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Sample records for cell cycle-related mrnas

  1. Cell cycle related /sup 125/IUDR-induced-division delay

    International Nuclear Information System (INIS)

    A series of experiments were run to determine if /sup 125/I-decays, in /sup 125/IUdR labeled DNA, specifically accumulated at 1, 3, 5, 7 and 9 hours after plating labeled mitotic cells caused a change in the rate or time of cell entry into mitosis. To accomplish this, a pool of labeled mitotic cells was selected in mitosis and plated in replicate flasks. /sup 125/I decays were accumulated in groups of cells by cooling (40C) for 2 hours starting at the designated times. After rewarding, colcemid was added to arrest cells in mitosis. The rate of cell progression into mitosis for each cell cycle time of accumulation was determined by scoring the mitotic index of cells sampled as a function of time after addition of the colcemid. The results are summarized: (1) Decays from /sup 125/I in /sup 125/I(UdR) labeled DNA reduced the rate of cell progression into mitosis and delayed the time of initiation of mitosis. (2) The reduced rate of progression and the delayed time of initiation of mitosis were independent of the cell cycle time that /sup 125/I-decays were accumulated. (3) The reduced rate of progression after cell cycle accumulation of /sup 125/I decay was statistically indistinguishable from the corresponding controls. (4) The delayed initiation of mitosis after specific cell cycle accumulation of /sup 125/I- decays was greater than the corresponding control. The relationship of these data to DNA and non-DNA division delay target(s) is emphasized

  2. Expression of cell cycle related genes in HL60 cells undergoing apoptosis by X-irradiation

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    Kim, Jin Hee [College of Medicine, Keimyung Univ., Taegu (Korea, Republic of); Park, In Kyu [College of Medicine, Kyungpook National Univ., Taegu (Korea, Republic of)

    1998-12-01

    To evaluate changes in expression of cell cycle related genes during apoptosis induced in HL60 cells by X-irradiation to understand molecular biologic aspects in mechanism of radiation therapy. HL-60 cell line (promyelocytic leukemia cell line was grown in culture media and irradiated with 8 Gy by linear accelerator (6 MV X-ray). At various times after irradiation, ranging from 3 to 48 hours were analyzed apoptotic DNA fragmentation assay for apoptosis and by western blot analysis and semi-quantitative RT-PCR for expression of cell cycle related genes (cyclin A, cyclin B, cyclin C, cyclin D1, cyclin E, cdc2, CDK2, CDK4, p16{sup INK4a}, p21{sup WAF1}, p27K{sup IP1}, E2F, PCNA and Rb). X-irradiation (8 Gy) induced apoptosis in HL-60 cell line. Cycline A protein increased after reaching its peak 48 h after radiation delivery and cyclin E, E2F, CDK2 and RB protein increased then decreased after radiation. Radiation induced up-regulation of the expression of E2F is due to mostly increase of phosphorylated retinoblastoma proteins (ppRb). Cyclin D1, PCNA, CDC1, CDK4 and p16{sup INK4a} protein underwent no significant change at any times after irradiation. There was not detected p21{sup WAF1} and p27{sup KIP1} protein. Cyclin A, B, C, mRNA decreased immediately after radiation and then increased at 12 h after radiation. Cyclin D1 mRNA increased immediately and then decreased with the lapse of time. CDK2 mRNA decreased at 3 h and increased at 6 h after radiation. CDK4 mRNA rapidly increased at 6 to 12 h after radiation. There was no change of expression of p16{sup INK4a} and not detected in expressin of p21{sup WAF1} and p27{sup KIP1} mRNA. We suggest that entry into S phaso may contribute to apoptosis of HL60 cells induced by irradiation. Increase of ppRb and decrease of pRb protein are related with radiation induced apoptosis of HL60 cells and tosis of HL60 cells induced by irradiation. Increase of ppRb and decrease of pRb protein are related with radiation induced

  3. Cell Cycle Related Differentiation of Bone Marrow Cells into Lung Cells

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    Dooner, Mark; Aliotta, Jason M.; Pimental, Jeffrey; Dooner, Gerri J.; Abedi, Mehrdad; Colvin, Gerald; Liu, Qin; Weier, Heinz-Ulli; Dooner, Mark S.; Quesenberry, Peter J.

    2007-12-31

    Green-fluorescent protein (GFP) labeled marrow cells transplanted into lethally irradiated mice can be detected in the lungs of transplanted mice and have been shown to express lung specific proteins while lacking the expression of hematopoietic markers. We have studied marrow cells induced to transit cell cycle by exposure to IL-3, IL-6, IL-11 and steel factor at different times of culture corresponding to different phases of cell cycle. We have found that marrow cells at the G1/S interface have a 3-fold increase in cells which assume a lung phenotype and that this increase is no longer seen in late S/G2. These cells have been characterized as GFP{sup +} CD45{sup -} and GFP{sup +} cytokeratin{sup +}. Thus marrow cells with the capacity to convert into cells with a lung phenotype after transplantation show a reversible increase with cytokine induced cell cycle transit. Previous studies have shown the phenotype of bone marrow stem cells fluctuates reversibly as these cells traverse cell cycle, leading to a continuum model of stem cell regulation. The present studies indicate that marrow stem cell production of nonhematopoietic cells also fluctuates on a continuum.

  4. Translation dynamics of single mRNAs in live cells and neurons.

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    Wu, Bin; Eliscovich, Carolina; Yoon, Young J; Singer, Robert H

    2016-06-17

    Translation is the fundamental biological process converting mRNA information into proteins. Single-molecule imaging in live cells has illuminated the dynamics of RNA transcription; however, it is not yet applicable to translation. Here, we report single-molecule imaging of nascent peptides (SINAPS) to assess translation in live cells. The approach provides direct readout of initiation, elongation, and location of translation. We show that mRNAs coding for endoplasmic reticulum (ER) proteins are translated when they encounter the ER membrane. Single-molecule fluorescence recovery after photobleaching provides direct measurement of elongation speed (5 amino acids per second). In primary neurons, mRNAs are translated in proximal dendrites but repressed in distal dendrites and display "bursting" translation. This technology provides a tool with which to address the spatiotemporal translation mechanism of single mRNAs in living cells. PMID:27313041

  5. Ghrelin regulates cell cycle-related gene expression in cultured hippocampal neural stem cells.

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    Chung, Hyunju; Park, Seungjoon

    2016-08-01

    We have previously demonstrated that ghrelin stimulates the cellular proliferation of cultured adult rat hippocampal neural stem cells (NSCs). However, little is known about the molecular mechanisms by which ghrelin regulates cell cycle progression. The purpose of this study was to investigate the potential effects of ghrelin on cell cycle regulatory molecules in cultured hippocampal NSCs. Ghrelin treatment increased proliferation assessed by CCK-8 proliferation assay. The expression levels of proliferating cell nuclear antigen and cell division control 2, well-known cell-proliferating markers, were also increased by ghrelin. Fluorescence-activated cell sorting analysis revealed that ghrelin promoted progression of cell cycle from G0/G1 to S phase, whereas this progression was attenuated by the pretreatment with specific inhibitors of MEK/extracellular signal-regulated kinase 1/2, phosphoinositide 3-kinase/Akt, mammalian target of rapamycin, and janus kinase 2/signal transducer and activator of transcription 3. Ghrelin-induced proliferative effect was associated with increased expression of E2F1 transcription factor in the nucleus, as determined by Western blotting and immunofluorescence. We also found that ghrelin caused an increase in protein levels of positive regulators of cell cycle, such as cyclin A and cyclin-dependent kinase (CDK) 2. Moreover, p27(KIP1) and p57(KIP2) protein levels were reduced when cell were exposed to ghrelin, suggesting downregulation of CDK inhibitors may contribute to proliferative effect of ghrelin. Our data suggest that ghrelin targets both cell cycle positive and negative regulators to stimulate proliferation of cultured hippocampal NSCs. PMID:27325242

  6. PARP-2 regulates cell cycle-related genes through histone deacetylation and methylation independently of poly(ADP-ribosyl)ation

    International Nuclear Information System (INIS)

    Highlights: ► PARP-2 acts as a transcription co-repressor independently of PARylation activity. ► PARP-2 recruits HDAC5, 7, and G9a and generates repressive chromatin. ► PARP-2 is recruited to the c-MYC promoter by DNA-binding factor YY1. ► PARP-2 represses cell cycle-related genes and alters cell cycle progression. -- Abstract: Poly(ADP-ribose) polymerase-2 (PARP-2) catalyzes poly(ADP-ribosyl)ation (PARylation) and regulates numerous nuclear processes, including transcription. Depletion of PARP-2 alters the activity of transcription factors and global gene expression. However, the molecular action of how PARP-2 controls the transcription of target promoters remains unclear. Here we report that PARP-2 possesses transcriptional repression activity independently of its enzymatic activity. PARP-2 interacts and recruits histone deacetylases HDAC5 and HDAC7, and histone methyltransferase G9a to the promoters of cell cycle-related genes, generating repressive chromatin signatures. Our findings propose a novel mechanism of PARP-2 in transcriptional regulation involving specific protein–protein interactions and highlight the importance of PARP-2 in the regulation of cell cycle progression

  7. PARP-2 regulates cell cycle-related genes through histone deacetylation and methylation independently of poly(ADP-ribosyl)ation

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    Liang, Ya-Chen; Hsu, Chiao-Yu [Institute of Molecular Biology, National Chung Hsing University, Taichung 40227, Taiwan (China); Yao, Ya-Li [Department of Biotechnology, Asia University, Taichung 41354, Taiwan (China); Yang, Wen-Ming, E-mail: yangwm@nchu.edu.tw [Institute of Molecular Biology, National Chung Hsing University, Taichung 40227, Taiwan (China)

    2013-02-01

    Highlights: ► PARP-2 acts as a transcription co-repressor independently of PARylation activity. ► PARP-2 recruits HDAC5, 7, and G9a and generates repressive chromatin. ► PARP-2 is recruited to the c-MYC promoter by DNA-binding factor YY1. ► PARP-2 represses cell cycle-related genes and alters cell cycle progression. -- Abstract: Poly(ADP-ribose) polymerase-2 (PARP-2) catalyzes poly(ADP-ribosyl)ation (PARylation) and regulates numerous nuclear processes, including transcription. Depletion of PARP-2 alters the activity of transcription factors and global gene expression. However, the molecular action of how PARP-2 controls the transcription of target promoters remains unclear. Here we report that PARP-2 possesses transcriptional repression activity independently of its enzymatic activity. PARP-2 interacts and recruits histone deacetylases HDAC5 and HDAC7, and histone methyltransferase G9a to the promoters of cell cycle-related genes, generating repressive chromatin signatures. Our findings propose a novel mechanism of PARP-2 in transcriptional regulation involving specific protein–protein interactions and highlight the importance of PARP-2 in the regulation of cell cycle progression.

  8. Cell cycle-related transformation of the E2F4-p130 repressor complex

    International Nuclear Information System (INIS)

    During G0 phase the p130, member of the pRb tumor suppressor protein family, forms a repressor complex with E2F4 which is inactivated in G1/S by hyperphosphorylation of the p130. The role of p130 after G1/S remains poorly investigated. We found that in nuclear extracts of T98G cells, the p130-E2F4-DNA (pp-E2F4) complex does not dissociate at G1/S transition, but instead reverts to the p130-E2F4-cyclin E/A-cdk2 (cyc/cdk-pp-E2F4) complex, which is detected in S and G2/M phases of the cell cycle. Hyperphosphorylation of the p130 at G1/S transition is associated with a decrease of its total amount; however, this protein is still detected during the rest of the cell cycle, and it is increasingly hyperphosphorylated in the cytosol, but continuously dephosphorylated in the nucleus. Both nuclear and cytosol cell fractions in T98G cells contain a hyperphosphorylated form of p130 in complex with E2F4 at S and G2/M cell cycle phases. In contrast to T98G cells, transformation of the p130 containing cyc/cdk-pp-E2F4 complex into the p130-pp-E2F4 repressor does not occur in HeLa cells under growth restriction conditions

  9. Interplay of noncoding RNAs, mRNAs, and proteins during the growth of eukaryotic cells

    Science.gov (United States)

    Zhdanov, V. P.

    2010-10-01

    Numerous biological functions of noncoding RNAs (ncRNAs) in eukaryotic cells are based primarily on their ability to pair with target mRNAs and then either to prevent translation or to result in rapid degradation of the mRNA-ncRNA complex. Using a general model describing this scenario, we show that ncRNAs may help to maintain constant mRNA and protein concentrations during the growth of cells. The possibility of observation of this effect on the global scale is briefly discussed.

  10. Interplay of noncoding RNAs, mRNAs, and proteins during the growth of eukaryotic cells

    International Nuclear Information System (INIS)

    Numerous biological functions of noncoding RNAs (ncRNAs) in eukaryotic cells are based primarily on their ability to pair with target mRNAs and then either to prevent translation or to result in rapid degradation of the mRNA-ncRNA complex. Using a general model describing this scenario, we show that ncRNAs may help to maintain constant mRNA and protein concentrations during the growth of cells. The possibility of observation of this effect on the global scale is briefly discussed.

  11. A versatile method for cell-specific profiling of translated mRNAs in Drosophila.

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    Amanda Thomas

    Full Text Available In Drosophila melanogaster few methods exist to perform rapid cell-type or tissue-specific expression profiling. A translating ribosome affinity purification (TRAP method to profile actively translated mRNAs has been developed for use in a number of multicellular organisms although it has only been implemented to examine limited sets of cell- or tissue-types in these organisms. We have adapted the TRAP method for use in the versatile GAL4/UAS system of Drosophila allowing profiling of almost any tissue/cell-type with a single genetic cross. We created transgenic strains expressing a GFP-tagged ribosomal protein, RpL10A, under the control of the UAS promoter to perform cell-type specific translatome profiling. The GFP::RpL10A fusion protein incorporates efficiently into ribosomes and polysomes. Polysome affinity purification strongly enriches mRNAs from expected genes in the targeted tissues with sufficient sensitivity to analyze expression in small cell populations. This method can be used to determine the unique translatome profiles in different cell-types under varied physiological, pharmacological and pathological conditions.

  12. Coptis japonica Makino extract suppresses angiogenesis through regulation of cell cycle-related proteins.

    Science.gov (United States)

    Kim, Seo Ho; Kim, Eok-Cheon; Kim, Wan-Joong; Lee, Myung-Hun; Kim, Sun-Young; Kim, Tack-Joong

    2016-06-01

    Angiogenesis, neovascularization from pre-existing vessels, is a key step in tumor growth and metastasis, and anti-angiogenic agents that can interfere with these essential steps of cancer development are a promising strategy for human cancer treatment. In this study, we characterized the anti-angiogenic effects of Coptis japonica Makino extract (CJME) and its mechanism of action. CJME significantly inhibited the proliferation, migration, and invasion of vascular endothelial growth factor (VEGF)-stimulated HUVECs. Furthermore, CJME suppressed VEGF-induced tube formation in vitro and VEGF-induced microvessel sprouting ex vivo. According to our study, CJME blocked VEGF-induced cell cycle transition in G1. CJME decreased expression of cell cycle-regulated proteins, including Cyclin D, Cyclin E, Cdk2, and Cdk4 in response to VEGF. Taken together, the results of our study indicate that CJME suppresses VEGF-induced angiogenic events such as proliferation, migration, and tube formation via cell cycle arrest in G1. PMID:26924430

  13. Reversible regulation of cell cycle-related genes by epigallocatechin gallate for hibernation of neonatal human tarsal fibroblasts.

    Science.gov (United States)

    Bae, Jung Yoon; Kanamune, Jun; Han, Dong-Wook; Matsumura, Kazuaki; Hyon, Suong-Hyu

    2009-01-01

    We investigated the hibernation effect of epigallocatechin-3-O-gallate (EGCG) on neonatal human tarsal fibroblasts (nHTFs) by analyzing the expression of cell cycle-related genes. EGCG application to culture media moderately inhibited the growth of nHTFs, and the removal of EGCG from culture media led to complete recovery of cell growth. EGCG resulted in a slight decrease in the cell population of the S and G(2)/M phases of cell cycle with concomitant increase in that of the G(0)/G(1) phase, but this cell cycle profile was restored to the initial level after EGCG removal. The expression of cyclin D1 (CCND1), CCNE2, CCN-dependent kinase 6 (CDK6), and CDK2 was restored, whereas that of CCNA, CCNB1, and CDK1 was irreversibly attenuated. The expression of a substantial number of genes analyzed by cDNA microarray was affected by EGCG application, and these affected expression levels were restored to the normal levels after EGCG removal. We also found the incorporation of FITC-EGCG into the cytosol of nHTFs and its further nuclear translocation, which might lead to the regulation of the exogenous signals directed to genes for cellular responses including proliferation and cell cycle progression. These results suggest that EGCG temporarily affects not only genes related to the cell cycle but also various other cellular functions. PMID:19622233

  14. Effects of HIV-1 Tat protein on expression of cell cycle-related genes and radiation-induced cell cycle arrest

    International Nuclear Information System (INIS)

    Objective: To explore effects of HIV-1 Tat protein on the expression of cell cycle-related genes and cell cycle arrest induced by ionizing radiation. Methods: A human rhabdomyosarcoma cell line TE671 and TT2 cells generated from TE671 cells by transfecting with tat gene of the HIV-1 strain were employed. Microarray, which contained the oligonucleotide probes corresponding to 102 human DNA damage response related genes, was used to analyze transcriptional changes. Cell cycle changes were analyzed by flow cytometry. Results: Microarray assay demonstrated that cell cycle-related genes Cdc20, Cdc25C, KIF2C, CTS1 and Wee1 were down-regulated in Tat-expressing TT2 cells. Tat-expressing cells exhibited a noticeable delay of the initiation and elimination of radiation-induced G2/M arrest and a prolonged S phase arrest as compared with parental cells. Moreover, overexpression of cyclinB1 was also observed in Tat-expressing TT2 cells. Conclusion: Dysregulated cell cycle checkpoint in Tat-expressing cells can provide new information for understanding the radiation responsiveness of AIDS patients with cancer to radiotherapy. (authors)

  15. AB109. Downregulation of tNASP inhibits proliferation through regulating cell cycle-related proteins and inactive ERK/MAPK signal pathway in renal cell carcinoma cells

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    Fang, Jianzheng; Wang, Hainan; Cheng, Gong; Wang, Shangqian; Deng, Yunfei; Song, Zhen; Xu, Aiming; Liu, Bianjiang; Wang, Zengjun

    2016-01-01

    Objective Nuclear auto-antigenic sperm protein (NASP), initially described as a highly auto-immunogenic testis and sperm-specific protein, is a histone chaperone that is proved to present in all dividing cells. NASP has two splice variants: testicular NASP (tNASP) and somatic form of NASP (sNASP). Only cancer, germ, transformed, and embryonic cells have a high level of expression of the tNASP. Up to now, little has been known about tNASP in renal cell carcinoma (RCC). In the present study, the molecular mechanism of tNASP in RCC was explored. Methods The expression level of tNASP in 16 paired human RCC specimens was determined. Downregulation of tNASP by small interfering RNA (siRNA) was transfected in RCC cell lines. The effect of downregulation of tNASP by siRNA on cell colony formation and proliferation was examined by colony formation assay and CCK-8 assay, cell cycle was analyzed by flow cytometry, and the expression of cyclin D1 and P21 were detected by Western blotting. ERK/MAPK signaling was also analyzed. Results tNASP has a relative high expression level in human RCC tissues. Via upregulation of P21 and downregulation of cyclinD1, silence of tNASP can inhibit cell proliferation, which induces cell cycle arrest. Furthermore, ERK signaling pathway is confirmed to mediate the regulation of cell cycle-related proteins caused by silence of tNASP. Conclusions Our research demonstrates that knockdown of tNASP effectively inhibits the proliferation and causes G1 phase arrest through ERK/MAPK signal pathway.

  16. Undifferentiated embryonic stem cells express ionotropic glutamate receptor mRNAs

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    Svenja Pachernegg

    2013-12-01

    Full Text Available Ionotropic glutamate receptors (iGluRs do not only mediate the majority of excitatory neurotransmission in the vertebrate CNS, but also modulate pre- and postnatal neurogenesis. Most of the studies on the developmental role of iGluRs are performed on neural progenitors and neural stem cells. We took a step back in our study by examining the role of iGluRs in the earliest possible cell type, embryonic stem cells (ESCs, by looking at the mRNA expression of the major iGluR subfamilies in undifferentiated mouse ESCs. For that, we used two distinct murine ES cell lines, 46C ESCs and J1 ESCs. Regarding 46C ESCs, we found transcripts of kainate receptors (GluK2 to GluK5, AMPA receptors (GluA1, GluA3, and GluA4, and NMDA receptors (GluN1, and GluN2A to GluN2D. Analysis of 46C-derived cells of later developmental stages, namely neuroepithelial precursor cells (NEPs and neural stem cells (NSCs, revealed that the mRNA expression of KARs is significantly upregulated in NEPs and, subsequently, downregulated in NSCs. However, we could not detect any protein expression of any of the KAR subunits present on the mRNA level either in ESCs, NEPs, or NSCs. Regarding AMPARs and NMDARs, GluN2A is weakly expressed at the protein level only in NSCs. Matching our findings for GluRs, all three cell types were found to weakly express pre- and postsynaptic markers of glutamatergic synapses only at the mRNA level. Finally, we performed patch-clamp recordings of 46C ESCs and could not detect any current upon iGluR agonist application. Similar to 46C ESCs, J1 ESCs express kainate receptors (GluK2 to GluK5, AMPA receptors (GluA3, and NMDA receptors (GluN1, and GluN2A to GluN2D at the mRNA level, but these transcripts are not translated into receptor proteins either. Thus, we conclude that ESCs do not contain functional iGluRs, although they do express an almost complete set of iGluR subunit mRNAs.

  17. Aubergine iCLIP Reveals piRNA-Dependent Decay of mRNAs Involved in Germ Cell Development in the Early Embryo

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    Bridlin Barckmann

    2015-08-01

    Full Text Available The Piwi-interacting RNA (piRNA pathway plays an essential role in the repression of transposons in the germline. Other functions of piRNAs such as post-transcriptional regulation of mRNAs are now emerging. Here, we perform iCLIP with the PIWI protein Aubergine (Aub and identify hundreds of maternal mRNAs interacting with Aub in the early Drosophila embryo. Gene expression profiling reveals that a proportion of these mRNAs undergo Aub-dependent destabilization during the maternal-to-zygotic transition. Strikingly, Aub-dependent unstable mRNAs encode germ cell determinants. iCLIP with an Aub mutant that is unable to bind piRNAs confirms piRNA-dependent binding of Aub to mRNAs. Base pairing between piRNAs and mRNAs can induce mRNA cleavage and decay that are essential for embryonic development. These results suggest general regulation of maternal mRNAs by Aub and piRNAs, which plays a key developmental role in the embryo through decay and localization of mRNAs encoding germ cell determinants.

  18. Identification of microRNAs and mRNAs associated with multidrug resistance of human laryngeal cancer Hep-2 cells

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    Yin, Wanzhong; Wang, Ping; Wang, Xin [Department of Otorhinolaryngology, Head and Neck Surgery, The First Clinical Hospital, Norman Bethune College of Medicine, Jilin University, Changchun (China); Song, Wenzhi [Department of Stomatology, China-Japan Friendship Hospital, Jilin University, Changchun (China); Cui, Xiangyan; Yu, Hong; Zhu, Wei [Department of Otorhinolaryngology, Head and Neck Surgery, The First Clinical Hospital, Norman Bethune College of Medicine, Jilin University, Changchun (China)

    2013-06-12

    Multidrug resistance (MDR) poses a serious impediment to the success of chemotherapy for laryngeal cancer. To identify microRNAs and mRNAs associated with MDR of human laryngeal cancer Hep-2 cells, we developed a multidrug-resistant human laryngeal cancer subline, designated Hep-2/v, by exposing Hep-2 cells to stepwise increasing concentrations of vincristine (0.02-0.96'µM). Microarray assays were performed to compare the microRNA and mRNA expression profiles of Hep-2 and Hep-2/v cells. Compared to Hep-2 cells, Hep-2/v cells were more resistant to chemotherapy drugs (∼45-fold more resistant to vincristine, 5.1-fold more resistant to cisplatin, and 5.6-fold more resistant to 5-fluorouracil) and had a longer doubling time (42.33±1.76 vs 28.75±1.12'h, P<0.05), higher percentage of cells in G0/G1 phase (80.98±0.52 vs 69.14±0.89, P<0.05), increased efflux of rhodamine 123 (95.97±0.56 vs 12.40±0.44%, P<0.01), and up-regulated MDR1 expression. A total of 7 microRNAs and 605 mRNAs were differentially expressed between the two cell types. Of the differentially expressed mRNAs identified, regulator of G-protein signaling 10, high-temperature requirement protein A1, and nuclear protein 1 were found to be the putative targets of the differentially expressed microRNAs identified. These findings may open a new avenue for clarifying the mechanisms responsible for MDR in laryngeal cancer.

  19. Identification of microRNAs and mRNAs associated with multidrug resistance of human laryngeal cancer Hep-2 cells

    International Nuclear Information System (INIS)

    Multidrug resistance (MDR) poses a serious impediment to the success of chemotherapy for laryngeal cancer. To identify microRNAs and mRNAs associated with MDR of human laryngeal cancer Hep-2 cells, we developed a multidrug-resistant human laryngeal cancer subline, designated Hep-2/v, by exposing Hep-2 cells to stepwise increasing concentrations of vincristine (0.02-0.96'µM). Microarray assays were performed to compare the microRNA and mRNA expression profiles of Hep-2 and Hep-2/v cells. Compared to Hep-2 cells, Hep-2/v cells were more resistant to chemotherapy drugs (∼45-fold more resistant to vincristine, 5.1-fold more resistant to cisplatin, and 5.6-fold more resistant to 5-fluorouracil) and had a longer doubling time (42.33±1.76 vs 28.75±1.12'h, P<0.05), higher percentage of cells in G0/G1 phase (80.98±0.52 vs 69.14±0.89, P<0.05), increased efflux of rhodamine 123 (95.97±0.56 vs 12.40±0.44%, P<0.01), and up-regulated MDR1 expression. A total of 7 microRNAs and 605 mRNAs were differentially expressed between the two cell types. Of the differentially expressed mRNAs identified, regulator of G-protein signaling 10, high-temperature requirement protein A1, and nuclear protein 1 were found to be the putative targets of the differentially expressed microRNAs identified. These findings may open a new avenue for clarifying the mechanisms responsible for MDR in laryngeal cancer

  20. Gene activation by UV light, fungal elicitor or fungal infection in Petroselinum crispum is correlated with repression of cell cycle-related genes

    International Nuclear Information System (INIS)

    The effects of UV light or fungal elicitors on plant cells have so far been studied mostly with respect to defense-related gene activation. Here, an inverse correlation of these stimulatory effects with the activities of several cell cycle-related genes is demonstrated. Concomitant with the induction of flavonoid biosynthetic enzymes in UV-irradiated cell suspension cultures of parsley (Petroselinum crispum), total histone synthesis declined to about half the initial rate. A subclass of the histone H3 gene family was selected to demonstrate the close correlation of its expression with cell division, both in intact plants and cultured cells. Using RNA-blot and run-on transcription assays, it was shown that one arbitrarily selected subclass of each of the histone H2A, H2B, H3 and H4 gene families and of the genes encoding a p34cdc2 protein kinase and a mitotic cyclin were transcriptionally repressed in UV-irradiated as well as fungal elicitor-treated parsley cells. The timing and extent of repression differed between the two stimuli; the response to light was more transient and smaller in magnitude. These differential responses to light and elicitor were inversely correlated with the induction of phenylalanine ammonia-lyase, a key enzyme of phenylpropanoid metabolism. Essentially the same result was obtained with a defined oligopeptide elicitor, indicating that the same signaling pathway is responsible for defense-related gene activation and cell cycle-related gene repression. A temporary (UV light) or long-lasting (fungal elicitor) cessation of cell culture growth is most likely due to an arrest of cell division which may be a prerequisite for full commitment of the cells to transcriptional activation of full commitment of the cells to transcriptional activation of pathways involved in UV protection or pathogen defense. This conclusion is corroborated by the observation that the histone H3 mRNA level greatly declined around fungal infection sites in young parsley

  1. The CCR4-NOT complex mediates deadenylation and degradation of stem cell mRNAs and promotes planarian stem cell differentiation.

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    Solana, Jordi; Gamberi, Chiara; Mihaylova, Yuliana; Grosswendt, Stefanie; Chen, Chen; Lasko, Paul; Rajewsky, Nikolaus; Aboobaker, A Aziz

    2013-01-01

    Post-transcriptional regulatory mechanisms are of fundamental importance to form robust genetic networks, but their roles in stem cell pluripotency remain poorly understood. Here, we use freshwater planarians as a model system to investigate this and uncover a role for CCR4-NOT mediated deadenylation of mRNAs in stem cell differentiation. Planarian adult stem cells, the so-called neoblasts, drive the almost unlimited regenerative capabilities of planarians and allow their ongoing homeostatic tissue turnover. While many genes have been demonstrated to be required for these processes, currently almost no mechanistic insight is available into their regulation. We show that knockdown of planarian Not1, the CCR4-NOT deadenylating complex scaffolding subunit, abrogates regeneration and normal homeostasis. This abrogation is primarily due to severe impairment of their differentiation potential. We describe a stem cell specific increase in the mRNA levels of key neoblast genes after Smed-not1 knock down, consistent with a role of the CCR4-NOT complex in degradation of neoblast mRNAs upon the onset of differentiation. We also observe a stem cell specific increase in the frequency of longer poly(A) tails in these same mRNAs, showing that stem cells after Smed-not1 knock down fail to differentiate as they accumulate populations of transcripts with longer poly(A) tails. As other transcripts are unaffected our data hint at a targeted regulation of these key stem cell mRNAs by post-transcriptional regulators such as RNA-binding proteins or microRNAs. Together, our results show that the CCR4-NOT complex is crucial for stem cell differentiation and controls stem cell-specific degradation of mRNAs, thus providing clear mechanistic insight into this aspect of neoblast biology. PMID:24367277

  2. The CCR4-NOT complex mediates deadenylation and degradation of stem cell mRNAs and promotes planarian stem cell differentiation.

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    Jordi Solana

    Full Text Available Post-transcriptional regulatory mechanisms are of fundamental importance to form robust genetic networks, but their roles in stem cell pluripotency remain poorly understood. Here, we use freshwater planarians as a model system to investigate this and uncover a role for CCR4-NOT mediated deadenylation of mRNAs in stem cell differentiation. Planarian adult stem cells, the so-called neoblasts, drive the almost unlimited regenerative capabilities of planarians and allow their ongoing homeostatic tissue turnover. While many genes have been demonstrated to be required for these processes, currently almost no mechanistic insight is available into their regulation. We show that knockdown of planarian Not1, the CCR4-NOT deadenylating complex scaffolding subunit, abrogates regeneration and normal homeostasis. This abrogation is primarily due to severe impairment of their differentiation potential. We describe a stem cell specific increase in the mRNA levels of key neoblast genes after Smed-not1 knock down, consistent with a role of the CCR4-NOT complex in degradation of neoblast mRNAs upon the onset of differentiation. We also observe a stem cell specific increase in the frequency of longer poly(A tails in these same mRNAs, showing that stem cells after Smed-not1 knock down fail to differentiate as they accumulate populations of transcripts with longer poly(A tails. As other transcripts are unaffected our data hint at a targeted regulation of these key stem cell mRNAs by post-transcriptional regulators such as RNA-binding proteins or microRNAs. Together, our results show that the CCR4-NOT complex is crucial for stem cell differentiation and controls stem cell-specific degradation of mRNAs, thus providing clear mechanistic insight into this aspect of neoblast biology.

  3. Characterization of a Novel Cardiac Isoform of the Cell Cycle-related Kinase That Is Regulated during Heart Failure*S⃞

    OpenAIRE

    Qiu, Hongyu; Dai, Huacheng; Jain, Komal; Shah, Rina; Hong, Chull; Pain, Jayashree; Tian, Bin; Vatner, Dorothy E.; Vatner, Stephen F.; Depre, Christophe

    2008-01-01

    Myocardial infarction (MI) is often followed by heart failure (HF), but the mechanisms precipitating the transition to HF remain largely unknown. A genomic profile was performed in a monkey model of MI, from the myocardium adjacent to chronic (2-month) MI followed by 3 weeks of pacing to develop HF. The transcript of the gene encoding the cell cycle-related kinase (CCRK) was down-regulated by 50% in HF heart compared with control (p < 0.05), which was confirmed by quan...

  4. Differential stability of host mRNAs in Friend erythroleukemia cells infected with herpes simplex virus type 1

    International Nuclear Information System (INIS)

    The consequences of herpes simplex virus type 1 infection on cellular macromolecules were investigated in Friend erythroleukemia cells. The patterns of protein synthesis, examined by polyacrylamide gel electrophoresis, demonstrated that by 4 h postinfection the synthesis of many host proteins, with the exception of histones, was inhibited. Examination of the steady-state level of histone H3 mRNA by molecular hybridization of total RNA to a cloned mouse histone H3 complementary DNA probe demonstrated that the ratio of histone H3 mRNA to total RNA remained unchanged for the first 4 h postinfection. In contrast, the steady-state levels of globin and actin mRNAs decreased progressively at early intervals postinfection. Studies on RNA synthesis in isolated nuclei demonstrated that the transcription of the histone H3 gene was inhibited to approximately the same extent as that of actin gene. It was concluded that the stabilization of preexisting histone H3 mRNA was responsible for the persistence of H3 mRNA and histone protein synthesis in herpes simplex virus type 1-infected Friend erythroleukemia cells. The possible mechanisms influencing the differential stability of host mRNAs during the course of productive infection with herpes simplex virus type 1 are discussed

  5. Reprogramming of Pancreatic Exocrine Cells AR42J Into Insulin-producing Cells Using mRNAs for Pdx1, Ngn3, and MafA Transcription Factors.

    Science.gov (United States)

    Koblas, Tomas; Leontovyc, Ivan; Loukotova, Sarka; Kosinova, Lucie; Saudek, Frantisek

    2016-01-01

    Direct reprogramming of pancreatic nonendocrine cells into insulin-producing β-cells represents a promising approach for the treatment of insulin-dependent diabetes. However, its clinical application is limited by the potential for insertional mutagenesis associated with the viral vectors currently used for cell reprogramming. With the aim of developing a nonintegrative reprogramming strategy for derivation of insulin-producing cells, here, we evaluated a new approach utilizing synthetic messenger RNAs encoding reprogramming transcription factors. Administration of synthetic mRNAs encoding three key transcription regulators of β-cell differentiation-Pdx1, Neurogenin3, and MafA-efficiently reprogrammed the pancreatic exocrine cells into insulin-producing cells. In addition to the insulin genes expression, the synthetic mRNAs also induced the expressions of genes important for proper pancreatic β-cell function, including Sur1, Kir6.2, Pcsk1, and Pcsk2. Pretreating cells with the chromatin-modifying agent 5-Aza-2'-deoxycytidine further enhanced reprogramming efficiency, increasing the proportion of insulin-producing cells from 3.5 ± 0.9 to 14.3 ± 1.9% (n = 4). Moreover, 5-Aza-2'-deoxycytidine pretreatment enabled the reprogrammed cells to respond to glucose challenge with increased insulin secretion. In conclusion, our results support that the reprogramming of pancreatic exocrine cells into insulin-producing cells, induced by synthetic mRNAs encoding pancreatic transcription factors, represents a promising approach for cell-based diabetes therapy. PMID:27187823

  6. Neurotrophins and Neurotrophin Receptors mRNAs Expression in Pancreatic Islets and Insulinoma Cell Lines

    Directory of Open Access Journals (Sweden)

    Bonini P

    2001-05-01

    Full Text Available CONTEXT: It is worth noting that islets and betaTC6-F7 cells share a common pattern of expression of neurotrophins and neurotrophin receptors. Recently, several studies have hypothesized a role for nerve growth factor in pancreatic development and maturation, suggesting that nerve growth factor may be a survival factor for pancreatic beta-cells. OBJECTIVE: The aim of the present study was to investigate the pattern of expression of neurotrophins and their relative receptors both in rat pancreatic islets and in a wide panel of insulinoma cell lines. MAIN OUTCOME MEASURES: A semi-quantitative reverse-transcription polymerase chain reaction analysis was performed on ribonucleic acids extracted from these cells. RESULTS: Reverse transcription-polymerase chain reaction analysis demonstrates that brain-derived neurotrophic factor, as well as neurotrophins 3 and 4, are expressed both in islets and in all insulinoma cells, while nerve growth factor is expressed only in islets, betaTC6-F7 cells and, at a low level, in RIN 1046-38 cells. Receptors protein tyrosine kinase A and C are ubiquitously expressed both in islets and insulinoma cells. Tyrosine kinase B is absent in HIT-T15 cells. CONCLUSIONS: These data indicate that betaTC6-F7 cells are a suitable model for studying the role of neurotrophins in the survival of beta-cells.

  7. Temporal changes in glycogenolytic enzyme mRNAs during myogenesis of primary porcine satellite cells

    DEFF Research Database (Denmark)

    Henckel, Poul; Theil, Peter Kappel; Sørensen, Inge Lise; Oksbjerg, Niels

    2007-01-01

    , phosphorylase kinase, phosphorylase and glycogen debranching enzyme, and no alterations of the transporter molecule GLUT4, clearly indicate that glycogenolytic enzymes of potential importance to meat quality development are regulated at the gene level during myogenesis, and are heavily involved in muscle cell...... and muscle fibre development. The genes, however, are not influenced by insulin, and the lack of response to insulin of expression of gene-encoding enzymes involved in the formation and degradation of glycogen may question the applicability of porcine cell culture systems, like the one applied, as a...

  8. Smoking-related microRNAs and mRNAs in human peripheral blood mononuclear cells.

    Science.gov (United States)

    Su, Ming-Wei; Yu, Sung-Liang; Lin, Wen-Chang; Tsai, Ching-Hui; Chen, Po-Hua; Lee, Yungling Leo

    2016-08-15

    Teenager smoking is of great importance in public health. Functional roles of microRNAs have been documented in smoke-induced gene expression changes, but comprehensive mechanisms of microRNA-mRNA regulation and benefits remained poorly understood. We conducted the Teenager Smoking Reduction Trial (TSRT) to investigate the causal association between active smoking reduction and whole-genome microRNA and mRNA expression changes in human peripheral blood mononuclear cells (PBMC). A total of 12 teenagers with a substantial reduction in smoke quantity and a decrease in urine cotinine/creatinine ratio were enrolled in genomic analyses. In Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA), differentially expressed genes altered by smoke reduction were mainly associated with glucocorticoid receptor signaling pathway. The integrative analysis of microRNA and mRNA found eleven differentially expressed microRNAs negatively correlated with predicted target genes. CD83 molecule regulated by miR-4498 in human PBMC, was critical for the canonical pathway of communication between innate and adaptive immune cells. Our data demonstrated that microRNAs could regulate immune responses in human PBMC after habitual smokers quit smoking and support the potential translational value of microRNAs in regulating disease-relevant gene expression caused by tobacco smoke. PMID:27321975

  9. Peroxisome proliferator-activated receptors (PPAR) agonists affect cell viability, apoptosis and expression of cell cycle related proteins in cell lines of glial brain tumors

    Czech Academy of Sciences Publication Activity Database

    Straková, N.; Ehrmann, J.; Bartoš, Jan; Malíková, J.; Doležel, Jaroslav; Kolář, Z.

    2005-01-01

    Roč. 52, - (2005), s. 126-136. ISSN 0028-2685 Institutional research plan: CEZ:AV0Z50380511; CEZ:AV0Z5038910 Keywords : PPAR * glioplasma * cell cycle Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.731, year: 2005

  10. Expressed miRNAs target feather related mRNAs involved in cell signaling, cell adhesion and structure during chicken epidermal development.

    Science.gov (United States)

    Bao, Weier; Greenwold, Matthew J; Sawyer, Roger H

    2016-10-15

    MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at the post-transcriptional level. Previous studies have shown that miRNA regulation contributes to a diverse set of processes including cellular differentiation and morphogenesis which leads to the creation of different cell types in multicellular organisms and is thus key to animal development. Feathers are one of the most distinctive features of extant birds and are important for multiple functions including flight, thermal regulation, and sexual selection. However, the role of miRNAs in feather development has been woefully understudied despite the identification of cell signaling pathways, cell adhesion molecules and structural genes involved in feather development. In this study, we performed a microarray experiment comparing the expression of miRNAs and mRNAs among three embryonic stages of development and two tissues (scutate scale and feather) of the chicken. We combined this expression data with miRNA target prediction tools and a curated list of feather related genes to produce a set of 19 miRNA-mRNA duplexes. These targeted mRNAs have been previously identified as important cell signaling and cell adhesion genes as well as structural genes involved in feather and scale morphogenesis. Interestingly, the miRNA target site of the cell signaling pathway gene, Aldehyde Dehydrogenase 1 Family, Member A3 (ALDH1A3), is unique to birds indicating a novel role in Aves. The identified miRNA target site of the cell adhesion gene, Tenascin C (TNC), is only found in specific chicken TNC splice variants that are differentially expressed in developing scutate scale and feather tissue indicating an important role of miRNA regulation in epidermal differentiation. Additionally, we found that β-keratins, a major structural component of avian and reptilian epidermal appendages, are targeted by multiple miRNA genes. In conclusion, our work provides quantitative expression data on miRNAs and mRNAs

  11. mRNAs containing the unstructured 5' leader sequence of alfalfa mosaic virus RNA 4 translate inefficiently in lysates from poliovirus-infected HeLa cells.

    OpenAIRE

    Hann, L E; Gehrke, L

    1995-01-01

    Poliovirus infection is accompanied by translational control that precludes translation of 5'-capped mRNAs and facilitates translation of the uncapped poliovirus RNA by an internal initiation mechanism. Previous reports have suggested that the capped alfalfa mosaic virus coat protein mRNA (AIMV CP RNA), which contains an unstructured 5' leader sequence, is unusual in being functionally active in extracts prepared from poliovirus-infected HeLa cells (PI-extracts). To identify the cis-acting nu...

  12. Lewis y Regulate Cell Cycle Related Factors in Ovarian Carcinoma Cell RMG-I in Vitro via ERK and Akt Signaling Pathways

    OpenAIRE

    Shulan Zhang; Qing Liu; Yingying Hao; Rui Hou; Bei Lin; Shuice Liu; Juanjuan Liu; Masao Iwamori; Dawo Liu

    2012-01-01

    Objective: To investigate the effect of Lewis y overexpression on the expression of proliferation-related factors in ovarian cancer cells. Methods: mRNA levels of cyclins, CDKs, and CKIs were measured in cells before and after transfection with the α1,2-fucosyltransferase gene by real-time PCR, and protein levels of cyclins, CDKs and CKIs were determined in cells before and after gene transfection by Western blot. Results: Lewis y overexpression led to an increase in both mRNA and protein exp...

  13. Heat shock causes destabilization of specific mRNAs and destruction of endoplasmic reticulum in barley aleurone cells.

    OpenAIRE

    Belanger, F. C.; Brodl, M R; Ho, T H

    1986-01-01

    In response to a phytohormone, gibberellic acid, the aleurone layers of barley seeds synthesize and secrete alpha-amylases, which are coded by a set of stable mRNAs. When aleurone layers are subjected to heat shock treatment, the synthesis of alpha-amylase is suppressed while heat shock proteins are induced. The suppression of alpha-amylase synthesis is not the result of translational control as reported in several other systems. Rather, the sequences of alpha-amylase mRNA are rapidly degrade...

  14. Effect of anticancer drugs on breast cancer cells sensitive and resistant to doxorubicin: expression of mRNAs of TGF-β and its receptors

    Directory of Open Access Journals (Sweden)

    Chorna I. V.

    2014-01-01

    Full Text Available Aim. Comparative study of the effect of chemotherapeutic drugs (doxorubicin, methotrexate and cisplatin and TGF-β on the human breast carcinoma MCF-7 cells, sensitive (wt and resistant (DOX/R to the doxorubicin action. Methods. Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR was used for the estimation of expression of mRNAs coding for the TGF-β isoforms (TGF-β1 and TGF-β2 and the TGF-β type I and II receptors (TRI and TRII. Trypan blue exclusion method was used for measuring cell number and cell viability. Results. The MCF-7(DOX/R cells were more refractory to the TGF1-dependent growth inhibition than the MCF-7(wt cells. The level of mRNAs coding for TGF and its receptors was higher in the untreated MCF-7 (DOX/R cells comparing to the MCF-7(wt cells. The expression of mRNA coding for TRII was decreased in both cell lines treated with doxorubicin, methotrexate and cisplatin, while the down-regulation of mRNA coding for TRI was revealed only in the MCF-7(DOX/R cells upon the treatment with doxorubicin and methotrexate. Conclusions. The differential effects of studied anticancer drugs and TGF-β on the doxorubicin-sensitive and -resistant cells have been demonstrated. The elucidation of the molecular mechanisms of escape of the MCF-7 (DOX/R cells from the growth inhibition by TGF-β requires further investigation.

  15. Effect of anticancer drugs on breast cancer cells sensitive and resistant to doxorubicin: expression of mRNAs of TGF-β and its receptors

    OpenAIRE

    Chorna I. V.; Fedorenko O. V.; Stoika R. S.

    2014-01-01

    Aim. Comparative study of the effect of chemotherapeutic drugs (doxorubicin, methotrexate and cisplatin) and TGF-β on the human breast carcinoma MCF-7 cells, sensitive (wt) and resistant (DOX/R) to the doxorubicin action. Methods. Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was used for the estimation of expression of mRNAs coding for the TGF-β isoforms (TGF-β1 and TGF-β2) and the TGF-β type I and II receptors (TRI and TRII). Trypan blue exclusion method was use...

  16. pp32 (ANP32A expression inhibits pancreatic cancer cell growth and induces gemcitabine resistance by disrupting HuR binding to mRNAs.

    Directory of Open Access Journals (Sweden)

    Timothy K Williams

    Full Text Available The expression of protein phosphatase 32 (PP32, ANP32A is low in poorly differentiated pancreatic cancers and is linked to the levels of HuR (ELAV1, a predictive marker for gemcitabine response. In pancreatic cancer cells, exogenous overexpression of pp32 inhibited cell growth, supporting its long-recognized role as a tumor suppressor in pancreatic cancer. In chemotherapeutic sensitivity screening assays, cells overexpressing pp32 were selectively resistant to the nucleoside analogs gemcitabine and cytarabine (ARA-C, but were sensitized to 5-fluorouracil; conversely, silencing pp32 in pancreatic cancer cells enhanced gemcitabine sensitivity. The cytoplasmic levels of pp32 increased after cancer cells are treated with certain stressors, including gemcitabine. pp32 overexpression reduced the association of HuR with the mRNA encoding the gemcitabine-metabolizing enzyme deoxycytidine kinase (dCK, causing a significant reduction in dCK protein levels. Similarly, ectopic pp32 expression caused a reduction in HuR binding of mRNAs encoding tumor-promoting proteins (e.g., VEGF and HuR, while silencing pp32 dramatically enhanced the binding of these mRNA targets. Low pp32 nuclear expression correlated with high-grade tumors and the presence of lymph node metastasis, as compared to patients' tumors with high nuclear pp32 expression. Although pp32 expression levels did not enhance the predictive power of cytoplasmic HuR status, nuclear pp32 levels and cytoplasmic HuR levels associated significantly in patient samples. Thus, we provide novel evidence that the tumor suppressor function of pp32 can be attributed to its ability to disrupt HuR binding to target mRNAs encoding key proteins for cancer cell survival and drug efficacy.

  17. Genome-wide analysis reveals selective modulation of microRNAs and mRNAs by histone deacetylase inhibitor in B cells induced to undergo class switch DNA recombination and plasma cell differentiation

    Directory of Open Access Journals (Sweden)

    Tian eShen

    2015-12-01

    Full Text Available As we have suggested, epigenetic factors, such as microRNAs (miRNAs, can interact with genetic programs to regulate B cell functions, thereby informing antibody and autoantibody responses. We have shown that histone deacetylase inhibitors (HDI inhibit the differentiation events critical to the maturation of the antibody response: class-switch DNA recombination (CSR, somatic hypermutation (SHM and plasma cell differentiation, by modulating intrinsic B cell mechanisms. HDI repress the expression of AID and Blimp-1, which are critical for CSR/SHM and plasma cell differentiation, respectively, in mouse and human B cells by upregulating selected miRNAs that silenced AICDA/Aicda and PRDM1/Prdm1 mRNAs, as demonstrated by multiple qRT-PCRs (J. Immunol. 193:5933-5950, 2014. To further define the selectivity of HDI-mediated modulation of miRNA and gene expression, we performed genome-wide miRNA-Seq and mRNA-Seq analysis in B cells stimulated by LPS plus IL-4 and treated with HDI or nil. Consistent with what we have shown using qRT-PCR, these HDI-treated B cells displayed reduced expression of Aicda and Prdm1, and increased expression of miR-155, miR-181b and miR-361, which target Aicda, and miR-23b, miR-30a and miR-125b, which target Prdm1. In B cells induced to undergo CSR and plasma cell differentiation, about 23% of over 22,000 mRNAs analyzed were expressed at a significantly high copy number (more than 20 copies/cell. Only 18 (0.36% of these highly expressed mRNAs, including Aicda, Prdm1 and Xbp1, were downregulated by HDI by 50% or more. Further, only 16 (0.30% of the highly expressed mRNAs were upregulated (more than twofold by HDI. The selectivity of HDI-mediated modulation of gene expression was emphasized by unchanged expression of the genes that are involved in regulation, targeting or DNA repair processes of CSR, as well as unchanged expression of the genes encoding epigenetic regulators and factors that are important for cell signaling or

  18. Genome-Wide Analysis Reveals Selective Modulation of microRNAs and mRNAs by Histone Deacetylase Inhibitor in B Cells Induced to Undergo Class-Switch DNA Recombination and Plasma Cell Differentiation.

    Science.gov (United States)

    Shen, Tian; Sanchez, Helia N; Zan, Hong; Casali, Paolo

    2015-01-01

    As we have suggested, epigenetic factors, such as microRNAs (miRNAs), can interact with genetic programs to regulate B cell functions, thereby informing antibody and autoantibody responses. We have shown that histone deacetylase (HDAC) inhibitors (HDI) inhibit the differentiation events critical to the maturation of the antibody response: class-switch DNA recombination (CSR), somatic hypermutation (SHM), and plasma cell differentiation, by modulating intrinsic B cell mechanisms. HDI repress the expression of AID and Blimp-1, which are critical for CSR/SHM and plasma cell differentiation, respectively, in mouse and human B cells by upregulating selected miRNAs that silenced AICDA/Aicda and PRDM1/Prdm1 mRNAs, as demonstrated by multiple qRT-PCRs (J Immunol 193:5933-5950, 2014). To further define the selectivity of HDI-mediated modulation of miRNA and gene expression, we performed genome-wide miRNA-Seq and mRNA-Seq analysis in B cells stimulated by LPS plus IL-4 and treated with HDI or nil. Consistent with what we have shown using qRT-PCR, these HDI-treated B cells displayed reduced expression of Aicda and Prdm1, and increased expression of miR-155, miR-181b, and miR-361, which target Aicda, and miR-23b, miR-30a, and miR-125b, which target Prdm1. In B cells induced to undergo CSR and plasma cell differentiation, about 23% of over 22,000 mRNAs analyzed were expressed at a significantly high copy number (more than 20 copies/cell). Only 18 (0.36%) of these highly expressed mRNAs, including Aicda, Prdm1, and Xbp1, were downregulated by HDI by 50% or more. Further, only 16 (0.30%) of the highly expressed mRNAs were upregulated (more than twofold) by HDI. The selectivity of HDI-mediated modulation of gene expression was emphasized by unchanged expression of the genes that are involved in regulation, targeting, or DNA repair processes of CSR, as well as unchanged expression of the genes encoding epigenetic regulators and factors that are important for cell signaling or

  19. Translation of ferritin light and heavy subunit mRNAs is regulated by intracellular chelatable iron levels in rat hepatoma cells

    International Nuclear Information System (INIS)

    Acute administration of iron to rats has been previously shown to induce liver ferritin synthesis by increasing the translation of inactive cytoplasmic ferritin mRNAs for both heavy (H) and light (L) subunits by mobilizing them onto polyribosomes. In this report rat hepatoma cells in culture are used to explore the relationship of this response to intracellular iron levels. After adding iron as ferric ammonium citrate to the medium, latent ferritin H- and L-mRNAs were extensively transferred to polyribosomes, accompanied by increased uptake of [35S]methionine into ferritin protein. Because total cellular levels of L- and H-mRNA were not significantly changed by exposure to iron, the increased ferritin mRNAs on polyribosomes most probably come from an inactive cytoplasmic pool, consistent with the inability of actinomycin-D and of cordycepin to inhibit iron-induced ferritin synthesis. When deferoxamine mesylate, an intracellular iron chelator, was added after the addition of iron to the medium, ferritin mRNA on the polyribosomes was reduced, while the free messenger pool increased, and ferritin synthesis diminished. In contrast, the extracellular iron chelator diethylenetriaminepentaacetic acid failed to inhibit the induction of ferritin protein synthesis. Addition of iron in the form of hemin also caused translocation of mRNA to polyribosomes, a response that could be similarly quenched by deferoxamine. Because hemin does not release chelatable iron extracellularly, they conclude that the level of chelatable iron within the cell has a regulatory role in ferritin synthesis through redistribution of the messenger RNAs between the free mRNA pool and the polyribosomes

  20. Analysis of cell cycle-related proteins in gastric intramucosal differentiated-type cancers based on mucin phenotypes: a novel hypothesis of early gastric carcinogenesis based on mucin phenotype

    Directory of Open Access Journals (Sweden)

    Matsushita Hiroo

    2010-06-01

    Full Text Available Abstract Background Abnormalities of cell cycle regulators are common features in human cancers, and several of these factors are associated with the early development of gastric cancers. However, recent studies have shown that gastric cancer tumorigenesis was characterized by mucin expression. Thus, expression patterns of cell cycle-related proteins were investigated in the early phase of differentiated-type gastric cancers to ascertain any mechanistic relationships with mucin phenotypes. Methods Immunostaining for Cyclins D1, A, E, and p21, p27, p53 and β-catenin was used to examine impairments of the cell cycle in 190 gastric intramucosal differentiated-type cancers. Mucin phenotypes were determined by the expressions of MUC5AC, MUC6, MUC2 and CD10. A Ki-67 positive rate (PR was also examined. Results Overexpressions of p53, cyclin D1 and cyclin A were significantly more frequent in a gastric phenotype than an intestinal phenotype. Cyclin A was overexpressed in a mixed phenotype compared with an intestinal phenotype, while p27 overexpression was more frequent in an intestinal phenotype than in a mixed phenotype. Reduction of p21 was a common feature of the gastric intramucosal differentiated-type cancers examined. Conclusions Our results suggest that the levels of some cell cycle regulators appear to be associated with mucin phenotypes of early gastric differentiated-type cancers.

  1. An integrative analysis of cellular contexts, miRNAs and mRNAs reveals network clusters associated with antiestrogen-resistant breast cancer cells

    Directory of Open Access Journals (Sweden)

    Nam Seungyoon

    2012-12-01

    Full Text Available Abstract Background A major goal of the field of systems biology is to translate genome-wide profiling data (e.g., mRNAs, miRNAs into interpretable functional networks. However, employing a systems biology approach to better understand the complexities underlying drug resistance phenotypes in cancer continues to represent a significant challenge to the field. Previously, we derived two drug-resistant breast cancer sublines (tamoxifen- and fulvestrant-resistant cell lines from the MCF7 breast cancer cell line and performed genome-wide mRNA and microRNA profiling to identify differential molecular pathways underlying acquired resistance to these important antiestrogens. In the current study, to further define molecular characteristics of acquired antiestrogen resistance we constructed an “integrative network”. We combined joint miRNA-mRNA expression profiles, cancer contexts, miRNA-target mRNA relationships, and miRNA upstream regulators. In particular, to reduce the probability of false positive connections in the network, experimentally validated, rather than prediction-oriented, databases were utilized to obtain connectivity. Also, to improve biological interpretation, cancer contexts were incorporated into the network connectivity. Results Based on the integrative network, we extracted “substructures” (network clusters representing the drug resistant states (tamoxifen- or fulvestrant-resistance cells compared to drug sensitive state (parental MCF7 cells. We identified un-described network clusters that contribute to antiestrogen resistance consisting of miR-146a, -27a, -145, -21, -155, -15a, -125b, and let-7s, in addition to the previously described miR-221/222. Conclusions By integrating miRNA-related network, gene/miRNA expression and text-mining, the current study provides a computational-based systems biology approach for further investigating the molecular mechanism underlying antiestrogen resistance in breast cancer cells. In

  2. Expression of mRNAs for PPT, CGRP, NF-200, and MAP-2 in cocultures of dissociated DRG neurons and skeletal muscle cells in administration of NGF or NT-3

    Directory of Open Access Journals (Sweden)

    Weiwei Zhang

    2012-07-01

    Full Text Available Both neurotrophins (NTs and target skeletal muscle (SKM cells are essential for the maintenance of the function of neurons and nerve-muscle communication. However, much less is known about the association of target SKM cells with distinct NTs on the expression of mRNAs for preprotachykinin (PPT, calcitonin-gene related peptide (CGRP, neurofilament 200 (NF-200, and microtubule associated protein 2 (MAP-2 in dorsal root ganglion (DRG sensory neurons. In the present study, a neuromuscular coculture model of dissociated dorsal root ganglion (DRG neurons and SKM cells was established. The morphology of DRG neurons and SKM cells in coculture was observed with an inverted phase contrast microscope. The effects of nerve growth factor (NGF or neurotrophin-3 (NT-3 on the expression of mRNAs for PPT, CGRP, NF-200, and MAP-2 was analyzed by real time-PCR assay. The morphology of DRG neuronal cell bodies and SKM cells in neuromuscular coculture at different conditions was similar. The neurons presented evidence of dense neurite outgrowth in the presence of distinct NTs in neuromuscular cocultures. NGF and NT-3 increased mRNA levels of PPT, CGRP, and NF-200, but not MAP-2, in neuromuscular cocultures. These results offer new clues towards a better understanding of the association of target SKM cells with distinct NTs on the expression of mRNAs for PPT, CGRP, NF-200 and MAP-2, and implicate the association of target SKM cells and NTs with DRG sensory neuronal phenotypes.

  3. Monocistronic mRNAs containing defective hepatitis C virus-like picornavirus internal ribosome entry site elements in their 5 ' untranslated regions are efficiently translated in cells by a cap-dependent mechanism

    DEFF Research Database (Denmark)

    Belsham, Graham; Nielsen, Inge; Normann, Preben; Royall, E.; Roberts, L.O.

    2008-01-01

    unable to direct internal initiation, are not significant barriers to efficient translation of capped monocistronic mRNAs that contain these defective elements within their 5'UTRs. Moreover, the translation of these mRNAs is highly sensitive to the expression of an enterovirus 2A protease (which induces...

  4. FRET-based identification of mRNAs undergoing translation.

    Science.gov (United States)

    Stevens, Benjamin; Chen, Chunlai; Farrell, Ian; Zhang, Haibo; Kaur, Jaskiran; Broitman, Steven L; Smilansky, Zeev; Cooperman, Barry S; Goldman, Yale E

    2012-01-01

    We present proof-of-concept in vitro results demonstrating the feasibility of using single molecule fluorescence resonance energy transfer (smFRET) measurements to distinguish, in real time, between individual ribosomes programmed with several different, short mRNAs. For these measurements we use either the FRET signal generated between two tRNAs labeled with different fluorophores bound simultaneously in adjacent sites to the ribosome (tRNA-tRNA FRET) or the FRET signal generated between a labeled tRNA bound to the ribosome and a fluorescent derivative of ribosomal protein L1 (L1-tRNA FRET). With either technique, criteria were developed to identify the mRNAs, taking into account the relative activity of the mRNAs. These criteria enabled identification of the mRNA being translated by a given ribosome to within 95% confidence intervals based on the number of identified FRET traces. To upgrade the approach for natural mRNAs or more complex mixtures, the stoichiometry of labeling should be enhanced and photobleaching reduced. The potential for porting these methods into living cells is discussed. PMID:22693619

  5. Changes in proteolytic enzymes mRNAs and proteins relevant for meat quality during myogenesis and hypoxia of primary bovine satellite cells.

    Science.gov (United States)

    Yang, You Bing; Pandurangan, Muthuraman; Hwang, InHo

    2012-06-01

    The current study was conducted to evaluate the functions of μ-calpain (CAPN1), calpastatin, HSPs (heat shock proteins), and caspases during myogenesis and cell death induced by sodium azide (NaN(3)) hypoxia. The cell samples were divided into three groups: satellite cells formed at confluent monolayer (stage 1), stage 1 cells fusion into myotubes on d eight post-differentiation (stage 2), and stage 2 cells treated with 1 mM NaN(3) for 24 h (stage 3). Real-time RT-PCR showed that stage 2 cells had increased CAPN1, calpastatin, caspase 7, and CARD9 (Caspase activation and recruitment domain 9) mRNA expressions compared to stage 1 cells (*p tenderization. PMID:22648733

  6. Identification of altered microRNAs and mRNAs in the cumulus cells of PCOS patients: miRNA-509-3p promotes oestradiol secretion by targeting MAP3K8.

    Science.gov (United States)

    Huang, Xin; Liu, Chang; Hao, Cuifang; Tang, Qianqing; Liu, Riming; Lin, Shaoxia; Zhang, Luping; Yan, Wei

    2016-06-01

    Polycystic ovary syndrome (PCOS) is a common endocrine and metabolic disorder in women and is characterised by polycystic ovaries, hyperandrogenism and chronic anovulation. Although the clinical and biochemical signs of PCOS are typically heterogeneous, abnormal folliculogenesis is considered a common characteristic of PCOS. Our aim is to identify the altered miRNA and mRNA expression profiles in the cumulus cells of PCOS patients to investigate their molecular function in the aetiology and pathophysiology of PCOS. In this study, the miRNA expression profiles of the cumulus cell samples isolated from five PCOS and five control patients were determined by an miRNA microarray. At the same time, the altered mRNA profiles of the same cumulus cell samples were also identified by a cDNA microarray. From the microarray data, 17 miRNAs and 1263 mRNAs showed significantly different expression in the PCOS cumulus cells. The differentially expressed miRNA-509-3p and its potential target gene (MAP3K8) were identified from the miRNA and mRNA microarrays respectively. The expression of miRNA-509-3p was up-regulated and MAP3K8 was down-regulated in the PCOS cumulus cells. The direct interaction between miRNA-509-3p and MAP3K8 was confirmed by a luciferase activity assay in KGN cells. In addition, miRNA-509-3p mimics or inhibitor transfection tests in KGN cells further confirmed that miRNA-509-3p improved oestradiol (E2) secretion by inhibiting the expression of MAP3K8 These results help to characterise the pathogenesis of anovulation in PCOS, especially the regulation of E2 production. PMID:27001999

  7. Tristetraprolin and Its Family Members Can Promote the Cell-Free Deadenylation of AU-Rich Element-Containing mRNAs by Poly(A) Ribonuclease

    OpenAIRE

    Lai, Wi S.; Kennington, Elizabeth A; Blackshear, Perry J.

    2003-01-01

    Eukaryotic mRNA stability can be influenced by AU-rich elements (AREs) within mRNA primary sequences. Tristetraprolin (TTP) is a CCCH tandem zinc finger protein that binds to ARE-containing transcripts and destabilizes them, apparently by first promoting the removal of their poly(A) tails. We developed a cell-free system in which TTP and its related proteins stimulated the deadenylation of ARE-containing, polyadenylated transcripts. Transcript deadenylation was not stimulated when a mutant TT...

  8. Drosha, DGCR8, and Dicer mRNAs are down-regulated in human cells infected with dengue virus 4, and play a role in viral pathogenesis.

    Science.gov (United States)

    Casseb, S M M; Simith, D B; Melo, K F L; Mendonça, M H; Santos, A C M; Carvalho, V L; Cruz, A C R; Vasconcelos, P F C

    2016-01-01

    Dengue virus (DENV) and its four serotypes (DENV1-4) belong to the Flavivirus genus of the Flaviviridae family. DENV infection is a life-threatening disease, which results in up to 20,000 deaths each year. Viruses have been shown to encode trans-regulatory small RNAs, or microRNAs (miRNAs), which bind to messenger RNA and negatively regulate host or viral gene expression. During DENV infections, miRNAs interact with proteins in the RNAi pathway, and are processed by ribonucleases such as Dicer and Drosha. This study aims to investigate Drosha, DGCR8, and Dicer expression levels in human A-549 cells following DENV4 infection. DENV4 infected A-549 cells were collected daily for 5 days, and RNA was extracted to quantify viral load. Gene expression of Drosha, Dicer, and DGCR8 was determined using quantitative PCR (RT-qPCR). We found that DENV4 infection exhibited the highest viral load 3 days post-infection. Dicer, Drosha, and DGCR8 showed reduced expression following DENV4 infection as compared with negative controls. In addition, we hypothesize that reduced expression of DGCR8 may not only be related to miRNA biogenesis, but also other small RNAs. This study may change our understanding regarding the relationship between host cells and the dengue virus. PMID:27173348

  9. Proteomic profiling of glucocorticoid-exposed myogenic cells: Time series assessment of protein translocation and transcription of inactive mRNAs

    Directory of Open Access Journals (Sweden)

    Hoffman Eric P

    2009-07-01

    Full Text Available Abstract Background Prednisone, one of the most highly prescribed drugs, has well characterized effects on gene transcription mediated by the glucocorticoid receptor. These effects are typically occurring on the scale of hours. Prednisone also has a number of non-transcriptional effects (occurring on minutes scale on protein signaling, yet these are less well studied. We sought to expand the understanding of acute effects of prednisone action on cell signaling using a combination of SILAC strategy and subcellular fractionations from C2C12 myotubes. Results De novo translation of proteins was inhibited in both SILAC labeled and unlabeled C2C12 myotubes. Unlabeled cells were exposed to prednisone while SILAC labeled cells remained untreated. After 0, 5, 15, and 30 minutes of prednisone exposure, labeled and unlabeled cells were mixed at 1:1 ratios and fractionated into cytosolic and nuclear fractions. A total of 534 proteins in the cytosol and 626 proteins in the nucleus were identified and quantitated, using 3 or more peptides per protein with peptide based probability ≤ 0.001. We identified significant increases (1.7- to 3.1- fold in cytoplasmic abundance of 11 ribosomal proteins within 5 minutes of exposure, all of which returned to baseline by 30 min. We hypothesized that these drug-induced acute changes in the subcellular localization of the cell's protein translational machinery could lead to altered translation of quiescent RNAs. To test this, de novo protein synthesis was assayed after 15 minutes of drug exposure. Quantitative fluorography identified 16 2D gel spots showing rapid changes in translation; five of these were identified by MS/MS (pyruvate kinase, annexin A6 isoform A and isoform B, nasopharyngeal epithelium specific protein 1, and isoform 2 of Replication factor C subunit 1, and all showed the 5' terminal oligopyrimidine motifs associated with mRNA sequestration to and from inactive mRNA pools. Conclusion We describe novel

  10. Properties of human liver cytosolic aspartate aminotransferase mRNAs generated by alternative polyadenylation site selection

    International Nuclear Information System (INIS)

    Human cytosolic aspartate aminotransferase (cAspAT) cDNA clones have been isolated from an adult human liver cDNA library. Among the clones, two cDNAs of 1,550 and 1,950 base pairs, respectively, have been characterized. These two cDNAs differ only in the lengths of their 3' noncoding regions and by the presence of one or two putative polyadenylation signals AATAAA. Northern blot analysis revealed two different mRNAs of 2.1 and 1.8 kbp in several human tissues, whereas Southern blot analysis suggested the existence of a single gene for the human cAspAT. The two mRNA species result from the alternative use of two polyadenylation signals. In the liver, the relative ratio of these mRNAs varies among different species and, in humans at least, during development. The properties of the two mRNAs were compared. The half-lives of the 2.1 and 1.8 kbp mRNAs, in the HepG2 cell line, are 8 and 12 h, respectively. The two mRNAs have similar and rather short poly(A) tracts of 20-50 nucleotides. Both mRNAs are capable of directing the in vitro synthesis of the cAspAT protein. The authors conclude that both the 2.1 and 1.8 kbp cAspAT mRNAs are functional and exhibit similar properties

  11. SARS coronavirus nsp1 protein induces template-dependent endonucleolytic cleavage of mRNAs: viral mRNAs are resistant to nsp1-induced RNA cleavage.

    Directory of Open Access Journals (Sweden)

    Cheng Huang

    2011-12-01

    Full Text Available SARS coronavirus (SCoV nonstructural protein (nsp 1, a potent inhibitor of host gene expression, possesses a unique mode of action: it binds to 40S ribosomes to inactivate their translation functions and induces host mRNA degradation. Our previous study demonstrated that nsp1 induces RNA modification near the 5'-end of a reporter mRNA having a short 5' untranslated region and RNA cleavage in the encephalomyocarditis virus internal ribosome entry site (IRES region of a dicistronic RNA template, but not in those IRES elements from hepatitis C or cricket paralysis viruses. By using primarily cell-free, in vitro translation systems, the present study revealed that the nsp1 induced endonucleolytic RNA cleavage mainly near the 5' untranslated region of capped mRNA templates. Experiments using dicistronic mRNAs carrying different IRESes showed that nsp1 induced endonucleolytic RNA cleavage within the ribosome loading region of type I and type II picornavirus IRES elements, but not that of classical swine fever virus IRES, which is characterized as a hepatitis C virus-like IRES. The nsp1-induced RNA cleavage of template mRNAs exhibited no apparent preference for a specific nucleotide sequence at the RNA cleavage sites. Remarkably, SCoV mRNAs, which have a 5' cap structure and 3' poly A tail like those of typical host mRNAs, were not susceptible to nsp1-mediated RNA cleavage and importantly, the presence of the 5'-end leader sequence protected the SCoV mRNAs from nsp1-induced endonucleolytic RNA cleavage. The escape of viral mRNAs from nsp1-induced RNA cleavage may be an important strategy by which the virus circumvents the action of nsp1 leading to the efficient accumulation of viral mRNAs and viral proteins during infection.

  12. Prevention, Evaluation, and Rehabilitation of Cycling-Related Injury.

    Science.gov (United States)

    Kotler, Dana H; Babu, Ashwin N; Robidoux, Greg

    2016-01-01

    The unique quality of the bicycle is its ability to accommodate a wide variety of injuries and disabilities. Cycling for recreation, transportation, and competition is growing nationwide, and has proven health and societal benefits. The demands of each type of cycling dictate the necessary equipment, as well as potential for injury. Prevention of cycling-related injury in both the athlete and the recreational cyclist involves understanding the common mechanisms for both traumatic and overuse injury, and early correction of strength and flexibility imbalances, technique errors, and bicycle fit. PMID:27172085

  13. TTP and BRF proteins nucleate processing body formation to silence mRNAs with AU-rich elements

    OpenAIRE

    Franks, Tobias M.; Lykke-Andersen, Jens

    2007-01-01

    In mammalian cells, mRNAs with AU-rich elements (AREs) are targeted for translational silencing and rapid degradation. Here we present evidence that in human cells the proteins Tristetraprolin (TTP) and BRF-1 deliver ARE-mRNAs to processing bodies (PBs), cytoplasmic assemblies of mRNAs, and associated factors that promote translational silencing and mRNA decay. First, depletion of endogenous TTP and BRF proteins, or overexpression of dominant-negative mutant TTP proteins, impairs the localiza...

  14. Expression of mRNAs coding for the alpha 1 chain of type XIII collagen in human fetal tissues: comparison with expression of mRNAs for collagen types I, II, and III

    OpenAIRE

    1989-01-01

    This paper describes the topographic distribution of the multiple mRNAs coding for a novel human short-chain collagen, the alpha 1 chain of type XIII collagen. To identify the tissues and cells expressing these mRNAs, human fetal tissues of 15-19 gestational wk were studied by Northern and in situ hybridizations. The distribution pattern of the type XIII collagen mRNAs was compared with that of fibrillar collagen types I, II, and III using specific human cDNA probes for each collagen type. No...

  15. ncRNA-mediated bistability in the synthesis of hundreds of distinct mRNAs and proteins

    Science.gov (United States)

    Zhdanov, Vladimir P.

    2010-02-01

    The kinetics of gene expression can be bistable due to the feedback between the mRNA and protein formation. In eukaryotic cells, the interplay between mRNAs and proteins can be influenced by non-coding RNAs. Some of these RNAs, e.g., microRNAs, may target hundreds of distinct mRNAs. The model presented here shows how a non-coding RNA can be used as a mediator in order to involve numerous mRNAs and proteins into a bistable network.

  16. Differential nonsense mediated decay of mutated mRNAs in mismatch repair deficient colorectal cancers.

    Science.gov (United States)

    El-Bchiri, Jamila; Buhard, Olivier; Penard-Lacronique, Virginie; Thomas, Gilles; Hamelin, Richard; Duval, Alex

    2005-08-15

    The nonsense-mediated decay (NMD) system normally targets mRNAs with premature termination codons (PTCs) for rapid degradation. We investigated for a putative role of NMD in cancers with microsatellite instability (MSI-H cancers), because numerous mutant mRNAs containing PTC are generated in these tumors as a consequence of their mismatch repair deficiency. Using a quantitative RT-PCR approach in a large series of colorectal cancer cell lines, we demonstrate a significantly increased rate of degradation of mutant mRNAs containing a PTC compared with wild-type. A specific siRNA strategy was used to inhibit RENT-1 and/or RENT-2 activity, two major genes in the NMD system. This allowed us to show that increased degradation of PTC-containing mRNAs in MSI-H tumors was partly dependent upon NMD activity. The efficiency of NMD for the degradation of mutant mRNAs from target genes was highly variable in these cancers. NMD degraded some of them (TGFssRII, MSH3, GRK4), although allowing the persistent expression of others (BAX, TCF-4). This is of particular interest within the context of a proposed conservation of biological activity for the corresponding mutated proteins. We thus propose that NMD might play an important role in the selection of target gene mutations with a functional role in MSI-H carcinogenesis. PMID:16000315

  17. Single particle imaging of mRNAs crossing the nuclear pore: Surfing on the edge.

    Science.gov (United States)

    Palazzo, Alexander F; Truong, Mathew

    2016-08-01

    Six years ago, the Singer lab published a landmark paper which described how individual mRNA particles cross the nuclear pore complex in mammalian tissue culture cells. This involved the simultaneous imaging of mRNAs, each labeled by a large number of tethered fluorescent proteins and fluorescently tagged nuclear pore components. Now two groups have applied this technique to the budding yeast Saccharomyces cerevisiae. Their results indicate that in the course of nuclear export, mRNAs likely engage complexes that are present on either side of the pore and that these interactions are modulated by proteins present in the messenger ribonucleoprotein (mRNP) complex. These findings lend support to the notion that just before and/or after the completion of nuclear export, mRNPs undergo one or more maturation steps that prepare the packaged mRNAs for translation. These results represent new and exciting insights into the mechanism of mRNA nuclear export. PMID:27276446

  18. Acute and chronic glucocorticoid treatments regulate astrocyte-enriched mRNAs in multiple brain regions in vivo

    Directory of Open Access Journals (Sweden)

    BradleyS.Carter

    2013-08-01

    Full Text Available Previous studies have primarily interpreted gene expression regulation by glucocorticoids in the brain in terms of impact on neurons; however, less is known about the corresponding impact of glucocorticoids on glia and specifically astrocytes in vivo. Recent microarray experiments have identified glucocorticoid-sensitive mRNAs in primary astrocyte cell culture, including a number of mRNAs that have reported astrocyte-enriched expression patterns relative to other brain cell types. Here, we have tested whether elevations of glucocorticoids regulate a subset of these mRNAs in vivo following acute and chronic corticosterone exposure in adult mice. Acute corticosterone exposure was achieved by a single injection of 10 mg/kg corticosterone, and tissue samples were harvested two hours post-injection. Chronic corticosterone exposure was achieved by administering 10 mg/mL corticosterone via drinking water for two weeks. Gene expression was then assessed in two brain regions associated with glucocorticoid action (prefrontal cortex and hippocampus by qPCR and by in situ hybridization. The majority of measured mRNAs regulated by glucocorticoids in astrocytes in vitro were similarly regulated by acute and/or chronic glucocorticoid exposure in vivo. In addition, the expression levels for mRNAs regulated in at least one corticosterone exposure condition (acute/chronic demonstrated moderate positive correlation between the two conditions by brain region. In situ hybridization analyses suggest that select mRNAs are regulated by chronic corticosterone exposure specifically in astroctyes based on (1 similar general expression patterns between corticosterone-treated and vehicle-treated animals and (2 similar expression patterns to the pan-astrocyte marker Aldh1l1. Our findings demonstrate that glucocorticoids regulate astrocyte-enriched mRNAs in vivo and suggest that glucocorticoids regulate gene expression in the brain in a cell type-dependent fashion.

  19. A beacon in the cytoplasm: Tracking translation of single mRNAs.

    Science.gov (United States)

    Pingali, Hema V; Hilliker, Angela K

    2016-09-12

    Translation is carefully regulated to control protein levels and allow quick responses to changes in the environment. Certain questions about translation in vivo have been unattainable until now. In this issue, Pichon et al. (2016. J. Cell Biol http://dx.doi.org/10.1083/jcb.201605024) describe a new technique to allow real-time monitoring of translation on single mRNAs. PMID:27597752

  20. Pluripotent State Induction in Mouse Embryonic Fibroblast Using mRNAs of Reprogramming Factors

    Directory of Open Access Journals (Sweden)

    Ahmed Kamel El-Sayed

    2014-11-01

    Full Text Available Reprogramming of somatic cells has great potential to provide therapeutic treatments for a number of diseases as well as provide insight into mechanisms underlying early embryonic development. Improvement of induced Pluripotent Stem Cells (iPSCs generation through mRNA-based methods is currently an area of intense research. This approach provides a number of advantages over previously used methods such as DNA integration and insertional mutagenesis. Using transfection of specifically synthesized mRNAs of various pluripotency factors, we generated iPSCs from mouse embryonic fibroblast (MEF cells. The genetic, epigenetic and functional properties of the iPSCs were evaluated at different times during the reprogramming process. We successfully introduced synthesized mRNAs, which localized correctly inside the cells and exhibited efficient and stable translation into proteins. Our work demonstrated a robust up-regulation and a gradual promoter de-methylation of the pluripotency markers, including non-transfected factors such as Nanog, SSEA-1 (stage-specific embryonic antigen 1 and Rex-1 (ZFP-42, zinc finger protein 42. Using embryonic stem cells (ESCs conditions to culture the iPS cells resulted in formation of ES-like colonies after approximately 12 days with only five daily repeated transfections. The colonies were positive for alkaline phosphatase and pluripotency-specific markers associated with ESCs. This study revealed the ability of pluripotency induction and generation of mouse mRNA induced pluripotent stem cells (mRNA iPSCs using transfection of specifically synthesized mRNAs of various pluripotency factors into mouse embryonic fibroblast (MEF cells. These generated iPSCs exhibited molecular and functional properties similar to ESCs, which indicate that this method is an efficient and viable alternative to ESCs and can be used for further biological, developmental and therapeutic investigations.

  1. Red1 promotes the elimination of meiosis-specific mRNAs in vegetatively growing fission yeast

    OpenAIRE

    Sugiyama, Tomoyasu; Sugioka-Sugiyama, Rie

    2011-01-01

    In mitotic fission yeast cells, expression of meiosis-specific mRNAs is prevented by their selective degradation in nuclear bodies. A novel required factor, Red1, leaves these nuclear bodies during meiosis, offering first hints on the regulation of this process.

  2. Granules Harboring Translationally Active mRNAs Provide a Platform for P-Body Formation following Stress

    Directory of Open Access Journals (Sweden)

    Jennifer Lui

    2014-11-01

    Full Text Available The localization of mRNA to defined cytoplasmic sites in eukaryotic cells not only allows localized protein production but also determines the fate of mRNAs. For instance, translationally repressed mRNAs localize to P-bodies and stress granules where their decay and storage, respectively, are directed. Here, we find that several mRNAs are localized to granules in unstressed, actively growing cells. These granules play a key role in the stress-dependent formation of P-bodies. Specific glycolytic mRNAs are colocalized in multiple granules per cell, which aggregate during P-body formation. Such aggregation is still observed under conditions or in mutants where P-bodies do not form. In unstressed cells, the mRNA granules appear associated with active translation; this might enable a coregulation of protein expression from the same pathways or complexes. Parallels can be drawn between this coregulation and the advantage of operons in prokaryotic systems.

  3. Polycistronic trypanosome mRNAs are a target for the exosome

    Science.gov (United States)

    Kramer, Susanne; Piper, Sophie; Estevez, Antonio; Carrington, Mark

    2016-01-01

    Eukaryotic cells have several mRNA quality control checkpoints to avoid the production of aberrant proteins. Intron-containing mRNAs are actively degraded by the nuclear exosome, prevented from nuclear exit and, if these systems fail, degraded by the cytoplasmic NMD machinery. Trypanosomes have only two introns. However, they process mRNAs from long polycistronic precursors by trans-splicing and polycistronic mRNA molecules frequently arise from any missed splice site. Here, we show that RNAi depletion of the trypanosome exosome, but not of the cytoplasmic 5′-3′ exoribonuclease XRNA or the NMD helicase UPF1, causes accumulation of oligocistronic mRNAs. We have also revisited the localization of the trypanosome exosome by expressing eYFP-fusion proteins of the exosome subunits RRP44 and RRP6. Both proteins are significantly enriched in the nucleus. Together with published data, our data suggest a major nuclear function of the trypanosome exosome in rRNA, snoRNA and mRNA quality control. PMID:26946399

  4. Death of a dogma: eukaryotic mRNAs can code for more than one protein.

    Science.gov (United States)

    Mouilleron, Hélène; Delcourt, Vivian; Roucou, Xavier

    2016-01-01

    mRNAs carry the genetic information that is translated by ribosomes. The traditional view of a mature eukaryotic mRNA is a molecule with three main regions, the 5' UTR, the protein coding open reading frame (ORF) or coding sequence (CDS), and the 3' UTR. This concept assumes that ribosomes translate one ORF only, generally the longest one, and produce one protein. As a result, in the early days of genomics and bioinformatics, one CDS was associated with each protein-coding gene. This fundamental concept of a single CDS is being challenged by increasing experimental evidence indicating that annotated proteins are not the only proteins translated from mRNAs. In particular, mass spectrometry (MS)-based proteomics and ribosome profiling have detected productive translation of alternative open reading frames. In several cases, the alternative and annotated proteins interact. Thus, the expression of two or more proteins translated from the same mRNA may offer a mechanism to ensure the co-expression of proteins which have functional interactions. Translational mechanisms already described in eukaryotic cells indicate that the cellular machinery is able to translate different CDSs from a single viral or cellular mRNA. In addition to summarizing data showing that the protein coding potential of eukaryotic mRNAs has been underestimated, this review aims to challenge the single translated CDS dogma. PMID:26578573

  5. 肌醇5'磷酸酶基因突变对K562细胞周期蛋白和Akt磷酸化的影响%Effects of SHIP gene mutation on cell cycle related proteins and phosphorylated Akt in K562 cells

    Institute of Scientific and Technical Information of China (English)

    杨琳; 罗建民; 刘小军; 温树鹏; 杨敬慈; 张敬宇

    2009-01-01

    Objective To investigate the effect of SHIP gene mutation on the cell cycle and its related gene expression in K562 cells.Methods The recombinated green fluorescent protein (GFP) containing F1V-SHIP gene was transfected into K562 cells.The transfection efficiency and cell cycle of K562/SHIP were assessed by flow cytometry (FCM).The proliferation of K562 ceils was detected by MTT assay,the mRNA levels of SHIP by real-time fluorescent relative-quantification reverse transcriptional PCR(FQ-PCR),and the protein levels of SHIP,CyclinDl,p21WAF1/CIPI and p27KIP1 by Western blot.Results Wild type SHIP inhibited K562 cell proliferation and caused a G0/G1 arrest [(34.2 ± 7.8) % vs (0.7 ± 8.3) % (P0.05).Conclusion ①wtSHIP gene can downregulate Akt phosphorylation and result in inhibition of cyclin D1 expression,up-regulating p27KIP1 and p21WIF1/CIPI expression,finally leading to the reduction of K562 cell proliferation,and inducing G0/G1 phase arrest.②SHIP gene suppresses the proliferation of K562,being dependent on its intact structure and function.%目的 从分子水平探讨肌醇5'磷酸酶(SHIP)基因突变对人白血病细胞系K562细胞周期及其相关基因表达的影响.方法 应用携带野生型和突变型SHIP及绿色荧光蛋白的慢病毒及空载体慢病毒质粒转染K562细胞,通过流式细胞术检测K562/SHIP细胞转染效率、细胞增殖指数及细胞周期变化;MTT法检测细胞增殖活性改变,实时荧光定量PCR(FQ-PCR)检测SHIP mRNA水平变化,Western blot检测各组K562细胞SHIP、细胞周期蛋白(cyclin)D1、p21WAF1/CIPI、P27KIP1蛋白表达水平及Akt磷酸化变化.结果 野生型SHIP基因能明显抑制K562细胞增殖,并产生明显的G0/G1期阻滞[G0/G1期细胞分别为(34.2±7.8)%和(0.7±8.3)%,P0.05].Western blot结果发现转染野生型SHIP基因后K562细胞Akt磷酸化和cyclin D1表达水平明显下降(P0.05).结论 ①野生型SHIP基因通过下调K562细胞Akt磷酸化

  6. Birthing histone mRNAs by CSR-1 section

    OpenAIRE

    Pasquinelli, Amy E.

    2012-01-01

    Histone pre-mRNAs undergo maturation through a mechanism distinct from all other RNA Pol II transcripts. In C. elegans, 3′-end processing of histone mRNAs depends on the RNAi pathway involving the Argonaute protein CSR-1.

  7. FRET-Based Identification of mRNAs Undergoing Translation

    OpenAIRE

    Stevens, Benjamin; Chen, Chunlai; Farrell, Ian; Zhang, Haibo; Kaur, Jaskiran; Broitman, Steven L.; Smilansky, Zeev; Cooperman, Barry S.; Goldman, Yale E.

    2012-01-01

    We present proof-of-concept in vitro results demonstrating the feasibility of using single molecule fluorescence resonance energy transfer (smFRET) measurements to distinguish, in real time, between individual ribosomes programmed with several different, short mRNAs. For these measurements we use either the FRET signal generated between two tRNAs labeled with different fluorophores bound simultaneously in adjacent sites to the ribosome (tRNA-tRNA FRET) or the FRET signal generated between a l...

  8. Bacterial translational regulations: high diversity between all mRNAs and major role in gene expression

    Directory of Open Access Journals (Sweden)

    Picard Flora

    2012-10-01

    Full Text Available Abstract Background In bacteria, the weak correlations at the genome scale between mRNA and protein levels suggest that not all mRNAs are translated with the same efficiency. To experimentally explore mRNA translational level regulation at the systemic level, the detailed translational status (translatome of all mRNAs was measured in the model bacterium Lactococcus lactis in exponential phase growth. Results Results demonstrated that only part of the entire population of each mRNA species was engaged in translation. For transcripts involved in translation, the polysome size reached a maximum of 18 ribosomes. The fraction of mRNA engaged in translation (ribosome occupancy and ribosome density were not constant for all genes. This high degree of variability was analyzed by bioinformatics and statistical modeling in order to identify general rules of translational regulation. For most of the genes, the ribosome density was lower than the maximum value revealing major control of translation by initiation. Gene function was a major translational regulatory determinant. Both ribosome occupancy and ribosome density were particularly high for transcriptional regulators, demonstrating the positive role of translational regulation in the coordination of transcriptional networks. mRNA stability was a negative regulatory factor of ribosome occupancy and ribosome density, suggesting antagonistic regulation of translation and mRNA stability. Furthermore, ribosome occupancy was identified as a key component of intracellular protein levels underlining the importance of translational regulation. Conclusions We have determined, for the first time in a bacterium, the detailed translational status for all mRNAs present in the cell. We have demonstrated experimentally the high diversity of translational states allowing individual gene differentiation and the importance of translation-level regulation in the complex process linking gene expression to protein

  9. The fragile X protein binds mRNAs involved in cancer progression and modulates metastasis formation.

    Science.gov (United States)

    Lucá, Rossella; Averna, Michele; Zalfa, Francesca; Vecchi, Manuela; Bianchi, Fabrizio; La Fata, Giorgio; Del Nonno, Franca; Nardacci, Roberta; Bianchi, Marco; Nuciforo, Paolo; Munck, Sebastian; Parrella, Paola; Moura, Rute; Signori, Emanuela; Alston, Robert; Kuchnio, Anna; Farace, Maria Giulia; Fazio, Vito Michele; Piacentini, Mauro; De Strooper, Bart; Achsel, Tilmann; Neri, Giovanni; Neven, Patrick; Evans, D Gareth; Carmeliet, Peter; Mazzone, Massimiliano; Bagni, Claudia

    2013-10-01

    The role of the fragile X mental retardation protein (FMRP) is well established in brain, where its absence leads to the fragile X syndrome (FXS). FMRP is almost ubiquitously expressed, suggesting that, in addition to its effects in brain, it may have fundamental roles in other organs. There is evidence that FMRP expression can be linked to cancer. FMR1 mRNA, encoding FMRP, is overexpressed in hepatocellular carcinoma cells. A decreased risk of cancer has been reported in patients with FXS while a patient-case with FXS showed an unusual decrease of tumour brain invasiveness. However, a role for FMRP in regulating cancer biology, if any, remains unknown. We show here that FMRP and FMR1 mRNA levels correlate with prognostic indicators of aggressive breast cancer, lung metastases probability and triple negative breast cancer (TNBC). We establish that FMRP overexpression in murine breast primary tumours enhances lung metastasis while its reduction has the opposite effect regulating cell spreading and invasion. FMRP binds mRNAs involved in epithelial mesenchymal transition (EMT) and invasion including E-cadherin and Vimentin mRNAs, hallmarks of EMT and cancer progression. PMID:24092663

  10. Structural and evolutionary analysis of the two chimpanzee alpha-globin mRNAs.

    OpenAIRE

    Liebhaber, S A; Begley, K A

    1983-01-01

    Two distinct alpha-globin mRNAs were detected in chimpanzee reticulocyte mRNA using a primer extension assay. DNA copies of these two mRNAs were cloned in the bacterial plasmid pBR322, and their sequence was determined. The two alpha-globin mRNAs have obvious structural homology to the two human alpha-globin mRNAs, alpha 1 and alpha 2. Comparison of the two chimpanzee alpha-globin mRNAs to each other and to their corresponding human counterparts revealed evidence of a recent gene conversion i...

  11. A Novel Role of Vimentin Filaments: Binding and Stabilization of Collagen mRNAs

    OpenAIRE

    Azariyas A Challa; Stefanovic, Branko

    2011-01-01

    The stem-loop in the 5′ untranslated region (UTR) of collagen α1(I) and α2(I) mRNAs (5′SL) is the key element regulating their stability and translation. Stabilization of collagen mRNAs is the predominant mechanism for high collagen expression in fibrosis. LARP6 binds the 5′SL of α1(I) and α2(I) mRNAs with high affinity. Here, we report that vimentin filaments associate with collagen mRNAs in a 5′SL- and LARP6-dependent manner and stabilize collagen mRNAs. LARP6 interacts with vimentin filame...

  12. Drosophila Pumilio Protein Contains Multiple Autonomous Repression Domains That Regulate mRNAs Independently of Nanos and Brain Tumor

    OpenAIRE

    Weidmann, Chase A.; Goldstrohm, Aaron C.

    2012-01-01

    Drosophila melanogaster Pumilio is an RNA-binding protein that potently represses specific mRNAs. In developing embryos, Pumilio regulates a key morphogen, Hunchback, in collaboration with the cofactor Nanos. To investigate repression by Pumilio and Nanos, we created cell-based assays and found that Pumilio inhibits translation and enhances mRNA decay independent of Nanos. Nanos robustly stimulates repression through interactions with the Pumilio RNA-binding domain. We programmed Pumilio to r...

  13. RNase-L control of cellular mRNAs: roles in biologic functions and mechanisms of substrate targeting.

    Science.gov (United States)

    Brennan-Laun, Sarah E; Ezelle, Heather J; Li, Xiao-Ling; Hassel, Bret A

    2014-04-01

    RNase-L is a mediator of type 1 interferon-induced antiviral activity that has diverse and critical cellular roles, including the regulation of cell proliferation, differentiation, senescence and apoptosis, tumorigenesis, and the control of the innate immune response. Although RNase-L was originally shown to mediate the endonucleolytic cleavage of both viral and ribosomal RNAs in response to infection, more recent evidence indicates that RNase-L also functions in the regulation of cellular mRNAs as an important mechanism by which it exerts its diverse biological functions. Despite this growing body of work, many questions remain regarding the roles of mRNAs as RNase-L substrates. This review will survey known and putative mRNA substrates of RNase-L, propose mechanisms by which it may selectively cleave these transcripts, and postulate future clinical applications. PMID:24697205

  14. Use of in situ hybridization to identify collagen and albumin mRNAs in isolated mouse hepatocytes.

    OpenAIRE

    Saber, M A; Zern, M A; Shafritz, D. A.

    1983-01-01

    We present a simple and improved method for in situ localization of albumin and collagen mRNAs in isolated mouse hepatocytes. The cells were isolated by collagenase perfusion, mincing, and differential centrifugation. Nick-translated 3H-labeled mouse albumin cDNA (pmalb-2) and chicken pro-alpha 2(I) collagen cDNA (pCg45) probes were then hybridized with the cells in silane-treated microcentrifuge tubes. The cells were transferred and fixed to a microscope slide and hybridization was evaluated...

  15. Expression and relationship of microRNA-31 and FOXP3 mRNAs in CD4 + T cells from a mouse asthma model%哮喘小鼠CD4+T细胞中microRNA-31和FOXP3mRNA的表达及相互关系的研究

    Institute of Scientific and Technical Information of China (English)

    酆孟洁; 史菲; 邱晨

    2012-01-01

    Objective To determine the expression levels of microRNA-31 and FOXP3 mRNAs in the spleen CD4 + T cells from asthma mice, and identify their relationship. Methods A total of 15 Balb/c mice were randomly divided into 3 groups, normal control group, model group, and dexamethasone treatment group. The murine asthma model was sensitized and challenged by ovalbumin ( OVA). The inflammatory cytokines IL-4 and IFN-γ in the bronchoalveolar lavage fluid ( BALF) in the 3 groups were measured by ELISA. Expression of microRNA-31 and FOXP3 mRNAs in spleen CD4 + T cells from the 3 groups were detected by quantitative real time-PCR assay. The relationship of the expression were also analyzed. Results The BALF level of IL-4 was increased in the model group (228.29 ±66. 18 pg/ml) compared with the normal control group (66.63 ± 17.33 pg/ml, P 0.05). There was no significant difference in levels of IFN-γ among the three groups( P > 0.05). The expression of FOXP3 Mrna in the spleen CD4 + T cells in the model group was 0. 10 times as great as in the normal control group (P 0. 05 ). The expressions of microRNA-31 Mrna in the splenn CD4+ T lymphocytes in the model group were 4.79 times as great as in the normal control (P 0. 05). Pearson correlation coefficient between microRNA-31 and FOXP3 Mrna was -0.609 (P<0.05). Conclusion There is a negative linear correlation between microRNA-31 and FOXP3 Mrna in asthma, and microRNA-31 and dexamethasone mayaffect the expression of FOXP3 Mrna via different pathways.%目的 初步探讨哮喘CD4+T细胞中microRNA-31和FOXP3的表达水平、两者的相关性及激素对它们的影响.方法 以卵蛋白(OVA)致敏激发建立哮喘小鼠模型.将15只BALB/c小鼠分为正常对照组、OVA组和地塞米松组,EHSA检测小鼠支气管肺泡灌洗液(BALF)中IL-4和IFN-γ水平;实时荧光定量PCR检测小鼠脾脏CD4+T细胞的microRNA-31和FOXP3 mRNA的水平,并分析二者之间的相关性.结果 OVA组BALF中IL-4

  16. The Expression of Insulin-like Growth Factor and Insulin-like Growth Factor Binding Protein mRNAs in Mouse Placenta

    DEFF Research Database (Denmark)

    Carter, Anthony M.; Nygard, K.; Mazzuca, D.M.; Han, V.K.M.

    Insulin-like growth factors (IGFs) and IGF binding proteins (IGFBPs) are paracrine regulators of tissue growth and development, and are expressed at the sites of biological action. To study the role of the IGFs and IGFBPs in mouse placental development, we determined the temporal and spatial....... Strong expression of IGF-II mRNA in glycogen cells suggests a role in the autocrine/paracrine regulation of invasion. Similar to rat and guinea pig, but in contrast to man and primates, IGFBP mRNAs, except IGFBP-4, were not expressed in mouse decidua. However, IGFBP-3, -4 and -5 mRNAs were expressed in...... endothelium of maternal blood vessels, and IGFBP-2 and -6 mRNAs in myometrium, where IGFBPs may play a critical role in regulating trophoblast invasion. These findings suggest possible biological roles of the peptides at the feto-maternal interface....

  17. Regulation of maternal mRNAs in early development.

    Science.gov (United States)

    Farley, Brian M; Ryder, Sean P

    2008-01-01

    Most sexually reproducing metazoans are anisogamous, meaning that the two gametes that combine during fertilization differ greatly in size. By convention, the larger gametes are considered female and are called ova, while the smaller gametes are male and are called sperm. In most cases, both gametes contribute similarly to the chromosomal content of the new organism. In contrast, the maternal gamete contributes nearly all of the cytoplasm. This cytoplasmic contribution is crucial to patterning early development; it contains the maternal proteins and transcripts that guide the early steps of development prior to the activation of zygotic transcription. This review compares and contrasts early development in common laboratory model organisms in order to highlight the similarities and differences in the regulation of maternal factors. We will focus on the production and reversible silencing of maternal mRNAs during oogenesis, their asymmetric activation after fertilization, and their subsequent clearance at the midblastula transition. Where possible, insights from mechanistic studies are presented. PMID:18365862

  18. Kid cleaves specific mRNAs at UUACU sites to rescue the copy number of plasmid R1

    OpenAIRE

    Pimentel, Belén; Madine, Mark A.; de la Cueva-Méndez, Guillermo

    2005-01-01

    Stability and copy number of extra-chromosomal elements are tightly regulated in prokaryotes and eukaryotes. Toxin Kid and antitoxin Kis are the components of the parD stability system of prokaryotic plasmid R1 and they can also function in eukaryotes. In bacteria, Kid was thought to become active only in cells that lose plasmid R1 and to cleave exclusively host mRNAs at UA(A/C/U) trinucleotide sites to eliminate plasmid-free cells. Instead, we demonstrate here that Kid becomes active in plas...

  19. Possible Cis-acting signal that could be involved in the localization of different mRNAs in neuronal axons

    Directory of Open Access Journals (Sweden)

    Manzo Jorge

    2005-08-01

    Full Text Available Abstract Background Messenger RNA (mRNA comprises three major parts: a 5'-UTR (UnTranslated Region, a coding region, and a 3'-UTR. The 3'-UTR contains signal sequences involved in polyadenylation, degradation and localization/stabilization processes. Some sequences in the 3'-UTR are involved in the localization of mRNAs in (e.g. neurons, epithelial cells, oocytes and early embryos, but such localization has been most thoroughly studied in neurons. Neuronal polarity is maintained by the microtubules (MTs found along both dendrites and axon and is partially influenced by sub-cellular mRNA localization. A widely studied mRNA is that for Tau protein, which is located in the axon hillock and growth cone; its localization depends on the well-characterized cis-acting signal (U-rich region in the 3'-UTR. Methods We compared the cis-acting signal of Tau with mRNAs in the axonal regions of neurons using the ClustalW program for alignment of sequences and the Mfold program for analysis of secondary structures. Results We found that at least 3 out of 12 mRNA analyzed (GRP75, cofilin and synuclein have a sequence similar to the cis-acting signal of Tau in the 3'-UTR. This could indicate that these messengers are localized specifically in the axon. The Mfold program showed that these mRNAs have a similar "bubble" structure in the putative sequence signal. Conclusion Hence, we suggest that a U-rich sequence in the 3'-UTR region of the mRNA could act as a signal for its localization in the axon in neuronal cells. Sequences homologous to the DTE sequence of BC1 mRNA could direct the messenger to the dendrites. Messengers with homologues of both types of sequence, e.g. β-actin, might be located in both dendrites and axon.

  20. Accumulation of long-lived mRNAs associated with germination in embryos during seed development of rice

    OpenAIRE

    Sano, Naoto; Ono, Hanako; Murata, Kazumasa; Yamada, Tetsuya; Hirasawa, Tadashi; Kanekatsu, Motoki

    2015-01-01

    Highlight Long-lived mRNAs stored in dry seed are translated after imbibition for germination. We report accumulation of long-lived mRNAs in developing rice embryos and candidates of the mRNAs required for germination.

  1. Microarray-based identification and RT-PCR test screening for epithelial-specific mRNAs in peripheral blood of patients with colon cancer

    Directory of Open Access Journals (Sweden)

    Coppola Domenico

    2006-10-01

    Full Text Available Abstract Background The efficacy of screening for colorectal cancer using a simple blood-based assay for the detection of tumor cells disseminated in the circulation at an early stage of the disease is gaining positive feedback from several lines of research. This method seems able to reduce colorectal cancer mortality and may replace colonoscopy as the most effective means of detecting colonic lesions. Methods In this work, we present a new microarray-based high-throughput screening method to identifying candidate marker mRNAs for the early detection of epithelial cells diluted in peripheral blood cells. This method includes 1. direct comparison of different samples of colonic mucosa and of blood cells to identify consistent epithelial-specific mRNAs from among 20,000 cDNA assayed by microarray slides; 2. identification of candidate marker mRNAs by data analysis, which allowed selection of only 10 putative differentially expressed genes; 3. Selection of some of the most suitable mRNAs (TMEM69, RANBP3 and PRSS22 that were assayed in blood samples from normal subjects and patients with colon cancer as possible markers for the presence of epithelial cells in the blood, using reverse transcription – polymerase chain reaction (RT-PCR. Results Our present results seem to provide an indication, for the first time obtained by genome-scale screening, that a suitable and consistent colon epithelium mRNA marker may be difficult to identify. Conclusion The design of new approaches to identify such markers is warranted.

  2. Differential distribution of ELMO1 and ELMO2 mRNAs in the developing mouse brain.

    Science.gov (United States)

    Katoh, Hironori; Fujimoto, Satoshi; Ishida, Chisaki; Ishikawa, Yukio; Negishi, Manabu

    2006-02-16

    ELMO is an upstream regulator of the Rho family small GTPase Rac. We investigated the distributions of mRNAs of two subtypes of ELMO, ELMO1 and ELMO2, in the developing mouse brain. Both ELMO1 and ELMO2 mRNAs are widely distributed in the developing mouse brain, but they were expressed in different neuronal populations in the cerebral cortex, thalamus, and cerebellum. Thus, ELMO1 and ELMO2 may play different roles during brain development. PMID:16443196

  3. Stress and Withdrawal from Chronic Ethanol Induce Selective Changes in Neuroimmune mRNAs in Differing Brain Sites.

    Science.gov (United States)

    Knapp, Darin J; Harper, Kathryn M; Whitman, Buddy A; Zimomra, Zachary; Breese, George R

    2016-01-01

    Stress is a strong risk factor in alcoholic relapse and may exert effects that mimic aspects of chronic alcohol exposure on neurobiological systems. With the neuroimmune system becoming a prominent focus in the study of the neurobiological consequences of stress, as well as chronic alcohol exposure proving to be a valuable focus in this regard, the present study sought to compare the effects of stress and chronic ethanol exposure on induction of components of the neuroimmune system. Rats were exposed to either 1 h exposure to a mild stressor (restraint) or exposure to withdrawal from 15 days of chronic alcohol exposure (i.e., withdrawal from chronic ethanol, WCE) and assessed for neuroimmune mRNAs in brain. Restraint stress alone elevated chemokine (C-C motif) ligand 2 (CCL2), interleukin-1-beta (IL-1β), tumor necrosis factor alpha (TNFα) and toll-like receptor 4 (TLR4) mRNAs in the cerebral cortex within 4 h with a return to a control level by 24 h. These increases were not accompanied by an increase in corresponding proteins. Withdrawal from WCE also elevated cytokines, but did so to varying degrees across different cytokines and brain regions. In the cortex, stress and WCE induced CCL2, TNFα, IL-1β, and TLR4 mRNAs. In the hypothalamus, only WCE induced cytokines (CCL2 and IL-1β) while in the hippocampus, WCE strongly induced CCL2 while stress and WCE induced IL-1β. In the amygdala, only WCE induced CCL2. Finally-based on the previously demonstrated role of corticotropin-releasing factor 1 (CRF1) receptor inhibition in blocking WCE-induced cytokine mRNAs-the CRF1 receptor antagonist CP154,526 was administered to a subgroup of stressed rats and found to be inactive against induction of CCL2, TNFα, or IL-1β mRNAs. These differential results suggest that stress and WCE manifest broad neuroimmune effects in brain depending on the cytokine and brain region, and that CRF inhibition may not be a relevant mechanism in non-alcohol exposed animals. Overall, these

  4. The RNA Binding Zinc Finger Protein Tristetraprolin Regulates AU-Rich mRNAs Involved in Breast Cancer-Related Processes

    OpenAIRE

    Al-Souhibani, Norah; Al-Ahmadi, Wijdan; Hesketh, John E.; Blackshear, Perry J.; Khabar, Khalid S.A.

    2010-01-01

    Tristetraprolin (TTP or ZFP36) is a tandem CCCH zinc finger RNA binding protein that regulates the stability of certain AU-rich mRNAs. Recent work suggests that TTP is deficient in cancer cells when compared to normal cell types. Here we found that TTP expression was lower in invasive breast cancer cells (MDA-MB-231) compared to normal breast cell lines, MCF12A and MCF-10. TTP targets were probed using a novel approach by expressing the C124R zinc finger TTP mutant that act as dominant negati...

  5. Expression analysis of miRNA and target mRNAs in esophageal cancer

    International Nuclear Information System (INIS)

    We aimed to investigate miRNAs and related mRNAs through a network-based approach in order to learn the crucial role that they play in the biological processes of esophageal cancer. Esophageal squamous-cell carcinoma (ESCC) and adenocarcinoma (EAC)-related miRNA and gene expression data were downloaded from the Gene Expression Omnibus database, and differentially expressed miRNAs and genes were selected. Target genes of differentially expressed miRNAs were predicted and their regulatory networks were constructed. Differentially expressed miRNA analysis selected four miRNAs associated with EAC and ESCC, among which hsa-miR-21 and hsa-miR-202 were shared by both diseases. hsa-miR-202 was reported for the first time to be associated with esophageal cancer in the present study. Differentially expressed miRNA target genes were mainly involved in cancer-related and signal-transduction pathways. Functional categories of these target genes were related to transcriptional regulation. The results may indicate potential target miRNAs and genes for future investigations of esophageal cancer

  6. Drosophila Pumilio protein contains multiple autonomous repression domains that regulate mRNAs independently of Nanos and brain tumor.

    Science.gov (United States)

    Weidmann, Chase A; Goldstrohm, Aaron C

    2012-01-01

    Drosophila melanogaster Pumilio is an RNA-binding protein that potently represses specific mRNAs. In developing embryos, Pumilio regulates a key morphogen, Hunchback, in collaboration with the cofactor Nanos. To investigate repression by Pumilio and Nanos, we created cell-based assays and found that Pumilio inhibits translation and enhances mRNA decay independent of Nanos. Nanos robustly stimulates repression through interactions with the Pumilio RNA-binding domain. We programmed Pumilio to recognize a new binding site, which garners repression of new target mRNAs. We show that cofactors Brain Tumor and eIF4E Homologous Protein are not obligatory for Pumilio and Nanos activity. The conserved RNA-binding domain of Pumilio was thought to be sufficient for its function. Instead, we demonstrate that three unique domains in the N terminus of Pumilio possess the major repressive activity and can function autonomously. The N termini of insect and vertebrate Pumilio and Fem-3 binding factors (PUFs) are related, and we show that corresponding regions of human PUM1 and PUM2 have repressive activity. Other PUF proteins lack these repression domains. Our findings suggest that PUF proteins have evolved new regulatory functions through protein sequences appended to their conserved PUF repeat RNA-binding domains. PMID:22064486

  7. Role of gonadotropin regulated testicular RNA helicase (GRTH/Ddx25 on polysomal associated mRNAs in mouse testis.

    Directory of Open Access Journals (Sweden)

    Chon-Hwa Tsai-Morris

    Full Text Available Gonadotropin Regulated Testicular RNA Helicase (GRTH/Ddx25 is a testis-specific multifunctional RNA helicase and an essential post-transcriptional regulator of spermatogenesis. GRTH transports relevant mRNAs from nucleus to cytoplasmic sites of meiotic and haploid germ cells and associates with actively translating polyribosomes. It is also a negative regulator of steroidogenesis in Leydig cells. To obtain a genome-wide perspective of GRTH regulated genes, in particularly those associated with polyribosomes, microarray differential gene expression analysis was performed using polysome-bound RNA isolated from testes of wild type (WT and GRTH KO mice. 792 genes among the entire mouse genome were found to be polysomal GRTH-linked in WT. Among these 186 were down-regulated and 7 up-regulated genes in GRTH null mice. A similar analysis was performed using total RNA extracted from purified germ cell populations to address GRTH action in individual target cells. The down-regulation of known genes concerned with spermatogenesis at polysomal sites in GRTH KO and their association with GRTH in WT coupled with early findings of minor or unchanged total mRNAs and abolition of their protein expression in KO underscore the relevance of GRTH in translation. Ingenuity pathway analysis predicted association of GRTH bound polysome genes with the ubiquitin-proteasome-heat shock protein signaling network pathway and NFκB/TP53/TGFB1 signaling networks were derived from the differentially expressed gene analysis. This study has revealed known and unexplored factors in the genome and regulatory pathways underlying GRTH action in male reproduction.

  8. Glucose-Regulated Phosphorylation of the PUF Protein Puf3 Regulates the Translational Fate of Its Bound mRNAs and Association with RNA Granules

    Directory of Open Access Journals (Sweden)

    Chien-Der Lee

    2015-06-01

    Full Text Available PUF proteins are post-transcriptional regulators that bind to the 3′ UTRs of mRNA transcripts. Herein, we show how a yeast PUF protein, Puf3p, responds to glucose availability to switch the fate of its bound transcripts that encode proteins required for mitochondrial biogenesis. Upon glucose depletion, Puf3p becomes heavily phosphorylated within its N-terminal region of low complexity, associates with polysomes, and promotes translation of its target mRNAs. Such nutrient-responsive phosphorylation toggles the activity of Puf3p to promote either degradation or translation of these mRNAs according to the needs of the cell. Moreover, activation of translation of pre-existing mRNAs might enable rapid adjustment to environmental changes without the need for de novo transcription. Strikingly, a Puf3p phosphomutant no longer promotes translation but becomes trapped in intracellular foci in an mRNA-dependent manner. Our findings suggest that the inability to properly resolve Puf3p-containing RNA-protein granules via a phosphorylation-based mechanism might be toxic to a cell.

  9. Quantitation of muscle-specific mRNAs by using cDNA probes during chicken embryonic muscle development in ovo.

    Science.gov (United States)

    Saidapet, C R; Munro, H N; Valgeirsdóttir, K; Sarkar, S

    1982-05-01

    The emergence of abundant-class mRNAs specific for contractile muscle proteins and their distribution between polysomal and free mRNP fractions were studied in skeletal muscle excised from chicken embryos during the transition from myoblasts (day 9) to myotubes (day 18). Muscle-specific cDNA was selectively prepared by hybridizing cDNA to template RNA (polysomal poly(A)+ mRNA) from day-14 embryos followed by isolation of the abundant class, which represents approximately 20% of total mRNA. The specificity of the cDNA probe for this class was confirmed by the differential degree of hybridization to cytoplasmic RNA from cultured myotube and myoblast cells and by its inability to hybridize with mRNA from nonmuscle cells such as liver. Except for muscle from day-9 embryos, the concentrations of the abundant-class muscle-specific mRNAs were higher in polysomes than in free mRNP fractions. Furthermore, the levels of these mRNAs in polysomes increased 12-fold from day 9 (myoblast) to day 14 (intermediate) with a further 3.6-fold increase from day 14 to day 18 (myotube). In contrast to this 45-fold net increase in the polysomal level of these mRNAs from day 9 to day 18, the levels in the free mRNP fraction showed only a 3-fold decrease during this period. Because the amount of mRNA lost from the mRNP fraction is much less than the net increase in the polysome fraction, mRNP does not serve as a reservoir of untranslated muscle-specific mRNA for transfer to polysomes. Consequently, the emergence of muscle-specific polysomal mRNA for contractile proteins during myogenesis in ovo appears to be regulated primarily by transcriptional control. PMID:6954459

  10. Localization of nuclear retained mRNAs in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Thomsen, Rune; Libri, Domenico; Boulay, Jocelyne;

    2003-01-01

    In the yeast Saccharomyces cerevisiae, a common conditional phenotype associated with deletion or mutation of genes encoding mRNA export factors is the rapid accumulation of mRNAs in intranuclear foci, suggested to be near transcription sites. The nuclear RNA exosome has been implicated in retain......In the yeast Saccharomyces cerevisiae, a common conditional phenotype associated with deletion or mutation of genes encoding mRNA export factors is the rapid accumulation of mRNAs in intranuclear foci, suggested to be near transcription sites. The nuclear RNA exosome has been implicated...... in retaining RNAs in these foci; on deletion of the exosome component Rrp6p, the RNA is released. To determine the exact nuclear location of retained as well as released mRNAs, we have used mRNA export mutant strains to analyze the spatial relationship between newly synthesized heat shock mRNA, the chromosomal...

  11. Identification and analysis of pig chimeric mRNAs using RNA sequencing data

    Directory of Open Access Journals (Sweden)

    Ma Lei

    2012-08-01

    Full Text Available Abstract Background Gene fusion is ubiquitous over the course of evolution. It is expected to increase the diversity and complexity of transcriptomes and proteomes through chimeric sequence segments or altered regulation. However, chimeric mRNAs in pigs remain unclear. Here we identified some chimeric mRNAs in pigs and analyzed the expression of them across individuals and breeds using RNA-sequencing data. Results The present study identified 669 putative chimeric mRNAs in pigs, of which 251 chimeric candidates were detected in a set of RNA-sequencing data. The 618 candidates had clear trans-splicing sites, 537 of which obeyed the canonical GU-AG splice rule. Only two putative pig chimera variants whose fusion junction was overlapped with that of a known human chimeric mRNA were found. A set of unique chimeric events were considered middle variances in the expression across individuals and breeds, and revealed non-significant variance between sexes. Furthermore, the genomic region of the 5′ partner gene shares a similar DNA sequence with that of the 3′ partner gene for 458 putative chimeric mRNAs. The 81 of those shared DNA sequences significantly matched the known DNA-binding motifs in the JASPAR CORE database. Four DNA motifs shared in parental genomic regions had significant similarity with known human CTCF binding sites. Conclusions The present study provided detailed information on some pig chimeric mRNAs. We proposed a model that trans-acting factors, such as CTCF, induced the spatial organisation of parental genes to the same transcriptional factory so that parental genes were coordinatively transcribed to give birth to chimeric mRNAs.

  12. Genome-scale identification of membrane-associated human mRNAs.

    Directory of Open Access Journals (Sweden)

    Maximilian Diehn

    2006-01-01

    Full Text Available The subcellular localization of proteins is critical to their biological roles. Moreover, whether a protein is membrane-bound, secreted, or intracellular affects the usefulness of, and the strategies for, using a protein as a diagnostic marker or a target for therapy. We employed a rapid and efficient experimental approach to classify thousands of human gene products as either "membrane-associated/secreted" (MS or "cytosolic/nuclear" (CN. Using subcellular fractionation methods, we separated mRNAs associated with membranes from those associated with the soluble cytosolic fraction and analyzed these two pools by comparative hybridization to DNA microarrays. Analysis of 11 different human cell lines, representing lymphoid, myeloid, breast, ovarian, hepatic, colon, and prostate tissues, identified more than 5,000 previously uncharacterized MS and more than 6,400 putative CN genes at high confidence levels. The experimentally determined localizations correlated well with in silico predictions of signal peptides and transmembrane domains, but also significantly increased the number of human genes that could be cataloged as encoding either MS or CN proteins. Using gene expression data from a variety of primary human malignancies and normal tissues, we rationally identified hundreds of MS gene products that are significantly overexpressed in tumors compared to normal tissues and thus represent candidates for serum diagnostic tests or monoclonal antibody-based therapies. Finally, we used the catalog of CN gene products to generate sets of candidate markers of organ-specific tissue injury. The large-scale annotation of subcellular localization reported here will serve as a reference database and will aid in the rational design of diagnostic tests and molecular therapies for diverse diseases.

  13. Dietary sucrose enhances processing of mRNA-S14 nuclear precursor.

    Science.gov (United States)

    Burmeister, L A; Mariash, C N

    1991-12-01

    The rapid response of rat hepatic mRNA-S14 to hormonal or dietary manipulation makes it an excellent model to study the control of lipogenic enzyme mRNA. The mechanism of regulation of this mRNA by triiodothyronine (T3) or sucrose remains controversial. Although initial studies suggested that T3 stabilized the nuclear precursor, subsequent studies suggest that T3 acts by increasing the transcriptional rate of this gene. More recently, the induction of mRNA-S14 by sucrose administration was shown to be associated with an increase in transcriptional "run-on" activity. Because T3 and carbohydrate feeding synergistically regulate this mRNA, we studied the response to short and long term high carbohydrate feeding in hypothyroid and euthyroid rats. We found the response to the lipogenic diet was rapid in hypothyroid rats, with maximal levels of mRNA-S14 attained by 4 h (2.2 +/- 0.6 chow fed versus 13.5 +/- 2.5 pg/micrograms RNA on lipogenic diet). The rapid induction by the lipogenic diet contrasts with the diminished response to sucrose by gastric gavage (4.6 +/- 1.2 pg/micrograms RNA) over the same time interval. Despite the large increase in the mature mRNA induced by the lipogenic diet, the rise in the nuclear precursor was small and not different from that observed after sucrose gavage (0.14 +/- 0.01 chow, 0.26 +/- 0.03 sucrose gavage, 0.25 +/- 0.04 pg/micrograms RNA lipogenic diet). The molar ratio of the mature to precursor mRNA-S14 showed progressive increases with the smallest level in the fasting rat, an intermediate level in the chow-fed and sucrose gavaged rats, and the highest level in the animals fed a lipogenic diet (2.1, 16.5, 16.3, 62.7, respectively). Based on the previously reported half-life for the mature mRNA-S14, these data show that feeding sucrose by gavage or by a lipogenic diet leads to enhanced fractional conversion of precursor to mature mRNA-S14 with a simultaneous stabilization of the precursor mRNA-S14. PMID:1744084

  14. Expression of gap junction genes connexin 32 and connexin 43 mRNAs and proteins, and their role in hepatocarcinogenesis

    Institute of Scientific and Technical Information of China (English)

    Xiang-Dong Ma; Xing Ma; Yan-Fang Sui; Wen-Liang Wang

    2002-01-01

    AIM: To investigate the relationship betweenhepatocarcinogenesis and the expression of connexin32(cx32), connexin43 (cx43) mRNAs and proteins in vitro.METHODS Gap junction genes cx32 and cx43 mRNA inhepatocellular carcinoma call lines HHCC, SMMC-7721 andnormal liver call line QZG were detected by in situhybridization (ISH) with digoxin-labeled cx32, and cx43cDNA probes. Expression of Cx32 and Cx43 proteins in thecell lines was revealed by indirect immuno-fluorescence andflow cytornetry (FCM).RESULTS Blue positive hybridization signals of cx32 andcx43 mRNAs detected by ISH with cx32 and cx43 cDNAprobes respectively were located in cytoplasm of cells ofHHCC, SMMC-7721 and QZG. No significant difference ofeither cx32 mRNA or cx43 mRNA was tested among HHCC,SMMC-7721 and QZG (P = 2.673, HHCC vs QZG; P =1.375, SMMC-7721 vs QZG). FCM assay showed that thepositive rates of Cx32 protein in HHCC, SMMC-7721 and QZGwere 0.7%, 1.7% and 99.0%, and the positive rates of Cx43protein in HHCC, SMMC-7721 and QZG were 7.3%, 26.5%and 99.1% respectively. Significant differences of both Cx32and Cx43 protein expression existed between hepatocellularcarcinoma cell lines and normal liver cell line ( P = 0.0069,HHCC vs QZG; P = 0.0087, SMMC-7721 vs QZG).Moreover, the fluorescent intensities of Cx32 and Cx43proteins in HHCC, SMMC-7721 were lower than that in QZG.CONCLUSION Hepatocellular carcinoma cell lines HHCCand SMMC-7721 exhibited lower positive rates andfluorescent intensities of Cx32, Cx43 proteins compared withthat of normal liver cell line QZG. lt is suggested that lowerexpression of both Cx32 and Cx43 proteins in hepatocellularcarcinoma cells could play pivotal roles in thehepatocarcinogenesis. Besides, genetic defects of cx32 andcx43 in post-translational processing should be considered.

  15. Identification of two DNA helicases UvrD and DinG as suppressors for lethality caused by mutant cspA mRNAs.

    Science.gov (United States)

    Hwang, Jihwan; Lee, Kangseok; Phadtare, Sangita; Inouye, Masayori

    2012-01-01

    CspA is a major cold shock-inducible protein (70 aa), and its major role in the cold shock response was shown to be as an RNA chaperone destabilizing secondary structure of mRNAs at low temperature. Previously, we showed that the overexpression of mutant cspA containing premature non-sense codons at various positions led to stalled ribosomes on mutant cspA transcripts, ultimately leading to cell death. This lethality is primarily due to the highly translatable cspA 5'-UTR that recruits most of the ribosomes from other mRNAs, which are then stalled at the abnormal stop codon. This was called the 'LACE' effect. We show here that non-sense mutation even at the 67th position as well as substitutions of aromatic amino acid residues present on the RNA-binding surface of CspA protein to alanine caused the LACE effect by trapping a substantial amount of ribosomes on cspA mRNAs. In an attempt to identify a suppressor(s), which may help the cells to recover from the inhibitory LACE effect, genetic screening of an Escherichia coli genomic library was performed. We isolated suppressors that contained the genomic fragments encoding uvrD and dinG, respectively, whose gene products are ATP-dependent DNA helicases. The nucleic acid-binding and ATPase activities of these two helicases were found to be essential for their suppression activity. This genomic screening offers an approach to shed light on the mechanistic of 5'-UTR of cspA mRNA and novel roles of E. coli helicases that function in DNA repair. PMID:22832783

  16. The RNA binding protein HuR differentially regulates unique subsets of mRNAs in estrogen receptor negative and estrogen receptor positive breast cancer

    International Nuclear Information System (INIS)

    The discordance between steady-state levels of mRNAs and protein has been attributed to posttranscriptional control mechanisms affecting mRNA stability and translation. Traditional methods of genome wide microarray analysis, profiling steady-state levels of mRNA, may miss important mRNA targets owing to significant posttranscriptional gene regulation by RNA binding proteins (RBPs). The ribonomic approach, utilizing RNA immunoprecipitation hybridized to microarray (RIP-Chip), provides global identification of putative endogenous mRNA targets of different RBPs. HuR is an RBP that binds to the AU-rich elements (ARE) of labile mRNAs, such as proto-oncogenes, facilitating their translation into protein. HuR has been shown to play a role in cancer progression and elevated levels of cytoplasmic HuR directly correlate with increased invasiveness and poor prognosis for many cancers, including those of the breast. HuR has been described to control genes in several of the acquired capabilities of cancer and has been hypothesized to be a tumor-maintenance gene, allowing for cancers to proliferate once they are established. We used HuR RIP-Chip as a comprehensive and systematic method to survey breast cancer target genes in both MCF-7 (estrogen receptor positive, ER+) and MDA-MB-231 (estrogen receptor negative, ER-) breast cancer cell lines. We identified unique subsets of HuR-associated mRNAs found individually or in both cell types. Two novel HuR targets, CD9 and CALM2 mRNAs, were identified and validated by quantitative RT-PCR and biotin pull-down analysis. This is the first report of a side-by-side genome-wide comparison of HuR-associated targets in wild type ER+ and ER- breast cancer. We found distinct, differentially expressed subsets of cancer related genes in ER+ and ER- breast cancer cell lines, and noted that the differential regulation of two cancer-related genes by HuR was contingent upon the cellular environment

  17. HuR-Regulated mRNAs Associated with Nuclear hnRNP A1-RNP Complexes

    Directory of Open Access Journals (Sweden)

    Apostolia Guialis

    2013-10-01

    Full Text Available Post-transcriptional regulatory networks are dependent on the interplay of many RNA-binding proteins having a major role in mRNA processing events in mammals. We have been interested in the concerted action of the two RNA-binding proteins hnRNP A1 and HuR, both stable components of immunoselected hnRNP complexes and having a major nuclear localization. Specifically, we present here the application of the RNA-immunoprecipitation (RIP-Chip technology to identify a population of nuclear transcripts associated with hnRNP A1-RNPs as isolated from the nuclear extract of either HuR WT or HuR-depleted (KO mouse embryonic fibroblast (MEF cells. The outcome of this analysis was a list of target genes regulated via HuR for their association (either increased or reduced with the nuclear hnRNP A1-RNP complexes. Real time PCR analysis was applied to validate a selected number of nuclear mRNA transcripts, as well as to identify pre-spliced transcripts (in addition to their mature mRNA counterpart within the isolated nuclear hnRNP A1-RNPs. The differentially enriched mRNAs were found to belong to GO categories relevant to biological processes anticipated for hnRNP A1 and HuR (such as transport, transcription, translation, apoptosis and cell cycle indicating their concerted function in mRNA metabolism.

  18. The rotaviral NSP3 protein stimulates translation of polyadenylated target mRNAs independently of its RNA-binding domain

    International Nuclear Information System (INIS)

    The non-structural protein 3 (NSP3) of rotaviruses is an RNA-binding protein that specifically recognises a 4 nucleotide sequence at the 3' extremity of the non-polyadenylated viral mRNAs. NSP3 also has a high affinity for eIF4G. These two functions are clearly delimited in separate domains the structures of which have been determined. They are joined by a central domain implicated in the dimerisation of the full length protein. The bridging function of NSP3 between the 3' end of the viral mRNA and eIF4G has been proposed to enhance the synthesis of viral proteins. However, this role has been questioned as knock-down of NSP3 did not impair viral protein synthesis. We show here using a MS2/MS2-CP tethering assay that a C-terminal fragment of NSP3 containing the eIF4G binding domain and the dimerisation domain can increase the expression of a protein encoded by a target reporter mRNA in HEK 293 cells. The amount of reporter mRNA in the cells is not significantly affected by the presence of the NSP3 derived fusion protein showing that the enhanced protein expression is due to increased translation. These results show that NSP3 can act as a translational enhancer even on a polyadenylated mRNA that should be a substrate for PABP1.

  19. The study on the induction of specific immune cytotoxic T lymphocyte responses against pancreatic cancer by transfected dendritic cells with common tumor antigen survivin mRNAs in vitro%Survivin mRNA转染树突细胞诱导特异性抗胰腺癌免疫反应的体外研究

    Institute of Scientific and Technical Information of China (English)

    郭晓钟; 陈江

    2011-01-01

    Objective To investigate the induction of specific anti-tumor immune response by transfected dendritic cells (DCs) with survivin mRNA of human pancreatic cancer, and to provide the experimental evidences for the treatment of human pancreatic cancer with DCs vaccine. Methods DCs were isolated and cultured from peripheral blood mononuclear cells (PBMCs). After being transcripted and amplified, survivin mRNA was transfected into DCs by electroporation. The expression of survivin in DCs at different time points was detected by quantitative real-time PCR. The survival rate of DCs before and after transfection was determined by MTT method. The induction of specific cytotoxic T lymphocyte (CTL) response by survivin mRNA transfected DCs was measured by 51Cr standard cytotoxicity test. The induction of specific CTL activation by survivin mRNA transfected DCs was evaluated through testing released IFN-γ by ELISA method. Results After survivin mRNA transfection for 48h, the expression of survivin mRNA in DCs reached the highest point (46.09±6.57). After transfection, the survival rate of DCs was stabilized around 80%. The DCs transfected with survivin mRNA could effectively induce HLA-A2+ / survivin+ specific CTL immune responses. Stimulated with pancreatic cancer cell line Capan-2 cells or SCL-1 cells as control group, the IFN-γ released in 24 hours by survivin specific CTL were (28.79±5.70) U/ml and (25.12±2.13) U/ml respectively, there was no significant difference (P=0.761). Conclusion The induction of CTLs by DCs transfected with human pancreatic cancer survivin mRNA could produce specific anti-tumor immunity.%目的 研究人胰腺癌survivin mRNA转染树突细胞(DC)诱导的特异性抗肿瘤免疫反应,为DC疫苗治疗胰腺癌提供实验依据.方法 自样本外周血单核细胞中分离和培养DC.体外转录和PCR扩增survivin mRNA后使用电穿孔法将其转染DC.采用实时定量PCR技术检测不同时间点DC中survivin的表达.用四甲基

  20. Polycistronic mRNAs code for polypeptides of the Vibrio harveyi luminescence system

    Energy Technology Data Exchange (ETDEWEB)

    Miyamoto, C.M.; Graham, A.D.; Boylan, M.; Evans, J.F.; Hasel, K.W.; Meighen, E.A.; Graham, A.F.

    1985-03-01

    DNA coding for the ..cap alpha.. and ..beta.. subunits of Vibrio harveyi luciferase, the luxA and luxB genes, and the adjoining chromosomal regions on both sides of these genes (total of 18 kilobase pairs) was cloned into Escherichia coli. Using labeled DNA coding for the ..cap alpha.. subunit as a hybridization probe, the authors identified a set of polycistronic mRNAs (2.6, 4, 7, and 8 kilobases) by Northern blotting; the most prominent of these was the one 4 kilobases long. This set of mRNAs was induced during the development of bioluminescence in V. harveyi. Furthermore, the same set of mRNAs was synthesized in E. coli by a recombinant plasmid that contained a 12-kilobase pair length of V. harveyi DNA and expressed the genes for the luciferase subunits. A cloned DNA segment corresponding to the major 4-kilobase mRNA coded for the ..cap alpha.. and ..beta.. subunits of luciferase, as well as a 32,000-dalton protein upstream from these genes that could be specifically modified by acyl-coenzyme A and is a component of the bioluminescence system. V. harveyi mRNA that was hybridized to the released from cloned DNA encompassing the luxA and luxB genes was translated in vitro. Luciferase ..cap alpha.. and ..beta.. subunits and the 32,000-dalton polypeptide were detected among the products, along with 42,000- and 55,000-dalton polypeptides, which are encoded downstream from the lux genes and are thought to be involved in luminescence.

  1. SR proteins SRp20 and 9G8 contribute to efficient export of herpes simplex virus 1 mRNAs

    International Nuclear Information System (INIS)

    Herpes simplex virus 1 (HSV-1) mRNAs are exported to the cytoplasm through the export receptor TAP/NFX1. HSV-1 multifunctional protein ICP27 interacts with TAP/NXF1, binds viral RNAs, and is required for efficient viral RNA export. In ICP27 mutant infections, viral RNA export is reduced but not ablated, indicating that other export adaptors can aid in viral RNA export. Export adaptor protein Aly/REF is recruited to viral replication compartments, however, Aly/REF knockdown has little effect on viral RNA export. SR proteins SRp20 and 9G8 interact with TAP/NXF1 and mediate export of some cellular RNAs. We report that siRNA knockdown of SRp20 or 9G8 resulted in about a 10 fold decrease in virus yields and in nuclear accumulation of poly(A+) RNA. In infected cells depleted of SRp20, newly transcribed Bromouridine-labeled RNA also accumulated in the nucleus. We conclude that SRp20 and 9G8 contribute to HSV-1 RNA export.

  2. Imaging single mRNAs to study dynamics of mRNA export in the yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Bensidoun, Pierre; Raymond, Pascal; Oeffinger, Marlene; Zenklusen, Daniel

    2016-04-01

    Regulation of mRNA and protein expression occurs at many levels, initiated at transcription and followed by mRNA processing, export, localization, translation and mRNA degradation. The ability to study mRNAs in living cells has become a critical tool to study and analyze how the various steps of the gene expression pathway are carried out. Here we describe a detailed protocol for real time fluorescent RNA imaging using the PP7 bacteriophage coat protein, which allows mRNA detection with high spatial and temporal resolution in the yeast Saccharomyces cerevisiae, and can be applied to study various stages of mRNA metabolism. We describe the different parameters required for quantitative single molecule imaging in yeast, including strategies for genomic integration, expression of a PP7 coat protein GFP fusion protein, microscope setup and analysis strategies. We illustrate the method's use by analyzing the behavior of nuclear mRNA in yeast and the role of the nuclear basket in mRNA export. PMID:26784711

  3. Determining exon connectivity in complex mRNAs by nanopore sequencing.

    Science.gov (United States)

    Bolisetty, Mohan T; Rajadinakaran, Gopinath; Graveley, Brenton R

    2015-01-01

    Short-read high-throughput RNA sequencing, though powerful, is limited in its ability to directly measure exon connectivity in mRNAs that contain multiple alternative exons located farther apart than the maximum read length. Here, we use the Oxford Nanopore MinION sequencer to identify 7,899 'full-length' isoforms expressed from four Drosophila genes, Dscam1, MRP, Mhc, and Rdl. These results demonstrate that nanopore sequencing can be used to deconvolute individual isoforms and that it has the potential to be a powerful method for comprehensive transcriptome characterization. PMID:26420219

  4. Testosterone regulation of sex steroid-related mRNAs and dopamine-related mRNAs in adolescent male rat substantia nigra

    Directory of Open Access Journals (Sweden)

    Purves-Tyson Tertia D

    2012-08-01

    Full Text Available Abstract Background Increased risk of schizophrenia in adolescent males indicates that a link between the development of dopamine-related psychopathology and testosterone-driven brain changes may exist. However, contradictions as to whether testosterone increases or decreases dopamine neurotransmission are found and most studies address this in adult animals. Testosterone-dependent actions in neurons are direct via activation of androgen receptors (AR or indirect by conversion to 17β-estradiol and activation of estrogen receptors (ER. How midbrain dopamine neurons respond to sex steroids depends on the presence of sex steroid receptor(s and the level of steroid conversion enzymes (aromatase and 5α-reductase. We investigated whether gonadectomy and sex steroid replacement could influence dopamine levels by changing tyrosine hydroxylase (TH protein and mRNA and/or dopamine breakdown enzyme mRNA levels [catechol-O-methyl transferase (COMT and monoamine oxygenase (MAO A and B] in the adolescent male rat substantia nigra. We hypothesized that adolescent testosterone would regulate sex steroid signaling through regulation of ER and AR mRNAs and through modulation of aromatase and 5α-reductase mRNA levels. Results We find ERα and AR in midbrain dopamine neurons in adolescent male rats, indicating that dopamine neurons are poised to respond to circulating sex steroids. We report that androgens (T and DHT increase TH protein and increase COMT, MAOA and MAOB mRNAs in the adolescent male rat substantia nigra. We report that all three sex steroids increase AR mRNA. Differential action on ER pathways, with ERα mRNA down-regulation and ERβ mRNA up-regulation by testosterone was found. 5α reductase-1 mRNA was increased by AR activation, and aromatase mRNA was decreased by gonadectomy. Conclusions We conclude that increased testosterone at adolescence can shift the balance of sex steroid signaling to favor androgenic responses through promoting

  5. Expression of type I and type V collagen mRNAs in the elasmoid scales of a teleost fish as revealed by in situ hybridization.

    Science.gov (United States)

    Le Guellec, D; Zylberberg, L

    1998-01-01

    The ability of scale-forming cells to produce both type I and type V collagens was investigated by in situ hybridization at the light and electron microscope levels. Biochemical analyses reported that type I collagen, the predominant component, was associated with the minor type V collagen in the collagenous matrix of the teleost scales where, thin and thick collagen fibrils formed distinct layers. Thin collagen fibrils of the external layer were produced by the episquamal scleroblasts scattered on the outer scale surface, while thick collagen fibrils forming the compact basal plate were produced by the hyposquamal scleroblasts lining the inner surface of the scale. We demonstrated that episquamal and hyposquamal scleroblasts contained mRNAs for alpha1(I) and alpha1(V) collagens. Quantification by image analysis of the relative amount of alpha1(I) and alpha1(V) mRNAs in episquamal and hyposquamal scleroblasts suggests that the gene expression of type V collagen was proportionally higher in episquamal scleroblasts. These results support our hypothesis that the diameter of the thin fibrils of the external layer is regulated by the significant amount of type V collagen that interacts with type I collagen. PMID:11063006

  6. Accumulation of long-lived mRNAs associated with germination in embryos during seed development of rice.

    Science.gov (United States)

    Sano, Naoto; Ono, Hanako; Murata, Kazumasa; Yamada, Tetsuya; Hirasawa, Tadashi; Kanekatsu, Motoki

    2015-07-01

    Mature dry seeds contain translatable mRNAs called long-lived mRNAs. Early studies have shown that protein synthesis during the initial phase of seed germination occurs from long-lived mRNAs, without de novo transcription. However, the gene expression systems that generate long-lived mRNAs in seeds are not well understood. To examine the accumulation of long-lived mRNAs in developing rice embryos, germination tests using the transcriptional inhibitor actinomycin D (Act D) were performed with the Japonica rice cultivar Nipponbare. Although over 70% of embryos at 10 days after flowering (DAF) germinated in the absence of the inhibitor, germination was remarkably impaired in embryos treated with Act D. In contrast, more than 70% of embryos at 20, 25, 30 and 40 DAF germinated in the presence of Act D. The same results were obtained when another cultivar, Koshihikari, was used, indicating that the long-lived mRNAs required for germination predominantly accumulate in embryos between 10 and 20 DAF during seed development. RNA-Seq identified 529 long-lived mRNA candidates, encoding proteins such as ABA, calcium ion and phospholipid signalling-related proteins, and HSP DNA J, increased from 10 to 20 DAF and were highly abundant in 40 DAF embryos of Nipponbare and Koshihikari. We also revealed that these long-lived mRNA candidates are clearly up-regulated in 10 DAF germinating embryos after imbibition, suggesting that the accumulation of these mRNAs in embryos is indispensable for the induction of germination. The findings presented here may facilitate in overcoming irregular seed germination or producing more vigorous seedlings. PMID:25941326

  7. Role of LARP6 and nonmuscle myosin in partitioning of collagen mRNAs to the ER membrane.

    Directory of Open Access Journals (Sweden)

    Hao Wang

    Full Text Available Type I collagen is extracellular matrix protein composed of two α1(I and one α2(I polypeptides that fold into triple helix. Collagen polypeptides are translated in coordination to synchronize the rate of triple helix folding to the rate of posttranslational modifications of individual polypeptides. This is especially important in conditions of high collagen production, like fibrosis. It has been assumed that collagen mRNAs are targeted to the membrane of the endoplasmic reticulum (ER after translation of the signal peptide and by signal peptide recognition particle (SRP. Here we show that collagen mRNAs associate with the ER membrane even when translation is inhibited. Knock down of LARP6, an RNA binding protein which binds 5' stem-loop of collagen mRNAs, releases a small amount of collagen mRNAs from the membrane. Depolimerization of nonmuscle myosin filaments has a similar, but stronger effect. In the absence of LARP6 or nonmuscle myosin filaments collagen polypeptides become hypermodified, are poorly secreted and accumulate in the cytosol. This indicates lack of coordination of their synthesis and retro-translocation due to hypermodifications and misfolding. Depolimerization of nonmuscle myosin does not alter the secretory pathway through ER and Golgi, suggesting that the role of nonmuscle myosin is primarily to partition collagen mRNAs to the ER membrane. We postulate that collagen mRNAs directly partition to the ER membrane prior to synthesis of the signal peptide and that LARP6 and nonmuscle myosin filaments mediate this process. This allows coordinated initiation of translation on the membrane bound collagen α1(I and α2(I mRNAs, a necessary step for proper synthesis of type I collagen.

  8. Differential display of skin mRNAs regulated under varying environmental conditions in a mudskipper.

    Science.gov (United States)

    Sakamoto, T; Yasunaga, H; Yokota, S; Ando, M

    2002-07-01

    To understand the molecular mechanisms underlying the terrestrial adaptation, as well as adaptation to different salinities, of the euryhaline and amphibious mudskipper ( Periophthalmus modestus), we have looked for the skin mRNAs that change during varying environmental conditions. Using differential mRNA display polymerase chain reaction, we compared skin mRNAs in mudskipper transferred from isotonic 30% seawater to fresh water or to seawater for 1 day and 7 days, as well as those kept out of water for 1 day. At the end of these periods, poly(A(+))RNA was prepared from the Cl(-)-secreting pectoral skins and also from the outer opercular skins where ion transport is negligible, and analyzed by differential display. We identified four cDNA products expressed differently under various environments as homologues of known genes. A further 34 cDNAs were expressed differentially, but they have no significant homology to identified sequences in GenBank. Northern blots demonstrate that mRNA levels of the actin-binding protein and the platelet-activating factor acetylhydrolase increased in the pectoral skins during seawater acclimation. The mRNA of the 90 kDa heat shock protein was down-regulated in water-deprived and freshwater fish, whose plasma cortisol levels were high. The aldolase mRNA was induced in both skins after desiccation. These four genes may be involved in the environmental adaptations. PMID:12122461

  9. Genome-wide identification of mRNAs associated with the translational regulator PUMILIO in Drosophila melanogaster

    Science.gov (United States)

    Gerber, André P.; Luschnig, Stefan; Krasnow, Mark A.; Brown, Patrick O.; Herschlag, Daniel

    2006-01-01

    Genome-wide identification of RNAs associated with RNA-binding proteins is crucial for deciphering posttranscriptional regulatory systems. PUMILIO is a member of the evolutionary conserved Puf-family of RNA-binding proteins that repress gene expression posttranscriptionally. We generated transgenic flies expressing affinity-tagged PUMILIO under the control of an ovary-specific promoter, and we purified PUMILIO from whole adult flies and embryos and analyzed associated mRNAs by using DNA microarrays. Distinct sets comprising hundreds of mRNAs were associated with PUMILIO at the two developmental stages. Many of these mRNAs encode functionally related proteins, supporting a model for coordinated regulation of posttranscriptional modules by specific RNA-binding proteins. We identified a characteristic sequence motif in the 3′-untranslated regions of mRNAs associated with PUMILIO, and the sufficiency of this motif for interaction with PUMILIO was confirmed by RNA pull-down experiments with biotinylated synthetic RNAs. The RNA motif strikingly resembles the one previously identified for Puf3p, one of five Saccharomyces cerevisiae Puf proteins; however, proteins encoded by the associated mRNAs in yeast and Drosophila do not appear to be related. The results suggest extensive posttranscriptional regulation by PUMILIO and uncover evolutionary features of this conserved family of RNA-binding proteins. PMID:16537387

  10. Induction of stilbene synthase and cinnamyl alcohol dehydrogenase mRNAs in Scots pine (Pinus sylvestris L.) seedlings

    International Nuclear Information System (INIS)

    Pine is known to respond to ozone by the induction of stilbene synthase (STS) and cinnamyl alcohol dehydrogenase (CAD) activities. Here we describe the influence of ozone on STS and CAD transcript levels, as well as on the amounts of actin mRNA and chlorophyll a/b-binding protein (cab) mRNA in needles of young Scots pine (Pinus sylvestris L.) seedlings. A single ozone pulse of 0.3 μL · L−1 for 8 h resulted in transient increases in STS, and CAD mRNA levels. In contrast, actin and cab transcript levels were reduced. Treatment of Scots pine seedlings with ozone (0.3 μL · L−1, 8 h · d−1) over a period of 12.5 d resulted in a constant high CAD mRNA level. In contrast STS transcripts were transiently induced over 6 d under these conditions. These results indicate selective ozone responses by the two genes. Compared with results for ozone fumigation alone, combined ozone/UV-B treatment led to a slightly higher increase in STS mRNA in primary needles, as well as in cotyledons. This points to an additive effect by the two stressors. In-situ hybridization with STS and CAD antisense mRNAs revealed an enhanced uniform labeling of mesophyll cells in tissue cross-sections of ozone-treated needles, whereas in the epidermal cell layer the amount of silver grains was unaltered in comparison with controls. (author)

  11. Transcriptome-wide mapping of pseudouridines: pseudouridine synthases modify specific mRNAs in S. cerevisiae.

    Directory of Open Access Journals (Sweden)

    Alexander F Lovejoy

    Full Text Available We developed a novel technique, called pseudouridine site identification sequencing (PSI-seq, for the transcriptome-wide mapping of pseudouridylation sites with single-base resolution from cellular RNAs based on the induced termination of reverse transcription specifically at pseudouridines following CMCT treatment. PSI-seq analysis of RNA samples from S. cerevisiae correctly detected all of the 43 known pseudouridines in yeast 18S and 25S ribosomal RNA with high specificity. Moreover, application of PSI-seq to the yeast transcriptome revealed the presence of site-specific pseudouridylation within dozens of mRNAs, including RPL11a, TEF1, and other genes implicated in translation. To identify the mechanisms responsible for mRNA pseudouridylation, we genetically deleted candidate pseudouridine synthase (Pus enzymes and reconstituted their activities in vitro. These experiments demonstrated that the Pus1 enzyme was necessary and sufficient for pseudouridylation of RPL11a mRNA, whereas Pus4 modified TEF1 mRNA, and Pus6 pseudouridylated KAR2 mRNA. Finally, we determined that modification of RPL11a at Ψ -68 was observed in RNA from the related yeast S. mikitae, and Ψ -239 in TEF1 mRNA was maintained in S. mikitae as well as S. pombe, indicating that these pseudouridylations are ancient, evolutionarily conserved RNA modifications. This work establishes that site-specific pseudouridylation of eukaryotic mRNAs is a genetically programmed RNA modification that naturally occurs in multiple yeast transcripts via distinct mechanisms, suggesting that mRNA pseudouridylation may provide an important novel regulatory function. The approach and strategies that we report here should be generally applicable to the discovery of pseudouridylation, or other RNA modifications, in diverse biological contexts.

  12. Deep sequencing shows multiple oligouridylations are required for 3' to 5' degradation of histone mRNAs on polyribosomes.

    Science.gov (United States)

    Slevin, Michael K; Meaux, Stacie; Welch, Joshua D; Bigler, Rebecca; Miliani de Marval, Paula L; Su, Wei; Rhoads, Robert E; Prins, Jan F; Marzluff, William F

    2014-03-20

    Histone mRNAs are rapidly degraded when DNA replication is inhibited during S phase with degradation initiating with oligouridylation of the stem loop at the 3' end. We developed a customized RNA sequencing strategy to identify the 3' termini of degradation intermediates of histone mRNAs. Using this strategy, we identified two types of oligouridylated degradation intermediates: RNAs ending at different sites of the 3' side of the stem loop that resulted from initial degradation by 3'hExo and intermediates near the stop codon and within the coding region. Sequencing of polyribosomal histone mRNAs revealed that degradation initiates and proceeds 3' to 5' on translating mRNA and that many intermediates are capped. Knockdown of the exosome-associated exonuclease PM/Scl-100, but not the Dis3L2 exonuclease, slows histone mRNA degradation consistent with 3' to 5' degradation by the exosome containing PM/Scl-100. Knockdown of No-go decay factors also slowed histone mRNA degradation, suggesting a role in removing ribosomes from partially degraded mRNAs. PMID:24656133

  13. RanBP2/Nup358 potentiates the translation of a subset of mRNAs encoding secretory proteins.

    Directory of Open Access Journals (Sweden)

    Kohila Mahadevan

    Full Text Available In higher eukaryotes, most mRNAs that encode secreted or membrane-bound proteins contain elements that promote an alternative mRNA nuclear export (ALREX pathway. Here we report that ALREX-promoting elements also potentiate translation in the presence of upstream nuclear factors. These RNA elements interact directly with, and likely co-evolved with, the zinc finger repeats of RanBP2/Nup358, which is present on the cytoplasmic face of the nuclear pore. Finally we show that RanBP2/Nup358 is not only required for the stimulation of translation by ALREX-promoting elements, but is also required for the efficient global synthesis of proteins targeted to the endoplasmic reticulum (ER and likely the mitochondria. Thus upon the completion of export, mRNAs containing ALREX-elements likely interact with RanBP2/Nup358, and this step is required for the efficient translation of these mRNAs in the cytoplasm. ALREX-elements thus act as nucleotide platforms to coordinate various steps of post-transcriptional regulation for the majority of mRNAs that encode secreted proteins.

  14. The optional long 5'-untranslated region of human ACAT1 mRNAs impairs the production of ACAT1 protein by promoting its mRNA decay

    Institute of Scientific and Technical Information of China (English)

    Xiaonan Zhao; Baoliang Song; Tayuan Chang; Boliang Li; Jia Chen; Lei Lei; Guangjing Hu; Ying Xiong; Jiajia Xu; Qin Li; Xinying Yang; Catherine C.Y.Chang

    2009-01-01

    We have previously reported that human ACAT1 mRNAs produce the 50 kDa protein using the AUG1397-1399 initiation codon,and also a minor 56 kDa isoform using the upstream in-frame GGC1274-1276initiation codon.The GGC1274-1276 codon is located at the optional long 5'-untranslated region(5'-UTR,nt 1-1396)of the mRNAs.The DNA sequences corresponding to this 5'-UTR are located in two different chromosomes,7 and 1.In the current work,we report that the optional long 5'-UTR significantly impairs the production of human ACAT1 protein initiated from the AUG1397-1399 codon,mainly by promoting its mRNA decay.The western blot analyses indicated that the optional long 5'-UTR potently impaired the production of different proteins initiated from the AUG1397-1399codon,meaning that this impairing effect was not influenced by the 3'-UTR or the coding sequence of ACAT1 mRNA.The results of reverse transcription-quantitative polymerase chain reaction demonstrated that this 5'-UTR dramatically reduced the contents of its linked mRNAs.Analyses of the protein to mRNA ratios showed that this 5'-UTR mainly decreased its mRNA stability rather than altering its translational efficiency.We next performed the plasmid transfection experiments and used actinomycin D to inhibit transcription.The results showed that this 5'-UTR promoted its mRNA decay.Additional transfection and nucleofection experiments using RNAs prepared in vitro illustrated that,in both the cytoplasm and the nucleus of cells,the optional long 5'-UTR-linked mRNAs decayed faster than those without the link.Overall,our study brings new insight to the regulation of the human ACAT1 gene expression at the post-transcription level.

  15. Human thyroid peroxidase: complete cDNA and protein sequence, chromosome mapping, and identification of two alternately spliced mRNAs

    International Nuclear Information System (INIS)

    Two forms of human thyroid peroxidase cDNAs were isolated from a λgt11 cDNA library, prepared from Graves disease thyroid tissue mRNA, by use of oligonucleotides. The longest complete cDNA, designated phTPO-1, has 3048 nucleotides and an open reading frame consisting of 933 amino acids, which would encode a protein with a molecular weight of 103,026. Five potential asparagine-linked glycosylation sites are found in the deduced amino acid sequence. The second peroxidase cDNA, designated phTPO-2, is almost identical to phTPO-1 beginning 605 base pairs downstream except that it contains 1-base-pair difference and lacks 171 base pairs in the middle of the sequence. This results in a loss of 57 amino acids corresponding to a molecular weight of 6282. Interestingly, this 171-nucleotide sequence has GT and AG at its 5' and 3' boundaries, respectively, that are in good agreement with donor and acceptor splice site consensus sequences. Using specific oligonucleotide probes for the mRNAs derived from the cDNA sequences hTOP-1 and hTOP-2, the authors show that both are expressed in all thyroid tissues examined and the relative level of two mRNAs is different in each sample. The results suggest that two thyroid peroxidase proteins might be generated through alternate splicing of the same gene. By using somatic cell hybrid lines, the thyroid peroxidase gene was mapped to the short arm of human chromosome 2

  16. The BTG4 and CAF1 complex prevents the spontaneous activation of eggs by deadenylating maternal mRNAs.

    Science.gov (United States)

    Pasternak, Michał; Pfender, Sybille; Santhanam, Balaji; Schuh, Melina

    2016-09-01

    Once every menstrual cycle, eggs are ovulated into the oviduct where they await fertilization. The ovulated eggs are arrested in metaphase of the second meiotic division, and only complete meiosis upon fertilization. It is crucial that the maintenance of metaphase arrest is tightly controlled, because the spontaneous activation of the egg would preclude the development of a viable embryo (Zhang et al. 2015 J. Genet. Genomics 42, 477-485. (doi:10.1016/j.jgg.2015.07.004); Combelles et al. 2011 Hum. Reprod. 26, 545-552. (doi:10.1093/humrep/deq363); Escrich et al. 2011 J. Assist. Reprod. Genet. 28, 111-117. (doi:10.1007/s10815-010-9493-5)). However, the mechanisms that control the meiotic arrest in mammalian eggs are only poorly understood. Here, we report that a complex of BTG4 and CAF1 safeguards metaphase II arrest in mammalian eggs by deadenylating maternal mRNAs. As a follow-up of our recent high content RNAi screen for meiotic genes (Pfender et al. 2015 Nature 524, 239-242. (doi:10.1038/nature14568)), we identified Btg4 as an essential regulator of metaphase II arrest. Btg4-depleted eggs progress into anaphase II spontaneously before fertilization. BTG4 prevents the progression into anaphase by ensuring that the anaphase-promoting complex/cyclosome (APC/C) is completely inhibited during the arrest. The inhibition of the APC/C relies on EMI2 (Tang et al. 2010 Mol. Biol. Cell 21, 2589-2597. (doi:10.1091/mbc.E09-08-0708); Ohe et al. 2010 Mol. Biol. Cell 21, 905-913. (doi:10.1091/mbc.E09-11-0974)), whose expression is perturbed in the absence of BTG4. BTG4 controls protein expression during metaphase II arrest by forming a complex with the CAF1 deadenylase and we hypothesize that this complex is recruited to the mRNA via interactions between BTG4 and poly(A)-binding proteins. The BTG4-CAF1 complex drives the shortening of the poly(A) tails of a large number of transcripts at the MI-MII transition, and this wave of deadenylation is essential for the arrest in

  17. Role of 5'- and 3'-untranslated regions of mRNAs in human diseases.

    Science.gov (United States)

    Chatterjee, Sangeeta; Pal, Jayanta K

    2009-05-01

    Protein synthesis is often regulated at the level of initiation of translation, making it a critical step. This regulation occurs by both the cis-regulatory elements, which are located in the 5'- and 3'-UTRs (untranslated regions), and trans-acting factors. A breakdown in this regulation machinery can perturb cellular metabolism, leading to various physiological abnormalities. The highly structured UTRs, along with features such as GC-richness, upstream open reading frames and internal ribosome entry sites, significantly influence the rate of translation of mRNAs. In this review, we discuss how changes in the cis-regulatory sequences of the UTRs, for example, point mutations and truncations, influence expression of specific genes at the level of translation. Such modifications may tilt the physiological balance from healthy to diseased states, resulting in conditions such as hereditary thrombocythaemia, breast cancer, fragile X syndrome, bipolar affective disorder and Alzheimer's disease. This information tends to establish the crucial role of UTRs, perhaps as much as that of coding sequences, in health and disease. PMID:19275763

  18. Unproductive alternative splicing and nonsense mRNAs: A widespread phenomenon among plant circadian clock genes

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    Filichkin Sergei A

    2012-07-01

    Full Text Available Abstract Background Recent mapping of eukaryotic transcriptomes and spliceomes using massively parallel RNA sequencing (RNA-seq has revealed that the extent of alternative splicing has been considerably underestimated. Evidence also suggests that many pre-mRNAs undergo unproductive alternative splicing resulting in incorporation of in-frame premature termination codons (PTCs. The destinies and potential functions of the PTC-harboring mRNAs remain poorly understood. Unproductive alternative splicing in circadian clock genes presents a special case study because the daily oscillations of protein expression levels require rapid and steep adjustments in mRNA levels. Results We conducted a systematic survey of alternative splicing of plant circadian clock genes using RNA-seq and found that many Arabidopsis thaliana circadian clock-associated genes are alternatively spliced. Results were confirmed using reverse transcription polymerase chain reaction (RT-PCR, quantitative RT-PCR (qRT-PCR, and/or Sanger sequencing. Intron retention events were frequently observed in mRNAs of the CCA1/LHY-like subfamily of MYB transcription factors. In contrast, the REVEILLE2 (RVE2 transcript was alternatively spliced via inclusion of a "poison cassette exon" (PCE. The PCE type events introducing in-frame PTCs are conserved in some mammalian and plant serine/arginine-rich splicing factors. For some circadian genes such as CCA1 the ratio of the productive isoform (i.e., a representative splice variant encoding the full-length protein to its PTC counterpart shifted sharply under specific environmental stress conditions. Conclusions Our results demonstrate that unproductive alternative splicing is a widespread phenomenon among plant circadian clock genes that frequently generates mRNA isoforms harboring in-frame PTCs. Because LHY and CCA1 are core components of the plant central circadian oscillator, the conservation of alternatively spliced variants between CCA1 and LHY

  19. Genome-wide identification of mRNAs associated with the translational regulator PUMILIO in Drosophila melanogaster

    OpenAIRE

    Gerber, André P.; Luschnig, Stefan; Krasnow, Mark A; Brown, Patrick O; Herschlag, Daniel

    2006-01-01

    Genome-wide identification of RNAs associated with RNA-binding proteins is crucial for deciphering posttranscriptional regulatory systems. PUMILIO is a member of the evolutionary conserved Puf-family of RNA-binding proteins that repress gene expression posttranscriptionally. We generated transgenic flies expressing affinity-tagged PUMILIO under the control of an ovary-specific promoter, and we purified PUMILIO from whole adult flies and embryos and analyzed associated mRNAs by using DNA micro...

  20. Genome-wide RIP-Chip analysis of translational repressor-bound mRNAs in the Plasmodium gametocyte

    KAUST Repository

    Guerreiro, Ana

    2014-11-03

    Background Following fertilization, the early proteomes of metazoans are defined by the translation of stored but repressed transcripts; further embryonic development relies on de novo transcription of the zygotic genome. During sexual development of Plasmodium berghei, a rodent model for human malaria species including P. falciparum, the stability of repressed mRNAs requires the translational repressors DOZI and CITH. When these repressors are absent, Plasmodium zygote development and transmission to the mosquito vector is halted, as hundreds of transcripts become destabilized. However, which mRNAs are direct targets of these RNA binding proteins, and thus subject to translational repression, is unknown. Results We identify the maternal mRNA contribution to post-fertilization development of P. berghei using RNA immunoprecipitation and microarray analysis. We find that 731 mRNAs, approximately 50% of the transcriptome, are associated with DOZI and CITH, allowing zygote development to proceed in the absence of RNA polymerase II transcription. Using GFP-tagging, we validate the repression phenotype of selected genes and identify mRNAs relying on the 5′ untranslated region for translational control. Gene deletion reveals a novel protein located in the ookinete crystalloid with an essential function for sporozoite development. Conclusions Our study details for the first time the P. berghei maternal repressome. This mRNA population provides the developing ookinete with coding potential for key molecules required for life-cycle progression, and that are likely to be critical for the transmission of the malaria parasite from the rodent and the human host to the mosquito vector.

  1. Two Chlamydomonas OPR proteins stabilize chloroplast mRNAs encoding small subunits of photosystem II and cytochrome b6 f.

    Science.gov (United States)

    Wang, Fei; Johnson, Xenie; Cavaiuolo, Marina; Bohne, Alexandra-Viola; Nickelsen, Joerg; Vallon, Olivier

    2015-06-01

    In plants and algae, chloroplast gene expression is controlled by nucleus-encoded proteins that bind to mRNAs in a specific manner, stabilizing mRNAs or promoting their splicing, editing, or translation. Here, we present the characterization of two mRNA stabilization factors of the green alga Chlamydomonas reinhardtii, which both belong to the OctotricoPeptide Repeat (OPR) family. MCG1 is necessary to stabilize the petG mRNA, encoding a small subunit of the cytochrome b6 f complex, while MBI1 stabilizes the psbI mRNA, coding for a small subunit of photosystem II. In the mcg1 mutant, the small RNA footprint corresponding to the 5'-end of the petG transcript is reduced in abundance. In both cases, the absence of the small subunit perturbs assembly of the cognate complex. Whereas PetG is essential for formation of a functional cytochrome b6 f dimer, PsbI appears partly dispensable as a low level of PSII activity can still be measured in its absence. Thus, nuclear control of chloroplast gene expression is not only exerted on the major core subunits of the complexes, but also on small subunits with a single transmembrane helix. While OPR proteins have thus far been involved in translation or trans-splicing of plastid mRNAs, our results expand the potential roles of this repeat family to their stabilization. PMID:25898982

  2. Translation by ribosome shunting on adenovirus and hsp70 mRNAs facilitated by complementarity to 18S rRNA

    OpenAIRE

    Yueh, Andrew; Schneider, Robert J.

    2000-01-01

    Translation initiation on eukaryotic mRNAs involves 40S ribosome association with mRNA caps (m7GpppN), mediated by initiation factor eIF4F. 40S eukaryotic ribosomes and initiation factors undergo 5′ scanning to the initiation codon, with no known role for complementarity between eukaryotic 18S rRNA and the 5′ noncoding region of mRNAs. We demonstrate that the 5′ noncoding region of human adenovirus late mRNAs, known as the tripartite leader, utilizes a striking complementarity to 18S rRNA to ...

  3. The ribosome as a missing link in prebiotic evolution II: Ribosomes encode ribosomal proteins that bind to common regions of their own mRNAs and rRNAs.

    Science.gov (United States)

    Root-Bernstein, Robert; Root-Bernstein, Meredith

    2016-05-21

    We have proposed that the ribosome may represent a missing link between prebiotic chemistries and the first cells. One of the predictions that follows from this hypothesis, which we test here, is that ribosomal RNA (rRNA) must have encoded the proteins necessary for ribosomal function. In other words, the rRNA also functioned pre-biotically as mRNA. Since these ribosome-binding proteins (rb-proteins) must bind to the rRNA, but the rRNA also functioned as mRNA, it follows that rb-proteins should bind to their own mRNA as well. This hypothesis can be contrasted to a "null" hypothesis in which rb-proteins evolved independently of the rRNA sequences and therefore there should be no necessary similarity between the rRNA to which rb-proteins bind and the mRNA that encodes the rb-protein. Five types of evidence reported here support the plausibility of the hypothesis that the mRNA encoding rb-proteins evolved from rRNA: (1) the ubiquity of rb-protein binding to their own mRNAs and autogenous control of their own translation; (2) the higher-than-expected incidence of Arginine-rich modules associated with RNA binding that occurs in rRNA-encoded proteins; (3) the fact that rRNA-binding regions of rb-proteins are homologous to their mRNA binding regions; (4) the higher than expected incidence of rb-protein sequences encoded in rRNA that are of a high degree of homology to their mRNA as compared with a random selection of other proteins; and (5) rRNA in modern prokaryotes and eukaryotes encodes functional proteins. None of these results can be explained by the null hypothesis that assumes independent evolution of rRNA and the mRNAs encoding ribosomal proteins. Also noteworthy is that very few proteins bind their own mRNAs that are not associated with ribosome function. Further tests of the hypothesis are suggested: (1) experimental testing of whether rRNA-encoded proteins bind to rRNA at their coding sites; (2) whether tRNA synthetases, which are also known to bind to their

  4. Regulación de la traducción de mRNAs virales: efecto de proteasas virales

    OpenAIRE

    Moral López, Pablo

    2016-01-01

    Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular Success of viral infection relays on the viral ability to overcome host antiviral response at the same time that their mRNAs hijack components of the translation machinery including ribosomes. Some viruses have developed the ability to bypass cellular antiviral response and optimize viral protein synthesis by inhibiting host translation. In the case of pic...

  5. Identification of RNA sequences and structures involved in site-specific cleavage of IGF-II mRNAs.

    OpenAIRE

    van Dijk, E L; Sussenbach, J S; Holthuizen, P E

    1998-01-01

    Insulin-like growth factor-II (IGF-II) mRNAs are subject to site-specific endonucleolytic cleavage in the 3' untranslated region (UTR), rendering an unstable 5' cleavage product containing the coding region and a very stable 3' cleavage product of 1.8 kb consisting of the 3'-UTR sequence and the poly(A) tail. Previously, it was established that two widely separated elements in the 3'-UTR (elements I and II), that can form a duplex structure, are necessary and sufficient for cleavage. To furth...

  6. Somatomedin-C/insulin-like growth factor-I and Insulin-like growth factor-II mRNAs in rate fetal and adult tissues

    International Nuclear Information System (INIS)

    Somatomedin-C or insulin-like growth factor I (Sm-C/IGF-I) and insulin-like growth factor II (IGF-II) have been implicated in the regulation of fetal growth and development. In the present study 32P-labeled complementary DNA probes encoding human and mouse Sm-C/IGF-I and human IGF-II were used in Northern blot hybridizations to analyze rat Sm-C/IGF-I and IGF-II mRNAs in poly(A+) RNAs from intestine, liver, lung, and brain of adult rats and fetal rats between day 14 and 17 of gestation. In fetal rats, all four tissues contained a major mRNA of 1.7 kilobase (kb) that hybridized with the human Sm-C/IGF-I cDNA and mRNAs of 7.5, 4.7, 1.7, and 1.2 kb that hybridized with the mouse Sm-C/IGF-I cDNA. Adult rat intestine, liver, and lung also contained these mRNAs but Sm-C/IGF-I mRNAs were not detected in adult rat brain. These findings provide direct support for prior observations that multiple tissues in the fetus synthesize immunoreactive Sm-C/IGF-I and imply a role for Sm-C/IGF-I in fetal development as well as postnatally. Multiple IGF-II mRNAs of estimated sizes 4.7, 3.9, 2.2, 1.75, and 1.2 kb were observed in fetal rat intestine, liver, lung, and brain. The 4.7- and 3.9-kb mRNAs were the major hybridizing IGF-II mRNAs in all fetal tissues. Higher abundance of IGF-II mRNAs in rat fetal tissues compared with adult tissues supports prior hypotheses, based on serum IGF-II concentrations, that IGF-II is predominantly a fetal somatomedin. IGF-II mRNAs are present, however, in some poly(A+) RNAs from adult rat tissues. The brain was the only tissue in the adult rat where the 4.7- and 3.9-kb IGF-II mRNAs were consistently detected. These findings suggest that a role for IGF-II in the adult rat, particularly in the central nervous system, cannot be excluded

  7. The Exon Junction Complex Controls the Efficient and Faithful Splicing of a Subset of Transcripts Involved in Mitotic Cell-Cycle Progression.

    Science.gov (United States)

    Fukumura, Kazuhiro; Wakabayashi, Shunichi; Kataoka, Naoyuki; Sakamoto, Hiroshi; Suzuki, Yutaka; Nakai, Kenta; Mayeda, Akila; Inoue, Kunio

    2016-01-01

    The exon junction complex (EJC) that is deposited onto spliced mRNAs upstream of exon-exon junctions plays important roles in multiple post-splicing gene expression events, such as mRNA export, surveillance, localization, and translation. However, a direct role for the human EJC in pre-mRNA splicing has not been fully understood. Using HeLa cells, we depleted one of the EJC core components, Y14, and the resulting transcriptome was analyzed by deep sequencing (RNA-Seq) and confirmed by RT-PCR. We found that Y14 is required for efficient and faithful splicing of a group of transcripts that is enriched in short intron-containing genes involved in mitotic cell-cycle progression. Tethering of EJC core components (Y14, eIF4AIII or MAGOH) to a model reporter pre-mRNA harboring a short intron showed that these core components are prerequisites for the splicing activation. Taken together, we conclude that the EJC core assembled on pre-mRNA is critical for efficient and faithful splicing of a specific subset of short introns in mitotic cell cycle-related genes. PMID:27490541

  8. Highly efficient gene knockout by injection of TALEN mRNAs into oocytes and host transfer in Xenopus laevis

    Directory of Open Access Journals (Sweden)

    Keisuke Nakajima

    2015-01-01

    Full Text Available Zinc-finger nucleases, transcription activator-like effector nucleases (TALENs and the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins system are potentially powerful tools for producing tailor-made knockout animals. However, their mutagenic activity is not high enough to induce mutations at all loci of a target gene throughout an entire tadpole. In this study, we present a highly efficient method for introducing gene modifications at almost all target sequences in randomly selected embryos. The gene modification activity of TALEN is enhanced by adopting the host-transfer technique. In our method, the efficiency is further improved by injecting TALEN mRNAs fused to the 3′UTR of the Xenopus DEADSouth gene into oocytes, which are then transferred into a host female frog, where they are ovulated and fertilized. The addition of the 3′UTR of the DEADSouth gene promotes mRNA translation in the oocytes and increases the expression of TALEN proteins to near-maximal levels three hours post fertilization (hpf. In contrast, TALEN mRNAs without this 3′UTR are translated infrequently in oocytes. Our data suggest that genomic DNA is more sensitive to TALEN proteins from fertilization to the midblastula (MBT stage. Our method works by increasing the levels of TALEN proteins during the pre-MBT stages.

  9. Putrescine-Mediated Changes in Mammalian Intracellular Polyamine Levels Increase Spermidine/Spermine-N1-Acetyltransferase Activity and Increase Gene Expression of Several Cell Cycle-Related Genes

    OpenAIRE

    Ancheta, Allan Atienza

    2010-01-01

    Polyamines are aliphatic polycations existing in all living organisms and are essential for life. Most organisms synthesize three types of polyamines: putrescine, spermidine, and spermine. Putrescine is the product of the rate-limiting reaction of ornithine decarboxylase (ODC) and the amino acid ornithine. Spermidine and spermine are downstream metabolites sequentially derived from putrescine. In a recent landmark colon cancer chemopreventative clinical trial researchers found that combin...

  10. In vivo monitoring of mRNA movement in Drosophila body wall muscle cells reveals the presence of myofiber domains.

    Directory of Open Access Journals (Sweden)

    Alice M C van Gemert

    Full Text Available BACKGROUND: In skeletal muscle each muscle cell, commonly called myofiber, is actually a large syncytium containing numerous nuclei. Experiments in fixed myofibers show that mRNAs remain localized around the nuclei in which they are produced. METHODOLOGY/PRINCIPAL FINDINGS: In this study we generated transgenic flies that allowed us to investigate the movement of mRNAs in body wall myofibers of living Drosophila embryos. We determined the dynamic properties of GFP-tagged mRNAs using in vivo confocal imaging and photobleaching techniques and found that the GFP-tagged mRNAs are not free to move throughout myofibers. The restricted movement indicated that body wall myofibers consist of three domains. The exchange of mRNAs between the domains is relatively slow, but the GFP-tagged mRNAs move rapidly within these domains. One domain is located at the centre of the cell and is surrounded by nuclei while the other two domains are located at either end of the fiber. To move between these domains mRNAs have to travel past centrally located nuclei. CONCLUSIONS/SIGNIFICANCE: These data suggest that the domains made visible in our experiments result from prolonged interactions with as yet undefined structures close to the nuclei that prevent GFP-tagged mRNAs from rapidly moving between the domains. This could be of significant importance for the treatment of myopathies using regenerative cell-based therapies.

  11. The 3' region of Human Papillomavirus type 16 early mRNAs decrease expression

    DEFF Research Database (Denmark)

    Vinther, J.; Rosenstierne, M.W.; Kristiansen, Karen; Norrild, B.

    2005-01-01

    Background: High risk human papillomavirus (HR-HPV) infects mucosal surfaces and HR-HPV infection is required for development of cervical cancer. Accordingly, enforced expression of the early HR-HPV proteins can induce immortalisation of human cells. In most cervical cancers and cervical cancer...... cell lines the HR-HPV double stranded DNA genome has been integrated into the host cell genome. Methods: We have used a retroviral GUS reporter system to generate pools of stably transfected HaCaT and SiHa cells. The HPV-16 early sequences that are deleted upon integration of the HPV-16 genome was...... inserted into the 3' UTR of the reporter mRNA. Pools containing thousands of independent integrations were tested for the steady state levels of the reporter mRNA by Real Time PCR and reporter protein by a GUS enzymatic activity assays. In addition, we tested the cellular distribution and half lives of the...

  12. BRCA1-Dependent Translational Regulation in Breast Cancer Cells.

    Directory of Open Access Journals (Sweden)

    Estelle Dacheux

    Full Text Available BRCA1 (Breast Cancer 1 has been implicated in a number of cellular processes, including transcription regulation, DNA damage repair and protein ubiquitination. We previously demonstrated that BRCA1 interacts with PABP1 (Poly(A-Binding Protein 1 and that BRCA1 modulates protein synthesis through this interaction. To identify the mRNAs that are translationally regulated by BRCA1, we used a microarray analysis of polysome-bound mRNAs in BRCA1-depleted and non-depleted MCF7 cells. Our findings show that BRCA1 modifies the translational efficiency of approximately 7% of the mRNAs expressed in these cells. Further analysis revealed that several processes contributing to cell surveillance such as cell cycle arrest, cell death, cellular growth and proliferation, DNA repair and gene expression, are largely enriched for the mRNAs whose translation is impacted by BRCA1. The BRCA1-dependent translation of these species of mRNAs therefore uncovers a novel mechanism through which BRCA1 exerts its onco-suppressive role. In addition, the BRCA1-dependent translation of mRNAs participating in unexpected functions such as cellular movement, nucleic acid metabolism or protein trafficking is indicative of novel functions for BRCA1. Finally, this study contributes to the identification of several markers associated with BRCA1 deficiency and to the discovery of new potential anti-neoplastic therapeutic targets.

  13. Microarray profiling and co-expression network analysis of circulating lncRNAs and mRNAs associated with major depressive disorder.

    Directory of Open Access Journals (Sweden)

    Zhifen Liu

    Full Text Available LncRNAs, which represent one of the most highly expressed classes of ncRNAs in the brain, are becoming increasingly interesting with regard to brain functions and disorders. However, changes in the expression of regulatory lncRNAs in Major Depressive Disorder (MDD have not yet been reported. Using microarrays, we profiled the expression of 34834 lncRNAs and 39224 mRNAs in peripheral blood sampled from MDD patients as well as demographically-matched controls. Among these, we found that 2007 lncRNAs and 1667 mRNAs were differentially expressed, 17 of which were documented as depression-related gene in previous studies. Gene Ontology (GO and pathway analyses indicated that the biological functions of differentially expressed mRNAs were related to fundamental metabolic processes and neurodevelopment diseases. To investigate the potential regulatory roles of the differentially expressed lncRNAs on the mRNAs, we also constructed co-expression networks composed of the lncRNAs and mRNAs, which shows significant correlated patterns of expression. In the MDD-derived network, there were a greater number of nodes and connections than that in the control-derived network. The lncRNAs located at chr10:874695-874794, chr10:75873456-75873642, and chr3:47048304-47048512 may be important factors regulating the expression of mRNAs as they have previously been reported associations with MDD. This study is the first to explore genome-wide lncRNA expression and co-expression with mRNA patterns in MDD using microarray technology. We identified circulating lncRNAs that are aberrantly expressed in MDD and the results suggest that lncRNAs may contribute to the molecular pathogenesis of MDD.

  14. No evidence that mRNAs have lower folding free energies than random sequences with the same dinucleotide distribution

    DEFF Research Database (Denmark)

    Workman, Christopher; Krogh, Anders Stærmose

    1999-01-01

    This work investigates whether mRNA has a lower estimated folding free energy than random sequences. The free energy estimates are calculated by the mfold program for prediction of RNA secondary structures. For a set of 46 mRNAs it is shown that the predicted free energy is not significantly...... different from random sequences with the same dinucleotide distribution. For random sequences with the same mononucleotide distribution it has previously been shown that the native mRNA sequences have a lower predicted free energy, which indicates a more stable structure than random sequences. However......, dinucleotide content is important when assessing the significance of predicted free energy as the physical stability of RNA secondary structure is known to depend on dinucleotide base stacking energies. Even known RNA secondary structures, like tRNAs, can be shown to have predicted free energies...

  15. Integrated analysis of noncoding RNAs and mRNAs reveals their potential roles in the biological activities of the growth hormone receptor.

    Science.gov (United States)

    Chang, Lei; Qi, Haolong; Xiao, Yusha; Li, Changsheng; Wang, Yitao; Guo, Tao; Liu, Zhisu; Liu, Quanyan

    2016-08-01

    Accumulating evidence has indicated that noncoding RNAs (ncRNAs) have important regulatory potential in various biological processes. The molecular mechanisms by which growth hormone receptor (GHR) deficiency protects against age-related pathologies, reduces the incidence and delays the occurrence of fatal neoplasms are unclear. The aim of this study was to investigate miRNA, lncRNA and mRNA expression profiles and the potential functional roles of these RNA molecules in GHR knockout (GHR-KO) mice. Microarray expression profiles of miRNAs, lncRNAs and mRNAs were determined in wild type control mice and in GHR-KO mice. Differential expression, pathway and gene network analyses were developed to identify the possible biological roles of functional RNA molecules. Compared to wild type control mice, 1695 lncRNAs, 914 mRNAs and 9 miRNAs were upregulated and 1747 lncRNAs, 786 mRNAs and 21 miRNAs were downregulated in female GHR-KO mice. Moreover, 1265 lncRNAs, 724 mRNAs and 41 miRNAs were upregulated and 1377 lncRNAs, 765 mRNAs and 16 miRNAs were downregulated in male GHR-KO mice compared to wild type mice. Co-expression analysis of mRNAs, lncRNAs, and miRNAs showed that mRNAs including Hemxi2, Ero1Ib, 4933434i20RIK, Pde7a and Lgals1, lncRNAs including ASMM9PARTA014848, EL605414-P1, ASMM9PARTA051724, ASMM9PARTA045378 and ASMM9PARTA049185, and miRNAs including miR-188-3p, miR-690, miR-709 and miR-710 are situated at the core position of a three-dimensional lncRNA-mRNA-miRNA regulatory network. KEGG analysis showed that the most significantly regulated pathway was steroid hormone biosynthesis. We identified a set of lncRNAs, miRNAs and mRNAs that were aberrantly expressed in GHR-KO mice. Our results provide a foundation and an expansive view of the biological activities of the GHR. PMID:27064376

  16. Integration of human papillomavirus type 16 DNA into the human genome leads to increased stability of E6 and E7 mRNAs: implications for cervical carcinogenesis.

    OpenAIRE

    S. Jeon; Lambert, P F

    1995-01-01

    In many cervical cancers, human papillomavirus type 16 (HPV-16) DNA genomes are found to be integrated into the host chromosome. In this study, we demonstrate that integration of HPV-16 DNA leads to increased steady-state levels of mRNAs encoding the viral oncogenes E6 and E7. This increase is shown to result, at least in part, from an increased stability of E6 and E7 mRNAs that arise specifically from those integrated viral genomes disrupted in the 3' untranslated region of the viral early r...

  17. Long-range RNA interaction of two sequence elements required for endonucleolytic cleavage of human insulin-like growth factor II mRNAs.

    OpenAIRE

    Scheper, W; Meinsma, D; Holthuizen, P E; Sussenbach, J S

    1995-01-01

    Human insulin-like growth factor II (IGF-II) mRNAs are subject to site-specific endonucleolytic cleavage in the 3' untranslated region, leading to an unstable 5' cleavage product containing the IGF-II coding region and a very stable 3' cleavage product of 1.8 kb. This endonucleolytic cleavage is most probably the first and rate-limiting step in degradation of IGF-II mRNAs. Two sequence elements within the 3' untranslated region are required for cleavage: element I, located approximately 2 kb ...

  18. Preferential translation of cold-shock mRNAs during cold adaptation

    OpenAIRE

    GIULIODORI, ANNA MARIA; Brandi, Anna; GUALERZI, CLAUDIO O.; Pon, Cynthia L.

    2004-01-01

    Upon temperature downshift below the lower threshold of balanced growth (~20°C), the Escherichia coli translational apparatus undergoes modifications allowing the selective translation of the transcripts of cold shock-induced genes, while bulk protein synthesis is drastically reduced. Here we were able to reproduce this translational bias in E. coli cell-free extracts prepared at various times during cold adaptation which were found to display different capacities to translate different types...

  19. Research Progress on Centrosome Cycle Related Protein Phosphorylation /Dephosphorylation and the Involved Functions%中心体周期相关蛋白磷酸化/去磷酸化及其功能的研究进展

    Institute of Scientific and Technical Information of China (English)

    谭锬; 梁前进

    2012-01-01

    or abnormal cells ( e. g. tumour cells) , protein modifications, especially the phosphorylation modification, dominate in cell regulation. As one kind of the major organelles cells, centrosome contains many centrosomal proteins depend on phosphorylation regulation. To provide references for future research, the centrosome cycle related protein phosphorylation and function were reviewed.

  20. The Fragile X Protein binds mRNAs involved in cancer progression and modulates metastasis formation

    OpenAIRE

    Lucá, Rossella; Averna, Michele; Zalfa, Francesca; Vecchi, Manuela; Bianchi, Fabrizio; Fata, Giorgio La; Del Nonno, Franca; Nardacci, Roberta; Bianchi, Marco; Nuciforo, Paolo; Munck, Sebastian; Parrella, Paola; Moura, Rute; Signori, Emanuela; Alston, Robert

    2013-01-01

    The role of the fragile X mental retardation protein (FMRP) is well established in brain, where its absence leads to the fragile X syndrome (FXS). FMRP is almost ubiquitously expressed, suggesting that, in addition to its effects in brain, it may have fundamental roles in other organs. There is evidence that FMRP expression can be linked to cancer. FMR1 mRNA, encoding FMRP, is overexpressed in hepatocellular carcinoma cells. A decreased risk of cancer has been reported in patients with FXS wh...

  1. Effect of plant species on P cycle-related microorganisms associated with litter decomposition and P soil availability: implications for agroforestry management

    Directory of Open Access Journals (Sweden)

    Correa E

    2016-04-01

    Full Text Available Cutting dry deciduous forest (preserved site for wood supply in semi-arid Brazil has led to invasion of a pioneer shrub vegetation called “Carrasco” (disturbed site, which inhibits the sprouting of native species. A land restoration project was undertaken in a cleared Carrasco area where a mixed plantation of native species and Eucalyptus spp. (experimental site was established to preserve the forest and ensure wood supply for the local population. We considered phosphorus as a limiting soil nutrient to plant growth, and we addressed the roles of litter decomposition and microbial activity on phosphorus release in the disturbed, preserved and experimental sites. The phosphorus released from leaf litter was affected by the vegetation type, which favored specific soil microbial populations during decomposition. The Carrasco vegetation predominantly favored arbuscular mycorrhizal fungi (AMF, as shown by root colonization in the litter bags; the Eucalyptus plants favored AMF and ectomycorrhizal fungi (EM, as well as phosphate solubilizing microorganisms (PSM, and the intercropping system favored AMF and PSM groups. In contrast, the preserved site favored the PSM population. High phosphatase activity was found in the preserved and experimental sites in contrast to the Carrasco soil. Principal component analysis showed that AMF root colonization and phosphatase activity were the main parameters influencing the increase in soil phosphorus. Based on the above results, rehabilitation appeared to be underway in the experimental site, since the samples were more similar to the preserved site than to the disturbed site. This effect was attributed to Eucalyptus camaldulensis that promote the establishment of all phosphorus cycle-related microorganisms (AMF, EM and PSF. E. camaldulensis associated with mycorrhizal fungi and PSM are recommended for inclusion in agroforestry systems.

  2. Isolation of bovine milk-derived microvesicles carrying mRNAs and microRNAs.

    Science.gov (United States)

    Hata, Taketoshi; Murakami, Kosuke; Nakatani, Hajime; Yamamoto, Yasunari; Matsuda, Tsukasa; Aoki, Naohito

    2010-05-28

    By a series of centrifugation and ultracentrifugation, we could isolate microvesicles with approximately 100 nm in diameter from bovine milk. We also found that approximately 1700 and 1000 ng of total RNA, in which small RNAs were major components, was contained inside the microvesicles isolated from 6 ml of colostrum and mature milk, respectively, despite high RNase activity in the milk. Polyadenylated gene transcripts for major milk proteins and translation elongation factor-1alpha (EF-1alpha) were present in the microvesicles, and integrity of some transcripts was confirmed by real-time PCR targeting 5'- and 3'-ends of mRNA and by in vitro translation analysis. Moreover, a considerable amount of mammary gland and immune-related microRNAs were present in the milk-derived microvesicles. Acidification of milk to mimic gastrointestinal tract did not mostly affected RNA yield and quality. The milk related gene transcripts were detected in cultured cells when incubated with milk-derived microvesicles, suggesting cellular uptake of the microvesicle contents including RNA. Our findings suggest that bovine breast milk contains RNAs capable for being transferred to living cells and involved in the development of calf's gastrointestinal and immune systems. PMID:20434431

  3. Deep sequencing shows multiple oligouridylations are required for 3′ to 5′ degradation of histone mRNAs on polyribosomes

    Science.gov (United States)

    Slevin, Michael K.; Meaux, Stacie; Welch, Joshua D.; Bigler, Rebecca; Miliani de Marval, Paula L.; Su, Wei; Rhoads, Robert E.; Prins, Jan F.; Marzluff, William F.

    2014-01-01

    SUMMARY Histone mRNAs are rapidly degraded when DNA replication is inhibited during S-phase with degradation initiating with oligouridylation of the stemloop at the 3′ end. We developed a customized RNA-Seq strategy to identify the 3′ termini of degradation intermediates of histone mRNAs. Using this strategy, we identified two types of oligouridylated degradation intermediates: RNAs ending at different sites of the 3′ side of the stemloop that resulted from initial degradation by 3′hExo and intermediates near the stop codon and within the coding region. Sequencing of polyribosomal histone mRNAs revealed that degradation initiates and proceeds 3′ to 5′ on translating mRNA and many intermediates are capped. Knockdown of the exosome-associated exonuclease Pml/Scl-100, but not the Dis3L2 exonuclease, slows histone mRNA degradation, consistent with 3′ to 5′ degradation by the exosome containing PM/Scl-100. Knockdown of No-go decay factors also slowed histone mRNA degradation, suggesting a role in removing ribosomes from partially degraded mRNAs. PMID:24656133

  4. Developmental expression of the G protein-coupled receptor 54 and three GnRH mRNAs in the teleost fish cobia.

    Science.gov (United States)

    Mohamed, J Shaik; Benninghoff, Abby D; Holt, G Joan; Khan, Izhar A

    2007-02-01

    The cDNAs of the G protein-coupled receptor 54 (GPR54) and three prepro-gonadotropin-releasing hormones, GnRH-I (seabream GnRH), GnRH-II (chicken GnRH-II), and GnRH-III (salmon GnRH) were isolated and cloned from the brain of the teleost fish cobia, Rachycentron canadum. The cobia GPR54 cDNA was 95 and 51-56% identical to those of tilapia and mammalian models respectively. The GnRH cDNA sequences of cobia showed strong identities to those of tilapia, Atlantic croaker, red drum, and the seabass and seabream species. The real-time quantitative RT-PCR methods allowed detection of all three GnRH mRNAs on the first day after hatching (DAH). The GnRH-I mRNA levels, which were the lowest among the three GnRHs, increased gradually with two distinct peaks in larvae at 3 and 4 DAH. On the other hand, GnRH-II and GnRH-III mRNAs were significantly higher in larvae at 2 and 6 DAH compared with those on the preceding days. In addition, significant peaks of all the three GnRH mRNAs were observed in the brains of 26-day-old fish. The finding of higher GnRH-I and GnRH-II mRNAs in males than females at 153 DAH may be related to early puberty observed during the first year in laboratory-reared male cobia. Moreover, this study demonstrates for the first time the expression of GPR54 mRNA during larval development in a vertebrate species. The concomitant expression patterns of GPR54 and GnRH mRNAs during different stages of larval and juvenile developments, and during early puberty in male cobia suggest a potential relationship between GPR54 and multiple GnRHs during these stages of development consistent with the role of GPR54 in controlling GnRH release in mammals. The increase in GPR54 and GnRH mRNAs observed during early puberty in cobia is consistent with a similar change reported in pubertal rats. This finding together with the localization of GPR54 mRNAs on GnRH neurons in fish and mammals suggests that the GPR54-GnRH interactions may be conserved in different vertebrate groups

  5. Functional genomic characterization of mRNAs associated with TcPUF6, a pumilio-like protein from Trypanosoma cruzi.

    Science.gov (United States)

    Dallagiovanna, Bruno; Correa, Alejandro; Probst, Christian M; Holetz, Fabiola; Smircich, Pablo; de Aguiar, Alessandra Melo; Mansur, Fernanda; da Silva, Claudio Vieira; Mortara, Renato A; Garat, Beatriz; Buck, Gregory A; Goldenberg, Samuel; Krieger, Marco A

    2008-03-28

    Trypanosoma cruzi is the protozoan parasite that causes Chagas disease or American trypanosomiasis. Kinetoplastid parasites could be considered as model organisms for studying factors involved in posttranscriptional regulation because they control gene expression almost exclusively at this level. The PUF (Pumilio/FBF1) protein family regulates mRNA stability and translation in eukaryotes, and several members have been identified in trypanosomatids. We used a ribonomic approach to identify the putative target mRNAs associated with TcPUF6, a member of the T. cruzi PUF family. TcPUF6 is expressed in discrete sites in the cytoplasm at various stages of the parasite life cycle and is not associated with the translation machinery. The overexpression of a tandem affinity purification-tagged TcPUF6 protein allowed the identification of associated mRNAs by affinity purification assays and microarray hybridization yielding nine putative target mRNAs. Whole expression analysis of transfected parasites showed that the mRNAs associated with TcPUF6 were down-regulated in populations overexpressing TcPUF6. The association of TcPUF6 with the TcDhh1 helicase in vivo and the cellular co-localization of these proteins in epimastigote forms suggest that TcPUF6 promotes degradation of its associated mRNAs through interaction with RNA degradation complexes. Analysis of the mRNA levels of the putative TcPUF6-regulated genes during the parasite life cycle showed that their transcripts were up-regulated in metacyclic trypomastigotes. In these infective forms no co-localization between TcPUF6 and TcDhh1 was observed. Our results suggest that TcPUF6 regulates the half-lives of its associated transcripts via differential association with mRNA degradation complexes throughout its life cycle. PMID:18056709

  6. Functional Genomic Characterization of mRNAs Associated with TcPUF6, a Pumilio-like Protein from Trypanosoma cruzi*S⃞

    Science.gov (United States)

    Dallagiovanna, Bruno; Correa, Alejandro; Probst, Christian M.; Holetz, Fabiola; Smircich, Pablo; de Aguiar, Alessandra Melo; Mansur, Fernanda; da Silva, Claudio Vieira; Mortara, Renato A.; Garat, Beatriz; Buck, Gregory A.; Goldenberg, Samuel; Krieger, Marco A.

    2008-01-01

    Trypanosoma cruzi is the protozoan parasite that causes Chagas disease or American trypanosomiasis. Kinetoplastid parasites could be considered as model organisms for studying factors involved in posttranscriptional regulation because they control gene expression almost exclusively at this level. The PUF (Pumilio/FBF1) protein family regulates mRNA stability and translation in eukaryotes, and several members have been identified in trypanosomatids. We used a ribonomic approach to identify the putative target mRNAs associated with TcPUF6, a member of the T. cruzi PUF family. TcPUF6 is expressed in discrete sites in the cytoplasm at various stages of the parasite life cycle and is not associated with the translation machinery. The overexpression of a tandem affinity purification-tagged TcPUF6 protein allowed the identification of associated mRNAs by affinity purification assays and microarray hybridization yielding nine putative target mRNAs. Whole expression analysis of transfected parasites showed that the mRNAs associated with TcPUF6 were down-regulated in populations overexpressing TcPUF6. The association of TcPUF6 with the TcDhh1 helicase in vivo and the cellular co-localization of these proteins in epimastigote forms suggest that TcPUF6 promotes degradation of its associated mRNAs through interaction with RNA degradation complexes. Analysis of the mRNA levels of the putative TcPUF6-regulated genes during the parasite life cycle showed that their transcripts were up-regulated in metacyclic trypomastigotes. In these infective forms no co-localization between TcPUF6 and TcDhh1 was observed. Our results suggest that TcPUF6 regulates the half-lives of its associated transcripts via differential association with mRNA degradation complexes throughout its life cycle. PMID:18056709

  7. Time-dependent expression profiles of microRNAs and mRNAs in rat milk whey.

    Directory of Open Access Journals (Sweden)

    Hirohisa Izumi

    Full Text Available Functional RNAs, such as microRNA (miRNA and mRNA, are present in milk, but their roles are unknown. To clarify the roles of milk RNAs, further studies using experimental animals such as rats are needed. However, it is unclear whether rat milk also contains functional RNAs and what their time dependent expression profiles are. Thus, we prepared total RNA from whey isolated from rat milk collected on days 2, 9, and 16 postpartum and analyzed using microarrays and quantitative PCR. The concentration of RNA in colostrum whey (day 2 was markedly higher than that in mature milk whey (days 9 and 16. Microarray analysis detected 161 miRNAs and 10,948 mRNA transcripts. Most of the miRNAs and mRNA transcripts were common to all tested milks. Finally, we selected some immune- and development-related miRNAs and mRNAs, and analysed them by quantitative PCR (in equal sample volumes to determine their time-dependent changes in expression in detail. Some were significantly more highly expressed in colostrum whey than in mature milk whey, but some were expressed equally. And mRNA expression levels of some cytokines and hormones did not reflect the protein levels. It is still unknown whether RNAs in milk play biological roles in neonates. However, our data will help guide future in vivo studies using experimental animals such as rats.

  8. Expression Pattern of Myogenic Regulatory Transcription Factor mRNAs in the Embryo and Adult Labeo rohita (Hamilton, 1822

    Directory of Open Access Journals (Sweden)

    Archya Sengupta

    2014-01-01

    Full Text Available Understanding the regulation of skeletal muscle development is important to meet the increasing demand of Indian major carp Labeo rohita. Myogenic regulatory factors (MRFs along with myocyte specific enhancer factor 2 (MEF2 play the pivotal role in the determination and differentiation of skeletal muscle. The majority of skeletal muscle genes require both MRFs and MEF2 family members to activate their transcription. In this study, the expression pattern of MyoD, myf-5, myogenin, and MEF2A was observed from 6 h after fertilization to 12 months of age using semiquantitative RT-PCR as well as real-time PCR method. MyoD and myf-5 mRNAs were expressed at high level at the early embryonic stages. Myogenin and MEF2A were expressed after MyoD and myf-5 and remained active up to adult stage. Expression of MyoD was lower than that of Myf-5 after the 5th month. Partial sequencing of MyoD, myf-5, and MEF2A was done to draw phylogeny. In phylogenetic study, Labeo MyoD, MEF2A and myf-5 were found to be closely related to those of common carp. The present investigation suggests that the four transcription factors play pivotal role in the regulation of muscle growth of Labeo rohita in an overlapping and interconnected way.

  9. Effective DNA/RNA co-extraction for analysis of microRNAs, mRNAs, and genomic DNA from formalin-fixed paraffin-embedded specimens.

    Directory of Open Access Journals (Sweden)

    Adam Kotorashvili

    Full Text Available BACKGROUND: Retrospective studies of archived human specimens, with known clinical follow-up, are used to identify predictive and prognostic molecular markers of disease. Due to biochemical differences, however, formalin-fixed paraffin-embedded (FFPE DNA and RNA have generally been extracted separately from either different tissue sections or from the same section by dividing the digested tissue. The former limits accurate correlation whilst the latter is impractical when utilizing rare or limited archived specimens. PRINCIPAL FINDINGS: For effective recovery of genomic DNA and total RNA from a single FFPE specimen, without splitting the proteinase-K digested tissue solution, we optimized a co-extraction method by using TRIzol and purifying DNA from the lower aqueous and RNA from the upper organic phases. Using a series of seven different archived specimens, we evaluated the total amounts of genomic DNA and total RNA recovered by our TRIzol-based co-extraction method and compared our results with those from two commercial kits, the Qiagen AllPrep DNA/RNA FFPE kit, for co-extraction, and the Ambion RecoverAll™ Total Nucleic Acid Isolation kit, for separate extraction of FFPE-DNA and -RNA. Then, to accurately assess the quality of DNA and RNA co-extracted from a single FFPE specimen, we used qRT-PCR, gene expression profiling and methylation assays to analyze microRNAs, mRNAs, and genomic DNA recovered from matched fresh and FFPE MCF10A cells. These experiments show that the TRIzol-based co-extraction method provides larger amounts of FFPE-DNA and -RNA than the two other methods, and particularly provides higher quality microRNAs and genomic DNA for subsequent molecular analyses. SIGNIFICANCE: We determined that co-extraction of genomic DNA and total RNA from a single FFPE specimen is an effective recovery approach to obtain high-quality material for parallel molecular and high-throughput analyses. Our optimized approach provides the option of

  10. Ribosome traffic on mRNAs maps to gene ontology: genome-wide quantification of translation initiation rates and polysome size regulation.

    Science.gov (United States)

    Ciandrini, Luca; Stansfield, Ian; Romano, M Carmen

    2013-01-01

    To understand the complex relationship governing transcript abundance and the level of the encoded protein, we integrate genome-wide experimental data of ribosomal density on mRNAs with a novel stochastic model describing ribosome traffic dynamics during translation elongation. This analysis reveals that codon arrangement, rather than simply codon bias, has a key role in determining translational efficiency. It also reveals that translation output is governed both by initiation efficiency and elongation dynamics. By integrating genome-wide experimental data sets with simulation of ribosome traffic on all Saccharomyces cerevisiae ORFs, mRNA-specific translation initiation rates are for the first time estimated across the entire transcriptome. Our analysis identifies different classes of mRNAs characterised by their initiation rates, their ribosome traffic dynamics, and by their response to ribosome availability. Strikingly, this classification based on translational dynamics maps onto key gene ontological classifications, revealing evolutionary optimisation of translation responses to be strongly influenced by gene function. PMID:23382661

  11. The differential expression of mRNAs and long noncoding RNAs between ectopic and eutopic endometria provides new insights into adenomyosis.

    Science.gov (United States)

    Zhou, Cheng; Zhang, Ting; Liu, Fei; Zhou, Ji; Ni, Xiaobei; Huo, Ran; Shi, Zhonghua

    2016-02-01

    Adenomyosis, defined as ectopic endometrial tissue within the myometrium, can often be misdiagnosed as multiple uterine leiomyomata or endometrial thickening. We therefore performed a combined mRNA and long noncoding (lnc)RNA microarray and bioinformatic analysis of eutopic and ectopic endometria in women with adenomyosis to better understand its pathogenesis and help in the development of a semi-invasive diagnostic test. A total of 586 mRNAs were increased and 305 mRNAs decreased in the ectopic endometrium of adenomyosis compared with the eutopic endometrium, while 388 lncRNA transcripts were up-regulated and 188 down-regulated in ectopic compared with paired eutopic endometrial tissue. Bioinformatic analysis suggested a series of metabolic and molecular abnormalities in adenomyosis, which have many similarities with endometriosis. Furthermore, our study constitutes the first known report of lncRNA expression patterns in human adenomyosis ectopic and eutopic endometrial tissue. PMID:26662114

  12. Effect of Mifepristone and Anordrin Compound on Lev-els of Estrogen and Progesterone Receptor mRNAs in Human Decidua of Early Pregnancy

    Institute of Scientific and Technical Information of China (English)

    张翔; 孙志达; 沈维雄; 江德琦; 朱月华; 王寒正; 金力

    2000-01-01

    Objective To provide the theoretical fundation for the further clinical application of mifepristone and anordrin compound.Materials & Methods Ribonuclease protection assay was used for the detection and quantitation of estrogen and progesterone receptor mRNAs in human decidua from the termination of early pregnancy. Three groups, each of which had 6~8 cases, were studied.Results Compared to the normal control group, estrogen and progesterone receptor mRNAs increased significantly (P <0. 05) in the mifepristone group, whereas the changes in the group administrated mifepristone compound which contains anordrin were not obvious.Conclusions The result suggests that with the similar clinical effect, mifepristone compound has less side effect on the patients, thus being more suitable for the anti-ear-ly pregnancy drug.

  13. Expression of hepatic mRNAs for insulin-like growth factors-I and -II during the development of hypothyroid rats.

    Science.gov (United States)

    Gallo, G; de Marchis, M; Voci, A; Fugassa, E

    1991-12-01

    The effect of thyroid status on the expression of insulin-like growth factors-I and -II mRNAs in the liver of developing rats has been investigated. Northern blot analyses of the specific mRNA demonstrated the presence of four IGF-II mRNA species which were strongly expressed in fetal liver and progressively declined after birth, becoming undetectable after week 3. This decrease was markedly delayed in the liver of hypothyroid rats. In addition, expression of IGF-I mRNA, absent in fetal liver, began during week 1 after birth and progressively increased with age. This increase was markedly delayed in the liver of hypothyroid rats. The data suggest that thyroid hormones regulate rat development via the co-ordinate expression of hepatic IGF-II and IGF-I mRNAs. PMID:1783883

  14. Ribosome Traffic on mRNAs Maps to Gene Ontology: Genome-wide Quantification of Translation Initiation Rates and Polysome Size Regulation

    OpenAIRE

    Ciandrini, Luca; Stansfield, Ian; Romano, M. Carmen

    2013-01-01

    To understand the complex relationship governing transcript abundance and the level of the encoded protein, we integrate genome-wide experimental data of ribosomal density on mRNAs with a novel stochastic model describing ribosome traffic dynamics during translation elongation. This analysis reveals that codon arrangement, rather than simply codon bias, has a key role in determining translational efficiency. It also reveals that translation output is governed both by initiation efficiency and...

  15. The effect of alpha-amanitin on the Arabidopsis seed proteome highlights the distinct toles of stored and neosynthesizes mRNAs during germination

    OpenAIRE

    Rajjou, Loïc; Gallardo, Karine; Debeaujon, Isabelle; Vandekerckhove, Joël; Job, Claudette; Job, Dominique

    2004-01-01

    To investigate the role of stored and neosynthesized mRNAs in seed germination, we examined the effect of alpha-amanitin, a transcriptional inhibitor targeting RNA polymerase II, on the germination of nondormant Arabidopsis seeds. We used transparent testa mutants, of which seed coat is highly permeable, to better ascertain that the drug can reach the embryo during seed imbibition. Even with the most permeable mutant (tt2-1), germination (radicle protrusion) occurred in the absence of transcr...

  16. Similar Source of Differential Blood mRNAs in Lung Cancer and Pulmonary Inflammatory Diseases: Calls for Improved Strategy for Identifying Cancer-Specific Biomarkers

    OpenAIRE

    Hong, Guini; Chen, Beibei; Li, Hongdong; Zhang, Wenjing; Zheng, Tingting; Li, Shan; Shi, Tongwei; Ao, Lu; Guo, Zheng

    2014-01-01

    Background Many studies try to identify cancer diagnostic biomarkers by comparing peripheral whole blood (PWB) of cancer samples and healthy controls, explicitly or implicitly assuming that such biomarkers are potential candidate biomarkers for distinguishing cancer from nonmalignant inflammation-associated diseases. Methods Multiple PWB gene expression profiles for lung cancer/inflammation-associated pulmonary diseases were used for differential mRNAs identification and comparison and for pr...

  17. Towards simultaneous individual and tissue identification: A proof-of-principle study on parallel sequencing of STRs, amelogenin, and mRNAs with the Ion Torrent PGM

    OpenAIRE

    Zubakov, Dmitry; Kokmeijer, I.; Ralf, Arwin; N Rajagopalan; Calandro, L.; Wootton, S; Langit, R.; C. CHANG; Lagace, R.; Kayser, Manfred

    2015-01-01

    textabstractAbstract DNA-based individual identification and RNA-based tissue identification represent two commonly-used tools in forensic investigation, aiming to identify crime scene sample donors and helping to provide links between DNA-identified sample donors and criminal acts. Currently however, both analyses are typically performed separately. In this proof-of-principle study, we developed an approach for the simultaneous analysis of forensic STRs, amelogenin, and forensic mRNAs based ...

  18. Profiles of nuclear and mitochondrial encoded mRNAs in developing and quiescent embryos of Artemia franciscana.

    Science.gov (United States)

    Hardewig, I; Anchordoguy, T J; Crawford, D L; Hand, S C

    1996-05-24

    Embryos of the brine shrimp Artemia franciscana are able to withstand long bouts of environmental anoxia by entering a quiescent state during which metabolism is greatly depressed. Recent evidence supports a global arrest of protein synthesis during quiescence. In this study we measured the amounts of mRNA for a mitochondrial-encoded subunit of cytochrome c oxidase (COX I) and for nuclear-encoded actin during aerobic development, anaerobiosis, and aerobic acidosis (artificial quiescence imposed by intracellular acidification under aerobic conditions). The levels of both COX I and actin transcripts increased significantly during aerobic development. COX I mRNA levels were tightly correlated with previous measures of COX catalytic activity, which suggests that COX synthesis could be regulated by message concentration during aerobic development. The ontogenetic increase for these mRNAs was blocked by anoxia and aerobic acidosis. Importantly, the levels of COX I and actin mRNA did not decline appreciably during the 6 h bouts of quiescence, even though protein synthesis is acutely arrested by these same treatments. Thus, the constancy of mRNA levels during quiescence indicate that reduced protein synthesis is not caused by message limitation, but rather, is likely controlled at the translational level. One advantage of this regulatory mechanism is the conservation of mRNA molecules during quiescence, which would potentially favor a quick resumption of translation as soon as oxygen is returned to the embryos. Finally, because anoxia and aerobic acidosis are both characterized by acidic intracellular pH, the reduction in pH may serve, directly or indirectly, as one signal regulating levels of mRNA in this embryo during quiescence. PMID:8817476

  19. UTRdb and UTRsite: specialized databases of sequences and functional elements of 5′ and 3′ untranslated regions of eukaryotic mRNAs. Update 2002

    Science.gov (United States)

    Pesole, Graziano; Liuni, Sabino; Grillo, Giorgio; Licciulli, Flavio; Mignone, Flavio; Gissi, Carmela; Saccone, Cecilia

    2002-01-01

    The 5′- and 3′-untranslated regions (5′- and 3′-UTRs) of eukaryotic mRNAs are known to play a crucial role in post-transcriptional regulation of gene expression modulating nucleo-cytoplasmic mRNA transport, translation efficiency, subcellular localization and stability. UTRdb is a specialized database of 5′ and 3′ untranslated sequences of eukaryotic mRNAs cleaned from redundancy. UTRdb entries are enriched with specialized information not present in the primary databases including the presence of nucleotide sequence patterns already demonstrated by experimental analysis to have some functional role. All these patterns have been collected in the UTRsite database so that it is possible to search any input sequence for the presence of annotated functional motifs. Furthermore, UTRdb entries have been annotated for the presence of repetitive elements. All Internet resources we implemented for retrieval and functional analysis of 5′- and 3′-UTRs of eukaryotic mRNAs are accessible at http://bighost.area.ba.cnr.it/BIG/UTRHome/. PMID:11752330

  20. UTRdb and UTRsite: specialized databases of sequences and functional elements of 5′ and 3′ untranslated regions of eukaryotic mRNAs

    Science.gov (United States)

    Pesole, Graziano; Liuni, Sabino; Grillo, Giorgio; Licciulli, Flavio; Larizza, Alessandra; Makalowski, Wojciech; Saccone, Cecilia

    2000-01-01

    The 5′ and 3′ untranslated regions of eukaryotic mRNAs may play a crucial role in the regulation of gene expression controlling mRNA localization, stability and translational efficiency. For this reason we developed UTRdb, a specialized database of 5′ and 3′ untranslated sequences of eukaryotic mRNAs cleaned from redundancy. UTRdb entries are enriched with specialized information not present in the primary databases including the presence of nucleotide sequence patterns already demonstrated by experimental analysis to have some functional role. All these patterns have been collected in the UTRsite database so that it is possible to search any input sequence for the presence of annotated functional motifs. Furthermore, UTRdb entries have been annotated for the presence of repetitive elements. All internet resources implemented for retrieval and functional analysis of 5′ and 3′ untranslated regions of eukaryotic mRNAs are accessible at http://bigarea.area.ba.cnr.it:8000/EmbIT/UTRHome/ PMID:10592223

  1. Phenol emulsion-enhanced DNA-driven subtractive cDNA cloning: isolation of low-abundance monkey cortex-specific mRNAs

    International Nuclear Information System (INIS)

    To isolate cDNA clones of low-abundance mRNAs expressed in monkey cerebral cortex but absent from cerebellum, the authors developed an improved subtractive cDNA cloning procedure that requires only modest quantities of mRNA. Plasmid DNA from a monkey cerebellum cDNA library was hybridized in large excess to radiolabeled monkey cortex cDNA in a phenol emulsion-enhanced reaction. The unhybridized cortex cDNA was isolated by chromatography on hydroxyapatite and used to probe colonies from a monkey cortex cDNA library. Of 60,000 colonies screened, 163 clones were isolated and confirmed by colony hybridization or RNA blotting to represent mRNAs, ranging from 0.001% to 0.1% abundance, specific to or highly enriched in cerebral cortex relative to cerebellum. Clones of one medium-abundance mRNA were recovered almost quantitatively. Two of the lower-abundance mRNAs were expressed at levels reduced by a factor of 10 in Alzheimer disease relative to normal human cortex. One of these was identified as the monkey preprosomatostatin I mRNA

  2. Widespread uncoupling between transcriptome and translatome variations after a stimulus in mammalian cells

    OpenAIRE

    Tebaldi Toma; Re Angela; Viero Gabriella; Pegoretti Ilaria; Passerini Andrea; Blanzieri Enrico; Quattrone Alessandro

    2012-01-01

    Abstract Background The classical view on eukaryotic gene expression proposes the scheme of a forward flow for which fluctuations in mRNA levels upon a stimulus contribute to determine variations in mRNA availability for translation. Here we address this issue by simultaneously profiling with microarrays the total mRNAs (the transcriptome) and the polysome-associated mRNAs (the translatome) after EGF treatment of human cells, and extending the analysis to other 19 different transcriptome/tran...

  3. Deep Sequencing of HIV-Infected Cells: Insights into Nascent Transcription and Host-Directed Therapy

    OpenAIRE

    Peng, Xinxia; Sova, Pavel; Green, Richard R.; Thomas, Matthew J.; Korth, Marcus J.; Proll, Sean; Xu, Jiabao; Cheng, Yanbing; Yi, Kang; Chen, Li; Peng, Zhiyu; Wang, Jun; Palermo, Robert E.; Katze, Michael G.

    2014-01-01

    Polyadenylated mature mRNAs are the focus of standard transcriptome analyses. However, the profiling of nascent transcripts, which often include nonpolyadenylated RNAs, can unveil novel insights into transcriptional regulation. Here, we separately sequenced total RNAs (Total RNAseq) and mRNAs (mRNAseq) from the same HIV-1-infected human CD4+ T cells. We found that many nonpolyadenylated RNAs were differentially expressed upon HIV-1 infection, and we identified 8 times more differentially expr...

  4. Cell-free translation systems prepared from starfish oocytes faithfully reflect in vivo activity; mRNA and initiation factors stimulate supernatants from immature oocytes.

    OpenAIRE

    Xu, Z.; Hille, M B

    1990-01-01

    Meiotic maturation stimulates a change in the translation of stored mRNAs: mRNAs encoding proteins needed for growth of oocytes are translated before meiotic maturation, whereas those encoding proteins required for cleavage are translated after meiotic maturation. Studies of translational regulation during meiotic maturation have been limited by the lack of translationally active cell-free supernatants. Starfish oocytes are ideal for preparing cell-free translation systems because experimenta...

  5. Exosomes Released from Breast Cancer Carcinomas Stimulate Cell Movement

    OpenAIRE

    Dinari A Harris; Patel, Sajni H.; Gucek, Marjan; Hendrix, An; Westbroek, Wendy; Taraska, Justin W.

    2015-01-01

    For metastasis to occur cells must communicate with to their local environment to initiate growth and invasion. Exosomes have emerged as an important mediator of cell-to-cell signalling through the transfer of molecules such as mRNAs, microRNAs, and proteins between cells. Exosomes have been proposed to act as regulators of cancer progression. Here, we study the effect of exosomes on cell migration, an important step in metastasis. We performed cell migration assays, endocytosis assays, and e...

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

    Science.gov (United States)

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

    2016-01-01

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

  7. Zar1 represses translation in Xenopus oocytes and binds to the TCS in maternal mRNAs with different characteristics than Zar2.

    Science.gov (United States)

    Yamamoto, Tomomi M; Cook, Jonathan M; Kotter, Cassandra V; Khat, Terry; Silva, Kevin D; Ferreyros, Michael; Holt, Justin W; Knight, Jefferson D; Charlesworth, Amanda

    2013-10-01

    Maternal mRNAs are translationally regulated during early development. Zar1 and its closely related homolog, Zar2, are both crucial in early development. Xenopus laevis Zygote arrest 2 (Zar2) binds to the Translational Control Sequence (TCS) in maternal mRNAs and regulates translation. The molecular mechanism of Zar1 has not been described. Here we report similarities and differences between Xenopus Zar1 and Zar2. Analysis of Zar sequences in vertebrates revealed two Zar family members with conserved, characteristic amino acid differences in the C-terminal domain. The presence of only two vertebrate Zar proteins was supported by analyzing Zar1 synteny. We propose that the criteria for naming Zar sequences are based on the characteristic amino acids and the chromosomal context. We also propose reclassification of some Zar sequences. We found that Zar1 is expressed throughout oogenesis and is stable during oocyte maturation. The N-terminal domain of Zar1 repressed translation of a reporter construct in immature oocytes. Both Zar1 and Zar2 bound to the TCS in the Wee1 and Mos 3' UTRs using a zinc finger in the C-terminal domain. However, Zar1 had much higher affinity for RNA than Zar2. To show the functional significance of the conserved amino acid substitutions, these residues in Zar2 were mutated to those found in Zar1. We show that these residues contributed to the different RNA binding characteristics of Zar1 compared to Zar2. Our study shows that Zar proteins have generally similar molecular functions in the translational regulation of maternal mRNAs, but they may have different roles in early development. PMID:23827238

  8. Obesity-Dependent Increases in Oocyte mRNAs Are Associated With Increases in Proinflammatory Signaling and Gut Microbial Abundance of Lachnospiraceae in Female Mice.

    Science.gov (United States)

    Xie, Fang; Anderson, Christopher L; Timme, Kelsey R; Kurz, Scott G; Fernando, Samodha C; Wood, Jennifer R

    2016-04-01

    RNAs stored in the metaphase II-arrested oocyte play important roles in successful embryonic development. Their abundance is defined by transcriptional activity during oocyte growth and selective degradation of transcripts during LH-induced oocyte maturation. Our previous studies demonstrated that mRNA abundance is increased in mature ovulated oocytes collected from obese humans and mice and therefore may contribute to reduced oocyte developmental competence associated with metabolic dysfunction. In the current study mouse models of diet-induced obesity were used to determine whether obesity-dependent increases in proinflammatory signaling regulate ovarian abundance of oocyte-specific mRNAs. The abundance of oocyte-specific Bnc1, Dppa3, and Pou5f1 mRNAs as well as markers of proinflammatory signaling were significantly increased in ovaries of obese compared with lean mice which were depleted of fully grown preovulatory follicles. Chromatin-immunoprecipitation analyses also demonstrated increased association of phosphorylated signal transducer and activator of transcription 3 with the Pou5f1 promoter in ovaries of obese mice suggesting that proinflammatory signaling regulates transcription of this gene in the oocyte. The cecum microbial content of lean and obese female mice was subsequently examined to identify potential relationships between microbial composition and proinflammatory signaling in the ovary. Multivariate Association with Linear Models identified significant positive correlations between cecum abundance of the bacterial family Lachnospiraceae and ovarian abundance of Tnfa as well as Dppa3, Bnc1, and Pou5f1 mRNAs. Together, these data suggest that diet-induced changes in gut microbial composition may be contributing to ovarian inflammation which in turn alters ovarian gene expression and ultimately contributes to obesity-dependent reduction in oocyte quality and development of infertility in obese patients. PMID:26881311

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

    Science.gov (United States)

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

    2016-01-01

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

  10. Antagonism of microRNA-122 in mice by systemically administered LNA-antimiR leads to up-regulation of a large set of predicted target mRNAs in the liver

    DEFF Research Database (Denmark)

    Elmen, Joachim; Lindow, Morten; Silahtaroglu, Asli; Bak, Mads; Christensen, Mette; Lind-Thomsen, Allan; Hedtjarn, Maj; hansen, jens bo; Hansen, Henrik Freydenlund; Straarup, Ellen Marie; McCullagh, Keith; Keraney, Phil; Kauppinen, Markus Sakari

    2008-01-01

    the LNA-antimiR in mouse liver cells, which was accompanied by markedly reduced hybridization signals for mature miR-122 in treated mice. Functional antagonism of miR-122 was inferred from a low cholesterol phenotype and derepression within 24 h of 199 liver mRNAs showing significant enrichment for mi......MicroRNA-122 (miR-122) is an abundant liver-specific miRNA, implicated in fatty acid and cholesterol metabolism as well as hepatitis C viral replication. Here, we report that a systemically administered 16-nt, unconjugated LNA (locked nucleic acid)- antimiR oligonucleotide complementary to the 5......’end of miR-122 leads to specific, dose-dependent silencing of miR-122 and shows no hepatotoxicity in mice. Antagonism of miR-122 is due to formation of stable heteroduplexes between the LNA-antimiR and miR-122 as detected by northern analysis. Fluorescence in situ hybridization demonstrated uptake of...

  11. Tristetraprolin Recruits Eukaryotic Initiation Factor 4E2 To Repress Translation of AU-Rich Element-Containing mRNAs

    OpenAIRE

    Tao, Xianzun; Gao, Guangxia

    2015-01-01

    Tristetraprolin (TTP) regulates the expression of AU-rich element-containing mRNAs through promoting the degradation and repressing the translation of target mRNA. While the mechanism for promoting target mRNA degradation has been extensively studied, the mechanism underlying translational repression is not well established. Here, we show that TTP recruits eukaryotic initiation factor 4E2 (eIF4E2) to repress target mRNA translation. TTP interacted with eIF4E2 but not with eIF4E. Overexpressio...

  12. Initiation-mediated mRNA decay in yeast affects heat-shock mRNAs, and works through decapping and 5′-to-3′ hydrolysis

    OpenAIRE

    Heikkinen, Heather L.; Llewellyn, Sara A.; Barnes, Christine A.

    2003-01-01

    The degradation of mRNA in the yeast Saccharomyces cerevisiae takes place through several related pathways. In the most general mRNA-decay pathway, that of poly(A)-dependent decay, the normal shortening of the poly(A) tail on an mRNA molecule by deadenylation triggers mRNA decapping by the enzyme Dcp1p, followed by exonucleolytic digestion by Xrn1p. A specialized mRNA-decay pathway, termed nonsense-mediated decay, comes into play for mRNAs that contain an early nonsense codon. This pathway op...

  13. Quantitation of muscle-specific mRNAs by using cDNA probes during chicken embryonic muscle development in ovo.

    OpenAIRE

    Saidapet, C R; Munro, H N; Valgeirsdóttir, K; Sarkar, S.

    1982-01-01

    The emergence of abundant-class mRNAs specific for contractile muscle proteins and their distribution between polysomal and free mRNP fractions were studied in skeletal muscle excised from chicken embryos during the transition from myoblasts (day 9) to myotubes (day 18). Muscle-specific cDNA was selectively prepared by hybridizing cDNA to template RNA (polysomal poly(A)+ mRNA) from day-14 embryos followed by isolation of the abundant class, which represents approximately 20% of total mRNA. Th...

  14. EXPRESSION OF mRNAS FOR CHEMOKINES AND CHEMOKINE RECEPTORS IN THE SKIN FROM PATIENTS WITH PSORIASIS

    Directory of Open Access Journals (Sweden)

    A. S. Beltiukova

    2014-07-01

    /MIP-1α (r = –0,94, p = 0.005, CCL4/MIP-1β (r = –0,85, p = 0.03 and their receptors: ССR5 (r = –0,79, p = 0.005 and CXCR2 (r = –0,94, p = 0.005 in visually normal skin of the patients with psoriasis and PASI values < 10.A direct correlation was found between expression of mRNAs for CCL11/eotaxin (r = 0.69, p = 0.04 in visually healthy skin from psoriatic patients, and CCR5 (r = 0.82, p = 0.006 in affected skin of the patients with psoriasis and PASI values of 10 to 20. In the group of patients with PASI values over 20, no correlations were detectable. These data allow us of concluding aboutan important contribution of chemokine system to pathogenesis of psoriasis. (Med. Immunol., vol. 10, N 4-5, pp 337-346.

  15. Ribosome traffic on mRNAs maps to gene ontology: genome-wide quantification of translation initiation rates and polysome size regulation.

    Directory of Open Access Journals (Sweden)

    Luca Ciandrini

    Full Text Available To understand the complex relationship governing transcript abundance and the level of the encoded protein, we integrate genome-wide experimental data of ribosomal density on mRNAs with a novel stochastic model describing ribosome traffic dynamics during translation elongation. This analysis reveals that codon arrangement, rather than simply codon bias, has a key role in determining translational efficiency. It also reveals that translation output is governed both by initiation efficiency and elongation dynamics. By integrating genome-wide experimental data sets with simulation of ribosome traffic on all Saccharomyces cerevisiae ORFs, mRNA-specific translation initiation rates are for the first time estimated across the entire transcriptome. Our analysis identifies different classes of mRNAs characterised by their initiation rates, their ribosome traffic dynamics, and by their response to ribosome availability. Strikingly, this classification based on translational dynamics maps onto key gene ontological classifications, revealing evolutionary optimisation of translation responses to be strongly influenced by gene function.

  16. Gibberellin-induced changes in the populations of translatable mRNAs and accumulated polypeptides in dwarfs of maize and pea

    International Nuclear Information System (INIS)

    Two-dimensional gel electrophoresis was used to characterize the molecular mechanism of gibberellin-induced stem elongation in maize and pea. Dwarf mutants of maize and pea lack endogenous gibberellin (GA1) but become phenotypically normal with exogenous applications of this hormone. Sections from either etiolated maize or green pea seedlings were incubated in the presence of [35S] methionine for 3 hours with or without gibberellin. Labeled proteins from soluble and particulate fractions were analyzed by two-dimensional gel electrophoresis and specific changes in the patterns of protein synthesis were observed upon treatment with gibberellin. Polyadenylated mRNAs from etiolated or green maize shoots and green pea epicotyls treated or not with gibberellin (a 0.5 to 16 hour time course) were assayed by translation in a rabbit reticulocyte extract and separation of products by two-dimensional gel electrophoresis. Both increases and decreases in the levels of specific polypeptides were seen for pea and corn, and these changes were observed within 30 minutes of treatment with gibberellin. Together, these data indicate that gibberellin induces changes in the expression of a subset of gene products within elongating dwarfs. This may be due to changes in transcription rate, mRNA stability, or increased efficiency of translation of certain mRNAs

  17. Changes in dopamine D2 and GluR-1 glutamate receptor mRNAs in the rat brain after treatment with phencyclidine.

    Directory of Open Access Journals (Sweden)

    Tomita,Hiroaki

    1995-04-01

    Full Text Available In situ hybridization of slide-mounted brain sections from rats subjected to acute and chronic phencyclidine treatment was carried out using synthetic oligonucleotides complementary to dopamine D2-receptor and non-N-methyl-D-aspartate (NMDA glutamate-receptor-subunit (GluR-1 mRNAs. There was no significant difference in either the D2-receptor or the GluR-1 mRNA levels in any brain region of the acute phencyclidine (10 mg/kg-treated and control groups. However, chronic administration of phencyclidine (10 mg/kg/day, 14 days significantly decreased the dopamine D2-receptor mRNA level in the caudate-putamen (by 27%, P < 0.01 and significantly increased the GluR-1 mRNA level in the prefrontal cortex (by 29%, P < 0.001. These results suggest that the chronic pharmaco-behavioral effects of phencyclidine may involve expression of both dopamine- and non-NMDA glutamate-receptor mRNAs.

  18. Patterns of cell division revealed by transcriptional regulation of genes during the cell cycle in plants.

    OpenAIRE

    Fobert, P R; Coen, E S; Murphy, G. J.; Doonan, J H

    1994-01-01

    Transcripts from five cell cycle related genes accumulate in isolated cells dispersed throughout the actively dividing regions of plant meristems. We propose that this pattern reflects gene expression during particular phases of the cell division cycle. The high proportion of isolated cells suggests that synchrony between daughter cells is rapidly lost following mitosis. This is the first time that such a cell specific expression pattern has been described in a higher organism. Counterstainin...

  19. Metabolism and expression of RNA polymerase II transcripts in Influenza virus-infected cells

    Energy Technology Data Exchange (ETDEWEB)

    Katze, M.G.; Krug, R.M.

    1984-10-01

    Influenza virus infection has adverse effects on the metabolism of two representative RNA polymerase II transcripts in chicken embryo fibroblasts, those coding for BETA-actin and for avian leukosis virus (ALV) proteins. Proviral ALV DNA was integrated into host cell DNA by prior infection with ALV. By S1 endonuclease assay, it was confirmed that nuclear ALV transcripts disappeared very early after infection, already decreasing ca. 80% by 1 h postinfection. A plausible explanation for this nuclear degradation is that the viral cap-dependent endonuclease in the nucleas cleaves the 5' ends of new polymerase II transcripts, rendering the resulting decapped RNAs susceptible to hydrolysis by cellular nucleases. Similar stability of cytoplasmic host cell mRNAs was observed in infected HeLa cells, in which the levels of actin mRNA and two HeLa cell mRNAs (pHe 7 and pHe 28) remained at undiminished levels for 3 h of infection and decreased only slightly by 4.5 h postinfection. The cytoplamic actin and pHe 7 mRNAs isolated from infected HeLa cells were shown to be translated in reticulocyte extracts in biro, indicating that host mRNAs were not inactivated by a virus-induced modification. Despite the continued presence of high levels of functional host cell mRNAs, host cell protein synthesis was effectively shut off by about 3 h postinfection in both chicken embryo fibroblasts and HeLa cells. These results are consistent with the establishment of an influenza virus-specific translational system that selectively translates viral and not host mRNAs.

  20. MicroRNA-126–mediated control of cell fate in B-cell myeloid progenitors as a potential alternative to transcriptional factors

    OpenAIRE

    Okuyama, Kazuki; Ikawa, Tomokatsu; Gentner, Bernhard; Hozumi, Katsuto; Harnprasopwat, Ratanakanit; Lu, Jun; Yamashita, Riu; Ha, Daon; Toyoshima, Takae; Chanda, Bidisha; Kawamata, Toyotaka; Yokoyama, Kazuaki; Wang, Shusheng; Ando, Kiyoshi; Lodish, Harvey F.

    2013-01-01

    Lineage specification is thought to be largely regulated at the level of transcription, where lineage-specific transcription factors drive specific cell fates. MicroRNAs (miR), vital to many cell functions, act posttranscriptionally to decrease the expression of target mRNAs. MLL-AF4 acute lymphocytic leukemia exhibits both myeloid and B-cell surface markers, suggesting that the transformed cells are B-cell myeloid progenitor cells. Through gain- and loss-of-function experiments, we demonstra...

  1. Isolation and characterization of the human tyrosine hydroxylase gene: identification of 5' alternative splice sites responsible for multiple mRNAs

    International Nuclear Information System (INIS)

    A full-length genomic clone for human tyrosine hydroxylase (L-tyrosine, tetrahydropteridine:oxygen oxidoreductase, EC 1.14.16.2) has been isolated. A human brain genomic library constructed in EMBL3 was screened by using a rat cDNA for tyrosine hydroxylase as a probe. Out of one million recombinant phage, one clone was identified that hybridized to both 5' and 3' rat cDNA probes. Restriction endonuclease mapping, Southern blotting, and sequence analysis revealed that, like its rodent counterpart, the human gene is single copy, contains 13 primary exons, and spans approximately 8 kilobases (kb). In contrast to the rat gene, human tyrosine hydroxylase undergoes alternative RNA processing within intron 1, generating at least three distinct mRNAs. A comparison of the human tyrosine hydroxylase and phenylalanine hydroxylase genes indicates that although both probably evolved from a common ancestral gene, major changes in the size of introns have occurred since their divergence

  2. Extracellular protease mRNAs are predominantly expressed in the stromal areas of microdissected mouse breast carcinomas

    DEFF Research Database (Denmark)

    Pedersen, Tanja Xenia; Pennington, Caroline J; Almholt, Kasper;

    2005-01-01

    cells. We have now used laser capture microdissection and real-time PCR to quantify the mRNA expression of components of matrix-degrading proteolytic systems in cancer and stromal areas of mouse mammary tumors genetically induced by the polyoma virus middle T (PyMT) antigen. We examined the mRNA levels...

  3. High-throughput sequencing of partially edited trypanosome mRNAs reveals barriers to editing progression and evidence for alternative editing.

    Science.gov (United States)

    Simpson, Rachel M; Bruno, Andrew E; Bard, Jonathan E; Buck, Michael J; Read, Laurie K

    2016-05-01

    Uridine insertion/deletion RNA editing in kinetoplastids entails the addition and deletion of uridine residues throughout the length of mitochondrial transcripts to generate translatable mRNAs. This complex process requires the coordinated use of several multiprotein complexes as well as the sequential use of noncoding template RNAs called guide RNAs. The majority of steady-state mitochondrial mRNAs are partially edited and often contain regions of mis-editing, termed junctions, whose role is unclear. Here, we report a novel method for sequencing entire populations of pre-edited partially edited, and fully edited RNAs and analyzing editing characteristics across populations using a new bioinformatics tool, the Trypanosome RNA Editing Alignment Tool (TREAT). Using TREAT, we examined populations of two transcripts, RPS12 and ND7-5', in wild-typeTrypanosoma brucei We provide evidence that the majority of partially edited sequences contain junctions, that intrinsic pause sites arise during the progression of editing, and that the mechanisms that mediate pausing in the generation of canonical fully edited sequences are distinct from those that mediate the ends of junction regions. Furthermore, we identify alternatively edited sequences that constitute plausible alternative open reading frames and identify substantial variability in the 5' UTRs of both canonical and alternatively edited sequences. This work is the first to use high-throughput sequencing to examine full-length sequences of whole populations of partially edited transcripts. Our method is highly applicable to current questions in the RNA editing field, including defining mechanisms of action for editing factors and identifying potential alternatively edited sequences. PMID:26908922

  4. Knockdown of Carboxypeptidase A6 in Zebrafish Larvae Reduces Response to Seizure-Inducing Drugs and Causes Changes in the Level of mRNAs Encoding Signaling Molecules

    Science.gov (United States)

    Lopes, Mark William; Sapio, Matthew R.; Leal, Rodrigo B.; Fricker, Lloyd D.

    2016-01-01

    Carboxypeptidase A6 (CPA6) is an extracellular matrix metallocarboxypeptidase that modulates peptide and protein function by removal of hydrophobic C-terminal amino acids. Mutations in the human CPA6 gene that reduce enzymatic activity in the extracellular matrix are associated with febrile seizures, temporal lobe epilepsy, and juvenile myoclonic epilepsy. The characterization of these human mutations suggests a dominant mode of inheritance by haploinsufficiency through loss of function mutations, however the total number of humans with pathologic mutations in CPA6 identified to date remains small. To better understand the relationship between CPA6 and seizures we investigated the effects of morpholino knockdown of cpa6 mRNA in zebrafish (Danio rerio) larvae. Knockdown of cpa6 mRNA resulted in resistance to the effect of seizure-inducing drugs pentylenetetrazole and pilocarpine on swimming behaviors. Knockdown of cpa6 mRNA also reduced the levels of mRNAs encoding neuropeptide precursors (bdnf, npy, chga, pcsk1nl, tac1, nts, edn1), a neuropeptide processing enzyme (cpe), transcription factor (c-fos), and molecules implicated in glutamatergic signaling (grin1a and slc1a2b). Treatment of zebrafish embryos with 60 mM pilocarpine for 1 hour led to reductions in levels of many of the same mRNAs when measured 1 day after pilocarpine exposure, except for c-fos which was elevated 1 day after pilocarpine treatment. Pilocarpine treatment, like cpa6 knockdown, led to a reduced sensitivity to pentylenetetrazole when tested 1 day after pilocarpine treatment. Taken together, these results add to mounting evidence that peptidergic systems participate in the biological effects of seizure-inducing drugs, and are the first in vivo demonstration of the molecular and behavioral consequences of cpa6 insufficiency. PMID:27050163

  5. Knockdown of Carboxypeptidase A6 in Zebrafish Larvae Reduces Response to Seizure-Inducing Drugs and Causes Changes in the Level of mRNAs Encoding Signaling Molecules.

    Science.gov (United States)

    Lopes, Mark William; Sapio, Matthew R; Leal, Rodrigo B; Fricker, Lloyd D

    2016-01-01

    Carboxypeptidase A6 (CPA6) is an extracellular matrix metallocarboxypeptidase that modulates peptide and protein function by removal of hydrophobic C-terminal amino acids. Mutations in the human CPA6 gene that reduce enzymatic activity in the extracellular matrix are associated with febrile seizures, temporal lobe epilepsy, and juvenile myoclonic epilepsy. The characterization of these human mutations suggests a dominant mode of inheritance by haploinsufficiency through loss of function mutations, however the total number of humans with pathologic mutations in CPA6 identified to date remains small. To better understand the relationship between CPA6 and seizures we investigated the effects of morpholino knockdown of cpa6 mRNA in zebrafish (Danio rerio) larvae. Knockdown of cpa6 mRNA resulted in resistance to the effect of seizure-inducing drugs pentylenetetrazole and pilocarpine on swimming behaviors. Knockdown of cpa6 mRNA also reduced the levels of mRNAs encoding neuropeptide precursors (bdnf, npy, chga, pcsk1nl, tac1, nts, edn1), a neuropeptide processing enzyme (cpe), transcription factor (c-fos), and molecules implicated in glutamatergic signaling (grin1a and slc1a2b). Treatment of zebrafish embryos with 60 mM pilocarpine for 1 hour led to reductions in levels of many of the same mRNAs when measured 1 day after pilocarpine exposure, except for c-fos which was elevated 1 day after pilocarpine treatment. Pilocarpine treatment, like cpa6 knockdown, led to a reduced sensitivity to pentylenetetrazole when tested 1 day after pilocarpine treatment. Taken together, these results add to mounting evidence that peptidergic systems participate in the biological effects of seizure-inducing drugs, and are the first in vivo demonstration of the molecular and behavioral consequences of cpa6 insufficiency. PMID:27050163

  6. RNase-L Control of Cellular mRNAs: Roles in Biologic Functions and Mechanisms of Substrate Targeting

    OpenAIRE

    Brennan-Laun, Sarah E.; Ezelle, Heather J.; Li, Xiao-Ling; Hassel, Bret A.

    2014-01-01

    RNase-L is a mediator of type 1 interferon-induced antiviral activity that has diverse and critical cellular roles, including the regulation of cell proliferation, differentiation, senescence and apoptosis, tumorigenesis, and the control of the innate immune response. Although RNase-L was originally shown to mediate the endonucleolytic cleavage of both viral and ribosomal RNAs in response to infection, more recent evidence indicates that RNase-L also functions in the regulation of cellular mR...

  7. Synthesis of coronavirus mRNAs: kinetics of inactivation of infectious bronchitis virus RNA synthesis by UV light

    International Nuclear Information System (INIS)

    Infection of cells with the avian coronavirus infectious bronchitis virus results in the synthesis of five major subgenomic RNAs. These RNAs and the viral genome form a 3' coterminal nested set. We found that the rates of inactivation of synthesis of the RNAs by UV light were different and increased with the length of the transcript. These results show that each RNA is transcribed from a unique promoter and that extensive processing of the primary transcripts probably does not occur

  8. Roles of estrogen receptor α and β in modulating urothelial cell proliferation

    OpenAIRE

    Teng, Jian; Wang, Zun-Yi; Jarrard, David F; Bjorling, Dale E.

    2008-01-01

    We reported previously that both subtypes of estrogen receptors, ERα and ERβ, are expressed by human urothelial cells and mediate estrogen-induced cell proliferation in these cells. The aim of this study was to determine the extent to which each ER subtype contributes to urothelial cell proliferation and their possible involvement in the regulation of the cell cycle. We compared the expression of ERα and ERβ mRNAs and protein quantitatively in primarily cultured human bladder urothelial cells...

  9. RNA-binding proteins in mouse male germline stem cells: a mammalian perspective

    OpenAIRE

    Qi, Huayu

    2016-01-01

    Adult stem cells that reside in particular types of tissues are responsible for tissue homeostasis and regeneration. Cellular functions of adult stem cells are intricately related to the gene expression programs in those cells. Past research has demonstrated that regulation of gene expression at the transcriptional level can decisively alter cell fate of stem cells. However, cellular contents of mRNAs are sometimes not equivalent to proteins, the functional units of cells. It is increasingly ...

  10. RNase-L regulates the stability of mitochondrial DNA-encoded mRNAs in mouse embryo fibroblasts

    International Nuclear Information System (INIS)

    Accelerated decrease in the levels of mitochondrial DNA-encoded mRNA (mt-mRNA) occurs in neuronal cells exposed either to the excitatory amino acid, glutamate or to the sodium ionophore, monensin, suggesting a role of mitochondrial RNase(s) on the stability of mt-mRNAs. Here we report that in mouse embryo fibroblasts that are devoid of the interferon-regulated RNase, RNase-L, the monensin-induced decrease in the half-life of mt-mRNA was reduced. In monensin (250 nM)-treated RNase-L+/+ cells the average half-life of mt-mRNA, determined after termination of transcription with actinomycin D, was found to be 3 h, whereas in monensin-treated RNase-L-/- cells the half-life of mt-mRNA was >6 h. In contrast, the stability of nuclear DNA-encoded β-actin mRNA was unaffected. Induction of RNase-L expression in mouse 3T3 fibroblasts further decreased the monensin-induced reduction in mt-mRNA half-life to 1.5 h. The results indicate that the RNase-L-dependent decrease in mtDNA-encoded mRNA transcript levels occurs through a decrease in the half-life of mt-mRNA, and that RNase-L may play a role in the stability of mt-mRNA

  11. Next generation sequencing analysis of human platelet PolyA+ mRNAs and rRNA-depleted total RNA.

    Directory of Open Access Journals (Sweden)

    Antheia Kissopoulou

    Full Text Available BACKGROUND: Platelets are small anucleate cells circulating in the blood vessels where they play a key role in hemostasis and thrombosis. Here, we compared platelet RNA-Seq results obtained from polyA+ mRNA and rRNA-depleted total RNA. MATERIALS AND METHODS: We used purified, CD45 depleted, human blood platelets collected by apheresis from three male and one female healthy blood donors. The Illumina HiSeq 2000 platform was employed to sequence cDNA converted either from oligo(dT isolated polyA+ RNA or from rRNA-depleted total RNA. The reads were aligned to the GRCh37 reference assembly with the TopHat/Cufflinks alignment package using Ensembl annotations. A de novo assembly of the platelet transcriptome using the Trinity software package and RSEM was also performed. The bioinformatic tools HTSeq and DESeq from Bioconductor were employed for further statistical analyses of read counts. RESULTS: Consistent with previous findings our data suggests that mitochondrially expressed genes comprise a substantial fraction of the platelet transcriptome. We also identified high transcript levels for protein coding genes related to the cytoskeleton function, chemokine signaling, cell adhesion, aggregation, as well as receptor interaction between cells. Certain transcripts were particularly abundant in platelets compared with other cell and tissue types represented by RNA-Seq data from the Illumina Human Body Map 2.0 project. Irrespective of the different library preparation and sequencing protocols, there was good agreement between samples from the 4 individuals. Eighteen differentially expressed genes were identified in the two sexes at 10% false discovery rate using DESeq. CONCLUSION: The present data suggests that platelets may have a unique transcriptome profile characterized by a relative over-expression of mitochondrially encoded genes and also of genomic transcripts related to the cytoskeleton function, chemokine signaling and surface components

  12. A cell-free extract from yeast cells for studying mRNA turnover.

    OpenAIRE

    Vreken, P.; Buddelmeijer, N.; Raué, H A

    1992-01-01

    We have isolated a cell-free extract from yeast cells that reproduces the differences observed in vivo in the rate of turnover of individual yeast mRNAs. Detailed analysis of the degradation of yeast phosphoglycerate kinase (PGK) mRNA in this system demonstrated that both natural and synthetically prepared PGK transcripts are degraded by the same pathway previously established by us in vivo, consisting of endonucleolytic cleavage at a number of 5'-GGUG-3' sequence motifs within a short target...

  13. The DYW Subgroup PPR Protein MEF35 Targets RNA Editing Sites in the Mitochondrial rpl16, nad4 and cob mRNAs in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Nadja Brehme

    Full Text Available RNA editing in plant mitochondria and plastids alters specific nucleotides from cytidine (C to uridine (U mostly in mRNAs. A number of PLS-class PPR proteins have been characterized as RNA recognition factors for specific RNA editing sites, all containing a C-terminal extension, the E domain, and some an additional DYW domain, named after the characteristic C-terminal amino acid triplet of this domain. Presently the recognition factors for more than 300 mitochondrial editing sites are still unidentified. In order to characterize these missing factors, the recently proposed computational prediction tool could be of use to assign target RNA editing sites to PPR proteins of yet unknown function. Using this target prediction approach we identified the nuclear gene MEF35 (Mitochondrial Editing Factor 35 to be required for RNA editing at three sites in mitochondria of Arabidopsis thaliana. The MEF35 protein contains eleven PPR repeats and E and DYW extensions at the C-terminus. Two T-DNA insertion mutants, one inserted just upstream and the other inside the reading frame encoding the DYW domain, show loss of editing at a site in each of the mRNAs for protein 16 in the large ribosomal subunit (site rpl16-209, for cytochrome b (cob-286 and for subunit 4 of complex I (nad4-1373, respectively. Editing is restored upon introduction of the wild type MEF35 gene in the reading frame mutant. The MEF35 protein interacts in Y2H assays with the mitochondrial MORF1 and MORF8 proteins, mutation of the latter also influences editing at two of the three MEF35 target sites. Homozygous mutant plants develop indistinguishably from wild type plants, although the RPL16 and COB/CYTB proteins are essential and the amino acids encoded after the editing events are conserved in most plant species. These results demonstrate the feasibility of the computational target prediction to screen for target RNA editing sites of E domain containing PLS-class PPR proteins.

  14. Expression and regulation of mRNAs for insulin-like growth factor-I receptor and LH receptor in corpora lutea

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Relationship between insulin-like growth factor-I receptor (IGF-IR) and luteinizing hormone receptor (LHR) mRNA expression as well as their regulation was determined in rat corpora lutea (CL) . In the CL of estrous cycle rat, LHR mRNA positive CL expressed high level of mRNA of IGF-IR. While the expression of LHR mRNA decreased on estrus, the CL still expressed relatively high level of IGF-IR mRNA. In pseudopregnant rat CL, the expression level of LHR mRNA was low on day 1, the most intense signals were detected on day 8, the signals of LHR mRNA became undetectable on day 14. In contrast to LHR expression, the high level of IGF-IR mRNA was observed in pseudopregnant CL of day 1, and thereafter its signals were detected from day 2 to day 14. Pregnant rat CL expressed both LHR and IGF-IR mRNAs. IGF-I stimulated LHR expression in CL. PGF2 inhibited expression of IGF-IR and LHR. PGE2 negated the inhibiting effects of PGF2. These data suggest that IGF-I may be involved in regulating CL function, and maintaining CL structure through changes in expression of its receptors. Inhibited expression of IGF-IR by PGF2 may be part of mechanisms for regression of CL.

  15. Expression and regulation of mRNAs for insulin-like growth factor-I receptor and LH receptor in corpora lutea

    Institute of Scientific and Technical Information of China (English)

    罗文祥; 祝诚

    2000-01-01

    Relationship between insulin-like growth factor-l receptor (IGF-IR) and luteinizing hormone receptor (LHR) mRNA expression as well as their regulation was determined in rat corpora lutea (CL) . In the CL of estrous cycle rat, LHR mRNA positive CL expressed high level of mRNA of IGF-IR. While the expression of LHR mRNA decreased on estrus, the CL still expressed relatively high level of IGF-IR mRNA. In pseudopregnant rat CL, the expression level of LHR mRNA was low on day 1, the most intense signals were detected on day 8, the signals of LHR mRNA became undetectable on day 14. In contrast to LHR expression, the high level of IGF-IR mRNA was observed in pseudopregnant CL of day 1, and thereafter its signals were detected from day 2 to day 14. Pregnant rat CL expressed both LHR and IGF-IR mRNAs. IGF-I stimulated LHR expression in CL. PGF2ainhibited expression of IGF-IR and LHR. PGE2 negated the inhibiting effects of PGF2α. These data suggest that IGF-I may be involved in regulating CL function, and maintai

  16. DEAD-box protein DDX3 associates with eIF4F to promote translation of selected mRNAs.

    Science.gov (United States)

    Soto-Rifo, Ricardo; Rubilar, Paulina S; Limousin, Taran; de Breyne, Sylvain; Décimo, Didier; Ohlmann, Théophile

    2012-09-12

    Here, we have characterized a step in translation initiation of viral and cellular mRNAs that contain RNA secondary structures immediately at the vicinity of their m(7)GTP cap. This is mediated by the DEAD-box helicase DDX3 which can directly bind to the 5' of the target mRNA where it clamps the entry of eIF4F through an eIF4G and Poly A-binding protein cytoplasmic 1 (PABP) double interaction. This could induce limited local strand separation of the secondary structure to allow 43S pre-initiation complex attachment to the 5' free extremity of the mRNA. We further demonstrate that the requirement for DDX3 is highly specific to some selected transcripts, cannot be replaced or substituted by eIF4A and is only needed in the very early steps of ribosome binding and prior to 43S ribosomal scanning. Altogether, these data define an unprecedented role for a DEAD-box RNA helicase in translation initiation. PMID:22872150

  17. Transcriptome-wide mapping of 5-methylcytidine RNA modifications in bacteria, archaea, and yeast reveals m5C within archaeal mRNAs.

    Directory of Open Access Journals (Sweden)

    Sarit Edelheit

    2013-06-01

    Full Text Available The presence of 5-methylcytidine (m(5C in tRNA and rRNA molecules of a wide variety of organisms was first observed more than 40 years ago. However, detection of this modification was limited to specific, abundant, RNA species, due to the usage of low-throughput methods. To obtain a high resolution, systematic, and comprehensive transcriptome-wide overview of m(5C across the three domains of life, we used bisulfite treatment on total RNA from both gram positive (B. subtilis and gram negative (E. coli bacteria, an archaeon (S. solfataricus and a eukaryote (S. cerevisiae, followed by massively parallel sequencing. We were able to recover most previously documented m(5C sites on rRNA in the four organisms, and identified several novel sites in yeast and archaeal rRNAs. Our analyses also allowed quantification of methylated m(5C positions in 64 tRNAs in yeast and archaea, revealing stoichiometric differences between the methylation patterns of these organisms. Molecules of tRNAs in which m(5C was absent were also discovered. Intriguingly, we detected m(5C sites within archaeal mRNAs, and identified a consensus motif of AUCGANGU that directs methylation in S. solfataricus. Our results, which were validated using m(5C-specific RNA immunoprecipitation, provide the first evidence for mRNA modifications in archaea, suggesting that this mode of post-transcriptional regulation extends beyond the eukaryotic domain.

  18. Endogenous short RNAs generated by Dicer 2 and RNA-dependent RNA polymerase 1 regulate mRNAs in the basal fungus Mucor circinelloides

    Energy Technology Data Exchange (ETDEWEB)

    Grigoriev, Igor; Nicolas, Francisco; Moxon, Simon; Haro, Juan de; Calo, Silvia; Torres-Martinez, Santiago; Moulton, Vincent; Ruiz-Vazquez, Rosa; Dalmay, Tamas

    2011-09-01

    Endogenous short RNAs (esRNAs) play diverse roles in eukaryotes and usually are produced from double-stranded RNA (dsRNA) by Dicer. esRNAs are grouped into different classes based on biogenesis and function but not all classes are present in all three eukaryotic kingdoms. The esRNA register of fungi is poorly described compared to other eukaryotes and it is not clear what esRNA classes are present in this kingdom and whether they regulate the expression of protein coding genes. However, evidence that some dicer mutant fungi display altered phenotypes suggests that esRNAs play an important role in fungi. Here, we show that the basal fungus Mucor circinelloides produces new classes of esRNAs that map to exons and regulate the expression of many protein coding genes. The largest class of these exonic-siRNAs (ex-siRNAs) are generated by RNA-dependent RNA Polymerase 1 (RdRP1) and dicer-like 2 (DCL2) and target the mRNAs of protein coding genes from which they were produced. Our results expand the range of esRNAs in eukaryotes and reveal a new role for esRNAs in fungi

  19. Differential expression of exons 1a and 1c in mRNAs for sterol regulatory element binding protein-1 in human and mouse organs and cultured cells.

    OpenAIRE

    Shimomura, I; Shimano, H; Horton, J D; Goldstein, J L; Brown, M S

    1997-01-01

    The 5' end of the mRNA-encoding sterol regulatory element binding protein-1 (SREBP-1) exists in two forms, designated 1a and 1c. The divergence results from the use of two transcription start sites that produce two separate 5' exons, each of which is spliced to a common exon 2. Here we show that the ratio of SREBP-1c to 1a transcripts varies markedly among organs of the adult mouse. At one extreme is the liver, in which the 1c transcript predominates by a 9:1 ratio. High 1c:1a ratios are also...

  20. RNA-binding proteins in mouse male germline stem cells: a mammalian perspective.

    Science.gov (United States)

    Qi, Huayu

    2016-01-01

    Adult stem cells that reside in particular types of tissues are responsible for tissue homeostasis and regeneration. Cellular functions of adult stem cells are intricately related to the gene expression programs in those cells. Past research has demonstrated that regulation of gene expression at the transcriptional level can decisively alter cell fate of stem cells. However, cellular contents of mRNAs are sometimes not equivalent to proteins, the functional units of cells. It is increasingly realized that post-transcriptional and translational regulation of gene expression are also fundamental for stem cell functions. Compared to differentiated somatic cells, effects on cellular status manifested by varied expression of RNA-binding proteins and global protein synthesis have been demonstrated in several stem cell systems. Through the cooperation of both cis-elements of mRNAs and trans-acting RNA-binding proteins that are intimately associated with them, regulation of localization, stability, and translational status of mRNAs directly influences the self-renewal and differentiation of stem cells. Previous studies have uncovered some of the molecular mechanisms that underlie the functions of RNA-binding proteins in stem cells in invertebrate species. However, their roles in adult stem cells in mammals are just beginning to be unveiled. This review highlights some of the RNA-binding proteins that play important functions during the maintenance and differentiation of mouse male germline stem cells, the adult stem cells in the male reproductive organ. PMID:26839690

  1. Collagen content, but not the ratios of collagen type III/I mRNAs, differs among hypertensive, alcoholic, and idiopathic dilated cardiomyopathy

    Directory of Open Access Journals (Sweden)

    H.N. Soufen

    2008-12-01

    Full Text Available Cardiac interstitial fibrosis may contribute to ventricular dysfunction and the prognosis of patients with dilated cardiomyopathy. The objective of the present study was to determine if total myocardial collagen content and collagen type III/I (III/I ratio mRNAs differ in hypertensive, alcoholic, and idiopathic dilated cardiomyopathy subjects. Echocardiography and exercise cardiopulmonary testing were performed in patients with idiopathic (N = 22, hypertensive (N = 12, and alcoholic (N = 11 dilated cardiomyopathy. Morphometric analysis of collagen was performed in fragments obtained by endomyocardial biopsy with picrosirius red staining. The collagen III/I ratio was determined by reverse transcription polymerase chain reaction. Samples of controls (N = 10 were obtained from autopsy. Echocardiographic variables and maximal oxygen uptake were not different among dilated cardiomyopathy groups. Collagen was higher in all dilated cardiomyopathy groups (idiopathic, hypertensive and alcoholic, 7.36 ± 1.09% versus controls (1.12 ± 0.18%, P < 0.05. Collagen was lower in idiopathic dilated cardiomyopathy (4.97 ± 0.83% than hypertensive (8.50 ± 1.11% and alcoholic (10.77 ± 2.09% samples (P < 0.005 for both. The collagen III/I ratio in all samples from dilated cardiomyopathy patients was higher compared to that in controls (0.29 ± 0.04, P < 0.05 but was the same in the samples from idiopathic (0.77 ± 0.07, hypertensive (0.75 ± 0.07, and alcoholic (0.81 ± 0.16 dilated cardiomyopathy groups. Because of the different physical properties of the types of collagen, the higher III/I ratio may contribute to progressive ventricular dilation and dysfunction in dilated cardiomyopathy patients.

  2. Identification of reference genes for quantitative RT-PCR analysis of microRNAs and mRNAs in castor bean (Ricinus communis L.) under drought stress.

    Science.gov (United States)

    Cassol, Daniela; Cruz, Fernanda P; Espindola, Kauê; Mangeon, Amanda; Müller, Caroline; Loureiro, Marcelo Ehlers; Corrêa, Régis L; Sachetto-Martins, Gilberto

    2016-09-01

    Quantitative real-time PCR (RT-qPCR) is one of the most powerful and sensitive techniques to the study of gene expression. Several factors influence RT-qPCR performance though, including the stability of the reference genes used for data normalization. While the selection of appropriate reference genes is crucial for accurate and reliable gene expression analysis, no suitable reference genes have been previously identified in castor bean under drought stress. In this study, the expression stability of eleven mRNAs, thirteen microRNAs (miRNAs) and one small nuclear RNA were analyzed in roots and leaves across different levels of water deficit. Three different algorithms were employed to analyze the RT-qPCR data, and the resulting outputs were merged using a non-weighted unsupervised rank aggregation method. Our analysis indicated that the Elongation factor 1-beta (EF1B), Protein phosphatase 2A (PP2A) and ADP-ribosylation factor (ADP) ranked as the best candidates across diverse samples submitted to different levels of drought conditions. EF1B and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and EF1B and SKP1/ASK-interacting protein 16 (SKIP16) were found as the most suitable reference genes for expression analysis in roots and leaves, respectively. In addition, miRNAs miR168, miR160 and miR397 were selected as optimal reference genes across all tissues and treatments. miR168 and miR156 were recommended as reference for roots, while miR168 and miR160 were recommended for leaves. Together, our results constitute the first attempt to identify and validate the most suitable reference genes for accurate normalization of gene expression in castor bean under drought stress. PMID:27156134

  3. Evidence for variation in the optimal translation initiation complex: plant eIF4B, eIF4F, and eIF(iso)4F differentially promote translation of mRNAs.

    Science.gov (United States)

    Mayberry, Laura K; Allen, M Leah; Dennis, Michael D; Browning, Karen S

    2009-08-01

    Eukaryotic initiation factor (eIF) 4B is known to interact with multiple initiation factors, mRNA, rRNA, and poly(A) binding protein (PABP). To gain a better understanding of the function of eIF4B, the two isoforms from Arabidopsis (Arabidopsis thaliana) were expressed and analyzed using biophysical and biochemical methods. Plant eIF4B was found by ultracentrifugation and light scattering analysis to most likely be a monomer with an extended structure. An extended structure would facilitate the multiple interactions of eIF4B with mRNA as well as other initiation factors (eIF4A, eIF4G, PABP, and eIF3). Eight mRNAs, barley (Hordeum vulgare) alpha-amylase mRNA, rabbit beta-hemoglobin mRNA, Arabidopsis heat shock protein 21 (HSP21) mRNA, oat (Avena sativa) globulin, wheat (Triticum aestivum) germin, maize (Zea mays) alcohol dehydrogenase, satellite tobacco necrosis virus RNA, and alfalfa mosaic virus (AMV) 4, were used in wheat germ in vitro translation assays to measure their dependence on eIF4B and eIF4F isoforms. The two Arabidopsis eIF4B isoforms, as well as native and recombinant wheat eIF4B, showed similar responses in the translation assay. AMV RNA 4 and Arabidopsis HSP21 showed only a slight dependence on the presence of eIF4B isoforms, whereas rabbit beta-hemoglobin mRNA and wheat germin mRNA showed modest dependence. Barley alpha-amylase, oat globulin, and satellite tobacco necrosis virus RNA displayed the strongest dependence on eIF4B. These results suggest that eIF4B has some effects on mRNA discrimination during initiation of translation. Barley alpha-amylase, oat globulin, and rabbit beta-hemoglobin mRNA showed the highest activity with eIF4F, whereas Arabidopsis HSP21 and AMV RNA 4 used both eIF4F and eIF(iso)4F equally well. These results suggest that differential or optimal translation of mRNAs may require initiation complexes composed of specific isoforms of initiation factor gene products. Thus, individual mRNAs or classes of mRNAs may respond to the

  4. Towards simultaneous individual and tissue identification: A proof-of-principle study on parallel sequencing of STRs, amelogenin, and mRNAs with the Ion Torrent PGM.

    Science.gov (United States)

    Zubakov, D; Kokmeijer, I; Ralf, A; Rajagopalan, N; Calandro, L; Wootton, S; Langit, R; Chang, C; Lagace, R; Kayser, M

    2015-07-01

    DNA-based individual identification and RNA-based tissue identification represent two commonly-used tools in forensic investigation, aiming to identify crime scene sample donors and helping to provide links between DNA-identified sample donors and criminal acts. Currently however, both analyses are typically performed separately. In this proof-of-principle study, we developed an approach for the simultaneous analysis of forensic STRs, amelogenin, and forensic mRNAs based on parallel targeted DNA/RNA sequencing using the Ion Torrent Personal Genome Machine(®) (PGM™) System coupled with the AmpliSeq™ targeted amplification. We demonstrated that 9 autosomal STRs commonly used for individual identification (CSF1PO, D16S539, D3S1358, D5S818, D7S820, D8S1179, TH01, TPOX, and vWA), the AMELX/AMELY system widely applied for sex identification, and 12 mRNA markers previously established for forensic tissue identification (ALAS2 and SPTB for peripheral blood, MMP10 and MMP11 for menstrual blood, HTN3 and STATH for saliva, PRM1 and TGM4 for semen, CYP2B7P1 and MUC4 for vaginal secretion, CCL27 and LCE1C for skin) together with two candidate reference mRNA markers (HPRT1 and SDHA) can all be successfully combined. Unambiguous mRNA-based tissue identification was achieved in all samples from all forensically relevant tissues tested, and STR sequencing analysis of the tissue sample donors was 100% concordant with conventional STR profiling using a commercial kit. Successful STR analysis was obtained from 1ng of genomic DNA and mRNA analysis from 10ng total RNA; however, sensitivity limits were not investigated in this proof-of-principle study and are expected to be much lower. Since dried materials with noticeable RNA degradation and small DNA/RNA amplicons with high-coverage sequencing were used, the achieved correct individual and tissue identification demonstrates the suitability of this approach for analyzing degraded materials in future forensic applications. Overall

  5. Sensitization to the behavioural effects of cocaine: alterations in tyrosine hydroxylase or endogenous opioid mRNAs are not necessarily involved.

    Science.gov (United States)

    Alvarez Fischer, D; Schäfer, M K; Ferger, B; Gross, S; Westermann, R; Weihe, E; Kuschinsky, K

    2001-03-01

    After repeated administration of cocaine at intervals, sensitization phenomena can be observed, so that its behavioural effects are enhanced. Since this phenomenon is long-lasting, it was of interest to study which persistent alterations in the activity of dopaminergic neurones or of endogenous opioid systems downstream of dopaminergic synapses in the basal ganglia are involved in the sensitization. Cocaine (10 mg/kg i.p.) was administered to rats on days 1, 3, 5 and 7 and saline on days 2, 4 and 6 ("repeated cocaine"), or saline was injected on days 1-6 and cocaine on day 7 ("acute cocaine"), or saline was injected on days 1-7 ("saline group"). The "repeated cocaine" schedule led to a significant sensitization to the locomotor activation produced by cocaine on day 7 or on day 17, 10 days after the end of sensitization protocol. Microdialysis in the nucleus accumbens which was performed after administration of cocaine (10 mg/kg i.p.) on day 7, or after an administration of the same dose 10 days after the last administration of cocaine, respectively, revealed significant acute increases of extracellular dopamine to about 200% of basal values. These increases were similar in "acute cocaine" and in "repeated cocaine" animals both after 7 days and after 17 days. For in situ hybridization studies, rats were sacrificed on day 7, 4.5 h after the last cocaine or saline administration. The mRNA for tyrosine hydroxylase (TH) in substantia nigra + ventral tegmental area was significantly elevated to about 140% of saline controls both in the "repeated cocaine" and the "acute cocaine" group as compared with the "saline group". In contrast, there were no differences between the three groups in the mRNAs of preprodynorphin or preproenkephalin levels measured in the nucleus accumbens (core and shell). These results suggest that sensitization phenomena to cocaine are not necessarily connected with alterations in the dopaminergic activity in the mesolimbic system or in the

  6. A genetic screen for asymmetrically localized RNAs in Drosophila tracheal cells

    OpenAIRE

    Nair, Jayan Nandanan

    2008-01-01

    Asymmetrical localization of mRNAs and localized protein synthesis have an important role in establishing and maintaining polarity in cells such as neurons or the Drosophila oocyte and in the regulation of developmental plasticity. In Drosophila a subset of highly branched cells of the respiratory system exhibits both a high degree of polarity and developmental plasticity. These tracheal cells respond to the need for oxygen in the surrounding tissue by outgrowth of branches, often at sites ve...

  7. Molecular beacon – tool for real time studying gene activity in stem cells.

    OpenAIRE

    Ilieva, Mirolyuba; Dufva, Martin

    2012-01-01

    Cells respond to their internal genetic programs and external stimuli by modulating the synthesis of specific mRNAs. Direct observation of mRNA expression in living cells can provide valuable information with regards to understanding fundamental processes such cell differentiation, regeneration and cancerogenesis. Molecular beacon technology is based on fluorescence resonance energy transfer (FRET) and the complementary pairing principles. These fluorescent molecular probes are highly specifi...

  8. Hemoglobin is Expressed in Alveolar Epithelial Type II Cells

    OpenAIRE

    Bhaskaran, Manoj; Chen, Haifeng; Chen, Zhongmong; Liu, Lin

    2005-01-01

    Hemoglobin is the main oxygen carrying heme protein in erythrocytes. In an effort to study the differential gene expression of alveolar epithelial type I and type II cells using DNA microarray technique, we found that the mRNAs of hemoglobin α- and β-chains were expressed in type II cells, but not in type I cells. The microarray data were confirmed by RT-PCR. The mRNA expression of both chains decreased when type II cells trans-differentiated into type I-like cells. Immunocyto/histochemistry ...

  9. Differential gene expression in wild-type and X-ray-sensitive mutants of Chinese hamster ovary cell lines

    International Nuclear Information System (INIS)

    Complementary DNA cloning, differential screening and Northern hybridization techniques were used to study differential gene expression in wild-type Chinese hamster ovary (CHO) K1 cell line and its two X-ray sensitive mutants, xrs-5 and xrs-6. Eleven species of mRNAs were found under-expressed in two independently isolated mutants. The steady-state levels of those mRNAs are 3-26-fold less in the 2 mutants, depending on the particular species. Of underexpressed mRNAs, 6 have been identified by comparing sequences of cloned cDNAs to the known sequences in GenBank 4 of them code for the structural proteins of ferritin heavy chain, non- muscle myosin light chain 3nm, ribosomal protein S17 and L7, resp. The other 2 have strong homology with mouse B2 or retroviral sequences. The remaining 5 mRNAs did not show significant homology with any of the known sequences and apparently represent newly isolated species. Effects of 137Cs γ-rays on the expression of the 11 mRNAs has been studied. Radiation inhibited expression of the B2-like gene in mutants but not in the wild type CHO cells. Levels of the other 10 mRNAs were not affected by radiation. The underexpression of this group of genes in both xrs-5 and xrs-6 mutants seems to be related to their radiation- sensitive phenotype, although the specific gene responsible has not been identified. Two models are proposed to explain the mechanism of under- expression. It is suggested that a cellular factor or/and chromosome structural changes are involved. (author). 33 refs.; 4 figs.; 1 tab

  10. Immunohistochemical study of the expression of cell cycle regulating proteins at different stages of bladder cancer

    DEFF Research Database (Denmark)

    Primdahl, Hanne; Maase, Hans von der; Sørensen, Flemming B.; Wolf, Hans; Ørntoft, Torben Falck

    2002-01-01

    PURPOSE: The cell cycle is known to be deregulated in cancer. We therefore analyzed the expression of the cell cycle related proteins p21, p27, p16, Rb, and L-myc by immunohistochemical staining of bladder tumors. METHODS: The tissue material consisted of bladder tumors from three groups of...

  11. Increased expression of alpha- and beta-globin mRNAs at the pituitary following exposure to estrogen during the critical period of neonatal sex differentiation in the rat

    DEFF Research Database (Denmark)

    Leffers, H; Navarro, V M; Nielsen, John E; Mayen, A; Pinilla, L; Dalgaard, M; Malagon, M M; Castaño, J P; Skakkebaek, N E; Aguilar, E; Tena-Sempere, M

    2006-01-01

    neuroendocrine system controlling development and function of the reproductive axis; the HP unit being highly sensitive to the organizing effects of endogenous and exogenous sex steroids. To gain knowledge on the molecular mode of action and potential biomarkers of exposure to estrogenic compounds at the HP unit...... was not detected at the hypothalamus, cortex, cerebellum, liver and testis. Finally, enhanced levels of alpha- and beta-globin mRNAs at the pituitary were also demonstrated after neonatal administration of the anti-androgen flutamide. In summary, alpha- and beta-globin genes may prove as sensitive...

  12. The downstream atpE cistron is efficiently translated via its own cis-element in partially overlapping atpB–atpE dicistronic mRNAs in chloroplasts

    OpenAIRE

    Suzuki, Haruka; Kuroda, Hiroshi; Yukawa, Yasushi; Sugiura, Masahiro

    2011-01-01

    The chloroplast atpB and atpE genes encode subunits β and ε of the ATP synthase, respectively. They are co-transcribed as dicistronic mRNAs in flowering plants. An unusual feature is an overlap (AUGA) of the atpB stop codon (UGA) with the atpE start codon (AUG). Hence, atpE translation has been believed to depend on atpB translation (i.e. translational coupling). Using an in vitro translation system from tobacco chloroplasts, we showed that both atpB and atpE cistrons are translated from the ...

  13. HIPSTR and thousands of lncRNAs are heterogeneously expressed in human embryos, primordial germ cells and stable cell lines

    Science.gov (United States)

    Yunusov, Dinar; Anderson, Leticia; DaSilva, Lucas Ferreira; Wysocka, Joanna; Ezashi, Toshihiko; Roberts, R. Michael; Verjovski-Almeida, Sergio

    2016-01-01

    Eukaryotic genomes are transcribed into numerous regulatory long non-coding RNAs (lncRNAs). Compared to mRNAs, lncRNAs display higher developmental stage-, tissue-, and cell-subtype-specificity of expression, and are generally less abundant in a population of cells. Despite the progress in single-cell-focused research, the origins of low population-level expression of lncRNAs in homogeneous populations of cells are poorly understood. Here, we identify HIPSTR (Heterogeneously expressed from the Intronic Plus Strand of the TFAP2A-locus RNA), a novel lncRNA gene in the developmentally regulated TFAP2A locus. HIPSTR has evolutionarily conserved expression patterns, its promoter is most active in undifferentiated cells, and depletion of HIPSTR in HEK293 and in pluripotent H1BP cells predominantly affects the genes involved in early organismal development and cell differentiation. Most importantly, we find that HIPSTR is specifically induced and heterogeneously expressed in the 8-cell-stage human embryos during the major wave of embryonic genome activation. We systematically explore the phenomenon of cell-to-cell variation of gene expression and link it to low population-level expression of lncRNAs, showing that, similar to HIPSTR, the expression of thousands of lncRNAs is more highly heterogeneous than the expression of mRNAs in the individual, otherwise indistinguishable cells of totipotent human embryos, primordial germ cells, and stable cell lines. PMID:27605307

  14. Resveratrol Induces Glioma Cell Apoptosis through Activation of Tristetraprolin

    OpenAIRE

    Ryu, Jinhyun; Yoon, Nal Ae; Seong, Hyemin; Jeong, Joo Yeon; Kang, Seokmin; Park, Nammi; Choi, Jungil; Lee, Dong Hoon; Roh, Gu Seob; Kim, Hyun Joon; Cho, Gyeong Jae; Choi, Wan Sung; Park, Jae-Yong; Park, Jeong Woo; Kang, Sang Soo

    2015-01-01

    Tristetraprolin (TTP) is an AU-rich elements (AREs)-binding protein, which regulates the decay of AREs-containing mRNAs such as proto-oncogenes, anti-apoptotic genes and immune regulatory genes. Despite the low expression of TTP in various human cancers, the mechanism involving suppressed expression of TTP is not fully understood. Here, we demonstrate that Resveratrol (3,5,4′-trihydroxystilbene, Res), a naturally occurring compound, induces glioma cell apoptosis through activation of tristetr...

  15. Analysis of the c-myc IRES; a potential role for cell-type specific trans-acting factors and the nuclear compartment

    OpenAIRE

    Stoneley, Mark; Subkhankulova, Tatyana; Le Quesne, John P.C.; Coldwell, Mark J; Jopling, Catherine L; Belsham, Graham J.; Willis, Anne E.

    2000-01-01

    The 5′ UTR of c-myc mRNA contains an internal ribosome entry segment (IRES) and consequently, c-myc mRNAs can be translated by the alternative mechanism of internal ribosome entry. However, there is also some evidence suggesting that c-myc mRNA translation can occur via the conventional cap-dependent scanning mechanism. Using both bicistronic and monocistronic mRNAs containing the c-myc 5′ UTR, we demonstrate that both mechanisms can contribute to c-myc protein synthesis. A wide range of cell...

  16. The emerging role of extracellular vesicle-derived miRNAs: implication in cancer progression and stem cell related diseases

    OpenAIRE

    Yang, Qiwei; Diamond, Michael P.; Al-Hendy, Ayman

    2016-01-01

    Cells release into the extracellular environment, diverse types of membrane vesicles of endosomal and plasma membrane origin called exosomes and microvesicles. A number of studies indicate that these extracellular vehicles (EVs) mediate the interaction between cancer cells and their microenvironment; and thereby, play a critical role in the development of cancers. EVs contain cargo which consist of proteins, lipids, mRNAs, and miRNAs that can be delivered to different types of cells in nascen...

  17. Depletion of eIF4G from yeast cells narrows the range of translational efficiencies genome-wide

    Directory of Open Access Journals (Sweden)

    Hinnebusch Alan G

    2011-01-01

    Full Text Available Abstract Background Eukaryotic translation initiation factor 4G (eIF4G is thought to influence the translational efficiencies of cellular mRNAs by its roles in forming an eIF4F-mRNA-PABP mRNP that is competent for attachment of the 43S preinitiation complex, and in scanning through structured 5' UTR sequences. We have tested this hypothesis by determining the effects of genetically depleting eIF4G from yeast cells on global translational efficiencies (TEs, using gene expression microarrays to measure the abundance of mRNA in polysomes relative to total mRNA for ~5900 genes. Results Although depletion of eIF4G is lethal and reduces protein synthesis by ~75%, it had small effects (less than a factor of 1.5 on the relative TE of most genes. Within these limits, however, depleting eIF4G narrowed the range of translational efficiencies genome-wide, with mRNAs of better than average TE being translated relatively worse, and mRNAs with lower than average TE being translated relatively better. Surprisingly, the fraction of mRNAs most dependent on eIF4G display an average 5' UTR length at or below the mean for all yeast genes. Conclusions This finding suggests that eIF4G is more critical for ribosome attachment to mRNAs than for scanning long, structured 5' UTRs. Our results also indicate that eIF4G, and the closed-loop mRNP it assembles with the m7 G cap- and poly(A-binding factors (eIF4E and PABP, is not essential for translation of most (if not all mRNAs but enhances the differentiation of translational efficiencies genome-wide.

  18. Metabolic properties of chicken embryonic stem cells

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    Cellular energy metabolism correlates with cell fate,but the metabolic properties of chicken embryonic stem (chES) cells are poorly understood.Using a previously established chES cell model and electron microscopy (EM),we found that undifferentiated chES cells stored glycogen.Additionally,undifferentiated chES cells expressed lower levels of glucose transporter 1 (GLUT1) and phosphofructokinase (PFK) mRNAs but higher levels of hexokinase 1 (HK1) and glycogen synthase (GYS) mRNAs compared with control primary chicken embryonic fibroblast (CEF) cells,suggesting that chES cells direct glucose flux towards the glycogenic pathway.Moreover,we demonstrated that undifferentiated chES cells block gluconeogenic outflow and impede the accumulation of glucose-6-phosphate (G6P) from this pathway,as evidenced by the barely detectable levels of pyruvate carboxylase (PCX) and mitochondrial phosphoenolpyruvate carboxykinase (PCK2) mRNAs.Additionally,cell death occurred in undifferentiated chES cells as shown by Hoechst 33342 and propidium iodide (PI) double staining,but it could be rescued by exogenous G6P.However,we found that differentiated chES cells decreased the glycogen reserve through the use of PAS staining.Moreover,differentiated chES cells expressed higher levels of GLUT1,HK1 and PFK mRNAs,while the level of GYS mRNA remained similar in control CEF cells.These data indicate that undifferentiated chES cells continue to synthesize glycogen from glucose at the expense of G6P,while differentiated chES cells have a decreased glycogen reserve,which suggests that the amount of glycogen is indicative of the chES cell state.

  19. Tracking single mRNA molecules in live cells

    Science.gov (United States)

    Moon, Hyungseok C.; Lee, Byung Hun; Lim, Kiseong; Son, Jae Seok; Song, Minho S.; Park, Hye Yoon

    2016-06-01

    mRNAs inside cells interact with numerous RNA-binding proteins, microRNAs, and ribosomes that together compose a highly heterogeneous population of messenger ribonucleoprotein (mRNP) particles. Perhaps one of the best ways to investigate the complex regulation of mRNA is to observe individual molecules. Single molecule imaging allows the collection of quantitative and statistical data on subpopulations and transient states that are otherwise obscured by ensemble averaging. In addition, single particle tracking reveals the sequence of events that occur in the formation and remodeling of mRNPs in real time. Here, we review the current state-of-the-art techniques in tagging, delivery, and imaging to track single mRNAs in live cells. We also discuss how these techniques are applied to extract dynamic information on the transcription, transport, localization, and translation of mRNAs. These studies demonstrate how single molecule tracking is transforming the understanding of mRNA regulation in live cells.

  20. Two Virus-Induced MicroRNAs Known Only from Teleost Fishes Are Orthologues of MicroRNAs Involved in Cell Cycle Control in Humans

    DEFF Research Database (Denmark)

    Schyth, Brian Dall; Bela-Ong, Dennis; Jalali, Seyed Amir Hossein;

    2015-01-01

    MicroRNAs (miRNAs) are similar to 22 base pair-long non-coding RNAs which regulate gene expression in the cytoplasm of eukaryotic cells by binding to specific target regions in mRNAs to mediate transcriptional blocking or mRNA cleavage. Through their fundamental roles in cellular pathways, gene r...

  1. Dysfunctional miRNA-Mediated Regulation in Chromophobe Renal Cell Carcinoma

    Science.gov (United States)

    Sun, Xiaohan; Zhang, Junying

    2016-01-01

    Past research on pathogenesis of a complex disease suggests that differentially expressed message RNAs (mRNAs) can be noted as biomarkers of a disease. However, significant miRNA-mediated regulation change might also be more deep underlying cause of a disease. In this study, a miRNA-mediated regulation module is defined based on GO terms (Gene Ontology terms) from which dysfunctional modules are identified as the suspected cause of a disease. A miRNA-mediated regulation module contains mRNAs annotated to a GO term and MicroRNAs (miRNAs) which regulate the mRNAs. Based on the miRNA-mediated regulation coefficients estimated from the expression profiles of the mRNA and the miRNAs, a SW (single regulation-weight) value is then designed to evaluate the miRNA-mediated regulation change of an mRNA, and the modules with significantly differential SW values are thus identified as dysfunctional modules. The approach is applied to Chromophobe renal cell carcinoma and it identifies 70 dysfunctional miRNA-mediated regulation modules from initial 4381 modules. The identified dysfunctional modules are detected to be comprehensive reflection of chromophobe renal cell carcinoma. The proposed approach suggests that accumulated alteration in miRNA-mediated regulation might cause functional alterations, which further cause a disease. Moreover, this approach can also be used to identify diffentially miRNA-mediated regulated mRNAs showing more comprehensive underlying association with a disease than differentially expressed mRNAs. PMID:27258182

  2. Short-term weightlessness produced by parabolic flight maneuvers altered gene expression patterns in human endothelial cells.

    Science.gov (United States)

    Grosse, Jirka; Wehland, Markus; Pietsch, Jessica; Ma, Xiao; Ulbrich, Claudia; Schulz, Herbert; Saar, Katrin; Hübner, Norbert; Hauslage, Jens; Hemmersbach, Ruth; Braun, Markus; van Loon, Jack; Vagt, Nicole; Infanger, Manfred; Eilles, Christoph; Egli, Marcel; Richter, Peter; Baltz, Theo; Einspanier, Ralf; Sharbati, Soroush; Grimm, Daniela

    2012-02-01

    This study focused on the effects of short-term microgravity (22 s) on the gene expression and morphology of endothelial cells (ECs) and evaluated gravisensitive signaling elements. ECs were investigated during four German Space Agency (Deutsches Zentrum für Luft- und Raumfahrt) parabolic flight campaigns. Hoechst 33342 and acridine orange/ethidium bromide staining showed no signs of cell death in ECs after 31 parabolas (P31). Gene array analysis revealed 320 significantly regulated genes after the first parabola (P1) and P31. COL4A5, COL8A1, ITGA6, ITGA10, and ITGB3 mRNAs were down-regulated after P1. EDN1 and TNFRSF12A mRNAs were up-regulated. ADAM19, CARD8, CD40, GSN, PRKCA (all down-regulated after P1), and PRKAA1 (AMPKα1) mRNAs (up-regulated) provide a very early protective mechanism of cell survival induced by 22 s microgravity. The ABL2 gene was significantly up-regulated after P1 and P31, TUBB was slightly induced, but ACTA2 and VIM mRNAs were not changed. β-Tubulin immunofluorescence revealed a cytoplasmic rearrangement. Vibration had no effect. Hypergravity reduced CARD8, NOS3, VASH1, SERPINH1 (all P1), CAV2, ADAM19, TNFRSF12A, CD40, and ITGA6 (P31) mRNAs. These data suggest that microgravity alters the gene expression patterns and the cytoskeleton of ECs very early. Several gravisensitive signaling elements, such as AMPKα1 and integrins, are involved in the reaction of ECs to altered gravity. PMID:22024737

  3. Increased expression of system large amino acid transporter (LAT)-1 mRNA is associated with invasive potential and unfavorable prognosis of human clear cell renal cell carcinoma

    International Nuclear Information System (INIS)

    The system L amino acid transporter (LAT) has an important role in the transport of various amino acids, and there have been reports about the relation of this system to cancer. Although LATs are highly expressed in the kidneys, little is known about their influence on human renal cancer. To clarify the role of LATs in human clear cell renal cell carcinoma (RCC), we investigated the expression of mRNAs for LAT1, LAT2, LAT3, LAT4, and 4F2hc in clear cell RCC tissues. The mRNAs of these five genes were analyzed by the real-time reverse transcription polymerase chain reaction in matched sets of tumor and non-tumor tissues obtained at operation from 82 Japanese patients with clear cell RCC. We also measured phosphorylated S6 ribosomal protein (Ser-235/236) proteins levels in 18 paired tumor and non-tumor tissues of the patients by Western blotting. Expression of LAT1 mRNA was significantly increased in tumor tissue compared with non-tumor tissue, while expression of LAT2 and LAT3 mRNAs was reduced. There was no difference in the expression of LAT4 and 4F2hc mRNAs between tumor and non-tumor tissues. Increased expression of LAT1 mRNA was associated with less differentiated tumors, local invasion, microscopic vascular invasion, and metastasis. Kaplan-Meier survival analysis showed that a higher serum LAT1 mRNA level was associated with a shorter overall survival time. Phosphorylated S6 ribosomal protein levels were associated with metastatic potential. LAT1 mRNA levels positively correlated with phosphorylated S6 ribosomal protein proteins levels in primary tumors. These findings suggest that LAT1 mRNA is related to the invasive and progressive potential of clear cell RCC

  4. A quantitative comparison of cell-type-specific microarray gene expression profiling methods in the mouse brain.

    Directory of Open Access Journals (Sweden)

    Benjamin W Okaty

    Full Text Available Expression profiling of restricted neural populations using microarrays can facilitate neuronal classification and provide insight into the molecular bases of cellular phenotypes. Due to the formidable heterogeneity of intermixed cell types that make up the brain, isolating cell types prior to microarray processing poses steep technical challenges that have been met in various ways. These methodological differences have the potential to distort cell-type-specific gene expression profiles insofar as they may insufficiently filter out contaminating mRNAs or induce aberrant cellular responses not normally present in vivo. Thus we have compared the repeatability, susceptibility to contamination from off-target cell-types, and evidence for stress-responsive gene expression of five different purification methods--Laser Capture Microdissection (LCM, Translating Ribosome Affinity Purification (TRAP, Immunopanning (PAN, Fluorescence Activated Cell Sorting (FACS, and manual sorting of fluorescently labeled cells (Manual. We found that all methods obtained comparably high levels of repeatability, however, data from LCM and TRAP showed significantly higher levels of contamination than the other methods. While PAN samples showed higher activation of apoptosis-related, stress-related and immediate early genes, samples from FACS and Manual studies, which also require dissociated cells, did not. Given that TRAP targets actively translated mRNAs, whereas other methods target all transcribed mRNAs, observed differences may also reflect translational regulation.

  5. Expression Profiling of Exosomal miRNAs Derived from Human Esophageal Cancer Cells by Solexa High-Throughput Sequencing

    OpenAIRE

    Juan Liao; Ran Liu; Lihong Yin; Yuepu Pu

    2014-01-01

    Cellular genetic materials, such as microRNAs (miRNAs), mRNAs and proteins, are packaged inside exosomes, small membrane vesicles of endocytic origin that are released into the extracellular environment. These cellular genetic materials can be delivered into recipient cells, where they exert their respective biological effects. However, the miRNA profiles and biological functions of exosomes secreted by cancer cells remain unknown. The present study explored the miRNA expression profile and d...

  6. Change of the cell cycle after flutamide treatment in prostate cancer cells and its molecular mechanism

    Institute of Scientific and Technical Information of China (English)

    Yong Wang; Wei-Jun Qin; He Wang; Guo-Xing Shao; Chen Shao; Chang-Hong Shi; Lei Zhang; Hong-Hong Yue; Peng-Fei Wang; Bo Yang; Yun-Tao Zhang; Fan Liu

    2005-01-01

    Aim: To explore the effect of androgen receptor (AR) on the expression of the cell cycle-related genes, such as CDKN1A and BTG1, in prostate cancer cell line LNCaP. Methods: After AR antagonist flutamide treatment and confirmation of its effect by phase contrast microscope and flow cytometry, the differential expression of the cell cycle-related genes was analyzed by a cDNA microarray. The flutamide treated cells were set as the experimental group and the LNCaP cells as the control. We labeled cDNA probes of the experimental group and control group with Cy5 and Cy3 dyes, respectively, through reverse transcription. Then we hybridized the cDNA probes with cDNA microarrays, which contained 8 126 unique human cDNA sequences and the chip was scanned to get the fluorescent values of Cy5 and Cy3 on each spot. After primary analysis, reverse transcription polymerase chain reaction (RTPCR) tests were carried out to confirm the results of the chips. Results:After AR antagonist flutamide treatment,three hundred and twenty-six genes (3.93 %) expressed differentially, 97 down-regulated and 219 up-regulated.Among them, eight up-regulated genes might be cell cycle-related, namely CDC10, NRAS, BTG1, Weel, CLK3,DKFZP564A122, CDKN1A and BTG2. The CDKN1A and BTG1 gene mRNA expression was confirmed to be higher in the experimental group by RT-PCR, whilep53 mRNA expression had no significant changes. Conclusion: Flutamide treatment might up-regulate CDKN1A and BTG1 expression in prostate cancer cells. The protein expressions of CDKN1A and BTG1 play an important role in inhibiting the proliferation of cancer cells. CDKN1A has a great impact on the cell cycle of prostate cancer cells and may play a role in the cancer cells in a p53-independent pathway. The prostate cancer cells might affect the cell cycle-related genes by activating AR and thus break the cell cycle control.

  7. The CCR4-NOT deadenylase activity contributes to generation of induced pluripotent stem cells.

    Science.gov (United States)

    Zukeran, Ari; Takahashi, Akinori; Takaoka, Shohei; Mohamed, Haytham Mohamed Aly; Suzuki, Toru; Ikematsu, Shinya; Yamamoto, Tadashi

    2016-05-27

    Somatic cells can be reprogrammed as induced pluripotent stem cells (iPSCs) by introduction of the transcription factors, OCT3/4, KLF4, SOX2, and c-MYC. The CCR4-NOT complex is the major deadenylase in eukaryotes. Its subunits Cnot1, Cnot2, and Cnot3 maintain pluripotency and self-renewal of mouse and human embryonic stem (ES) cells and contribute to the transition from partial to full iPSCs. However, little is known about how the CCR4-NOT complex post-transcriptionally regulates the reprogramming process. Here, we show that the CCR4-NOT deadenylase subunits Cnot6, Cnot6l, Cnot7, and Cnot8, participate in regulating iPSC generation. Cnot1 knockdown suppresses expression levels of Cnot6, Cnot6l, Cnot7, and Cnot8 in mouse embryonic fibroblasts (MEFs) and decreases the number of alkaline phosphatase (ALP)-positive colonies after iPSC induction. Intriguingly, Cnot1 depletion allows Eomes and p21 mRNAs to persist, increasing their expression levels. Both mRNAs have longer poly(A) tails in Cnot1-depleted cells. Conversely, forced expression of a combination of Cnot6, Cnot6l, Cnot7, and Cnot8 increases the number of ALP-positive colonies after iPSC induction and decreases expression levels of Eomes and p21 mRNAs. Based on these observations, we propose that the CCR4-NOT deadenylase activity contributes to iPSC induction. PMID:27037025

  8. In Vitro and In Vivo Relevance of Infectious Laryngotracheitis Virus gJ Proteins That Are Expressed from Spliced and Nonspliced mRNAs

    Science.gov (United States)

    Fuchs, Walter; Wiesner, Dorothee; Veits, Jutta; Teifke, Jens P.; Mettenleiter, Thomas C.

    2005-01-01

    The positional homologue in the infectious laryngotracheitis virus (ILTV) genome of the glycoprotein gJ gene of herpes simplex virus and the gp2 gene of equine herpesvirus 1 is expressed into four proteins of 85, 115, 160, and 200 kDa (J. Veits, B. Köllner, J. P. Teifke, H. Granzow, T. C. Mettenleiter, and W. Fuchs, Avian Dis. 47:330-342, 2003). RNA analyses revealed that these proteins are expressed from two different late (γ2) transcripts, an unspliced 5.5-kb and a spliced 4.3-kb mRNA that are translated into proteins of 985 and 611 amino acids, respectively. ILTV gJ is incorporated into virions and is modified by N- and O-linked glycosylation. After cotransfection of chicken cells with genomic DNA of a pathogenic ILTV strain and transfer plasmids, gJ-negative ILTV mutants could be isolated. In vitro growth studies demonstrated that deletion of the gJ gene has only minor effects on direct cell-to-cell spread as measured by plaque size. However, progeny virus titers of ILTV-ΔgJ were significantly reduced in comparison to those of the parental virus and a gJ rescue mutant. After experimental infection of chickens the gJ rescue mutant, like wild-type ILTV, caused severe disease and considerable mortality, whereas ILTV-ΔgJ was significantly attenuated. All immunized animals were protected against subsequent challenge infection with virulent ILTV. In sera collected after immunization with the gJ-rescue mutant or with wild-type ILTV, gJ-specific antibodies were detectable by immunofluorescence on cells that had been transfected with a gJ expression plasmid. As expected, no gJ-specific antibodies were found in sera obtained from chickens immunized with ILTV-ΔgJ. Thus, gJ deletion mutants of ILTV might be usable as attenuated live-virus vaccines. Furthermore, the gJ gene might constitute a reliable marker for serological discrimination between vaccinated and field virus-infected chickens. PMID:15613298

  9. MiR-107 and MiR-185 Can Induce Cell Cycle Arrest in Human Non Small Cell Lung Cancer Cell Lines

    OpenAIRE

    Takahashi, Yukari; Forrest, Alistair R. R.; Maeno, Emi; Hashimoto, Takehiro; Daub, Carsten O.; Yasuda, Jun

    2009-01-01

    Background MicroRNAs (miRNAs) are short single stranded noncoding RNAs that suppress gene expression through either translational repression or degradation of target mRNAs. The annealing between messenger RNAs and 5′ seed region of miRNAs is believed to be essential for the specific suppression of target gene expression. One miRNA can have several hundred different targets in a cell. Rapidly accumulating evidence suggests that many miRNAs are involved in cell cycle regulation and consequentia...

  10. Structure and expression of ferritin genes in a human promyelocytic cell line that differentiates in vitro.

    OpenAIRE

    Chou, C. C.; Gatti, R A; Fuller, M L; Concannon, P; Wong, A.; Chada, S; Davis, R C; Salser, W A

    1986-01-01

    HL-60 is a human promyelocytic cell line with the capability of differentiating in vitro to give neutrophils, macrophages, or eosinophils. We screened libraries of HL-60 cDNA clones representing different time points during these differentiation processes to isolate clones corresponding to mRNAs whose expression is regulated during terminal differentiation. Upon sequencing this group of regulated clones, one clone encoding the heavy subunit and two clones encoding the light subunit of human f...

  11. Effective Alu Repeat Based RT-Qpcr Normalization in Cancer Cell Perturbation Experiments

    OpenAIRE

    Ali Rihani; Tom Van Maerken; Filip Pattyn; Gert Van Peer; Anneleen Beckers; Sara De Brouwer; Candy Kumps; Evelien Mets; Joni Van der Meulen; Pieter Rondou; Carina Leonelli; Pieter Mestdagh; Frank Speleman; Jo Vandesompele

    2013-01-01

    BACKGROUND: Measuring messenger RNA (mRNA) levels using the reverse transcription quantitative polymerase chain reaction (RT-qPCR) is common practice in many laboratories. A specific set of mRNAs as internal control reference genes is considered as the preferred strategy to normalize RT-qPCR data. Proper selection of reference genes is a critical issue, especially in cancer cells that are subjected to different in vitro manipulations. These manipulations may result in dramatic alterations in ...

  12. Comparison of myosin heavy chain mRNAs, protein isoforms and fiber type proportions in the rat slow and fast muscles

    Czech Academy of Sciences Publication Activity Database

    Žurmanová, J.; Soukup, Tomáš

    2013-01-01

    Roč. 62, č. 4 (2013), s. 445-453. ISSN 0862-8408 R&D Projects: GA ČR(CZ) GA304/08/0256; GA ČR(CZ) GA305/09/1228; GA MŠk(CZ) 7AMB12SK158; GA MŠk(CZ) LH12058 Grant ostatní: EC(XE) LSH-CT-2004-511978 Institutional research plan: CEZ:AV0Z50110509 Institutional support: RVO:67985823 Keywords : rat * soleus * extensor digitorum longus * myosin heavy chain isoforms * muscle gene expression * quantitative Real Time RT-PCR * SDS-PAGE * fiber type analysis Subject RIV: EA - Cell Biology Impact factor: 1.487, year: 2013

  13. Imaging of mRNA-protein interactions in live cells using novel mCherry trimolecular fluorescence complementation systems.

    Directory of Open Access Journals (Sweden)

    Juan Yin

    Full Text Available Live cell imaging of mRNA-protein interactions makes it possible to study posttranscriptional processes of cellular and viral gene expression under physiological conditions. In this study, red color mCherry-based trimolecular fluorescence complementation (TriFC systems were constructed as new tools for visualizing mRNA-protein interaction in living cells using split mCherry fragments and HIV REV-RRE and TAT-TAR peptide-RNA interaction pairs. The new mCherry TriFC systems were successfully used to image RNA-protein interactions such as that between influenza viral protein NS1 and the 5' UTR of influenza viral mRNAs NS, M, and NP. Upon combination of an mCherry TriFC system with a Venus TriFC system, multiple mRNA-protein interactions could be detected simultaneously in the same cells. Then, the new mCherry TriFC system was used for imaging of interactions between influenza A virus mRNAs and some of adapter proteins in cellular TAP nuclear export pathway in live cells. Adapter proteins Aly and UAP56 were found to associate with three kinds of viral mRNAs. Another adapter protein, splicing factor 9G8, only interacted with intron-containing spliced M2 mRNA. Co-immunoprecipitation assays with influenza A virus-infected cells confirmed these interactions. This study provides long-wavelength-spectrum TriFC systems as new tools for visualizing RNA-protein interactions in live cells and help to understand the nuclear export mechanism of influenza A viral mRNAs.

  14. MAPK Signal Transduction Pathway Regulation: A Novel Mechanism of Rat HSC-T6 Cell Apoptosis Induced by FUZHENGHUAYU Tablet

    Directory of Open Access Journals (Sweden)

    Qi Wang

    2013-01-01

    Full Text Available FUZHENGHUAYU Tablets have been widely used in the treatment of liver fibrosis in China. Here, we investigate the apoptotic effect of FUZHENGHUAYU Tablet in rat liver stellate cell line HSC-T6. HSC-T6 cells were incubated with control serum or drug serum from rats fed with 0.9% NaCl or FUZHENGHUAYU Tablet, respectively. Cells exposed to drug serum showed higher proportions of early and late apoptotic cells than controls. The mRNA levels of collagens I and III, TGF-β1 and α-SMA were reduced by drug serum compared to control serum. Differentially expressed mRNAs and miRNAs were analyzed by microarray and sequencing, respectively. We identified 334 differentially expressed mRNAs and also 60 GOs and two pathways related to the mRNAs. Seventy-five differentially expressed miRNAs were down-regulated by drug serum and 1963 target genes were predicted. 134 GOs up-regulated in drug serum group were linked to miRNA targets, and drug serum also regulated 43 miRNA signal transduction pathways. Protein levels were evaluated by Western blot. Drug serum down-regulated (phospho-SAPK/JNK/(SAPK/JNK and up-regulated phospho-p38/p38 ratios. The study showed that FUZHENGHUAYU Tablet induced apoptosis in rat HSC-T6 cells possibly in part by activating p38 and inhibiting SAPK/JNK.

  15. Fluorescent probes for imaging endogenous β-actin mRNA in living cells using fluorescent protein-tagged pumilio.

    Science.gov (United States)

    Yoshimura, Hideaki; Inaguma, Asumi; Yamada, Toshimichi; Ozawa, Takeaki

    2012-06-15

    Subcellular localization and dynamics of mRNAs control various physiological functions in living cells. A novel technique for visualizing endogenous mRNAs in living cells is necessary for investigation of the spatiotemporal movement of mRNAs. A pumilio homology domain of human pumilio 1 (PUM-HD) is a useful RNA binding protein as a tool for mRNA recognition because the domain can be modified to bind a specific 8-base sequence of target mRNA. In this study, we designed PUM-HD to match the sequence of β-actin mRNA and developed an mRNA probe consisting of two PUM-HD mutants flanking full-length enhanced green fluorescent protein (EGFP). Fluorescence microscopy with the probe in living cells revealed that the probe was labeled precisely with the β-actin mRNA in cytosol. Fluorescent spots from the probe were colocalized with microtubules and moved directionally in living cells. The PUM-HD mutants conjugated with full-length EGFP can enable visualization of β-actin mRNA localization and dynamics in living cells. PMID:22387832

  16. Genome-wide identification and analysis of mRNA expression in fibroblasts, ES cells, and iPS cells.

    Science.gov (United States)

    Hirai, Hiroyuki; Kikyo, Nobuaki

    2016-03-01

    Genome-wide expression patterns of mRNA were compared between mouse embryonic fibroblasts (MEFs), embryonic stem cells (ESCs), and various types of induced pluripotent stem cells (iPSCs). iPSCs were established and maintained using modified Oct4 with or without exogenous leukemia inhibitory factor (LIF) and used to identify mRNAs that were potentially involved in the LIF-independence. The data have been deposited in the NCBI's Gene Expression Omnibus (GEO) database with the accession number GSE65563. PMID:26981399

  17. Expression of Transforming Growth Factor-β1 by Pancreatic Stellate Cells and Its Implications for Matrix Secretion and Turnover in Chronic Pancreatitis

    OpenAIRE

    Shek, Fanny Wai-Tsing; Benyon, Robert Christopher; Walker, Fiona Mairi; McCrudden, Peter Raymond; Pender, Sylvia Lin Foon; Williams, Elizabeth Jean; Johnson, Penelope Ann; Johnson, Colin David; Bateman, Adrian Calvin; Fine, David Roger; Iredale, John Peter

    2002-01-01

    Pancreatic stellate cells mediate fibrosis in chronic pancreatitis. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs)-1 and -2 are crucial modulators of fibrosis. Transforming growth factor-β (TGF-β) is a key regulator of extracellular matrix production and myofibroblast proliferation. We have examined MMP and TIMP synthesis by transformed cultured pancreatic stellate cells and their regulation by TGF-β1. By Northern analysis they expressed mRNAs for procoll...

  18. Evaluating the SERCA2 and VEGF mRNAs as Potential Molecular Biomarkers of the Onset and Progression in Huntington's Disease.

    Directory of Open Access Journals (Sweden)

    Federica Cesca

    Full Text Available Abnormalities of intracellular Ca2+ homeostasis and signalling as well as the down-regulation of neurotrophic factors in several areas of the central nervous system and in peripheral tissues are hallmarks of Huntington's disease (HD. As there is no therapy for this hereditary, neurodegenerative fatal disease, further effort should be made to slow the progression of neurodegeneration in patients through the definition of early therapeutic interventions. For this purpose, molecular biomarker(s for monitoring disease onset and/or progression and response to treatment need to be identified. In the attempt to contribute to the research of peripheral candidate biomarkers in HD, we adopted a multiplex real-time PCR approach to analyse the mRNA level of targeted genes involved in the control of cellular calcium homeostasis and in neuroprotection. For this purpose we recruited a total of 110 subjects possessing the HD mutation at different clinical stages of the disease and 54 sex- and age-matched controls. This study provides evidence of reduced transcript levels of sarco-endoplasmic reticulum-associated ATP2A2 calcium pump (SERCA2 and vascular endothelial growth factor (VEGF in peripheral blood mononuclear cells (PBMCs of manifest and pre-manifest HD subjects. Our results provide a potentially new candidate molecular biomarker for monitoring the progression of this disease and contribute to understanding some early events that might have a role in triggering cellular dysfunctions in HD.

  19. Human adrenodoxin reductase: Two mRNAs encoded by a single gene on chromosome 17cen → q25 are expressed in steroidogenic tissues

    International Nuclear Information System (INIS)

    Adrenodoxin reductase is a mitochondrial flavoprotein that receives electrons from NADPH, thus initiating the electron-transport chain serving mitochondrial cytochromes P450. The authors have cloned and sequenced two human adrenodoxin reductase cDNAs that differ by the presence of six additional codons in the middle of one clone. The sequence in this region indicates that these six extra codons arise by alternative splicing of the pre-mRNA. Southern blot hybridization patterns of human genomic DNA cut with four restriction enzymes indicate that the human genome has only one gene for adrenodoxin reductase. Analysis of a panel of mouse-human somatic cell hybrids localized this gene to chromosome 17cen → q25. The alternatively spliced mRNA containing the six extra codons represents 10-20% of all adrenodoxin reductase mRNA. The expression of the adrenodoxin reductase gene may be stimulated by pituitary tropic hormones acting through cAMP, but its response is quantitatively much less than the responses of P450scc and adrenodoxin

  20. MicroRNA screen of human embryonic stem cell differentiation reveals miR-105 as an enhancer of megakaryopoiesis from adult CD34+ cells

    OpenAIRE

    Kamat, Viraj; Paluru, Prasuna; Myint, Melissa; French, Deborah L.; Gadue, Paul; Diamond, Scott L.

    2014-01-01

    MicroRNAs (miRNAs) can control stem cell differentiation by targeting mRNAs. Using 96-well plate electroporation, we screened 466 human miRNA mimics by 4-color flow cytometry to explore differentiation of common myeloid progenitors (CMP) derived from human embryonic stem cells (hESCs). The transfected cells were then cultured in a cytokine cocktail that supported multiple hematopoietic lineages. At 4-5 days post-transfection, flow cytometry of erythroid (CD235+CD41−), megakaryocyte (CD41+CD42...

  1. Gene profiling of postnatal Mfrprd6 mutant eyes reveals differential accumulation of Prss56, visual cycle and phototransduction mRNAs.

    Directory of Open Access Journals (Sweden)

    Ramani Soundararajan

    Full Text Available Mutations in the membrane frizzled-related protein (MFRP/Mfrp gene, specifically expressed in the retinal pigment epithelium (RPE and ciliary body, cause nanophthalmia or posterior microphthalmia with retinitis pigmentosa in humans, and photoreceptor degeneration in mice. To better understand MFRP function, microarray analysis was performed on eyes of homozygous Mfrprd6 and C57BL/6J mice at postnatal days (P 0 and P14, prior to photoreceptor loss. Data analysis revealed no changes at P0 but significant differences in RPE and retina-specific transcripts at P14, suggesting a postnatal influence of the Mfrprd6 allele. A subset of these transcripts was validated by quantitative real-time PCR (qRT-PCR. In Mfrprd6 eyes, a significant 1.5- to 2.0-fold decrease was observed among transcripts of genes linked to retinal degeneration, including those involved in visual cycle (Rpe65, Lrat, Rgr, phototransduction (Pde6a, Guca1b, Rgs9, and photoreceptor disc morphogenesis (Rpgrip1 and Fscn2. Levels of RPE65 were significantly decreased by 2.0-fold. Transcripts of Prss56, a gene associated with angle-closure glaucoma, posterior microphthalmia and myopia, were increased in Mfrprd6 eyes by 17-fold. Validation by qRT-PCR indicated a 3.5-, 14- and 70-fold accumulation of Prss56 transcripts relative to controls at P7, P14 and P21, respectively. This trend was not observed in other RPE or photoreceptor mutant mouse models with similar disease progression, suggesting that Prss56 upregulation is a specific attribute of the disruption of Mfrp. Prss56 and Glul in situ hybridization directly identified Müller glia in the inner nuclear layer as the cell type expressing Prss56. In summary, the Mfrprd6 allele causes significant postnatal changes in transcript and protein levels in the retina and RPE. The link between Mfrp deficiency and Prss56 up-regulation, together with the genetic association of human MFRP or PRSS56 variants and ocular size, raises the possibility that

  2. Isolation of Genes that Are Preferentially Expressed at the G1/S Boundary during the Cell Cycle in Synchronized Cultures of Catharanthus roseus Cells 1

    Science.gov (United States)

    Kodama, Hiroaki; Ito, Masaki; Hattori, Tsukaho; Nakamura, Kenzo; Komamine, Atsushi

    1991-01-01

    A cDNA library was screened for genes that may be involved in the progression of the cell cycle of cells of higher plants. The Catharanthus roseus L. (G) Don. cells were synchronized by the double phosphate starvation method, and a λgt11 cDNA library was prepared using poly(A)+ RNA from cells in the S phase of the cell cycle. Two independent sequences, cyc02 and cyc07, were identified by differential screening. The levels of cyc02 and cyc07 mRNAs increased dramatically, but transiently, at the G1/S boundary of the cell cycle. High levels of cyc02 mRNA, but not of cyc07 mRNA, were also present in cells arrested at the G1 phase by phosphate starvation. In an asynchronous batch culture, cyc02 and cyc07 mRNAs accumulated transiently at different stages of the growth cycle, cyc02 mRNA early in the stationary phase, and cyc07 mRNA in the midlogarithmic phase. When the proliferation of cells was arrested by nutrient starvation, i.e. by sucrose or nitrogen starvation, the relative amounts of the cyc02 and cyc07 mRNAs decreased. These results indicate that cyc02 and cyc07 contain nucleotide sequences from growth-related genes. The analysis of nucleotide sequence of cyc02 shows that the predicted product of this gene is basic and is composed of 101 amino acids. No significant homology to other known proteins was detected. Images Figure 1 Figure 4 Figure 5 PMID:16667998

  3. The PUF binding landscape in metazoan germ cells

    Science.gov (United States)

    Prasad, Aman; Porter, Douglas F.; Kroll-Conner, Peggy L.; Mohanty, Ipsita; Ryan, Anne R.; Crittenden, Sarah L.; Wickens, Marvin; Kimble, Judith

    2016-01-01

    PUF (Pumilio/FBF) proteins are RNA-binding proteins and conserved stem cell regulators. The Caenorhabditis elegans PUF proteins FBF-1 and FBF-2 (collectively FBF) regulate mRNAs in germ cells. Without FBF, adult germlines lose all stem cells. A major gap in our understanding of PUF proteins, including FBF, is a global view of their binding sites in their native context (i.e., their “binding landscape”). To understand the interactions underlying FBF function, we used iCLIP (individual-nucleotide resolution UV crosslinking and immunoprecipitation) to determine binding landscapes of C. elegans FBF-1 and FBF-2 in the germline tissue of intact animals. Multiple iCLIP peak-calling methods were compared to maximize identification of both established FBF binding sites and positive control target mRNAs in our iCLIP data. We discovered that FBF-1 and FBF-2 bind to RNAs through canonical as well as alternate motifs. We also analyzed crosslinking-induced mutations to map binding sites precisely and to identify key nucleotides that may be critical for FBF–RNA interactions. FBF-1 and FBF-2 can bind sites in the 5′UTR, coding region, or 3′UTR, but have a strong bias for the 3′ end of transcripts. FBF-1 and FBF-2 have strongly overlapping target profiles, including mRNAs and noncoding RNAs. From a statistically robust list of 1404 common FBF targets, 847 were previously unknown, 154 were related to cell cycle regulation, three were lincRNAs, and 335 were shared with the human PUF protein PUM2. PMID:27165521

  4. Epigallocatechin-3-gallate regulates cell growth, cell cycle and phosphorylated nuclear factor-KB in human dermal fibroblasts

    Institute of Scientific and Technical Information of China (English)

    Dong-Wook HAN; Mi Hee LEE; Hak Hee KIM; Suong-Hyu HYON; Jong-Chul PARK

    2011-01-01

    Aim: To investigate the effects of (-)epigallocatechin-3-gallate (EGCG), the main polyphenol in green tea, on cell growth, cell cycle and phosphorylated nuclear factor-kB (pNF-KB) expression in neonatal human dermal fibroblasts (nHDFs).Methods: The proliferation and cell-cycle of nHDFs were determined using WST-8 cell growth assay and flow cytometry, respectively. The apoptosis was examined using DNA ladder and Annexin V-FITC assays. The expression levels of pNF-kB and cell cycle-related genes and proteins in nHDFs were measured using cDNA microarray analyses and Western blot. The cellular uptake of EGCG was examined using fluorescence (FITC)-Iabeled EGCG (FITC-EGCG) in combination with confocal microscopy.Results: The effect of EGCG on the growth of nHDFs depended on the concentration tested. At a low concentration (200 μmol/L), EGCG resulted in a slight decrease in the proportion of ceils in the S and G/M phases of cell cycle with a concomitant increase in the proportion of cells in G/G phase. At the higher doses (400 and 800 pmol/L), apoptosis was induced. The regulation of EGCG on the expression of pNF-kB was also concentration-dependent, whereas it did not affect the unphosphorylated NF-kB expression, cDNA microarray analysis showed that cell cycle-related genes were down-regulated by EGCG (200 μmol/L). The expression of cyclins A/B and cyclin-dependent kinase 1 was reversibly regulated by EGCG (200 μmol/L). FITC-EGCG was found to be internalized into the cyto-plasm and translocated into the nucleus of nHDFs.Conclusion: EGCG, through uptake into cytoplasm, reversibly regulated the cell growth and expression of cell cycle-related proteins and genes in normal fibroblasts.

  5. Mechanism of T cell regulation by microRNAs

    International Nuclear Information System (INIS)

    MicroRNAs (miRNAs) are small, non-coding single-stranded RNAs that can modulate target gene expression at post-transcriptional level and participate in cell proliferation, differentiation, and apoptosis. T cells have important functions in acquired immune response; miRNAs regulate this immune response by targeting the mRNAs of genes involved in T cell development, proliferation, differentiation, and function. For instance, miR-181 family members function in progression by targeting Bcl2 and CD69, among others. MiR-17 to miR-92 clusters function by binding to CREB1, PTEN, and Bim. Considering that the suppression of T cell-mediated immune responses against tumor cells is involved in cancer progression, we should investigate the mechanism by which miRNA regulates T cells to develop new approaches for cancer treatment

  6. There is a steady-state transcriptome in exponentially growing yeast cells

    OpenAIRE

    Pelechano García, Vicente José; Pérez Ortín, José Enrique

    2010-01-01

    The growth of yeast cells in batches in glucose-based media is a standard condition in most yeast laboratories. Most gene expression experiments are done by taking this condition as a reference. Presumably, cells are in a stable physiological condition that can be easily reproduced in other laboratories. With this assumption, however, it is necessary to consider that the average amount of the mRNAs per cell for most genes does not change during exponential growth. That is to say, there is a s...

  7. IRES-mediated translation of utrophin A is enhanced by glucocorticoid treatment in skeletal muscle cells.

    Directory of Open Access Journals (Sweden)

    Pedro Miura

    Full Text Available Glucocorticoids are currently the only drug treatment recognized to benefit Duchenne muscular dystrophy (DMD patients. The nature of the mechanisms underlying the beneficial effects remains incompletely understood but may involve an increase in the expression of utrophin. Here, we show that treatment of myotubes with 6alpha-methylprednisolone-21 sodium succinate (PDN results in enhanced expression of utrophin A without concomitant increases in mRNA levels thereby suggesting that translational regulation contributes to the increase. In agreement with this, we show that PDN treatment of cells transfected with monocistronic reporter constructs harbouring the utrophin A 5'UTR, causes an increase in reporter protein expression while leaving levels of reporter mRNAs unchanged. Using bicistronic reporter assays, we further demonstrate that PDN enhances activity of an Internal Ribosome Entry Site (IRES located within the utrophin A 5'UTR. Analysis of polysomes demonstrate that PDN causes an overall reduction in polysome-associated mRNAs indicating that global translation rates are depressed under these conditions. Importantly, PDN causes an increase in the polysome association of endogenous utrophin A mRNAs and reporter mRNAs harbouring the utrophin A 5'UTR. Additional experiments identified a distinct region within the utrophin A 5'UTR that contains the inducible IRES activity. Together, these studies demonstrate that a translational regulatory mechanism involving increased IRES activation mediates, at least partially, the enhanced expression of utrophin A in muscle cells treated with glucocorticoids. Targeting the utrophin A IRES may thus offer an important and novel therapeutic avenue for developing drugs appropriate for DMD patients.

  8. Massive parallel gene expression profiling of RINm5F pancreatic islet beta-cells stimulated with interleukin-1beta

    DEFF Research Database (Denmark)

    Rieneck, K; Bovin, L F; Josefsen, K;

    2000-01-01

    Interleukin 1 (IL-1) is a pleiotropic cytokine with the potential to kill pancreatic beta-cells, and this unique property is thought to be involved in the pathogenesis of type I diabetes mellitus. We therefore determined the quantitative expression of 24,000 mRNAs of RINm5F, an insulinoma cell line...... derived from rat pancreatic beta-cells, before and after challenge with 30 and 1,000 pg/ml of recombinant human IL-1beta. The highest concentration resulted in decreased insulin production and cell death over a period of 4 days. Using three different time points, 2, 4 and 24 hours after challenge, we...

  9. Regulation of collagen biosynthesis in cultured bovine aortic smooth muscle cells

    International Nuclear Information System (INIS)

    Aortic smooth muscles cells have been implicated in the etiology of lesions which occur in atherosclerosis and hypertension. Both diseases involve proliferation of smooth muscle cells and accumulation of excessive amounts of extracellular matrix proteins, including collagen type I and type III produced by the smooth muscle cells. To better understand the sites of regulation of collagen biosynthesis and to correlate these with the growth rate of the cells, cultured bovine aortic smooth muscle cells were studied as a function of the number of days (3 to 14) in second passage. Cells grew rapidly up to day 6 when confluence was reached. The total incorporation of [3H]-proline into proteins was highest at day 3 and decreased to a constant level after the cultures reached confluence. In contrast, collagen protein production was lowest before confluence and continued to increase over the entire time course of the experiments. cDNA clones for the α1 and α2 chains of type I and the α1 chain of type III collagen were used to quantitate the steady state level of collagen mRNAs. RNA was tested in a cell-free translation system. Changes in the translational activity of collagen mRNAs parallelled the observed increases in collagen protein production. Thus, at later time points, collagen mRNAs are more active in directing synthesis of preprocollagens, even though less collagen mRNA is present. The conclusion is that the site of regulation of the expression of collagen genes is a function of the growth rate of cultured smooth muscle cells

  10. Poly(A) binding protein abundance regulates eukaryotic translation initiation factor 4F assembly in human cytomegalovirus-infected cells.

    Science.gov (United States)

    McKinney, Caleb; Perez, Cesar; Mohr, Ian

    2012-04-10

    By commandeering cellular translation initiation factors, or destroying those dispensable for viral mRNA translation, viruses often suppress host protein synthesis. In contrast, cellular protein synthesis proceeds in human cytomegalovirus (HCMV)-infected cells, forcing viral and cellular mRNAs to compete for limiting translation initiation factors. Curiously, inactivating the host translational repressor 4E-BP1 in HCMV-infected cells stimulates synthesis of the cellular poly(A) binding protein (PABP), significantly increasing PABP abundance. Here, we establish that new PABP synthesis is translationally controlled by the HCMV-encoded UL38 mammalian target of rapamycin complex 1-activator. The 5' UTR within the mRNA encoding PABP contains a terminal oligopyrimidine (TOP) element found in mRNAs, the translation of which is stimulated in response to mitogenic, growth, and nutritional stimuli, and proteins encoded by TOP-containing mRNAs accumulated in HCMV-infected cells. Furthermore, UL38 expression was necessary and sufficient to regulate expression of a PABP TOP-containing reporter. Remarkably, preventing the rise in PABP abundance by RNAi impaired eIF4E binding to eIF4G, thereby reducing assembly of the multisubunit initiation factor eIF4F, viral protein production, and replication. This finding demonstrates that viruses can increase host translation initiation factor concentration to foster their replication and defines a unique mechanism whereby control of PABP abundance regulates eIF4F assembly. PMID:22431630

  11. Studies of globin gene expression in differentiating erythroid cells

    International Nuclear Information System (INIS)

    The author has addressed questions concerning globin gene expression and the loss of protein synthesis in the terminal stages of erythroid development. (1) The hypothesis that the rate of cell division affects the relative synthesis of γ and β globin in erythroid cells was investigated. The effect of hydroxyurea, aminopterin, or low culture temperature on the in vitro growth of erythroid progenitor cells and on the relative synthesis of γ and β globin was measured. No consistent change in γ globin synthesis was detected. (2) The hypothesis that the ratio of γ and β globin synthesis decreases during erythroid maturation because of differential mRNA stability was investigated. The half-lives of γ and β globin mRNAs and γ and β globin protein synthesis were measured in cultured reticulocytes. γ and β globin mRNAs were assayed by solution hybridization and by in vitro translation. Globin synthesis was determined by 3H-leucine incorporation into the γ and β globin chains. γ and β globin mRNAs decay with similar half-lives in cultured reticulocytes. Therefore, the change in the ratio of γ and β globin synthesis during erythroid maturation cannot be explained by differences in mRNA stability and is likely to result from asynchronous transcription of the genes. These data suggest that protein synthesis in maturing reticulocytes is not limited by the quantity of mRNA but by the availability of translation factors. (3) The hypothesis was tested that the initiation factor GEF becomes limiting for protein synthesis during reticulocyte maturation

  12. Positive selection at codon 38 of the human KCNE1 (= minK gene and sporadic absence of 38Ser-coding mRNAs in Gly38Ser heterozygotes

    Directory of Open Access Journals (Sweden)

    Pfeufer Arne

    2009-08-01

    Full Text Available Abstract Background KCNE1 represents the regulatory beta-subunit of the slowly activating delayed rectifier potassium channel (IKs. Variants of KCNE1 have repeatedly been linked to the long-QT syndrome (LQTS, a disorder which predisposes to deafness, ventricular tachyarrhythmia, syncope, and sudden cardiac death. Results We here analyze the evolution of the common Gly38Ser variant (rs1805127, using genomic DNAs, complementary DNAs, and HEK293-expressed variants of altogether 19 mammalian species. The between species comparison reveals that the human-specific Gly38Ser polymorphism evolved under strong positive Darwinian selection, probably in adaptation to specific challenges in the fine-tuning of IKs channels. The involved amino acid exchanges (Asp > Gly, Gly > Ser are moderately radical and do not induce apparent changes in posttranslational modification. According to population genetic analyses (HapMap phase II a heterozygote advantage accounts for the maintenance of the Gly38Ser polymorphism in humans. On the other hand, the expression of the 38Ser allele seems to be disadvantageous under certain conditions, as suggested by the sporadic deficiency of 38Ser-coding mRNAs in heterozygote Central Europeans and the depletion of homozygotes 38Ser in the Yoruban sample. Conclusion We speculate that individual differences in genomic imprinting or genomic recoding might have contributed to conflicting results of recent association studies between Gly38Ser polymorphism and QT phenotype. The findings thus highlight the relevance of mRNA data in future association studies of genotypes and clinical disorders. To the best of our knowledge, they moreover provide first time evidence for a unique pattern; i.e. coincidence of positive Darwinian selection and polymorphism with a sporadically suppressed expression of one allele.

  13. Translation in cell-free systems

    International Nuclear Information System (INIS)

    The simplest, unambiguous identification of a particular mRNA is the identification of its protein product. This can be established by translation of the mRNA of interest in a cell-free protein-synthesizing system. Messenger RNA protein product identification is important in the isolation of a particular mRNA species for cDNA cloning and in the identification of positive cDNA clones. The two high-activity translation systems in common use are those prepared from rabbit reticulocytes and from wheat germ. Both systems are easy to prepare, and both are available commercially. Each has advantages and disadvantages over the other and a choice between the two will depend on the type of mRNAs to be translated, the prejudices of experience, and availability. The main disadvantage of the reticulocyte system is that it requires removal of endogenous mRNA. However, this is a relatively simple procedure. The wheat germ system does not require removal of endogenous mRNA and may translate weakly initiating mRNAs more efficiently. However, ionic optima for translation in the wheat germ system are more sensitive to the nature and concentration of mRNA and may need to be determined for each template. The biggest problem with the use of the wheat germ system is its tendency to produce incomplete translation products due to premature termination

  14. Effects of celecoxib on proliferation and tenocytic differentiation of tendon-derived stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Kairui; Zhang, Sheng [Department of Orthopaedics and Traumatology, Nanfang Hospital, Southern Medical University, Guangzhou 510515 (China); Li, Qianqian [Cancer Research Institute, Southern Medical University, Guangzhou 510515 (China); Yang, Jun [Department of Orthopaedics and Traumatology, Nanfang Hospital, Southern Medical University, Guangzhou 510515 (China); Department of Orthopaedics, 421 Hospital of PLA, Guangzhou 510318 (China); Dong, Weiqiang [Department of Orthopaedics and Traumatology, Nanfang Hospital, Southern Medical University, Guangzhou 510515 (China); Department of Orthopaedics, The First Affiliated Hospital to Guangzhou Medical University, Guangzhou 510120 (China); Wang, Shengnan; Cheng, Yirong; Al-Qwbani, Mohammed [Department of Orthopaedics and Traumatology, Nanfang Hospital, Southern Medical University, Guangzhou 510515 (China); Wang, Qiang, E-mail: 1780468505@qq.com [Department of Orthopaedics, Subei People’s Hospital of Jiangsu Province (Clinical Medical College of Yangzhou University), Yangzhou, Jiangsu Province 225001 (China); Yu, Bin, E-mail: carryzhang1985@live.com [Department of Orthopaedics and Traumatology, Nanfang Hospital, Southern Medical University, Guangzhou 510515 (China)

    2014-07-18

    Highlights: • Celecoxib has no effects on TDSCs cell proliferation in various concentrations. • Celecoxib reduced mRNAs levels of tendon associated transcription factor. • Celecoxib reduced mRNAs levels of main tendon associated collagen. • Celecoxib reduced mRNAs levels of tendon associated molecules. - Abstract: NSAIDs are often ingested to reduce the pain and improve regeneration of tendon after tendon injury. Although the effects of NSAIDs in tendon healing have been reported, the data and conclusions are not consistent. Recently, tendon-derived stem cells (TDSCs) have been isolated from tendon tissues and has been suggested involved in tendon repair. Our study aims to determine the effects of COX-2 inhibitor (celecoxib) on the proliferation and tenocytic differentiation of TDSCs. TDSCs were isolated from mice Achilles tendon and exposed to celecoxib. Cell proliferation rate was investigated at various concentrations (0.1, 1, 10 and 100 μg/ml) of celecoxib by using hemocytometer. The mRNA expression of tendon associated transcription factors, tendon associated collagens and tendon associated molecules were determined by reverse transcription-polymerase chain reaction. The protein expression of Collagen I, Collagen III, Scleraxis and Tenomodulin were determined by Western blotting. The results showed that celecoxib has no effects on TDSCs cell proliferation in various concentrations (p > 0.05). The levels of most tendon associated transcription factors, tendon associated collagens and tendon associated molecules genes expression were significantly decreased in celecoxib (10 μg/ml) treated group (p < 0.05). Collagen I, Collagen III, Scleraxis and Tenomodulin protein expression were also significantly decreased in celecoxib (10 μg/ml) treated group (p < 0.05). In conclusion, celecoxib inhibits tenocytic differentiation of tendon-derived stem cells but has no effects on cell proliferation.

  15. Effects of celecoxib on proliferation and tenocytic differentiation of tendon-derived stem cells

    International Nuclear Information System (INIS)

    Highlights: • Celecoxib has no effects on TDSCs cell proliferation in various concentrations. • Celecoxib reduced mRNAs levels of tendon associated transcription factor. • Celecoxib reduced mRNAs levels of main tendon associated collagen. • Celecoxib reduced mRNAs levels of tendon associated molecules. - Abstract: NSAIDs are often ingested to reduce the pain and improve regeneration of tendon after tendon injury. Although the effects of NSAIDs in tendon healing have been reported, the data and conclusions are not consistent. Recently, tendon-derived stem cells (TDSCs) have been isolated from tendon tissues and has been suggested involved in tendon repair. Our study aims to determine the effects of COX-2 inhibitor (celecoxib) on the proliferation and tenocytic differentiation of TDSCs. TDSCs were isolated from mice Achilles tendon and exposed to celecoxib. Cell proliferation rate was investigated at various concentrations (0.1, 1, 10 and 100 μg/ml) of celecoxib by using hemocytometer. The mRNA expression of tendon associated transcription factors, tendon associated collagens and tendon associated molecules were determined by reverse transcription-polymerase chain reaction. The protein expression of Collagen I, Collagen III, Scleraxis and Tenomodulin were determined by Western blotting. The results showed that celecoxib has no effects on TDSCs cell proliferation in various concentrations (p > 0.05). The levels of most tendon associated transcription factors, tendon associated collagens and tendon associated molecules genes expression were significantly decreased in celecoxib (10 μg/ml) treated group (p < 0.05). Collagen I, Collagen III, Scleraxis and Tenomodulin protein expression were also significantly decreased in celecoxib (10 μg/ml) treated group (p < 0.05). In conclusion, celecoxib inhibits tenocytic differentiation of tendon-derived stem cells but has no effects on cell proliferation

  16. Regulation of histone gene expression during the cell cycle.

    Science.gov (United States)

    Meshi, T; Taoka, K I; Iwabuchi, M

    2000-08-01

    The steady-state level of histone mRNAs fluctuates coordinately with chromosomal DNA synthesis during the cell cycle. Such an S phase-specific expression pattern results from transcriptional activation of histone genes coupled with the onset of replication and from transcriptional repression of the genes as well as specific destabilization of histone mRNAs around the end of the S phase. Proliferation-coupled and S phase-specific expression of histone genes is primarily achieved by the activities of the proximal promoter regions, where several conserved cis-acting elements have been identified. Among them, three kinds of Oct-containing composite elements (OCEs) play a pivotal role in S phase-specific transcriptional activation. Other ones, such as Nona, solo-Oct, and CCGTC motifs, appear to modulate the functions of OCEs to enhance or repress the transcriptional level, possibly depending on the state of the cells. Here, we review the growing evidence concerning the regulatory mechanisms by which plant histone genes are expressed S phase-specifically in proliferating cells. PMID:11089867

  17. Effect of polo-like kinase 1 gene silence on cell cycle and drug resistance in K562/A02 cell

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    @@ Polo-like kinase 1(PLK1) plays an important role in many cell-cycle-related events.1 At G2/M transition, PLK1 contributes to the activation of cyclinB/Cdc by phosphorylation of Cdc25C, centrosome functional maturation, bipolar spindle formation. In later stage of mitosis, PLK1 is involved in regulating components of the anaphase-promoting complex (APC) for mitotic exit and in the execution of cytokinesis.

  18. Peptidomic discovery of short open reading frame-encoded peptides in human cells

    OpenAIRE

    Slavoff, Sarah A.; Mitchell, Andrew J.; Schwaid, Adam G.; Cabili, Moran N; Ma, Jiao; Levin, Joshua Z.; Karger, Amir; Budnik, Bogdan A.; Rinn, John L; Saghatelian, Alan

    2012-01-01

    The amount of the transcriptome that is translated into polypeptides is of fundamental importance. We developed a peptidomic strategy to detect short ORF (sORF)-encoded polypeptides (SEPs) in human cells. We identified 90 SEPs, 86 of which are novel, the largest number of human SEPs ever reported. SEP abundances range from 10-1000 molecules per cell, identical to known proteins. SEPs arise from sORFs in non-coding RNAs as well as multi-cistronic mRNAs, and many SEPs initiate with non-AUG star...

  19. UV-induction of chalcone synthase mRNA in cell suspension cultures of Petroselinum hortense

    OpenAIRE

    Kreuzaler, Fritz; Ragg, Hermann; Fautz, Erich; David N Kuhn; Hahlbrock, Klaus

    1983-01-01

    DNAs complementary to poly(A)+ mRNAs from UV-irradiated cell suspension cultures of parsley (Petroselinum hortense) were inserted into pBR322 and used to transform Escherichia coli strain RR1. A clone containing a DNA complementary to chalcone synthase mRNA was identified by hybrid-selected and hybrid-arrested translation. Large and rapid changes in the amount of chalcone synthase mRNA in response to irradiation of the cells was detected by RNA blot hybridization experiments. The pattern of c...

  20. Enforcing temporal control of maternal mRNA translation during oocyte cell-cycle progression

    OpenAIRE

    Arumugam, Karthik; Wang, Yiying; Hardy, Linda L.; MacNicol, Melanie C.; MacNicol, Angus M

    2009-01-01

    Meiotic cell-cycle progression in progesterone-stimulated Xenopus oocytes requires that the translation of pre-existing maternal mRNAs occur in a strict temporal order. Timing of translation is regulated through elements within the mRNA 3′ untranslated region (3′ UTR), which respond to cell cycle-dependant signalling. One element that has been previously implicated in the temporal control of mRNA translation is the cytoplasmic polyadenylation element (CPE). In this study, we show that the CPE...

  1. Downregulation of HuR as a new mechanism of doxorubicin resistance in breast cancer cells

    OpenAIRE

    Latorre Elisa; Tebaldi Toma; Viero Gabriella; Spartà Antonino; Quattrone Alessandro; Provenzani Alessandro

    2012-01-01

    Abstract Background HuR, an RNA binding protein involved in the post-transcriptional regulation of a wide spectrum of mRNAs, has been demonstrated to be a determinant of carcinogenesis and tumor aggressiveness in several cancer types. In this study, we investigated the role of HuR in the apoptosis and in the chemoresistance induced by the widely used anticancer drug doxorubicin in human breast cancer cells (MCF-7). Results We showed that HuR acts in the early phase of cell response to doxorub...

  2. EIF3 p170, a Mediator of Mimosine Effect on Protein Synthesis and Cell Cycle Progression

    OpenAIRE

    Dong, Zizheng; Zhang, Jian-Ting

    2003-01-01

    l-Mimosine, a plant amino acid, can reversibly block mammalian cells at late G1 phase and has been suggested to affect translation of mRNAs such as p27, the CDK inhibitor. However, the mechanism of this effect is not known. Regulation of translation generally occurs at the initiation step that, in mammalian cells, is a complex process that requires multiple eukaryotic initiation factors (eIFs) and ribosome. The effects of mimosine on initiation factors or regulators consequently will influenc...

  3. The IRE1α/XBP1s Pathway Is Essential for the Glucose Response and Protection of β Cells

    Science.gov (United States)

    Hassler, Justin R.; Scheuner, Donalyn L.; Wang, Shiyu; Han, Jaeseok; Kodali, Vamsi K.; Li, Philip; Nguyen, Julie; George, Jenny S.; Davis, Cory; Wu, Shengyang P.; Bai, Yongsheng; Sartor, Maureen; Cavalcoli, James; Malhi, Harmeet; Baudouin, Gregory; Zhang, Yaoyang; Yates III, John R.; Itkin-Ansari, Pamela; Volkmann, Niels; Kaufman, Randal J.

    2015-01-01

    Although glucose uniquely stimulates proinsulin biosynthesis in β cells, surprisingly little is known of the underlying mechanism(s). Here, we demonstrate that glucose activates the unfolded protein response transducer inositol-requiring enzyme 1 alpha (IRE1α) to initiate X-box-binding protein 1 (Xbp1) mRNA splicing in adult primary β cells. Using mRNA sequencing (mRNA-Seq), we show that unconventional Xbp1 mRNA splicing is required to increase and decrease the expression of several hundred mRNAs encoding functions that expand the protein secretory capacity for increased insulin production and protect from oxidative damage, respectively. At 2 wk after tamoxifen-mediated Ire1α deletion, mice develop hyperglycemia and hypoinsulinemia, due to defective β cell function that was exacerbated upon feeding and glucose stimulation. Although previous reports suggest IRE1α degrades insulin mRNAs, Ire1α deletion did not alter insulin mRNA expression either in the presence or absence of glucose stimulation. Instead, β cell failure upon Ire1α deletion was primarily due to reduced proinsulin mRNA translation primarily because of defective glucose-stimulated induction of a dozen genes required for the signal recognition particle (SRP), SRP receptors, the translocon, the signal peptidase complex, and over 100 other genes with many other intracellular functions. In contrast, Ire1α deletion in β cells increased the expression of over 300 mRNAs encoding functions that cause inflammation and oxidative stress, yet only a few of these accumulated during high glucose. Antioxidant treatment significantly reduced glucose intolerance and markers of inflammation and oxidative stress in mice with β cell-specific Ire1α deletion. The results demonstrate that glucose activates IRE1α-mediated Xbp1 splicing to expand the secretory capacity of the β cell for increased proinsulin synthesis and to limit oxidative stress that leads to β cell failure. PMID:26469762

  4. The IRE1α/XBP1s Pathway Is Essential for the Glucose Response and Protection of β Cells.

    Directory of Open Access Journals (Sweden)

    Justin R Hassler

    2015-10-01

    Full Text Available Although glucose uniquely stimulates proinsulin biosynthesis in β cells, surprisingly little is known of the underlying mechanism(s. Here, we demonstrate that glucose activates the unfolded protein response transducer inositol-requiring enzyme 1 alpha (IRE1α to initiate X-box-binding protein 1 (Xbp1 mRNA splicing in adult primary β cells. Using mRNA sequencing (mRNA-Seq, we show that unconventional Xbp1 mRNA splicing is required to increase and decrease the expression of several hundred mRNAs encoding functions that expand the protein secretory capacity for increased insulin production and protect from oxidative damage, respectively. At 2 wk after tamoxifen-mediated Ire1α deletion, mice develop hyperglycemia and hypoinsulinemia, due to defective β cell function that was exacerbated upon feeding and glucose stimulation. Although previous reports suggest IRE1α degrades insulin mRNAs, Ire1α deletion did not alter insulin mRNA expression either in the presence or absence of glucose stimulation. Instead, β cell failure upon Ire1α deletion was primarily due to reduced proinsulin mRNA translation primarily because of defective glucose-stimulated induction of a dozen genes required for the signal recognition particle (SRP, SRP receptors, the translocon, the signal peptidase complex, and over 100 other genes with many other intracellular functions. In contrast, Ire1α deletion in β cells increased the expression of over 300 mRNAs encoding functions that cause inflammation and oxidative stress, yet only a few of these accumulated during high glucose. Antioxidant treatment significantly reduced glucose intolerance and markers of inflammation and oxidative stress in mice with β cell-specific Ire1α deletion. The results demonstrate that glucose activates IRE1α-mediated Xbp1 splicing to expand the secretory capacity of the β cell for increased proinsulin synthesis and to limit oxidative stress that leads to β cell failure.

  5. Simultaneous detection of mRNA and protein stem cell markers in live cells

    Directory of Open Access Journals (Sweden)

    Bao Gang

    2009-04-01

    Full Text Available Abstract Background Biological studies and medical application of stem cells often require the isolation of stem cells from a mixed cell population, including the detection of cancer stem cells in tumor tissue, and isolation of induced pluripotent stem cells after eliciting the expression of specific genes in adult cells. Here we report the detection of Oct-4 mRNA and SSEA-1 protein in live carcinoma stem cells using respectively molecular beacon and dye-labeled antibody, aiming to establish a new method for stem cells detection and isolation. Results Quantification of Oct-4 mRNA and protein in P19 mouse carcinoma stem cells using respectively RT-PCR and immunocytochemistry confirmed that their levels drastically decreased after differentiation. To visualize Oct-4 mRNA in live stem cells, molecular beacons were designed, synthesized and validated, and the detection specificity was confirmed using control studies. We found that the fluorescence signal from Oct-4-targeting molecular beacons provides a clear discrimination between undifferentiated and retinoic acid-induced differentiated cells. Using deconvolution fluorescence microscopy, Oct-4 mRNAs were found to reside on one side of the cytosol. We demonstrated that, using a combination of Oct-4 mRNA-targeting molecular beacon with SSEA-1 antibody in flow cytometric analysis, undifferentiated stem cells can be clearly distinguished from differentiated cells. We revealed that Oct-4 targeting molecular beacons do not seem to affect stem cell biology. Conclusion Molecular beacons have the potential to provide a powerful tool for highly specific detection and isolation of stem cells, including cancer stem cells and induced pluripotent stem (iPS cells without disturbing cell physiology. It is advantageous to perform simultaneous detection of intracellular (mRNA and cell-surface (protein stem cell markers in flow cytometric analysis, which may lead to high detection sensitivity and efficiency.

  6. Epithelial Cell Transforming 2 and Aurora Kinase B Modulate Formation of Stress Granule-Containing Transcripts from Diverse Cellular Pathways in Astrocytoma Cells.

    Science.gov (United States)

    Weeks, Adrienne; Agnihotri, Sameer; Lymer, Jennifer; Chalil, Alan; Diaz, Roberto; Isik, Semra; Smith, Christian; Rutka, James T

    2016-06-01

    Stress granules are small RNA-protein granules that modify the translational landscape during cellular stress to promote survival. The RhoGTPase RhoA is implicated in the formation of RNA stress granules. Our data demonstrate that the cytokinetic proteins epithelial cell transforming 2 and Aurora kinase B (AurkB) are localized to stress granules in human astrocytoma cells. AurkB and its downstream target histone-3 are phosphorylated during arsenite-induced stress. Chemical (AZD1152-HQPA) and siRNA inhibition of AurkB results in fewer and smaller stress granules when analyzed using high-throughput fluorescent-based cellomics assays. RNA immunoprecipitation with the known stress granule aggregates TIAR and G3BP1 was performed on astrocytoma cells, and subsequent analysis revealed that astrocytoma stress granules harbor unique mRNAs for various cellular pathways, including cellular migration, metabolism, translation, and transcriptional regulation. Human astrocytoma cell stress granules contain mRNAs that are known to be involved in glioma signaling and the mammalian target of rapamycin pathway. These data provide evidence that RNA stress granules are a novel form of epigenetic regulation in astrocytoma cells, which may be targetable by chemical inhibitors and enhance astrocytoma susceptibility to conventional therapy, such as radiation and chemotherapy. PMID:27106762

  7. The increased number of Leydig cells by di(2-ethylhexyl) phthalate comes from the differentiation of stem cells into Leydig cell lineage in the adult rat testis

    International Nuclear Information System (INIS)

    Highlights: ► DEHP increases rat Leydig cell number. ► DEHP induces the proliferation of stem Leydig cells. ► DEHP induces the formation of progenitor Leydig cells. - Abstract: The objective of the present study is to determine whether di(2-ethylhexyl) phthalate (DEHP) exposure at adulthood increases rat Leydig cell number and to investigate the possible mechanism. 90-day-old Long–Evans rats were randomly divided into 3 groups, and were gavaged with the corn oil (control) or 10 or 750 mg/kg DEHP daily for 7 days, and then received an intraperitoneal injection of 75 mg/kg ethane dimethanesulfonate (EDS) to eliminate Leydig cells. Serum testosterone concentrations were assessed by RIA, and the mRNA levels of Leydig cell genes were measured by qPCR. EDS eliminated all Leydig cells in the control testis on day 4 post-EDS, as judged by undetectable serum testosterone level and no 3β-hydroxysteroid dehydrogenase positive (3β-HSDpos) cells in the interstitium. However, in DEHP-treated groups, there were detectable serum testosterone concentrations and some oval-shaped 3β-HSDpos cells in the interstitium. These 3β-HSDpos cells were not stained by the antibody against 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1), a marker for Leydig cells at a more advanced stage. The disappearance of mRNAs of Leydig cell biomarkers including Lhcgr, Cyp11a1, Cyp17a1, Insl3 and Hsd11b1 in the control testis was observed on day 4 post-EDS. However, there were detectable concentrations of Lhcgr, Cyp11a1 and Cyp17a1 mRNAs but undetectable concentrations of Insl3, Hsd17b3 and Hsd11b1 in the DEHP-treated testes, indicating that these 3β-HSDpos cells were newly formed progenitor Leydig cells. The mRNA level for nestin (Nes, biomarker for stem Leydig cells) was significantly increased in the control testis on day 4 post-EDS, but not in the DEHP treated testes, suggesting that these nestin positive stem cells were differentiated into progenitor Leydig cells in the DEHP-treated testes

  8. Alterations of gene profiles in Leydig-cell-regenerating adult rat testis after ethane dimethane sulfonate-treatment

    Directory of Open Access Journals (Sweden)

    Yu-Fei Zhang

    2015-04-01

    Full Text Available Only occupying about 1%-5% of total testicular cells, the adult Leydig cell (ALC is a unique endocrine cell that produces androgens. Rat Leydig cells regenerate after these cells in the testis are eliminated with ethane dimethane sulfonate (EDS. In this study, we have characterized Leydig cell regeneration and messenger ribonucleic acids (mRNA profiles of EDS treated rat testes. Serum testosterone, testicular gene profiling and some steroidogenesis-related proteins were analyzed at 7, 21, 35 and 90 days after EDS treatment. Testicular testosterone levels declined to undetectable levels until 7 days after treatment and then started to recover. Seven days after treatment, 81 mRNAs were down-regulated greater than or equal to two-fold, with 48 becoming undetectable. These genes increased their expression 21 days and completely returned to normal levels 90 days after treatment. The undetectable genes include steroidogenic pathway proteins: steroidogenic acute regulatory protein, Scarb1, Cyp11a1, Cyp17a1, Hsd3b1, Cyp1b1 and Cyp2a1. Seven days after treatment, there were 89 mRNAs up-regulated two-fold or more including Pkib. These up-regulated mRNAs returned to normal 90 days after treatment. Cyp2a1 did not start to recover until 35 days after treatment, indicating that this gene is only expressed in ALCs not in the precursor cells. Quantitative polymerase chain reaction, western blotting and semi-quantitative immunohistochemical staining using tissue array confirmed the changes of several randomly picked genes and their proteins.

  9. Re-induction of cell differentiation and 131I uptake in dedifferentiated FTC-133 cell line by TSHR gene transfection

    International Nuclear Information System (INIS)

    Introduction: Radioiodine therapy is commonly used to treat differentiated thyroid cancer (DTC), but a major challenge is dedifferentiation of DTC with the loss of radioiodine uptake. TSHR is a key molecule regulating thyrocyte proliferation and function. This study aimed to test the therapeutic potential of TSHR in dedifferentiated DTC by gene transfection in order to restore cell differentiation and radioiodine uptake. Methods: Dedifferentiated FTC-133 (dFTC-133) cells were obtained by monoclonal culture of FTC-133 cell line after 131I radiation. Recombinant plasmid pcDNA3.1-hTSHR was transfected into dFTC-133 cells by using Lipofectamine 2000 reagent. Immunofluorescence analysis was carried out to confirm TSHR expression and its location. Radioiodine uptake assay was thereafter investigated. mRNAs and proteins of TSHR and other thyroid differentiated markers were detected by real-time PCR and western blot respectively. Results: Among the thyroid specific genes in dFTC-133 cells with stable low radioiodine uptake, TSHR was down-regulated most significantly compared with FTC-133. Then, after TSHR gene transfection, augmented expression of TSHR was observed in dFTC-133 cell surface and cytoplasm by immunofluorescence analysis. It was found that 125I uptake was 2.9 times higher (t = 28.63, P < .01) in cells with TSHR transfection than control. The mRNAs of TSHR, NIS, TPO and Tg were also significantly increased by 1.7 times (t = 13.8, P < .05), 4 times (t = 28.52, P < .05), 1.5 times (t = 14.43, P < .05) and 2.2 times (t = 19.83, P < .05) respectively compared with control group. Conclusion: Decreased TSHR expression correlated with FTC-133 ongoing dedifferentiation. TSHR transfection contributed to the re-differentiation of dedifferentiated thyroid follicular carcinoma cells.

  10. Exosomes released from breast cancer carcinomas stimulate cell movement.

    Directory of Open Access Journals (Sweden)

    Dinari A Harris

    Full Text Available For metastasis to occur cells must communicate with to their local environment to initiate growth and invasion. Exosomes have emerged as an important mediator of cell-to-cell signalling through the transfer of molecules such as mRNAs, microRNAs, and proteins between cells. Exosomes have been proposed to act as regulators of cancer progression. Here, we study the effect of exosomes on cell migration, an important step in metastasis. We performed cell migration assays, endocytosis assays, and exosome proteomic profiling on exosomes released from three breast cancer cell lines that model progressive stages of metastasis. Results from these experiments suggest: (1 exosomes promote cell migration and (2 the signal is stronger from exosomes isolated from cells with higher metastatic potentials; (3 exosomes are endocytosed at the same rate regardless of the cell type; (4 exosomes released from cells show differential enrichment of proteins with unique protein signatures of both identity and abundance. We conclude that breast cancer cells of increasing metastatic potential secrete exosomes with distinct protein signatures that proportionally increase cell movement and suggest that released exosomes could play an active role in metastasis.

  11. Cytosolic DNA Sensor Upregulation Accompanies DNA Electrotransfer in B16.F10 Melanoma Cells.

    Science.gov (United States)

    Znidar, Katarina; Bosnjak, Masa; Cemazar, Maja; Heller, Loree C

    2016-01-01

    In several preclinical tumor models, antitumor effects occur after intratumoral electroporation, also known as electrotransfer, of plasmid DNA devoid of a therapeutic gene. In mouse melanomas, these effects are preceded by significant elevation of several proinflammatory cytokines. These observations implicate the binding and activation of intracellular DNA-specific pattern recognition receptors or DNA sensors in response to DNA electrotransfer. In tumors, IFNβ mRNA and protein levels significantly increased. The mRNAs of several DNA sensors were detected, and DAI, DDX60, and p204 tended to be upregulated. These effects were accompanied with reduced tumor growth and increased tumor necrosis. In B16.F10 cells in culture, IFNβ mRNA and protein levels were significantly upregulated. The mRNAs for several DNA sensors were present in these cells; DNA-dependent activator of interferon regulatory factor (DAI), DEAD (Asp-Glu-Ala-Asp) box polypeptide 60 (DDX60), and p204 were significantly upregulated while DDX60 protein levels were coordinately upregulated. Upregulation of DNA sensors in tumors could be masked by the lower transfection efficiency compared to in vitro or to dilution by other tumor cell types. Mirroring the observation of tumor necrosis, cells underwent a significant DNA concentration-dependent decrease in proliferation and survival. Taken together, these results indicate that DNA electrotransfer may cause the upregulation of several intracellular DNA sensors in B16.F10 cells, inducing effects in vitro and potentially in vivo. PMID:27271988

  12. The Cytotoxic Role of Intermittent High Glucose on Apoptosis and Cell Viability in Pancreatic Beta Cells

    Directory of Open Access Journals (Sweden)

    Zhen Zhang

    2014-01-01

    Full Text Available Objectives. Glucose fluctuations are both strong predictor of diabetic complications and crucial factor for beta cell damages. Here we investigated the effect of intermittent high glucose (IHG on both cell apoptosis and proliferation activity in INS-1 cells and the potential mechanisms. Methods. Cells were treated with normal glucose (5.5 mmol/L, constant high glucose (CHG (25 mmol/L, and IHG (rotation per 24 h in 11.1 or 25 mmol/L for 7 days. Reactive oxygen species (ROS, xanthine oxidase (XOD level, apoptosis, cell viability, cell cycle, and expression of cyclinD1, p21, p27, and Skp2 were determined. Results. We found that IHG induced more significant apoptosis than CHG and normal glucose; intracellular ROS and XOD levels were more markedly increased in cells exposed to IHG. Cells treated with IHG showed significant decreased cell viability and increased cell proportion in G0/G1 phase. Cell cycle related proteins such as cyclinD1 and Skp2 were decreased significantly, but expressions of p27 and p21 were increased markedly. Conclusions. This study suggested that IHG plays a more toxic effect including both apoptosis-inducing and antiproliferative effects on INS-1 cells. Excessive activation of cellular stress and regulation of cyclins might be potential mechanism of impairment in INS-1 cells induced by IHG.

  13. An Envelope Glycoprotein of the Human Endogenous Retrovirus HERV-W Is Expressed in the Human Placenta and Fuses Cells Expressing the Type D Mammalian Retrovirus Receptor

    OpenAIRE

    Blond, Jean-Luc; Lavillette, Dimitri; Cheynet, Valérie; Bouton, Olivier; Oriol, Guy; Chapel-Fernandes, Sylvie; Mandrand, Bernard; Mallet, François; Cosset, François-Loïc

    2000-01-01

    A new human endogenous retrovirus (HERV) family, termed HERV-W, was recently described (J.-L. Blond, F. Besème, L. Duret, O. Bouton, F. Bedin, H. Perron, B. Mandrand, and F. Mallet, J. Virol. 73:1175–1185, 1999). HERV-W mRNAs were found to be specifically expressed in placenta cells, and an env cDNA containing a complete open reading frame was recovered. In cell-cell fusion assays, we demonstrate here that the product of the HERV-W env gene is a highly fusogenic membrane glycoprotein. Transfe...

  14. 78-kilodalton glucose-regulated protein is induced in Rous sarcoma virus-transformed cells independently of glucose deprivation.

    OpenAIRE

    Stoeckle, M Y; Sugano, S; Hampe, A; Vashistha, A; Pellman, D.; Hanafusa, H

    1988-01-01

    To identify mRNAs with altered expression in Rous sarcoma virus (RSV)-transformed cells, we screened a chicken embryo fibroblast (CEF) cDNA library by differential hybridization. One clone, designated R1H, showed markedly elevated mRNA expression in RSV-transformed cells. Nucleotide sequence analysis indicated that R1H mRNA encodes 78-kilodalton glucose-regulated protein (GRP78). Chicken GRP78 was found to be very highly conserved in comparison with rat GRP78 (96% identity between chicken and...

  15. FRTL-5 Rat Thyroid Cells Release Thyroglobulin Sequestered in Exosomes: A Possible Novel Mechanism for Thyroglobulin Processing in the Thyroid

    OpenAIRE

    Vlasov, Pavel; Doi, Sonia Q.; Sellitti, Donald F.

    2016-01-01

    Exosomes are 30–100 nm, membrane-bound vesicles containing specific cellular proteins, mRNAs, and microRNAs that take part in intercellular communication between cells. A possible role for exosomes in thyroid function has not been fully explored. In the present study, FRTL-5 rat thyroid cells were grown to confluence and received medium containing either thyroid stimulating hormone (TSH), exogenous bovine thyroglobulin (bTg), or neither additive for 24 or 48 hours followed by collection of sp...

  16. Human apolipoprotein B (apoB) mRNA: Identification of two distinct apoB mRNAs, an mRNA with the apoB-100 sequence and an apoB mRNA containing a premature in-frame translational stop codon, in both liver and intestine

    International Nuclear Information System (INIS)

    Human apolipoprotein B (apoB) is present in plasma as two separate isoproteins, designated apoB-100 (512 kDa) and apoB-48 (250 kDa). ApoB is encoded by a single gene on chromosome 2, and a single nuclear mRNA is edited and processed into two separate apoB mRNAs. A 14.1-kilobase apoB mRNA codes for apoB-100, and the second mRNA, which codes for apoB-48, contains a premature stop codon generated by a single base substitution of cytosine to uracil at nucleotide 6,538, which converts the translated CAA codon coding for the amino acid glutamine at residue 2,153 in apoB-100 to a premature in-frame stop codon (UAA). Two 30-base synthetic oligonucleotides, designated apoB-Stop and apoB-Gln, were synthesized containing the complementary sequence to the stop codon (UAA) and glutamine codon (CAA), respectively. The combined results from these studies establish that both human intestine and liver contain the two distinct apoB mRNAs, an mRNA that codes for apoB-100 and an apoB mRNA that contains the premature stop codon, which codes for apoB-48. The premature in-frame stop codon is not tissue specific and is present in both human liver and intestine

  17. Repression of Pumilio Protein Expression by Rbfox1 Promotes Germ Cell Differentiation.

    Science.gov (United States)

    Carreira-Rosario, Arnaldo; Bhargava, Varsha; Hillebrand, Jens; Kollipara, Rahul K; Ramaswami, Mani; Buszczak, Michael

    2016-03-01

    RNA-binding Fox (Rbfox) proteins have well-established roles in regulating alternative splicing, but specific Rbfox isoforms lack nuclear localization signals and accumulate in the cytoplasm. The potential splicing-independent functions of these proteins remain unknown. Here we demonstrate that cytoplasmic Drosophila Rbfox1 regulates germ cell development and represses the translation of mRNAs containing (U)GCAUG elements within their 3'UTRs. During germline cyst differentiation, Rbfox1 targets pumilio mRNA for destabilization and translational silencing, thereby promoting germ cell development. Mis-expression of pumilio results in the formation of germline tumors, which contain cysts that break down and dedifferentiate back to single, mitotically active cells. Together, these results reveal that cytoplasmic Rbfox family members regulate the translation of specific target mRNAs. In the Drosophila ovary, this activity provides a genetic barrier that prevents germ cells from reverting back to an earlier developmental state. The finding that Rbfox proteins regulate mRNA translation has implications for Rbfox-related diseases. PMID:26954550

  18. Widespread uncoupling between transcriptome and translatome variations after a stimulus in mammalian cells

    Directory of Open Access Journals (Sweden)

    Tebaldi Toma

    2012-06-01

    Full Text Available Abstract Background The classical view on eukaryotic gene expression proposes the scheme of a forward flow for which fluctuations in mRNA levels upon a stimulus contribute to determine variations in mRNA availability for translation. Here we address this issue by simultaneously profiling with microarrays the total mRNAs (the transcriptome and the polysome-associated mRNAs (the translatome after EGF treatment of human cells, and extending the analysis to other 19 different transcriptome/translatome comparisons in mammalian cells following different stimuli or undergoing cell programs. Results Triggering of the EGF pathway results in an early induction of transcriptome and translatome changes, but 90% of the significant variation is limited to the translatome and the degree of concordant changes is less than 5%. The survey of other 19 different transcriptome/translatome comparisons shows that extensive uncoupling is a general rule, in terms of both RNA movements and inferred cell activities, with a strong tendency of translation-related genes to be controlled purely at the translational level. By different statistical approaches, we finally provide evidence of the lack of dependence between changes at the transcriptome and translatome levels. Conclusions We propose a model of diffused independency between variation in transcript abundances and variation in their engagement on polysomes, which implies the existence of specific mechanisms to couple these two ways of regulating gene expression.

  19. Elevation of soluble guanylate cyclase suppresses proliferation and survival of human breast cancer cells.

    Directory of Open Access Journals (Sweden)

    Hui-Chin Wen

    Full Text Available Nitric oxide (NO is an essential signaling molecule in biological systems. Soluble guanylate cyclase (sGC, composing of α1 and β1 subunit, is the receptor for NO. Using radioimmunoassay, we discovered that activation of sGC by treatment with bradykinin or sodium nitroprusside (SNP is impaired in MCF-7 and MDA-MB-231 breast cancer cells as compared to normal breast epithelial 184A1 cells. The 184A1 cells expressed both sGC α1 and sGCβ1 mRNAs. However, levels of sGCβ1 mRNAs were relatively lower in MCF-7 cells while both mRNA of sGC subunits were absent in MDA-MB-231 cells. Treatment with DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-dC increased mRNA levels of both sGCα1 and sGCβ1 in MDA-MB-231 cells but only sGCβ1 mRNAs in MCF-7 cells. The 5-aza-dC treatment increased the SNP-induced cGMP production in MCF-7 and MDA-MB-231, but not in 184A1 cells. Bisulfite sequencing revealed that the promoter of sGCα1 in MDA-MB-231 cells and promoter of sGCβ1 in MCF-7 cells were methylated. Promoter hypermethylation of sGCα1 and sGCβ1 was found in 1 out of 10 breast cancer patients. Over-expression of both sGC subunits in MDA-MB-231 cells induced apoptosis and growth inhibition in vitro as well as reduced tumor incidence and tumor growth rate of MDA-MB-231 xenografts in nude mice. Elevation of sGC reduced protein abundance of Bcl-2, Bcl-xL, Cdc2, Cdc25A, Cyclin B1, Cyclin D1, Cdk6, c-Myc, and Skp2 while increased protein expression of p53. Our study demonstrated that down-regulation of sGC, partially due to promoter methylation, provides growth and survival advantage in human breast cancer cells.

  20. Differentiation of human embryonic stem cells into cells with corneal keratocyte phenotype.

    Directory of Open Access Journals (Sweden)

    Audrey A Chan

    Full Text Available Corneal transparency depends on a unique extracellular matrix secreted by stromal keratocytes, mesenchymal cells of neural crest lineage. Derivation of keratocytes from human embryonic stem (hES cells could elucidate the keratocyte developmental pathway and open a potential for cell-based therapy for corneal blindness. This study seeks to identify conditions inducing differentiation of pluripotent hES cells to the keratocyte lineage. Neural differentiation of hES cell line WA01(H1 was induced by co-culture with mouse PA6 fibroblasts. After 6 days of co-culture, hES cells expressing cell-surface NGFR protein (CD271, p75NTR were isolated by immunoaffinity adsorption, and cultured as a monolayer for one week. Keratocyte phenotype was induced by substratum-independent pellet culture in serum-free medium containing ascorbate. Gene expression, examined by quantitative RT-PCR, found hES cells co-cultured with PA6 cells for 6 days to upregulate expression of neural crest genes including NGFR, SNAI1, NTRK3, SOX9, and MSX1. Isolated NGFR-expressing cells were free of PA6 feeder cells. After expansion as a monolayer, mRNAs typifying adult stromal stem cells were detected, including BMI1, KIT, NES, NOTCH1, and SIX2. When these cells were cultured as substratum-free pellets keratocyte markers AQP1, B3GNT7, PTDGS, and ALDH3A1 were upregulated. mRNA for keratocan (KERA, a cornea-specific proteoglycan, was upregulated more than 10,000 fold. Culture medium from pellets contained high molecular weight keratocan modified with keratan sulfate, a unique molecular component of corneal stroma. These results show hES cells can be induced to differentiate into keratocytes in vitro. Pluripotent stem cells, therefore, may provide a renewable source of material for development of treatment of corneal stromal opacities.

  1. Nudt3 is an mRNA decapping enzyme that modulates cell migration.

    Science.gov (United States)

    Grudzien-Nogalska, Ewa; Jiao, Xinfu; Song, Man-Gen; Hart, Ronald P; Kiledjian, Megerditch

    2016-05-01

    Removal of the 5'-end 7-methylguanosine cap structure is a critical step in the highly regulated process of mRNA decay. The Nudix hydrolase, Dcp2, was identified as a first decapping enzyme and subsequently shown to preferentially modulate stability of only a subset of mRNAs. This observation led to the hypothesis that mammalian cells possess multiple decapping enzymes that may function in distinct pathways. Here we report Nudt3 is a Nudix protein that possesses mRNA decapping activity in cells and is a modulator of MCF-7 breast cancer cell migration. Reduction of Nudt3 protein levels in MCF-7 cells promotes increased cell migration and corresponding enhanced filopodia extensions. Importantly, this phenotype was reversed by complementation with wild type, but not catalytically inactive Nudt3 protein indicating Nudt3 decapping activity normally functions to control cell migration. Genome-wide analysis of Nudt3 compromised cells identified elevated levels of transcripts involved in cell motility including integrin β6, lipocalin-2, and fibronectin. The observed increase in mRNA abundance was dependent on Nudt3 decapping activity where integrin β6 and lipocalin-2 were modulated directly through mRNA stability, while fibronectin was indirectly controlled. Moreover, increased cell migration observed in Nudt3 knockdown cells was mediated through the extracellular integrin β6 and fibronectin protein nexus. We conclude that Nudt3 is an mRNA decapping enzyme that orchestrates expression of a subset of mRNAs to modulate cell migration and further substantiates the existence of multiple decapping enzymes functioning in distinct cellular pathways in mammals. PMID:26932476

  2. Overexpression of amyloid precursor protein inhibits neurite outgrowth and disrupts cytoskeleton in N2a cells

    Institute of Scientific and Technical Information of China (English)

    王泽芬; 王建枝

    2004-01-01

    @@ There is considerable evidence suggesting that altered metabolism of β-amyloid precursor protein (APP) and accumulation of its β-amyloid (Aβ) fragment are key features of Alzheimer's disease (AD). APP is a type Ⅰ integral membrane protein and consists of 695-770 amino acids encoded by differentially spliced mRNAs transcribed from a single gene located on human chromosome 21.1 The 695-amino acid APP is expressed preferentially in the brain. Aβ, the major component of senile plaques, is derived by proteolytic processing of APP by β-and γ-secretase and is constitutively released from most cells.

  3. Cell recognition molecule L1 promotes embryonic stem cell differentiation through the regulation of cell surface glycosylation

    Energy Technology Data Exchange (ETDEWEB)

    Li, Ying [Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian 116044 (China); Department of Clinical Laboratory, Second Affiliated Hospital of Dalian Medical University, Dalian 116023 (China); Huang, Xiaohua [Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian 116044 (China); Department of Clinical Biochemistry, College of Laboratory Medicine, Dalian Medical University, Dalian 116044 (China); An, Yue [Department of Clinical Laboratory, Second Affiliated Hospital of Dalian Medical University, Dalian 116023 (China); Ren, Feng [Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian 116044 (China); Yang, Zara Zhuyun; Zhu, Hongmei; Zhou, Lei [The Key Laboratory of Stem Cell and Regenerative Medicine, Institute of Molecular and Clinical Medicine, Kunming Medical University, Kunming 650228 (China); Department of Anatomy and Developmental Biology, Monash University, Clayton 3800 (Australia); He, Xiaowen; Schachner, Melitta [Keck Center for Collaborative Neuroscience and Department of Cell Biology and Neuroscience, Rutgers University, New Brunswick, NJ (United States); Xiao, Zhicheng, E-mail: zhicheng.xiao@monash.edu [The Key Laboratory of Stem Cell and Regenerative Medicine, Institute of Molecular and Clinical Medicine, Kunming Medical University, Kunming 650228 (China); Department of Anatomy and Developmental Biology, Monash University, Clayton 3800 (Australia); Ma, Keli, E-mail: makeli666@aliyun.com [Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian 116044 (China); Li, Yali, E-mail: yalilipaper@gmail.com [Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian 116044 (China); Department of Anatomy, National University of Singapore, Singapore 119078 (Singapore)

    2013-10-25

    Highlights: •Down-regulating FUT9 and ST3Gal4 expression blocks L1-induced neuronal differentiation of ESCs. •Up-regulating FUT9 and ST3Gal4 expression in L1-ESCs depends on the activation of PLCγ. •L1 promotes ESCs to differentiate into neuron through regulating cell surface glycosylation. -- Abstract: Cell recognition molecule L1 (CD171) plays an important role in neuronal survival, migration, differentiation, neurite outgrowth, myelination, synaptic plasticity and regeneration after injury. Our previous study has demonstrated that overexpressing L1 enhances cell survival and proliferation of mouse embryonic stem cells (ESCs) through promoting the expression of FUT9 and ST3Gal4, which upregulates cell surface sialylation and fucosylation. In the present study, we examined whether sialylation and fucosylation are involved in ESC differentiation through L1 signaling. RNA interference analysis showed that L1 enhanced differentiation of ESCs into neurons through the upregulation of FUT9 and ST3Gal4. Furthermore, blocking the phospholipase Cγ (PLCγ) signaling pathway with either a specific PLCγ inhibitor or knockdown PLCγ reduced the expression levels of both FUT9 and ST3Gal4 mRNAs and inhibited L1-mediated neuronal differentiation. These results demonstrate that L1 promotes neuronal differentiation from ESCs through the L1-mediated enhancement of FUT9 and ST3Gal4 expression.

  4. Cell recognition molecule L1 promotes embryonic stem cell differentiation through the regulation of cell surface glycosylation

    International Nuclear Information System (INIS)

    Highlights: •Down-regulating FUT9 and ST3Gal4 expression blocks L1-induced neuronal differentiation of ESCs. •Up-regulating FUT9 and ST3Gal4 expression in L1-ESCs depends on the activation of PLCγ. •L1 promotes ESCs to differentiate into neuron through regulating cell surface glycosylation. -- Abstract: Cell recognition molecule L1 (CD171) plays an important role in neuronal survival, migration, differentiation, neurite outgrowth, myelination, synaptic plasticity and regeneration after injury. Our previous study has demonstrated that overexpressing L1 enhances cell survival and proliferation of mouse embryonic stem cells (ESCs) through promoting the expression of FUT9 and ST3Gal4, which upregulates cell surface sialylation and fucosylation. In the present study, we examined whether sialylation and fucosylation are involved in ESC differentiation through L1 signaling. RNA interference analysis showed that L1 enhanced differentiation of ESCs into neurons through the upregulation of FUT9 and ST3Gal4. Furthermore, blocking the phospholipase Cγ (PLCγ) signaling pathway with either a specific PLCγ inhibitor or knockdown PLCγ reduced the expression levels of both FUT9 and ST3Gal4 mRNAs and inhibited L1-mediated neuronal differentiation. These results demonstrate that L1 promotes neuronal differentiation from ESCs through the L1-mediated enhancement of FUT9 and ST3Gal4 expression

  5. miR-145 induces caspase-dependent and -independent cell death in urothelial cancer cell lines with targeting of an expression signature present in Ta bladder tumors

    DEFF Research Database (Denmark)

    Ostenfeld, Marie Stampe; Bramsen, Jesper Bertram; Lamy, Philippe;

    2010-01-01

    Downregulation of miR-145 in a variety of cancers suggests a possible tumor suppressor function for this microRNA. Here, we show that miR-145 expression is reduced in bladder cancer and urothelial carcinoma in situ, compared with normal urothelium, using transcription profiling and in situ...... hybridization. Ectopic expression of miR-145 induced extensive apoptosis in urothelial carcinoma cell lines (T24 and SW780) as characterized by caspase activation, nuclear condensation and fragmentation, cellular shrinkage, and detachment. However, cell death also proceeded upon caspase inhibition by the...... pharmacological inhibitor zVAD-fmk and ectopic expression of anti-apoptotic Bcl-2, indicating the activation of an alternative caspase-independent death pathway. Microarray analysis of transcript levels in T24 cells, before the onset of cell death, showed destabilization of mRNAs enriched for miR-145 7mer target...

  6. Let-7b-mediated suppression of basigin expression and metastasis in mouse melanoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Fu, Tzu-Yen [Department of Animal Science, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan (China); Chang, Chia-Che [Institute of Biomedical Sciences, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan (China); Graduate Institute of Basic Medical Science, China Medical University, 91 Hsueh Shih Road, Taichung 40402, Taiwan (China); Lin, Chun-Ting [Department of Animal Science, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan (China); Lai, Cong-Hao [Institute of Biomedical Sciences, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan (China); Department of Life Sciences, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan (China); Peng, Shao-Yu; Ko, Yi-Ju [Department of Animal Science, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan (China); Tang, Pin-Chi, E-mail: pctang@dragon.nchu.edu.tw [Department of Animal Science, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan (China)

    2011-02-15

    Basigin (Bsg), also called extracellular matrix metalloproteinase inducer (EMMPRIN), is highly expressed on the surface of tumor cells and stimulates adjacent fibroblasts or tumor cells to produce matrix metalloproteinases (mmps). It has been shown that Bsg plays an important role in growth, development, cell differentiation, and tumor progression. MicroRNAs (miRNAs) are a class of short endogenous non-protein coding RNAs of 20-25 nucleotides (nt) that function as post-transcriptional regulators of gene expression by base-pairing to their target mRNAs and thereby mediate cleavage of target mRNAs or translational repression. In this study, let-7b, one of the let-7 family members, was investigated for its effect on the growth and invasiveness of the mouse melanoma cell line B16-F10. We have shown that let-7b can suppress the expression of Bsg in B16-F10 cells and also provided evidence that this suppression could result in the indirect suppression of mmp-9. The ability of B16-F10 cells transfected with let-7b to invade or migrate was significantly reduced. In addition, let-7b transfected B16-F10 cells displayed an inhibition of both cellular proliferation and colony formation. Furthermore, it was shown that the overexpression of let-7b in B16-F10 cells could reduce lung metastasis. Taken together, the present study identifies let-7b as a tumor suppressor that represses cancer cell proliferation and migration as well as tumor metastasis in mouse melanoma cells.

  7. Suppression of FAT/CD36 mRNA by human growth hormone in pancreatic ß-cells

    DEFF Research Database (Denmark)

    Dalgaard, Louise Torp; Thams, Peter Grevsen; Gaarn, Louise Winkel;

    2011-01-01

    this study was to examine the effect of human growth hormone (hGH) on mRNAs of fatty acid transport and binding proteins expressed in pancreatic ß-cells, and to examine this in relation to ß-cell survival after exposure to fatty acids. hGH decreased mRNA levels of FAT/CD36, whereas mRNAs of GPR40, FASN...... not bind directly to the FAT/CD36 promoter. The hGH-mediated suppression of FAT/CD36 mRNA was associated with a decrease in palmitate uptake and fatty acid-induced basal hyper-secretion of insulin resulting in improved glucose-stimulated insulin secretion. This study suggests that hGH can protect ß-cells......Fatty acid-induced damage in pancreatic ß-cells is assumed to play an important role in the development of type 2 diabetes. Lactogens (prolactin, placental lactogen and growth hormone) improve ß-cell survival via STAT5 activation but the molecular targets are incompletely characterized. The aim of...

  8. Suppression of FAT/CD36 mRNA by human growth hormone in pancreatic β-cells

    DEFF Research Database (Denmark)

    Dalgaard, Louise Torp; Thams, Peter Grevsen; Gaarn, Louise Winkel;

    2011-01-01

    this study was to examine the effect of human growth hormone (hGH) on mRNAs of fatty acid transport and binding proteins expressed in pancreatic β-cells, and to examine this in relation to β-cell survival after exposure to fatty acids. hGH decreased mRNA levels of FAT/CD36, whereas mRNAs of GPR40, FASN...... not bind directly to the FAT/CD36 promoter. The hGH-mediated suppression of FAT/CD36 mRNA was associated with a decrease in palmitate uptake and fatty acid-induced basal hyper-secretion of insulin resulting in improved glucose-stimulated insulin secretion. This study suggests that hGH can protect β-cells......Fatty acid-induced damage in pancreatic β-cells is assumed to play an important role in the development of type 2 diabetes. Lactogens (prolactin, placental lactogen and growth hormone) improve β-cell survival via STAT5 activation but the molecular targets are incompletely characterized. The aim of...

  9. Differential gene regulation under altered gravity conditions in follicular thyroid cancer cells: relationship between the extracellular matrix and the cytoskeleton.

    Science.gov (United States)

    Ulbrich, Claudia; Pietsch, Jessica; Grosse, Jirka; Wehland, Markus; Schulz, Herbert; Saar, Katrin; Hübner, Norbert; Hauslage, Jens; Hemmersbach, Ruth; Braun, Markus; van Loon, Jack; Vagt, Nicole; Egli, Marcel; Richter, Peter; Einspanier, Ralf; Sharbati, Soroush; Baltz, Theo; Infanger, Manfred; Ma, Xiao; Grimm, Daniela

    2011-01-01

    Extracellular matrix proteins, adhesion molecules, and cytoskeletal proteins form a dynamic network interacting with signalling molecules as an adaptive response to altered gravity. An important issue is the exact differentiation between real microgravity responses of the cells or cellular reactions to hypergravity and/or vibrations. To determine the effects of real microgravity on human cells, we used four DLR parabolic flight campaigns and focused on the effects of short-term microgravity (22 s), hypergravity (1.8 g), and vibrations on ML-1 thyroid cancer cells. No signs of apoptosis or necrosis were detectable. Gene array analysis revealed 2,430 significantly changed transcripts. After 22 s microgravity, the F-actin and cytokeratin cytoskeleton was altered, and ACTB and KRT80 mRNAs were significantly upregulated after the first and thirty-first parabolas. The COL4A5 mRNA was downregulated under microgravity, whereas OPN and FN were significantly upregulated. Hypergravity and vibrations did not change ACTB, KRT-80 or COL4A5 mRNA. MTSS1 and LIMA1 mRNAs were downregulated/slightly upregulated under microgravity, upregulated in hypergravity and unchanged by vibrations. These data indicate that the graviresponse of ML-1 cells occurred very early, within the first few seconds. Downregulated MTSS1 and upregulated LIMA1 may be an adaptive mechanism of human cells for stabilizing the cytoskeleton under microgravity conditions. PMID:21865726

  10. Quantitative spatial analysis of transcripts in multinucleate cells using single-molecule FISH.

    Science.gov (United States)

    Lee, ChangHwan; Roberts, Samantha E; Gladfelter, Amy S

    2016-04-01

    mRNA positioning in the cell is important for diverse cellular functions and proper development of multicellular organisms. Single-molecule RNA FISH (smFISH) enables quantitative investigation of mRNA localization and abundance at the level of individual molecules in the context of cellular features. Details about spatial mRNA patterning at various times, in different genetic backgrounds, at different developmental stages, and under varied environmental conditions provide invaluable insights into the mechanisms and functions of spatial regulation. Here, we describe detailed methods for performing smFISH along with immunofluorescence for two large, multinucleate cell types: the fungus Ashbya gossypii and cultured mouse myotubes. We also put forward a semi-automated image processing tool that systematically detects mRNAs from smFISH data and statistically analyzes the spatial pattern of mRNAs using a customized MATLAB code. These protocols and image analysis tools can be adapted to a wide variety of transcripts and cell types for systematically and quantitatively analyzing mRNA distribution in three-dimensional space. PMID:26690072

  11. Target-dependent biogenesis of cognate microRNAs in human cells.

    Science.gov (United States)

    Bose, Mainak; Bhattacharyya, Suvendra N

    2016-01-01

    Extensive research has established how miRNAs regulate target mRNAs by translation repression and/or endonucleolytic degradation in metazoans. However, information related to the effect of target mRNA on biogenesis and stability of corresponding miRNAs in animals is limited. Here we report regulated biogenesis of cognate miRNAs by their target mRNAs. Enhanced pre-miRNA processing by AGO-associated DICER1 contributes to this increased miRNP formation. The processed miRNAs are loaded onto AGO2 to form functionally competent miRISCs both in vivo and also in a cell-free in vitro system. Thus, we identify an additional layer of posttranscriptional regulation that helps the cell to maintain requisite levels of mature forms of respective miRNAs by modulating their processing in a target-dependent manner, a process happening for miR-122 during stress reversal in human hepatic cells. PMID:27448149

  12. Changes in tumor-antigen expression profile as human small-cell lung cancers progress

    International Nuclear Information System (INIS)

    Our group has previously observed that in patients with small-cell lung cancers (SCLCs), the expression of a tumor antigen, glioma big potassium (gBK) ion channel, is higher at the time of death than when the cancer is first treated by surgical resection. This study aimed to determine whether this dichotomy was common in other potential lung tumor antigens by examining the same patient samples using our more extensive profile analysis of tumor-antigen precursor protein (TAPP). We then tested the hypothesis that therapeutic intervention may inadvertently cause this increased gBK production. SCLC samples (eight surgical resections and three autopsy samples) and three control lungs were examined by quantitative real-time polymerase chain reaction for 42 potential TAPPs that represent potential T-cell-mediated immunological targets. Twenty-two TAPP mRNAs displayed the same profile as gBK, i.e., more mRNAs were expressed at autopsy than in their surgical counterparts. B-cyclin and mouse double minute 2, human homolog of P53-binding protein were elevated in both autopsy and surgical specimens above the normal-lung controls. When HTB119 cells were incubated with doxorubicin, gBK was strongly induced, as confirmed by intracellular flow cytometry with a gBK-specific antibody. Our findings suggested that more immunological targets became available as the tumor responded to chemotherapy and proceeded toward its terminal stages

  13. Global cDNA Amplification Combined with Real-Time RT–PCR: Accurate Quantification of Multiple Human Potassium Channel Genes at the Single Cell Level

    OpenAIRE

    Al-Taher, A.; Bashein, A.; Nolan, T.; Hollingsworth, M.; Brady, G

    2000-01-01

    We have developed a sensitive quantitative RT–PCR procedure suitable for the analysis of small samples, including single cells, and have used it to measure levels of potassium channel mRNAs in a panel of human tissues and small numbers of cells grown in culture. The method involves an initial global amplification of cDNA derived from all added polyadenylated mRNA followed by quantitative RT–PCR of individual genes using specific primers. In order to facilitate rapid and accurate processing of...

  14. Regulation of CYP11B1 and CYP11B2 steroidogenic genes by hypoxia-inducible miR-10b in H295R cells

    International Nuclear Information System (INIS)

    Highlights: • Identification of miR-10b as a hypoxia-inducible microRNA in H295R human adrenocortical cells. • Characterization of miR-10b as a negative regulator of the human CYP11B1 and CYP11B2 genes. • Evidence to support that CYP11B1 and and CYP11B2 mRNAs are likely targets of miR-10b. • miR-10b inhibits cortisol and aldosterone production in H295R cells. - Abstract: Although numerous studies have shown that hypoxia affects cortisol and aldosterone production in vivo, the underlying molecular mechanisms regulating the steroidogenic genes of these steroid hormones are still poorly known. MicroRNAs are post-transcriptional regulators that control diverse biological processes and this study describes the identification and validation of the hypoxia-inducible microRNA, miR-10b, as a negative regulator of the CYP11B1 and CYP11B2 steroidogenic genes in H295R human adrenocortical cells. Using the human TaqMan Low Density miRNA Arrays, we determined the miRNA expression patterns in H295R cells under normoxic and hypoxic conditions, and in cells overexpressing the human HIF-1α. Computer analysis using three in silico algorithms predicted that the hypoxia-inducible miR-10b molecule targets CYP11B1 and CYP11B2 mRNAs. Gene transfection studies of luciferase constructs containing the 3′-untranslated region of CYP11B1 or CYP11B2, combined with miRNA overexpression and knockdown experiments provide compelling evidence that CYP11B1 and CYP11B2 mRNAs are likely targets of miR-10b

  15. SHP1-mediated cell cycle redistribution inhibits radiosensitivity of non-small cell lung cancer

    International Nuclear Information System (INIS)

    Radioresistance is the common cause for radiotherapy failure in non-small cell lung cancer (NSCLC), and the degree of radiosensitivity of tumor cells is different during different cell cycle phases. The objective of the present study was to investigate the effects of cell cycle redistribution in the establishment of radioresistance in NSCLC, as well as the signaling pathway of SH2 containing Tyrosine Phosphatase (SHP1). A NSCLC subtype cell line, radioresistant A549 (A549S1), was induced by high-dose hypofractionated ionizing radiations. Radiosensitivity-related parameters, cell cycle distribution and expression of cell cycle-related proteins and SHP1 were investigated. siRNA was designed to down-regulate SHP1expression. Compared with native A549 cells, the proportion of cells in the S phase was increased, and cells in the G0/G1 phase were consequently decreased, however, the proportion of cells in the G2/M phase did not change in A549S1 cells. Moreover, the expression of SHP1, CDK4 and CylinD1 were significantly increased, while p16 was significantly down-regulated in A549S1 cells compared with native A549 cells. Furthermore, inhibition of SHP1 by siRNA increased the radiosensitivity of A549S1 cells, induced a G0/G1 phase arrest, down-regulated CDK4 and CylinD1expressions, and up-regulated p16 expression. SHP1 decreases the radiosensitivity of NSCLC cells through affecting cell cycle distribution. This finding could unravel the molecular mechanism involved in NSCLC radioresistance

  16. Peptide aptamer identified by molecular docking targeting translationally controlled tumor protein in leukemia cells.

    Science.gov (United States)

    Kadioglu, Onat; Efferth, Thomas

    2016-08-01

    Bioinformatics screening and molecular docking analyses were utilized to select high affinity peptides targeting translationally controlled tumor protein (TCTP). Selected peptide aptamers were tested towards cancer cell lines with different levels of TCTP expression. One peptide (WGQWPYHC) revealed specific cytotoxicity according to the TCTP expression in tumor cells without affecting normal cells. Western blot analysis showed peptide-induced down-regulation of TCTP as primary target as well as of cell-cycle related downstream proteins (CDK2, CDK6, Cyclin D3) in MOLT-4 leukemia cells. "WGQWPYHC" deserves further analysis for targeted therapy of TCTP-expressing tumor cells. Graphical abstract Molecular docking on TCTP, cytotoxicity toward MOLT-4 leukemia cell line and downregulation of CDK2, CDK6, CyclinD3 and TCTP proteins. PMID:26972431

  17. Effect of amlodipine on apoptosis of human breast carcinoma MDA-MB-231 cells

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Objective: To elucidate the effects of amlodipine on the proliferation and apoptosis of human breast carcinoma MDA-MB-231 cells. Methods: Light microscopy was used to determine the effects of amiodipine on cell morphology; Flow cytometry was used to quantitate cells undergoing apoptosis; the expression of a cell cycle-related protein, proliferating cell nuclear antigen (PCNA) and an antiapoptosis protein, Bcl-2 were assessed by immunocytochemistry. Results: Amlodipine concentration of 8.25 Ixmol/L (1/2 of IC50) affected the morphology, decreased the expression of PCNA and Bcl-2 and induced apoptosis of human breast carcinoma MDA-MB-231 cells. Conclusion: The effect of amlodipine on the antiproliferation of human breast carcinoma MDA-MB-231 cells is related to inducement of apoptosis, and the decrease of the expression of Bcl-2 and PCNA may be the possible mechanism for proliferation inhibitory and inducement of apoptosis.

  18. Cyclebase 3.0: a multi-organism database on cell-cycle regulation and phenotypes

    DEFF Research Database (Denmark)

    Santos Delgado, Alberto; Wernersson, Rasmus; Jensen, Lars Juhl

    2015-01-01

    Cyclebase version 3.0, we have updated the content of the database to reflect changes to genome annotation, added new mRNAand protein expression data, and integrated cell-cycle phenotype information from high-content screens and model-organism databases. The new version of Cyclebase also features a new web...... are not easily accessed, analyzed and combined due to their inherent heterogeneity. To address this, we have created Cyclebase-available at http://www.cyclebase.org-an online database that allows users to easily visualize and download results from genome-wide cell-cycle-related experiments. In...

  19. Dynamic response to peripheral nerve injury detected by in situ hybridization of IL-6 and its receptor mRNAs in the dorsal root ganglia is not strictly correlated with signs of neuropathic pain

    Czech Academy of Sciences Publication Activity Database

    Brázda, Václav; Klusáková, I.; Svíženská, I.; Dubový, P.

    2013-01-01

    Roč. 9, č. 42 (2013). ISSN 1744-8069 R&D Projects: GA MŠk(CZ) ED1.1.00/02.0068 Institutional research plan: CEZ:AV0Z50040702 Institutional support: RVO:68081707 Keywords : CHRONIC CONSTRICTION INJURY * SATELLITE GLIAL-CELLS * SCIATIC-NERVE Subject RIV: BO - Biophysics Impact factor: 3.531, year: 2013

  20. Molecular asymmetry in the 8-cell stage Xenopus tropicalis embryo described by single blastomere transcript sequencing.

    Science.gov (United States)

    De Domenico, Elena; Owens, Nick D L; Grant, Ian M; Gomes-Faria, Rosa; Gilchrist, Michael J

    2015-12-15

    Correct development of the vertebrate body plan requires the early definition of two asymmetric, perpendicular axes. The first axis is established during oocyte maturation, and the second is established by symmetry breaking shortly after fertilization. The physical processes generating the second asymmetric, or dorsal-ventral, axis are well understood, but the specific molecular determinants, presumed to be maternal gene products, are poorly characterized. Whilst enrichment of maternal mRNAs at the animal and vegetal poles in both the oocyte and the early embryo has been studied, little is known about the distribution of maternal mRNAs along either the dorsal-ventral or left-right axes during the early cleavage stages. Here we report an unbiased analysis of the distribution of maternal mRNA on all axes of the Xenopus tropicalis 8-cell stage embryo, based on sequencing of single blastomeres whose positions within the embryo are known. Analysis of pooled data from complete sets of blastomeres from four embryos has identified 908 mRNAs enriched in either the animal or vegetal blastomeres, of which 793 are not previously reported as enriched. In contrast, we find no evidence for asymmetric distribution along either the dorsal-ventral or left-right axes. We confirm that animal pole enrichment is on average distinctly lower than vegetal pole enrichment, and that considerable variation is found between reported enrichment levels in different studies. We use publicly available data to show that there is a significant association between genes with human disease annotation and enrichment at the animal pole. Mutations in the human ortholog of the most animally enriched novel gene, Slc35d1, are causative for Schneckenbecken dysplasia, and we show that a similar phenotype is produced by depletion of the orthologous protein in Xenopus embryos. PMID:26100918

  1. PUMILIO-2 is involved in the positive regulation of cellular proliferation in human adipose-derived stem cells.

    Science.gov (United States)

    Shigunov, Patrícia; Sotelo-Silveira, Jose; Kuligovski, Crisciele; de Aguiar, Alessandra Melo; Rebelatto, Carmen K; Moutinho, José A; Brofman, Paulo S; Krieger, Marco A; Goldenberg, Samuel; Munroe, David; Correa, Alejandro; Dallagiovanna, Bruno

    2012-01-20

    Stem cells can either differentiate into more specialized cells or undergo self-renewal. Several lines of evidence from different organisms suggest that these processes depend on the post-transcriptional regulation of gene expression. The presence of the PUF [Pumilio/FBF (fem-3 binding factor)] domain defines a conserved family of RNA binding proteins involved in repressing gene expression. It has been suggested that a conserved function of PUF proteins is to repress differentiation and sustain the mitotic proliferation of stem cells. In humans, Pumilio-2 (PUM2) is expressed in embryonic stem cells and adult germ cells. Here we show that PUM2 is expressed in a subpopulation of adipose-derived stem cell (ASC) cultures, with a granular pattern of staining in the cytoplasm. Protein levels of PUM2 showed no changes during the differentiation of ASCs into adipocytes. Moreover, RNAi knockdown of pum2 did not alter the rate of adipogenic differentiation compared with wild-type control cells. A ribonomic approach was used to identify PUM2-associated mRNAs. Microarray analysis showed that PUM2-bound mRNAs are part of gene networks involved in cell proliferation and gene expression control. We studied pum2 expression in cell cultures with low or very high levels of proliferation and found that changes in pum2 production were dependent on the proliferation status of the cell. Transient knockdown of pum2 expression by RNAi impaired proliferation of ASCs in vitro. Our results suggest that PUM2 does not repress differentiation of ASCs but rather is involved in the positive control of ASCs division and proliferation. PMID:21649561

  2. The effects of differential polyadenylation on expression of the dihydrofolate reductase-encoding gene in Chinese hamster lung cells.

    Science.gov (United States)

    Yang, H; Hussain, A; Melera, P W

    1995-10-01

    Three differently sized mRNAs are expressed from each of two DHFR (encoding dihydrofolate reductase) alleles present in the Chinese hamster lung (CHL) cell line, DC-3F. The relative abundancy of the transcripts produced from each allele differs dramatically as a result of differential utilization of the multiple poly(A) sites present in the DHFR DHFR gene and a genetic polymorphism located within the third poly(A) signal of one allele. We sought to determine whether such differences in polyadenylation affect the steady-state levels of DHFR and mRNAs expressed from either allele and, in a more general sense, to ask whether differences in 3' end RNA processing in a gene containing multiple poly(A) sites affects the final level of gene expression. An SV40 promoter-based transient expression system producing chimeric cat::DHFR transcripts was developed to regenerate the in vivo mRNA polyadenylation patterns associated with each of the two DHFR alleles. The results demonstrate that the total amount of polyadenylated RNA expressed from each of these constructs in vitro is the same regardless of the differential utilization of the poly(A) signals that occurs between them. Moreover, measurement of the individual turnover rates of the DHFR mRNAs expressed in vivo from each allele, as determined by pulse-chase labeling and actinomycin D inhibition studies, revealed no significant allele-specific differences in transcript half-lives. Finally, measuring the steady-state levels of DHFR poly(A)+ mRNA in parental DC-3F cells demonstrated that both alleles are expressed to the same extent during normal growth. Thus, even though dramatic allele-specific differences in 3' end processing of DHFR transcripts occur in vivo, such differences do not appear to influence the steady-state levels of DHFR gene expression. PMID:7590264

  3. Novel polysome messages and changes in translational activity appear after induction of adipogenesis in 3T3-L1 cells

    Directory of Open Access Journals (Sweden)

    Fromm-Dornieden Carolin

    2012-03-01

    Full Text Available Abstract Background Control of translation allows for rapid adaptation of the cell to stimuli, rather than the slower transcriptional control. We presume that translational control is an essential process in the control of adipogenesis, especially in the first hours after hormonal stimulation. 3T3-L1 preadipocytes were cultured to confluency and adipogenesis was induced by standard protocols using a hormonal cocktail. Cells were harvested before and 6 hours after hormonal induction. mRNAs attached to ribosomes (polysomal mRNAs were separated from unbound mRNAs by velocity sedimentation. Pools of polysomal and unbound mRNA fractions were analyzed by microarray analysis. Changes in relative abundance in unbound and polysomal mRNA pools were calculated to detect putative changes in translational activity. Changes of expression levels of selected genes were verified by qPCR and Western blotting. Results We identified 43 genes that shifted towards the polysomal fraction (up-regulated and 2 genes that shifted towards free mRNA fraction (down-regulated. Interestingly, we found Ghrelin to be down-regulated. Up-regulated genes comprise factors that are nucleic acid binding (eIF4B, HSF1, IRF6, MYC, POLR2a, RPL18, RPL27a, RPL6, RPL7a, RPS18, RPSa, TSC22d3, form part of ribosomes (RPL18, RPL27a, RPL6, RPL7a, RPS18, RPSa, act on the regulation of translation (eIF4B or transcription (HSF1, IRF6, MYC, TSC22d3. Others act as chaperones (BAG3, HSPA8, HSP90ab1 or in other metabolic or signals transducing processes. Conclusions We conclude that a moderate reorganisation of the functionality of the ribosomal machinery and translational activity are very important steps for growth and gene expression control in the initial phase of adipogenesis.

  4. Molecular biological mechanism II. Molecular mechanisms of cell cycle regulation

    International Nuclear Information System (INIS)

    The cell cycle in eukaryotes is regulated by central cell cycle controlling protein kinase complexes. These protein kinase complexes consist of a catalytic subunit from the cyclin-dependent protein kinase family (CDK), and a regulatory subunit from the cyclin family. Cyclins are characterised by their periodic cell cycle related synthesis and destruction. Each cell cycle phase is characterised by a specific set of CDKs and cyclins. The activity of CDK/cyclin complexes is mainly regulated on four levels. It is controlled by specific phosphorylation steps, the synthesis and destruction of cyclins, the binding of specific inhibitor proteins, and by active control of their intracellular localisation. At several critical points within the cell cycle, named checkpoints, the integrity of the cellular genome is monitored. If damage to the genome or an unfinished prior cell cycle phase is detected, the cell cycle progression is stopped. These cell cycle blocks are of great importance to secure survival of cells. Their primary importance is to prevent the manifestation and heritable passage of a mutated genome to daughter cells. Damage sensing, DNA repair, cell cycle control and apoptosis are closely linked cellular defence mechanisms to secure genome integrity. Disregulation in one of these defence mechanisms are potentially correlated with an increased cancer risk and therefore in at least some cases with an increased radiation sensitivity. (orig.)

  5. Oxidative stress regulated heme-oxygenase-1 and glutathione S-transferase-m1 gene expression changes in cell lines exposed to melanins

    Institute of Scientific and Technical Information of China (English)

    Jie Li; Peng Zhao; Junfeng Yang; Renyun Zhang; Shen Li; Dan Liu

    2011-01-01

    To investigate the effects of oxidative stress on substantia nigra neuronal degeneration and death in patients with Parkinson's disease, we treated neuroblastoma cells (SK-N-SH) and glioma cells with Fenton's reagent, iron chelating agent, neuromelanin and dopamine melanin. We investigated the changes in expression of nine oxidative stress-related genes and proteins. The levels of mRNAs for heme-oxygenase-1 and glutathione S-transferase-m1 were significantly reduced in SK-N-SH cells exposed to oxidative stress, and increased in glial cells treated with deferoxamine. These results revealed that SK-N-SH neurons react sensitively to oxidative stress, which implies different outcomes between these two types of cells in the substantia nigra. Moreover, the influences of neuromelanin and dopamine melanin on cell function are varied, and dopamine melanin is not a good model for neuromelanin.

  6. Comparing the immunosuppressive potency of naïve marrow stromal cells and Notch-transfected marrow stromal cells

    Directory of Open Access Journals (Sweden)

    Dao Mo A

    2011-10-01

    Full Text Available Abstract Background SB623 cells are expanded from marrow stromal cells (MSCs transfected with a Notch intracellular domain (NICD-expressing plasmid. In stroke-induced animals, these cells reduce infarct size and promote functional recovery. SB623 cells resemble the parental MSCs with respect to morphology and cell surface markers despite having been in extended culture. MSCs are known to have immunosuppressive properties; whether long-term culture of MSCs impact their immunomodulatory activity has not been addressed. Methods To assess the possible senescent properties of SB623 cells, we performed cell cycle related assays and beta-galactosidase staining. To assess the immunomodulatory activity of these expanded NICD-transfected MSCs, we performed co-cultures of SB623 cells or MSCs with either enriched human T cells or monocytes and assessed cytokine production by flow cytometry. In addition, we monitored the immunosuppressive activity of SB623 cells in both allogenic and xenogenic mixed lymphocyte reaction (MLR. Results Compared to MSCs, we showed that a small number of senescent-like cells appear in each lot of SB623 cells. Nevertheless, we demonstrated that these cells suppress human T cell proliferation in both the allogeneic and xenogeneic mixed lymphocyte reaction (MLR in a manner comparable to MSCs. IL-10 producing T cells were generated and monocyte-dendritic cell differentiation was dampened by co-culture with SB623 cells. Compared to the parental MSCs, SB623 cells appear to exert a greater inhibitory impact on the maturation of dendritic cells as demonstrated by a greater reduction in the surface expression of the co-stimulatory molecule, CD86. Conclusion The results demonstrated that the immunosuppressive activity of the expanded NICD-transfected MSCs is comparable to the parental MSCs, in spite of the appearance of a small number of senescent-like cells.

  7. CTCFL (BORIS) mRNA Expression in a Peripheral Giant Cell Granuloma of the Oral Cavity

    Science.gov (United States)

    Zambrano-Galván, Graciela; Reyes-Romero, Miguel; Bologna-Molina, Ronell; Almeda-Ojeda, Oscar Eduardo; Lemus-Rojero, Obed

    2014-01-01

    Peripheral giant cell granuloma (PGCG) is a relatively common benign reactive lesion of the oral cavity which can occur at any age. CTCFL/BORIS (CTCF like/Brother of the Regulator of Imprinted Sites) and CTCF (CCCTC-binding factor) are paralogous genes with an important role in the regulation of gene expression, genomic imprinting, and nuclear chromatin insulators regulation. BORIS expression promotes cell immortalization and growth while CTCF has tumor suppressor activity; the expression pattern may reflect the reverse transcription silencing of BORIS. The aim of this work was to describe a histopathological and molecular approach of an 8-year-old pediatric male patient with PGCG diagnosis. It was observed that the PGCG under study expressed CTCF as well as BORIS mRNAs alongside with the housekeeping gene GAPDH, which may be related to possible genetic and epigenetic changes in normal cells of oral cavity. PMID:25114808

  8. Extracellular Membrane Vesicles as Vehicles for Brain Cell-to-Cell Interactions in Physiological as well as Pathological Conditions

    Directory of Open Access Journals (Sweden)

    Gabriella Schiera

    2015-01-01

    Full Text Available Extracellular vesicles are involved in a great variety of physiological events occurring in the nervous system, such as cross talk among neurons and glial cells in synapse development and function, integrated neuronal plasticity, neuronal-glial metabolic exchanges, and synthesis and dynamic renewal of myelin. Many of these EV-mediated processes depend on the exchange of proteins, mRNAs, and noncoding RNAs, including miRNAs, which occurs among glial and neuronal cells. In addition, production and exchange of EVs can be modified under pathological conditions, such as brain cancer and neurodegeneration. Like other cancer cells, brain tumours can use EVs to secrete factors, which allow escaping from immune surveillance, and to transfer molecules into the surrounding cells, thus transforming their phenotype. Moreover, EVs can function as a way to discard material dangerous to cancer cells, such as differentiation-inducing proteins, and even drugs. Intriguingly, EVs seem to be also involved in spreading through the brain of aggregated proteins, such as prions and aggregated tau protein. Finally, EVs can carry useful biomarkers for the early diagnosis of diseases. Herein we summarize possible roles of EVs in brain physiological functions and discuss their involvement in the horizontal spreading, from cell to cell, of both cancer and neurodegenerative pathologies.

  9. Effect of neurturin on multipotent cells isolated from the adult skeletal muscle

    International Nuclear Information System (INIS)

    Ligands of the glial cell line-derived neurotrophic factors (GDNF)-family are trophic factors for the development and survival of multiple cell types, however their effects on non-neuronal stem cells are unknown. We examined the action of neurturin on a candidate stem cell population isolated from adult skeletal muscles. When grown as spheres, these cells expressed mRNAs for GDNF, persephin, GFR-α2, GFR-α4 (neurturin receptor), and Ret. Exposure of these cells to neurturin significantly augmented cell numbers via increased cell proliferation. After addition of retinoic acid, the cells exited the cell cycle, developed thin processes, and became immunoreactive for βIII-tubulin, while Ret mRNA expression decreased, without changes in the level of GFR-α2 mRNA. Neurturin induced an outgrowth of processes on these βIII-tubulin positive cells. Neurturin may therefore be beneficial in the use of these multipotent cells isolated from adult muscles for autologous transplants in neurological applications

  10. Correction of Down syndrome and Edwards syndrome aneuploidies in human cell cultures.

    Science.gov (United States)

    Amano, Tomokazu; Jeffries, Emiko; Amano, Misa; Ko, Akihiro C; Yu, Hong; Ko, Minoru S H

    2015-10-01

    Aneuploidy, an abnormal number of chromosomes, has previously been considered irremediable. Here, we report findings that euploid cells increased among cultured aneuploid cells after exposure to the protein ZSCAN4, encoded by a mammalian-specific gene that is ordinarily expressed in preimplantation embryos and occasionally in stem cells. For footprint-free delivery of ZSCAN4 to cells, we developed ZSCAN4 synthetic mRNAs and Sendai virus vectors that encode human ZSCAN4. Applying the ZSCAN4 biologics to established cultures of mouse embryonic stem cells, most of which had become aneuploid and polyploid, dramatically increased the number of euploid cells within a few days. We then tested the biologics on non-immortalized primary human fibroblast cells derived from four individuals with Down syndrome—the most frequent autosomal trisomy of chromosome 21. Within weeks after ZSCAN4 application to the cells in culture, fluorescent in situ hybridization with a chromosome 21-specific probe detected the emergence of up to 24% of cells with only two rather than three copies. High-resolution G-banded chromosomes further showed up to 40% of cells with a normal karyotype. These findings were confirmed by whole-exome sequencing. Similar results were obtained for cells with the trisomy 18 of Edwards syndrome. Thus a direct, efficient correction of aneuploidy in human fibroblast cells seems possible in vitro using human ZSCAN4. PMID:26324424

  11. RNA-binding IMPs promote cell adhesion and invadopodia formation

    DEFF Research Database (Denmark)

    Vikesaa, Jonas; Hansen, Thomas V O; Jønson, Lars; Borup, Rehannah; Wewer, Ulla M; Christiansen, Jan; Nielsen, Finn C

    2006-01-01

    Oncofetal RNA-binding IMPs have been implicated in mRNA localization, nuclear export, turnover and translational control. To depict the cellular actions of IMPs, we performed a loss-of-function analysis, which showed that IMPs are necessary for proper cell adhesion, cytoplasmic spreading and...... invadopodia formation. Loss of IMPs was associated with a coordinate downregulation of mRNAs encoding extracellular matrix and adhesion proteins. The transcripts were present in IMP RNP granules, implying that IMPs were directly involved in the post-transcriptional control of the transcripts. In particular......-mediated invadopodia formation. Taken together, our results indicate that RNA-binding proteins exert profound effects on cellular adhesion and invasion during development and cancer formation....

  12. Co-regulation of pituitary tumor cell adhesion and prolactin gene expression by glucocorticoid.

    Science.gov (United States)

    Spangler, P R; Delidow, B C

    1998-01-01

    Rat 235-1 pituitary tumor cells are lactotrophs producing high levels of prolactin (PRL). Dexamethasone (Dex, 100 nM) inhibits PRL gene expression in 235-1 cells by 50%, while simultaneously decreasing cell replication and cell-cell aggregation. To determine the time course of Dex action, we used a quantitative assay for cell-cell interaction, based on the number of single cells present before and after re-aggregation of dispersed cells. 235-1 cells were cultured in growth medium or medium plus 100 nM Dex for 1-4 days before assay. Control cells had 90% re-aggregation on all days of assay. Aggregation of Dex-treated cells decreased to 55% by day 4. Dex treatment also reduced cell numbers by 40%, but this decrease did not contribute to reduced aggregation. To determine the mechanism of Dex-inhibited cell-cell adhesion, we examined the expression of cadherins and catenins. Cadherin-related mRNAs (P- and N-cadherin probes) were detectable in 235-1 cells, but their levels were unchanged by Dex. A pancadherin antibody was unable to detect classical cadherins in these cells. Both alpha- and beta-catenins were detected by Western blotting and their levels were decreased by Dex. Unlike control aggregates, aggregates of Dex-treated cells were able to inhibit expression of PRL mRNA when added to monolayers of 235-1 cells. These data suggest that Dex influences cadherin function by inhibiting catenin expression and that this has the functional consequence of altering 235-1 cell-cell interactions. Overall the data show that Dex affects important aspects of lactotroph function other than PRL gene expression. These changes may include physical alterations in pituitary cell contacts that further support a change in functional state. PMID:9397162

  13. 2-Methoxy-4-vinylphenol can induce cell cycle arrest by blocking the hyper-phosphorylation of retinoblastoma protein in benzo[a]pyrene-treated NIH3T3 cells

    Energy Technology Data Exchange (ETDEWEB)

    Jeong, Jin Boo [Bioresource Sciences, Andong National University, Andong 760749 (Korea, Republic of); Jeong, Hyung Jin, E-mail: jhj@andong.ac.kr [Bioresource Sciences, Andong National University, Andong 760749 (Korea, Republic of)

    2010-10-01

    Research highlights: {yields} 2M4VP activated the expression of p21 and p15 protein, and down-regulated the expression of cyclin D1 and cyclin E. {yields} 2M4VP inhibited hyper-phosphorylation of Rb protein. {yields} 2M4VP induced cell cycle arrest from G1 to S. {yields} 2M4VP inhibited hyper-proliferation of the cells in BaP-treated cells. {yields} 2M4VP induces growth arrest of BaP-treated cells by blocking hyper-phosphorylation of Rb via regulating the expression of cell cycle-related proteins. -- Abstract: Benzo[a]pyrene (BaP) is an environment carcinogen that can enhance cell proliferation by disturbing the signal transduction pathways in cell cycle regulation. In this study, the effects of 2M4VP on cell proliferation, cell cycle and cell cycle regulatory proteins were studied in BaP-treated NIH 3T3 cells to establish the molecular mechanisms of 2M4VP as anti-proliferative agents. 2M4VP exerted a dose-dependent inhibitory effect on cell growth correlated with a G1 arrest. Analysis of G1 cell cycle regulators expression revealed 2M4VP increased expression of CDK inhibitor, p21Waf1/Cip1 and p15 INK4b, decreased expression of cyclin D1 and cyclin E, and inhibited kinase activities of CDK4 and CDK2. However, 2M4VP did not affect the expression of CDK4 and CDK2. Also, 2M4VP inhibited the hyper-phosphorylation of Rb induced by BaP. Our results suggest that 2M4VP induce growth arrest of BaP-treated NIH 3T3 cells by blocking the hyper-phosphorylation of Rb via regulating the expression of cell cycle-related proteins.

  14. Suppression of hepatic stellate cell activation by microRNA-29b

    International Nuclear Information System (INIS)

    Highlights: → Expression of miR-29b was found to be down-regulated during the activation of hepatic stellate cells in primary culture. → Transfection of a miR-29b precursor markedly attenuated the expression of Col1a1 and Col1a2 mRNAs. → It blunted the increased expression of α-SMA, DDR2, FN1, ITGB1, and PDGFR-b mRNAs essential for stellate cell activation. → miR-29b overexpression led stellate cells to remain in a quiescent state, as evidenced by their star-like morphology. → miR-29b overexpression suppressed the expression of c-fos mRNA. -- Abstract: MicroRNAs (miRNAs) participate in the regulation of cellular functions including proliferation, apoptosis, and migration. It has been previously shown that the miR-29 family is involved in regulating type I collagen expression by interacting with the 3'UTR of its mRNA. Here, we investigated the roles of miR-29b in the activation of mouse primary-cultured hepatic stellate cells (HSCs), a principal collagen-producing cell in the liver. Expression of miR-29b was found to be down-regulated during HSC activation in primary culture. Transfection of a miR-29b precursor markedly attenuated the expression of Col1a1 and Col1a2 mRNAs and additionally blunted the increased expression of α-SMA, DDR2, FN1, ITGB1, and PDGFR-β, which are key genes involved in the activation of HSCs. Further, overexpression of miR-29b led HSCs to remain in a quiescent state, as evidenced by their quiescent star-like cell morphology. Although phosphorylation of FAK, ERK, and Akt, and the mRNA expression of c-jun was unaffected, miR-29b overexpression suppressed the expression of c-fos mRNA. These results suggested that miR-29b is involved in the activation of HSCs and could be a candidate molecule for suppressing their activation and consequent liver fibrosis.

  15. Signaling factors and pathways of α-particle irradiation induced bilateral bystander responses between Beas-2B and U937 cells.

    Science.gov (United States)

    Fu, Jiamei; Wang, Juan; Wang, Xiangdong; Wang, Ping; Xu, Jinping; Zhou, Cuiping; Bai, Yang; Shao, Chunlin

    2016-07-01

    Although radiation induced bystander effects (RIBE) have been investigated for decades for their potential health risk, the underlying gene regulation is still largely unclear, especially the roles of immune system and inflammatory response in RIBE. In the present study, macrophage U937 cells and epithelial Beas-2B cells were co-cultured to disclose the cascades of bystander signaling factors and intercellular communications. After α-particle irradiation, both ERK and p38 pathways were activated in Beas-2B cells and were associated with the autocrine and paracrine signaling of TNF-α and IL-8, resulting in direct damage to the irradiated cells. Similar upregulation of TNF-α and IL-8 was induced in the bystander U937 cells after co-culture with α-irradiated Beas-2B cells. This upregulation was dependent on the activation of NF-κB pathway and was responsible for the enhanced damage of α-irradiated Beas-2B cells. Interestingly, the increased expressions of TNF-α and IL-8 mRNAs in the bystander U937 cells were clearly relayed on the activated ERK and p38 pathways in the irradiated Beas-2B cells, and the upregulation of TNF-α and IL-8 mRNAs in co-cultured Beas-2B cells was also partly due to the activated NF-κB pathway in the bystander U937 cells. With the pretreatment of U0126 (MEK1/2 inhibitor), SB203580 (p38 inhibitor) or BAY 11-7082 (NF-κB inhibitor), the aggravated damage in the α-irradiated Beas-2B cells could be largely alleviated. Our results disclosed novel signaling cascades of macrophage-mediated bilateral bystander responses that the release of TNF-α and IL-8 regulated by MAPK and NF-κB pathways synergistically increased cellular injury after α-particle irradiation. PMID:27155559

  16. Proportions of myosin heavy chain mRNAs, protein isoforms and fiber types in the slow and fast skeletal muscles are maintained after alterations of thyroid status in rats

    Czech Academy of Sciences Publication Activity Database

    Soukup, Tomáš; Diallo, Michael

    2015-01-01

    Roč. 64, č. 1 (2015), s. 111-118. ISSN 0862-8408 R&D Projects: GA ČR(CZ) GA304/08/0256; GA MŠk(CZ) 7AMB12SK158; GA MŠk(CZ) 7AMB14SK123; GA MŠk(CZ) EE2.3.30.0025 Grant ostatní: EC(XE) LSH-CT-2004-511978 Institutional support: RVO:67985823 Keywords : thyroid hormones * muscle gene expression * MyHC isoforms and muscle fiber types * quantitative real time RT-PCR * SDS-PAGE and 2-D Stereological analysis Subject RIV: EA - Cell Biology Impact factor: 1.293, year: 2014

  17. Histone Deacetylase Inhibitors Activate Tristetraprolin Expression through Induction of Early Growth Response Protein 1 (EGR1 in Colorectal Cancer Cells

    Directory of Open Access Journals (Sweden)

    Cyril Sobolewski

    2015-08-01

    Full Text Available The RNA-binding protein tristetraprolin (TTP promotes rapid decay of mRNAs bearing 3' UTR AU-rich elements (ARE. In many cancer types, loss of TTP expression is observed allowing for stabilization of ARE-mRNAs and their pathologic overexpression. Here we demonstrate that histone deacetylase (HDAC inhibitors (Trichostatin A, SAHA and sodium butyrate promote TTP expression in colorectal cancer cells (HCA-7, HCT-116, Moser and SW480 cells and cervix carcinoma cells (HeLa. We found that HDAC inhibitors-induced TTP expression, promote the decay of COX-2 mRNA, and inhibit cancer cell proliferation. HDAC inhibitors were found to promote TTP transcription through activation of the transcription factor Early Growth Response protein 1 (EGR1. Altogether, our findings indicate that loss of TTP in tumors occurs through silencing of EGR1 and suggests a therapeutic approach to rescue TTP expression in colorectal cancer.

  18. Massive parallel gene expression profiling of RINm5F pancreatic islet beta-cells stimulated with interleukin-1beta

    DEFF Research Database (Denmark)

    Rieneck, K; Bovin, L F; Josefsen, K;

    2000-01-01

    derived from rat pancreatic beta-cells, before and after challenge with 30 and 1,000 pg/ml of recombinant human IL-1beta. The highest concentration resulted in decreased insulin production and cell death over a period of 4 days. Using three different time points, 2, 4 and 24 hours after challenge, we......Interleukin 1 (IL-1) is a pleiotropic cytokine with the potential to kill pancreatic beta-cells, and this unique property is thought to be involved in the pathogenesis of type I diabetes mellitus. We therefore determined the quantitative expression of 24,000 mRNAs of RINm5F, an insulinoma cell line......, e.g. alpha-endosulfine and K+ channel Kir6.2 are differentially regulated. A number of transcripts in the biosynthesis pathway for cholesterol are also differentially regulated....

  19. Gut microbes promote colonic serotonin production through an effect of short-chain fatty acids on enterochromaffin cells.

    Science.gov (United States)

    Reigstad, Christopher S; Salmonson, Charles E; Rainey, John F; Szurszewski, Joseph H; Linden, David R; Sonnenburg, Justin L; Farrugia, Gianrico; Kashyap, Purna C

    2015-04-01

    Gut microbiota alterations have been described in several diseases with altered gastrointestinal (GI) motility, and awareness is increasing regarding the role of the gut microbiome in modulating GI function. Serotonin [5-hydroxytryptamine (5-HT)] is a key regulator of GI motility and secretion. To determine the relationship among gut microbes, colonic contractility, and host serotonergic gene expression, we evaluated mice that were germ-free (GF) or humanized (HM; ex-GF colonized with human gut microbiota). 5-HT reduced contractile duration in both GF and HM colons. Microbiota from HM and conventionally raised (CR) mice significantly increased colonic mRNAs Tph1 [(tryptophan hydroxylase) 1, rate limiting for mucosal 5-HT synthesis; P cell numbers (cells producing 5-HT) were unchanged. Short-chain fatty acids (SCFAs) promoted TPH1 transcription in BON cells (human EC cell model). Thus, gut microbiota acting through SCFAs are important determinants of enteric 5-HT production and homeostasis. PMID:25550456

  20. Nonsense mutations in the rhodopsin gene that give rise to mild phenotypes trigger mRNA degradation in human cells by nonsense-mediated decay.

    Science.gov (United States)

    Roman-Sanchez, Ramon; Wensel, Theodore G; Wilson, John H

    2016-04-01

    Eight different nonsense mutations in the human rhodopsin gene cause retinitis pigmentosa (RP), an inherited degenerative disease of the retina that can lead to complete blindness. Although all these nonsense mutations lead to premature termination codons (PTCs) in rhodopsin mRNA, some display dominant inheritance, while others are recessive. Because nonsense-mediated decay (NMD) can degrade mRNAs containing PTCs and modulate the inheritance patterns of genetic diseases, we asked whether any of the nonsense mutations in the rhodopsin gene generated mRNAs that were susceptible to degradation by NMD. We hypothesized that nonsense mutations that caused mild RP phenotypes would trigger NMD, whereas those that did not engage NMD would cause more severe RP phenotypes-presumably due to the toxicity of the truncated protein. To test our hypothesis, we transfected human rhodopsin nonsense mutants into HEK293 and HT1080 human cells and measured transcript levels by qRT-PCR. In both cell lines, rhodopsin mutations Q64X and Q344X, which cause severe phenotypes that are dominantly inherited, yielded the same levels of rhodopsin mRNA as wild type. By contrast, rhodopsin mutations W161X and E249X, which cause recessive RP, showed decreased rhodopsin mRNA levels, consistent with NMD. Rhodopsin mutant Y136X, a dominant mutation that causes late-onset RP with a very mild pathology, also gave lower mRNA levels. Treatment of cells with Wortmannin, an inhibitor of NMD, eliminated the degradation of Y136X, W161X, and E249X rhodopsin mRNAs. These results suggest that NMD modulates the severity of RP in patients with nonsense mutations in the rhodopsin gene. PMID:26416182

  1. Stochastic mRNA synthesis in mammalian cells.

    Science.gov (United States)

    Raj, Arjun; Peskin, Charles S; Tranchina, Daniel; Vargas, Diana Y; Tyagi, Sanjay

    2006-10-01

    Individual cells in genetically homogeneous populations have been found to express different numbers of molecules of specific proteins. We investigated the origins of these variations in mammalian cells by counting individual molecules of mRNA produced from a reporter gene that was stably integrated into the cell's genome. We found that there are massive variations in the number of mRNA molecules present in each cell. These variations occur because mRNAs are synthesized in short but intense bursts of transcription beginning when the gene transitions from an inactive to an active state and ending when they transition back to the inactive state. We show that these transitions are intrinsically random and not due to global, extrinsic factors such as the levels of transcriptional activators. Moreover, the gene activation causes burst-like expression of all genes within a wider genomic locus. We further found that bursts are also exhibited in the synthesis of natural genes. The bursts of mRNA expression can be buffered at the protein level by slow protein degradation rates. A stochastic model of gene activation and inactivation was developed to explain the statistical properties of the bursts. The model showed that increasing the level of transcription factors increases the average size of the bursts rather than their frequency. These results demonstrate that gene expression in mammalian cells is subject to large, intrinsically random fluctuations and raise questions about how cells are able to function in the face of such noise. PMID:17048983

  2. Regulation of Meristem Morphogenesis by Cell Wall Synthases in Arabidopsis.

    Science.gov (United States)

    Yang, Weibing; Schuster, Christoph; Beahan, Cherie T; Charoensawan, Varodom; Peaucelle, Alexis; Bacic, Antony; Doblin, Monika S; Wightman, Raymond; Meyerowitz, Elliot M

    2016-06-01

    The cell walls of the shoot apical meristem (SAM), containing the stem cell niche that gives rise to the above-ground tissues, are crucially involved in regulating differentiation. It is currently unknown how these walls are built and refined or their role, if any, in influencing meristem developmental dynamics. We have combined polysaccharide linkage analysis, immuno-labeling, and transcriptome profiling of the SAM to provide a spatiotemporal plan of the walls of this dynamic structure. We find that meristematic cells express only a core subset of 152 genes encoding cell wall glycosyltransferases (GTs). Systemic localization of all these GT mRNAs by in situ hybridization reveals members with either enrichment in or specificity to apical subdomains such as emerging flower primordia, and a large class with high expression in dividing cells. The highly localized and coordinated expression of GTs in the SAM suggests distinct wall properties of meristematic cells and specific differences between newly forming walls and their mature descendants. Functional analysis demonstrates that a subset of CSLD genes is essential for proper meristem maintenance, confirming the key role of walls in developmental pathways. PMID:27212401

  3. Curcumin attenuates quinocetone induced apoptosis and inflammation via the opposite modulation of Nrf2/HO-1 and NF-kB pathway in human hepatocyte L02 cells.

    Science.gov (United States)

    Dai, Chongshan; Li, Bin; Zhou, Yan; Li, Daowen; Zhang, Shen; Li, Hui; Xiao, Xilong; Tang, Shusheng

    2016-09-01

    The potential toxicity of quinocetone (QCT) has raised widely concern, but its mechanism is still unclear. This study aimed to investigate the protective effect of curcumin on QCT induced apoptosis and the underlying mechanism in human hepatocyte L02 cells. The results showed that QCT treatment significantly decreased the cell viability of L02 cell and increased the release of lactate dehydrogenase (LDH), which was attenuated by curcumin pre-treatment at 1.25, 2.5 and 5 μM. Compared to the QCT alone group, curcumin pre-treatment significantly attenuated QCT induced oxidative stress, mitochondrial dysfunction and apoptosis. In addition, curcumin pretreatment markedly attenuated QCT-induced increase of iNOS activity and NO production in a dose-dependent manner. Meanwhile, curcumin pretreatment markedly down-regulated the expression of nuclear factor -kB (NF-kB) and iNOS mRNAs, but up-regulated the expressions of Nrf2 and HO-1 mRNAs, compared to the QCT alone group. Zinc protoporphyrin IX, a HO-1 inhibitor, markedly partly abolished the cytoprotective effect of curcumin against QCT-induced caspase activation, NF-kB mRNA expression. These results indicate that curcumin could effectively inhibit QCT induced apoptosis and inflammatory response in L02 cells, which may involve the activation of Nrf2/HO-1 and inhibition of NF-kB pathway. PMID:27375190

  4. Knockdown of Pokemon protein expression inhibits hepatocellular carcinoma cell proliferation by suppression of AKT activity.

    Science.gov (United States)

    Zhu, Xiaosan; Dai, Yichen; Chen, Zhangxin; Xie, Junpei; Zeng, Wei; Lin, Yuanyuan

    2013-01-01

    Overexpression of Pokemon, which is an erythroid myeloid ontogenic factor protein, occurs in different cancers, including hepatocellular carcinoma (HCC). Pokemon is also reported to have an oncogenic activity in various human cancers. This study investigated the effect of Pokemon knockdown on the regulation of HCC growth. POK shRNA suppressed the expression of Pokemon protein in HepG2 cells compared to the negative control vector-transfected HCC cells. Pokemon knockdown also reduced HCC cell viability and enhanced cisplatin-induced apoptosis in HCC cells. AKT activation and the expression of various cell cycle-related genes were inhibited following Pokemon knockdown. These data demonstrate that Pokemon may play a role in HCC progression, suggesting that inhibition of Pokemon expression using Pokemon shRNA should be further evaluated as a novel target for the control of HCC. PMID:23924858

  5. Spiegelzymes® mirror-image hammerhead ribozymes and mirror-image DNAzymes, an alternative to siRNAs and microRNAs to cleave mRNAs in vivo?

    Directory of Open Access Journals (Sweden)

    Eliza Wyszko

    Full Text Available With the discovery of small non-coding RNA (ncRNA molecules as regulators for cellular processes, it became intriguing to develop technologies by which these regulators can be applied in molecular biology and molecular medicine. The application of ncRNAs has significantly increased our knowledge about the regulation and functions of a number of proteins in the cell. It is surprising that similar successes in applying these small ncRNAs in biotechnology and molecular medicine have so far been very limited. The reasons for these observations may lie in the high complexity in which these RNA regulators function in the cells and problems with their delivery, stability and specificity. Recently, we have described mirror-image hammerhead ribozymes and DNAzymes (Spiegelzymes® which can sequence-specifically hydrolyse mirror-image nucleic acids, such as our mirror-image aptamers (Spiegelmers discovered earlier. In this paper, we show for the first time that Spiegelzymes are capable of recognising complementary enantiomeric substrates (D-nucleic acids, and that they efficiently hydrolyse them at submillimolar magnesium concentrations and at physiologically relevant conditions. The Spiegelzymes are very stable in human sera, and do not require any protein factors for their function. They have the additional advantages of being non-toxic and non-immunogenic. The Spiegelzymes can be used for RNA silencing and also as therapeutic and diagnostic tools in medicine. We performed extensive three-dimensional molecular modelling experiments with mirror-image hammerhead ribozymes and DNAzymes interacting with D-RNA targets. We propose a model in which L/D-double helix structures can be formed by natural Watson-Crick base pairs, but where the nucleosides of one of the two strands will occur in an anticlinal conformation. Interestingly enough, the duplexes (L-RNA/D-RNA and L-DNA/D-RNA in these models can show either right- or left-handedness. This is a very new

  6. MiR-107 and MiR-185 can induce cell cycle arrest in human non small cell lung cancer cell lines.

    Directory of Open Access Journals (Sweden)

    Yukari Takahashi

    Full Text Available BACKGROUND: MicroRNAs (miRNAs are short single stranded noncoding RNAs that suppress gene expression through either translational repression or degradation of target mRNAs. The annealing between messenger RNAs and 5' seed region of miRNAs is believed to be essential for the specific suppression of target gene expression. One miRNA can have several hundred different targets in a cell. Rapidly accumulating evidence suggests that many miRNAs are involved in cell cycle regulation and consequentially play critical roles in carcinogenesis. METHODOLOGY/PRINCIPAL FINDINGS: Introduction of synthetic miR-107 or miR-185 suppressed growth of the human non-small cell lung cancer cell lines. Flow cytometry analysis revealed these miRNAs induce a G1 cell cycle arrest in H1299 cells and the suppression of cell cycle progression is stronger than that by Let-7 miRNA. By the gene expression analyses with oligonucleotide microarrays, we find hundreds of genes are affected by transfection of these miRNAs. Using miRNA-target prediction analyses and the array data, we listed up a set of likely targets of miR-107 and miR-185 for G1 cell cycle arrest and validate a subset of them using real-time RT-PCR and immunoblotting for CDK6. CONCLUSIONS/SIGNIFICANCE: We identified new cell cycle regulating miRNAs, miR-107 and miR-185, localized in frequently altered chromosomal regions in human lung cancers. Especially for miR-107, a large number of down-regulated genes are annotated with the gene ontology term 'cell cycle'. Our results suggest that these miRNAs may contribute to regulate cell cycle in human malignant tumors.

  7. Deletion of Tristetraprolin (TTP) caused spontaneous reactive granulopoiesis by a non-cell autonomous mechanism without disturbing LT-HSC quiescence1

    OpenAIRE

    Ian M Kaplan; Morisot, Sebastien; Heiser, Diane; Cheng, Wen-Chih; Kim, Min Jung; Civin, Curt I.

    2011-01-01

    Tristetraprolin (TTP, Zfp36, Nup475, Tis11) dramatically reduces the stability of target mRNAs by binding to AU-rich elements (AREs) in their 3′UTRs. Through this mechanism, TTP functions as a rheostatic, temporal regulator of gene expression. TTP KO mice exhibit completely penetrant granulocytic hyperplasia. We have shown that the hematopoietic stem-progenitor cell (HSPC) compartment in TTP KO mice is also altered. Although no change was detected in long-term HSC (LT-HSC) frequency or functi...

  8. A Novel Missense Mutation in the Thyroid Peroxidase Gene, R175Q, Resulting in Insufficient Cell Surface Enzyme in Two Siblings

    OpenAIRE

    Kotani, Tomio; Umeki, Kazumi; Kawano, Jun-ichi; Suganuma, Tatsuo; Yamamoto, Ikuo; Aratake, Yatsuki; Ichiba, Yozo; Furujo, Mahoko

    2004-01-01

    Thyroid peroxidase (TPO) abnormality is one of the causes of congenital hypothyroidism. Two missense mutations were found as a compound heterozygous mutation in two siblings with congenital goitrous hypothyroidism. One of these mutations, G614A (R175Q), was a novel mutation. Characterization of the novel mutation and a cotransfection experiment with two mutated TPO mRNAs were carried out. G614A-mRNA introduced into CHO-K1 cells expressed TPO protein with the same molecular weight as that of w...

  9. Gene expression in the pulp of ripening bananas. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of in vitro translation products and cDNA cloning of 25 different ripening-related mRNAs.

    Science.gov (United States)

    Medina-Suárez, R; Manning, K; Fletcher, J; Aked, J; Bird, C R; Seymour, G B

    1997-10-01

    mRNA was extracted from the pulp and peel of preclimacteric (d 0) bananas (Musa AAA group, cv Grand Nain) and those exposed to ethylene gas for 24 h and stored in air alone for a further 1 (d 2) and 4 d (d 5). Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of in vitro translation products from the pulp and peel of these fruits revealed significant up-regulation of numerous transcripts during ripening. The majority of the changes were initiated by d 2, with the level of these messages increasing during the remainder of the ripening period. Pulp tissue from d 2 was used for the construction of a cDNA library. This library was differentially screened for ripening-related clones using cDNA from d-0 and d-2 pulp by a novel microtiter plate method. In the primary screen 250 up- and down-regulated clones were isolated. Of these, 59 differentially expressed clones were obtained from the secondary screen. All of these cDNAs were partially sequenced and grouped into families after database searches. Twenty-five nonredundant groups of pulp clones were identified. These encoded enzymes were involved in ethylene biosynthesis, respiration, starch metabolism, cell wall degradation, and several other key metabolic events. We describe the analysis of these clones and their possible involvement in ripening. PMID:9342865

  10. Regulatory mechanism of radiation-induced cancer cell death by the change of cell cycle

    Energy Technology Data Exchange (ETDEWEB)

    Jeong, Soo Jin; Jeong, Min Ho; Jang, Ji Yeon [College of Medicine, Donga Univ., Pusan (Korea, Republic of)

    2003-09-01

    In our previous study, we have shown the main cell death pattern induced by irradiation or protein tyrosine kinase (PTK) inhibitors in K562 human myelogenous leukemic cell line. Death of the cells treated with irradiation alone was characterized by mitotic catastrophe and typical radiation-induced apoptosis was accelerated by herbimycin A (HMA). Both types of cell death were inhibited by genistein. In this study, we investigated the effects of HMA and genistein on cell cycle regulation and its correlation with the alterations of radiation-induced cell death. K562 cells in exponential growth phase were used for this study. The cells were irradiated with 10 Gy using 6 MeV Linac (200-300 cGy/min). Immediately after irradiation, cells were treated with 250 nM of HMA or 25{mu}M of genistein. The distributions of cell cycle, the expressions of cell cycle-related protein, the activities of cyclin-dependent kinase, and the yield of senescence and differentiation were analyzed. X-irradiated cells were arrested in the G2 phase of the cell cycle but unlike the p53-positive cells, they were not able to sustain the cell cycle arrest. An accumulation of cells in G2 phase of first cell-cycle post-treatment and an increase of cyclin B1 were correlated with spontaneous, premature, chromosome condensation and mitotic catastrophe. HMA induced rapid G2 checkpoint abrogation and concomitant p53-independent G1 accumulation HMA-induced cell cycle modifications correlated with the increase of cdc2 kinase activity, the decrease of the expressions of cyclins E and A and of CDK2 kinase activity, and the enhancement of radiation-induced apoptosis. Genistein maintained cells that were arrested in the G2-phase, decreased the expressions of cyclin B1 and cdc25C and cdc2 kinase activity, increased the expression of p16, and sustained senescence and megakaryocytic differentiation. The effects of HMA and genistein on the radiation-induced cell death of K562 cells were closely related to the cell

  11. Molecular interactions between the specialist herbivore Manduca sexta (lepidoptera, sphingidae) and its natural host Nicotiana attenuata: V. microarray analysis and further characterization of large-scale changes in herbivore-induced mRNAs.

    Science.gov (United States)

    Hui, Dequan; Iqbal, Javeed; Lehmann, Katja; Gase, Klaus; Saluz, Hans Peter; Baldwin, Ian T

    2003-04-01

    We extend our analysis of the transcriptional reorganization that occurs when the native tobacco, Nicotiana attenuata, is attacked by Manduca sexta larvae by cloning 115 transcripts by mRNA differential display reverse transcription-polymerase chain reaction and subtractive hybridization using magnetic beads (SHMB) from the M. sexta-responsive transcriptome. These transcripts were spotted as cDNA with eight others, previously confirmed to be differentially regulated by northern analysis on glass slide microarrays, and hybridized with Cy3- and Cy5-labeled probes derived from plants after 2, 6, 12, and 24 h of continuous attack. Microarray analysis proved to be a powerful means of verifying differential expression; 73 of the cloned genes (63%) were differentially regulated (in equal proportions from differential display reverse transcription-polymerase chain reaction and SHMB procedures), and of these, 24 (32%) had similarity to known genes or putative proteins (more from SHMB). The analysis provided insights into the signaling and transcriptional basis of direct and indirect defenses used against herbivores, suggesting simultaneous activation of salicylic acid-, ethylene-, cytokinin-, WRKY-, MYB-, and oxylipin-signaling pathways and implicating terpenoid-, pathogen-, and cell wall-related transcripts in defense responses. These defense responses require resources that could be made available by decreases in four photosynthetic-related transcripts, increases in transcripts associated with protein and nucleotide turnover, and increases in transcripts associated with carbohydrate metabolism. This putative up-regulation of defense-associated and down-regulation of growth-associated transcripts occur against a backdrop of altered transcripts for RNA-binding proteins, putative ATP/ADP translocators, chaperonins, histones, and water channel proteins, responses consistent with a major metabolic reconfiguration that underscores the complexity of response to herbivore attack

  12. Dexamethasone acutely regulates endocrine parameters in stallions and subsequently affects gene expression in testicular germ cells.

    Science.gov (United States)

    Ing, N H; Brinsko, S P; Curley, K O; Forrest, D W; Love, C C; Hinrichs, K; Vogelsang, M M; Varner, D D; Welsh, T H

    2015-01-01

    Testicular steroidogenesis and spermatogenesis are negatively impacted by stress-related hormones such as glucocorticoids. The effects of two injections of a therapeutic dose of dexamethasone (a synthetic glucocorticoid, 0.1mg/kg; i.v.) given 24h apart to each of three stallions were investigated and compared to three saline-injected control stallions. Dexamethasone decreased circulating concentrations of cortisol by 50% at 24h after the initial injection. Serum testosterone decreased by a maximum of 94% from 4 to 20h after the initial injection of dexamethasone. Semen parameters of the dexamethasone-treated stallions were unchanged in the subsequent two weeks. Two weeks after treatment, stallions were castrated. Functional genomic analyses of the testes revealed that, of eight gene products analyzed, dexamethasone depressed concentrations of heat shock protein DNAJC4 and sperm-specific calcium channel CATSPER1 mRNAs by more than 60%. Both genes are expressed in germ cells during spermiogenesis and have been related to male fertility in other species, including humans. This is the first report of decreased DNAJC4 and CATSPER1 mRNA concentrations in testes weeks after dexamethasone treatment. Concentrations of these mRNAs in sperm may be useful as novel markers of fertility in stallions. PMID:25487569

  13. Increased interferon alpha receptor 2 mRNA levels is associated with renal cell carcinoma metastasis

    Directory of Open Access Journals (Sweden)

    Yamanishi Tomonori

    2007-08-01

    Full Text Available Abstract Background Interferon-α (IFN-α is one of the central agents in immunotherapy for renal cell carcinoma (RCC and binds to the IFN-α receptor (IFNAR. We investigated the role of IFNAR in RCC. Methods We quantified IFNAR mRNA expression in paired tumor and non-tumor samples from the surgical specimens of 103 consecutive patients with RCC using a real-time reverse transcription polymerase chain reaction (RT-PCR, and IFNAR2 protein using Western blotting. Results The absolute level of IFNAR1 and IFNAR2 mRNAs in tumor and non-tumor tissues did not correlate with the malignant and metastatic profiles. The relative yields of the PCR product from the tumor tissue to that from the corresponding non-tumor tissue (T/N for the expression of IFNAR mRNAs were calculated. While the T/N ratio of IFNAR1 did not correlate with any factor, a high T/N ratio of IFNAR2 correlated with poor differentiation (P P P P P Conclusion IFNAR2 is associated with the progression of RCC.

  14. Integrative analysis of microRNA and mRNA expression profiles in non-small-cell lung cancer.

    Science.gov (United States)

    Yang, C; Sun, C; Liang, X; Xie, S; Huang, J; Li, D

    2016-04-01

    Non-small-cell lung cancer (NSCLC) represents the most common deadly disease. Emerging evidences suggest that abnormal epigenetic modulation via mRNAs and microRNAs (miRNAs) might be involved in the tumorigenesis. To explore novel therapeutic target of NSCLC, a more detailed mRNAs and miRNA expression profiling study is needed. High-quality total RNA including miRNA was isolated from NSCLC tissue and para-carcinoma tissue and used for RNA and small RNA sequencing. Results were analyzed bioinformatically and validated using quantitative real-time (qRT)-PCR. A total of 3530 genes (1977 up-regulated and 1553 down-regulated) and 211 miRNAs (171 up-regulated and 30 down-regulated) were differentially expressed (DE) in NSCLC tissue versus adjacent normal tissues. Furthermore, 157 novel miRNAs were predicted in our samples. Of these, 918 significant miRNA-mRNA pairs were identified, consisting of 100 miRNAs and 443 mRNAs. Gene ontology analysis revealed that most of the target genes were enriched in the terms of plasma membrane, binding, and multiple biological-molecular signaling processes. Pathway analysis of these miRNA signatures highlights their critical roles in calcium signaling pathway. Using qRT-PCR, the expression of several DE genes (KRAS and RBM5) and miRNAs (miR-1-5p, let-7b-5p, miR-21-5p, miR-1290, miR-149-5p, chr8_28846, chrX_31594, and chr9_29897) were confirmed. The integrative analysis based on mRNA and miRNA profiling may provide more potential molecular for the tumorigenesis and development of NSCLC. PMID:26964645

  15. Alphastatin downregulates vascular endothelial cells sphingosine kinase activity and suppresses tumor growth in nude mice bearing human gastric cancer xenografts

    Institute of Scientific and Technical Information of China (English)

    Lin Chen; Tao Li; Rong Li; Bo Wei; Zheng Peng

    2006-01-01

    AIM: To investigate whether alphastatin could inhibit human gastric cancer growth and furthermore whether sphingosine kinase (SPK) activity is involved in this process.METHODS: Using migration assay, MTT assay and Matrigel assay, the effect of alphastatin on vascular endothelial cells (ECs) was evaluated in vitro. SPK and endothelial differentiation gene (EDG)-1, -3, -5 mRNAs were detected by reverse transcription-polymerase chain reaction (RT-PCR). SPK activity assay was used to evaluate the effect of alphastatin on ECs. Matrigel plug assay in nude mice was used to investigate the effect of alphastatin on angiogenesis in vivo. Female nude mice were subcutaneously implanted with human gastric cancer cells (BGC823) for the tumor xenografts studies.Micro vessel density was analyzed in Factor Ⅷ-stained tumor sections by the immunohistochemical SP method.RESULTS: In vitro, alphastatin inhibited the migration and tube formation of ECs, but had no effect on proliferation of ECs. RT-PCR analysis demonstrated that ECs expressed SPK and EDG-1, -3, -5 mRNAs. In vivo,alphastatin sufficiently suppressed neovascularization of the tumor in the nude mice. Daily administration of alphastatin produced significant tumor growth suppression. Immunohistochemical studies of tumor tissues revealed decreased micro vessel density in alphastatin-treated animals as compared with controls.CONCLUSION: Downregulating ECs SPK activity may be one of the mechanisms that alphastatin inhibits gastric cancer angiogenesis. Alphastatin might be a useful and relatively nontoxic adjuvant therapy in the treatment of gastric cancer.

  16. A quantitative inverse relationship between connexin32 expression and cell proliferation in a rat hepatoma cell line

    International Nuclear Information System (INIS)

    Gap junctions comprised of connexin proteins are involved in direct intercellular communication and the regulation of cell behaviour and homeostasis. Reduced connexin expression and loss of gap junction function is a characteristic of many cancer cells and of the effect of many non-genotoxic carcinogens that induce cell proliferation. Moreover, when certain cancer cell lines are transfected with specific connexin genes, cells can regain control over proliferation. We have employed RNA interference and dexamethasone to modulate connexin32 expression in MH1C1 cells to a range of concentrations. This allowed the determination of the quantitative relationship between connexin32 protein expression and cell proliferation. The magnitude of cell proliferation, measured by bromodeoxyuridine incorporation, was inversely proportional to the level of connexin32 expression. Q-PCR indicated a lack of change of expression of a range of cell cycle-related genes at 24 h. The inverse relationship between Cx32 expression and proliferation was continuous, and a threshold level of reduction of connexin32 was not observable for an influence on proliferation

  17. TMC function in hair cell transduction.

    Science.gov (United States)

    Holt, Jeffrey R; Pan, Bifeng; Koussa, Mounir A; Asai, Yukako

    2014-05-01

    Transmembrane channel-like (TMC) proteins 1 and 2 are necessary for hair cell mechanotransduction but their precise function is controversial. A growing body of evidence supports a direct role for TMC1 and TMC2 as components of the transduction complex. However, a number of important questions remain and alternate hypotheses have been proposed. Here we present an historical overview of the identification and cloning of Tmc genes, a discussion of mutations in TMC1 that cause deafness in mice and humans and a brief review of other members of the Tmc gene superfamily. We also examine expression of Tmc mRNAs and localization of the protein products. The review focuses on potential functions of TMC proteins and the evidence from Beethoven mice that suggests a direct role for TMC1 in hair cell mechanotransduction. Data that support alternate interpretations are also considered. The article concludes with a discussion of outstanding questions and future directions for TMC research. This article is part of a Special Issue entitled . PMID:24423408

  18. Lineage-restricted expression of homeobox-containing genes in human hematopoietic cell lines

    International Nuclear Information System (INIS)

    The authors investigated the role of homeobox-containing genes in human hematopoiesis because homeobox genes (i) control cell fate in the Drosophila embryo, (ii) are expressed in specific patterns in human embryos, and (iii) appear to function as transcription factors that control cell phenotype in other mammalian organs. Using four homeobox probes from the HOX2 locus and a previously undescribed homeobox cDNA (PL1), they screened mRNAs from 18 human leukemic cell lines representing erythroid, myeloid, and T- and B-cell lineages. Complex patterns of lineage-restricted expression are observed. No single homeobox gene is expressed in all types of hematopoietic cells, but each cell type exhibits homeobox gene expression. They have demonstrated (i) lineage-restricted expression of five homeobox genes in erythroid and monocytic cell lines; (ii) expression of additional homeobox genes in other cell lineages (HL-60 and lymphoid cells); (iii) expression of one homeobox gene in normal marrow cells; and (iv) modulation of expression during differentiation. These data suggest that these genes play a role in human hematopoietic development and lineage commitment

  19. Coordinate turnover of nuclear and cytoplasmic histone messenger RNA following inhibition of DNA replication of HeLa S3 cells

    International Nuclear Information System (INIS)

    The authors have examined the metabolism of human H4 histone mRNA in the nucleus and cytoplasm of HeLa S3 cells following inhibition of DNA synthesis to address the extent to which histone mRNA stability in these cellular compartments is coupled to DNA replication. The nuclear and cytoplasmic levels of histone mRNAs encoded by the pF0108A human H4 histone gene were determined by S1 nuclease analysis using a 32P-labeled probe that could distinguish pF0108A transcripts from those of other members of the H4 histone multigene family. Hydroxyurea treatment resulted within 15 min in a 75% reduction in the level of histone H4 mRNA in the nucleus, which corresponds to the 85% decrease observed for H4 histone mRNA in the cytoplasm. The kinetics of nuclear and cytoplasmic H4 mRNA turnover following hydroxyurea treatment were also similar. Northern blot analysis using a 32P-labeled mitochondrial cytochrome b probe indicated that the association of cytoplasmic RNA with the nuclear fraction was less than 0.5%. Treatment of cells with a protein synthesis inhibitor resulted in a 1.3-fold increase in nuclear H4 histone mRNA levels and a 1.5-fold increase of H4 mRNA in the cytoplasm after 45 min. Together, these results indicate that nuclear and cytoplasmic H4 histone mRNAs respond similarly to metabolic perturbations that influence message stability and that mechanisms operative in the turnover of histone mRNAs in the nucleus and cytoplasm may be similar

  20. Abnormal structural luteolysis in ovaries of the senescence accelerated mouse (SAM): expression of Fas ligand/Fas-mediated apoptosis signaling molecules in luteal cells.

    Science.gov (United States)

    Kiso, Minako; Manabe, Noboru; Komatsu, Kohji; Shimabe, Munetake; Miyamoto, Hajime

    2003-12-01

    Senescence accelerated mouse-prone (SAMP) mice with a shortened life span show accelerated changes in many of the signs of aging and a shorter reproductive life span than SAM-resistant (SAMR) controls. We previously showed that functional regression (progesterone dissimilation) occurs in abnormally accumulated luteal bodies (aaLBs) of SAMP mice, but structural regression of luteal cells in aaLB is inhibited. A deficiency of luteal cell apoptosis causes the abnormal accumulation of LBs in SAMP ovaries. In the present study, to show the abnormality of Fas ligand (FasL)/Fas-mediated apoptosis signal transducing factors in the aaLBs of the SAMP ovaries, we assessed the changes in the expression of FasL, Fas, caspase-8 and caspase-3 mRNAs by reverse transcription-polymerase chain reaction, and in the expression and localization of FasL, Fas and activated caspase-3 proteins by Western blotting and immunohistochemistry, respectively, during the estrus cycle/luteolysis. These mRNAs and proteins were expressed in normal LBs of both SAMP and SAMR ovaries, but not at all or only in trace amounts in aaLBs of SAMP, indicating that structural regression is inhibited by blockage of the expression of these transducing factors in luteal cells of aaLBs in SAMP mice. PMID:14967896

  1. Bombesin stimulation of c-fos and c-myc gene expression in cultured of Swiss 3T3 cells

    International Nuclear Information System (INIS)

    Bombesin has been show to be a potent mitogen for Swiss 3T3 cells. At nanomolar concentrations it stimulates DNA synthesis in quiescent cultures of 3T3 cells and also induces the expression of c-fos and c-myc mRNA. c-fos mRNA transcripts dramatically increase 15 min after the addition of bombesin, are still abundant after 30-60 min and then decrease. c-myc mRNA induction is detectable later, 1 h after bombesin treatment. Conversely, no changes in c-Ki-ras expression are observed after stimulation with bombesin. These results demonstrate that the increased expression of c-fos and c-myc mRNAs appears to be a common response to diverse agents that induce DNA synthesis and cell proliferation

  2. Differential regulation of polysome mRNA levels in mouse Hepa-1C1C7 cells exposed to dioxin.

    Science.gov (United States)

    Thornley, Jessica A; Trask, Heidi W; Ridley, Christian J A; Korc, Murray; Gui, Jiang; Ringelberg, Carol S; Wang, Sinny; Tomlinson, Craig R

    2011-10-01

    The environmental agent 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin) causes a multitude of human illnesses. In order to more fully understand the underlying biology of TCDD toxicity, we tested the hypothesis that new candidate genes could be identified using polysome RNA from TCDD-treated mouse Hepa-1c1c7 cells. We found that (i) differentially expressed whole cell and cytoplasm RNA levels are both poor predictors of polysome RNA levels; (ii) for a majority of RNAs, differential RNA levels are regulated independently in the nucleus, cytoplasm, and polysomes; (iii) for the remaining polysome RNAs, levels are regulated via several different mechanisms, including a "tagging" of mRNAs in the nucleus for immediate polysome entry; and (iv) most importantly, a gene list derived from differentially expressed polysome RNA generated new genes and cell pathways potentially related to TCDD biology. PMID:21570461

  3. The anti-canine distemper virus activities of ex vivo-expanded canine natural killer cells.

    Science.gov (United States)

    Park, Ji-Yun; Shin, Dong-Jun; Lee, Soo-Hyeon; Lee, Je-Jung; Suh, Guk-Hyun; Cho, Duck; Kim, Sang-Ki

    2015-04-17

    Natural killer (NK) cells play critical roles in induction of antiviral effects against various viruses of humans and animals. However, few data on NK cell activities during canine distemper virus (CDV) infections are available. Recently, we established a culture system allowing activation and expansion of canine non-B, non-T, large granular NK lymphocytes from PBMCs of normal dogs. In the present study, we explored the ability of such expanded NK cells to inhibit CDV infection in vitro. Cultured CD3-CD5-CD21- NK cells produced large amounts of IFN-γ, exhibited highly upregulated expression of mRNAs encoding NK-cell-associated receptors, and demonstrated strong natural killing activity against canine tumor cells. Although the expanded NK cells were dose-dependently cytotoxic to both normal and CDV-infected Vero cells, CDV infection rendered Vero cells more susceptible to NK cells. Pretreatment with anti-CDV serum from hyperimmunized dogs enhanced the antibody-dependent cellular cytotoxicity (ADCC) of NK cells against CDV-infected Vero cells. The culture supernatants of NK cells, added before or after infection, dose-dependently inhibited both CDV replication and development of CDV-induced cytopathic effects (CPEs) in Vero cells. Anti-IFN-γ antibody neutralized the inhibitory effects of NK cell culture supernatants on CDV replication and CPE induction in Vero cells. Such results emphasize the potential significance of NK cells in controlling CDV infection, and indicate that NK cells may play roles both during CDV infection and in combating such infections, under certain conditions. PMID:25680810

  4. Involvement of cdc25c in cell cycle alteration of a radioresistant lung cancer cell line established with fractionated ionizing radiation.

    Science.gov (United States)

    Li, Jie; Yang, Chun-Xu; Mei, Zi-Jie; Chen, Jing; Zhang, Shi-Min; Sun, Shao-Xing; Zhou, Fu-Xiang; Zhou, Yun-Feng; Xie, Cong-Hua

    2013-01-01

    Cancer patients often suffer from local tumor recurrence after radiation therapy. Cell cycling, an intricate sequence of events which guarantees high genomic fidelity, has been suggested to affect DNA damage responses and eventual radioresistant characteristics of cancer cells. Here, we established a radioresistant lung cancer cell line, A549R , by exposing the parental A549 cells to repeated γ-ray irradiation with a total dose of 60 Gy. The radiosensitivity of A549 and A549R was confirmed using colony formation assays. We then focused on examination of the cell cycle distribution between A549 and A549R and found that the proportion of cells in the radioresistant S phase increased, whereas that in the radiosensitive G1 phase decreased. When A549 and A549R cells were exposed to 4 Gy irradiation the total differences in cell cycle redistribution suggested that G2-M cell cycle arrest plays a predominant role in mediating radioresistance. In order to further explore the possible mechanisms behind the cell cycle related radioresistance, we examined the expression of Cdc25 proteins which orchestrate cell cycle transitions. The results showed that expression of Cdc25c increased accompanied by the decrease of Cdc25a and we proposed that the quantity of Cdc25c, rather than activated Cdc25c or Cdc25a, determines the radioresistance of cells. PMID:24289569

  5. Long-noncoding RNAs in basal cell carcinoma.

    Science.gov (United States)

    Sand, Michael; Bechara, Falk G; Sand, Daniel; Gambichler, Thilo; Hahn, Stephan A; Bromba, Michael; Stockfleth, Eggert; Hessam, Schapoor

    2016-08-01

    Long noncoding RNAs (lncRNAs) are fundamental regulators of pre- and post-transcriptional gene regulation. Over 35,000 different lncRNAs have been described with some of them being involved in cancer formation. The present study was initiated to describe differentially expressed lncRNAs in basal cell carcinoma (BCC). Patients with BCC (n = 6) were included in this study. Punch biopsies were harvested from the tumor center and nonlesional epidermal skin (NLES, control, n = 6). Microarray-based lncRNA and mRNA expression profiles were identified through screening for 30,586 lncRNAs and 26,109 protein-coding transcripts (mRNAs). The microarray data were validated by RT-PCR in a second set of BCC versus control samples. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of mRNAs were performed to assess biologically relevant pathways. A total of 1851 lncRNAs were identified as being significantly up-regulated, whereas 2165 lncRNAs were identified as being significantly down-regulated compared to nonlesional skin (p < 0.05). Oncogenic and/or epidermis-specific lncRNAs, such as CASC15 or ANRIL, were among the differentially expressed sequences. GO analysis showed that the highest enriched GO targeted by up-regulated transcripts was "extracellular matrix." KEGG pathway analysis showed the highest enrichment scores in "Focal adhesion." BCC showed a significantly altered lncRNA and mRNA expression profile. Dysregulation of previously described lncRNAs may play a role in the molecular pathogenesis of BCC and should be subject of further analysis. PMID:26861560

  6. Long noncoding RNAs are rarely translated in two human cell lines.

    Science.gov (United States)

    Bánfai, Balázs; Jia, Hui; Khatun, Jainab; Wood, Emily; Risk, Brian; Gundling, William E; Kundaje, Anshul; Gunawardena, Harsha P; Yu, Yanbao; Xie, Ling; Krajewski, Krzysztof; Strahl, Brian D; Chen, Xian; Bickel, Peter; Giddings, Morgan C; Brown, James B; Lipovich, Leonard

    2012-09-01

    Data from the Encyclopedia of DNA Elements (ENCODE) project show over 9640 human genome loci classified as long noncoding RNAs (lncRNAs), yet only ~100 have been deeply characterized to determine their role in the cell. To measure the protein-coding output from these RNAs, we jointly analyzed two recent data sets produced in the ENCODE project: tandem mass spectrometry (MS/MS) data mapping expressed peptides to their encoding genomic loci, and RNA-seq data generated by ENCODE in long polyA+ and polyA- fractions in the cell lines K562 and GM12878. We used the machine-learning algorithm RuleFit3 to regress the peptide data against RNA expression data. The most important covariate for predicting translation was, surprisingly, the Cytosol polyA- fraction in both cell lines. LncRNAs are ~13-fold less likely to produce detectable peptides than similar mRNAs, indicating that ~92% of GENCODE v7 lncRNAs are not translated in these two ENCODE cell lines. Intersecting 9640 lncRNA loci with 79,333 peptides yielded 85 unique peptides matching 69 lncRNAs. Most cases were due to a coding transcript misannotated as lncRNA. Two exceptions were an unprocessed pseudogene and a bona fide lncRNA gene, both with open reading frames (ORFs) compromised by upstream stop codons. All potentially translatable lncRNA ORFs had only a single peptide match, indicating low protein abundance and/or false-positive peptide matches. We conclude that with very few exceptions, ribosomes are able to distinguish coding from noncoding transcripts and, hence, that ectopic translation and cryptic mRNAs are rare in the human lncRNAome. PMID:22955977

  7. Identifying functional cancer-specific miRNA-mRNA interactions in testicular germ cell tumor.

    Science.gov (United States)

    Sedaghat, Nafiseh; Fathy, Mahmood; Modarressi, Mohammad Hossein; Shojaie, Ali

    2016-09-01

    Testicular cancer is the most common cancer in men aged between 15 and 35 and more than 90% of testicular neoplasms are originated at germ cells. Recent research has shown the impact of microRNAs (miRNAs) in different types of cancer, including testicular germ cell tumor (TGCT). MicroRNAs are small non-coding RNAs which affect the development and progression of cancer cells by binding to mRNAs and regulating their expressions. The identification of functional miRNA-mRNA interactions in cancers, i.e. those that alter the expression of genes in cancer cells, can help delineate post-regulatory mechanisms and may lead to new treatments to control the progression of cancer. A number of sequence-based methods have been developed to predict miRNA-mRNA interactions based on the complementarity of sequences. While necessary, sequence complementarity is, however, not sufficient for presence of functional interactions. Alternative methods have thus been developed to refine the sequence-based interactions using concurrent expression profiles of miRNAs and mRNAs. This study aims to find functional cancer-specific miRNA-mRNA interactions in TGCT. To this end, the sequence-based predicted interactions are first refined using an ensemble learning method, based on two well-known methods of learning miRNA-mRNA interactions, namely, TaLasso and GenMiR++. Additional functional analyses were then used to identify a subset of interactions to be most likely functional and specific to TGCT. The final list of 13 miRNA-mRNA interactions can be potential targets for identifying TGCT-specific interactions and future laboratory experiments to develop new therapies. PMID:27235586

  8. Pfaffosidic Fraction from Hebanthe paniculata Induces Cell Cycle Arrest and Caspase-3-Induced Apoptosis in HepG2 Cells

    Directory of Open Access Journals (Sweden)

    Tereza Cristina da Silva

    2015-01-01

    Full Text Available Hebanthe paniculata roots (formerly Pfaffia paniculata and popularly known as Brazilian ginseng show antineoplastic, chemopreventive, and antiproliferative properties. Functional properties of these roots and their extracts are usually attributed to the pfaffosidic fraction, which is composed mainly by pfaffosides A–F. However, the therapeutic potential of this fraction in cancer cells is not yet entirely understood. This study aimed to analyze the antitumoral effects of the purified pfaffosidic fraction or saponinic fraction on the human hepatocellular carcinoma HepG2 cell line. Cellular viability, proliferation, and apoptosis were evaluated, respectively, by MTT assay, BrdU incorporation, activated caspase-3 immunocytochemistry, and DNA fragmentation assay. Cell cycle was analyzed by flow cytometry and the cell cycle-related proteins were analyzed by quantitative PCR and Western blot. The cells exposed to pfaffosidic fraction had reduced viability and cellular growth, induced G2/M at 48 h or S at 72 h arrest, and increased sub-G1 cell population via cyclin E downregulation, p27KIP1 overexpression, and caspase-3-induced apoptosis, without affecting the DNA integrity. Antitumoral effects of pfaffosidic fraction from H. paniculata in HepG2 cells originated by multimechanisms of action might be associated with cell cycle arrest in the S phase, by CDK2 and cyclin E downregulation and p27KIP1 overexpression, besides induction of apoptosis through caspase-3 activation.

  9. Stem Cells

    Science.gov (United States)

    Stem cells are cells with the potential to develop into many different types of cells in the body. ... the body. There are two main types of stem cells: embryonic stem cells and adult stem cells. Stem ...

  10. Differential long non‑coding RNA and mRNA expression in differentiated human glioblastoma stem cells.

    Science.gov (United States)

    Li, Hao; Li, Haowen; Hao, Yajing; Jiao, Yuming; Li, Zhicen; Yue, Haiyan; Xu, Zhe; Wang, Shuo; Cao, Yong; Zhao, Jizong

    2016-09-01

    Differentiation of glioblastoma stem cells (GSCs) may lead to inhibition of their self‑renewing ability and tumorigenic potential, as well as increasing their sensitivity to treatment. The critical role of long non‑coding RNAs (lncRNAs) in numerous biological processes has been revealed. However, the involvement of lncRNAs in GSC differentiation remains to be elucidated. In the present study, GSCs were isolated from patient samples and differentiation was induced. Using a high‑throughput microarray, the present study identified a profile of 1,545 lncRNAs and 2,729 mRNAs that differed between GSCs and their non‑differentiated counterparts. To ascertain the association between the altered lncRNAs and mRNAs, a co‑expression network was constructed in which 1,087 lncRNAs and 1,928 mRNAs altered upon GSC differentiation formed a total of 19,642 lncRNA‑mRNA pairs. Based on the co‑expression network, the lncRNA functions were additionally predicted by a cis‑ or trans‑ targeting program. Furthermore, three pairs of lncRNAs and their nearby target mRNAs were selected [ENSG00000261924.1‑regulatory associated protein of MTOR complex 1, ENSG00000235427.1‑caveolin 1 and Tax1 binding protein 3 (TAX1BP3)‑purinergic receptor P2X 5 (P2RX5)‑TAX1BP3] and their expression levels were validated by reverse transcription‑quantitative polymerase chain reaction. The altered lncRNAs were also regulated by various pluripotency transcription factors (POU domain, class 3, transcription factor, sex determining region Y‑box 2, spalt‑like transcription factor 2 and oligodendrocyte lineage transcription factor 2). In conclusion, the results of the present study revealed that lncRNAs may function in GSC differentiation by regulating their target mRNAs, and a set of lncRNAs were identified as candidates for further study concerning the future treatment of GSCs. PMID:27432080

  11. [miR-126 knockdown enhances the activity of murine CD4(+);T cells in vivo and promotes their differentiation into Th1 cells].

    Science.gov (United States)

    Cui, Panpan; Hu, Yan; Tao, Yijing; Chen, Chao; Zhao, Juanjuan; Guo, Mengmeng; Zhou, Ya; Xu, Lin

    2016-03-01

    Objective To investigate the change of CD4(+);T cell activity in microRNA-126 (miR-126) knockdown (KD) mice and explore its significance. Methods The expression level of mature miR-126 in CD4(+);CD62L(+);T cells purified by magnetic-activated cell sorting (MACS) was analyzed by real-time PCR using specific probe. Furthermore, the expression levels of CD69, CD62L and CD44 molecules, as well as intracellular proliferating nuclear antigen Ki-67, in CD4(+);T cells in miR-126 KD mice were detected by fluorescence-activated cell sorting (FACS). Moreover, the apoptosis of CD4(+);T cells was analyzed by annexin V/PI staining assay combined with flow cytometry. Finally, the relative expressions of function-related cytokines including interleukine 4 (IL-4), IL-10, IL-12, transforming growth factor (TGF-β), interferon (IFN-γ) and tumor necrosis factor (TNF-α) in CD4(+);T cells were determined by real-time PCR. Results Compared with wild-type (WT) mice, the expression level of mature miR-126 in CD4(+);T cells in miR-126 KD mice was dramatically reduced. Furthermore, the proportion of CD62L(+); in CD4(+);T cells also decreased significantly, while the proportions of CD69(+);, CD44(+); and Ki-67(+); cells were remarkably elevated. Meanwhile, the apoptosis proportion of CD4(+);T cells in vivo dropped dramatically in miR-126 KD mice. Finally, the mRNA expressions of IL-4 and IL-10 in CD4(+);T cells were significantly downregulated, but IL-12, TGF-β, TNF-α and IFN-γ mRNAs were obviously up-regulated. Conclusion miR-126 knockdown could significantly enhance the functional activity of CD4(+); T cells in vivo and promote cell differentiation into Th1 cells. PMID:26927555

  12. Inferring yeast cell cycle regulators and interactions using transcription factor activities

    Directory of Open Access Journals (Sweden)

    Galbraith Simon J

    2005-06-01

    Full Text Available Abstract Background Since transcription factors are often regulated at the post-transcriptional level, their activities, rather than expression levels may provide valuable information for investigating functions and their interactions. The recently developed Network Component Analysis (NCA and its generalized form (gNCA provide a robust framework for deducing the transcription factor activities (TFAs from various types of DNA microarray data and transcription factor-gene connectivity. The goal of this work is to demonstrate the utility of TFAs in inferring transcription factor functions and interactions in Saccharomyces cerevisiae cell cycle regulation. Results Using gNCA, we determined 74 TFAs from both wild type and fkh1 fkh2 deletion mutant microarray data encompassing 1529 ORFs. We hypothesized that transcription factors participating in the cell cycle regulation exhibit cyclic activity profiles. This hypothesis was supported by the TFA profiles of known cell cycle factors and was used as a basis to uncover other potential cell cycle factors. By combining the results from both cluster analysis and periodicity analysis, we recovered nearly 90% of the known cell cycle regulators, and identified 5 putative cell cycle-related transcription factors (Dal81, Hap2, Hir2, Mss11, and Rlm1. In addition, by analyzing expression data from transcription factor knockout strains, we determined 3 verified (Ace2, Ndd1, and Swi5 and 4 putative interaction partners (Cha4, Hap2, Fhl1, and Rts2 of the forkhead transcription factors. Sensitivity of TFAs to connectivity errors was determined to provide confidence level of these predictions. Conclusion By subjecting TFA profiles to analyses based upon physiological signatures we were able to identify cell cycle related transcription factors consistent with current literature, transcription factors with potential cell cycle dependent roles, and interactions between transcription factors.

  13. Functional dissection of Caenorhabditis elegans CLK-2/TEL2 cell cycle defects during embryogenesis and germline development.

    Directory of Open Access Journals (Sweden)

    Sandra C Moser

    2009-04-01

    Full Text Available CLK-2/TEL2 is essential for viability from yeasts to vertebrates, but its essential functions remain ill defined. CLK-2/TEL2 was initially implicated in telomere length regulation in budding yeast, but work in Caenorhabditis elegans has uncovered a function in DNA damage response signalling. Subsequently, DNA damage signalling defects associated with CLK-2/TEL2 have been confirmed in yeast and human cells. The CLK-2/TEL2 interaction with the ATM and ATR DNA damage sensor kinases and its requirement for their stability led to the proposal that CLK-2/TEL2 mutants might phenocopy ATM and/or ATR depletion. We use C. elegans to dissect developmental and cell cycle related roles of CLK-2. Temperature sensitive (ts clk-2 mutants accumulate genomic instability and show a delay of embryonic cell cycle timing. This delay partially depends on the worm p53 homolog CEP-1 and is rescued by co-depletion of the DNA replication checkpoint proteins ATL-1 (C. elegans ATR and CHK-1. In addition, clk-2 ts mutants show a spindle orientation defect in the eight cell stages that lead to major cell fate transitions. clk-2 deletion worms progress through embryogenesis and larval development by maternal rescue but become sterile and halt germ cell cycle progression. Unlike ATL-1 depleted germ cells, clk-2-null germ cells do not accumulate DNA double-strand breaks. Rather, clk-2 mutant germ cells arrest with duplicated centrosomes but without mitotic spindles in an early prophase like stage. This germ cell cycle arrest does not depend on cep-1, the DNA replication, or the spindle checkpoint. Our analysis shows that CLK-2 depletion does not phenocopy PIKK kinase depletion. Rather, we implicate CLK-2 in multiple developmental and cell cycle related processes and show that CLK-2 and ATR have antagonising functions during early C. elegans embryonic development.

  14. Radiation-responsive transcriptome analysis in human lymphoid cells

    International Nuclear Information System (INIS)

    Ionising radiation (IR) causes DNA (deoxyribonucleic acid) injury and activates intracellular signal pathways including the regulation of DNA repair and cell cycle. However, the further knowledge of molecular events involved in radiation exposure is essential to more comprehensively understand the effects of irradiation. Therefore, the gene expressions of mRNA (messenger ribonucleic acid) by X-ray irradiation in human B lymphoblasts cell line (IM-9) using a microarray were investigated. The mRNA expressions of 65 genes were shown to be up-regulated at >2.0-fold in irradiated cells (4 Gy) when compared with non-irradiated cells (0 Gy) by microarray analysis. Among 65 genes, a large number of genes were up-regulated with an X-ray dose-dependent change. These results indicate that the up-regulation of their mRNAs is the effects of irradiation and may be due to biological dosimetric markers for the evaluation of radiation exposure in the future. (authors)

  15. Paris chinensis dioscin induces G2/M cell cycle arrest and apoptosis in human gastric cancer SGC-7901 cells

    Institute of Scientific and Technical Information of China (English)

    Lin-Lin Gao; Fu-Rong Li; Peng Jiao; Ming-Feng Yang; Xiao-Jun Zhou; Yan-Hong Si; Wen-Jian Jiang; Ting-Ting Zheng

    2011-01-01

    AIM: To investigate the anti-tumor effects of Paris chinensis dioscin (PCD) and mechanisms regarding cell cycle regulation and apoptosis in human gastric cancer SGC-7901 cells.METHODS: Cell viability was analyzed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay. Cell apoptosis was evaluated by flow cytometry and laser scanning confocal microscope (LSCM) using Annexin-V/propidium iodide (PI) staining, and the cell cycle was evaluated using PI staining with flow cytometry. Intracellular calcium ions were detected under fluorescence microscope. The expression of cell cycle and apoptosis-related proteins cyclin B1, CDK1, cytochrome C and caspase-3 was measured by immunohistochemical staining. RESULTS: PCD had an anti-proliferation effect on human gastric cancer SGC-7901 cells in a dose- and time-dependent manner. After treatment of SGC-7901 cells with PCD, apoptosis appeared in SGC-7901 cells. Morphological changes typical of apoptosis were also observed with LSCM by Annexin V/PI staining, and the cell number of the G0/G1 phase was decreased, while the number of cells in the G2/M phase was increased. Cell cycle-related proteins, such as cyclin B1 and CDK1, were all down-regulated, but caspase-3 and cytochrome C were up-regulated. Moreover, intracellular calcium accumulation occurred in PCD-treated cells. CONCLUSION: G2/M phase arrest and apoptosis induced by PCD are associated with the inhibition of CDK-activating kinase activity and the activation of Ca2+-related mitochondrion pathway in SGC-7901 cells.

  16. A systematic study on drug-response associated genes using baseline gene expressions of the Cancer Cell Line Encyclopedia

    Science.gov (United States)

    Liu, Xiaoming; Yang, Jiasheng; Zhang, Yi; Fang, Yun; Wang, Fayou; Wang, Jun; Zheng, Xiaoqi; Yang, Jialiang

    2016-03-01

    We have studied drug-response associated (DRA) gene expressions by applying a systems biology framework to the Cancer Cell Line Encyclopedia data. More than 4,000 genes are inferred to be DRA for at least one drug, while the number of DRA genes for each drug varies dramatically from almost 0 to 1,226. Functional enrichment analysis shows that the DRA genes are significantly enriched in genes associated with cell cycle and plasma membrane. Moreover, there might be two patterns of DRA genes between genders. There are significantly shared DRA genes between male and female for most drugs, while very little DRA genes tend to be shared between the two genders for a few drugs targeting sex-specific cancers (e.g., PD-0332991 for breast cancer and ovarian cancer). Our analyses also show substantial difference for DRA genes between young and old samples, suggesting the necessity of considering the age effects for personalized medicine in cancers. Lastly, differential module and key driver analyses confirm cell cycle related modules as top differential ones for drug sensitivity. The analyses also reveal the role of TSPO, TP53, and many other immune or cell cycle related genes as important key drivers for DRA network modules. These key drivers provide new drug targets to improve the sensitivity of cancer therapy.

  17. Regulation and function of the IL-1 family cytokine IL-1F9 in human bronchial epithelial cells.

    Science.gov (United States)

    Chustz, Regina T; Nagarkar, Deepti R; Poposki, Julie A; Favoreto, Silvio; Avila, Pedro C; Schleimer, Robert P; Kato, Atsushi

    2011-07-01

    The IL-1 family of cytokines, which now includes 11 members, is well known to participate in inflammation. Although the most recently recognized IL-1 family cytokines (IL-1F5-11) have been shown to be expressed in airway epithelial cells, the regulation of their expression and function in the epithelium has not been extensively studied. We investigated the regulation of IL-1F5-11 in primary normal human bronchial epithelial cells. Messenger (m)RNAs for IL-1F6 and IL-1F9, but not IL-1F5, IL-1F8 or IL-1F10, were significantly up-regulated by TNF, IL-1β, IL-17 and the Toll-like receptor (TLR)3 ligand double-stranded (ds)RNA. mRNAs for IL-1F7 and IL-1F11 (IL-33) were weakly up-regulated by some of the cytokines tested. Notably, mRNAs for IL-1F6 and IL-1F9 were synergistically enhanced by the combination of TNF/IL-17 or dsRNA/IL-17. IL-1F9 protein was detected in the supernatant following stimulation with dsRNA or a combination of dsRNA and IL-17. IL-1F6 protein was detected in the cell lysate but was not detected in the supernatant. We screened for the receptor for IL-1F9 and found that lung fibroblasts expressed this receptor. We found that IL-1F9 activated mitogen-activated protein kinases and the transcription factor NF-κB in primary normal human lung fibroblasts. IL-1F9 also stimulated the expression of the neutrophil chemokines IL-8 and CXCL3 and the Th17 chemokine CCL20 in lung fibroblasts. These results suggest that epithelial activation by TLR3 (e.g., by respiratory viral infection) and exposure to cytokines from Th17 cells (IL-17) and inflammatory cells (TNF) may amplify neutrophilic inflammation in the airway via induction of IL-1F9 and activation of fibroblasts. PMID:20870894

  18. 1α,25-Dihydroxyvitamin D3 Regulates Mitochondrial Oxygen Consumption and Dynamics in Human Skeletal Muscle Cells.

    Science.gov (United States)

    Ryan, Zachary C; Craig, Theodore A; Folmes, Clifford D; Wang, Xuewei; Lanza, Ian R; Schaible, Niccole S; Salisbury, Jeffrey L; Nair, K Sreekumaran; Terzic, Andre; Sieck, Gary C; Kumar, Rajiv

    2016-01-15

    Muscle weakness and myopathy are observed in vitamin D deficiency and chronic renal failure, where concentrations of the active vitamin D3 metabolite, 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), are low. To evaluate the mechanism of action of 1α,25(OH)2D3 in skeletal muscle, we examined mitochondrial oxygen consumption, dynamics, and biogenesis and changes in expression of nuclear genes encoding mitochondrial proteins in human skeletal muscle cells following treatment with 1α,25(OH)2D3. The mitochondrial oxygen consumption rate (OCR) increased in 1α,25(OH)2D3-treated cells. Vitamin D3 metabolites lacking a 1α-hydroxyl group (vitamin D3, 25-hydroxyvitamin D3, and 24R,25-dihydroxyvitamin D3) decreased or failed to increase OCR. 1α-Hydroxyvitamin D3 did not increase OCR. In 1α,25(OH)2D3-treated cells, mitochondrial volume and branching and expression of the pro-fusion protein OPA1 (optic atrophy 1) increased, whereas expression of the pro-fission proteins Fis1 (fission 1) and Drp1 (dynamin 1-like) decreased. Phosphorylated pyruvate dehydrogenase (PDH) (Ser-293) and PDH kinase 4 (PDK4) decreased in 1α,25(OH)2D3-treated cells. There was a trend to increased PDH activity in 1α,25(OH)2D3-treated cells (p = 0.09). 83 nuclear mRNAs encoding mitochondrial proteins were changed following 1α,25(OH)2D3 treatment; notably, PDK4 mRNA decreased, and PDP2 mRNA increased. MYC, MAPK13, and EPAS1 mRNAs, which encode proteins that regulate mitochondrial biogenesis, were increased following 1α,25(OH)2D3 treatment. Vitamin D receptor-dependent changes in the expression of 1947 mRNAs encoding proteins involved in muscle contraction, focal adhesion, integrin, JAK/STAT, MAPK, growth factor, and p53 signaling pathways were observed following 1α,25(OH)2D3 treatment. Five micro-RNAs were induced or repressed by 1α,25(OH)2D3. 1α,25(OH)2D3 regulates mitochondrial function, dynamics, and enzyme function, which are likely to influence muscle strength. PMID:26601949

  19. Nanoscale bio-platforms for living cell interrogation: current status and future perspectives

    Science.gov (United States)

    Chang, Lingqian; Hu, Jiaming; Chen, Feng; Chen, Zhou; Shi, Junfeng; Yang, Zhaogang; Li, Yiwen; Lee, Ly James

    2016-02-01

    The living cell is a complex entity that dynamically responds to both intracellular and extracellular environments. Extensive efforts have been devoted to the understanding intracellular functions orchestrated with mRNAs and proteins in investigation of the fate of a single-cell, including proliferation, apoptosis, motility, differentiation and mutations. The rapid development of modern cellular analysis techniques (e.g. PCR, western blotting, immunochemistry, etc.) offers new opportunities in quantitative analysis of RNA/protein expression up to a single cell level. The recent entries of nanoscale platforms that include kinds of methodologies with high spatial and temporal resolution have been widely employed to probe the living cells. In this tutorial review paper, we give insight into background introduction and technical innovation of currently reported nanoscale platforms for living cell interrogation. These highlighted technologies are documented in details within four categories, including nano-biosensors for label-free detection of living cells, nanodevices for living cell probing by intracellular marker delivery, high-throughput platforms towards clinical current, and the progress of microscopic imaging platforms for cell/tissue tracking in vitro and in vivo. Perspectives for system improvement were also discussed to solve the limitations remains in current techniques, for the purpose of clinical use in future.

  20. Signaling pathways involved in PDGF-evoked cellular responses in human RPE cells

    International Nuclear Information System (INIS)

    We examined whether PDGF may directly stimulate the expression of VEGF by retinal pigment epithelial (RPE) cells in vitro, and the involvement of three signal transduction pathways in the regulation of PDGF-evoked cell proliferation, migration, and production of VEGF-A was investigated. PDGF stimulated the gene and protein expression of VEGF-A by RPE cells, and increased cell proliferation and chemotaxis. PDGF activated all signaling pathways investigated, as determined by increased phosphorylation levels of ERK1/2, p38, and Akt proteins. The three signaling pathways were involved in the mediation of PDGF-evoked cell proliferation, while p38 and PI3K mediated cell migration, and PI3K mediated secretion of VEGF-A. In addition to VEGF-A, the cells expressed mRNAs for various members of the VEGF family and for their receptors, including VEGF-B, -C, -D, flt-1, and KDR. The data indicate that PDGF selectively stimulates the expression of VEGF-A in RPE cells. PDGF evokes at least three signal transduction pathways which are differentially involved in various cellular responses

  1. Energy metabolism and ATP turnover time during the cell cycle in roentgen irradiated Ehrlich ascites tumour cells

    International Nuclear Information System (INIS)

    The cell-cycle related energy metabolism after roentgen irradiation of in vivo growing Ehrlich ascites tumour cells was investigated in cell fractions obtained by elutriator centrifuging. The oxygen consumption and the lactate and pyruvate production, measured in vitro after 4.5, 5 and 9 Gy up to 24 h, were undisturbed, while the decrease in the in vivo ATP content was dose-independent in all parts of the cell cycle. On the basis of these data the ATP turnover time was found to be decreased. The decrease in the ATP content is considered to be less likely to be due to membrane leakage or increased ATP consumption than to reduced ATP production. Since in vitro incubation normalizes the ATP content, it is suggested that environmental factors in the ascites liquid after irradiation cause a decrease in the ATP production. Low ATP contents of the cells do not appear to influence the irradiation-induced changes in the cell flow through the cell cycle. (Auth.)

  2. Asparanin A induces G(2)/M cell cycle arrest and apoptosis in human hepatocellular carcinoma HepG2 cells.

    Science.gov (United States)

    Liu, Wei; Huang, Xue-Feng; Qi, Qi; Dai, Qin-Sheng; Yang, Li; Nie, Fei-Fei; Lu, Na; Gong, Dan-Dan; Kong, Ling-Yi; Guo, Qing-Long

    2009-04-17

    We recently established that asparanin A, a steroidal saponin extracted from Asparagus officinalis L., is an active cytotoxic component. The molecular mechanisms by which asparanin A exerts its cytotoxic activity are currently unknown. In this study, we show that asparanin A induces G(2)/M phase arrest and apoptosis in human hepatocellular carcinoma HepG2 cells. Following treatment of HepG2 cells with asparanin A, cell cycle-related proteins such as cyclin A, Cdk1 and Cdk4 were down-regulated, while p21(WAF1/Cip1) and p-Cdk1 (Thr14/Tyr15) were up-regulated. Additionally, we observed poly (ADP-ribose) polymerase (PARP) cleavage and activation of caspase-3, caspase-8 and caspase-9. The expression ratio of Bax/Bcl-2 was increased in the treated cells, where Bax was also up-regulated. We also found that the expression of p53, a modulator of p21(WAF1/Cip1) and Bax, was not affected in asparanin A-treated cells. Collectively, our findings demonstrate that asparanin A induces cell cycle arrest and triggers apoptosis via a p53-independent manner in HepG2 cells. These data indicate that asparanin A shows promise as a preventive and/or therapeutic agent against human hepatoma. PMID:19254688

  3. Molecular characterization of clear cell renal cell carcinoma identifies CSNK2A1, SPP1 and DEFB1 as promising novel prognostic markers.

    Science.gov (United States)

    Rabjerg, Maj; Bjerregaard, Henriette; Halekoh, Ulrich; Jensen, Boye L; Walter, Steen; Marcussen, Niels

    2016-05-01

    The prognosis associated with clear cell renal carcinoma (ccRCC) can vary widely and novel molecular prognostic markers are needed to assess prognosis at an earlier stage. Several gene products have been investigated for this purpose, but none of them have been implemented in clinical practice. Here we hypothesized that we, using TaqMan(®) Array, could identify superior prognostic messenger RNA (mRNA)s in long-term follow-up. Messenger RNA level of 19 candidate genes was investigated in 97 patients with ccRCC. Three genes impacted significantly on prognosis in both univariate and multivariate analysis. In univariate analysis, CSNK2A1 was a strong indicator of a poor overall survival (OS) (HR = 5.01, p renal cancer development. PMID:26876164

  4. Single-cell transcriptomics and functional target validation of brown adipocytes show their complex roles in metabolic homeostasis.

    Science.gov (United States)

    Spaethling, Jennifer M; Sanchez-Alavez, Manuel; Lee, JaeHee; Xia, Feng C; Dueck, Hannah; Wang, Wenshan; Fisher, Stephen A; Sul, Jai-Yoon; Seale, Patrick; Kim, Junhyong; Bartfai, Tamas; Eberwine, James

    2016-01-01

    Brown adipocytes (BAs) are specialized for adaptive thermogenesis and, upon sympathetic stimulation, activate mitochondrial uncoupling protein (UCP)-1 and oxidize fatty acids to generate heat. The capacity for brown adipose tissue (BAT) to protect against obesity and metabolic disease is recognized, yet information about which signals activate BA, besides β3-adrenergic receptor stimulation, is limited. Using single-cell transcriptomics, we confirmed the presence of mRNAs encoding traditional BAT markers (i.e., UCP1, expressed in 100% of BAs Adrb3, expressed in 1000-fold) in their expression at both the mRNA and protein levels. We further identified mRNAs encoding novel markers, orphan GPCRs, and many receptors that bind the classic neurotransmitters, neuropeptides, chemokines, cytokines, and hormones. The transcriptome variability between BAs suggests a much larger range of responsiveness of BAT than previously recognized and that not all BAs function identically. We examined the in vivo functional expression of 12 selected receptors by microinjecting agonists into live mouse BAT and analyzing the metabolic response. In this manner, we expanded the number of known receptors on BAs at least 25-fold, while showing that the expression of classic BA markers is more complex and variable than previously thought. PMID:26304220

  5. Prevention of Simvastatin-Induced Inhibition of Tendon Cell Proliferation and Cell Cycle Progression by Geranylgeranyl Pyrophosphate.

    Science.gov (United States)

    Tsai, Wen-Chung; Yu, Tung-Yang; Lin, Li-Ping; Cheng, Mei-Ling; Chen, Cheng-Lun; Pang, Jong-Hwei S

    2016-02-01

    Statins have been reported to induce tendinopathy and even tendon rupture. The present study was designed to investigate the potential molecular mechanism underlying the adverse effect of simvastatin on tendon cells. An in vitro tendon healing model was performed using tendon cells isolated from rat Achilles tendons. The viability of tendon cells and cell cycle progression were examined by the MTT assay and flow cytometric analysis, respectively. Immunofluorescent staining for Ki-67 was used to assess the proliferation activity of tendon cells. Western blot analysis and coimmunoprecipitation was used to determine the protein expression of cell cycle-related proteins. To investigate the potential mechanism underlying the effect of statins on tendon cells, mevalonate, farnesyl pyrophosphate (FPP), or geranylgeranyl pyrophosphate (GGPP) was added to simvastatin-treated tendon cells. Simvastatin inhibited the in vitro tendon healing model and tendon cell proliferation in a dose-dependent manner. Immunofluorescent staining demonstrated reduced ki-67 expression in simvastatin-treated tendon cells. Furthermore, simvastatin induced cell cycle arrest at the G1 phase. The expression levels of cdk1, cdk2, cyclin A, and cyclin E were downregulated by simvastatin in a dose-dependent manner. The inhibitory effect of simvastatin was proved to mediate the reduction of mevalonate, and the addition of exogenous GGPP completely prevented the inhibitory effect of simvastatin on tendon cells. The present study demonstrated, for the first time, the molecular mechanism underlying simvastatin-induced tendinopathy or tendon rupture. GGPP was shown to prevent the adverse effect of simvastatin in tendon cells without interfering with its cholesterol-reducing efficacy. PMID:26577051

  6. Differentiation-Associated MicroRNA Alterations in Mouse Heart-Derived Sca-1+CD31− and Sca-1+CD31+ Cells

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    Qiong Wu

    2016-01-01

    Full Text Available Cardiac resident stem/progenitor cells (CSC/CPCs are critical to the cellular and functional integrity of the heart because they maintain myocardial cell homeostasis. Several populations of CSC/CPCs have been identified based on expression of different stem cell-associated antigens. Sca-1+ cells in the cardiac tissue may be the most common CSC/CPCs. However, they are a heterogeneous cell population and, in transplants, clinicians might transplant more endothelial cells, cardiomyocytes, or other cells than stem cells. The purposes of this study were to (1 isolate CSC/CPCs with Lin−CD45−Sca-1+CD31− and Lin−CD45−Sca-1+CD31+ surface antigens using flow-activated cell sorting; (2 investigate their differentiation potential; and (3 determine the molecular basis for differences in stemness characteristics between cell subtypes. The results indicated that mouse heart-derived Sca-1+CD31− cells were multipotent and retained the ability to differentiate into different cardiac cell lineages, but Sca-1+CD31+ cells did not. Integrated analysis of microRNA and mRNA expression indicated that 20 microRNAs and 49 mRNAs were inversely associated with Sca-1+CD31− and Sca-1+CD31+ subtype stemness characteristics. In particular, mmu-miR-322-5p had more targeted and inversely associated genes and transcription factors and might have higher potential for CSC/CPCs differentiation.

  7. Comparison of phenotype characteristics of rat annulus fibrosus cells cultured on flexible silicone membrane and in plastic plate

    Institute of Scientific and Technical Information of China (English)

    GUO Zhi-liang; CHENG Min; CAO Guo-yong; LI Hua-zhuang; TENG Hai-jun; ZHOU Yue

    2006-01-01

    Objective:To compare the phenotype characteristics of rat annulus fibrosus (AF) cells cultured on flexible silicone membranes and those in plastic plates. Methods :The morphology of AF cells cultured in different substrates was examined. Proteoglycan was stained by toluidine blue. Contents of collagen type I , collagen type Ⅱ and aggrecan mRNAs were determined by reverse transcription-polymerase chain reaction (RT-PCR). The expression of integrin β1 was monitored by flow cytometry. By using propidium iodide (PI), the cell cycle in AF cells was analyzed. Cell adhesion to silicone membrane was also measured. Results:The AF cells cultured on different substrates were morphologically undistinguishable.Toluidine blue staining showed that there was also no difference between AF cells cultured on these 2 substrates. They still had the same expression levels of collagen type Ⅰ , collagen type Ⅱ , aggrecan mRNAs,and integrin β1. No significant difference was observed in the distribution of the cell cycle. AF cells grew well on silicone membrane. Conclusion:AF cells cultured on flexible silicone membrane maintain the stability of phenotype and may be appropriate for further studying the metabolic responses to mechanical stimuli at the cellular level.

  8. Graft-infiltrating cells expressing a CD200 transgene prolong allogeneic skin graft survival in association with local increases in Foxp3(+)Treg and mast cells.

    Science.gov (United States)

    Gorczynski, Reginald M; Chen, Zhiqi; Khatri, Ismat; Yu, Kai

    2011-12-01

    Expression of the molecule CD200 has been reported to increase allograft survival by suppression of inflammation and acquired immunity. In previous studies we have shown that increased skin and cardiac allograft survival in transgenic mice over-expressing CD200 (CD200(tg)) occurs in association with increased intra-graft expression of mRNAs for genes associated with altered T cell subset differentiation. We investigated changes in graft-infiltrating cells, Treg and mast cells in skin grafts post transplantation into control or CD200(tg) mice, using focused gene array and real-time PCR to assess altered gene expression, and FACS, immunohistology and MLC to determine numbers/function of those cells. Graft-infiltrating cells isolated from CD200(tg) recipients suppressed induction of CTL from control lymph node cells in vitro, and contained increased numbers of infiltrating, non-degranulating, mast cells and Foxp3(+)Treg. Mast cells were also evident in graft tissue of control animals, but there these cells showed evidence for degranulation, and fewer Foxp3(+)Treg were present than was the case of CD200(tg) mice. The infusion of a competitive inhibitor of CD200:CD200R interactions, CD200(tr), at high concentrations (50μg/mouse iv) caused rapid rejection of grafts in CD200(tg) mice, mast cell degranulation within graft tissue, and a decrease in Treg infiltrates. These effects were attenuated by simultaneous infusion of the mast cell stabilizer, sodium cromoglycate. We conclude that CD200 expression contributes to graft prolongation through local suppression of mast cell degranulation, attraction/expansion of Treg, and attenuation of T cell effector activation. PMID:21801836

  9. Bone marrow-derived cultured mast cells and peritoneal mast cells as targets of a growth activity secreted by BALB/3T3 fibroblasts

    International Nuclear Information System (INIS)

    When fibroblast cell lines were cultured in contact with bone marrow-derived cultured mast cells (CMC), both NIH/3T3 and BALB/3T3 cell lines supported the proliferation of CMC. In contrast, when contact between fibroblasts and CMC was prohibited by Biopore membranes or soft agar, only BALB/3T3 fibroblasts supported CMC proliferation, suggesting that BALB/3T3 but not NIH/3T3 cells secreted a significant amount of a mast cell growth activity. Moreover, the BALB/3T3-derived growth activity induced the incorporation of [3H]thymidine by CMC and the clonal growth of peritoneal mast cells in methylcellulose. The mast cell growth activity appeared to be different from interleukin 3 (IL-3) and interleukin 4 (IL-4), because mRNAs for these interleukins were not detectable in BALB/3T3 fibroblasts. Although mast cells are genetically deficient in tissues of W/Wv mice, CMC did develop when bone marrow cells of W/Wv mice were cultured with pokeweed mitogen-stimulated spleen cell-conditioned medium. Because BALB/3T3 fibroblast-conditioned medium (BALB-FCM) did not induce the incorporation of [3H]thymidine by W/Wv CMC, the growth activity in BALB-FCM appeared to be a ligand for the receptor encoded by the W (c-kit) locus. Because CMC and peritoneal mast cells are obtained as homogeneous suspensions rather easily, these cells may be potentially useful as targets for the fibroblast-derived mast cell growth activity

  10. In vivo robustness analysis of cell division cycle genes in Saccharomyces cerevisiae.

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    Hisao Moriya

    2006-07-01

    Full Text Available Intracellular biochemical parameters, such as the expression level of gene products, are considered to be optimized so that a biological system, including the parameters, works effectively. Those parameters should have some permissible range so that the systems have robustness against perturbations, such as noise in gene expression. However, little is known about the permissible range in real cells because there has been no experimental technique to test it. In this study, we developed a genetic screening method, named "genetic tug-of-war" (gTOW that evaluates upper limit copy numbers of genes in a model eukaryote Saccharomyces cerevisiae, and we applied it for 30 cell-cycle related genes (CDC genes. The experiment provided unique quantitative data that could be used to argue the system-level properties of the cell cycle such as robustness and fragility. The data were used to evaluate the current computational model, and refinements to the model were suggested.

  11. Phase-specific polypeptides and poly(A) sup + RNAs during the cell cycle in synchronous cultures of Catharanthus roseus cells

    Energy Technology Data Exchange (ETDEWEB)

    Kodama, Hiroaki; Komamine, Atsushi (Tohoku Univ., Sendai (Japan)); Kawakami, Naoto; Watanabe, Akira (Nagoya Univ. (Japan))

    1989-03-01

    This study shows an overall analysis of gene expression during the cell cycle in synchronous suspension cultures of Catharanthus roseus cells. First, the cellular cytoplasmic proteins were fractionated by two-dimensional gel electrophoresis and visualized by staining with silver. Seventeen polypeptides showed qualitative or quantitative changes during the cell cycle. Second, the rates of synthesis of cytoplasmic proteins were also investigated by autoradiography by labeling cells with ({sup 35}S)methionine at each phase of the cell cycle. The rates of synthesis of 13 polypeptides were found to vary during the cell cycle. The silver-stained electrophoretic pattern of proteins in the G{sub 2} phase in particular showed characteristic changes in levels of polypeptides, while the rates of synthesis of polypeptides synthesized during the G{sub 2} phase did not show such phase-specific changes. This result suggest that posttranslational processing of polypeptides occurs during or prior to the G{sub 2} phase. In the G{sub 1} and S phases and during cytokinesis, several other polypeptides were specifically synthesized. Finally, the variation of mRNAs was analyzed from the autoradiograms of in vitro translation products of poly(A){sup +} RNA isolated at each phase. Three poly(A){sup +} RNAs increased in amount from the G{sub 1} to the S phase and one poly(A){sup +} RNA increased preferentially from the G{sub 2} phase to cytokinesis.

  12. Phase-Specific Polypeptides and Poly(A)+ RNAs during the Cell Cycle in Synchronous Cultures of Catharanthus roseus Cells 1

    Science.gov (United States)

    Kodama, Hiroaki; Kawakami, Naoto; Watanabe, Akira; Komamine, Atsushi

    1989-01-01

    This study shows an overall analysis of gene expression during the cell cycle in synchronous suspension cultures of Catharanthus roseus cells. First, the cellular cytoplasmic proteins were fractionated by two-dimensional gel electrophoresis and visualized by staining with silver. Seventeen polypeptides showed qualitative or quantitative changes during the cell cycle. Second, the rates of synthesis of cytoplasmic proteins were also investigated by autoradiography by labeling cells with [35S]methionine at each phase of the cell cycle. The rates of synthesis of 13 polypeptides were found to vary during the cell cycle. The silverstained electrophoretic pattern of proteins in the G2 phase in particular showed characteristic changes in levels of polypeptides, while the rates of synthesis of polypeptides synthesized during the G2 phase did not show such phase-specific changes. This result suggests that posttranslational processing of polypeptides occurs during or prior to the G2 phase. In the G1 and S phases and during cytokinesis, several other polypeptides were specifically synthesized. Finally, the variation of mRNAs was analyzed from the autoradiograms of in vitro translation products of poly(A)+ RNA isolated at each phase. Three poly(A)+ RNAs increased in amount from the G1 to the S phase and one poly (A)+ RNA increased preferentially from the G2 phase to cytokinesis. Images Figure 1 Figure 3 Figure 4 Figure 6 Figure 7 Figure 8 Figure 10 Figure 11 Figure 12 PMID:16666641

  13. Menstrual Cycle-Related Changes of Functional Cerebral Asymmetries in Fine Motor Coordination

    Science.gov (United States)

    Bayer, Ulrike; Hausmann, Markus

    2012-01-01

    Fluctuating sex hormone levels during the menstrual cycle have been shown to affect functional cerebral asymmetries in cognitive domains. These effects seem to result from the neuromodulatory properties of sex hormones and their metabolites on interhemispheric processing. The present study was carried out to investigate whether functional cerebral…

  14. CO 2 degassing and trapping during hydrothermal cycles related to Gondwana rifting in eastern Australia

    Science.gov (United States)

    Uysal, I. Tonguç; Golding, Suzanne D.; Bolhar, Robert; Zhao, Jian-xin; Feng, Yue-xing; Baublys, Kim A.; Greig, Alan

    2011-10-01

    Intensive carbonate and clay mineral authigenesis took place throughout the Late Permian Bowen-Gunnedah-Sydney basin system in eastern Australia. We conducted isotopic and trace element analyses of carbonate and clay minerals from clastic sedimentary rocks of the Gunnedah Basin and the Denison Trough in the Bowen Basin. Rb-Sr isochron age data of the illitic clays are consistent with episodic hydrothermal fluid flow events that occurred in association with Gondwana rifting accompanied by alkaline magmatism at ˜85 Ma and ˜95 Ma. Stable isotope data of carbonate and clay minerals from the Gunnedah Basin are indicative of meteoric waters from a high-latitude environment as the main fluid source, whereas trace element, Sr and Nd isotope data highlight mixing of meteoric fluids with magmatic and/or crustal components, with a possible input from marine carbonates for some samples. Trace metals, oxygen and strontium isotopes of dawsonites from the Denison Trough are interpreted to have been mobilised by fluids that interacted with evolved clastic sedimentary and marine carbonate end members. According to the carbon isotope data, CO 2 for calcite and ankerite precipitation was sourced mainly from thermal degradation of organic matter and magmatism, whereas the CO 2 used for dawsonite formation is inferred to have been derived from magmatic and marine sources. In the low permeability environments (particularly in coal seams), the increasing accumulation and oversaturation of CO 2 particularly promote the precipitation of dawsonite.

  15. Fuel cycle related parametric study considering long lived actinide production, decay heat and fuel cycle performances

    International Nuclear Information System (INIS)

    One of the very attractive HTGR reactor characteristics is its highly versatile and flexible core that can fulfil a wide range of diverse fuel cycles. Based on a GTMHR-600 MWth reactor, analyses of several fuel cycles were carried out without taking into account common fuel particle performance limits (burnup, fast fluence, temperature). These values are, however, indicated in each case. Fuel derived from uranium, thorium and a wide variety of plutonium grades has been considered. Long-lived actinide production and total residual decay heat were evaluated for the various types of fuel. The results presented in this papers provide a comparison of the potential and limits of each fuel cycle and allow to define specific cycles offering lowest actinide production and residual heat associated with a long life cycle. (author)

  16. Global translation variations in host cells upon attack of lytic and sublytic Staphylococcus aureus α-haemolysin.

    Science.gov (United States)

    Clamer, Massimiliano; Tebaldi, Toma; Marchioretto, Marta; Bernabò, Paola; Bertini, Efrem; Guella, Graziano; Dalla Serra, Mauro; Quattrone, Alessandro; Viero, Gabriella

    2015-11-15

    Genome-wide analyses of translation can provide major contributions in our understanding of the complex interplay between virulent factors and host cells. So far, the activation of host translational control mechanisms by bacterial toxins, owing to specific recruitment of mRNAs, RNA-binding proteins (RBPs) and ncRNAs (non-coding RNAs), are far from being understood. In the present study, we characterize for the first time the changes experienced by the translational control system of host cells in response to the well-known Staphylococcus aureus α-haemolysin (AHL) under both sublytic and lytic conditions. By comparing variations occurring in the cellular transcriptome and translatome, we give evidence that global gene expression is primarily rewired at the translational level, with the contribution of the RBP ELAVL1 (HuR) in the sublytic response. These results reveal the importance of translational control during host-pathogen interaction, opening new approaches for AHL-induced diseases. PMID:26371376

  17. c-Myc affects mRNA translation, cell proliferation and progenitor cell function in the mammary gland

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    Trumpp Andreas

    2009-09-01

    Full Text Available Abstract Background The oncoprotein c-Myc has been intensely studied in breast cancer and mouse mammary tumor models, but relatively little is known about the normal physiological role of c-Myc in the mammary gland. Here we investigated functions of c-Myc during mouse mammary gland development using a conditional knockout approach. Results Generation of c-mycfl/fl mice carrying the mammary gland-specific WAPiCre transgene resulted in c-Myc loss in alveolar epithelial cells starting in mid-pregnancy. Three major phenotypes were observed in glands of mutant mice. First, c-Myc-deficient alveolar cells had a slower proliferative response at the start of pregnancy, causing a delay but not a block of alveolar development. Second, while milk composition was comparable between wild type and mutant animals, milk production was reduced in mutant glands, leading to slower pup weight-gain. Electron microscopy and polysome fractionation revealed a general decrease in translational efficiency. Furthermore, analysis of mRNA distribution along the polysome gradient demonstrated that this effect was specific for mRNAs whose protein products are involved in milk synthesis. Moreover, quantitative reverse transcription-polymerase chain reaction analysis revealed decreased levels of ribosomal RNAs and ribosomal protein-encoding mRNAs in mutant glands. Third, using the mammary transplantation technique to functionally identify alveolar progenitor cells, we observed that the mutant epithelium has a reduced ability to repopulate the gland when transplanted into NOD/SCID recipients. Conclusion We have demonstrated that c-Myc plays multiple roles in the mouse mammary gland during pregnancy and lactation. c-Myc loss delayed, but did not block proliferation and differentiation in pregnancy. During lactation, lower levels of ribosomal RNAs and proteins were present and translation was generally decreased in mutant glands. Finally, the transplantation studies suggest a role

  18. Tyk2 expression and its signaling enhances the invasiveness of prostate cancer cells

    International Nuclear Information System (INIS)

    Protein tyrosine kinase plays a central role in the proliferation and differentiation of various types of cells. One of these protein kinases, Tyk2, a member of the Jak family kinases, is known to play important roles in receptor signal transduction by interferons, interleukins, growth factors, and other hormones. In the present study, we investigated Tyk2 expression and its role in the growth and invasiveness of human prostate cancer cells. We used a small interfering RNA targeting Tyk2 and an inhibitor of Tyk2, tyrphostin A1, to suppress the expression and signaling of Tyk2 in prostate cancer cells. We detected mRNAs for Jak family kinases in prostate cancer cell lines by RT-PCR and Tyk2 protein in human prostate cancer specimens by immunohistochemistry. Inhibition of Tyk2 signaling resulted in attenuation of the urokinase-type plasminogen activator-enhanced invasiveness of prostate cancer cells in vitro without affecting the cellular growth rate. These results suggest that Tyk2 signaling in prostate cancer cells facilitate invasion of these cells, and interference with this signaling may be a potential therapeutic pathway

  19. Development of disease preventive method using radiated pathogenic microorganisms, cell lines and animals

    International Nuclear Information System (INIS)

    The effects of radiation were investigated on pathogenic plasmid aiming at a development of a method to induce mutagenesis in plasmid DNA by radiation. To construct an experimental system which allows to detect a plasmid-segregated cell, kanamycin-resistant casette was inserted into pX02, a capsule plasmid in Bacillus anthracis to produce acpA:: Kmr by homologous recombination. This plasmid is thought available for analyzing the rate of plasmid segregation caused by radiation. Next, developments of detection and determination methods for various cytokines were attempted by RT-PCR method with an aim to investigate the expression changes of cytokine mRNA in calf immunocytes by radiation. In calf peripheral monocytes and alveolar macrophages, expressions of cytokine mRNAs such as IL-4, IFNα and GM-CSF mRNA as well as IL-1α, IL-1β, IL-2 and IL-6 were detected by RT-PCR method. (M.N.)

  20. Interleukin mRNA changes in mast cells stimulated by TSL-1 antigens

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    Arizmendi N.

    2001-06-01

    Full Text Available In this work we analyzed by RT-PCR, the mRNA changes for IL-4, IL-10, TNF and IFN ( induced by TSL-1 antigens in a rat mast cell line (HRMC with mucosal characteristics. The data obtained showed an increase of 65 and 52 % in mRNA expression for IL-4 and TNF respectively and a decrease of 59 and 55 % in mRNAs for IFNγ and IL-10. Our results suggest that TSL-1 antigens induce the release from MC of regulatory molecules, such as IL-4 by an IgE independent mechanism. Our data also provides important information related to the ability of MC to participate not only in the effector phase against the infectious agents, but also in the orchestration of the immune response by the host against parasites.

  1. Differentiation of human bronchial epithelial cells: role of hydrocortisone in development of ion transport pathways involved in mucociliary clearance.

    Science.gov (United States)

    Zaidman, Nathan A; Panoskaltsis-Mortari, Angela; O'Grady, Scott M

    2016-08-01

    Glucocorticoids strongly influence the mucosal-defense functions performed by the bronchial epithelium, and inhaled corticosteroids are critical in the treatment of patients with inflammatory airway diseases such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis. A common pathology associated with these diseases is reduced mucociliary clearance, a defense mechanism involving the coordinated transport of salt, water, and mucus by the bronchial epithelium, ultimately leading to retention of pathogens and particles in the airways and to further disease progression. In the present study we investigated the role of hydrocortisone (HC) in differentiation and development of the ion transport phenotype of normal human bronchial epithelial cells under air-liquid interface conditions. Normal human bronchial epithelial cells differentiated in the absence of HC (HC0) showed significantly less benzamil-sensitive short-circuit current than controls, as well as a reduced response after stimulation with the selective β2-adrenergic receptor agonist salbutamol. Apical membrane localization of epithelial Na(+) channel α-subunits was similarly reduced in HC0 cells compared with controls, supporting a role of HC in the trafficking and density of Na(+) channels in the plasma membrane. Additionally, glucocorticoid exposure during differentiation regulated the transcription of cystic fibrosis transmembrane conductance regulator and β2-adrenergic receptor mRNAs and appeared to be necessary for the expression of cystic fibrosis transmembrane conductance regulator-dependent anion secretion in response to β2-agonists. HC had no significant effect on surface cell differentiation but did modulate the expression of mucin mRNAs. These findings indicate that glucocorticoids support mucosal defense by regulating critical transport pathways essential for effective mucociliary clearance. PMID:27306366

  2. Transcriptomic-Wide Discovery of Direct and Indirect HuR RNA Targets in Activated CD4+ T Cells.

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    Patsharaporn Techasintana

    Full Text Available Due to poor correlation between steady state mRNA levels and protein product, purely transcriptomic profiling methods may miss genes posttranscriptionally regulated by RNA binding proteins (RBPs and microRNAs (miRNAs. RNA immunoprecipitation (RIP methods developed to identify in vivo targets of RBPs have greatly elucidated those mRNAs which may be regulated via transcript stability and translation. The RBP HuR (ELAVL1 and family members are major stabilizers of mRNA. Many labs have identified HuR mRNA targets; however, many of these analyses have been performed in cell lines and oftentimes are not independent biological replicates. Little is known about how HuR target mRNAs behave in conditional knock-out models. In the present work, we performed HuR RIP-Seq and RNA-Seq to investigate HuR direct and indirect targets using a novel conditional knock-out model of HuR genetic ablation during CD4+ T activation and Th2 differentiation. Using independent biological replicates, we generated a high coverage RIP-Seq data set (>160 million reads that was analyzed using bioinformatics methods specifically designed to find direct mRNA targets in RIP-Seq data. Simultaneously, another set of independent biological replicates were sequenced by RNA-Seq (>425 million reads to identify indirect HuR targets. These direct and indirect targets were combined to determine canonical pathways in CD4+ T cell activation and differentiation for which HuR plays an important role. We show that HuR may regulate genes in multiple canonical pathways involved in T cell activation especially the CD28 family signaling pathway. These data provide insights into potential HuR-regulated genes during T cell activation and immune mechanisms.

  3. Cyclosporine A inhibits transcription of cytokine genes and decreases the frequencies of IL-2 producing cells in feline mononuclear cells.

    Science.gov (United States)

    Kuga, Kazufumi; Nishifuji, Koji; Iwasaki, Toshiroh

    2008-10-01

    Cyclosporine A (CsA) has been widely used for suppression of transplant rejection and controlling pruritus in allergic dermatitis in humans, dogs and cats. CsA is known to suppress the expression of inflammatory cytokines, including IL-2, IL-4, IFN-gamma and TNF-alpha in humans, dogs and experimental mice. However, little is known about the immunomodulating effect of CsA in cats. The aim of this study was to evaluate the effects of CsA on the expression of inflammatory cytokines in feline peripheral blood mononuclear cells (PBMC). Real-time PCR analyses with Concanavalin A (ConA)-stimulated PBMC obtained from 5 cats revealed that the expression of mRNAs for IL-2, IL-4, IFN- gamma and TNF-alpha was inhibited by CsA in a dose-dependent manner. Moreover, an enzyme-linked immunospot (ELISPOT) assay, which is capable of detecting IL-2 secreting cells as single spots, revealed that the frequency of IL-2 secreting cells in ConA-stimulated feline PBMC was significantly reduced in the presence of CsA. These results might provide an explanation for the mechanisms of action of CsA in the suppression of transplant rejection and the control of pruritus in cats. PMID:18981654

  4. Interactions between exosomes from breast cancer cells and primary mammary epithelial cells leads to generation of reactive oxygen species which induce DNA damage response, stabilization of p53 and autophagy in epithelial cells.

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    Sujoy Dutta

    Full Text Available Exosomes are nanovesicles originating from multivesicular bodies and are released by all cell types. They contain proteins, lipids, microRNAs, mRNAs and DNA fragments, which act as mediators of intercellular communications by inducing phenotypic changes in recipient cells. Tumor-derived exosomes have been shown to play critical roles in different stages of tumor development and metastasis of almost all types of cancer. One of the ways by which exosomes affect tumorigenesis is to manipulate the tumor microenvironments to create tumor permissive "niches". Whether breast cancer cell secreted exosomes manipulate epithelial cells of the mammary duct to facilitate tumor development is not known. To address whether and how breast cancer cell secreted exosomes manipulate ductal epithelial cells we studied the interactions between exosomes isolated from conditioned media of 3 different breast cancer cell lines (MDA-MB-231, T47DA18 and MCF7, representing three different types of breast carcinomas, and normal human primary mammary epithelial cells (HMECs. Our studies show that exosomes released by breast cancer cell lines are taken up by HMECs, resulting in the induction of reactive oxygen species (ROS and autophagy. Inhibition of ROS by N-acetyl-L-cysteine (NAC led to abrogation of autophagy. HMEC-exosome interactions also induced the phosphorylation of ATM, H2AX and Chk1 indicating the induction of DNA damage repair (DDR responses. Under these conditions, phosphorylation of p53 at serine 15 was also observed. Both DDR responses and phosphorylation of p53 induced by HMEC-exosome interactions were also inhibited by NAC. Furthermore, exosome induced autophagic HMECs were found to release breast cancer cell growth promoting factors. Taken together, our results suggest novel mechanisms by which breast cancer cell secreted exosomes manipulate HMECs to create a tumor permissive microenvironment.

  5. High Glucose Causes Human Cardiac Progenitor Cell Dysfunction by Promoting Mitochondrial Fission: Role of a GLUT1 Blocker

    Science.gov (United States)

    Choi, He Yun; Park, Ji Hye; Jang, Woong Bi; Ji, Seung Taek; Jung, Seok Yun; Kim, Da Yeon; Kang, Songhwa; Kim, Yeon Ju; Yun, Jisoo; Kim, Jae Ho; Baek, Sang Hong; Kwon, Sang-Mo

    2016-01-01

    Cardiovascular disease is the most common cause of death in diabetic patients. Hyperglycemia is the primary characteristic of diabetes and is associated with many complications. The role of hyperglycemia in the dysfunction of human cardiac progenitor cells that can regenerate damaged cardiac tissue has been investigated, but the exact mechanism underlying this association is not clear. Thus, we examined whether hyperglycemia could regulate mitochondrial dynamics and lead to cardiac progenitor cell dysfunction, and whether blocking glucose uptake could rescue this dysfunction. High glucose in cardiac progenitor cells results in reduced cell viability and decreased expression of cell cycle-related molecules, including CDK2 and cyclin E. A tube formation assay revealed that hyperglycemia led to a significant decrease in the tube-forming ability of cardiac progenitor cells. Fluorescent labeling of cardiac progenitor cell mitochondria revealed that hyperglycemia alters mitochondrial dynamics and increases expression of fission-related proteins, including Fis1 and Drp1. Moreover, we showed that specific blockage of GLUT1 improved cell viability, tube formation, and regulation of mitochondrial dynamics in cardiac progenitor cells. To our knowledge, this study is the first to demonstrate that high glucose leads to cardiac progenitor cell dysfunction through an increase in mitochondrial fission, and that a GLUT1 blocker can rescue cardiac progenitor cell dysfunction and downregulation of mitochondrial fission. Combined therapy with cardiac progenitor cells and a GLUT1 blocker may provide a novel strategy for cardiac progenitor cell therapy in cardiovascular disease patients with diabetes. PMID:27350339

  6. High Glucose Causes Human Cardiac Progenitor Cell Dysfunction by Promoting Mitochondrial Fission: Role of a GLUT1 Blocker.

    Science.gov (United States)

    Choi, He Yun; Park, Ji Hye; Jang, Woong Bi; Ji, Seung Taek; Jung, Seok Yun; Kim, Da Yeon; Kang, Songhwa; Kim, Yeon Ju; Yun, Jisoo; Kim, Jae Ho; Baek, Sang Hong; Kwon, Sang-Mo

    2016-07-01

    Cardiovascular disease is the most common cause of death in diabetic patients. Hyperglycemia is the primary characteristic of diabetes and is associated with many complications. The role of hyperglycemia in the dysfunction of human cardiac progenitor cells that can regenerate damaged cardiac tissue has been investigated, but the exact mechanism underlying this association is not clear. Thus, we examined whether hyperglycemia could regulate mitochondrial dynamics and lead to cardiac progenitor cell dysfunction, and whether blocking glucose uptake could rescue this dysfunction. High glucose in cardiac progenitor cells results in reduced cell viability and decreased expression of cell cycle-related molecules, including CDK2 and cyclin E. A tube formation assay revealed that hyperglycemia led to a significant decrease in the tube-forming ability of cardiac progenitor cells. Fluorescent labeling of cardiac progenitor cell mitochondria revealed that hyperglycemia alters mitochondrial dynamics and increases expression of fission-related proteins, including Fis1 and Drp1. Moreover, we showed that specific blockage of GLUT1 improved cell viability, tube formation, and regulation of mitochondrial dynamics in cardiac progenitor cells. To our knowledge, this study is the first to demonstrate that high glucose leads to cardiac progenitor cell dysfunction through an increase in mitochondrial fission, and that a GLUT1 blocker can rescue cardiac progenitor cell dysfunction and downregulation of mitochondrial fission. Combined therapy with cardiac progenitor cells and a GLUT1 blocker may provide a novel strategy for cardiac progenitor cell therapy in cardiovascular disease patients with diabetes. PMID:27350339

  7. Evidence for ovarian granulosa stem cells: telomerase activity and localization of the telomerase ribonucleic acid component in bovine ovarian follicles.

    Science.gov (United States)

    Lavranos, T C; Mathis, J M; Latham, S E; Kalionis, B; Shay, J W; Rodgers, R J

    1999-08-01

    We have previously postulated that granulosa cells of developing follicles arise from a population of stem cells. Stem cells and cancer cells can divide indefinitely partly because they express telomerase. Telomerase is a ribonucleoprotein enzyme that repairs the ends of telomeres that otherwise shorten progressively upon each successive cell division. In this study we carried out cell cycle analyses and examined telomerase expression to examine our hypothesis. Preantral (60-100 microm) and small (1 mm) follicles, as well as granulosa cells from medium-sized (3 mm) and large (6-8 mm) follicles, were isolated. Cell cycle analyses and expression of Ki-67, a cell cycle-related protein, were undertaken on follicles of each size (n = 3) by flow cytometry; 12% to 16% of granulosa cells in all follicles were in the S phase, and less than 2% were in the G(2)/M phase. Telomerase activity (n = 3) was highest in the small preantral follicles, declining at the 1-mm stage and even further at the 3-mm stage. In situ hybridization histochemistry was carried out on bovine ovaries, and telomerase RNA was detected in the granulosa cells of growing follicles but not primordial follicles. Two major patterns of staining were observed in the membrana granulosa of antral follicles: staining in the middle and antral layers, and staining in the middle and basal layers. No staining was detected in oocytes. Our results strongly support our hypothesis that granulosa cells arise from a population of stem cells. PMID:10411512

  8. The NMDA receptor NR2A subunit regulates proliferation of MKN45 human gastric cancer cells.

    Science.gov (United States)

    Watanabe, Kanako; Kanno, Takeshi; Oshima, Tadayuki; Miwa, Hiroto; Tashiro, Chikara; Nishizaki, Tomoyuki

    2008-03-01

    The present study investigated proliferation of MKN28 and MKN45 human gastric cancer cells regulated by the N-methyl-d-aspartate (NMDA) receptor subunit. The NMDA receptor antagonist dl-2-amino-5-phosphonovaleric acid (AP5) inhibited proliferation of MKN45 cells, but not MKN28 cells. Of the NMDA subunits such as NR1, NR2 (2A, 2B, 2C, and 2D), and NR3 (3A and 3B), all the NMDA subunit mRNAs except for the NR2B subunit mRNA were expressed in both MKN28 and MKN45 cells. MKN45 cells were characterized by higher expression of the NR2A subunit mRNA and lower expression of the NR1 subunit mRNA, but MKN28 otherwise by higher expression of the NR1 subunit mRNA and lower expression of the NR2A subunit mRNA. MKN45 cell proliferation was also inhibited by silencing the NR2A subunit-targeted gene. For MKN45 cells, AP5 or knocking-down the NR2A subunit increased the proportion of cells in the G(1) phase of cell cycling and decreased the proportion in the S/G(2) phase. The results of the present study, thus, suggest that blockage of NMDA receptors including the NR2A subunit suppresses MKN45 cell proliferation due to cell cycle arrest at the G(1) phase; in other words, the NR2A subunit promotes MKN45 cell proliferation by accelerating cell cycling. PMID:18178157

  9. The NMDA receptor NR2A subunit regulates proliferation of MKN45 human gastric cancer cells

    International Nuclear Information System (INIS)

    The present study investigated proliferation of MKN28 and MKN45 human gastric cancer cells regulated by the N-methyl-D-aspartate (NMDA) receptor subunit. The NMDA receptor antagonist DL-2-amino-5-phosphonovaleric acid (AP5) inhibited proliferation of MKN45 cells, but not MKN28 cells. Of the NMDA subunits such as NR1, NR2 (2A, 2B, 2C, and 2D), and NR3 (3A and 3B), all the NMDA subunit mRNAs except for the NR2B subunit mRNA were expressed in both MKN28 and MKN45 cells. MKN45 cells were characterized by higher expression of the NR2A subunit mRNA and lower expression of the NR1 subunit mRNA, but MKN28 otherwise by higher expression of the NR1 subunit mRNA and lower expression of the NR2A subunit mRNA. MKN45 cell proliferation was also inhibited by silencing the NR2A subunit-targeted gene. For MKN45 cells, AP5 or knocking-down the NR2A subunit increased the proportion of cells in the G1 phase of cell cycling and decreased the proportion in the S/G2 phase. The results of the present study, thus, suggest that blockage of NMDA receptors including the NR2A subunit suppresses MKN45 cell proliferation due to cell cycle arrest at the G1 phase; in other words, the NR2A subunit promotes MKN45 cell proliferation by accelerating cell cycling

  10. Variant G6PD levels promote tumor cell proliferation or apoptosis via the STAT3/5 pathway in the human melanoma xenograft mouse model

    International Nuclear Information System (INIS)

    Glucose-6-phosphate dehydrogenase (G6PD), elevated in tumor cells, catalyzes the first reaction in the pentose-phosphate pathway. The regulation mechanism of G6PD and pathological change in human melanoma growth remains unknown. HEM (human epidermal melanocyte) cells and human melanoma cells with the wild-type G6PD gene (A375-WT), G6PD deficiency (A375-G6PD∆), G6PD cDNA overexpression (A375-G6PD∆-G6PD-WT), and mutant G6PD cDNA (A375-G6PD∆-G6PD-G487A) were subcutaneously injected into 5 groups of nude mice. Expressions of G6PD, STAT3, STAT5, cell cycle-related proteins, and apoptotic proteins as well as mechanistic exploration of STAT3/STAT5 were determined by quantitative real-time PCR (qRT-PCR), immunohistochemistry and western blot. Delayed formation and slowed growth were apparent in A375-G6PD∆ cells, compared to A375-WT cells. Significantly decreased G6PD expression and activity were observed in tumor tissues induced by A375-G6PD∆, along with down-regulated cell cycle proteins cyclin D1, cyclin E, p53, and S100A4. Apoptosis-inhibited factors Bcl-2 and Bcl-xl were up-regulated; however, apoptosis factor Fas was down-regulated, compared to A375-WT cells. Moderate protein expressions were observed in A375-G6PD∆-G6PD-WT and A375-G6PD∆-G6PD-G487A cells. G6PD may regulate apoptosis and expression of cell cycle-related proteins through phosphorylation of transcription factors STAT3 and STAT5, thus mediating formation and growth of human melanoma cells. Further study will, however, be required to determine potential clinical applications

  11. Utility of Lymphoblastoid Cell Lines for Induced Pluripotent Stem Cell Generation

    Directory of Open Access Journals (Sweden)

    Satish Kumar

    2016-01-01

    Full Text Available A large number of EBV immortalized LCLs have been generated and maintained in genetic/epidemiological studies as a perpetual source of DNA and as a surrogate in vitro cell model. Recent successes in reprograming LCLs into iPSCs have paved the way for generating more relevant in vitro disease models using this existing bioresource. However, the overall reprogramming efficiency and success rate remain poor and very little is known about the mechanistic changes that take place at the transcriptome and cellular functional level during LCL-to-iPSC reprogramming. Here, we report a new optimized LCL-to-iPSC reprogramming protocol using episomal plasmids encoding pluripotency transcription factors and mouse p53DD (p53 carboxy-terminal dominant-negative fragment and commercially available reprogramming media. We achieved a consistently high reprogramming efficiency and 100% success rate using this optimized protocol. Further, we investigated the transcriptional changes in mRNA and miRNA levels, using FC-abs ≥ 2.0 and FDR ≤ 0.05 cutoffs; 5,228 mRNAs and 77 miRNAs were differentially expressed during LCL-to-iPSC reprogramming. The functional enrichment analysis of the upregulated genes and activation of human pluripotency pathways in the reprogrammed iPSCs showed that the generated iPSCs possess transcriptional and functional profiles very similar to those of human ESCs.

  12. Utility of Lymphoblastoid Cell Lines for Induced Pluripotent Stem Cell Generation

    Science.gov (United States)

    Kumar, Satish; Curran, Joanne E.; Glahn, David C.; Blangero, John

    2016-01-01

    A large number of EBV immortalized LCLs have been generated and maintained in genetic/epidemiological studies as a perpetual source of DNA and as a surrogate in vitro cell model. Recent successes in reprograming LCLs into iPSCs have paved the way for generating more relevant in vitro disease models using this existing bioresource. However, the overall reprogramming efficiency and success rate remain poor and very little is known about the mechanistic changes that take place at the transcriptome and cellular functional level during LCL-to-iPSC reprogramming. Here, we report a new optimized LCL-to-iPSC reprogramming protocol using episomal plasmids encoding pluripotency transcription factors and mouse p53DD (p53 carboxy-terminal dominant-negative fragment) and commercially available reprogramming media. We achieved a consistently high reprogramming efficiency and 100% success rate using this optimized protocol. Further, we investigated the transcriptional changes in mRNA and miRNA levels, using FC-abs ≥ 2.0 and FDR ≤ 0.05 cutoffs; 5,228 mRNAs and 77 miRNAs were differentially expressed during LCL-to-iPSC reprogramming. The functional enrichment analysis of the upregulated genes and activation of human pluripotency pathways in the reprogrammed iPSCs showed that the generated iPSCs possess transcriptional and functional profiles very similar to those of human ESCs.

  13. Phase II enzyme induction by a carotenoid, lutein, in a PC12D neuronal cell line

    Energy Technology Data Exchange (ETDEWEB)

    Miyake, Seiji [Laboratory of Retinal Cell Biology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan); Department of Ophthalmology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan); Wakasa Seikatsu Co., Ltd., 134 Chudoujiminami-cho, Shimogyo-ku, Kyoto 600-8813 (Japan); Kobayashi, Saori [Wakasa Seikatsu Co., Ltd., 134 Chudoujiminami-cho, Shimogyo-ku, Kyoto 600-8813 (Japan); Tsubota, Kazuo [Laboratory of Retinal Cell Biology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan); Ozawa, Yoko, E-mail: ozawa@a5.keio.jp [Laboratory of Retinal Cell Biology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan); Department of Ophthalmology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan)

    2014-04-04

    Highlights: • Lutein reduced ROS levels in a PC12D neuronal cell line. • Lutein induced mRNAs of phase II antioxidative enzymes in PC12D neuronal cells. • Lutein increased protein levels of HO-1, SOD2, and NQO-1 in PC12D neuronal cells. • Lutein had no effect on intranuclear Nrf2 levels in PC12D neuronal cells. • Lutein did not activate potential upstream Nrf2 nuclear translocation pathways. - Abstract: The mechanism by which lutein, a carotenoid, acts as an antioxidant in retinal cells is still not fully understood. Here, lutein treatment of a neuronal cell line (PC12D) immediately resulted in reduced intracellular ROS levels, implying that it has a direct role in ROS scavenging. Significantly, lutein treatment also induced phase II antioxidative enzyme expression, probably via a nuclear factor-like 2 (Nrf2) independent pathway. This latter mechanism could explain why lutein acts diversely to protect against oxidative/cytotoxic stress, and why it is physiologically involved in the human neural tissue, such as the retina.

  14. Complementation of radiation-sensitive Ataxia telangiectasia cells after transfection of cDNA expression libraries and cosmid clones from wildtype cells

    International Nuclear Information System (INIS)

    In this Ph.D.-thesis, phenotypic complementation of AT-cells (AT5BIVA) by transfection of cDNA-expression-libraries was adressed: After stable transfection of cDNA-expression-libraries G418 resistant clones were selected for enhanced radioresistance by a fractionated X-ray selection. One surviving transfectant clone (clone 514) exhibited enhanced radiation resistance in dose-response experiments and further X-ray selections. Cell cycle analysis revealed complementation of untreated and irradiated 514-cells in cell cycle progression. The rate of DNA synthesis, however, is not diminished after irradiation but shows the reverse effect. A transfected cDNA-fragment (AT500-cDNA) was isolated from the genomic DNA of 514-cells and proved to be an unknown DNA sequence. A homologous sequence could be detected in genomic DNA from human cell lines, but not in DNA from other species. The cDNA-sequence could be localized to human chromosome 11. In human cells the cDNA sequence is part of two large mRNAs. 4 different cosmid clones containing high molecular genomic DNA from normal human cells could be isolated from a library, each hybridizing to the AT500-cDNA. After stable transfection into AT-cells, one cosmid-clone was able to confer enhanced radiation resistance both in X-ray selections and dose-response experiments. The results indicate that the cloned cDNA-fragment is based on an unknown gene from human chromosome 11 which partially complements the radiosensitivity and the defective cell cycle progression in AT5BIVA cells. (orig.)

  15. S14 protein in breast cancer cells: Direct evidence of regulation by SREBP-1c, superinduction with progestin, and effects on cell growth

    International Nuclear Information System (INIS)

    Most breast cancers exhibit brisk lipogenesis, and require it for growth. S14 is a lipogenesis-related nuclear protein that is overexpressed in most breast cancers. Sterol response element-binding protein-1c (SREBP-1c) is required for induction of lipogenesis-related genes, including S14 and fatty acid synthase (FAS), in hepatocytes, and correlation of SREBP-1c and FAS expression suggested that SREBP-1c drives lipogenesis in tumors as well. We directly tested the hypothesis that SREBP-1c drives S14 expression and mediates lipogenic effects of progestin in T47D breast cancer cells. Dominant-negative SREBP-1c inhibited induction of S14 and FAS mRNAs by progestin, while active SREBP-1c induced without hormone and superinduced in its presence. Changes in S14 mRNA were reflected in protein levels. A lag time and lack of progestin response elements indicated that S14 and FAS gene activation by progestin is indirect. Knockdown of S14 reduced, whereas overexpression stimulated, T47D cell growth, while nonlipogenic MCF10a mammary epithelial cells were not growth-inhibited. These data directly demonstrate that SREBP-1c drives S14 gene expression in breast cancer cells, and progestin magnifies that effect via an indirect mechanism. This supports the prediction, based on S14 gene amplification and overexpression in breast tumors, that S14 augments breast cancer cell growth and survival

  16. Profile of differentially expressed genes mediated by the type III epidermal growth factor receptor mutation expressed in a small-cell lung cancer cell line

    DEFF Research Database (Denmark)

    Pedersen, M.W.; Andersen, Thomas Thykjær; Ørntoft, Torben Falck;

    2001-01-01

    understanding of how the EGFRvIII contributes to the malignant phenotype is of major importance for future therapy. The GeneChip Hu6800Set developed by Affymetrix was used to identify changes in gene expression caused by the expression of EGFRvIII. The cell line selected for the study was an EGF receptor...... negative small-cell-lung cancer cell line, GLC3, stably transfected with the EGFRvIII gene in a Tet-On system. By comparison of mRNA levels in EGFRvIII-GLC3 with those of Tet-On-GLC3, it was found that the levels of mRNAs encoding several transcription factors (ATF-3, JunD, and c-Myb), cell adhesion......Previous studies have shown a correlation between expression of the EGF receptor type III mutation (EGFRvIII) and a more malignant phenotype of various cancers including: non-small-cell lung cancer, glioblastoma multiforme, prostate cancer and breast cancer. Thus, a detailed molecular genetic...

  17. miR-93 suppresses proliferation and colony formation of human colon cancer stem cells

    Institute of Scientific and Technical Information of China (English)

    Xiao-Feng Yu; Jian Zou; Zhi-Jun Bao; Jie Dong

    2011-01-01

    AIM: To identify differentially expressed microRNAs (miRNAs) in human colon cancer stem cells (SW1116csc) and study their function in SW1116csc proliferation.METHODS: SW1116csc were isolated from the human colon cancer cell line, SW1116 and cultured in serum free medium. A miRNA microarray was used to detect differential expression profiles of miRNAs in SW1116csc and SW1116 cells. Real-time quantitative polymerase chain reaction (PCR) was performed to verify the dif ferential expression of candidate miRNAs obtained from the microarray. Target mRNAs of differentially expressed miRNAs were predicted with target predic tion tools. miRNA expression plasmids were transfected into SW1116csc using Lipofectamine 2000 reagent. Cell proliferation curves were generated with trypan blue staining, and the colony formation rate of transfected cells was measured with the soft agar colony formation assay. Expression of target mRNAs and proteins from differentially expressed miRNAs were detected using reverse transcription (RT)-PCR and western blotting.RESULTS: Compared with expression in SW1116 cells, 35 miRNAs (including hsa-miR-192, hsa-miR-29b, hsa-miR-215, hsa-miR-194, hsa-miR-33a and hsa-miR-32) were upregulated more than 1.5-fold, and 11 miRNAs (including hsa-miR-93, hsa-miR-1231, hsa-miRPIus-F1080, hsa-miR-524-3p, hsa-miR-886-3p and hsa-miR-561) were downregulated in SW1116csc. The miRNA microarray results were further validated with quantitative RT-PCR. miR-93 was downregulated, and its predicted mRNA targets included BAMBI, CCND2, CDKN1A, HDAC8, KIF23, MAP3K9, MAP3K11, MYCN, PPARD, TLE4 and ZDHHC1. Overexpressed miR-93 sig nificantly inhibited cell proliferation and colony forma tion by SW1116CSC. Furthermore, miR-93 negatively regulated the mRNA and protein levels of HDAC8 and TLE4.CONCLUSION: Some miRNAs were differentially ex pressed during differentiation of SW1116csc into SW1116 cells. miR-93 may inhibit SW1116csc proliferation and colony formation.

  18. Cell proliferation-associated nuclear antigen defined by antibody Ki-67: a new kind of cell cycle-maintaining proteins

    International Nuclear Information System (INIS)

    A decade of studies on the human nuclear antigen defined by monoclonal antibody Ki-67 (the 'Ki-67 proteins') has made it abundantly clear that this structure is strictly associated with human cell proliferation and the expression of this protein can be used to access the growth fraction of a given cell population. Until recently the Ki-67 protein was described as a nonhistone protein that is highly susceptible to protease treatment. We have isolated and sequenced cDNAs encoding for this antigen and found two isoforms of the full length cDNA of 11.5 and 12.5 kb, respectively, sequence and structure of which are thus far unique. The gene encoding the Ki-67 protein is organized in 15 exons and is localized on chromosome 10. The center of this gene is formed by an extraordinary 6845 bp exon containing 16 successively repeated homologous segments of 366 bp ('Ki-67 repeats'), each containing a highly conserved new motif of 66 bp ('Ki-67 motif'). The deduced peptide sequence of this central exon possesses 10 ProGluSerThr (PEST) motifs which are associated with high turnover proteins such as other cell cycle-related proteins, oncogenes and transcription factors, etc. Like the latter proteins the Ki-67 antigen plays a pivotal role in maintaining cell proliferation because Ki-67 protein antisense oligonucleotides significantly inhibit 3H-thymidine incorporation in permanent human tumor cell lines in a dose-dependent manner. (author). 30 refs, 2 figs

  19. Oral keratinocytes support non-replicative infection and transfer of harbored HIV-1 to permissive cells

    Directory of Open Access Journals (Sweden)

    Giacaman Rodrigo A

    2008-07-01

    Full Text Available Abstract Background Oral keratinocytes on the mucosal surface are frequently exposed to HIV-1 through contact with infected sexual partners or nursing mothers. To determine the plausibility that oral keratinocytes are primary targets of HIV-1, we tested the hypothesis that HIV-1 infects oral keratinocytes in a restricted manner. Results To study the fate of HIV-1, immortalized oral keratinocytes (OKF6/TERT-2; TERT-2 cells were characterized for the fate of HIV-specific RNA and DNA. At 6 h post inoculation with X4 or R5-tropic HIV-1, HIV-1gag RNA was detected maximally within TERT-2 cells. Reverse transcriptase activity in TERT-2 cells was confirmed by VSV-G-mediated infection with HIV-NL4-3Δenv-EGFP. AZT inhibited EGFP expression in a dose-dependent manner, suggesting that viral replication can be supported if receptors are bypassed. Within 3 h post inoculation, integrated HIV-1 DNA was detected in TERT-2 cell nuclei and persisted after subculture. Multiply spliced and unspliced HIV-1 mRNAs were not detectable up to 72 h post inoculation, suggesting that HIV replication may abort and that infection is non-productive. Within 48 h post inoculation, however, virus harbored by CD4 negative TERT-2 cells trans infected co-cultured peripheral blood mononuclear cells (PBMCs or MOLT4 cells (CD4+ CCR5+ by direct cell-to-cell transfer or by releasing low levels of infectious virions. Primary tonsil epithelial cells also trans infected HIV-1 to permissive cells in a donor-specific manner. Conclusion Oral keratinocytes appear, therefore, to support stable non-replicative integration, while harboring and transmitting infectious X4- or R5-tropic HIV-1 to permissive cells for up to 48 h.

  20. Capsosiphon fulvescens glycoprotein inhibits AGS gastric cancer cell proliferation by downregulating Wnt-1 signaling.

    Science.gov (United States)

    Kim, Young-Min; Kim, In-Hye; Nam, Taek-Jeong

    2013-11-01

    Previously, we examined various apoptosis pathways in the AGS gastric cancer cell line using Capsosiphon fulvescens glycoprotein (Cf-GP). In this study, we focused on the downregulation of the Wnt-1 signaling pathway and cell cycle arrest. Upregulation of the Wnt signaling pathway has been observed in various cancer cells. The Wnt signal ligand acts in both canonical and non-canonical pathways. Among them, Wnt-1 was dependent on the canonical pathway. Here, we show inhibition of Wnt-1 signaling, β-catenin and transcription factors in AGS cells via Cf-GP. First, we examined the Frizzled receptor and Wnt-1 signal-related proteins including Axin, LRP, β-catenin, APC and GSK-3β. In addition, the expression levels of transcription factors Tcf/LEF were determined by western blot analysis and RT-PCR. Based on the data, we confirmed downregulation of the Wnt-1 signaling pathway by Cf-GP. Also, we determined the expression levels of cell cycle-related proteins cyclin D and c-myc, and looked for cell cycle arrest by cell cycle test analysis. We found that AGS cells arrested in the G0/G1 phase by Cf-GP. These results provide a mechanism of AGS cell inhibition through the downregulation of Wnt-1 signaling by Cf-GP. PMID:23982808

  1. Co-expression networks in generation of induced pluripotent stem cells.

    Science.gov (United States)

    Paul, Sharan; Pflieger, Lance; Dansithong, Warunee; Figueroa, Karla P; Gao, Fuying; Coppola, Giovanni; Pulst, Stefan M

    2016-01-01

    We developed an adenoviral vector, in which Yamanaka's four reprogramming factors (RFs) were controlled by individual CMV promoters in a single cassette (Ad-SOcMK). This permitted coordinated expression of RFs (SOX2, OCT3/4, c-MYC and KLF4) in a cell for a transient period of time, synchronizing the reprogramming process with the majority of transduced cells assuming induced pluripotent stem cell (iPSC)-like characteristics as early as three days post-transduction. These reprogrammed cells resembled human embryonic stem cells (ESCs) with regard to morphology, biomarker expression, and could be differentiated into cells of the germ layers in vitro and in vivo. These iPSC-like cells, however, failed to expand into larger iPSC colonies. The short and synchronized reprogramming process allowed us to study global transcription changes within short time intervals. Weighted gene co-expression network analysis (WGCNA) identified sixteen large gene co-expression modules, each including members of gene ontology categories involved in cell differentiation and development. In particular, the brown module contained a significant number of ESC marker genes, whereas the turquoise module contained cell-cycle-related genes that were downregulated in contrast to upregulation in human ESCs. Strong coordinated expression of all four RFs via adenoviral transduction may constrain stochastic processes and lead to silencing of genes important for cellular proliferation. PMID:26892236

  2. Co-expression networks in generation of induced pluripotent stem cells

    Directory of Open Access Journals (Sweden)

    Sharan Paul

    2016-03-01

    Full Text Available We developed an adenoviral vector, in which Yamanaka's four reprogramming factors (RFs were controlled by individual CMV promoters in a single cassette (Ad-SOcMK. This permitted coordinated expression of RFs (SOX2, OCT3/4, c-MYC and KLF4 in a cell for a transient period of time, synchronizing the reprogramming process with the majority of transduced cells assuming induced pluripotent stem cell (iPSC-like characteristics as early as three days post-transduction. These reprogrammed cells resembled human embryonic stem cells (ESCs with regard to morphology, biomarker expression, and could be differentiated into cells of the germ layers in vitro and in vivo. These iPSC-like cells, however, failed to expand into larger iPSC colonies. The short and synchronized reprogramming process allowed us to study global transcription changes within short time intervals. Weighted gene co-expression network analysis (WGCNA identified sixteen large gene co-expression modules, each including members of gene ontology categories involved in cell differentiation and development. In particular, the brown module contained a significant number of ESC marker genes, whereas the turquoise module contained cell-cycle-related genes that were downregulated in contrast to upregulation in human ESCs. Strong coordinated expression of all four RFs via adenoviral transduction may constrain stochastic processes and lead to silencing of genes important for cellular proliferation.

  3. Laminin-8 (alpha4beta1gamma1) is synthesized by lymphoid cells, promotes lymphocyte migration and costimulates T cell proliferation.

    Science.gov (United States)

    Geberhiwot, T; Assefa, D; Kortesmaa, J; Ingerpuu, S; Pedraza, C; Wondimu, Z; Charo, J; Kiessling, R; Virtanen, I; Tryggvason, K; Patarroyo, M

    2001-01-01

    Laminins are a growing family of large heterotrimeric proteins with cell adhesive and signalling functions. They are major components of basement membranes and are found in many organs, including the vasculature and other compartments of bone marrow, thymus, lymph nodes and spleen. However, expression, recognition and use of laminin isoforms by lymphoid cells are poorly understood. In the present study, lymphoid T cells (Jurkat) were found to synthesize laminin alpha4, beta1 and gamma1 mRNAs and polypeptides and to assemble the chains into laminin-8. Lymphoblastoid B (NAD-20) cells, lymphoid NK (NKL) cells and blood lymphocytes also contained laminin-8 and, after cell permeabilization, practically all blood lymphocytes reacted with mAbs to laminin beta1 and gamma1 chains. Following stimulation, blood lymphocytes secreted laminin-8, and this laminin isoform, but not laminin-10/11(alpha5beta1gamma1/alpha5beta2gamma1), promoted chemokine-induced migration of the cells. In an activation-dependent manner, purified blood CD4 T cells adhered to immobilized laminin-8 and laminin-10/11 by using alpha6beta1 integrin, but minimally to laminin-1 (alpha1beta1gamma1). Accordingly, laminin-8 and laminin-10/11, but not laminin-1, strongly costimulated proliferation of the T cells via the same integrin. Thus, lymphoid cells are able to synthesize and secrete complete laminin molecules. In addition, synthesis of laminin-8 and recognition of laminin-8 and -10/11 by lymphocytes indicate relevance of these laminin isoforms in lymphocyte physiology. PMID:11148143

  4. LncRNA-uc.167 influences cell proliferation, apoptosis and differentiation of P19 cells by regulating Mef2c.

    Science.gov (United States)

    Song, Guixian; Shen, Yahui; Ruan, Zhongbao; Li, Xing; Chen, Yumei; Yuan, Wei; Ding, Xiangwei; Zhu, Li; Qian, Lingmei

    2016-09-15

    In our previous study we screened thousands of lncRNAs for their relationship with ventricular septal defect. Among these lncRNAs, uc.167 attracted our attention for its high level of conservation and that it was antisense to the Mef2c gene, which encodes myocyte enhancer factor 2C. This study aims to investigate the role of uc.167 during cardiomyocyte maturation in P19 cells induction and possible mechanism. The uc.167 expression level in human heart tissue of ventricular septum defect (VSD) was evaluated by qRT-PCR. The UCSC database was searched to investigate the bioinformatics of uc.167. We constructed overexpression vector of uc.167 and Mef2c. To detect proliferation and apoptosis, we combined cell cycle analysis and CCK8, Hoechst staining, flow cytometry and caspase-3 assays, respectively. The cardiomyogenesis related RNAs (cTnT, GATA4, and Mef2c) and proteins were detected by qRT-PCR and Western blotting. In this study, we found that uc.167 expression was significantly increased in VSD heart tissues. uc.167 is on the opposite strand to the coding gene Mef2c. The expression model of Mef2c and uc.167 showed an opposite correlation in the embryonic development and process of differentiation of P19 cells into cardiomyocytes. Overexpression of uc.167 inhibited proliferation but promoted apoptosis in P19 cells compared with the vector group, and those relative mRNAs and proteins decreased during the differentiation process. Whereas, co-expression of Mef2c and uc.167 can partially reverse the negative effects of uc.167 on proliferation, apoptosis and differentiation. Taken together, our findings suggest that uc.167 contributes to the development potential of VSD and may constitute a potential therapeutic target in this disease. uc.167 influences cell proliferation, apoptosis and differentiation of P19 cell by regulating Mef2c. PMID:27268728

  5. Stimulatory interactions between human coronary smooth muscle cells and dendritic cells.

    Directory of Open Access Journals (Sweden)

    Sara Paccosi

    Full Text Available Despite inflammatory and immune mechanisms participating to atherogenesis and dendritic cells (DCs driving immune and non-immune tissue injury response, the interactions between DCs and vascular smooth muscle cells (VSMCs possibly relevant to vascular pathology including atherogenesis are still unclear. To address this issue, immature DCs (iDCs generated from CD14+ cells isolated from healthy donors were matured either with cytokines (mDCs, or co-cultured (ccDCs with human coronary artery VSMCs (CASMCs using transwell chambers. Co-culture induced DC immunophenotypical and functional maturation similar to cytokines, as demonstrated by flow cytometry and mixed lymphocyte reaction. In turn, factors from mDCs and ccDCs induced CASMC migration. MCP-1 and TNFα, secreted from DCs, and IL-6 and MCP-1, secreted from CASMCs, were primarily involved. mDCs adhesion to CASMCs was enhanced by CASMC pre-treatment with IFNγ and TNFα ICAM-1 and VCAM-1 were involved, since the expression of specific mRNAs for these molecules increased and adhesion was inhibited by neutralizing antibodies to the counter-receptors CD11c and CD18. Adhesion was also inhibited by CASMC pre-treatment with the HMG-CoA-reductase inhibitor atorvastatin and the PPARγ agonist rosiglitazone, which suggests a further mechanism for the anti-inflammatory action of these drugs. Adhesion of DCs to VSMCs was shown also in vivo in rat carotid 7 to 21 days after crush and incision injury. The findings indicate that DCs and VSMCs can interact with reciprocal stimulation, possibly leading to perpetuate inflammation and vascular wall remodelling, and that the interaction is enhanced by a cytokine-rich inflammatory environment and down-regulated by HMGCoA-reductase inhibitors and PPARγ agonists.

  6. Mitochondrial regulation of cell cycle progression through SLC25A43.

    Science.gov (United States)

    Gabrielson, Marike; Reizer, Edwin; Stål, Olle; Tina, Elisabet

    2016-01-22

    An increasing body of evidence is pointing towards mitochondrial regulation of the cell cycle. In a previous study of HER2-positive tumours we could demonstrate a common loss in the gene encoding for the mitochondrial transporter SLC25A43 and also a significant relation between SLC25A43 protein expression and S-phase fraction. Here, we investigated the consequence of suppressed SLC25A43 expression on cell cycle progression and proliferation in breast epithelial cells. In the present study, we suppressed SLC25A43 using siRNA in immortalised non-cancerous breast epithelial MCF10A cells and HER2-positive breast cancer cells BT-474. Viability, apoptosis, cell proliferation rate, cell cycle phase distribution, and nuclear Ki-67 and p21, were assessed by flow cytometry. Cell cycle related gene expressions were analysed using real-time PCR. We found that SLC25A43 knockdown in MCF10A cells significantly inhibited cell cycle progression during G1-to-S transition, thus significantly reducing the proliferation rate and fraction of Ki-67 positive MCF10A cells. In contrast, suppressed SLC25A43 expression in BT-474 cells resulted in a significantly increased proliferation rate together with an enhanced G1-to-S transition. This was reflected by an increased fraction of Ki-67 positive cells and reduced level of nuclear p21. In line with our previous results, we show a role for SLC25A43 as a regulator of cell cycle progression and proliferation through a putative mitochondrial checkpoint. These novel data further strengthen the connection between mitochondrial function and the cell cycle, both in non-malignant and in cancer cells. PMID:26721434

  7. Inhibition of Hec1 expression enhances the sensitivity of human ovarian cancer cells to paclitaxel

    Institute of Scientific and Technical Information of China (English)

    Qing-qing MO; Ping-bo CHEN; Xin JIN; Qian CHEN; Lan TANG; Bei-bei WANG; Ke-zhen LI

    2013-01-01

    Aim:Hec1,a member of the Ndc80 kinetochore complex,is highly expressed in cancers.The aim of this study was to explore the role and mechanism of action of Hec1 with respect to the cytotoxicity of paclitaxel in ovarian cancer.Methods:Thirty ovarian cancer samples and 6 normal ovarian samples were collected.Hec1 expression in these samples was determined with immunohistochemistry.Ovarian cancer cell lines A2780,OV2008,C13K,SKOV3,and CAOV3 and A2780/Taxol were examined.Cell apoptosis and cell cycle analysis were detected with flow cytometric technique.siRNA was used to delete Hec1 in the cells.The expression of related mRNAs and proteins was measured using RT-PCR and Western blot analysis,respectively.Results:Hec1 expression was significantly higher in ovarian cancer samples than in normal ovarian samples,and was associated with paclitaxel-resistance and poor prognosis.Among the 6 ovarian cancer cell lines examined,Hec1 expression was highest in paclitaxelresistant A2780/Taxol cells,and lowest in A2780 cells.Depleting Hec1 in A2780/Taxol cells with siRNA decreased the IC5o value of paclitaxel by more than 10-fold (from 590±26.7 to 45.6±19.4 nmol/L).Depleting Hec1 in A2780 cells had no significant effect on the paclitaxel sensitivity.In paclitaxel-treated A2780/Taxol cells,depleting Hec1 significantly increased the cleaved PARP and Bax protein levels,and decreased the Bcl-xL protein level.Conclusion:Hec1 overexpression is associated with the progression and poor prognosis of ovarian cancer.Inhibition of Hec1 expression can sensitize ovarian cancer cells to paclitaxel.

  8. DAZL Limits Pluripotency, Differentiation, and Apoptosis in Developing Primordial Germ Cells

    Directory of Open Access Journals (Sweden)

    Hsu-Hsin Chen

    2014-11-01

    Full Text Available The scarcity of primordial germ cells (PGCs in the developing mammalian embryo hampers robust biochemical analysis of the processes that underlie early germ cell formation. Here, we demonstrate that DAZL, a germ cell-specific RNA binding protein, is a robust PGC marker during in vitro germ cell development. Using Dazl-GFP reporter ESCs, we demonstrate that DAZL plays a central role in a large mRNA/protein interactive network that blocks the translation of core pluripotency factors, including Sox2 and Sall4, as well as of Suz12, a polycomb family member required for differentiation of pluripotent cells. Thus, DAZL limits both pluripotency and somatic differentiation in nascent PGCs. In addition, we observed that DAZL associates with mRNAs of key Caspases and similarly inhibits their translation. This elegant fail-safe mechanism ensures that, whereas loss of DAZL results in prolonged expression of pluripotency factors, teratoma formation is avoided due to the concomitant activation of the apoptotic cascade.

  9. MicroRNA expression profiling in neurogenesis of adipose tissue-derived stem cells

    Indian Academy of Sciences (India)

    Jung Ah Cho; Ho Park; Eun Hye Lim; Kyo Won Lee

    2011-04-01

    Adipose tissue-derived stem cells (ADSCs) are one population of adult stem cells that can self renew and differentiate into multiple lineages. Because of advantages in method and quantity of acquisition, ADSCs are gaining attention as an alternative source of bone marrow mesenchymal stem cells. In this study, we performed microRNA profiling of undifferentiated and of neurally-differentiated ADSCs to identify the responsible microRNAs in neurogenesis using this type of stem cell. MicroRNAs from four different donors were analysed by microarray. Compared to the undifferentiation control, we identified 39–101 microRNAs with more than two-fold higher expression and 3–9 microRNAs with two-fold lower expression. The identified microRNAs were further analysed in terms of gene ontology (GO) in relation with neurogenesis, based on their target mRNAs predicted by computational analysis. This study revealed the specific microRNAs involved in neurogenesis via microRNA microarray, and may provide the basic information for genetic induction of adult stem cell differentiation using microRNAs.

  10. Suppressive effects of liquid crystal compounds on the growth of U937 human leukemic monocyte lymphoma cells

    Directory of Open Access Journals (Sweden)

    Ishikawa Junya

    2012-02-01

    Full Text Available Abstract Background The aim of this study was to evaluate the biological and pharmaceutical activities of 14 amphiphilic liquid-crystalline compounds (LCs, i.e, phenylpyrimidine derivatives possessing D-glucamine and cyanobiphenyl derivatives with a terminal hydroxyl unit. Results The cytotoxic properties of the LCs on the cell growth, cell cycle distribution, and cell signaling pathway of U937 human leukemic monocyte lymphoma cells were assessed by flow cytometry and western blot analysis. Some LCs showed cytostatic effects, suppressing cell growth via S-phase arrest and without apoptosis in U937 cells. To investigate the mechanisms of the LC-induced S-phase arrest, proteins relevant to cell cycle regulation were investigated by western blot analysis. The rate of LC-induced S-phase arrest was congruent with the decreased expression of MCM2, cyclin A, cyclin B, CDK2, phospho-CDK1 and Cdc25C. Observed changes in cell cycle distribution by LC treated might be caused by insufficient preparation for G2/M transition. Considering the structure of the LCs, the rod-like molecules displaying cytotoxicity against U937 cells possessed flexible spacers with no bulky polar group attached via the flexible spacer. Conclusions Our results revealed that some LCs showed cytotoxic properties against non-solid type tumor human leukemic cells via LC-induced S-phase arrest and decreasing expression of several cell cycle related proteins.

  11. Increased UV resistance in xeroderma pigmentosum group A cells after transformation with a human genomic DNA clone

    International Nuclear Information System (INIS)

    Xeroderma pigmentosum (XP) is an autosomal recessive disease in which the major clinical manifestation is a 2,000-fold enhanced probability of developing sunlight-induced skin tumors, and the molecular basis for the disease is a defective DNA excision repair system. To clone the gene defective XP complementation group A (XP-A), cDNA clones were isolated by a competition hybridization strategy in which the corresponding mRNAs were more abundant in cells of the obligately heterozygous parents relative to cells to the homozygous proband affected with the disease. In this report, a human genomic DNA clone that contains this cDNA was transformed into two independent homozygous XP-A cell lines, and these transformants displayed partial restoration of resistance to the killing effects of UV irradiation. The abundance of mRNA corresponding to this cDNA appears to correlate well with the observed UV cell survival. The results of unscheduled DNA synthesis after UV exposure indicate that the transformed cells are repair proficient relative to that of the control XP-A cells. However, using this same genomic DNA, transformation of an XP-F cell line did not confer any enhancement of UV survival or promote unscheduled DNA synthesis after UV exposure

  12. The human mineral dust-induced gene, mdig, is a cell growth regulating gene associated with lung cancer

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Y.D.; Lu, Y.J.; Yuan, B.Z.; Castranova, V.; Shi, X.L.; Stauffer, J.L.; Demers, L.M.; Chen, F. [NIOSH, Morgantown, WV (US). Health Effects Laboratory Division

    2005-07-21

    Environmental or occupational exposure to mineral dusts, mainly silica and asbestos, is associated with an increased incidence of lung inflammation, fibrosis, and/or cancer. To better understand the molecular events associated with these pulmonary diseases, we attempted to identify genes that are regulated by mineral dusts. Using a differential display reverse transcription polymerase chain reaction technique and mRNAs of alveolar macrophages from both normal individuals and coal miners, we identified a novel mineral dust-induced gene named mdig, which had not been fully characterized. The expression of mdig mRNA was detected in alveolar macrophages from coal miners but not from normal subjects. The inducible expression of mdig could be observed in A549 cells exposed to silica particles in a time-dependent manner. The full-length mdig mRNA was expressed in human lung cancer tissues but was barely detectable in the adjacent normal tissues. In addition, a number of lung cancer cell lines constitutively express mdig. Alternative spliced transcripts of mdig were detected in some lung cancer cell lines. Silencing mdig mRNA expression in A549 lung cancer cells by siRNA-mediated RNA interference inhibits cell proliferation and sensitizes the cells to silica-induced cytotoxicity. These results suggest that the mdig gene may be involved in the regulation of cell growth and possibly the development of cancer.

  13. Oxidative Nanopatterning of Titanium Surface Influences mRNA and MicroRNA Expression in Human Alveolar Bone Osteoblastic Cells

    Science.gov (United States)

    Wimmers Ferreira, Maidy Rehder; Rodrigo Fernandes, Roger; Freire Assis, Amanda; Dernowsek, Janaína A.; Passos, Geraldo A.; Variola, Fabio; Fittipaldi Bombonato-Prado, Karina

    2016-01-01

    Titanium implants have been extensively used in orthopedic and dental applications. It is well known that micro- and nanoscale surface features of biomaterials affect cellular events that control implant-host tissue interactions. To improve our understanding of how multiscale surface features affect cell behavior, we used microarrays to evaluate the transcriptional profile of osteoblastic cells from human alveolar bone cultured on engineered titanium surfaces, exhibiting the following topographies: nanotexture (N), nano+submicrotexture (NS), and rough microtexture (MR), obtained by modulating experimental parameters (temperature and solution composition) of a simple yet efficient chemical treatment with a H2SO4/H2O2 solution. Biochemical assays showed that cell culture proliferation augmented after 10 days, and cell viability increased gradually over 14 days. Among the treated surfaces, we observed an increase of alkaline phosphatase activity as a function of the surface texture, with higher activity shown by cells adhering onto nanotextured surfaces. Nevertheless, the rough microtexture group showed higher amounts of calcium than nanotextured group. Microarray data showed differential expression of 716 mRNAs and 32 microRNAs with functions associated with osteogenesis. Results suggest that oxidative nanopatterning of titanium surfaces induces changes in the metabolism of osteoblastic cells and contribute to the explanation of the mechanisms that control cell responses to micro- and nanoengineered surfaces. PMID:27200092

  14. Revealing the cellular localization of STAT1 during the cell cycle by super-resolution imaging

    Science.gov (United States)

    Gao, Jing; Wang, Feng; Liu, Yanhou; Cai, Mingjun; Xu, Haijiao; Jiang, Junguang; Wang, Hongda

    2015-03-01

    Signal transducers and activators of transcription (STATs) can transduce cytokine signals and regulate gene expression. The cellular localization and nuclear trafficking of STAT1, a representative of the STAT family with multiple transcriptional functions, is tightly related with transcription process, which usually happens in the interphase of the cell cycle. However, these priority questions regarding STAT1 distribution and localization at the different cell-cycle stages remain unclear. By using direct stochastic optical reconstruction microscopy (dSTORM), we found that the nuclear expression level of STAT1 increased gradually as the cell cycle carried out, especially after EGF stimulation. Furthermore, STAT1 formed clusters in the whole cell during the cell cycle, with the size and the number of clusters also increasing significantly from G1 to G2 phase, suggesting that transcription and other cell-cycle related activities can promote STAT1 to form more and larger clusters for fast response to signals. Our work reveals that the cellular localization and clustering distribution of STAT1 are associated with the cell cycle, and further provides an insight into the mechanism of cell-cycle regulated STAT1 signal transduction.

  15. Cholesterol esterification during differentiation of mouse erythroleukemia (Friend) cells.

    Science.gov (United States)

    Mulas, Maria Franca; Mandas, Antonella; Abete, Claudia; Dessì, Sandra; Mocali, Alessandra; Paoletti, Francesco

    2011-08-31

    Cholesterol is an essential constituent of all mammalian cell membranes and its availability is therefore a prerequisite for cellular growth and other functions. Several lines of evidence are now indicating an association between alterations of cholesterol homeostasis and cell cycle progression. However, the role of cholesterol in cell differentiation is still largely unknown. To begin to address this issue, in this study we examined changes in cholesterol metabolism and in the mRNA levels of proteins involved in cholesterol import and esterification (multi-drug resistance, MDR-3) and acylCoA: cholesterol acyltransferase (ACAT) and cholesterol export (caveolin-1) in Friend virus-induced erythroleukemia cells (MELC), in the absence or in the presence of the chemical inducer of differentiation, hexamethylene bisacetamide (HMBA). FBS-stimulated growth of MELC was accompanied by an immediate elevation of cholesterol synthesis and cholesterol esterification, and by an increase in the levels of MDR-3 and ACAT mRNAs. A decrease in caveolin-1 expression was also observed. However, when MELC were treated with HMBA, the inhibition of DNA synthesis caused by HMBA treatment, was associated with a decrease in cholesterol esterification and in ACAT and MDR-3 mRNA levels and an increase in caveolin-1 mRNA. Detection of cytoplasmic neutral lipids by staining MELC with oil red O, a dye able to evidence CE but not FC, revealed that HMBA-treatment also reduced growth-stimulated accumulation of cholesterol ester to approximately the same extent as the ACAT inhibitor, SaH. Overall, these results indicate for the first time a role of cholesterol esterification and of some related genes in differentiation of erythroid cells. PMID:22184540

  16. Cholesterol esterification during differentiation of mouse erythroleukemia (Friend) cells

    Science.gov (United States)

    Mulas, Maria Franca; Mandas, Antonella; Abete, Claudia; Dessì, Sandra; Mocali, Alessandra; Paoletti, Francesco

    2011-01-01

    Cholesterol is an essential constituent of all mammalian cell membranes and its availability is therefore a prerequisite for cellular growth and other functions. Several lines of evidence are now indicating an association between alterations of cholesterol homeostasis and cell cycle progression. However, the role of cholesterol in cell differentiation is still largely unknown. To begin to address this issue, in this study we examined changes in cholesterol metabolism and in the mRNA levels of proteins involved in cholesterol import and esterification (multi-drug resistance, MDR-3) and acylCoA: cholesterol acyltransferase (ACAT) and cholesterol export (caveolin-1) in Friend virus-induced erythroleukemia cells (MELC), in the absence or in the presence of the chemical inducer of differentiation, hexamethylene bisacetamide (HMBA). FBS-stimulated growth of MELC was accompanied by an immediate elevation of cholesterol synthesis and cholesterol esterification, and by an increase in the levels of MDR-3 and ACAT mRNAs. A decrease in caveolin-1 expression was also observed. However, when MELC were treated with HMBA, the inhibition of DNA synthesis caused by HMBA treatment, was associated with a decrease in cholesterol esterification and in ACAT and MDR-3 mRNA levels and an increase in caveolin-1 mRNA. Detection of cytoplasmic neutral lipids by staining MELC with oil red O, a dye able to evidence CE but not FC, revealed that HMBA-treatment also reduced growth-stimulated accumulation of cholesterol ester to approximately the same extent as the ACAT inhibitor, SaH. Overall, these results indicate for the first time a role of cholesterol esterification and of some related genes in differentiation of erythroid cells. PMID:22184540

  17. Cholesterol esterification during differentiation of mouse erythroleukemia (Friend cells

    Directory of Open Access Journals (Sweden)

    Maria Franca Mulas

    2011-10-01

    Full Text Available Cholesterol is an essential constituent of all mammalian cell membranes, and its availability is therefore a prerequisite for cellular growth and other functions. Several lines of evidence are now indicating an association between alterations of cholesterol homeostasis and cell cycle progression. However, the role of cholesterol in cell differentiation is still largely unknown. To begin to address this issue, in this study we examined changes in cholesterol metabolism and in the mRNA levels of proteins involved in cholesterol import and esterification (multi-drug resistance, MDR-3 and acylCoA:cholesterol acyltransferase (ACAT and cholesterol export (caveolin-1 in Friend virus-induced erythroleukemia cells (MELC, in the absence or in the presence of the chemical inducer of differentiation, hexamethylene bisacetamide (HMBA. FBS-stimulated growth of MELC was accompanied by an immediate elevation of cholesterol synthesis and cholesterol esterification, and by an increase in the levels of MDR-3 and ACAT mRNAs. A decrease in caveolin-1 expression was also observed. However, when MELC were treated with HMBA, the inhibition of DNA synthesis caused by HMBA treatment, was associated with a decrease in cholesterol esterification and in ACAT and MDR-3 mRNA levels and an increase in caveolin-1 mRNA. Detection of cytoplasmic neutral lipids by staining MELC with oil red O, a dye able to evidence CE but not FC, revealed that HMBA-treatment also reduced growth-stimulated accumulation of cholesterol ester to approximately the same extent as the ACAT inhibitor, SaH. Overall, these results indicate for the first time a role of cholesterol esterification and of some related genes in differentiation of erythroid cells.

  18. Circulating angiogenic cell dysfunction in patients with hereditary hemorrhagic telangiectasia.

    Directory of Open Access Journals (Sweden)

    Liana Zucco

    Full Text Available Hereditary hemorrhagic telangiectasia (HHT is an autosomal dominant vascular disorder. Circulating angiogenic cells (CACs play an important role in vascular repair and regeneration. This study was designed to examine the function of CACs derived from patients with HHT. Peripheral blood mononuclear cells (PBMNCs isolated from patients with HHT and age- and gender-matched healthy volunteers were assessed for expression of CD34, CD133 and VEGF receptor 2 by flow cytometry. PBMNCs were cultured to procure early outgrowth CACs. Development of endothelial cell (EC phenotype in CACs was analyzed by fluorescence microscopy. CAC apoptosis was assayed with Annexin V staining, and CAC migration assessed by a modified Boyden chamber assay. mRNA expression of endoglin (ENG, activin receptor-like kinase-1 (ACVLR1 or ALK1 and endothelial nitric oxide synthase (eNOS in CACs was measured by real time RT-PCR. The percentage of CD34+ cells in PBMNCs from HHT patients was significantly higher than in PBMNCs of healthy controls. CACs derived from patients with HHT not only showed a significant reduction in EC-selective surface markers following 7-day culture, but also a significant increase in the rate of basal apoptosis and blunted migration in response to vascular endothelial growth factor and stromal cell-derived factor-1. CACs from HHT patients expressed significantly lower levels of ENG, ALK1 and eNOS mRNAs. In conclusion, CACs from patients with HHT exhibited various functional impairments, suggesting a reduced regenerative capacity of CACs to repair the vascular lesions seen in HHT patients.

  19. Neural differentiation potential of human bone marrow-derived mesenchymal stromal cells: misleading marker gene expression

    Directory of Open Access Journals (Sweden)

    Montzka Katrin

    2009-03-01

    Full Text Available Abstract Background In contrast to pluripotent embryonic stem cells, adult stem cells have been considered to be multipotent, being somewhat more restricted in their differentiation capacity and only giving rise to cell types related to their tissue of origin. Several studies, however, have reported that bone marrow-derived mesenchymal stromal cells (MSCs are capable of transdifferentiating to neural cell types, effectively crossing normal lineage restriction boundaries. Such reports have been based on the detection of neural-related proteins by the differentiated MSCs. In order to assess the potential of human adult MSCs to undergo true differentiation to a neural lineage and to determine the degree of homogeneity between donor samples, we have used RT-PCR and immunocytochemistry to investigate the basal expression of a range of neural related mRNAs and proteins in populations of non-differentiated MSCs obtained from 4 donors. Results The expression analysis revealed that several of the commonly used marker genes from other studies like nestin, Enolase2 and microtubule associated protein 1b (MAP1b are already expressed by undifferentiated human MSCs. Furthermore, mRNA for some of the neural-related transcription factors, e.g. Engrailed-1 and Nurr1 were also strongly expressed. However, several other neural-related mRNAs (e.g. DRD2, enolase2, NFL and MBP could be identified, but not in all donor samples. Similarly, synaptic vesicle-related mRNA, STX1A could only be detected in 2 of the 4 undifferentiated donor hMSC samples. More significantly, each donor sample revealed a unique expression pattern, demonstrating a significant variation of marker expression. Conclusion The present study highlights the existence of an inter-donor variability of expression of neural-related markers in human MSC samples that has not previously been described. This donor-related heterogeneity might influence the reproducibility of transdifferentiation protocols as

  20. Nestin-positive mesenchymal stem cells favour the astroglial lineage in neural progenitors and stem cells by releasing active BMP4

    Directory of Open Access Journals (Sweden)

    Leprince Pierre

    2004-09-01

    Full Text Available Abstract Background Spontaneous repair is limited after CNS injury or degeneration because neurogenesis and axonal regrowth rarely occur in the adult brain. As a result, cell transplantation has raised much interest as potential treatment for patients with CNS lesions. Several types of cells have been considered as candidates for such cell transplantation and replacement therapies. Foetal brain tissue has already been shown to have significant effects in patients with Parkinson's disease. Clinical use of the foetal brain tissue is, however, limited by ethical and technical problems as it requires high numbers of grafted foetal cells and immunosuppression. Alternatively, several reports suggested that mesenchymal stem cells, isolated from adult bone marrow, are multipotent cells and could be used in autograft approach for replacement therapies. Results In this study, we addressed the question of the possible influence of mesenchymal stem cells on neural stem cell fate. We have previously reported that adult rat mesenchymal stem cells are able to express nestin in defined culture conditions (in the absence of serum and after 25 cell population doublings and we report here that nestin-positive (but not nestin-negative mesenchymal stem cells are able to favour the astroglial lineage in neural progenitors and stem cells cultivated from embryonic striatum. The increase of the number of GFAP-positive cells is associated with a significant decrease of the number of Tuj1- and O4-positive cells. Using quantitative RT-PCR, we demonstrate that mesenchymal stem cells express LIF, CNTF, BMP2 and BMP4 mRNAs, four cytokines known to play a role in astroglial fate decision. In this model, BMP4 is responsible for the astroglial stimulation and oligodendroglial inhibition, as 1 this cytokine is present in a biologically-active form only in nestin-positive mesenchymal stem cells conditioned medium and 2 anti-BMP4 antibodies inhibit the nestin-positive mesenchymal

  1. Effects of Ellagic Acid on Angiogenic Factors in Prostate Cancer Cells

    International Nuclear Information System (INIS)

    Background: Several natural antioxidants, including ellagic acid (EA), have been reported to have chemotherapeutic activity in vivo and in vitro settings. Cytochrome P450 (CYP) activity and synthesis of both epoxyeicosatrienoic acids (EETs) and 20-hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE), together with vascular endothelial growth factor (VEGF) and heme oxygenase system (HO) have emerged as important modulators of tumor growth and metastasis. Methods: The anti-angiogenic effects of EA were investigated in the human prostatic cancer cell line LnCap. HO-1, HO-2, CYP2J2 and soluble epoxyde hydrolase (sEH) expressions were evaluated by western blotting. Levels of VEGF and osteoprotegerin (OPG) were determined in the culture supernatant using an ELISA assay, while CYP mRNAs were determined by qRT-PCR. Results: EA treatment induced a significant decrease (p < 0.05) in HO-1, HO-2 and CYP2J2 expression, and in VEGF and OPG levels. Similarly CYP2J2, CYP4F2 and CYPA22 mRNAs were significantly (p < 0.05) down-regulated by EA treatment. The decrease in CYP2J2 mRNA was associated with an increase in sEH expression. Conclusions: Results reported in the present study highlighted the ability of EA to modulate a new pathway, in addition to anti-proliferative and pro-differentiation properties, via a mechanism that involves a decrease in eicosanoid synthesis and a down-regulation of the HO system in prostate cancer

  2. Cyclebase.org: version 2.0, an updated comprehensive, multi-species repository of cell cycle experiments and derived analysis results

    DEFF Research Database (Denmark)

    Jensen, Lars Juhl; Wernersson, Rasmus; Brunak, Søren;

    2010-01-01

    -cycle-related experiments. This database provides an easy-to-use web interface that facilitates visualization and download of genome-wide cell-cycle data and analysis results. Data from different experiments are normalized to a common timescale and are complimented with key cell-cycle information and derived analysis...... results. In Cyclebase version 2.0, we have updated the entire database to reflect changes to genome annotations, included information on cyclin-dependent kinase (CDK) substrates, predicted degradation signals and loss-of-function phenotypes from genome-wide screens. The web interface has been improved and...... provides a single, gene-centric graph summarizing the available cell-cycle experiments. Finally, key information and links to orthologous and paralogous genes are now included to further facilitate comparison of cell-cycle regulation across species. Cyclebase version 2.0 is available at http://www.cyclebase.org....

  3. T Cells

    Science.gov (United States)

    T Cells - National Multiple Sclerosis Society Skip to navigation Skip to content Menu Navigation National Multiple Sclerosis Society Sign ... Is MS? Definition of MS T Cells T Cells Share Smaller Text Larger Text Print In this ...

  4. Cell counting.

    Science.gov (United States)

    Phelan, M C; Lawler, G

    2001-05-01

    This unit presents protocols for counting cells using either a hemacytometer or electronically using a Coulter counter. Cell counting with a hemacytometer permits effective discrimination of live from dead cells using trypan blue exclusion. In addition, the procedure is less subject to errors arising from cell clumping or size heterogeneity. Counting cells is more quickly and easily performed using an electronic counter, but live-dead discrimination is unreliable. Cell populations containing large numbers of dead cells and/or cell clumps are difficult to count accurately. In addition, electronic counting requires resetting of the instrument for cell populations of different sizes; heterogeneous populations can give rise to inaccurate counts, and resting and activated cells may require counting at separate settings. In general, electronic cell counting is best performed on fresh peripheral blood cells. PMID:18770655

  5. Conversion of mononuclear cells from human umbilical cord blood into hepatocyte-like cells

    Institute of Scientific and Technical Information of China (English)

    ZHANG Fang-ting; FANG Jia-zhi; YU Jie; WAN Hui-juan; YE Jing; LONG Xia; YIN Mei-jun; HUANG Chun-qiao

    2006-01-01

    Objective:To evaluate the differentiation of human umbilical cord blood cells into hepatocyte-like cells. Methods: Mononuclear cells (MNCs) derived from human umbilical cord blood were isolated using Ficoll. The experiment was derived into 3 categories: (1) MNCs co-cultured with 50 mg minced liver tissue separated by a trans-well membrane and then collected at 0 h,24 h,48 h and 72 h; (2) MNCs cultured along supplemented with 100 ml/L FBS, 100 μ/ml penicillin, 100 μg/ml streptomycin, 4. 7 μg/ml linoleic acid, 1×ITS, 10-4 mol/L L-Ascorbic acid 2-P and a combination of FGF4 (100 ng/ml) and HGF (20 ng/Ml). Cells were then collected at 0 d and 16 d to examine the expression profile of hepatocyte correlating markers; (3) 0.2-0.3 ml of MNCs with a cell density of 2×107/ml were transplanted into prepared recipient mice [n= 12, injected with 0.4 ml/kg (20%) CCl4 and 150 ng/kg 5-fluorouracil (5-Fu) prior the transplant 24 h and 48 h, respectively] via injection through tail vein. Mice were sacrificed 4 weeks after transplantation. The hepatocyte correlating mRNAs and proteins were determined by RTPCR, immunohistochemical analysis and immunoflurence technique. Results: (1) After 72 h, a number of glycogen positive stained cells were observed with MNCs co-cultured with damaged mouse liver tissues.The expression of hepatocyte markers, human albumin (ALB), α-fetal protein (AFP) and human GATA4 Mrna and proteins were detected by RT-PCR and immunohistochemistry as well. For the confirmation,the DNA sequencing of PCR products was performed. In control groups, MNCs co-cultured with normal mouse hepatocytes or MNCs cultured alone, all markers remained negative. (2) In growth factor supplemented culture system, MNCs developed into larger volume with richer cytoplasm and binucleation after 16 d. Positive expression of ALB, AFP, CK18 and CK19 Mrna were detected with RT-PCR, and ALB positive staining was observed by immunocytochemistry as well. In contrast, MNCs cultured without

  6. Activation of ERK mitogen-activated protein kinase in human cells by the mycotoxin patulin

    International Nuclear Information System (INIS)

    Patulin (PAT), a mycotoxin produced by certain species of Penicillium and Aspergillus, is often detectable in moldy fruits and their derivative products. PAT led to a concentration-dependent and time-dependent increase in phosphorylation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) in human embryonic kidney (HEK293) cells, human peripheral blood mononuclear cells (PBMCs), and Madin-Darby canine kidney (MDCK) cells. Exposure of HEK293 cells to concentrations above 5 μM PAT for 30 min induced ERK1/2 phosphorylation; activation of ERK1/2 was also observed after 24 h incubation with 0.05 μM of PAT. Treatment of human PBMCs for 30 min with 30 μM PAT dramatically increased the phosphorylated ERK1/2 levels. Both MEK1/2 inhibitors, U0126 and PD98059, suppressed ERK1/2 activation in either HEK293 or MDCK cells. In HEK293 cells, U0126-mediated inhibition of PAT-induced ERK1/2 phosphorylation resulted in a significant decrease in levels of DNA damage, expressed as tail moment values, in the single cell gel electrophoresis assay. Conversely, U0126 did not affect cell viability, lactate dehydrogenase release, and the DNA synthesis rate in PAT-treated cultures. Exposure of HEK293 cells for 90 min to 15 μM PAT elevated the levels of early growth response gene-1 (egr-1) mRNA, but not of c-fos, fosB, and junB mRNAs. These results indicate that in human cells, PAT causes a rapid and persistent activation of ERK1/2 and this signaling pathway plays an important role in mediating PAT-induced DNA damage and egr-1 gene expression

  7. The effect of calcium phosphate nanoparticles on hormone production and apoptosis in human granulosa cells

    Directory of Open Access Journals (Sweden)

    Gao Li

    2010-04-01

    Full Text Available Abstract Objectives Although many nanomaterials are being used in academia, industry and daily life, there is little understanding about the effects of nanoparticles on the reproductive health of vertebral animals, including human beings. An experimental study was therefore performed here to explore the effect of calcium phosphate nanoparticles on both steroid hormone production and apoptosis in human ovarian granulosa cells. Methods Calcium phosphate nanoparticles uptaking was evaluated by transmission electron microscopy (TEM. The cell cycle was assessed with propidium iodide-stained cells (distribution of cells in G0/G1, S, and G2/M phases by flow cytometry. The pattern of cell death (necrosis and apoptosis was analyzed by flow cytometry with annexin V-FITC/PI staining. The expression of mRNAs encoding P450scc, P450arom and StAR were determined by RT-PCR. Progesterone and estradiol levels were measured by radioimmunoassay. Results TEM results confirmed that calcium phosphate nanoparticles could enter into granulosa cells, and distributed in the membranate compartments, including lysosome and mitochondria and intracellular vesicles. The increased percentage of cells in S phase when cultured with nanoparticles indicated that there was an arrest at the checkpoint from phase S-to-G2/M (from 6.28 +/- 1.55% to 11.18 +/- 1.73%, p Conclusion Calcium phosphate nanoparticles interfered with cell cycle of cultured human ovarian granulosa cells thus increasing cell apoptosis. This pilot study suggested that effects of nanoparticles on ovarian function should be extensively investigated.

  8. Responses of genes involved in cell cycle control to diverse DNA damaging chemicals in human lung adenocarcinoma A549 cells

    Directory of Open Access Journals (Sweden)

    Gooderham Nigel J

    2005-08-01

    Full Text Available Abstract Background Many anticancer agents and carcinogens are DNA damaging chemicals and exposure to such chemicals results in the deregulation of cell cycle progression. The molecular mechanisms of DNA damage-induced cell cycle alteration are not well understood. We have studied the effects of etoposide (an anticancer agent, cryptolepine (CLP, a cytotoxic alkaloid, benzo [a]pyrene (BaP, a carcinogenic polycyclic aromatic hydrocarbon and 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP, a cooked-meat derived carcinogen on the expression of cell cycle regulatory genes to understand the molecular mechanisms of the cell cycle disturbance. Results A549 cells were treated with DMSO or chemicals for up to 72 h and periodically sampled for cell cycle analysis, mRNA and protein expression. DMSO treated cells showed a dominant G1 peak in cell cycle at all times examined. Etoposide and CLP both induced G2/M phase arrest yet the former altered the expression of genes functioning at multiple phases, whilst the latter was more effective in inhibiting the expression of genes in G2-M transition. Both etoposide and CLP induced an accumulation of p53 protein and upregulation of p53 transcriptional target genes. Neither BaP nor PhIP had substantial phase-specific cell cycle effect, however, they induced distinctive changes in gene expression. BaP upregulated the expression of CYP1B1 at 6–24 h and downregulated many cell cycle regulatory genes at 48–72 h. By contrast, PhIP increased the expression of many cell cycle regulatory genes. Changes in the expression of key mRNAs were confirmed at protein level. Conclusion Our experiments show that DNA damaging agents with different mechanisms of action induced distinctive changes in the expression pattern of a panel of cell cycle regulatory genes. We suggest that examining the genomic response to chemical exposure provides an exceptional opportunity to understand the molecular mechanism involved in cellular

  9. Costunolide causes mitotic arrest and enhances radiosensitivity in human hepatocellular carcinoma cells

    Directory of Open Access Journals (Sweden)

    Chen Chih-Jen

    2011-05-01

    Full Text Available Abstract Purpose This work aimed to investigate the effect of costunolide, a sesquiterpene lactone isolated from Michelia compressa, on cell cycle distribution and radiosensitivity of human hepatocellular carcinoma (HCC cells. Methods The assessment used in this study included: cell viability assay, cell cycle analysis by DNA histogram, expression of phosphorylated histone H3 (Ser 10 by flow cytometer, mitotic index by Liu's stain and morphological observation, mitotic spindle alignment by immunofluorescence of alpha-tubulin, expression of cell cycle-related proteins by Western blotting, and radiation survival by clonogenic assay. Results Our results show that costunolide reduced the viability of HA22T/VGH cells. It caused a rapid G2/M arrest at 4 hours shown by DNA histogram. The increase in phosphorylated histone H3 (Ser 10-positive cells and mitotic index indicates costunolide-treated cells are arrested at mitosis, not G2, phase. Immunofluorescence of alpha-tubulin for spindle formation further demonstrated these cells are halted at metaphase. Costunolide up-regulated the expression of phosphorylated Chk2 (Thr 68, phosphorylated Cdc25c (Ser 216, phosphorylated Cdk1 (Tyr 15 and cyclin B1 in HA22T/VGH cells. At optimal condition causing mitotic arrest, costunolide sensitized HA22T/VGH HCC cells to ionizing radiation with sensitizer enhancement ratio up to 1.9. Conclusions Costunolide could reduce the viability and arrest cell cycling at mitosis in hepatoma cells. Logical exploration of this mitosis-arresting activity for cancer therapeutics shows costunolide enhanced the killing effect of radiotherapy against human HCC cells.

  10. Costunolide causes mitotic arrest and enhances radiosensitivity in human hepatocellular carcinoma cells

    International Nuclear Information System (INIS)

    This work aimed to investigate the effect of costunolide, a sesquiterpene lactone isolated from Michelia compressa, on cell cycle distribution and radiosensitivity of human hepatocellular carcinoma (HCC) cells. The assessment used in this study included: cell viability assay, cell cycle analysis by DNA histogram, expression of phosphorylated histone H3 (Ser 10) by flow cytometer, mitotic index by Liu's stain and morphological observation, mitotic spindle alignment by immunofluorescence of alpha-tubulin, expression of cell cycle-related proteins by Western blotting, and radiation survival by clonogenic assay. Our results show that costunolide reduced the viability of HA22T/VGH cells. It caused a rapid G2/M arrest at 4 hours shown by DNA histogram. The increase in phosphorylated histone H3 (Ser 10)-positive cells and mitotic index indicates costunolide-treated cells are arrested at mitosis, not G2, phase. Immunofluorescence of alpha-tubulin for spindle formation further demonstrated these cells are halted at metaphase. Costunolide up-regulated the expression of phosphorylated Chk2 (Thr 68), phosphorylated Cdc25c (Ser 216), phosphorylated Cdk1 (Tyr 15) and cyclin B1 in HA22T/VGH cells. At optimal condition causing mitotic arrest, costunolide sensitized HA22T/VGH HCC cells to ionizing radiation with sensitizer enhancement ratio up to 1.9. Costunolide could reduce the viability and arrest cell cycling at mitosis in hepatoma cells. Logical exploration of this mitosis-arresting activity for cancer therapeutics shows costunolide enhanced the killing effect of radiotherapy against human HCC cells

  11. Different molecular responses of bronchoalveolar lavage and interstitial cells in c57bl/6j mice following thoracic irradiation

    International Nuclear Information System (INIS)

    Full text: To determine the contribution of intra-alveolar cells, as opposed to cells fixed in the interstitium in the development of radiation-induced fibrosis. C57BL/6J mice were irradiated to the thorax with various doses of radiation. The cellular composition and cytokine production were assessed in the two sites by histological staining and RNase protection assay (RPA). Following thoracic irradiation, there was an initial decrease in the number of bronchial alveolar lavage (BAL) cells that was followed after 2 months by a dose-dependent increase up to 6 months. Foamy Mac1+ve macrophages were present early and persistent to 6 months in the BAL populations, which also expressed the pro-inflammatory cytokine IL-1β. In contrast, in whole lung tissue there was a steady increase in Mac-1+ve cells and increased expression of TNF-αand IL-1β mRNAs to maximum levels at 2 months, subsided at 3 - 4 months, and further increased at 5 - 6 months when mice began to die from fibrosis. These data indicate distinct temporal and spatial changes in pro-inflammatory cytokine gene expression in different cellular compartments of the irradiated lung. BAL cells became inflammatory early on, but interstitial cells became involved later and are probably more involved in contributing to the fibrosis

  12. Human decidual macrophages and NK cells differentially express Toll-like receptors and display distinct cytokine profiles upon TLR stimulation.

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    ElisabethMenu

    2014-07-01

    We report for the first time that both human dMs and dNK cells express mRNAs encoding TLRs 1-9, albeit with a higher expression level in dMs. TLR2, TLR3 and TLR4 protein expression checked by flow cytometry was positive for both dMs and dNK cells. In vitro treatment of primary dMs and dNK cells with specific TLR2, TLR3, TLR4, TLR7/8 and TLR9 agonists enhanced their secretion of pro- and anti-inflammatory cytokines, as well as cytokines and chemokines involved in immune cell crosstalk. Only dNK cells released IFN-γ, whereas only dMs released IL-1β, IL-10 and IL-12. TLR9 activation of dMs resulted in a distinct pattern of cytokine expression compared to the other TLRs. The cytokine profiles expressed by dMs and dNK cells upon TLR activation are compatible with maintenance of the fetotolerant immune environment during initiation of immune responses to pathogens at the maternofetal interface.

  13. Expression and localization of prohormone convertase PC1 in the calcitonin-producing cells of the bullfrog ultimobranchial gland.

    Science.gov (United States)

    Yaoi, Yuichi; Suzuki, Masakazu; Tomura, Hideaki; Kurabuchi, Shingo; Sasayama, Yuichi; Tanaka, Shigeyasu

    2003-11-01

    We examined the expression and localization of the prohormone convertases, PC1 and PC2, in the ultimobranchial gland of the adult bullfrog using immunohistochemical (IHC) and in situ hybridization (ISH) techniques. In the ultimobranchial gland, PC1-immunoreactive cells were columnar, and were present in the follicular epithelium. When serial sections were immunostained with anti-calcitonin, anti-CGRP, anti-PC1, and anti-PC2 sera, PC1 was found only in the calcitonin/CGRP-producing cells. No PC2-immunopositive cells were detected. In the ISH, PC1 mRNA-positive cells were detected in the follicle cells in the ultimobranchial gland. No PC2 mRNA-positive cells were detected. RT-PCR revealed expression of the mRNAs of PC1 and the PC2 in the ultimobranchial gland. However, very little of the PC2 mRNA is probably translated because no PC2 protein was detected either by IHC staining or by Western blotting analysis. We conclude that the main prohormone convertase that is involved in the proteolytic cleavage of procalcitonin in the bullfrog is PC1. PMID:14566018

  14. The long non-coding RNA HOTAIR promotes the proliferation of serous ovarian cancer cells through the regulation of cell cycle arrest and apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Qiu, Jun-jun [Department of Gynecology, Obstetrics and Gynecology Hospital, Fudan University, 419 Fangxie Road, Shanghai 200011 (China); Department of Obstetrics and Gynecology of Shanghai Medical College, Fudan University, 138 Yixueyuan Road, Shanghai 200032 (China); Shanghai Key Laboratory of Female Reproductive Endocrine-Related Diseases, 413 Zhaozhou Road, Shanghai 200011 (China); Wang, Yan [Cancer Institute, Fudan University Shanghai Cancer Center, 270 Dong' an Road, Shanghai 200032 (China); Department of Oncology, Shanghai Medical College, Fudan University, 130 Dong' an Road, Shanghai 200032 (China); Ding, Jing-xin; Jin, Hong-yan [Department of Gynecology, Obstetrics and Gynecology Hospital, Fudan University, 419 Fangxie Road, Shanghai 200011 (China); Department of Obstetrics and Gynecology of Shanghai Medical College, Fudan University, 138 Yixueyuan Road, Shanghai 200032 (China); Shanghai Key Laboratory of Female Reproductive Endocrine-Related Diseases, 413 Zhaozhou Road, Shanghai 200011 (China); Yang, Gong, E-mail: yanggong@fudan.edu.cn [Cancer Institute, Fudan University Shanghai Cancer Center, 270 Dong' an Road, Shanghai 200032 (China); Department of Oncology, Shanghai Medical College, Fudan University, 130 Dong' an Road, Shanghai 200032 (China); Hua, Ke-qin, E-mail: huakeqin@126.com [Department of Gynecology, Obstetrics and Gynecology Hospital, Fudan University, 419 Fangxie Road, Shanghai 200011 (China); Department of Obstetrics and Gynecology of Shanghai Medical College, Fudan University, 138 Yixueyuan Road, Shanghai 200032 (China); Shanghai Key Laboratory of Female Reproductive Endocrine-Related Diseases, 413 Zhaozhou Road, Shanghai 200011 (China)

    2015-05-01

    HOX transcript antisense RNA (HOTAIR) is a well-known long non-coding RNA (lncRNA) whose dysregulation correlates with poor prognosis and malignant progression in many forms of cancer. Here, we investigate the expression pattern, clinical significance, and biological function of HOTAIR in serous ovarian cancer (SOC). Clinically, we found that HOTAIR levels were overexpressed in SOC tissues compared with normal controls and that HOTAIR overexpression was correlated with an advanced FIGO stage and a high histological grade. Multivariate analysis revealed that HOTAIR is an independent prognostic factor for predicting overall survival in SOC patients. We demonstrated that HOTAIR silencing inhibited A2780 and OVCA429 SOC cell proliferation in vitro and that the anti-proliferative effects of HOTAIR silencing also occurred in vivo. Further investigation into the mechanisms responsible for the growth inhibitory effects by HOTAIR silencing revealed that its knockdown resulted in the induction of cell cycle arrest and apoptosis through certain cell cycle-related and apoptosis-related proteins. Together, these results highlight a critical role of HOTAIR in SOC cell proliferation and contribute to a better understanding of the importance of dysregulated lncRNAs in SOC progression. - Highlights: • HOTAIR overexpression correlates with an aggressive tumour phenotype and a poor prognosis in SOC. • HOTAIR promotes SOC cell proliferation both in vitro and in vivo. • The proliferative role of HOTAIR is associated with regulation of the cell cycle and apoptosis.

  15. A phthalide derivative isolated from endophytic fungi Pestalotiopsis photiniae induces G1 cell cycle arrest and apoptosis in human HeLa cells

    Directory of Open Access Journals (Sweden)

    C. Chen

    2013-08-01

    Full Text Available MP [4-(3′,3′-dimethylallyloxy-5-methyl-6-methoxyphthalide] was obtained from liquid culture of Pestalotiopsis photiniae isolated from the Chinese Podocarpaceae plant Podocarpus macrophyllus. MP significantly inhibited the proliferation of HeLa tumor cell lines. After treatment with MP, characteristic apoptotic features such as DNA fragmentation and chromatin condensation were observed in DAPI-stained HeLa cells. Flow cytometry showed that MP induced G1 cell cycle arrest and apoptosis in a dose-dependent manner. Western blotting and real-time reverse transcription-polymerase chain reaction were used to investigate protein and mRNA expression. MP caused significant cell cycle arrest by upregulating the cyclin-dependent kinase inhibitor p27KIP1 protein and p21CIP1 mRNA levels in HeLa cells. The expression of p73 protein was increased after treatment with various MP concentrations. mRNA expression of the cell cycle-related genes, p21CIP1 , p16INK4a and Gadd45α, was significantly upregulated and mRNA levels demonstrated significantly increased translation of p73, JunB, FKHR, and Bim. The results indicate that MP may be a potential treatment for cervical cancer.

  16. A phthalide derivative isolated from endophytic fungi Pestalotiopsis photiniae induces G1 cell cycle arrest and apoptosis in human HeLa cells

    Energy Technology Data Exchange (ETDEWEB)

    Chen, C. [College of Life Science, Hebei University, Baoding (China); Yang, R.L. [Key Laboratory of Microbial Diversity Research and Application of Hebei Province, Baoding, China, Key Laboratory of Microbial Diversity Research and Application of Hebei Province, Baoding (China)

    2013-07-30

    MP [4-(3′,3′-dimethylallyloxy)-5-methyl-6-methoxyphthalide] was obtained from liquid culture of Pestalotiopsis photiniae isolated from the Chinese Podocarpaceae plant Podocarpus macrophyllus. MP significantly inhibited the proliferation of HeLa tumor cell lines. After treatment with MP, characteristic apoptotic features such as DNA fragmentation and chromatin condensation were observed in DAPI-stained HeLa cells. Flow cytometry showed that MP induced G1 cell cycle arrest and apoptosis in a dose-dependent manner. Western blotting and real-time reverse transcription-polymerase chain reaction were used to investigate protein and mRNA expression. MP caused significant cell cycle arrest by upregulating the cyclin-dependent kinase inhibitor p27{sup KIP1} protein and p21{sup CIP1} mRNA levels in HeLa cells. The expression of p73 protein was increased after treatment with various MP concentrations. mRNA expression of the cell cycle-related genes, p21{sup CIP1}, p16{sup INK4a} and Gadd45α, was significantly upregulated and mRNA levels demonstrated significantly increased translation of p73, JunB, FKHR, and Bim. The results indicate that MP may be a potential treatment for cervical cancer.

  17. The long non-coding RNA HOTAIR promotes the proliferation of serous ovarian cancer cells through the regulation of cell cycle arrest and apoptosis

    International Nuclear Information System (INIS)

    HOX transcript antisense RNA (HOTAIR) is a well-known long non-coding RNA (lncRNA) whose dysregulation correlates with poor prognosis and malignant progression in many forms of cancer. Here, we investigate the expression pattern, clinical significance, and biological function of HOTAIR in serous ovarian cancer (SOC). Clinically, we found that HOTAIR levels were overexpressed in SOC tissues compared with normal controls and that HOTAIR overexpression was correlated with an advanced FIGO stage and a high histological grade. Multivariate analysis revealed that HOTAIR is an independent prognostic factor for predicting overall survival in SOC patients. We demonstrated that HOTAIR silencing inhibited A2780 and OVCA429 SOC cell proliferation in vitro and that the anti-proliferative effects of HOTAIR silencing also occurred in vivo. Further investigation into the mechanisms responsible for the growth inhibitory effects by HOTAIR silencing revealed that its knockdown resulted in the induction of cell cycle arrest and apoptosis through certain cell cycle-related and apoptosis-related proteins. Together, these results highlight a critical role of HOTAIR in SOC cell proliferation and contribute to a better understanding of the importance of dysregulated lncRNAs in SOC progression. - Highlights: • HOTAIR overexpression correlates with an aggressive tumour phenotype and a poor prognosis in SOC. • HOTAIR promotes SOC cell proliferation both in vitro and in vivo. • The proliferative role of HOTAIR is associated with regulation of the cell cycle and apoptosis

  18. A phthalide derivative isolated from endophytic fungi Pestalotiopsis photiniae induces G1 cell cycle arrest and apoptosis in human HeLa cells

    International Nuclear Information System (INIS)

    MP [4-(3′,3′-dimethylallyloxy)-5-methyl-6-methoxyphthalide] was obtained from liquid culture of Pestalotiopsis photiniae isolated from the Chinese Podocarpaceae plant Podocarpus macrophyllus. MP significantly inhibited the proliferation of HeLa tumor cell lines. After treatment with MP, characteristic apoptotic features such as DNA fragmentation and chromatin condensation were observed in DAPI-stained HeLa cells. Flow cytometry showed that MP induced G1 cell cycle arrest and apoptosis in a dose-dependent manner. Western blotting and real-time reverse transcription-polymerase chain reaction were used to investigate protein and mRNA expression. MP caused significant cell cycle arrest by upregulating the cyclin-dependent kinase inhibitor p27KIP1 protein and p21CIP1 mRNA levels in HeLa cells. The expression of p73 protein was increased after treatment with various MP concentrations. mRNA expression of the cell cycle-related genes, p21CIP1, p16INK4a and Gadd45α, was significantly upregulated and mRNA levels demonstrated significantly increased translation of p73, JunB, FKHR, and Bim. The results indicate that MP may be a potential treatment for cervical cancer

  19. Effect of Hangeshashinto on calprotectin expression in human oral epithelial cells.

    Science.gov (United States)

    Hiroshima, Yuka; Bando, Mika; Inagaki, Yuji; Kido, Reiko; Kataoka, Masatoshi; Nagata, Toshihiko; Kido, Jun-Ichi

    2016-05-01

    Oral epithelial cells produce antimicrobial peptides (AMPs) to prevent microbial infection. Calprotectin (S100A8/S100A9) is one of these AMPs in oral epithelial cells, the expression of which is up-regulated by interleukin-1α (IL-1α). Hangeshashinto (HST) is a traditional Japanese herbal medicine that has anti-inflammatory effects. The purpose of this study was to investigate the effect of HST on the expression of calprotectin through the regulation of IL-1α in oral epithelial cells. Human oral epithelial cells (TR146) were cultured with HST in the presence or absence of anti-IL-1α antibody or IL-1 receptor antagonist, or with six major components of HST (3,4-dihydroxybenzaldehyde, baicalin, ginsenoside Rb1, glycyrrhizin, oleanolic acid and berberine). The expression of S100A8, S100A9, other AMPs and cytokine mRNAs was examined by RT-PCR and quantitative real-time PCR. Calprotectin expression and IL-1α secretion were investigated by ELISA. HST (6 μg/ml) increased the expression of S100A8/S100A9 mRNAs and calprotectin protein, and also up-regulated β-defensin 2 (DEFB4) and S100A7 expression. The expression of IL-1α mRNA and its protein was slightly but significantly increased by HST. A neutralizing antibody against IL-1α and IL-1 receptor antagonist inhibited HST-up-regulated S100A8/S100A9 mRNA expression. Although 3,4-dihydroxybenzaldehyde, baicalin and ginsenoside Rb1 as HST components increased S100A8/S100A9 expression, oleanolic acid and berberine decreased their expression. These results suggest that HST increases the expression of calprotectin, DEFB4 and S100A7 in oral epithelial cells. In response to HST, up-regulation of calprotectin expression may be partially induced via IL-1α. PMID:25649126

  20. Enterovirus 71 transmission by exosomes establishes a productive infection in human neuroblastoma cells.

    Science.gov (United States)

    Mao, Lingxiang; Wu, Jing; Shen, Li; Yang, Jing; Chen, Jianguo; Xu, Huaxi

    2016-04-01

    Exosomes are small secreted cellular vesicles for intercellular communications which contain proteins, mRNAs, and microRNAs (miRNAs). Recent studies have shown that exosomes play an important role in the transmission of infectious agents including hepatitis C virus, human immunodeficiency virus, and so on. However, the role of exosomes in the transfer of enterovirus 71 (EV71) between host cells remains unknown. In this study, we show that the exosomes derived from EV71-infected rhabdomyosarcoma cells contain EV71 RNA and capsid protein VP1, determined by quantitative reverse transcription-PCR (QRT-PCR) and Western blot analysis. The shedding of exosomes containing virus can establish a productive infection in human neuroblastoma cell line (SK-N-SH). A comparative analysis of neutralization by EV71-specific immunoglobulins showed different levels of neutralization of exosomes-mediated infection compared with free virus. In conclusion, exosomes from EV71-infected cells may play an important role in virus dissemination and are partially resisted to antibody neutralization. Our results suggest that there is an exosomal route of EV71 transmission infection. PMID:26837894

  1. AKAP4 is a circulating biomarker for non-small cell lung cancer

    Science.gov (United States)

    Gumireddy, Kiranmai; Li, Anping; Chang, David H.; Liu, Qin; Kossenkov, Andrew V.; Yan, Jinchun; Korst, Robert J.; Nam, Brian T.; Xu, Hua; Zhang, Lin; Ganepola, Ganepola A.P.; Showe, Louise C.; Huang, Qihong

    2015-01-01

    Cancer testis antigens (CTAs) are widely expressed in tumor tissues, circulating tumor cells (CTCs) and in cancer derived exosomes that are frequently engulfed by lymphoid cells. To determine whether tumor derived CTA mRNAs could be detected in RNA from purified peripheral blood mononuclear cells (PBMC) of non-small cell lung cancer (NSCLC) patients, we assayed for the expression of 116 CTAs in PBMC RNA in a discovery set and identified AKAP4 as a potential NSCLC biomarker. We validated AKAP4 as a highly accurate biomarker in a cohort of 264 NSCLCs and 135 controls from 2 different sites including a subset of controls with high risk lung nodules. When all (264) lung cancers were compared with all (135) controls the area under the ROC curve (AUC) was 0.9714. When 136 stage I NSCLC lung cancers are compared with all controls the AUC is 0.9795 and when all lung cancer patients were compared to 27 controls with histologically confirmed benign lung nodules, a comparison of significant clinical importance, the AUC was 0.9825. AKAP4 expression increases significantly with tumor stage, but independent of age, gender, smoking history or cancer subtype. Follow-up studies in a small number of resected NSCLC patients revealed a decrease of AKAP4 expression post-surgical resection that remained low in patients in remission and increased with tumor recurrence. AKAP4 is a highly accurate biomarker for the detection of early stage lung cancer. PMID:26160834

  2. Effective Alu repeat based RT-Qpcr normalization in cancer cell perturbation experiments.

    Directory of Open Access Journals (Sweden)

    Ali Rihani

    Full Text Available BACKGROUND: Measuring messenger RNA (mRNA levels using the reverse transcription quantitative polymerase chain reaction (RT-qPCR is common practice in many laboratories. A specific set of mRNAs as internal control reference genes is considered as the preferred strategy to normalize RT-qPCR data. Proper selection of reference genes is a critical issue, especially in cancer cells that are subjected to different in vitro manipulations. These manipulations may result in dramatic alterations in gene expression levels, even of assumed reference genes. In this study, we evaluated the expression levels of 11 commonly used reference genes as internal controls for normalization of 19 experiments that include neuroblastoma, T-ALL, melanoma, breast cancer, non small cell lung cancer (NSCL, acute myeloid leukemia (AML, prostate cancer, colorectal cancer, and cervical cancer cell lines subjected to various perturbations. RESULTS: The geNorm algorithm in the software package qbase+ was used to rank the candidate reference genes according to their expression stability. We observed that the stability of most of the candidate reference genes varies greatly in perturbation experiments. Expressed Alu repeats show relatively stable expression regardless of experimental condition. These Alu repeats are ranked among the best reference assays in all perturbation experiments and display acceptable average expression stability values (M<0.5. CONCLUSIONS: We propose the use of Alu repeats as a reference assay when performing cancer cell perturbation experiments.

  3. Analysis of miR-302 host RNA as a stem cell marker

    DEFF Research Database (Denmark)

    Rahimi, Karim

    2016-01-01

    Stem cells have unique properties including self-renewal and multipotency that are shared with cancer stem cells (CSCs). MicroRNAs are short non-coding RNAs that posttranscriptionally regulate gene expression either by degradation or by translation inhibition of their mRNA targets. MiRNAs are...... involved in many biological processes. The MiR-302s/367 gene is a polycistronic miRNA cluster including miR-302b/c/a/d and miR-367, that is expressed under the contol of Oct4, Sox2 and Nanog and is a direct target of the Wnt/β- catenin signaling pathway. MiR-302 has been shown to induce pluripotency in...... somatic cells and to repress mRNAs required for differentiation. In this study, we explored the possibility to use the miR-302 promoter/enhancer to drive stem cell specific expression of reporter genes. We first performed 'Rapid Amplification of cDNA Ends' (RACE) for the 5’ and 3’ ends of mmiR-302 and...

  4. PEG10 promotes human breast cancer cell proliferation, migration and invasion.

    Science.gov (United States)

    Li, Xinran; Xiao, Ruijing; Tembo, Kingsley; Hao, Ling; Xiong, Meng; Pan, Shan; Yang, Xiangyong; Yuan, Wen; Xiong, Jie; Zhang, Qiuping

    2016-05-01

    Paternally expressed imprinted gene 10 (PEG10), derived from the Ty3/Gypsy family of retrotransposons, has been implicated as a genetic imprinted gene. Accumulating evidence suggests that PEG10 plays an important role in tumor growth in various cancers, including hepatocellular carcinoma, lung cancer and prostate cancer. However, the correlation between PEG10 and breast cancer remains unclear. In the present study, we evaluated and characterized the role of PEG10 in human breast cancer proliferation, cell cycle, clone formation, migration and invasion. The expression level of PEG10 was significantly elevated in breast cancer tissues and associated with distant metastasis and poor clinical outcome. Gene set enrichment analysis indicated that high expression of PEG10 could enrich cell cycle-related processes in breast cancer tissues. Ectopic overexpression of PEG10 in breast cancer cells enhanced cell proliferation, cell cycle, clone formation along with migration and invasion. Cell-to-cell junction molecule E-cadherin was downregulated and matrix degradation proteases MMP-1, MMP-2, MMP-9 were up-regulated after PEG10 overexpression. Our results demonstrated that PEG10 is a crucial oncogene and has prognostic value for breast cancer, which could be applied in breast cancer diagnosis and targeting therapy in future. PMID:26934961

  5. Atrazine promotes RM1 prostate cancer cell proliferation by activating STAT3 signaling.

    Science.gov (United States)

    Hu, Kebang; Tian, Yong; Du, Yanwei; Huang, Liandi; Chen, Junyu; Li, Na; Liu, Wei; Liang, Zuowen; Zhao, Lijing

    2016-05-01

    Atrazine, a widely used pesticide, is frequently detected in soil and surface water, which alarms epidemiologists and medical professionals because of its potential deleterious effects on health. Indeed, atrazine is a potent endocrine disruptor that increases aromatase expression in some human cancer cell lines. Both animal and human studies have suggested that atrazine is possibly carcinogenic, although discrepant results have been reported. In this study, RM1 cells were used to explore the atrazine effects on prostate cancer. Proliferation, migration and invasion of RM1 cells were assessed by colony formation, wound-healing and invasion assays, respectively, after in vitro exposure to atrazine. In addition, an RM1 cell xenograft model was generated to evaluate the effects of atrazine in vivo. To explore the molecular mechanisms, qRT‑PCR, immunohistochemistry, and western blot analyses were employed to detect mRNA and protein levels of STAT3 signaling and cell cycle related proteins, including p53, p21, cyclin B1 and cyclin D1. Interestingly, RM1 cell proliferation was increased after treatment with atrazine, concomitantly with STAT3 signaling activation. These results suggest that atrazine promotes RM1 cell growth in vitro and in vivo by activating STAT3 signaling. PMID:26984284

  6. Induction of apoptosis and inhibition of cell growth by tbx5 knockdown contribute to dysmorphogenesis in Zebrafish embryos

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    Tang Renbing

    2011-10-01

    Full Text Available Abstract Background The tbx5 mutation in human causes Holt-Oram syndrome, an autosomal dominant condition characterized by a familial history of congenital heart defects and preaxial radial upper-limb defects. We report aberrant apoptosis and dormant cell growth over head, heart, trunk, fin, and tail of zebrafish embryos with tbx5 deficiency correspond to the dysmorphogenesis of tbx5 morphants. Methods Wild-type zebrafish embryos at the 1-cell stage were injected with 4.3 nl of 19.4 ng of tbx5 morpholino or mismatch-tbx5-MO respectively in tbx5 morphants and mismatched control group. Semi-quantitative RT-PCR was used to for expression analysis of apoptosis and cell cycle-related genes. TUNEL and immunohistochemical assay showed the apoptosis spots within the local tissues. Ultra-structure of cardiac myocardium was examined by transmission electron microscope. Results Apoptosis-related genes (bad, bax, and bcl2, and cell cycle-related genes (cdk2, pcna, p27, and p57 showed remarkable increases in transcriptional level by RT-PCR. Using a TUNEL and immnuohistochemical assay, apoptosis was observed in the organs including the head, heart, pectoral fins, trunk, and tail of tbx5 knockdown embryos. Under transmission electron microscopic examination, mitochondria in cardiomyocytes became swollen and the myocardium was largely disorganized with a disarrayed appearance, compatible with reduced enhancement of myosin in the cardiac wall. The ATP level was reduced, and the ADP/ATP ratio as an apoptotic index significantly increased in the tbx5 deficient embryos. Conclusion Our study highlighted that tbx5 deficiency evoked apoptosis, distributed on multiple organs corresponding to dysmorphogenesis with the shortage of promising maturation, in tbx5 knockdown zebrafish embryos. We hypothesized that mesenchymal cell apoptosis associated with altered TBX5 level may subsequently interfered with organogenesis and contributed to dysmorphogenesis in tbx5 deficiency

  7. Expansion of intestinal epithelial stem cells during murine development.

    Directory of Open Access Journals (Sweden)

    Jeffrey J Dehmer

    Full Text Available Murine small intestinal crypt development is initiated during the first postnatal week. Soon after formation, overall increases in the number of crypts occurs through a bifurcating process called crypt fission, which is believed to be driven by developmental increases in the number of intestinal stem cells (ISCs. Recent evidence suggests that a heterogeneous population of ISCs exists within the adult intestine. Actively cycling ISCs are labeled by Lgr5, Ascl2 and Olfm4; whereas slowly cycling or quiescent ISC are marked by Bmi1 and mTert. The goal of this study was to correlate the expression of these markers with indirect measures of ISC expansion during development, including quantification of crypt fission and side population (SP sorting. Significant changes were observed in the percent of crypt fission and SP cells consistent with ISC expansion between postnatal day 14 and 21. Quantitative real-time polymerase chain reaction (RT-PCR for the various ISC marker mRNAs demonstrated divergent patterns of expression. mTert surged earliest, during the first week of life as crypts are initially being formed, whereas Lgr5 and Bmi1 peaked on day 14. Olfm4 and Ascl2 had variable expression patterns. To assess the number and location of Lgr5-expressing cells during this period, histologic sections from intestines of Lgr5-EGFP mice were subjected to quantitative analysis. There was attenuated Lgr5-EGFP expression at birth and through the first week of life. Once crypts were formed, the overall number and percent of Lgr5-EGFP positive cells per crypt remain stable throughout development and into adulthood. These data were supported by Lgr5 in situ hybridization in wild-type mice. We conclude that heterogeneous populations of ISCs are expanding as measured by SP sorting and mRNA expression at distinct developmental time points.

  8. MicroRNA expression and regulation in human ovarian carcinoma cells by luteinizing hormone.

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    Juan Cui

    Full Text Available BACKGROUND: MicroRNAs have been widely-studied with regard to their aberrant expression and high correlation with tumorigenesis and progression in various solid tumors. With the major goal of assessing gonadotropin (luteinizing hormone, LH contributions to LH receptor (LHR-positive ovarian cancer cells, we have conducted a genome-wide transcriptomic analysis on human epithelial ovarian cancer cells to identify the microRNA-associated cellular response to LH-mediated activation of LHR. METHODS: Human ovarian cancer cells (SKOV3 were chosen as negative control (LHR- and stably transfected to express functional LHR (LHR+, followed by incubation with LH (0-20 h. At different times of LH-mediated activation of LHR the cancer cells were analyzed by a high-density Ovarian Cancer Disease-Specific-Array (DSA, ALMAC™, which profiled ∼ 100,000 transcripts with ∼ 400 non-coding microRNAs. FINDINGS: In total, 65 microRNAs were identified to exhibit differential expression in either LHR expressing SKOV3 cells or LH-treated cells, a few of which have been found in the genomic fragile regions that are associated with abnormal deletion or amplification in cancer, such as miR-21, miR-101-1, miR-210 and miR-301a. By incorporating the dramatic expression changes observed in mRNAs, strong microRNA/mRNA regulatory pairs were predicted through statistical analyses coupled with collective computational prediction. The role of each microRNA was then determined through a functional analysis based on the highly-confident microRNA/mRNA pairs. CONCLUSION: The overall impact on the transcriptome-level expression indicates that LH may regulate apoptosis and cell growth of LHR+ SKOV3 cells, particularly by reducing cancer cell proliferation, with some microRNAs involved in regulatory roles.

  9. Acute ablation of PERK results in ER dysfunctions followed by reduced insulin secretion and cell proliferation

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    McGrath Barbara C

    2009-09-01

    Full Text Available Abstract Background A deficiency in Perk (EIF2AK3 causes multiple neonatal defects in humans known as the Wolcott Rallison syndrome. Perk KO mice exhibit the same array of defects including permanent neonatal diabetes (PND. PND in mice was previously shown by us to be due to a decrease in beta cell proliferation and insulin secretion. The aim of this study was to determine if acute ablation of PERK in the 832/13 beta cells recapitulates these defects and to identify the primary molecular basis for beta cell dysfunction. Results The INS1 832/13 transformed rat beta cell line was transduced with a dominant-negative Perk transgene via an adenoviral vector. AdDNPerk-832/13 beta cells exhibited reduced expression of insulin and MafA mRNAs, reduced insulin secretion, and reduced cell proliferation. Although proinsulin content was reduced in AdDNPerk-832/13 beta cells, proinsulin was abnormally retained in the endoplasmic reticulum. A temporal study of the acute ablation of Perk revealed that the earliest defect seen was induced expression of two ER chaperone proteins, GRP78/BiP and ERp72. The oxidized states of ERp72 and ERp57 were also increased suggesting an imbalance in the redox state of the ER. Conclusion Acute ablation of Perk in INS 832/13 beta cells exhibited all of the major defects seen in Perk KO mice and revealed abnormal expression and redox state of key ER chaperone proteins. Dysregulation of ER chaperone/folding enzymes ERp72 and GRP78/BiP occurred early after ablation of PERK function suggesting that changes in ER secretory functions may give rise to the other defects including reduced insulin gene expression, secretion, and cell proliferation.

  10. A novel marker for terminal Schwann cells, homocysteine-responsive ER-resident protein, as isolated by a single cell PCR-differential display.

    Science.gov (United States)

    Oda, Ryo; Yaoi, Takeshi; Okajima, Seiichiro; Kobashi, Hiroaki; Kubo, Toshikazu; Fushiki, Shinji

    2003-09-01

    Terminal Schwann cells (TSCs) that cover motor neuron terminals are known to play important roles in maintaining neuromuscular junctions, as well as in the repair process after nerve injury. However, molecular characteristics of TSCs remain unknown, because of the difficulties in analyzing them due to their paucity. We have established a method of selectively and efficiently collecting TSCs so that cDNA analysis can be done properly. The expression of 1-2% of whole mRNAs was compared between myelinating Schwann cells (MSCs) and TSCs, and it turned out that approximately one-third of the bands could be categorized as cell-type-specific bands. TSCs thus constitute a distinct entity from the viewpoint of gene expression. As one of the cDNA clones belonging to TSC-specific bands was identified homocysteine-responsive ER-resident protein (Herp), and in situ hybridization confirmed that Herp mRNA is expressed in TSCs on motor nerve terminals but not in MSCs, both in developing and adult rats. In conclusion, we have been able to identify Herp as a novel molecular marker for TSCs. PMID:12927800

  11. Galvanic Cells

    Science.gov (United States)

    Young, I. G.

    1973-01-01

    Many standard physical chemistry textbooks contain ambiguities which lead to confusion about standard electrode potentials, calculating cell voltages, and writing reactions for galvanic cells. This article shows how standard electrode potentials can be used to calculate cell voltages and deduce cell reactions. (Author/RH)

  12. The fibrate decreases radiation sensitivity via peroxisome proliferator-activated receptor {alpha}-mediated superoxide dismutase induction in HeLa cells

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Xianguang; An, Zhengzhe; Song, Hye Jin; Kim, Won Dong; Park, Woo Yoon [Chungbuk National University College of Medicine, Cheongju (Korea, Republic of); Jang, Seong Soon [The Catholic University of Korea College of Medicine, Seoul (Korea, Republic of); Yu, Jae Ran [Konkuk University College of Medicine, Chungju (Korea, Republic of)

    2012-06-15

    The fibrates are ligands for peroxisome proliferator-activated receptor (PPAR) {alpha} and used clinically as hypolipidemic drugs. The fibrates are known to cause peroxisome proliferation, enhance superoxide dismutase (SOD) expression and catalase activity. The antioxidant actions of the fibrates may modify radiation sensitivity. Here, we investigated the change of the radiation sensitivity in two cervix cancer cell lines in combination with fenofi brate (FF). Activity and protein expression of SOD were measured according to the concentration of FF. The mRNA expressions were measured by using real time reverse-transcription polymerase chain reaction. Combined cytotoxic effect of FF and radiation was measured by using clonogenic assay. In HeLa cells total SOD activity was increased with increasing FF doses up to 30 {mu}M. In the other hand, the catalase activity was increased a little. As with activity the protein expression of SOD1 and SOD2 was increased with increasing doses of FF. The mRNAs of SOD1, SOD2, PPAR{alpha} and PPAR{gamma} were increased with increasing doses of FF. The reactive oxygen species (ROS) produced by radiation was decreased by preincubation with FF. The surviving fractions (SF) by combining FF and radiation was higher than those of radiation alone. In Me180 cells SOD and catalase activity were not increased with FF. Also, the mRNAs of SOD1, SOD2, and PPAR{alpha} were not increased with FF. However, the mRNA of PPAR{gamma} was increased with FF. FF can reduce radiation sensitivity by ROS scavenging via SOD induction in HeLa. SOD induction by FF is related with PPAR{alpha}.

  13. Stem Cells

    OpenAIRE

    Madhukar Thakur

    2009-01-01

    Objective: The objective of this presentation is to create awareness of stem cell applications in the ISORBE community and to foster a strategy of how the ISORBE community can disseminate information and promote the use of radiolabeled stem cells in biomedical applications. Methods: The continued excitement in Stem Cells, in many branches of basic and applied biomedical science, stems from the remarkable ability of stem cells to divide and develop into different types of cells in ...

  14. Cell Wall

    OpenAIRE

    Jamet, Elisabeth; Canut, Hervé; Boudart, Georges; Albenne, Cécile; Pont-Lezica, Rafael F

    2008-01-01

    This chapter covers our present knowledge of cell wall proteomics highlighting the distinctive features of cell walls and cell wall proteins in relation to problems encountered for protein extraction, separation and identification. It provides clues to design strategies for efficient cell wall proteomic studies. It gives an overview of the kinds of proteins that have yet been identified: the expected proteins vs the identified proteins. Finally, the new vision of the cell wall proteome, and t...

  15. Most microRNAs in the single-cell alga Chlamydomonas reinhardtii are produced by Dicer-like 3-mediated cleavage of introns and untranslated regions of coding RNAs.

    Science.gov (United States)

    Valli, Adrian A; Santos, Bruno A C M; Hnatova, Silvia; Bassett, Andrew R; Molnar, Attila; Chung, Betty Y; Baulcombe, David C

    2016-04-01

    We describe here a forward genetic screen to investigate the biogenesis, mode of action, and biological function of miRNA-mediated RNA silencing in the model algal species,Chlamydomonas reinhardtii Among the mutants from this screen, there were three atDicer-like 3that failed to produce both miRNAs and siRNAs and others affecting diverse post-biogenesis stages of miRNA-mediated silencing. The DCL3-dependent siRNAs fell into several classes including transposon- and repeat-derived siRNAs as in higher plants. The DCL3-dependent miRNAs differ from those of higher plants, however, in that many of them are derived from mRNAs or from the introns of pre-mRNAs. Transcriptome analysis of the wild-type anddcl3mutant strains revealed a further difference from higher plants in that the sRNAs are rarely negative switches of mRNA accumulation. The few transcripts that were more abundant indcl3mutant strains than in wild-type cells were not due to sRNA-targeted RNA degradation but to direct DCL3 cleavage of miRNA and siRNA precursor structures embedded in the untranslated (and translated) regions of the mRNAs. Our analysis reveals that the miRNA-mediated RNA silencing inC. reinhardtiidiffers from that of higher plants and informs about the evolution and function of this pathway in eukaryotes. PMID:26968199

  16. Inferring RBP-Mediated Regulation in Lung Squamous Cell Carcinoma

    Science.gov (United States)

    Lafzi, Atefeh; Kazan, Hilal

    2016-01-01

    RNA-binding proteins (RBPs) play key roles in post-transcriptional regulation of mRNAs. Dysregulations in RBP-mediated mechanisms have been found to be associated with many steps of cancer initiation and progression. Despite this, previous studies of gene expression in cancer have ignored the effect of RBPs. To this end, we developed a lasso regression model that predicts gene expression in cancer by incorporating RBP-mediated regulation as well as the effects of other well-studied factors such as copy-number variation, DNA methylation, TFs and miRNAs. As a case study, we applied our model to Lung squamous cell carcinoma (LUSC) data as we found that there are several RBPs differentially expressed in LUSC. Including RBP-mediated regulatory effects in addition to the other features significantly increased the Spearman rank correlation between predicted and measured expression of held-out genes. Using a feature selection procedure that accounts for the adaptive search employed by lasso regularization, we identified the candidate regulators in LUSC. Remarkably, several of these candidate regulators are RBPs. Furthermore, majority of the candidate regulators have been previously found to be associated with lung cancer. To investigate the mechanisms that are controlled by these regulators, we predicted their target gene sets based on our model. We validated the target gene sets by comparing against experimentally verified targets. Our results suggest that the future studies of gene expression in cancer must consider the effect of RBP-mediated regulation. PMID:27186987

  17. Different Donor Cell Culture Methods Can Influence the Developmental Ability of Cloned Sheep Embryos.

    Directory of Open Access Journals (Sweden)

    LiBing Ma

    Full Text Available It was proposed that arresting nuclear donor cells in G0/G1 phase facilitates the development of embryos that are derived from somatic cell nuclear transfer (SCNT. Full confluency or serum starvation is commonly used to arrest in vitro cultured somatic cells in G0/G1 phase. However, it is controversial as to whether these two methods have the same efficiency in arresting somatic cells in G0/G1 phase. Moreover, it is unclear whether the cloned embryos have comparable developmental ability after somatic cells are subjected to one of these methods and then used as nuclear donors in SCNT. In the present study, in vitro cultured sheep skin fibroblasts were divided into four groups: (1 cultured to 70-80% confluency (control group, (2 cultured to full confluency, (3 starved in low serum medium for 4 d, or (4 cultured to full confluency and then further starved for 4 d. Flow cytometry was used to assay the percentage of fibroblasts in G0/G1 phase, and cell counting was used to assay the viability of the fibroblasts. Then, real-time reverse transcription PCR was used to determine the levels of expression of several cell cycle-related genes. Subsequently, the four groups of fibroblasts were separately used as nuclear donors in SCNT, and the developmental ability and the quality of the cloned embryos were compared. The results showed that the percentage of fibroblasts in G0/G1 phase, the viability of fibroblasts, and the expression levels of cell cycle-related genes was different among the four groups of fibroblasts. Moreover, the quality of the cloned embryos was comparable after these four groups of fibroblasts were separately used as nuclear donors in SCNT. However, cloned embryos derived from fibroblasts that were cultured to full confluency combined with serum starvation had the highest developmental ability. The results of the present study indicate that there are synergistic effects of full confluency and serum starvation on arresting fibroblasts in

  18. A systems biology approach identifies a regulatory network in parotid acinar cell terminal differentiation.

    Directory of Open Access Journals (Sweden)

    Melissa A Metzler

    Full Text Available The transcription factor networks that drive parotid salivary gland progenitor cells to terminally differentiate, remain largely unknown and are vital to understanding the regeneration process.A systems biology approach was taken to measure mRNA and microRNA expression in vivo across acinar cell terminal differentiation in the rat parotid salivary gland. Laser capture microdissection (LCM was used to specifically isolate acinar cell RNA at times spanning the month-long period of parotid differentiation.Clustering of microarray measurements suggests that expression occurs in four stages. mRNA expression patterns suggest a novel role for Pparg which is transiently increased during mid postnatal differentiation in concert with several target gene mRNAs. 79 microRNAs are significantly differentially expressed across time. Profiles of statistically significant changes of mRNA expression, combined with reciprocal correlations of microRNAs and their target mRNAs, suggest a putative network involving Klf4, a differentiation inhibiting transcription factor, which decreases as several targeting microRNAs increase late in differentiation. The network suggests a molecular switch (involving Prdm1, Sox11, Pax5, miR-200a, and miR-30a progressively decreases repression of Xbp1 gene transcription, in concert with decreased translational repression by miR-214. The transcription factor Xbp1 mRNA is initially low, increases progressively, and may be maintained by a positive feedback loop with Atf6. Transfection studies show that Xbp1 activates the Mist1 promoter [corrected]. In addition, Xbp1 and Mist1 each activate the parotid secretory protein (Psp gene, which encodes an abundant salivary protein, and is a marker of terminal differentiation.This study identifies novel expression patterns of Pparg, Klf4, and Sox11 during parotid acinar cell differentiation, as well as numerous differentially expressed microRNAs. Network analysis identifies a novel stemness arm, a

  19. The PPARgamma-selective ligand BRL-49653 differentially regulates the fate choices of rat calvaria versus rat bone marrow stromal cell populations

    Directory of Open Access Journals (Sweden)

    Yoshiko Yuji

    2008-07-01

    Full Text Available Abstract Background Osteoblasts and adipocytes are derived from a common mesenchymal progenitor and an inverse relationship between expression of the two lineages is seen with certain experimental manipulations and in certain diseases, i.e., osteoporosis, but the cellular pathway(s and developmental stages underlying the inverse relationship is still under active investigation. To determine which precursor mesenchymal cell types can differentiate into adipocytes, we compared the effects of BRL-49653 (BRL, a selective ligand for peroxisome proliferators-activated receptor (PPARγ, a master transcription factor of adipogenesis, on osteo/adipogeneis in two different osteoblast culture models: the rat bone marrow (RBM versus the fetal rat calvaria (RC cell system. Results BRL increased the number of adipocytes and corresponding marker expression, such as lipoprotein lipase, fatty acid-binding protein (aP2, and adipsin, in both culture models, but affected osteoblastogenesis only in RBM cultures, where a reciprocal decrease in bone nodule formation and osteoblast markers, e.g., osteopontin, alkaline phosphatase (ALP, bone sialoprotein, and osteocalcin was seen, and not in RC cell cultures. Even though adipocytes were histologically undetectable in RC cultures not treated with BRL, RC cells expressed PPAR and CCAAT/enhancer binding protein (C/EBP mRNAs throughout osteoblast development and their expression was increased by BRL. Some single cell-derived BRL-treated osteogenic RC colonies were stained not only with ALP/von Kossa but also with oil red O and co-expressed the mature adipocyte marker adipsin and the mature osteoblast marker OCN, as well as PPAR and C/EBP mRNAs. Conclusion The data show that there are clear differences in the capacity of BRL to alter the fate choices of precursor cells in stromal (RBM versus calvarial (RC cell populations and that recruitment of adipocytes can occur from multiple precursor cell pools (committed preadipocyte

  20. Comparison of transcript levels and mRNA half-lives for the subunits of the branched-chain {alpha}-keto acid dehydrogenase (BCKD) complex in two human cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Bowers, B.A.; Danner, D.J. [Emory Univ., Atlanta, GA (United States)

    1994-09-01

    BCKD is a mitochondrial multienzyme complex that catalyzes the committed step in catabolism of the keto acid derivatives of leucine, isoleucine and valine. Three subunits, El{alpha}, E1{beta} and E2 are specific to the complex. The subunits are nuclearly encoded from genes located on separate chromosomes, and it is not yet understood how gene expression of the components is regulated to maintain proper stoichiometry of the complex. The focus of the present study is to establish mRNA half-lives for the BCKD subunits in two human cell lines and to examine whether expression of transcripts for the subunits is similar in different cell types. HepG2 cells, a hepatocarcinoma cell line, and DG75 cells, a Burkitt`s lymphoma cell line, express comparable levels of BCKD complex based on total enzyme activity. Half-lives of the mRNAs for each subunit have been determined in HepG2 cells and are presently being defined in DG75 cells. mRNA half-lives were calculated by quantifying message levels over a 24 hour period following an actinomycin D block. Transcripts for the BCKD subunits are relatively stable in HepG2 cells with mRNA half-lives for the E1{alpha} of 11 hours, E1{beta}, 24 hours and E2, 22 hours. Steady-state message levels have been analyzed in both cell lines by RNase protection and quantified as a percentage of total RNA. mRNA levels for all three subunits are higher in DG75 cells than in HepG2 cells (E1{alpha}, 4-fold; E1{beta}, 1.9-fold; E2, 1.8-fold). Preliminary data indicates that the half-life of the E1{alpha} transcript in DG75 cells is approximately 29 hours, and it is possible that differences in steady-state levels of the mRNAs are achieved through different half-lives of the transcripts. The relationship between transcript levels and protein levels for the three subunits is being examined in both cell types.

  1. Downregulation of HuR as a new mechanism of doxorubicin resistance in breast cancer cells

    Directory of Open Access Journals (Sweden)

    Latorre Elisa

    2012-03-01

    Full Text Available Abstract Background HuR, an RNA binding protein involved in the post-transcriptional regulation of a wide spectrum of mRNAs, has been demonstrated to be a determinant of carcinogenesis and tumor aggressiveness in several cancer types. In this study, we investigated the role of HuR in the apoptosis and in the chemoresistance induced by the widely used anticancer drug doxorubicin in human breast cancer cells (MCF-7. Results We showed that HuR acts in the early phase of cell response to doxorubicin, being induced to translocate into the cytoplasm upon phosphorylation. Reducing HuR levels diminished the apoptotic response to doxorubicin. Doxorubicin-induced apoptosis was also correlated with the presence of HuR in the cytoplasm. Rottlerin, which was able to block HuR nuclear export, had correspondingly antagonistic effects with doxorubicin on cell toxicity. The proapoptotic activity of HuR was not due to cleavage to an active form, as was previously reported. In in vitro selected doxorubicin resistant MCF-7 cells (MCF-7/doxoR overexpressing the multidrug resistance (MDR related ABCG2 transporter, we observed a significant HuR downregulation that was paralleled by a corresponding downregulation of HuR targets and by loss of rottlerin toxicity. Restoration of HuR expression in these cells resensitized MCF-7/doxoR cells to doxorubicin, reactivating the apoptotic response. Conclusions The present study shows that HuR is necessary to elicit the apoptotic cell response to doxorubicin and that restoration of HuR expression in resistant cells resensitizes them to the action of this drug, thereby identifying HuR as a key protein in doxorubicin pharmacology.

  2. The transcriptome of corona radiata cells from individual MII oocytes that after ICSI developed to embryos selected for transfer: PCOS women compared to healthy women

    DEFF Research Database (Denmark)

    Wissing, Marie Louise; Sonne, Si Brask; Westergaard, David;

    2014-01-01

    from individual oocytes developing into embryos selected for transfer. CRCs were isolated in a two-step denudation procedure, separating outer cumulus cells from the inner CRCs. Extracted RNA was amplified and transcriptome profiling was performed with Human Agilent® arrays. The transcriptomes of CRCs...... cell cycle-related genes and cell cycle pathways in PCOS CRCs could indicate a disturbed or delayed final maturation and differentiation of the CRCs in response to the human chorionic gonadotropin (hCG) surge. However, this had no effect on the in vitro development of the corresponding embryos. Future...... is controversial whether PCOS associate with diminished oocyte quality. The purpose of this study was to compare individual human CRC samples between PCOS patients and controls. All patients were stimulated by the long gonadotropin-releasing hormone (GnRH) agonist protocol. The CRC samples originated...

  3. Normal HC11 and ras-transformed mouse mammary cells are resistant to the antiproliferative effects of retinoic acid

    Directory of Open Access Journals (Sweden)

    Snitcovsky I.

    2003-01-01

    Full Text Available The objective of the present study was to determine the effects of retinoic acid on the growth of the mouse mammary cells HC11 and HC11ras, which are a model for in vitro breast cancer progression. The expression of the two classes (RARs and RXRs of retinoic acid receptor mRNAs was determined by Northern blot analysis. Receptor functional integrity was determined by testing whether RAR ß mRNA could be induced by retinoic acid. The effects of a 72-h exposure to 50 µM 13-cis retinoic acid on HC11 and HC11ras cell proliferation and HC11 cell differentiation were investigated by flow cytometric cell cycle analysis, and by determination of ß-casein mRNA expression, respectively. The possibility that retinoic acid would induce the expression of the vitamin D receptor and synergize with vitamin D, a known inhibitor of HC11 cell growth, was also investigated. HC11 cells expressed higher mRNA levels of both RAR a and RAR g when compared to HC11ras cells. In contrast, RAR ß, as well as RXR a, ß and g expression was low in both HC11 and HC11ras cells. In addition, RAR ß mRNA was induced by retinoic acid treatment in both cells. In spite of these observations, no effects were seen on cell proliferation or differentiation upon exposure to retinoic acid. Neither vitamin D receptor induction nor synergy with vitamin D on growth inhibition was observed. We conclude that the RAR expression profile could be related to the transformed state in HC11ras cells and that the retinoic acid resistance observed merits further investigation.

  4. FRTL-5 Rat Thyroid Cells Release Thyroglobulin Sequestered in Exosomes: A Possible Novel Mechanism for Thyroglobulin Processing in the Thyroid

    Science.gov (United States)

    Vlasov, Pavel; Doi, Sonia Q.; Sellitti, Donald F.

    2016-01-01

    Exosomes are 30–100 nm, membrane-bound vesicles containing specific cellular proteins, mRNAs, and microRNAs that take part in intercellular communication between cells. A possible role for exosomes in thyroid function has not been fully explored. In the present study, FRTL-5 rat thyroid cells were grown to confluence and received medium containing either thyroid stimulating hormone (TSH), exogenous bovine thyroglobulin (bTg), or neither additive for 24 or 48 hours followed by collection of spent medium and ultracentrifugation to isolate small vesicles. Transmission electron microscopy and Western blotting for CD9 indicated the presence of exosomes. Western blotting of exosome extract using a monoclonal anti-Tg antibody revealed a Tg-positive band at ~330 kDa (the expected size of monomeric Tg) with a higher density in TSH-treated cells compared to that in untreated cells. These results are the first to show that normal thyroid cells in culture produce exosomes containing undegraded Tg. PMID:27379194

  5. miR-10b*, a master inhibitor of the cell cycle, is down-regulated in human breast tumours

    Science.gov (United States)

    Biagioni, Francesca; Bossel Ben-Moshe, Noa; Fontemaggi, Giulia; Canu, Valeria; Mori, Federica; Antoniani, Barbara; Di Benedetto, Anna; Santoro, Raffaela; Germoni, Sabrina; De Angelis, Fernanda; Cambria, Anna; Avraham, Roi; Grasso, Giuseppe; Strano, Sabrina; Muti, Paola; Mottolese, Marcella; Yarden, Yosef; Domany, Eytan; Blandino, Giovanni

    2012-01-01

    Deregulated proliferation is a hallmark of cancer cells. Here, we show that microRNA-10b* is a master regulator of breast cancer cell proliferation and is downregulated in tumoural samples versus matched peritumoural counterparts. Two canonical CpG islands (5 kb) upstream from the precursor sequence are hypermethylated in the analysed breast cancer tissues. Ectopic delivery of synthetic microRNA-10b* in breast cancer cell lines or into xenograft mouse breast tumours inhibits cell proliferation and impairs tumour growth in vivo, respectively. We identified and validated in vitro and in vivo three novel target mRNAs of miR-10b* (BUB1, PLK1 and CCNA2), which play a remarkable role in cell cycle regulation and whose high expression in breast cancer patients is associated with reduced disease-free survival, relapse-free survival and metastasis-free survival when compared to patients with low expression. This also suggests that restoration of microRNA-10b* expression might have therapeutic promise. PMID:23125021

  6. Antitumor activity of rhein lysinate against human glioma U87 cells in vitro and in vivo.

    Science.gov (United States)

    Liu, Jin; Zhang, Ke; Zhen, Yong-Zhan; Wei, Jie; Hu, Gang; Gao, Jun-Ling; Tian, Yan-Xia; Lin, Ya-Jun

    2016-03-01

    In previous studies, we demonstrated that rhein lysinate (RHL), the salt of rhein and lysine that is easily dissolved in water, inhibited the growth of tumor cells derived from breast and ovarian cancer, hepatocellular carcinoma, cervical cancer and lung carcinoma. Based on these observations, human glioma U87 cells and a xenograft model in BALB/c nude mice were used to examine the antitumor activity of RHL against human glioma. Notably, RHL statistically significantly suppressed the growth of human glioma U87 xenografts in BALB/c nude mice. In vitro, there was a significant reduction in cell proliferation after treatment with RHL in a dose- and time-dependent manner. The overall growth inhibition was correlated with the increase in reactive oxygen species (ROS) production and cell apoptosis. The apoptosis- and cell cycle-related proteins including BAX and Bim were increased, whereas Bcl-2 and cyclin D were decreased in the RHL-treated cells. The results demonstrated that RHL is highly effective against the growth of human glioma U87 xenografts in BALB/c nude mice. The potent antitumor activity of RHL may be mediated through downregulation of Bcl-2 and cyclin D expression and upregulation of BAX and Bim expression. PMID:26707131

  7. Hypersensitivity of mouse NEIL1-knockdown cells to hydrogen peroxide during S phase

    International Nuclear Information System (INIS)

    Oxidative base damage occurs spontaneously due to reactive oxygen species generated as byproducts of respiration and other pathological processes in mammalian cells. Many oxidized bases are mutagenic and/or toxic, and most are repaired through the base excision repair pathway. Human endonuclease VIII-like protein 1 (hNEIL1) is thought to play an important role during the S phase of the cell cycle by removing oxidized bases in DNA replication fork-like (bubble) structures, and the protein level of hNEIL1 is increased in S phase. Compared with hNEIL1, there is relatively little information on the properties of the mouse ortholog mNEIL1. Since mouse cell nuclei lack endonuclease III-like protein (NTH) activity, in contrast to human cell nuclei, mNEIL1 is a major DNA glycosylase for repair of oxidized pyrimidines in mouse nuclei. In this study, we made mNEIL1-knockdown cells using an shRNA expression vector and examined the cell cycle-related variation in hydrogen peroxide (H2O2) sensitivity. Hypersensitivity to H2O2 caused by mNEIL1 knockdown was more significant in S phase than in G1 phase, suggesting that mNEIL1 has an important role during S phase, similarly to hNEIL1

  8. CD5 expression is regulated during human T-cell activation by alternative polyadenylation, PTBP1, and miR-204.

    Science.gov (United States)

    Domingues, Rita G; Lago-Baldaia, Inês; Pereira-Castro, Isabel; Fachini, Joseph M; Oliveira, Liliana; Drpic, Danica; Lopes, Nair; Henriques, Telmo; Neilson, Joel R; Carmo, Alexandre M; Moreira, Alexandra

    2016-06-01

    T lymphocytes stimulated through their antigen receptor (TCR) preferentially express mRNA isoforms with shorter 3´ untranslated regions (3´-UTRs) derived from alternative pre-mRNA cleavage and polyadenylation (APA). However, the physiological relevance of APA programs remains poorly understood. CD5 is a T-cell surface glycoprotein that negatively regulates TCR signaling from the onset of T-cell activation. CD5 plays a pivotal role in mediating outcomes of cell survival or apoptosis, and may prevent both autoimmunity and cancer. In human primary T lymphocytes and Jurkat cells we found three distinct mRNA isoforms encoding CD5, each derived from distinct poly(A) signals (PASs). Upon T-cell activation, there is an overall increase in CD5 mRNAs with a specific increase in the relative expression of the shorter isoforms. 3´-UTRs derived from these shorter isoforms confer higher reporter expression in activated T cells relative to the longer isoform. We further show that polypyrimidine tract binding protein (PTB/PTBP1) directly binds to the proximal PAS and PTB siRNA depletion causes a decrease in mRNA derived from this PAS, suggesting an effect on stability or poly(A) site selection to circumvent targeting of the longer CD5 mRNA isoform by miR-204. These mechanisms fine-tune CD5 expression levels and thus ultimately T-cell responses. PMID:27005442

  9. Protective effects of components of the Chinese herb grassleaf sweetlfag rhizome on PC12 cells incubated with amyloid-beta42

    Institute of Scientific and Technical Information of China (English)

    Zi-hao Liang; Xiao-hui Cheng; Zhi-gang Ruan; Han Wang; Shan-shan Li; Jing Liu; Guo-ying Li; Su-min Tian

    2015-01-01

    The major ingredients of grassleaf sweetlfag rhizome areβ-asarone and eugenol, which can cross the blood-brain barrier and protect neurons. This study aimed to observe the neuroprotective effects and mechanisms ofβ-asarone and eugenol, components of the Chinese herb grassleaf sweetlfag rhizome, on PC12 cells. First, PC12 cells were cultured with different concentrations (between 1 × 10–10 M and 1 × 10–5 M) ofβ-asarone and eugenol. Survival rates of PC12 cells were not significantly affected. Second, PC12 cells incubated with amyloid-beta42, which reduced cell survival, were cultured under the same conditions (1 × 10–6 Mβ-asarone and eugenol). The survival rates of PC12 cells significantly increased, while expression levels of the mRNAs for the pro-apoptotic protein Bax decreased, and those for the anti-apoptotic protein Bcl mRNA increased. In addition, the combination ofβ-asarone with eugenol achieved better results than either component alone. Our experimental ifndings indicate that bothβ-asarone and eugenol protect PC12 cells through inhibiting apoptosis, and that the combination of the two is better than either alone.

  10. Protective effects of components of the Chinese herb grassleaf sweetflag rhizome on PC12 cells incubated with amyloid-beta42

    Directory of Open Access Journals (Sweden)

    Zi-hao Liang

    2015-01-01

    Full Text Available The major ingredients of grassleaf sweetflag rhizome are β-asarone and eugenol, which can cross the blood-brain barrier and protect neurons. This study aimed to observe the neuroprotective effects and mechanisms of β-asarone and eugenol, components of the Chinese herb grassleaf sweetflag rhizome, on PC12 cells. First, PC12 cells were cultured with different concentrations (between 1 × 10 -10 M and 1 × 10 -5 M of β-asarone and eugenol. Survival rates of PC12 cells were not significantly affected. Second, PC12 cells incubated with amyloid-beta42, which reduced cell survival, were cultured under the same conditions (1 × 10 -6 M β-asarone and eugenol. The survival rates of PC12 cells significantly increased, while expression levels of the mRNAs for the pro-apoptotic protein Bax decreased, and those for the anti-apoptotic protein Bcl mRNA increased. In addition, the combination of β-asarone with eugenol achieved better results than either component alone. Our experimental findings indicate that both β-asarone and eugenol protect PC12 cells through inhibiting apoptosis, and that the combination of the two is better than either alone.

  11. Pituitary adenylate cyclase-activating polypeptide (PACAP) contributes to the proliferation of hematopoietic progenitor cells in murine bone marrow via PACAP-specific receptor.

    Science.gov (United States)

    Xu, Zhifang; Ohtaki, Hirokazu; Watanabe, Jun; Miyamoto, Kazuyuki; Murai, Norimitsu; Sasaki, Shun; Matsumoto, Minako; Hashimoto, Hitoshi; Hiraizumi, Yutaka; Numazawa, Satoshi; Shioda, Seiji

    2016-01-01

    Pituitary adenylate cyclase-activating polypeptide (PACAP, encoded by adcyap1) plays an important role in ectodermal development. However, the involvement of PACAP in the development of other germ layers is still unclear. This study assessed the expression of a PACAP-specific receptor (PAC1) gene and protein in mouse bone marrow (BM). Cells strongly expressing PAC1(+) were large in size, had oval nuclei, and merged with CD34(+) cells, suggesting that the former were hematopoietic progenitor cells (HPCs). Compared with wild-type mice, adcyap1(-/-) mice exhibited lower multiple potential progenitor cell populations and cell frequency in the S-phase of the cell cycle. Exogenous PACAP38 significantly increased the numbers of colony forming unit-granulocyte/macrophage progenitor cells (CFU-GM) with two peaks in semi-solid culture. PACAP also increased the expression of cyclinD1 and Ki67 mRNAs. These increases were completely and partially inhibited by the PACAP receptor antagonists, PACAP6-38 and VIP6-28, respectively. Little or no adcyap1 was expressed in BM and the number of CFU-GM colonies was similar in adcyap1(-/-) and wild-type mice. However, PACAP mRNA and protein were expressed in paravertebral sympathetic ganglia, which innervate tibial BM, and in the sympathetic fibers of BM cavity. These results suggested that sympathetic nerve innervation may be responsible for PACAP-regulated hematopoiesis in BM, mainly via PAC1. PMID:26925806

  12. Targeted Knockdown of RNA-Binding Protein TIAR for Promoting Self-Renewal and Attenuating Differentiation of Mouse Embryonic Stem Cells.

    Science.gov (United States)

    Geng, Zhe; Li, Ping; Tan, Li; Song, Houyan

    2015-01-01

    RNA-binding protein TIAR has been suggested to mediate the translational silencing of ARE-containing mRNAs. To analyze the functions of TIAR, we established RNAi and genetic rescue assays. We evaluated the expression of neuroectoderm markers Pax6 and nestin, mesoderm markers brachyury and Flk1, and hypoblast and definitive endoderm markers Sox17 and Gata6 during EB differentiation and found that knockdown TIAR expression restrained the differentiation of E14 cells. We assessed gene expression levels of Flk-1 and VE-cadherin and observed attenuated differentiation of E14 cells into endothelial cells upon downregulation of TIAR gene expression. As such, we hypothesized an essential role of TIAR related to EB differentiation. As TIAR inhibits the translation of c-myc, we proposed that downregulation of TIAR results in restrained differentiation of E14 cells, due in part to the function of c-myc. We found that TIAR inhibited c-myc expression at the translational level in E14 cells; accordingly, a reduction of TIAR expression promoted self-renewal of pluripotent cells and attenuated differentiation. Additionally, we established that TIAR inhibited TIA-1 expression at the translational level in E14 cells. Taken together, we have contributed to the understanding of the regulatory relationships between TIAR and both c-myc and TIA-1. PMID:25918534

  13. Musashi1 regulates breast tumor cell proliferation and is a prognostic indicator of poor survival

    Directory of Open Access Journals (Sweden)

    Wang Xiao-Yang

    2010-08-01

    Full Text Available Abstract Background Musashi1 (Msi1 is a conserved RNA-binding protein that regulates the Notch and Wnt pathways, and serves as a stem cell marker in the breast and other tissues. It is unknown how Msi1 relates to other breast cancer markers, whether it denotes tumor initiating cells (TICs, and how it affects gene expression and tumor cell survival in breast cancer cells. Results Msi1 expression was analyzed in 20 breast cancer cell lines and in 140 primary breast tumors by western blotting and immunohistochemistry, respectively. Lentivirus RNA interference was used to reduce Msi1 expression in breast cancer cell lines MCF-7 and T47D grown as spheroid cultures and to assess stem cell gene expression and the growth of these cell lines as xenografts. In normal human breast tissue, Msi1 was expressed in 10.6% of myoepithelum and 1.2% of ductal epithelium in the terminal ductal lobular unit (TDLU, whereas, less than 0.05% of ductal epithelium and myoepithelium in large ducts outside the TDLU expressed Msi1. Msi1 was expressed in 55% of the breast cancer cell lines and correlated with ErbB2 expression in 50% of the cell lines. Msi1 was expressed in 68% of primary tumors and in 100% of lymph node metastases, and correlated with 5 year survival. Msi1 was enriched in CD133+ MCF-7 and T47D cells and in spheroid cultures of these cells, and Msi1 'knockdown' (KD with a lentivirus-expressed shRNA decreased the number and size of spheroid colonies. Msi1 KD reduced Notch1, c-Myc, ErbB2 and pERK1/2 expression, and increased p21CIP1 expression, which is consistent with known Msi1 target mRNAs. Msi1 KD also reduced the expression of the somatic and embryonic stem cell markers, CD133, Bmi1, Sox2, Nanog and Oct4. Xenografts of MCF-7 and T47D Msi1 KD cells resulted in a marked reduction of tumor growth, reduced Msi1 and Notch1 expression and increased p21CIP1 expression. Conclusion Msi1 is a negative prognostic indicator of breast cancer patient survival, and is

  14. Wogonin induced G1 cell cycle arrest by regulating Wnt/β-catenin signaling pathway and inactivating CDK8 in human colorectal cancer carcinoma cells

    International Nuclear Information System (INIS)

    Highlights: • Wogonin inhibited HCT116 cells growth and arrested at G1 phase of the cell cycle. • Wogonin down-regulated the canonical Wnt/β-catenin signaling pathway. • Wogonin interfered in the combination of β-catenin and TCF/Lef. • Wogonin limited the kinase activity of CDK8. - Abstract: Wogonin, a naturally occurring mono-flavonoid, has been reported to have tumor therapeutic potential and good selectivity both in vitro and in vivo. Herein, we investigated the anti-proliferation effects and associated mechanisms of wogonin in human colorectal cancer in vitro. The flow-cytometric analysis showed that wogonin induced a G1 phase cell cycle arrest in HCT116 cells in a concentration- and time-dependent manner. Meanwhile, the cell cycle-related proteins, such as cyclin A, E, D1, and CDK2, 4 were down-regulated in wogonin-induced G1 cell cycle arrest. Furthermore, we showed that the anti-proliferation and G1 arrest effect of wogonin on HCT116 cells was associated with deregulation of Wnt/β-catenin signaling pathway. Wogonin-treated cells showed decreased intracellular levels of Wnt proteins, and activated degradation complex to phosphorylated and targeted β-catenin for proteasomal degradation. Wogonin inhibited β-catenin-mediated transcription by interfering in the transcriptional activity of TCF/Lef, and repressing the kinase activity of CDK8 which has been considered as an oncogene involving in the development of colorectal cancers. Moreover, CDK8 siRNA-transfected HCT116 cells showed similar results to wogonin treated cells. Thus, our data suggested that wogonin induced anti-proliferation and G1 arrest via Wnt/β-catenin signaling pathway and it can be developed as a therapeutic agent against human colorectal cancer

  15. Ethanolamine enhances the proliferation of intestinal epithelial cells via the mTOR signaling pathway and mitochondrial function.

    Science.gov (United States)

    Yang, Huansheng; Xiong, Xia; Li, Tiejun; Yin, Yulong

    2016-05-01

    Ethanolamine (Etn), which is the base constituent of phosphatidylethanolamine, a major phospholipid in animal cell membranes, is required for the proliferation of many types of mammalian epithelial cells. However, it is not clear whether the proliferation of intestinal epithelial cells requires Etn. The present study was conducted to examine the effects of Etn on the proliferation of intestinal epithelial cells and to elucidate the underlying mechanisms. The addition of Etn at 100 or 200 μM was found to enhance the proliferation of IPEC-1 cells. The expression of cell cycle-related proteins CDK4, RB3, cyclin A, and PCNA was also enhanced by Etn. Moreover, the expression or phosphorylation levels of the mammalian target of rapamycin (mTOR) signaling pathway protein and the expression of proteins related to mitochondrial function were also affected by Etn in IPEC-1 cells. These results indicate that Etn promotes the proliferation of intestinal epithelial cells by exerting effects on mTOR signaling pathway and mitochondrial function. PMID:27083163

  16. Increased ATP generation in the host cell is required for efficient vaccinia virus production

    Directory of Open Access Journals (Sweden)

    Hsu Che-Fang

    2009-09-01

    Full Text Available Abstract To search for cellular genes up-regulated by vaccinia virus (VV infection, differential display-reverse transcription-polymerase chain reaction (ddRT-PCR assays were used to examine the expression of mRNAs from mock-infected and VV-infected HeLa cells. Two mitochondrial genes for proteins that are part of the electron transport chain that generates ATP, ND4 and CO II, were up-regulated after VV infection. Up-regulation of ND4 level by VV infection was confirmed by Western blotting analysis. Up-regulation of ND4 was reduced by the MAPK inhibitor, apigenin, which has been demonstrated elsewhere to inhibit VV replication. The induction of ND4 expression occurred after viral DNA replication since ara C, an inhibitor of poxviral DNA replication, could block this induction. ATP production was increased in the host cells after VV infection. Moreover, 4.5 μM oligomycin, an inhibitor of ATP production, reduced the ATP level 13 hr after virus infection to that of mock-infected cells and inhibited viral protein expression and virus production, suggesting that increased ATP production is required for efficient VV production. Our results further suggest that induction of ND4 expression is through a Bcl-2 independent pathway.

  17. GATA Factor-Dependent Positive-Feedback Circuit in Acute Myeloid Leukemia Cells

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    Koichi R. Katsumura

    2016-08-01

    Full Text Available The master regulatory transcription factor GATA-2 triggers hematopoietic stem and progenitor cell generation. GATA2 haploinsufficiency is implicated in myelodysplastic syndrome (MDS and acute myeloid leukemia (AML, and GATA2 overexpression portends a poor prognosis for AML. However, the constituents of the GATA-2-dependent genetic network mediating pathogenesis are unknown. We described a p38-dependent mechanism that phosphorylates GATA-2 and increases GATA-2 target gene activation. We demonstrate that this mechanism establishes a growth-promoting chemokine/cytokine circuit in AML cells. p38/ERK-dependent GATA-2 phosphorylation facilitated positive autoregulation of GATA2 transcription and expression of target genes, including IL1B and CXCL2. IL-1β and CXCL2 enhanced GATA-2 phosphorylation, which increased GATA-2-mediated transcriptional activation. p38/ERK-GATA-2 stimulated AML cell proliferation via CXCL2 induction. As GATA2 mRNA correlated with IL1B and CXCL2 mRNAs in AML-M5 and high expression of these genes predicted poor prognosis of cytogenetically normal AML, we propose that the circuit is functionally important in specific AML contexts.

  18. Urinary cell mRNA profiles predictive of human kidney allograft status.

    Science.gov (United States)

    Lee, John R; Muthukumar, Thangamani; Dadhania, Darshana; Ding, Ruchuang; Sharma, Vijay K; Schwartz, Joseph E; Suthanthiran, Manikkam

    2014-03-01

    Kidney allograft status is currently characterized using the invasive percutaneous needle core biopsy procedure. The procedure has become safer over the years, but challenges and complications still exist including sampling error, interobserver variability, bleeding, arteriovenous fistula, graft loss, and even death. Because the most common type of acute rejection is distinguished by inflammatory cells exiting the intravascular compartment and gaining access to the renal tubular space, we reasoned that a kidney allograft may function as an in vivo flow cytometer and sort cells involved in rejection into urine. To test this idea, we developed quantitative polymerase chain reaction (PCR) assays for absolute quantification of mRNA and pre-amplification protocols to overcome the low RNA yield from urine. Here, we review our single center urinary cell mRNA profiling studies that led to the multicenter Clinical Trials in Organ Transplantation (CTOT-04) study and the discovery and validation of a 3-gene signature of 18S rRNA-normalized measures of CD3ε mRNA and IP-10 mRNA and 18S rRNA that is diagnostic and predictive of acute cellular rejection in the kidney allograft. We also review our development of a 4-gene signature of mRNAs for vimentin, NKCC2, E-cadherin, and 18S rRNA diagnostic of interstitial fibrosis/tubular atrophy (IF/TA). PMID:24517436

  19. Analysis of Gene Regulation in Rabbit Corneal Epithelial Cells Induced by Ultraviolet Radiation

    Directory of Open Access Journals (Sweden)

    Lai-Man Chan

    2005-04-01

    Full Text Available Ultraviolet (UV-induced cataracts are becoming a major environmental health concern because of the possible decrease in the stratospheric ozone layer. Experiments were designed to isolate gene(s affected by UV irradiation in rabbit cornea tissues using fluorescent differential display-reverse transcription-polymerase chain reaction (FDDRT-PCR. The epithelial cells were grown in standard medium for 2 or 4 hours post treatment. Cornea epithelial cells were irradiated with UVB for 20 minutes. RNA was extracted and amplified by reverse transcriptase-polymerase chain reaction using poly A+ specific anchoring primers and random arbitrary primers. Polyacrylamide gel electrophoresis revealed several differentially expressed genes in untreated versus UV irradiated cells. Complimentary DNA (cDNA fragments resulting from fluorescent differentially expressed mRNAs were eluted from the gel and re-amplified. The re-amplified PCR products were cloned directly into the PCR-TRAP cloning system. These data showed that FDDRT-PCR is a useful technique to elucidate UV-regulated gene expressions. Future experiments will involve sequence analysis of cloned inserts. The identification of these genes through sequence analysis could lead to a better understanding of cataract formation via DNA damage and mechanisms of prevention.

  20. Analysis of gene regulation in rabbit corneal epithelial cells induced by ultraviolet radiation.

    Science.gov (United States)

    Stevens, Jacqueline J; Rogers, Christian; Howard, Carolyn B; Moore, Caronda; Chan, Lai-Man

    2005-04-01

    Ultraviolet (UV)-induced cataracts are becoming a major environmental health concern because of the possible decrease in the stratospheric ozone layer. Experiments were designed to isolate gene(s) affected by UV irradiation in rabbit cornea tissues using fluorescent differential display-reverse transcription-polymerase chain reaction (FDDRT-PCR). The epithelial cells were grown in standard medium for 2 or 4 hours post treatment. Cornea epithelial cells were irradiated with UVB for 20 minutes. RNA was extracted and amplified by reverse transcriptase-polymerase chain reaction using poly A+ specific anchoring primers and random arbitrary primers. Polyacrylamide gel electrophoresis revealed several differentially expressed genes in untreated versus UV irradiated cells. Complimentary DNA (cDNA) fragments resulting from fluorescent differentially expressed mRNAs were eluted from the gel and re-amplified. The re-amplified PCR products were cloned directly into the PCR-TRAP cloning system. These data showed that FDDRT-PCR is a useful technique to elucidate UV-regulated gene expressions. Future experiments will involve sequence analysis of cloned inserts. The identification of these genes through sequence analysis could lead to a better understanding of cataract formation via DNA damage and mechanisms of prevention. PMID:16705801