatin-14 (5 nM) partially inhibited the GRF induced c-fos expression. Forskolin and phorbol 12, 13 dibutyrate induced c-fos gene in cultured primary pituitary cells with similar...

  7. Apoptosis induced by radionuclide 153Sm and expression of relevant genes in three different cancer cells

    International Nuclear Information System (INIS)

    Zou Baomin; Duan Xiaoyi; Chen Wei; Hu Guoying

    2003-01-01

    To study apoptosis of PC-3, ER-75-30 and A549 cells induced by radionuclide 153 Sm and the expression of bcl-2, bax in apoptosis cells, MTT assay was used to detect the anti-tumor effect, light microscope, transmission electron microscope, flow cytometer were used to detect apoptosis, while image analysis was used to detect the expression of bcl-2 and bax. 153 Sm showed anti-tumor effect and could induce tumor cell apoptosis. Both bcl-2 and bax played an important role in apoptosis. Different kind of cells had different sensitivity to 153 Sm

  8. Estimation of Ship Long-term Wave-induced Bending Moment using Closed-Form Expressions

    DEFF Research Database (Denmark)

    Jensen, Jørgen Juncher; Mansour, A. E.

    2002-01-01

    A semi-analytical approach is used to derive frequency response functions and standard deviations for the wave-induced bending moment amidships for mono-hull ships. The results are given as closed-form expressions and the required input information for the procedure is restricted to the main......-empirical closed-form expression for the skewness. The effect of whipping is included by assuming that whipping and wave-induced responses are conditionally independent given Hs. The procedure is simple and can be used to make quick estimates of the design wave bending moment at the conceptual design phase...

  9. The effect of streptozotocin-induced diabetes on phenylalanine hydroxylase expression in rat liver.

    OpenAIRE

    Taylor, D S; Dahl, H H; Mercer, J F; Green, A K; Fisher, M J

    1989-01-01

    The impact of experimentally induced diabetes on the expression of rat liver phenylalanine hydroxylase has been investigated. A significant elevation in maximal enzymic activity was observed in diabetes. This was associated with significant increases in the amount of enzyme, the phenylalanine hydroxylase-specific translational activity of hepatic RNA and the abundance of phenylalanine hydroxylase-specific mRNA. These changes in phenylalanine hydroxylase expression were not observed when diabe...

  10. Cigarette smoke modulates expression of human rhinovirus-induced airway epithelial host defense genes.

    Directory of Open Access Journals (Sweden)

    David Proud

    Full Text Available Human rhinovirus (HRV infections trigger acute exacerbations of chronic obstructive pulmonary disease (COPD and asthma. The human airway epithelial cell is the primary site of HRV infection and responds to infection with altered expression of multiple genes, the products of which could regulate the outcome to infection. Cigarette smoking aggravates asthma symptoms, and is also the predominant risk factor for the development and progression of COPD. We, therefore, examined whether cigarette smoke extract (CSE modulates viral responses by altering HRV-induced epithelial gene expression. Primary cultures of human bronchial epithelial cells were exposed to medium alone, CSE alone, purified HRV-16 alone or to HRV-16+ CSE. After 24 h, supernatants were collected and total cellular RNA was isolated. Gene array analysis was performed to examine mRNA expression. Additional experiments, using real-time RT-PCR, ELISA and/or western blotting, validated altered expression of selected gene products. CSE and HRV-16 each induced groups of genes that were largely independent of each other. When compared to gene expression in response to CSE alone, cells treated with HRV+CSE showed no obvious differences in CSE-induced gene expression. By contrast, compared to gene induction in response to HRV-16 alone, cells exposed to HRV+CSE showed marked suppression of expression of a number of HRV-induced genes associated with various functions, including antiviral defenses, inflammation, viral signaling and airway remodeling. These changes were not associated with altered expression of type I or type III interferons. Thus, CSE alters epithelial responses to HRV infection in a manner that may negatively impact antiviral and host defense outcomes.

  11. 2-Chlorohexadecanal and 2-chlorohexadecanoic acid induce COX-2 expression in human coronary artery endothelial cells

    OpenAIRE

    Messner, Maria C.; Albert, Carolyn J.; Ford, David A.

    2008-01-01

    2-Chlorohexadecanal (2-ClHDA), a 16-carbon chain chlorinated fatty aldehyde that is produced by reactive chlorinating species attack of plasmalogens, is elevated in atherosclerotic plaques, infarcted myocardium, and activated leukocytes. We tested the hypothesis that 2-ClHDA and its metabolites, 2-chlorohexadecanoic acid (2-ClHA) and 2-chlorohexadecanol (2-ClHOH), induce COX-2 expression in human coronary artery endothelial cells (HCAEC). COX-2 protein expression increased in response to 2-Cl...

  12. Uterine and placental expression of canine oxytocin receptor during pregnancy and normal and induced parturition.

    OpenAIRE

    Gram A Boos A Kowalewski MP.

    2014-01-01

    Abstract Oxytocin (OT) plays an important role as an inducer of uterine contractility acting together with its receptor (OTR) to increase synthesis of prostaglandins. Although OT is commonly used in the treatment for dystocia and uterine inertia in the bitch little attention has been paid to the role of OT in mechanisms regulating parturition in the dog so that knowledge about the expression of OTR in the canine uterus and placenta is sparse. Consequently the expression and cellular localizat...

  13. Dux4 induces cell cycle arrest at G1 phase through upregulation of p21 expression

    International Nuclear Information System (INIS)

    Xu, Hongliang; Wang, Zhaoxia; Jin, Suqin; Hao, Hongjun; Zheng, Lemin; Zhou, Boda; Zhang, Wei; Lv, He; Yuan, Yun

    2014-01-01

    Highlights: • Dux4 induced TE671 cell proliferation defect and G1 phase arrest. • Dux4 upregulated p21 expression without activating p53. • Silencing p21 rescued Dux4 mediated proliferation defect and cell cycle arrest. • Sp1 binding site was required for Dux4-induced p21 promoter activation. - Abstract: It has been implicated that Dux4 plays crucial roles in development of facioscapulohumeral dystrophy. But the underlying myopathic mechanisms and related down-stream events of this retrogene were far from clear. Here, we reported that overexpression of Dux4 in a cell model TE671 reduced cell proliferation rate, and increased G1 phase accumulation. We also determined the impact of Dux4 on p53/p21 signal pathway, which controls the checkpoint in cell cycle progression. Overexpression of Dux4 increased p21 mRNA and protein level, while expression of p53, phospho-p53 remained unchanged. Silencing p21 rescued Dux4 mediated proliferation defect and cell cycle arrest. Furthermore, we demonstrated that enhanced Dux4 expression increased p21 promoter activity and elevated expression of Sp1 transcription factor. Mutation of Sp1 binding site decreased dux4 induced p21 promoter activation. Chromatin immunoprecipitation (ChIP) assays confirmed the Dux4-induced binding of Sp1 to p21 promoter in vivo. These results suggest that Dux4 might induce proliferation inhibition and G1 phase arrest through upregulation of p21

  14. Dux4 induces cell cycle arrest at G1 phase through upregulation of p21 expression

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Hongliang; Wang, Zhaoxia; Jin, Suqin; Hao, Hongjun [Department of Neurology, Peking University First Hospital, Beijing 100034 (China); Zheng, Lemin [The Institute of Cardiovascular Sciences, Peking University Health Science Center, Key Laboratory of Molecular Cardiovascular Sciences of Education Ministry, Key Laboratory of Cardiovascular Molecular Biology and Regulatory Peptides of Health Ministry, Beijing 100191 (China); Zhou, Boda [The Department of Cardiology, Peking University Third Hospital, Beijing 100191 (China); Zhang, Wei; Lv, He [Department of Neurology, Peking University First Hospital, Beijing 100034 (China); Yuan, Yun, E-mail: yuanyun2002@sohu.com [Department of Neurology, Peking University First Hospital, Beijing 100034 (China)

    2014-03-28

    Highlights: • Dux4 induced TE671 cell proliferation defect and G1 phase arrest. • Dux4 upregulated p21 expression without activating p53. • Silencing p21 rescued Dux4 mediated proliferation defect and cell cycle arrest. • Sp1 binding site was required for Dux4-induced p21 promoter activation. - Abstract: It has been implicated that Dux4 plays crucial roles in development of facioscapulohumeral dystrophy. But the underlying myopathic mechanisms and related down-stream events of this retrogene were far from clear. Here, we reported that overexpression of Dux4 in a cell model TE671 reduced cell proliferation rate, and increased G1 phase accumulation. We also determined the impact of Dux4 on p53/p21 signal pathway, which controls the checkpoint in cell cycle progression. Overexpression of Dux4 increased p21 mRNA and protein level, while expression of p53, phospho-p53 remained unchanged. Silencing p21 rescued Dux4 mediated proliferation defect and cell cycle arrest. Furthermore, we demonstrated that enhanced Dux4 expression increased p21 promoter activity and elevated expression of Sp1 transcription factor. Mutation of Sp1 binding site decreased dux4 induced p21 promoter activation. Chromatin immunoprecipitation (ChIP) assays confirmed the Dux4-induced binding of Sp1 to p21 promoter in vivo. These results suggest that Dux4 might induce proliferation inhibition and G1 phase arrest through upregulation of p21.

  15. Pseudogene PHBP1 promotes esophageal squamous cell carcinoma proliferation by increasing its cognate gene PHB expression.

    Science.gov (United States)

    Feng, Feiyue; Qiu, Bin; Zang, Ruochuan; Song, Peng; Gao, Shugeng

    2017-04-25

    Natural antisense transcripts (NATs) as one of the most diverse classes of long noncoding RNAs (lncRNAs), have been demonstrated involved in fundamental biological processes in human. Here, we reported that human prohibitin gene pseudogene 1 (PHBP1) was upregulated in ESCC, and increased PHBP1 expression in ESCC was associated with clinical advanced stage. Functional experiments showed that PHBP1 knockdown inhibited ESCC cells proliferation, colony formation and xenograft tumor growth in vitro and in vivo by causing cell-cycle arrest at the G1-G0 phase. Mechanisms analysis revealed that PHBP1 transcript as an antisense transcript of PHB is partially complementary to PHB mRNA and formed an RNA-RNA hybrid with PHB, consequently inducing an increase of PHB expression at both the mRNA and protein levels. Furthermore, PHBP1 expression is strongly correlated with PHB expression in ESCC tissues. Collectively, this study elucidates an important role of PHBP1 in promoting ESCC partly via increasing PHB expression.

  16. Ketamine induces brain-derived neurotrophic factor expression via phosphorylation of histone deacetylase 5 in rats.

    Science.gov (United States)

    Choi, Miyeon; Lee, Seung Hoon; Park, Min Hyeop; Kim, Yong-Seok; Son, Hyeon

    2017-08-05

    Ketamine shows promise as a therapeutic agent for the treatment of depression. The increased expression of brain-derived neurotrophic factor (BDNF) has been associated with the antidepressant-like effects of ketamine, but the mechanism of BDNF induction is not well understood. In the current study, we demonstrate that the treatment of rats with ketamine results in the dose-dependent rapid upregulation of Bdnf promoter IV activity and expression of Bdnf exon IV mRNAs in rat hippocampal neurons. Transfection of histone deacetylase 5 (HDAC5) into rat hippocampal neurons similarly induces Bdnf mRNA expression in response to ketamine, whereas transfection of a HDAC5 phosphorylation-defective mutant (Ser259 and Ser498 replaced by Ala259 and Ala498), results in the suppression of ketamine-mediated BDNF promoter IV transcriptional activity. Viral-mediated hippocampal knockdown of HDAC5 induces Bdnf mRNA and protein expression, and blocks the enhancing effects of ketamine on BDNF expression in both unstressed and stressed rats, and thereby providing evidence for the role of HDAC5 in the regulation of Bdnf expression. Taken together, our findings implicate HDAC5 in the ketamine-induced transcriptional regulation of Bdnf, and suggest that the phosphorylation of HDAC5 regulates the therapeutic actions of ketamine. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Construction of Egr1-mediated human truncated apoptosis inducing factor expression vector and its expression regularity induced by radiation in breast cancer MCF-7 cells

    International Nuclear Information System (INIS)

    Wang Jianfeng; Gong Shouliang; Wang Zhicheng; Fang Fang; Liu Yang; Wu Jiahui

    2012-01-01

    Objective: To clone human truncated apoptosis inducing factor (AIF) cDNA sequence, and to construct early growth response 1 (Egr1)-mediated recombinant expression vector pcDNA 3.1-Egr1-AIF Δ1-480 (pEgr1-AIFΔ 1-480 ), and to observe its regularity induced by radiation in human breast cancer MCF-7 cells. Methods: The total mRNA extracted from human leukemia Jurkat cells used as template, and the human AIFΔ 1-480 was acquired by RT-PCR, and it was linked to pMD18T vector for sequencing. Egr1 fragment was digested from pMD19T-Egr1 by restrictive enzyme, and the Egr1-mediated expression plasmid pEgr1-AIFΔ 1-480 was constructed by gene recombination. There were control group, pcDNA3.1 group, pAIFΔ 1-480 group and pEgr1-AIFΔ 1-480 group in the experiment. After the plasmids in various groups were transfected into human breast cancer MCF-7 cells, the AIF and AIFΔ 1-480 protein expression time-effect (0, 2, 4, 12, 24 and 48 h after 2.0 Gy irradiation) and dose-effect (24 h after 0, 0.2, 0.5, 1.0, 2.0 and 5.0 Gy irradiation) regularity were measured by Western blotting method. Results: The sequencing results showed that the AIFΔ 1-480 acquired by RT-PCR was consistent with the sequence expected, the pEgr-AIFΔ 1-480 was confirmed by PCR and restrictive enzyme digestion. After 0-48 h the MCF-7 cells were irradiated by 2.0 Gy, and the AIF protein expressed in the cells in each group, and it increased significantly from 4 h and the AIF expressions in 4, 12, 24 and 48 h groups were higher than that in 0 h group (P<0.05), and it reached to maximum value at 48 h. But the AIFΔ 1-480 protein expressed in the cells in pAIFΔ 1-480 and pEgr1-AIFΔ 1-480 groups from 2 h (P<0.05), and it reached to peak value at 24 h. The AIFΔ 1-480 expressions in pEgr1-AIFΔ 1-480 group were higher than those in pAIFΔ 1-480 group at and 48 h (P<0.05). After the MCF-7 cells were irradiated by 0-5 Gy for 24 h, the AIF protein expressed in the cells in each group, but the AIFΔ 1-480 protein

  18. Sialoadhesin expressed on IFN-induced monocytes binds HIV-1 and enhances infectivity.

    Directory of Open Access Journals (Sweden)

    Hans Rempel

    2008-04-01

    Full Text Available HIV-1 infection dysregulates the immune system and alters gene expression in circulating monocytes. Differential gene expression analysis of CD14(+ monocytes from subjects infected with HIV-1 revealed increased expression of sialoadhesin (Sn, CD169, Siglec 1, a cell adhesion molecule first described in a subset of macrophages activated in chronic inflammatory diseases.We analyzed sialoadhesin expression on CD14(+ monocytes by flow cytometry and found significantly higher expression in subjects with elevated viral loads compared to subjects with undetectable viral loads. In cultured CD14(+ monocytes isolated from healthy individuals, sialoadhesin expression was induced by interferon-alpha and interferon-gamma but not tumor necrosis factor-alpha. Using a stringent binding assay, sialoadhesin-expressing monocytes adsorbed HIV-1 through interaction with the sialic acid residues on the viral envelope glycoprotein gp120. Furthermore, monocytes expressing sialoadhesin facilitated HIV-1 trans infection of permissive cells, which occurred in the absence of monocyte self-infection.Increased sialoadhesin expression on CD14(+ monocytes occurred in response to HIV-1 infection with maximum expression associated with high viral load. We show that interferons induce sialoadhesin in primary CD14(+ monocytes, which is consistent with an antiviral response during viremia. Our findings suggest that circulating sialoadhesin-expressing monocytes are capable of binding HIV-1 and effectively delivering virus to target cells thereby enhancing the distribution of HIV-1. Sialoadhesin could disseminate HIV-1 to viral reservoirs during monocyte immunosurveillance or migration to sites of inflammation and then facilitate HIV-1 infection of permissive cells.

  19. 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

  20. Wolbachia-induced cytoplasmic incompatibility is associated with decreased Hira expression in male Drosophila.

    Directory of Open Access Journals (Sweden)

    Ya Zheng

    Full Text Available BACKGROUND: Wolbachia are obligate endosymbiotic bacteria that infect numerous species of arthropods and nematodes. Wolbachia can induce several reproductive phenotypes in their insect hosts including feminization, male-killing, parthenogenesis and cytoplasmic incompatibility (CI. CI is the most common phenotype and occurs when Wolbachia-infected males mate with uninfected females resulting in no or very low numbers of viable offspring. However, matings between males and females infected with the same strain of Wolbachia result in viable progeny. Despite substantial scientific effort, the molecular mechanisms underlying CI are currently unknown. METHODOLOGY/PRINCIPAL FINDINGS: Gene expression studies were undertaken in Drosophila melanogaster and D. simulans which display differential levels of CI using quantitative RT-PCR. We show that Hira expression is correlated with the induction of CI and occurs in a sex-specific manner. Hira expression is significantly lower in males which induce strong CI when compared to males inducing no CI or Wolbachia-uninfected males. A reduction in Hira expression is also observed in 1-day-old males that induce stronger CI compared to 5-day-old males that induce weak or no CI. In addition, Hira mutated D. melanogaster males mated to uninfected females result in significantly decreased hatch rates comparing with uninfected crosses. Interestingly, wMel-infected females may rescue the hatch rates. An obvious CI phenotype with chromatin bridges are observed in the early embryo resulting from Hira mutant fertilization, which strongly mimics the defects associated with CI. CONCLUSIONS/SIGNIFICANCE: Our results suggest Wolbachia-induced CI in Drosophila occurs due to a reduction in Hira expression in Wolbachia-infected males leading to detrimental effects on sperm fertility resulting in embryo lethality. These results may help determine the underlying mechanism of CI and provide further insight in to the important role

  1. Inhibition of lipopolysaccharide-induced inducible nitric oxide synthase and cyclooxygenase-2 expression by xanthanolides isolated from Xanthium strumarium.

    Science.gov (United States)

    Yoon, Jeong Hoon; Lim, Hyo Jin; Lee, Hwa Jin; Kim, Hee-Doo; Jeon, Raok; Ryu, Jae-Ha

    2008-03-15

    Three sesquiterpenoids, xanthatin (1), xanthinosin (2), and 4-oxo-bedfordia acid (3) were isolated from Xanthium strumarium as inhibitors of nitric oxide synthesis in activated microglia (IC(50) values: 0.47, 11.2, 136.5 microM, respectively). Compounds 1 and 2 suppressed the expression of iNOS and COX-2 and the activity of NF-kappaB through the inhibition of LPS-induced I-kappaB-alpha degradation in microglia.

  2. Bifidobacterium bifidum Actively Changes the Gene Expression Profile Induced by Lactobacillus acidophilus in Murine Dendritic Cells

    DEFF Research Database (Denmark)

    Weiss, Gudrun Margarethe; Rasmussen, Simon; Fink, Lisbeth Nielsen

    2010-01-01

    Dendritic cells (DC) play a pivotal regulatory role in activation of both the innate as well as the adaptive immune system by responding to environmental microorganisms. We have previously shown that Lactobacillus acidophilus induces a strong production of the pro-inflammatory and Th1 polarizing...... cytokine IL-12 in DC, whereas bifidobacteria do not induce IL-12 but inhibit the IL-12 production induced by lactobacilli. In the present study, genome-wide microarrays were used to investigate the gene expression pattern of murine DC stimulated with Lactobacillus acidophilus NCFM and Bifidobacterium...

  3. Hypoglycemia induced changes in cholinergic receptor expression in the cerebellum of diabetic rats

    Directory of Open Access Journals (Sweden)

    Anju TR

    2010-02-01

    Full Text Available Abstract Glucose homeostasis in humans is an important factor for the functioning of nervous system. Hypoglycemia and hyperglycemia is found to be associated with central and peripheral nerve system dysfunction. Changes in acetylcholine receptors have been implicated in the pathophysiology of many major diseases of the central nervous system (CNS. In the present study we showed the effects of insulin induced hypoglycemia and streptozotocin induced diabetes on the cerebellar cholinergic receptors, GLUT3 and muscle cholinergic activity. Results showed enhanced binding parameters and gene expression of Muscarinic M1, M3 receptor subtypes in cerebellum of diabetic (D and hypoglycemic group (D + IIH and C + IIH. α7nAchR gene expression showed a significant upregulation in diabetic group and showed further upregulated expression in both D + IIH and C + IIH group. AchE expression significantly upregulated in hypoglycemic and diabetic group. ChAT showed downregulation and GLUT3 expression showed a significant upregulation in D + IIH and C + IIH and diabetic group. AchE activity enhanced in the muscle of hypoglycemic and diabetic rats. Our studies demonstrated a functional disturbance in the neuronal glucose transporter GLUT3 in the cerebellum during insulin induced hypoglycemia in diabetic rats. Altered expression of muscarinic M1, M3 and α7nAchR and increased muscle AchE activity in hypoglycemic rats in cerebellum is suggested to cause cognitive and motor dysfunction. Hypoglycemia induced changes in ChAT and AchE gene expression is suggested to cause impaired acetycholine metabolism in the cerebellum. Cerebellar dysfunction is associated with seizure generation, motor deficits and memory impairment. The results shows that cerebellar cholinergic neurotransmission is impaired during hyperglycemia and hypoglycemia and the hypoglycemia is causing more prominent imbalance in cholinergic neurotransmission which is suggested to be a cause of cerebellar

  4. 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

  5. Microarray-based screening of differentially expressed genes in glucocorticoid-induced avascular necrosis

    Science.gov (United States)

    Huang, Gangyong; Wei, Yibing; Zhao, Guanglei; Xia, Jun; Wang, Siqun; Wu, Jianguo; Chen, Feiyan; Chen, Jie; Shi, Jingshen

    2017-01-01

    The underlying mechanisms of glucocorticoid (GC)-induced avascular necrosis of the femoral head (ANFH) have yet to be fully understood, in particular the mechanisms associated with the change of gene expression pattern. The present study aimed to identify key genes with a differential expression pattern in GC-induced ANFH. E-MEXP-2751 microarray data were downloaded from the ArrayExpress database. Differentially expressed genes (DEGs) were identified in 5 femoral head samples of steroid-induced ANFH rats compared with 5 placebo-treated rat samples. Gene Ontology (GO) and pathway enrichment analyses were performed upon these DEGs. A total 93 DEGs (46 upregulated and 47 downregulated genes) were identified in GC-induced ANFH samples. These DEGs were enriched in different GO terms and pathways, including chondrocyte differentiation and detection of chemical stimuli. The enrichment map revealed that skeletal system development was interconnected with several other GO terms by gene overlap. The literature mined network analysis revealed that 5 upregulated genes were associated with femoral necrosis, including parathyroid hormone receptor 1 (PTHR1), vitamin D (1,25-Dihydroxyvitamin D3) receptor (VDR), collagen, type II, α1, proprotein convertase subtilisin/kexin type 6 and zinc finger protein 354C (ZFP354C). In addition, ZFP354C and VDR were identified to transcription factors. Furthermore, PTHR1 was revealed to interact with VDR, and α-2-macroglobulin (A2M) interacted with fibronectin 1 (FN1) in the PPI network. PTHR1 may be involved in GC-induced ANFH via interacting with VDR. A2M may also be involved in the development of GC-induced ANFH through interacting with FN1. An improved understanding of the molecular mechanisms underlying GC-induced ANFH may provide novel targets for diagnostics and therapeutic treatment. PMID:28393228

  6. Microarray‑based screening of differentially expressed genes in glucocorticoid‑induced avascular necrosis.

    Science.gov (United States)

    Huang, Gangyong; Wei, Yibing; Zhao, Guanglei; Xia, Jun; Wang, Siqun; Wu, Jianguo; Chen, Feiyan; Chen, Jie; Shi, Jingshen

    2017-06-01

    The underlying mechanisms of glucocorticoid (GC)‑induced avascular necrosis of the femoral head (ANFH) have yet to be fully understood, in particular the mechanisms associated with the change of gene expression pattern. The present study aimed to identify key genes with a differential expression pattern in GC‑induced ANFH. E‑MEXP‑2751 microarray data were downloaded from the ArrayExpress database. Differentially expressed genes (DEGs) were identified in 5 femoral head samples of steroid‑induced ANFH rats compared with 5 placebo‑treated rat samples. Gene Ontology (GO) and pathway enrichment analyses were performed upon these DEGs. A total 93 DEGs (46 upregulated and 47 downregulated genes) were identified in GC‑induced ANFH samples. These DEGs were enriched in different GO terms and pathways, including chondrocyte differentiation and detection of chemical stimuli. The enrichment map revealed that skeletal system development was interconnected with several other GO terms by gene overlap. The literature mined network analysis revealed that 5 upregulated genes were associated with femoral necrosis, including parathyroid hormone receptor 1 (PTHR1), vitamin D (1,25‑Dihydroxyvitamin D3) receptor (VDR), collagen, type II, α1, proprotein convertase subtilisin/kexin type 6 and zinc finger protein 354C (ZFP354C). In addition, ZFP354C and VDR were identified to transcription factors. Furthermore, PTHR1 was revealed to interact with VDR, and α‑2‑macroglobulin (A2M) interacted with fibronectin 1 (FN1) in the PPI network. PTHR1 may be involved in GC‑induced ANFH via interacting with VDR. A2M may also be involved in the development of GC‑induced ANFH through interacting with FN1. An improved understanding of the molecular mechanisms underlying GC‑induced ANFH may provide novel targets for diagnostics and therapeutic treatment.

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

    International Nuclear Information System (INIS)

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

    2005-01-01

    Osteosarcoma is a common malignant bone tumor associated with childhood and adolescence. The results of numerous studies have suggested that hyaluronan plays an important role in regulating the aggressive behavior of various types of cancer cells. However, no studies have addressed hyaluronan with respect to osteosarcomas. In this investigation, the mRNA expression copy number of three mammalian hyaluronan synthases (HAS) was determined using competitive RT-PCR in the osteoblastic osteosarcoma cell line, MG-63. MG-63 are highly malignant osteosarcoma cells with an abundant hyaluronan-rich matrix. The results demonstrated that HAS-2 is the predominant HAS in MG-63. Accumulation of intracellular hyaluronan increased in association with the proliferative phase of these cells. The selective inhibition of HAS-2 mRNA in MG-63 cells by antisense phosphorothioate oligonucleotides resulted in reduced hyaluronan accumulation by these cells. As expected, the reduction in hyaluronan disrupted the assembly of cell-associated matrices. However, of most interest, coincident with the reduction in hyaluronan, there was a substantial decrease in cell proliferation, a decrease in cell motility and a decrease in cell invasiveness. These data suggest that hyaluronan synthesized by HAS-2 in MG-63 plays a crucial role in osteosarcoma cell proliferation, motility, and invasion

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

    Science.gov (United States)

    Dawid, Alexandre; Cayrol, Bastien; Isambert, Hervé

    2009-07-01

    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.

  9. Development of Multiexon Skipping Antisense Oligonucleotide Therapy for Duchenne Muscular Dystrophy

    Science.gov (United States)

    Yokota, Toshifumi; Wood, Matthew J. A.

    2013-01-01

    Duchenne muscular dystrophy (DMD) is an incurable, X-linked progressive muscle degenerative disorder that results from the absence of dystrophin protein and leads to premature death in affected individuals due to respiratory and/or cardiac failure typically by age of 30. Very recently the exciting prospect of an effective oligonucleotide therapy has emerged which restores dystrophin protein expression to affected tissues in DMD patients with highly promising data from a series of clinical trials. This therapeutic approach is highly mutation specific and thus is personalised. Therefore DMD has emerged as a model genetic disorder for understanding and overcoming of the challenges of developing personalised genetic medicines. One of the greatest weaknesses of the current oligonucleotide approach is that it is a mutation-specific therapy. To address this limitation, we have recently demonstrated that exons 45–55 skipping therapy has the potential to treat clusters of mutations that cause DMD, which could significantly reduce the number of compounds that would need to be developed in order to successfully treat all DMD patients. Here we discuss and review the latest preclinical work in this area as well as a variety of accompanying issues, including efficacy and potential toxicity of antisense oligonucleotides, prior to human clinical trials. PMID:23984357

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

    International Nuclear Information System (INIS)

    Dawid, Alexandre; Cayrol, Bastien; Isambert, Hervé

    2009-01-01

    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

  11. A long natural-antisense RNA is accumulated in the conidia of Aspergillus oryzae.

    Science.gov (United States)

    Tsujii, Masaru; Okuda, Satoshi; Ishi, Kazutomo; Madokoro, Kana; Takeuchi, Michio; Yamagata, Youhei

    2016-01-01

    Analysis of expressed sequence tag libraries from various culture conditions revealed the existence of conidia-specific transcripts assembled to putative conidiation-specific reductase gene (csrA) in Aspergillus oryzae. However, the all transcripts were transcribed with opposite direction to the gene csrA. The sequence analysis of the transcript revealed that the RNA overlapped mRNA of csrA with 3'-end, and did not code protein longer than 60 amino acid residues. We designated the transcript Conidia Specific Long Natural-antisense RNA (CSLNR). The real-time PCR analysis demonstrated that the CSLNR is conidia-specific transcript, which cannot be transcribed in the absence of brlA, and the amount of CSLNR was much more than that of the transcript from csrA in conidia. Furthermore, the csrA deletion, also lacking coding region of CSLNR in A. oryzae reduced the number of conidia. Overexpression of CsrA demonstrated the inhibition of growth and conidiation, while CSLNR did not affect conidiation.

  12. Wnt/β-catenin signaling changes C2C12 myoblast proliferation and differentiation by inducing Id3 expression

    International Nuclear Information System (INIS)

    Zhang, Long; Shi, Songting; Zhang, Juan; Zhou, Fangfang; Dijke, Peter ten

    2012-01-01

    Highlights: ► Expression of Id3 but not Id1 is induced by Wnt3a stimulation in C2C12 cells. ► Wnt3a induces Id3 expression via canonical Wnt/β-catenin pathway. ► Wnt3a-induced Id3 expression does not depend on BMP signaling activation. ► Induction of Id3 expression is critical determinant in Wnt3a-induced cell proliferation and differentiation. -- Abstract: Canonical Wnt signaling plays important roles in regulating cell proliferation and differentiation. In this study, we report that inhibitor of differentiation (Id)3 is a Wnt-inducible gene in mouse C2C12 myoblasts. Wnt3a induced Id3 expression in a β-catenin-dependent manner. Bone morphogenetic protein (BMP) also potently induced Id3 expression. However, Wnt-induced Id3 expression occurred independent of the BMP/Smad pathway. Functional studies showed that Id3 depletion in C2C12 cells impaired Wnt3a-induced cell proliferation and alkaline phosphatase activity, an early marker of osteoblast cells. Id3 depletion elevated myogenin induction during myogenic differentiation and partially impaired Wnt3a suppressed myogenin expression in C2C12 cells. These results suggest that Id3 is an important Wnt/β-catenin induced gene in myoblast cell fate determination.

  13. Expressions of matrix metalloproteinase-2 and extracellular matrix metalloproteinase inducer in retinoblastoma

    Directory of Open Access Journals (Sweden)

    Yu-Hong Cheng

    2015-07-01

    Full Text Available AIM: To investigate expressions of matrix metalloproteinase-2(MMP-2and extracellular matrix metalloproteinase inducer(EMMPRINin retinoblastoma(Rband the relationships between MMP-2, EMMPRIN and tumor development.METHODS:Immunohistochemical technique was used to detect expressions of MMP-2 and EMMPRIN in 39 cases of paraffin embedded Rb samples. Quantitative analysis of expressions of MMP-2 and EMMPRIN were assessed by measuring the mean gray scale of Rb tissue with LEICA IM50 Color Pathologic Analysis System. The differences of expressions of MMP-2 and EMMPRIN in each clinical and pathological stage were statistically analyzed, and the same step was also undertaken to study the relationship between Rb with MMP-2 positive expression and that with EMMPRIN positive expression.RESULTS: The positive expression rate of MMP-2 was 90%(Gray value: 109.64±14.52; 35/39, and that of EMMPRIN was 85%(Gray value: 108.01±13.60; 33/39. The expressions of MMP-2 and EMMPRIN were significantly higher in tumors of glaucomatous stage(Gray value: 108.21±11.47 and 107.56±14.32than those in intraocular stage(Gray value: 121.13±11.32 and 119.34±12.66; PPPPPPCONCLUSION: The positive expression levels of MMP-2 and EMMPRIN may correlate with tumor infiltration and metastasis.

  14. Ozone-induced gene expression occurs via ethylene-dependent and -independent signalling.

    Science.gov (United States)

    Grimmig, Bernhard; Gonzalez-Perez, Maria N; Leubner-Metzger, Gerhard; Vögeli-Lange, Regina; Meins, Fred; Hain, Rüdiger; Penuelas, Josep; Heidenreich, Bernd; Langebartels, Christian; Ernst, Dieter; Sandermann, Heinrich

    2003-03-01

    Recent studies suggest that ethylene is involved in signalling ozone-induced gene expression. We show here that application of ozone increased glucuronidase (GUS) expression of chimeric reporter genes regulated by the promoters of the tobacco class I beta-1,3-glucanases (GLB and Gln2) and the grapevine resveratrol synthase (Vst1) genes in transgenic tobacco leaves. 5'-deletion analysis of the class I beta-1,3-glucanase promoter revealed that ozone-induced gene regulation is mainly mediated by the distal enhancer region containing the positively acting ethylene-responsive element (ERE). In addition, application of 1-methylcyclopropene (1-MCP), an inhibitor of ethylene action, blocked ozone-induced class I beta-1,3-glucanase promoter activity. Enhancer activity and ethylene-responsiveness depended on the integrity of the GCC boxes, cis-acting elements present in the ERE of the class I beta-1,3-glucanase and the basic-type pathogenesis-related PR-1 protein (PRB-1b) gene promoters. The minimal PRB-1b promoter containing only the ERE with intact GCC boxes, was sufficient to confer 10-fold ozone inducibility to a GUS-reporter gene, while a substitution mutation in the GCC box abolished ozone responsiveness. The ERE region of the class I beta-1,3-glucanase promoter containing two intact GCC boxes confered strong ozone inducibility to a minimal cauliflower mosaic virus (CaMV) 35S RNA promoter, whereas two single-base substitution in the GCC boxes resulted in a complete loss of ozone inducibility. Taken together, these datastrongly suggest that ethylene is signalling ozone-induced expression of class I beta-l,3-glucanase and PRB-1b genes. Promoter analysis of the stilbene synthase Vst1 gene unravelled different regions for ozone and ethylene-responsiveness. Application of 1-MCP blocked ethylene-induced Vst1 induction, but ozone induction was not affected. This shows that ozone-induced gene expression occurs via at least two different signalling mechanisms and suggests an

  15. Molecular imaging of atherosclerotic plaques with technetium-99m-labelled antisense oligonucleotides

    International Nuclear Information System (INIS)

    Qin Guangming; Zhang Yongxue; Cao Wei; An Rui; Gao Zairong; Xu Wendai; Zhang Kaijun; Li Guiling; Li Shuren

    2005-01-01

    The purpose of this study was to visualise experimental atherosclerotic lesions using radiolabelled antisense oligonucleotides (ASONs). Atherosclerosis was induced in New Zealand White rabbits fed 1% cholesterol for approximately 60 days. In vivo and ex vivo imaging was performed in atherosclerotic rabbits and normal control rabbits after i.v. injection of 92.5±18.5 MBq 99m Tc-labelled ASON or 99m Tc-labelled sense oligonucleotides. Immediately after the in vivo imaging, the animals were sacrificed and ex vivo imaging of the aortic specimens was performed. Biodistribution of radiolabelled c-mycASON was evaluated in vivo in atherosclerotic rabbits. Planar imaging revealed accumulation of 99m Tc-labelled c-mycASON in atherosclerotic lesions along the artery wall. Ex vivo imaging further demonstrated that the area of activity accumulation matched the area of atherosclerotic lesions. In contrast, no atherosclerotic lesions were found in the vessel wall and no positive imaging results were obtained in animals of the control group. This molecular imaging approach has potential for non-invasive imaging of atherosclerotic plaques at an early stage. (orig.)

  16. Severe hypoxia induces chemo-resistance in clinical cervical tumors through MVP over-expression.

    Science.gov (United States)

    Lara, Pedro C; Lloret, Marta; Clavo, Bernardino; Apolinario, Rosa M; Henríquez-Hernández, Luis Alberto; Bordón, Elisa; Fontes, Fausto; Rey, Agustín

    2009-08-06

    Oxygen molecule modulates tumour response to radiotherapy. Higher radiation doses are required under hypoxic conditions to induce cell death. Hypoxia may inhibit the non-homologous end-joining DNA repair through down regulating Ku70/80 expression. Hypoxia induces drug resistance in clinical tumours, although the mechanism is not clearly elucidated. Vaults are ribonucleoprotein particles with a hollow barrel-like structure composed of three proteins: major vault protein (MVP), vault poly(ADP-ribose) polymerase, and telomerase associated protein-1 and small untranslated RNA. Over-expression of MVP has been associated with chemotherapy resistance. Also, it has been related to poor outcome in patients treated with radiotherapy alone. The aim of the present study was to assess the relation of Major Vault Protein expression and tumor hypoxia in clinical cervical tumors. MVP, p53 and angiogenesis, together with tumor oxygenation, were determined in forty-three consecutive patients suffering from localized cervix carcinoma. High MVP expression was related to severe hypoxia compared to low MVP expressing tumors (p = 0.022). Tumors over-expressing MVP also showed increased angiogenesis (p = 0.003). Besides it, in this study we show for the first time that severe tumor hypoxia is associated with high MVP expression in clinical cervical tumors. Up-regulation of MVP by hypoxia is of critical relevance as chemotherapy is currently a standard treatment for those patients. From our results it could be suggested that hypoxia not only induces increased genetic instability, oncogenic properties and metastatization, but through the correlation observed with MVP expression, another pathway of chemo and radiation resistance could be developed.

  17. Severe hypoxia induces chemo-resistance in clinical cervical tumors through MVP over-expression

    International Nuclear Information System (INIS)

    Lara, Pedro C; Lloret, Marta; Clavo, Bernardino; Apolinario, Rosa M; Henríquez-Hernández, Luis Alberto; Bordón, Elisa; Fontes, Fausto; Rey, Agustín

    2009-01-01

    Oxygen molecule modulates tumour response to radiotherapy. Higher radiation doses are required under hypoxic conditions to induce cell death. Hypoxia may inhibit the non-homologous end-joining DNA repair through down regulating Ku70/80 expression. Hypoxia induces drug resistance in clinical tumours, although the mechanism is not clearly elucidated. Vaults are ribonucleoprotein particles with a hollow barrel-like structure composed of three proteins: major vault protein (MVP), vault poly(ADP-ribose) polymerase, and telomerase associated protein-1 and small untranslated RNA. Over-expression of MVP has been associated with chemotherapy resistance. Also, it has been related to poor outcome in patients treated with radiotherapy alone. The aim of the present study was to assess the relation of Major Vault Protein expression and tumor hypoxia in clinical cervical tumors. MVP, p53 and angiogenesis, together with tumor oxygenation, were determined in forty-three consecutive patients suffering from localized cervix carcinoma. High MVP expression was related to severe hypoxia compared to low MVP expressing tumors (p = 0.022). Tumors over-expressing MVP also showed increased angiogenesis (p = 0.003). Besides it, in this study we show for the first time that severe tumor hypoxia is associated with high MVP expression in clinical cervical tumors. Up-regulation of MVP by hypoxia is of critical relevance as chemotherapy is currently a standard treatment for those patients. From our results it could be suggested that hypoxia not only induces increased genetic instability, oncogenic properties and metastatization, but through the correlation observed with MVP expression, another pathway of chemo and radiation resistance could be developed

  18. Heme-Oxygenase-1 Expression Contributes to the Immunoregulation Induced by Fasciola hepatica and Promotes Infection

    Directory of Open Access Journals (Sweden)

    Paula Carasi

    2017-07-01

    Full Text Available Fasciola hepatica, also known as the liver fluke, is a trematode that infects livestock and humans causing fasciolosis, a zoonotic disease of increasing importance due to its worldwide distribution and high economic losses. This parasite immunoregulates the host immune system by inducing a strong Th2 and regulatory T immune response by immunomodulating dendritic cell (DC maturation and alternative activation of macrophages. In this paper, we show that F. hepatica infection in mice induces the upregulation of heme-oxygenase-1 (HO-1, the rate-limiting enzyme in the catabolism of free heme that regulates the host inflammatory response. We show and characterize two different populations of antigen presenting cells that express HO-1 during infection in the peritoneum of infected animals. Cells that expressed high levels of HO-1 expressed intermediate levels of F4/80 but high expression of CD11c, CD38, TGFβ, and IL-10 suggesting that they correspond to regulatory DCs. On the other hand, cells expressing intermediate levels of HO-1 expressed high levels of F4/80, CD68, Ly6C, and FIZZ-1, indicating that they might correspond to alternatively activated macrophages. Furthermore, the pharmacological induction of HO-1 with the synthetic metalloporphyrin CoPP promoted F. hepatica infection increasing the clinical signs associated with the disease. In contrast, treatment with the HO-1 inhibitor SnPP protected mice from parasite infection, indicating that HO-1 plays an essential role during F. hepatica infection. Finally, HO-1 expression during F. hepatica infection was associated with TGFβ and IL-10 levels in liver and peritoneum, suggesting that HO-1 controls the expression of these immunoregulatory cytokines during infection favoring parasite survival in the host. These results contribute to the elucidation of the immunoregulatory mechanisms induced by F. hepatica in the host and provide alternative checkpoints to control fasciolosis.

  19. HEAT INDUCIBLE EXPRESSION OF ANTIFREEZE PROTEIN GENES FROM THE BEETLES Tenebrio molitor AND Microdera punctipennis.

    Science.gov (United States)

    Li, Jieqiong; Ma, Wenjing; Ma, Ji

    2016-01-01

    Antifreeze proteins (AFPs) play important roles in protecting poikilothermic organisms from cold damage. The expression of AFP genes (afps) is induced by low temperature. However, it is reported that heat can influence the expression of afps in the desert beetle Microdera punctipennis. To further detect whether heat also induce the expression of afps in other insects, and to determine the expression profiling of insect afps at different temperatures. The expression of antifreeze protein genes in the two beetles, Microdera punctipennis and Tenebrio molitor that have quite different living environment, under different temperatures were studied by using real-time quantitative PCR. Mild low temperatures (5~15 degree C), high temperature (38~47 degree C for M. punctipennis, or 37~42 degree C for T. molitor) and temperature difference (10~30 degree C) all stimulated strongly to the expression of AFP genes (Mpafps) in M. punctipennis which lives in the wild filed in desert. The mRNA level of Mpafps after M. punctipennis were exposed to these temperatures for 1h~5h was at least 30-fold of the control at 25 degree C. For T. molitor which is breeding in door with wheat bran all these temperatures stimulated significantly to the expression of Tmafps, while the extent and degree of the temperature stimulation on Tmafps expression were much lower than on Mpafps. After T. molitor were exposed to 5 degree C and 15 degree C for 1h~5h, the mRNA level of Tmafps was over 6-fold and 45-fold of the control at 25 degree C. High temperature (37~42 degree C) for 1h~3h treatments increased Tmafps mRNA level 4.8-fold of the control. Temperature difference of 10 degree C was effective in stimulating Tmafps expression. The expression of insect antifreeze protein genes both in M. punctipennis and T. molitor was induced by heat, suggesting that this phenomenon may be common in insects; the extent and degree of the influence differ in species that have different living conditions. The heat

  20. Low density lipoprotein induces upregulation of vasoconstrictive endothelin type B receptor expression

    DEFF Research Database (Denmark)

    Xu, Cang-Bao; Zheng, Jian-Pu; Zhang, Wei

    2014-01-01

    Vasoconstrictive endothelin type B (ET(B)) receptors promote vasospasm and ischemic cerebro- and cardiovascular diseases. The present study was designed to examine if low density lipoprotein (LDL) induces upregulation of vasoconstrictive ET(B) receptor expression and if extracellular signal...

  1. Pregnancy-induced gene expression changes in vivo among women with rheumatoid arthritis

    DEFF Research Database (Denmark)

    Goin, Dana E; Smed, Mette Kiel; Pachter, Lior

    2017-01-01

    BACKGROUND: Little is known about gene expression changes induced by pregnancy in women with rheumatoid arthritis (RA) and healthy women because the few studies previously conducted did not have pre-pregnancy samples available as baseline. We have established a cohort of women with RA and healthy...

  2. Elicitor and fusarium-induced expression of NPR-1 like genes in banana

    CSIR Research Space (South Africa)

    Endah, R

    2008-11-01

    Full Text Available NPR1 is an essential positive regulator of salicylic acid-induced PR gene expression and systemic acquired resistance. Two novel full-length NPR1-like genes; MNPR1A and MNPR1B, were isolated by application of the PCR and RACE techniques. The two...

  3. Heme oxygenase-1 expression protects melanocytes from stress-induced cell death: implications for vitiligo

    NARCIS (Netherlands)

    Elassiuty, Yasser E.; Klarquist, Jared; Speiser, Jodi; Yousef, Randa M.; El Refaee, Abdelaziz A.; Hunter, Nahla S.; Shaker, Olfat G.; Gundeti, Mohan; Nieuweboer-Krobotova, Ludmila; Caroline Le Poole, I.

    2011-01-01

    To study protection of melanocytes from stress-induced cell death by heme oxygenases during depigmentation and repigmentation in vitiligo, expression of isoforms 1 and 2 was studied in cultured control and patient melanocytes and normal skin explants exposed to UV or bleaching agent 4-TBP.

  4. Increased hepatic CD36 expression contributes to dyslipidemia associated with diet-induced obesity

    Science.gov (United States)

    The etiology of type 2 diabetes often involves diet-induced obesity (DIO), which is associated with elevated plasma fatty acids and lipoprotein associated triglycerides. Since aberrant hepatic fatty acid uptake may contribute to this, we investigated whether increased expression of a fatty acid tran...

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

    Directory of Open Access Journals (Sweden)

    John Benedict Readman

    2016-03-01

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

  6. Epidermal growth factor receptor expression in radiation-induced dog lung tumors by immunocytochemical localization

    Energy Technology Data Exchange (ETDEWEB)

    Leung, F.L.; Park, J.F.; Dagle, G.E.

    1993-06-01

    In studies to determine the role of growth factors in radiation-induced lung cancer, epidermal growth factor (EGFR) expression was examined by immunocytochemistry in 51 lung tumors from beagle dogs exposed to inhaled plutonium; 21 of 51 (41%) tumors were positive for EGFR. The traction of tumors positive for EGFR and the histological type of EGFR-positive tumors in the plutonium-exposed dogs were not different from spontaneous dog lung tumors, In which 36% were positive for EGFR. EGFR involvement in Pu-induced lung tumors appeared to be similar to that in spontaneous lung tumors. However, EGFR-positive staining was observed in only 1 of 16 tumors at the three lowest Pu exposure levels, compared to 20 of 35 tumors staining positive at the two highest Pu exposure levels. The results in dogs were in good agreement with the expression of EGFR reported in human non-small cell carcinoma of the lung, suggesting that Pu-induced lung tumors in the dog may be a suitable animal model to investigate the role of EGFR expression in lung carcinogenesis. In humans, EGFR expression in lung tumors has been primarily related to histological tumor types. In individual dogs with multiple primary lung tumors, the tumors were either all EGFR positive or EGFR negative, suggesting that EGFR expression may be related to the response of the individual dog as well as to the histological type of tumor.

  7. Drug-loaded nanoparticles induce gene expression in human pluripotent stem cell derivatives

    Science.gov (United States)

    Gajbhiye, Virendra; Escalante, Leah; Chen, Guojun; Laperle, Alex; Zheng, Qifeng; Steyer, Benjamin; Gong, Shaoqin; Saha, Krishanu

    2013-12-01

    Tissue engineering and advanced manufacturing of human stem cells requires a suite of tools to control gene expression spatiotemporally in culture. Inducible gene expression systems offer cell-extrinsic control, typically through addition of small molecules, but small molecule inducers typically contain few functional groups for further chemical modification. Doxycycline (DXC), a potent small molecule inducer of tetracycline (Tet) transgene systems, was conjugated to a hyperbranched dendritic polymer (Boltorn H40) and subsequently reacted with polyethylene glycol (PEG). The resulting PEG-H40-DXC nanoparticle exhibited pH-sensitive drug release behavior and successfully controlled gene expression in stem-cell-derived fibroblasts with a Tet-On system. While free DXC inhibited fibroblast proliferation and matrix metalloproteinase (MMP) activity, PEG-H40-DXC nanoparticles maintained higher fibroblast proliferation levels and MMP activity. The results demonstrate that the PEG-H40-DXC nanoparticle system provides an effective tool to controlling gene expression in human stem cell derivatives.Tissue engineering and advanced manufacturing of human stem cells requires a suite of tools to control gene expression spatiotemporally in culture. Inducible gene expression systems offer cell-extrinsic control, typically through addition of small molecules, but small molecule inducers typically contain few functional groups for further chemical modification. Doxycycline (DXC), a potent small molecule inducer of tetracycline (Tet) transgene systems, was conjugated to a hyperbranched dendritic polymer (Boltorn H40) and subsequently reacted with polyethylene glycol (PEG). The resulting PEG-H40-DXC nanoparticle exhibited pH-sensitive drug release behavior and successfully controlled gene expression in stem-cell-derived fibroblasts with a Tet-On system. While free DXC inhibited fibroblast proliferation and matrix metalloproteinase (MMP) activity, PEG-H40-DXC nanoparticles maintained

  8. Arsenite enhances tumor necrosis factor-α-induced expression of vascular cell adhesion molecule-1

    International Nuclear Information System (INIS)

    Tsou, T.-C.; Yeh, Szu Ching; Tsai, E.-M.; Tsai, F.-Y.; Chao, H.-R.; Chang, Louis W.

    2005-01-01

    Epidemiological studies demonstrated a high association of vascular diseases with arsenite exposure. We hypothesize that arsenite potentiates the effect of proinflammatory cytokines on vascular endothelial cells, and hence contributes to atherosclerosis. In this study, we investigated the effect of arsenite and its induction of glutathione (GSH) on vascular cell adhesion molecule-1 (VCAM-1) protein expression in human umbilical vein endothelial cells (HUVECs) in response to tumor necrosis factor-α (TNF-α), a typical proinflammatory cytokine. Our study demonstrated that arsenite pretreatment potentiated the TNF-α-induced VCAM-1 expression with up-regulations of both activator protein-1 (AP-1) and nuclear factor-κB (NF-κB). To elucidate the role of GSH in regulation of AP-1, NF-κB, and VCAM-1 expression, we employed L-buthionine (S,R)-sulfoximine (BSO), a specific γ-glutamylcysteine synthetase (γ-GCS) inhibitor, to block intracellular GSH synthesis. Our investigation revealed that, by depleting GSH, arsenite attenuated the TNF-α-induced VCAM-1 expression as well as a potentiation of AP-1 and an attenuation of NF-κB activations by TNF-α. Moreover, we found that depletion of GSH would also attenuate the TNF-α-induced VCAM-1 expression with a down-regulation of the TNF-α-induced NF-κB activation and without significant effect on AP-1. On the other hand, the TNF-α-induced VCAM-1 expression could be completely abolished by inhibition of AP-1 or NF-κB activity, suggesting that activation of both AP-1 and NF-κB was necessary for VCAM-1 expression. In summary, we demonstrate that arsenite enhances the TNF-α-induced VCAM-1 expression in HUVECs via regulation of AP-1 and NF-κB activities in a GSH-sensitive manner. Our present study suggested a potential mechanism for arsenite in the induction of vascular inflammation and vascular diseases via modulating the actions of proinflammatory cytokines

  9. GATA-dependent regulation of TPO-induced c-mpl gene expression during megakaryopoiesis.

    Science.gov (United States)

    Sunohara, Masataka; Morikawa, Shigeru; Fuse, Akira; Sato, Iwao

    2014-01-01

    Thrombopoietin (TPO) and its receptor, c-Mpl, play the crucial role during megakaryocytopoiesis. Previously, we have shown that the promoter activity of c-mpl induced by TPO is modulated by transcription through a PKC-dependent pathway and that GATA(-77) is involved as a positive regulatory element in TPO-induced c-mpl gene expression in the megakaryoblastic CMK cells. In this research, to examine participating possibility of GATA promoter element in TPO- induced c-mpl gene expression through a PKC-independent pathway, the promoter activity of site-directed mutagenesis and the effect of potein kinase C modulator were measured by a transient transfection assay system. Together with our previous results on the TPO-induced c-mpl promoter, this study indicates destruction of -77GATA in c-mpl promoter decreased the activity by 47.3% under existence of GF109203. These results suggest that GATA promoter element plays significant role in TPO-induced c-mpl gene expression through a PKC-independent pathway.

  10. Antisense Phosphorodiamidate Morpholino Oligomers as Novel Antiviral Compounds

    Directory of Open Access Journals (Sweden)

    Yuchen Nan

    2018-04-01

    Full Text Available Phosphorodiamidate morpholino oligomers (PMO are short single-stranded DNA analogs that are built upon a backbone of morpholine rings connected by phosphorodiamidate linkages. As uncharged nucleic acid analogs, PMO bind to complementary sequences of target mRNA by Watson–Crick base pairing to block protein translation through steric blockade. PMO interference of viral protein translation operates independently of RNase H. Meanwhile, PMO are resistant to a variety of enzymes present in biologic fluids, a characteristic that makes them highly suitable for in vivo applications. Notably, PMO-based therapy for Duchenne muscular dystrophy (DMD has been approved by the United States Food and Drug Administration which is now a hallmark for PMO-based antisense therapy. In this review, the development history of PMO, delivery methods for improving cellular uptake of neutrally charged PMO molecules, past studies of PMO antagonism against RNA and DNA viruses, PMO target selection, and remaining questions of PMO antiviral strategies are discussed in detail and new insights are provided.

  11. [Expression of various matrix metalloproteinases in mice with hyperoxia-induced acute lung injury].

    Science.gov (United States)

    Zhang, Xiang-feng; Ding, Shao-fang; Gao, Yuan-ming; Liang, Ying; Foda, Hussein D

    2006-08-01

    To investigate the role of matrix metalloproteinases (MMPs) and extracellular matrix metalloproteinase inducer (EMMPRIN) in the pathogenesis of acute lung injury induced by hyperoxia. Fifty four mice were exposed in sealed cages to >98% oxygen (for 24-72 hours), and another 18 mice to room air. The severity of lung injury was assessed, and the expression of mRNA and protein of MMP-2, MMP-9 and EMMPRIN in lung tissue, after exposure for 24, 48 and 72 hours of hyperoxia were studied by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. Hyperoxia caused acute lung injury; this was accompanied by increased expression of an upregulation of MMP-2, MMP-9 and EMMPRIN mRNA and protein in lung tissues. Hyperoxia causes acute lung injury in mice; increases in MMP-2, MMP-9 and EMMPRIN may play an important role in the development of hyperoxia induced lung injury in mice.

  12. The molecular mechanism of leptin secretion and expression induced by aristolochic acid in kidney fibroblast.

    Directory of Open Access Journals (Sweden)

    Tsung-Chieh Lin

    Full Text Available BACKGROUND: Leptin is a peptide hormone playing pivotal role in regulating food intake and energy expenditure. Growing evidence has suggested the pro-inflammatory and fibrogenic properties of leptin. In addition, patients with renal fibrosis have higher level of plasma leptin, which was due to the increased leptin production. Aristolochic acid (AA is a botanical toxin characterized to associate with the development of renal fibrosis including tubulointerstitial fibrosis. However, whether leptin is upregulated to participate in AA-induced kidney fibrosis remain completely unknown. METHODOLOGY/PRINCIPAL FINDINGS: In this study, leptin expression was increased by sublethal dose of AA in kidney fibroblast NRK49f determined by enzyme-linked immunosorbent assay and Western blot. Data from real-time reverse transcriptase-polymerase chain reaction revealed that leptin was upregulated by AA at transcriptional level. DNA binding activity of CCAAT enhancer binding protein α (C/EBP α, one of the transcription factors for leptin gene, was enhanced in DNA affinity precipitation assay and chromatin immunoprecipitation experiments. Knockdown of C/EBP α expression by small interfering RNA markedly reduced AA-induced leptin expression. Moreover, AA promoted Akt interaction with p-PDK1, and increased phosphorylated activation of Akt. Akt knockdown, and inhibition of Akt signaling by LY294002 and mTOR inhibitor rapamycin reduced leptin expression. Furthermore, treatment of LY294002 or rapamycin significantly suppressed AA-induced C/EBP α DNA-binding activity. These results suggest that Akt and C/EBP α activation were involved in AA-regulated leptin expression. CONCLUSIONS/SIGNIFICANCE: Our findings demonstrate the first that AA could induce secretion and expression of fibrogenic leptin in kidney fibroblasts, which reveal potential involvement of leptin in the progression of kidney fibrosis in aristolochic acid nephropathy.

  13. Cannabidivarin (CBDV suppresses pentylenetetrazole (PTZ-induced increases in epilepsy-related gene expression

    Directory of Open Access Journals (Sweden)

    Naoki Amada

    2013-11-01

    Full Text Available To date, anticonvulsant effects of the plant cannabinoid, cannabidivarin (CBDV, have been reported in several animal models of seizure. However, these behaviourally observed anticonvulsant effects have not been confirmed at the molecular level. To examine changes to epilepsy-related gene expression following chemical convulsant treatment and their subsequent control by phytocannabinoid administration, we behaviourally evaluated effects of CBDV (400 mg/kg, p.o. on acute, pentylenetetrazole (PTZ: 95 mg/kg, i.p.-induced seizures, quantified expression levels of several epilepsy-related genes (Fos, Casp 3, Ccl3, Ccl4, Npy, Arc, Penk, Camk2a, Bdnf and Egr1 by qPCR using hippocampal, neocortical and prefrontal cortical tissue samples before examining correlations between expression changes and seizure severity. PTZ treatment alone produced generalised seizures (median: 5.00 and significantly increased expression of Fos, Egr1, Arc, Ccl4 and Bdnf. Consistent with previous findings, CBDV significantly decreased PTZ-induced seizure severity (median: 3.25 and increased latency to the first sign of seizure. Furthermore, there were correlations between reductions of seizure severity and mRNA expression of Fos, Egr1, Arc, Ccl4 and Bdnf in the majority of brain regions in the CBDV+PTZ treated group. When CBDV treated animals were grouped into CBDV responders (criterion: seizure severity ≤3.25 and non-responders (criterion: seizure severity >3.25, PTZ-induced increases of Fos, Egr1, Arc, Ccl4 and Bdnf expression were suppressed in CBDV responders. These results provide the first molecular confirmation of behaviourally observed effects of the non-psychoactive, anticonvulsant cannabinoid, CBDV, upon chemically-induced seizures and serve to underscore its suitability for clinical development.

  14. RpoS induces expression of the Vibrio anguillarum quorum-sensing regulator VanT.

    Science.gov (United States)

    Weber, Barbara; Croxatto, Antony; Chen, Chang; Milton, Debra L

    2008-03-01

    In vibrios, regulation of the Vibrio harveyi-like LuxR transcriptional activators occurs post-transcriptionally via small regulatory RNAs (sRNAs) that destabilize the luxR mRNA at a low cell population, eliminating expression of LuxR. Expression of the sRNAs is modulated by the vibrio quorum-sensing phosphorelay systems. However, vanT mRNA, which encodes a LuxR homologue in Vibrio anguillarum, is abundant at low and high cell density, indicating that VanT expression may be regulated via additional mechanisms. In this study, Western analyses showed that VanT was expressed throughout growth with a peak of expression during late exponential growth. VanO induced partial destabilization of vanT mRNA via activation of at least one Qrr sRNA. Interestingly, the sigma factor RpoS significantly stabilized vanT mRNA and induced VanT expression during late exponential growth. This induction was in part due to RpoS repressing expression of Hfq, an RNA chaperone. RpoS is not part of the quorum-sensing regulatory cascade since RpoS did not regulate expression or activity of VanO, and RpoS was not regulated by VanO or VanT. VanT and RpoS were needed for survival following UV irradiation and for pigment and metalloprotease production, suggesting that RpoS works with the quorum-sensing systems to modulate expression of VanT, which regulates survival and stress responses.

  15. Hypoxia induced expression of endogenous markers in vitro is highly influenced by pH

    International Nuclear Information System (INIS)

    Sorensen, Brita Singers; Alsner, Jan; Overgaard, Jens; Horsman, Michael R.

    2007-01-01

    Background: Genes such as carbonic anhydrase IX (Ca9), glucose transporter 1 (Glut1), lactate dehydrogenase A (LDH-A), osteopontin (OPN) and lysyl oxidase (LOX) have been suggested as hypoxic markers, but inconsistent results suggest that factors other than oxygen influence their expression. The current study is a detailed investigation using a range of pH values from 6.3 to 7.5 in two human cell lines to establish the pH dependency of hypoxia induced gene expression. Methods: Human tumour cell lines (uterine cervix squamous cell carcinoma (SiHa) and pharyngeal squamous cell carcinoma [FaDu DD ]) were used. Hypoxia was induced by gassing cells in airtight chambers with various oxygen concentrations (21%, 1%, 0.1%, 0.01% and 0%) for up to 24 h. The media were titrated to a range of pH values (7.5, 7.0, 6.7, 6.5 and 6.3). Gene expression was determined by real-time PCR. Results: In both SiHa and FaDu DD cells Ca9 and LOX reached the highest level of expression at 1% oxygen. In FaDu DD cells, a pH of 6.5 had a medium suppression effect on the hypoxia induced expression of Ca9. pH 6.3 resulted in severe suppression of expression for Ca9 and LOX in both SiHa and FaDu DD . Glut1 and LDH-A had a similar expression pattern to each other, with a maximum expression at 0.01% oxygen, in both cell lines. For these genes pH 6.5 and 6.3 changed the expression pattern in SiHa cells. OPN was up regulated at low oxygen in SiHa cells, but was not induced by hypoxia in FaDu DD cells. Conclusion: As tumour hypoxia occurs in a deprived microenvironment, other environmental factors, for example low pH, might interact with the effect of low oxygen concentration on gene expression. This study shows that pH in two cell lines has a profound influence on the oxygen dependent induction of certain endogenous hypoxic markers

  16. Intermedilysin induces EGR-1 expression through calcineurin/NFAT pathway in human cholangiocellular carcinoma cells

    International Nuclear Information System (INIS)

    Susilowati, Heni; Okamura, Hirohiko; Hirota, Katsuhiko; Shono, Masayuki; Yoshida, Kaya; Murakami, Keiji; Tabata, Atsushi; Nagamune, Hideaki; Haneji, Tatsuji; Miyake, Yoichiro

    2011-01-01

    Research highlights: → ILY leads to the accumulation of [Ca 2+ ]i in the nucleus in HuCCT1 cells. → ILY induced activation of NFAT1 through a calcineurin-dependent pathway. → Calcineuri/NFAT pathway is involved in EGR-1 expression in response to ILY treatment. -- Abstract: Intermedilysin (ILY) is a cholesterol-dependent cytolysin produced by Streptococcus intermedius, which is associated with human brain and liver abscesses. Although intrahepatic bile duct cells play a valuable role in the pathogenesis of liver abscess, the molecular mechanism of ILY-treated intrahepatic bile duct cells remains unknown. In this study, we report that ILY induced a nuclear accumulation of intracellular calcium ([Ca 2+ ]i) in human cholangiocellular cells HuCCT1. We also demonstrate that 10 ng/ml ILY induced NFAT1 dephosphorylation and its nuclear translocation in HuCCT1 cells. In contrast to the result that ILY induced NF-κB translocation in human hepatic HepG2 cells, ILY did not affect NF-κB localization in HuCCT1 cells. Dephosphorylation and nuclear translocation of NFAT1 caused by ILY were prevented by [Ca 2+ ]i calcium chelator, BAPTA/AM, and calcineurin inhibitors, cyclosporine A and tacrolimus. ILY induced early growth response-1 (EGR-1) expression and it was inhibited by the pre-treatment with cyclosporine A, indicating that the calcineurin/NFAT pathway was involved in EGR-1 expression in response to ILY. ILY-induced calcineurin/NFAT1 activation and sequential EGR-1 expression might be related to the pathogenesis of S. intermedius in human bile duct cells.

  17. Intermedilysin induces EGR-1 expression through calcineurin/NFAT pathway in human cholangiocellular carcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Susilowati, Heni [Department of Oral Microbiology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima 770-8504 (Japan); Okamura, Hirohiko [Department of Histology and Oral Histology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima 770-8504 (Japan); Hirota, Katsuhiko, E-mail: hirota@dent.tokushima-u.ac.jp [Department of Oral Microbiology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima 770-8504 (Japan); Shono, Masayuki [Support Center for Advanced Medical Sciences, The University of Tokushima, Tokushima 770-8504 (Japan); Yoshida, Kaya [Department of Fundamental Oral Health Science, School of Oral Health and Welfare, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima 770-8504 (Japan); Murakami, Keiji [Department of Oral Microbiology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima 770-8504 (Japan); Tabata, Atsushi; Nagamune, Hideaki [Department of Biological Science and Technology, Life System, Institute of Technology and Science, The University of Tokushima Graduate School, Tokushima 770-8506 (Japan); Haneji, Tatsuji [Department of Histology and Oral Histology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima 770-8504 (Japan); Miyake, Yoichiro [Department of Oral Microbiology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima 770-8504 (Japan)

    2011-01-07

    Research highlights: {yields} ILY leads to the accumulation of [Ca{sup 2+}]i in the nucleus in HuCCT1 cells. {yields} ILY induced activation of NFAT1 through a calcineurin-dependent pathway. {yields} Calcineuri/NFAT pathway is involved in EGR-1 expression in response to ILY treatment. -- Abstract: Intermedilysin (ILY) is a cholesterol-dependent cytolysin produced by Streptococcus intermedius, which is associated with human brain and liver abscesses. Although intrahepatic bile duct cells play a valuable role in the pathogenesis of liver abscess, the molecular mechanism of ILY-treated intrahepatic bile duct cells remains unknown. In this study, we report that ILY induced a nuclear accumulation of intracellular calcium ([Ca{sup 2+}]i) in human cholangiocellular cells HuCCT1. We also demonstrate that 10 ng/ml ILY induced NFAT1 dephosphorylation and its nuclear translocation in HuCCT1 cells. In contrast to the result that ILY induced NF-{kappa}B translocation in human hepatic HepG2 cells, ILY did not affect NF-{kappa}B localization in HuCCT1 cells. Dephosphorylation and nuclear translocation of NFAT1 caused by ILY were prevented by [Ca{sup 2+}]i calcium chelator, BAPTA/AM, and calcineurin inhibitors, cyclosporine A and tacrolimus. ILY induced early growth response-1 (EGR-1) expression and it was inhibited by the pre-treatment with cyclosporine A, indicating that the calcineurin/NFAT pathway was involved in EGR-1 expression in response to ILY. ILY-induced calcineurin/NFAT1 activation and sequential EGR-1 expression might be related to the pathogenesis of S. intermedius in human bile duct cells.

  18. Suppressor of cytokine signaling 1 expression during LPS-induced inflammation and bone loss in rats

    Directory of Open Access Journals (Sweden)

    João Antonio Chaves de SOUZA

    2017-10-01

    Full Text Available Abstract This study aimed to characterize the dynamics of suppressor of cytokine signaling (SOCS1 expression in a rat model of lipopolysaccharide-induced periodontitis. Wistar rats in the experimental groups were injected three times/week with LPS from Escherichia coli on the palatal aspect of the first molars, and control animals were injected with vehicle (phosphate-buffered saline. Animals were sacrificed 7, 15, and 30 days after the first injection to analyze inflammation (stereometric analysis, bone loss (macroscopic analysis, gene expression (qRT-PCR, and protein expression/activation (Western blotting. The severity of inflammation and bone loss associated with LPS-induced periodontitis increased from day 7 to day 15, and it was sustained through day 30. Significant (p < 0.05 increases in SOCS1, RANKL, OPG, and IFN-γ gene expression were observed in the experimental group versus the control group at day 15. SOCS1 protein expression and STAT1 and NF-κB activation were increased throughout the 30-day experimental period. Gingival tissues affected by experimental periodontitis express SOCS1, indicating that this protein may potentially downregulate signaling events involved in inflammatory reactions and bone loss and thus may play a relevant role in the development and progression of periodontal disease.

  19. Alpha-lipoic acid induces sodium iodide symporter expression in TPC-1 thyroid cancer cell line

    International Nuclear Information System (INIS)

    Choi, Hyun-Jeung; Kim, Tae Yong; Ruiz-Llorente, Sergio; Jeon, Min Ji; Han, Ji Min; Kim, Won Gu; Shong, Young Kee; Kim, Won Bae

    2012-01-01

    Introduction: Patients with metastatic thyroid cancers that do not uptake iodine need effective therapeutic option. Differentiation-inducing agents have been tried to restore functional expression of sodium iodide symporter (NIS) without success. Our objective was to assess the effect of alpha-lipoic acid (ALA), known as potential antioxidant, on expression of sodium iodide symporter in thyroid cancer cells. Methods: Human thyroid cancer-derived cell lines, TPC-1, were treated with ALA, and changes in NIS mRNA and protein expression were measured. ALA's effect on NIS gene promoter was evaluated, and functional NIS expression was assessed by iodide uptake assay. Results: Treatment with ALA increased NIS mRNA expression up to ten folds of control dose-dependently after 24 h of exposure. ALA increased NIS promoter activity, and increased iodide uptake by 1.6 fold. ALA induced expression of NIS protein, but had no significant effect on the plasma membrane trafficking. ALA increased phosphorylation of CREB and nuclear translocation of pCREB, and co-treatment of ALA and trichostatin A increased iodide uptake by three folds in TPC-1 cells. Conclusions: ALA is a potential agent to increase NIS transcription in TPC-1. It could be used as an adjunctive agent to increase efficacy of radioiodine therapy if combined with a strategy to increase NIS protein trafficking to cell membrane.

  20. Halobenzoquinone-Induced Alteration of Gene Expression Associated with Oxidative Stress Signaling Pathways.

    Science.gov (United States)

    Li, Jinhua; Moe, Birget; Liu, Yanming; Li, Xing-Fang

    2018-06-05

    Halobenzoquinones (HBQs) are emerging disinfection byproducts (DBPs) that effectively induce reactive oxygen species and oxidative damage in vitro. However, the impacts of HBQs on oxidative-stress-related gene expression have not been investigated. In this study, we examined alterations in the expression of 44 genes related to oxidative-stress-induced signaling pathways in human uroepithelial cells (SV-HUC-1) upon exposure to six HBQs. The results show the structure-dependent effects of HBQs on the studied gene expression. After 2 h of exposure, the expression levels of 9 to 28 genes were altered, while after 8 h of exposure, the expression levels of 29 to 31 genes were altered. Four genes ( HMOX1, NQO1, PTGS2, and TXNRD1) were significantly upregulated by all six HBQs at both exposure time points. Ingenuity pathway analysis revealed that the Nrf2 pathway was significantly responsive to HBQ exposure. Other canonical pathways responsive to HBQ exposure included GSH redox reductions, superoxide radical degradation, and xenobiotic metabolism signaling. This study has demonstrated that HBQs significantly alter the gene expression of oxidative-stress-related signaling pathways and contributes to the understanding of HBQ-DBP-associated toxicity.

  1. Matrix rigidity induces osteolytic gene expression of metastatic breast cancer cells.

    Directory of Open Access Journals (Sweden)

    Nazanin S Ruppender

    Full Text Available Nearly 70% of breast cancer patients with advanced disease will develop bone metastases. Once established in bone, tumor cells produce factors that cause changes in normal bone remodeling, such as parathyroid hormone-related protein (PTHrP. While enhanced expression of PTHrP is known to stimulate osteoclasts to resorb bone, the environmental factors driving tumor cells to express PTHrP in the early stages of development of metastatic bone disease are unknown. In this study, we have shown that tumor cells known to metastasize to bone respond to 2D substrates with rigidities comparable to that of the bone microenvironment by increasing expression and production of PTHrP. The cellular response is regulated by Rho-dependent actomyosin contractility mediated by TGF-ß signaling. Inhibition of Rho-associated kinase (ROCK using both pharmacological and genetic approaches decreased PTHrP expression. Furthermore, cells expressing a dominant negative form of the TGF-ß receptor did not respond to substrate rigidity, and inhibition of ROCK decreased PTHrP expression induced by exogenous TGF-ß. These observations suggest a role for the differential rigidity of the mineralized bone microenvironment in early stages of tumor-induced osteolysis, which is especially important in metastatic cancer since many cancers (such as those of the breast and lung preferentially metastasize to bone.

  2. Selective Inducible Nitric Oxide Synthase Inhibitor Reversed Zinc Chloride-Induced Spatial Memory Impairment via Increasing Cholinergic Marker Expression.

    Science.gov (United States)

    Tabrizian, Kaveh; Azami, Kian; Belaran, Maryam; Soodi, Maliheh; Abdi, Khosrou; Fanoudi, Sahar; Sanati, Mehdi; Mottaghi Dastjerdi, Negar; Soltany Rezaee-Rad, Mohammad; Sharifzadeh, Mohammad

    2016-10-01

    Zinc, an essential micronutrient and biochemical element of the human body, plays structural, catalytic, and regulatory roles in numerous physiological functions. In the current study, the effects of a pretraining oral administration of zinc chloride (10, 25, and 50 mg/kg) for 14 consecutive days and post-training bilateral intra-hippocampal infusion of 1400W as a selective inducible nitric oxide synthase (iNOS) inhibitor (10, 50, and 100 μM/side), alone and in combination, on the spatial memory retention in Morris water maze (MWM) were investigated. Animals were trained for 4 days and tested 48 h after completion of training. Also, the molecular effects of these compounds on the expression of choline acetyltransferase (ChAT), as a cholinergic marker in the CA1 region of the hippocampus and medial septal area (MSA), were evaluated. Behavioral and molecular findings of this study showed that a 2-week oral administration of zinc chloride (50 mg/kg) impaired spatial memory retention in MWM and decreased ChAT expression. Immunohistochemical analysis of post-training bilateral intra-hippocampal infusion of 1400W revealed a significant increase in ChAT immunoreactivity. Furthermore, post-training bilateral intra-hippocampal infusion of 1400W into the CA1 region of the hippocampus reversed zinc chloride-induced spatial memory impairment in MWM and significantly increased ChAT expression in comparison with zinc chloride-treated animals. Taken together, these results emphasize the role of selective iNOS inhibitors in reversing zinc chloride-induced spatial memory deficits via modulation of cholinergic marker expression.

  3. 1,25-Dihydroxyvitamin-D3 Induces Avian β-Defensin Gene Expression in Chickens.

    Directory of Open Access Journals (Sweden)

    Long Zhang

    Full Text Available Host defense peptides (HDPs play a critical role in innate immunity. Specific modulation of endogenous HDP synthesis by dietary compounds has been regarded as a novel approach to boost immunity and disease resistance in animal production. 1,25-dihydroxy vitamin D3 (1,25D3 is well known as a powerful HDP inducer in humans, but limited information about the effect of 1,25D3 on HDPs in poultry is available. Here, we sought to examine whether 1,25D3 could stimulate avian β-defensin (AvBD expression in chickens. We used chicken embryo intestinal epithelial cells (CEIEPCs and peripheral blood mononuclear cells (PBMCs to study the effect of 1,25D3 on the expression of AvBDs. We observed that 1,25D3 is able to up-regulate the expression of several AvBDs in CEIEPCs and PBMCs, whereas it increased the amounts of AvBD4 mRNA in CEIEPCs only in the presence of lipopolysaccharide (LPS. On the other hand, LPS treatment not only inhibited the expression of CYP24A1 but also altered the expression pattern of VDR in CEIEPCs. Furthermore, AvBDs were not directly regulated by 1,25D3, as cycloheximide completely blocked 1,25D3-induced expression of AvBDs. Our observations suggest that 1,25D3 is capable of inducing AvBD gene expression and is a potential antibiotic alternative through augmentation of host innate immunity as well as disease control in chickens.

  4. 14-3-3σ induces heat shock protein 70 expression in hepatocellular carcinoma

    International Nuclear Information System (INIS)

    Liu, Chia-Chia; Wang, John; Shyue, Song-Kun; Sung, Li-Ying; Liou, Jun-Yang; Jan, Yee-Jee; Ko, Bor-Sheng; Wu, Yao-Ming; Liang, Shu-Man; Chen, Shyh-Chang; Lee, Yen-Ming; Liu, Tzu-An; Chang, Tzu-Ching

    2014-01-01

    14-3-3σ is implicated in promoting tumor development of various malignancies. However, the clinical relevance of 14-3-3σ in hepatocellular carcinoma (HCC) tumor progression and modulation and pathway elucidation remain unclear. We investigated 14-3-3σ expression in 109 HCC tissues by immunohistochemistry. Overexpression and knockdown experiments were performed by transfection with cDNA or siRNA. Protein expression and cell migration were determined by Western blot and Boyden chamber assay. In this study, we found that 14-3-3σ is abundantly expressed in HCC tumors. Stable or transient overexpression of 14-3-3σ induces the expression of heat shock factor-1α (HSF-1α) and heat shock protein 70 (HSP70) in HCC cells. Moreover, expression of 14-3-3σ significantly correlates with HSF-1α/HSP70 in HCC tumors and both 14-3-3σ and HSP70 overexpression are associated with micro-vascular thrombi in HCC patients, suggesting that 14-3-3σ/HSP70 expression is potentially involved in cell migration/invasion. Results of an in vitro migration assay indicate that 14-3-3σ promotes cell migration and that 14-3-3σ-induced cell migration is impaired by siRNA knockdown of HSP70. Finally, 14-3-3σ-induced HSF-1α/HSP70 expression is abolished by the knockdown of β-catenin or activation of GSK-3β. Our findings indicate that 14-3-3σ participates in promoting HCC cell migration and tumor development via β-catenin/HSF-1α/HSP70 pathway regulation. Thus, 14-3-3σ alone or combined with HSP70 are potential prognostic biomarkers for HCC

  5. Clinicopathologic significance of fascin, extracellular matrix metalloproteinase inducer, and ezrin expressions in colorectal adenocarcinoma

    Directory of Open Access Journals (Sweden)

    Eun-Joo Jung

    2011-01-01

    Full Text Available Background: The over expression of fascin, extracellular matrix metalloproteinase inducer (EMMPRIN, and ezrin proteins has been associated with poor prognosis in various carcinomas and sarcomas. However, very few studies have reported the relationship between the expression of fascin, EMMPRIN, and ezrin proteins and the clinico-pathologic parameters of colorectal carcinomas. Aims: The aim was to investigate the relationship between fascin, EMMPRIN, and ezrin proteins in colorectal adenocarcinomas and their correlation with clinico-pathologic parameters. Settings and Design: The expression of fascin, EMMPRIN, and ezrin proteins was studied in 210 colorectal adenocarcinoma patients through immunohistochemical staining. Materials and Methods: Immunohistochemical staining by the avidin-biotin peroxidase method was done. The scoring of each protein expression was done and divided into three groups (negative, low-, and high-expression groups. Statistical Analysis: A chi-square test, and Kendall′s tau-b correlation test were used for comparing. Survival analysis was performed using the Kaplan-Meier method with log-rank tests and the Cox proportional hazard model. Results: The percentages of the high-expression group of fascin, EMMPRIN, and ezrin proteins in colorectal adenocarcinomas were 24%, 73%, and 62%, respectively. Weak positive correlations were observed among these protein expressions. An increased expression of the fascin protein was significantly associated with advanced tumor depth and shorter survival times, and a high expression of fascin protein was an independent prognostic factor in univariate and multivariate survival analyses. EMMPRIN and ezrin protein expressions were not associated with the clinico-pathologic parameters. Conclusions: The high expression of fascin protein may be an unfavorable prognostic marker for individual colorectal cancer patients.

  6. Characterization of human septic sera induced gene expression modulation in human myocytes

    Science.gov (United States)

    Hussein, Shaimaa; Michael, Paul; Brabant, Danielle; Omri, Abdelwahab; Narain, Ravin; Passi, Kalpdrum; Ramana, Chilakamarti V.; Parrillo, Joseph E.; Kumar, Anand; Parissenti, Amadeo; Kumar, Aseem

    2009-01-01

    To gain a better understanding of the gene expression changes that occurs during sepsis, we have performed a cDNA microarray study utilizing a tissue culture model that mimics human sepsis. This study utilized an in vitro model of cultured human fetal cardiac myocytes treated with 10% sera from septic patients or 10% sera from healthy volunteers. A 1700 cDNA expression microarray was used to compare the transcription profile from human cardiac myocytes treated with septic sera vs normal sera. Septic sera treatment of myocytes resulted in the down-regulation of 178 genes and the up-regulation of 4 genes. Our data indicate that septic sera induced cell cycle, metabolic, transcription factor and apoptotic gene expression changes in human myocytes. Identification and characterization of gene expression changes that occur during sepsis may lead to the development of novel therapeutics and diagnostics. PMID:19684886

  7. Expression of hepatitis C virus envelope protein 2 induces apoptosis in cultured mammalian cells

    Institute of Scientific and Technical Information of China (English)

    Li-Xin Zhu; Jing Liu; You-Hua Xie; Yu-Ying Kong; Ye Ye; Chun-Lin Wang; Guang-Di Li; Yuan Wang

    2004-01-01

    AIM: To explore the role of hepatitis C virus (HCV) envelope protein 2 (E2) in the induction of apoptosis.METHODS: A carboxyterminal truncated E2 (E2-661) was transiently expressed in several cultured mammalian cell lines or stably expressed in Chinese hamster ovary (CHO)cell line. Cell proliferation was assessed by 3H thymidine uptake. Apoptosis was examined by Hoechst 33258staining, flow cytometry and DNA fragmentation analysis.RESULTS: Reduced proliferation was readily observed in the E2-661 expressing cells. These cells manifested the typical features of apoptosis, including cell shrinkage,chromatin condensation and hypodiploid genomic DNA content. Similar apoptotic cell death was observed in an E2-661 stably expressing cell line.CONCLUSION: HCV E2 can induce apoptosis in cultured mammalian cells.

  8. Direct evidence that radiation induced micronuclei of early embryos require a mitosis for expression

    International Nuclear Information System (INIS)

    Mueller, W.U.; Schlusen, I.; Streffer, C.

    1991-01-01

    The naturally synchronous development of early mouse embryos was exploited to address the question, whether micronuclei require a mitosis for expression or whether they can be expressed in the same cell cycle, in which exposure to X-rays or caffeine took place. Experiments with 2-cell and with 4-cell embryos showed that micronulcei are expressed only if a mitosis is completed. There was no indication, even after doses up to 20 Gy, that micronuclei can be expressed before the mitosis was reached, which followed exposure. Furthermore, no nuclear fragmentation pointing to apoptosis could be detected in the cycle, in which cells were exposed. The same results were obtained when caffeine (5 mM) was used as micronucleus inducing agent. (orig.)

  9. 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.

    , while both cytokines are over-expressed within the first day following injury, CTGF protein levels could not be correlated with observed adhesion development. In addition, we synthesized linear triblock copolymers of polyethylene glycol (PEG) and poly(D,L-lactide-co-glycolide) (PLGA), two of the most widely studied biodegradable polymers in use today. Bulk gels and microparticles of the copolymers were then evaluated for gelling behavior, temperature stability, and drug loading and release kinetics in order assess their suitability as potential carriers of antisense therapeutics. A novel approach to affecting the antisense oligonucleotide release kinetics by varying the relative concentrations of co-encapsulated cationic lipid transfection agents was also presented.

  10. Regulation of an antisense RNA with the transition of neonatal to IIb myosin heavy chain during postnatal development and hypothyroidism in rat skeletal muscle.

    Science.gov (United States)

    Pandorf, Clay E; Jiang, Weihua; Qin, Anqi X; Bodell, Paul W; Baldwin, Kenneth M; Haddad, Fadia

    2012-04-01

    Postnatal development of fast skeletal muscle is characterized by a transition in expression of myosin heavy chain (MHC) isoforms, from primarily neonatal MHC at birth to primarily IIb MHC in adults, in a tightly coordinated manner. These isoforms are encoded by distinct genes, which are separated by ∼17 kb on rat chromosome 10. The neonatal-to-IIb MHC transition is inhibited by a hypothyroid state. We examined RNA products [mRNA, pre-mRNA, and natural antisense transcript (NAT)] of developmental and adult-expressed MHC genes (embryonic, neonatal, I, IIa, IIx, and IIb) at 2, 10, 20, and 40 days after birth in normal and thyroid-deficient rat neonates treated with propylthiouracil. We found that a long noncoding antisense-oriented RNA transcript, termed bII NAT, is transcribed from a site within the IIb-Neo intergenic region and across most of the IIb MHC gene. NATs have previously been shown to mediate transcriptional repression of sense-oriented counterparts. The bII NAT is transcriptionally regulated during postnatal development and in response to hypothyroidism. Evidence for a regulatory mechanism is suggested by an inverse relationship between IIb MHC and bII NAT in normal and hypothyroid-treated muscle. Neonatal MHC transcription is coordinately expressed with bII NAT. A comparative phylogenetic analysis also suggests that bII NAT-mediated regulation has been a conserved trait of placental mammals for most of the eutherian evolutionary history. The evidence in support of the regulatory model implicates long noncoding antisense RNA as a mechanism to coordinate the transition between neonatal and IIb MHC during postnatal development.

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

    Science.gov (United States)

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

    2016-04-15

    Defects in mRNA 3'end formation have been described to alter transcription termination, transport of the mRNA from the nucleus to the cytoplasm, stability of the mRNA and translation efficiency. Therefore, inhibition of polyadenylation may lead to gene silencing. Here, we choose facioscapulohumeral dystrophy (FSHD) as a model to determine whether or not targeting key 3' end elements involved in mRNA processing using antisense oligonucleotide drugs can be used as a strategy for gene silencing within a potentially therapeutic context. FSHD is a gain-of-function disease characterized by the aberrant expression of the Double homeobox 4 (DUX4) transcription factor leading to altered pathogenic deregulation of multiple genes in muscles. Here, we demonstrate that targeting either the mRNA polyadenylation signal and/or cleavage site is an efficient strategy to down-regulate DUX4 expression and to decrease the abnormally high-pathological expression of genes downstream of DUX4. We conclude that targeting key functional 3' end elements involved in pre-mRNA to mRNA maturation with antisense drugs can lead to efficient gene silencing and is thus a potentially effective therapeutic strategy for at least FSHD. Moreover, polyadenylation is a crucial step in the maturation of almost all eukaryotic mRNAs, and thus all mRNAs are virtually eligible for this antisense-mediated knockdown strategy. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  12. Expression Profile of Cationic Amino Acid Transporters in Rats with Endotoxin-Induced Uveitis

    Directory of Open Access Journals (Sweden)

    Yung-Ray Hsu

    2016-01-01

    Full Text Available Purpose. The transcellular arginine transportation via cationic amino acid transporter (CAT is the rate-limiting step in nitric oxide (NO synthesis, which is crucial in intraocular inflammation. In this study, CAT isoforms and inducible nitric oxide synthase (iNOS expression was investigated in endotoxin-induced uveitis (EIU. Methods. EIU was induced in Lewis rats by lipopolysaccharide (LPS injection. In the treatment group, the rats were injected intraperitoneally with the proteasome inhibitor bortezomib before EIU induction. After 24 hours, leukocyte quantification, NO measurement of the aqueous humor, and histopathological examination were evaluated. The expression of CAT isoforms and iNOS was determined by reverse transcription-polymerase chain reaction, western blotting, and immunofluorescence staining. Nuclear factor-kappa B (NF-κB binding activity was evaluated by electrophoretic mobility shift assay. The mouse macrophage cell line RAW 264.7 was used to validate the in vivo findings. Results. LPS significantly stimulated iNOS, CAT-2A, and CAT-2B mRNA and protein expression but did not affect CAT-1 in EIU rats and RAW 264.7 cells. Bortezomib attenuated inflammation and inhibited iNOS, CAT-2A, and CAT-2B expression through NF-κB inhibition. Conclusions. CAT-2 and iNOS, but not CAT-1, are specifically involved in EIU. NF-κB is essential in the induction of CAT-2 and iNOS in EIU.

  13. Transcript-specific effects of adrenalectomy on seizure-induced BDNF expression in rat hippocampus

    DEFF Research Database (Denmark)

    Lauterborn, J C; Poulsen, F R; Stinis, C T

    1998-01-01

    Activity-induced brain-derived neurotrophic factor (BDNF) expression is negatively modulated by circulating adrenal steroids. The rat BDNF gene gives rise to four major transcript forms that each contain a unique 5' exon (I-IV) and a common 3' exon (V) that codes for BDNF protein. Exon-specific i......Activity-induced brain-derived neurotrophic factor (BDNF) expression is negatively modulated by circulating adrenal steroids. The rat BDNF gene gives rise to four major transcript forms that each contain a unique 5' exon (I-IV) and a common 3' exon (V) that codes for BDNF protein. Exon...... and in exon II-containing mRNA with 30-days survival. In the dentate gyrus granule cells, adrenalectomy markedly potentiated increases in exon I and II cRNA labeling, but not increases in exon III and IV cRNA labeling, elicited by one hippocampal afterdischarge. Similarly, for the granule cells and CA1...... no effect on exon IV-containing mRNA content. These results demonstrate that the negative effects of adrenal hormones on activity-induced BDNF expression are by far the greatest for transcripts containing exons I and II. Together with evidence for region-specific transcript expression, these results suggest...

  14. Xenotropic type C virus expression in murine thymomas induced by radiation or 3-methylcholanthrene

    International Nuclear Information System (INIS)

    Mayer, A.; Duran-Reynals, M.L.

    1981-01-01

    Thymic lymphoma incidence and thymic expression of MuLV with xenotropic infectivity was monitored in AKR, RF, and reciprocal F 1 mice of the AKR X RF cross after treatment with either γ radiation or the chemical carcinogen 3-methylcholanthrene (MCA). These two inbred strains and the F 1 hybrids developed similary high incidences of thymoma, and lymphomatous cells from AKR mice and (ARK] X RF∫)F 1 mice were observed to be expressing MuLV with xenotropic host range. However, lymphoma cells from RF mice and (RF] X AKR∫)F 1 mice did not shed xenotropic MuLV. Thymic xenotropic virus expression was therefore not correlated with a high incidence of radiation or chemically induced thymoma, but rather appeared to be a phenotype genetically transmitted by AKR mice to F 1 mice of the AKR X RF cross as a dominant trait in induced thymomas. In addition, a maternal effect on thymic xenotropic virus expression in induced thymomas was observed by the comparison of reciprocal F 1 hybrids in this cross

  15. VCC-1 over-expression inhibits cisplatin-induced apoptosis in HepG2 cells

    International Nuclear Information System (INIS)

    Zhou, Zhitao; Lu, Xiao; Zhu, Ping; Zhu, Wei; Mu, Xia; Qu, Rongmei; Li, Ming

    2012-01-01

    Highlights: ► VCC-1 is hypothesized to be associated with carcinogenesis. ► Levels of VCC-1 are increased significantly in HCC. ► Over-expression of VCC-1 could promotes cellular proliferation rate. ► Over-expression of VCC-1 inhibit the cisplatin-provoked apoptosis in HepG2 cells. ► VCC-1 plays an important role in control the tumor growth and apoptosis. -- Abstract: Vascular endothelial growth factor-correlated chemokine 1 (VCC-1), a recently described chemokine, is hypothesized to be associated with carcinogenesis. However, the molecular mechanisms by which aberrant VCC-1 expression determines poor outcomes of cancers are unknown. In this study, we found that VCC-1 was highly expressed in hepatocellular carcinoma (HCC) tissue. It was also associated with proliferation of HepG2 cells, and inhibition of cisplatin-induced apoptosis of HepG2 cells. Conversely, down-regulation of VCC-1 in HepG2 cells increased cisplatin-induced apoptosis of HepG2 cells. In summary, these results suggest that VCC-1 is involved in cisplatin-induced apoptosis of HepG2 cells, and also provides some evidence for VCC-1 as a potential cellular target for chemotherapy.

  16. Hepatocyte growth factor limits autoimmune neuroinflammation via glucocorticoid-induced leucine zipper expression in dendritic cells.

    Science.gov (United States)

    Benkhoucha, Mahdia; Molnarfi, Nicolas; Dunand-Sauthier, Isabelle; Merkler, Doron; Schneiter, Gregory; Bruscoli, Stefano; Riccardi, Carlo; Tabata, Yasuhiko; Funakoshi, Hiroshi; Nakamura, Toshikazu; Reith, Walter; Santiago-Raber, Marie-Laure; Lalive, Patrice H

    2014-09-15

    Autoimmune neuroinflammation, including multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), a prototype for T cell-mediated autoimmunity, is believed to result from immune tolerance dysfunction leading to demyelination and substantial neurodegeneration. We previously showed that CNS-restricted expression of hepatocyte growth factor (HGF), a potent neuroprotective factor, reduced CNS inflammation and clinical deficits associated with EAE. In this study, we demonstrate that systemic HGF treatment ameliorates EAE through the development of tolerogenic dendritic cells (DCs) with high expression levels of glucocorticoid-induced leucine zipper (GILZ), a transcriptional repressor of gene expression and a key endogenous regulator of the inflammatory response. RNA interference-directed neutralization of GILZ expression by DCs suppressed the induction of tolerance caused by HGF. Finally, adoptive transfer of HGF-treated DCs from wild-type but not GILZ gene-deficient mice potently mediated functional recovery in recipient mice with established EAE through effective modulation of autoaggressive T cell responses. Altogether, these results show that by inducing GILZ in DCs, HGF reproduces the mechanism of immune regulation induced by potent immunomodulatory factors such as IL-10, TGF-β1, and glucocorticoids and therefore that HGF therapy may have potential in the treatment of autoimmune dysfunctions. Copyright © 2014 by The American Association of Immunologists, Inc.

  17. Caffeic acid phenethyl ester inhibits 3-MC-induced CYP1A1 expression through induction of hypoxia-inducible factor-1α

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Hyung Gyun [Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon (Korea, Republic of); Han, Eun Hee [Division of Life Science, Korea Basic Science Institute, Daejeon (Korea, Republic of); Im, Ji Hye; Lee, Eun Ji; Jin, Sun Woo [Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon (Korea, Republic of); Jeong, Hye Gwang, E-mail: hgjeong@cnu.ac.kr [Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon (Korea, Republic of)

    2015-09-25

    Caffeic acid phenethyl ester (CAPE), a natural component of propolis, is reported to have anticarcinogenic properties, although its precise chemopreventive mechanism remains unclear. In this study, we examined the effects of CAPE on 3-methylcholanthrene (3-MC)-induced CYP1A1 expression and activities. CAPE reduced the formation of the benzo[a]pyrene-DNA adduct. Moreover, CAPE inhibited 3-MC-induced CYP1A1 activity, mRNA expression, protein level, and promoter activity. CAPE treatment also decreased 3-MC-inducible xenobiotic-response element (XRE)-linked luciferase, aryl hydrocarbons receptor (AhR) transactivation and nuclear localization. CAPE induced hypoxia inducible factor-1α (HIF-1α) protein level and HIF-1α responsible element (HRE) transcriptional activity. CAPE-mediated HIF-1α reduced 3-MC-inducible CYP1A1 protein expression. Taken together, CAPE decreases 3-MC-mediated CYP1A1 expression, and this inhibitory response is associated with inhibition of AhR and HIF-1α induction. - Highlights: • CAPE reduced the formation of the benzo[a]pyrene-DNA adduct. • CAPE inhibited 3-MC-induced CYP1A1 expression. • CAPE induced HIF-1α induction. • CAPE-mediated HIF-1α reduced 3-MC-inducible CYP1A1 expression.

  18. Expression of MDM2 in an acute lymphocytic leukemia mice model induced by γ-radiation

    International Nuclear Information System (INIS)

    Huang Yuecheng; Cai Jianming; Han Ling; Gao Fu; Cui Jianguo; Gao Jianguo

    2004-01-01

    Objective: To investigate the role of the MDM 2 in the process of carcinogenesis induced by γ-rays and its molecular mechanisms. Methods: Animal model of radiation-induced leukemia was established by γ-irradiation. According to the histological and morphological results, mice tissues were divided into three groups: cancerization group, incancerization group and control group. Expression of MDM 2 protein and mRNA in thymus/bone marrow was detected with Western blot and in situ hybridization (ISH), respectively. The authors also examined the protein phosphorylation level of MDM 2 protein by immunoprecipitation (IP). PCR-SSCP was performed to detect gene mutation. Results: A mice leukemia model was successfully established as verified by pathological findings and confirmed by transplantation test in nude mice. The protein expression in thymus/bone marrow in irradiation groups was significantly higher than that in controls (P 2 was found to be hyper-phosphorylated in the cancerization group as compared with other groups. No gene mutation was detected by SSCP/silver-staining assay in the tumor samples. Conclusion: MDM 2 may be involved in the development and progression of leukemia induced by γ-irradiation. The over-expression but not gene mutation may be responsible for malignant transformation induced by radiation. Phosphorylation is at least partly attributed to activation of MDM 2

  19. Baclofen blocks the acquisition and expression of mitragynine-induced conditioned place preference in rats.

    Science.gov (United States)

    Yusoff, Nurul H M; Mansor, Sharif M; Müller, Christian P; Hassan, Zurina

    2018-06-01

    Mitragynine is the major alkaloid found in the leaves of M. speciosa Korth (Rubiaceae), a plant that is native to Southeast Asia. This compound has been used, either traditionally or recreationally, due to its psychostimulant and opioid-like effects. Recently, mitragynine has been shown to exert conditioned place preference (CPP), indicating the rewarding and motivational properties of M. speciosa. Here, the involvement of GABA B receptors in mediating mitragynine reward is studied using a CPP paradigm in rats. First, we examined the effects of GABA B receptor agonist baclofen (1.25, 2.5 and 5 mg/kg) on the acquisition of mitragynine (10 mg/kg)-induced CPP. Second, the involvement of GABA B receptors in the expression of mitragynine-induced CPP was tested. We found that the acquisition of mitragynine-induced CPP could be blocked by higher doses (2.5 and 5 mg/kg) of baclofen. Baclofen at a high dose inhibited locomotor activity and caused a CPP. Furthermore, we found that baclofen (2.5 and 5 mg/kg) also blocked the expression of mitragynine-induced CPP. These findings suggest that both, the acquisition and expression of mitragynine's reinforcing properties is controlled by the GABA B receptor. Copyright © 2018 Elsevier B.V. All rights reserved.

  20. Acute Vhl gene inactivation induces cardiac HIF-dependent erythropoietin gene expression.

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    Marta Miró-Murillo

    Full Text Available Von Hippel Lindau (Vhl gene inactivation results in embryonic lethality. The consequences of its inactivation in adult mice, and of the ensuing activation of the hypoxia-inducible factors (HIFs, have been explored mainly in a tissue-specific manner. This mid-gestation lethality can be also circumvented by using a floxed Vhl allele in combination with an ubiquitous tamoxifen-inducible recombinase Cre-ER(T2. Here, we characterize a widespread reduction in Vhl gene expression in Vhl(floxed-UBC-Cre-ER(T2 adult mice after dietary tamoxifen administration, a convenient route of administration that has yet to be fully characterized for global gene inactivation. Vhl gene inactivation rapidly resulted in a marked splenomegaly and skin erythema, accompanied by renal and hepatic induction of the erythropoietin (Epo gene, indicative of the in vivo activation of the oxygen sensing HIF pathway. We show that acute Vhl gene inactivation also induced Epo gene expression in the heart, revealing cardiac tissue to be an extra-renal source of EPO. Indeed, primary cardiomyocytes and HL-1 cardiac cells both induce Epo gene expression when exposed to low O(2 tension in a HIF-dependent manner. Thus, as well as demonstrating the potential of dietary tamoxifen administration for gene inactivation studies in UBC-Cre-ER(T2 mouse lines, this data provides evidence of a cardiac oxygen-sensing VHL/HIF/EPO pathway in adult mice.

  1. PTEN gene and phosphorylation of Akt protein expression in the LPS-induced lung fibroblast

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    Mao-lin HUANG

    2014-09-01

    Full Text Available Objective: To investigate PTEN gene expression and the Akt phosphorylation of protein expression in the LPS-induced lung fibroblast, to initially reveal the relation between PTEN gene and the Akt phosphorylated proteins to LPS-induced lung fibroblast proliferation mechanism. Methods: BrdU experiments was performed to evaluate the LPS-induced lung fibroblast proliferation,  RT-PCR and Western Blot analysis were used to analyze the PTEN gene expression and Western blot was performed to analyze Akt phosphorylated protein expression. Results: PTEN mRNA level of the experimental group were significantly lower than the control group (P<0.05 with LPS simulation for 24h and 72h , and there were no significant difference between the experimental group and control group the experimental group and control group (P>0.05 . PTEN protein expression levels of the experimental group were significantly lower than the control group (P<0.05 , at 72h, and PTEN mRNA levels had no significant differences between these of the experimental and control group at 6h,12h and 24h(p>0.05. Phosphorylation Akt protein level (relative to total Akt protein was significantly higer than the control group (P<0.05 at 24h and 72h, and phosphorylation Akt protein levels had no significant differences between these of the experimental and control group at 6h and 12h (P>0.05 .Conclusion: PTEN gene and phosphorylation Akt protein involve in LPS-induced lung fibroblast proliferation signal transduction pathway.

  2. Changes in gene expression linked to methamphetamine-induced dopaminergic neurotoxicity.

    Science.gov (United States)

    Xie, Tao; Tong, Liqiong; Barrett, Tanya; Yuan, Jie; Hatzidimitriou, George; McCann, Una D; Becker, Kevin G; Donovan, David M; Ricaurte, George A

    2002-01-01

    The purpose of these studies was to examine the role of gene expression in methamphetamine (METH)-induced dopamine (DA) neurotoxicity. First, the effects of the mRNA synthesis inhibitor, actinomycin-D, and the protein synthesis inhibitor, cycloheximide, were examined. Both agents afforded complete protection against METH-induced DA neurotoxicity and did so independently of effects on core temperature, DA transporter function, or METH brain levels, suggesting that gene transcription and mRNA translation play a role in METH neurotoxicity. Next, microarray technology, in combination with an experimental approach designed to facilitate recognition of relevant gene expression patterns, was used to identify gene products linked to METH-induced DA neurotoxicity. This led to the identification of several genes in the ventral midbrain associated with the neurotoxic process, including genes for energy metabolism [cytochrome c oxidase subunit 1 (COX1), reduced nicotinamide adenine dinucleotide ubiquinone oxidoreductase chain 2, and phosphoglycerate mutase B], ion regulation (members of sodium/hydrogen exchanger and sodium/bile acid cotransporter family), signal transduction (adenylyl cyclase III), and cell differentiation and degeneration (N-myc downstream-regulated gene 3 and tau protein). Of these differentially expressed genes, we elected to further examine the increase in COX1 expression, because of data implicating energy utilization in METH neurotoxicity and the known role of COX1 in energy metabolism. On the basis of time course studies, Northern blot analyses, in situ hybridization results, and temperature studies, we now report that increased COX1 expression in the ventral midbrain is linked to METH-induced DA neuronal injury. The precise role of COX1 and other genes in METH neurotoxicity remains to be elucidated.

  3. TNFα-Induced Mucin 4 Expression Elicits Trastuzumab Resistance in HER2-Positive Breast Cancer.

    Science.gov (United States)

    Mercogliano, María F; De Martino, Mara; Venturutti, Leandro; Rivas, Martín A; Proietti, Cecilia J; Inurrigarro, Gloria; Frahm, Isabel; Allemand, Daniel H; Deza, Ernesto Gil; Ares, Sandra; Gercovich, Felipe G; Guzmán, Pablo; Roa, Juan C; Elizalde, Patricia V; Schillaci, Roxana

    2017-02-01

    Although trastuzumab administration improved the outcome of HER2-positive breast cancer patients, resistance events hamper its clinical benefits. We demonstrated that TNFα stimulation in vitro induces trastuzumab resistance in HER2-positive breast cancer cell lines. Here, we explored the mechanism of TNFα-induced trastuzumab resistance and the therapeutic strategies to overcome it. Trastuzumab-sensitive breast cancer cells, genetically engineered to stably overexpress TNFα, and de novo trastuzumab-resistant tumors, were used to evaluate trastuzumab response and TNFα-blocking antibodies effectiveness respectively. Immunohistochemistry and antibody-dependent cell cytotoxicity (ADCC), together with siRNA strategy, were used to explore TNFα influence on the expression and function of its downstream target, mucin 4 (MUC4). The clinical relevance of MUC4 expression was studied in a cohort of 78 HER2-positive breast cancer patients treated with adjuvant trastuzumab. TNFα overexpression turned trastuzumab-sensitive cells and tumors into resistant ones. Histopathologic findings revealed mucin foci in TNFα-producing tumors. TNFα induced upregulation of MUC4 that reduced trastuzumab binding to its epitope and impaired ADCC. Silencing MUC4 enhanced trastuzumab binding, increased ADCC, and overcame trastuzumab and trastuzumab-emtansine antiproliferative effects in TNFα-overexpressing cells. Accordingly, administration of TNFα-blocking antibodies downregulated MUC4 and sensitized de novo trastuzumab-resistant breast cancer cells and tumors to trastuzumab. In HER2-positive breast cancer samples, MUC4 expression was found to be an independent predictor of poor disease-free survival (P = 0.008). We identified TNFα-induced MUC4 expression as a novel trastuzumab resistance mechanism. We propose MUC4 expression as a predictive biomarker of trastuzumab efficacy and a guide to combination therapy of TNFα-blocking antibodies with trastuzumab. Clin Cancer Res; 23(3); 636-48.

  4. Slow food: insect prey and chitin induce phytohormone accumulation and gene expression in carnivorous Nepenthes plants.

    Science.gov (United States)

    Yilamujiang, Ayufu; Reichelt, Michael; Mithöfer, Axel

    2016-08-01

    Carnivorous Nepenthes plants use modified leaves forming pitfall traps to capture and digest prey, mainly insects, for additional nutrient supply. These traps, so called pitchers, contain a plant-derived fluid composed of many hydrolytic enzymes and defence-related proteins. In this study, the prey-induced induction of corresponding genes of those proteins and a role for phytohormones in this process was analysed. Tissue from insect prey-fed, chitin- and phytohormone-challenged pitchers was harvested and analysed for selected gene expressions by a quantitative PCR technique. Phytohormone levels were determined by LC-MS/MS. Nepenthesin proteolytic activities were measured in the digestive fluid using a fluorescence substrate. Insect prey in the pitchers induced the accumulation of phytohormones such as jasmonates as well as the transcription of studied genes encoding a chitinase 3 and a protease (nepenthesin I), whereas a defence-related protein (PR-1) gene was not induced. Treatment with chitin as a component of the insects' exoskeleton triggered the accumulation of jasmonates, the expression of nepenthesin I and chitinase 3 genes similar to jasmonic acid treatment, and induced protease activity in the fluid. All detectable responses were slowly induced. The results suggest that upon insect prey catch a sequence of signals is initiated: (1) insect-derived chitin, (2) jasmonate as endogenous phytohormone signal, (3) the induction of digestive gene expression and (4) protein expression. This resembles a similar hierarchy of events as described from plant pathogen/herbivore interactions, supporting the idea that carnivory evolved from plant defences. © The Author 2016. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  5. The level of CD147 expression correlates with cyclophilin-induced signalling and chemotaxis

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    Constant Stephanie

    2011-10-01

    Full Text Available Abstract Background Previous studies identified CD147 as the chemotactic receptor on inflammatory leukocytes for extracellular cyclophilins (eCyp. However, CD147 is not known to associate with signal transducing molecules, so other transmembrane proteins, such as proteoglycans, integrins, and CD98, were suggested as receptor or co-receptor for eCyp. CD147 is ubiquitously expressed on many cell types, but relationship between the level of CD147 expression and cellular responses to eCyp has never been analyzed. Given the role of eCyp in pathogenesis of many diseases, it is important to know whether cellular responses to eCyp are regulated at the level of CD147 expression. Results Here, we manipulated CD147 expression levels on HeLa cells using RNAi and investigated the signalling and chemotactic responses to eCypA. Both Erk activation and chemotaxis correlated with the level of CD147 expression, with cells exhibiting low level expression being practically unresponsive to eCypA. Conclusions Our results provide the first demonstration of a chemotactic response of HeLa cells to eCypA, establish a correlation between the level of CD147 expression and the magnitude of cellular responses to eCypA, and indicate that CD147 may be a limiting factor in the receptor complex determining cyclophilin-induced Erk activation and cell migration.

  6. Expression of intracisternal A-particles in radiation-induced murine osteosarcomas

    International Nuclear Information System (INIS)

    Seidel, E.

    1989-01-01

    The expression of intracisternal A-type particles (IAPs) and the distribution of the corresponding type I and type II genes was investigated in radiation induced osteosarcomas of Balb/c, NMRI and CBA mice. The results can be summarized as follows: 1. Slot blot analysis showed that 9 out of 23 (i.e. 39%) of the examined tumours had enhanced IAP expression as compared to normal tissues. None of the CBA tumours were enhanced in IAP expression - only tumours of the strains Balb/c and NMRI expressed higher levels of IAP RNA. 2. Northern blot analysis of RNA from osteosarcomas revealed that the two IAP transcrips (7.2 kb and 5.4 kb) had the same mobility in these tissues as in normal tissue. Some tumours showed a higher level of expression of the 7.2 kb transcript as compared to normal tissues. This 7.2 kb transcript encodes the major structural protein p 73 of IAPs. 3. Hybridization of Southern blots of mouse DNA using the IAP type I specific probe revealed a unique fragment in three of the investigated Balb/c osteosarcomas. 4. Although it could not directly be shown that IAPs were involved in the genesis of osteosarcomas, the changes in IAP expression and the unique fragments suggest a possible correlation with tumour progression. In this light the level of IAP expression may serve as a marker for the degree of the malignancy of the tumour. (orig.) [de

  7. Dynamic, mating-induced gene expression changes in female head and brain tissues of Drosophila melanogaster

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    Stirling Emma J

    2010-10-01

    Full Text Available Abstract Background Drosophila melanogaster females show changes in behavior and physiology after mating that are thought to maximize the number of progeny resulting from the most recent copulation. Sperm and seminal fluid proteins induce post-mating changes in females, however, very little is known about the resulting gene expression changes in female head and central nervous system tissues that contribute to the post-mating response. Results We determined the temporal gene expression changes in female head tissues 0-2, 24, 48 and 72 hours after mating. Females from each time point had a unique post-mating gene expression response, with 72 hours post-mating having the largest number of genes with significant changes in expression. At most time points, genes expressed in the head fat body that encode products involved in metabolism showed a marked change in expression. Additional analysis of gene expression changes in dissected brain tissues 24 hours post-mating revealed changes in transcript abundance of many genes, notably, the reduced transcript abundance of genes that encode ion channels. Conclusions Substantial changes occur in the regulation of many genes in female head tissues after mating, which might underlie aspects of the female post-mating response. These results provide new insights into the physiological and metabolic changes that accompany changes in female behaviors.

  8. Phaleria macrocarpa reduces glomerular growth factor expression in alloxan-induced diabetic rats

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    Evy Sulistyoningrum

    2013-08-01

    Full Text Available Background Diabetic nephropathy (DN is the most serious complication of diabetes, causing end-stage renal disease throughout the world. Recent studies have reported a direct role of vascular endothelial growth factor (VEGF and transforming growth factor-â (TGF-â in DN pathogenesis. VEGF and TGF-â are expressed early in glomeruli in response to hyperglycemia. Active substances of Phaleria macrocarpa (PM pericarp are known to have nephroprotective effects. This study aimed to evaluate the effects of Phaleria macrocarpa (Scheff. Boerl pericarp extract on VEGF and TGF-â expression in alloxan-induced diabetic rats. Methods An experimental study was conducted on twenty five male albino (Sprague Dawley rats divided into five groups (of five each: normal control; diabetic; diabetic + metformin 100 mg/kgBW; diabetic + methanolic PM extract 250 mg/kgBW; and diabetic + aqueous PM extract 250 mg/kgBW. Diabetes was induced by alloxan monohydrate 150 mg/BW intraperitoneally. Treatment was given for 3 weeks. VEGF and TGF-â expression analysis was performed by means of immunohistochemical technique. Differences between groups were assessed by one-way ANOVA. Results VEGF expression in the PM extract group was significantly lower than that in the diabetic group and even metformin group (p<0.01. TGF-â expression in methanolic PM extract group was significantly lower than in diabetic and metformin group (p<0.01, but aqueous PM extract group only showed significancy when compared with diabetic group (p< 0.01. Conclusions Phaleria macrocarpa pericarp extract reduces glomerular expression of TGF-â and VEGF in alloxan-induced diabetic rats.

  9. Podoplanin expression in primary brain tumors induces platelet aggregation and increases risk of venous thromboembolism.

    Science.gov (United States)

    Riedl, Julia; Preusser, Matthias; Nazari, Pegah Mir Seyed; Posch, Florian; Panzer, Simon; Marosi, Christine; Birner, Peter; Thaler, Johannes; Brostjan, Christine; Lötsch, Daniela; Berger, Walter; Hainfellner, Johannes A; Pabinger, Ingrid; Ay, Cihan

    2017-03-30

    Venous thromboembolism (VTE) is common in patients with brain tumors, and underlying mechanisms are unclear. We hypothesized that podoplanin, a sialomucin-like glycoprotein, increases the risk of VTE in primary brain tumors via its ability to induce platelet aggregation. Immunohistochemical staining against podoplanin and intratumoral platelet aggregates was performed in brain tumor specimens of 213 patients (mostly high-grade gliomas [89%]) included in the Vienna Cancer and Thrombosis Study, a prospective observational cohort study of patients with newly diagnosed cancer or progressive disease aimed at identifying patients at risk of VTE. Platelet aggregation in response to primary human glioblastoma cells was investigated in vitro. During 2-year follow-up, 29 (13.6%) patients developed VTE. One-hundred fifty-one tumor specimens stained positive for podoplanin (33 high expression, 47 medium expression, 71 low expression). Patients with podoplanin-positive tumors had lower peripheral blood platelet counts ( P < .001) and higher D-dimer levels ( P < .001). Podoplanin staining intensity was associated with increasing levels of intravascular platelet aggregates in tumor specimens ( P < .001). High podoplanin expression was associated with an increased risk of VTE (hazard ratio for high vs no podoplanin expression: 5.71; 95% confidence interval, 1.52-21.26; P = 010), independent of age, sex, and tumor type. Podoplanin-positive primary glioblastoma cells induced aggregation of human platelets in vitro, which could be abrogated by an antipodoplanin antibody. In conclusion, high podoplanin expression in primary brain tumors induces platelet aggregation, correlates with hypercoagulability, and is associated with increased risk of VTE. Our data indicate novel insights into the pathogenesis of VTE in primary brain tumors. © 2017 by The American Society of Hematology.

  10. Regulation of human heme oxygenase in endothelial cells by using sense and antisense retroviral constructs.

    Science.gov (United States)

    Quan, S; Yang, L; Abraham, N G; Kappas, A

    2001-10-09

    Our objective was to determine whether overexpression and underexpression of human heme oxygenase (HHO)-1 could be controlled on a long-term basis by introduction of the HO-1 gene in sense (S) and antisense (AS) orientation with an appropriate vector into endothelial cells. Retroviral vector (LXSN) containing viral long terminal repeat promoter-driven human HO-1 S (LSN-HHO-1) and LXSN vectors containing HHO-1 promoter (HOP)-controlled HHO-1 S and AS (LSN-HOP-HHO-1 and LSN-HOP-HHO-1-AS) sequences were constructed and used to transfect rat lung microvessel endothelial cells (RLMV cells) and human dermal microvessel endothelial cells (HMEC-1 cells). RLMV cells transduced with HHO-1 S expressed human HO-1 mRNA and HO-1 protein associated with elevation in total HO activity compared with nontransduced cells. Vector-mediated expression of HHO-1 S or AS under control of HOP resulted in effective production of HO-1 or blocked induction of endogenous human HO-1 in HMEC-1 cells, respectively. Overexpression of HO-1 AS was associated with a long-term decrease (45%) of endogenous HO-1 protein and an increase (167%) in unmetabolized exogenous heme in HMEC-1 cells. Carbon monoxide (CO) production in HO-1 S- or AS-transduced HMEC-1 cells after heme treatment was increased (159%) or decreased (50%), respectively, compared with nontransduced cells. HO-2 protein levels did not change. These findings demonstrate that HHO-1 S and AS retroviral constructs are functional in enhancing and reducing HO activity, respectively, and thus can be used to regulate cellular heme levels, the activity of heme-dependent enzymes, and the rate of heme catabolism to CO and bilirubin.

  11. Radiation and desiccation response motif mediates radiation induced gene expression in D. radiodurans

    International Nuclear Information System (INIS)

    Anaganti, Narasimha; Basu, Bhakti; Apte, Shree Kumar

    2015-01-01

    Deinococcus radiodurans is an extremophile that withstands lethal doses of several DNA damaging agents such as gamma irradiation, UV rays, desiccation and chemical mutagens. The organism responds to DNA damage by inducing expression of several DNA repair genes. At least 25 radiation inducible gene promoters harbour a 17 bp palindromic sequence known as radiation and desiccation response motif (RDRM) implicated in gamma radiation inducible gene expression. However, mechanistic details of gamma radiation-responsive up-regulation in gene expression remain enigmatic. The promoters of highly radiation induced genes ddrB (DR0070), gyrB (DR0906), gyrA (DR1913), a hypothetical gene (DR1143) and recA (DR2338) from D. radiodurans were cloned in a green fluorescence protein (GFP)-based promoter probe shuttle vector pKG and their promoter activity was assessed in both E. coli as well as in D. radiodurans. The gyrA, gyrB and DR1143 gene promoters were active in E. coli although ddrB and recA promoters showed very weak activity. In D. radiodurans, all the five promoters were induced several fold following 6 kGy gamma irradiation. Highest induction was observed for ddrB promoter (25 fold), followed by DR1143 promoter (15 fold). The induction in the activity of gyrB, gyrA and recA promoters was 5, 3 and 2 fold, respectively. To assess the role of RDRM, the 17 bp palindromic sequence was deleted from these promoters. The promoters devoid of RDRM sequence displayed increase in the basal expression activity, but the radiation-responsive induction in promoter activity was completely lost. The substitution of two conserved bases of RDRM sequence yielded decreased radiation induction of PDR0070 promoter. Deletion of 5 bases from 5'-end of PDR0070 RDRM increased basal promoter activity, but radiation induction was completely abolished. Replacement of RDRM with non specific sequence of PDR0070 resulted in loss of basal expression and radiation induction. The results demonstrate that

  12. Expression of telomerase reverse transcriptase in radiation-induced chronic human skin ulcer

    International Nuclear Information System (INIS)

    Zhao Po; Li Zhijun; Lu Yali; Zhong Mei; Gu Qingyang; Wang Dewen

    2001-01-01

    Objective: To investigate the expression of the catalytic subunit of telomerase, telomerase reverse transcriptase (TRT) and the possible relationship between the TRT and cancer transformation or poor healing in radiation-induced chronic ulcer of human skin. Methods: Rabbit antibody against human TRT and SP immunohistochemical method were used to detect TRT expression in 24 cases of formalin-fixed, paraffin-embed human skin chronic ulcer tissues induced by radiation, 5 cases of normal skin, 2 of burned skin, and 8 of carcinoma. Results: The positive rate for TRT was 58.3%(14/24) in chronic radiation ulcers, of which the strongly positive rate was 41.7%(10/24) and the weakly positive 16.7%(4/24), 0% in normal (0/5) and burned skin (0/2), and 100% in carcinoma (8/8). The strongly positive expression of TRT was observed almost always in the cytoplasm and nucleus of squamous epithelial cells of proliferative epidermis but the negative and partly weakly positive expression in the smooth muscles, endothelia of small blood vessels and capillaries, and fibroblasts. Chronic inflammtory cells, plasmacytes and lymphocytes also showed weakly positive for TRT. Conclusion: TRT expression could be involved in the malignant transformation of chronic radiation ulcer into squamous carcinoma, and in the poor healing caused by sclerosis of small blood vessels and lack of granulation tissue consisting of capillaries and fibroblasts

  13. Anti-inflammatory effect of resveratrol on TNF-α-induced MCP-1 expression in adipocytes

    International Nuclear Information System (INIS)

    Zhu Jian; Yong Wei; Wu Xiaohong; Yu Ying; Lv Jinghuan; Liu Cuiping; Mao Xiaodong; Zhu Yunxia; Xu Kuanfeng; Han Xiao; Liu Chao

    2008-01-01

    Chronic low-grade inflammation characterized by adipose tissue macrophage accumulation and abnormal cytokine production is a key feature of obesity and type 2 diabetes. Adipose-tissue-derived monocyte chemoattractant protein (MCP)-1, induced by cytokines, has been shown to play an essential role in the early events during macrophage infiltration into adipose tissue. In this study we investigated the effects of resveratrol upon both tumor necrosis factor (TNF)-α-induced MCP-1 gene expression and its underlying signaling pathways in 3T3-L1 adipoctyes. Resveratrol was found to inhibit TNF-α-induced MCP-1 secretion and gene transcription, as well as promoter activity, which based on down-regulation of TNF-α-induced MCP-1 transcription. Nuclear factor (NF)-κB was determined to play a major role in the TNF-α-induced MCP-1 expression. Further analysis showed that resveratrol inhibited DNA binding activity of the NF-κB complex and subsequently suppressed NF-κB transcriptional activity in TNF-α-stimulated cells. Finally, the inhibition of MCP-1 may represent a novel mechanism of resveratrol in preventing obesity-related pathologies

  14. Axotomy induces MHC class I antigen expression on rat nerve cells

    DEFF Research Database (Denmark)

    Maehlen, J; Schröder, H D; Klareskog, L

    1988-01-01

    Immunomorphological staining demonstrates that class I major histocompatibility complex (MHC)-coded antigen expression can be selectively induced on otherwise class I-negative rat nerve cells by peripheral axotomy. Induction of class I as well as class II antigen expression was simultaneously seen...... on non-neural cells in the immediate vicinity of the injured nerve cells. As nerve regeneration after axotomy includes growth of new nerve cell processes and formation of new nerve cell contacts, the present findings raise the question of a role for MHC-coded molecules in cell-cell interactions during...... nerve cell growth....

  15. PIDDosome Expression and the Role of Caspase-2 Activation for Chemotherapy-Induced Apoptosis in RCCs

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    Sebastian Heikaus

    2010-01-01

    Full Text Available Background: The importance of caspase-2 activation for mediating apoptosis in cancer is not clear and seems to differ between different tumour types. Furthermore, only few data have been obtained concerning the expression of caspase-2, which can be alternatively spliced into caspase-2L and caspase-2S, and the other PIDDosome members PIDD and RAIDD in human tumours in vivo. We, therefore, investigated their expression in renal cell carcinomas (RCCs of the clear cell type in vivo and analysed the role of caspase-2 in chemotherapy-induced apoptosis in RCCs in vitro.

  16. Mongersen, an oral SMAD7 antisense oligonucleotide, and Crohn's disease.

    Science.gov (United States)

    Monteleone, Giovanni; Neurath, Markus F; Ardizzone, Sandro; Di Sabatino, Antonio; Fantini, Massimo C; Castiglione, Fabiana; Scribano, Maria L; Armuzzi, Alessandro; Caprioli, Flavio; Sturniolo, Giacomo C; Rogai, Francesca; Vecchi, Maurizio; Atreya, Raja; Bossa, Fabrizio; Onali, Sara; Fichera, Maria; Corazza, Gino R; Biancone, Livia; Savarino, Vincenzo; Pica, Roberta; Orlando, Ambrogio; Pallone, Francesco

    2015-03-19

    Crohn's disease-related inflammation is characterized by reduced activity of the immunosuppressive cytokine transforming growth factor β1 (TGF-β1) due to high levels of SMAD7, an inhibitor of TGF-β1 signaling. Preclinical studies and a phase 1 study have shown that an oral SMAD7 antisense oligonucleotide, mongersen, targets ileal and colonic SMAD7. In a double-blind, placebo-controlled, phase 2 trial, we evaluated the efficacy of mongersen for the treatment of persons with active Crohn's disease. Patients were randomly assigned to receive 10, 40, or 160 mg of mongersen or placebo per day for 2 weeks. The primary outcomes were clinical remission at day 15, defined as a Crohn's Disease Activity Index (CDAI) score of less than 150, with maintenance of remission for at least 2 weeks, and the safety of mongersen treatment. A secondary outcome was clinical response (defined as a reduction of 100 points or more in the CDAI score) at day 28. The proportions of patients who reached the primary end point were 55% and 65% for the 40-mg and 160-mg mongersen groups, respectively, as compared with 10% for the placebo group (P<0.001). There was no significant difference in the percentage of participants reaching clinical remission between the 10-mg group (12%) and the placebo group. The rate of clinical response was significantly greater among patients receiving 10 mg (37%), 40 mg (58%), or 160 mg (72%) of mongersen than among those receiving placebo (17%) (P=0.04, P<0.001, and P<0.001, respectively). Most adverse events were related to complications and symptoms of Crohn's disease. We found that study participants with Crohn's disease who received mongersen had significantly higher rates of remission and clinical response than those who received placebo. (Funded by Giuliani; EudraCT number, 2011-002640-27.).

  17. The new pLAI (lux regulon based auto-inducible expression system for recombinant protein production in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Nocadello Salvatore

    2012-01-01

    Full Text Available Abstract Background After many years of intensive research, it is generally assumed that no universal expression system can exist for high-level production of a given recombinant protein. Among the different expression systems, the inducible systems are the most popular for their tight regulation. However, induction is in many cases less favorable due to the high cost and/or toxicity of inducers, incompatibilities with industrial scale-up or detrimental growth conditions. Expression systems using autoinduction (or self-induction prove to be extremely versatile allowing growth and induction of recombinant proteins without the need to monitor cell density or add inducer. Unfortunately, almost all the actual auto inducible expression systems need endogenous or induced metabolic changes during the growth to trigger induction, both frequently linked to detrimental condition to cell growth. In this context, we use a simple modular approach for a cell density-based genetic regulation in order to assemble an autoinducible recombinant protein expression system in E. coli. Result The newly designed pLAI expression system places the expression of recombinant proteins in Escherichia coli under control of the regulatory genes of the lux regulon of Vibrio fischeri's Quorum Sensing (QS system. The pLAI system allows a tight regulation of the recombinant gene allowing a negligible basal expression and expression only at high cell density. Sequence optimization of regulative genes of QS of V. fischeri for expression in E. coli upgraded the system to high level expression. Moreover, partition of regulative genes between the plasmid and the host genome and introduction of a molecular safety lock permitted tighter control of gene expression. Conclusion Coupling gene expression to cell density using cell-to-cell communication provides a promising approach for recombinant protein production. The system allows the control of expression of the target recombinant gene

  18. Positron emission tomography imaging of gene expression

    International Nuclear Information System (INIS)

    Tang Ganghua

    2001-01-01

    The merging of molecular biology and nuclear medicine is developed into molecular nuclear medicine. Positron emission tomography (PET) of gene expression in molecular nuclear medicine has become an attractive area. Positron emission tomography imaging gene expression includes the antisense PET imaging and the reporter gene PET imaging. It is likely that the antisense PET imaging will lag behind the reporter gene PET imaging because of the numerous issues that have not yet to be resolved with this approach. The reporter gene PET imaging has wide application into animal experimental research and human applications of this approach will likely be reported soon

  19. Arsenite induced oxidative damage in mouse liver is associated with increased cytokeratin 18 expression

    Energy Technology Data Exchange (ETDEWEB)

    Gonsebatt, M.E. [UNAM, Ciudad Universitaria, Dept. Medicina Genomica y Toxicologia Ambiental, Instituto de Investigaciones Biomedicas, Mexico (Mexico); Razo, L.M. del; Sanchez-Pena, L.C. [Seccion de Toxicologia, CINVESTAV, Mexico (Mexico); Cerbon, M.A. [Facultad de Quimica, UNAM, Departamento de Biologia, Mexico (Mexico); Zuniga, O.; Ramirez, P. [Facultad de Estudios Superiores Cuautitlan, UNAM, Laboratorio de Toxicologia Celular, Coordinacion General de Estudios de Posgrado e Investigacion, Cuautitlan Izcalli, Estado de Mexico (Mexico)

    2007-09-15

    Cytokeratins (CK) constitute a family of cytoskeletal intermediate filament proteins that are typically expressed in epithelial cells. An abnormal structure and function are effects that are clearly related to liver diseases as non-alcoholic steatohepatitis, cirrhosis and hepatocellular carcinoma. We have previously observed that sodium arsenite (SA) induced the synthesis of CK18 protein and promotes a dose-related disruption of cytoplasmic CK18 filaments in a human hepatic cell line. Both abnormal gene expression and disturbance of structural organization are toxic effects that are likely to cause liver disease by interfering with normal hepatocyte function. To investigate if a disruption in the CK18 expression pattern is associated with arsenite liver damage, we investigated CK18 mRNA and protein levels in liver slices treated with low levels of SA. Organotypic cultures were incubated with 0.01, 1 and 10 {mu}M of SA in the absence and presence of N-acetyl cysteine (NAC). Cell viability and inorganic arsenic metabolism were determined. Increased expression of CK18 was observed after exposure to SA. The addition of NAC impeded the oxidative effects of SA exposure, decreasing the production of thiobarbituric acid-reactive substances and significantly diminishing the up regulation of CK18 mRNA and protein. Liver arsenic levels correlated with increased levels of mRNA. Mice treated with intragastric single doses of 2.5 and 5 mg/kg of SA showed an increased expression of CK18. Results suggest that CK18 expression may be a sensible early biomarker of oxidative stress and damage induced by arsenite in vitro and in vivo. Then, during SA exposure, altered CK expression may compromise liver function. (orig.)

  20. UDP-Glucuronosyltransferase Expression in Mouse Liver Is Increased in Obesity- and Fasting-Induced Steatosis

    Science.gov (United States)

    Xu, Jialin; Kulkarni, Supriya R.; Li, Liya

    2012-01-01

    UDP-glucuronosyltransferases (Ugt) catalyze phase II conjugation reactions with glucuronic acid, which enhances chemical polarity and the elimination from the body. Few studies have addressed whether Ugt expression and activity are affected by liver disease, such as steatosis. The purpose of this study was to determine whether steatosis induced by obesity or fasting could affect liver Ugt mRNA expression and activity. Male C57BL/6J and Lepob/ob (ob/ob) mice were fed ad libitum or food was withheld for 24 h. In steatotic livers of ob/ob mice, Ugt1a1, -1a6, -1a9, -2a3, -3a1, and -3a2 mRNA expression increased. Fasting, which also induced steatosis, increased hepatic Ugt1a1, -1a6, -1a7, -1a9, -2b1, -2b5, -2a3, -3a1, and -3a2 mRNA expression in mouse liver. Likewise, acetaminophen glucuronidation increased by 47% in hepatic microsomes from ob/ob mice compared with that in C57BL/6J mice, but not after fasting. In both steatosis models, Ugt induction was accompanied by increased aryl hydrocarbon receptor, constitutive androstane receptor (CAR), peroxisome proliferator-activated receptor (PPAR)-α, pregnane X receptor, nuclear factor (erythroid-derived 2)-like 2 (Nrf2), and peroxisome proliferator-activated receptor-γ coactivator-1α mRNA expression. In addition, fasting increased CAR, PPAR, and Nrf2 binding activity. The work points to hepatic triglyceride concentrations corresponding with nuclear receptor and Ugt expression. The findings indicate that steatosis significantly alters hepatic Ugt expression and activity, which could have a significant impact on determining circulating hormone levels, drug efficacy, and environmental chemical clearance. PMID:22031624

  1. UDP-glucuronosyltransferase expression in mouse liver is increased in obesity- and fasting-induced steatosis.

    Science.gov (United States)

    Xu, Jialin; Kulkarni, Supriya R; Li, Liya; Slitt, Angela L

    2012-02-01

    UDP-glucuronosyltransferases (Ugt) catalyze phase II conjugation reactions with glucuronic acid, which enhances chemical polarity and the elimination from the body. Few studies have addressed whether Ugt expression and activity are affected by liver disease, such as steatosis. The purpose of this study was to determine whether steatosis induced by obesity or fasting could affect liver Ugt mRNA expression and activity. Male C57BL/6J and Lep(ob/ob) (ob/ob) mice were fed ad libitum or food was withheld for 24 h. In steatotic livers of ob/ob mice, Ugt1a1, -1a6, -1a9, -2a3, -3a1, and -3a2 mRNA expression increased. Fasting, which also induced steatosis, increased hepatic Ugt1a1, -1a6, -1a7, -1a9, -2b1, -2b5, -2a3, -3a1, and -3a2 mRNA expression in mouse liver. Likewise, acetaminophen glucuronidation increased by 47% in hepatic microsomes from ob/ob mice compared with that in C57BL/6J mice, but not after fasting. In both steatosis models, Ugt induction was accompanied by increased aryl hydrocarbon receptor, constitutive androstane receptor (CAR), peroxisome proliferator-activated receptor (PPAR)-α, pregnane X receptor, nuclear factor (erythroid-derived 2)-like 2 (Nrf2), and peroxisome proliferator-activated receptor-γ coactivator-1α mRNA expression. In addition, fasting increased CAR, PPAR, and Nrf2 binding activity. The work points to hepatic triglyceride concentrations corresponding with nuclear receptor and Ugt expression. The findings indicate that steatosis significantly alters hepatic Ugt expression and activity, which could have a significant impact on determining circulating hormone levels, drug efficacy, and environmental chemical clearance.

  2. Construction of permanently inducible miRNA-based expression vectors using site-specific recombinases

    Directory of Open Access Journals (Sweden)

    Garwick-Coppens Sara E

    2011-11-01

    Full Text Available Abstract Background RNA interference (RNAi is a conserved gene silencing mechanism mediated by small inhibitory microRNAs (miRNAs. Promoter-driven miRNA expression vectors have emerged as important tools for delivering natural or artificially designed miRNAs to eukaryotic cells and organisms. Such systems can be used to query the normal or pathogenic functions of natural miRNAs or messenger RNAs, or to therapeutically silence disease genes. Results As with any molecular cloning procedure, building miRNA-based expression constructs requires a time investment and some molecular biology skills. To improve efficiency and accelerate the construction process, we developed a method to rapidly generate miRNA expression vectors using recombinases instead of more traditional cut-and-paste molecular cloning techniques. In addition to streamlining the construction process, our cloning strategy provides vectors with added versatility. In our system, miRNAs can be constitutively expressed from the U6 promoter, or inducibly expressed by Cre recombinase. We also engineered a built-in mechanism to destroy the vector with Flp recombinase, if desired. Finally, to further simplify the construction process, we developed a software package that automates the prediction and design of optimal miRNA sequences using our system. Conclusions We designed and tested a modular system to rapidly clone miRNA expression cassettes. Our strategy reduces the hands-on time required to successfully generate effective constructs, and can be implemented in labs with minimal molecular cloning expertise. This versatile system provides options that permit constitutive or inducible miRNA expression, depending upon the needs of the end user. As such, it has utility for basic or translational applications.

  3. Extracellular matrix metalloproteinase inducer (EMMPRIN) expression correlates positively with active angiogenesis and negatively with basic fibroblast growth factor expression in epithelial ovarian cancer.

    Science.gov (United States)

    Szubert, Sebastian; Szpurek, Dariusz; Moszynski, Rafal; Nowicki, Michal; Frankowski, Andrzej; Sajdak, Stefan; Michalak, Slawomir

    2014-03-01

    The primary aim of this paper was to evaluate the expression of extracellular matrix metalloproteinase inducer (EMMPRIN) and its relationship with proangiogenic factors and microvessel density (MVD) in ovarian cancer. The study group included 58 epithelial ovarian cancers (EOCs), 35 benign ovarian tumors, and 21 normal ovaries. The expression of EMMPRIN, vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF) was assessed by ELISA of tissue homogenates. Antibodies against CD105, CD31, and CD34 were used to immunohistochemically assess MVD. We have found significantly higher EMMPRIN expression in EOC than in benign ovarian tumors and normal ovaries. Similarly, the VEGF expression was higher in EOC than in benign ovarian tumors and normal ovaries. By contrast, bFGF expression was lower in EOC than in benign ovarian tumors and ovary samples. EMMPRIN expression in EOC was directly correlated with VEGF expression and CD105-MVD, but inversely correlated with bFGF expression. Grade 2/3 ovarian cancers had increased expression of EMMPRIN and VEGF, increased CD105-MVD, and lowered expression of bFGF compared to grade 1 ovarian cancers. Moreover, EMMPRIN expression was higher in advanced (FIGO III and IV) ovarian cancer. The upregulation of EMMPRIN and VEGF expression is correlated with increased CD105-MVD and silenced bFGF, which suggests early and/or reactivated angiogenesis in ovarian cancer. Aggressive EOC is characterized by the following: high expression of EMMPRIN and VEGF, high CD105-MVD, and low expression of bFGF.

  4. Mechanism of inhibitory effect of atorvastatin on resistin expression induced by tumor necrosis factor-α in macrophages

    Directory of Open Access Journals (Sweden)

    Chua Su-Kiat

    2009-05-01

    Full Text Available Abstract Atorvastatin has been shown to reduce resistin expression in macrophages after pro-inflammatory stimulation. However, the mechanism of reducing resistin expression by atorvastatin is not known. Therefore, we sought to investigate the molecular mechanisms of atorvastatin for reducing resistin expression after proinflammatory cytokine, tumor necrosis factor-α (TNF-α stimulation in cultured macrophages. Cultured macrophages were obtained from human peripheral blood mononuclear cells. TNF-α stimulation increased resistin protein and mRNA expression and atorvastatin inhibited the induction of resistin by TNF-α. Addition of mevalonate induced resistin protein expression similar to TNF-α stimulation. However, atorvastatin did not have effect on resistin protein expression induced by mevalonate. SP600125 and JNK small interfering RNA (siRNA completely attenuated the resistin protein expression induced by TNF-α and mevalonate. TNF-α induced phosphorylation of Rac, while atorvastatin and Rac-1 inhibitor inhibited the phosphorylation of Rac induced by TNF-α. The gel shift and promoter activity assay showed that TNF-α increased AP-1-binding activity and resistin promoter activity, while SP600125 and atorvastatin inhibited the AP-1-binding activity and resistin promoter activity induced by TNF-α. Recombinant resistin and TNF-α significantly reduced glucose uptake in cultured macrophages, while atorvastatin reversed the reduced glucose uptake by TNF-α. In conclusion, JNK and Rac pathway mediates the inhibitory effect of atorvastatin on resistin expression induced by TNF-α.

  5. Changes in Gene Expression during G-CSF-Induced Emergency Granulopoiesis in Humans

    DEFF Research Database (Denmark)

    Pedersen, Corinna C.; Borup, Rehannah; Fischer-Nielsen, Anne

    2016-01-01

    Emergency granulopoiesis refers to the increased production of neutrophils in bone marrow and their release into circulation induced by severe infection. Several studies point to a critical role for G-CSF as the main mediator of emergency granulopoiesis. However, the consequences of G-CSF...... stimulation on the transcriptome of neutrophils and their precursors have not yet been investigated in humans. In this work, we examine the changes in mRNA expression induced by administration of G-CSF in vivo, as a model of emergency granulopoiesis in humans. Blood samples were collected from healthy...... individuals after 5 d of G-CSF administration. Neutrophil precursors were sorted into discrete stages of maturation by flow cytometry, and RNA was subjected to microarray analysis. mRNA levels were compared with previously published expression levels in corresponding populations of neutrophil precursors...

  6. Sarcosine induces increase in HER2/neu expression in androgen-dependent prostate cancer cells

    DEFF Research Database (Denmark)

    Dahl, Malin; Bouchelouche, Pierre; Kramer-Marek, Gabriela

    2011-01-01

    Increasing evidence suggests that Human epidermal growth factor receptor 2 (HER2/neu) is involved in progression of prostate cancer. Recently, sarcosine was reported to be highly increased during prostate cancer progression, and exogenous sarcosine induces an invasive phenotype in benign prostate...... epithelial cells. The aim of this work was to investigate the effect of sarcosine on HER2/neu expression in prostate cancer cell lines LNCaP (androgen dependent), PC-3 and DU145 (both androgen independent). Relative amounts of HER2/neu and androgen receptor (AR) transcripts were determined using RT...... that sarcosine is involved in the regulation of the oncoprotein HER2/neu. Thus, sarcosine may induce prostate cancer progression by increased HER2/neu expression. However, detailed information on cellular mechanisms remains to be elucidated....

  7. UVB-induced gene expression in the skin of Xiphophorus maculatus Jp 163 B☆

    Science.gov (United States)

    Yang, Kuan; Boswell, Mikki; Walter, Dylan J.; Downs, Kevin P.; Gaston-Pravia, Kimberly; Garcia, Tzintzuni; Shen, Yingjia; Mitchell, David L.; Walter, Ronald B.

    2014-01-01

    Xiphophorus fish and interspecies hybrids represent long-standing models to study the genetics underlying spontaneous and induced tumorigenesis. The recent release of the Xiphophorus maculatus genome sequence will allow global genetic regulation studies of genes involved in the inherited susceptibility to UVB-induced melanoma within select backcross hybrids. As a first step toward this goal, we report results of an RNA-Seq approach to identify genes and pathways showing modulated transcription within the skin of X. maculatus Jp 163 B upon UVB exposure. X. maculatus Jp 163 B were exposed to various doses of UVB followed by RNA-Seq analysis at each dose to investigate overall gene expression in each sample. A total of 357 genes with a minimum expression change of 4-fold (p-adj fish skin to UVB exposure. PMID:24556253

  8. Ethylene-induced senescence-related gene expression requires protein synthesis

    International Nuclear Information System (INIS)

    Lawton, K.A.; Raghothama, K.G.; Woodson, W.R.

    1990-01-01

    We have investigated the effects of inhibiting protein synthesis on the ethylene-induced expression of 3 carnation senescence-related genes, pSR5, pSR8, and pSR12. Treatment of preclimacteric carnation petal discs with 1μg/ml of cycloheximide, a cytoplasmic protein synthesis inhibitor, for 3h inhibited protein synthesis by >80% as quantitated by the incorporation of [35S]methionine into protein. Pre-treatment of petal discs with cycloheximide prevented ethylene-induced SR transcript accumulation. Cycloheximide treatment of petal discs held in air did not result in increased levels of SR mRNA. These results indicate that ethylene does not interact with pre-formed factors but rather that the activation of SR gene expression by ethylene is mediated by labile protein factor(s) synthesized on cytoplasmic ribosomes. Experiments are currently underway to determine if cycloheximide exerts its effect at the transcriptional or post-transcriptional level

  9. Targeting receptor for advanced glycation end products (RAGE) expression induces apoptosis and inhibits prostate tumor growth

    International Nuclear Information System (INIS)

    Elangovan, Indira; Thirugnanam, Sivasakthivel; Chen, Aoshuang; Zheng, Guoxing; Bosland, Maarten C.; Kajdacsy-Balla, André; Gnanasekar, Munirathinam

    2012-01-01

    Highlights: ► Targeting RAGE by RNAi induces apoptosis in prostate cancer cells. ► Silencing RAGE expression abrogates rHMGB1 mediated cell proliferation. ► Down regulation of RAGE by RNAi inhibits PSA secretion of prostate cancer cells. ► Knock down of RAGE abrogates prostate tumor growth in vivo. ► Disruption of RAGE expression in prostate tumor activates death receptors. -- Abstract: Expression of receptor for advanced glycation end products (RAGE) plays a key role in the progression of prostate cancer. However, the therapeutic potential of targeting RAGE expression in prostate cancer is not yet evaluated. Therefore in this study, we have investigated the effects of silencing the expression of RAGE by RNAi approach both in vitro and in vivo. The results of this study showed that down regulation of RAGE expression by RNAi inhibited the cell proliferation of androgen-dependent (LNCaP) and androgen-independent (DU-145) prostate cancer cells. Furthermore, targeting RAGE expression resulted in apoptotic elimination of these prostate cancer cells by activation of caspase-8 and caspase-3 death signaling. Of note, the levels of prostate specific antigen (PSA) were also reduced in LNCaP cells transfected with RAGE RNAi constructs. Importantly, the RAGE RNAi constructs when administered in nude mice bearing prostate tumors, inhibited the tumor growth by targeting the expression of RAGE, and its physiological ligand, HMGB1 and by up regulating death receptors DR4 and DR5 expression. Collectively, the results of this study for the first time show that targeting RAGE by RNAi may be a promising alternative therapeutic strategy for treating prostate cancer.

  10. Radiation-induced VEGF-C expression and endothelial cell proliferation in lung cancer

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Yu-Hsuan [National Taiwan University Hospital, Department of Oncology, Taipei (China); National Taiwan University, Pharmacological Institute, College of Medicine, Taipei (China); Pan, Shiow-Lin; Wang, Jing-Chi; Teng, Che-Ming [National Taiwan University, Pharmacological Institute, College of Medicine, Taipei (China); Kuo, Sung-Hsin [National Taiwan University Hospital, Department of Oncology, Taipei (China); National Taiwan University College of Medicine, Department of Internal Medicine, Taipei (China); Cheng, Jason Chia-Hsien [National Taiwan University Hospital, Department of Oncology, Taipei (China); National Taiwan University College of Medicine, Graduate Institute of Clinical Medicine, Taipei (China)

    2014-12-15

    The present study was undertaken to investigate whether radiation induces the expression of vascular endothelial growth factor C (VEGF-C) through activation of the PI3K/Akt/mTOR pathway,subsequently affecting endothelial cells. Radiotherapy-induced tumor micro-lymphatic vessel density (MLVD) was determined in a lung cancer xenograft model established in SCID mice. The protein expression and phosphorylation of members of the PI3K/Akt/mTOR pathway and VEGF-C secretion and mRNA expression in irradiated lung cancer cells were assessed by Western blot analysis, enzyme-linked immunosorbent assays (ELISAs), and reverse transcriptase-polymerase chain reaction (RT-PCR). Moreover, specific chemical inhibitors were used to evaluate the role of the PI3K/Akt/mTOR signaling pathway. Conditioned medium (CM) from irradiated control-siRNA or VEGF-C-siRNA-expressing A549 cells was used to evaluate the proliferation of endothelial cells by the MTT assay. Radiation increased VEGF-C expression in a dose-dependent manner over time at the protein but not at the mRNA level. Radiation also up-regulated the phosphorylation of Akt, mTOR, 4EBP, and eIF4E, but not of p70S6K. Radiation-induced VEGF-C expression was down-regulated by LY294002 and rapamycin (both p < 0.05). Furthermore, CM from irradiated A549 cells enhanced human umbilical vein endothelial cell (HUVEC) and lymphatic endothelial cell (LEC) proliferation, which was not observed with CM from irradiated VEGF-C-siRNA-expressing A549 cells. Radiation-induced activation of the PI3K/Akt/mTOR signaling pathway increases VEGF-C expression in lung cancer cells, thereby promoting endothelial cell proliferation. (orig.) [German] Die vorliegende Studie untersucht, ob die Strahlung die Expression von VEGF-C (vascular endothelial growth factor C) mittels Aktivierung des PI3K/Akt/mTOR-Signalwegs induziert und anschliessend die endothelialen Zellen beeinflusst. Die durch Strahlentherapie induzierte Mikrolymphgefaessdichte (MLVD) im Tumor wurde in

  11. The relationship between radiation-induced apoptosis and the expression of cytokines in the rat's liver

    International Nuclear Information System (INIS)

    An, Eun Joo; Lee, Kyung Ja; Rhee, Chung Sik

    2000-01-01

    To determine the role of cytokines in the apoptosis of rat's liver following irradiation. Sprague-Dawley rats were irradiated to entire body with a single dose of 8 Gy. The rats were divided into 5 groups according to the sacrifice day after irradiation. The liver and blood after 1, 3, 5, 7, and 14 days irradiation were sampled for evaluation of mechanism of apoptosis and role of cytokine in relation to radiation-induced tissue damage. The study was composed of microscopic evaluation of liver tissue, in situ detection method for apoptosis, immunohistochemical stain of IL-1, IL-4, IL-6 and TNF, bioassay and radioimmunoassay of IL-6 in liver tissue and blood. Radiation-induced liver damage was noted from first day of radiation, and most severe parenchymal damage associated with infiltration of chronic inflammatory cells was seen in the groups of 5 days after radiation. A number of apoptosis were observed 1 day after radiation on both light microscope and in situ method. Afterwards, the number of apoptosis was gradually diminished. On immunohistochemical study, IL-1 and TNF were expressed 1, 3 days after radiation, but not expressed after that. IL-4 was not expressed in the entire groups. IL-6 was expressed with strong positivity in 1, 3 days after radiation. Bioassay and RIA of IL-6 in liver tissue and blood showed the highest value in 1 day after radiation, and the value is diminished after then. Apoptosis seemed to be the important mechanism of radiation-induced liver damage, and is possibly induced by the release of cytokines, such as IL-1, IL-6, TNF in view the simultaneously increased appearance of apoptosis and cytokines

  12. Expressions of Uroplakins in the Mouse Urinary Bladder with Cyclophosphamide-Induced Cystitis

    OpenAIRE

    Choi, Seong Hoo; Byun, Youngmin; Lee, Gilho

    2009-01-01

    Even though uroplakins (UPs) are believed to serve a strong protective barrier against toxic materials, cyclophosphamide (CP) causes extensive cystitis. We investigated the expression of UPs in the urothelium in CP induced mouse cystitis. A total of 27 ICR female mice received a single intraperitoneal injection of 200 mg CP/kg. Nine CP-treated mice and 6 controls were sequentially killed at 12, 24, and 72 hr post injection. Extensive cystitis and an increased vesical weight were seen. These a...

  13. Smad, but not MAPK, pathway mediates the expression of type I collagen in radiation induced fibrosis

    International Nuclear Information System (INIS)

    Yano, Hiroyuki; Hamanaka, Ryoji; Nakamura, Miki; Sumiyoshi, Hideaki; Matsuo, Noritaka; Yoshioka, Hidekatsu

    2012-01-01

    Highlights: ► We examine how radiation affects the expression level and signal pathway of collagen. ► TGF-β1 mRNA is elevated earlier than those of collagen genes after irradiation. ► Smad pathway mediates the expression of collagen in radiation induced fibrosis. ► MAPK pathways are not affected in the expression of collagen after irradiation. -- Abstract: Radiation induced fibrosis occurs following a therapeutic or accidental radiation exposure in normal tissues. Tissue fibrosis is the excessive accumulation of collagen and other extracellular matrix components. This study investigated how ionizing radiation affects the expression level and signal pathway of type I collagen. Real time RT-RCR showed that both α1and α2 chain of type I collagen mRNA were elevated from 48 h after irradiation with 10 Gy in NIH3T3 cells. The relative luciferase activities of both genes and type I collagen marker were elevated at 72 h. TGF-β1 mRNA was elevated earlier than those of type I collagen genes. A Western blot analysis showed the elevation of Smad phosphorylation at 72 h. Conversely, treatment with TGF-β receptor inhibitor inhibited the mRNA and relative luciferase activity of type I collagen. The phosphorylation of Smad was repressed with the inhibitor, and the luciferase activity was cancelled using a mutant construct of Smad binding site of α2(I) collagen gene. However, the MAPK pathways, p38, ERK1/2 and JNK, were not affected with specific inhibitors or siRNA. The data showed that the Smad pathway mediated the expression of type I collagen in radiation induced fibrosis.

  14. Oleocanthal Modulates Estradiol-Induced Gene Expression Involving Estrogen Receptor α.

    Science.gov (United States)

    Keiler, Annekathrin Martina; Djiogue, Sefirin; Ehrhardt, Tino; Zierau, Oliver; Skaltsounis, Leandros; Halabalaki, Maria; Vollmer, Günter

    2015-09-01

    Oleocanthal is a bioactive compound from olive oil. It has attracted considerable attention as it is anti-inflammatory, antiproliferative, and has been shown to possess neuroprotective properties in vitro and in vivo. Delineated from its polyphenolic structure, the aim of this study was to characterize oleocanthal towards estrogenic properties. This might contribute to partly explain the beneficial effects described for the Mediterranean diet. Estrogenic properties of oleocanthal were assessed by different methods: a) stimulation of reporter gene activity in MVLN or RNDA cells either expressing estrogen receptor α or β, b) stimulation of luciferase reporter gene activity in U2OS osteosarcoma cells expressing estrogen receptor α or β, and c) elucidation of the impact on estradiol-induced gene expression in U2OS cells transduced with both estrogen receptors. Depending on the cell line origin, oleocanthal inhibited luciferase activity (MVLN, U2OS-estrogen receptor β) or weakly induced reporter gene activity at 10 µM in U2OS-estrogen receptor α cells. However, oleocanthal inhibited stimulation of luciferase activity by estradiol from both estrogen receptors. Oleocanthal, if given alone, did not stimulate gene expression in U2OS cells, but it significantly modulated the response of estradiol. Oleocanthal enhanced the effect of estradiol on the regulation of those genes, which are believed to be regulated through heterodimeric estrogen receptors. As the estrogenic response pattern of oleocanthal is rather unique, we compared the results obtained with oleacein. Oleocanthal binds to both estrogen receptors inducing estradiol-agonistic or antiagonistic effects depending on the cell line. Regarding regulation of gene expression in U2OS-estrogen receptor α/β cells, oleocanthal and oleacein enhanced estradiol-mediated regulation of heterodimer-regulated genes. Georg Thieme Verlag KG Stuttgart · New York.

  15. Smad, but not MAPK, pathway mediates the expression of type I collagen in radiation induced fibrosis

    Energy Technology Data Exchange (ETDEWEB)

    Yano, Hiroyuki [Department of Matrix Medicine, Oita University, 1-1 Idaigaoka Hasama-machi, Yufu, Oita 879-5593 (Japan); Division of Radioisotope Research, Department of Research Support, Research Promotion Project, Oita University, 1-1 Idaigaoka Hasama-machi, Yufu, Oita 879-5593 (Japan); Hamanaka, Ryoji; Nakamura, Miki [Cell Biology, Faculty of Medicine, Oita University, 1-1 Idaigaoka Hasama-machi, Yufu, Oita 879-5593 (Japan); Sumiyoshi, Hideaki; Matsuo, Noritaka [Department of Matrix Medicine, Oita University, 1-1 Idaigaoka Hasama-machi, Yufu, Oita 879-5593 (Japan); Yoshioka, Hidekatsu, E-mail: hidey@oita-u.ac.jp [Department of Matrix Medicine, Oita University, 1-1 Idaigaoka Hasama-machi, Yufu, Oita 879-5593 (Japan)

    2012-02-17

    Highlights: Black-Right-Pointing-Pointer We examine how radiation affects the expression level and signal pathway of collagen. Black-Right-Pointing-Pointer TGF-{beta}1 mRNA is elevated earlier than those of collagen genes after irradiation. Black-Right-Pointing-Pointer Smad pathway mediates the expression of collagen in radiation induced fibrosis. Black-Right-Pointing-Pointer MAPK pathways are not affected in the expression of collagen after irradiation. -- Abstract: Radiation induced fibrosis occurs following a therapeutic or accidental radiation exposure in normal tissues. Tissue fibrosis is the excessive accumulation of collagen and other extracellular matrix components. This study investigated how ionizing radiation affects the expression level and signal pathway of type I collagen. Real time RT-RCR showed that both {alpha}1and {alpha}2 chain of type I collagen mRNA were elevated from 48 h after irradiation with 10 Gy in NIH3T3 cells. The relative luciferase activities of both genes and type I collagen marker were elevated at 72 h. TGF-{beta}1 mRNA was elevated earlier than those of type I collagen genes. A Western blot analysis showed the elevation of Smad phosphorylation at 72 h. Conversely, treatment with TGF-{beta} receptor inhibitor inhibited the mRNA and relative luciferase activity of type I collagen. The phosphorylation of Smad was repressed with the inhibitor, and the luciferase activity was cancelled using a mutant construct of Smad binding site of {alpha}2(I) collagen gene. However, the MAPK pathways, p38, ERK1/2 and JNK, were not affected with specific inhibitors or siRNA. The data showed that the Smad pathway mediated the expression of type I collagen in radiation induced fibrosis.

  16. Lung inflammation induces IL-1β expression in hypoglossal neurons in rat brainstem

    Science.gov (United States)

    Jafri, Anjum; Belkadi, Abdelmadjid; Zaidi, Syed I. A.; Getsy, Paulina; Wilson, Christopher G.; Martin, Richard J.

    2013-01-01

    Perinatal inflammation is associated with respiratory morbidity. Immune modulation of brainstem respiratory control centers may provide a link for this pathobiology. We exposed 11-day old rats to intratracheal lipopolysaccharide (LPS, 0.5 µg/g) to test the hypothesis that intrapulmonary inflammation increases expression of the proinflammatory cytokine IL-1β within respiratory-related brainstem regions. Intratracheal LPS resulted in a 32% increase in IL-1β protein expression in the medulla oblongata. In situ hybridization showed increased intensity of IL-1β mRNA but no change in neuronal numbers. Co-localization experiments showed that hypoglossal neurons express IL-1β mRNA and immunostaining showed a 43% increase in IL-1β protein-expressing cells after LPS exposure. LPS treatment also significantly increased microglial cell numbers though they did not express IL-1β mRNA. LPS-induced brainstem expression of neuronal IL-1β mRNA and protein may have implications for our understanding of the vulnerability of neonatal respiratory control in response to a peripheral pro-inflammatory stimulus. PMID:23648475

  17. A BAC-based transgenic mouse specifically expresses an inducible Cre in the urothelium.

    Directory of Open Access Journals (Sweden)

    Tian Huai Shen

    Full Text Available Cre-loxp mediated conditional knockout strategy has played critical roles for revealing functions of many genes essential for development, as well as the causal relationships between gene mutations and diseases in the postnatal adult mice. One key factor of this strategy is the availability of mice with tissue- or cell type-specific Cre expression. However, the success of the traditional molecular cloning approach to generate mice with tissue specific Cre expression often depends on luck. Here we provide a better alternative by using bacterial artificial chromosome (BAC-based recombineering to insert iCreERT2 cDNA at the ATG start of the Upk2 gene. The BAC-based transgenic mice express the inducible Cre specifically in the urothelium as demonstrated by mRNA expression and staining for LacZ expression after crossing with a Rosa26 reporter mouse. Taking into consideration the size of the gene of interest and neighboring genes included in a BAC, this method should be widely applicable for generation of mice with tissue specific gene expression or deletions in a more specific manner than previously reported.

  18. Effect of bcl-2 antisense oligodexynucleotides on chemotherapy efficacy of Vp-16 on human small cell lung cancer cell line NCI-H69

    International Nuclear Information System (INIS)

    He Wenqian; Liu Zhonghua

    2007-01-01

    Objective: To study the effect of bcl-2 antisense oligodexynucleotides on chemotherapy efficacy of Vp-16 on human small cell lung cancer cell line NCI-H69. Methods: Cultured NCI-H69 cells were derided into 4 groups: bcl-2 antisense oligodexynucleotides (ASODN) added, sense oligodexynucleotides (SODN) added, nonsense oligodexynucleotides (NSODN) added and control (no nucleotides added), the oligodexynucleotides were transfected into the cultured cells with oligofectamine. The cellular expression of Bcl-2 protein 72h later was examined with Western-Blot. The four different groups of cultured tumor cells were treated with etopside(Vp-16) at different concentrations (0, 0.25, 0.5, 1.0, 2.0 and 4.0 μg/ml) for 48hr then the cell survival fraction was assessed with MTY test. Results: The apoptotic rate of cells in the ASODN group was significantly higher than that of the control group, also, the survival fraction of cells in ASODN group was significantly lower than that of the control group. The Bcl-2 protein expression in ASODN group was significantly lower than that in the control group, but no inhibition was observed in SODN and NSODN groups. Conclusion: The bcl-2 ASODN could enhance the sensitivity to chemotherapy with Vp-16 in small cell lung cancer cell line NCI-H69 by effectively blocking bcl-2 gene expression. (authors)

  19. Lymphocytes from wasted mice express enhanced spontaneous and {gamma}-ray-induced apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Woloschak, G.E. [Argonne National Lab., IL (United States)]|[Loyola Univ. Medical Center, Maywood, IL (United States); Chang-Liu, Chin-Mei [Argonne National Lab., IL (United States); Chung, Jen; Libertin, C.R. [Loyola Univ. Medical Center, Maywood, IL (United States)

    1993-09-01

    Mice bearing the autosomal recessive mutation wasted (wst/wst) display a disease pattern including faulty repair of DNA damage in lymphocytes after radiation exposure, neurologic abnormalities, and immunodeficiency. Many of the features of this mouse model have suggested a premature or increased spontaneous frequency of apoptosis in thymocytes; past work has shown an inability to establish cultured T cell lines, an abnormally high death rate of stimulated T cells in culture, and an increased sensitivity of T cells to the killing effects of ionizing radiations in wst/wst mice relative to controls. The experiments reported here were designed to examine splenic and thymic lymphocytes from wasted and control mice for signs of early apoptosis. Our results revealed enhanced expression of Rp-8 mRNA (associated with apoptosis) in thymic lymphocytes and reduced expression in splenic lymphocytes of wst/wst mice relative to controls; expression of Rp-2 and Td-30 mRNA (induced during apoptosis) were not detectable in spleen or thymus. Higher spontaneous DNA fragmentation was observed in wasted mice than in controls; however, {gamma}-ray-induced DNA fragmentation peaked at a lower dose and occurred to a greater extent in wasted mice relative to controls. These results provide evidence for high spontaneous and {gamma}-ray-induced apoptosis in T cells of wasted mice as a mechanism underlying the observed lymphocyte and DNA repair abnormalities.

  20. Effect of ethylene action inhibitors upon wound-induced gene expression in tomato pericarp

    International Nuclear Information System (INIS)

    Henstrand, J.M.; Handa, A.K.

    1989-01-01

    The contribution of wound-ethylene to wound-induced gene expression was investigated in unripe tomato pericarp using inhibitors of ethylene action. Wounded unripe tomato pericarp was treated with 2,5-norbornadiene or silver thiosulfate to inhibit specifically the induction of ethylene-dependent mRNA species. Poly(A) + RNAs isolated from these tissues after 12 hours of wounding were translated in vitro in a rabbit reticulocyte lysate system and [ 35 S]methionine-labeled polypeptides were compared to unwounded controls after separation by one and two-dimensional polyacrylamide gel electrophoresis. Results show that mechanical wounding induces a dramatic shift in gene expression (over 50 mRNA species) but expression of less than 15% of these genes is affected by the treatment with ethylene action inhibitors. A selective decrease in mRNAs coding for a 37 kilodalton doublet and 75 kilodalton polypeptides is observed in 2,5-norbornadiene and silver thiosulfate treated wounded pericarp. Levels of hydroxyproline-rich glycoprotein mRNAs induced in wounded tissue were not influenced by inhibitors of ethylene action

  1. Bifunctional effects of fucoidan on the expression of inducible nitric oxide synthase

    International Nuclear Information System (INIS)

    Yang, Jin Won; Yoon, Se Young; Oh, Soo Jin; Kim, Sang Kyum; Kang, Keon Wook

    2006-01-01

    Algal fucoidan is a marine sulfated polysaccharide with a wide variety of biological activities including anti-thrombotic and anti-inflammatory effects. This study evaluated the effect of fucoidan on the expression of inducible nitric oxide synthase (iNOS) in a macrophage cell line, RAW264.7. Low concentration range of fucoidan (10 μg/ml) increased the basal expression level of iNOS in quiescent macrophages. However, we found for the first time that fucoidan inhibited the release of nitric oxide (NO) in RAW264.7 cells stimulated with lipopolysaccharide (LPS). Western blot analysis revealed that fucoidan suppressed the LPS-induced expression of the inducible nitric oxide synthase (iNOS) gene. Moreover, the activation of both nuclear factor-κB (NF-κB) and activator protein 1 (AP-1) are key steps in the transcriptional activation of the iNOS gene. Here, it was revealed that fucoidan selectively suppressed AP-1 activation, and that the activation of AP-1 appears to be essential for the induction of iNOS in activated macrophages. This inhibitory effect on AP-1 activation by fucoidan might be associated with its NO blocking and anti-inflammatory effects

  2. Nontypeable Haemophilus influenzae induces sustained lung oxidative stress and protease expression.

    Directory of Open Access Journals (Sweden)

    Paul T King

    Full Text Available Nontypeable Haemophilus influenzae (NTHi is a prevalent bacterium found in a variety of chronic respiratory diseases. The role of this bacterium in the pathogenesis of lung inflammation is not well defined. In this study we examined the effect of NTHi on two important lung inflammatory processes 1, oxidative stress and 2, protease expression. Bronchoalveolar macrophages were obtained from 121 human subjects, blood neutrophils from 15 subjects, and human-lung fibroblast and epithelial cell lines from 16 subjects. Cells were stimulated with NTHi to measure the effect on reactive oxygen species (ROS production and extracellular trap formation. We also measured the production of the oxidant, 3-nitrotyrosine (3-NT in the lungs of mice infected with this bacterium. NTHi induced widespread production of 3-NT in mouse lungs. This bacterium induced significantly increased ROS production in human fibroblasts, epithelial cells, macrophages and neutrophils; with the highest levels in the phagocytic cells. In human macrophages NTHi caused a sustained, extracellular production of ROS that increased over time. The production of ROS was associated with the formation of macrophage extracellular trap-like structures which co-expressed the protease metalloproteinase-12. The formation of the macrophage extracellular trap-like structures was markedly inhibited by the addition of DNase. In this study we have demonstrated that NTHi induces lung oxidative stress with macrophage extracellular trap formation and associated protease expression. DNase inhibited the formation of extracellular traps.

  3. Increased expression of PPARγ in high fat diet-induced liver steatosis in mice

    International Nuclear Information System (INIS)

    Inoue, Mitsutaka; Ohtake, Takaaki; Motomura, Wataru; Takahashi, Nobuhiko; Hosoki, Yayoi; Miyoshi, Shigeki; Suzuki, Yasuaki; Saito, Hiroyuki; Kohgo, Yutaka; Okumura, Toshikatsu

    2005-01-01

    The present study was performed to examine a hypothesis that peroxisome proliferator-activated receptor γ (PPARγ) is implicated in high fat diet-induced liver steatosis. Mice were fed with control or high fat diet containing approximately 10% or 80% cholesterol, respectively. Macroscopic and microscopic findings demonstrated that lipid accumulation in the liver was observed as early as 2 weeks after high fat diet and that high fat diet for 12 weeks developed a fatty liver phenotype, establishing a novel model of diet-induced liver steatosis. Gene profiling with microarray and real-time PCR studies demonstrated that among genes involved in lipid metabolism, adipogenesis-related genes, PPARγ and its targeted gene, CD36 mRNA expression was specifically up-regulated in the liver by high fat diet for 2 weeks. Immunohistochemical study revealed that PPARγ protein expression is increased in the nuclei of hepatocytes by high fat diet. It was also shown that protein expression of cAMP response element-binding protein (CREB), an upstream molecule of PPARγ, in the liver was drastically suppressed by high fat diet. All these results suggest for the first time that the CREB-PPARγ signaling pathway may be involved in the high fat diet-induced liver steatosis

  4. Substrate Stiffness Influences Doxorubicin-Induced p53 Activation via ROCK2 Expression

    Directory of Open Access Journals (Sweden)

    Takahiro Ebata

    2017-01-01

    Full Text Available The physical properties of the extracellular matrix (ECM, such as stiffness, are involved in the determination of the characteristics of cancer cells, including chemotherapy sensitivity. Resistance to chemotherapy is often linked to dysfunction of tumor suppressor p53; however, it remains elusive whether the ECM microenvironment interferes with p53 activation in cancer cells. Here, we show that, in MCF-7 breast cancer cells, extracellular stiffness influences p53 activation induced by the antitumor drug doxorubicin. Cell growth inhibition by doxorubicin was increased in response to ECM rigidity in a p53-dependent manner. The expression of Rho-associated coiled coil-containing protein kinase (ROCK 2, which induces the activation of myosin II, was significantly higher when cells were cultured on stiffer ECM substrates. Knockdown of ROCK2 expression or pharmacological inhibition of ROCK decreased doxorubicin-induced p53 activation. Our results suggest that a soft ECM causes downregulation of ROCK2 expression, which drives resistance to chemotherapy by repressing p53 activation.

  5. Reversing multidrug resistance in Caco-2 by silencing MDR1, MRP1, MRP2, and BCL-2/BCL-xL using liposomal antisense oligonucleotides.

    Directory of Open Access Journals (Sweden)

    Yu-Li Lo

    Full Text Available Multidrug resistance (MDR is a major impediment to chemotherapy. In the present study, we designed antisense oligonucleotides (ASOs against MDR1, MDR-associated protein (MRP1, MRP2, and/or BCL-2/BCL-xL to reverse MDR transporters and induce apoptosis, respectively. The cationic liposomes (100 nm composed of N-[1-(2,3-dioleyloxypropyl]-n,n,n-trimethylammonium chloride and dioleoyl phosphotidylethanolamine core surrounded by a polyethylene glycol (PEG shell were prepared to carry ASOs and/or epirubicin, an antineoplastic agent. We aimed to simultaneously suppress efflux pumps, provoke apoptosis, and enhance the chemosensitivity of human colon adenocarcinoma Caco-2 cells to epirubicin. We evaluated encapsulation efficiency, particle size, cytotoxicity, intracellular accumulation, mRNA levels, cell cycle distribution, and caspase activity of these formulations. We found that PEGylated liposomal ASOs significantly reduced Caco-2 cell viability and thus intensified epirubicin-mediated apoptosis. These formulations also decreased the MDR1 promoter activity levels and enhanced the intracellular retention of epirubicin in Caco-2 cells. Epirubicin and ASOs in PEGylated liposomes remarkably decreased mRNA expression levels of human MDR1, MRP1, MRP2, and BCL-2. The combined treatments all significantly increased the mRNA expressions of p53 and BAX, and activity levels of caspase-3, -8, and -9. The formulation of epirubicin and ASOs targeting both pump resistance of MDR1, MRP1, and MRP2 and nonpump resistance of BCL-2/BCL-xL demonstrated more superior effect to all the other formulations used in this study. Our results provide a novel insight into the mechanisms by which PEGylated liposomal ASOs against both resistance types act as activators to epirubicin-induced apoptosis through suppressing MDR1, MRP1, and MRP2, as well as triggering intrinsic mitochondrial and extrinsic death receptor pathways. The complicated regulation of MDR highlights the necessity

  6. Fisetin Induces Apoptosis Through p53-Mediated Up-Regulation of DR5 Expression in Human Renal Carcinoma Caki Cells

    Directory of Open Access Journals (Sweden)

    Kyoung-jin Min

    2017-08-01

    Full Text Available Fisetin is a natural compound found in fruits and vegetables such as strawberries, apples, cucumbers, and onions. Since fisetin can elicit anti-cancer effects, including anti-proliferation and anti-migration, we investigated whether fisetin induced apoptosis in human renal carcinoma (Caki cells. Fisetin markedly induced sub-G1 population and cleavage of poly (ADP-ribose polymerase (PARP, which is a marker of apoptosis, and increased caspase activation. We found that pan-caspase inhibitor (z-VAD-fmk inhibited fisetin-induced apoptosis. In addition, fisetin induced death receptor 5 (DR5 expression at the transcriptional level, and down-regulation of DR5 by siRNA blocked fisetin-induced apoptosis. Furthermore, fisetin induced p53 protein expression through up-regulation of protein stability, whereas down-regulation of p53 by siRNA markedly inhibited fisetin-induced DR5 expression. In contrast, fisetin induced up-regulation of CHOP expression and reactive oxygen species production, which had no effect on fisetin-induced apoptosis. Taken together, our study demonstrates that fisetin induced apoptosis through p53 mediated up-regulation of DR5 expression at the transcriptional level.

  7. Fisetin Induces Apoptosis Through p53-Mediated Up-Regulation of DR5 Expression in Human Renal Carcinoma Caki Cells.

    Science.gov (United States)

    Min, Kyoung-Jin; Nam, Ju-Ock; Kwon, Taeg Kyu

    2017-08-02

    Fisetin is a natural compound found in fruits and vegetables such as strawberries, apples, cucumbers, and onions. Since fisetin can elicit anti-cancer effects, including anti-proliferation and anti-migration, we investigated whether fisetin induced apoptosis in human renal carcinoma (Caki) cells. Fisetin markedly induced sub-G1 population and cleavage of poly (ADP-ribose) polymerase (PARP), which is a marker of apoptosis, and increased caspase activation. We found that pan-caspase inhibitor (z-VAD-fmk) inhibited fisetin-induced apoptosis. In addition, fisetin induced death receptor 5 (DR5) expression at the transcriptional level, and down-regulation of DR5 by siRNA blocked fisetin-induced apoptosis. Furthermore, fisetin induced p53 protein expression through up-regulation of protein stability, whereas down-regulation of p53 by siRNA markedly inhibited fisetin-induced DR5 expression. In contrast, fisetin induced up-regulation of CHOP expression and reactive oxygen species production, which had no effect on fisetin-induced apoptosis. Taken together, our study demonstrates that fisetin induced apoptosis through p53 mediated up-regulation of DR5 expression at the transcriptional level.

  8. Bcl-2 antisense therapy in B-cell malignant proliferative disorders.

    Science.gov (United States)

    Chanan-Khan, Asher; Czuczman, Myron S

    2004-08-01

    Overexpression of Bcl-2 oncogene has been clinically associated with an aggressive clinical course, chemotherapy and radiotherapy resistance, and poor survival in patients with malignant B-cell disorders. Patients with relapsed or refractory chronic lymphocytic leukemia, multiple myeloma, or non-Hodgkin's lymphoma have limited therapeutic options. Preclinical and early clinical data have shown that Bcl-2 oncoprotein can be decreased by Bcl-2 antisense therapy. Also, downregulation of Bcl-2 protein can result in reversal of chemotherapy resistance and improved antitumor activity of biologic agents. Various clinical trials are evaluating the role of targeting Bcl-2 as a mechanism to enhance the antitumor potential of chemotherapy and immunotherapy. Early results from these clinical studies are encouraging and confirm the proof of principle for antisense therapy. As current data mature, these trials will hopefully validate preliminary results and establish Bcl-2 antisense as an important addition to the current armamentarium used in the treatment of patients with B-cell neoplasms.

  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...... 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...... is an efficient vector for PNA delivery yielding significantly higher (up to 10-fold) antisense activity than an analogous PNA-octaarginine conjugate, even in the presence of chloroquine, which only slightly enhances the PEI-PNA activity. The PEI-PEG conjugates are preferred due to lower acute cellular toxicity...

  10. Hypoxia-inducible factor 1 alpha expression increases during colorectal carcinogenesis and tumor progression

    International Nuclear Information System (INIS)

    Simiantonaki, Nektaria; Taxeidis, Marios; Jayasinghe, Caren; Kurzik-Dumke, Ursula; Kirkpatrick, Charles James

    2008-01-01

    Hypoxia-inducible factor 1 alpha (HIF-1α) is involved in processes promoting carcinogenesis of many tumors. However, its role in the development of colorectal cancer is unknown. To investigate the significance of HIF-1α during colorectal carcinogenesis and progression we examined its expression in precursor lesions constituting the conventional and serrated pathways, as well as in non-metastatic and metastatic adenocarcinomas. Immunohistochemistry and Western blot is used to analyse HIF-1α expression in normal colonic mucosa, hyperplastic polyps (HPP), sessile serrated adenomas (SSA), low-grade (TA-LGD) and high-grade (TA-HGD) traditional adenomas as well as in non-metastatic and metastatic colorectal adenocarcinomas. Eight colorectal carcinoma cell lines are tested for their HIF-1α inducibility after lipopolysaccharide (LPS) stimulation using western blot and immunocytochemistry. In normal mucosa, HPP and TA-LGD HIF-1α was not expressed. In contast, perinuclear protein accumulation and nuclear expression of HIF-1α were shown in half of the examined SSA and TA-HGD. In all investigated colorectal carcinomas a significant nuclear HIF-1α overexpression compared to the premalignant lesions was observed but a significant correlation with the metastatic status was not found. Nuclear HIF-1α expression was strongly accumulated in perinecrotic regions. In these cases HIF-1α activation was seen in viable cohesive tumor epithelia surrounding necrosis and in dissociated tumor cells, which subsequently die. Enhanced distribution of HIF-1α was also seen in periiflammatory regions. In additional in vitro studies, treatment of diverse colorectal carcinoma cell lines with the potent pro-inflammatory factor lipopolysaccharide (LPS) led to HIF-1α expression and nuclear translocation. We conclude that HIF-1α expression occurs in early stages of colorectal carcinogenesis and achieves a maximum in the invasive stage independent of the metastatic status. Perinecrotic

  11. Expression of inducible nitric oxide synthase in trigeminal ganglion cells during culture

    DEFF Research Database (Denmark)

    Jansen-Olesen, Inger; Zhou, MingFang; Zinck, Tina Jovanovic

    2005-01-01

    RNA and protein could be detected. The data suggest that iNOS expression may be a molecular mechanism mediating the adaptive response of trigeminal ganglia cells to the serum free stressful stimulus the culture environment provides. It may act as a cellular signalling molecule that is expressed after cell......Nitric oxide (NO) is an important signalling molecule that has been suggested to be a key molecule for induction and maintenance of migraine attacks based on clinical studies, animal experimental studies and the expression of nitric oxide synthase (NOS) immunoreactivity within the trigeminovascular......, reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting. In trigeminal ganglia cells not subjected to culture, endothelial (e) and neuronal (n) but not inducible (i) NOS mRNA and protein were detected. Culture of rat neurones resulted in a rapid axonal outgrowth of NOS positive...

  12. Regulation of endogenous human gene expression by ligand-inducible TALE transcription factors.

    Science.gov (United States)

    Mercer, Andrew C; Gaj, Thomas; Sirk, Shannon J; Lamb, Brian M; Barbas, Carlos F

    2014-10-17

    The construction of increasingly sophisticated synthetic biological circuits is dependent on the development of extensible tools capable of providing specific control of gene expression in eukaryotic cells. Here, we describe a new class of synthetic transcription factors that activate gene expression in response to extracellular chemical stimuli. These inducible activators consist of customizable transcription activator-like effector (TALE) proteins combined with steroid hormone receptor ligand-binding domains. We demonstrate that these ligand-responsive TALE transcription factors allow for tunable and conditional control of gene activation and can be used to regulate the expression of endogenous genes in human cells. Since TALEs can be designed to recognize any contiguous DNA sequence, the conditional gene regulatory system described herein will enable the design of advanced synthetic gene networks.

  13. Role of promoter element in c-mpl gene expression induced by TPO.

    Science.gov (United States)

    Sunohara, Masataka; Morikawa, Shigeru; Fuse, Akira; Sato, Iwao

    2013-01-01

    Thrombopoietin (TPO) and its receptor, c-Mpl, play the crucial role for the development of megakaryocyte and considered to regulate megakaryocytopoiesis. Previously we reported that TPO increased the c-mpl promoter activity determined by a transient expression system using a vector containing the luciferase gene as a reporter and the expression of the c-mpl gene is modulated by transcription through a protein kinase C (PKC)-dependent pathway in the megakaryoblastic cells. In this research, to elucidate the required elements in c-mpl promoter, the promoter activity of the deletion constructs and site-directed mutagenesis were measured by a transient transfection assay system. Destruction of -77GATA in c-mpl promoter decreased the activity by 22.8%. Our study elucidated that -77GATA involved in TPO-induced c-mpl gene expression in a human megakaryoblastic cell line, CMK.

  14. Nuclear antigen expression by ultraviolet light irradiation - a contribution to the UV-induced autoimmunity

    Energy Technology Data Exchange (ETDEWEB)

    Wollina, U

    1986-05-01

    A review is given of nuclear antigen expression due to UVB, UVA, and PUVA. UVB alters DNA resulting in strong immunogenic UVDNA and complementary antibodies. Antibodies to UVDNA cross react with double-stranded DNA. UVDNA plays a (hypothetical) role in the induction of cutaneous lesions in lupus erythematosus (LE). Investigations of SS-A/Ro expression due to UVB seem to be more important under this view. Antibodies against SS-A/Ro are related to an increased photosensitivity in LE. PUVA and UVA are able to induce antinuclear antibodies of unknown specificity. It is likely that PUVA enhances SS-A/Ro expression in vitro. The results are discussed in sense of LE photobiology and unwanted side effects of photo(chemo)therapy in psoriasis. 54 references.

  15. Human Cementum Protein 1 induces expression of bone and cementum proteins by human gingival fibroblasts

    International Nuclear Information System (INIS)

    Carmona-Rodriguez, Bruno; Alvarez-Perez, Marco Antonio; Narayanan, A. Sampath; Zeichner-David, Margarita; Reyes-Gasga, Jose; Molina-Guarneros, Juan; Garcia-Hernandez, Ana Lilia; Suarez-Franco, Jose Luis; Chavarria, Ivet Gil; Villarreal-Ramirez, Eduardo; Arzate, Higinio

    2007-01-01

    We recently presented evidence showing that a human cementoblastoma-derived protein, named Cementum Protein 1 (CEMP1) may play a role as a local regulator of cementoblast differentiation and cementum-matrix mineralization. This protein was shown to be expressed by cementoblasts and progenitor cells localized in the periodontal ligament. In this study we demonstrate that transfection of CEMP1 into human gingival fibroblasts (HGF) induces mineralization and expression of bone and cementum-matrix proteins. The transfected HGF cells had higher alkaline phosphatase activity and proliferation rate and they expressed genes for alkaline phosphatase, bone sialoprotein, osteocalcin, osteopontin, the transcription factor Runx2/Cbfa1, and cementum attachment protein (CAP). They also produced biological-type hydroxyapatite. These findings indicate that the CEMP1 might participate in differentiation and mineralization of nonosteogenic cells, and that it might have a potential function in cementum and bone formation

  16. Nuclear antigen expression by ultraviolet light irradiation - a contribution to the UV-induced autoimmunity

    International Nuclear Information System (INIS)

    Wollina, U.

    1986-01-01

    A review is given about nuclear antigen expression due to UVB, UVA, and PUVA. UVB alters DNA resulting in strong immunogenic UVDNA and complementary antibodies. Antibodies to UVDNA cross react with double-stranded DNA. UVDNA plays a (hypothetical) role in the induction of cutaneous lesions in lupus erythematosus (LE). Investigations about SS-A/Ro expression due to UVB seem to be more important under this view. Antibodies against SS-A/Ro are related to an increased photosensitivity in LE. PUVA and UVA are able to induce antinuclear antibodies of unknown specificity. It is likely that PUVA enhances SS-A/Ro expression in vitro. The results are discussed in sense of LE photobiology and unwanted side effects of photo(chemo)therapy in psoriasis. (author)

  17. Expression of FSH receptor in ovary tissue of rats with letrozole-induced polycystic ovary syndrome

    International Nuclear Information System (INIS)

    Guo Hongsheng; An Changxin; Chen Dong

    2009-01-01

    Objective: To investigate the expressions of FSH receptor mRNA and protein in ovary tissue in rats with letrozole-induced polycystic ovary syndrome (PCOS), and to provide experimental data for the model application. Methods: Forty rats were randomly divided into two groups (n=20), in PCOS model group letrozole was administered once daily during 21 d, and in control group without any treatment. The gonadal hormone concentrations in serum were determined by radioimmunoassay, the histologic changes in ovaries were observed by HE staining, the expression of FSH receptor gene in ovary tissue was detected by realtime -PCR, Western blotting and immunohistochemistry. Results: Compared with control group, estradiol (E 2 ) and progesterone in model group showed a considerable reduction (P 0.05). Compared with control group, the ovaries from model group showed high incidence of subcapsular ovarian cyst and capsular thickening and decreased number of corpora lute a. The expressions of FSH receptor mRNA and protein were significantly higher in model group than those in control group (P<0.05). Conclusion: The expression of FSH receptor gene in letrozole-induced polycystic ovaries is similar with that of PCOS women, the rat model is proved to be an ideal PCOS animal model to study the pathophysiology of PCOS. (authors)

  18. Expression of HSF2 decreases in mitosis to enable stress-inducible transcription and cell survival

    Science.gov (United States)

    Elsing, Alexandra N.; Aspelin, Camilla; Björk, Johanna K.; Bergman, Heidi A.; Himanen, Samu V.; Kallio, Marko J.; Roos-Mattjus, Pia

    2014-01-01

    Unless mitigated, external and physiological stresses are detrimental for cells, especially in mitosis, resulting in chromosomal missegregation, aneuploidy, or apoptosis. Heat shock proteins (Hsps) maintain protein homeostasis and promote cell survival. Hsps are transcriptionally regulated by heat shock factors (HSFs). Of these, HSF1 is the master regulator and HSF2 modulates Hsp expression by interacting with HSF1. Due to global inhibition of transcription in mitosis, including HSF1-mediated expression of Hsps, mitotic cells are highly vulnerable to stress. Here, we show that cells can counteract transcriptional silencing and protect themselves against proteotoxicity in mitosis. We found that the condensed chromatin of HSF2-deficient cells is accessible for HSF1 and RNA polymerase II, allowing stress-inducible Hsp expression. Consequently, HSF2-deficient cells exposed to acute stress display diminished mitotic errors and have a survival advantage. We also show that HSF2 expression declines during mitosis in several but not all human cell lines, which corresponds to the Hsp70 induction and protection against stress-induced mitotic abnormalities and apoptosis. PMID:25202032

  19. Vibration induced hearing loss in guinea pig cochlea: expression of TNF-alpha and VEGF.

    Science.gov (United States)

    Zou, Jing; Pyykkö, Ilmari; Sutinen, Päivi; Toppila, Esko

    2005-04-01

    Transcranial vibration was applied for seven animals at a frequency of 250 Hz for 15 min, and five animals were used as normal controls to investigate cellular and molecular mechanism linked to vibration-induced hearing loss in animal model. Compound action potential (CAP) thresholds were measured by round window niche electrode. The expression of tumour necrosis factor alpha (TNF-alpha) and its receptors (TNF R1, TNF R2), vascular endothelium growth factor (VEGF) and its receptors (VEGF R1, VEGF R2) were analysed by immunohistochemistry. Transcranial vibration caused expression of TNF-alpha, TNF R1 and TNF R2 in the cochlea and the expression of TNF R2 was stronger than that of TNF R1. Vibration also induced VEGF and VEGF R2 expression in the cochlea. The average immediate hearing loss was 62 dB and after three days still 48 dB. It is concluded that transcranial vibration as during temporal bone drilling produces cochlear shear stress that is connected with up-regulation of TNF-alpha and its receptors. Also VEGF and VEGF R2 are up-regulated. These responses may be linked to both the damage and repair process of the cochlea.

  20. Translational control is a major contributor to hypoxia induced gene expression

    International Nuclear Information System (INIS)

    Beucken, Twan van den; Magagnin, Michael G.; Jutten, Barry; Seigneuric, Renaud; Lambin, Philippe; Koritzinsky, Marianne; Wouters, Bradly G.

    2011-01-01

    Background and purpose: Hypoxia is a common feature of solid tumors that is associated with an aggressive phenotype, resistance to therapy and poor prognosis. Major contributors to these adverse effects are the transcriptional program activated by the HIF family of transcription factors as well as the translational response mediated by PERK-dependent phosphorylation of eIF2α and inhibition of mTORC1 activity. In this study we determined the relative contribution of both transcriptional and translational responses to changes in hypoxia induced gene expression. Material and methods: Total and efficiently translated (polysomal) mRNA was isolated from DU145 prostate carcinoma cells that were exposed for up to 24 h of hypoxia ( 2 ). Changes in transcription and translation were assessed using affymetrix microarray technology. Results: Our data reveal an unexpectedly large contribution of translation control on both induced and repressed gene expression at all hypoxic time points, particularly during acute hypoxia (2-4 h). Gene ontology analysis revealed that gene classes like transcription and signal transduction are stimulated by translational control whereas expression of genes involved in cell growth and protein metabolism are repressed during hypoxic conditions by translational control. Conclusions: Our data indicate that translation influences gene expression during hypoxia on a scale comparable to that of transcription.

  1. The tobacco smoke component acrolein induces glucocorticoid resistant gene expression via inhibition of histone deacetylase.

    Science.gov (United States)

    Randall, Matthew J; Haenen, Guido R M M; Bouwman, Freek G; van der Vliet, Albert; Bast, Aalt

    2016-01-05

    Chronic obstructive pulmonary disease (COPD) is the leading cause of cigarette smoke-related death worldwide. Acrolein, a crucial reactive electrophile found in cigarette smoke mimics many of the toxic effects of cigarette smoke-exposure in the lung. In macrophages, cigarette smoke is known to hinder histone deacetylases (HDACs), glucocorticoid-regulated enzymes that play an important role in the pathogenesis of glucocorticoid resistant inflammation, a common feature of COPD. Thus, we hypothesize that acrolein plays a role in COPD-associated glucocorticoid resistance. To examine the role of acrolein on glucocorticoid resistance, U937 monocytes, differentiated with PMA to macrophage-like cells were treated with acrolein for 0.5h followed by stimulation with hydrocortisone for 8h, or treated simultaneously with LPS and hydrocortisone for 8h without acrolein. GSH and nuclear HDAC activity were measured, or gene expression was analyzed by qPCR. Acrolein-mediated TNFα gene expression was not suppressed by hydrocortisone whereas LPS-induced TNFα expression was suppressed. Acrolein also significantly inhibited nuclear HDAC activity in macrophage-like cells. Incubation of recombinant HDAC2 with acrolein led to the formation of an HDAC2-acrolein adduct identified by mass spectrometry. Therefore, these results suggest that acrolein-induced inflammatory gene expression is resistant to suppression by the endogenous glucocorticoid, hydrocortisone. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  2. [The humoral immune response in mice induced by recombinant Lactococcus lactis expressing HIV-1 gag].

    Science.gov (United States)

    Zhao, Xiaofei; Zhang, Cairong; Liu, Xiaojuan; Ma, Zhenghai

    2014-11-01

    To analyze the humoral immune response induced by recombinant Lactococcus lactis expressing HIV-1 gag in mice immunized orally, intranasally, subcutaneously or in the combined way of above three. Fifty BALB/c mice were randomly divided into 5 groups, 10 mice per group. The mice were immunized consecutively three times at two week intervals with 10(9) CFU of recombinant Lactococcus lactis expressing gag through oral, intranasal, subcutaneous administration or the mix of them. The mice that were immunized orally with Lactococcus lactis containing PMG36e served as a control group. The sera of mice were collected before primary immunization and 2 weeks after each immunization to detect the gag specific IgG by ELISA. Compared with the control group, the higher titer of serum gag specific IgG was detected in the four groups immunized with recombinant Lactococcus lactis expressing gag, and it was the highest in the mixed immunization group (PLactococcus lactis expressing gag can induce humoral immune response in mice by oral, intranasal, subcutaneous injection or the mix of them, and the mixed immunization can enhance the immune effects of Lactococcus lactis vector vaccine.

  3. Expression of immediate-early genes in the dorsal cochlear nucleus in salicylate-induced tinnitus.

    Science.gov (United States)

    Hu, Shou-Sen; Mei, Ling; Chen, Jian-Yong; Huang, Zhi-Wu; Wu, Hao

    2016-02-01

    Spontaneous neuronal activity in dorsal cochlear nucleus (DCN) may be involved in the physiological processes underlying salicylate-induced tinnitus. As a neuronal activity marker, immediate-early gene (IEG) expression, especially activity-dependent cytoskeletal protein (Arc/Arg3.1) and the early growth response gene-1 (Egr-1), appears to be highly correlated with sensory-evoked neuronal activity. However, their relationships with tinnitus induced by salicylate have rarely been reported in the DCN. In this study, we assessed the effect of acute and chronic salicylate treatment on the expression of N-methyl D-aspartate receptor subunit 2B (NR2B), Arg3.1, and Egr-1. We also observed ultrastructural alterations in the DCN synapses in an animal model of tinnitus. Levels of mRNA and protein expression of NR2B and Arg3.1 were increased in rats that were chronically administered salicylate (200 mg/kg, twice daily for 3, 7, or 14 days). These levels returned to baseline 14 days after cessation of treatment. However, no significant changes were observed in Egr-1 gene expression in any groups. Furthermore, rats subjected to long-term salicylate administration showed more presynaptic vesicles, thicker and longer postsynaptic densities, and increased synaptic interface curvature. Alterations of Arg3.1 and NR2B may be responsible for the changes in the synaptic ultrastructure. These changes confirm that salicylate can cause neural plasticity changes at the DCN level.

  4. Prolactin, TNF alpha and nitric oxide expression in nitroso-N-methylurea-induced-mammary tumours

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    Vegh Irene

    2007-11-01

    Full Text Available Abstract Background The N-Nitrosomethylurea breast cancer model induced in rats is used for the study of carcinogenesis in mammary cancer, prostate, pancreas, etc. This model is very similar to human neoplastic disease. Methods The present experimental study was designed to assess whether metoclopramide administration has any effect on development of MNU-induced tumours, and evaluate the treatment of goserelin acetate on PRL, TNF alpha and NO expression. NMU was administered to female Wistar rats on 2 occasions (5 mg/100 g body w/rat. PRL and TNF alpha were performed by immune-assay. Nitric Oxide by semi automated-assay and ploidy analyses by flow cytometry. Results The administration of metoclopramide made the induction time shorter and increased the incidence and average of tumours per rat. Tumours development was inhibited by a goserelin chronic administration. The ploidy of adenocarcinoma was polyploid-aneuploid type (average S = 60%. It was higher basal PRL plasma levels in rats with NMU induced tumours than in basal controls without tumour (p Conclusion The increase of blood PRL levels in NMU-induced rats may be an indicator of a poor prognosis of mammary cancer evolution. The metoclopramide administration accelerates tumour growth. However goserelin administration achieves regression in tumour development associated to inhibition PRL, TNF alpha and NO expression.

  5. Nucleotide Pool Depletion Induces G-Quadruplex-Dependent Perturbation of Gene Expression

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    Charikleia Papadopoulou

    2015-12-01

    Full Text Available Nucleotide pool imbalance has been proposed to drive genetic instability in cancer. Here, we show that slowing replication forks by depleting nucleotide pools with hydroxyurea (HU can also give rise to both transient and permanent epigenetic instability of a reporter locus, BU-1, in DT40 cells. HU induces stochastic formation of Bu-1low variants in dividing cells, which have lost the H3K4me3 present in untreated cells. This instability is potentiated by an intragenic G quadruplex, which also promotes local H2Ax phosphorylation and transient heterochromatinization. Genome-wide, gene expression changes induced by HU significantly overlap with those resulting from loss of the G4-helicases FANCJ, WRN, and BLM. Thus, the effects of global replication stress induced by nucleotide pool depletion can be focused by local replication impediments caused by G quadruplex formation to induce epigenetic instability and changes in gene expression, a mechanism that may contribute to selectable transcriptional changes in cancer.

  6. The NALP3/Cryopyrin-Inflammasome Complex is Expressed in LPS-Induced Ocular Inflammation

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    José F. González-Benítez

    2008-01-01

    Full Text Available In the inflammosome complex, NALP3 or NALP1 binds to ASC and activates caspase-1 which induces IL-1β. In murine LPS-induced ocular inflammation, the production of IL-1β is increased. We suggest that NALP3- or NALP1-inflammasome complex can be participating in the LPS-induced ocular inflammation. In this work, eye, brain, testis, heart, spleen, and lung were obtained from C3H/HeN mice treated with LPS for 3 to 48 hours, and the expression of NALP1b,