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Sample records for human htt mrna

  1. Platelet serotonin transporter (5HTt): physiological influences on kinetic characteristics in a large human population.

    Science.gov (United States)

    Banović, Miroslav; Bordukalo-Niksić, Tatjana; Balija, Melita; Cicin-Sain, Lipa; Jernej, Branimir

    2010-01-01

    The present study had two goals: first, to give a detailed description of a reliable method for full kinetic analysis of serotonin transporter (5HTt) on the membrane of human platelets, and second, as a main issue, to report on physiological influences on kinetic characteristics of this transmembrane transport on a large population of healthy individuals. Full kinetic analyses of platelet serotonin uptake were performed on 334 blood donors of both sexes by the use of 14C-radioisotopic method, which was first optimized according to assumptions of enzyme kinetic analyses, with regard to platelet concentration, duration of uptake, concentration of substrate as well as important technical parameters (underpressure of filtration, blanks, incubating temperature, etc). Kinetic parameters of platelet serotonin uptake in the whole population were for V(max): 142 +/- 25.3 pmol 5HT/10(8) platelets/minute and for K(m): 0.404 +/- 0.089 microM 5HT. Besides the report on kinetic values of 5HT transporter protein, we have also described major physiological influences on the mentioned parameters, V(max), K(m) and their derivative, V(max)/K(m) (transporter efficiency): range and frequency distribution of normal values, intraindividual stability over time, lack of age influence, gender dependence and seasonal variations. The report on kinetic values and main physiological influences on platelet serotonin transport kinetics, obtained by the use of thoroughly reassessed methodology, and on by far the largest human population studied until now, offers a reliable frame of reference for pathophysiological studies of this parameter in various clinical fields.

  2. Generation of KCL028 research grade human embryonic stem cell line carrying a mutation in the HTT gene

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    Laureen Jacquet

    2016-03-01

    Full Text Available The KCL028 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomal dominant mutation affecting one allele of the HTT gene encoding huntingtin (43 trinucleotide repeats; 21 for the normal allele. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro and in vivo assays.

  3. pARIS-htt: an optimised expression platform to study huntingtin reveals functional domains required for vesicular trafficking

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    Pardo Raúl

    2010-06-01

    Full Text Available Abstract Background Huntingtin (htt is a multi-domain protein of 350 kDa that is mutated in Huntington's disease (HD but whose function is yet to be fully understood. This absence of information is due in part to the difficulty of manipulating large DNA fragments by using conventional molecular cloning techniques. Consequently, few studies have addressed the cellular function(s of full-length htt and its dysfunction(s associated with the disease. Results We describe a flexible synthetic vector encoding full-length htt called pARIS-htt (Adaptable, RNAi Insensitive &Synthetic. It includes synthetic cDNA coding for full-length human htt modified so that: 1 it is improved for codon usage, 2 it is insensitive to four different siRNAs allowing gene replacement studies, 3 it contains unique restriction sites (URSs dispersed throughout the entire sequence without modifying the translated amino acid sequence, 4 it contains multiple cloning sites at the N and C-ter ends and 5 it is Gateway compatible. These modifications facilitate mutagenesis, tagging and cloning into diverse expression plasmids. Htt regulates dynein/dynactin-dependent trafficking of vesicles, such as brain-derived neurotrophic factor (BDNF-containing vesicles, and of organelles, including reforming and maintenance of the Golgi near the cell centre. We used tests of these trafficking functions to validate various pARIS-htt constructs. We demonstrated, after silencing of endogenous htt, that full-length htt expressed from pARIS-htt rescues Golgi apparatus reformation following reversible microtubule disruption. A mutant form of htt that contains a 100Q expansion and a htt form devoid of either HAP1 or dynein interaction domains are both unable to rescue loss of endogenous htt. These mutants have also an impaired capacity to promote BDNF vesicular trafficking in neuronal cells. Conclusion We report the validation of a synthetic gene encoding full-length htt protein that will facilitate

  4. pARIS-htt: an optimised expression platform to study huntingtin reveals functional domains required for vesicular trafficking.

    Science.gov (United States)

    Pardo, Raúl; Molina-Calavita, Maria; Poizat, Ghislaine; Keryer, Guy; Humbert, Sandrine; Saudou, Frédéric

    2010-06-01

    Huntingtin (htt) is a multi-domain protein of 350 kDa that is mutated in Huntington's disease (HD) but whose function is yet to be fully understood. This absence of information is due in part to the difficulty of manipulating large DNA fragments by using conventional molecular cloning techniques. Consequently, few studies have addressed the cellular function(s) of full-length htt and its dysfunction(s) associated with the disease. We describe a flexible synthetic vector encoding full-length htt called pARIS-htt (Adaptable, RNAi Insensitive &Synthetic). It includes synthetic cDNA coding for full-length human htt modified so that: 1) it is improved for codon usage, 2) it is insensitive to four different siRNAs allowing gene replacement studies, 3) it contains unique restriction sites (URSs) dispersed throughout the entire sequence without modifying the translated amino acid sequence, 4) it contains multiple cloning sites at the N and C-ter ends and 5) it is Gateway compatible. These modifications facilitate mutagenesis, tagging and cloning into diverse expression plasmids. Htt regulates dynein/dynactin-dependent trafficking of vesicles, such as brain-derived neurotrophic factor (BDNF)-containing vesicles, and of organelles, including reforming and maintenance of the Golgi near the cell centre. We used tests of these trafficking functions to validate various pARIS-htt constructs. We demonstrated, after silencing of endogenous htt, that full-length htt expressed from pARIS-htt rescues Golgi apparatus reformation following reversible microtubule disruption. A mutant form of htt that contains a 100Q expansion and a htt form devoid of either HAP1 or dynein interaction domains are both unable to rescue loss of endogenous htt. These mutants have also an impaired capacity to promote BDNF vesicular trafficking in neuronal cells. We report the validation of a synthetic gene encoding full-length htt protein that will facilitate analyses of its structure/function. This may help

  5. Truncated N-terminal huntingtin fragment with expanded-polyglutamine (htt552-100Q)suppresses brain-derived neurotrophic factor transcription in astrocytes

    Institute of Scientific and Technical Information of China (English)

    Linhui Wang; Fang Lin; Jin Wang; Junchao Wu; Rong Han; Lujia Zhu; Guoxing Zhang; Marian DiFiglia; Zhenghong Qin

    2012-01-01

    Although huntingtin (htt) can be cleaved at many sites by caspases,calpains,and aspartyl proteases,amino acid (aa) 552 was defined as a preferred site for cleavage in human Huntington disease (HD) brains in vivo.To date,the normal function of wild-type N-terminal htt fragment 1-552 aa (htt552) and its pathological roles of mutant htt552 are still unknown.Although mutant htt (mhtt) is also expressed in astrocytes,whether and how mhtt contributes to the neurodegeneration through astrocytes in HD remains largely unknown.In this study,a glia HD model,using an adenoviral vector to express wild-type htt552 (htt552-18Q) and its mutation (htt552-100Q) in rat primary cortical astrocytes,was generated to investigate the influence of htt552 on the transcription of brainderived neurotrophic factor (BDNF). Results from enzyme linked immunosorbent assay showed that the level of BDNF in astrocyte-conditioned medium was decreased in the astrocytes expressing htt552-100Q.Quantitative real-time polymerase chain reaction demonstrated that htt552-100Q reduced the transcripts of the BDNF Ⅲ and Ⅳ, hence, repressed the transcription of BDNF.Furthermore,immunofluorescence showed that aggregates formed by htt552-100Q entrapped transcription factors cAMP-response element-binding protein and stimulatory protein 1,which might account for the reduction of BDNF transcription.These findings suggest that mhtt552 reduces BDNF transcription in astrocytes,which might contribute to the neuronal dysfunction in HD.

  6. Selenoprotein P mRNA expression in human hepatic tissues

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    Chun-Li Li; Ke-Jun Nan; Tao Tian; Chen-Guang Sui; Yan-Fang Liu

    2007-01-01

    AIM: To investigate the expression of Selenoprotein P mRNA (SePmRNA) in tissues of normal liver, liver cirrhosis and hepatocellular carcinoma (HCC), and its relationship with HCC occurrence and development.METHODS: The expression of SePmRNA in tissues of normal liver, liver cirrhosis and HCC were detected by in situ hybridization using a cDNA probe.RESULTS: The enzyme digesting products of pBluescript-Human Selenoprotein P were evaluated by electrophoresis.The positive expression of SePmRNA was found in the tissues of normal liver,liver cirrhosis and HCC.The expression of SeP mRNA was found in hepatic interstitial substance,especially in endothelial cells and lymphocytes of vasculature.The positive rate of SePmRNA in normal liver tissue was 84.6% (11/13) and the positive signals appeared in the nucleus and cytoplasm,mostly in the nucleolus,and the staining granules were larger in the nucleolus and around the nucleus.The positive rate of SePmRNA in liver cirrhosis tissue was 45.O% (9/20) and the positive signals were mainly in the nucleolus and cytoplasm,being less around the nucleus and inner nucleus than that in normal liver tissue. The positive rate of SePmRNA in HCC tissue was 30.0% (9/30) and the positive signals were in the cytoplasm, but less in the nucleus.CONCLUSION: SePmRNA expression in the tissues of normal liver and HCC is significantly different (84.6% vs 30.0%, P = 0.003), suggesting that SeP might play a role in the occurrence and development of HCC.

  7. mRNA related to insulin family in human placenta

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    Younes, M.A.; D' Agostino, J.B.; Frazier, M.L.; Besch, P.K.

    1986-03-01

    The authors have previously reported that human term placenta contains mRNA displaying sequence homology to a rat preproinsulin I cDNA clone (p119). When placental poly(A/sup +/) RNA was analyzed for homology to p119 by RNA/DNA blot hybridization, prominent hybridization was observed which was found by densitometric analysis to be three-fold higher than control. To further characterize this insulin-like message, a cDNA library was generated (approx.7000 transformants) using normal term cesarean-sectioned tissue to prepare placental poly(A/sup +/) RNA templates. Five hundred transformants were initially screened by colony hybridization using a /sup 32/P-labeled rat preproinsulin I cDNA as probe. Of the ten initial positives obtained, three were found to be true positives based on Southern hybridization analyses of the recombinant plasmids. Using Taq I digested pBr322 as a size marker, the cDNAs were found to be approximately 300 bp in length. Preliminary DNA sequencing using the Sanger dideoxy chain termination method has revealed that one of these clones displays significant homology to the 5' region of human insulin-like growth factors I and II.

  8. Human cytomegalovirus induces alteration of (-actin mRNA and microfilaments in human embryo fibroblast cells

    Institute of Scientific and Technical Information of China (English)

    林茂芳; 魏国庆; 黄河; 蔡真

    2004-01-01

    Objective: To investigate the infection of human embryo fibroblast cell line HF cells by CMV as well as the effects of CMV on β-actin mRNA and microfilaments. Methods: HF cells shape was observed after the infection of CMV. RT-PCR assay was used to detect the mRNA expression of CMV immediate early (IE) gene, β-actin and GAPDH genes of HF cells infected by CMV. CMV particles and cell microfilaments were detected with electron microscope. Results: Shape of HF cell changed after the infection by CMV. HF cells infected by CMV could express IE mRNA and the expression of β-actin mRNA decreased in a time- and titer-dependent manner compared with the uninfected HF cells whose expression of GAPDH mRNA did not change much. CMV particles were found with electron microscope in the cells. Microfilaments were ruptured and shortened after the infection of CMV. Conclusion: CMV can not only infect human embryo fibroblast cells line HF cells and replicate in the cells, but can also affect the expression of β-actin mRNA and the microfilaments.

  9. Serotonin transporter gene (5-HTT) polymorphisms and compulsive buying.

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    Devor, E J; Magee, H J; Dill-Devor, R M; Gabel, J; Black, D W

    1999-04-16

    We examined a panel of 21 patients diagnosed with compulsive buying for two DNA sequence polymorphisms found in the gene that encodes the serotonin transport (5-HTT). One polymorphism, found in the promoter region of the 5-HTT gene, involves a 44-base pair (bp) deletion, and the other, found in the second intron, is due to variable numbers of a repeat sequence. We also typed a panel of 38 psychiatrically normal controls for both 5-HH markers. When compared to this control panel, no significant differences were seen for either 5-HTT marker among the compulsive buyers.

  10. Astrocyte cultures derived from human brain tissue express angiotensinogen mRNA

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    Milsted, A.; Barna, B.P.; Ransohoff, R.M.; Brosnihan, K.B.; Ferrario, C.M. (Cleveland Clinic Foundation, OH (USA))

    1990-08-01

    The authors have identified human cultured cell lines that are useful for studying angiotensinogen gene expression and its regulation in the central nervous system. A model cell system of human central nervous system origin expressing angiotensinogen has not previously been available. Expression of angiotensinogen mRNA appears to be a basal property of noninduced human astrocytes, since astrocytic cell lines derived from human glioblastomas or nonneoplastic human brain tissue invariably produced angiotensinogen mRNA. In situ hybridization histochemistry revealed that angiotensinogen mRNA production was not limited to a subpopulation of astrocytes because >99% of cells in these cultures contained angiotensinogen mRNA. These cell lines will be useful in studies of the molecular mechanisms controlling angiotensin synthesis and the role of biologically active angiotensin in the human brain by allowing the authors to examine regulation of expression of the renin-angiotensin system in human astrocyte cultures.

  11. Oxytocin receptor (OXTR) and serotonin transporter (5-HTT) genes associated with observed parenting.

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    Bakermans-Kranenburg, Marian J; van Ijzendoorn, Marinus H

    2008-06-01

    Both oxytocin and serotonin modulate affiliative responses to partners and offspring. Animal studies suggest a crucial role of oxytocin in mammalian parturition and lactation but also in parenting and social interactions with offspring. The serotonergic system may also be important through its influence on mood and the release of oxytocin. We examined the role of serotonin transporter (5-HTT) and oxytocin receptor (OXTR) genes in explaining differences in sensitive parenting in a community sample of 159 Caucasian, middle-class mothers with their 2-year-old toddlers at risk for externalizing behavior problems, taking into account maternal educational level, maternal depression and the quality of the marital relationship. Independent genetic effects of 5-HTTLPR SCL6A4 and OXTR rs53576 on observed maternal sensitivity were found. Controlling for differences in maternal education, depression and marital discord, parents with the possibly less efficient variants of the serotonergic (5-HTT ss) and oxytonergic (AA/AG) system genes showed lower levels of sensitive responsiveness to their toddlers. Two-way and three-way interactions with marital discord or depression were not significant. This first study on the role of both OXTR and 5-HTT genes in human parenting points to molecular genetic differences that may be implicated in the production of oxytocin explaining differences in sensitive parenting.

  12. Stability of Human Follicle-Stimulating Hormone Receptor mRNA in Stably Transfected Cells

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    朱长虹; 田红

    2001-01-01

    In order to assess the impact of mRNA degradation on steady state levels of follicle-stimulating hormone receptor (FSHR) mRNA and on regulation of FSHR gene expression, the stability and half-life of FSHR mRNA were determined in transfected cells expressing recombinant FSHR. Time-dependent changes in FSHR mRNA content were determined by nuclease protection-solution hybridization assay (NPA) or by qualitative reverse transcription-competitive polymerase chain reaction (RT-PCR) in cultured hFSHR-YI cells, cell lines stably transfected with a human FSHR cDNA. FSHR mRNA content remained constant during 8 h control incubations of hFSHR-Y1 cells (NPA, 2.9±0.3 μg/mg RNA; RT-PCR, 2.7±0.3 μg/mg RNA). Actinomycin D (ActD, 5 μg/ml) inhibited mRNA synthesis, as assessed by incorporation of [3 H]uridine into total RNA, by 90 % within 1 h in hFSHR-Y1 cells. No effect of ActD on cellular morphology or viability was observed. ActD caused a time-dependent decrease in FSHR mRNA content in hFSHR-Y1 cell lines with a lag time of 1 h. There were no significant differences in the rate of FSHR mRNA degradation between the two methods of mRNA quantification. The half-life of hFSHR mRNA was 3.6±0.2 h by NPA and 3.1±0.1 h by RT-PCR. The results indicated that degradation of mRNA was an important process in maintenance of steady state expression of the FSHR gene in cells stably expressing recombinant receptor.

  13. Cloning of human brevican cDNA and expression of its mRNA in human glioma

    Institute of Scientific and Technical Information of China (English)

    韩唏; 董艳; 由振东; 何成; 卢亦成

    2003-01-01

    Objective:To clone the cDNA of human brevican secreting isoform and to investigate its mRNA expression in human glioma.Methods:The full-length cDNA of human brevican secreted isoform was cloned from a human ahaplastic astrocytoma by RT-PCR,and the expression of human brevican mRNA in 22 cases of human glioma and 13 cases of non-glial brain tumors were investigated by in situ hybridization.Results:The cDNA which including the whole open reading frame of human brevican secreted isoform was obtained.In situ hybridization showed that brevican positive cells were present in all of the 22 cases of gliomas(100%),whereas none were found in the 13 cases of non-glial and metastasis brain tumors examined.Conclusion:The results suggest that brevican mRNA is highly and specifically expressed in human glioma.

  14. Stable RNA hairpins in 88 coding regions of human mRNA

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    PAN Min; WANG Chuanming; LIU Ciquan

    2004-01-01

    RNA hairpins containing UNCG, GNRA, CUUG (N=A, U, C or G, R=G or A) loops are unusually thermodynamic stable and conserved structures. The structural features of these hairpin loops are very special, and they play very important roles in vivo. They are prevalent in rRNA, catalytic RNA and non-coding mRNA. However, the 5′ C(UUCG)G 3′ hairpin is not found in the folding structure of 88 human mRNA coding regions. It is also different from rRNA in that there is no preference for certain sequences among tetraloops in these 88 mRNA folding structures.

  15. Endothelin converting-enzyme-1 mRNA expression in human cardiovascular disease

    NARCIS (Netherlands)

    Bohnemeier, H; Pinto, YM; Horkay, F; Toth, M; Juhasz-Nagy, A; Orzechowski, HD; Bohm, M; Paul, M

    1998-01-01

    Endothelin-1 converting-enzyme (ECE-1) cleaves the precursor, big-endothelin-1, to the active peptide endothelin-1. The aim of this study was to investigate whether ECE-1 mRNA expression is modified in human cardiovascular disease. Tissue samples from the left human atrium were analyzed for ECE-1 ex

  16. mRNA transfection of mouse and human neural stem cell cultures.

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    Samuel McLenachan

    Full Text Available The use of synthetic mRNA as an alternative gene delivery vector to traditional DNA-based constructs provides an effective method for inducing transient gene expression in cell cultures without genetic modification. Delivery of mRNA has been proposed as a safer alternative to viral vectors in the induction of pluripotent cells for regenerative therapies. Although mRNA transfection of fibroblasts, dendritic and embryonic stem cells has been described, mRNA delivery to neurosphere cultures has not been previously reported. Here we sought to establish an efficient method for delivering mRNA to primary neurosphere cultures. Neurospheres derived from the subventricular zone of adult mice or from human embryonic stem cells were transfected with EGFP mRNA by lipofection and electroporation. Transfection efficiency and expression levels were monitored by flow cytometry. Cell survival following transfection was examined using live cell counting and the MTT assay. Both lipofection and electroporation provided high efficiency transfection of neurospheres. In comparison with lipofection, electroporation resulted in increased transfection efficiencies, but lower expression per cell and shorter durations of expression. Additional rounds of lipofection renewed EGFP expression in neurospheres, suggesting this method may be suitable for reprogramming applications. In summary, we have developed a protocol for achieving high efficiency transfection rates in mouse and human neurosphere cell culture that can be applied for future studies of gene function studies in neural stem cells, such as defining efficient differentiation protocols for glial and neuronal linages.

  17. Involvement of hGLD-2 in cytoplasmic polyadenylation of human p53 mRNA

    DEFF Research Database (Denmark)

    Glahder, Jacob-Andreas Harald; Norrild, Bodil

    2011-01-01

    Cytoplasmic polyadenylation is a post-transcriptional mechanism regulating mRNA stability and translation. The human p53 3'-untranslated region (3'-UTR) contains two regions similar to cytoplasmic polyadenylation elements (CPEs) just upstream of the poly(A) hexanucleotide. Evaluation of the p53 CPE...... cytoplasmic poly(A) polymerase] is overexpressed instead. The stability of a luciferase mRNA containing the p53 3'-UTR downstream, is decreased when hCPEB1 is overexpressed as seen by qPCR. Expression of hGLD-2 restores the mRNA stability. This is due to elongation of the poly(A) tail as seen by a PCR......-based poly(A) test and in vitro poly(A) assay. Taken together, our results suggest that hCPEB1 and hGLD-2 are antagonizing factors regulating p53 mRNA stability....

  18. In human granulosa cells from small antral follicles, androgen receptor mRNA and androgen levels in follicular fluid correlate with FSH receptor mRNA

    DEFF Research Database (Denmark)

    Nielsen, M. E.; Rasmussen, I. A.; Kristensen, Stine Gry

    2011-01-01

    concentrations of AMH, inhibin-B, progesterone and estradiol. Androgen Receptor mRNA expression in granulosa cells, and the FF content of androgens, both showed a highly significant positive association with to the expression of FSHR mRNA in granulosa cells. AR mRNA expression also correlated significantly...... with the expression of AMHR2, but did not correlate with any of the hormones in the FF. These data demonstrate an intimate association between AR expression in immature granulosa cells, and the expression of FSHR in normal small human antral follicles and between the FF levels of androgen and FSHR expression...

  19. Detection of histidine decarboxylase mRNA in human vascular smooth muscle and endothelial cells.

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    Tippens, A S; Gruetter, C A

    2004-06-01

    The objective of this study was to investigate histamine synthesis capability of human vascular smooth muscle and endothelial cells by detecting histidine decarboxylase (HDC) mRNA. HDC catalyzes exclusively the formation of histamine in mammalian cells. Experiments utilizing nested reverse transcription-polymerase chain reaction (nRT-PCR) were conducted to detect the presence of HDC mRNA. Human aortic smooth muscle cells (HAoSMC) and human aortic endothelial cells (HAEC) were cultured and RNA was extracted and amplified using two sets of HDC-specific primers. Rat liver and kidney RNA were isolated and amplified to serve as positive and negative controls, respectively. Gel electrophoresis of HAoSMC, HAEC and liver mRNA revealed bands coinciding with an expected product size of 440 base pairs. Sequence analysis revealed that the observed bands were the appropriate HDC amplicons. These findings are the first to indicate the presence of HDC mRNA in vascular smooth muscle cells and confirm the presence of HDC mRNA in endothelial cells which is consistent with an ability of these cell types to synthesize histamine in the vascular wall.

  20. Tissue-specific mRNA expression profiles of human solute carrier 35 transporters.

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    Nishimura, Masuhiro; Suzuki, Satoshi; Satoh, Tetsuo; Naito, Shinsaku

    2009-01-01

    Pairs of forward and reverse primers and TaqMan probes specific to each of 23 human solute carrier 35 (SLC35) transporters were prepared. The mRNA expression level of each target transporter was analyzed in total RNA from single and pooled specimens of adult human tissues (adipose tissue, adrenal gland, bladder, bone marrow, brain, cerebellum, colon, heart, kidney, liver, lung, mammary gland, ovary, pancreas, peripheral leukocytes, placenta, prostate, retina, salivary gland, skeletal muscle, small intestine, smooth muscle, spinal cord, spleen, stomach, testis, thymus, thyroid gland, tonsil, trachea, and uterus), from pooled specimens of fetal human tissues (brain, heart, kidney, liver, spleen, and thymus), and from three human cell lines (HeLa cell line ATCC#: CCL-2, human cell line Hep G2, and human breast carcinoma cell line MDA-435) by real-time reverse transcription PCR using an Applied Biosystems 7500 Fast Real-Time PCR System. The mRNA expression of SLC35As, SLC35Bs, SLC35Cs, SLC35D1, SLC35D2, SLC35Es, and SLC35F5 was found to be ubiquitous in both adult and fetal tissues. SLC35D3 mRNA was expressed at the highest levels in the adult retina. SLC35F1 mRNA was expressed at high levels in the adult and fetal brain. SLC35F2 mRNA was expressed at the highest levels in the adult salivary gland. Both SLC35F3 and SLC35F4 mRNAs were expressed at the highest levels in the adult cerebellum. Further, individual differences in the mRNA expression levels of human SLC35 transporters in the liver were also evaluated. Our newly determined expression profiles were used to study the gene expression in 31 adult human tissues, 6 fetal human tissues, and 3 cell lines, and tissues with high transcriptional activity for human SLC35 transporters were identified. These results are expected to be valuable for research concerning the clinical diagnosis of disease.

  1. The rules and impact of nonsense-mediated mRNA decay in human cancers.

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    Lindeboom, Rik G H; Supek, Fran; Lehner, Ben

    2016-10-01

    Premature termination codons (PTCs) cause a large proportion of inherited human genetic diseases. PTC-containing transcripts can be degraded by an mRNA surveillance pathway termed nonsense-mediated mRNA decay (NMD). However, the efficiency of NMD varies; it is inefficient when a PTC is located downstream of the last exon junction complex (EJC). We used matched exome and transcriptome data from 9,769 human tumors to systematically elucidate the rules of NMD targeting in human cells. An integrated model incorporating multiple rules beyond the canonical EJC model explains approximately three-fourths of the non-random variance in NMD efficiency across thousands of PTCs. We also show that dosage compensation may sometimes mask the effects of NMD. Applying the NMD model identifies signatures of both positive and negative selection on NMD-triggering mutations in human tumors and provides a classification for tumor-suppressor genes.

  2. Angiotensin II receptor mRNA expression and vasoconstriction in human coronary arteries

    DEFF Research Database (Denmark)

    Wackenfors, Angelica; Pantev, Emil; Emilson, Malin;

    2004-01-01

    Angiotensin II is a potent vasoconstrictor that is implicated in the pathogenesis of hypertension, heart failure and atherosclerosis. In the present study, angiotensin II receptor mRNA expression levels were quantified by real-time polymerase chain reaction and the vasocontractile responses...... to angiotensin II were characterised by in vitro pharmacology in endothelium-denuded human coronary arteries. Angiotensin II type 1 (AT(1)) and type 2 (AT(2)) receptor mRNA expression levels were significantly down-regulated in arteries from patients with heart failure as compared to controls. The angiotensin II...

  3. Ischemic heart disease induces upregulation of endothelin receptor mRNA in human coronary arteries

    DEFF Research Database (Denmark)

    Wackenfors, Angelica; Emilson, Malin; Ingemansson, Richard;

    2004-01-01

    and controls using real-time polymerase chain reaction (real-time PCR). In addition, the suitability of organ culture as a model mimicking endothelin receptor changes in cardiovascular disease was evaluated by in vitro pharmacology and real-time PCR. Endothelin ETA and ETB receptor mRNA levels were......Endothelin has been implicated in the pathogenesis of ischemic heart disease and congestive heart failure. The aims were to quantify endothelin type A (ETA) and type B (ETB) receptor mRNA levels in human coronary arteries from patients with ischemic heart disease, congestive heart failure...

  4. Human cytomegalovirus induces alteration of β-actin mRNA and microfilaments in human embryo fibroblast cells

    Institute of Scientific and Technical Information of China (English)

    林茂芳; 魏国庆; 黄河; 蔡真

    2004-01-01

    Objective: To investigate the infection of human embryo fibroblast cell line HF cells by CMV as well as the effects of CMV on β-actin mRNA and microfilaments. Methods: HF cells shape was observed after the infection of CMV.RT-PCR assay was used to detect the mRNA expression of CMV immediate early (IE) gene, β-actin and GAPDH genes of HF cells infected by CMV. CMV particles and cell microfilaments were detected with electron microscope. Results: Shape of HF cell changed after the infection by CMV. HF cells infected by CMV could express IE mRNA and the expression of β-actin mRNA decreased in a time-and titer-dependent manner compared with the uninfected HF cells whose expression of GAPDH mRNA did not change much. CMV particles were found with electron microscope in the cells. Microfilaments were ruptured and shortened after the infection of CMV. Conclusion: CMV can not only infect human embryo fibroblast cells line HF cells and replicate in the cells, but can also affect the expression of β-actin mRNA and the microfilaments.

  5. Reduced fructosamine-3-kinase activity and its mRNA in human distal colorectal carcinoma.

    Science.gov (United States)

    Notarnicola, M; Caruso, Maria G; Tutino, V; Guerra, V; Frisullo, S; Altomare, D F; Misciagna, G

    2010-09-01

    Fructosamine-3-Kinase (FN3K) is an enzyme phosphorilating fructoselysine (FL) residues on glycated proteins, resulting in the production of protein-bound FL-3-phosphate. The pathological role of the non-enzymatic modification of proteins by reducing sugars has become increasingly evident in various types of disorders, including the cancer. In this study, our aim was to study FN3K enzyme activity, as well as its mRNA in human colorectal cancer (CRC). Thirty consecutive CRC patients undergoing surgery of the colon were enrolled in the study. FN3K enzymatic activity and gene expression were analyzed using a radiometric assay and quantitative RT-PCR, respectively. FN3K is a functionally active enzyme in human colon tissue, without significant differences between normal mucosa and cancer. The mean level of FN3K mRNA was significantly lower in cancer than in the corresponding normal colorectal mucosa The colorectal tumors located on the left side showed lower levels of both enzymatic activity and mRNA FN3K than tumors located in the right side of colon. This paper is the first studying FN3K enzyme activity in human CRC, showing a significant relationship between enzymatic activity, its mRNA and tumor side.

  6. Pseudouridine profiling reveals regulated mRNA pseudouridylation in yeast and human cells.

    Science.gov (United States)

    Carlile, Thomas M; Rojas-Duran, Maria F; Zinshteyn, Boris; Shin, Hakyung; Bartoli, Kristen M; Gilbert, Wendy V

    2014-11-01

    Post-transcriptional modification of RNA nucleosides occurs in all living organisms. Pseudouridine, the most abundant modified nucleoside in non-coding RNAs, enhances the function of transfer RNA and ribosomal RNA by stabilizing the RNA structure. Messenger RNAs were not known to contain pseudouridine, but artificial pseudouridylation dramatically affects mRNA function--it changes the genetic code by facilitating non-canonical base pairing in the ribosome decoding centre. However, without evidence of naturally occurring mRNA pseudouridylation, its physiological relevance was unclear. Here we present a comprehensive analysis of pseudouridylation in Saccharomyces cerevisiae and human RNAs using Pseudo-seq, a genome-wide, single-nucleotide-resolution method for pseudouridine identification. Pseudo-seq accurately identifies known modification sites as well as many novel sites in non-coding RNAs, and reveals hundreds of pseudouridylated sites in mRNAs. Genetic analysis allowed us to assign most of the new modification sites to one of seven conserved pseudouridine synthases, Pus1-4, 6, 7 and 9. Notably, the majority of pseudouridines in mRNA are regulated in response to environmental signals, such as nutrient deprivation in yeast and serum starvation in human cells. These results suggest a mechanism for the rapid and regulated rewiring of the genetic code through inducible mRNA modifications. Our findings reveal unanticipated roles for pseudouridylation and provide a resource for identifying the targets of pseudouridine synthases implicated in human disease.

  7. 5-HT(1A) receptor and 5-HTT binding during the menstrual cycle in healthy women examined with [(11)C] WAY100635 and [(11)C] MADAM PET.

    Science.gov (United States)

    Jovanovic, Hristina; Karlsson, Per; Cerin, Asta; Halldin, Christer; Nordström, Anna-Lena

    2009-04-30

    The aim of the present study was to explore the effects of the menstrual cycle phases on 5-HT(1A) receptor and 5-HTT binding potentials (BPs) in healthy women by using positron emission tomography (PET). Women were investigated in the follicular and luteal phase of the menstrual cycle with radioligands [(11)C]WAY10035 (n=13) and [(11)C]MADAM (n=8) to study 5-HT(1A) and 5-HTT BPs. The BPs values were quantified using the simplified reference tissue model. The phases of the menstrual cycle were characterized by transvaginal ultrasound (TSV) and plasma levels of hormones estradiol (E(2)), progesterone (P(4)), follicle stimulating hormone (FSH) and luteinizing hormone (LH).The 5-HT(1A) receptor and 5-HTT BPs did not significantly differ between follicular and luteal phases in any of the investigated regions. There were no significant correlations between the change in E(2) or P(4) values with the change in 5-HT(1A) receptor or 5-HTT BPs. The results provide principally a new in vivo finding in human female biology, suggesting the absence of influence of menstrual cycle phase on 5-HT(1A) receptors or 5-HTT. The finding however does not preclude that gonadal hormones differentially influence central serotonin system inwomen and men, which might contribute to gender differences in serotonin-associated disorders.

  8. The human mRNA encoding the Goodpasture antigen is alternatively spliced.

    Science.gov (United States)

    Bernal, D; Quinones, S; Saus, J

    1993-06-05

    The noncollagenous (NC1) domain of the human collagen alpha 3(IV)-chain is the primary target of autoantibodies produced in Goodpasture syndrome and, therefore, has been designated as the Goodpasture antigen. In this report, we show that Goodpasture antigen mRNA undergoes processing to at least two alternatively spliced forms in a variety of human tissues, resulting in the exclusion of sequence encoded by either one or two exons. Interestingly, no alternatively spliced forms were observed in bovine or rat tissues. The derived amino acid sequences of the two variant mRNA forms are identical and significantly shorter than that arising from the complete Goodpasture antigen mRNA. They lack the carboxyl-terminal region contributing to the formation of the Goodpasture epitope and all but one of the cysteines found in the complete form. These sequence characteristics suggest that, if translated, the variant Goodpasture antigen is likely to be defective in triple helix formation and no longer reactive with Goodpasture autoantibodies. Although each tissue expressing Goodpasture antigen displayed a specific mRNA pattern, the complete form was always the most abundant and was present at levels apparently unrelated to whether or not the organ of origin is a potential target in Goodpasture syndrome. Furthermore, the antigen sequence was identical in the kidneys of normal and Goodpasture-affected individuals, and no major differences in the expression of the complete and spliced forms were observed.

  9. Real-time determination of human telomerase reverse transcriptase mRNA in gastric cancer

    Institute of Scientific and Technical Information of China (English)

    Li-Hua Hu; Feng-Hua Chen; Yi-Rong Li; Lin Wang

    2004-01-01

    AIM: To set up a real-time fluorescent quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay,to detect human telomerase reverse transcriptase (hTERT)messenger RNA in gastric carcinomas, and to evaluate quantitative determination of hTERT mRNA in the diagnostic value of gastric carcinomas, and to analyze the correlation between the expression level of hTERT mRNA and dinicopathological parameters in patients with gastric cancer.METHODS: A real-time quantitative RT-PCR (RQ-PCR)based on TaqMan fluorescence methodoloogy and the LightCyder system was used to quantify the full range of hTERT mRNA copy numbers in 35 samples of gastric carcinomas and corresponding adjacent non-cancerous tissues. The normalized hTERT (NhTERT) was standardized by quantifying the number of GAPDH transcripts as internal control and expressed as 100× (hTERT/GAPDH) ratio. Variables were analyzed by the Student's t-test, χ2 test and Fisher's exact test.RESULTS: NhTERT from gastric carcinomas and corresponding adjacent non-cancerous tissues was 6.27±0.89 and 0.93±0.18,respectively (t= 12.76, P<0.001). There was no significant association between gastric cancer hTERT mRNA expression level and patient's age, gender, tumor size, location and stage (pTNM), but a significant correlation was found between hTERT mRNA expression level in gastric carcinomas and the degree of differentiation.CONCLUSION: Quantitative determination of hTERT mRNA by RQ-PCR is a rapid and sensitive method. hTERT might be a potential biomarker for the early detection of gastric cancer.

  10. Native Mutant Huntingtin in Human Brain

    Science.gov (United States)

    Sapp, Ellen; Valencia, Antonio; Li, Xueyi; Aronin, Neil; Kegel, Kimberly B.; Vonsattel, Jean-Paul; Young, Anne B.; Wexler, Nancy; DiFiglia, Marian

    2012-01-01

    Huntington disease (HD) is caused by polyglutamine expansion in the N terminus of huntingtin (htt). Analysis of human postmortem brain lysates by SDS-PAGE and Western blot reveals htt as full-length and fragmented. Here we used Blue Native PAGE (BNP) and Western blots to study native htt in human postmortem brain. Antisera against htt detected a single band broadly migrating at 575–850 kDa in control brain and at 650–885 kDa in heterozygous and Venezuelan homozygous HD brains. Anti-polyglutamine antisera detected full-length mutant htt in HD brain. There was little htt cleavage even if lysates were pretreated with trypsin, indicating a property of native htt to resist protease cleavage. A soluble mutant htt fragment of about 180 kDa was detected with anti-htt antibody Ab1 (htt-(1–17)) and increased when lysates were treated with denaturants (SDS, 8 m urea, DTT, or trypsin) before BNP. Wild-type htt was more resistant to denaturants. Based on migration of in vitro translated htt fragments, the 180-kDa segment terminated ≈htt 670–880 amino acids. If second dimension SDS-PAGE followed BNP, the 180-kDa mutant htt was absent, and 43–50 kDa htt fragments appeared. Brain lysates from two HD mouse models expressed native full-length htt; a mutant fragment formed if lysates were pretreated with 8 m urea + DTT. Native full-length mutant htt in embryonic HD140Q/140Q mouse primary neurons was intact during cell death and when cell lysates were exposed to denaturants before BNP. Thus, native mutant htt occurs in brain and primary neurons as a soluble full-length monomer. PMID:22375012

  11. Resistance artery creatine kinase mRNA and blood pressure in humans.

    Science.gov (United States)

    Karamat, Fares A; Oudman, Inge; Ris-Stalpers, Carrie; Afink, Gijs B; Keijser, Remco; Clark, Joseph F; van Montfrans, Gert A; Brewster, Lizzy M

    2014-01-01

    Hypertension remains the main risk factor for cardiovascular death. Environmental and biological factors are known to contribute to the condition, and circulating creatine kinase was reported to be the main predictor of blood pressure in the general population. This was proposed to be because of high resistance artery creatine kinase-BB rapidly regenerating ATP for vascular contractility. Therefore, we assessed whether creatine kinase isoenzyme mRNA levels in human resistance arteries are associated with blood pressure. We isolated resistance-sized arteries from omental fat donated by consecutive women undergoing uterine fibroid surgery. Blood pressure was measured in the sitting position. Vessels of 13 women were included, 6 normotensive and 7 hypertensive, mean age 42.9 years (SE, 1.6) and mean systolic/diastolic blood pressure, 144.8 (8.0)/86.5 (4.3) mm Hg. Arteriolar creatine kinase isoenzyme mRNA was assessed using quantitative real-time polymerase chain reaction. Normalized creatine kinase B mRNA copy numbers, ranging from 5.2 to 24.4 (mean, 15.0; SE, 1.9), showed a near-perfect correlation with diastolic blood pressure (correlation coefficient, 0.9; 95% confidence interval, 0.6-1.0) and were well correlated with systolic blood pressure, with a 90% relative increase in resistance artery creatine kinase B mRNA in hypertensives compared with normotensives, normalized copy numbers were, respectively, 19.3 (SE, 2.0) versus 10.1 (SE, 2.1), P=0.0045. To our knowledge, this is the first direct evidence suggesting that resistance artery creatine kinase mRNA expression levels concur with blood pressure levels, almost doubling with hypertension. These findings add to the evidence that creatine kinase might be involved in the vasculature's pressor responses.

  12. Transcription pattern of UL131A-128 mRNA in clinical strains of human cytomegalovirus

    Indian Academy of Sciences (India)

    Zhengrong Sun; Gaowei Ren; Yanping Ma; Ning Wang; Yaohua Ji; Ying Qi; Mali Li; Rong He; Qiang Ruan

    2010-09-01

    Human cytomegalovirus (HCMV) mRNA was obtained from human embryonic lung fibroblast cells infected by HCMV clinical strains from urine samples of infants at different kinetic periods. The cDNA of UL131A-128 mRNAs was amplified using reverse transcription-polymerase chain reaction (RT-PCR) and analysed by sequencing. Mean while, clones containing UL131A-128 transcripts in an HCMV cDNA library of a clinical strain were selected and sequenced. It was demonstrated that UL131A-128 mRNA was expressed with immediately early, early and late kinetics. Sequences obtained by RT-PCR showed that the UL131A gene consisted of two exons and the coding region of the UL130 gene was not interrupted by any intron in the region as reported earlier. However, the transcript of the UL128 gene showed two patterns: one pattern consisted of three exons as reported earlier; the other contained the three exons and also the first intron. Moreover, the above characteristics of UL131A-128 spliced transcripts were confirmed by the sequences of clones selected from the HCMV cDNA library. Our results demonstrated that the UL131A, UL130 and UL128 genes were transcribed with the 3′-coterminal, although the initiation points of their mRNA may be different. The variation in the transcripts found in our study indicated the complex nature of transcription of UL131A-128 genes in clinical strains of HCMV.

  13. Induction of human spermine oxidase SMO(PAOh1) is regulated at the levels of new mRNA synthesis, mRNA stabilization and newly synthesized protein.

    Science.gov (United States)

    Wang, Yanlin; Hacker, Amy; Murray-Stewart, Tracy; Fleischer, Jennifer G; Woster, Patrick M; Casero, Robert A

    2005-03-15

    The oxidation of polyamines induced by antitumour polyamine analogues has been associated with tumour response to specific agents. The human spermine oxidase, SMO(PAOh1), is one enzyme that may play a direct role in the cellular response to the antitumour polyamine analogues. In the present study, the induction of SMO(PAOh1) enzyme activity by CPENSpm [N1-ethyl-N11-(cyclopropyl)methyl-4,8,diazaundecane] is demonstrated to be a result of newly synthesized mRNA and protein. Inhibition of new RNA synthesis by actinomycin D inhibits both the appearance of SMO(PAOh1) mRNA and enzyme activity. Similarly, inhibition of newly synthesized protein with cycloheximide prevents analogue-induced enzyme activity. Half-life determinations indicate that stabilization of SMO(PAOh1) protein does not play a significant role in analogue-induced activity. However, half-life experiments using actinomycin D indicate that CPENSpm treatment not only increases mRNA expression, but also leads to a significant increase in mRNA half-life (17.1 and 8.8 h for CPENSpm-treated cells and control respectively). Using reporter constructs encompassing the SMO(PAOh1) promoter region, a 30-90% increase in transcription is observed after exposure to CPENSpm. The present results are consistent with the hypothesis that analogue-induced expression of SMO(PAOh1) is a result of increased transcription and stabilization of SMO(PAOh1) mRNA, leading to increased protein production and enzyme activity. These data indicate that the major level of control of SMO(PAOh1) expression in response to polyamine analogues exposure is at the level of mRNA.

  14. Studies on P1, P2 Protamine mRNA in Sperms of Human,Rat and Mouse

    Institute of Scientific and Technical Information of China (English)

    吴小芳; 陈晖; 费仁仁; 陈松; 曹坚

    2001-01-01

    Objective To investigate the existence of the protamine Mrna in sperms of human,rat and mouse Methods By means of RT-PCR technique, protamine cDNA fragments were obtained from total RNA of the mature sperms of human, rat and mouse respectively.Results mRNA of protamine gene was found in the mature sperm of human, rat and mouse. The protamine cDNA with an abnormal head obtained by PT-TCR in rat sperm was much less tn number than that in the normal rat sperm.Conclusion mRNA in the sperms might represent the condition of corresponding gene expression during spermatogenesis.

  15. Dendritic transport element of human arc mRNA confers RNA degradation activity in a translation-dependent manner.

    Science.gov (United States)

    Ninomiya, Kensuke; Ohno, Mutsuhito; Kataoka, Naoyuki

    2016-11-01

    Localization of mRNA in neuronal cells is a critical process for spatiotemporal regulation of gene expression. Cytoplasmic localization of mRNA is often conferred by transport elements in 3' untranslated region (UTR). Activity-regulated cytoskeleton-associated protein (arc) mRNA is one of the localizing mRNAs in neuronal cells, and its localization is mediated by dendritic targeting element (DTE). As arc mRNA has introns in its 3' UTR, it was thought that arc mRNA is a natural target of nonsense-mediated mRNA decay (NMD). Here, we show that DTE in human arc 3' UTR has destabilizing activity of RNA independent of NMD pathway. DTE alone was able to cause instability of the reporter mRNA and this degradation was dependent on translation. Our results indicate that DTE has dual activity in mRNA transport and degradation, which suggests the novel spatiotemporal regulation mechanism of activity-dependent degradation of the mRNA.

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

    Science.gov (United States)

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

    1996-11-01

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

  17. Combined sequencing of mRNA and DNA from human embryonic stem cells.

    Science.gov (United States)

    Mertes, Florian; Kuhl, Heiner; Wruck, Wasco; Lehrach, Hans; Adjaye, James

    2016-06-01

    Combined transcriptome and whole genome sequencing of the same ultra-low input sample down to single cells is a rapidly evolving approach for the analysis of rare cells. Besides stem cells, rare cells originating from tissues like tumor or biopsies, circulating tumor cells and cells from early embryonic development are under investigation. Herein we describe a universal method applicable for the analysis of minute amounts of sample material (150 to 200 cells) derived from sub-colony structures from human embryonic stem cells. The protocol comprises the combined isolation and separate amplification of poly(A) mRNA and whole genome DNA followed by next generation sequencing. Here we present a detailed description of the method developed and an overview of the results obtained for RNA and whole genome sequencing of human embryonic stem cells, sequencing data is available in the Gene Expression Omnibus (GEO) database under accession number GSE69471.

  18. Activation of pluripotency genes in human fibroblast cells by a novel mRNA based approach.

    Directory of Open Access Journals (Sweden)

    Jordan R Plews

    Full Text Available BACKGROUND: Several methods have been used to induce somatic cells to re-enter the pluripotent state. Viral transduction of reprogramming genes yields higher efficiency but involves random insertions of viral sequences into the human genome. Although induced pluripotent stem (iPS cells can be obtained with the removable PiggyBac transposon system or an episomal system, both approaches still use DNA constructs so that resulting cell lines need to be thoroughly analyzed to confirm they are free of harmful genetic modification. Thus a method to change cell fate without using DNA will be very useful in regenerative medicine. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we synthesized mRNAs encoding OCT4, SOX2, cMYC, KLF4 and SV40 large T (LT and electroporated them into human fibroblast cells. Upon transfection, fibroblasts expressed these factors at levels comparable to, or higher than those in human embryonic stem (ES cells. Ectopically expressed OCT4 localized to the cell nucleus within 4 hours after mRNA introduction. Transfecting fibroblasts with a mixture of mRNAs encoding all five factors significantly increased the expression of endogenous OCT4, NANOG, DNMT3β, REX1 and SALL4. When such transfected fibroblasts were also exposed to several small molecules (valproic acid, BIX01294 and 5'-aza-2'-deoxycytidine and cultured in human embryonic stem cell (ES medium they formed small aggregates positive for alkaline phosphatase activity and OCT4 protein within 30 days. CONCLUSION/SIGNIFICANCE: Our results demonstrate that mRNA transfection can be a useful approach to precisely control the protein expression level and short-term expression of reprogramming factors is sufficient to activate pluripotency genes in differentiated cells.

  19. Increase in uncoupling protein-2 mRNA expression by BRL49653 and bromopalmitate in human adipocytes.

    Science.gov (United States)

    Viguerie-Bascands, N; Saulnier-Blache, J S; Dandine, M; Dauzats, M; Daviaud, D; Langin, D

    1999-03-05

    Uncoupling protein-2 (UCP2) is a novel mitochondrial protein that may be involved in the control of energy expenditure. We have previously reported an upregulation of adipose tissue UCP2 mRNA expression during fasting in humans. Analysis of changes in metabolic parameters suggested that fatty acids may be associated with the increased UCP2 mRNA level. Culture of human adipose tissue explants was used to study in vitro regulation of adipocyte UCP2 gene expression. A 48-h treatment with BRL49653 and bromopalmitate, two potent activators of PPARgamma, resulted in a dose-dependent increase in UCP2 mRNA levels. The induction by BRL49653 was rapid (from 6 h) and maintained up to 5 days. TNFalpha provoked a 2-fold decrease in UCP2 mRNA levels. Human recombinant leptin did not affect UCP2 mRNA expression. The data support the hypothesis that fatty acids are involved in the control of adipocyte UCP2 mRNA expression in humans. Copyright 1999 Academic Press.

  20. Heritability in the efficiency of nonsense-mediated mRNA decay in humans

    KAUST Repository

    Seoighe, Cathal

    2010-07-21

    Background: In eukaryotes mRNA transcripts of protein-coding genes in which an intron has been retained in the coding region normally result in premature stop codons and are therefore degraded through the nonsense-mediated mRNA decay (NMD) pathway. There is evidence in the form of selective pressure for in-frame stop codons in introns and a depletion of length three introns that this is an important and conserved quality-control mechanism. Yet recent reports have revealed that the efficiency of NMD varies across tissues and between individuals, with important clinical consequences. Principal Findings: Using previously published Affymetrix exon microarray data from cell lines genotyped as part of the International HapMap project, we investigated whether there are heritable, inter-individual differences in the abundance of intron-containing transcripts, potentially reflecting differences in the efficiency of NMD. We identified intronic probesets using EST data and report evidence of heritability in the extent of intron expression in 56 HapMap trios. We also used a genome-wide association approach to identify genetic markers associated with intron expression. Among the top candidates was a SNP in the DCP1A gene, which forms part of the decapping complex, involved in NMD. Conclusions: While we caution that some of the apparent inter-individual difference in intron expression may be attributable to different handling or treatments of cell lines, we hypothesize that there is significant polymorphism in the process of NMD, resulting in heritable differences in the abundance of intronic mRNA. Part of this phenotype is likely to be due to a polymorphism in a decapping enzyme on human chromosome 3. © 2010 Seoighe, Gehring.

  1. Heritability in the efficiency of nonsense-mediated mRNA decay in humans.

    LENUS (Irish Health Repository)

    Seoighe, Cathal

    2010-01-01

    BACKGROUND: In eukaryotes mRNA transcripts of protein-coding genes in which an intron has been retained in the coding region normally result in premature stop codons and are therefore degraded through the nonsense-mediated mRNA decay (NMD) pathway. There is evidence in the form of selective pressure for in-frame stop codons in introns and a depletion of length three introns that this is an important and conserved quality-control mechanism. Yet recent reports have revealed that the efficiency of NMD varies across tissues and between individuals, with important clinical consequences. PRINCIPAL FINDINGS: Using previously published Affymetrix exon microarray data from cell lines genotyped as part of the International HapMap project, we investigated whether there are heritable, inter-individual differences in the abundance of intron-containing transcripts, potentially reflecting differences in the efficiency of NMD. We identified intronic probesets using EST data and report evidence of heritability in the extent of intron expression in 56 HapMap trios. We also used a genome-wide association approach to identify genetic markers associated with intron expression. Among the top candidates was a SNP in the DCP1A gene, which forms part of the decapping complex, involved in NMD. CONCLUSIONS: While we caution that some of the apparent inter-individual difference in intron expression may be attributable to different handling or treatments of cell lines, we hypothesize that there is significant polymorphism in the process of NMD, resulting in heritable differences in the abundance of intronic mRNA. Part of this phenotype is likely to be due to a polymorphism in a decapping enzyme on human chromosome 3.

  2. Heritability in the efficiency of nonsense-mediated mRNA decay in humans.

    Directory of Open Access Journals (Sweden)

    Cathal Seoighe

    Full Text Available BACKGROUND: In eukaryotes mRNA transcripts of protein-coding genes in which an intron has been retained in the coding region normally result in premature stop codons and are therefore degraded through the nonsense-mediated mRNA decay (NMD pathway. There is evidence in the form of selective pressure for in-frame stop codons in introns and a depletion of length three introns that this is an important and conserved quality-control mechanism. Yet recent reports have revealed that the efficiency of NMD varies across tissues and between individuals, with important clinical consequences. PRINCIPAL FINDINGS: Using previously published Affymetrix exon microarray data from cell lines genotyped as part of the International HapMap project, we investigated whether there are heritable, inter-individual differences in the abundance of intron-containing transcripts, potentially reflecting differences in the efficiency of NMD. We identified intronic probesets using EST data and report evidence of heritability in the extent of intron expression in 56 HapMap trios. We also used a genome-wide association approach to identify genetic markers associated with intron expression. Among the top candidates was a SNP in the DCP1A gene, which forms part of the decapping complex, involved in NMD. CONCLUSIONS: While we caution that some of the apparent inter-individual difference in intron expression may be attributable to different handling or treatments of cell lines, we hypothesize that there is significant polymorphism in the process of NMD, resulting in heritable differences in the abundance of intronic mRNA. Part of this phenotype is likely to be due to a polymorphism in a decapping enzyme on human chromosome 3.

  3. Molecular mechanisms of early-life stress in 5-Htt deficient mice: Gene x environment interactions and epigenetic programming

    OpenAIRE

    2012-01-01

    Early-life stress has been shown to influence the development of the brain and to increase the risk for psychiatric disorders later in life. Furthermore, variation in the human serotonin transporter (5-HTT, SLC6A4) gene is suggested to exert a modulating effect on the association between early-life stress and the risk for depression. At the basis of these gene x environment (G x E) interactions, epigenetic mechanisms, such as DNA-methylation, seem to represent the primary biological processes...

  4. Two Human ACAT2 mRNA Variants Produced by Alternative Splicing and Coding for Novel Isoenzymes

    Institute of Scientific and Technical Information of China (English)

    Xiao-Min YAO; Bo-Liang LI; Can-Hua WANG; Bao-Liang SONG; Xin-Ying YANG; Zhen-Zhen WANG; Wei QI; Zhi-Xin LIN; Catherine C. Y. CHANG; Ta-Yuan CHANG

    2005-01-01

    Acyl coenzyme A:cholesterol acyltransferase 2 (ACAT2) plays an important role in cholesterol absorption. Human ACAT2 is highly expressed in small intestine and fetal liver, but its expression is greatly diminished in adult liver. The full-length human ACAT2 mRNA encodes a protein, designated ACAT2a, with 522 amino acids. We have previously reported the organization of the human ACAT2 gene and the differentiation-dependent promoter activity in intestinal Caco-2 cells. In the current work, two human ACAT2 mRNA variants produced by alternative splicing are cloned and predicted to encode two novel ACAT2 isoforms,named ACAT2b and ACAT2c, with 502 and 379 amino acids, respectively. These mRNA variants differ from ACAT2a mRNA by lack of the exon 4 (ACAT2b mRNA) and exons 4-5 plus 8-9-10 (ACAT2c mRNA).Significantly, comparable amounts of the alternatively spliced ACAT2 mRNA variants were detected by RTPCR, and Western blot analysis confirmed the presence of their corresponding proteins in human liver and intestine cells. Furthermore, phosphorylation and enzymatic activity analyses demonstrated that the novel isoenzymes ACAT2b and ACAT2c lacked the phosphorylatable site SLLD, and their enzymatic activities reduced to 25%-35% of that of ACAT2a. These evidences indicate that alternative splicing produces two human ACAT2 mRNA variants that encode the novel ACAT2 isoenzymes. Our findings might help to understand the regulation of the ACAT2 gene expression under certain physiological and pathological conditions.

  5. Inhibin mRNA in Human Corpora Lutea during the Menstrual Cycle

    Institute of Scientific and Technical Information of China (English)

    孙志达; 沈卫雄; 王寒正; 任佩锋; 张永莲; 龚岳亭

    1994-01-01

    The steady state of mRNAs for the α-and βA-subunits of inhibin was investigated in human corpora lutea(CL) during the menstrual cycle by Northern blot analysis with more sensitive cRNA probes. It revealed α 1.6kb mRNA for a subunit and 6.0,4.0, 2.8 and 1.8kb transcripts for βA subunit, Both of α-and βA inhibin mRNAs were detectable in CLs during early, mid and late luteal phases, but not in retrogressive CL after day-27 of the cycle or in tissues of ovarian stroma. An overall profile of the

  6. cDNA cloning, mRNA distribution and heterogeneity, chromosomal location, and RFLP analysis of human osteopontin (OPN)

    DEFF Research Database (Denmark)

    Young, M F; Kerr, J M; Termine, J D

    1990-01-01

    A human osteopontin (OP) cDNA was isolated from a library made from primary cultures of human bone cells. The distribution of osteopontin mRNA in human tissues was investigated by Northern analysis and showed that the human message was predominant in cultures of bone cells and in decidua cells...... osteopontin cDNA indicated that the gene is a single copy with an approximate length of 5.4-8.2 kb....

  7. A classical brown adipose tissue mRNA signature partly overlaps with brite in the supraclavicular region of adult humans

    DEFF Research Database (Denmark)

    Jespersen, Naja Zenius; Larsen, Therese Juhlin; Peijs, Lone

    2013-01-01

    . A similar mRNA expression profile was observed when comparing isolated human adipocytes from BAT and white adipose tissue (WAT) depots, differentiated in vitro. In conclusion, our data suggest that human BAT might consist of both classical brown and recruitable brite adipocytes, an observation important...

  8. CBFA1 and topoisomerase I mRNA levels decline during cellular aging of human trabecular osteoblasts

    DEFF Research Database (Denmark)

    Christiansen, M; Kveiborg, Marie; Kassem, M

    2000-01-01

    reverse transcriptase-polymerase chain reaction (RT-PCR). There is a progressive and significant reduction of the CBFA1 steady-state mRNA level down to 50% during cellular aging of human osteoblasts. In comparison to the normal cells, human osteosarcoma cell lines SaOS-2 and KHOS/NP, and the SV40...

  9. Chaperone protein HYPK interacts with the first 17 amino acid region of Huntingtin and modulates mutant HTT-mediated aggregation and cytotoxicity

    Energy Technology Data Exchange (ETDEWEB)

    Choudhury, Kamalika Roy [Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, 1/AF Bidhannagar, Kolkata 700064 (India); Centre for Neuroscience, Indian Institute of Science, Bangalore 560012 (India); Bhattacharyya, Nitai P., E-mail: nitai_sinp@yahoo.com [Biomedical Genomics Centre, PG Polyclinic Building, 5, Suburbun Hospital Road, Kolkata 700020 (India)

    2015-01-02

    Highlights: • HYPK reduces mutant HTT-mediated aggregate formation and cytotoxicity. • Interaction of HYPK with HTT requires N-terminal 17 amino acid of HTT (HTT-N17). • Deletion of HTT-N17 leads to SDS-soluble, smaller, nuclear aggregates. • These smaller aggregates do not associate with HYPK and are more cytotoxic. • Maybe, interaction of HYPK with amphipathic HTT-N17 block HTT aggregate formation. - Abstract: Huntington’s disease is a polyglutamine expansion disorder, characterized by mutant HTT-mediated aggregate formation and cytotoxicity. Many reports suggests roles of N-terminal 17 amino acid domain of HTT (HTT-N17) towards subcellular localization, aggregate formation and subsequent pathogenicity induced by N-terminal HTT harboring polyQ stretch in pathogenic range. HYPK is a HTT-interacting chaperone which can reduce N-terminal mutant HTT-mediated aggregate formation and cytotoxicity in neuronal cell lines. However, how HYPK interacts with N-terminal fragment of HTT remained unknown. Here we report that specific interaction of HYPK with HTT-N17 is crucial for the chaperone activity of HYPK. Deletion of HTT-N17 leads to formation of tinier, SDS-soluble nuclear aggregates formed by N-terminal mutant HTT. The increased cytotoxicity imparted by these tiny aggregates might be contributed due to loss of interaction with HYPK.

  10. HTT-lowering reverses Huntington's disease immune dysfunction caused by NFκB pathway dysregulation.

    Science.gov (United States)

    Träger, Ulrike; Andre, Ralph; Lahiri, Nayana; Magnusson-Lind, Anna; Weiss, Andreas; Grueninger, Stephan; McKinnon, Chris; Sirinathsinghji, Eva; Kahlon, Shira; Pfister, Edith L; Moser, Roger; Hummerich, Holger; Antoniou, Michael; Bates, Gillian P; Luthi-Carter, Ruth; Lowdell, Mark W; Björkqvist, Maria; Ostroff, Gary R; Aronin, Neil; Tabrizi, Sarah J

    2014-03-01

    Huntington's disease is an inherited neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. The peripheral innate immune system contributes to Huntington's disease pathogenesis and has been targeted successfully to modulate disease progression, but mechanistic understanding relating this to mutant huntingtin expression in immune cells has been lacking. Here we demonstrate that human Huntington's disease myeloid cells produce excessive inflammatory cytokines as a result of the cell-intrinsic effects of mutant huntingtin expression. A direct effect of mutant huntingtin on the NFκB pathway, whereby it interacts with IKKγ, leads to increased degradation of IκB and subsequent nuclear translocation of RelA. Transcriptional alterations in intracellular immune signalling pathways are also observed. Using a novel method of small interfering RNA delivery to lower huntingtin expression, we show reversal of disease-associated alterations in cellular function-the first time this has been demonstrated in primary human cells. Glucan-encapsulated small interfering RNA particles were used to lower huntingtin levels in human Huntington's disease monocytes/macrophages, resulting in a reversal of huntingtin-induced elevated cytokine production and transcriptional changes. These findings improve our understanding of the role of innate immunity in neurodegeneration, introduce glucan-encapsulated small interfering RNA particles as tool for studying cellular pathogenesis ex vivo in human cells and raise the prospect of immune cell-directed HTT-lowering as a therapeutic in Huntington's disease.

  11. Quantification of human telomerase RNA (Htr) and human telomerase reverse transcriptase (Htert)Mrna in testicular tissue of infertile patients

    Institute of Scientific and Technical Information of China (English)

    Mark Schrader; Markus Miller; Ridiger Heicappell; Bernd Straub; Kurt Miller

    2001-01-01

    Aim: To evaluate the quantitative detection of human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT) mRNA as diagnostic parameters in the workup of testicular tissue specimens from patients presenting with non-obstructive azoospemia. Methods: hTR and hTERT mRNA expression were quantified in 38 cryopreserved testicular tissue specimens by fluorescence real-time reverse transcription- polymerase chain reaction (RT-PCR)in a LightCycler(r). This was paralleled by conventional histological workup in all tissue specimens and additional semithin sectioning preparation in cases with maturation arrest ( n = 12) and Sertoli-cell-only syndrome ( n = 12). Results: The average normalized hTERT expression (NhTERT) Was 131.9 ± 48.0 copies (mean ± SD) in tissue specimens with full spermatogenesis, NhTERT = 51.2 ± 17.2 copies in those with maturation arrest and NhTERT = 2.7 ± 2.4 copies in those with Sertoli-cell-only syndrome (SCOS). The discriminant analysis showed that detection of NhTERT (NhTR) had a predictive value of 86.8 % (55.3 % ) for correct classification in one of the three histological subgroups.Conclusion: Our results demonstrate that quantitative detection of hTERT mRNA expression in testicular tissue enables a molecular-diagnostic classification of gametogenesis. Quantitative detection of hTERT in testicular biopsies is thus well suited for supplementing the histopathological evaluation.

  12. Distinct regulation of vasoactive intestinal peptide (VIP) expression at mRNA and peptide levels in human neuroblastoma cells.

    Science.gov (United States)

    Agoston, D V; Colburn, S; Krajniak, K G; Waschek, J A

    1992-05-25

    Neuronal differentiation was induced in cultures of the human neuroblastoma cell line subclone SH-SY5Y by 14-day treatment with dibutyryl cAMP (dBcAMP), retinoic acid, and phorbol 12-myristate 13-acetate (PMA). An approximate 4-fold increase in vasoactive intestinal peptide (VIP) mRNA concentration was observed after differentiation with retinoic acid, whereas no change in VIP mRNA concentration was observed after differentiation with dBcAMP or PMA. A short-term treatment of cells with PMA did however result in a 5-fold transient increase in VIP mRNA; prior differentiation with retinoic acid or dBcAMP diminished this effect. Observed increases in VIP mRNA were in all cases accompanied by increases in VIP immunoreactivity. Remarkably, however, long-term treatment of cells with dBcAMP, which caused no change in mRNA levels, resulted in a six-fold increase in VIP immunoreactivity. Acute (36-h) treatment with carbachol also caused an increase in VIP immunoreactivity (about 2-fold, and blocked by atropine) without an increase in VIP mRNA level. Thus, a quantitative change in gene transcription or mRNA stability appears not to be a prerequisite for increased VIP expression, indicating that regulation can occur at translational or post-translational steps.

  13. Preferential germline usage and VH/VL pairing observed in human antibodies selected by mRNA display.

    Science.gov (United States)

    Chen, Lei; Kutskova, Yuliya A; Hong, Feng; Memmott, John E; Zhong, Suju; Jenkinson, Megan D; Hsieh, Chung-Ming

    2015-10-01

    Since the invention of phage display, in vitro antibody display technologies have revolutionized the field of antibody discovery. In combination with antibody libraries constructed with sequences of human origin, such technologies enable accelerated therapeutic antibody discovery while bypassing the laborious animal immunization and hybridoma generation processes. Many in vitro display technologies developed since aim to differentiate from phage display by displaying full-length IgG proteins, utilizing eukaryotic translation system and codons, increasing library size or real-time kinetic selection by fluorescent activated cell sorting. We report here the development of an mRNA display technology and an accompanying HCDR3 size spectratyping monitor for human antibody discovery. Importantly, the mRNA display technology maintains a monovalent linkage between the mRNA (genotype) and display binding protein (phenotype), which minimizes avidity effect common in other display systems and allows for a stringent affinity and off-rate selection. The mRNA display technology successfully identified 100 human antibodies in 15 different selections against various targets from naïve human antibody libraries. These antibodies in general have high affinity and diversity. By analyzing the germline usage and combination of antibodies selected by the mRNA display technology, we identified trends and determined the productivity of each germline subgroup in the libraries that could serve as the knowledge base for constructing fully synthetic, next generation antibody libraries.

  14. Human low molecular weight neurofilament (NFL) mRNA interacts with a predicted p190RhoGEF homologue (RGNEF) in humans.

    Science.gov (United States)

    Volkening, Kathryn; Leystra-Lantz, Cheryl; Strong, Michael J

    2010-01-01

    In the mouse, p190RhoGEF is a low molecular weight neurofilament (NFL) mRNA stability factor that is involved in NF aggregate formation in neurons. A human homologue of this protein has not been described. Our objective was to identify a human homologue of p190RhoGEF, and to determine its interaction with human NFL mRNA. We used sequence homology searches to predict a human homologue (RGNEF), and RT-PCR to determine the expression of mRNA in ALS and neuropathologically normal control tissues. Gel shift assays determined the interaction of RGNEF with human NFL mRNA in vitro, while IP-RT-PCR and gel shift assays were used to confirm the interaction in tissue lysates. We determined that RGNEF is a human homologue of p190RhoGEF, and that its RNA is expressed in both brain and spinal cord. While RGNEF and NFL mRNA interact directly in vitro, interestingly they only appear to interact in ALS lysates and not in controls. These data add another player to the family of NFL mRNA stability regulators, and raise the intriguing possibility that the mechanism by which p190RhoGEF contributes to murine neuronal NF aggregate formation may be important to human ALS NF aggregate formation.

  15. Expression of ET(A) and ET(B) receptor mRNA in human cerebral arteries

    DEFF Research Database (Denmark)

    Hansen-Schwartz, J; Szok, D; Edvinsson, L

    2002-01-01

    The vascular effects of endothelins (ET) are in mammals mediated via two receptor subtypes, endothelin A (ET(A), mainly constrictive) and endothelin B (ET(B), mainly dilating) receptors. We have examined the presence of ET(A) and ET(B) receptor mRNA using the reverse transcription polymerase chain...... reaction (RT-PCR) in both normal human cerebral arteries and cerebral arteries from patients with cerebrovascular disease. Two vessel preparations were studied: macroscopic arteries and microvessels, the latter obtained through a sensitive separation method. In endothelial cells both ET(A) and ET......(B) receptor mRNA was detected. In almost all samples from normal cerebral arteries only ET(A) receptor mRNA was detected, whereas in vessel samples from patients with cerebrovascular disease as well as cerebral neoplasms, additional ET(B) receptor mRNA was detected significantly more frequently...

  16. Specificity of recognition of mRNA 5' cap by human nuclear cap-binding complex.

    Science.gov (United States)

    Worch, Remigiusz; Niedzwiecka, Anna; Stepinski, Janusz; Mazza, Catherine; Jankowska-Anyszka, Marzena; Darzynkiewicz, Edward; Cusack, Stephen; Stolarski, Ryszard

    2005-09-01

    The heterodimeric nuclear cap-binding complex (CBC) binds to the mono-methylated 5' cap of eukaryotic RNA polymerase II transcripts such as mRNA and U snRNA. The binding is important for nuclear maturation of mRNAs and possibly in the first round of translation and nonsense-mediated decay. It is also essential for nuclear export of U snRNAs in metazoans. We report characterization by fluorescence spectroscopy of the recognition of 5' capped RNA by human CBC. The association constants (K(as)) for 17 mono- and dinucleotide cap analogs as well as for the oligomer m7GpppA(m2') pU(m2')pA(m2') cover the range from 1.8 x 10(6) M(-1) to 2.3 x 10(8) M(-1). Higher affinity for CBC is observed for the dinucleotide compared with mononucleotide analogs, especially for those containing a purine nucleoside next to m7G. The mRNA tetramer associates with CBC as tightly as the dinucleotide analogs. Replacement of Tyr138 by alanine in the CBP20 subunit of CBC reduces the cap affinity except for the mononucleotide analogs, consistent with the crystallographic observation of the second base stacking on this residue. Our spectroscopic studies showed that contrary to the other known cap-binding proteins, the first two nucleotides of a capped-RNA are indispensable for its specific recognition by CBC. Differences in the cap binding of CBC compared with the eukaryotic translation initiation factor 4E (eIF4E) are analyzed and discussed regarding replacement of CBC by eIF4E.

  17. Ischemic heart disease down-regulates angiotensin type 1 receptor mRNA in human coronary arteries

    DEFF Research Database (Denmark)

    Wackenfors, Angelica; Emilson, Malin; Ingemansson, Richard;

    2004-01-01

    Angiotensin II is important in the development of cardiovascular disease. In the present study, angiotensin II receptor mRNA levels were quantified by real-time polymerase chain reaction (real-time PCR) in human coronary arteries from patients with ischemic heart disease and controls. Furthermore......, the suitability of artery culture for studying angiotensin receptor changes was evaluated by in vitro pharmacology and real-time PCR. The angiotensin type 1 (AT1) receptor mRNA levels were down-regulated in human coronary arteries from patients with ischemic heart disease as compared to controls (P

  18. A SNP in the HTT promoter alters NF-κB binding and is a bidirectional genetic modifier of Huntington disease

    DEFF Research Database (Denmark)

    Bečanović, Kristina; Nørremølle, Anne; Neal, Scott J

    2015-01-01

    . HTT promoter analysis identified a NF-κB binding site that regulates HTT promoter transcriptional activity. A non-coding SNP, rs13102260:G > A, in this binding site impaired NF-κB binding and reduced HTT transcriptional activity and HTT protein expression. The presence of the rs13102260 minor (A...

  19. Multiple correlation analyses revealed complex relationship between DNA methylation and mRNA expression in human peripheral blood mononuclear cells.

    Science.gov (United States)

    Xie, Fang-Fei; Deng, Fei-Yan; Wu, Long-Fei; Mo, Xing-Bo; Zhu, Hong; Wu, Jian; Guo, Yu-Fan; Zeng, Ke-Qin; Wang, Ming-Jun; Zhu, Xiao-Wei; Xia, Wei; Wang, Lan; He, Pei; Bing, Peng-Fei; Lu, Xin; Zhang, Yong-Hong; Lei, Shu-Feng

    2017-07-22

    DNA methylation is an important regulator on the mRNA expression. However, a genome-wide correlation pattern between DNA methylation and mRNA expression in human peripheral blood mononuclear cells (PBMCs) is largely unknown. The comprehensive relationship between mRNA and DNA methylation was explored by using four types of correlation analyses and a genome-wide methylation-mRNA expression quantitative trait locus (eQTL) analysis in PBMCs in 46 unrelated female subjects. An enrichment analysis was performed to detect biological function for the detected genes. Single pair correlation coefficient (r T1) between methylation level and mRNA is moderate (-0.63-0.62) in intensity, and the negative and positive correlations are nearly equal in quantity. Correlation analysis on each gene (T4) found 60.1% genes showed correlations between mRNA and gene-based methylation at P correlation (R T4 > 0.8). Methylation sites have regulation effects on mRNA expression in eQTL analysis, with more often observations in region of transcription start site (TSS). The genes under significant methylation regulation both in correlation analysis and eQTL analysis tend to cluster to the categories (e.g., transcription, translation, regulation of transcription) that are essential for maintaining the basic life activities of cells. Our findings indicated that DNA methylation has predictive regulation effect on mRNA with a very complex pattern in PBMCs. The results increased our understanding on correlation of methylation and mRNA and also provided useful clues for future epigenetic studies in exploring biological and disease-related regulatory mechanisms in PBMC.

  20. Effect of in vitro estrogenic pesticides on human oestrogen receptor α and β mRNA levels

    DEFF Research Database (Denmark)

    Theander Grünfeld, Heidi; Bonefeld-Jørgensen, Eva Cecilie

    2004-01-01

    Nine widely distributed pesticides were recently demonstrated to posses potential estrogenic properties in oestrogen receptor (ER) transactivation and/or E-screen assays. We tested the effect of these nine pesticides on the human ERα and ERβ mRNA steady state levels in the mamma cancer fibroblast...

  1. Human papillomavirus mRNA and DNA testing in women with atypical squamous cells of undetermined significance

    DEFF Research Database (Denmark)

    Thomsen, Louise T; Dehlendorff, Christian; Junge, Jette

    2016-01-01

    In this prospective cohort study, we compared the performance of human papillomavirus (HPV) mRNA and DNA testing of women with atypical squamous cells of undetermined significance (ASC-US) during cervical cancer screening. Using a nationwide Danish pathology register, we identified women aged 30...

  2. Determination of the human cardiomyocyte mRNA and miRNA differentiation network by fine-scale profiling.

    Science.gov (United States)

    Babiarz, Joshua E; Ravon, Morgane; Sridhar, Sriram; Ravindran, Palanikumar; Swanson, Brad; Bitter, Hans; Weiser, Thomas; Chiao, Eric; Certa, Ulrich; Kolaja, Kyle L

    2012-07-20

    To gain insight into the molecular regulation of human heart development, a detailed comparison of the mRNA and miRNA transcriptomes across differentiating human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes and biopsies from fetal, adult, and hypertensive human hearts was performed. Gene ontology analysis of the mRNA expression levels of the hiPSCs differentiating into cardiomyocytes revealed 3 distinct groups of genes: pluripotent specific, transitional cardiac specification, and mature cardiomyocyte specific. Hierarchical clustering of the mRNA data revealed that the transcriptome of hiPSC cardiomyocytes largely stabilizes 20 days after initiation of differentiation. Nevertheless, analysis of cells continuously cultured for 120 days indicated that the cardiomyocytes continued to mature toward a more adult-like gene expression pattern. Analysis of cardiomyocyte-specific miRNAs (miR-1, miR-133a/b, and miR-208a/b) revealed an miRNA pattern indicative of stem cell to cardiomyocyte specification. A biostatistitical approach integrated the miRNA and mRNA expression profiles revealing a cardiomyocyte differentiation miRNA network and identified putative mRNAs targeted by multiple miRNAs. Together, these data reveal the miRNA network in human heart development and support the notion that overlapping miRNA networks re-enforce transcriptional control during developmental specification.

  3. Two type XVII collagen (BP180) mRNA transcripts in human keratinocytes : a long and a short form

    NARCIS (Netherlands)

    Molnar, K; van der Steege, G; Jonkman, MF; Nijenhuis, Albertine; Husz, S; van der Meer, JB; Pas, HH

    We have analysed BP180 mRNA expression in normal human keratinocytes. Here we report the presence in normal keratinocytes of two COL17A1 transcripts which differ by 0.6 kb in length. Both mRNAs hybridized on Northern blot with probes directed to sequences encoding intracellular and extracellular

  4. mRNA Transcriptomics of Galectins Unveils Heterogeneous Organization in Mouse and Human Brain

    Directory of Open Access Journals (Sweden)

    Sebastian John

    2016-12-01

    Full Text Available Background: Galectins, a family of non-classically secreted, β-galactoside binding proteins is involved in several brain disorders; however no systematic knowledge on the normal neuroanatomical distribution and functions of galectins exits. Hence, the major purpose of this study was to understand spatial distribution and predict functions of galectins in brain and also compare the degree of conservation vs. divergence between mouse and human species. The latter objective was required to determine the relevance and appropriateness of studying galectins in mouse brain which may ultimately enable us to extrapolate the findings to human brain physiology and pathologies.Results: In order to fill this crucial gap in our understanding of brain galectins, we analyzed the in situ hybridization (ISH and microarray data of adult mouse and human brain respectively, from the Allen Brain Atlas, to resolve each galectin-subtype’s spatial distribution across brain distinct cytoarchitecture. Next, transcription factors (TFs that may regulate galectins were identified using TRANSFAC software and the list obtained was further curated to sort TFs on their confirmed transcript expression in the adult brain. Galectin-TF cluster analysis, gene-ontology annotations and co-expression networks were then extrapolated to predict distinct functional relevance of each galectin in the neuronal processes. Data shows that galectins have highly heterogeneous expression within and across brain sub-structures and are predicted to be the crucial targets of brain enriched TFs. Lgals9 had maximal spatial distribution across mouse brain with inferred predominant roles in neurogenesis while LGALS1 was ubiquitously expressed in human. Limbic region associated with learning, memory and emotions and substantia nigra associated with motor movements showed strikingly high expression of LGALS1 and LGALS8 in human vs. mouse brain. The overall expression profile of galectin-8 was most

  5. Quantifiable mRNA transcripts for tamoxifen-metabolising enzymes in human endometrium.

    Science.gov (United States)

    Singh, Maneesh N; Stringfellow, Helen F; Walsh, Michael J; Ashton, Kate M; Paraskevaidis, Evangelos; Abdo, Khalil R; Martin-Hirsch, Pierre L; Phillips, David H; Martin, Francis L

    2008-07-10

    Tamoxifen has been used in the management of receptor-positive breast cancer for >20 years. Usage confers an elevated risk of developing endometrial carcinoma. Its mechanism of carcinogenicity remains unresolved with controversy as to whether or not this is mediated through a genotoxic mechanism. Usage is not only associated with an elevated occurrence of endometrioid endometrial carcinoma, but also type 2 and mixed epithelial-stromal tumours (MESTs) that have a poorer prognosis. Following hysterectomy, endometrial tissues (n=18) classified as benign (n=6), non-tamoxifen-associated carcinoma (n=6) and tamoxifen-associated carcinoma (n=6) were obtained; quantitative gene expression was performed. Employing real-time RT-PCR, the relative gene expressions of phase I/II metabolic enzymes CYP1A2, CYP1B1 and CYP3A4, cathechol-O-methyltransferase (COMT) and SULT2A1 were ascertained. Measurable mRNA transcripts, especially for those genes associated with tamoxifen bioactivation, were quantifiable in all the tissues examined. Whether this is evidence that generation of genotoxic tamoxifen metabolites may occur in human endometrial tissue remains to be ascertained.

  6. The mRNA expression of SATB1 and SATB2 in human breast cancer

    Directory of Open Access Journals (Sweden)

    Mansel Robert

    2009-07-01

    increasing tumour grade (grade 1 vs. grade 3 p = 0.035. SATB2 was associated with ER positivity (ER(- vs. ER(+ p = 0.0283 within ductal carcinomas. Higher transcript levels showed a significant association with poorer OS (p = 0.0433. Conclusion SATB1 mRNA expression is significantly associated with poor prognostic parameters in breast cancer, including increasing tumour grade, TNM stage and NPI. SATB2 mRNA expression is significantly associated with increasing tumour grade and poorer OS. These results are consistent with the notion that SATB1 acts as a 'master genome organizer' in human breast carcinogenesis.

  7. FUCOIDIN INHIBITS OXIDIZED LOW DENSITY LIPOPROTEIN FROM INDUCING HUMAN PERIPHERAL BLOOD MONOCYTE EXPRESSION OF PROINFLAMMATORY CYTOKINES mRNA

    Institute of Scientific and Technical Information of China (English)

    雷新军; 马爱群; 任冰稳; 耿涛; 张葳; 白玲

    2003-01-01

    Objective To study the significance of scavenger receptor class A(SR-A)in mediating human peripheral blood monocyte to uptake oxidized low density lipoprotein(OxLDL) and promoting the atherosclerotic immuno-pathological lesion in the local blood vessel. Methods With the Digoxenin-labeled Oligonucleotide-probes In situ Hybridization, this research investigated the effects of OxLDL on the mRNA expression of proinflammatory cytokines including MCP-1, bFGF, PDGF and IL-10 in the human peripheral blood monocyte and whether fucoidin, a peculiarly inhibitory ligand for SR-A, would influence this process. Results Monocyte was significantly increased the mRNA expression of MCP-1, bFGF, PDGF and IL-10 in a dose-dependent manner after incubating with OxLDL (10,15,20,25,30·mg·L-1, respectively)for 24 hours(P<0.001). Fucoidin(50,100,150,200,250·mg·mL-1, respectively)completely inhibited OxLDL(20·mg·L-1)from inducing monocyte the mRNA expression of above proinflammatory cytokines(P<0.001). Conclusion OxLDL can stimulate human peripheral blood monocyte to give expression to proinflammatory cytokines mRNA in a dose-dependent manner, while a peculiarly inhibitory ligand for SR-A-fucoidin has an obviously opposed role.

  8. Concordant regulation of translation and mRNA abundance for hundreds of targets of a human microRNA.

    Directory of Open Access Journals (Sweden)

    David G Hendrickson

    2009-11-01

    Full Text Available MicroRNAs (miRNAs regulate gene expression posttranscriptionally by interfering with a target mRNA's translation, stability, or both. We sought to dissect the respective contributions of translational inhibition and mRNA decay to microRNA regulation. We identified direct targets of a specific miRNA, miR-124, by virtue of their association with Argonaute proteins, core components of miRNA effector complexes, in response to miR-124 transfection in human tissue culture cells. In parallel, we assessed mRNA levels and obtained translation profiles using a novel global approach to analyze polysomes separated on sucrose gradients. Analysis of translation profiles for approximately 8,000 genes in these proliferative human cells revealed that basic features of translation are similar to those previously observed in rapidly growing Saccharomyces cerevisiae. For approximately 600 mRNAs specifically recruited to Argonaute proteins by miR-124, we found reductions in both the mRNA abundance and inferred translation rate spanning a large dynamic range. The changes in mRNA levels of these miR-124 targets were larger than the changes in translation, with average decreases of 35% and 12%, respectively. Further, there was no identifiable subgroup of mRNA targets for which the translational response was dominant. Both ribosome occupancy (the fraction of a given gene's transcripts associated with ribosomes and ribosome density (the average number of ribosomes bound per unit length of coding sequence were selectively reduced for hundreds of miR-124 targets by the presence of miR-124. Changes in protein abundance inferred from the observed changes in mRNA abundance and translation profiles closely matched changes directly determined by Western analysis for 11 of 12 proteins, suggesting that our assays captured most of miR-124-mediated regulation. These results suggest that miRNAs inhibit translation initiation or stimulate ribosome drop-off preferentially near the

  9. How the cerebral serotonin homeostasis predicts environmental changes: a model to explain seasonal changes of brain 5-HTT as intermediate phenotype of the 5-HTTLPR.

    Science.gov (United States)

    Kalbitzer, Jan; Kalbitzer, Urs; Knudsen, Gitte Moos; Cumming, Paul; Heinz, Andreas

    2013-12-01

    Molecular imaging studies with positron emission tomography have revealed that the availability of serotonin transporter (5-HTT) in the human brain fluctuates over the course of the year. This effect is most pronounced in carriers of the short allele of the 5-HTT promoter region (5-HTTLPR), which has in several previous studies been linked to an increased risk to develop mood disorders. We argue that long-lasting fluctuations in the cerebral serotonin transmission, which is regulated via the 5-HTT, are responsible for mediating responses to environmental changes based on an assessment of the expected "safety" of the environment; this response is obtained in part through serotonergic modulation of the hypothalamic-pituitary-adrenal (HPA) axis. We posit that the intermediate phenotype of the s-allele may properly be understood as mediating a trade-off, wherein increased responsiveness of cerebral serotonin transmission to seasonal and other forms of environmental change imparts greater behavioral flexibility, at the expense of increased vulnerability to stress. This model may explain the somewhat higher prevalence of the s-allele in some human populations dwelling at geographic latitudes with pronounced seasonal climatic changes, while this hypothesis does not rule out that genetic drift plays an additional or even exclusive role. We argue that s-allele manifests as an intermediate phenotype in terms of an increased responsiveness of the 5-HTT expression to number of daylight hours, which may serve as a stable surrogate marker of other environmental factors, such as availability of food and safety of the environment in populations that live closer to the geographic poles.

  10. Social defeat: impact on fear extinction and amygdala-prefrontal cortical theta synchrony in 5-HTT deficient mice.

    Directory of Open Access Journals (Sweden)

    Venu Narayanan

    Full Text Available Emotions, such as fear and anxiety, can be modulated by both environmental and genetic factors. One genetic factor is for example the genetically encoded variation of the serotonin transporter (5-HTT expression. In this context, the 5-HTT plays a key role in the regulation of central 5-HT neurotransmission, which is critically involved in the physiological regulation of emotions including fear and anxiety. However, a systematic study which examines the combined influence of environmental and genetic factors on fear-related behavior and the underlying neurophysiological basis is missing. Therefore, in this study we used the 5-HTT-deficient mouse model for studying emotional dysregulation to evaluate consequences of genotype specific disruption of 5-HTT function and repeated social defeat for fear-related behaviors and corresponding neurophysiological activities in the lateral amygdala (LA and infralimbic region of the medial prefrontal cortex (mPFC in male 5-HTT wild-type (+/+, homo- (-/- and heterozygous (+/- mice. Naive males and experienced losers (generated in a resident-intruder paradigm of all three genotypes, unilaterally equipped with recording electrodes in LA and mPFC, underwent a Pavlovian fear conditioning. Fear memory and extinction of conditioned fear was examined while recording neuronal activity simultaneously with fear-related behavior. Compared to naive 5-HTT+/+ and +/- mice, 5-HTT-/- mice showed impaired recall of extinction. In addition, 5-HTT-/- and +/- experienced losers showed delayed extinction learning and impaired recall of extinction. Impaired behavioral responses were accompanied by increased theta synchronization between the LA and mPFC during extinction learning in 5-HTT-/- and +/- losers. Furthermore, impaired extinction recall was accompanied with increased theta synchronization in 5-HTT-/- naive and in 5-HTT-/- and +/- loser mice. In conclusion, extinction learning and memory of conditioned fear can be modulated

  11. Association of VNTR polymorphisms in DRD4, 5-HTT and DAT1 genes with obesity.

    Science.gov (United States)

    Uzun, Mustafa; Saglar, Emel; Kucukyildirim, Sibel; Erdem, Beril; Unlu, Hande; Mergen, Hatice

    2015-05-01

    To investigate the association between VNTR polymorphisms of DRD4, DAT1 and 5-HTT genes and obesity. Peripheral blood samples of 234 obese (BMI ≥ 30) and 148 healthy individuals (BMI ≤ 25) were objected to PCR to detect the VNTR of the 2nd intron of 5-HTT, 3rd exon of DRD4 and 3'UTR of DAT1 genes. The association between obesity and genotype distributions of 5-HTT, DAT1 and DRD4 genes and between obesity and distributions of allele frequencies were tested by Chi Square (χ(2)) test and were not found statistically significant. BMI values for genotype of obese and morbidly obese (BMI > 40) individuals were analyzed by Kruskal-Wallis and not found statistically significant differences between BMI values for the most frequent genotypes of 5-HTT, DAT1 and DRD4 genes. As a conclusion, there was no association between 5-HTT, DAT1 and DRD4 genes VNTR polymorphisms and obesity.

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

    Institute of Scientific and Technical Information of China (English)

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

    2005-01-01

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

  13. Administration of nucleoside-modified mRNA encoding broadly neutralizing antibody protects humanized mice from HIV-1 challenge

    Science.gov (United States)

    Pardi, Norbert; Secreto, Anthony J.; Shan, Xiaochuan; Debonera, Fotini; Glover, Joshua; Yi, Yanjie; Muramatsu, Hiromi; Ni, Houping; Mui, Barbara L.; Tam, Ying K.; Shaheen, Farida; Collman, Ronald G.; Karikó, Katalin; Danet-Desnoyers, Gwenn A.; Madden, Thomas D.; Hope, Michael J.; Weissman, Drew

    2017-01-01

    Monoclonal antibodies are one of the fastest growing classes of pharmaceutical products, however, their potential is limited by the high cost of development and manufacturing. Here we present a safe and cost-effective platform for in vivo expression of therapeutic antibodies using nucleoside-modified mRNA. To demonstrate feasibility and protective efficacy, nucleoside-modified mRNAs encoding the light and heavy chains of the broadly neutralizing anti-HIV-1 antibody VRC01 are generated and encapsulated into lipid nanoparticles. Systemic administration of 1.4 mg kg−1 of mRNA into mice results in ∼170 μg ml−1 VRC01 antibody concentrations in the plasma 24 h post injection. Weekly injections of 1 mg kg−1 of mRNA into immunodeficient mice maintain trough VRC01 levels above 40 μg ml−1. Most importantly, the translated antibody from a single injection of VRC01 mRNA protects humanized mice from intravenous HIV-1 challenge, demonstrating that nucleoside-modified mRNA represents a viable delivery platform for passive immunotherapy against HIV-1 with expansion to a variety of diseases. PMID:28251988

  14. Human Platelet Antigen Genotyping and Expression of CD109 (Human Platelet Antigen 15 mRNA in Various Human Cell Types

    Directory of Open Access Journals (Sweden)

    Sang Mee Hwang

    2013-01-01

    Full Text Available CD109 gene encodes a glycosylphosphatidylinositol-linked glycoprotein found in a subset of platelets and endothelial cell, and human platelet antigen (HPA 15 is found on CD109. We evaluated the HPA genotype and/or the CD109 mRNA expression on two peripheral blood stem cells (PBSC, two peripheral bloods (PB, 12 granulocyte products, natural killer (NK-92, B-lymphocyte (CO88BV59-1, K-562 leukemia cell line, human embryonic stem cell (hESC, and human fibroblasts (HF. HPA genotyping was performed by SNaPshot assay and CD109 mRNA expression was evaluated by real-time PCR with SYBR green and melting curve analysis. Genotype HPA-15a/-15a was found in PBSC#1 and two granulocyte products, and HPA-15a/-15b was found in PBSC#2, eight granulocyte products, NK-92, K-562, hESC, and HF, and HPA-15b/-15b was found in two granulocyte products. CD109 mRNA expression was highly increased in HF and increased in CD34+ and CD34− PBSCs and some granulocyte products, compared to the PB. However, the increase of expression level varied among the PBSC and granulocyte products. The CD109 mRNA expression of NK-92, K-562, hESC, and CO 88BV59-1 was not detected. HPA genotype was evaluated in various cells and the expression of CD109, which contains HPA 15, was different among cell lines and high in HF and PBSCs.

  15. Gene Manipulation of Human Embryonic Stem Cells by In Vitro-Synthesized mRNA for Gene Therapy.

    Science.gov (United States)

    Wang, Xiao Li; Yu, Li; Ding, Yan; Guo, Xing Rong; Yuan, Ya Hong; Li, Dong Sheng

    2015-01-01

    The difficulty in producing genetically modified human embryonic stem cells (hESCs) limits research on their applications. Virus-based gene transfer is not safe for clinical use, whereas DNAbased non-viral methods are not efficient or safe, and mRNA-based methods are useful for genetic manipulation. In this study, we easily obtained multiple types and large amounts of in vitro-synthesized mRNA by PCR. The efficiency of different transfection methods was studied by flow cytometry. The effect of different mRNA modifications on protein translation efficiency and dynamics of luciferase mRNA expression in hESCs were studied using a bioluminescence imaging system. The pluripotency of hESCs after transfection was studied by immunofluorescence. In vitro-synthesized pancreatic-duodenal homeobox 1 (PDX1) mRNA was used to induce the differentiation of hESCs into insulin-producing cells. We found that electroporation is the most efficient transfection method, and it produces more than 95% transgene expression in multiple hESC lines. Synthesized mRNA with a combination of a polyA tail, cap and base analogues is more efficiently translated into protein in hESCs compared with single-modified mRNA. Transfection of mRNA into hESCs by trypsinizing the cells into single-cell suspensions did not affect their pluripotency, and multiple types of mRNAs can be transfected into hESCs efficiently. We found that PDX-1 mRNA transfection significantly improved the expression level of genes related to beta cells and differentiated cells that express insulin and C-peptide. ELISA analysis validate the insulin secretion of islet-like cell clusters in response to glucose stimulation. Our results indicate that electroporation of in vitro-synthesized mRNA is useful for genetic manipulation of hESCs and differentiation of hESCs into particular cell types, and this finding will pave the way for clinical applications of this method.

  16. [The expression of human telomerase reverse transcriptase mRNA and its significance in acute leukemia].

    Science.gov (United States)

    Meng, Xiao-Li; Lin, Mao-Fang; Jin, Jie

    2003-02-01

    To investigate the expression of hTERT mRNA in bone marrow mononuclear cells (MNCs) from acute leukemia patients, the method of semi-quntitative RT-PCR was used to examine the expression of hTERT mRNA in marrow MNCs, and the telomerase activity of marrow MNCs was determined with the method of TRAP-PCR-ELISA by using a commercial kit. The results indicated that the expression of hTERT mRNA of marrow MNCs in 30 untreated AL patients was markedly higher than that in 12 CR cases (0.71 +/- 0.34 vs 0.43 +/- 0.25, P < 0.05) and 6 normal volunteers (0.71 +/- 0.34 vs 0.22 +/- 0.21, P < 0.01), respectively. Telomerase activity of marrow MNCs in 30 untreated AL patients was significantly higher than that in 12 CR cases (0.235 +/- 0.395 vs 0.012 +/- 0.015, P = 0.007). Moreover, there was a positive correlation between the hTERT mRNA synthesis and telomerase activity in AL cells (r = 0.421, P < 0.01). The pencentage of blast cells in marrow smear of the untreated AL patients was positively correlated with both the expression of hTERT mRNA and the telomerase activity of bone marrow MNCs (r = 0.457, P < 0.05 and r = 0.411, P < 0.05), respectively. It is concluded that the expression of hTERT mRNA in bone marrow MNCs from untreated AL patients was correlated with their telomerase activity. It is suggested that the expression of hTERT mRNA leukemic cells indicates their higher proliferation ability.

  17. Oxidative stress induced Interleukin-32 mRNA expression in human bronchial epithelial cells

    Directory of Open Access Journals (Sweden)

    Kudo Megumi

    2012-03-01

    Full Text Available Abstract Background Chronic obstructive pulmonary disease (COPD is characterized by airflow obstruction and persistent inflammation in the airways and lung parenchyma. Oxidative stress contributes to the pathogenesis of COPD. Interleukin (IL-32 expression has been reported to increase in the lung tissue of patients with COPD. Here, we show that IFNγ upregulated IL-32 expression and that oxidative stress augmented IFNγ-induced-IL-32 expression in airway epithelial cells. We further investigated transcriptional regulation responsible for IFNγ induced IL-32 expression in human airway epithelial cells. Methods Human bronchial epithelial (HBE cells were stimulated with H2O2 and IFNγ, and IL-32 expression was evaluated. The cell viability was confirmed by MTT assay. The intracellular signaling pathways regulating IL-32 expression were investigated by examining the regulatory effects of MAPK inhibitors and JAK inhibitor after treatment with H2O2 and IFNγ, and by using a ChIP assay to identify transcription factors (i.e. c-Jun, CREB binding to the IL-32 promoter. Promoter activity assays were conducted after mutations were introduced into binding sites of c-Jun and CREB in the IL-32 promoter. IL-32 expression was also examined in HBE cells in which the expression of either c-Jun or CREB was knocked out by siRNA of indicated transcription factors. Results There were no significant differences of cell viability among groups. After stimulation with H2O2 or IFNγ for 48 hours, IL-32 expression in HBE cells was increased by IFNγ and synergistically upregulated by the addition of H2O2. The H2O2 augmented IFNγ induced IL-32 mRNA expression was suppressed by a JNK inhibitor, but not by MEK inhibitor, p38 inhibitor, and JAK inhibitor I. Significant binding of c-Jun and CREB to the IL-32 promoter was observed in the IFNγ + H2O2 stimulated HBE cells. Introducing mutations into the c-Jun/CREB binding sites in the IL-32 promoter prominently suppressed its

  18. Aggression and 5HTT polymorphism in females: study of synchronized swimming and control groups.

    Science.gov (United States)

    Sysoeva, Olga V; Maluchenko, Natalia V; Timofeeva, Marina A; Portnova, Galina V; Kulikova, Maria A; Tonevitsky, Alexandr G; Ivanitsky, Alexey M

    2009-05-01

    Aggression is a heterogeneous heritable psychological trait, also influenced by environmental factors. Previous studies, mostly conducted on male population, have found some associations of the aggression with the polymorphisms of genes, regulating the activity of serotonin (5-HT) in the brain. However, psychological as well as biochemical manifestations of the aggression are different in males and females. Our study aimed to investigate the association of 5-HTT gene polymorphism with different facets of aggression (BDHI) in females. Two groups: the synchronized swimming and non-athlete control, - were examined to study the possible modulation effect of sport on the association between 5-HTT gene polymorphism and aggression. It was found that in both groups the low-active 5-HTT polymorphism (SS) was associated with increased scores on Indirect Hostility scale and decreased scores on Negativism scale, compared to LL genotype. No interaction effect between sport and 5-HTT polymorphism was found. The higher percentage of LL-carriers and lower of LS-carriers in the synchronized swimming group compared to the control one was observed. This may be the sign of the importance of LL polymorphism of 5-HTT gene, previously associated with higher resistance to stress factors, for being an athlete, although this result has to be taken cautiously keeping in mind the stratification problem. Synchronized swimmers had lower scores on Assault, Negativism, Irritability and Verbal Hostility compared to age-matched control girls (in general and for each 5-HTT genotype separately), suggesting that they may have more matured emotional system (older control group has also lower scores on these scales).

  19. The mRNA expression profile of metabolic genes relative to MHC isoform pattern in human skeletal muscles.

    Science.gov (United States)

    Plomgaard, Peter; Penkowa, Milena; Leick, Lotte; Pedersen, Bente K; Saltin, Bengt; Pilegaard, Henriette

    2006-09-01

    The metabolic profile of rodent muscle is generally reflected in the myosin heavy chain (MHC) fiber-type composition. The present study was conducted to test the hypothesis that metabolic gene expression is not tightly coupled with MHC fiber-type composition for all genes in human skeletal muscle. Triceps brachii, vastus lateralis quadriceps, and soleus muscle biopsies were obtained from normally physically active, healthy, young male volunteers, because these muscles are characterized by different fiber-type compositions. As expected, citrate synthase and 3-hydroxyacyl dehydrogenase activity was more than twofold higher in soleus and vastus than in triceps. Contrary, phosphofructokinase and total lactate dehydrogenase (LDH) activity was approximately three- and twofold higher in triceps than in both soleus and vastus. Expression of metabolic genes was assessed by determining the mRNA content of a broad range of metabolic genes. The triceps muscle had two- to fivefold higher MHC IIa, phosphofructokinase, and LDH A mRNA content and two- to fourfold lower MHC I, lipoprotein lipase, CD36, hormone-sensitive lipase, and LDH B and hexokinase II mRNA than vastus lateralis or soleus. Interestingly, such mRNA differences were not evident for any of the genes encoding mitochondrial oxidative proteins, 3-hydroxyacyl dehydrogenase, carnitine palmitoyl transferase I, citrate synthase, alpha-ketogluterate dehydrogenase, and cytochrome c, nor for the transcriptional regulators peroxisome proliferator activator receptor gamma coactivator-1alpha, forkhead box O1, or peroxisome proliferator activator receptor-alpha. Thus the mRNA expression of genes encoding mitochondrial proteins and transcriptional regulators does not seem to be fiber type specific as the genes encoding glycolytic and lipid metabolism genes, which suggests that basal mRNA regulation of genes encoding mitochondrial proteins does not match the wide differences in mitochondrial content of these muscles.

  20. Normal and mutant HTT interact to affect clinical severity and progression in Huntington disease

    DEFF Research Database (Denmark)

    Aziz, N A; Jurgens, C K; Landwehrmeyer, G B;

    2009-01-01

    OBJECTIVE: Huntington disease (HD) is an autosomal dominant neurodegenerative disorder caused by a CAG repeat expansion in the HD gene (HTT). We aimed to assess whether interaction between CAG repeat sizes in the mutant and normal allele could affect disease severity and progression. METHODS: Using...... with less severe symptoms and pathology. CONCLUSIONS: Increasing CAG repeat size in normal HTT diminishes the association between mutant CAG repeat size and disease severity and progression in Huntington disease. The underlying mechanism may involve interaction of the polyglutamine domains of normal...

  1. Visfatin mRNA expression in human subcutaneous adipose tissue is regulated by exercise

    DEFF Research Database (Denmark)

    Frydelund-Larsen, Lone; Åkerström, Thorbjörn; Nielsen, Søren

    2006-01-01

    Visfatin [pre-beta-cell colony-enhancing factor (PBEF)] is a novel adipokine that is produced by adipose tissue, skeletal muscle, and liver and has insulin-mimetic actions. Regular exercise enhances insulin sensitivity. In the present study, we therefore examined visfatin mRNA expression...... in abdominal subcutaneous adipose tissue and skeletal muscle biopsies obtained from healthy young men at time points 0, 3, 4.5, 6, 9, and 24 h in relation to either 3 h of ergometer cycle exercise at 60% of Vo(2 max) or rest. Adipose tissue visfatin mRNA expression increased threefold at the time points 3, 4.......5, and 6 h in response to exercise (n = 8) compared with preexercise samples and compared with the resting control group (n = 7, P = 0.001). Visfatin mRNA expression in skeletal muscle was not influenced by exercise. The exercise-induced increase in adipose tissue visfatin was, however, not accompanied...

  2. How is mRNA expression predictive for protein expression? A correlation study on human circulating monocytes

    Institute of Scientific and Technical Information of China (English)

    Yanfang Guo; Yuan Chen; Hui Jiang; Lijun Tan; Jingyun Xie; Xuezhen Zhu; Songping Liang; Hongwen Deng; Peng Xiao; Shufeng Lei; Feiyan Deng; Gary Guishan Xiao; Yaozhong Liu; Xiangding Chen; Liming Li; Shan Wu

    2008-01-01

    A key assumption in studying mRNA expression is that it is informative in the prediction of protein expression. However,only limited studies have explored the mRNA-protein expression correlation in yeast or human tissues and the results have been relatively inconsistent. We carried out correlation analyses on mRNA-protein expressions in freshly isolated human circulating monocytes from 30 unrelated women. The expressed proteins for 71 genes were quantified and identified by 2-D electrophoresis coupled with mass spectrometry. The corresponding mRNA expressions were quantified by Affymetrix gene chips. Significant correlation (r=0.235, P<0.0001) was observed for the whole dataset including all studied genes and all samples. The correlations varied in different biological categories of gene ontology. For example, the highest correlation was achieved for genes of the extracellular region in terms of cellular component (r=0.643, P<0.0001) and the lowest correlation was obtained for genes of regulation (r=0.099, P=0.213) in terms of biological process. In the genome, half of the samples showed significant positive correlation for the 71 genes and significant correlation was found between the average mRNA and the average protein expression levels in all samples (r=0.296, P<0.01). However, at the study group level, only five studied genes had significant positive correlation across all the samples. Our results showed an overall positive correlation between mRNA and protein expression levels.However, the moderate and varied correlations suggest that mRNA expression might be sometimes useful, but certainly far from perfect, in predicting protein expression levels.

  3. Expression of cholinesterase gene(s) in human brain tissues: translational evidence for multiple mRNA species.

    Science.gov (United States)

    Soreq, H; Zevin-Sonkin, D; Razon, N

    1984-06-01

    To resolve the origin(s) of the molecular heterogeneity of human nervous system cholinesterases (ChEs), we used Xenopus oocytes, which produce biologically active ChE when microinjected with unfractionated brain mRNA. The RNA was prepared from primary gliomas, meningiomas and embryonic brain, each of which expresses ChE activity with distinct substrate specificities and molecular forms. Sucrose gradient fractionation of DMSO-denatured mRNA from these sources revealed three size classes of ChE-inducing mRNAs, sedimenting at approximately 32S, 20S and 9S. The amounts of these different classes of ChE-inducing mRNAs varied between the three tissue sources examined. To distinguish between ChEs produced in oocytes and having different substrate specificities, their activity was determined in the presence of selective inhibitors. Both 'true' (acetylcholine hydrolase, EC 3.1.1.7) and 'pseudo' (acylcholine acylhydrolase, EC 3.1.1.8) multimeric cholinesterase activities were found in the mRNA-injected oocytes. Moreover, human brain mRNAs inducing 'true' and 'pseudo' ChE activities had different size distribution, indicating that different mRNAs might be translated into various types of ChEs. These findings imply that the heterogeneity of ChEs in the human nervous system is not limited to the post-translational level, but extends to the level of mRNA.

  4. Caffeine Bitterness is Related to Daily Caffeine Intake and Bitter Receptor mRNA Abundance in Human Taste Tissue.

    Science.gov (United States)

    Lipchock, Sarah V; Spielman, Andrew I; Mennella, Julie A; Mansfield, Corrine J; Hwang, Liang-Dar; Douglas, Jennifer E; Reed, Danielle R

    2017-01-01

    We investigated whether the abundance of bitter receptor mRNA expression from human taste papillae is related to an individual's perceptual ratings of bitter intensity and habitual intake of bitter drinks. Ratings of the bitterness of caffeine and quinine and three other bitter stimuli (urea, propylthiouracil, and denatonium benzoate) were compared with relative taste papilla mRNA abundance of bitter receptors that respond to the corresponding bitter stimuli in cell-based assays ( TAS2R4, TAS2R10, TAS2R38, TAS2R43, and TAS2R46). We calculated caffeine and quinine intake from a food frequency questionnaire. The bitterness of caffeine was related to the abundance of the combined mRNA expression of these known receptors, r = 0.47, p = .05, and self-reported daily caffeine intake, t(18) = 2.78, p = .012. The results of linear modeling indicated that 47% of the variance among subjects in the rating of caffeine bitterness was accounted for by these two factors (habitual caffeine intake and taste receptor mRNA abundance). We observed no such relationships for quinine but consumption of its primary dietary form (tonic water) was uncommon. Overall, diet and TAS2R gene expression in taste papillae are related to individual differences in caffeine perception.

  5. MicroRNA-mediated target mRNA cleavage and 3'-uridylation in human cells.

    Science.gov (United States)

    Xu, Kai; Lin, Jing; Zandi, Roza; Roth, Jack A; Ji, Lin

    2016-07-21

    MicroRNAs (miRNAs) play an important role in targeted gene silencing by facilitating posttranscriptional and translational repression. However, the precise mechanism of mammalian miRNA-mediated gene silencing remains to be elucidated. Here, we used a stem-loop array reverse-transcription polymerase chain reaction assay to analyse miRNA-induced mRNA recognition, cleavage, posttranscriptional modification, and degradation. We detected endogenous let-7 miRNA-induced and Argonaute-catalysed endonucleolytic cleavage on target mRNAs at various sites within partially paired miRNA:mRNA sequences. Most of the cleaved mRNA 5'-fragments were 3'-oligouridylated by activities of terminal uridylyl transferases (TUTases) in miRNA-induced silencing complexes and temporarily accumulated in the cytosol for 5'-3' degradation or other molecular fates. Some 3'-5' decayed mRNA fragments could also be captured by the miRNA-induced silencing complex stationed at the specific miRNA:mRNA target site and oligouridylated by other TUTases at its proximity without involving Argonaute-mediated RNA cleavage. Our findings provide new insights into the molecular mechanics of mammalian miRNA-mediated gene silencing by coordinated target mRNA recognition, cleavage, uridylation and degradation.

  6. Ammonium Chloride Ingestion Attenuates Exercise-Induced mRNA Levels in Human Muscle.

    Directory of Open Access Journals (Sweden)

    Johann Edge

    Full Text Available Minimizing the decrease in intracellular pH during high-intensity exercise training promotes greater improvements in mitochondrial respiration. This raises the intriguing hypothesis that pH may affect the exercise-induced transcription of genes that regulate mitochondrial biogenesis. Eight males performed 10x2-min cycle intervals at 80% VO2speak intensity on two occasions separated by ~2 weeks. Participants ingested either ammonium chloride (ACID or calcium carbonate (PLA the day before and on the day of the exercise trial in a randomized, counterbalanced order, using a crossover design. Biopsies were taken from the vastus lateralis muscle before and after exercise. The mRNA level of peroxisome proliferator-activated receptor co-activator 1α (PGC-1α, citrate synthase, cytochome c and FOXO1 was elevated at rest following ACID (P0.05; the difference in PGC-1α mRNA content 2 h post-exercise between ACID and PLA was not significant (P = 0.08. Thus, metabolic acidosis abolished the early post-exercise increase of PGC-1α mRNA and the mRNA of downstream mitochondrial and glucose-regulating proteins. These findings indicate that metabolic acidosis may affect mitochondrial biogenesis, with divergent responses in resting and post-exercise skeletal muscle.

  7. Sex differences in global mRNA content of human skeletal muscle.

    Directory of Open Access Journals (Sweden)

    Amy C Maher

    Full Text Available Women oxidize more fat as compared to men during endurance exercise and several groups have shown that the mRNA content of selected genes related to fat oxidation are higher in women (e.g. hormone sensitive lipase, beta-hydroxyacyl-CoA dehydrogenase, CD36. One of the possible mechanisms is that women tend to have a higher area percentage of type I skeletal muscle fibers as compared with men. Consequently, we hypothesized that sex would influence the basal mRNA and protein content for genes involved in metabolism and the determination of muscle fiber type. Muscle biopsies from the vastus lateralis were collected from healthy men and women. We examined mRNA content globally using Affymetrix GeneChips, and selected genes were examined and/or confirmed by RT-PCR. Furthermore, we examined protein content by Western blot analysis. Stringent gene array analysis revealed 66 differentially expressed genes representing metabolism, mitochondrial function, transport, protein biosynthesis, cell proliferation, signal transduction pathways, transcription and translation. Stringent gene array analysis and RT-PCR confirmed that mRNA for; acyl-coenzyme A acyltransferase 2 (ACAA2, trifunctional protein beta (HADHB, catalase, lipoprotein lipase (LPL, and uncoupling protein-2 (UCP-2 were higher in women. Targeted gene analysis revealed that myosin heavy chain I (MHCI, peroxisome proliferator-activated receptor (PPARdelta were higher in women compared with men. Surprisingly, there were no significant sex based differences in protein content for HADHB, ACAA2, catalase, PPARdelta, and MHC1. In conclusion, the differences in the basal mRNA content in resting skeletal muscle suggest that men and women are transcriptionally "primed" for known physiological differences in metabolism however the mechanism behind sex differences in fiber type remains to be determined.

  8. mRNA expression levels in failing human hearts predict cellular electrophysiological remodeling: a population-based simulation study.

    Directory of Open Access Journals (Sweden)

    John Walmsley

    Full Text Available Differences in mRNA expression levels have been observed in failing versus non-failing human hearts for several membrane channel proteins and accessory subunits. These differences may play a causal role in electrophysiological changes observed in human heart failure and atrial fibrillation, such as action potential (AP prolongation, increased AP triangulation, decreased intracellular calcium transient (CaT magnitude and decreased CaT triangulation. Our goal is to investigate whether the information contained in mRNA measurements can be used to predict cardiac electrophysiological remodeling in heart failure using computational modeling. Using mRNA data recently obtained from failing and non-failing human hearts, we construct failing and non-failing cell populations incorporating natural variability and up/down regulation of channel conductivities. Six biomarkers are calculated for each cell in each population, at cycle lengths between 1500 ms and 300 ms. Regression analysis is performed to determine which ion channels drive biomarker variability in failing versus non-failing cardiomyocytes. Our models suggest that reported mRNA expression changes are consistent with AP prolongation, increased AP triangulation, increased CaT duration, decreased CaT triangulation and amplitude, and increased delay between AP and CaT upstrokes in the failing population. Regression analysis reveals that changes in AP biomarkers are driven primarily by reduction in I[Formula: see text], and changes in CaT biomarkers are driven predominantly by reduction in I(Kr and SERCA. In particular, the role of I(CaL is pacing rate dependent. Additionally, alternans developed at fast pacing rates for both failing and non-failing cardiomyocytes, but the underlying mechanisms are different in control and heart failure.

  9. Effect of gamma radiation on the expression of mRNA growth factors in glycerol cryopreserved human amniotic membrane.

    Science.gov (United States)

    Yatim, Rusidah Mat; Kannan, Thirumulu Ponnuraj; Ab Hamid, Suzina Sheikh

    2016-12-01

    Human amniotic membrane (HAM) due to its high biocompatibility, low immunogenicity, anti-microbial, anti-viral properties as well as the presence of growth factors has been used in various clinical applications. The growth factors play an important role in wound healing. The current study aimed to explore the effect of 15 kGy gamma radiation dose on selected growth factors and receptors mRNA present in HAM. Eight growth factors, namely, EGF, HGF, KGF, TGF-α, TGF-β1, TGF-β2, TGF-β3 and bFGF and two growth factor receptors, HGFR and KGFR were evaluated in this study. The total RNA was extracted and converted to complimentary DNA using commercial kits. Subsequently, the mRNA expressions of these growth factors were evaluated using real-time PCR and the results were statistically analyzed using REST-MCS software. This study confirmed the presence of these mRNA growth factors and receptors in fresh, glycerol cryopreserved and irradiated glycerol cryopreserved HAM. In glycerol cryopreserved HAM, the results showed up-regulation of HGF and bFGF and down-regulation of EGF, HGFR, KGF, KGFR, TGF-α, TGF-β1, TGF-β2 and TGF-β3 relative to the fresh HAM which acted as the control, whereas in irradiated glycerol cryopreserved HAM, the results showed up-regulation of EGF, HGF, KGF, KGFR, TGF-β1, TGF-β2 and TGF-β3 and down-regulation of HGFR, TGF-α and bFGF relative to the glycerol cryopreserved HAM which acted as the control. However, these mRNA expressions did not show any statistical significant difference compared to the control groups. This study concluded that a dose of 15 kGy of gamma radiation did not affect the mRNA expression for the growth factors' and receptors' in the glycerol cryopreserved HAM.

  10. Negative and positive mRNA splicing elements act competitively to regulate human immunodeficiency virus type 1 vif gene expression.

    Science.gov (United States)

    Exline, C M; Feng, Z; Stoltzfus, C M

    2008-04-01

    Over 40 different human immunodeficiency virus type 1 (HIV-1) mRNAs are produced by alternative splicing of the primary HIV-1 RNA transcripts. In addition, approximately half of the viral RNA remains unspliced and is used as genomic RNA and as mRNA for the Gag and Pol gene products. Regulation of splicing at the HIV-1 3' splice sites (3'ss) requires suboptimal polypyrimidine tracts, and positive or negative regulation occurs through the binding of cellular factors to cis-acting splicing regulatory elements. We have previously shown that splicing at HIV-1 3'ss A1, which produces single-spliced vif mRNA and promotes the inclusion of HIV exon 2 into both completely and incompletely spliced viral mRNAs, is increased by optimizing the 5' splice site (5'ss) downstream of exon 2 (5'ss D2). Here we show that the mutations within 5'ss D2 that are predicted to lower or increase the affinity of the 5'ss for U1 snRNP result in reduced or increased Vif expression, respectively. Splicing at 5'ss D2 was not necessary for the effect of 5'ss D2 on Vif expression. In addition, we have found that mutations of the GGGG motif proximal to the 5'ss D2 increase exon 2 inclusion and Vif expression. Finally, we report the presence of a novel exonic splicing enhancer (ESE) element within the 5'-proximal region of exon 2 that facilitates both exon inclusion and Vif expression. This ESE binds specifically to the cellular SR protein SRp75. Our results suggest that the 5'ss D2, the proximal GGGG silencer, and the ESE act competitively to determine the level of vif mRNA splicing and Vif expression. We propose that these positive and negative splicing elements act together to allow the accumulation of vif mRNA and unspliced HIV-1 mRNA, compatible with optimal virus replication.

  11. Viperin mRNA is a novel target for the human RNase MRP/RNase P endoribonuclease.

    Science.gov (United States)

    Mattijssen, Sandy; Hinson, Ella R; Onnekink, Carla; Hermanns, Pia; Zabel, Bernhard; Cresswell, Peter; Pruijn, Ger J M

    2011-07-01

    RNase MRP is a conserved endoribonuclease, in humans consisting of a 267-nucleotide RNA associated with 7-10 proteins. Mutations in its RNA component lead to several autosomal recessive skeletal dysplasias, including cartilage-hair hypoplasia (CHH). Because the known substrates of mammalian RNase MRP, pre-ribosomal RNA, and RNA involved in mitochondrial DNA replication are not likely involved in CHH, we analyzed the effects of RNase MRP (and the structurally related RNase P) depletion on mRNAs using DNA microarrays. We confirmed the upregulation of the interferon-inducible viperin mRNA by RNAi experiments and this appeared to be independent of the interferon response. We detected two cleavage sites for RNase MRP/RNase P in the coding sequence of viperin mRNA. This is the first study providing direct evidence for the cleavage of a mRNA by RNase MRP/RNase P in human cells. Implications for the involvement in the pathophysiology of CHH are discussed.

  12. Enzymatically stable 5' mRNA cap analogs: synthesis and binding studies with human DcpS decapping enzyme.

    Science.gov (United States)

    Kalek, Marcin; Jemielity, Jacek; Darzynkiewicz, Zbigniew M; Bojarska, Elzbieta; Stepinski, Janusz; Stolarski, Ryszard; Davis, Richard E; Darzynkiewicz, Edward

    2006-05-01

    Four novel 5' mRNA cap analogs have been synthesized with one of the pyrophosphate bridge oxygen atoms of the triphosphate linkage replaced with a methylene group. The analogs were prepared via reaction of nucleoside phosphor/phosphon-1-imidazolidates with nucleoside phosphate/phosphonate in the presence of ZnCl2. Three of the new cap analogs are completely resistant to degradation by human DcpS, the enzyme responsible for hydrolysis of free cap resulting from 3' to 5' cellular mRNA decay. One of the new analogs has very high affinity for binding to human DcpS. Two of these analogs are Anti Reverse Cap Analogs which ensures that they are incorporated into mRNA chains exclusively in the correct orientation. These new cap analogs should be useful in a variety of biochemical studies, in the analysis of the cellular function of decapping enzymes, and as a basis for further development of modified cap analogs as potential anti-cancer and anti-parasite drugs.

  13. High efficiency adenovirus-mediated expression of truncated N-terminal huntingtin fragment (htt552) in primary rat astrocytes

    Institute of Scientific and Technical Information of China (English)

    Linhui Wang; Fang Lin; Junchao Wu; Zhenghong Qin

    2009-01-01

    Huntington's disease (HD) is caused by an expansion of polyglutamine tract in N-terminus of huntingtin (htt).The mutation of htt leads to dysfunction and premature death of striatal and cortical neurons. However, the effects of htt mutation on glia remain largely unknown.This study aimed to establish a glia HD model using an adenoviral vector to express wild-type and mutant N-terminal huntingtin fragment 1-552 amino acids (htt552) in rat primary cortical astrocytes. We have eval-uated optimal conditions for the infection of astrocytes with adenovirai vectors, and the kinetics of the expression of htt552 in astrocytes. The majority of astroeytes expressed the transgene after infection. At 24 h post-infection, the highest rate of infection was 89 + 3% for the wild-type (htt552-18Q) with a multiplicity of infection (m.o.i.) of 80, and the highest rate of infection was 91 +4% for the mutant type (htt552-100Q) with the same viral dose. The duration of expression of htt552 lasted for about 7 days with a relatively high level from 1 to 4 days post-infection. Mutant huntingtin (htt552-100Q) pro-duced the characteristic HD pathology after 3 days by the appearance of cytoplasmic aggregates and intranue-lear inclusions. The result of MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliu mbromide)assay showed that the inhibition of viability by virus on astrocytes was also dose-dependent. To obtain high infection rate and low toxicity, the viral dose with an m.o.i, of 40 was optimal to our cell model. The present study demonstrates that adenovirai-mediated expression of mutant htt provides an advantageous system for his-tological and biochemical analysis of HD pathogenesis in primary cortical astrocyte cultures.

  14. Living in a dangerous world: the shaping of behavioral profile by early environment and 5-HTT genotype

    Directory of Open Access Journals (Sweden)

    Rebecca S Heiming

    2009-09-01

    Full Text Available Anxiety and anxiety disorders are influenced by both, environmental and genetic factors. One genetic factor under scrutiny for anxiety disorders is the genetically encoded variation of the serotonin transporter (5-HTT. The aim of this study was to elucidate the effects of a threatening environment during early phases of life on anxiety-like (ANX and exploratory behavior (EXP in adult mice, varying in serotonin transporter (5-HTT genotype. For this purpose, pregnant and lactating 5-HTT +/- dams were repeatedly exposed to olfactory cues of unfamiliar adult males by introducing small amounts of soiled bedding to their home cage. These stimuli signal the danger of infanticide and simulate a threatening environment. Control females were treated with neutral bedding. The offspring (5-HTT +/+, +/-, -/- were examined for their ANX and EXP. The main results were: (1 a main effect of genotype existed, with 5-HTT -/- showing higher levels of ANX and lower levels of EXP than 5-HTT +/- and wildtypes. (2 When mothers had lived in a threatening environment, their offspring showed increased ANX and reduced EXP compared to controls. (3 These effects were most pronounced in 5-HTT -/- mice. By applying a new ecologically relevant paradigm we conclude: If 5-HTT +/- mothers live in a threatening environment during pregnancy and lactation, their offspring behavioral profile will, in principle, be shaped in an adaptive way preparing the young for an adverse environment. This process is, however, modulated by 5-HTT genotype, bearing the risk that individuals with impaired serotonergic neurotransmission (5-HTT -/- will develop an exaggerated, potentially pathological level of anxiety from gene x environment interactions.

  15. Effect of acute resistance exercise and sex on human patellar tendon structural and regulatory mRNA expression

    DEFF Research Database (Denmark)

    Sullivan, B.E.; Carroll, C.C.; Jemiolo, B.;

    2009-01-01

    (6 men and 6 women). Collagen type I, collagen type III, and MMP-2 were downregulated (P 0.05) 24 h after RE. All other genes remained unchanged (P > 0.05) after RE. Women had higher resting mRNA expression (P ... = 0.08) toward lower resting expression of MMP-3 than men. All other genes were not influenced (P > 0.05) by sex. Acute RE appears to stimulate a change in collagen type I, collagen type III, and MMP-2 gene regulation in the human patellar tendon. Sex influences the structural and regulatory m...... and mechanical properties, it is uncertain what structural and regulatory components contribute to these responses. We measured the mRNA expression of tendon's main fibrillar collagens (type I and type III) and the main proteoglycans (decorin, biglycan, fibromodulin, and versican) and the regulatory enzymes MMP...

  16. Expression of LL-37, Human beta Defensin-2, and CCR6 mRNA in Patients with Psoriasis Vulgaris

    Institute of Scientific and Technical Information of China (English)

    李东升; 李家文; 段逸群; 周小勇

    2004-01-01

    To investigate whether LL-37 and human beta defensin-2 (hBD-2) is related to the patients with psoriasis seldom having skin infections and explore the role of the two peptides and CCR6 (the receptor of hBD-2) in the pathogenesis of psoriasis, the expression levels of mRNA of LL-37, hBD-2, and CCR6 in skin lesions of patients with psoriasis vulgaris were detected by using RT-PCR. The results showed that the mRNA expression levels of the two peptides and CCR6 in psoriatic lesions all increased compared with the normal skin (P<0. 001). It was suggested that upregulated expression of LL-37 and hBD-2 might be the main reason that result in the the skin of patients with psoriasis being seldom infected, and the two peptides and CCR6 might play crucial roles in the pathogenesis of psoriasis.

  17. Distinctive properties of the 5'-untranslated region of human hsp70 mRNA.

    Science.gov (United States)

    Rubtsova, Maria P; Sizova, Daria V; Dmitriev, Sergei E; Ivanov, Dmitri S; Prassolov, Vladimir S; Shatsky, Ivan N

    2003-06-20

    A relaxed cap-dependence of translation of the mRNA-encoding mammalian heat shock protein Hsp70 may suggest that its 5'-untranslated region (UTR) possesses an internal ribosome entry site (IRES). In this study, this possibility has been tested in transfected cells using plasmids that express dicistronic mRNAs. Using a reporter gene construct, Renilla luciferase/Photinus pyralis luciferase, we show that the 216-nt long 5'-UTR of Hsp70 mRNA acts as an IRES that directs ribosomes to the downstream start codon by a cap-independent mechanism. The relative activity of this IRES (100-fold over the empty vector) is similar to that of the classical picornaviral IRESs. Additional controls indicate that this high expression of the downstream reporter is not due to readthrough from the upstream cistron, nor is it due to translation of cryptic monocistronic transcripts. The effect of small deletions within the 5'-UTR of Hsp70 mRNA on the IRES activity varies in dependence on their position within the 5'-UTR sequence. With the exception of deletion of nt 33-50, it is small for the 5'-terminal half of the 5'-UTR and rather strong for the 3'-terminal section. However, neither of these small deletions abolishes the IRES activity completely. Excision of larger sections (>50 nt) by truncation of the 5'-UTR from the 5'-end or by internal deleting results in a dramatic impairment of the IRES function. Taken together, these data suggest that the IRES activity of the 5'-UTR of Hsp70 mRNA requires integrity of almost the entire sequence of the 5'-UTR. The data are discussed in terms of a model that allows a three-dimensional rather than linear mode of selection of the initiation region surrounding the start codon of Hsp70 mRNA.

  18. Induction of mRNA expression of osteogenesis-related genes by guaiacol in human dental pulp cells.

    Science.gov (United States)

    Kato, Takashi; Shirayama, Kumiko; Tsutsui, Takeo W; Tsutsui, Takeki

    2010-07-01

    To investigate the stimulating effect of endodontic medications on the mRNA expression of some osteogenesis-related genes associated with reparative dentinogenesis and hard-tissue formation, human dental pulp cells (D824 cells) were treated with calcium hydroxide (Ca (OH)(2)), formocresol, or guaiacol. The effect on growth was determined by growth curves of D824 cells treated for 1-3 days with 0.03-0.3 mM Ca (OH)(2), 0.0007%-0.0014% formocresol, or 0.24-2.43 mM guaiacol. The mitotic activity of individual cells and the mRNA expression of the osteogenesis-related genes for alkaline phosphatase (ALP), type I collagen (COL-1), and bone sialoprotein (BSP) in the cells treated for 24 h with the same concentrations of the medications as described above were determined by colony-forming efficiency and by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis, respectively. Cellular growth and mitotic activity were scarcely affected by Ca (OH)(2), but were significantly reduced by formocresol or guaiacol. The mRNA expression of the osteogenesis-related genes was little affected by Ca (OH)(2) or formocresol, but was significantly enhanced by guaiacol. The results indicate that guaiacol may stimulate the mRNA expression of genes associated with reparative dentinogenesis and hard-tissue formation in human dental pulp cells, suggesting that the novel property of guaiacol provides new insights into the utilization of guaiacol in endodontic therapy.

  19. Integrated Analysis of Dysregulated ncRNA and mRNA Expression Profiles in Humans Exposed to Carbon Nanotubes.

    Directory of Open Access Journals (Sweden)

    Anna A Shvedova

    Full Text Available As the application of carbon nanotubes (CNT in consumer products continues to rise, studies have expanded to determine the associated risks of exposure on human and environmental health. In particular, several lines of evidence indicate that exposure to multi-walled carbon nanotubes (MWCNT could pose a carcinogenic risk similar to asbestos fibers. However, to date the potential markers of MWCNT exposure are not yet explored in humans.In the present study, global mRNA and ncRNA expression profiles in the blood of exposed workers, having direct contact with MWCNT aerosol for at least 6 months (n = 8, were compared with expression profiles of non-exposed (n = 7 workers (e.g., professional and/or technical staff from the same manufacturing facility.Significant changes in the ncRNA and mRNA expression profiles were observed between exposed and non-exposed worker groups. An integrative analysis of ncRNA-mRNA correlations was performed to identify target genes, functional relationships, and regulatory networks in MWCNT-exposed workers. The coordinated changes in ncRNA and mRNA expression profiles revealed a set of miRNAs and their target genes with roles in cell cycle regulation/progression/control, apoptosis and proliferation. Further, the identified pathways and signaling networks also revealed MWCNT potential to trigger pulmonary and cardiovascular effects as well as carcinogenic outcomes in humans, similar to those previously described in rodents exposed to MWCNTs.This study is the first to investigate aberrant changes in mRNA and ncRNA expression profiles in the blood of humans exposed to MWCNT. The significant changes in several miRNAs and mRNAs expression as well as their regulatory networks are important for getting molecular insights into the MWCNT-induced toxicity and pathogenesis in humans. Further large-scale prospective studies are necessary to validate the potential applicability of such changes in mRNAs and miRNAs as prognostic markers

  20. cDNA cloning of human myeloperoxidase: decrease in myeloperoxidase mRNA upon induction of HL-60 cells

    Energy Technology Data Exchange (ETDEWEB)

    Weil, S.C.; Rosner, G.L.; Reid, M.S.; Chisholm, R.L.; Farber, N.M.; Spitznagel, J.K.; Swanson, M.S.

    1987-04-01

    Myeloperoxidase (MPO), the most abundant neutrophil protein, is a bacteriocidal component of the primary granules and a critical marker in distinguishing acute myelogenous leukemia from acute lymphoid leukemia. A cDNA clone for human MPO was isolated by immunologic screening of human hematopoietic lambdagt11 expression vector libraries with specific anti-MPO antibody. The identity of the cDNA clone was confirmed by finding that (i) epitope-selected antibody against this clone recognizes purified MPO and MPO in human promyelocytic (HL-60) cell lysates by immunoblot analysis, and that (ii) hybrid section of HL-60 mRNA with this cDNA clone and translation in vitro results in the synthesis of an 80-kDa protein recognized by the anti-MPO antiserum. RNA blot analysis with this MPO cDNA clone detects hybridization to two polyadenylylated transcripts of approx. = 3.6 and approx. = 2.9 kilobases in HL-60 cells. No hybridization is detected to human placenta mRNA. Upon induction of HL-60 cells to differentiate by incubation for 4 days with dimethyl sulfoxide, a drastic decrease in the hybridization intensity of these two bands is seen. This is consistent with previous data suggesting a decrease in MPO synthesis upon such induction of these cells. The MPO cDNA should be useful for further molecular and genetic characterization of MPO and its expression and biosynthesis in normal and leukemic granulocytic differentiation.

  1. Induction of cyclo-oxygenase-2 mRNA by prostaglandin E2 in human prostatic carcinoma cells

    Science.gov (United States)

    Tjandrawinata, R. R.; Dahiya, R.; Hughes-Fulford, M.

    1997-01-01

    Prostaglandins are synthesized from arachidonic acid by the enzyme cyclo-oxygenase. There are two isoforms of cyclooxygenases: COX-1 (a constitutive form) and COX-2 (an inducible form). COX-2 has recently been categorized as an immediate-early gene and is associated with cellular growth and differentiation. The purpose of this study was to investigate the effects of exogenous dimethylprostaglandin E2 (dmPGE2) on prostate cancer cell growth. Results of these experiments demonstrate that administration of dmPGE2 to growing PC-3 cells significantly increased cellular proliferation (as measured by the cell number), total DNA content and endogenous PGE2 concentration. DmPGE2 also increased the steady-state mRNA levels of its own inducible synthesizing enzyme, COX-2, as well as cellular growth to levels similar to those seen with fetal calf serum and phorbol ester. The same results were observed in other human cancer cell types, such as the androgen-dependent LNCaP cells, breast cancer MDA-MB-134 cells and human colorectal carcinoma DiFi cells. In PC-3 cells, the dmPGE2 regulation of the COX-2 mRNA levels was both time dependent, with maximum stimulation seen 2 h after addition, and dose dependent on dmPGE2 concentration, with maximum stimulation seen at 5 microg ml(-1). The non-steroidal anti-inflammatory drug flurbiprofen (5 microM), in the presence of exogenous dmPGE2, inhibited the up-regulation of COX-2 mRNA and PC-3 cell growth. Taken together, these data suggest that PGE2 has a specific role in the maintenance of human cancer cell growth and that the activation of COX-2 expression depends primarily upon newly synthesized PGE2, perhaps resulting from changes in local cellular PGE2 concentrations.

  2. Induction of cyclo-oxygenase-2 mRNA by prostaglandin E2 in human prostatic carcinoma cells

    Science.gov (United States)

    Tjandrawinata, R. R.; Dahiya, R.; Hughes-Fulford, M.

    1997-01-01

    Prostaglandins are synthesized from arachidonic acid by the enzyme cyclo-oxygenase. There are two isoforms of cyclooxygenases: COX-1 (a constitutive form) and COX-2 (an inducible form). COX-2 has recently been categorized as an immediate-early gene and is associated with cellular growth and differentiation. The purpose of this study was to investigate the effects of exogenous dimethylprostaglandin E2 (dmPGE2) on prostate cancer cell growth. Results of these experiments demonstrate that administration of dmPGE2 to growing PC-3 cells significantly increased cellular proliferation (as measured by the cell number), total DNA content and endogenous PGE2 concentration. DmPGE2 also increased the steady-state mRNA levels of its own inducible synthesizing enzyme, COX-2, as well as cellular growth to levels similar to those seen with fetal calf serum and phorbol ester. The same results were observed in other human cancer cell types, such as the androgen-dependent LNCaP cells, breast cancer MDA-MB-134 cells and human colorectal carcinoma DiFi cells. In PC-3 cells, the dmPGE2 regulation of the COX-2 mRNA levels was both time dependent, with maximum stimulation seen 2 h after addition, and dose dependent on dmPGE2 concentration, with maximum stimulation seen at 5 microg ml(-1). The non-steroidal anti-inflammatory drug flurbiprofen (5 microM), in the presence of exogenous dmPGE2, inhibited the up-regulation of COX-2 mRNA and PC-3 cell growth. Taken together, these data suggest that PGE2 has a specific role in the maintenance of human cancer cell growth and that the activation of COX-2 expression depends primarily upon newly synthesized PGE2, perhaps resulting from changes in local cellular PGE2 concentrations.

  3. CCR4-NOT Deadenylates mRNA Associated with RNA-Induced Silencing Complexes in Human Cells▿

    Science.gov (United States)

    Piao, Xianghua; Zhang, Xue; Wu, Ligang; Belasco, Joel G.

    2010-01-01

    MicroRNAs (miRNAs) repress gene expression posttranscriptionally by inhibiting translation and by expediting deadenylation so as to trigger rapid mRNA decay. Their regulatory influence is mediated by the protein components of the RNA-induced silencing complex (RISC), which deliver miRNAs and siRNAs to their mRNA targets. Here, we present evidence that CCR4-NOT is the deadenylase that removes poly(A) from messages destabilized by miRNAs in human cells. Overproducing a mutationally inactivated form of either of the catalytic subunits of this deadenylase (CCR4 or CAF1/POP2) significantly impedes the deadenylation and decay of mRNA targeted by a partially complementary miRNA. The same deadenylase initiates the degradation of “off-target” mRNAs that are bound by an imperfectly complementary siRNA introduced by transfection. The greater inhibitory effect of inactive CAF1 or POP2 (versus inactive CCR4) suggests a predominant role for this catalytic subunit of CCR4-NOT in miRNA- or small interfering RNA (siRNA)-mediated deadenylation. These effects of mi/siRNAs and CCR4-NOT can be fully reproduced by directly tethering RISC to mRNA without the guidance of a small RNA, indicating that the ability of RISC to accelerate deadenylation is independent of RNA base pairing. Despite its importance for mi/siRNA-mediated deadenylation, CCR4-NOT appears not to associate significantly with RISC, as judged by the failure of CAF1 and POP2 to coimmunoprecipitate detectably with either the Ago or TNRC6 subunit of RISC, a finding at odds with deadenylase recruitment as the mechanism by which RISC accelerates poly(A) removal. PMID:20065043

  4. Cytochrome c oxidase subunit 1-based human RNA quantification to enhance mRNA profiling in forensic biology

    Directory of Open Access Journals (Sweden)

    Dong Zhao

    2017-01-01

    Full Text Available RNA analysis offers many potential applications in forensic science, and molecular identification of body fluids by analysis of cell-specific RNA markers represents a new technique for use in forensic cases. However, due to the nature of forensic materials that often admixed with nonhuman cellular components, human-specific RNA quantification is required for the forensic RNA assays. Quantification assay for human RNA has been developed in the present study with respect to body fluid samples in forensic biology. The quantitative assay is based on real-time reverse transcription-polymerase chain reaction of mitochondrial RNA cytochrome c oxidase subunit I and capable of RNA quantification with high reproducibility and a wide dynamic range. The human RNA quantification improves the quality of mRNA profiling in the identification of body fluids of saliva and semen because the quantification assay can exclude the influence of nonhuman components and reduce the adverse affection from degraded RNA fragments.

  5. Epigenetic Regulation of Dopamine Transporter mRNA Expression in Human Neuroblastoma Cells

    Science.gov (United States)

    Green, Ashley L.; Hossain, Muhammad M.; Tee, Siew C.; Zarbl, Helmut; Guo, Grace L.; Richardson, Jason R.

    2016-01-01

    The dopamine transporter (DAT) is a key regulator of dopaminergic neurotransmission. As such, proper regulation of DAT expression is important to maintain homeostasis, and disruption of DAT expression can lead to neurobehavioral dysfunction. Based on genomic features within the promoter of the DAT gene, there is potential for DAT expression to be regulated through epigenetic mechanisms, including DNA methylation and histone acetylation. However, the relative contribution of these mechanisms to DAT expression has not been empirically determined. Using pharmacologic and genetic approaches, we demonstrate that inhibition of DNA methyltransferase (DNMT) activity increased DAT mRNA approximately 1.5–2 fold. This effect was confirmed by siRNA knockdown of DNMT1. Likewise, the histone deacetylase (HDAC) inhibitors valproate and butyrate also increased DAT mRNA expression, but the response was much more robust with expression increasing over tenfold. Genetic knockdown of HDAC1 by siRNA also increased DAT expression, but not to the extent seen with pharmacological inhibition, suggesting additional isoforms of HDAC or other targets may contribute to the observed effect. Together, these data identify the relative contribution of DNMTs and HDACs in regulating expression. These finding may aid in understanding the mechanistic basis for changes in DAT expression in normal and pathophysiological states. PMID:25963949

  6. Regulation of CD163 mRNA and soluble CD163 protein in human adipose tissue in vitro.

    Science.gov (United States)

    Fjeldborg, Karen; Møller, Holger J; Richelsen, Bjørn; Pedersen, Steen B

    2014-10-01

    CD163-positive macrophages are highly expressed in the human adipose tissue (AT) particularly from obese individuals. However, little is known about the regulation of CD163 mRNA and the protein level of sCD163 in human AT. We aimed to examine the regulation of CD163 and sCD163 in AT. Human s.c. AT samples (n=5) were stimulated with dexamethasone (DEX; 200  nmol/l), lipopolysaccharide (LPS; 100  ng/ml), or DEX+LPS for various time periods up to 24  h. Gene expressions of CD163, ADAM17, IL10, and TNFA (TNF) were measured by RT-PCR. Protein levels of sCD163, IL10, and TNFα (TNF) were measured by ELISA. Furthermore, AT was separated into stromal and adipocyte fraction. We found that CD163 mRNA was strongly expressed in the stromal vascular fraction but hardly detectable in the isolated adipocytes. Incubating whole AT with DEX significantly up-regulated CD163 (PCD163 (P>0.05). By contrast, the protein level of sCD163 was not affected by DEX (P>0.05), but LPS significantly increased the level of sCD163 and TNFα (PCD163 and the release of sCD163 are differentially regulated in human AT. Moreover, similar to studies on differentiated blood monocytes, TNFα and sCD163 are concomitantly released in human AT by LPS, which also up-regulate ADAM17. © 2014 Society for Endocrinology.

  7. Negative and Positive mRNA Splicing Elements Act Competitively To Regulate Human Immunodeficiency Virus Type 1 Vif Gene Expression▿

    Science.gov (United States)

    Exline, C. M.; Feng, Z.; Stoltzfus, C. M.

    2008-01-01

    Over 40 different human immunodeficiency virus type 1 (HIV-1) mRNAs are produced by alternative splicing of the primary HIV-1 RNA transcripts. In addition, approximately half of the viral RNA remains unspliced and is used as genomic RNA and as mRNA for the Gag and Pol gene products. Regulation of splicing at the HIV-1 3′ splice sites (3′ss) requires suboptimal polypyrimidine tracts, and positive or negative regulation occurs through the binding of cellular factors to cis-acting splicing regulatory elements. We have previously shown that splicing at HIV-1 3′ss A1, which produces single-spliced vif mRNA and promotes the inclusion of HIV exon 2 into both completely and incompletely spliced viral mRNAs, is increased by optimizing the 5′ splice site (5′ss) downstream of exon 2 (5′ss D2). Here we show that the mutations within 5′ss D2 that are predicted to lower or increase the affinity of the 5′ss for U1 snRNP result in reduced or increased Vif expression, respectively. Splicing at 5′ss D2 was not necessary for the effect of 5′ss D2 on Vif expression. In addition, we have found that mutations of the GGGG motif proximal to the 5′ss D2 increase exon 2 inclusion and Vif expression. Finally, we report the presence of a novel exonic splicing enhancer (ESE) element within the 5′-proximal region of exon 2 that facilitates both exon inclusion and Vif expression. This ESE binds specifically to the cellular SR protein SRp75. Our results suggest that the 5′ss D2, the proximal GGGG silencer, and the ESE act competitively to determine the level of vif mRNA splicing and Vif expression. We propose that these positive and negative splicing elements act together to allow the accumulation of vif mRNA and unspliced HIV-1 mRNA, compatible with optimal virus replication. PMID:18272582

  8. Human intronless genes: Functional groups, associated diseases, evolution, and mRNA processing in absence of splicing

    Energy Technology Data Exchange (ETDEWEB)

    Grzybowska, Ewa A., E-mail: ewag@coi.waw.pl [Cancer Center Institute, Roentgena 5, 02-781 Warsaw (Poland)

    2012-07-20

    Highlights: Black-Right-Pointing-Pointer Functional characteristics of intronless genes (IGs). Black-Right-Pointing-Pointer Diseases associated with IGs. Black-Right-Pointing-Pointer Origin and evolution of IGs. Black-Right-Pointing-Pointer mRNA processing without splicing. -- Abstract: Intronless genes (IGs) constitute approximately 3% of the human genome. Human IGs are essentially different in evolution and functionality from the IGs of unicellular eukaryotes, which represent the majority in their genomes. Functional analysis of IGs has revealed a massive over-representation of signal transduction genes and genes encoding regulatory proteins important for growth, proliferation, and development. IGs also often display tissue-specific expression, usually in the nervous system and testis. These characteristics translate into IG-associated diseases, mainly neuropathies, developmental disorders, and cancer. IGs represent recent additions to the genome, created mostly by retroposition of processed mRNAs with retained functionality. Processing, nuclear export, and translation of these mRNAs should be hampered dramatically by the lack of splice factors, which normally tightly cover mature transcripts and govern their fate. However, natural IGs manage to maintain satisfactory expression levels. Different mechanisms by which IGs solve the problem of mRNA processing and nuclear export are discussed here, along with their possible impact on reporter studies.

  9. The personality trait openness is related to cerebral 5-HTT levels

    DEFF Research Database (Denmark)

    Kalbitzer, Jan; Frøkjær, Vibe G.; Erritzøe, David

    2009-01-01

    Potentiation of serotonergic transmission increases cognitive flexibility, but can in other circumstances increase sensitivity to stressful environmental cues. The personality trait Openness to Experience reflects and is also associated with an increased risk for mood disorders. We hypothesized...... that the personality trait has an association with a biomarker of serotonergic transmission, the plasma membrane serotonin transporter (5-HTT). In 50 healthy volunteers, we tested for correlations between scores on the NEO-PI-R scale Openness to Experience and its subscales, and cerebral binding of the 5-HTT selective...... and Openness to Values. The latter subscore was negatively correlated with [11C]DASB binding in all brain regions in which [11C]DASB binding was quantified. Genetic analysis showed that homozygote LA carriers had significantly higher [11C]DASB binding in the caudate nucleus, but no significant differences...

  10. HTT-lowering reverses Huntington’s disease immune dysfunction caused by NFκB pathway dysregulation

    Science.gov (United States)

    Träger, Ulrike; Andre, Ralph; Lahiri, Nayana; Magnusson-Lind, Anna; Weiss, Andreas; Grueninger, Stephan; McKinnon, Chris; Sirinathsinghji, Eva; Kahlon, Shira; Pfister, Edith L.; Moser, Roger; Hummerich, Holger; Antoniou, Michael; Bates, Gillian P.; Luthi-Carter, Ruth; Lowdell, Mark W.; Björkqvist, Maria; Ostroff, Gary R.; Aronin, Neil

    2014-01-01

    Huntington’s disease is an inherited neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. The peripheral innate immune system contributes to Huntington’s disease pathogenesis and has been targeted successfully to modulate disease progression, but mechanistic understanding relating this to mutant huntingtin expression in immune cells has been lacking. Here we demonstrate that human Huntington’s disease myeloid cells produce excessive inflammatory cytokines as a result of the cell-intrinsic effects of mutant huntingtin expression. A direct effect of mutant huntingtin on the NFκB pathway, whereby it interacts with IKKγ, leads to increased degradation of IκB and subsequent nuclear translocation of RelA. Transcriptional alterations in intracellular immune signalling pathways are also observed. Using a novel method of small interfering RNA delivery to lower huntingtin expression, we show reversal of disease-associated alterations in cellular function–the first time this has been demonstrated in primary human cells. Glucan-encapsulated small interfering RNA particles were used to lower huntingtin levels in human Huntington’s disease monocytes/macrophages, resulting in a reversal of huntingtin-induced elevated cytokine production and transcriptional changes. These findings improve our understanding of the role of innate immunity in neurodegeneration, introduce glucan-encapsulated small interfering RNA particles as tool for studying cellular pathogenesis ex vivo in human cells and raise the prospect of immune cell-directed HTT-lowering as a therapeutic in Huntington’s disease. PMID:24459107

  11. 5HTT is associated with the phenotype psychological flexibility: results from a randomized clinical trial.

    Science.gov (United States)

    Gloster, Andrew T; Gerlach, Alexander L; Hamm, Alfons; Höfler, Michael; Alpers, Georg W; Kircher, Tilo; Ströhle, Andreas; Lang, Thomas; Wittchen, Hans-Ulrich; Deckert, Jürgen; Reif, Andreas

    2015-08-01

    Adaption to changing environments is evolutionarily advantageous. Studies that link genetic and phenotypic expression of flexible adjustment to one's context are largely lacking. In this study, we tested the importance of psychological flexibility, or goal-related context sensitivity, in an interaction between psychotherapy outcome for panic disorder with agoraphobia (PD/AG) and a genetic polymorphism. Given the established role of the 5HTT-LPR polymorphism in behavioral flexibility, we tested whether this polymorphism (short group vs. long group) impacted therapy response as a function of various endophenotypes (i.e., psychological flexibility, panic, agoraphobic avoidance, and anxiety sensitivity). Patients with PD/AG were recruited from a large multicenter randomized controlled clinical trial on cognitive-behavioral therapy. Pre- to post-treatment changes by 5HTT polymorphism were analyzed. 5HTT polymorphism status differentiated pre- to post-treatment changes in the endophenotype psychological flexibility (effect size difference d = 0.4, p < 0.05), but none of the specific symptom-related endophenotypes consistently for both the intent-to-treat sample (n = 228) and the treatment completers (n = 194). Based on the consistency of these findings with existing theory on behavioral flexibility, the specificity of the results across phenotypes, and the consistency of results across analyses (i.e., completer and intent to treat), we conclude that 5HTT polymorphism and the endophenotype psychological flexibility are important variables for the treatment of PD/AG. The endophenotype psychological flexibility may help bridge genetic and psychological literatures. Despite the limitation of the post hoc nature of these analyses, further study is clearly warranted.

  12. Prevalence of Human Papillomavirus Infection in Unselected SurePath Samples Using the APTIMA HPV mRNA Assay

    DEFF Research Database (Denmark)

    Rebolj, Matejka; Preisler, Sarah; Ejegod, Ditte M;

    2013-01-01

    The APTIMA Human Papillomavirus (HPV) Assay detects E6/E7 mRNA from 14 human papillomavirus genotypes. Horizon was a population-based split-sample study among well-screened women, with an aim to compare APTIMA, Hybrid Capture 2 (HC2), and liquid-based cytology (LBC) using SurePath samples. APTIMA...... agreement between APTIMA and HC2. This is the first APTIMA study using SurePath samples on the PANTHER platform. The trends in positivity rates on SurePath samples for APTIMA, HC2, and LBC were consistent with studies based on PreservCyt samples, and the agreement between the two HPV assays was substantial....... The high proportions of women testing positive suggest that in countries with a high HPV prevalence, caution will be needed if HPV tests, including mRNA-based tests, are to replace LBC....

  13. Three new alternative splicing variants of human cytochrome P450 2D6 mRNA in human extratumoral liver tissue

    Institute of Scientific and Technical Information of China (English)

    Jian Zhuge; Ying-Nian Yu

    2004-01-01

    AIM: To identify the new alternative splicing variants of human CYP2D6 in human extratumoral liver tissue with RT-PCR and sequencing.METHODS: Full length of human CYP2D6 cDNAs was amplificated by reverse transcription-polymerase chain reaction (RT-PCR) from a human extratumoral liver tissue and cloned into pGEM-T vector. The cDNA was sequenced.Exons from 1 to 4 of human CYP2D6 cDNAs were also amplificated by RT-PCR from extratumoral liver tissues of17 human hepatocellular carcinomas. Some RT-PCR products were sequenced. Exons 1 to 4 of CYP2D6 gene were amplified by PCR from extratumoral liver tissue DNA.Two PCR products from extratumoral liver tissues expressing skipped mRNA were partially sequenced.RESULTS: One of the CYP2D6cDNAs had 470 nucleotides from 79 to 548 (3' portion of exons 1 to 5' portion of exon 4),and was skipped. Exons 1 to 4 of CYP2D6 cDNA were assayed with RT-PCR in 17 extratumoral liver tissues. Both wild type and skipped mRNAs were expressed in 4 samples,only wild type mRNA was expressed in 5 samples, and only skipped mRNA was expressed in 8 samples. Two more variants were identified by sequencing the RT-PCR products of exons 1 to 4 of CYP2D6cDNA. The second variant skipped 411 nucleotides from 175 to 585. This variant was identified in 4 different liver tissues by sequencing the RT-PCR products. We sequenced partially 2 of the PCR products amplified of CYP2D6 exon 1 to exon 4 from extratumoral liver tissue genomic DNA that only expressed skipped mRNA by RT-PCR. No point mutations around exon 1, intron 1, and exon 4, and no deletion in CYP2D6gene were detected. The third variant was the skipped exon 3, and 153 bp was lost.CONCLUSION: Three new alternative splicing variants of CYP2D6 mRNA have been identified. They may not be caused by gene mutation and may lose CYP2D6 activity and act as a down-regulator of CYP2D6.

  14. Human 8-oxoguanine DNA glycosylase 1 mRNA expression as an oxidative stress exposure biomarker of cooking oil fumes.

    Science.gov (United States)

    Cherng, Shur-Hueih; Huang, Kuo Hao; Yang, Sen-Chih; Wu, Tzu-Chin; Yang, Jia-Ling; Lee, Huei

    2002-02-01

    Epidemiological studies have indicated that the exposure to carcinogenic components formed during the cooking of food might be associated with lung cancer risk of Chinese women. Previous studies have confirmed that cooking oil fumes from frying fish (COF) contained relatively high amount of benzo[a]pyrene, 2-methyl-3,8-dimethylimidazo[4,5-f] qunoxaline, benzene, and 1,3-butadiene, reported in fumes from heated soybean oil. Thus, we consider that oxidative stress induced by COF may play a role in lung cancer development among Chinese women. To verify whether the oxidative DNA damage was induced by COF, high-performance liquid chromatography (HPLC) analysis data showed that the levels of 8-hydroxydeoxyguanine (8-OH dG) were increased in a dose-dependent manner when calf thymus DNA reacted with various concentrations of COF. Since human 8-oxoguanine DNA glycosylase 1 (hOGG1) was a repair enzyme for removing 8- OH dG from damaged DNA, we hypothesized that hOGG1 mRNA may be used to assess the risk of oxidative damage induced by the exposure of COF. The results from reverse-transcription polymerase chain reaction showed that the hOGG1 mRNA expression was induced by hydrogen peroxide (H2O2) and COF in human lung adenocarcinoma CL-3 cells. To elucidate whether hOGG1 mRNA expression was an exposure biomarker of COF, a cross-sectional study of 238 subjects including 94 professional cooks, 43 housewives, and 101 COF-nonexposed control subjects was conducted. The hOGG1 mRNA expression frequencies of COF-exposed cooks (27 of 94, 28.7%) and housewives (6 of 43, 14%) were significantly higher than those of control subjects (4 of 101, 4%). After adjusting for age, sex, and smoking and drinking status, the odds risks (ORs) of housewives versus control and cooks versus control were 3.94 (95% confidence interval [CI] = 0.95-16.62) and 10.12 (95% CI = 2.83-36.15), respectively. These results indicated that hOGG1 may be adequate to act as an exposure biomarker to assess the oxidative

  15. Hepatic lipase mRNA is expressed in rat and human steroidogenic organs

    NARCIS (Netherlands)

    A.J.M. Verhoeven (Adrie); H. Jansen (Hans)

    1994-01-01

    textabstractRat and human steroidogenic organs contain an enzyme activity that is indistinguishable from hepatic lipase present in liver. Using primers that recognize exons 5 and 8 of the rat and human HL gene, a 596-bp product was found by RT-PCR in rat liver, adrenal, ovaries and testes, but not i

  16. Association of serotonin transporter gene (5HTT) polymorphism and juvenile myoclonic epilepsy: a case-control study.

    Science.gov (United States)

    Esmail, Eman H; Labib, Dalia M; Rabie, Walaa A

    2015-09-01

    Serotonin levels might alter susceptibility to seizures. Serotonin transporter (5HTT) gene polymorphisms were found to be associated with some forms of epilepsy. Here, we attempted to examine an association between 5HTT VNTR allele variants in a serotonin transporter gene and epileptogenesis in juvenile myoclonic epilepsy (JME) cases. We conducted a case-control candidate gene study evaluating the frequencies of 5HTT VNTR allele variants using SYBR green real-time PCR with melting curve analysis in JME patients and healthy subjects. Forty patients with JME were selected from the Epilepsy Outpatient Clinic of Kasr Al Ainy Hospital, Cairo University, who had been classified according to the electroclinical classification of the ILAE. The control group consisted of 40 healthy Egyptian subjects. The less efficient transcriptional genotypes for 5-HTT polymorphisms were more frequent in JME patients (OR 9.33, CI 2.85-30.60; p value epileptogenesis in JME.

  17. Weak binding affinity of human 4EHP for mRNA cap analogs.

    Science.gov (United States)

    Zuberek, Joanna; Kubacka, Dorota; Jablonowska, Agnieszka; Jemielity, Jacek; Stepinski, Janusz; Sonenberg, Nahum; Darzynkiewicz, Edward

    2007-05-01

    Ribosome recruitment to the majority of eukaryotic mRNAs is facilitated by the interaction of the cap binding protein, eIF4E, with the mRNA 5' cap structure. eIF4E stimulates translation through its interaction with a scaffolding protein, eIF4G, which helps to recruit the ribosome. Metazoans also contain a homolog of eIF4E, termed 4EHP, which binds the cap structure, but not eIF4G, and thus cannot stimulate translation, but it instead inhibits the translation of only one known, and possibly subset mRNAs. To understand why 4EHP does not inhibit general translation, we studied the binding affinity of 4EHP for cap analogs using two methods: fluorescence titration and stopped-flow measurements. We show that 4EHP binds cap analogs m(7)GpppG and m(7)GTP with 30 and 100 lower affinity than eIF4E. Thus, 4EHP cannot compete with eIF4E for binding to the cap structure of most mRNAs.

  18. C3a Increases VEGF and Decreases PEDF mRNA Levels in Human Retinal Pigment Epithelial Cells

    Science.gov (United States)

    Long, Qin; Cao, Xiaoguang; Bian, Ailing

    2016-01-01

    Complement activation, specifically complement 3 (C3) activation and C3a generation, contributes to an imbalance between angiogenic stimulation by vascular endothelial growth factor (VEGF) and angiogenic inhibition by pigment epithelial derived factor (PEDF), leading to pathological angiogenesis. This study aimed to investigate the effects of C3a and small interfering RNA (siRNA) targeting C3 on the levels of VEGF and PEDF mRNAs in human retinal pigment epithelial (RPE) cells. ARPE-19 cells were cultured in the presence of exogenous C3a at 0.1 μM and 0.3 μM C3a for 24, 48, and 72 hours. 0.1 pmol/μL duplexes of siRNA targeting C3 were applied for C3a inhibition by transfecting ARPE-19 cells for 48 hours. RT-PCR was performed to examine the level of VEGF and PEDF mRNA. A random siRNA duplex was set for control siRNA. Results demonstrated that exogenous C3a significantly upregulated VEGF and downregulated PEDF mRNA levels in cultured ARPE-19 cells, and siRNA targeting C3 transfection reversed the above changes, significantly reducing VEGF and enhancing PEDF mRNAs level in ARPE-19 cells compared to the control. The present data provided evidence that reducing C3 activation can decreases VEGF and increase PEDF mRNA level in RPE and may serve as a potential therapy in pathological angiogenesis.

  19. Social Defeat: Impact on Fear Extinction and Amygdala-Prefrontal Cortical Theta Synchrony in 5-HTT Deficient Mice

    OpenAIRE

    Venu Narayanan; Heiming, Rebecca S.; Friederike Jansen; Jörg Lesting; Norbert Sachser; Hans-Christian Pape; Thomas Seidenbecher

    2011-01-01

    Emotions, such as fear and anxiety, can be modulated by both environmental and genetic factors. One genetic factor is for example the genetically encoded variation of the serotonin transporter (5-HTT) expression. In this context, the 5-HTT plays a key role in the regulation of central 5-HT neurotransmission, which is critically involved in the physiological regulation of emotions including fear and anxiety. However, a systematic study which examines the combined influence of environmental and...

  20. Interaction of human decapping scavenger with 5' mRNA cap analogues: structural requirements for catalytic activity

    Energy Technology Data Exchange (ETDEWEB)

    Darzynkiewicz, Zbigniew M [Department of Biophysics, Institute of Experimental Physics, Warsaw University, 93 Zwirki and Wigury Street, 02-089 Warsaw (Poland); Bojarska, Elzbieta [Department of Biophysics, Institute of Experimental Physics, Warsaw University, 93 Zwirki and Wigury Street, 02-089 Warsaw (Poland); Kowalska, Joanna [Department of Biophysics, Institute of Experimental Physics, Warsaw University, 93 Zwirki and Wigury Street, 02-089 Warsaw (Poland); Lewdorowicz, Magdalena [Department of Biophysics, Institute of Experimental Physics, Warsaw University, 93 Zwirki and Wigury Street, 02-089 Warsaw (Poland); Jemielity, Jacek [Department of Biophysics, Institute of Experimental Physics, Warsaw University, 93 Zwirki and Wigury Street, 02-089 Warsaw (Poland); Kalek, Marcin [Department of Biophysics, Institute of Experimental Physics, Warsaw University, 93 Zwirki and Wigury Street, 02-089 Warsaw (Poland); Stepinski, Janusz [Department of Biophysics, Institute of Experimental Physics, Warsaw University, 93 Zwirki and Wigury Street, 02-089 Warsaw (Poland); Davis, Richard E [Department of Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, 12801 East 17th Avenue, Aurora, CO 80045 (United States); Darzynkiewicz, Edward [Department of Biophysics, Institute of Experimental Physics, Warsaw University, 93 Zwirki and Wigury Street, 02-089 Warsaw (Poland)

    2007-07-18

    The cap structure is a specific feature of the 5' end of mRNA which plays an important role in the post-transcriptional control in gene expression. A major step of gene regulation occurs at the level of mRNA turnover. Degradation of most eukaryotic mRNAs entails the removal of the cap structure in the various pathways. A human scavenger decapping enzyme (hDcpS) catalyses the cleavage of the residual cap structure m{sup 7}GpppN and/or short oligonucleotides after the 3' to 5' exosom mediated digestion. In this paper we report a fluorescence study of association process of hDcpS with m{sup 7}GMP, m{sup 7}GDP and selected dinucleotide cap analogues resistant to enzymatic hydrolysis. The calculated values of association constants (K{sub as}) and corresponding Gibbs free energies ({delta}G{sup 0}) depend on the type of substituents and their positions in the cap molecule, indicating which structural modifications are crucial for the catalysis.

  1. Real-time RT-PCR analysis of mRNA decay: half-life of Beta-actin mRNA in human leukemia CCRF-CEM and Nalm-6 cell lines

    Directory of Open Access Journals (Sweden)

    Barredo Julio C

    2002-03-01

    Full Text Available Abstract Background We describe an alternative method to determine mRNA half-life (t1/2 based on the Real-Time RT-PCR procedure. This approach was evaluated by using the β-actin gene as a reference molecule for measuring of mRNA stability. Results Human leukemia Nalm-6 and CCRF-CEM cells were treated with various concentrations of Actinomycin D to block transcription and aliquots were removed periodically. Total RNA was isolated and quantified using the RiboGreen® fluorescent dye with the VersaFluor Fluorometer System. One μg of total RNA was reverse transcribed and used as template for the amplification of a region of the β-actin gene (231 bp. To generate the standard curve, serial ten-fold dilutions of the pBactin-231 vector containing the cDNA amplified fragment were employed, β-actin mRNAs were quantified by Real-Time RT-PCR using the SYBR® Green I fluorogenic dye and data analyzed using the iCycle iQ system software. Using this method, the β-actin mRNA exhibited a half-life of 6.6 h and 13.5 h in Nalm-6 and CCRF-CEM cells, respectively. The t1/2 value obtained for Nalm-6 is comparable to those estimated from Northern blot studies, using normal human leukocytes (5.5 h. Conclusions We have developed a rapid, sensitive, and reliable method based on Real-Time RT-PCR for measuring mRNA half-life. Our results confirm that β-actin mRNA half-life can be affected by the cellular growth rate.

  2. Presence of. beta. hCG mRNA in the human pituitary

    Energy Technology Data Exchange (ETDEWEB)

    van Strien, A.; van Wezenbeek, P. (Organon International BV, Oss (Netherlands))

    1989-07-11

    A strong structural and biological similarity exist between human chorionic gonadotropin (hCG) and luteinizing hormone (LH). They share the same {alpha} subunit and have evolutionary related {beta} subunits. hCG is synthesized in first trimester placenta, whereas LH is synthesized in the pituitary. Substances with immunological, physical and biological properties of hCG have, however, been observed in postmenopausal urinary gonadotropin preparations as well as in pituitary extracts. Despite this finding it has not yet been proven that the hCG-like material is identical to placental hCG. This report describes the presence of a minor messenger RNA species in the human pituitary. RNA was isolated from five human pituitary glands and an amplified cDNA library was prepared.

  3. Analysis of Several PLA2 mRNA in Human Meningiomas

    OpenAIRE

    Yves Denizot; Rafael De Armas; Karine Durand; Sandrine Robert; Jean-Jacques Moreau; François Caire; Nicolas Weinbreck; François Labrousse

    2009-01-01

    In view of the important oncogenic action of phospholipase A2(PLA2) we investigated PLA2 transcripts in human meningiomas. Real-time PCR was used to investigate PLA2 transcripts in 26 human meningioma tumors. Results indicated that three Ca2+-dependent high molecular weight PLA2 (PLA2-IVA, PLA2-IVB, PLA2-IVC), one Ca2+-independent high molecular weight PLA2 (PLA2-VI) and five low molecular weight secreted forms of PLA2 (PLA2-IB, PLA2-IIA, PLA2-III, PLA2-V, and PLA2-XII) are expressed with PLA...

  4. Upregulation of human telomerase reverse transcriptase mRNA expression by in vitro transfection of hepatitis B virus X gene into human hepatocarcinoma and cholangiocarcinoma cells

    Institute of Scientific and Technical Information of China (English)

    Zhen-Liang Qu; Sheng-Quan Zou; Nai-Qiang Cui; Xian-Zhong Wu; Ming-Fang Qin; Di Kong; Zhen-Li Zhou

    2005-01-01

    AIM: To study the changes of human telomerase reverse transcriptase (hTERT) mRNA expression in human hepatocarcinoma cell lines (HepG2) and cholangiocarcinoma cell lines (QBC939) after HBx gene transfection and to illustrate the significance of transcriptional regulation of hTERT gene by HBx gene in the carcinogenesis.METHODS: HepG2 and QBC939 cell lines were cultured and co-transfected with eukaryotic expression vector containing the HBx coding region and cloning vector containing enhanced green fluorescent protein (EGFP) coding sequence using lipid-mediated gene transduction technique. Thirty-six hours after transfection, EGFP expression in cells was used as the indicator of successful transfection. Flow cytometry was performed to determine the transfection efficiency.Cells were harvested and total RNA was extracted using TRIzol() reagent. The expression of hTERT mRNA in HepG2and QBC939 cell lines was assayed by reverse transcriptionpolymerase chain reaction. The expression of HBx protein in both cell lines was detected by immunocytochemical staining and Western blotting.RESULTS: Flow cytometry showed that the transfection efficiency was 46.4% in HepG2 cells and 29.6% in QBC939cells for both HBx gene expression vector and blank vector. The expression of hTERT mRNA was meaningfully increased in HepG2 and QBC939 cell lines when transfected with HBx gene expression vector compared to those transfected with OPTI-MEM medium and blank vector.Immunocytochemical staining and Western blotting revealed HBx protein expression in HepG2 and QBC939cells only when transfected with HBx gene.CONCLUSION: HBx gene transfection can upregulate the transcriptional expression of hTERT mRNA. The transactivation of hTERT gene by HBx gene is a newfound mechanism for pathogenesis of hepatocarcinomas and cholangiocarcinomas after HBV infection.

  5. Cloning, characterization, and mRNA expression analysis of novel human fetal cochlear cDNAs.

    NARCIS (Netherlands)

    Luijendijk, M.W.J.; Pol, T.J.R. van de; Duijnhoven, G.C.F. van; Hollander, A.I. den; Caat, J. ten; Limpt, V. van; Brunner, H.G.; Kremer, J.M.J.; Cremers, F.P.M.

    2003-01-01

    To identify novel genes that are expressed specifically or preferentially in the cochlea, we constructed a cDNA library enriched for human cochlear cDNAs using a suppression subtractive hybridization technique. We analyzed 2640 clones by sequencing and BLAST similarity searches. One hundred and fift

  6. Increased levels of endothelin ETB receptor mRNA in human omental arteries after organ culture

    DEFF Research Database (Denmark)

    Möller, S; Adner, M; Edvinsson, L

    1998-01-01

    1. Using competitive reverse transcription-polymerase chain reaction (RT-PCR) and in vitro pharmacology, smooth muscle endothelin ETB receptor expression was studied in segments of human omental artery, fresh and after organ culture for 1 and 5 days. 2. The competitive RT-PCR assay used in the pr...

  7. 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

    of 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, FABP......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...

  8. 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

    of 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, FABP......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...

  9. Bed rest reduces metabolic protein content and abolishes exercise-induced mRNA responses in human skeletal muscle

    DEFF Research Database (Denmark)

    Jørgensen, Stine Ringholm; Biensø, Rasmus S; Kiilerich, Kristian

    2011-01-01

    Background: The aim was to test the hypothesis that one week of bed rest will reduce mitochondrial number and expression and activity of oxidative proteins in human skeletal muscle, but that exercise-induced intracellular signaling as well as mRNA and microRNA (miR) responses are maintained after......-legged knee extensor exercise performed before and after bed rest. Results: Maximal oxygen uptake decreased 5% and exercise endurance decreased non-significantly 25% by bed rest. Bed rest reduced skeletal muscle mitochondrial DNA/nuclear DNA content 15%, hexokinase II and sirtuin 1 protein content ~45%, 3...... bed rest. Research Design and Methods: Twelve young, healthy, male subjects completed 7 days of bed rest with vastus lateralis muscle biopsies taken before and after bed rest. In addition, muscle biopsies were obtained from 6 of the subjects prior to, immediately after and 3h after 45 min one...

  10. Study on the immune responses against pancreatic cancer induced by mucin 4 and human telomerase reverse transcriptase mRNA co-transfected dendritic cells in vitro

    Institute of Scientific and Technical Information of China (English)

    陈江

    2014-01-01

    Objective To investigate the anti-tumor immune response induced by human pancreatic cancer mucin 4mRNA and human telomerase reverse transcriptase(hTERT)mRNA cotransfected dendritic cells(DC),and to provide the experimental evidences for the treatment of pancreatic cancer with multi-epitope loaded DC vaccine.Methods DC were isolated from peripheral DC.

  11. Exonuclease hDIS3L2 specifies an exosome-independent 3'-5' degradation pathway of human cytoplasmic mRNA

    DEFF Research Database (Denmark)

    Lubas, Michal Szymon; Damgaard, Christian Kroun; Tomecki, Rafal

    2013-01-01

    Turnover of mRNA in the cytoplasm of human cells is thought to be redundantly conducted by the monomeric 5'-3' exoribonuclease hXRN1 and the 3'-5' exoribonucleolytic RNA exosome complex. However, in addition to the exosome-associated 3'-5' exonucleases hDIS3 and hDIS3L, the human genome encodes a...

  12. Effect of experimental treatment on GAPDH mRNA expression as a housekeeping gene in human diploid fibroblasts

    Directory of Open Access Journals (Sweden)

    Zainuddin Azalina

    2010-08-01

    Full Text Available Abstract Background Several genes have been used as housekeeping genes and choosing an appropriate reference gene is important for accurate quantitative RNA expression in real time RT-PCR technique. The expression levels of reference genes should remain constant between the cells of different tissues and under different experimental conditions. The purpose of this study was to determine the effect of different experimental treatments on the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH mRNA so that the reliability of GAPDH as reference gene for quantitative real time RT-PCR in human diploid fibroblasts (HDFs can be validated. HDFs in 4 different treatment groups viz; young (passage 4, senescent (passage 30, H2O2-induced oxidative stress and γ-tocotrienol (GTT-treated groups were harvested for total RNA extraction. Total RNA concentration and purity were determined prior to GAPDH mRNA quantification. Standard curve of GAPDH expression in serial diluted total RNA, melting curve analysis and agarose gel electrophoresis were used to determine the reliability of GAPDH as reference gene. Results HDFs with different experimental treatments exhibited diverse cell morphology with different expression of senescence-associated β-galactosidase (SA β-gal activity. However the expression level of GAPDH was consistent in all treatment groups. Conclusion The study demonstrated that GAPDH is reliable as reference gene for quantitative gene expression analysis in HDFs. Therefore it can be used as housekeeping gene for quantitative real time RT-PCR technique in human diploid fibroblasts particularly in studying cellular senescence.

  13. Evaluation of Existing Methods for Human Blood mRNA Isolation and Analysis for Large Studies

    OpenAIRE

    Meyer, Anke; Paroni, Federico; Günther, Kathrin; Dharmadhikari, Gitanjali; Ahrens, Wolfgang; Kelm, Sørge; Maedler, Kathrin

    2016-01-01

    Aims Prior to implementing gene expression analyses from blood to a larger cohort study, an evaluation to set up a reliable and reproducible method is mandatory but challenging due to the specific characteristics of the samples as well as their collection methods. In this pilot study we optimized a combination of blood sampling and RNA isolation methods and present reproducible gene expression results from human blood samples. Methods The established PAXgeneTM blood collection method (Qiagen)...

  14. Expression of a novel pyridoxal kinase mRNA splice variant, PKH-T, in human testis

    Institute of Scientific and Technical Information of China (English)

    XingFang; Zuo-MinZhou; LiLu; Lan-LanYin; Jian-MinLi; YinZhen; HuiWang; Jia-HaoSha

    2004-01-01

    Aim: To identify the genes specifically expressed in human adult and fetal testes and spermatozoa.Methods: A human testis cDNA microarray was established. Then mRNAs of human adult and fetal testis and spermatozoa were purified and probes were prepared by a reverse transcription reaction with mRNA as the template.The microarray was hybridized with probes of adult and fetal testes and spermatozoa. The nucleic acid sequences of differentially expressed genes were determined and homologies were searched in the databases of GenBank. Results:A novel human testis-specific gene, PKH-T, was identified by hybridizing adult and fetal testis and spermatozoa probes with a human testis cDNA microarray. The cDNA of PKH-T was 1069 bp in length. The cDNA sequence of this clone was deposited in the Genbank (AY303972) and PKH-T was also determined as Interim GenSymbol (Unigene,HS.38041). PKH-T contained most PKH conserved motif. The 239 amino acid sequences deduced from the 719 bp open reading frame (ORF) had a homology with the gene PKH (U89606). PKH-T was specifically and strongly expressed in the testis. Comparison of the differential expressions of PKH and PKH-T in testes of different develop-mental stages indicated that PKH-T was expressed in the adult testis and spermatozoa, while PKH, in the adult, fetal and aged testes. PKH-T had no expression in the testis of Sertoli cell only and partially spermatogenic arrest patients.Conclusion: PKH-T is a gene highly expressed in adult human testis and spermatozoa. It may play an important role in spermatogenesis and could be related to male infertility.

  15. BCFA suppresses LPS induced IL-8 mRNA expression in human intestinal epithelial cells.

    Science.gov (United States)

    Yan, Y; Wang, Z; Greenwald, J; Kothapalli, K S D; Park, H G; Liu, R; Mendralla, E; Lawrence, P; Wang, X; Brenna, J T

    2017-01-01

    Branched chain fatty acids (BCFA) are components of common food fats and are major constituents of the normal term human newborn GI tract. Polyunsaturated fatty acids (PUFA) have been suggested to reduce the risk and development of inflammatory bowel diseases (IBD); however, little is known about the influence of BCFA on inflammation. We investigated the effect of BCFA on interleukin (IL)-8 and NF-κB production in a human intestinal epithelial cell line (Caco-2). Cells were pre-treated with specific BCFA, or DHA, or EPA, and then activated with lipopolysaccharide (LPS). Both anteiso- and iso- BCFA reduce IL-8. Anteiso-BCFA more effectively suppressed IL-8 than iso-BCFA in LPS stimulated Caco-2 cells. However BCFA in general were less effective than DHA or EPA. Activated BCFA-treated cells expressed less of the cell surface Toll-like receptor 4 (TLR-4) compared to controls. These are the first data to show the reduction of pro-inflammatory markers in human cells mediated by BCFA. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Analysis of Several PLA2 mRNA in Human Meningiomas

    Directory of Open Access Journals (Sweden)

    Yves Denizot

    2009-01-01

    Full Text Available In view of the important oncogenic action of phospholipase A2(PLA2 we investigated PLA2 transcripts in human meningiomas. Real-time PCR was used to investigate PLA2 transcripts in 26 human meningioma tumors. Results indicated that three Ca2+-dependent high molecular weight PLA2 (PLA2-IVA, PLA2-IVB, PLA2-IVC, one Ca2+-independent high molecular weight PLA2 (PLA2-VI and five low molecular weight secreted forms of PLA2 (PLA2-IB, PLA2-IIA, PLA2-III, PLA2-V, and PLA2-XII are expressed with PLA2-IVA, PLA2-IVB, PLA2-VI, and PLA2-XIIA as the major expressed forms. PLA2-IIE, PLA2-IIF, PLA2-IVD, and PLA2-XIIB are not detected. Plasma (PLA2-VIIA and intracellular (PLA2-VIIB platelet-activating factor acetylhydrolase transcripts are expressed in human meningiomas. However no difference was found for PLA2 transcript amounts in relation to the tumor grade, the subtype of meningiomas, the presence of inflammatory infiltrated cells, of an associated edema, mitosis, brain invasion, vascularisation or necrosis. In conclusion numerous genes encoding multiples forms of PLA2 are expressed in meningiomas where they might act on the phospholipid remodeling and on the local eicosanoid and/or cytokine networks.

  17. Novel way of capping mRNA trimer and studies of its interaction with human nuclear cap-binding complex.

    Science.gov (United States)

    Worch, Remigiusz; Stepinski, Janusz; Niedzwiecka, Anna; Jankowska-Anyszka, Marzena; Mazza, Catherine; Cusack, Stephen; Stolarski, Ryszard; Darzynkiewicz, Edward

    2005-01-01

    Binding of mRNA 5' cap by the nuclear cap-binding complex (CBC) is crucial for a wide variety of mRNA metabolic events. The interaction involving the CBP20 subunit of CBC is mediated by numerous hydrogen bonds and by stacking of the tyrosine sidechains with two first bases of the capped mRNA. To examine a possible role of a longer mRNA chain in the CBC-cap recognition, we have synthesized an mRNA tetramer using a novel way of capping an RNA trimer and determined its affinity for CBC by fluorescence titration.

  18. Clinical performance of human papillomavirus E6, E7 mRNA flow cytometric assay compared to human papillomavirus DNA typing.

    Science.gov (United States)

    Kottaridi, Christine; Tsiodras, Sotirios; Spathis, Aris; Chranioti, Aikaterini; Pappas, Asimakis; Kassanos, Dimitrios; Panayiotides, Ioannis; Karakitsos, Petros

    2011-12-01

    To use flow cytometry to screen cervical samples for the overexpression of human papillomavirus (HPV) E6 and E7 mRNA and compare the performance of this assay with an HPV DNA array for the detection of high-grade cervical lesions. Cervical samples were analyzed for HPV DNA by clinical arrays, and the overexpression of E6 and E7 viral oncogenes was monitored using an HPV mRNA detection kit that quantifies the intracellular HPV E6 and E7 mRNA on a cell-by-cell basis. HPV positivity increased with severity of histologic lesions. On the basis of histology-confirmed CIN 2+ cases the specificity of HPV assay was 73.9% (95% CI 66.07, 80.88), whereas it was 39.3% (95% CI 31.85, 47.1) for the DNA assay. The HPV assay provides an early predictor of persistent HPV infection and may improve cervical cancer screening by increasing the specificity of detecting high-grade lesions.

  19. Direct RNA sequencing mediated identification of mRNA localized in protrusions of human MDA-MB-231 metastatic breast cancer cells

    DEFF Research Database (Denmark)

    Jakobsen, Kristine Raaby; Sørensen, Emilie; Brøndum, Karin Kathrine;

    2013-01-01

    Background Protrusions of cancer cells conferrers a vital function for cell migration and metastasis. Protein and RNA localization mechanisms have been extensively examined and shown to play pivotal roles for the functional presence of specific protein components in cancer cell protrusions. Methods...... To describe genome wide RNA localized in protrusions of the metastatic human breast cancer cell line MDA-MB-231 we used Boyden chamber based methodology followed by direct mRNA sequencing. Results In the hereby identified group of protrusion localized mRNA some previously were described to be localized...... exemplified by mRNA for Ras-Related protein 13 (RAB13) and p0071 (Plakophilin-4/PKP4). For other transcripts, exemplified by mRNA for SH3PXD2A/TKS5 and PPFIA1/Liprin-α1, only the corresponding proteins previously were described to have protrusion localization. Finally, a cohort of MDA-MB-231 protrusion...

  20. Effect of Simvastatin on 5-HT and 5-HTT in a Rat Model of Pulmonary Artery Hypertension

    Directory of Open Access Journals (Sweden)

    Xue Jiang

    2015-11-01

    Full Text Available Background/Aims: To investigaterole of serotonin (5-HT and serotonin transporter (5-HTT in a rat model of cigarette smoke-induced pulmonary artery hypertension (PAH and the effect of statins on regulating 5HT and 5-HTT. Methods: A rat model of COPD comorbid with PAH was established by cigarette smoke exposure with or without simvastatin administration. The smoking and the simvastatin plus smoking groups were exposed to cigarette smoke daily, and the latter received simvastatin at 5mg/kg, once a day. After 16 weeks of cigarette smoke exposure, body weight and mean pulmonary arterial pressure (mPAP were measured, bronchoalveolar lavage (BAL was performed, and lung tissues and blood samples were collected to determine cardiopulmonary pathology, physiological indices, blood levelof 5-HT and expression of 5-HTT in the lung. Results: In addition to alveolar structural damage (COPD-like injury, chronic cigarette smoke exposure lead to pulmonary artery remodeling and PAH as evidenced by significant elevation of mPAP, RVHI, WT%and WA%. Cigarette smoke exposure resulted in significant reduction in animal body weight, and simvastatin significantly prevented smoke-induced weight loss. The number of inflammatory cells in BALF was dramatically increased in smoke exposed rats, and simvastatin dampened the number of leukocytes, neutrophils, lymphocytes, and macrophages. In addition, circulating 5-HTand expression of 5-HTT in the lung were significantly increased in the smoked rats compared to control rats, and it was significantly reduced by simvastatin. Alteration of BALF inflammatory cells, 5-HT and 5-HTT was significantly correlated with changes of mPAP, RVHI, WT% and WA%. Conclusions: Cigarette smoke exposure could result in not only COPD, but also PAH, which may attribute to the alteration of blood 5-HT and lung tissue 5-HTT. Simvastatin could significantly inhibited 5-HT and 5-HTT expression, and by which mechanism, it may protect animals from development

  1. Expression of a novel beta adaptin subunit mRNA splice variant in human testes

    Institute of Scientific and Technical Information of China (English)

    Xin-Dong Zhang; Lan-Lan Yin; Ying Zheng; Li Lu; Zuo-Min Zhou; Jia-Hao Sha

    2005-01-01

    Aim: To identify a novel isoform of adaptin 2 beta subunit (named Ap2β-NY) and to investigate its relationship with testicular development and spermatogenesis. Methods: Using a human testis cDNA microarray, a clone (Ap2β-NY),which was strongly expressed in adult testes but weakly expressed in embryo testes, was sequenced and analyzed.Using polymerase chain reaction (PCR), the tissue distribution and expression time pattern of Ap2β-NY were determined.Results: Ap2β-NY was identified and has been deposited in the GenBank (AY341427). The expression level of Ap2β-NY in the adult testis was about 3-fold higher than that in the embryo testis. PCR analysis using multi-tissue cDNA indicated that Ap2β-NY was highly expressed in the testis, spleen, thymus, prostate, ovary, blood leukocyte and brain, but not in the heart, placenta, lung, liver, skeletal muscle, kidney and pancreas. In addition, Ap2β-NY was variably expressed in the testes of patients with spermatogenesis-disturbance and spermatogenesis-arrest but not expressed in those of Sertoli-cell-only syndrome, which implied that, in the testis, Ap2β-NY was restrictively expressed in germ cells. Conclusion: Ap2β-NY is an isoform of Ap2β and may be involved in regulating the process of spermatogenesis and testis development.

  2. Reprogramming of human fibroblasts to pluripotent stem cells using mRNA of four transcription factors

    Energy Technology Data Exchange (ETDEWEB)

    Yakubov, Eduard [Department of Molecular Cell Biology, Weizmann Institute of Science, 76100 Rehovot (Israel); Rechavi, Gidi [Cancer Research Center, Chaim Sheba Medical Center, Tel-Hashomer and Sackler School of Medicine, Tel-Aviv University, Tel-Aviv (Israel); Rozenblatt, Shmuel [Department of Molecular Microbiology and Biotechnology, Tel-Aviv University, Tel-Aviv (Israel); Givol, David, E-mail: david.givol@weizmann.ac.il [Department of Molecular Cell Biology, Weizmann Institute of Science, 76100 Rehovot (Israel)

    2010-03-26

    Reprogramming of differentiated cells into induced pluripotent cells (iPS) was accomplished in 2006 by expressing four, or less, embryonic stem cell (ESC)-specific transcription factors. Due to the possible danger of DNA damage and the potential tumorigenicity associated with such DNA damage, attempts were made to minimize DNA integration by the vectors involved in this process without complete success. Here we present a method of using RNA transfection as a tool for reprogramming human fibroblasts to iPS. We used RNA synthesized in vitro from cDNA of the same reprogramming four transcription factors. After transfection of the RNA, we show intracellular expression and nuclear localization of the respective proteins in at least 70% of the cells. We used five consecutive transfections to support continuous protein expression resulting in the formation of iPS colonies that express alkaline phosphatase and several ESC markers and that can be expanded. This method completely avoids DNA integration and may be developed to replace the use of DNA vectors in the formation of iPS.

  3. Expression of Heat Shock Protein 70 mRNA in Epithelial Cells of Human Lens

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Objective:To try to find out the pathogenesis of the cataract , effects of beat and oxidative stresson heat shock proteins of tissue cultured human lens epithelial cells (LEC-B3) were investigate& Methods:Cells were exposed to heat shock (45℃) and oxidative stress(5OmMH2O2 for 30 min, and then allowed to recoverat different intervals (Oh, 2h, 4h, 6h, 16h, 24h) in physiological medium Reverse transcription polymerasechain reaction (RT-PCR) were used to determined the level of HSP70. Results: HSPs existed in both physiologicaland stressful situation. The level of HSP7OmRNA increased 2h later after both stresses. The expression of HSP70got to the summit during 2h to 6h in each group. Subsequently it decreased gradually in each group, maintaininga high level at 16h. Conclusion: HSP70 exists in lens epithelial cells and can be induced after stress. Thedata suggested it may play an important protective role in lens epithelial cells in respond to cellular stress.

  4. IMPROVING P-gp EXPRESSION IN HUMAN MONONUCLEAR CELLS IN VITRO TRANSFECTED BY MULTIDRUG RESISTANCE-1 mRNA

    Institute of Scientific and Technical Information of China (English)

    Yang Xiang; Lei Li; Fang Tian; Xiu-yu Yang

    2005-01-01

    Objective To evaluate the expression and function activity of P-glycoprotein (P-gp) in human mononuclear cells (MNCs) in vitro transfected by multidrug resistance-1 (MDR1) mRNA.Methods Two MDR1 cDNA vectors, pT7TS_MDR1 and pGEM5Zf(+)_MDR1, were constructed and transcripted in vitro. Vector pGEM5Zf(+)_MDR1 only contained the coding region ofmdrl cDNA, and pT7TS_MDR1 also included Xeponus β-globin 5' and 3' untranslated region. MNCs were prepared from peripheral blood of parvicellular lung cancer patient. The two human mdrl mRNAs were then transferred into human MNCs in vitro by DOTAP. And the expression efficiency and pump functon of P-gp were measured with flow cytometry.Results Expression of P-gp significantly elevated in both transferred cells compared with untransferred cells (P< 0.01).And pT7TS_MDR1 showed higher capability in elevating the expression of P-gp than pGEM5Zf(+)_MDR1 (P < 0.01). The P-gp function was elevated in both pT7TS_MDR1 and pGEM5Zf(+)_MDR1 groups. The survival ratio of MNCs in erythrocyte-lysis-solution (ELS, 86.07%) and lymphocyte-isolation-solution (LIS, 83.67%) had no significant difference.The CD34+ cells content of the MNCs used for transfection was 2.65% and 1.01% in ELS and LIS group, respectively (P <0.01).Conclusions It is a feasible approach to improve P-gp expression in human MNCs by transfection of MDR-1 mRNA.And the ELS may be more suitable for purifing MNCs for mRNA transfection than LIS.

  5. 5-HTT deficiency affects neuroplasticity and increases stress sensitivity resulting in altered spatial learning performance in the Morris water maze but not in the Barnes maze.

    Directory of Open Access Journals (Sweden)

    Margherita M Karabeg

    Full Text Available The purpose of this study was to evaluate whether spatial hippocampus-dependent learning is affected by the serotonergic system and stress. Therefore, 5-HTT knockout (-/-, heterozygous (+/- and wildtype (+/+ mice were subjected to the Barnes maze (BM and the Morris water maze (WM, the latter being discussed as more aversive. Additionally, immediate early gene (IEG expression, hippocampal adult neurogenesis (aN, and blood plasma corticosterone were analyzed. While the performance of 5-HTT-/- mice in the BM was undistinguishable from both other genotypes, they performed worse in the WM. However, in the course of the repeated WM trials 5-HTT-/- mice advanced to wildtype level. The experience of a single trial of either the WM or the BM resulted in increased plasma corticosterone levels in all genotypes. After several trials 5-HTT-/- mice exhibited higher corticosterone concentrations compared with both other genotypes in both tests. Corticosterone levels were highest in 5-HTT-/- mice tested in the WM indicating greater aversiveness of the WM and a greater stress sensitivity of 5-HTT deficient mice. Quantitative immunohistochemistry in the hippocampus revealed increased cell counts positive for the IEG products cFos and Arc as well as for proliferation marker Ki67 and immature neuron marker NeuroD in 5-HTT-/- mice compared to 5-HTT+/+ mice, irrespective of the test. Most differences were found in the suprapyramidal blade of the dentate gyrus of the septal hippocampus. Ki67-immunohistochemistry revealed a genotype x environment interaction with 5-HTT genotype differences in naïve controls and WM experience exclusively yielding more Ki67-positive cells in 5-HTT+/+ mice. Moreover, in 5-HTT-/- mice we demonstrate that learning performance correlates with the extent of aN. Overall, higher baseline IEG expression and increased an in the hippocampus of 5-HTT-/- mice together with increased stress sensitivity may constitute the neurobiological correlate of

  6. 5-HTT deficiency affects neuroplasticity and increases stress sensitivity resulting in altered spatial learning performance in the Morris water maze but not in the Barnes maze.

    Science.gov (United States)

    Karabeg, Margherita M; Grauthoff, Sandra; Kollert, Sina Y; Weidner, Magdalena; Heiming, Rebecca S; Jansen, Friederike; Popp, Sandy; Kaiser, Sylvia; Lesch, Klaus-Peter; Sachser, Norbert; Schmitt, Angelika G; Lewejohann, Lars

    2013-01-01

    The purpose of this study was to evaluate whether spatial hippocampus-dependent learning is affected by the serotonergic system and stress. Therefore, 5-HTT knockout (-/-), heterozygous (+/-) and wildtype (+/+) mice were subjected to the Barnes maze (BM) and the Morris water maze (WM), the latter being discussed as more aversive. Additionally, immediate early gene (IEG) expression, hippocampal adult neurogenesis (aN), and blood plasma corticosterone were analyzed. While the performance of 5-HTT-/- mice in the BM was undistinguishable from both other genotypes, they performed worse in the WM. However, in the course of the repeated WM trials 5-HTT-/- mice advanced to wildtype level. The experience of a single trial of either the WM or the BM resulted in increased plasma corticosterone levels in all genotypes. After several trials 5-HTT-/- mice exhibited higher corticosterone concentrations compared with both other genotypes in both tests. Corticosterone levels were highest in 5-HTT-/- mice tested in the WM indicating greater aversiveness of the WM and a greater stress sensitivity of 5-HTT deficient mice. Quantitative immunohistochemistry in the hippocampus revealed increased cell counts positive for the IEG products cFos and Arc as well as for proliferation marker Ki67 and immature neuron marker NeuroD in 5-HTT-/- mice compared to 5-HTT+/+ mice, irrespective of the test. Most differences were found in the suprapyramidal blade of the dentate gyrus of the septal hippocampus. Ki67-immunohistochemistry revealed a genotype x environment interaction with 5-HTT genotype differences in naïve controls and WM experience exclusively yielding more Ki67-positive cells in 5-HTT+/+ mice. Moreover, in 5-HTT-/- mice we demonstrate that learning performance correlates with the extent of aN. Overall, higher baseline IEG expression and increased an in the hippocampus of 5-HTT-/- mice together with increased stress sensitivity may constitute the neurobiological correlate of raised

  7. Evaluation of Existing Methods for Human Blood mRNA Isolation and Analysis for Large Studies

    Science.gov (United States)

    Meyer, Anke; Paroni, Federico; Günther, Kathrin; Dharmadhikari, Gitanjali; Ahrens, Wolfgang; Kelm, Sørge; Maedler, Kathrin

    2016-01-01

    Aims Prior to implementing gene expression analyses from blood to a larger cohort study, an evaluation to set up a reliable and reproducible method is mandatory but challenging due to the specific characteristics of the samples as well as their collection methods. In this pilot study we optimized a combination of blood sampling and RNA isolation methods and present reproducible gene expression results from human blood samples. Methods The established PAXgeneTM blood collection method (Qiagen) was compared with the more recent TempusTM collection and storing system. RNA from blood samples collected by both systems was extracted on columns with the corresponding Norgen and PAX RNA extraction Kits. RNA quantity and quality was compared photometrically, with Ribogreen and by Real-Time PCR analyses of various reference genes (PPIA, β-ACTIN and TUBULIN) and exemplary of SIGLEC-7. Results Combining different sampling methods and extraction kits caused strong variations in gene expression. The use of PAXgeneTM and TempusTM collection systems resulted in RNA of good quality and quantity for the respective RNA isolation system. No large inter-donor variations could be detected for both systems. However, it was not possible to extract sufficient RNA of good quality with the PAXgeneTM RNA extraction system from samples collected by TempusTM collection tubes. Comparing only the Norgen RNA extraction methods, RNA from blood collected either by the TempusTM or PAXgeneTM collection system delivered sufficient amount and quality of RNA, but the TempusTM collection delivered higher RNA concentration compared to the PAXTM collection system. The established Pre-analytix PAXgeneTM RNA extraction system together with the PAXgeneTM blood collection system showed lowest CT-values, i.e. highest RNA concentration of good quality. Expression levels of all tested genes were stable and reproducible. Conclusions This study confirms that it is not possible to mix or change sampling or

  8. Herbal compound 861 regulates mRNA expression of collagen synthesis- and degradation-related genes in human hepatic stellate cells

    Institute of Scientific and Technical Information of China (English)

    Lin Wang; Bao-En Wang; Jian Wang; Pei-Gen Xiao; Xue-Hai Tan

    2008-01-01

    AIM: To identify the role of herbal compound 861 (Cpd 861) in the regulation of mRNA expression of collagen synthesis- and degradation-related genes in human hepatic stellate cells (HSCs).METHODS: mRNA levels of collagen types I and III, matrix metalloproteinase 1 (MMP-1), matrix metalloproteinase 2 (MMP-2), membrane type-1 matrix metalloproteinase (MT1-MMP), tissue inhibitor of metalloproteinase 1 (TIMP-1), and transforming growth factor β1 (TGF-βi) in cultured-activated HSCs treated with Cpd 861 or interferon-γ (IFN-γ) were determined by real-time PCR.RESULTS: Both Cpd 861 and IFN-γ reduced the mRNA levels of collagen type Ⅲ, MMP-2 and TGF-βl. Moreover, Cpd 861 significantly enhanced the MMP-1 mRNA levels while down-regulated the TIMP-1 mRNA expression, increasing the ratio of MMP-1 to TIMP-1 to (6.3 + 0.3)-fold compared to the control group.CONCLUSION: The anti-fibrosis function of Cpd 861 may be mediated by both decreased interstitial collagen sythesis by inhibiting the transcription of collagen type in and TGF-pi and increased degradation of these collagens by up-regulating MMP-1 and down-regulating TIMP-1 mRNA levels.

  9. mRNA Expression of Vimentin Gene in Lens of Transgenic Mouse and DNA Amplification in Human Cataracts

    Institute of Scientific and Technical Information of China (English)

    YanLi; XienpingLiu; 等

    1995-01-01

    Purpose:To investigate the role of vimentin gene in cataractogenesis.Methods:The12.7kb chicken vimentin genes were microinjected into the male pronuclei of 918 fertilized mice eggs.841injected embryos were transferred into oviducts of pseudopregnant recipient females.of which 12pregnant mice gave birth to 49offsping mice.The integration and expression of exogenous gene in the offsping were analysed by Southern and Northern blot byhridizations,In the human senile cataract,the lens vimentin gene was analyzed with the chicken vi-mentin gene probe.Results:It showed that four of F1offspring were transgenic mice in which the chicken vimenttin gene was integrated in their genomes.The transgenic band was12kb,similar to the12.7kb chicken vimentin fragment injected.One2kbvi-mentin mRNAwas visualized on E2 mouse lens blot.which revealed that the chicken vimentin gene was efficiently expressed in this transgenic mouse.In the humansenile cataract lens,12kb BamHI-restricted vimentin fragments displayed a stronger hybridization signal than that of the control lens in Southern blot anal-ysis,It implies that the Formation of human senile cataract may be associated with the amplification of vimentin gene.Conclusions:We have successfully developed four transgenic mice bearing chicken vimentin gene and having mRNA expression which can be used for further study.It is to be observed if the normal lens cell function is affected by the expressed product and cataract occurs in our transgenic mice.The cause of the gene ampli-fication in human ctaract remains for further investigation.Eye Science 1995;11:113-116.

  10. The miRNA and mRNA Signatures of Peripheral Blood Cells in Humans Infected with Trypanosoma brucei gambiense.

    Directory of Open Access Journals (Sweden)

    Smiths Lueong

    Full Text Available Simple, reliable tools for diagnosis of human African Trypanosomiases could ease field surveillance and enhance patient care. In particular, current methods to distinguish patients with (stage II and without (stage I brain involvement require samples of cerebrospinal fluid. We describe here an exploratory study to find out whether miRNAs from peripheral blood leukocytes might be useful in diagnosis of human trypanosomiasis, or for determining the stage of the disease. Using microarrays, we measured miRNAs in samples from Trypanosoma brucei gambiense-infected patients (9 stage I, 10 stage II, 8 seronegative parasite-negative controls and 12 seropositive, but parasite-negative subjects. 8 miRNAs (out of 1205 tested showed significantly lower expression in patients than in seronegative, parasite-negative controls, and 1 showed increased expression. There were no clear differences in miRNAs between patients in different disease stages. The miRNA profiles could not distinguish seropositive, but parasitologically negative samples from controls and results within this group did not correlate with those from the trypanolysis test. Some of the regulated miRNAs, or their predicted mRNA targets, were previously reported changed during other infectious diseases or cancer. We conclude that the changes in miRNA profiles of peripheral blood lymphocytes in human African trypanosomiasis are related to immune activation or inflammation, are probably disease-non-specific, and cannot be used to determine the disease stage. The approach has little promise for diagnostics but might yield information about disease pathology.

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

    Institute of Scientific and Technical Information of China (English)

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

    2005-01-01

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

  12. Impact of adrenaline and metabolic stress on exercise-induced intracellular signaling and PGC-1α mRNA response in human skeletal muscle

    DEFF Research Database (Denmark)

    Brandt, Nina; Gunnarsson, Thomas Gunnar Petursson; Hostrup, Morten

    2016-01-01

    This study tested the hypothesis that elevated plasma adrenaline or metabolic stress enhances exercise-induced PGC-1α mRNA and intracellular signaling in human muscle. Trained (VO2-max: 53.8 ± 1.8 mL min(-1) kg(-1)) male subjects completed four different exercise protocols (work load of the legs...

  13. Potential Osteoinductive Effects of Calcitriol on the m-RNA of Mesenchymal Stem Cells Derived from Human Alveolar Periosteum

    Directory of Open Access Journals (Sweden)

    Hsiang-Hsi Hong

    2016-01-01

    Full Text Available This study characterized alveolar periosteum-derived mesenchymal stem cells (P-MSCs and examined the hypothesis that 1,25-(OH2D3 (calcitriol exerts osteoinductive effects on P-MSCs. The mRNA expressions of alkaline phosphatase (ALP, bone sialoprotein (BSP, core-binding factor alpha-1 (CBFA1, collagen-1 (Col-1, osteocalcin (OCN, and vitamin D3 receptor (VDR were assessed after incubation with calcitriol for 2 weeks. Vitamin C as positive control (Vit. C-p increased ALP and CBFA1 mRNA expression at both 1 and 2 weeks and increased BSP and Col-1 mRNA expression only at the first week. A concentration of 10−8 M calcitriol enhanced ALP, CBFA1, Col-1, and OCN mRNA expression at both weeks and BSP mRNA expression at the first week. Furthermore, 10−7 M calcitriol increased the mRNA expressions of all compounds at both weeks, except that of CBFA1 at the first week. 10−8 M calcitriol and Vit. C-p enhanced ALP activity at the second and third weeks. The results revealed that 10−9, 10−8, and 10−7 M calcitriol induced osteoinduction in alveolar P-MSCs by increasing ALP, CBFA1, Col-1, and OCN mRNA expression. A 10−7 M calcitriol yielded a higher mRNA expression than Vit. Cp on VDR and OCN mRNA expression at both weeks and on Col-1 mRNA at the second week.

  14. Potential Osteoinductive Effects of Calcitriol on the m-RNA of Mesenchymal Stem Cells Derived from Human Alveolar Periosteum

    Science.gov (United States)

    Hong, Hsiang-Hsi; Hong, Adrienne

    2016-01-01

    This study characterized alveolar periosteum-derived mesenchymal stem cells (P-MSCs) and examined the hypothesis that 1,25-(OH)2D3 (calcitriol) exerts osteoinductive effects on P-MSCs. The mRNA expressions of alkaline phosphatase (ALP), bone sialoprotein (BSP), core-binding factor alpha-1 (CBFA1), collagen-1 (Col-1), osteocalcin (OCN), and vitamin D3 receptor (VDR) were assessed after incubation with calcitriol for 2 weeks. Vitamin C as positive control (Vit. C-p) increased ALP and CBFA1 mRNA expression at both 1 and 2 weeks and increased BSP and Col-1 mRNA expression only at the first week. A concentration of 10−8 M calcitriol enhanced ALP, CBFA1, Col-1, and OCN mRNA expression at both weeks and BSP mRNA expression at the first week. Furthermore, 10−7 M calcitriol increased the mRNA expressions of all compounds at both weeks, except that of CBFA1 at the first week. 10−8 M calcitriol and Vit. C-p enhanced ALP activity at the second and third weeks. The results revealed that 10−9, 10−8, and 10−7 M calcitriol induced osteoinduction in alveolar P-MSCs by increasing ALP, CBFA1, Col-1, and OCN mRNA expression. A 10−7 M calcitriol yielded a higher mRNA expression than Vit. Cp on VDR and OCN mRNA expression at both weeks and on Col-1 mRNA at the second week. PMID:28105418

  15. Acute simulated soccer-specific training increases PGC-1α mRNA expression in human skeletal muscle.

    Science.gov (United States)

    Jeong, Tae-Seok; Bartlett, Jonathan D; Joo, Chang-Hwa; Louhelainen, Jari; Close, Graeme L; Morton, James P; Drust, Barry

    2015-01-01

    The aim of the current study was to quantify oxygen uptake, heart rate and molecular responses of human skeletal muscle associated with mitochondrial biogenesis following an acute bout of simulated soccer training. Muscle biopsies (vastus lateralis) were obtained from nine active men immediately pre-completion, post-completion and 3 h post-completion of a laboratory-based soccer-specific training simulation on a motorised treadmill. The soccer-specific simulation was a similar intensity (55 ± 6% [Formula: see text]) and duration (60 min) as that observed in professional soccer training (e.g. standing 41%, walking 37%, jogging 11%, high-speed running 9% and sprinting 2%). Post-exercise, muscle glycogen decreased (Pre; 397 ± 86 mmol∙kg(-1) dw, Post; 344 ± 64 mmol∙kg(-1) dw; P = 0.03), plasma lactate increased (P soccer-specific training is associated with increases in PGC-1α mRNA. These data may have implications for practitioners in better understanding the metabolic and muscle responses to soccer-specific training protocols in the field.

  16. X-ray irradiation affects Gadd45 mRNA expression in human peripheral blood lymphocytes%X 射线对人周围血淋巴细胞 Gadd45 mRNA 表达影响

    Institute of Scientific and Technical Information of China (English)

    王恰; 李明芳; 杨爱初; 杨宇华; 梁晓阳; 严茂胜

    2015-01-01

    Objective To analyze the relative expression level of Growth arrest and DNA damage gene (Gadd)45 mRNA of human peripheral blood lymphocytes after X-ray irradiation with different doses and time,and to explore the possibility of Gadd45 gene as a biological dosimeter.Methods i)Dose-effect experiment.The healthy human peripheral blood were irradiated by 0.00,0.10,0.25,0.50,1.00,2.00,3.00 and 5.00 Gy X-ray and the lymphocytes were separated for measurement.ii)Time-effect experiment.The healthy human peripheral blood were irradiated by 2.00 Gy X-ray and the lymphocytes were separated and then cultivate for measurement after cultivation time of 0,3,6,12,24 and 48 hours.The real-time fluorescent quantitative polymerase chain reaction method was used to detect Gadd45 mRNA expression level. Results i)The relative expression level of Gadd45 mRNA increased with the increasing dose from 0.00 to 0.50 Gy.The linear regression equation was ^y =1.056 9 +7.132 2 x (determination coefficient was 0.992,P <0.01 ).The relative expression level of Gadd45 mRNA achieved the peak value when the irradiation dose was 0.50 Gy,which was 4.53 times of that with 0.00 Gy dose irradiation.The relative expression level of Gadd45 mRNA gradually decreased in 0.50-5.00 Gy.ii)In the period of 3-12 hours after 2.00 Gy of X-ray irradiation,the relative expression level of Gadd45 mRNA increased with the increasing exposure time.The linear regression equation was ^y =-6.366 0 +4.965 0 x (determination coefficient was 0.932,P <0.05).It achieved the peak value at the time of 12 hours,which was 57.68 times of that with 0-hour time group.During the time of 24-48 hours after irradiation,the relative expression level of Gadd45 mRNA decreased sharply,the 48-hour group was 2.08 times of 0-hour time group.Conclusion X-ray irradiation up-regulated the expression level of Gadd45 mRNA.There was a good dose-effect and time-effect relation in specific dose and time range.Further study is needed to explore if Gadd45

  17. Low-level lasers on microRNA and uncoupling protein 2 mRNA levels in human breast cancer cells

    Science.gov (United States)

    Canuto, K. S.; Teixeira, A. F.; Rodrigues, J. A.; Paoli, F.; Nogueira, E. M.; Mencalha, A. L.; Fonseca, A. S.

    2017-06-01

    MicroRNA is short non-coding RNA and is a mediator of post-transcriptional regulation of gene expression. In addition, uncoupling proteins (UCPs) regulate thermogenesis, metabolic and energy balance, and decrease reactive oxygen species production. Both microRNA and UCP2 expression can be altered in cancer cells. At low power, laser wavelength, frequency, fluence and emission mode deternube photobiological responses, which are the basis of low-level laser therapy. There are few studies on miRNA and UCP mRNA levels after low-level laser exposure on cancer cells. In this work, we evaluate the micrRNA (mir-106b and mir-15a) and UCP2 mRNA levels in human breast cancer cells exposed to low-level lasers. MDA-MB-231 human breast cancer cells were exposed to low-level red and infrared lasers, total RNA was extracted for cDNA synthesis and mRNA levels by real time quantitative polymerase chain reaction were evaluated. Data show that mir-106b and mir-15a relative levels are not altered, but UCP2 mRNA relative levels are increased in MDA-MB-231 human breast cancer cells exposed to low-level red and infrared lasers at fluences used in therapeutic protocols.

  18. Regulation and function of FTO mRNA expression in human skeletal muscle and subcutaneous adipose tissue

    DEFF Research Database (Denmark)

    Grunnet, Louise G; Nilsson, Emma; Ling, Charlotte

    2009-01-01

    and adipose tissue, and their influence on in vivo glucose and fat metabolism. Research Design and Methods. The FTO rs9939609 polymorphism was genotyped in two twin cohorts: 1) 298 elderly twins aged 62-83 years with glucose tolerance ranging from normal to type 2 diabetes and 2) 196 young (25-32 years...... expression was not influenced by FTO rs9939609 genotype. FTO mRNA expression in skeletal muscle was regulated by age and sex, whereas age and BMI were predictors of adipose tissue FTO mRNA expression. FTO mRNA expression in adipose tissue was associated with an atherogenic lipid profile. In skeletal muscle...

  19. The mRNA expression profile of metabolic genes relative to MHC isoform pattern in human skeletal muscles

    DEFF Research Database (Denmark)

    Plomgaard, Peter; Penkowa, Milena; Leick, Lotte;

    2006-01-01

    was more than twofold higher in soleus and vastus than in triceps. Contrary, phosphofructokinase and total lactate dehydrogenase (LDH) activity was approximately three- and twofold higher in triceps than in both soleus and vastus. Expression of metabolic genes was assessed by determining the mRNA content...... of a broad range of metabolic genes. The triceps muscle had two- to fivefold higher MHC IIa, phosphofructokinase, and LDH A mRNA content and two- to fourfold lower MHC I, lipoprotein lipase, CD36, hormone-sensitive lipase, and LDH B and hexokinase II mRNA than vastus lateralis or soleus. Interestingly...

  20. Comparative expression profile of miRNA and mRNA in primary peripheral blood mononuclear cells infected with human immunodeficiency virus (HIV-1.

    Directory of Open Access Journals (Sweden)

    Ankit Gupta

    Full Text Available Host cells respond to exogenous infectious agents such as viruses, including HIV-1. Studies have evaluated the changes associated with virus infection at the transcriptional and translational levels of the cellular genes involved in specific pathways. While this approach is useful, in our view it provides only a partial view of genome-wide changes. Recently, technological advances in the expression profiling at the microRNA (miRNA and mRNA levels have made it possible to evaluate the changes in the components of multiple pathways. To understand the role of miRNA and its interplay with host cellular gene expression (mRNA during HIV-1 infection, we performed a comparative global miRNA and mRNA microarray using human PBMCs infected with HIV-1. The PBMCs were derived from multiple donors and were infected with virus generated from the molecular clone pNL4-3. The results showed that HIV-1 infection led to altered regulation of 21 miRNAs and 444 mRNA more than 2-fold, with a statistical significance of p<0.05. Furthermore, the differentially regulated miRNA and mRNA were shown to be associated with host cellular pathways involved in cell cycle/proliferation, apoptosis, T-cell signaling, and immune activation. We also observed a number of inverse correlations of miRNA and mRNA expression in infected PBMCs, further confirming the interrelationship between miRNA and mRNA regulation during HIV-1 infection. These results for the first time provide evidence that the miRNA profile could be an early indicator of host cellular dysfunction induced by HIV-1.

  1. Human papillomavirus (HPV) E6/E7 mRNA detection in cervical exfoliated cells: a potential triage for HPV-positive women.

    Science.gov (United States)

    Yao, Ye-Li; Tian, Qi-Fang; Cheng, Bei; Cheng, Yi-Fan; Ye, Jing; Lu, Wei-Guo

    Cytology triage has been generally recommended for human papillomavirus (HPV)-positive women, but is highly dependent on well-trained cytologists. The present study was designed to explore whether HPV E6/E7 mRNA detection in cervical exfoliated cells can be a potential triage for HPV-positive women from a clinic-based population. Both the primary HPV testing and Papanicolaou (Pap) test were performed on all eligible HPV-positive women. HPV E6/E7 mRNA was detected by QuantiVirus(®) HPV E6/E7 mRNA assay in cervical exfoliated cells. All HPV-positive women underwent colposcopy and further biopsy if indicated. The data were assessed by Pearson's Chi-squared test and the receiver operating characteristic curve. A total of 404 eligible HPV-positive women were enrolled. Positive rate of E6/E7 mRNA in high-grade squamous intraepithelial lesion (HSIL) cases was higher than that in low-grade squamous intraepithelial lesion (LSIL) or normal cases. There was no statistical difference found between mRNA and cytological testing with sensitivity (89.52% vs. 86.67%, P=0.671), specificity (48.96% vs. 48.96%, P=1.000), positive predictive value (39.00% vs. 38.24%, P=1.000), and negative predictive value (92.76% vs. 90.97%, P=0.678) for detecting ≥HSIL. HPV E6/E7 mRNA detection in cervical exfoliated cells shows the same performance as Pap triage for HSIL identification for HPV-positive women. Detection of HPV E6/E7 mRNA may be used as a new triage option for HPV-positive women.

  2. Rational Design of Human Metapneumovirus Live Attenuated Vaccine Candidates by Inhibiting Viral mRNA Cap Methyltransferase

    Science.gov (United States)

    Zhang, Yu; Wei, Yongwei; Zhang, Xiaodong; Cai, Hui; Niewiesk, Stefan

    2014-01-01

    ABSTRACT The paramyxoviruses human respiratory syncytial virus (hRSV), human metapneumovirus (hMPV), and human parainfluenza virus type 3 (hPIV3) are responsible for the majority of pediatric respiratory diseases and inflict significant economic loss, health care costs, and emotional burdens. Despite major efforts, there are no vaccines available for these viruses. The conserved region VI (CR VI) of the large (L) polymerase proteins of paramyxoviruses catalyzes methyltransferase (MTase) activities that typically methylate viral mRNAs at positions guanine N-7 (G-N-7) and ribose 2′-O. In this study, we generated a panel of recombinant hMPVs carrying mutations in the S-adenosylmethionine (SAM) binding site in CR VI of L protein. These recombinant viruses were specifically defective in ribose 2′-O methylation but not G-N-7 methylation and were genetically stable and highly attenuated in cell culture and viral replication in the upper and lower respiratory tracts of cotton rats. Importantly, vaccination of cotton rats with these recombinant hMPVs (rhMPVs) with defective MTases triggered a high level of neutralizing antibody, and the rats were completely protected from challenge with wild-type rhMPV. Collectively, our results indicate that (i) amino acid residues in the SAM binding site in the hMPV L protein are essential for 2′-O methylation and (ii) inhibition of mRNA cap MTase can serve as a novel target to rationally design live attenuated vaccines for hMPV and perhaps other paramyxoviruses, such as hRSV and hPIV3. IMPORTANCE Human paramyxoviruses, including hRSV, hMPV, and hPIV3, cause the majority of acute upper and lower respiratory tract infections in humans, particularly in infants, children, the elderly, and immunocompromised individuals. Currently, there is no licensed vaccine available. A formalin-inactivated vaccine is not suitable for these viruses because it causes enhanced lung damage upon reinfection with the same virus. A live attenuated vaccine

  3. The human insulin mRNA is partly translated via a cap- and eIF4A-independent mechanism.

    Science.gov (United States)

    Fred, Rikard G; Sandberg, Monica; Pelletier, Jerry; Welsh, Nils

    2011-09-09

    The aim of this study was to investigate whether cap-independent insulin mRNA translation occurs in human pancreatic islets at basal conditions, during stimulation at a high glucose concentration and at conditions of nitrosative stress. We also aimed at correlating cap-independent insulin mRNA translation with binding of the IRES trans-acting factor polypyrimidine tract binding protein (PTB) to the 5'-UTR of insulin mRNA. For this purpose, human islets were incubated for 2h in the presence of low (1.67 mM) or high glucose (16.7 mM). Nitrosative stress was induced by addition of 1 mM DETA/NO and cap-dependent mRNA translation was inhibited with hippuristanol. Insulin biosynthesis rates were determined by radioactive labeling and immunoprecipitation. PTB affinity to insulin mRNA 5'-UTR was assessed by a magnetic micro bead pull-down procedure. We observed that in the presence of 1.67 mM glucose, approximately 70% of the insulin mRNA translation was inhibited by hippuristanol. Corresponding value from islets incubated at 16.7 mM glucose was 93%. DETA/NO treatment significantly decreased the translation of insulin by 85% in high glucose incubated islets, and by 50% at a low glucose concentration. The lowered insulin biosynthesis rates of DETA/NO-exposed islets were further suppressed by hippuristanol with 55% at 16.7 mM glucose but not at 1.67 mM glucose. Thus, hippuristanol-induced inhibition of insulin biosynthesis was less pronounced in DETA/NO-treated islets as compared to control islets. We observed also that PTB bound specifically to the insulin mRNA 5'-UTR in vitro, and that this binding corresponded well with rates of cap-independent insulin biosynthesis at the different conditions. In conclusion, our studies show that insulin biosynthesis is mainly cap-dependent at a high glucose concentration, but that the cap-independent biosynthesis of insulin can constitute as much as 40-100% of all insulin biosynthesis during conditions of nitrosative stress. These data

  4. Saturated fatty acids induce post-transcriptional regulation of HAMP mRNA via AU-rich element-binding protein, human antigen R (HuR).

    Science.gov (United States)

    Lu, Sizhao; Mott, Justin L; Harrison-Findik, Duygu Dee

    2015-10-02

    Iron is implicated in fatty liver disease pathogenesis. The human hepcidin gene, HAMP, is the master switch of iron metabolism. The aim of this study is to investigate the regulation of HAMP expression by fatty acids in HepG2 cells. For these studies, both saturated fatty acids (palmitic acid (PA) and stearic acid (SA)) and unsaturated fatty acid (oleic acid (OA)) were used. PA and, to a lesser extent, SA, but not OA, up-regulated HAMP mRNA levels, as determined by real-time PCR. To understand whether PA regulates HAMP mRNA at the transcriptional or post-transcriptional level, the transcription inhibitor actinomycin D was employed. PA-mediated induction of HAMP mRNA expression was not blocked by actinomycin D. Furthermore, PA activated HAMP 3'-UTR, but not promoter, activity, as shown by reporter assays. HAMP 3'-UTR harbors a single AU-rich element (ARE). Mutation of this ARE abolished the effect of PA, suggesting the involvement of ARE-binding proteins. The ARE-binding protein human antigen R (HuR) stabilizes mRNA through direct interaction with AREs on 3'-UTR. HuR is regulated by phosphorylation-mediated nucleo-cytoplasmic shuttling. PA activated this process. The binding of HuR to HAMP mRNA was also induced by PA in HepG2 cells. Silencing of HuR by siRNA abolished PA-mediated up-regulation of HAMP mRNA levels. PKC is known to phosphorylate HuR. Staurosporine, a broad-spectrum PKC inhibitor, inhibited both PA-mediated translocation of HuR and induction of HAMP expression. Similarly, rottlerin, a novel class PKC inhibitor, abrogated PA-mediated up-regulation of HAMP expression. In conclusion, lipids mediate post-transcriptional regulation of HAMP throughPKC- and HuR-dependent mechanisms.

  5. mRNA and DNA detection of human papillomaviruses in women of all ages attending two colposcopy clinics.

    Directory of Open Access Journals (Sweden)

    Aris Spathis

    Full Text Available OBJECTIVE: HPV infection is a common finding, especially in young women while the majority of infections are cleared within a short time interval. The aim of this study was to examine the efficacy of HPV DNA and mRNA testing in a population attending colposcopy units of two University hospitals. METHODS: 1173 liquid based cervical samples from two colposcopy clinics were tested for HPV DNA positivity using a commercial typing kit and HPV E6/E7 mRNA positivity with a flow cytometry based commercial kit. Statistic measures were calculated for both molecular tests and morphological cytology and colposcopy diagnosis according to histology results. RESULTS: HPV DNA, high-risk HPV DNA, HPV16 or 18 DNA and HPV mRNA was detected in 55.5%, 50.6%, 20.1% and 29.7% of the cervical smears respectively. Concordance between the DNA and the mRNA test was 71.6% with their differences being statistically significant. Both tests' positivity increased significantly as lesion grade progressed and both displayed higher positivity rates in samples from women under 30 years old. mRNA testing displayed similar NPV, slightly lower sensitivity but significantly higher specificity and PPV than DNA testing, except only when DNA positivity for either HPV16 or 18 was used. CONCLUSIONS: Overall mRNA testing displayed higher clinical efficacy than DNA testing, either when used as a reflex test or as an ancillary test combined with morphology. Due to enhanced specificity of mRNA testing and its comparable sensitivity in ages under 25 or 30 years old, induction of mRNA testing in young women could be feasible if a randomized trial verifies these results.

  6. Morphine and endomorphins differentially regulate micro-opioid receptor mRNA in SHSY-5Y human neuroblastoma cells.

    Science.gov (United States)

    Yu, Xin; Mao, Xin; Blake, Allan D; Li, Wen Xin; Chang, Sulie L

    2003-08-01

    A sensitive quantitative-competitive reverse transcriptase-polymerase chain reaction method was developed to measure micro-opioid receptor (MOR) mRNA expression in SHSY-5Y neuroblastoma cells. Differentiation of SHSY-5Y cells with either retinoic acid (RA) or 12-o-tetradecanoyl-phorbol-13-acetate (TPA) significantly increased MOR mRNA levels. Morphine treatment (10 microM) for 24 h decreased MOR mRNA levels in control, as well as RA- and TPA-differentiated cells. In contrast, chronic exposure to the opioid peptides endomorphin-1 or endomorphin-2 significantly increased MOR mRNA levels in undifferentiated and RA-differentiated cells. An opioid antagonist, naloxone, reversed the morphine and endomorphin-1 and -2 effects on MOR mRNA levels in undifferentiated SHSY-5Y cells, but naloxone had differential reversing effects on the agonists' regulation of MOR mRNA in RA- or TPA-differentiated cells. To investigate whether the changes in MOR mRNA expression paralleled changes in MOR receptor function, intracellular cAMP accumulation in SHSY-5Y cells was measured. After chronic treatment with morphine, forskolin-induced cAMP levels in SHSY-5Y cells were significantly higher than those of untreated control cells. In contrast, forskolin-induced cAMP accumulation levels were lower in cells treated with endomorphin-1 or -2 than in untreated control cells. Together, our studies indicate that the opioid alkaloid morphine and the opioid peptides endomorphin-1 and -2 differentially regulate MOR mRNA expression and MOR function in SHSY-5Y cells.

  7. Human IFIT1 Inhibits mRNA Translation of Rubulaviruses but Not Other Members of the Paramyxoviridae Family

    Science.gov (United States)

    Young, D. F.; Andrejeva, J.; Li, X.; Inesta-Vaquera, F.; Dong, C.; Cowling, V. H.; Goodbourn, S.

    2016-01-01

    ABSTRACT We have previously shown that IFIT1 is primarily responsible for the antiviral action of interferon (IFN) alpha/beta against parainfluenza virus type 5 (PIV5), selectively inhibiting the translation of PIV5 mRNAs. Here we report that while PIV2, PIV5, and mumps virus (MuV) are sensitive to IFIT1, nonrubulavirus members of the paramyxoviridae such as PIV3, Sendai virus (SeV), and canine distemper virus (CDV) are resistant. The IFIT1 sensitivity of PIV5 was not rescued by coinfection with an IFIT1-resistant virus (PIV3), demonstrating that PIV3 does not specifically inhibit the antiviral activity of IFIT1 and that the inhibition of PIV5 mRNAs is regulated by cis-acting elements. We developed an in vitro translation system using purified human IFIT1 to further investigate the mechanism of action of IFIT1. While the translations of PIV2, PIV5, and MuV mRNAs were directly inhibited by IFIT1, the translations of PIV3, SeV, and CDV mRNAs were not. Using purified human mRNA-capping enzymes, we show biochemically that efficient inhibition by IFIT1 is dependent upon a 5′ guanosine nucleoside cap (which need not be N7 methylated) and that this sensitivity is partly abrogated by 2′O methylation of the cap 1 ribose. Intriguingly, PIV5 M mRNA, in contrast to NP mRNA, remained sensitive to inhibition by IFIT1 following in vitro 2′O methylation, suggesting that other structural features of mRNAs may influence their sensitivity to IFIT1. Thus, surprisingly, the viral polymerases (which have 2′-O-methyltransferase activity) of rubulaviruses do not protect these viruses from inhibition by IFIT1. Possible biological consequences of this are discussed. IMPORTANCE Paramyxoviruses cause a wide variety of diseases, and yet most of their genes encode structural proteins and proteins involved in their replication cycle. Thus, the amount of genetic information that determines the type of disease that paramyxoviruses cause is relatively small. One factor that will influence

  8. Targeted expression of human FSH receptor Asp567Gly mutant mRNA in testis of transgenic mice: role of human FSH receptor promoter

    Institute of Scientific and Technical Information of China (English)

    VerenaNordhoff; JorgGromoll; LucaFoppiani; C.MarcLuetjens; StefanSchlatt; ElenaKostova; IlpoHuhtaniemi; EberhardNieschlag; ManuelaSimoni

    2003-01-01

    Aim:To specifically express the Asp567Gly human follicle-stimulating hormone receptor (FSHR) under the control of its promoter to evaluate the phenotypic consequences in the presence of normal pituitary function.Methods:We produced transgenic mice overexpressing the Asp567Gly human FSHR under the control of a 1.5kb 5’-flanking region fragment of its promoter.Results: Mice were phenotypically normal and fertile.In males,mRNA could be detected in the testis and the brain, indicating that the 1.5kb promoter fragment drives expression not only in the gonads. The testis weight/body weight ratio and the testosterone levels in transgenic and non-transgenic littermates were similar. By in situ hybridisation we found that the transgenic FSHR was highly expressed in Sertoli cells,spermatocytes and round spermatids. However, a radioligand receptor assay failed to show a significant difference in total FSHR binding sites in testis homogenates of transgenic and wild type animals, suggesting that the transgenic FSHR is probably not translated into functional receptor protein. Conclusion: A 1.5kb 5"-region of the human FSHR drives mRNA expression of the transgene in the testis but leads to ectopic expression in germ cells and in the brain. No phenotypic consequences could be documented due to the lack of protein expression.

  9. Hydrothermal Treatment (HTT) of Microalgae: Evaluation of the Process As Conversion Method in an Algae Biorefinery Concept

    NARCIS (Netherlands)

    Garcia Alba, L.; Torri, C.; Samori, C.; Spek, van der J.J.; Fabbri, D.; Kersten, S.R.A.; Brilman, D.W.F.

    2012-01-01

    The hydrothermal treatment (HTT) technology is evaluated for its potential as a process to convert algae and algal debris into a liquid fuel, within a sustainable algae biorefinery concept in which, next to fuels (gaseous and liquid), high value products are coproduced, nutrients and water are recyc

  10. Hydrothermal Treatment (HTT) of Microalgae: Evaluation of the Process As Conversion Method in an Algae Biorefinery Concept

    NARCIS (Netherlands)

    Garcia Alba, Laura; Torri, C.; Samori, C.; van der Spek, J.J.; Fabbri, D.; Kersten, Sascha R.A.; Brilman, Derk Willem Frederik

    2012-01-01

    The hydrothermal treatment (HTT) technology is evaluated for its potential as a process to convert algae and algal debris into a liquid fuel, within a sustainable algae biorefinery concept in which, next to fuels (gaseous and liquid), high value products are coproduced, nutrients and water are

  11. Human Epidermal Growth Factor Receptor-3 mRNA Expression as a Prognostic Marker for Invasive Duct Carcinoma not Otherwise Specified

    Science.gov (United States)

    Hammoda, Ghada Ezat; El-Hefnawy, Sally Mohammed; Abdallah, Rania Abdallah

    2017-01-01

    Introduction Breast cancer is the most common cancer in women and the Erythroblastosis Oncogene B(ErbB) receptor family holds crucial role in its pathogenesis. Human Epidermal Growth Factor Receptor 3 (HER-3) gene over expression in breast tissue has been associated with aggressive clinical behaviour and bad prognosis. Aim To evaluate HER-3 mRNA expression level as a prognostic marker for breast cancer and to correlate its level with other established prognostic parameters. Materials and Methods This study was carried out on specimens of 100 cases that were divided into 40 patients presented with fibroadenoma and 60 patients presented with Invasive Ductal Carcinoma (IDC) not otherwise specified and underwent modified radical mastectomy. All specimens were investigated for HER-2/neu, ER and PR expression by Immunohistochemistry (IHC) and quantitative assay of HER-3 mRNA expression using real time PCR technique. Results There was a significant high HER3 mRNA level in carcinoma cases compared to fibroadenoma. In malignant cases, HER3 mRNA level was significantly associated with advanced T stage, advanced N stage, number of positive lymph nodes, large tumour size and cases associated with an adjacent in situ component. Moreover, HER-3 mRNA level was of highest values in Her-2/neu positive group followed by triple negative cases with the lowest level in luminal group (p<0.05). Conclusion HER-3 gene is upregulated in IDC especially those carrying poor prognostic features. HER-3 mRNA level may identify a subset of patients with a poor prognosis, and who could undergo further evaluation for the efficacy of HER3 targeted anticancer therapy. PMID:28384967

  12. mRNA levels of BACE1 and its interacting proteins, RTN3 and PPIL2, correlate in human post mortem brain tissue.

    Science.gov (United States)

    Beyer, N; Coulson, D T R; Quinn, J G; Brockbank, S; Hellemans, J; Irvine, G B; Ravid, R; Johnston, J A

    2014-08-22

    β-Site amyloid precursor protein cleaving enzyme (BACE1) is the rate-limiting enzyme for production of Aβ peptides, proposed to drive the pathological changes found in Alzheimer's disease (AD). Reticulon 3 (RTN3) is a negative modulator of BACE1 (β-secretase) proteolytic activity, while peptidylprolyl isomerase (cyclophilin)-like 2 (PPIL2) positively regulated BACE1 gene expression in a cell-based assay. This study aimed to analyze RTN3 and PPIL2 mRNA levels in four brain regions from individuals with AD and controls. BACE1 mRNA had been previously quantified in the samples, as had glial fibrillary acidic protein (GFAP) and neuron-specific enolase (NSE), to track changing cell populations in the tissue. mRNA levels in the human post mortem brain tissue were assayed using quantitative real-time polymerase chain reaction (qPCR) and qbase(PLUS), employing validated stably expressed reference genes. No differences in RTN3 or PPIL2 mRNA levels were found in individuals with AD, compared to controls. Both RTN3 and PPIL2 mRNA levels correlated significantly with BACE1 mRNA and all three showed similar disease stage-dependent changes with respect to NSE and GFAP. These findings indicated that the in vitro data demonstrating an effect of PPIL2 on BACE1 expression have functional relevance in vivo. Further research into BACE1-interacting proteins could provide a fruitful approach to the modulation of this protease and consequently Aβ production.

  13. Inhibitory effect of theobromine on induction of angiogenesis and VEGF mRNA expression in v-raf transfectants of human urothelial cells HCV-29.

    Science.gov (United States)

    Skopinska-Rózewska, E; Janik, P; Przybyszewska, M; Sommer, E; Bialas-Chromiec, B

    1998-12-01

    Neovascularisation plays a crucial role in solid tumor growth and metastasis formation. Our previous studies showed that theophylline and theobromine suppressed cutaneous neovascular reaction induced in mice by human blood leukocytes, and lung as well as ovarian cancer cells. Here, we investigated the in vivo effect of theobromine on angiogenic activity of human urothelial cell line HCV-29, v-raf transfected (mouse cutaneous assay), and the in vitro effect of this drug on VEGF, tPA, uPA and PAI mRNA expression in these cells (RT-PCR method). Theobromine suppressed angiogenesis induced in mice by HCV-29-v-raf cells, inhibited VEGF mRNA expression, and had no effect on transcription of uPA and tPA in these cells. HCV-29-v-raf transfectants do not display transcripts of PAI, in the presence or the absence of theobromine.

  14. Intronic SNP in ESR1 encoding human estrogen receptor alpha is associated with brain ESR1 mRNA isoform expression and behavioral traits.

    Science.gov (United States)

    Pinsonneault, Julia K; Frater, John T; Kompa, Benjamin; Mascarenhas, Roshan; Wang, Danxin; Sadee, Wolfgang

    2017-01-01

    Genetic variants of ESR1 have been implicated in multiple diseases, including behavioral disorders, but causative variants remain uncertain. We have searched for regulatory variants affecting ESR1 expression in human brain, measuring allelic ESR1 mRNA expression in human brain tissues with marker SNPs in exon4 representing ESR1-008 (or ESRα-36), and in the 3'UTR of ESR1-203, two main ESR1 isoforms in brain. In prefrontal cortex from subjects with bipolar disorder, schizophrenia, and controls (n = 35 each; Stanley Foundation brain bank), allelic ESR1 mRNA ratios deviated from unity up to tenfold at the exon4 marker SNP, with large allelic ratios observed primarily in bipolar and schizophrenic subjects. SNP scanning and targeted sequencing identified rs2144025, associated with large allelic mRNA ratios (p = 1.6E10-6). Moreover, rs2144025 was significantly associated with ESR1 mRNA levels in the Brain eQTL Almanac and in brain regions in the Genotype-Tissue Expression project. In four GWAS cohorts, rs2104425 was significantly associated with behavioral traits, including: hypomanic episodes in female bipolar disorder subjects (GAIN bipolar disorder study; p = 0.0004), comorbid psychological symptoms in both males and females with attention deficit hyperactivity disorder (GAIN ADHD, p = 0.00002), psychological diagnoses in female children (eMERGE study of childhood health, subject age ≥9, p = 0.0009), and traits in schizophrenia (e.g., grandiose delusions, GAIN schizophrenia, p = 0.0004). The first common ESR1 variant (MAF 12-33% across races) linked to regulatory functions, rs2144025 appears conditionally to affect ESR1 mRNA expression in the brain and modulate traits in behavioral disorders.

  15. Fas ligand expression in human and mouse cancer cell lines; a caveat on over-reliance on mRNA data

    Directory of Open Access Journals (Sweden)

    Ryan Aideen E

    2006-02-01

    Full Text Available Abstract Background During carcinogenesis, tumors develop multiple mechanisms for evading the immune response, including upregulation of Fas ligand (FasL/CD95L expression. Expression of FasL may help to maintain tumor cells in a state of immune privilege by inducing apoptosis of anti-tumor immune effector cells. Recently this idea has been challenged by studies reporting that tumor cells of varying origin do not express FasL. In the present study, we aimed to comprehensively characterize FasL expression in tumors of both murine and human origin over a 72 hour time period. Methods RNA and protein was extracted from six human (SW620, HT29, SW480, KM12SM, HCT116, Jurkat and three mouse (CMT93, CT26, B16F10 cancer cell lines at regular time intervals over a 72 hour time period. FasL expression was detected at the mRNA level by RT-PCR, using intron spanning primers, and at the protein level by Western Blotting and immunofluorescence, using a polyclonal FasL- specific antibody. Results Expression of FasL mRNA and protein was observed in all cell lines analysed. However, expression of FasL mRNA varied dramatically over time, with cells negative for FasL mRNA at many time points. In contrast, 8 of the 9 cell lines constitutively expressed FasL protein. Thus, cells can abundantly express FasL protein at times when FasL mRNA is absent. Conclusion These findings demonstrate the importance of complete analysis of FasL expression by tumor cells in order to fully characterize its biological function and may help to resolve the discrepancies present in the literature regarding FasL expression and tumor immune privilege.

  16. Age-dependent decrease and alternative splicing of methionine synthase mRNA in human cerebral cortex and an accelerated decrease in autism.

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    Christina R Muratore

    Full Text Available The folate and vitamin B12-dependent enzyme methionine synthase (MS is highly sensitive to cellular oxidative status, and lower MS activity increases production of the antioxidant glutathione, while simultaneously decreasing more than 200 methylation reactions, broadly affecting metabolic activity. MS mRNA levels in postmortem human cortex from subjects across the lifespan were measured and a dramatic progressive biphasic decrease of more than 400-fold from 28 weeks of gestation to 84 years was observed. Further analysis revealed alternative splicing of MS mRNA, including deletion of folate-binding domain exons and age-dependent deletion of exons from the cap domain, which protects vitamin B12 (cobalamin from oxidation. Although three species of MS were evident at the protein level, corresponding to full-length and alternatively spliced mRNA transcripts, decreasing mRNA levels across the lifespan were not associated with significant changes in MS protein or methionine levels. MS mRNA levels were significantly lower in autistic subjects, especially at younger ages, and this decrease was replicated in cultured human neuronal cells by treatment with TNF-α, whose CSF levels are elevated in autism. These novel findings suggest that rather than serving as a housekeeping enzyme, MS has a broad and dynamic role in coordinating metabolism in the brain during development and aging. Factors adversely affecting MS activity, such as oxidative stress, can be a source of risk for neurological disorders across the lifespan via their impact on methylation reactions, including epigenetic regulation of gene expression.

  17. Human retina-specific amine oxidase: genomic structure of the gene (AOC2), alternatively spliced variant, and mRNA expression in retina.

    Science.gov (United States)

    Imamura, Y; Noda, S; Mashima, Y; Kudoh, J; Oguchi, Y; Shimizu, N

    1998-07-15

    Previously, we reported the isolation of cDNA for human retina-specific amine oxidase (RAO) and the expression of RAO exclusively in retina. Bacterial artificial chromosome clones containing the human RAO gene (AOC2) were mapped to human chromosome 17q21 (Imamura et al., 1997, Genomics 40: 277-283). Here, we report the complete genomic structure of the RAO gene, including 5' flanking sequence, and mRNA expression in retina. The human RAO gene spans 6 kb and is composed of four exons corresponding to the amino acid sequence 1-530, 530-598, 598-641, and 642-729 separated by three introns of 3000, 310, and 351 bp. Screening of a human retina cDNA library revealed the existence of an alternatively spliced cDNA variant with an additional 81 bp at the end of exon 2. The sizes of exons and the locations of exon/intron boundaries in the human RAO gene showed remarkable similarity to those of the human kidney diamine oxidase gene (AOC1). In situ hybridization revealed that mRNA coding for RAO is expressed preferentially in the ganglion cell layer of the mouse retina. We designed four sets of PCR primers to amplify four exons, which will be valuable for analyzing mutations in patients with ocular diseases affecting the retinal ganglion cell layer.

  18. Nup42 and IP6 coordinate Gle1 stimulation of Dbp5/DDX19B for mRNA export in yeast and human cells.

    Science.gov (United States)

    Adams, Rebecca L; Mason, Aaron C; Glass, Laura; Aditi; Wente, Susan R

    2017-09-04

    The mRNA lifecycle is driven through spatiotemporal changes in the protein composition of mRNA particles (mRNPs) that are triggered by RNA-dependent DEAD-box protein (Dbp) ATPases. As mRNPs exit the nuclear pore complex (NPC) in Saccharomyces cerevisiae, this remodeling occurs through activation of Dbp5 by inositol hexakisphosphate (IP6 )-bound Gle1. At the NPC, Gle1 also binds Nup42, but Nup42's molecular function is unclear. Here we employ the power of structure-function analysis in S. cerevisiae and human (h) cells, and find that the high-affinity Nup42-Gle1 interaction is integral to Dbp5 (hDDX19B) activation and efficient mRNA export. The Nup42 carboxy-terminal domain (CTD) binds Gle1/hGle1B at an interface distinct from the Gle1-Dbp5/hDDX19B interaction site. A nup42-CTD/gle1-CTD/Dbp5 trimeric complex forms in the presence of IP6 . Deletion of NUP42 abrogates Gle1-Dbp5 interaction, and disruption of the Nup42 or IP6 binding interfaces on Gle1/hGle1B leads to defective mRNA export in S. cerevisiae and human cells. In vitro, Nup42-CTD and IP6 stimulate Gle1/hGle1B activation of Dbp5 and DDX19B recombinant proteins in similar, non-additive manners, demonstrating complete functional conservation between humans and S. cerevisiae. Together, a highly conserved mechanism governs spatial coordination of mRNP remodeling during export. This has implications for understanding human disease mutations that perturb the Nup42-hGle1B interaction. This article is protected by copyright. All rights reserved.

  19. Dominant effects of the Huntington's disease HTT CAG repeat length are captured in gene-expression data sets by a continuous analysis mathematical modeling strategy.

    Science.gov (United States)

    Lee, Jong-Min; Galkina, Ekaterina I; Levantovsky, Rachel M; Fossale, Elisa; Anne Anderson, Mary; Gillis, Tammy; Srinidhi Mysore, Jayalakshmi; Coser, Kathryn R; Shioda, Toshi; Zhang, Bin; Furia, Matthew D; Derry, Jonathan; Kohane, Isaac S; Seong, Ihn Sik; Wheeler, Vanessa C; Gusella, James F; MacDonald, Marcy E

    2013-08-15

    In Huntington's disease (HD), the size of the expanded HTT CAG repeat mutation is the primary driver of the processes that determine age at onset of motor symptoms. However, correlation of cellular biochemical parameters also extends across the normal repeat range, supporting the view that the CAG repeat represents a functional polymorphism with dominant effects determined by the longer allele. A central challenge to defining the functional consequences of this single polymorphism is the difficulty of distinguishing its subtle effects from the multitude of other sources of biological variation. We demonstrate that an analytical approach based upon continuous correlation with CAG size was able to capture the modest (∼21%) contribution of the repeat to the variation in genome-wide gene expression in 107 lymphoblastoid cell lines, with alleles ranging from 15 to 92 CAGs. Furthermore, a mathematical model from an iterative strategy yielded predicted CAG repeat lengths that were significantly positively correlated with true CAG allele size and negatively correlated with age at onset of motor symptoms. Genes negatively correlated with repeat size were also enriched in a set of genes whose expression were CAG-correlated in human HD cerebellum. These findings both reveal the relatively small, but detectable impact of variation in the CAG allele in global data in these peripheral cells and provide a strategy for building multi-dimensional data-driven models of the biological network that drives the HD disease process by continuous analysis across allelic panels of neuronal cells vulnerable to the dominant effects of the HTT CAG repeat.

  20. Sensitivity, specificity, and clinical value of human papillomavirus (HPV) E6/E7 mRNA assay as a triage test for cervical cytology and HPV DNA test.

    Science.gov (United States)

    Benevolo, Maria; Vocaturo, Amina; Caraceni, Donatella; French, Deborah; Rosini, Sandra; Zappacosta, Roberta; Terrenato, Irene; Ciccocioppo, Lucia; Frega, Antonio; Giorgi Rossi, Paolo

    2011-07-01

    There is evidence that testing for human papillomavirus (HPV) E6/E7 mRNA is more specific than testing for HPV DNA. A retrospective study was carried out to evaluate the performance of the PreTect HPV-Proofer E6/E7 mRNA assay (Norchip) as a triage test for cytology and HPV DNA testing. This study analyzed 1,201 women, 688 of whom had a colposcopy follow-up and 195 of whom had histology-confirmed high-grade intraepithelial neoplasia or worse (CIN2+). The proportion of positive results and the sensitivity and specificity for CIN2+ were determined for HPV mRNA in comparison to HPV DNA and cytology. All data were adjusted for follow-up completeness. Stratified by cytological grades, the HPV mRNA sensitivity was 83% (95% confidence interval [CI] = 63 to 94%) in ASC-US (atypical squamous cells of undetermined significance), 62% (95% CI = 47 to 75%) in L-SIL (low-grade squamous intraepithelial lesion), and 67% (95% CI = 57 to 76%) in H-SIL (high-grade squamous intraepithelial lesion). The corresponding figures were 99, 91, and 96%, respectively, for HPV DNA. The specificities were 82, 76, and 45%, respectively, for HPV mRNA and 29, 13, and 4%, respectively, for HPV DNA. Used as a triage test for ASC-US and L-SIL, mRNA reduced colposcopies by 79% (95% CI = 74 to 83%) and 69% (95% CI = 65 to 74%), respectively, while HPV DNA reduced colposcopies by 38% (95% CI = 32 to 44%) and by 15% (95% CI = 12 to 19%), respectively. As a HPV DNA positivity triage test, mRNA reduced colposcopies by 63% (95% CI = 60 to 66%), having 68% sensitivity (95% CI = 61 to 75%), whereas cytology at the ASC-US+ threshold reduced colposcopies by 23% (95% CI = 20 to 26%), showing 92% sensitivity (95% CI = 87 to 95%). In conclusion, PreTect HPV-Proofer mRNA can serve as a better triage test than HPV DNA to reduce colposcopy referral in both ASC-US and L-SIL. It is also more efficient than cytology for the triage of HPV DNA-positive women. Nevertheless, its low sensitivity demands a strict follow-up of

  1. Molecular Detection of Human Telomerase mRNA (hTERT-mRNA in Egyptian Patients with Hepatocellular Carcinoma

    Directory of Open Access Journals (Sweden)

    Gahan Kamal El-Saeed

    2009-06-01

    Full Text Available Background and Aims: Diagnostic modalities for hepatocellular carcinoma (HCC as markers, sonography, and CT have contributed to the early detection of HCC but are still not sensitive enough. Human telomerase RNA subunit (hTERT-mRNA has been identified in many cancers and claimed to be reactivated in HCC. To investigate hTERT-mRNA in the peripheral blood of HCC and chronic liver disease (CLD patients and correlate its level with alpha feto protein (AFP, the serological marker for HCC.Methods: The study was conducted on 44 patients selected from the National Liver Institute. Patients included Group I (22 patients diagnosed to have HCC, Group II (22 patients with CLD, and 12 apparently healthy volunteers as controls (Group III. All selected individuals were subjected to history taking, a clinical examination, abdominal sonography and laboratory investigations as liver function tests (LFTs, cell blood count (CBC, hepatitis viral markers, AFP, and real-time polymerase chain reaction (PCR Quantitative detection of -mRNA expression, encoding for telomerase catalytic subunit.Results: There was a significant elevation of AFP levels in the HCC group compared to both the CLD and control groups (P < 0.00, P < 0.001. The mean hTERT-mRNA expression in HCC patients was significantly higher than both CLD patients and controls (P < 0.001, P < 0.001. hTERT-mRNA was correlated with AFP and tumor size (P < 0.05, P < 0.001. The AFP cutoff level (185 ng/ml resulted in a 63.6% sensitivity, a 85.3% specificity; a 89.3% positive predictive value (PPV level, a 76.2 % negative predictive value (NPV level and a 83.4% accuracy for HCC prediction. The hTERT-mRNA cutoff level (112.5 copies/ml showed a 77.3% sensitivity, a 97.1% specificity, a 98% PPV level, a 79.2 % NPV level, and an accuracy of 84% for HCC prediction. Combining hTERT-mRNA and AFP increased diagnostic accuracy to 90.5%. Both markers had a 84.1% sensitivity, a 86.4% specificity, a 86.4% PPV level, and a 88

  2. Urokinase receptor mRNA level and gene transcription are strongly and rapidly increased by phorbol myristate acetate in human monocyte-like U937 cells

    DEFF Research Database (Denmark)

    Lund, L R; Rønne, E; Roldan, A L;

    1991-01-01

    We have studied the effect of the tumor promotor phorbol myristate acetate (PMA) on the level of mRNA for the receptor for urokinase-type plasminogen activator (u-PAR) in the human monocyte-like cell line U937. PMA causes an early increase in the u-PAR mRNA level which reaches a maximal 50-fold...... enhancement after 24 h of treatment. Half-maximal stimulation occurs at approximately 5 nM PMA. The effect is observed only with phorbol esters that also act as tumor promotors. The protein synthesis inhibitor cycloheximide (10 micrograms/ml) also increases the level of u-PAR mRNA. Nuclear run-on experiments...... show a time-dependent increase in the u-PAR gene transcription rate after exposure of the cells to PMA. The PMA-induced increase in u-PAR mRNA is paralleled by a time-dependent increase in u-PAR protein as detected by cross-linking studies with radiolabeled ligand. We conclude that PMA stimulates...

  3. Electroporated Antigen-Encoding mRNA Is Not a Danger Signal to Human Mature Monocyte-Derived Dendritic Cells

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    Stefanie Hoyer

    2015-01-01

    Full Text Available For therapeutic cancer vaccination, the adoptive transfer of mRNA-electroporated dendritic cells (DCs is frequently performed, usually with monocyte-derived, cytokine-matured DCs (moDCs. However, DCs are rich in danger-sensing receptors which could recognize the exogenously delivered mRNA and induce DC activation, hence influencing the DCs’ immunogenicity. Therefore, we examined whether electroporation of mRNA with a proper cap and a poly-A tail of at least 64 adenosines had any influence on cocktail-matured moDCs. We used 16 different RNAs, encoding tumor antigens (MelanA, NRAS, BRAF, GNAQ, GNA11, and WT1, and variants thereof. None of those RNAs induced changes in the expression of CD25, CD40, CD83, CD86, and CD70 or the secretion of the cytokines IL-8, IL-6, and TNFα of more than 1.5-fold compared to the control condition, while an mRNA encoding an NF-κB-activation protein as positive control induced massive secretion of the cytokines. To determine whether mRNA electroporation had any effect on the whole transcriptome of the DCs, we performed microarray analyses of DCs of 6 different donors. None of 60,000 probes was significantly different between mock-electroporated DCs and MelanA-transfected DCs. Hence, we conclude that no transcriptional programs were induced within cocktail-matured DCs by electroporation of single tumor-antigen-encoding mRNAs.

  4. Endurance exercise induces mRNA expression of oxidative enzymes in human skeletal muscle late in recovery

    DEFF Research Database (Denmark)

    Leick, Lotte; Plomgaard, Peter S.; Grønløkke, L.

    2010-01-01

    exercise. To test the hypothesis that mRNA expression of many oxidative enzymes is up-regulated late in recovery (10-24 h) after exercise, male subjects (n=8) performed a 90-min cycling exercise (70% VO(2-max)), with muscle biopsies obtained before exercise (pre), and after 10, 18 and 24 h of recovery...

  5. OIL FLY ASH AND VANADIUM DIMINISH NRAMP-2MRNA AND PROTEIN EXPRESSION IN HUMAN BRONCHIAL EPITHELIAL CELLS

    Science.gov (United States)

    The capacity of Nramp2 to transport iron and its ubiquitous expression make it a likely candidate for transferrin-independent uptake of iron in peripheral tissues. Airway epithelial cells increase both mRNA and expression of that isoform of Nramp-2 without an iron response ele...

  6. Comparative mRNA Expression Profiles of Riboflavin Biosynthesis Genes in Lactobacilli Isolated from Human Feces and Fermented Bamboo Shoots

    Science.gov (United States)

    Thakur, Kiran; Tomar, Sudhir K.; Wei, Zhao-Jun

    2017-01-01

    With the aim to bioprospect potent riboflavin producing lactobacilli, the present study was carried out to evaluate the relative mRNA expression of riboflavin biosynthesis genes namely Rib 1, Rib 2, Rib 3, and Rib 4 from potent riboflavin producers obtained from our previous studies. All the four genes were successfully cloned and sequenced for further analysis by in silico procedures. As studied by non-denaturing Polyacrylamide gel electrophoresis, no difference in size of all the four genes among those of various lactobacilli was observed. The relative fold increase in mRNA expression in Rib 1, Rib 2, Rib 3, and Rib 4 genes has been observed to be 10-, 1-, 0.7-, and 8.5-fold, respectively. Due to increase in relative mRNA expression for all the Rib genes as well as phenotypic production attribute, KTLF1 strain was used further for expression studies in milk and whey. The fold increase in mRNA expression for all the four Rib genes was higher at 12 and 18 h in milk and whey respectively. After exposure to roseoflavin, resistant variant of KTLF1 showed considerable increase in expression of all the targets genes. This is the first ever study to compare the mRNA expression of riboflavin biosynthesis pathway genes in lactobacilli and it also under lines the effect of media and harvesting time which significantly affect the expression of rib genes. The use of roseoflavin-resistant strains capable of synthesizing riboflavin in milk and whey paves a way for an exciting and economically viable biotechnological approach to develop novel riboflavin bio-enriched functional foods. PMID:28367143

  7. 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.

  8. Quantitative Real-Time Reverse Transcription-PCR Assay for the Expression of Tob mRNA in Human Colorectal Cancer

    Institute of Scientific and Technical Information of China (English)

    Dian-chao WU

    2010-01-01

    OBJECTIVE Tob is a member of Tob/BTG antiproliferative family. To date, Tob expression in human carcinoma using clinical specimens has not been studied in depth except for lung carcinoma and thyroid carcinoma. This study is the first to investigate the expression levels of Tob gene in human colorectal cancer tissues,and their corresponding para-cancerous tissues. The correlation of expression of the Tob gene with clinicopathological characteristics of colorectal cancer was also analyzed.METHODS Quantitative real time RT-PCR was used to detect the expression of Tob mRNA in 31 colorectal cancers.RESULTS Compared with normal tissues, up-regulation of Tob mRNA was observed in 31 colorectal cancer tissues (P = 0.020).The expression level of Tob at Dukes C + D phase was higher than Dukes A + B phase, and the difference was signifi cant (P < 0.05).However, in this study, it was found that the expression of Tob mRNA was not related with age, gender, and pathological type of colorectal cancer.CONCLUSION The up-regulation of Tob may be closely associated with tumorigenesis of colorectal carcinoma.

  9. Human papillomavirus mRNA and DNA testing in women with atypical squamous cells of undetermined significance: A prospective cohort study.

    Science.gov (United States)

    Thomsen, Louise T; Dehlendorff, Christian; Junge, Jette; Waldstrøm, Marianne; Schledermann, Doris; Frederiksen, Kirsten; Kjaer, Susanne K

    2016-10-15

    In this prospective cohort study, we compared the performance of human papillomavirus (HPV) mRNA and DNA testing of women with atypical squamous cells of undetermined significance (ASC-US) during cervical cancer screening. Using a nationwide Danish pathology register, we identified women aged 30-65 years with ASC-US during 2005-2011 who were tested for HPV16/18/31/33/45 mRNA using PreTect HPV-Proofer (n = 3,226) or for high-risk HPV (hrHPV) DNA using Hybrid Capture 2 (HC2) (n = 9,405) or Linear Array HPV-Genotyping test (LA) (n = 1,533). Women with ≥1 subsequent examination in the register (n = 13,729) were followed for up to 9.5 years for high-grade cervical intraepithelial neoplasia (CIN) or cancer. After 3 years' follow-up, mRNA testing had higher specificity for CIN3 or worse (CIN3+) than HC2 testing (88.1% [95% confidence interval (CI): 86.8-89.6%] versus 59.3% [95% CI: 58.1-60.4%]) and higher positive predictive value (PPV) (38.2% [95% CI: 33.8%-43.1%] versus 19.5% [95% CI: 17.8-20.9%]). However, the sensitivity of mRNA testing was lower than that of HC2 testing (66.7% [95% CI: 59.3-74.5%] versus 97.0% [95% CI: 95.5-98.4%]), and women testing mRNA negative had higher 3-year risk for CIN3+ than those testing HC2 negative (3.2% [95% CI: 2.2-4.2%] versus 0.5% [95% CI: 0.3-0.7%]). Patterns were similar after 18 months and 5 years'; follow-up; for CIN2+ and cancer as outcomes; across all age groups; and when comparing mRNA testing to hrHPV DNA testing using LA. In conclusion, the HPV16/18/31/33/45 mRNA test is not optimal for ASC-US triage due to its low sensitivity and the substantial risk for precancer following a negative test.

  10. Calcium and Vitamin D increase mRNA levels for the growth control hIK1 channel in human epidermal keratinocytes but functional channels are not observed

    Directory of Open Access Journals (Sweden)

    Rossie Sandra

    2004-06-01

    Full Text Available Abstract Background Intermediate-conductance, calcium-activated potassium channels (IKs modulate proliferation and differentiation in mesodermal cells by enhancing calcium influx, and they contribute to the physiology of fluid movement in certain epithelia. Previous reports suggest that IK channels stimulate proliferative growth in a keratinocyte cell line; however, because these channels indirectly promote calcium influx, a critically unique component of the keratinocyte differentiation program, an alternative hypothesis is that they would be anti-proliferative and pro-differentiating. This study addresses these hypotheses. Methods Real-time PCR, patch clamp electrophysiology, and proliferation assays were used to determine if human IK1 (hIK1 expression and function are correlated with either proliferation or differentiation in cultured human skin epidermal keratinocytes, and skin biopsies grown in explant culture. Results hIK1 mRNA expression in human keratinocytes and skin was increased in response to anti-proliferative/pro-differentiating stimuli (elevated calcium and Vitamin D. Correspondingly, the hIK1 agonist 1-EBIO inhibited keratinocyte proliferation suggesting that the channel could be anti-proliferative and pro-differentiating. However, this proliferative inhibition by 1-EBIO was not reversed by a panel of hIK1 blockers, calling into question the mechanism of 1-EBIO action. Subsequent patch clamp electrophysiological analysis failed to detect hIK1 channel currents in keratinocytes, even those expressing substantial hIK1 mRNA in response to calcium and Vitamin D induced differentiation. Identical electrophysiological recording conditions were then used to observe robust IK1 currents in fibroblasts which express IK1 mRNA levels comparable to those of keratinocytes. Thus, the absence of observable hIK1 currents in keratinocytes was not a function of the electrophysiological techniques. Conclusion Human keratinocyte differentiation is

  11. Myogenic, matrix and growth factor mRNA expression in human skeletal muscle: effect of contraction intensity and feeding

    DEFF Research Database (Denmark)

    Agergaard, Jakob; Reitelseder, Søren; Pedersen, T.G.

    2013-01-01

    exercise. No major differences were seen in atrophy-related genes between HL and LL resistance exercise. No changes were seen over 12-week training for any of the targets. CONCLUSIONS: Resistance exercise at LL and HL elevated the expression of genes involved in skeletal muscle hypertrophy, although......INTRODUCTION: We examined short-term (3-hour) and long-term (12-week) training effects after heavy load [HL; 70% 1RM] and light load (LL; 16% 1RM) exercise. METHODS: mRNA expression of genes involved in skeletal muscle remodeling were analyzed and muscle activity (EMG measurements) was measured....... RESULTS: Relative muscle activity differed between HL and LL resistance exercise, whereas median power frequency was even, suggesting an equal muscle-fiber-type recruitment distribution. mRNA expression of Myf6, myogenin, and p21 was mostly increased, and myostatin was mostly depressed by HL resistance...

  12. Scalable production in human cells and biochemical characterization of full-length normal and mutant huntingtin.

    Directory of Open Access Journals (Sweden)

    Bin Huang

    Full Text Available Huntingtin (Htt is a 350 kD intracellular protein, ubiquitously expressed and mainly localized in the cytoplasm. Huntington's disease (HD is caused by a CAG triplet amplification in exon 1 of the corresponding gene resulting in a polyglutamine (polyQ expansion at the N-terminus of Htt. Production of full-length Htt has been difficult in the past and so far a scalable system or process has not been established for recombinant production of Htt in human cells. The ability to produce Htt in milligram quantities would be a prerequisite for many biochemical and biophysical studies aiming in a better understanding of Htt function under physiological conditions and in case of mutation and disease. For scalable production of full-length normal (17Q and mutant (46Q and 128Q Htt we have established two different systems, the first based on doxycycline-inducible Htt expression in stable cell lines, the second on "gutless" adenovirus mediated gene transfer. Purified material has then been used for biochemical characterization of full-length Htt. Posttranslational modifications (PTMs were determined and several new phosphorylation sites were identified. Nearly all PTMs in full-length Htt localized to areas outside of predicted alpha-solenoid protein regions. In all detected N-terminal peptides methionine as the first amino acid was missing and the second, alanine, was found to be acetylated. Differences in secondary structure between normal and mutant Htt, a helix-rich protein, were not observed in our study. Purified Htt tends to form dimers and higher order oligomers, thus resembling the situation observed with N-terminal fragments, although the mechanism of oligomer formation may be different.

  13. MicroRNA-mediated target mRNA cleavage and 3′-uridylation in human cells

    OpenAIRE

    Kai Xu; Jing Lin; Roza Zandi; Jack A Roth; Lin Ji

    2016-01-01

    MicroRNAs (miRNAs) play an important role in targeted gene silencing by facilitating posttranscriptional and translational repression. However, the precise mechanism of mammalian miRNA-mediated gene silencing remains to be elucidated. Here, we used a stem-loop array reverse-transcription polymerase chain reaction assay to analyse miRNA-induced mRNA recognition, cleavage, posttranscriptional modification, and degradation. We detected endogenous let-7 miRNA-induced and Argonaute-catalysed endon...

  14. mRNA expression and localization of bNOS, eNOS and iNOS in human cervix at preterm and term labour

    Directory of Open Access Journals (Sweden)

    Byström Birgitta

    2005-08-01

    Full Text Available Abstract Background Preterm birth is the primary cause of the neonatal mortality and morbidity. There will be no preterm birth without a cervical softening. Nitric oxide (NO is shown to be a mediator of term cervical ripening. The aim of this study was to investigate mRNA expression of the three isomers of NO synthases (NOS and to identify them by immunohistochemistry in the human cervix at preterm birth compared to term. Methods The three isomers of NOS- inducible (iNOS, endothelial (eNOS and neuronal (bNOS – were investigated in the human cervix. The expression of mRNA was determined using Real-Time Multiplex RT-PCR. The localisation of synthases in the cervical tissue was analysed using immunohistochemistry. Cervical biopsies were obtained from 4 groups of women without clinical signs of infection: preterm (PTL, term labour (TL, preterm not in labour (PTnotL and term not in labour (TnotL patients. One-Way ANOVA, Kruskal-Wallis, Student t-test or Mann-Whitney test were applied as appropriate to determine statistically significant differences among the groups. Results Patients in preterm labour had significantly (p Conclusion The mRNA levels were higher in the preterm labour group compared to the women at term labour. The significant increase of the eNOS mRNA expression, from the unripe to the favourable cervical state during labour, may indicate a role of eNOS and supports the role of NO in the cervical ripening process. All the three synthases were identified by immunohistochemistry in all the groups of study.

  15. Epidemiology of Huntington disease: first post-HTT gene analysis of prevalence in Italy.

    Science.gov (United States)

    Squitieri, F; Griguoli, A; Capelli, G; Porcellini, A; D'Alessio, B

    2016-03-01

    Huntington disease (HD) prevalence shows geographic variability and has been recently updated by taking into account the mutation diagnostic test. In Italy, the last epidemiological estimation was reported well before the HTT gene discovery and the availability of the corresponding genetic test. It reported a prevalence of affected subjects ranging between 2.3 and 4.8/100,000 in some restricted areas of Northern Italy. We have performed a service-based epidemiological analysis in a very restricted geographic area named Molise, where our institutions currently operate and represent the only point of reference for rare neuropsychiatric diseases. The estimated prevalence rate found was 10.85/100,000 (95% confidence interval (CI): 7.20-14.50), remarkably higher than that previously described before the gene test analysis was available, and expected to an increase of an additional 17% by 2030, because of Italian population aging. According to our analysis, we estimate that about 6500 subjects are currently affected by HD in Italy, and that this number will further increase in the next decades because of population aging, variable phenotype penetrance and improved life expectancy.

  16. 5-HTT VNTR多态性与偏头痛关系的系统评价%A systematic review with meta-analysis on the relationship of 5-HTT VNTR and migraine

    Institute of Scientific and Technical Information of China (English)

    王贤琦; 李光明; 柳华; 冯胜刚

    2011-01-01

    背景5羟色胺(5-HT)在偏头痛的发病机制中起着重要作用,但是研究5-HT转运体(5-HTT)基因多态性和偏头痛关系的单个遗传关联研究的结果却不一致.目的使用系统评价方法评价5-HTT可变数目串联重复序列(VNTR)多态性和偏头痛的关系.方法 广泛检索中英文数据库以发现合格研究,使用随机或固定效应模型计算合并比值比(OR值),使用Q检验评估研究之间异质性,Egger's(埃格)检验和漏斗图评估发表偏倚.以家族为基础的关联研究则进行描述性分析.结果 总共4个研究纳入meta分析,发现在所有人群中,5-HTT VNTR Stin2.12等位基因或12/12基因型增加了偏头痛的发病风险(Stin2.12等位基因:OR:1.34,95%CI:1.09~1.64,P=0.006; 12/12基因型:OR:1.55,95%CI:1.17~2.05,P=0.002).结论 现有证据表明,5-HTT VNTR多态性(主要是Stin2.12基因型)增加了偏头痛的发病风险,该结论需大样本研究进一步验证.%Objective Serotonin is known to play an important role in the pathogenesis of migraine, but individual genetic association studies that examine the relationship between polymorphisms of serotonin transporter (5-HTT) gene and migraine have yielded inconsistent results. This study aimed to evaluate the association between 5-HTT VNTR polymorphism and migraine using systematic review with meta-analysis. Methods Relevant studies were identified by searching English and Chinese databases extensively. Allele and genotype frequencies for each included study were extracted. The odds ratio (OR) was calculated using a random-effects or fixed-effects model. Q statistic was used to evaluate homogeneity, and Egger's test and Funnel plot were used to assess publication bias. For family-based association studies. A descriptive analysis was carried out) Results A total of 4 studies were identified for meta-analysis. It was found that the 5-HTT VNTR Stin2.12 allele or 12/12 genotype had an increased risk for migraine in the

  17. Effect of hepatitis C virus infection on the mRNA expression of drug transporters and cytochrome p450 enzymes in chimeric mice with humanized liver.

    Science.gov (United States)

    Kikuchi, Ryota; McCown, Matthew; Olson, Pamela; Tateno, Chise; Morikawa, Yoshio; Katoh, Yumiko; Bourdet, David L; Monshouwer, Mario; Fretland, Adrian J

    2010-11-01

    The expression of drug transporters and metabolizing enzymes is a primary determinant of drug disposition. Chimeric mice with humanized liver, including PXB mice, are an available model that is permissive to the in vivo infection of hepatitis C virus (HCV), thus being a promising tool for investigational studies in development of new antiviral molecules. To investigate the potential of HCV infection to alter the pharmacokinetics of small molecule antiviral therapeutic agents in PXB mice, we have comprehensively determined the mRNA expression profiles of human ATP-binding cassette (ABC) transporters, solute carrier (SLC) transporters, and cytochrome P450 (P450) enzymes in the livers of these mice under noninfected and HCV-infected conditions. Infection of PXB mice with HCV resulted in an increase in the mRNA expression levels of a series of interferon-stimulated genes in the liver. For the majority of genes involved in drug disposition, minor differences in the mRNA expression of ABC and SLC transporters as well as P450s between the noninfected and HCV-infected groups were observed. The exceptions were statistically significantly higher expression of multidrug resistance-associated protein 4 and organic anion-transporting polypeptide 2B1 and lower expression of organic cation transporter 1 and CYP2D6 in HCV-infected mice. Furthermore, the enzymatic activities of the major human P450s were, in general, comparable in the two experimental groups. These data suggest that the pharmacokinetic properties of small molecule antiviral therapies in HCV-infected PXB mice are likely to be similar to those in noninfected PXB mice. However, caution is needed in the translation of this relationship to HCV-infected patients as the PXB mouse model does not accurately reflect the pathology of patients with chronic HCV infection.

  18. Effect of Nuclear Factor-kappa B on Vascular Endothelial Growth Factor mRNA Expression of Human Pulmonary Artery Smooth Muscle Cells in Hypoxia

    Institute of Scientific and Technical Information of China (English)

    张焕萍; 徐永健; 张珍祥; 许淑云; 倪望; 陈士新

    2004-01-01

    Summary: In order to investigate the effect of nuclear factor-kappa B (NF-κB) on vascular endothelial growth factor (VEGF) mRNA expression of human pulmonary artery smooth muscle cells (HPASMCs) in hypoxia, the cultured HPASMCs in vitro were stimulated with pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB. The NF-κB p65 nuclei positive expression was detected by immunocytochemical technique. The IκBa protein expression was measured by Western blot.RT-PCR was used to detect the VEGF mRNA expression of HPASMCs. The results showed that no significant change was observed in the NF-κB p65 nuclei positive expression of cultured HPASMCs during 6 h-24 h in normoxia, but the levels of NF-κB p65 nuclei positive expression of cultured HPASMCs were significantly increased in hypoxia groups as compared with those in all normoxia groups (P<0.05). The IκBα protein expression of cultured HPASMCs showed no significant change during 6 h-24 h in normoxia, but significantly decreased in hypoxia as comapred with that in normoxia groups (P<0.05). PDTC (1 to 100 μmol/L) could inhibit the VEGF mRNA expression of HPASMCs in a concentration-dependent manner in hypoxia. In conclusion, NF-κB can be partly translocation activated from cytoplasm into nuclei in the cultured HPASMCs under hypoxia. The inhibition of NF-κB activation can decrease the VEGF mRNA expression. h is suggested that the activation of NF-κB is involved in the VEGFmRNA expression of HPASMCs under hypoxia.

  19. Regular endurance training reduces the exercise induced HIF-1alpha and HIF-2alpha mRNA expression in human skeletal muscle in normoxic conditions

    DEFF Research Database (Denmark)

    Lundby, Carsten; Gassmann, Max; Pilegaard, Henriette

    2005-01-01

    Regular exercise induces a variety of adaptive responses that enhance the oxidative and metabolic capacity of human skeletal muscle. Although the physiological adjustments of regular exercise have been known for decades, the underlying mechanisms are still unclear. The hypoxia inducible factors 1...... with a single exercise bout, and that this response is blunted with training. We obtained muscle biopsies from a trained (5 days/week during 4 weeks) and untrained leg from the same human subject before, immediately after, and during the recovery from a 3 h two-legged knee extensor exercise bout, where the two......alpha and HIF-2alpha mRNA levels are transiently increased in untrained human skeletal muscle in response to an acute exercise bout, but this response is blunted after exercise training. We propose that HIFs expression is upregulated with exercise and that it may be an important transcription factor...

  20. Comprehensive Analysis of MicroRNA and mRNA Expression in Normal and Tumorous Human Esophageal Squamous Cell Lines Using Microarray Datasets

    Directory of Open Access Journals (Sweden)

    Ichiro Akagi

    2014-01-01

    Full Text Available Despite the undisputed importance of altered microRNA (miRNA expression in various cancers, there is limited information on the clinicopathologic significance of cancer-related miRNAs in esophageal squamous cell carcinoma (ESCC. Previously, it was reported that the expression of several miRNAs was dysregulated in ESCC. However, the target genes of these miRNAs have not been identified. Furthermore, additional miRNAs in humans have been discovered recently, indicating that revised miRNA and gene expression profiling for ESCC are necessary. Here, we provide datasets from microarray analyses to identify miRNA and mRNA expression comprehensively in Het-1A, a normal human esophageal squamous cell line, and three human ESCC cell lines.

  1. Human glia can both induce and rescue aspects of disease phenotype in Huntington disease

    DEFF Research Database (Denmark)

    Benraiss, Abdellatif; Wang, Su; Herrlinger, Stephanie

    2016-01-01

    (hGPCs), derived from either human embryonic stem cells or mHTT-transduced fetal hGPCs. Here we show that mHTT glia can impart disease phenotype to normal mice, since mice engrafted intrastriatally with mHTT hGPCs exhibit worse motor performance than controls, and striatal neurons in mHTT glial......The causal contribution of glial pathology to Huntington disease (HD) has not been heavily explored. To define the contribution of glia to HD, we established human HD glial chimeras by neonatally engrafting immunodeficient mice with mutant huntingtin (mHTT)-expressing human glial progenitor cells...... survival in R6/2 HD mice. These observations suggest a causal role for glia in HD, and further suggest a cell-based strategy for disease amelioration in this disorder....

  2. Downregulation of VEGF mRNA expression by triamcinolone acetonide acetate-loaded chitosan derivative nanoparticles in human retinal pigment epithelial cells

    Directory of Open Access Journals (Sweden)

    Zhou H

    2012-08-01

    Full Text Available Huaisheng Zhou,1 Liqun Yang,2,* Huajie Li,2 Haijun Gong,1 Liangzheng Cheng,2 Haisheng Zheng,1 Li-Ming Zhang,2 Yuqing Lan1,*1Department of Ophthalmology, Sun Yat-sen Memorial Hospital of Sun Yat-sen University, 2Institute of Polymer Science, School of Chemistry and Chemical Engineering, Key Laboratory of Designed Synthesis and Application of Polymer Material, Key Laboratory for Polymeric Composite and Functional Materials of Ministry of Education, Sun Yat-sen University, Guangzhou, China*Both corresponding authors contributed equally to this workBackground: The purpose of this study was to investigate the downregulation of mRNA expression of vascular endothelial growth factor (VEGF by triamcinolone acetonide acetate (TAA-loaded chitosan nanoparticles in human retinal pigment epithelial cells.Methods: TAA-loaded deoxycholic acid-modified chitosan (TAA/DA-Chit nanoparticles were prepared via a self-assembly mechanism, and their morphology and zeta potential were examined by transmission electron microscopy and zeta potential analysis, respectively. DA-Chit and TAA/DA-Chit nanoparticle toxicity was evaluated using a Cell Counting Kit-8 assay. The efficiency of cellular uptake was determined using fluorescein isothiocyanate-labeled DA-Chit nanoparticles, in place of TAA/DA-Chit nanoparticles, assessed by both inverted fluorescence microscopy and flow cytometry. Downregulation of VEGF mRNA expression by TAA/DA-Chit nanoparticles was further investigated by real-time reverse transcription polymerase chain reaction (RT-PCR assay of the treated human retinal pigment epithelial cells.Results: TAA/DA-Chit nanoparticles were prepared with a TAA-loading capacity in the range of 12%–82%, which increased the water solubility of TAA from 0.3 mg/mL to 2.1 mg/mL. These nanoparticles showed oblate shapes 100–550 nm in size in transmission electron microscopic images and had positive zeta potentials. The Cell Counting Kit-8 assay indicated that the DA-Chit and

  3. Effects of prostaglandin E2 on p53 mRNA transcription and p53 mutagenesis during T-cell-independent human B-cell clonal expansion

    Science.gov (United States)

    Haque, Shabirul; Yan, Xiao Jie; Rosen, Lisa; McCormick, Steven; Chiorazzi, Nicholas; Mongini, Patricia K. A.

    2014-01-01

    Within T-cell-dependent germinal centers, p53 gene transcription is repressed by Bcl-6 and is thus less vulnerable to mutation. Malignant lymphomas within inflamed extranodal sites exhibit a relatively high incidence of p53 mutations. The latter might originate from normal B-cell clones manifesting activation-induced cytosine deaminase (AID) and up-regulated p53 following T-cell-independent (TI) stimulation. We here examine p53 gene transcription in such TI clones, with a focus on modulatory effects of prostaglandin E2 (PGE2), and evaluate progeny for p53 mutations. Resting IgM+IgD+CD27− B cells from human tonsils were labeled with CFSE and stimulated in vitro with complement-coated antigen surrogate, IL-4, and BAFF ± exogenous PGE2 (50 nM) or an analog specific for the EP2 PGE2 receptor. We use flow cytometry to measure p53 and AID protein within variably divided blasts, qRT-PCR of p53 mRNA from cultures with or without actinomycin D to monitor mRNA transcription/stability, and single-cell p53 RT-PCR/sequencing to assess progeny for p53 mutations. We report that EP2 signaling triggers increased p53 gene transcriptional activity in AID+ cycling blasts (P<0.01). Progeny exhibit p53 mutations at a frequency (8.5×10−4) greater than the baseline error rate (<0.8×10−4). We conclude that, devoid of the repressive influences of Bcl-6, dividing B lymphoblasts in inflamed tissues should display heightened p53 transcription and increased risk of p53 mutagenesis.—Haque, S., Yan, X. J., Rosen, L., McCormick, S., Chiorazzi, N., Mongini, P. K. A. Effects of prostaglandin E2 on p53 mRNA transcription and p53 mutagenesis during T-cell-independent human B-cell clonal expansion. PMID:24145719

  4. Quantitative analysis of the mRNA expression levels of BCL2 and BAX genes in human osteoarthritis and normal articular cartilage: An investigation into their differential expression.

    Science.gov (United States)

    Karaliotas, Georgios I; Mavridis, Konstantinos; Scorilas, Andreas; Babis, George C

    2015-09-01

    Osteoarthritis (OA) is primarily characterized by articular cartilage degeneration and chondrocyte loss. Although the role of apoptosis in cartilage pathobiology remains to be elucidated, the apoptotic B‑cell CLL/lymphoma 2 (BCL2) gene family is considered to be involved in OA. The purpose of the present study was to quantitatively analyze the mRNA expression profiles of the BCL2‑associated X protein (BAX) and BCL2 genes in human OA and in normal cartilage. Cartilage tissue samples were obtained from 78 patients undergoing total knee arthroplasty for OA (OA group) and orthopedic interventions for causes other than OA (control group). Total RNA was isolated from the cartilage tissue specimens and reverse transcribed into cDNA. A highly sensitive and specific reverse transcription quantitative polymerase chain reaction assay was developed for quantification of the mRNA levels of BAX and BCL2, using beta‑2 microglobulin as an endogenous control for normalization purposes. Gene expression analysis was performed using the comparative Ct (2(‑ΔΔCt)) method. The mRNA expression of BAX presented an increasing trend in the OA group compared with the control group, although without statistically significace (P=0.099). By contrast, the expression ratio of BCL2/BAX was found to be significantly decreased (2.76‑fold) in the OA group compared with the normal cartilage control group (P=0.022). A notable 4.6‑fold overexpression of median mRNA levels of BAX was also observed in patients with stage III OA compared with the control (P=0.034), while the BCL2/BAX ratio was markedly (2.5‑fold) decreased (P=0.024). A marked positive correlation was observed between the mRNA levels of BAX and BCL2 in the control group (r(s)=0.728; P<0.001), which was also present in the OA group, although to a lesser degree (r(s)=0.532; P<0.001). These results further implicate apoptosis in the pathogenesis of OA, through molecular mechanisms, which include the aberrant expression of the

  5. MHC2TA mRNA levels and human herpesvirus 6 in multiple sclerosis patients treated with interferon beta along two-year follow-up

    Directory of Open Access Journals (Sweden)

    Dominguez-Mozo Maria Inmaculada

    2012-09-01

    Full Text Available Abstract Background In previous studies we found that MHC2TA +1614 genotype frequency was very different when MS patients with and without human herpesvirus 6 (HHV-6 in serum samples were compared; a different clinical behavior was also described. The purpose of the study was: 1. To evaluate if MHC2TA expression in MS patients was influenced by interferon beta (IFN-beta treatment. 2. To study MHC2TA expression in MS patients with and without minor allele C. 3. To analyze the relation between MHC2TA mRNA levels and HHV-6 active infection in MS patients. Methods Blood and serum samples of 154 MS patients were collected in five programmed visits: basal (prior to beginning IFN-beta treatment, six, twelve, eighteen and twenty-four months later. HHV-6 in serum and MHC2TA mRNA levels were evaluated by PCR and RT-PCR, respectively. Neutralizing antibodies (NAbs against IFN-beta were analyzed by the cytopathic effect assay. Results We found that MHC2TA mRNA levels were significantly lower among MS patients with HHV-6 active infection at the basal visit (without treatment than in those MS patients without HHV-6 active infection at the basal visit (p = 0.012; in all the positive samples we only found variant A. Furthermore, 58/99 (58.6% MS patients without HHV-6 along the five programmed visits and an increase of MHC2TA expression after two-years of IFN-beta treatment were clinical responders vs. 5/21 (23.8% among those MS patients with HHV-6 and a decrease of MHC2TA mRNA levels along the two-years with IFN-beta treatment (p = 0.004; no differences were found between patients with and without NAbs. Conclusions MHC2TA mRNA levels could be decreased by the active replication of HHV-6; the absence of HHV-6 in serum and the increase of MHC2TA expression could be further studied as markers of good clinical response to IFN-beta treatment.

  6. Comparison of GLUT1, GLUT3, and GLUT4 mRNA and the subcellular distribution of their proteins in normal human muscle

    Science.gov (United States)

    Stuart, C. A.; Wen, G.; Gustafson, W. C.; Thompson, E. A.

    2000-01-01

    Basal, "insulin-independent" glucose uptake into skeletal muscle is provided by glucose transporters positioned at the plasma membrane. The relative amount of the three glucose transporters expressed in muscle has not been previously quantified. Using a combination of qualitative and quantitative ribonuclease protection assay (RPA) methods, we found in normal human muscle that GLUT1, GLUT3, and GLUT4 mRNA were expressed at 90 +/- 10, 46 +/- 4, and 156 +/- 12 copies/ng RNA, respectively. Muscle was fractionated by DNase digestion and differential sedimentation into membrane fractions enriched in plasma membranes (PM) or low-density microsomes (LDM). GLUT1 and GLUT4 proteins were distributed 57% to 67% in LDM, whereas GLUT3 protein was at least 88% in the PM-enriched fractions. These data suggest that basal glucose uptake into resting human muscle could be provided in part by each of these three isoforms.

  7. Comparison of GLUT1, GLUT3, and GLUT4 mRNA and the subcellular distribution of their proteins in normal human muscle

    Science.gov (United States)

    Stuart, C. A.; Wen, G.; Gustafson, W. C.; Thompson, E. A.

    2000-01-01

    Basal, "insulin-independent" glucose uptake into skeletal muscle is provided by glucose transporters positioned at the plasma membrane. The relative amount of the three glucose transporters expressed in muscle has not been previously quantified. Using a combination of qualitative and quantitative ribonuclease protection assay (RPA) methods, we found in normal human muscle that GLUT1, GLUT3, and GLUT4 mRNA were expressed at 90 +/- 10, 46 +/- 4, and 156 +/- 12 copies/ng RNA, respectively. Muscle was fractionated by DNase digestion and differential sedimentation into membrane fractions enriched in plasma membranes (PM) or low-density microsomes (LDM). GLUT1 and GLUT4 proteins were distributed 57% to 67% in LDM, whereas GLUT3 protein was at least 88% in the PM-enriched fractions. These data suggest that basal glucose uptake into resting human muscle could be provided in part by each of these three isoforms.

  8. Relationships between hepatic stearoyl-CoA desaturase-1 activity and mRNA expression with liver fat content in humans.

    Science.gov (United States)

    Peter, Andreas; Cegan, Alexander; Wagner, Silvia; Elcnerova, Michaela; Königsrainer, Alfred; Königsrainer, Ingmar; Häring, Hans-Ulrich; Schleicher, Erwin D; Stefan, Norbert

    2011-02-01

    Stearoyl-CoA desaturase-1 (SCD1) has gained much interest as a future drug target to treat fatty liver and its consequences. However, there are few and inconsistent human data about expression and activity of this important enzyme. We investigated activity and expression of SCD1 and their relationships with liver fat (LF) content in human liver samples. Fifty subjects undergoing liver surgery were studied. SCD1 activity was estimated from the ratio of oleate (C18:1) to stearate (C18:0) within lipid subfractions. Furthermore, SCD1 mRNA expression and LF content were measured. Similarly to previous studies, we observed a strong positive correlation between LF content and the C18:1/C18:0 ratio in the combined fatty acid (FA) fractions (r = 0.96, P < 0.0001), which could be interpreted as higher SCD1 activity with increasing LF. However, hepatic SCD1 mRNA expression did not correlate with LF (r = 0.16, P = 0.13). To solve these conflicting data, we analyzed the FA composition of hepatic lipid subfractions. With increasing LF content the amount of FAs from the triglyceride (TG) fraction increased (r = 0.96, P < 0.0001), whereas the FAs from the phospholipid (PL) fraction remained unchanged (r = -0.17, P = 0.19). Of these two major lipid fractions, the C18:1/C18:0 ratio in TG was 16-fold higher than in PL. Supporting the SCD1 mRNA expression data, the C18:1/C18:0 ratio of the TG or PL fraction did not correlate with LF (r = 0.26, P = 0.12 and r = 0.08, P = 0.29). We provide novel information that SCD1 activity and mRNA expression appear not to be elevated in subjects with high LF content. We suggest that the FA composition of lipid subclasses, rather than of mixed lipids, should be analyzed to estimate SCD1 activity.

  9. Human DDX3 interacts with the HIV-1 Tat protein to facilitate viral mRNA translation.

    Directory of Open Access Journals (Sweden)

    Ming-Chih Lai

    Full Text Available Nuclear export and translation of intron-containing viral mRNAs are required for HIV-1 gene expression and replication. In this report, we provide evidence to show that DDX3 regulates the translation of HIV-1 mRNAs. We found that knockdown of DDX3 expression effectively inhibited HIV-1 production. Translation of HIV-1 early regulatory proteins, Tat and rev, was impaired in DDX3-depleted cells. All HIV-1 transcripts share a highly structured 5' untranslated region (UTR with inhibitory elements on translation of viral mRNAs, yet DDX3 promoted translation of reporter mRNAs containing the HIV-1 5' UTR, especially with the transactivation response (TAR hairpin. Interestingly, DDX3 directly interacts with HIV-1 Tat, a well-characterized transcriptional activator bound to the TAR hairpin. HIV-1 Tat is partially targeted to cytoplasmic stress granules upon DDX3 overexpression or cell stress conditions, suggesting a potential role of Tat/DDX3 complex in translation. We further demonstrated that HIV-1 Tat remains associated with translating mRNAs and facilitates translation of mRNAs containing the HIV-1 5' UTR. Taken together, these findings indicate that DDX3 is recruited to the TAR hairpin by interaction with viral Tat to facilitate HIV-1 mRNA translation.

  10. Human DDX3 interacts with the HIV-1 Tat protein to facilitate viral mRNA translation.

    Science.gov (United States)

    Lai, Ming-Chih; Wang, Shainn-Wei; Cheng, Lie; Tarn, Woan-Yuh; Tsai, Shaw-Jenq; Sun, H Sunny

    2013-01-01

    Nuclear export and translation of intron-containing viral mRNAs are required for HIV-1 gene expression and replication. In this report, we provide evidence to show that DDX3 regulates the translation of HIV-1 mRNAs. We found that knockdown of DDX3 expression effectively inhibited HIV-1 production. Translation of HIV-1 early regulatory proteins, Tat and rev, was impaired in DDX3-depleted cells. All HIV-1 transcripts share a highly structured 5' untranslated region (UTR) with inhibitory elements on translation of viral mRNAs, yet DDX3 promoted translation of reporter mRNAs containing the HIV-1 5' UTR, especially with the transactivation response (TAR) hairpin. Interestingly, DDX3 directly interacts with HIV-1 Tat, a well-characterized transcriptional activator bound to the TAR hairpin. HIV-1 Tat is partially targeted to cytoplasmic stress granules upon DDX3 overexpression or cell stress conditions, suggesting a potential role of Tat/DDX3 complex in translation. We further demonstrated that HIV-1 Tat remains associated with translating mRNAs and facilitates translation of mRNAs containing the HIV-1 5' UTR. Taken together, these findings indicate that DDX3 is recruited to the TAR hairpin by interaction with viral Tat to facilitate HIV-1 mRNA translation.

  11. EXPRESSION OF mRNA FOR MEMBRANE-TYPE 1, 2, AND 3 MATRIX METALLOPROTEINASES IN HUMAN LARYNGEAL CANCER

    Institute of Scientific and Technical Information of China (English)

    Ya-nan Sun; Yuan Li

    2004-01-01

    Objective To investigate correlation of expressions of membrane-type 1, 2, and 3 matrix metalloproteinases (MT1, MT2,and MT3-MMP) to the invasion and metastases in laryngeal cancer.Methods Reverse transcription-polymerase chain reaction (RT-PCR) was used to examine the mRNA level of MT1,MT2, and MT3-MMP in 24 patients with laryngeal cancer. The relationships of these three MT-MMP expressions to clinicopathology were analyzed by statistics.Results The expressions of MT1, MT2, and MT3-MMP were significantly higher in laryngeal cancer tissues than those in para-tumorous tissues (P < 0.01) and had a close relationship with invasive depth (P < 0.05). But no significantly different expressions of these three MT-MMPs were found in different primary location and different histological grade of laryngeal cancer (P > 0.05). The expression of MT1-MMP was obviously higher in patients with metastatic lymph nodes than that in patients without metastatic lymph nodes (P < 0.05).Conclusion MT1, MT2, and MT3-MMP play an important role in the progression of laryngeal cancer, and MT1-MMP may serve as a reliable marker in estimating invasive and metastatic potency of laryngeal cancer. Suppressing expressions of MT 1, MT2, and MT3-MMP early may inhibit the invasion and metastases of laryngeal cancer.

  12. Recombinant growth hormone enhances muscle myosin heavy-chain mRNA accumulation and amino acid accrual in humans.

    OpenAIRE

    Fong, Y; M. Rosenbaum; Tracey, K J; Raman, G.; Hesse, D G; Matthews, D. E.; Leibel, R. L.; Gertner, J M; Fischman, D. A.; Lowry, S F

    1989-01-01

    A potentially lethal complication of trauma, malignancy, and infection is a progressive erosion of muscle protein mass that is not readily reversed by nutritional support. Growth hormone is capable of improving total body nitrogen balance, but its role in myofibrillar protein synthesis in humans is unknown. The acute, in situ muscle protein response to an infusion of methionyl human growth hormone was investigated in the limbs of nutritionally depleted subjects during a period of intravenous ...

  13. Pattern-Recognition Receptor Signaling Regulator mRNA Expression in Humans and Mice, and in Transient Inflammation or Progressive Fibrosis

    Directory of Open Access Journals (Sweden)

    Maciej Lech

    2013-09-01

    Full Text Available The cell type-, organ-, and species-specific expression of the pattern-recognition receptors (PRRs are well described but little is known about the respective expression profiles of their negative regulators. We therefore determined the mRNA expression levels of A20, CYLD, DUBA, ST2, CD180, SIGIRR, TANK, SOCS1, SOCS3, SHIP, IRAK-M, DOK1, DOK2, SHP1, SHP2, TOLLIP, IRF4, SIKE, NLRX1, ERBIN, CENTB1, and Clec4a2 in human and mouse solid organs. Humans and mice displayed significant differences between their respective mRNA expression patterns of these factors. Additionally, we characterized their expression profiles in mononuclear blood cells upon bacterial endotoxin, which showed a consistent induction of A20, SOCS3, IRAK-M, and Clec4a2 in human and murine cells. Furthermore, we studied the expression pattern in transient kidney ischemia-reperfusion injury versus post-ischemic atrophy and fibrosis in mice. A20, CD180, ST2, SOCS1, SOCS3, SHIP, IRAK-M, DOK1, DOK2, IRF4, CENTB1, and Clec4a2 were all induced, albeit at different times of injury and repair. Progressive fibrosis was associated with a persistent induction of these factors. Thus, the organ- and species-specific expression patterns need to be considered in the design and interpretation of studies related to PRR-mediated innate immunity, which seems to be involved in tissue injury, tissue regeneration and in progressive tissue scarring.

  14. Increased Levels of Cell-Free Human Placental Lactogen mRNA at 28-32 Gestational Weeks in Plasma of Pregnant Women With Placenta Previa and Invasive Placenta

    OpenAIRE

    2014-01-01

    We compared the levels of cell-free human placental lactogen (hPL) messenger RNA (mRNA) in maternal plasma at 28 to 32 weeks of gestation between women with diagnosis of placenta previa or invasive placenta and women with an uneventful pregnancy. Sensitivity and specificity of hPL mRNA for the prediction of invasive placenta were further explored. Plasma hPL mRNA were quantified by real-time reverse-transcriptase polymerase chain reaction in women with placenta previa (n = 13), invasive place...

  15. Manganese superoxide dismutase: effect of the ala16val polymorphism on protein, activity, and mRNA levels in human breast cancer cell lines and stably transfected mouse embryonic fibroblasts.

    Science.gov (United States)

    McAtee, Britt L; Yager, James D

    2010-02-01

    The manganese superoxide dismutase (MnSOD) ala16val polymorphism has been associated with various diseases including breast cancer. In the present study, we investigated levels of MnSOD protein, enzymatic activity, and mRNA with respect to MnSOD genotype in several human breast carcinoma cell lines and in mouse embryonic fibroblasts (MEF), developed from the MnSOD knockout mouse, stably expressing human MnSOD-ala and MnSOD-val. In human breast cell lines, the MnSOD-ala allele was associated with increased levels of MnSOD protein and MnSOD protein per unit mRNA. In the MEF transformants, MnSOD activity correlated fairly well with MnSOD protein levels. MnSOD mRNA expression was significantly lower in MnSOD-ala versus MnSOD-val lines. MnSOD protein and activity levels were not related to MnSOD genotype in the transformed MEF, although, as observed in the human breast cell lines, the MEF human MnSOD-ala lines produced significantly more human MnSOD protein per unit mRNA than the human MnSOD-val lines. This suggests that there is more efficient production of MnSOD-ala protein compared to MnSOD-val protein. Examination of several indicators of reactive oxygen species levels, including superoxide and hydrogen peroxide, in wild-type MEF and in MEF expressing similar elevated amounts of MnSOD-ala or val activity did not show differences related to the levels of MnSOD protein expression. In conclusion, in both human breast carcinoma cell lines and MEF cell lines stably transfected with human MnSOD, the MnSOD-ala allele was associated with increased production of MnSOD protein per unit mRNA indicating a possible imbalance in MnSOD protein production from the MnSOD-val mRNA.

  16. Effect of insulin on human skeletal muscle mitochondrial ATP production, protein synthesis, and mRNA transcripts

    Science.gov (United States)

    Stump, Craig S.; Short, Kevin R.; Bigelow, Maureen L.; Schimke, Jill M.; Sreekumaran Nair, K.

    2003-06-01

    Mitochondria are the primary site of skeletal muscle fuel metabolism and ATP production. Although insulin is a major regulator of fuel metabolism, its effect on mitochondrial ATP production is not known. Here we report increases in vastus lateralis muscle mitochondrial ATP production capacity (32-42%) in healthy humans (P oxidative phosphorylation in skeletal muscle along with synthesis of gene transcripts and mitochondrial protein in human subjects. Skeletal muscle of type 2 diabetic patients has a reduced capacity to increase ATP production with high insulin levels. cytochrome c oxidase | NADH dehydrogenase subunit IV | amino acids | citrate synthase

  17. Expression of Cyclooxygenase-2 mRNA and Identification of Its Splice Variant in Human Myometrium Obtained from Women in Labor

    Institute of Scientific and Technical Information of China (English)

    HUANG Yinping; WAN Jingyuan; YE Duyun; WU Ping; HUANG Yanjun; ZHANG Li; ZHOU Xiaoyan; HUANG Yunfeng; YUAN Ping; ZHANG Daijuan

    2005-01-01

    In order to investigate the expression of cyclooxygenase-2 (COX-2) in human lower segments of myometrium obtained from women in labor and those not in labor and identify the splicing variant of COX-2, reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the expression of COX-2. The primers were designed and synthesized according to the sequence of rat COX-2 splice variant which was discovered firstly by us. Then the splicing variant of COX-2 in human myometrium from woman in labor was identified, cloned into vector and sequenced. The results showed that the expression of COX-2 mRNA was lower in human myometrium obtained from women who were not in labor than that in labor women and a new band of COX-2 was obtained in myometrium from labor woman. The fragment included an unspliced intron, which pitched between exons 7 and 8. It was suggested that COX-2 gene was not only expressed highly in human myometrium from woman in labor, but also produced splicing variant by alternative splicing.

  18. Comparison of three human papillomavirus DNA assays and one mRNA assay in women with abnormal cytology

    DEFF Research Database (Denmark)

    Rebolj, Matejka; Lynge, Elsebeth; Ejegod, Ditte;

    2014-01-01

    OBJECTIVE: To compare the clinical characteristics of four human papillomavirus (HPV) assays: hybrid capture 2 (HC2), cobas, CLART, and APTIMA in Danish women with abnormal cytology. METHODS: SurePath samples from 367 consecutive women from Copenhagen, with atypical squamous cells of undetermined...

  19. Motivational, proteostatic and transcriptional deficits precede synapse loss, gliosis and neurodegeneration in the B6.HttQ111/+ model of Huntington’s disease

    Science.gov (United States)

    Bragg, Robert M.; Coffey, Sydney R.; Weston, Rory M.; Ament, Seth A.; Cantle, Jeffrey P.; Minnig, Shawn; Funk, Cory C.; Shuttleworth, Dominic D.; Woods, Emily L.; Sullivan, Bonnie R.; Jones, Lindsey; Glickenhaus, Anne; Anderson, John S.; Anderson, Michael D.; Dunnett, Stephen B.; Wheeler, Vanessa C.; MacDonald, Marcy E.; Brooks, Simon P.; Price, Nathan D.; Carroll, Jeffrey B.

    2017-01-01

    We investigated the appearance and progression of disease-relevant signs in the B6.HttQ111/+ mouse, a genetically precise model of the mutation that causes Huntington’s disease (HD). We find that B6.HttQ111/+ mice are healthy, show no overt signs of central or peripheral inflammation, and no gross motor impairment as late as 12 months of age. Behaviorally, we find that 4–9 month old B6.HttQ111/+ mice have normal activity levels and show no clear signs of anxiety or depression, but do show clear signs of reduced motivation. The neuronal density, neuronal size, synaptic density and number of glia is normal in B6.HttQ111/+ striatum, the most vulnerable brain region in HD, up to 12 months of age. Despite this preservation of the synaptic and cellular composition of the striatum, we observe clear progressive, striatal-specific transcriptional dysregulation and accumulation of neuronal intranuclear inclusions (NIIs). Simulation studies suggest these molecular endpoints are sufficiently robust for future preclinical studies, and that B6.HttQ111/+ mice are a useful tool for modeling disease-modifying or neuroprotective strategies for disease processes before the onset of overt phenotypes. PMID:28176805

  20. Investigating Mutations to Reduce Huntingtin Aggregation by Increasing Htt-N-Terminal Stability and Weakening Interactions with PolyQ Domain

    Science.gov (United States)

    Mazza-Anthony, Cody; Waldispühl, Jérôme

    2016-01-01

    Huntington's disease is a fatal autosomal genetic disorder characterized by an expanded glutamine-coding CAG repeat sequence in the huntingtin (Htt) exon 1 gene. The Htt protein associated with the disease misfolds into toxic oligomers and aggregate fibril structures. Competing models for the misfolding and aggregation phenomena have suggested the role of the Htt-N-terminal region and the CAG trinucleotide repeats (polyQ domain) in affecting aggregation propensities and misfolding. In particular, one model suggests a correlation between structural stability and the emergence of toxic oligomers, whereas a second model proposes that molecular interactions with the extended polyQ domain increase aggregation propensity. In this paper, we computationally explore the potential to reduce Htt aggregation by addressing the aggregation causes outlined in both models. We investigate the mutation landscape of the Htt-N-terminal region and explore amino acid residue mutations that affect its structural stability and hydrophobic interactions with the polyQ domain. Out of the millions of 3-point mutation combinations that we explored, the (L4K E12K K15E) was the most promising mutation combination that addressed aggregation causes in both models. The mutant structure exhibited extreme alpha-helical stability, low amyloidogenicity potential, a hydrophobic residue replacement, and removal of a solvent-inaccessible intermolecular side chain that assists oligomerization. PMID:28096892

  1. Characterization of HTT inclusion size, location, and timing in the zQ175 mouse model of Huntington's disease: an in vivo high-content imaging study.

    Directory of Open Access Journals (Sweden)

    Nikisha Carty

    Full Text Available Huntington's disease (HD is an autosomal dominant neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the huntingtin gene. Major pathological hallmarks of HD include inclusions of mutant huntingtin (mHTT protein, loss of neurons predominantly in the caudate nucleus, and atrophy of multiple brain regions. However, the early sequence of histological events that manifest in region- and cell-specific manner has not been well characterized. Here we use a high-content histological approach to precisely monitor changes in HTT expression and characterize deposition dynamics of mHTT protein inclusion bodies in the recently characterized zQ175 knock-in mouse line. We carried out an automated multi-parameter quantitative analysis of individual cortical and striatal cells in tissue slices from mice aged 2-12 months and confirmed biochemical reports of an age-associated increase in mHTT inclusions in this model. We also found distinct regional and subregional dynamics for inclusion number, size and distribution with subcellular resolution. We used viral-mediated suppression of total HTT in the striatum of zQ175 mice as an example of a therapeutically-relevant but heterogeneously transducing strategy to demonstrate successful application of this platform to quantitatively assess target engagement and outcome on a cellular basis.

  2. Integration Analysis of MicroRNA and mRNA Expression Profiles in Human Peripheral Blood Lymphocytes Cultured in Modeled Microgravity

    Directory of Open Access Journals (Sweden)

    C. Girardi

    2014-01-01

    Full Text Available We analyzed miRNA and mRNA expression profiles in human peripheral blood lymphocytes (PBLs incubated in microgravity condition, simulated by a ground-based rotating wall vessel (RWV bioreactor. Our results show that 42 miRNAs were differentially expressed in MMG-incubated PBLs compared with 1 g incubated ones. Among these, miR-9-5p, miR-9-3p, miR-155-5p, miR-150-3p, and miR-378-3p were the most dysregulated. To improve the detection of functional miRNA-mRNA pairs, we performed gene expression profiles on the same samples assayed for miRNA profiling and we integrated miRNA and mRNA expression data. The functional classification of miRNA-correlated genes evidenced significant enrichment in the biological processes of immune/inflammatory response, signal transduction, regulation of response to stress, regulation of programmed cell death, and regulation of cell proliferation. We identified the correlation of miR-9-3p, miR-155-5p, miR-150-3p, and miR-378-3p expression with that of genes involved in immune/inflammatory response (e.g., IFNG and IL17F, apoptosis (e.g., PDCD4 and PTEN, and cell proliferation (e.g., NKX3-1 and GADD45A. Experimental assays of cell viability and apoptosis induction validated the results obtained by bioinformatics analyses demonstrating that in human PBLs the exposure to reduced gravitational force increases the frequency of apoptosis and decreases cell proliferation.

  3. Structural analysis of human 2'-O-ribose methyltransferases involved in mRNA cap structure formation

    Science.gov (United States)

    Smietanski, Miroslaw; Werner, Maria; Purta, Elzbieta; Kaminska, Katarzyna H.; Stepinski, Janusz; Darzynkiewicz, Edward; Nowotny, Marcin; Bujnicki, Janusz M.

    2014-01-01

    The 5' cap of human messenger RNA contains 2'-O-methylation of the first and often second transcribed nucleotide that is important for its processing, translation and stability. Human enzymes that methylate these nucleotides, termed CMTr1 and CMTr2, respectively, have recently been identified. However, the structures of these enzymes and their mechanisms of action remain unknown. In the present study, we solve the crystal structures of the active CMTr1 catalytic domain in complex with a methyl group donor and a capped oligoribonucleotide, thereby revealing the mechanism of specific recognition of capped RNA. This mechanism differs significantly from viral enzymes, thus providing a framework for their specific targeting. Based on the crystal structure of CMTr1, a comparative model of the CMTr2 catalytic domain is generated. This model, together with mutational analysis, leads to the identification of residues involved in RNA and methyl group donor binding.

  4. Immunolabelling, histochemistry and in situ hybridisation in human skeletal muscle fibres to detect myosin heavy chain expression at the protein and mRNA level

    Science.gov (United States)

    SERRANO, A. L.; PÉREZ, MARGARITA; LUCÍA, A.; CHICHARRO, J. L.; QUIROZ-ROTHE, E.; RIVERO, J. L. L.

    2001-01-01

    The distribution of muscle fibres classified on the basis of their content of different myosin heavy chain (MHC) isoforms was analysed in vastus lateralis muscle biopsies of 15 young men (with an average age of 22 y) by correlating immunohistochemistry with specific anti-MHC monoclonal antibodies, myofibrillar ATPase (mATPase) histochemistry and in situ hybridisation with probes specific for MHC β-slow, MHC-IIA and MHC-IIX. The characterisation of a large number of individual fibres was compared and correlated on a fibre-to-fibre basis. The panel of monoclonal antibodies used in the study allowed classification of human skeletal muscle fibres into 5 categories according to the MHC isoform they express at the protein level, types I, I+IIA, IIA, IIAX and IIX. Hybrid fibres coexpressing two isoforms represented a considerable proportion of the fibre composition (about 14%) and were clearly underestimated by mATPase histochemistry. For a very high percentage of fibres there was a precise correspondence between the MHC protein isoforms and mRNA transcripts. The integrated methods used demonstrate a high degree of precision of the immunohistochemical procedure used for the identification and quantification of human skeletal muscle fibre types. The monoclonal antibody S5-8H2 is particularly useful for identifying hybrid IIAX fibres. This protocol offers new prospects for muscle fibre classification in human experimental studies. PMID:11554510

  5. VHL Frameshift Mutation as Target of Nonsense-Mediated mRNA Decay in Drosophila melanogaster and Human HEK293 Cell Line

    Directory of Open Access Journals (Sweden)

    Lucia Micale

    2009-01-01

    Full Text Available There are many well-studied examples of human phenotypes resulting from nonsense or frameshift mutations that are modulated by Nonsense-Mediated mRNA Decay (NMD, a process that typically degrades transcripts containing premature termination codons (PTCs in order to prevent translation of unnecessary or aberrant transcripts. Different types of germline mutations in the VHL gene cause the von Hippel-Lindau disease, a dominantly inherited familial cancer syndrome with a marked phenotypic variability and age-dependent penetrance. By generating the Drosophila UAS:Upf1D45B line we showed the possible involvement of NMD mechanism in the modulation of the c.172delG frameshift mutation located in the exon 1 of Vhl gene. Further, by Quantitative Real-time PCR (QPCR we demonstrated that the corresponding c.163delG human mutation is targeted by NMD in human HEK 293 cells. The UAS:Upf1D45B line represents a useful system to identify novel substrates of NMD pathway in Drosophila melanogaster. Finally, we suggest the possible role of NMD on the regulation of VHL mutations.

  6. Immunoflourescence and mRNA analysis of human embryonic stem cells (hESCs) grown under feeder-free conditions

    DEFF Research Database (Denmark)

    Awan, Aashir; Oliveri, Roberto S; Jensen, Pernille L

    2010-01-01

    This chapter describes the procedures in order to do immunofluorescence (IF) microscopy and quantitative PCR (qPCR) analysis of human embryonic stem cells (hESCs) grown specifically under feeder-free conditions. A detailed protocol outlining the steps from initially growing the cells, passaging...... onto 16-well glass chambers, and continuing with the general IF and qPCR steps will be provided. The techniques will be illustrated with new results on cellular localization of transcriptional factors and components of the Hedgehog, Wnt, and PDGF signaling pathways to primary cilia in stem cell...

  7. Highly purified, multi-wall carbon nanotubes induce light-chain 3B expression in human lung cells

    Energy Technology Data Exchange (ETDEWEB)

    Tsukahara, Tamotsu, E-mail: ttamotsu@kanazawa-med.ac.jp [Department of Hematology and Immunology, Kanazawa Medical University, 1-1 Daigaku, Uchinada, Ishikawa 920-0293 (Japan); Matsuda, Yoshikazu [Clinical Pharmacology Educational Center, Nihon Pharmaceutical University, Ina-machi, Saitama 362-0806 (Japan); Usui, Yuki [Research Center for Exotic Nanocarbons, Shinshu University, 4-17-1 Wakasato, Nagano-shi, Nagano 380-8553 (Japan); Haniu, Hisao [Department of Orthopaedic Surgery, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621 (Japan)

    2013-10-18

    Highlights: •HTT2800-treated BEAS-2B cells induced LC3B in a time-dependent manner. •HTT2800-treated BEAS-2B cells showed decreased cell proliferation that was both time- and dose-dependent. •Addition of 3-MA, LC3B-II protein and mRNA levels were significantly decreased. •3-MA and E64-d + pepstatin A, but not brefeldin A, provided protection against HTT2800-induced cell death. •These results suggest that HTT2800 predominantly causes autophagy rather than apoptotic cell death in BEAS-2B cells. -- Abstract: Bronchial epithelial cells are targets of inhalation and play a critical role in the maintenance of mucosal integrity as mechanical barriers against various particles. Our previous result suggest that vapor-grown carbon fiber, HTT2800, which is one of the most highly purified multi-wall carbon nanotubes (MWCNT) showed cellular uptake of the carbon nanotube, increased cell death, enhanced DNA damage, and induced cytokine release. Increasing evidence suggests that autophagy may critically influence vital cellular processes such as apoptosis, cell proliferation and inflammation and thereby may play a critical role in pulmonary diseases. Autophagy was recently recognized as a critical cell death pathway, and autophagosome accumulation has been found to be associated with the exposure of various nanoparticles. In this study, the authors focus on the autophagic responses of HTT2800 exposure. The HTT2800-exposed cells induced LC3B expression and induced cell growth inhibition.

  8. Regulation of the stability of poly(I)xpoly(C)-induced human fibroblast interferon mRNA: selective inactivation of interferon mRNA and lack of involvement of 2',5'-oligo(A) synthetase activation during the shutoff of interferon production.

    Science.gov (United States)

    Sehgal, P B; Gupta, S L

    1980-06-01

    The inactivation of interferon mRNA during the shutoff phase of interferon production in poly(I)xpoly(C)-induced human fibroblast cultures is selective. We have determined that the shutoff of interferon production, which takes place from 3 to 8 hr after the beginning of induction, is not associated with an appreciable declined in the rate of bulk cellular protein synthesis or of cellular protein secretion. While the amount of translatable interferon mRNA declined markedly during the shutoff phase, the level of translatable bulk cellular mRNA and the stability of [3H]uridine-labeled mRNA were unaffected. Superinduction with actinomycin D selectively stabilized interferon mRNA with no apparent effect on the stability of bulk cellular mRNA. Furthermore, an activation of the 2',5'-oligo(A) synthetase/endonuclease system does not appear to be involved in the shutoff phenomenon. Uninduced FS-4 cells contained a low basal level of 2'5'-oligo(A) synthetase activity, which was unchanged in poly(I)xpoly(C)-induced cells during the shutoff phase. Treatment of FS-4 cells with interferon for 16-18 hr prior to induction increased the enzyme activity by approximately 200-fold. However, this did not inhibit interferon production after induction with poly(I)xpoly(C) alone or after superinduction with cycloheximide or actinomycin D or both. Furthermore, the rates of decay of interferon production were comparable in cells with either a basal or an increased level of 2',5'-oligo(A) synthetase. Thus a 200-fold increase in 2',5'-oligo(A) synthetase level did not affect either the stability of interferon mRNA or the efficacy of interferon superinduction by metabolic inhibitors.

  9. TIME- AND DOSE-DEPENDENT UP-REGULATION OF TNF-α mRNA AFTER IRRADIATION OF HUMAN NSCLC CELL LINES IN VITRO

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Objective: Even though radiotherapy plays a major role in the local treatment of non-small cell lung cancer (NSCLC), little is known about the molecular effects of irradiation in this tumor. In the present study, we examined two NSCLC cell lines for their endogenous production of TNF-α after irradiation. To investigate the radiation-induced TNF-α production in NSCLC cell lines. Methods: Two human NSCLC cell lines (A549: squamous; NCI-H596: adenosquamous) were investigated for their TNF-α mRNA (real-time RT-PCR) after exposure to different irradiation doses (2, 5, 10, 20, 30, 40 Gy) and time intervals (1, 3, 6, 12, 24, 48 or 72 h). The TNF-α mRNA expression was quantified by real-time RT-PCR. The clonogenic survival was evaluated after irradiation with 2, 4, 6 and 8 Gy. Results: Non-irradiated NSCLC cells exhibited no or very low TNF-α expression. For the NCI-H596 cell line, TNF-α expression was significantly elevated 1~12 h (maximum 6h: 568fold increase relative to unirradiated cells) in a time-dependent manner. The radiation-induced increase could be observed after irradiation with 2 Gy reaching maximal at 40 Gy, with 83 times higher than normal controls. The clonogenic survival of these cell lines was nearly identical. Conclusion: NCI-H596 cells produce significant quantities of TNF-α following irradiation in a time- and dose-dependent manner. The pro-inflammatory cytokine TNF-α is a key mediator for the pathogenesis of radiation pneumonitis. Radiation-induced endogenous TNF-α expression in NSCLC cells may affect the normal lung adjacent to the tumor and may be associated with an adverse clinical outcome of the patient.

  10. Lipopolysaccharides and the Enhanced Expression of TACE Mrna in HL-60 Cells and Adhesive Cells from Human Spleen

    Institute of Scientific and Technical Information of China (English)

    DING; Tingbo(

    2001-01-01

    [1]Flad H D Loppnow H.Agonists and antagonists for LPS-induced cytokines.Immunology 1993 187:303[2]Robache-Gallea S Morand V Bruneau J H et al.In vitro processing of human tumor necrosis factor-α.J Bio Chem 1995 270(40):23 688[3]Kim K U Kwon O J Jue D M.Pro-tumor necrosis factor cleaving enzyme in macrophage membrane/particulate.Immunology 1993 80(2) :134[4]Moss M L.Cloning of a disintegrin metalloproteinase that processes precursor tumor necrosis factor-α.Nature 1997 385 (6):733[5]Flick D A Gilford G E.The detection of biological activity of tumor necrosis factor-α.J Immunol Methods 1984 68:167[6]Sambrook J Fritsch E F Maniatis F.In Vitro Amplification of DNA by the Polymerase Chain Reaction.In:Molecular Cloning A Laboratory Manual.2nd edition.New York:Cold Spring Harbor Laboratory Press 1989.10.1-10.70[7]吴朝栋 李明珍 张艳萍等.中药热毒清对内毒素DIC家兔血浆肿瘤坏死因子α白细胞介素水平影响的研究.中国中西医结合杂志 1995 15:365

  11. RNA secondary structures located in the interchromosomal region of human ACAT1 chimeric mRNA are re-quired to produce the 56-kDa isoform

    Institute of Scientific and Technical Information of China (English)

    Jia Chen; Ta-Yuan Chang; Bo-Liang Li; Xiao-Nan Zhao; Li Yang; Guang-Jing Hu; Ming Lu; Ying Xiong; Xin-Ying Yang; Catherine CY Chang; Bao-Liang Song

    2008-01-01

    We have previously reported that the human ACAT1 gene produces a chimeric mRNAthrough the interchromosomal processing of two discontinuous RNAs transcribed from chromosomes 1 and 7. The chimeric mRNA uses AUG1397-1399 and GGC1274-1276 as translation initiation codons to produce normal 50-kDa ACATI and a novel enzymatically active 56-kDa isoform,respectively,with the latter being authentically present in human cells,including human monocyte derived macrophages. In this work,we report that RNA secondary structures located in the vicinity,of the GGC1274-1276 codon are required for production of the 56-kDa isoform. The effects of the three predicted stem-loops (nt 1255-1268,1286-1342 and 1355-1384) were tested individually by transfecting expression plasmids into cells that contained the wild-type,deleted or mutant stem-loop sequences linked to a partial ACAT1 AUG open reading frame (ORF) or to theORFs of other genes. The expression patterns were monitored by western blot analyses. We found that the upstream stem-loop1255-1268 from chromosome 7 and downstream stem-loop1286-1342 from chromosome I were needed for production of the 56-kDa isoform,whereas the last stem-ioop1355-1384 from chromosome I was dispensable. The results of experi ments using both monocistronic and bicistronic vectors with a stable hairpin showed that translation initiation from the GGC1274-1276 codon was mediated by an internal ribosome entry site (IRES). Further experiments revealed that translation initiation from the GGC1274-1276 codon requires the upstream AU-constituted RNA secondary structure and the downstream GC-rich structure. This mechanistic work provides further support for the biological significance of the chimeric nature of the human ACATI transcript.

  12. Towards a unified biological hypothesis for the BDNF Val66Met-associated memory deficits in humans: a model of impaired dendritic mRNA trafficking

    Directory of Open Access Journals (Sweden)

    Gabriele eBaj

    2013-10-01

    Full Text Available Brain-derived neurotrophic factor (BDNF represents promotesa key molecule for the survival and differentiation of specific populations of neurons in the central nervous system. BDNF also regulates plasticity-related processes underlying memory and learning. A common single nucleotide polymorphism (SNP rs6265 has been identified on the coding sequence of human BDNF located at 11p13. The SNP rs6265 is a single base mutation with an adenine instead of a guanine at position 196 (G196A, resulting in the amino acid substitution Val66Met. This polymorphism only exists in humans and has been associated with a plethora of effects ranging from molecular, cellular and brain structural modifications in association with deficits in social and cognitive functions. To date, the literature on Val66Met polymorphism describes a complex and often conflicting pattern of effects. In this review, we attempt to provide a unifying model of the Val66Met effects. We discuss the clinical evidence of the association between Val66Met and memory deficits, as well as the molecular mechanisms involved including the reduced transport of BDNF mRNA to the dendrites as well as the reduced processing and secretion of BDNF protein through the regulated secretory pathway.

  13. Primary cervical cancer screening and triage using an mRNA human papillomavirus assay and visual inspection.

    Science.gov (United States)

    Nieves, Lucybeth; Enerson, Christine L; Belinson, Suzanne; Brainard, Jennifer; Chiesa-Vottero, Andres; Nagore, Norma; Booth, Christine; Pérez, Areli Gutierrez; Chávez-Avilés, Mauricio N; Belinson, Jerome

    2013-03-01

    Mexican Cervical Cancer Screening Study II (MECCS II) seeks to develop a highly sensitive and highly specific screening program able to be adapted to all socioeconomic levels in Mexico. The objectives of MECCS II are (1) to compare the sensitivity and specificity for cervical intraepithelial neoplasia (CIN) 3 or cancer of self-collected vaginal specimens tested for high-risk types of the human papillomavirus (HR-HPV) by APTIMA with those tested for HR-HPV by Hybrid Capture 2 (HC2); and (2) determine the efficacy of cryotherapy in the treatment of HR-HPV-positive and acetic acid-aided visual inspection (VIA)-positive and -negative women after VIA triage. The study was conducted in rural Mexico. Women aged 30 to 50 years, nonpregnant, with no history of hysterectomy or pelvic irradiation and varied histories of screening, participated. A direct endocervical sample was tested for cytology, HC2, and APTIMA assay (AHPV). Subjects positive on any test were recalled for triage VIA, biopsies, and immediate cryotherapy. Tests were compared using McNemar test. Two thousand forty-nine patients have complete results. Mean age of the patients was 39.2 years; 7.7% presented with ≥atypical squamous cells of uncertain significance (ASCUS), 1.8% ≥low-grade squamous intraepithelial neoplasia, and 0.5% ≥high-grade squamous intraepithelial neoplasia. Two percent of patients had ≥CIN2, and 0.78% had ≥CIN3 (including 2 with invasive disease). The sensitivity of ThinPrep (>ASCUS), HC2, and AHPV for >CIN3 for direct endocervical collection was 87.5%, 100%, and 100%, respectively. The specificity of ThinPrep (>ASCUS), HC2, and AHPV for >CIN3 was 94.1%, 92.2%, and 93.5%, respectively. Specificities of HC2 and AHPV differed significantly. The overall percentage of agreement among HPV assays (HC2 vs APTIMA) is 97%. Four hundred sixty-nine women returned for VIA. Two hundred ninety-one women were treated with cryotherapy. The specificity of the APTIMA assay along with high

  14. Studies on SSTR2 mRNA expression and its correlation to steroid receptors in human benign and malignant breast lesions

    Institute of Scientific and Technical Information of China (English)

    ZENG Xizhi(曾希志); YAO Zhenxiang(姚榛祥)

    2002-01-01

    Objective:This sudy was designed to observe somatostatin receptor subtype 2 (SSTR2) Mrna expression, and investigate the correlations between SSTR2 Mrna expression and steroid receptors in benign and malignant lesions of the breast. Methods: A total of 23 breast carcinomas,16 mammary hyperplasia and 9 mammary adenofibroma samples were analysed. The SSTR2 Mrna expression was examined by in situ hybridization using multiphase oligoprobes.The ER and PR were detected by immunohistochemical staining. A computerized image analysis system was utilized to estimate the relative contents of SSTR2 Mrna. Results: The positive rates of expression (87.0%) and relative contents (0.47) of SSTR2 Mrna in breast cancer were higher than those in benign breast lesions(64%,0.26) respectively( P<0.05). SSTR2 Mrna expression was closely correlated with ER and PR in breast cancer( P<0. 05), A positive correlation between SSTR2 Mrna expression and ER was also found in benign breast lesions. Conclusions: SSTR2 Mrna expressed both in benign and in malignant breast lesions, but higher in malignant than in benign ones. There was a significant positive correlation of SSTR2 Mrna expression with ER or PR. The results suggest that conbined treatment with an antiestrogen and a somatostatin analogue for ER-positive breast cancer is feasible.

  15. Quantification of human telomerase reverse transcriptase mRNA in testicular germ cell tumors by quantitative fluorescence real-time RT-PCR.

    Science.gov (United States)

    Schrader, Mark; Burger, Angelika M; Müller, Markus; Krause, Hans; Straub, Bernd; Smith, Gilian L; Newlands, Eward S; Miller, Kurt

    2002-01-01

    Telomerase is a ribonucleoprotein enzyme which is endogenously expressed in germ, stem and tumor cells, but absent in benign somatic cells. The two major telomerase components are human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT). It has been shown that hTERT is rate-limiting for telomerase activity and that it plays a central role in human carcinogenesis. Here, we investigated the potential of hTERT and hTR gene expression as diagnostic markers in testicular germ cell tumors (TGCT). hTERT mRNA and hTR expression were quantified in 55 testicular germ cell tumors comprising 36 primary and 19 germ cell tumors from retroperitonal sides by fluorescence real-time RT-PCR using the LightCycler technology. Porphobilinogen deaminase (PBGD) was used as housekeeping gene and to enable relative quantification. For comparison to TGCTs, 38 benign testicular biopsies from patients with fertility disorders were assayed. hTERT expression was detected in all examined undifferentiated TGCTs and in the benign testicular tissue specimens with germ cell content (N(hTERT) 38-127). In contrast, mature teratomas from primary and post-chemotherapy masses, which are characterized by well-differentiated tissue components showed a nearly complete downregulation of hTERT expression (N(hTERT) 2-4, pmRNA is expressed in all undifferentiated TGCTs but repressed in mature teratomas. This suggests an inverse correlation between the differentiation status of germ cell tumors and hTERT expression. Thus, detection of hTERT expression in tumors histopathologically classified as mature teratomas enables a molecular-diagnostic confirmation and might aid decision making for treatment of patients presenting with this tumor subtype.

  16. Development of highly sensitive and specific mRNA multiplex system (XCYR1) for forensic human body fluids and tissues identification.

    Science.gov (United States)

    Xu, Yan; Xie, Jianhui; Cao, Yu; Zhou, Huaigu; Ping, Yuan; Chen, Liankang; Gu, Lihua; Hu, Wei; Bi, Gang; Ge, Jianye; Chen, Xin; Zhao, Ziqin

    2014-01-01

    The identification of human body fluids or tissues through mRNA-based profiling is very useful for forensic investigations. Previous studies have shown mRNA biomarkers are effective to identify the origin of biological samples. In this study, we selected 16 tissue specific biomarkers to evaluate their specificities and sensitivities for human body fluids and tissues identification, including porphobilinogen deaminase (PBGD), hemoglobin beta (HBB) and Glycophorin A (GLY) for circulatory blood, protamine 2 (PRM2) and transglutaminase 4 (TGM4) for semen, mucin 4 (MUC4) and human beta defensin 1(HBD1) for vaginal secretion, matrix metalloproteinases 7 and 11 (MMP7 and MMP11) for menstrual blood, keratin 4(KRT4) for oral mucosa, loricrin (LOR) and cystatin 6 (CST6) for skin, histatin 3(HTN3) for saliva, statherin (STATH) for nasal secretion, dermcidin (DCD) for sweat and uromodulin (UMOD) for urine. The above mentioned ten common forensic body fluids or tissues were used in the evaluation. Based on the evaluation, a reverse transcription (RT) PCR multiplex assay, XCYR1, which includes 12 biomarkers (i.e., HBB, GLY, HTN3, PRM2, KRT4, MMP11, MUC4, DCD, UMOD, MMP7, TGM4, and STATH) and 2 housekeeping genes [i.e., glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 18SrRNA], was developed. This assay was further validated with real casework samples and mock samples (with both single source and mixture) and it was approved that XCYR1 is effective to identify common body fluids or tissues (i.e., circulatory blood, saliva, semen, vaginal secretion, menstrual blood, oral mucosa, nasal secretion, sweat and urine) in forensic casework samples.

  17. Analytic and Diagnostic Performances of Human Papillomavirus E6/E7 mRNA Test on up-to 11-Year-Old Liquid-Based Cervical Samples. A Biobank-Based Longitudinal Study

    Directory of Open Access Journals (Sweden)

    Roberta Zappacosta

    2017-07-01

    Full Text Available Human Papillomavirus (HPV E6/E7 mRNA test demonstrated high specificity in detecting HPV infections, but studies assessing its efficacy in terms of cancer risk stratification are lacking. Follow-up studies are arduous and expensive. Biobank would be the answer to the problem, although data investigating the effects of long-term storage on RNA preservation are still needed. We addressed these issues by retrieving 202 residual liquid-based cervical specimens, collected from 149 women attending cervical cancer screening during the years 2001–2012. Samples were stored in Adriatic Biobank at room temperature and without any handing. After calculation of RNA yield and purity, E6/E7 mRNA test was retrospectively performed on each samples, to assess analytic and diagnostic performances. Using automated extraction procedures, RNA of good quantity and quality was obtained. The mean value of RNA concentration was 27.5 ng/μL. The mean A260/A280 ratio was 2.1. An invalid mRNA test result was found in 11.9% of the specimens. Neither RNA integrity, nor analytic performances of mRNA test were influenced by the year of sample collection. In total, 62.4% of the specimens tested as mRNA positive; among these, 89.2% were CIN2+. E6/E7 mRNA was detected in all Squamous Cervical Cancer (SCC cases. Percentage of positive samples increased with the severity of histological diagnosis. mRNA testing, showing specificity and predictive values of 75.6% and 84.4%, respectively, significantly improved the corresponding values for DNA testing. Thus, the reflex mRNA test was demonstrated to be suitable to triage women with persistent cervical lesions. A “one sample for all” approach is possible, with practical benefits for Biobank-based long-term longitudinal studies, diseases prevention, prediction, diagnosis and treatment.

  18. The HTT CAG-Expansion Mutation Determines Age at Death but Not Disease Duration in Huntington Disease.

    Science.gov (United States)

    Keum, Jae Whan; Shin, Aram; Gillis, Tammy; Mysore, Jayalakshmi Srinidhi; Abu Elneel, Kawther; Lucente, Diane; Hadzi, Tiffany; Holmans, Peter; Jones, Lesley; Orth, Michael; Kwak, Seung; MacDonald, Marcy E; Gusella, James F; Lee, Jong-Min

    2016-02-04

    Huntington disease (HD) is caused by an expanded HTT CAG repeat that leads in a length-dependent, completely dominant manner to onset of a characteristic movement disorder. HD also displays early mortality, so we tested whether the expanded CAG repeat exerts a dominant influence on age at death and on the duration of clinical disease. We found that, as with clinical onset, HD age at death is determined by expanded CAG-repeat length and has no contribution from the normal CAG allele. Surprisingly, disease duration is independent of the mutation's length. It is also unaffected by a strong genetic modifier of HD motor onset. These findings suggest two parsimonious alternatives. (1) HD pathogenesis is driven by mutant huntingtin, but before or near motor onset, sufficient CAG-driven damage occurs to permit CAG-independent processes and then lead to eventual death. In this scenario, some pathological changes and their clinical correlates could still worsen in a CAG-driven manner after disease onset, but these CAG-related progressive changes do not themselves determine duration. Alternatively, (2) HD pathogenesis is driven by mutant huntingtin acting in a CAG-dependent manner with different time courses in multiple cell types, and the cellular targets that lead to motor onset and death are different and independent. In this scenario, processes driven by HTT CAG length lead directly to death but not via the striatal pathology associated with motor manifestations. Each scenario has important ramifications for the design and testing of potential therapeutics, especially those aimed at preventing or delaying characteristic motor manifestations.

  19. Impact of Institutional Care on Attachment Disorganization and Insecurity of Ukrainian Preschoolers: Protective Effect of the Long Variant of the Serotonin Transporter Gene (5HTT)

    Science.gov (United States)

    Bakermans-Kranenburg, Marian J.; Dobrova-Krol, Natasha; van IJzendoorn, Marinus

    2012-01-01

    Institutional care has been shown to lead to insecure and disorganized attachments and indiscriminate friendliness. Some children, however, are surprisingly resilient to the adverse environment. Here the protective role of the long variant of the serotonin receptor gene (5HTT) is explored in a small hypothesis-generating study of 37 Ukrainian…

  20. Tumor necrosis factor induces phosphorylation of a 28-kDa mRNA cap-binding protein in human cervical carcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Marino, M.W.; Guidon, P.T. Jr.; Donner, D.B. (Cornell Univ. Graduate School of Medical Sciences, New York, NY (USA)); Pfeffer, L.M. (Rockefeller Univ., New York, NY (USA))

    1989-11-01

    Tumor necrosis factor {alpha} (TNF-{alpha}) stimulated the phosphorylation of a 28-kDa protein (p28) in the ME-180 line of human cervical carcinoma cells. The effect of TNF-{alpha} on the phosphorylation state of p28 was rapid (4-fold increase within 15 min) and persistent, remaining above the basal level for at least 2 hr. The specific binding of {sup 125}I-labeled TNF-{alpha} to cell-surface binding sites, the stimulation of p28 phosphorylation by TNF-{alpha}, and the inhibition of cell proliferation by TNF-{alpha} occurred with nearly identical dose-response relationships. Two-dimensional SDS/PAGE resolved p28 into two isoforms having pI values of 6.2 and 6.1. A phosphorylated cap-binding protein was substantially enriched from lysates of control or TNF-{alpha}-treated ME-180 cells by affinity chromatography with 7-methylguanosine 5{prime}-triposphate-Sepharose. The phosphoprotein recovered from this procedure was the substrate for TNF-{alpha}-promoted phosphorylation, p28. Thus, TNF-{alpha} stimulates the phosphorylation of this mRNA cap-binding protein, which may be involved in the transduction of TNF-{alpha}-receptor binding into cellular responses.

  1. Tumor necrosis factor induces phosphorylation of a 28-kDa mRNA cap-binding protein in human cervical carcinoma cells.

    Science.gov (United States)

    Marino, M W; Pfeffer, L M; Guidon, P T; Donner, D B

    1989-11-01

    Tumor necrosis factor alpha (TNF-alpha) stimulated the phosphorylation of a 28-kDa protein (p28) in the ME-180 line of human cervical carcinoma cells. The effect of TNF-alpha on the phosphorylation state of p28 was rapid (4-fold increase within 15 min) and persistent, remaining above the basal level for at least 2 hr. The specific binding of 125I-labeled TNF-alpha to cell-surface binding sites, the stimulation of p28 phosphorylation by TNF-alpha, and the inhibition of cell proliferation by TNF-alpha occurred with nearly identical dose-response relationships. Two-dimensional SDS/PAGE resolved p28 into two isoforms having pI values of 6.2 and 6.1. A phosphorylated cap-binding protein was substantially enriched from lysates of control or TNF-alpha-treated ME-180 cells by affinity chromatography with 7-methylguanosine 5'-triphosphate-Sepharose. The phosphoprotein recovered from this procedure was the substrate for TNF-alpha-promoted phosphorylation, p28. Thus, TNF-alpha stimulates the phosphorylation of this mRNA cap-binding protein, which may be involved in the transduction of TNF-alpha-receptor binding into cellular responses.

  2. Detection of Human Papilloma Virus Type 16 E6 mRNA in Laryngeal Squamous Cell Carcinoma by In Situ Hybridization

    Institute of Scientific and Technical Information of China (English)

    Hai-rong Jiang; Peng Wang; Yong Li; Tao Ning; Xiao-song Rao; Bao-guoLiu

    2010-01-01

    Objective:Laryngeal squamous cell carcinoma(LSCC)is a common malignant tumor in Northeast China and is frequently associated with well-established risk factors like smoking and alcohol abuse.Human papilloma virus(HPV)is an epitheliotropic oncogenic virus that has been detected in a variety of head and neck tumors including LSCC.This retrospective study was to investigate the prevalence of HPV infection in patients with LSCC.Methods:In situ hybridization was performed in 99 patients with LSCC to detect the expression of HPV-16 E6mRNA.Results:The positive rate of HPV16 E6 mRNA was 36.36%(36/99)in laryngeal squamous cell carcinoma(LSCC),whereas only 3 of 50(6%)specimens of the normal laryngeal mucosa as a control group showed positive results(P0.05).Conclusion:The results suggest that the increased prevalence of HPV infection compared to normal laryngeal mucosa and the fact that high-risk HPV types(especially type 16)were the most frequently identified do not allow the exclusion of HPV as a risk factor in laryngeal squamous cell carcinoma.However,their clinical value remains to be further investigated.

  3. Detection of Human Papilloma Virus Type 16 E6 mRNA in Laryngeal Squamous Cell Carcinoma by In Situ Hybridization

    Institute of Scientific and Technical Information of China (English)

    Hai-rong Jiang; Peng Wang; Yong Li; Tao Ning; Xiao-song Rao; Bao-guoLiu

    2011-01-01

    Objective: Laryngeal squamous cell carcinoma(LSCC) is a common malignant tumor in Northeast China and is frequently associated with well-established risk factors like smoking and alcohol abuse.Human papilloma virus (HPV) is an epitheliotropic oncogenic virus that has been detected in a variety of head and neck tumors including LSCC.This retrospective study was to investigate the prevalence of HPV infection in patients with LSCC.Methods: In situ hybridization was performed in 99 patients with LSCC to detect the expression of HPV-16 E6 mRNA.Results: The positive rate of HPV 16 E6 mRNA was 36.36%(36/99) in laryngeal squamous cell carcinoma (LSCC),whereas only 3 of 50(6%) specimens of the normal laryngeal mucosa as a control group showed positive results(P0.05).Conclusion: The results suggest that the increased prevalence of HPV infection compared to normal laryngeal mucosa and the fact that high-risk HPV types(especially type 16) were the most frequently identified do not allow the exclusion of HPV as a risk factor in laryngeal squamous cell carcinoma.However,their clinical value remains to be further investigated.

  4. mRNA sequencing of novel cell lines from human papillomavirus type-16 related vulval intraepithelial neoplasia: consequences of expression of HPV16 E4 and E5.

    Science.gov (United States)

    Bryant, Dean; Onions, Tiffany; Raybould, Rachel; Flynn, Áine; Tristram, Amanda; Meyrick, Sian; Giles, Peter; Ashelford, Kevin; Hibbitts, Samantha; Fiander, Alison; Powell, Ned

    2014-09-01

    Vulval intraepithelial neoplasia is a precursor of vulval cancer and is commonly caused by infection with Human Papillomavirus (HPV). Development of topical treatments for vulval intraepithelial neoplasia requires appropriate in vitro models. This study evaluated the feasibility of primary culture of vulval intraepithelial neoplasia biopsy tissue to produce cell lines for use as in vitro models. A potentially immortal cell line was produced which gave rise to three monoclonal lines. These lines were characterized for HPV genomic integration and for viral gene expression using ligation-mediated PCR and quantitative PCR. Distinct patterns of viral integration and gene expression were observed among the three lines. Integration and expression data were validated using deep sequencing of mRNA. Gene ontology analyses of these data also demonstrated that expression of the HPV16 E4 and E5 proteins resulted in substantial changes in the composition of the cell membrane and extracellular space, associated with alterations in cell adhesion and differentiation. These data illustrate the diverse patterns of HPV gene expression potentially present within a single lesion. The derived cell lines provide useful models to investigate the biology of vulval intraepithelial neoplasia and the interactions between different HPV gene products and potential therapeutic agents.

  5. Human cyclophilin 33 (hCyP33) in T-cell binds specifically to poly(A)~+RNA (mRNA)

    Institute of Scientific and Technical Information of China (English)

    张万起; 袁直; 宓怀风; 元云峰; 何炳林; 王亦农

    2002-01-01

    Human cyclophilin 33 (hCyP33), found in 1996, consists of an RNA-binding domain in N-terminus, a cyclophilin domain in C-terminus and a connected part between the two domains. RNA-binding proteins concern functions, such as splicing, modification and transport, after transcription in eukaryotic cells. Cyclophilins (CyPs) possess enzymatic activity, namely peptidyl-proryl cis-trans isomerase (PPlase). They are involved in folding, transport and interaction of proteins. Cyclosporin A (CsA), an immunosuppressant used by organ transplantation, binds to CyPs and suppresses their enzymatic activity. However, up to now it is unknown that which cellular and physiological roles hCyP33, which possesses the above-mentioned both functions, plays. In this paper the binding specificity of hCyP33 to different cellular RNA is investigated by means of ion-exchange chromatography and affinity adsorption. The results show that it binds specifically to poly(A) tailed mRNA, namely poly(A)+RNA.

  6. Human cyclophilin 33 (hCyP33) in T-cell binds specifically to poly(A)+RNA (mRNA)

    Institute of Scientific and Technical Information of China (English)

    张万起; 元云峰; 王亦农; 袁直; 宓怀风; 何炳林

    2002-01-01

    Human cyclophilin 33 (hCyP33), found in 1996, consists of an RNA-binding domain in N-terminus, a cyclophilin domain in C-terminus and a connected part between the two domains. RNA-binding proteins concern functions, such as splicing, modification and transport, after transcription in eukaryotic cells. Cyclophilins (CyPs) possess enzymatic activity, namely peptidyl-proryl cis-trans isomerase (PPIase). They are involved in folding, transport and interaction of proteins. Cyclosporin A (CsA), an immunosuppressant used by organ transplantation, binds to CyPs and suppresses their enzymatic activity. However, up to now it is unknown that which cellular and physiological roles hCyP33, which possesses the above-mentioned both functions, plays. In this paper the binding specificity of hCyP33 to different cellular RNA is investigated by means of ion-exchange chromatography and affinity adsorption. The results show that it binds specifically to poly(A) tailed mRNA, namely poly(A)+RNA.

  7. The stoichiometric production of IL-2 and IFN-γ mRNA defines memory T cells that can self-renew after adoptive transfer in humans.

    Science.gov (United States)

    Wang, Anran; Chandran, Smita; Shah, Syed A; Chiu, Yu; Paria, Biman C; Aghamolla, Tamara; Alvarez-Downing, Melissa M; Lee, Chyi-Chia Richard; Singh, Sanmeet; Li, Thomas; Dudley, Mark E; Restifo, Nicholas P; Rosenberg, Steven A; Kammula, Udai S

    2012-08-29

    Adoptive immunotherapy using ex vivo-expanded tumor-reactive lymphocytes can mediate durable cancer regression in selected melanoma patients. Analyses of these trials have associated the in vivo engraftment ability of the transferred cells with their antitumor efficacy. Thus, there is intensive clinical interest in the prospective isolation of tumor-specific T cells that can reliably persist after transfer. Animal studies have suggested that central memory CD8(+) T cells (T(CM)) have divergent capabilities including effector differentiation to target antigen and stem cell-like self-renewal that enable long-term survival after adoptive transfer. We sought to isolate human melanoma-specific T(CM) to define their in vivo fate and function after autologous therapeutic transfer to metastatic patients. To facilitate the high-throughput identification of these rare cells from patients, we report that T(CM) have a defined stoichiometric production of interleukin-2 (IL-2) and interferon-γ (IFN-γ) mRNA after antigen stimulation. Melanoma-specific T cells screened for high relative IL-2 production had a T(CM) phenotype and superior in vitro proliferative capacity compared to cells with low IL-2 production. To investigate in vivo effector function and self-renewal capability, we allowed melanoma-specific T(CM) to undergo in vitro expansion and differentiation into lytic effector clones and then adoptively transferred them back into their hosts. These clones targeted skin melanocytes in all five patients and persisted long term and reacquired parental T(CM) attributes in four patients after transfer. These findings demonstrate the favorable engraftment fitness for human T(CM)-derived clones, but further efforts to improve their antitumor efficacy are still necessary.

  8. Comparative analysis of mRNA targets for human PUF-family proteins suggests extensive interaction with the miRNA regulatory system.

    Directory of Open Access Journals (Sweden)

    Alessia Galgano

    Full Text Available Genome-wide identification of mRNAs regulated by RNA-binding proteins is crucial to uncover post-transcriptional gene regulatory systems. The conserved PUF family RNA-binding proteins repress gene expression post-transcriptionally by binding to sequence elements in 3'-UTRs of mRNAs. Despite their well-studied implications for development and neurogenesis in metazoa, the mammalian PUF family members are only poorly characterized and mRNA targets are largely unknown. We have systematically identified the mRNAs associated with the two human PUF proteins, PUM1 and PUM2, by the recovery of endogenously formed ribonucleoprotein complexes and the analysis of associated RNAs with DNA microarrays. A largely overlapping set comprised of hundreds of mRNAs were reproducibly associated with the paralogous PUM proteins, many of them encoding functionally related proteins. A characteristic PUF-binding motif was highly enriched among PUM bound messages and validated with RNA pull-down experiments. Moreover, PUF motifs as well as surrounding sequences exhibit higher conservation in PUM bound messages as opposed to transcripts that were not found to be associated, suggesting that PUM function may be modulated by other factors that bind conserved elements. Strikingly, we found that PUF motifs are enriched around predicted miRNA binding sites and that high-confidence miRNA binding sites are significantly enriched in the 3'-UTRs of experimentally determined PUM1 and PUM2 targets, strongly suggesting an interaction of human PUM proteins with the miRNA regulatory system. Our work suggests extensive connections between the RBP and miRNA post-transcriptional regulatory systems and provides a framework for deciphering the molecular mechanism by which PUF proteins regulate their target mRNAs.

  9. Comparative analysis of mRNA targets for human PUF-family proteins suggests extensive interaction with the miRNA regulatory system.

    Science.gov (United States)

    Galgano, Alessia; Forrer, Michael; Jaskiewicz, Lukasz; Kanitz, Alexander; Zavolan, Mihaela; Gerber, André P

    2008-09-08

    Genome-wide identification of mRNAs regulated by RNA-binding proteins is crucial to uncover post-transcriptional gene regulatory systems. The conserved PUF family RNA-binding proteins repress gene expression post-transcriptionally by binding to sequence elements in 3'-UTRs of mRNAs. Despite their well-studied implications for development and neurogenesis in metazoa, the mammalian PUF family members are only poorly characterized and mRNA targets are largely unknown. We have systematically identified the mRNAs associated with the two human PUF proteins, PUM1 and PUM2, by the recovery of endogenously formed ribonucleoprotein complexes and the analysis of associated RNAs with DNA microarrays. A largely overlapping set comprised of hundreds of mRNAs were reproducibly associated with the paralogous PUM proteins, many of them encoding functionally related proteins. A characteristic PUF-binding motif was highly enriched among PUM bound messages and validated with RNA pull-down experiments. Moreover, PUF motifs as well as surrounding sequences exhibit higher conservation in PUM bound messages as opposed to transcripts that were not found to be associated, suggesting that PUM function may be modulated by other factors that bind conserved elements. Strikingly, we found that PUF motifs are enriched around predicted miRNA binding sites and that high-confidence miRNA binding sites are significantly enriched in the 3'-UTRs of experimentally determined PUM1 and PUM2 targets, strongly suggesting an interaction of human PUM proteins with the miRNA regulatory system. Our work suggests extensive connections between the RBP and miRNA post-transcriptional regulatory systems and provides a framework for deciphering the molecular mechanism by which PUF proteins regulate their target mRNAs.

  10. 5-HTT and 5-HT(1A) receptor occupancy of the novel substance vortioxetine (Lu AA21004). A PET study in control subjects.

    Science.gov (United States)

    Stenkrona, Per; Halldin, Christer; Lundberg, Johan

    2013-10-01

    Vortioxetine (Lu AA21004) is a new potential substance for the treatment of anxiety and mood disorders. It has high affinity for the 5-HT transporter (5-HTT) and moderate affinity for the 5-HT1A receptor in vitro. Positron emission tomography (PET) has commonly been used to examine the relation between dose/plasma concentration and occupancy to predict relevant dose intervals in a clinical setting. In this study 11 control subjects were examined with PET and [¹¹C]MADAM at baseline, after a single dose and after 9 days of dosing with Lu AA21004 (2.5, 10 or 60 mg) for quantification of 5-HTT occupancy. Four subjects were examined with PET and [¹¹C]WAY 100635 at baseline, after a single dose and after 9 days of dosing of Lu AA21004 (30 mg) for quantification of 5-HT(1A) occupancy. To allow for quantification of binding in the raphe nuclei, PET data were analyzed using wavelet aided parametric imaging. 5-HTT occupancy ranged from 2 (mean, 2.5 mg day 1) to 97% (60 mg day 9). The apparent affinity of Lu AA21004 binding to 5-HTT (KD(ND)) was calculated to 16.7 nM (R=0.95), and the corresponding oral dose (KD(ND)-dose) to 8.5 mg (R=0.91). No significant occupancy of 5-HT(1A) receptors was found after dosing of 30 mg Lu AA21004. Based on the literature and the present [¹¹C]MADAM binding data, a dose of 20-30 mg Lu AA21004 is suggested to give clinically relevant occupancy of the 5-HTT.

  11. Increased levels of cell-free human placental lactogen mRNA at 28-32 gestational weeks in plasma of pregnant women with placenta previa and invasive placenta.

    Science.gov (United States)

    Kawashima, Akihiro; Sekizawa, Akihiko; Ventura, Walter; Koide, Keiko; Hori, Kyouko; Okai, Takashi; Masashi, Yoshida; Furuya, Kenichi; Mizumoto, Yoshifumi

    2014-02-01

    We compared the levels of cell-free human placental lactogen (hPL) messenger RNA (mRNA) in maternal plasma at 28 to 32 weeks of gestation between women with diagnosis of placenta previa or invasive placenta and women with an uneventful pregnancy. Sensitivity and specificity of hPL mRNA for the prediction of invasive placenta were further explored. Plasma hPL mRNA were quantified by real-time reverse-transcriptase polymerase chain reaction in women with placenta previa (n = 13), invasive placenta (n = 5), and normal pregnancies (n = 92). Median (range) hPL mRNA was significantly higher in women with placenta previa, 782 (10-2301) copies/mL of plasma, and in those with invasive placenta, 615 (522-2102) copies/mL of plasma, when compared to normal pregnancies, 90 (4-4407) copies/mL of plasma, P placenta among women with placenta previa. In conclusion, expression of hPL mRNA is increased in plasma of women with placenta previa and invasive placenta at 28 to 32 weeks of gestation.

  12. Aryl hydrocarbon hydroxylase represents CYP1B1, and not CYP1A1, in human freshly isolated white cells: trimodal distribution of Japanese population according to induction of CYP1B1 mRNA by environmental dioxins.

    Science.gov (United States)

    Toide, Kenji; Yamazaki, Hiroshi; Nagashima, Rikako; Itoh, Keisuke; Iwano, Shunsuke; Takahashi, Yoshiki; Watanabe, Shaw; Kamataki, Tetsuya

    2003-03-01

    The expression level of mRNAs for cytochrome P450 (CYP) 1A1 and 1B1 in freshly prepared white cells from 72 subjects exposed to dioxins at waste incinerators was investigated. The amounts of CYP1B1 mRNA ranged from 0.16 to 671 molecules/10(7) molecules of 18S rRNA, whereas the amounts of CYP1A1 mRNA were dioxins. The inducibility of CYP1B1 mRNA in leukocytes, defined as the ratio of CYP1B1 mRNA to the plasma concentration of dioxins, varied among the subjects. It was found that the subjects showed trimodal distribution according to inducibility: 39 (54.2%), 25 (34.7%), and 8 (11.1%) of 72 subjects were judged as poor, intermediate, and high responders to environmental dioxins, respectively. The amounts of CYP1B1 mRNA in leukocytes of the intermediate and high responders were highly correlated with the plasma concentrations of dioxins (P dioxins is involved in aromatic hydrocarbon hydroxylase activities in human lymphocytes.

  13. Detection of siRNA Mediated Target mRNA Cleavage Activities in Human Cells by a Novel Stem-Loop Array RT-PCR Analysis

    Science.gov (United States)

    2016-09-07

    modification or cleavages of transcripts through various RNA editing mechanisms , including siRNA targeted mRNA cleavages. Currently, 5′-RACE (rapid ampli... transcription polymerase chain reaction (SLA-RT-PCR) approach to detect and verify the siRNA- mediated target mRNA cleavage sites by determining precise...Reverse Transcription kit (Life Technologies) in combination with an array of stem-loop RT primers. The 20 ml of RT reaction con- tained 50 ng of total RNA

  14. Diagnostic and prognostic validity of the human papillomavirus E6/E7 mRNA test in cervical cytological samples of HC2-positive patients.

    Science.gov (United States)

    Benevolo, Maria; Terrenato, Irene; Mottolese, Marcella; Marandino, Ferdinando; Carosi, Mariantonia; Rollo, Francesca; Ronchetti, Livia; Muti, Paola; Mariani, Luciano; Sindico, Stefano; Vocaturo, Giuseppe; Vocaturo, Amina

    2011-06-01

    The study aimed to assess the clinical utility in identifying CIN2 or worse (CIN2+), of the Pretect HPV-Proofer test for E6/E7 mRNA detection in Hybrid Capture 2 (HC2)-positive patients, who underwent colposcopy. In particular, the study analyzed the mRNA test performance as the third test in a subgroup of HC2+ patients with less severe than high-grade squamous intraepithelial lesions (HSIL-). We analyzed 464 cervico-vaginal samples by liquid-based cytology (LBC) and PreTect HPV-Proofer. Moreover 231 patients also had a biopsy at baseline and 75, with HSIL-, were followed up within 2 years by LBC, colposcopy, and histology when indicated. The highest sensitivity for CIN2+ belonged to the mRNA compared to LBC, at the HSIL+ threshold (72% vs. 58%), whereas the LBC showed the highest specificity and positive predictive value (PPV) (99 and 93% vs. 73 and 39%, respectively). Focusing on the 408 HSIL- patients, the mRNA positivity was significantly more associated with CIN2+ than CIN2- lesions (p < 0.0001). Moreover, among the 75 HSIL- followed up patients, the mRNA displayed high longitudinal Specificity (89%), even if the sensitivity and the PPV were low (50 and 20%, respectively). The present data suggest that the mRNA test may have a diagnostic and a potentially prognostic role in HC2+/HSIL- patients.

  15. Molecular beacon-decorated polymethylmethacrylate core-shell fluorescent nanoparticles for the detection of survivin mRNA in human cancer cells.

    Science.gov (United States)

    Adinolfi, Barbara; Pellegrino, Mario; Giannetti, Ambra; Tombelli, Sara; Trono, Cosimo; Sotgiu, Giovanna; Varchi, Greta; Ballestri, Marco; Posati, Tamara; Carpi, Sara; Nieri, Paola; Baldini, Francesco

    2017-02-15

    One of the main goals of nanomedicine in cancer is the development of effective drug delivery systems, primarily nanoparticles. Survivin, an overexpressed anti-apoptotic protein in cancer, represents a pharmacological target for therapy and a Molecular Beacon (MB) specific for survivin mRNA is available. In this study, the ability of polymethylmethacrylate nanoparticles (PMMA-NPs) to promote survivin MB uptake in human A549 cells was investigated. Fluorescent and positively charged core PMMA-NPs of nearly 60nm, obtained through an emulsion co-polymerization reaction, and the MB alone were evaluated in solution, for their analytical characterization; then, the MB specificity and functionality were verified after adsorption onto the PMMA-NPs. The carrier ability of PMMA-NPs in A549 was examined by confocal microscopy. With the optimized protocol, a hardly detectable fluorescent signal was obtained after incubation of the cells with the MB alone (fluorescent spots per cell of 1.90±0.40 with a mean area of 1.04±0.20µm(2)), while bright fluorescent spots inside the cells were evident by using the MB loaded onto the PMMA-NPs. (27.50±2.30 fluorescent spots per cell with a mean area of 2.35±0.16µm(2)). These results demonstrate the ability of the PMMA-NPs to promote the survivin-MB internalization, suggesting that this complex might represent a promising strategy for intracellular sensing and for the reduction of cancer cell proliferation. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Performance of the Aptima High-Risk Human Papillomavirus mRNA Assay in a Referral Population in Comparison with Hybrid Capture 2 and Cytology▿

    Science.gov (United States)

    Clad, Andreas; Reuschenbach, Miriam; Weinschenk, Johanna; Grote, Ruth; Rahmsdorf, Janina; Freudenberg, Nikolaus

    2011-01-01

    This study compared the Aptima human papillomavirus (HPV) (AHPV; Gen-Probe Incorporated) assay, which detects E6/E7 mRNA from 14 high-risk types, the Hybrid Capture 2 HPV DNA (HC2; Qiagen Incorporated) test, and repeat cytology for their ability to detect high-grade cervical lesions (cervical intraepithelial neoplasia grade 2+ [CIN2+]) in women referred to colposcopy due to an abnormal Papanicolaou (Pap) smear. A total of 424 clinical specimens, stored in liquid-based cytology (LBC) vials at room temperature for up to 3 years, were tested by repeat cytology, the AHPV assay, and the HC2 test. Assay results were compared to each other and to histology results. The overall agreement between the AHPV assay and the HC2 test was 88.4%. The sensitivity (specificity) of cytology, the HC2 test, and the AHPV assay for the detection of CIN2+ was 84.9% (66.3%), 91.3% (61.0%), and 91.7% (75.0%) and for the detection of CIN3+ was 93.9% (54.4%), 95.7% (46.0%), and 98.2% (56.3%), respectively. Of the disease-positive specimens containing high-risk HPV (HR HPV) DNA as determined by Linear Array (Roche Diagnostics), the AHPV assay missed 3 CIN2 and 1 microfocal CIN3 specimen, while the HC2 test missed 6 CIN2, 4 CIN3, and 1 cervical carcinoma specimen. The AHPV assay had a sensitivity similar to but a specificity significantly higher (P < 0.0001) than the HC2 test for the detection of CIN2+. The AHPV assay was significantly more sensitive (P = 0.0041) and significantly more specific (P = 0.0163) than cytology for the detection of disease (CIN2+). PMID:21191046

  17. Performance of the Aptima high-risk human papillomavirus mRNA assay in a referral population in comparison with Hybrid Capture 2 and cytology.

    Science.gov (United States)

    Clad, Andreas; Reuschenbach, Miriam; Weinschenk, Johanna; Grote, Ruth; Rahmsdorf, Janina; Freudenberg, Nikolaus

    2011-03-01

    This study compared the Aptima human papillomavirus (HPV) (AHPV; Gen-Probe Incorporated) assay, which detects E6/E7 mRNA from 14 high-risk types, the Hybrid Capture 2 HPV DNA (HC2; Qiagen Incorporated) test, and repeat cytology for their ability to detect high-grade cervical lesions (cervical intraepithelial neoplasia grade 2+ [CIN2+]) in women referred to colposcopy due to an abnormal Papanicolaou (Pap) smear. A total of 424 clinical specimens, stored in liquid-based cytology (LBC) vials at room temperature for up to 3 years, were tested by repeat cytology, the AHPV assay, and the HC2 test. Assay results were compared to each other and to histology results. The overall agreement between the AHPV assay and the HC2 test was 88.4%. The sensitivity (specificity) of cytology, the HC2 test, and the AHPV assay for the detection of CIN2+ was 84.9% (66.3%), 91.3% (61.0%), and 91.7% (75.0%) and for the detection of CIN3+ was 93.9% (54.4%), 95.7% (46.0%), and 98.2% (56.3%), respectively. Of the disease-positive specimens containing high-risk HPV (HR HPV) DNA as determined by Linear Array (Roche Diagnostics), the AHPV assay missed 3 CIN2 and 1 microfocal CIN3 specimen, while the HC2 test missed 6 CIN2, 4 CIN3, and 1 cervical carcinoma specimen. The AHPV assay had a sensitivity similar to but a specificity significantly higher (P < 0.0001) than the HC2 test for the detection of CIN2+. The AHPV assay was significantly more sensitive (P = 0.0041) and significantly more specific (P = 0.0163) than cytology for the detection of disease (CIN2+).

  18. Prevalence of type-specific oncogenic human papillomavirus infection assessed by HPV E6/E7 mRNA among women with high-grade cervical lesions.

    Science.gov (United States)

    Wang, Hye-Young; Park, Sunyoung; Lee, Dongsup; Kim, Sunghyun; Kim, Geehyuk; Park, Kwang Hwa; Lee, Hyeyoung

    2015-08-01

    Human papillomavirus (HPV) infection is a major cause of premalignant dysplasia and cervical cancer. There are no data on the prevalence of genotype-specific HPV infection assessed by HPV E6/E7 mRNA in women representative of the Korean population across a broad age range. A total of 630 women aged 17-90 years were enrolled in this study. ThinPrep liquid-based cytology samples were evaluated using the CervicGen HPV RT-qDx assay, which detects 16 high-risk (HR) HPV genotypes (set 1: HPV 16, 31, 33, 35, 52, and 58; set 2: HPV 18, 39, 45, 51, 59, and 68; and set 3: HPV 53, 56, 66, and 69). The overall prevalence of HPV infection was 33.2% (n=209), and oncogenic high-risk HPV was detected in 75.9% (n=107) of 141 women with high-grade cervical lesions. HPV 16 was the most common HPV genotype among women with high-grade cervical lesions and histologically confirmed cervical intraepithelial neoplasia grade 2 and above (CIN2+) in the Republic of Korea (41.6%). Among women aged over 30 years, 182/329 (55%) had invasive cervical cancer and 135 (74%) of these were infected with oncogenic HR-HPV types (in particular 25% with HPV 16). Among patients diagnosed with CIN2+, the positivity rate of HR-HPV was the highest in women aged 40-49 years. These results suggest that the determination of specific HPV genotypes is very important for evaluating the potential impact of preventive measures, including the use of prophylactic vaccines, on reducing the burden of cervical cancer. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  19. Hydrophobically Modified siRNAs Silence Huntingtin mRNA in Primary Neurons and Mouse Brain

    Directory of Open Access Journals (Sweden)

    Julia F Alterman

    2015-01-01

    Full Text Available Applications of RNA interference for neuroscience research have been limited by a lack of simple and efficient methods to deliver oligonucleotides to primary neurons in culture and to the brain. Here, we show that primary neurons rapidly internalize hydrophobically modified siRNAs (hsiRNAs added directly to the culture medium without lipid formulation. We identify functional hsiRNAs targeting the mRNA of huntingtin, the mutation of which is responsible for Huntington's disease, and show that direct uptake in neurons induces potent and specific silencing in vitro. Moreover, a single injection of unformulated hsiRNA into mouse brain silences Htt mRNA with minimal neuronal toxicity. Thus, hsiRNAs embody a class of therapeutic oligonucleotides that enable simple and straightforward functional studies of genes involved in neuronal biology and neurodegenerative disorders in a native biological context.

  20. Differential susceptibility in spillover between interparental conflict and maternal parenting practices: evidence for OXTR and 5-HTT genes.

    Science.gov (United States)

    Sturge-Apple, Melissa L; Cicchetti, Dante; Davies, Patrick T; Suor, Jennifer H

    2012-06-01

    Guided by the affective spillover hypothesis and the differential susceptibility to environmental influence frameworks, the present study examined how associations between interparental conflict and mothers' parenting practices were moderated by serotonin transporter (5-HTT) and oxytocin receptor (OXTR) genes. A sample of 201 mothers and their 2-year old child participated in a laboratory-based research assessment. Results supported differential susceptibility hypotheses within spillover frameworks. With respect to OXTR rs53576, mothers with the GG genotype showed greater differential maternal sensitivity across varying levels of interparental conflict. Mothers with one or two copies of the 5-HTTLPR S allele demonstrated differential susceptibility for both sensitive and harsh/punitive caregiving behaviors. Finally, analyses examined whether maternal depressive symptoms and emotional closeness to their child mediated the moderating effects. Findings suggest that maternal emotional closeness with their child indirectly linked OXTR with maternal sensitivity. The results highlight how molecular genetics may explain heterogeneity in spillover models with differential implications for specific parenting behaviors. Implications for clinicians and therapists working with maritally distressed parents are discussed.

  1. Association between Neurocognitive Impairment and the Short Allele of the 5-HTT Promoter Polymorphism in Depression: A Pilot Study

    Directory of Open Access Journals (Sweden)

    Hely Kalska

    2013-01-01

    Full Text Available Depression has been shown to be associated with cognitive deficits in various cognitive domains. However, it is still unclear which factors contribute to cognitive impairment. The objective of this study was to find out whether a functional polymorphism in the promoter region of the serotonin transporter (5-HTTLPR gene is associated with the impairment of cognitive functioning among depressed patients. In a pilot study, a sample of 19 patients with major depressive disorder (MDD and 19 healthy controls was investigated with an extensive psychiatric and neuropsychological examination. All participants were genotyped for 5-HTTLPR. Depressed patients with the short allele of the 5-HTT promoter region exhibited inferior cognitive performance compared to patients with the long allele polymorphism. In healthy controls, no association between genotype and cognitive performance was found. The result suggests that in MDD patients with the short allele of the 5-HTTLPR polymorphism the vulnerability to cognitive impairment is increased compared to MDD patients without the short allele inheritance. These preliminary findings need to be confirmed in a larger cohort of MDD patients.

  2. Unaltered mRNA expression of calcitonin-like receptor and receptor activity modifying proteins in human arteries in stroke and myocardial infarction

    DEFF Research Database (Denmark)

    Eskesen, Karen; János, Tajti; Tibor, Hortobágyi;

    2007-01-01

    -dependent change in total RNA and level of mRNA for p-actin or GAPDH could be detected in vessels removed from 1 and 5 days post mortem. The expression of beta-actin appears lower in coronary artery than in pulmonary artery and middle cerebral artery with no significant difference for GAPDH; both worked well......-R and RAMPs in arteries from patients with hemorrhagic stroke, arteriosclerosis and acute myocardial infarction when compared to patients without these diagnoses. Thus the mRNA expression seems to be unaltered in these disorders....

  3. Serotonin Transporter (5-HTT) and gamma-Aminobutyric Acid Receptor Subunit beta3 (GABRB3) Gene Polymorphisms are not Associated with Autism in the IMGSA Families

    DEFF Research Database (Denmark)

    Maestrini, E.; Lai, C.; Marlow, A.;

    1999-01-01

    Previous studies have suggested that the serotonin transporter (5-HTT) gene and the gamma-aminobutyric acid receptor subunit beta3 (GABRB3) gene, or other genes in the 15q11-q13 region, are possibly involved in susceptibility to autism. To test this hypothesis we performed an association study...... and the GABRB3 genes are unlikely to play a major role in the aetiology of autism in our family data set....

  4. Sensitivity, Specificity, and Clinical Value of Human Papillomavirus (HPV) E6/E7 mRNA Assay as a Triage Test for Cervical Cytology and HPV DNA Test ▿

    Science.gov (United States)

    Benevolo, Maria; Vocaturo, Amina; Caraceni, Donatella; French, Deborah; Rosini, Sandra; Zappacosta, Roberta; Terrenato, Irene; Ciccocioppo, Lucia; Frega, Antonio; Rossi, Paolo Giorgi

    2011-01-01

    There is evidence that testing for human papillomavirus (HPV) E6/E7 mRNA is more specific than testing for HPV DNA. A retrospective study was carried out to evaluate the performance of the PreTect HPV-Proofer E6/E7 mRNA assay (Norchip) as a triage test for cytology and HPV DNA testing. This study analyzed 1,201 women, 688 of whom had a colposcopy follow-up and 195 of whom had histology-confirmed high-grade intraepithelial neoplasia or worse (CIN2+). The proportion of positive results and the sensitivity and specificity for CIN2+ were determined for HPV mRNA in comparison to HPV DNA and cytology. All data were adjusted for follow-up completeness. Stratified by cytological grades, the HPV mRNA sensitivity was 83% (95% confidence interval [CI] = 63 to 94%) in ASC-US (atypical squamous cells of undetermined significance), 62% (95% CI = 47 to 75%) in L-SIL (low-grade squamous intraepithelial lesion), and 67% (95% CI = 57 to 76%) in H-SIL (high-grade squamous intraepithelial lesion). The corresponding figures were 99, 91, and 96%, respectively, for HPV DNA. The specificities were 82, 76, and 45%, respectively, for HPV mRNA and 29, 13, and 4%, respectively, for HPV DNA. Used as a triage test for ASC-US and L-SIL, mRNA reduced colposcopies by 79% (95% CI = 74 to 83%) and 69% (95% CI = 65 to 74%), respectively, while HPV DNA reduced colposcopies by 38% (95% CI = 32 to 44%) and by 15% (95% CI = 12 to 19%), respectively. As a HPV DNA positivity triage test, mRNA reduced colposcopies by 63% (95% CI = 60 to 66%), having 68% sensitivity (95% CI = 61 to 75%), whereas cytology at the ASC-US+ threshold reduced colposcopies by 23% (95% CI = 20 to 26%), showing 92% sensitivity (95% CI = 87 to 95%). In conclusion, PreTect HPV-Proofer mRNA can serve as a better triage test than HPV DNA to reduce colposcopy referral in both ASC-US and L-SIL. It is also more efficient than cytology for the triage of HPV DNA-positive women. Nevertheless, its low sensitivity demands a strict follow-up of

  5. Human immunodeficiency virus type 1 Vif functionally interacts with diverse APOBEC3 cytidine deaminases and moves with them between cytoplasmic sites of mRNA metabolism.

    Science.gov (United States)

    Marin, Mariana; Golem, Sheetal; Rose, Kristine M; Kozak, Susan L; Kabat, David

    2008-01-01

    Vif(IIIB), which has been a standard model for the viral infectivity factor of human immunodeficiency virus type 1 (HIV-1), binds the cytidine deaminase APOBEC3G (A3G) and induces its degradation, thereby precluding its lethal incorporation into assembling virions. Additionally, Vif(IIIB) less efficiently degrades A3F, another potent anti-HIV-1 cytidine deaminase. Although the APOBEC3 paralogs A3A, A3B, and A3C have weaker anti-HIV-1 activities and are only partially degraded by Vif(IIIB), we found that Vif(IIIB) induces their emigration from the nucleus to the cytosol and thereby causes net increases in the cytosolic concentrations and anti-HIV-1 activities of A3A and A3B. In contrast, some other Vifs, exemplified by Vif(HXB2) and Vif(ELI-1), much more efficiently degrade and thereby neutralize all APOBEC3s. Studies focused mainly on A3F imply that it occurs associated with mRNA-PABP1 in translationally active polysomes and to a lesser extent in mRNA processing bodies (P-bodies). A3F appears to stabilize the P-bodies with which it is associated. A correspondingly small proportion of Vif(IIIB) also localizes in P-bodies in an A3F-dependent manner. Stress causes A3A, A3B, A3C, and A3F to colocalize efficiently with Vif(IIIB) and mRNA-PABP1 complexes in stress granules in a manner that is prevented by cycloheximide, an inhibitor of translational elongation. Coimmunoprecipitation studies suggest that Vifs from different HIV-1 isolates associate with all tested APOBEC3s. Thus, Vifs interact closely with structurally diverse APOBEC3s, with effects on their subcellular localization, degradation rates, and antiviral activities. Cytosolic APOBEC3-Vif complexes are predominantly bound to mRNAs that dynamically move between translationally active and storage or processing pools.

  6. Repression of microRNA-768-3p by MEK/ERK signalling contributes to enhanced mRNA translation in human melanoma.

    Science.gov (United States)

    Jiang, C C; Croft, A; Tseng, H-Y; Guo, S T; Jin, L; Hersey, P; Zhang, X D

    2014-05-15

    Increased global protein synthesis and selective translation of mRNAs encoding proteins contributing to malignancy is common in cancer cells. This is often associated with elevated expression of eukaryotic translation initiation factor 4 (eIF4E), the rate-limiting factor of cap-dependent translation initiation. We report here that in human melanoma downregulation of miR-768-3p as a result of activation of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway has an important role in the upregulation of eIF4E and enhancement in protein synthesis. Melanoma cells displayed increased nascent protein production and elevated eIF4E expression, which was associated with the downregulation of miR-768-3p that was predicted to target the 3'-untranslated region of the eIF4E mRNA. Overexpression of miR-768-3p led to the downregulation of the endogenous eIF4E protein, reduction in nascent protein synthesis and inhibition of cell survival and proliferation. These effects were efficiently reversed when eIF4E was co-overexpressed in melanoma cells. On the other hand, introduction of anti-miR-768-3p into melanocytes upregulated endogenous eIF4E protein expression and increased global protein synthesis. Downregulation of miR-768-3p appeared to be mediated by activation of the MEK/ERK pathway, in that treatment of BRAF(V600E) melanoma cells with the mutant BRAF inhibitor PLX4720 or exposure of either BRAF(V600E) or wild-type BRAF melanoma cells to the MEK inhibitor U0126 resulted in the upregulation of miR-768-3p and inhibition of nascent protein synthesis. This inhibition was partially blocked in cells cointroduced with anti-miR-768-3p. Significantly, miR-768-3p was similarly downregulated, which was inversely associated with the expression levels of eIF4E in fresh melanoma isolates. Taken together, these results identify downregulation of miR-768-3p and subsequent upregulation of eIF4E as an important mechanism in addition to

  7. 14-3-3 isoforms bind directly exon B of the 5'-UTR of human surfactant protein A2 mRNA.

    Science.gov (United States)

    Noutsios, Georgios T; Ghattas, Paul; Bennett, Stephanie; Floros, Joanna

    2015-07-15

    Human surfactant protein (SP) A (SP-A), an innate immunity molecule, is encoded by two genes, SFTPA1 and SFTPA2. The 5'-untranslated splice variant of SP-A2 (ABD), but not SP-A1 (AD), contains exon B (eB). eB is an enhancer for transcription and translation and contains cis-regulatory elements. Specific trans-acting factors, including 14-3-3, bind eB. The 14-3-3 protein family contains seven isoforms that have been found by mass spectrometry in eB electromobility shift assays (Noutsios et al. Am J Physiol Lung Cell Mol Physiol 304: L722-L735, 2013). We used four different approaches to investigate whether 14-3-3 isoforms bind directly to eB. 1) eB RNA pulldown assays showed that 14-3-3 isoforms specifically bind eB. 2) RNA electromobility shift assay complexes were formed using purified 14-3-3 isoforms β, γ, ε, η, σ, and τ, but not isoform ζ, with wild-type eB RNA. 3 and 4) RNA affinity chromatography assays and surface plasmon resonance analysis showed that 14-3-3 isoforms β, γ, ε, η, σ, and τ, but not isoform ζ, specifically and directly bind eB. Inhibition of 14-3-3 isoforms γ, ε, η, and τ/θ with shRNAs in NCI-H441 cells resulted in downregulation of SP-A2 levels but did not affect SP-A1 levels. However, inhibition of 14-3-3 isoform σ was correlated with lower levels of SP-A1 and SP-A2. Inhibition of 14-3-3 isoform ζ/δ, which does not bind eB, had no effect on expression levels of SP-A1 and SP-A2. In conclusion, the 14-3-3 protein family affects differential regulation of SP-A1 and SP-A2 by binding directly to SP-A2 5'-UTR mRNA.

  8. Urokinase receptor mRNA level and gene transcription are strongly and rapidly increased by phorbol myristate acetate in human monocyte-like U937 cells

    DEFF Research Database (Denmark)

    Lund, L R; Rønne, E; Roldan, A.L.

    1991-01-01

    enhancement after 24 h of treatment. Half-maximal stimulation occurs at approximately 5 nM PMA. The effect is observed only with phorbol esters that also act as tumor promotors. The protein synthesis inhibitor cycloheximide (10 micrograms/ml) also increases the level of u-PAR mRNA. Nuclear run-on experiments...

  9. Passive leg movement enhances interstitial VEGF protein, endothelial cell proliferation, and eNOS mRNA content in human skeletal muscle

    DEFF Research Database (Denmark)

    Hellsten, Ylva; Rufener, Nora; Nielsen, Jens J

    2008-01-01

    were analyzed for mRNA content of VEGF, endothelial nitric oxide synthase (eNOS), and matrix metalloproteinase-2 (MMP-2). The passive leg movement caused an increase (P dialysate...... to cultured endothelial cells revealed that dialysate obtained during leg movement induced a 3.2-fold higher proliferation rate (P dialysate obtained at rest. Passive movement also enhanced (P

  10. Human papillomavirus mRNA testing for the detection of anal high-grade squamous intraepithelial lesions in men who have sex with men infected with HIV.

    Science.gov (United States)

    Sendagorta, Elena; Romero, Maria P; Bernardino, Jose I; Beato, María J; Alvarez-Gallego, Mario; Herranz, Pedro

    2015-08-01

    Currently, screening for anal high-grade squamous intraepithelial lesions (anal HSIL) relies on anal cytology and high-resolution anoscopy. Since this approach has limited sensitivity and specificity for detecting anal HSIL, there is increasing interest in the role of biomarkers for predicting anal HSIL. The aim of this study is to evaluate the diagnostic accuracy of HPV E6/E7-mRNA expression for the detection of anal HSIL in MSM infected with HIV, in comparison to DNA-HR-HPV and anal cytology. This cross-sectional screening study included 101 MSM followed at the HIV-unit of La Paz University Hospital. Intra-anal swabs from patients participating in a screening program including cytology, high-resolution anoscopy and histology were analyzed. HR-HPV-DNA detection was performed by means of the CLART® HPV2 assay (GENOMICA S.A.U., Madrid, Spain). E6/E7-mRNA detection of HR-HPV-types 16, 18, 31, 33, and 45 was performed using the NucliSENS-EasyQ assay (BioMérieux, Marcy ĺEtoile, France). HR-HPV DNA and HPVE6/E7 mRNA were detected in 82% and 57% of the anal smears respectively. Anal cytology screening was abnormal in 70.3%. For the detection of HSIL sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were 71.7%, 55.6%, 57.9%, and 69.8% for E6/E7-mRNA testing, respectively, compared to 100%, 31.5%, 55.4%, and 100% for HR-HPV-DNA testing and to 83%, 40.7%, 54.9%, 73.3% of cytology testing. In comparison with the other tests, HPVE6/E7 mRNA testing yielded a lower clinical sensitivity but a higher clinical specificity and PPV for the detection of anal HSIL in MSM infected with HIV.

  11. Vimentin is a component of a complex that binds to the 5'-UTR of human heme-regulated eIF2α kinase mRNA and regulates its translation.

    Science.gov (United States)

    Chatterjee, Sangeeta; Panda, Amaresh C; Berwal, Sunil K; Sreejith, R K; Ritvika, Charu; Seshadri, Vasudevan; Pal, Jayanta K

    2013-03-01

    The human heme-regulated eIF2α kinase, also called the human heme-regulated inhibitor (hHRI) is significantly up-regulated particularly at the level of translation during stress. In this report we show that during lead-stress, the regulation of hHRI mRNA translation is mediated through its 5'-untranslated region (UTR) that interacts with specific trans-acting factors. Further, vimentin has been identified as one of the trans-acting factors that contribute to this regulation.

  12. Evaluation of the Relationship between 5-HTT and MAO Gene Polymorphisms, Mood and Level of Anxiety among Postmenopausal Women

    Directory of Open Access Journals (Sweden)

    Elżbieta Grochans

    2014-12-01

    Full Text Available Objective: The aim of this study was to analyze how mood and anxiety level are related to the functional genetic polymorphism in the promoter region of SLC6A4 (5-HTTLPR and the 30-bp VNTR polymorphism in the MAO A promoter region. Methods: The study involved 272 postmenopausal women from Poland. The authors employed the State-Trait Anxiety Inventory for measuring levels of anxiety, the Mood Adjective Check List for measuring mood, and genetic tests. Results: Analysis did not show any statistically significant differences in the mean levels of anxiety, and mood disorders in women in relation to genotypes of the 5-HTTLPR (SLC6A4 polymorphism and the 30-bp VNTR polymorphism in the MAO A promoter region. However, these problems were more severe among women with s/s genotype. In the case of MAO A gene polymorphism, the level of anxiety was higher in women with a 4/4 genotype. Conclusions: The study did not prove the possibility of the identification of homogeneous groups of women with an elevated risk of developing anxiety and mood disorders during the post-menopausal period. Nevertheless, it showed that respondents with s/s genotype of the 44-bp polymorphism in the 5-HTT (SLC6A4 promoter region had the highest average anxiety levels both as a state and as a trait. Furthermore, the analysis of the 30-bp VNTR polymorphism in the MAO A promoter region demonstrated slight differences in anxiety levels between the women, indicating that those with a 4/4 genotype had higher severity of anxiety symptoms.

  13. REVIEW-ARTICLE Intermediate alleles of Huntington's disease HTT gene in different populations worldwide: a systematic review.

    Science.gov (United States)

    Apolinário, T A; Paiva, C L A; Agostinho, L A

    2017-04-05

    Huntington's disease (HD) is an autosomal dominant progressive neurodegenerative disorder caused by a dynamic mutation due to the expansion of CAG repeats in the HTT gene (4p16.3). The considered normal alleles have less than 27 CAG repeats. Intermediate alleles (IAs) show 27 to 35 CAG repeats and expanded alleles have more than 35 repeats. The IAs apparently have shown a normal phenotype. However, there are some reported associations between individuals that bear an IA and clinical HD signs, such as behavioral disturbs. The association of IAs with the presence of clinical signs gives clinical relevance to these patients. We emphasized the importance of determining the frequency of IA alleles in the general population as well as in HD families. Therefore, the aim of this study was to conduct a systematic review, in order to investigate the frequency of IAs in the overall chromosomes of different ethnic groups and of families with HD history worldwide as well as the frequency of individuals who bear the intermediate alleles. We searched indexed articles from the following electronic databases: U.S. National Library of Medicine and the National Institutes of Health (PubMed), Pubmed Central (PMC) and Virtual Health Library (VHL). Therefore, 488 articles were obtained and, of these, 33 had been published in more than one database. We accepted the article of only one database and ended up with 455 articles for this review. The frequency of IAs within the chromosomes of the general population ranged from 0.45 to 8.7% and of individuals with family history of HD ranged from 0.05 to 5.1%. The higher frequency of IAs in the general population (8.7%) was found in one Brazilian cohort.

  14. Evaluation of the Relationship between 5-HTT and MAO Gene Polymorphisms, Mood and Level of Anxiety among Postmenopausal Women

    Science.gov (United States)

    Grochans, Elżbieta; Jurczak, Anna; Szkup, Małgorzata; Samochowiec, Agnieszka; Włoszczak-Szubzda, Anna; Karakiewicz, Beata; Grzywacz, Anna; Brodowska, Agnieszka; Samochowiec, Jerzy

    2014-01-01

    Objective: The aim of this study was to analyze how mood and anxiety level are related to the functional genetic polymorphism in the promoter region of SLC6A4 (5-HTTLPR) and the 30-bp VNTR polymorphism in the MAO A promoter region. Methods: The study involved 272 postmenopausal women from Poland. The authors employed the State-Trait Anxiety Inventory for measuring levels of anxiety, the Mood Adjective Check List for measuring mood, and genetic tests. Results: Analysis did not show any statistically significant differences in the mean levels of anxiety, and mood disorders in women in relation to genotypes of the 5-HTTLPR (SLC6A4) polymorphism and the 30-bp VNTR polymorphism in the MAO A promoter region. However, these problems were more severe among women with s/s genotype. In the case of MAO A gene polymorphism, the level of anxiety was higher in women with a 4/4 genotype. Conclusions: The study did not prove the possibility of the identification of homogeneous groups of women with an elevated risk of developing anxiety and mood disorders during the post-menopausal period. Nevertheless, it showed that respondents with s/s genotype of the 44-bp polymorphism in the 5-HTT (SLC6A4) promoter region had the highest average anxiety levels both as a state and as a trait. Furthermore, the analysis of the 30-bp VNTR polymorphism in the MAO A promoter region demonstrated slight differences in anxiety levels between the women, indicating that those with a 4/4 genotype had higher severity of anxiety symptoms. PMID:25547397

  15. In humans IL-6 is released from the brain during and after exercise and paralleled by enhanced IL-6 mRNA expression in the hippocampus of mice

    DEFF Research Database (Denmark)

    Rasmussen, Per; Vedel, J-C; Olesen, J

    2011-01-01

    . Additionally, it was evaluated in mice whether brain release of IL-6 reflected enhanced IL-6 mRNA expression in the brain as modulated by brain glycogen levels. Methods: Nine healthy male subjects completed 4 h of ergometer rowing while the arterio-jugular venous difference (a-v diff) for IL-6 was determined....... The IL-6 mRNA and the glycogen content were determined in mouse hippocampus, cerebellum and cortex before and after 2 h treadmill running (N = 8). Results: At rest, the IL-6 a-v diff was negligible but decreased to -2.2 ± 1.9 pg ml(-1) at the end of exercise and remained low (-2.1 ± 2.1 pg ml(-1) ) 1 h...

  16. 吗啡对人胃癌MGC-803细胞p53 mRNA和E2F-1 mRNA表达的影响%Effect of morphine on expression of p53 mRNA and E2F-1 mRNA in human gastric carcinoma cell line MGC-803

    Institute of Scientific and Technical Information of China (English)

    覃怡; 唐小曼; 廖淳杰; 谢玉波

    2010-01-01

    Objective To investigate the effect of morphine on the expression of p53 mRNA and E2F-1 mRNA in human gastric carcinoma cell line MGC-803 .Methods The human gastric cancer cell line MGC-803 was purchased from Cell Biology Research Institute, Chinese Academy of Sciences, and cultured in DMEM liquid culture mediun. The cells were seeded in 6-well plates (1 × 103/ml or 2 × 105/ml, 1 ml/well) and divided into 2 groups (n = 18 wells each):group Ⅰ normal control (group C); group Ⅱ was exposed to 10 μmol/L morphine (group M). The proliferation of the cells was determined by colony formation assay at 7 day of incubation with morphine. The expression of p53 mRNA and E2F-1 mRNA was detected and the ulrastructure of the cells examined with transmission electron microscope after being incubated with morphine for 24 h. Results The proliferation of the cells and E2F-1 mRNA expression were significantly lower and p53 mRNA expression was significantly higher in group M than in group C (P < 0.05). The nuclear evelope was intact and the nucleolus and chromosomes were clearly visible in group C, while in group M fragmentation of nuclear envelope and nucleolus and apoptotic bodies were observed. Conclusion Morphine can inhibit the proliferation of the cells and accelerate the cell apoptosis through up-regulating the expression of p53 gene and down-regulating the expression of E2F-1gene in human gastric carcinoma cell line MGC-803.%目的 探讨吗啡对人胃癌MGC-803细胞p53 mRNA和E2F-1 mRNA表达的影响.方法 人胃癌MGC-803细胞以1×103/ml或2×105/ml密度接种于6孔培养板中,1 ml/孔,随机分为2组(n=18),正常对照组(C组)不作任何处理,吗啡组(M组)加入吗啡,使其终浓度为10μmol/L.于吗啡孵育7 d时应用克隆形成实验测定细胞增殖情况,于吗啡孵育24 h时测定细胞中p53 mRNA、E2F-1 mRNA的表达情况,并采用透射电子显微镜观察细胞超微结构.结果 与C组比较,M组克隆形成率降低,p53 m

  17. mRNA Expression and DNA Methylation Analysis of Serotonin Receptor 2A (HTR2A in the Human Schizophrenic Brain

    Directory of Open Access Journals (Sweden)

    Sern-Yih Cheah

    2017-01-01

    Full Text Available Serotonin receptor 2A (HTR2A is an important signalling factor implicated in cognitive functions and known to be associated with schizophrenia. The biological significance of HTR2A in schizophrenia remains unclear as molecular analyses including genetic association, mRNA expression and methylation studies have reported inconsistent results. In this study, we examine HTR2A expression and methylation and the interaction with HTR2A polymorphisms to identify their biological significance in schizophrenia. Subjects included 25 schizophrenia and 25 control post-mortem brain samples. Genotype and mRNA data was generated by transcriptome sequencing. DNA methylation profiles were generated for CpG sites within promoter-exon I region. Expression, genotype and methylation data were examined for association with schizophrenia. HTR2A mRNA levels were reduced by 14% (p = 0.006 in schizophrenia compared to controls. Three CpG sites were hypermethylated in schizophrenia (cg5 p = 0.028, cg7 p = 0.021, cg10 p = 0.017 and HTR2A polymorphisms rs6314 (p = 0.008 and rs6313 (p = 0.026 showed genetic association with schizophrenia. Differential DNA methylation was associated with rs6314 and rs6313. There was a strong correlation between HTR2A DNA methylation and mRNA expression. The results were nominally significant but did not survive the rigorous Benjamini-Hochberg correction for multiple testing. Differential HTR2A expression in schizophrenia in our study may be the result of the combined effect of multiple differentially methylated CpG sites. Epigenetic HTR2A regulation may alter brain function, which contributes to the development of schizophrenia.

  18. mRNA Expression and DNA Methylation Analysis of Serotonin Receptor 2A (HTR2A) in the Human Schizophrenic Brain.

    Science.gov (United States)

    Cheah, Sern-Yih; Lawford, Bruce R; Young, Ross McD; Morris, Charles P; Voisey, Joanne

    2017-01-04

    Serotonin receptor 2A (HTR2A) is an important signalling factor implicated in cognitive functions and known to be associated with schizophrenia. The biological significance of HTR2A in schizophrenia remains unclear as molecular analyses including genetic association, mRNA expression and methylation studies have reported inconsistent results. In this study, we examine HTR2A expression and methylation and the interaction with HTR2A polymorphisms to identify their biological significance in schizophrenia. Subjects included 25 schizophrenia and 25 control post-mortem brain samples. Genotype and mRNA data was generated by transcriptome sequencing. DNA methylation profiles were generated for CpG sites within promoter-exon I region. Expression, genotype and methylation data were examined for association with schizophrenia. HTR2A mRNA levels were reduced by 14% (p = 0.006) in schizophrenia compared to controls. Three CpG sites were hypermethylated in schizophrenia (cg5 p = 0.028, cg7 p = 0.021, cg10 p = 0.017) and HTR2A polymorphisms rs6314 (p = 0.008) and rs6313 (p = 0.026) showed genetic association with schizophrenia. Differential DNA methylation was associated with rs6314 and rs6313. There was a strong correlation between HTR2A DNA methylation and mRNA expression. The results were nominally significant but did not survive the rigorous Benjamini-Hochberg correction for multiple testing. Differential HTR2A expression in schizophrenia in our study may be the result of the combined effect of multiple differentially methylated CpG sites. Epigenetic HTR2A regulation may alter brain function, which contributes to the development of schizophrenia.

  19. Studies on microsatellite instability in p16 gene and expression of hMSH2 mRNA in human gastric cancer tissues

    Institute of Scientific and Technical Information of China (English)

    Qin-Xian Zhang; Yi Ding; Xiao-Ping Le; Peng Du

    2003-01-01

    AIM: To detect the loss of heterozygosity (LOH) frequency of microsatellite sites D9s171, D9s1604 of p16 gene and expression of hMSH2 mRNA in various differentiated types of gastric cancer, adjacent cancer tissues and normal gastric mucosa.METHODS: LOH was detected by polymerase chain reaction (PCR)-denaturing polyacrylamide gel electrophoresis-silver staining. The expression of hMSH2 mRNA was examined with in situ hybridization.RESULTS: The frequency rate of LOH was significantly higher in gastric cancers than that in adjacent cancer tissues (P=0.032). No significant difference was noted among various differentiated types and various clinical stages of gastric cancers. The significantly reduced expression of hMSH2mRNA positive signal cells exhibited in gastric cancers, in comparison with that in the adjacent cancer tissues and normal gastric mucosa, respectively (P=0.001). No significant difference was noted among various clinical stages of gastric cancers (P>0.05). The difference of positive signal cells in poorly differentiated cancers and those in well and moderately differentiated cancers were significant (P<0.001).CONCLUSION: The frequencies of LOH in two microsatellite sites, D9s171 and D9s1604, in p16 genome were associated with development of gastric cancer and no significant correlation was demonstrated between the LOH frequency and the cell differentiated types of tumor cells or clinical stages. There was a positive relationship between the expression of hMSH2 mRNA and the differentiated types of gastric cancer.

  20. Dose-Dependent Pattern of Inducible mRNA Expression of PIG3 Gene in Normal Human Lymphoblastoid Cells by Thermal Neutron

    Institute of Scientific and Technical Information of China (English)

    MA; Nan-ru; SUI; Li; WANG; Xiao; KONG; Fu-quan; LIU; Xiao-dan; ZHOU; Ping-kun

    2012-01-01

    <正>Using the thermal neutron produced by the hospital neutron irradiator, AHH-1 cell was irradiated at the various dose, 0, 0.5, 2, 4, 6 and 8 Gy, respectively. After irradiation, cells were collected at 2, 6, 12, 24 and 48 h post-irradiation, and then cell cycle distribution was tested using flow cytometry, as well as PIG3 mRNA expression level was detected using real-time fluorescent quantitative PCR detection.

  1. Evidence for genetic regulation of mRNA expression of the dosage-sensitive gene retinoic acid induced-1 (RAI1) in human brain

    Science.gov (United States)

    Chen, Li; Tao, Yu; Song, Fan; Yuan, Xi; Wang, Jian; Saffen, David

    2016-01-01

    RAI1 (retinoic acid induced-1) is a dosage-sensitive gene that causes Smith-Magenis syndrome (SMS) when mutated or deleted and Potocki-Lupski Syndrome (PTLS) when duplicated, with psychiatric features commonly observed in both syndromes. How common genetic variants regulate this gene, however, is unknown. In this study, we found that RAI1 mRNA expression in Chinese prefrontal and temporal cortex correlate with genotypes of common single nucleotide polymorphisms (SNPs) located in the RAI1 5′-upstream region. Using genotype imputation, “R2-Δ2” analysis, and data from the RegulomeDB database, we identified SNPs rs4925102 and rs9907986 as possible regulatory variants, accounting for approximately 30–40% of the variance in RAI1 mRNA expression in both brain regions. Specifically, rs4925102 and rs9907986 are predicted to disrupt the binding of retinoic acid RXR-RAR receptors and the transcription factor DEAF1 (Deformed epidermal autoregulatory factor-1), respectively. Consistent with these predictions, we observed binding of RXRα and RARα to the predicted RAI1 target in chromatin immunoprecipitation assays. Retinoic acid is crucial for early development of the central neural system, and DEAF1 is associated with intellectual disability. The observation that a significant portion of RAI1 mRNA expression is genetically controlled raises the possibility that common RAI1 5′-region regulatory variants contribute more generally to psychiatric disorders. PMID:26743651

  2. A Stable Upstream Stem-loop Structure Enhances Selection of the First 5′-ORF-AUG as a Main Start Codon for Translation Initiation of Human ACAT1 mRNA

    Institute of Scientific and Technical Information of China (English)

    Li YANG; Jiang CHEN; Catherine C. Y. CHANG; Xin-Ying YANG; Zhen-Zhen WANG; Ta-Yuan CHANG; Bo-Liang LI

    2004-01-01

    Human ACAT1 cDNA K1 was first cloned and functionally expressed in 1993. There are two adjacent in-frame AUG codons, AUG1397-1399 and AUG1415-1417, at 5′-terminus of the open reading frame (ORF,nt 1397-3049) of human ACAT1 mRNA corresponding to cDNA K1. In current work, these two adjacent inframe AUGs at 5 ′-terminus of the predicted ORF (5′-ORF-AUGs) as start codons for translation initiation of human ACAT1 mRNA were characterized in detail. Codon mutations indicated that both of these two adjacent 5′-ORF-AUGs can be selected as start codons but the first 5′-ORF-AUG1397-1399 is a main start codon consistent with that of the predicted ORF of human ACAT1 mRNA. Further deletion and mutation analyses demonstrated that a stable upstream stem-loop structure enhanced the selection of the first 5 ′-ORF-AUG1397-1399 as a main start codon, in addition to upstream nucleotide A in the -3 position, which is a key site of Kozak sequence. In addition, result of ACAT1 enzymatic activity assay showed no obvious difference between these two ACAT1 proteins respectively initiated from the two adjacent 5′-ORF-AUGs. This work showed that a stable upstream stem-loop structure could modulate the start codon selection during translation initiation of mRNAs that contain adjacent multi-5′-ORF-AUGs.

  3. Effect of phytic acid ketone on expression of HPV16/18 E6/E7 mRNA and protein in human cervical cancer cell line%植酸酮对宫颈癌细胞株中 HPV16/18 E6/E7 mRNA 及蛋白表达的影响

    Institute of Scientific and Technical Information of China (English)

    王侃; 高晓丽; 田文艳; 岳天孚

    2016-01-01

    Objective To observe the effects of phytic acid ketone on expression of HPV 16/18 E6/E7 mRNA and protein in human cervical cancer cells.Methods The Caski cells ( containing HPV16 ) and Hela cells ( containing HPV18) were cultivated in vitro and then divided into the experimental groups 1, 2, 3, 4 and the control group ( each group contained Caski cell line and Hela cell line).The experimental groups 1, 2, 3, 4 were respectively treated with dif-ferent concentrations of phytic acid ketone (58.6 mg/L, 117 mg/L, 586 mg/L, 5 860 mg/L) for 72 h.The levels of HPV16/18 E6/E7 mRNA were determined with real-time PCR.Western blotting was used to measure the expression levels of E6 and E7 proteins.Results E6/E7 mRNA and protein expression in the same type of cells of the experimental groups 1, 2, 3 and 4 was lower than that of the control group, and the E6/E7 mRNA and protein expression showed a decreasing tendency in the experimental groups 1, 2, 3 and 4 (all P<0.05).In each experimental group, E6/E7 mRNA expression in Caski cells was lower than that in Hela cells (all P<0.05).In the same type of cells of the experiential groups, the E6 mRNA expression was lower than that of E7 mRNA (all P<0.05).Conclusions Phytic acid ketone could down-regulate the expression of E6/E7 mRNA and protein in human cervical cancer lines, which was in a dose-dependent manner.The inhibitory effect of phytic acid ketone on HPV16 E6/E7 mRNA was stronger than that on HPV18 E6/E7 mRNA and the suppression of E6 probably played a leading role.%目的:观察植酸酮对宫颈癌细胞株中HPV16/18 E6/E7 mRNA及蛋白表达的影响。方法体外培养Caski细胞株(含HPV16)与Hela细胞株(含HPV18),将所有细胞分成实验1、2、3、4组和对照组,每组均包含Caski细胞和Hela细胞。实验1、2、3、4组分别加入含58.6、117、586、5860 mg/L植酸酮的培养液,对照组加入不含植酸酮的培养液。各组培养72 h后,采用real-time PCR

  4. Non-secreted clusterin isoforms are translated in rare amounts from distinct human mRNA variants and do not affect Bax-mediated apoptosis or the NF-κB signaling pathway.

    Directory of Open Access Journals (Sweden)

    Hans Prochnow

    Full Text Available Clusterin, also known as apolipoprotein J, is expressed from a variety of tissues and implicated in pathological disorders such as neurodegenerative diseases, ischemia and cancer. In contrast to secretory clusterin (sCLU, which acts as an extracellular chaperone, the synthesis, subcellular localization and function(s of intracellular CLU isoforms is currently a matter of intense discussion. By investigating human CLU mRNAs we here unravel mechanisms leading to the synthesis of distinct CLU protein isoforms and analyze their subcellular localization and their impact on apoptosis and on NF-κB-activity. Quantitative PCR-analyses revealed the expression of four different stress-inducible CLU mRNA variants in non-cancer and cancer cell lines. In all cell lines variant 1 represents the most abundant mRNA, whereas all other variants collectively account for no more than 0.34% of total CLU mRNA, even under stressed conditions. Overexpression of CLU cDNAs combined with in vitro mutagenesis revealed distinct translational start sites including a so far uncharacterized non-canonical CUG start codon. We show that all exon 2-containing mRNAs encode sCLU and at least three non-glycosylated intracellular isoforms, CLU1‑449, CLU21‑449 and CLU34‑449, which all reside in the cytosol of unstressed and stressed HEK‑293 cells. The latter is the only form expressed from an alternatively spliced mRNA variant lacking exon 2. Functional analysis revealed that none of these cytosolic CLU forms modulate caspase-mediated intrinsic apoptosis or significantly affects TNF-α-induced NF-κB-activity. Therefore our data challenge some of the current ideas regarding the physiological functions of CLU isoforms in pathologies.

  5. Dominant-Negative Effects of Adult-Onset Huntingtin Mutations Alter the Division of Human Embryonic Stem Cells-Derived Neural Cells.

    Science.gov (United States)

    Lopes, Carla; Aubert, Sophie; Bourgois-Rocha, Fany; Barnat, Monia; Rego, Ana Cristina; Déglon, Nicole; Perrier, Anselme L; Humbert, Sandrine

    2016-01-01

    Mutations of the huntingtin protein (HTT) gene underlie both adult-onset and juvenile forms of Huntington's disease (HD). HTT modulates mitotic spindle orientation and cell fate in mouse cortical progenitors from the ventricular zone. Using human embryonic stem cells (hESC) characterized as carrying mutations associated with adult-onset disease during pre-implantation genetic diagnosis, we investigated the influence of human HTT and of an adult-onset HD mutation on mitotic spindle orientation in human neural stem cells (NSCs) derived from hESCs. The RNAi-mediated silencing of both HTT alleles in neural stem cells derived from hESCs disrupted spindle orientation and led to the mislocalization of dynein, the p150Glued subunit of dynactin and the large nuclear mitotic apparatus (NuMA) protein. We also investigated the effect of the adult-onset HD mutation on the role of HTT during spindle orientation in NSCs derived from HD-hESCs. By combining SNP-targeting allele-specific silencing and gain-of-function approaches, we showed that a 46-glutamine expansion in human HTT was sufficient for a dominant-negative effect on spindle orientation and changes in the distribution within the spindle pole and the cell cortex of dynein, p150Glued and NuMA in neural cells. Thus, neural derivatives of disease-specific human pluripotent stem cells constitute a relevant biological resource for exploring the impact of adult-onset HD mutations of the HTT gene on the division of neural progenitors, with potential applications in HD drug discovery targeting HTT-dynein-p150Glued complex interactions.

  6. Prediction of translation initiation sites in human mRNA sequences with AUG start codon in weak Kozak context: A neural network approach.

    Science.gov (United States)

    Tikole, Suhas; Sankararamakrishnan, Ramasubbu

    2008-05-16

    Translation of eukaryotic mRNAs is often regulated by nucleotides around the start codon. A purine at position -3 and a guanine at position +4 contribute significantly to enhance the translation efficiency. Algorithms to predict the translation initiation site often fail to predict the start site if the sequence context is not present. We have developed a neural network method to predict the initiation site of mRNA sequences that lack the preferred nucleotides at the positions -3 and +4 surrounding the translation initiation site. Neural networks of various architectures comprising different number of hidden layers were designed and tested for various sizes of windows of nucleotides surrounding translation initiation sites. We found that the neural network with two hidden layers showed a sensitivity of 83% and specificity of 73% indicating a vastly improved performance in successfully predicting the translation initiation site of mRNA sequences with weak Kozak context. WeakAUG server is freely available at http://bioinfo.iitk.ac.in/AUGPred/.

  7. EFFECTS OF ALVEOLAR MACROPHAGE CONDITIONED MEDIA FROM INTERSTITIAL LUNG DISEASEPATIENTS ON THE PROCOLLAGEN mRNA EXPRESSION IN HUMAN LUNG FIBROBLASTS

    Institute of Scientific and Technical Information of China (English)

    郭子健; 朱元珏; 刘秉慈; 朱亚玲; 赵文理; 陈勇

    1996-01-01

    Progressive inflammation and fibrosis are the central processez in the pathogenesis of pulmonary fibrosis. It is believed that macrophages in areas of chronically inflamed lung play a key role in fibrotic response. Therefore, we investigated the effects of alveolar macrophage (Amφ) conditioned media from interstitial lung disease (ILD) patients on lung fibroblast proliferation and procollagen mRNA expression, After stimulating with Amφ conditioned media from ILD pasients, the fibroblast proliferation increased 71.4% compared with the control, but for media from bronchial carcinoma (BC) patients, it just increased 14.3%. There is a significant dffference between the two groups (P<0. 05). The procollagen αl(I) mRNA in fibroblasts stimulated with Amφ conditioned media from ILD patients was increased 21.3% α1(Ⅲ)was 37.2 higher than control (P<0. 05). It increased 6. 8% and 12.8% fof media from BC patients respectively, but there was no difference when compared to the control. We considered that Amφ from ILD patients might be in an activated state and could release some growth factors to stimulate fibroblast proliferation and promote collagen DNA expression,

  8. 军团菌体外刺激对人PBMC TLR2 mRNA表达影响%Effects of Legionella on expression of TLR2 mRNA in human peripheral blood mononuclear cells

    Institute of Scientific and Technical Information of China (English)

    高向东; 张帆; 王素萍; 史晓红; 王伟刚; 王芳芳; 董洁敏; 雷智

    2013-01-01

    目的 探讨嗜肺军团菌活菌刺激对人外周血单个核细胞(PBMC)表达TLR2 mRNA的影响.方法 采用不同浓度的嗜肺军团菌活菌悬液刺激体外培养的健康人PBMC,收集细胞运用RT-PCR方法测定TLR2mRNA的表达水平.结果 析因分析结果显示,时间的主效应有统计学意义(F=26.06,P<0.05);时间与浓度的交互作用有统计学意义(F=11.39,P <0.05);24 h时,MOI 1 、MOI 10刺激组TLR2 mRNA的表达量均高于对照组(P<0.05),呈现随菌液浓度升高表达量也逐渐升高,48 h时,各组之间TLR2 mRNA的表达量差异虽无统计学意义(P>0.05),但表达量随浓度升高呈现下降趋势,72 h时,各组之间TLR2 mRNA的表达量差异有统计学意义(P<0.05),但随菌液浓度升高表达量反而降低.结论 用军团菌体外刺激人PBMC,其TLR2mRNA的表达在一定范围内呈一定的时效与量效关系,可为今后的研究提供有益的时间和剂量参考.%Objective To examine the expression of TLR2 in human peripheral blood mononuclear cells (PBMCs) stimulatedby Legionella. Methods Human peripheral blood mononuclear cells ( POMCs) were treated with different concentrations of Legionella(2×106/ml,2×107/ml) ,and real timel reverse transcriptase PCR(RT-PCR) was used to detect TLR2 mRNA expression. Results Factorial analyses showed that leading effect of time had statistical significance (F = 26. 06,P < 0. 05) and there was an interaction between the time and concentration ( F = 11. 39, P < 0. 05). In 24 hours,the expressions of TLR2 mRNA of PBMCs stimulated with 2×106/ml and 2×107/ml Legionella were obviously higher than TLR2 mRNA expression in control group. In 48 hours,there was no difference between stimulated group and control group. In 72 hours,the expressions of TLR2 mRNA of PBMCs stimulated with 2×106/ml and 2×107/ml Legionella , were obviously lower than TLR2 mRNA expression in the control group. When the PBMCs were stimulated at the level of 2×106/ml and 2×107/ml

  9. Suppression of testosterone does not blunt mRNA expression of myoD, myogenin, IGF, myostatin or androgen receptor post strength training in humans

    DEFF Research Database (Denmark)

    Kvorning, Thue; Andersen, Marianne; Brixen, Kim

    2007-01-01

    We hypothesized that suppression of endogenous testosterone blunts mRNA expression post strength training (ST). Twenty-two young men were randomized for treatment with the GnRH analogue goserelin (3.6 mg every 4 weeks) or placebo for a period of 12 weeks. The ST period of 8 weeks started at week ...... events were the same, despite divergent muscle hypertrophy and strength gains........ Strength test, blood sampling, muscle biopsies, and whole-body dual-energy X-ray absorptiometry (DXA) scan were performed at weeks 4 and 12. Muscle biopsies were taken during the final ST session (pre, post 4 h, and post 24 h). Resting serum testosterone decreased significantly (P ... group (P Muscle strength and muscle mass showed a tendency to increase more in the placebo group than in the goserelin group (P = 0...

  10. A promoter within the E6 ORF of human papillomavirus type 16 contributes to the expression of the E7 oncoprotein from a monocistronic mRNA

    DEFF Research Database (Denmark)

    Glahder, Jacob-Andreas Harald; Hansen, Christina N; Vinther, Jeppe

    2003-01-01

    RNA that encodes E7 as the first open reading frame (ORF) has been identified. We recently identified a transcription initiation site within the E6 ORF of HPV-16 at nt 542. In the present study we have characterized the P542 promoter, which putatively controls monocistronic expression of E7. The monocistronic m...... from nt 226 to 409. Furthermore, the translation initiation of E7 is most abundant from the monocistronic mRNA. We have also shown that the P542 promoter is downregulated by the transcription factor activator protein 4 (AP-4) and the differentiation-dependent factor hSkn-1a, both binding downstream...... of the transcription initiation site. In conclusion, we have found that P542 is a relatively weak promoter compared with P97 and may be downregulated in differentiated epithelial cells....

  11. Calciotrophic hormones and hyperglycemia modulate vitamin D receptor and 25 hydroxyy vitamin D 1-α hydroxylase mRNA expression in human vascular smooth muscle cells.

    Science.gov (United States)

    Somjen, D; Knoll, E; Sharon, O; Many, A; Stern, N

    2015-04-01

    Estrogen receptors (ERα and ERβ), the vitamin D receptor (VDR) and 25 hydroxyy vitamin D 1-α hydroxylase (1OHase) mRNA are expressed in vascular smooth muscle cells (VSMC). In these cells estrogenic hormones modulate cell proliferation as measured by DNA synthesis (DNA). In the present study we determined whether or not the calciotrophic hormones PTH 1-34 (PTH) and less- calcemic vitamin D analog QW as well as hyperglycemia can regulate DNA synthesis and CK. E2 had a bimodal effect on VSMC DNA synthesis, such that proliferation was inhibited at 30nM but stimulated at 0.3nM. PTH at 50nM increased, whereas QW at 10nM inhibited DNA synthesis. Hyperglycemia inhibited the effects on high E2, QW and PTH on DNA only. Both QW and PTH increased ERα mRNA expression, but only PTH increased ERβ expression. Likewise, both PTH and QW stimulated VDR and 1OHase expression and activity. ERβ, VDR and 1OHase expression and activity were inhibited by hyperglycemia, but ERα expression was unaffected by hyperglycemia. In conclusion, calcitrophic hormones modify VSMC growth and concomitantly affect ER expression in these cells as well as the endogenous VSMC vitamin D system elements, including VDR and 1OHase. Some of the later changes may likely participate in growth effects. Of importance in the observation is that several regulatory effects are deranged in the presence of hyperglycemia, particularly the PTH- and vitamin D-dependent up regulation of VDR and 1OHase in these cells. The implications of these effects require further studies. This article is part of a Special Issue entitled '17th Vitamin D Workshop'.

  12. Differential expression of glutathione S-transferases P1-1 and A1-1 at protein and mRNA levels in hepatocytes derived from human bone marrow mesenchymal stem cells.

    Science.gov (United States)

    Allameh, Abdolamir; Esmaeli, Shahnaz; Kazemnejad, Somaieh; Soleimani, Masoud

    2009-06-01

    The aim of this study was to find out the profile of cellular glutathione (GSH) and GSH S-transferase (GST) in hepatocytes differentiated from adult mesenchymal stem cells (MSC). For this purpose, we have derived functionally active hepatocyte-like cells from normal human multipotent adult MSC. Then the differentiated cells were characterized by specific hepatic markers. The cellular GSH and GST catalytic activity toward 1-chloro-2,4-dinitrobenzene (CDNB) were determined in hepatocyte-like cells differentiated from MSC compared with undifferentiated MSC. Reverse transcription polymerase chain reaction (RT-PCR) and immunoblotting techniques were used to study GST-P1-1 and GST-A1-1 expression in differentiated and undifferentiated cells. The results showed that there is more than threefold increase in GST catalytic activity in hepatocytes recovered by day 14 of differentiation. GST-P1-1 mRNA expression was detected in both differentiated hepatocyte-like cells and their undifferentiated progenitors. Under similar conditions, only differentiated hepatocyte-like cells expressed GST-A1-1 mRNA. These results were further confirmed by showing that the undifferentiated cells expressed both GST-A and GST-P proteins. Unlike GST, the level of cellular GSH was declined (approximately 20%) in hepatocytes derived from MSC as compared to that of undifferentiated cells. These data may suggest that hepatogenic differentiation of human bone marrow MSC is accompanied with the regulation of factors participating in GSH conjugation pathway.

  13. Self-amplifying mRNA vaccines.

    Science.gov (United States)

    Brito, Luis A; Kommareddy, Sushma; Maione, Domenico; Uematsu, Yasushi; Giovani, Cinzia; Berlanda Scorza, Francesco; Otten, Gillis R; Yu, Dong; Mandl, Christian W; Mason, Peter W; Dormitzer, Philip R; Ulmer, Jeffrey B; Geall, Andrew J

    2015-01-01

    This chapter provides a brief introduction to nucleic acid-based vaccines and recent research in developing self-amplifying mRNA vaccines. These vaccines promise the flexibility of plasmid DNA vaccines with enhanced immunogenicity and safety. The key to realizing the full potential of these vaccines is efficient delivery of nucleic acid to the cytoplasm of a cell, where it can amplify and express the encoded antigenic protein. The hydrophilicity and strong net negative charge of RNA impedes cellular uptake. To overcome this limitation, electrostatic complexation with cationic lipids or polymers and physical delivery using electroporation or ballistic particles to improve cellular uptake has been evaluated. This chapter highlights the rapid progress made in using nonviral delivery systems for RNA-based vaccines. Initial preclinical testing of self-amplifying mRNA vaccines has shown nonviral delivery to be capable of producing potent and robust innate and adaptive immune responses in small animals and nonhuman primates. Historically, the prospect of developing mRNA vaccines was uncertain due to concerns of mRNA instability and the feasibility of large-scale manufacturing. Today, these issues are no longer perceived as barriers in the widespread implementation of the technology. Currently, nonamplifying mRNA vaccines are under investigation in human clinical trials and can be produced at a sufficient quantity and quality to meet regulatory requirements. If the encouraging preclinical data with self-amplifying mRNA vaccines are matched by equivalently positive immunogenicity, potency, and tolerability in human trials, this platform could establish nucleic acid vaccines as a versatile new tool for human immunization. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. A large number of nuclear genes in the human parasite blastocystis require mRNA polyadenylation to create functional termination codons.

    Science.gov (United States)

    Klimeš, Vladimír; Gentekaki, Eleni; Roger, Andrew J; Eliáš, Marek

    2014-07-10

    Termination codons in mRNA molecules are typically specified directly by the sequence of the corresponding gene. However, in mitochondria of a few eukaryotic groups, some mRNAs contain the termination codon UAA deriving one or both adenosines from transcript polyadenylation. Here, we show that a similar phenomenon occurs for a substantial number of nuclear genes in Blastocystis spp., divergent unicellular eukaryote gut parasites. Our analyses of published genomic data from Blastocystis sp. subtype 7 revealed that polyadenylation-mediated creation of termination codons occurs in approximately 15% of all nuclear genes. As this phenomenon has not been noticed before, the procedure previously employed to annotate the Blastocystis nuclear genome sequence failed to correctly define the structure of the 3'-ends of hundreds of genes. From sequence data we have obtained from the distantly related Blastocystis sp. subtype 1 strain, we show that this phenomenon is widespread within the Blastocystis genus. Polyadenylation in Blastocystis appears to be directed by a conserved GU-rich element located four nucleotides downstream of the polyadenylation site. Thus, the highly precise positioning of the polyadenylation in Blastocystis has allowed reduction of the 3'-untranslated regions to the point that, in many genes, only one or two nucleotides of the termination codon are left. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  15. Inhibition of Mahkota Dewa (Phaleria macrocarpa bioactive fraction on proliferation of human retinoblastoma tumor cells Y-79 through suppression of mRNA level of cyclin E

    Directory of Open Access Journals (Sweden)

    Nugroho Trilaksana

    2017-04-01

    Conclusions: Our results suggest that DLBS1425 is a potential anticancer agent which targets genes involved in cell proliferation in human retinoblastoma cells which make it pharmacologically ideal for the prevention and/or treatment of retinoblastoma cancer.

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

    DEFF Research Database (Denmark)

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

    2010-01-01

    -life as longer oligonucleotides. Pharmacology studies in both mice and non-human primates were conducted with a 13-mer LNA oligonucleotide against apoB, and the data showed that repeated dosing of the 13-mer at 1-2 mg/kg/week was sufficient to provide a significant and long lasting lowering of non...... using the LNA chemistry. Conclusively, we present a 13-mer LNA oligonucleotide with therapeutic potential that produce beneficial cholesterol lowering effect in non-human primates....

  17. Nuclear phosphoinositide-specific phospholipase C β1 controls cytoplasmic CCL2 mRNA levels in HIV-1 gp120-stimulated primary human macrophages.

    Directory of Open Access Journals (Sweden)

    Francesca Spadaro

    Full Text Available HIV-1 envelope glycoprotein gp120 induces, independently of infection, the release of CCL2 from macrophages. In turn, this chemokine acts as an autocrine factor enhancing viral replication. In this study, we show for the first time that phosphoinositide-specific phospholipase C (PI-PLC is required for the production of CCL2 triggered by gp120 in macrophages. Using a combination of confocal laser-scanner microscopy, pharmacologic inhibition, western blotting and fluorescence-activated cell sorter analysis, we demonstrate that gp120 interaction with CCR5 leads to nuclear localization of the PI-PLC β1 isozyme mediated by mitogen-activated protein kinase ERK-1/2. Notably, phosphatidylcholine-specific phospholipase C (PC-PLC, previously reported to be required for NF-kB-mediated CCL2 production induced by gp120 in macrophages, drives both ERK1/2 activation and PI-PLC β1 nuclear localization induced by gp120. PI-PLC β1 activation through CCR5 is also triggered by the natural chemokine ligand CCL4, but independently of ERK1/2. Finally, PI-PLC inhibition neither blocks gp120-mediated NF-kB activation nor overall accumulation of CCL2 mRNA, whereas it decreases CCL2 transcript level in the cytoplasm. These results identify nuclear PI-PLC β1 as a new intermediate in the gp120-triggered PC-PLC-driven signal transduction pathway leading to CCL2 secretion in macrophages. The finding that a concerted gp120-mediated signaling involving both PC- and PI-specific PLCs is required for the expression of CCL2 in macrophages suggests that this signal transduction pathway may also be relevant for the modulation of viral replication in these cells. Thus, this study may contribute to identify novel targets for therapeutic intervention in HIV-1 infection.

  18. Characterization of novel alternative splicing sites in human telomerase reverse transcriptase (hTERT: analysis of expression and mutual correlation in mRNA isoforms from normal and tumour tissues

    Directory of Open Access Journals (Sweden)

    Gaudernack Gustav

    2006-08-01

    Full Text Available Abstract Background Human telomerase reverse transcriptase (hTERT is a key component for synthesis and maintenance of telomeres on chromosome ends and is required for the continued proliferation of cells. Estimation of hTERT expression therefore has broad relevance in oncology and stem cell research. Several splicing variants of hTERT have been described whose regulated expression contributes to the control of telomerase activity. Knowledge of the different hTERT mRNA isoforms and the ability to distinguish between them is an important issue when evaluating telomerase expression. Results By establishing cDNA-clone panels from lung and colon tissues, we could map hTERT clones individually for differences in DNA sequence. This made possible the identification of novel alternatively spliced sites as well as analysis of their frequency and mutual correlation in mRNA isoforms. Ten different alternatively spliced sites were detected, of which six were novel sites resulting from alternative splicing of intron 2 or 14. The majority of hTERT cDNA clones from normal and tumour lung and colon tissues encoded truncated proteins ending close after exon 2 or 6. Conclusion The increased complexity in telomerase expression revealed here has implications for our understanding of telomerase regulation and for the choice of suitable methods for addressing hTERT expression.

  19. {sup 68}Ga-DOTATOC PET/CT and somatostatin receptor (sst1-sst5) expression in normal human tissue: correlation of sst2 mRNA and SUV{sub max}

    Energy Technology Data Exchange (ETDEWEB)

    Boy, Christian; Poeppel, Thorsten D.; Jentzen, Walter; Brandau, Wolfgang; Bockisch, Andreas [University Hospital Essen, University of Duisburg-Essen, Department of Nuclear Medicine, Essen (Germany); Heusner, Till A.; Antoch, Gerald [University Hospital Essen, University of Duisburg-Essen, Department of Diagnostic and Interventional Radiology and Neuroradiology, Essen (Germany); Redmann-Bischofs, Anja; Unger, Nicole; Mann, Klaus; Petersenn, Stephan [University of Duisburg-Essen, Department of Endocrinology and Division of Laboratory Research, Essen (Germany)

    2011-07-15

    By targeting somatostatin receptors (sst) radiopeptides have been established for both diagnosis and therapy. For physiologically normal human tissues the study provides a normative database of maximum standardized uptake value (SUV{sub max}) and sst mRNA. A total of 120 patients were subjected to diagnostic {sup 68}Ga-DOTATOC positron emission tomography (PET)/CT (age range 19-83 years). SUV{sub max} values were measured in physiologically normal tissues defined by normal morphology, absence of surgical intervention and absence of metastatic spread during clinical follow-up. Expression of sst subtypes (sst1-sst5) was measured independently in pooled adult normal human tissue by real-time reverse transcriptase polymerase chain reaction (RT-PCR). SUV{sub max} revealed a region-specific pattern (e.g., mean {+-} SD, spleen 31.1 {+-} 10.9, kidney 16.9 {+-} 5.3, liver 12.8 {+-} 3.6, stomach 7.0 {+-} 3.1, head of pancreas 6.2 {+-} 2.3, small bowel 4.8 {+-} 1.8, thyroid 4.7 {+-} 2.2, bone 3.9 {+-} 1.3, large bowel 2.9 {+-} 0.8, muscle 2.1 {+-} 0.5, parotid gland 1.9 {+-} 0.6, axillary lymph node 0.8 {+-} 0.3 and lung 0.7 {+-} 0.3). SUV{sub max} was age independent. Gender differences were evident within the thyroid (female/male: 3.7 {+-} 1.6/5.5 {+-} 2.4, p < 0.001; Mann-Whitney U test) and the pancreatic head (5.5 {+-} 1.9/6.9 {+-} 2.2, p < 0.001). The sst mRNA was widely expressed and heterogeneous, showing sst1 to be most abundant. SUV{sub max} values exclusively correlated with sst2 expression (r = 0.846, p < 0.001; Spearman rank correlation analysis), whereas there was no correlation of SUV{sub max} with the expression of the other four subtypes. In normal human tissues {sup 68}Ga-DOTATOC imaging has been related to the expression of sst2 at the level of mRNA. The novel normative database may improve diagnostics, monitoring and therapy of sst-expressing tumours or inflammation on a molecular basis. (orig.)

  20. Exon B of human surfactant protein A2 mRNA, alone or within its surrounding sequences, interacts with 14-3-3; role of cis-elements and secondary structure.

    Science.gov (United States)

    Noutsios, Georgios T; Silveyra, Patricia; Bhatti, Faizah; Floros, Joanna

    2013-06-01

    Human surfactant protein A, an innate immunity molecule, is encoded by two genes: SFTPA1 (SP-A1) and SFTPA2 (SP-A2). The 5' untranslated (5'UTR) splice variant of SP-A2 (ABD), but not of SP-A1 (AD), contains exon B (eB), which is an enhancer for transcription and translation. We investigated whether eB contains cis-regulatory elements that bind trans-acting factors in a sequence-specific manner as well as the role of the eB mRNA secondary structure. Binding of cytoplasmic NCI-H441 proteins to wild-type eB, eB mutant, AD, and ABD 5'UTR mRNAs were studied by RNA electromobility shift assays (REMSAs). The bound proteins were identified by mass spectroscopy and specific antibodies (Abs). We found that 1) proteins bind eB mRNA in a sequence-specific manner, with two cis-elements identified within eB to be important; 2) eB secondary structure is necessary for binding; 3) mass spectroscopy and specific Abs in REMSAs identified 14-3-3 proteins to bind (directly or indirectly) eB and the natural SP-A2 (ABD) splice variant but not the SP-A1 (AD) splice variant; 4) other ribosomal and cytoskeletal proteins, and translation factors, are also present in the eB mRNA-protein complex; 5) knockdown of 14-3-3 β/α isoform resulted in a downregulation of SP-A2 expression. In conclusion, proteins including the 14-3-3 family bind two cis-elements within eB of hSP-A2 mRNA in a sequence- and secondary structure-specific manner. Differential regulation of SP-A1 and SP-A2 is mediated by the 14-3-3 protein family as well as by a number of other proteins that bind UTRs with or without eB mRNA.

  1. [The influence of parents personality and DRD4 and 5HTT genes polymorphisms on predisposition to alcohol dependence in their sons].

    Science.gov (United States)

    Samochowiec, Agnieszka; Horodnicki, Jan M; Samochowiec, Jerzy

    2011-01-01

    The aim of this work was to investigate differences in parents' personality and dopaminergic and serotoninergic gene polymorphisms which may affect certain predispositions to alcohol dependence as described in the typology developed by R. Cloninger and O.M. Lesch. Also the possibility of recognising their genotypes DRD4 (Gene ID: 1815A ) and 5HTT (Gene ID: 6532) could be helpful in predicting predisposition to addiction. A total number of 213 individuals (71 Polish trios), Caucasian families were investigated. Fathers' mean age was: 61.7 +/- 10.8 and mothers were 59 +/- 10 years old. None of the parents fulfilled the criteria of alcohol dependence. The alcohol dependent probands were male, with confirmed biological descent, mean age: 35.2 +/- 9.7 years. In all the participants TCI was performed. Characterisation of alcohol dependence and the course of withdrawal were obtained by SSAGA. Specially designed questionnaires based on Cloninger and Lesch typologies were used. The essential data on both parents was collected and AUDIT was performed. DRD4 and 5HTT gene polymorphisms were determined by PCR and TDT test was calculated. TDT analysis showed no differences in the transmission of alleles of 5HTT and DRD4 genes in the investigated families. The analysis of TCI personality profiles confirmed no statistically significant relations between Cloninger 1 and 2 subtypes of alcoholics. A statistically significant difference was recorded between the scores for groups I and II classified according to Lesch's typology in dimensions NS, NS2 and NS4. Fathers of probands characterised as type I according to Cloninger had statistically lower scores in dimension C and C5 in comparison to type II fathers. Fathers of type II alcoholics according to Lesch's typology had higher NS2. Mothers of type I alcoholics according to Cloninger had statistically lower scores in dimension HA2 in comparison with type II mothers. On the basis of the above presented findings it can be stated that

  2. The human Ago2 MC region does not contain an eIF4E-like mRNA cap binding motif

    Directory of Open Access Journals (Sweden)

    Grishin Nick V

    2009-01-01

    Full Text Available Abstract Background Argonaute (Ago proteins interact with small regulatory RNAs to mediate gene regulatory pathways. A recent report by Kiriakidou et al. 1 describes an MC sequence region identified in Ago2 that displays similarity to the cap-binding motif in translation initiation factor 4E (eIF4E. In a cap-bound eIF4E structure, two important aromatic residues of the motif stack on either side of a 7-methylguanosine 5'-triphosphate (m7Gppp base. The corresponding Ago2 aromatic residues (F450 and F505 were hypothesized to perform the same cap-binding function. However, the detected similarity between the MC sequence and the eIF4E cap-binding motif was questionable. Results A number of sequence-based and structure-based bioinformatics methods reveal the reported similarity between the Ago2 MC sequence region and the eIF4E cap-binding motif to be spurious. Alternatively, the MC sequence region is confidently assigned to the N-terminus of the Ago piwi module, within the mid domain of experimentally determined prokaryotic Ago structures. Confident mapping of the Ago2 MC sequence region to the piwi mid domain results in a homology-based structure model that positions the identified aromatic residues over 20 Å apart, with one of the aromatic side chains (F450 contributing instead to the hydrophobic core of the domain. Conclusion Correct functional prediction based on weak sequence similarity requires substantial evolutionary and structural support. The evolutionary context of the Ago mid domain suggested by multiple sequence alignment is limited to a conserved hydrophobicity profile required for the fold and a motif following the MC region that binds guide RNA. Mapping of the MC sequence to the mid domain structure reveals Ago2 aromatics that are incompatible with eIF4E-like mRNA cap-binding, yet display some limited local structure similarities that cause the chance sequence match to eIF4E. Reviewers This article was reviewed by Arcady Mushegian

  3. Regulation of mRNA translation by MID1: a common mechanism of expanded CAG repeat RNAs

    Directory of Open Access Journals (Sweden)

    Nadine Griesche

    2016-10-01

    Full Text Available Expansion of CAG repeats, which code for the disease-causing polyglutamine protein, is a common feature in polyglutamine diseases. RNA-mediated mechanisms that contribute to neuropathology in polyglutamine diseases are important. RNA-toxicity describes a phenomenon by which the mutant CAG repeat RNA recruits RNA-binding proteins, thereby leading to aberrant function. For example the MID1 protein binds to mutant huntingtin (HTT RNA, which is linked to Huntington’s disease (HD, at its CAG repeat region and induces protein synthesis of mutant protein. But is this mechanism specific to HD or is it a common mechanism in CAG repeat expansion disorders? To answer this question, we have analysed the interaction between MID1 and three other CAG repeat mRNAs, Ataxin2 (ATXN2, Ataxin3 (ATXN3, and Ataxin7 (ATXN7, that all differ in the sequence flanking the CAG repeat. We show that ATXN2, ATXN3 and ATXN7 bind to MID1 in a CAG repeat length-dependent manner. Furthermore, we show that functionally, in line with what we have previously observed for HTT, the binding of MID1 to ATXN2, ATXN3 and ATXN7 mRNA induces protein synthesis in a repeat length-dependent manner. Our data suggest that regulation of protein translation by the MID1 complex is a common mechanism for CAG repeat containing mRNAs.

  4. Life satisfaction in the new country: a multilevel longitudinal analysis of effects of culture and 5-HTT allele frequency distribution in country of origin.

    Science.gov (United States)

    Kashima, Emiko S; Kent, Stephen; Kashima, Yoshihisa

    2015-01-01

    Life satisfaction of migrants to Australia from 17 countries, assessed at 4-5 months, 16-17 months and 3½ years after arrival, was analyzed with a longitudinal, multilevel analysis. The results indicated that migrants were more satisfied, if the national average life satisfaction was higher in their country of origin, after adjustment for individual-level income, age, and sex and a linear temporal trend. Simultaneously, the migrants were also happier if people in their country of origin had a higher frequency of 5-HTT long allele, a genotype known to be associated with resilience under life stresses. These two relationships were independent, suggesting that both culture and gene matter in international transitions.

  5. Effects of tacrolimus (FK506) on human insulin gene expression, insulin mRNA levels, and insulin secretion in HIT-T15 cells.

    OpenAIRE

    1996-01-01

    FK506 (tacrolimus) is an immunosuppressive drug which interrupts Ca2+-calmodulin-calcineurin signaling pathways in T lymphocytes, thereby blocking antigen activation of T cell early activation genes. Regulation of insulin gene expression in the beta cell may also involve Ca2+-signaling pathways and FK506 has been associated with insulin-requiring diabetes mellitus during clinical use. The purpose of this study was to characterize the effects of FK506 on human insulin gene transcription, insul...

  6. Ascorbic Acid, Ultraviolet C Rays, and Glucose but not Hyperthermia Are Elicitors of Human β-Defensin 1 mRNA in Normal Keratinocytes

    Science.gov (United States)

    Cruz Díaz, Luis Antonio; Flores Miramontes, María Guadalupe; Allen, Kirk; Gonzalez Ávila, Marisela; Prado Montes de Oca, Ernesto

    2015-01-01

    Hosts' innate defense systems are upregulated by antimicrobial peptide elicitors (APEs). Our aim was to investigate the effects of hyperthermia, ultraviolet A rays (UVA), and ultraviolet C rays (UVC) as well as glucose and ascorbic acid (AA) on the regulation of human β-defensin 1 (DEFB1), cathelicidin (CAMP), and interferon-γ (IFNG) genes in normal human keratinocytes (NHK). The indirect in vitro antimicrobial activity against Staphylococcus aureus and Listeria monocytogenes of these potential APEs was tested. We found that AA is a more potent APE for DEFB1 than glucose in NHK. Glucose but not AA is an APE for CAMP. Mild hypo- (35°C) and hyperthermia (39°C) are not APEs in NHK. AA-dependent DEFB1 upregulation below 20 mM predicts in vitro antimicrobial activity as well as glucose- and AA-dependent CAMP and IFNG upregulation. UVC upregulates CAMP and DEFB1 genes but UVA only upregulates the DEFB1 gene. UVC is a previously unrecognized APE in human cells. Our results suggest that glucose upregulates CAMP in an IFN-γ-independent manner. AA is an elicitor of innate immunity that will challenge the current concept of late activation of adaptive immunity of this vitamin. These results could be useful in designing new potential drugs and devices to combat skin infections. PMID:25815330

  7. Ascorbic Acid, Ultraviolet C Rays, and Glucose but not Hyperthermia Are Elicitors of Human β-Defensin 1 mRNA in Normal Keratinocytes

    Directory of Open Access Journals (Sweden)

    Luis Antonio Cruz Díaz

    2015-01-01

    Full Text Available Hosts’ innate defense systems are upregulated by antimicrobial peptide elicitors (APEs. Our aim was to investigate the effects of hyperthermia, ultraviolet A rays (UVA, and ultraviolet C rays (UVC as well as glucose and ascorbic acid (AA on the regulation of human β-defensin 1 (DEFB1, cathelicidin (CAMP, and interferon-γ (IFNG genes in normal human keratinocytes (NHK. The indirect in vitro antimicrobial activity against Staphylococcus aureus and Listeria monocytogenes of these potential APEs was tested. We found that AA is a more potent APE for DEFB1 than glucose in NHK. Glucose but not AA is an APE for CAMP. Mild hypo- (35°C and hyperthermia (39°C are not APEs in NHK. AA-dependent DEFB1 upregulation below 20 mM predicts in vitro antimicrobial activity as well as glucose- and AA-dependent CAMP and IFNG upregulation. UVC upregulates CAMP and DEFB1 genes but UVA only upregulates the DEFB1 gene. UVC is a previously unrecognized APE in human cells. Our results suggest that glucose upregulates CAMP in an IFN-γ-independent manner. AA is an elicitor of innate immunity that will challenge the current concept of late activation of adaptive immunity of this vitamin. These results could be useful in designing new potential drugs and devices to combat skin infections.

  8. A High-Resolution In Vivo Atlas of the Human Brain's Serotonin System.

    Science.gov (United States)

    Beliveau, Vincent; Ganz, Melanie; Feng, Ling; Ozenne, Brice; Højgaard, Liselotte; Fisher, Patrick M; Svarer, Claus; Greve, Douglas N; Knudsen, Gitte M

    2017-01-04

    The serotonin (5-hydroxytryptamine, 5-HT) system modulates many important brain functions and is critically involved in many neuropsychiatric disorders. Here, we present a high-resolution, multidimensional, in vivo atlas of four of the human brain's 5-HT receptors (5-HT1A, 5-HT1B, 5-HT2A, and 5-HT4) and the 5-HT transporter (5-HTT). The atlas is created from molecular and structural high-resolution neuroimaging data consisting of positron emission tomography (PET) and magnetic resonance imaging (MRI) scans acquired in a total of 210 healthy individuals. Comparison of the regional PET binding measures with postmortem human brain autoradiography outcomes showed a high correlation for the five 5-HT targets and this enabled us to transform the atlas to represent protein densities (in picomoles per milliliter). We also assessed the regional association between protein concentration and mRNA expression in the human brain by comparing the 5-HT density across the atlas with data from the Allen Human Brain atlas and identified receptor- and transporter-specific associations that show the regional relation between the two measures. Together, these data provide unparalleled insight into the serotonin system of the human brain.

  9. Improvement of oxidative/anti-oxidative stress balance and expressions of NET and 5-HTT involved in the anti-depressive effect of reboxetine%改善氧化/抗氧化应激平衡及NET和5-HTT表达与瑞波西汀抗抑郁作用有关

    Institute of Scientific and Technical Information of China (English)

    李娜; 王涵; 文威; 周岐新

    2011-01-01

    Aim To investigate the relationship between the anti-depressive effect of reboxetine and the oxidative/anti-oxidative stress balance as well as expressions of NET and 5-HTT in rat depressive model induced by chronic unpredictable mild stress( CUMS ).Methods Sixty male SD rats were randomly divided into normal group( NG ). model group( MG ). reboxetine( 0.7 mg · kg-1 · d-1 )-treated normal group ( RNG), and reboxetine( 0. 7 mg·kg-1 · d-1 )-treated model group( RMG ). CUMS with solitary condition was taken to establish rat depression model. The openfield test and sucrose consumption were used to evaluate the depression behaviors of rats. Biochemistry methods were used to determine the content of MDA and activities of SOD and CAT in serum. Reverse transcription polymerase chain reaction( RT-PCR ) method was used to determine the mRNA expressions of NET and 5-HTT in either pons cerebelli or hippocampus tissues of rats. Hippocampal pathological change with HE staining slices was observed under microscope. Results It was shown that except MDA level,there were significant decreases of open-field scores( crossing, rearing, and preening activities ). sucrose consumption,SOD and CAT activities , and expressions of NET and 5-HTT, as well as obvious karyopyknosis and deep staining of hippocampal neurocytes in MG,in comparison with NG. The treatment of roboxetine obviously blunted the changes above caused by CUMS. However treatment of roboxetine had no influences on NG rats. Conclusions The treatment of reboxetine can improve depression behaviors of rats through reversing imbalance of oxidative/anti-oxidative stress systems and increasing expressions of NET and 5-HTT.%目的 探讨瑞波西汀抗慢性轻度不可预见性刺激(CUMS)致大鼠抑郁症行为与机体氧化/抗氧化应激平衡以及去甲肾上腺素转运体(NET)和5-羟色胺转运体(5-HTT)表达的关系.方法 ♂ SD大鼠60只,随机分为正常对照组(NG)、模型组(MG)、瑞波西汀(0.7 mg

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

    Science.gov (United States)

    Straarup, Ellen Marie; Fisker, Niels; Hedtjärn, Maj; Lindholm, Marie W; Rosenbohm, Christoph; Aarup, Vibeke; Hansen, Henrik Frydenlund; Ørum, Henrik; Hansen, Jens B Rode; Koch, Troels

    2010-11-01

    The potency and specificity of locked nucleic acid (LNA) antisense oligonucleotides was investigated as a function of length and affinity. The oligonucleotides were designed to target apolipoprotein B (apoB) and were investigated both in vitro and in vivo. The high affinity of LNA enabled the design of short antisense oligonucleotides (12- to 13-mers) that possessed high affinity and increased potency both in vitro and in vivo compared to longer oligonucleotides. The short LNA oligonucleotides were more target specific, and they exhibited the same biodistribution and tissue half-life as longer oligonucleotides. Pharmacology studies in both mice and non-human primates were conducted with a 13-mer LNA oligonucleotide against apoB, and the data showed that repeated dosing of the 13-mer at 1-2 mg/kg/week was sufficient to provide a significant and long lasting lowering of non-high-density lipoprotein (non-HDL) cholesterol without increasing serum liver toxicity markers. The data presented here show that oligonucleotide length as a parameter needs to be considered in the design of antisense oligonucleotide and that potent short oligonucleotides with sufficient target affinity can be generated using the LNA chemistry. Conclusively, we present a 13-mer LNA oligonucleotide with therapeutic potential that produce beneficial cholesterol lowering effect in non-human primates.

  11. Identification of a novel splice variant of human PD-L1 Mrna encoding an isoform-lacking Igv-like domain

    Institute of Scientific and Technical Information of China (English)

    Xian-hui HE; Li-hui XU; Yi LIU

    2005-01-01

    Aim: To investigate the expression and regulation of PD-1 ligand 1 (PD-L1) in peripheral blood mononuclear cells (PBMC). Methods: The cDNA encoding human PD-L1 precursor was cloned from the total RNA extracted from the resting and phorbol dibutyrate plus ionomycin- or phytohemagglutinin-activated PBMC, by reverse transcription polymerase chain reaction (RT-PCR), and independent clones were sequenced and analyzed. The expression and subcellular localization were examined in transiently transfected cells. The PD-L1 gene expression in different PBMC was also analyzed by RT-PCR. Results: A novel human PD-L1 splice variant was identified from the activated PBMC. It was generated by splicing out exon 2 encoding an immunoglobulin variable domain (Igv)-like domain but retaining all other exons without a frame-shift. Consequently, the putative translated protein contained all other domains including the transmembrane region except for the Igv-like domain. Furthermore, the conventional isoform was expressed on the plasma surface whereas the novel isoform showed a pattern of intmcellular membrane distribution in transiently transfected K562 cells. In addition, the expression pattern of the PD-L1 splice variant was variable in different individuals and in different cellular status. Conclusion: PD-L1 expression may be regulated at the posttranscriptional level through alternative splicing, and modulation of the PD-L1 isoform expression may influence the outcome of specific immune responses in the peripheral tissues.

  12. The anti-inflammatory pharmacology of Pycnogenol in humans involves COX-2 and 5-LOX mRNA expression in leukocytes.

    Science.gov (United States)

    Canali, Raffaella; Comitato, Raffaella; Schonlau, Frank; Virgili, Fabio

    2009-09-01

    We investigated the effects of Pycnogenol supplementation on the arachidonic acid pathway in human polymorphonuclear leukocytes (PMNL) in response to an inflammatory stimulus. Pycnogenol is a standardised extract of French maritime pine bark consisting of procyanidins and polyphenolic monomers. Healthy volunteers aged 35 to 50 years were supplemented with 150 mg Pycnogenol a day for five days. Before and after the final day of supplementation, blood was drawn and PMNL were isolated. PMNL were primed with lipopolysaccharide (LPS) and stimulated with the receptor-mediated agonist formyl-methionyl-leucyl-phenylalanine (fMLP) to activate the arachidonic acid pathway and the biosynthesis of leukotrienes, thromboxane and prostaglandins. Pycnogenol supplementation inhibited 5-lipoxygenase (5-LOX) and cyclooxygenase-2 (COX-2) gene expression and phospholipase A2 (PLA2) activity. This effect was associated with a compensatory up-regulation of COX-1 gene expression. Interestingly, Pycnogenol suspended the interdependency between 5-LOX and 5-lipoxygenase activating protein (FLAP) expression. Pycnogenol supplementation reduced leukotriene production but did not leave prostaglandins unaltered, which we attribute to a decline of COX-2 activity in favour of COX-1. Here we show for the first time that Pycnogenol supplementation simultaneously inhibits COX-2 and 5-LOX gene expression and reduces leukotriene biosynthesis in human PMNL upon pro-inflammatory stimulation ex vivo.

  13. IL-17A acts via p38 MAPK to increase stability of TNF-alpha-induced IL-8 mRNA in human ASM.

    Science.gov (United States)

    Henness, Sheridan; van Thoor, Eveline; Ge, Qi; Armour, Carol L; Hughes, J Margaret; Ammit, Alaina J

    2006-06-01

    Human airway smooth muscle (ASM) plays an immunomodulatory role in asthma. Recently, IL-17A has become of increasing interest in asthma, being found at elevated levels in asthmatic airways and emerging as playing an important role in airway neutrophilia. IL-17A predominantly exerts its neutrophil orchestrating role indirectly via the induction of cytokines by resident airway structural cells. Here, we perform an in vitro study to show that although IL-17A did not induce secretion of the CXC chemokine IL-8 from ASM cells, IL-17A significantly potentiates TNF-alpha-induced IL-8 protein secretion and gene expression in a concentration- and time-dependent manner (P ASM cells, acting via a p38 MAPK-dependent posttranscriptional pathway to augment TNF-alpha-induced secretion of the potent neutrophil chemoattractant IL-8 from ASM cells.

  14. Quantitative Electroencephalographic Biomarkers in Preclinical and Human Studies of Huntington's Disease: Are They Fit-for-Purpose for Treatment Development?

    Science.gov (United States)

    Leuchter, Michael K; Donzis, Elissa J; Cepeda, Carlos; Hunter, Aimee M; Estrada-Sánchez, Ana María; Cook, Ian A; Levine, Michael S; Leuchter, Andrew F

    2017-01-01

    A major focus in development of novel therapies for Huntington's disease (HD) is identification of treatments that reduce the burden of mutant huntingtin (mHTT) protein in the brain. In order to identify and test the efficacy of such therapies, it is essential to have biomarkers that are sensitive to the effects of mHTT on brain function to determine whether the intervention has been effective at preventing toxicity in target brain systems before onset of clinical symptoms. Ideally, such biomarkers should have a plausible physiologic basis for detecting the effects of mHTT, be measureable both in preclinical models and human studies, be practical to measure serially in clinical trials, and be reliably measurable in HD gene expansion carriers (HDGECs), among other features. Quantitative electroencephalography (qEEG) fulfills many of these basic criteria of a "fit-for-purpose" biomarker. qEEG measures brain oscillatory activity that is regulated by the brain structures that are affected by mHTT in premanifest and early symptom individuals. The technology is practical to implement in the laboratory and is well tolerated by humans in clinical trials. The biomarkers are measureable across animal models and humans, with findings that appear to be detectable in HDGECs and translate across species. We review here the literature on recent developments in both preclinical and human studies of the use of qEEG biomarkers in HD, and the evidence for their usefulness as biomarkers to help guide development of novel mHTT lowering treatments.

  15. Single base mismatches in the mRNA target site allow specific seed region-mediated off-target binding of siRNA targeting human coagulation factor 7.

    Science.gov (United States)

    Ravon, Morgane; Berrera, Marco; Ebeling, Martin; Certa, Ulrich

    2012-01-01

    We have analyzed the off-target activity of two siRNAs (F7-1, F7-2) that knock-down human blood coagulation factor 7 mRNA. F7-1 modulates a significant number of non-target transcripts while F7-2 shows high selectivity for the target transcript under various experimental conditions. The 3'-UTRs of all F7-1 off-target genes show statistically significant enrichment of the reverse complement of the F7-1 siRNA seed region located in the guide strand. Seed region enrichment was confirmed in off-target transcripts modulated by siRNA targeting the glucocorticoid receptor. To investigate how these sites contribute to off-target recognition of F7-1, we employed CXCL5 transcript as model system because it contains five F7-1 seed sequence motifs with single base mismatches. We show by transient transfection of reporter gene constructs into HEK293 cells that three out of five sites located in the 3'-UTR region are required for F7-1 off-target activity. For further mechanistic dissection, the sequences of these sites were synthesized and inserted either individually or joined in dimeric or trimeric constructs. Only the fusion constructs were silenced by F7-1 while the individual sites had no off-target activity. Based on F7-1 as a model, a single mismatch between the siRNA seed region and mRNA target sites is tolerated for target recognition and the CXCL5 data suggest a requirement for binding to multiple target sites in off-target transcripts.

  16. Microarray study reveals that HIV-1 induces rapid type-I interferon-dependent p53 mRNA up-regulation in human primary CD4+ T cells

    Directory of Open Access Journals (Sweden)

    Tremblay Michel J

    2009-01-01

    Full Text Available Abstract Background Infection with HIV-1 has been shown to alter expression of a large array of host cell genes. However, previous studies aimed at investigating the putative HIV-1-induced modulation of host gene expression have been mostly performed in established human cell lines. To better approximate natural conditions, we monitored gene expression changes in a cell population highly enriched in human primary CD4+ T lymphocytes exposed to HIV-1 using commercial oligonucleotide microarrays from Affymetrix. Results We report here that HIV-1 influences expression of genes related to many important biological processes such as DNA repair, cellular cycle, RNA metabolism and apoptosis. Notably, expression of the p53 tumor suppressor and genes involved in p53 homeostasis such as GADD34 were up-regulated by HIV-1 at the mRNA level. This observation is distinct from the previously reported p53 phosphorylation and stabilization at the protein level, which precedes HIV-1-induced apoptosis. We present evidence that the HIV-1-mediated increase in p53 gene expression is associated with virus-mediated induction of type-I interferon (i.e. IFN-α and IFN-β. Conclusion These observations have important implications for our understanding of HIV-1 pathogenesis, particularly in respect to the virus-induced depletion of CD4+ T cells.

  17. Correlation between high risk type human papillomavirus E6/E7 mRNA and cervical cancer%高危型HPV E6/E7 mRNA与宫颈癌相关性分析

    Institute of Scientific and Technical Information of China (English)

    王小红; 钱艺美; 缪铃; 乐瑶; 杜娟

    2016-01-01

    Objective To investigate the correlation between the positive rate of high risk human papillomavirus (HPV) mRNA E6/E7 and cervical cancer,and provide evidence for the prevention and treatment of cervical cancer.Methods A total of 100 cervical cancer cases and 100 healthy controls were selected in our hospital from January 2015 to December 2015.The fluorescence quantitative PCR and pathological examination on HPV E6/E7 mRNA were carried out.The correlation between HPV E6/E7 mRNA and cervical squamous epithelial lesions were analyzed.Results In case group,the positive rate ofHPV E6/E7 mRNA was 76.0% (76/100).In control group,the positive rate was 13.0% (13/100).The positive rate in case group was significantly higher than that in control group,and the difference was statistically significant (x2=24.522,P<0.001).The positive predictive value and negative predictive value of the two groups were compared,and the difference was not significant (P>0.05).The positive rate of HPV E6/E7 mRNA was significantly higher than high-grade squamous intraepithelial lesion (SIL) rate (26.1%),low-grade SIL rate (17.6%) and atypical squamous cell hyperplasia rate (6.7%),the difference was statistically significant (x2=7.615,P=0.001;x2=9.114,P=0.001;x2=18.241,P<0.001).Conclusions The detection rate ofHPV E6/E7 mRNA in cervical cancer patients was high.And with the increased severity of cervical squamous epithelial lesions,the positive rate of HPV E6/E7 mRNA increased.%目的 探讨高危型HPV E6/E7 mRNA检出率与宫颈癌的相关性,为临床防治宫颈癌提供依据.方法 选择2015年收治的100例宫颈癌患者为A组,同期100例健康体检者为B组,采用荧光定量PCR检测入组患者高危型HPV E6/E7 mRNA和病理学检查,比较两组患者HPV E6/E7感染率和荧光定量PCR检查效率,分析HPV E6/E7感染与宫颈鳞状上皮病变的相关性.结果 A组阳性76例,阳性率为76.0%;B组阳性13例,阳性率为13.0%;A组阳性率高于B

  18. Effects of intracellular zinc depletion on the abundance of VDAC mRNA in cultured human spermatozoa%Zn缺乏对人精子VDAC mRNA的影响

    Institute of Scientific and Technical Information of China (English)

    赵丹; 吴炜; 章轶; 王增军; 王心如

    2012-01-01

    目的:探讨Zn离子缺乏对成熟精子活动能力和电压依赖性离子通道(voltage-dependent anion channel,VDAC)的mRNA丰度的影响.方法:从3个正常生育者捐献所得的精液经非连续Percoll梯度离心获得精子样本,并将其分3组连续培养24h:对照组;加入Zn离子螯合剂的TPEN组(2 μmol/L);同时加入Zn离子螯合剂N,N,N',N'-tetrakis (2-pyridylmethy1)Ethylenediamine (TPEN)和Zn离子(5μmol/L)组.行CASA和荧光定量PCR(qPCR)分别分析不同时间的精子活动能力和24h后各VDAC亚型mRNA丰度.结果:CASA显示:培养3h,TPEN组与对照组比较,精子活力明显下降(P<0.05).qPCR结果显示:培养24h后,TPEN组VDAC3 mRNA丰度明显低于对照组(P<0.05).而同时加入TPEN和Zn离子组与对照组比较,差异无统计学意义.结论:Zn离子缺乏使VDAC3 mRNA丰度降低,可能影响到精子的正常功能.%Objective:To investigate whether intracellular zinc depletion can actually change spermatozoa motiJity and the abundance of voltage-dependent anion channel (VDAC) mRNA in cultured human spermatozoa. Methods; Spermatozoa were separated from ejaculates of three donors using a discontinuous Percoll gradient centrifugation. Each spermatozoa specimen were divided into 3 groups,one was exposed to a cell membrane-permeable zinc chelator N,N,N',N'-tetrakis (2-pyridylmethyl) Ethylenediamine (TPEN) (2 μmol/L) for 24 hours,one was to TPEN plus zinc sulfate (5 μmol/L) for 24 hours,and one was as control. CASA and quantitative real-time PCR (qPCR) were performed to detect sperm motility characteristics,mRNA abundance and difference of the three VDAC subtypes between three different treatment groups respectively. Results;The results of CASA demonstrated that exposure of spermatozoa to TPEN for three hours significantly decreased sperm motility compared with the control group (P = 0.049). qPCR demonstrated that VDAC3 mRNA level of TPEN group were significantly lower than that of control group,while no

  19. Clinical pharmacology of 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy": the influence of gender and genetics (CYP2D6, COMT, 5-HTT.

    Directory of Open Access Journals (Sweden)

    Ricardo Pardo-Lozano

    Full Text Available The synthetic psychostimulant MDMA (± 3,4-methylenedioxymethamphetamine, ecstasy acts as an indirect serotonin, dopamine, and norepinephrine agonist and as a mechanism-based inhibitor of the cytochrome P-450 2D6 (CYP2D6. It has been suggested that women are more sensitive to MDMA effects than men but no clinical experimental studies have satisfactorily evaluated the factors contributing to such observations. There are no studies evaluating the influence of genetic polymorphism on the pharmacokinetics (CYP2D6; catechol-O-methyltransferase, COMT and pharmacological effects of MDMA (serotonin transporter, 5-HTT; COMT. This clinical study was designed to evaluate the pharmacokinetics and physiological and subjective effects of MDMA considering gender and the genetic polymorphisms of CYP2D6, COMT, and 5-HTT. A total of 27 (12 women healthy, recreational users of ecstasy were included (all extensive metabolizers for CYP2D6. A single oral weight-adjusted dose of MDMA was administered (1.4 mg/kg, range 75-100 mg which was similar to recreational doses. None of the women were taking oral contraceptives and the experimental session was performed during the early follicular phase of their menstrual cycle. Principal findings show that subjects reached similar MDMA plasma concentrations, and experienced similar positive effects, irrespective of gender or CYP2D6 (not taking into consideration poor or ultra-rapid metabolizers or COMT genotypes. However, HMMA plasma concentrations were linked to CYP2D6 genotype (higher with two functional alleles. Female subjects displayed more intense physiological (heart rate, and oral temperature and negative effects (dizziness, sedation, depression, and psychotic symptoms. Genotypes of COMT val158met or 5-HTTLPR with high functionality (val/val or l/* determined greater cardiovascular effects, and with low functionality (met/* or s/s negative subjective effects (dizziness, anxiety, sedation. In conclusion, the contribution

  20. Paradoxical expression of IL-28B mRNA in peripheral blood in human T-cell leukemia virus Type-1 mono-infection and co-infection with hepatitis C Virus

    Directory of Open Access Journals (Sweden)

    Kamihira Shimeru

    2012-02-01

    Full Text Available Abstract Background Human T-cell leukemia virus type-1 (HTLV-1 carriers co-infected with and hepatitis C virus (HCV have been known to be at higher risk of their related diseases than mono-infected individuals. The recent studies clarified that IL-28B polymorphism rs8099917 is associated with not only the HCV therapeutic response by IFN, but also innate immunity and antiviral activity. The aim of our research was to clarify study whether IL-28B gene polymorphism (rs8099917 is associated with HTLV-1/HCV co-infection. Results The genotyping and viral-serological analysis for 340 individuals showed that IL-28B genotype distribution of rs8099917 SNP did not differ significantly by respective viral infection status. However, the IL-28B mRNA expression level was 3.8 fold higher in HTLV-1 mono-infection than HTLV-1/HCV co-infection. The high expression level was associated with TT (OR, 6.25, whiles the low expression was associated with co-infection of the two viruses (OR, 9.5. However, there was no association between down-regulation and ATL development (OR, 0.8. Conclusion HTLV-1 mono-infection up-regulates the expression of IL-28B transcripts in genotype-dependent manner, whiles HTLV-1/HCV co-infection down-regulates regardless of ATL development.

  1. 改善氧化应激平衡和逆转NET及5-HTT表达异常涉及文拉法辛的抗抑郁作用%Improving oxidative stress response and reversing abnormal expression of hippocampus NET and 5-HTT involved in anti-depressive effect of venlafaxine

    Institute of Scientific and Technical Information of China (English)

    胡小娅; 费慧芝; 蒋心惠; 刘丹; 邱红梅; 周岐新

    2014-01-01

    establish rat depression model. The force swimming test was used to evaluate the behavior chan-ges of experimental rats. The malondialdehyde ( MDA) level and activity of superoxide dismutase ( SOD ) in serum were determined by biochemical methods. The mRNA and protein expressions of NET and 5-HTT in hippocampus were determined by Real-Time Reverse transcription polymerase chain reaction ( real-time RT-PCR) and Western blot ( WB) , respectively. Results Compared with NG rats, obviously increasing immo-bile time of rats in force swimming test and serum MDA level, as well as significantly decreasing SOD activity in serum was observed with clearly decreasing 5-HTT expression and elevating NET expression in hippocam-pus of MG rats. The treatment of venlafaxine distinctly suppressed changes above from CUS-induced rats. However, significant changes failed to be found in VNG rats. Conclusion The anti-depressive effect of venlafaxine may at least partly involve in improving ox-idative stress/anti-oxidative stress balance and revers-ing abnormal expression of NET and 5-HTT.

  2. Regulation of Vif mRNA splicing by human immunodeficiency virus type 1 requires 5' splice site D2 and an exonic splicing enhancer to counteract cellular restriction factor APOBEC3G.

    Science.gov (United States)

    Mandal, Dibyakanti; Exline, Colin M; Feng, Zehua; Stoltzfus, C Martin

    2009-06-01

    The human immunodeficiency virus type 1 (HIV-1) accessory protein Vif is encoded by an incompletely spliced mRNA resulting from splicing of the major splice donor in the HIV-1 genome, 5' splice site (5'ss) D1, to the first splice acceptor, 3'ss A1. We have shown previously that splicing of HIV-1 vif mRNA is tightly regulated by suboptimal 5'ss D2, which is 50 nucleotides downstream of 3'ss A1; a GGGG silencer motif proximal to 5'ss D2; and an SRp75-dependent exonic splicing enhancer (ESEVif). In agreement with the exon definition hypothesis, mutations within 5'ss D2 that are predicted to increase or decrease U1 snRNP binding affinity increase or decrease the usage of 3'ss A1 (D2-up and D2-down mutants, respectively). In this report, the importance of 5'ss D2 and ESEVif for avoiding restriction of HIV-1 by APOBEC3G (A3G) was determined by testing the infectivities of a panel of mutant viruses expressing different levels of Vif. The replication of D2-down and ESEVif mutants in permissive CEM-SS cells was not significantly different from that of wild-type HIV-1. Mutants that expressed Vif in 293T cells at levels greater than 10% of that of the wild type replicated similarly to the wild type in H9 cells, and Vif levels as low as 4% were affected only modestly in H9 cells. This is in contrast to Vif-deleted HIV-1, whose replication in H9 cells was completely inhibited. To test whether elevated levels of A3G inhibit replication of D2-down and ESEVif mutants relative to wild-type virus replication, a Tet-off Jurkat T-cell line that expressed approximately 15-fold-higher levels of A3G than control Tet-off cells was generated. Under these conditions, the fitness of all D2-down mutant viruses was reduced relative to that of wild-type HIV-1, and the extent of inhibition was correlated with the level of Vif expression. The replication of an ESEVif mutant was also inhibited only at higher levels of A3G. Thus, wild-type 5'ss D2 and ESEVif are required for production of

  3. Influence of 5-HTT variation, childhood trauma and self-efficacy on anxiety traits: a gene-environment-coping interaction study.

    Science.gov (United States)

    Schiele, Miriam A; Ziegler, Christiane; Holitschke, Karoline; Schartner, Christoph; Schmidt, Brigitte; Weber, Heike; Reif, Andreas; Romanos, Marcel; Pauli, Paul; Zwanzger, Peter; Deckert, Jürgen; Domschke, Katharina

    2016-08-01

    Environmental vulnerability factors such as adverse childhood experiences in interaction with genetic risk variants, e.g., the serotonin transporter gene linked polymorphic region (5-HTTLPR), are assumed to play a role in the development of anxiety and affective disorders. However, positive influences such as general self-efficacy (GSE) may exert a compensatory effect on genetic disposition, environmental adversity, and anxiety traits. We, thus, assessed childhood trauma (Childhood Trauma Questionnaire, CTQ) and GSE in 678 adults genotyped for 5-HTTLPR/rs25531 and their interaction on agoraphobic cognitions (Agoraphobic Cognitions Questionnaire, ACQ), social anxiety (Liebowitz Social Anxiety Scale, LSAS), and trait anxiety (State-Trait Anxiety Inventory, STAI-T). The relationship between anxiety traits and childhood trauma was moderated by self-efficacy in 5-HTTLPR/rs25531 LALA genotype carriers: LALA probands maltreated as children showed high anxiety scores when self-efficacy was low, but low anxiety scores in the presence of high self-efficacy despite childhood maltreatment. Our results extend previous findings regarding anxiety-related traits showing an interactive relationship between 5-HTT genotype and adverse childhood experiences by suggesting coping-related measures to function as an additional dimension buffering the effects of a gene-environment risk constellation. Given that anxiety disorders manifest already early in childhood, this insight could contribute to the improvement of psychotherapeutic interventions by including measures strengthening self-efficacy and inform early targeted preventive interventions in at-risk populations, particularly within the crucial time window of childhood and adolescence.

  4. Gene regulation by mRNA editing

    Energy Technology Data Exchange (ETDEWEB)

    Ashkenas, J. [Univ. of Washington, Seattle, WA (United States)

    1997-02-01

    The commonly cited figure of 10{sup 5} genes in the human genome represents a tremendous underestimate of our capacity to generate distinct gene products with unique functions. Our cells possess an impressive collection of tools for altering the products of a single gene to create a variety of proteins. The different gene products may have related but distinct functions, allowing cells of different types or at different developmental stages to fine-tune their patterns of gene expression. These tools may act in the cytoplasm, as when proteins undergo post-translational modifications, or in the nucleus, in the processing of pre-mRNA. Two forms of intranuclear fine-tuning are well established and widely studied: alternative splicing of pre-mRNAs and alternative polyadenylation site selection. In recent years it has become clear that cells possess yet another tool to create RNA sequence diversity, mRNA editing. The term {open_quotes}editing{close_quotes} is applied to posttranscriptional modifications of a purine or pyrimidine, which alter an mRNA sequence as it is read, for example, by ribosomes. Covalent changes to the structure of nucleotide bases are well known to occur on tRNA and rRNA molecules, but such changes in mRNA sequence are novel in that they have the capacity to change specific protein sequences. 43 refs., 1 fig.

  5. A novel form of necrosis, TRIAD, occurs in human Huntington's disease.

    Science.gov (United States)

    Yamanishi, Emiko; Hasegawa, Kazuko; Fujita, Kyota; Ichinose, Shizuko; Yagishita, Saburo; Murata, Miho; Tagawa, Kazuhiko; Akashi, Takumi; Eishi, Yoshinobu; Okazawa, Hitoshi

    2017-03-08

    We previously reported transcriptional repression-induced atypical cell death of neuron (TRIAD), a new type of necrosis that is mainly regulated by Hippo pathway signaling and distinct from necroptosis regulated by RIP1/3 pathway. Here, we examined the ultrastructural and biochemical features of neuronal cell death in the brains of human HD patients in parallel with the similar analyses using mutant Htt-knock-in (Htt-KI) mice. LATS1 kinase, the critical regulator and marker of TRIAD, is actually activated in cortical neurons of postmortem human HD and of Htt-KI mouse brains, while apoptosis promoter kinase Plk1 was inactivated in human HD brains. Expression levels of YAP/YAPdeltaC were decreased in cortical neurons of human HD brains. Ultra-structural analyses revealed extreme enlargement of endoplasmic reticulum (ER), which characterizes TRIAD, in cortical neurons of human HD and those of Htt-KI mice. These biochemical and morphological results support that TRIAD occurs in human and mouse neurons under the HD pathology.

  6. Control of gene expression during T cell activation: alternate regulation of mRNA transcription and mRNA stability

    Directory of Open Access Journals (Sweden)

    Gorospe Myriam

    2005-05-01

    Full Text Available Abstract Background Microarray technology has become highly valuable for identifying complex global changes in gene expression patterns. The effective correlation of observed changes in gene expression with shared transcription regulatory elements remains difficult to demonstrate convincingly. One reason for this difficulty may result from the intricate convergence of both transcriptional and mRNA turnover events which, together, directly influence steady-state mRNA levels. Results In order to investigate the relative contribution of gene transcription and changes in mRNA stability regulation to standard analyses of gene expression, we used two distinct microarray methods which individually measure nuclear gene transcription and changes in polyA mRNA gene expression. Gene expression profiles were obtained from both polyA mRNA (whole-cell and nuclear run-on (newly transcribed RNA across a time course of one hour following the activation of human Jurkat T cells with PMA plus ionomycin. Comparative analysis revealed that regulation of mRNA stability may account for as much as 50% of all measurements of changes in polyA mRNA in this system, as inferred by the absence of any corresponding regulation of nuclear gene transcription activity for these groups of genes. Genes which displayed dramatic elevations in both mRNA and nuclear run-on RNA were shown to be inhibited by Actinomycin D (ActD pre-treatment of cells while large numbers of genes regulated only through altered mRNA turnover (both up and down were ActD-resistant. Consistent patterns across the time course were observed for both transcribed and stability-regulated genes. Conclusion We propose that regulation of mRNA stability contributes significantly to the observed changes in gene expression in response to external stimuli, as measured by high throughput systems.

  7. An interaction between a polymorphism of the serotonin transporter (5HTT gene and the clinical picture of adolescents with combined type of ADHD (hyperkinetic disorder and youth drinking

    Directory of Open Access Journals (Sweden)

    Gorzkowska, Izabela

    2014-06-01

    Full Text Available Introduction: The combined type of ADHD and alcohol dependence are two different disorders. Research demonstrate that 45-55% of patients diagnosed with ADHD also suffer from comorbid substance abuse, and 11-55% of patients diagnosed with substance abuse suffer from undiagnosed ADHD. Alcohol is by far the most widely used psychoactive substance in the European culture. The serotonin transporter (5HHT gene has been implicated as one of the candidate genes in both disorders in recent molecular genetic research. Aim: The aim of the present study was to seek a common clinical and biological marker for hyperkinetic disorder and youth drinking. Methods: The study was conducted between 2008 and 2012. The sample consisted of 100 combined type ADHD patients: 51 adolescents youth drinking and 100 individuals without mental disorders or addiction in a population-based group. The 5HHT gene polymorphism was examined using PCR (Polymerase Chain Reaction. Statistical analysis was conducted with STATISTICA.PL software (version 5.0.97 licensed by StatSoft, Inc. USA. Results: A preferential trend for the “s” short allele of the investigated 5HHT gene polymorphism was observed in all the groups of adolescents compared to the population-based group of adults without alcohol dependence (p=0.01. Conclusion: Based on the conducted study a provisional conclusion may be drawn that the presence of the short “s” allele of the 5HTT gene polymorphism may be a prognostic factor of impulsivity in ADHD and of predisposition to alcohol dependence.

  8. Expression and significance of Cystatin SN mRNA in human gastric cancer tissues%胃癌组织Cystatin SN mRNA表达及其临床意义的探讨

    Institute of Scientific and Technical Information of China (English)

    徐大方; 陈平; 石磊; 赵群; 顾学文

    2012-01-01

    OBJECTIVE! To investigate the expression and clinical significance of Cystatin SN in human gastric cancer. METHODS; The Cystatin SNmRNA expression was examined quantitatively by using real-time quantitative polymer-ase chain reaction on a cohort of human gastric cancer tissues (n=40) and corresponding adjacent tissues (w=40). RESULTS: The expression levels of Cystatin SN mRNA in gastric cancer were 5. 370 0±3. 034 5 and adjacent tissues were 1. 705 0±0. 738 8 respectively; Cystatin SN expression was dramatically increased in gastric cancer tissues compared with the corresponding adjacent tissues (?=8. 297, P = 0. 000). The expression of Cystatin SN was significantly associated with tumor size (t = - 0. 646, P = 0. 001), lymphnode metastasis (t = 3. 393 ,-P = 0. 002), gastric cancer stage (I = - 3. 798,P = 0. 001) and infiltration (£=4. 904,P=0. 000). However.it was not related to gastric differentiation,age and grader of patients (P>0. 05). CONCLUSION: Cystatin SN is overexpressed in gastric cancer tissues and significantly correlates with clinical prognosis.%目的:研究半胱氨酸蛋白酶抑制剂1 (Cystatin SN)在胃癌组织中的表达及临床意义.方法:荧光定量PCR方法检测40例胃癌组织和40例癌旁组织中Cystatin SN的表达.结果:胃癌组织中Cystatin SN mRNA的表达水平为5.370 0±3.034 5,相应的癌旁组织为1.705 0±0.738 8,癌组织显著高于癌旁组织,t=8.297,P=0.000.Cystatin SN的表达与肿瘤大小(t=-0.646,P=0.001)、淋巴转移(t=3.393,P=0.002)、TNM分期(t=-3.798,P=0.001)和浸润程度(t=4.904,P=0.000)相关,与肿瘤的分化程度、患者年龄和性别无关,P>0.05.结论:Cystatin SN在胃癌组织中的高表达与胃癌的发生发展密切相关.

  9. UIF, a New mRNA export adaptor that works together with REF/ALY, requires FACT for recruitment to mRNA.

    Science.gov (United States)

    Hautbergue, Guillaume M; Hung, Ming-Lung; Walsh, Matthew J; Snijders, Ambrosius P L; Chang, Chung-Te; Jones, Rachel; Ponting, Chris P; Dickman, Mark J; Wilson, Stuart A

    2009-12-01

    Messenger RNA (mRNA) export adaptors play an important role in the transport of mRNA from the nucleus to the cytoplasm. They couple early mRNA processing events such as 5' capping and 3' end formation with loading of the TAP/NXF1 export receptor onto mRNA. The canonical adaptor REF/ALY/Yra1 is recruited to mRNA via UAP56 and subsequently delivers the mRNA to NXF1 [1]. Knockdown of UAP56 [2, 3] and NXF1 [4-7] in higher eukaryotes efficiently blocks mRNA export, whereas knockdown of REF only causes a modest reduction, suggesting the existence of additional adaptors [8-10]. Here we identify a new UAP56-interacting factor, UIF, which functions as an export adaptor, binding NXF1 and delivering mRNA to the nuclear pore. REF and UIF are simultaneously found on the same mRNA molecules, and both proteins are required for efficient export of mRNA. We show that the histone chaperone FACT specifically binds UIF, but not REF, via the SSRP1 subunit, and this interaction is required for recruitment of UIF to mRNA. Together the results indicate that REF and UIF represent key human adaptors for the export of cellular mRNAs via the UAP56-NXF1 pathway.

  10. Recent innovations in mRNA vaccines.

    Science.gov (United States)

    Ulmer, Jeffrey B; Geall, Andrew J

    2016-08-01

    Nucleic acid-based vaccines are being developed as a means to combine the positive attributes of both live-attenuated and subunit vaccines. Viral vectors and plasmid DNA vaccines have been extensively evaluated in human clinical trials and have been shown to be safe and immunogenic, although none have yet been licensed for human use. Recently, mRNA based vaccines have emerged as an alternative approach. They promise the flexibility of plasmid DNA vaccines, without the need for electroporation, but with enhanced immunogenicity and safety. In addition, they avoid the limitations of anti-vector immunity seen with viral vectors, and can be dosed repeatedly. This review highlights the key papers published over the past few years and summarizes prospects for the near future.

  11. Expression of heat shock protein mRNA in epithelial cells of human lens%人晶状体上皮细胞内热休克蛋白mRNA的表达

    Institute of Scientific and Technical Information of China (English)

    张雪岩; 贾琳琳; 刘宏伟

    2007-01-01

    BACKGROUND: Special anatomical location makes eye lens expose to stressful situation in a long term, so it needs persistent protection to resist stress. Heat shock proteins (HSPs) exert self-protective defensive effect under the stress.OBJECTIVE: To observe the changes in mRNA expression of HSPs in the lens under the condition of exogenous stress.DESIGN: Repeated measurement, controlled observation trial.SETTING: Department of Ophthalmology, First Hospital Affiliated to Jiamusi University.PARTICIPANTS: Human lens endothelial cells B3 (LEC-B3) were obtained from the Department of Ophthalmology, First Hospital Affiliated to China Medical University. Reverse transcription-polymerase chain reaction (RT-PCR) kit was purchased from Bao Biotechnology Company, Japan.METHODS: This experiment was carried out in the Laboratory of Congenital Malformation, Ministry of Public Health,China Medical University between September 2003 and September 2004. LEC-B3 of the exponential growth was used for the experiment. Thermal or oxidative stress on LEC-B3 cells were produced by 30-minute exposure at 45 ℃ or incubated for 30 minutes in DMEM supplemented with 50 mmol/L H2O2. Some stressful cells were allowed to recuperate after exposure to stress in normal culture medium at 37 ℃ for 0, 2, 4, 6, 16 and 24 hours, separately. The expressions of HSP27 and HSP70 in the LEC-B3 were detected by RT-PCR.MAIN OUTCOME MEASURES: The expressions of HSP27 and HSP70 in LEC-B3.RESULTS: HSPs existed in both physiological and stressful situation. The levels of HSPmRNA were increased 2 hours after both stresses. But the expressions of HSP27 and HSP70 got to the summit at different intervals from 2 hours to 6 hours in each group. Subsequently, they were decreased gradually in each group, but they all could maintain a high level at 16 hours.CONCLUSION: Stressful environment induces the over-expressions of HSP27 mRNA and HSP70 mRNA in LEC-B3. This may play an important protective role in lens epithelial

  12. RNA Sequence Analysis of Human Huntington Disease Brain Reveals an Extensive Increase in Inflammatory and Developmental Gene Expression.

    Directory of Open Access Journals (Sweden)

    Adam Labadorf

    Full Text Available Huntington's Disease (HD is a devastating neurodegenerative disorder that is caused by an expanded CAG trinucleotide repeat in the Huntingtin (HTT gene. Transcriptional dysregulation in the human HD brain has been documented but is incompletely understood. Here we present a genome-wide analysis of mRNA expression in human prefrontal cortex from 20 HD and 49 neuropathologically normal controls using next generation high-throughput sequencing. Surprisingly, 19% (5,480 of the 28,087 confidently detected genes are differentially expressed (FDR<0.05 and are predominantly up-regulated. A novel hypothesis-free geneset enrichment method that dissects large gene lists into functionally and transcriptionally related groups discovers that the differentially expressed genes are enriched for immune response, neuroinflammation, and developmental genes. Markers for all major brain cell types are observed, suggesting that HD invokes a systemic response in the brain area studied. Unexpectedly, the most strongly differentially expressed genes are a homeotic gene set (represented by Hox and other homeobox genes, that are almost exclusively expressed in HD, a profile not widely implicated in HD pathogenesis. The significance of transcriptional changes of developmental processes in the HD brain is poorly understood and warrants further investigation. The role of inflammation and the significance of non-neuronal involvement in HD pathogenesis suggest anti-inflammatory therapeutics may offer important opportunities in treating HD.

  13. Contraction-induced increases in Na+-K+-ATPase mRNA levels in human skeletal muscle are not amplified by activation of additional muscle mass

    DEFF Research Database (Denmark)

    Nordsborg, Nikolai; Thomassen, Martin; Lundby, Carsten

    2005-01-01

    The present study tested the hypothesis that exercise with a large compared with a small active muscle mass results in a higher contraction-induced increase in Na+-K+-ATPase mRNA expression due to greater hormonal responses. Furthermore, the relative abundance of Na+-K+-ATPase subunit a1, a2, a3,...

  14. Splice variants of the human zinc transporter ZnT5 (SLC30A5) are differentially localized and regulated by zinc through transcription and mRNA stability.

    Science.gov (United States)

    Jackson, Kelly A; Helston, Rachel M; McKay, Jill A; O'Neill, Elaine D; Mathers, John C; Ford, Dianne

    2007-04-06

    Maintenance of cellular zinc homeostasis includes regulating the expression of cell membrane zinc transporters. Knowledge about the mechanisms underlying changes in mammalian zinc transporter mRNA abundance is poor. We demonstrated that when expressed in Chinese hamster ovary cells as N-terminal fusions to green fluorescent protein, two splice variants of ZnT5 adopt different subcellular locations (either in the Golgi apparatus or throughout the cell, including at the plasma membrane) indicating discrete roles in cellular zinc homeostasis. We demonstrated, using a beta-galactosidase reporter gene, that both splice variants were expressed from a promoter region that was transcriptionally repressed by increased extracellular zinc (150 microM compared with 3 mum; approximately 40%) and by extracellular zinc depletion, using the chelator N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine ( approximately 20%). We mapped the zinc-responsive element to the region -154 to +50, relative to the predicted start of transcription, and showed that a consensus metal response element sequence (-410 to -404) was not responsible for these effects. Changes in ZnT5 mRNA abundance in Caco-2 cells at different zinc concentrations were in parallel to the changes in promoter activity ( approximately 40% reduction at 150 microM zinc) but in the presence of actinomycin D, to prevent transcription, we observed a marked stabilization (1.7-2-fold accumulation over 24 h) of ZnT5 mRNA. We conclude that effects of zinc on ZnT5 transcription and mRNA stability act in opposition to balance mRNA abundance for cellular zinc homeostasis. To our knowledge, this is the first report that zinc affects the stability of a transcript with a direct role in cellular zinc homeostasis.

  15. Consequences of nigrostriatal denervation on the functioning of the basal ganglia in human and nonhuman primates: an in situ hybridization study of cytochrome oxidase subunit I mRNA.

    Science.gov (United States)

    Vila, M; Levy, R; Herrero, M T; Ruberg, M; Faucheux, B; Obeso, J A; Agid, Y; Hirsch, E C

    1997-01-15

    To examine the consequences of nigrostriatal denervation and chronic levodopa (L-DOPA) treatment on functional activity of the basal ganglia, we analyzed, using in situ hybridization, the cellular expression of the mRNA encoding for cytochrome oxidase subunit I (COI mRNA), a molecular marker for functional neuronal activity, in the basal ganglia. This analysis was performed in monkeys rendered parkinsonian by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) Intoxication, some of which had been receiving L-DOPA, and in patients with Parkinson's disease (PD). In MPTP-intoxicated monkeys compared with control animals, COI mRNA expression was increased in the subthalamic nucleus (STN) and in the output nuclei of the basal ganglia, i.e., the internal segment of the globus pallidus and the substantia nigra pars reticulata. This increase was partially reversed by L-DOPA treatment. COI mRNA expression remained unchanged in the external segment of the globus pallidus (GPe). In PD patients, all of whom had been treated chronically by L-DOPA, COI mRNA expression in the analyzed basal ganglia structures was similar to that in control subjects. These results are in agreement with the accepted model of basal ganglia organization, to the extent that the output nuclei of the basal ganglia are considered to be overactive after nigrostriatal denervation, partly because of increased activity of excitatory afferents from the STN. Yet, our results would also seem to contradict this model, because the overactivity of the STN does not seem to be attributable to a hypoactivation of the GPe.

  16. MRNA-based skin identification for forensic applications

    NARCIS (Netherlands)

    M. Visser (Mijke); D. Zubakov (Dmitry); K. Ballantyne (Kaye); M.H. Kayser (Manfred)

    2011-01-01

    textabstractAlthough the identification of human skin cells is of important relevance in many forensic cases, there is currently no reliable method available. Here, we present a highly specific and sensitive messenger RNA (mRNA) approach for skin identification, meeting the key requirements in

  17. 垂体生长激素腺瘤中gsp癌基因对GHSR-1a、Ghrelin mRNA表达的影响%Effect of gsp oncogene on expression of GHSR - 1a and Ghrelin mRNA in human GH - secreting pituitary adenomas

    Institute of Scientific and Technical Information of China (English)

    徐同江; 叶飞; 田春雷; 谢蕊繁; 舒凯; 郭东生; 雷霆

    2010-01-01

    目的 研究垂体生长激素(GH)腺瘤组织中Ghrelin、生长激素释放剂受体-1a亚型(GHSR-1a)mRNA的表达,并探讨其表达与gsp癌基因的关系.方法 采用PCR-DNA直接序列分析方法,观察43例垂体GH腺瘤组织中gsp癌基因的表达;应用实时荧光定量PCR检测Ghrelin和GHSR-1a mRNA的表达水平;统计学分析Ghrelin和GHSR-1a mRNA表达与gsp癌基因的关系.结果 Ghrelin mRNA表达在gsp癌基因阳性、阴性组织间差异无统计学意义(P>0.05);gsp阳性组织中GHSR-1a mRNA表达明显高于gsp阴性组织(P<0.05);gsp阳性、阴性腺瘤组织中GHSR-1amRNA与Ghrelin mRNA的表达均呈明显正相关(R=0.592或0.544,P<0.05).结论 垂体GH腺瘤中gsp癌基因上调GHSR-1a mRNA的表达;而对Ghrelin mRNA表达无明显影响.gsp阳性、阴性腺瘤中Ghrelin均可正向调控GHSR-1a mRNA表达.%Objective To investigate the relationship between the expression of growth hormone secretagogue receptor type 1a (GHSR- 1a) 、Ghrelin mRNA and gsp oncogene in human GH- secreting pituitary adenomas. Methods The gsp oncogene mutation in 43 cases of GH -secreting pituitary adenomas was detected by PCR - DNA direct sequencing analysis. The expression of GHSR - 1a、Ghrelin mRNA was determined by SYBR green real -time fluorescent quantitative PCR. Then the relationships between the expression of GHSR -1a、Ghrelin mRNA and gsp oncegene were studied with statistical methods. Results There was no significant difference in the expression of ghrelin mRNA between mutation - positive and mutation - negative adenomas. The expression of GHSR - 1a mRNA was significantly higher in gsp - positive adenomas than that in gsp - negative adenomas. There was significant direct correlation between the levels of ghrelin mRNA and GHSR mRNA expression in gsp - positive and gsp - negative adenomas. Conclusions Gsp oncogene may up - regulate the expression of GHSR - 1a mRNA and has no effect on the expression of ghrelin mRNA in human GH

  18. Main: MRNA3ENDTAH3 [PLACE

    Lifescience Database Archive (English)

    Full Text Available MRNA3ENDTAH3 S000069 17-May-1998 (last modified) kehi Cis element in 3' end region ...of wheat (T.a.) histone H3 mRNA; 3' end formation; Also found in histone genes of other plants, yeast, etc; histone H3; mRNA

  19. Influence of the Cyp1B1 L432V gene polymorphism and exposure to tobacco smoke on Cyp1B1 mRNA expression in human leukocytes.

    Science.gov (United States)

    Helmig, Simone; Hadzaad, Bahar; Döhrel, Juliane; Schneider, Joachim

    2009-07-01

    Cytochrome P450 1B1 (CYP1B1), a phase I enzyme, is involved in the activation of a broad spectrum of procarcinogens. An association of the Cyp1B1 L432V polymorphism with diverse types of cancer, as well as an impact on the catalytic activity of the enzyme, has been described. To show the functional impact of the allelic variant Cyp1B1*3, we investigated the quantitative Cyp1B1 mRNA expression in a population of smokers, nonsmokers, and ex-smokers and determined their genotypes. Detection of the L432V polymorphism in exon 3 of the Cyp1B1 gene was performed by rapid capillary polymerase chain reaction (PCR) with melting curve analysis. For quantitative comparison of Cyp1B1 mRNA levels, real-time PCR was performed using SYBR Green fluorescence in a LightCycler system. Calculations of expression were made with the 2(-DeltaDeltaCT) method. In comparing relative Cyp1B1 mRNA expression, highly significant differences between the two homozygote genotypes *1/*1 and *3/*3 (0.185 +/- 0.027, n = 118 versus 0.071 +/- 0.013, n = 56; p = 0.000), as well as between the heterozygote genotype *1/*3 and the homozygote genotype *3/*3 (0.178 +/- 0.025, n = 171 versus 0.071 +/- 0.013, n = 56; p = 0.000), were revealed. Significant differences between the genotypes were also detected within the subgroups of smokers, nonsmokers, and ex-smokers. No significant differences were determined in comparing the relative Cyp1B1 mRNA expression with regard to tobacco smoke exposure. Our results suggest that genotypes carrying the C allele (*1/*1 and *1/*3) at Cyp1B1 L432V polymorphism have a significantly higher Cyp1B1 mRNA expression compared with the genotype without the C allele (*3/*3). Gene expression of Cyp1B1 mRNA cannot be used as a biomarker for exposure of tobacco smoke.

  20. Sex differences in the serotonin 1A receptor and serotonin transporter binding in the human brain measured by PET.

    Science.gov (United States)

    Jovanovic, Hristina; Lundberg, Johan; Karlsson, Per; Cerin, Asta; Saijo, Tomoyuki; Varrone, Andrea; Halldin, Christer; Nordström, Anna-Lena

    2008-02-01

    Women and men differ in serotonin associated psychiatric conditions, such as depression, anxiety and suicide. Despite this, very few studies focus on sex differences in the serotonin system. Of the biomarkers in the serotonin system, serotonin(1A) (5-HT(1A)) receptor is implicated in depression, and anxiety and serotonin transporter (5-HTT) is a target for selective serotonin reuptake inhibitors, psychotropic drugs used in the treatment of these disorders. The objective of the present study was to study sex related differences in the 5-HT(1A) receptor and 5-HTT binding potentials (BP(ND)s) in healthy humans, in vivo. Positron emission tomography and selective radioligands [(11)C]WAY100635 and [(11)C]MADAM were used to evaluate binding potentials for 5-HT(1A) receptors (14 women and 14 men) and 5-HTT (8 women and 10 men). The binding potentials were estimated both on the level of anatomical regions and voxel wise, derived by the simplified reference tissue model and wavelet/Logan plot parametric image techniques respectively. Compared to men, women had significantly higher 5-HT(1A) receptor and lower 5-HTT binding potentials in a wide array of cortical and subcortical brain regions. In women, there was a positive correlation between 5-HT(1A) receptor and 5-HTT binding potentials for the region of hippocampus. Sex differences in 5-HT(1A) receptor and 5-HTT BP(ND) may reflect biological distinctions in the serotonin system contributing to sex differences in the prevalence of psychiatric disorders such as depression and anxiety. The result of the present study may help in understanding sex differences in drug treatment responses to drugs affecting the serotonin system.

  1. Regulation of P-glycoprotein expression in brain capillaries in Huntington's disease and its impact on brain availability of antipsychotic agents risperidone and paliperidone.

    Science.gov (United States)

    Kao, Yu-Han; Chern, Yijuang; Yang, Hui-Ting; Chen, Hui-Mei; Lin, Chun-Jung

    2016-08-01

    Huntington's disease (HD) is a neurodegenerative disease marked by an expanded polyglutamine (polyQ) tract on the huntingtin (HTT) protein that may cause transcriptional dysfunction. This study aimed to investigate the regulation and function of P-glycoprotein, an important efflux transporter, in brain capillaries in HD. The results showed that, compared with the littermate controls, R6/2 HD transgenic mice with the human mutant HTT gene had higher levels of P-glycoprotein mRNA and protein and enhanced NF-κB activity in their brain capillaries. Higher P-glycoprotein expression was also observed in the brain capillaries of human HD patients. Consistent with this enhanced P-glycoprotein expression, brain extracellular levels and brain-to-plasma ratios of the antipsychotic agents risperidone and paliperidone were significantly lower in R6/2 mice than in their littermate controls. Exogenous expression of human mutant HTT protein with expanded polyQ (mHTT-109Q) in HEK293T cells enhanced the levels of P-glycoprotein transcripts and NF-κB activity compared with cells expressing normal HTT-25Q. Treatment with the IKK inhibitor, BMS-345541, decreased P-glycoprotein mRNA level in cells transfected with mHTT-109Q or normal HTT-25Q In conclusion, mutant HTT altered the expression of P-glycoprotein through the NF-κB pathway in brain capillaries in HD and markedly affected the availability of P-glycoprotein substrates in the brain.

  2. HIF1A and EPAS1 mRNA and protein expression during in vitro culture of human primary term cytotrophoblasts and effect of oxygen tension on their expression.

    Science.gov (United States)

    Depoix, Christophe Louis; Flabat, Olivier; Debiève, Frédéric; Hubinont, Corinne

    2016-09-01

    During the first trimester of pregnancy, placenta formation probably occurs in a low-oxygen environment necessary to protect cytotrophoblasts from oxidative stress and to allow proper gene regulation. Transcription factors involved in gene regulation under low oxygen tension are the hypoxia-inducible factors, mainly HIF1A, EPAS1 and their dimerization partner HIF1B. Little is known about their expression during in vitro culture of cytotrophoblasts under chronic hypoxia. We assessed HIF1A and EPAS1 expression in a 4-day in vitro culture of primary term cytotrophoblasts under 21% O2 and 2.5% O2. Copy numbers and relative mRNA expression were assessed by real-time quantitative polymerase chain reaction. Protein levels were quantified by immunoblot and densitometric analysis. In undifferentiated cytotrophoblasts, EPAS1 transcripts were four times more abundant than HIF1A transcripts (2.14e(7) and 5e(6)copies/μg total RNA, respectively). During cell culture, HIF1A mRNA expression increased after 24h and then decreased to stay stable. The expression was even lower when cells were grown under 2.5% O2. EPAS1 mRNA expression increased during cytotrophoblast differentiation. The expression was higher when cells were under 21% O2 than when they were under 2.5% O2. Interestingly, HIF1A, but not EPAS1, was detected in the nuclei of undifferentiated cytotrophoblasts, and in the nuclei of cytotrophoblasts that grew under 21% O2. During cytotrophoblast differentiation, no variation in HIF1A protein levels was detected. To the contrary, EPAS1 protein level increased during differentiation, and oxygen tension had no effect on EPAS1 protein level. In conclusion, HIF1A and EPAS1 expression was not inhibited by chronic hypoxia during in vitro cytotrophoblast differentiation.

  3. Efficient Translation Initiation Directed by the 900-Nucleotide-Long and GC-Rich 5′ Untranslated Region of the Human Retrotransposon LINE-1 mRNA Is Strictly Cap Dependent Rather than Internal Ribosome Entry Site Mediated▿

    Science.gov (United States)

    Dmitriev, Sergey E.; Andreev, Dmitri E.; Terenin, Ilya M.; Olovnikov, Ivan A.; Prassolov, Vladimir S.; Merrick, William C.; Shatsky, Ivan N.

    2007-01-01

    Retrotransposon L1 is a mobile genetic element of the LINE family that is extremely widespread in the mammalian genome. It encodes a dicistronic mRNA, which is exceptionally rare among eukaryotic cellular mRNAs. The extremely long and GC-rich L1 5′ untranslated region (5′UTR) directs synthesis of numerous copies of RNA-binding protein ORF1p per mRNA. One could suggest that the 5′UTR of L1 mRNA contained a powerful internal ribosome entry site (IRES) element. Using transfection of cultured cells with the polyadenylated monocistronic (L1 5′UTR-Fluc) or bicistronic (Rluc-L1 5′UTR-Fluc) RNA constructs, capped or uncapped, it has been firmly established that the 5′UTR of L1 does not contain an IRES. Uncapping reduces the initiation activity of the L1 5′UTR to that of background. Moreover, the translation is inhibited by upstream AUG codons in the 5′UTR. Nevertheless, this cap-dependent initiation activity of the L1 5′UTR was unexpectedly high and resembles that of the beta-actin 5′UTR (84 nucleotides long). Strikingly, the deletion of up to 80% of the nucleotide sequence of the L1 5′UTR, with most of its stem loops, does not significantly change its translation initiation efficiency. These data can modify current ideas on mechanisms used by 40S ribosomal subunits to cope with complex 5′UTRs and call into question the conception that every long GC-rich 5′UTR working with a high efficiency has to contain an IRES. Our data also demonstrate that the ORF2 translation initiation is not directed by internal initiation, either. It is very inefficient and presumably based on a reinitiation event. PMID:17470553

  4. Efficient translation initiation directed by the 900-nucleotide-long and GC-rich 5' untranslated region of the human retrotransposon LINE-1 mRNA is strictly cap dependent rather than internal ribosome entry site mediated.

    Science.gov (United States)

    Dmitriev, Sergey E; Andreev, Dmitri E; Terenin, Ilya M; Olovnikov, Ivan A; Prassolov, Vladimir S; Merrick, William C; Shatsky, Ivan N

    2007-07-01

    Retrotransposon L1 is a mobile genetic element of the LINE family that is extremely widespread in the mammalian genome. It encodes a dicistronic mRNA, which is exceptionally rare among eukaryotic cellular mRNAs. The extremely long and GC-rich L1 5' untranslated region (5'UTR) directs synthesis of numerous copies of RNA-binding protein ORF1p per mRNA. One could suggest that the 5'UTR of L1 mRNA contained a powerful internal ribosome entry site (IRES) element. Using transfection of cultured cells with the polyadenylated monocistronic (L1 5'UTR-Fluc) or bicistronic (Rluc-L1 5'UTR-Fluc) RNA constructs, capped or uncapped, it has been firmly established that the 5'UTR of L1 does not contain an IRES. Uncapping reduces the initiation activity of the L1 5'UTR to that of background. Moreover, the translation is inhibited by upstream AUG codons in the 5'UTR. Nevertheless, this cap-dependent initiation activity of the L1 5'UTR was unexpectedly high and resembles that of the beta-actin 5'UTR (84 nucleotides long). Strikingly, the deletion of up to 80% of the nucleotide sequence of the L1 5'UTR, with most of its stem loops, does not significantly change its translation initiation efficiency. These data can modify current ideas on mechanisms used by 40S ribosomal subunits to cope with complex 5'UTRs and call into question the conception that every long GC-rich 5'UTR working with a high efficiency has to contain an IRES. Our data also demonstrate that the ORF2 translation initiation is not directed by internal initiation, either. It is very inefficient and presumably based on a reinitiation event.

  5. 人肝癌组织中MCT1,MCT2基因mRNA表达的半定量研究%Semi-quantitative analysis of MCT1 and MCT2 genes mRNA expression in human hepatocellular carcinoma

    Institute of Scientific and Technical Information of China (English)

    李靖; 韩本立

    2001-01-01

    Objective To study the mRNA expression of MCT1 and MCT2 genes in human hepatocellular carcinoma (HCC) and paracarcinoma liver tissue (PCLT). Methods The semi-quantitative analysis of MCT1 and MCT2 genes mRNA expression in human HCC and PCLT was conducted by RT-PCR method and electrophoresis band opacity density (OD) comparison analysis method in 25 patients with HCC. Results The mRNA expression of MCT1 was significantly higher than MCT2 in HCC and PCLT, in HCC the mRNA expression of MCT1 and MCT2 genes were significant higher than that in PCLT. Conclusions The high expression of mRNA of MCT1 and MCT2 genes in HCC indicates that these genes may take a significant role on lactate and other monocarboxylate transmembrane transportation and on pH regulation in tumor cells.%目的探讨单羧酸转运蛋白-1(MCT1)、单羧酸转运蛋白-2(MCT2) mRNA在人肝癌及癌旁肝组织中的表达及意义。方法应用RT-PCR及电泳条带光密度比较法对25例肝癌病人的肝癌及癌旁肝组织中MCT1,MCT2 mRNA的表达进行半定量分析。结果肝癌及癌旁肝组织的MCT1 mRNA的表达显著高于MCT2。肝癌组织中MCT1,MCT2 mRNA的表达显著高于癌旁肝组织。结论 MCT1,MCT2在肝癌细胞内乳酸等单羧酸的跨膜转运及细胞内pH的调节上可能起重要作用。

  6. Telomere length and DCC gene mRNA expression of human large intestine cancers%大肠癌中端粒长度与DCC基因mRNA表达的研究

    Institute of Scientific and Technical Information of China (English)

    张方信; 谢咏梅; 邓芝云; 贾忠建; 魏亚明; 樊俊杰; 吴汉平

    2001-01-01

    目的分析端粒长度及DCC基因mRNA表达在大肠癌及腺瘤发生发展中的作用。方法采用Sourthern印迹杂交及RT-PCR技术,分别检测46例大肠腺瘤及62例大肠癌组织中的端粒限制性片段(TRF)长度及DCC mRNA表达状态,并观察它们与肿瘤临床病理的关系。结果在大肠癌及大肠腺瘤中,TRF长度较正常组织明显缩短,其缩短者占53.2%和41.3%,而延长者仅占6.5%和4.4%,结肠癌的TRF长度也较直肠癌的TRF长度明显缩短。DCC mRNA表达缺失率在大肠癌及大肠腺瘤中则分别达62.9%和34.8%,显著高于正常组织(1.6%);同时,大肠癌患者的平均TRF长度还随患者年龄的增长而缩短,DCC mRNA表达缺失率则随肿瘤的分化程度下降及临床阶段的进展而升高,但DCC mRNA表达缺失与TRF长度缩短在大肠癌中未表现出明显的相关性。结论端粒缩短与DCC mRNA表达缺失可能是大肠腺瘤恶变及大肠癌形成过程中较具特征表现的生物学异常行为。%Objective To evaluate the role of telomere and DCC in tumor transformation and progression. Methods  Telomere length and DCC gene mRNA expression were examined by southern blot hybridization and RT-PCR analysis in 46 adenomas of large intestine, 62 cancers of large intestine and corresponding normal mucosa. Results  Shortening of the telomere was present in the tissues of 41.3% of the adenomas and 53.2% of the cancers, and their average TRF lengths were significantly shorter than those of corresponding normal mucosa(P<0.05, P<0.01), whereas the telomere elongation was only detected in 4.4% and 6.5% of the adenomas and cancers respectively. In addition, the average telomere length in colon carcinomas was also shorter than that in rectal carcinomas. Moreover, the average telomere lengths of the colorectal cancer mucosa became shorter with age. The rates of DCC mRNA expression deletion were 34.8% and 62.9% in the tissues of adenomas and cancers

  7. Reference: MRNA3ENDTAH3 [PLACE

    Lifescience Database Archive (English)

    Full Text Available MRNA3ENDTAH3 Ohtsubo N, Iwabuchi M The conserved 3'-flanking sequence, AATGGAAATG, ...of the wheat histone H3 gene is necessary for the accurate 3'-end formation of mRNA. Nucleic Acids Res 22:1052-1058 (1994) PubMed: 8152910; ...

  8. First-in-human Phase I study of EZN-4176, a locked nucleic acid antisense oligonucleotide to exon 4 of the androgen receptor mRNA in patients with castration-resistant prostate cancer.

    Science.gov (United States)

    Bianchini, D; Omlin, A; Pezaro, C; Lorente, D; Ferraldeschi, R; Mukherji, D; Crespo, M; Figueiredo, I; Miranda, S; Riisnaes, R; Zivi, A; Buchbinder, A; Rathkopf, D E; Attard, G; Scher, H I; de Bono, J; Danila, D C

    2013-11-12

    Prostate cancer remains dependent of androgen receptor (AR) signalling, even after emergence of castration resistance. EZN-4176 is a third-generation antisense oligonucleotide that binds to the hinge region (exon 4) of AR mRNA resulting in full-length AR mRNA degradation and decreased AR protein expression. This Phase I study aimed to evaluate EZN-4176 in men with castration-resistant prostate cancer (CRPC). Patients with progressing CRPC were eligible; prior abiraterone and enzalutamide treatment were allowed. EZN-4176 was administered as a weekly (QW) 1-h intravenous infusion. The starting dose was 0.5 mg kg(-1) with a 4-week dose-limiting toxicity (DLT) period and a 3+3 modified Fibonacci dose escalation design. After determination of the DLT for weekly administration, an every 2 weeks schedule was initiated. A total of 22 patients were treated with EZN-4176. At 10 mg kg(-1) QW, two DLTs were observed due to grade 3-4 ALT or AST elevation. No confirmed biochemical or soft tissue responses were observed. Of eight patients with <5 circulating tumour cells at baseline, a conversion to <5 was observed in three (38%) patients. The most common EZN-4176-related toxicities (all grades) were fatigue (59%), reversible abnormalities in liver function tests ALT (41%) and AST (41%) and infusion-related reactions including chills (36%) and pyrexia (14%). Activity of EZN-4176 at the doses and schedules explored was minimal. The highest dose of 10 mg kg(-1) QW was associated with significant but reversible transaminase elevation.

  9. Systems perspectives on mRNA processing

    Institute of Scientific and Technical Information of China (English)

    Adrienne E McKee; Pamela A Silver

    2007-01-01

    The application of genomic technologies to the study of mRNA processing is increasingly conducted in metazoan organisms in order to understand the complex events that occur during and after transcription. Large-scale systems analyses of mRNA-protein interactions and mRNA dynamics have revealed specificity in mRNA transcription, splicing, transport, translation, and turnover, and have begun to make connections between the different layers of mRNA processing. Here, we review global studies of post-transcriptional processes and discuss the challenges facing our understanding of mRNA regulation in metazoan organisms. In parallel, we examine genome-scale investigations that have expanded our knowledge of RNA-binding proteins and the networks of mRNAs that they regulate.

  10. Influenza virus mRNA trafficking through host nuclear speckles.

    Science.gov (United States)

    Mor, Amir; White, Alexander; Zhang, Ke; Thompson, Matthew; Esparza, Matthew; Muñoz-Moreno, Raquel; Koide, Kazunori; Lynch, Kristen W; García-Sastre, Adolfo; Fontoura, Beatriz M A

    2016-05-27

    Influenza A virus is a human pathogen with a genome composed of eight viral RNA segments that replicate in the nucleus. Two viral mRNAs are alternatively spliced. The unspliced M1 mRNA is translated into the matrix M1 protein, while the ion channel M2 protein is generated after alternative splicing. These proteins are critical mediators of viral trafficking and budding. We show that the influenza virus uses nuclear speckles to promote post-transcriptional splicing of its M1 mRNA. We assign previously unknown roles for the viral NS1 protein and cellular factors to an intranuclear trafficking pathway that targets the viral M1 mRNA to nuclear speckles, mediates splicing at these nuclear bodies and exports the spliced M2 mRNA from the nucleus. Given that nuclear speckles are storage sites for splicing factors, which leave these sites to splice cellular pre-mRNAs at transcribing genes, we reveal a functional subversion of nuclear speckles to promote viral gene expression.

  11. COX-2 mRNA expression in esophageal squamous cell carcinoma (ESCC) and effect by NSAID.

    Science.gov (United States)

    Liu, X; Li, P; Zhang, S-T; You, H; Jia, J-D; Yu, Z-L

    2008-01-01

    To investigate cyclooxygenase-2 (COX-2) mRNA expression in human esophageal squamous cell carcinoma and the effect of a non-steroidal anti-inflammatory drug (NSAID) on it, in order to explore the mechanism of COX-2 in esophageal squamous cell carcinoma (ESCC) carcinogenesis and the ability of NSAID to prevent or treat ESCC. Frozen specimens of human ESCC and adjacent normal esophageal squamous epithelium pairs (n = 22) were examined for COX-2 mRNA expression by reverse-transcription polymerase chain reaction (RT-PCR). After incubation with aspirin (a non-selective COX inhibitor) or Nimesulide (a selective COX-2 inhibitor), the proliferation status of two human esophageal squamous cancer cell lines, EC-9706 and EC-109, was quantified by 3-(4,5-dimethyl-thiazol-2yl)-2,5-diphenyltetrazolium bromide assay. The expression of COX-2 mRNA in these cells was detected by RT-PCR. COX-2 mRNA was expressed in 12 of 22 (54.5%) ESCC tissue samples, but it was undetectable in all the specimens of adjacent normal esophageal squamous epithelium COX-2 mRNA expression. Both aspirin (5-20 mmol/L) and Nimesulide (0.1-0.8 mmol/L) inhibited EC-9706 cell line proliferation and suppressed its COX-2 mRNA expression dose-dependently. However, only aspirin (5-20 mmol/L) could inhibit proliferation in the EC-109 cell line and suppress COX-2 mRNA expression. Nimesulide (0.1-0.8 mmol/L) could neither inhibit EC-109 cell growth nor suppress COX-2 mRNA expression. COX-2 mRNA expression is a frequent phenomenon in human ESCC tissue samples and plays an important role in the carcinogenesis of ESCC. NSAID may be useful in the chemoprevention and therapy of human ESCC and its effects are likely to be mediated by modulating COX-2 activity.

  12. Sequence of the human glycogen-associated regulatory subunit of type 1 protein phosphatase and analysis of its coding region and mRNA level in muscle from patients with NIDDM

    DEFF Research Database (Denmark)

    Chen, Y H; Hansen, L; Chen, Min

    1994-01-01

    of protein phosphatase 1 (PP1 G-subunit) plays a key role in the insulin stimulation of glycogen synthesis and the activity of PP1 is decreased in insulin-resistant subjects, we have now cloned the human G-subunit cDNA to search for abnormalities in the corresponding gene (designated PPP1R3 in the human...

  13. Promoter Region Hypermethylation and mRNA Expression of MGMT and p16 Genes in Tissue and Blood Samples of Human Premalignant Oral Lesions and Oral Squamous Cell Carcinoma

    Directory of Open Access Journals (Sweden)

    Vikram Bhatia

    2014-01-01

    Full Text Available Promoter methylation and relative gene expression of O6-methyguanine-DNA-methyltransferase (MGMT and p16 genes were examined in tissue and blood samples of patients with premalignant oral lesions (PMOLs and oral squamous cell carcinoma (OSCC. Methylation-specific PCR and reverse transcriptase PCR were performed in 146 tissue and blood samples from controls and patients with PMOLs and OSCC. In PMOL group, significant promoter methylation of MGMT and p16 genes was observed in 59% (P=0.0010 and 57% (P=0.0016 of tissue samples, respectively, and 39% (P=0.0135 and 33% (P=0.0074 of blood samples, respectively. Promoter methylation of both genes was more frequent in patients with OSCC, that is, 76% (P=0.0001 and 82% (P=0.0001 in tissue and 57% (P=0.0002 and 70% (P=0.0001 in blood, respectively. Significant downregulation of MGMT and p16 mRNA expression was observed in both tissue and blood samples from patients with PMOLs and OSCC. Hypermethylation-induced transcriptional silencing of MGMT and p16 genes in both precancer and cancer suggests important role of these changes in progression of premalignant state to malignancy. Results support use of blood as potential surrogate to tissue samples for screening or diagnosing PMOLs and early OSCC.

  14. Promoter region hypermethylation and mRNA expression of MGMT and p16 genes in tissue and blood samples of human premalignant oral lesions and oral squamous cell carcinoma.

    Science.gov (United States)

    Bhatia, Vikram; Goel, Madhu Mati; Makker, Annu; Tewari, Shikha; Yadu, Alka; Shilpi, Priyanka; Kumar, Sandeep; Agarwal, S P; Goel, Sudhir K

    2014-01-01

    Promoter methylation and relative gene expression of O(6)-methyguanine-DNA-methyltransferase (MGMT) and p16 genes were examined in tissue and blood samples of patients with premalignant oral lesions (PMOLs) and oral squamous cell carcinoma (OSCC). Methylation-specific PCR and reverse transcriptase PCR were performed in 146 tissue and blood samples from controls and patients with PMOLs and OSCC. In PMOL group, significant promoter methylation of MGMT and p16 genes was observed in 59% (P = 0.0010) and 57% (P = 0.0016) of tissue samples, respectively, and 39% (P = 0.0135) and 33% (P = 0.0074) of blood samples, respectively. Promoter methylation of both genes was more frequent in patients with OSCC, that is, 76% (P = 0.0001) and 82% (P = 0.0001) in tissue and 57% (P = 0.0002) and 70% (P = 0.0001) in blood, respectively. Significant downregulation of MGMT and p16 mRNA expression was observed in both tissue and blood samples from patients with PMOLs and OSCC. Hypermethylation-induced transcriptional silencing of MGMT and p16 genes in both precancer and cancer suggests important role of these changes in progression of premalignant state to malignancy. Results support use of blood as potential surrogate to tissue samples for screening or diagnosing PMOLs and early OSCC.

  15. BMP2作用下人牙乳头细胞内Smad1 mRNA表达的变化%Smad1 mRNA expression and modulation in the human dental papilla cells treated by BMP2

    Institute of Scientific and Technical Information of China (English)

    何文喜; 牛忠英; 赵守亮; 陈健

    2001-01-01

    目的:观察人牙乳头细胞内Smad1 mRNA 的表达及在BMP2作用下,细胞内Smad1 mRNA的表达变化,探讨人牙乳头细胞内Smad1 信号途径在BMP2调控牙乳头细胞分化中的作用.方法:原代培养的人牙乳头细胞用BMP2处理后,提取总RNA,采用Northern blot法,从mRNA水平观察Smad1基因的表达及含量变化.结果:从mRNA水平观察到Smad1基因在人牙乳头细胞内的表达,但在BMP2作用6、12、24h后,Smad1 mRNA表达量无显著变化.结论:人牙乳头细胞内存在Smad1信号转导途径,牙乳头细胞内Sma d1 mRNA表达量不受BMP2调控.

  16. Development of bcl-2 mRNA repressor of apoptosis in human fetal central nervous system%人胎儿中枢神经系统凋亡抑制因子bcl-2mRNA的发育

    Institute of Scientific and Technical Information of China (English)

    李泽桂; 蔡文琴

    2003-01-01

    目的研究人胎儿中枢神经系统凋亡抑制因子 bcl-2 mRNA 的发育表达.方法用地高辛标记的bcl-2 cRNA 探针原位杂交组织化学技术,检测了12~39周胎儿中枢神经系统内bcl-2 mRNA的表达情况. 结果①在所检测的各脑区均有bcl-2 mRNA表达.第12周,有强阳性的bcl-2 mRNA出现在脊髓、延髓的运动神经元、大脑额叶的皮质板; 小脑和大脑室层的bcl-2 mRNA表达较弱.bcl-2 mRNA的水平一般是随胎龄的增长而下降,至第39周表达最弱.②bcl-2 mRNA主要在神经元表达.结论在人胎儿神经系统发育中表达的bcl-2可能与编程性细胞死亡有关.

  17. Mapping the distribution of serotonin transporter in the human brainstem with high-resolution PET: Validation using postmortem autoradiography data.

    Science.gov (United States)

    Fazio, P; Schain, M; Varnäs, K; Halldin, C; Farde, L; Varrone, A

    2016-06-01

    The human brainstem is a complex structure with several small nuclei and neural pathways of interest in the pathophysiology of central nervous system (CNS) disorders. In common with other monoaminergic systems, serotoninergic neurons originate from a group of nuclei located in the brainstem. The present study was designed to validate a user-independent approach for a detailed in vivo quantification of serotonin transporter (5-HTT) availability in the human brainstem using a template-based approach that consisted of three steps. First, 3T-MR images and parametric binding potential (BPND) [(11)C]MADAM images of ten healthy subjects were used to generate a PET template of 5-HTT availability. In the second step, volumes of interest (VOIs) for different brainstem nuclei were obtained using a method in which VOIs are initially delineated on MRI images using anatomical landmarks and then are finally tailored on the distribution of 5-HTT binding using a thresholding approach applied to the 5-HTT template. In the final step, the VOIs were transformed and applied individually to BPND images of 16 healthy subjects (14M/2F, 20-64years). The in vivo distribution of BPND values obtained with the template-based method were in good agreement with an individual-based approach taken as gold standard. Results were also in agreement with 5-HTT quantification using in vitro binding data obtained with autoradiography (ARG) studies using [(3)H]MADAM. The proposed template-based method can be applied to PET data acquired in several CNS disorders in which serotonin neurons in the brainstem might be affected.

  18. Quantification of human telomerase reverse transcriptase mRNA in gastric cancer with real-time fluorescent quantitative RT-PCR%胃癌中人端粒酶逆转录酶mRNA的实时定量检测

    Institute of Scientific and Technical Information of China (English)

    胡丽华; 陈凤花; 李一荣; 王琳

    2004-01-01

    目的建立一种实时荧光定量逆转录-聚合酶链反应(RT-PCR)方法,检测胃癌组织中hTERT mRNA 的表达,探讨hTERT的表达水平与胃癌的关系及其在胃癌诊断中的价值.方法采用Taqman技术与LightCycler荧光定量PCR仪对35例胃癌及其相应切缘组织中hTERT mRNA的表达进行实时定量检测.将hTERT与GAPDH拷贝数之比的100倍作为标准化hTERT(NhTERT).结果 (1)胃癌及其相应切缘组织中NhTERT分别为6.27±0.89、0.93±0.18,两者之间差异有统计学意义(t=12.76,P<0.01).(2)胃癌组织中hTERT mRNA的表达水平与组织的分化程度密切相关(P<0.01),而与患者的年龄、性别、肿瘤的大小、定位以及pTNM分期无相关性.结论实时荧光定量RT-PCR 方法能对hTERT mRNA进行准确、高效的定量.hTERT mRNA的实时定量检测可能有助于胃癌的早期诊断.

  19. Differential mRNA expression of the human DNA methyltransferases (DNMTs) 1, 3a and 3b during the G0/G1 to S phase transition in normal and tumor cells

    Science.gov (United States)

    Robertson, Keith D.; Keyomarsi, Khandan; Gonzales, Felicidad A.; Velicescu, Mihaela; Jones, Peter A.

    2000-01-01

    DNA methylation is essential for mammalian development, X-chromosome inactivation, and imprinting yet aberrant methylation patterns are one of the most common features of transformed cells. One of the proposed causes for these defects in the methylation machinery is overexpression of one or more of the three known catalytically active DNA methyltransferases (DNMTs) 1, 3a and 3b, yet there are clearly examples in which overexpression is minimal or non-existent but global methylation anomalies persist. An alternative mechanism which could give rise to global methylation errors is the improper expression of one or more of the DNMTs during the cell cycle. To begin to study the latter possibility we examined the expression of the mRNAs for DNMT1, 3a and 3b during the cell cycle of normal and transformed cells. We found that DNMT1 and 3b levels were significantly downregulated in G0/G1 while DNMT3a mRNA levels were less sensitive to cell cycle alterations and were maintained at a slightly higher level in tumor lines compared to normal cell strains. Enzymatic activity assays revealed a similar decrease in the overall methylation capacity of the cells during G0/G1 arrest and again revealed that a tumor cell line maintained a higher methylation capacity during arrest than a normal cell strain. These results reveal a new level of control exerted over the cellular DNA methylation machinery, the loss of which provides an alternative mechanism for the genesis of the aberrant methylation patterns observed in tumor cells. PMID:10773079

  20. 阿托伐他汀对脂多糖诱导的人脐静脉内皮细胞肝X受体α及其靶基因表达的影响%Effect of Atorvastatin on mRNA Expression of Liver X Receptor α and Its Target Gene in Human Umbilical Vein Endothelial Cells Induced by Lipopolysacharide

    Institute of Scientific and Technical Information of China (English)

    顾文娟; 罗俊; 李江

    2012-01-01

    Aim To investigate the effect of atorvastatin on mRNA expression of liver X receptor a (LXRa) and its target gene ATP-binding cassette transporter Al (ABCA1), sterol regulatory element binding protein-1 ( SREBP-1) in human umbilical vein endothelial cells (HUVEC) after treated by lipopolysacharide(LPS). Methods Human umbilical vein endothelial cells were cultured in vitro with gibco 1640 medium and 10 percent fetal bovine serum and 1 percent double antibiotics, and then were plated in 6-well plates at a denisity of approximately 2 X 10! cells per milliliter of media to be intervened. (1) Control group: HUVEC were cultured with the completed medium involving phosphate buffered saline (PBS), and LPS treated group or control intervention group: HUVEC were treated with LPS (the terminal concentration was 100 |ig/L) for 24 hours; (2) Atorvastatin intervention group or control intervention group; HUVEC were first treated with atorvastatin at different concentrations (0. 1, 1.0, 10.0 (unol/L) or dimethyl sulfoxide (DMSO) for 2 hours, and then co-treated with LPS for 22 hours. The level of LXRa and its target genes mRNA expression were measured by realtime polymerase chain reaction. Results Compared with control group, LPS could inhibit the mRNA expression of LXRa and its target genes ABCA1, SREBP-1 in HUVEC. The differences were statistically significant (P<0. 05). Compared with LPS plus DMSO group, atorvastatin could upregulate the mRNA expression of LXRa and its target gene mRNA expression in a dose-dependent manner. The differences were statistically significant (P <0. 05). Meanwhile, atorvastatin could downregulate the mRNA expression of inflammatory factor and adhesion factors in a dose-dependent manner.Conclusion These results demonstrate that LPS may inhibit the mRNA expression of LXRa and its target genes in HU-VEC, and atorvastatin can upregulate the mRNA expression of LXRa and its target gene mRNA expression in a dose-dependent manner to inhibit the inflammatory

  1. IMP3 RNP safe houses prevent miRNA-directed HMGA2 mRNA decay in cancer and development

    DEFF Research Database (Denmark)

    Jønson, Lars; Christiansen, Jan; Hansen, Thomas van Overeem;

    2014-01-01

    The IMP3 RNA-binding protein is associated with metastasis and poor outcome in human cancer. Using solid cancer transcriptome data, we found that IMP3 correlates with HMGA2 mRNA expression. Cytoplasmic IMP3 granules contain HMGA2, and IMP3 dose-dependently increases HMGA2 mRNA. HMGA2 is regulated...... that IMP3 RNPs may function as cytoplasmic safe houses and prevent miRNA-directed mRNA decay of oncogenes during tumor progression....

  2. Myc regulates VEGF production in B cells by stimulating initiation of VEGF mRNA translation.

    Science.gov (United States)

    Mezquita, Pau; Parghi, Sean S; Brandvold, Kimberly A; Ruddell, Alanna

    2005-01-27

    Deregulated c-myc gene expression is associated with many human and animal cancers. Myc overexpression promotes the growth of blood and lymphatic vessels, which is due in part to induction of growth factors including vascular endothelial growth factor (VEGF). We determined that the P493-6 human B-cell line increases VEGF production 10-fold upon Myc overexpression. Myc overexpression in avian B cells similarly resulted in high level VEGF production. Real-time RT-PCR analyses showed that Myc did not alter the VEGF mRNA content of these cell lines, indicating that a post-transcriptional mechanism regulates VEGF production. VEGF mRNA translation was examined by RT-PCR analysis of monosome and polysome sucrose gradient fractions from Myc-on and Myc-off P493-6 cells. Myc increased VEGF mRNA translation initiation, as VEGF mRNA loading onto polysomes increased 14-fold in Myc-on cells, and the number of ribosomes loaded per VEGF mRNA increased threefold. This translational regulation is specific to VEGF mRNA, as total polysomes show the same sucrose gradient profile in Myc-on and Myc-off cells, with no change in the percent ribosomes in polysomes, or in the number of ribosomes per polysomal mRNA. Myc stimulates VEGF production by a rapamycin- and LY294002-sensitive pathway, which does not involve alteration of eIF4E activity.

  3. Visual detection of Akt mRNA in living cell using gold nanoparticle beacon

    Science.gov (United States)

    Ma, Yi; Tian, Caiping; Li, Siwen; Wang, Zhaohui; Gu, Yueqing

    2014-09-01

    PI3K-Akt signaling pathway plays the key role in cell apoptosis and survival, and the components of PI3K /Akt signaling pathway are often abnormally expressed in human tumors. Therefore, determination of the Akt (protein kinase B, PKB) messenger ribonucleic acid (mRNA) expression is significantly important in understanding the mechanism of tumor progression. In this study, we designed a special hairpin deoxyribonucleic acid (DNA) functionalized with gold nanoparticles and fluorescein isothiocyanate(FITC) as a beacon for detecting human Akt mRNA. Spectrofluorometer was used to detect the fluorescence quenching and recovery of the beacons, and laser confocal scanning microscopy was adopted to image Akt mRNA in cells. The results showed that this beacon could sensitively and quantitatively measure the Akt mRNA in living cells . This strategy is potentially useful for the cellular imaging of RNA or protein expression in living cells.

  4. Mutation of genes controlling mRNA metabolism and protein synthesis predisposes to neurodevelopmental disorders.

    Science.gov (United States)

    Sartor, Francesca; Anderson, Jihan; McCaig, Colin; Miedzybrodzka, Zosia; Müller, Berndt

    2015-12-01

    Brain development is a tightly controlled process that depends upon differentiation and function of neurons to allow for the formation of functional neural networks. Mutation of genes encoding structural proteins is well recognized as causal for neurodevelopmental disorders (NDDs). Recent studies have shown that aberrant gene expression can also lead to disorders of neural development. Here we summarize recent evidence implicating in the aetiology of NDDs mutation of factors acting at the level of mRNA splicing, mRNA nuclear export, translation and mRNA degradation. This highlights the importance of these fundamental processes for human health and affords new strategies and targets for therapeutic intervention.

  5. Cloning of a human insulin-stimulated protein kinase (ISPK-1) gene and analysis of coding regions and mRNA levels of the ISPK-1 and the protein phosphatase-1 genes in muscle from NIDDM patients

    DEFF Research Database (Denmark)

    Bjørbaek, C; Vik, T A; Echwald, S M

    1995-01-01

    with non-insulin-dependent diabetes mellitus (NIDDM). The human ISPK-1 cDNA was cloned from T-cell leukemia and placental cDNA libraries and mapped to the short arm of the human X chromosome. Single-strand conformation polymorphism (SSCP) analysis identified a total of six variations in the coding regions...... of the PP1 genes: two in PP1 alpha at codons 90 and 255; one in PP1 beta at codon 67; and three in PP1 gamma at codons 11,269, and 273, respectively. All were, however, silent single nucleotide substitutions. SSCP analysis of the ISPK-1 gene identified one silent polymorphism at codon 266 and one amino acid...

  6. Evaluation of p16INK4a/Ki-67 dual stain in comparison with an mRNA human papillomavirus test on liquid-based cytology samples with low-grade squamous intraepithelial lesion

    DEFF Research Database (Denmark)

    Waldstrom, M.; Christensen, R. K.; Ornskov, D.

    2013-01-01

    BACKGROUND: The objective of the current study was to investigate the clinical performance of detecting high-grade lesions with the CINtec PLUS p16INK4a/Ki-67 dual stain and the APTIMA human papillomavirus (HPV) Assay in a cohort of women with low-grade squamous intraepithelial lesion (LSIL...... INK4a/Ki-67 dual-staining test in LSIL cytology samples demonstrated high...

  7. Human CHAC1 Protein Degrades Glutathione, and mRNA Induction Is Regulated by the Transcription Factors ATF4 and ATF3 and a Bipartite ATF/CRE Regulatory Element*

    Science.gov (United States)

    Crawford, Rebecca R.; Prescott, Eugenia T.; Sylvester, Charity F.; Higdon, Ashlee N.; Shan, Jixiu; Kilberg, Michael S.; Mungrue, Imran N.

    2015-01-01

    Using an unbiased systems genetics approach, we previously predicted a role for CHAC1 in the endoplasmic reticulum stress pathway, linked functionally to activating transcription factor 4 (ATF4) following treatment with oxidized phospholipids, a model for atherosclerosis. Mouse and yeast CHAC1 homologs have been shown to degrade glutathione in yeast and a cell-free system. In this report, we further defined the ATF4-CHAC1 interaction by cloning the human CHAC1 promoter upstream of a luciferase reporter system for in vitro assays in HEK293 and U2OS cells. Mutation and deletion analyses defined two major cis DNA elements necessary and sufficient for CHAC1 promoter-driven luciferase transcription under conditions of ER stress or ATF4 coexpression: the −267 ATF/cAMP response element (CRE) site and a novel −248 ATF/CRE modifier (ACM) element. We also examined the ability of the CHAC1 ATF/CRE and ACM sequences to bind ATF4 and ATF3 using immunoblot-EMSA and confirmed ATF4, ATF3, and CCAAT/enhancer-binding protein β binding at the human CHAC1 promoter in the proximity of the ATF/CRE and ACM using ChIP. To further validate the function of CHAC1 in a human cell model, we measured glutathione levels in HEK293 cells with enhanced CHAC1 expression. Overexpression of CHAC1 led to a robust depletion of glutathione, which was alleviated in a CHAC1 catalytic mutant. These results suggest an important role for CHAC1 in oxidative stress and apoptosis with implications for human health and disease. PMID:25931127

  8. Human CHAC1 Protein Degrades Glutathione, and mRNA Induction Is Regulated by the Transcription Factors ATF4 and ATF3 and a Bipartite ATF/CRE Regulatory Element.

    Science.gov (United States)

    Crawford, Rebecca R; Prescott, Eugenia T; Sylvester, Charity F; Higdon, Ashlee N; Shan, Jixiu; Kilberg, Michael S; Mungrue, Imran N

    2015-06-19

    Using an unbiased systems genetics approach, we previously predicted a role for CHAC1 in the endoplasmic reticulum stress pathway, linked functionally to activating transcription factor 4 (ATF4) following treatment with oxidized phospholipids, a model for atherosclerosis. Mouse and yeast CHAC1 homologs have been shown to degrade glutathione in yeast and a cell-free system. In this report, we further defined the ATF4-CHAC1 interaction by cloning the human CHAC1 promoter upstream of a luciferase reporter system for in vitro assays in HEK293 and U2OS cells. Mutation and deletion analyses defined two major cis DNA elements necessary and sufficient for CHAC1 promoter-driven luciferase transcription under conditions of ER stress or ATF4 coexpression: the -267 ATF/cAMP response element (CRE) site and a novel -248 ATF/CRE modifier (ACM) element. We also examined the ability of the CHAC1 ATF/CRE and ACM sequences to bind ATF4 and ATF3 using immunoblot-EMSA and confirmed ATF4, ATF3, and CCAAT/enhancer-binding protein β binding at the human CHAC1 promoter in the proximity of the ATF/CRE and ACM using ChIP. To further validate the function of CHAC1 in a human cell model, we measured glutathione levels in HEK293 cells with enhanced CHAC1 expression. Overexpression of CHAC1 led to a robust depletion of glutathione, which was alleviated in a CHAC1 catalytic mutant. These results suggest an important role for CHAC1 in oxidative stress and apoptosis with implications for human health and disease. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Impact of age, BMI and HbA1c levels on the genome-wide DNA methylation and mRNA expression patterns in human adipose tissue and identification of epigenetic biomarkers in blood

    DEFF Research Database (Denmark)

    Rönn, Tina; Volkov, Petr; Gillberg, Linn

    2015-01-01

    Increased age, BMI and HbA1c levels are risk factors for several non-communicable diseases. However, the impact of these factors on the genome-wide DNA methylation pattern in human adipose tissue remains unknown. We analyzed the DNA methylation of ∼480 000 sites in human adipose tissue from 96 ma...... of candidate genes for obesity, type 2 diabetes and cancer in human adipose tissue. Importantly, we demonstrate that epigenetic biomarkers in blood can mirror age-related epigenetic signatures in target tissues for metabolic diseases such as adipose tissue....... males and 94 females and related methylation to age, BMI and HbA1c. We also compared epigenetic signatures in adipose tissue and blood. Age was significantly associated with both altered DNA methylation and expression of 1050 genes (e.g. FHL2, NOX4 and PLG). Interestingly, many reported epigenetic...... to, for example, RAB37, TICAM1 and HLA-DPB1. Pathway analyses demonstrated that methylation levels associated with age and BMI are overrepresented among genes involved in cancer, type 2 diabetes and cardiovascular disease. Our results highlight the impact of age, BMI and HbA1c on epigenetic variation...

  10. 重组人促红细胞生成素对大鼠视神经挫伤后GAP-43mRNA影响%Recombinant human erythropoietin on GAP-43mRNA of optic nerve after optic nerves crushed in rats

    Institute of Scientific and Technical Information of China (English)

    刘晓坤; 罗钢; 赵平

    2014-01-01

    目的 观察大鼠视神经夹挫伤后视神经生长相关蛋白-43mRNA(growth associated protein-43mRNA,GAP-43mRNA)的变化,观察玻璃体腔内注射重组人促红细胞生成素(recombinant human erythropoietin,rhEPO)对大鼠视神经不完全损伤后GAP-43mRNA的影响.方法 动物实验研究.于2011年5~10月在河北医科大学第三医院实验中心应用建立的外伤性视神经损伤动物模型,进行实验观察.此动物模型分为正常对照组、损伤组(视神经钳夹+生理盐水组)及rhEPO组(视神经钳夹+rhEPO组),于损伤后1、4、7、14和28 d应用反转录-聚合酶链反应(reversetranscription polymerase chain reaction,RT-PCR) 技术观察视神经GAP-43mRNA的变化.结果 RT-PCR结果显示伤后1d损伤组和rhEPO组均无表达;4d损伤组和rhEPO组GAP-43mRNA均表达阳性,组间差异无统计学意义(P>0.05);7、14、28 d rhEPO组GAP-43mRNA表达呈强阳性,损伤组表达呈弱阳性,rhEPO组GAP-43mRNA表达强于损伤组,半定量分析差异有统计学意义(P<0.05).结论 视神经夹挫伤能上调视神经GAP-43mRNA表达,玻璃体腔内注射rhEPO能增强视神经GAP-43 mRNA表达.%Objective To observe the growth associated protein-43 (GAP-43)mRNA expression in optic nerve after optic nerves crushed in rats,and to observe the effect of recombinant human erythropoietin (rhEPO) on the GAP-43mRNA expression.Methods The model of traumatic optic nerve was established,and experiments were conducted to observe during May to October of 2011 at the Third Hospital of Hebei Medical University experiment centre.Sixty-three healthy adult SD rats rule out eye diseases were divided into three groups randomly:control group,crush group and rhEPO group.GAP-43mRNA expressions were detected with reversetranscription polymerase chain reaction (RT-PCR) including 1,4,7,14 and 28th days after crush.Results RT-PCR results showed that there was no GAP-43mRNA expression at the 1st day.At the 4th days,there were no

  11. Impact of age, BMI and HbA1c levels on the genome-wide DNA methylation and mRNA expression patterns in human adipose tissue and identification of epigenetic biomarkers in blood.

    Science.gov (United States)

    Rönn, Tina; Volkov, Petr; Gillberg, Linn; Kokosar, Milana; Perfilyev, Alexander; Jacobsen, Anna Louisa; Jørgensen, Sine W; Brøns, Charlotte; Jansson, Per-Anders; Eriksson, Karl-Fredrik; Pedersen, Oluf; Hansen, Torben; Groop, Leif; Stener-Victorin, Elisabet; Vaag, Allan; Nilsson, Emma; Ling, Charlotte

    2015-07-01

    Increased age, BMI and HbA1c levels are risk factors for several non-communicable diseases. However, the impact of these factors on the genome-wide DNA methylation pattern in human adipose tissue remains unknown. We analyzed the DNA methylation of ∼480 000 sites in human adipose tissue from 96 males and 94 females and related methylation to age, BMI and HbA1c. We also compared epigenetic signatures in adipose tissue and blood. Age was significantly associated with both altered DNA methylation and expression of 1050 genes (e.g. FHL2, NOX4 and PLG). Interestingly, many reported epigenetic biomarkers of aging in blood, including ELOVL2, FHL2, KLF14 and GLRA1, also showed significant correlations between adipose tissue DNA methylation and age in our study. The most significant association between age and adipose tissue DNA methylation was found upstream of ELOVL2. We identified 2825 genes (e.g. FTO, ITIH5, CCL18, MTCH2, IRS1 and SPP1) where both DNA methylation and expression correlated with BMI. Methylation at previously reported HIF3A sites correlated significantly with BMI in females only. HbA1c (range 28-46 mmol/mol) correlated significantly with the methylation of 711 sites, annotated to, for example, RAB37, TICAM1 and HLA-DPB1. Pathway analyses demonstrated that methylation levels associated with age and BMI are overrepresented among genes involved in cancer, type 2 diabetes and cardiovascular disease. Our results highlight the impact of age, BMI and HbA1c on epigenetic variation of candidate genes for obesity, type 2 diabetes and cancer in human adipose tissue. Importantly, we demonstrate that epigenetic biomarkers in blood can mirror age-related epigenetic signatures in target tissues for metabolic diseases such as adipose tissue.

  12. Effects of cyclic-tension strain on the expression of matrix metalloproteinase-8/13 mRNA in human periodontal ligament cells%循环张应力对牙周膜细胞基质金属蛋白酶-8和13表达的影响

    Institute of Scientific and Technical Information of China (English)

    岑玉锋; 李雪; 韩光丽

    2011-01-01

    目的 以同一频率下不同力值、不同时间的循环张应力对人牙周膜细胞(HPDLC)中的基质金属蛋白酶(MMP)一8、13表达的影响,探讨生物力介导下牙周组织细胞外基质代谢调节的分子机制和牙周组织改建的分子生物学基础.方法 对培养在弹性硅胶膜6孔板上的HPDLC施加0.1Hz,硅胶膜形变率分别为6%、12%和18%的周期性循环张应力,分别在加载2、6、12h后检测HPDLC的MMP一8、13的表达.结果 体外培养的HPDLC的生长方向顺应力的方向改变,HPDLC表达MMP一8、13.MMP一8在加载6%、12%形变率循环张应力后,表达量随时间增加而明显增加;加载18%形变率组,各时间段的表达量都增加,但12h较6h表达水平低.MMP-13在6%形变率的循环张应力刺激下,表达量随时间增加而增加;在12%、18%形变率组,各时间段表达量都增加,12h较6h表达水平低,但差异没有统计学意义.结论 循环张应力可影响HPDLC中MMP一8、13的表达水平,MMP一8、13可能参与正畸力下牙周组织的改建.%Objective The purpose of this study was to investigate the mechanism of extracellular matrix (ECM)metabolism regulation and the molecular biological basis of periodontal tissue remodeling, by which MMP-8/13 expression in the condition of cyclic mechanical strain with the same frequency and different strength and time to human periodontal ligament cells (HPDLC). Methods HPDLC were cultured on flexible substrates and subjected to 6%, 12%, 18% elongation by strain unit at 0.1 Hz for 2, 6, 12h, expression and secretion of MMP-8/13 mRNA were determined after loading cyclic tension strain. Results After loading cyclic tension strain, the cell growth direction was changed to force direction, HPDLC expression of MMP-8 and MMP-13. After loading of 6% and 12% cyclic tensile strain, the expression of MMP-8 mRNA was increased significantly and it was raised with time increased, in 18% group with different time, the levels

  13. Human alpha 2-adrenergic receptor subtype distribution: widespread and subtype-selective expression of alpha 2C10, alpha 2C4, and alpha 2C2 mRNA in multiple tissues.

    Science.gov (United States)

    Eason, M G; Liggett, S B

    1993-07-01

    At present, molecular cloning and pharmacological studies have delineated three human alpha 2-adrenergic receptor (alpha 2AR) subtypes, alpha 2C10, alpha 2C4, and alpha 2C2. Assignment of the alpha 2AR subtypes to specific functions has been limited by an unclear definition of tissue alpha 2AR expression outside of the central nervous system. It has been suggested that alpha 2C4 expression is confined to the brain, that alpha 2C2 expression is only in the liver and kidney, and that there is nearly ubiquitous expression of alpha 2C10. However, this is based on studies of a limited number of rat tissues or on studies using non-species-specific approaches. Therefore, to define alpha 2C10, alpha 2C4, and alpha 2C2 tissue expression, we used reverse transcription of total RNA isolated from 20 human tissues, followed by amplification of alpha 2AR cDNA using the polymerase chain reaction. This technique provided two advantages: high sensitivity and, with the use of subtype-specific oligonucleotide primers and probes, differentiation between the alpha 2AR subtypes. The tissues studied were aorta, vena cava, heart (epicardium and endocardium), lung, skeletal muscle, liver, pancreas (head and tail), fat (perinephric and subcutaneous), kidney (cortex and medulla), prostate, stomach, ileum, jejunum, colon, adrenal gland, and spleen. We found that the majority of these tissues expressed alpha 2C10, with the exceptions being the head of the pancreas, subcutaneous fat, colon, and spleen. In marked distinction to other studies, however, we found a prolific expression of the alpha 2C4 and alpha 2C2 subtypes. Expression of alpha 2C4 was found in all tissues with the exception of liver, fat, stomach, and colon, and a virtually ubiquitous expression of alpha 2C2 was found, with the exception of epicardium. Of all tissues studied, only colon and subcutaneous fat expressed a single alpha 2AR subtype, which was alpha 2C2. Thus, the alpha 2AR subtypes do not have a confined expression but

  14. 肥胖及2型糖尿病患者脂肪组织视黄醇结合蛋白4 mRNA的表达及调控%Expression and regulation of retinol binding protein 4 mRNA in human adipose tissue in obese and type 2 diabetics

    Institute of Scientific and Technical Information of China (English)

    刘晓华; 魏丽; 王玲艳; 祝超瑜; 包玉倩; 贾伟平

    2010-01-01

    Objective To study the RBP4 mRNA expression between subcutaneous and visceral adipose tissue in obese and type 2 diabetic patients and to investigate the factors that influence RBP4 mRNA expression in Human visceral adipose tissue. Methods 9 individuals with normal weight normal glucose regulation subjects, 9 obesity subjects and 9 type 2 diabetes subjects were enrolled. All of the subjects were prepared to undergone an operation because of nondiabetes disease. Subcutaneous and visceral adipose tissue were taken out as soon as cultured. RT-PCR and Real-time PCR were used to assay the relative expression of RBP4 mRNA. Results RBP4 mRNA level in visceral adipose tissue of obesity group was (2. 10 ± 1.84),and that of type 2 diabetes group was ( 1.54 ± 0. 46 ), both were significantly higher than that in normal weight normal glucose group ( 0. 75 ± 0. 28, P < 0. 01 , P < 0. 05 ). RBP4 mRNA level in subcutaneous adipose tissue of the three groups were ( 1.05 ±0. 15 vs 0. 99 ±0. 14 vs 1.13 ±0. 07), no difference among them(P > 0. 05 ). Insulin, dexamethasone, pioglitazone, free fatty acids can significantly increase RBP4 mRNA expression, compared with the control group, respectively, have an increase of 2. 13 times, 0. 84times, 2. 04 times, 4. 88 times; however, tumor necrosis factor-αt can significantly lower RBP4 mRNA level, compared with the control group decreased by 38%. Conclusion RBP4 mRNA expression in visceral adipose tissue were significantly higher in obesity and type 2 diabetes subjects. In vitro system, RBP4 gene expression in visceral adipose tissue of normal weight normal glucose subjects was regulated by insulin,dexamethasone, pioglitazone, palmitic acid and TNF-α, such factors were also participated in the pathophysiological process of insulin resistance and type 2 diabetes.%目的 研究肥胖及2型糖尿病患者皮下和腹内脂肪组织视黄醇结合蛋白4(RBP4)mRNA的表达差异,并探讨影响其表达调控的因素.方法

  15. The effect of blue light on L-type calcium channel subunit mRNA expression of human retinal pigment epithelial cells cultured in vitro%蓝光照射对人视网膜色素上皮细胞L-型钙通道mRNA表达的影响

    Institute of Scientific and Technical Information of China (English)

    宫鑫; 蔡善君; 李海辉; 吕建平; 伍志鹏; 宿罡; 谢兵

    2013-01-01

    effect of blue light on mRNA expression of L-type calcium channel subtypes of human retinal pigment epithelial (RPE) cells in vitro.Methods The fourth-generation of human RPE cells were randomly divided into four groups including control group (no light group),light group,light + nifedipine group,and light + (-) BayK8644 group.The cells were exposed to blue light (2000± 500) lux for 6 hours,and then cultured for another 24 hours.Reverse transcription polymerase chain reaction real time (RT-PCR) and fluorescence quantitative PCR technologies were used to analyze mRNA expression of L-type calcium channel subunit of cardiac subtype (1C or CaV1.2),neuroendocrine subtype (1D or CaV1.3) and retinal subtypes (1F or CaV1.4) in each group.Results The length of PCR product of 1C,1D,1F subunit and-actin was 68,157,125 and 186 base pairs respectively.(1) 1C mRNA expression in light,light + nifedipine and light + (-) BayK8644 group was higher than that in control group,the difference was statistically significant (P<0.05).1C mRNA expression in light + nifedipine group and light + (-) BayK8644 group was higher than in light group (P<0.05).1C mRNA expression in light + (-) BayK8644 group was higher than that in light + nifedipine group (P< 0.05).(2)Comparing with control group,1D mRNA expression was higher in light,light +nifedipine and light +(-) BayK8644 group,the difference was statistically significant (P<0.05).Light + (-) BayK8644 group was higher than light group and light + nifedipine group (P<0.05),light group and the light +nifedipine group was not statistically significant (P>0.05).(3) 1F mRNA expression in light,light +nifedipine and light + (-) BayK8644 group was higher than those in control group,there was statistically significant (P<0.05),light +nifedipine group and light + (-) BayK8644 group was higher than light group (P<0.05),light + nifedipine group and the light + (-) BayK8644 group was not statistically significant (P>0.05).Conclusions The human

  16. Circulating hTERT mRNA as a tumor marker in cholangiocarcinoma patients

    Institute of Scientific and Technical Information of China (English)

    Kawin Leelawat; Surang Leelawat; Thawee Ratanachu-Ek; Somboon Trubwongchareon; Jerasak Wannaprasert; Saad Tripongkaruna; Suchart Chantawibul; Panadda Tepaksorn

    2006-01-01

    AIM: To investigate human telomerase reverse transcriptase (hTERT) mRNA in the serum of cholangiocarcinoma patients.METHODS: The serum of thirty three cholangiocarcinoma patients, forty one benign biliary tract disease patients and ten healthy volunteers were collected and analyzed for the expression of hTERT mRNA by real-time reverse transcriptase-polymerase chain reaction (RT-PCR).We then examined the correlation between values of serum hTERT mRNA and the pathological staging of cholangiocarcinoma.RESULTS: hTERT mRNA was detected in 28 of 33(84.85%) of serum obtained from cholangiocarcinoma patients and 9 of 41 (21.9%) of serum obtained from benign biliary tract disease patients. hTERT mRNA was not detected in any serum obtained from healthy volunteers. on the other hand the common tumor marker, CA19-9 was detected in 20 of 33 (60.6%) of serum obtained from cholangiocarcinoma patients and 8 of 41 (19.5%) of serum obtained from benign biliary tract disease patients. However, no correlation was found between the present of serum hTERT mRNA and tumor staging.CONCLUSION: These results indicate that the detection of circulating hTERT mRNA was identified in almost all cholangiocarcinoma patients. It offers anovel tumor marker, which can be used as a complementary study for diagnosis of cholangiocarcinoma.

  17. The expression variations of IL-1 beta and TNF-alpha mRNA in human spermatozoa%细胞因子IL-1β和TNF-α mRNA在人精子表达的变化及意义

    Institute of Scientific and Technical Information of China (English)

    白文俊; 朱积川; 邓庆平; 闫征; 何培英

    2001-01-01

    目的 了解IL-1β和TNF-α在人精子表达的变化及意义。方法 应用逆转录聚合酶链式反应(RT-PCR)检测13例正常生育者、30例不育者及30例慢性前列腺炎患者精子IL-1β和TNF-αmRNA表达。结果 不育者精子中IL-1β和TNF-αmRNA表达均显著低于正常生育者(P=0.018和P=0.046)。慢性前列腺炎患者精子中TNF-αmRNA的表达显著低于正常生育者(P=0.018),而IL-1βmRNA表达与正常生育者相比差别无显著性意义(P=0.42)。IL-1β和TNF-αmRNA在不育者与慢性前列腺炎患者精子中的表达差别无显著性意义(P=0.49和P=0.62)。结论 某些因素可能干扰不育症患者精子IL-1β和TNF-α的合成,导致精液质量下降及不育。生殖道炎症也可能影响精子TNF-α表达,其机制和意义有待进一步研究证实。%Objective To investigate the expression variations of IL-1 betaand TNF-alpha in human spermatozoa.Methods IL-1β and TNF-α mRNA expressions were studied by means of reverse transcription polymerase reaction (RT-PCR)in 13 fertile men,in 30 infertiles and in 30 chronic prostatitis patients.Results The expressions of IL-1β and TNF-α mRNA in infertile men were significantly lower than those in fertile men(P=0.018 and P=0.050),and the expressions TNF-α mRNA in chronic prostatitis patients were significantly lower than those in fertile men(P=0.018 ),but the expressions of IL-1β in these patients were not significantly lower than in fertile men(P=0.420). The expression differences of IL-1β and TNF-α mRNA between the infertile men and chronic prostatitis patients were not significantly(P=0.488 and P=0.616).Conclusions Some unknown factors might impair the production of IL-1β and TNF-α in infertile spermatozoa, and that may be responsible for the poor quality of semen and the infertile status in these patients. Furthermore, urogenital inflammation could also disturb the expression of TNF-α in human spermatozoa.

  18. Therapeutic efficacy in a hemophilia B model using a biosynthetic mRNA liver depot system

    Science.gov (United States)

    DeRosa, F; Guild, B; Karve, S; Smith, L; Love, K; Dorkin, J R; Kauffman, K J; Zhang, J; Yahalom, B; Anderson, D G; Heartlein, M W

    2016-01-01

    DNA-based gene therapy has considerable therapeutic potential, but the challenges associated with delivery continue to limit progress. Messenger RNA (mRNA) has the potential to provide for transient production of therapeutic proteins, without the need for nuclear delivery and without the risk of insertional mutagenesis. Here we describe the sustained delivery of therapeutic proteins in vivo in both rodents and non-human primates via nanoparticle-formulated mRNA. Nanoparticles formulated with lipids and lipid-like materials were developed for delivery of two separate mRNA transcripts encoding either human erythropoietin (hEPO) or factor IX (hFIX) protein. Dose-dependent protein production was observed for each mRNA construct. Upon delivery of hEPO mRNA in mice, serum EPO protein levels reached several orders of magnitude (>125 000-fold) over normal physiological values. Further, an increase in hematocrit (Hct) was established, demonstrating that the exogenous mRNA-derived protein maintained normal activity. The capacity of producing EPO in non-human primates via delivery of formulated mRNA was also demonstrated as elevated EPO protein levels were observed over a 72-h time course. Exemplifying the possible broad utility of mRNA drugs, therapeutically relevant amounts of human FIX (hFIX) protein were achieved upon a single intravenous dose of hFIX mRNA-loaded lipid nanoparticles in mice. In addition, therapeutic value was established within a hemophilia B (FIX knockout (KO)) mouse model by demonstrating a marked reduction in Hct loss following injury (incision) to FIX KO mice. PMID:27356951

  19. Cytokine mRNA quantification by real-time PCR.

    Science.gov (United States)

    Stordeur, Patrick; Poulin, Lionel F; Craciun, Ligia; Zhou, Ling; Schandené, Liliane; de Lavareille, Aurore; Goriely, Stanislas; Goldman, Michel

    2002-01-01

    Real-time PCR represents a new methodology that accurately quantifies nucleic acids. This has been made possible by the use of fluorogenic probes, which are presented in two forms, namely hydrolysis probes (also called TaqMan probes) and hybridisation probes. We decided to apply this methodology to cytokine mRNA quantification and this led us to the development of a protocol that provides an easy way to develop and perform rapidly real-time PCR on a Lightcycler instrument. It was made possible by the use of freely available software that permits a choice of both the hydrolysis probe and the primers. We firstly demonstrated that the reproducibility of the method using hydrolysis probes compares favourably with that obtained with hybridisation probes. We then applied this technique to determine the kinetics of IL-1ra, IL-1beta, IL-5, IL-13, TNF-alpha and IFN-gamma induction upon stimulation of human peripheral blood mononuclear cells (PBMC) by phytohaemagglutinin (PHA). Finally, the method was also used successfully to demonstrate that IFN-alpha induces IL-10 mRNA accumulation in human monocytes.

  20. Stable low-level expression of p21WAF1/CIP1 in A549 human bronchogenic carcinoma cell line-derived clones down-regulates E2F1 mRNA and restores cell proliferation control

    Directory of Open Access Journals (Sweden)

    Crawford Erin L

    2006-01-01

    Full Text Available Abstract Background Deregulated cell cycle progression and loss of proliferation control are key properties of malignant cells. In previous studies, an interactive transcript abundance index (ITAI comprising three cell cycle control genes, [MYC × E2F1]/p21 accurately distinguished normal from malignant bronchial epithelial cells (BEC, using a cut-off threshold of 7,000. This cut-off is represented by a line with a slope of 7,000 on a bivariate plot of p21 versus [MYC × E2F1], with malignant BEC above the line and normal BEC below the line. This study was an effort to better quantify, at the transcript abundance level, the difference between normal and malignant BEC. The hypothesis was tested that experimental elevation of p21 in a malignant BEC line would decrease the value of the [MYC × E2F1]/p21 ITAI to a level below this line, resulting in loss of immortality and limited cell population doubling capacity. In order to test the hypothesis, a p21 expression vector was transfected into the A549 human bronchogenic carcinoma cell line, which has low constitutive p21 TA expression relative to normal BEC. Results Following transfection of p21, four A549/p21 clones with stable two-fold up-regulated p21 expression were isolated and expanded. For each clone, the increase in p21 transcript abundance (TA was associated with increased total p21 protein level, more than 5-fold reduction in E2F1 TA, and 10-fold reduction in the [MYC × E2F1]/p21 ITAI to a value below the cut-off threshold. These changes in regulation of cell cycle control genes were associated with restoration of cell proliferation control. Specifically, each transfectant was capable of only 15 population doublings compared with unlimited population doublings for parental A549. This change was associated with an approximate 2-fold increase in population doubling time to 38.4 hours (from 22.3 hrs, resumption of contact-inhibition, and reduced dividing cell fraction as measured by flow

  1. Investigation of HSP60 gene expression in mRNA level in heart at dilated cardiomyopathy

    Directory of Open Access Journals (Sweden)

    Riabenko D. V.

    2009-02-01

    Full Text Available The expression of HSP60 in the mRNA level in human hearts at the end-stage of dilated cardiomyopathy (DCM as well as in the hearts of mice with disease model similar to human DCM was investigated. We observed a significant increase in the Hsp60 mRNA level at the beginning of the disease and decrease to a normal level at the end stage. As the Hsp60 level was increased during the disease up to the end stage we can presume some changes in the regulation of Hsp60 synthesis or its degradation at DCM progression

  2. Increased IL-10 mRNA and IL-23 mRNA expression in multiple sclerosis

    DEFF Research Database (Denmark)

    Krakauer, Martin; Sorensen, P; Khademi, M

    2008-01-01

    of the regulatory cytokine IL-10. The elevated IL-23 mRNA levels in MS patients are noteworthy in view of the newly discovered IL-23-driven Th17 T-cell subset, which is crucial in animal models of MS. Since IFN-beta therapy resulted in decreased IL-23 mRNA levels, the Th17 axis could be another target of IFN...

  3. Acute digoxin loading reduces ABCA8A mRNA expression in the mouse liver.

    Science.gov (United States)

    Wakaumi, Michi; Ishibashi, Kenichi; Ando, Hitoshi; Kasanuki, Hiroshi; Tsuruoka, Shuichi

    2005-12-01

    Human ABCA8, a new member of the ATP binding cassette (ABC) transporter family, transports certain lipophilic drugs, such as digoxin. To investigate the roles of this transporter, we cloned a mouse homologue of ABCA8, from a mouse heart cDNA library, named ABCA8a. The deduced mouse ABCA8a protein is 66% identical with that of human ABCA8 and possesses features common to the ABC superfamily. It was found that ABCA8a was mainly expressed in the liver and heart, similar to human ABCA8. We further evaluated the effect of acute digoxin (a substrate for ABCA8) intoxication on the mRNA expression of ABCA8 using northern blotting with a 3' non-coding region as a probe to avoid cross-hybridization with other ABCA genes. Following acute digoxin infusion, the mRNA expression of ABCA8 was significantly reduced in the liver 12-24 h after injection (14.7% of vehicle treatment), but not in the heart and kidney. Real-time quantitative polymerase chain reaction analysis confirmed the reduction in ABCA8a mRNA. Similar reductions in ABCA5, ABCA7, ABCA8b and ABCA9 mRNA were also observed. A comparable amount of digitoxin did not affect ABCA8a mRNA expression in the liver. The results suggest that ABCA8 may play a role in digoxin metabolism in the liver.

  4. GLUT3 protein and mRNA in autopsy muscle specimens

    Science.gov (United States)

    Stuart, C. A.; Wen, G.; Jiang, J.

    1999-01-01

    GLUT3 is expressed in rat muscle, but this glucose transporter protein has not been identified previously in adult human skeletal muscle. We quantified the rapidity of disappearance of mRNA and protein from human skeletal muscle at room temperature and at 4 degrees C. Fifty percent of the immunologically detectable GLUT3 protein disappeared by 1 hour at 20 degrees C and by 2 hours at 4 degrees C. mRNA for GLUT3 was decreased 50% by 2.2 hours at 20 degrees C and by 24 hours at 4 degrees C. Half of the measurable mRNAs for GLUT4, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), alpha-actin, and beta-myosin disappeared by 0.8 to 2.1 hours at 20 degrees C and by 5.0 to 16.6 hours at 4 degrees C. Previous conclusions that GLUT3 is not expressed in human muscle were likely drawn because of artifacts related to degradation of GLUT3 protein in the specimens prior to study. Because of the rapid degradation of protein and mRNA, autopsy specimens of muscle must be obtained within 6 hours of death, and even then, protein and mRNA data will likely dramatically underestimate their expression in fresh muscle. Some previously published conclusions and recommendations regarding autopsy specimens are not stringent enough to consistently yield useful protein and mRNA.

  5. Correlation between indoleamine 2,3 dioxygenase mRNA and CDKN2A/p16 mRNA: a combined strategy to cervical cancer diagnosis.

    Science.gov (United States)

    Saffi Junior, Mario Cezar; Duarte, Ivone da Silva; Brito, Rodrigo Barbosa de Oliveira; Prado, Giovana Garcia; Makabe, Sergio; Dellê, Humberto; Camacho, Cleber P

    2016-11-01

    Cervical cancer (CC) is one of the most common cancers among women worldwide. The relation of the human papillomavirus (HPV) with CC and its precursor lesions was first suspected for over 40 years. The indoleamine 2,3 dioxygenase (IDO) is an immune modulator enzyme responsible for the immune system tissue protection mechanism, which may be the key to the tumoural persistence. HPV oncoprotein E7 promotes the increase in cyclin-dependent kinase inhibitor p16 (CDKN2A/p16). The isolated and combined analysis of CDKN2A/p16 mRNA to CC diagnosis was done with promising results. The aim of this study is to evaluate the correlation between IDO mRNA and CDKN2A/p16 mRNA. We will explore the potential of both as diagnostic tools. RNA was extracted from tissue samples. cDNA was generated with High Capacity RNA-to-cDNA kit. The real-time PCR results were analysed using nonlinear curve estimation, ROC curve, Chi-squared test, the proportion of variance explained and Galen and Gambino formulas. From 270 patients attended, colposcopy examination was performed in 110 and the biopsy in 75 patients. We found a positive correlation in patients older than 28 years old with low-risk lesions, but the correlation is lost in high-risk lesions. Although cytology, IDO mRNA and CDKN2A/p16 mRNA could not differentiate the risk groups, IDO combined with CDKN2A/p16 mRNA results could (p = 0.028). The best diagnostic result was achieved by IDO coupled with CDKN2A/p16 mRNA, which may considerably increase the sensitivity of screening for CC.

  6. Association of the 5-HTT gene-linked promoter region (5-HTTLPR polymorphism with psychiatric disorders: review of psychopathology and pharmacotherapy

    Directory of Open Access Journals (Sweden)

    Kenna GA

    2012-01-01

    Full Text Available George A Kenna1, Nick Roder-Hanna2, Lorenzo Leggio3, William H Zywiak4, James Clifford5, Steven Edwards3, John A Kenna6, Jessica Shoaff1, Robert M Swift11Center for Alcohol and Addiction Studies, Department of Psychiatry and Human Behavior, Brown University, Providence; 2College of Pharmacy, University of Rhode Island, Kingston; 3Center for Alcohol and Addiction Studies, Department of Community Health, Brown University, Providence; 4Butler Hospital, Providence, RI; 5Virginia Institute for Psychiatric and Behavior Genetics, Virginia Commonwealth University, Richmond, VA; 6College of Nursing, University of Rhode Island, Kingston, RI, USAAbstract: Serotonin (5-HT regulates important biological and psychological processes including mood, and may be associated with the development of several psychiatric disorders. An association between psychopathology and genes that regulate 5-HT neurotransmission is a robust area of research. Identification of the genes responsible for the predisposition, development, and pharmacological response of various psychiatric disorders is crucial to the advancement of our understanding of their underlying neurobiology. This review highlights research investigating 5-HT transporter (5-HTTLPR polymorphism, because studies investigating the impact of the 5-HTTLPR polymorphism have demonstrated significant associations with many psychiatric disorders. Decreased transcriptional activity of the S allele (“risk allele” may be associated with a heightened amygdala response leading to anxiety-related personality traits, major depressive disorder, suicide attempts, and bipolar disorder. By contrast, increased transcriptional activity of the L allele is considered protective for depression but is also associated with completed suicide, nicotine dependence, and attention deficit hyperactivity disorder. For some disorders, such as post-traumatic stress disorder and major depressive disorder, the research suggests that treatment

  7. Prognostic relevance of circulating CK19 mRNA in advanced malignant biliary tract diseases

    Institute of Scientific and Technical Information of China (English)

    Kawin Leelawat; Siriluck Narong; Wandee Udomchaiprasertkul; Jerasak Wannaprasert; Sa-ard Treepongkaruna; Somboon Subwongcharoen; Tawee Ratanashu-ek

    2012-01-01

    AIM: To determine the role of circulating tumor cells (CTCs) in prediction of the overall survival of patients with advanced malignant biliary tract obstruction. METHODS: We investigated the prognostic value of CTCs by examining two markers, cytokeratin (CK) 19 and human telomerase reverse transcriptase (hTERT) mRNA, in 40 patients diagnosed with advanced malignant biliary tract diseases. Quantitative real-time reverse transcription polymerase chain reaction was used to detect CK19 and hTERT mRNA in the peripheral blood of these patients. Overall survival was analyzed using the Kaplan-Meier method and Cox regression modeling. RESULTS: Positive CK19 and hTERT mRNA expression was detected in 45% and 60%, respectively, of the 40 patients. Univariable analysis indicated that positive CK19 mRNA expression was significantly associated with worse overall survival (P = 0.009). Multivariable analysis determined that positive CK19 mRNA expression, patient's age and serum bilirubin were each independently associated with overall survival. CONCLUSION: CK19 mRNA expression levels in peripheral blood appear to provide a valuable marker to predict the overall survival of patients with advanced malignant biliary tract obstruction.

  8. The value of 5-HTT gene polymorphism for the assessment and prediction of male adolescence violence%5-羟色胺转运体基因多态性与青少年暴力犯罪行为的相关性研究

    Institute of Scientific and Technical Information of China (English)

    于跃; 刘祥; 杨振兴; 邱昌建; 马小红

    2012-01-01

    Objective To establish an adolescent violence crime prediction model,and to assess the value of serotonin transporter (5-HTT)gene polymorphism for the assessment and prediction of violent crime.Methods Investigative tools were used to analyze the difference in personality dimensions,social support,coping styles,aggressiveness,impulsivity,and family condition scale between 223 adolescents with violence behavior and 148 adolescents without violence behavior. The distribution of 5-HTT gene polymorphisms (5-HTTLPR and 5-HTTVNTR) was compared between the two groups.The role of 5-HTT gene polymorphism on adolescent personality,impulsion and aggression scale also was also analyzed.Stepwise logistic regression was used to establish a predictive model for adolescent violent crime.Results Significant difference was found between the violence group and the control group on multiple dimensions of psychology and environment scales.However,no statistical difference was found with regard to the 5-HTT genotypes and alleles between adolescents with violent behaviors and normal controls.The rate of prediction accuracy was not significantly improved when 5-HTT gene polymorphism was taken into the model.Conclusion The violent crime of adolescents was closely related with social and environmental factors.No association was found between 5-HTT polymorphisms and adolescent violence criminal behavior.%目的 通过建立青少年暴力犯罪模型,探讨5-羟色胺转运体(serotonin transporter,5-HTT)基因多态性在暴力犯罪评估预测中的作用.方法 使用调查量表分析223例暴力组和148名对照组青少年在人格、社会支持、应付方式、冲动性、攻击性和家庭情况等方面的差异.分别比较暴力组和正常对照组5-HTT基因多态位点5-HTTVNTR、5-HTTLPR基因型与等位基因的分布,以观察5-HTT基因多态性与青少年人格、冲动和攻击等分量表的关系.采用逐步Logistic回归建立青少年

  9. Methylation Inactivation Mechanism of Parkin Gene mRNA Expression in Nasopharyngeal Carcinoma

    Institute of Scientific and Technical Information of China (English)

    Ni Haifeng; Jiang Bo; Zhou Zhen; Li Yong; Huang Guangwu

    2014-01-01

    Objective:To investigate the methylation inactivation and the clinical signiifcance of Parkin gene mRNA expression in nasopharyngeal carcinoma (NPC). Methods: The methylation-speciifc PCR (MSP) and semi-quantitative reverse transcription PCR (RT-PCR) were used to detect methylation and the mRNA expression level of Parkin gene in 54 cases of NPC tissues and 16 cases of normal nasopharyngeal epithelial (NNE) tissues.The mRNA expression of Parkin gene in two NPC cell lines (CNE1 and CNE2) were detected before and after treatment with the methyltransferase inhibitor 5-aza-2-deoxycytidine so as to analyze the effects of Parkin gene methylation and demethylation on Parkin gene mRNA expression and the relationship between Parkin gene mRNA expression and clinical factors. Results: The methylation frequency of Parkin gene in human NPC tissues was 62.96% (34/54), but didn’t happen in any of 16 cases of NNE tissues. The mRNA expression level was (0.3430±0.4947) in 54 cases of NPC tissues and (1.0052±0.4911) in NNE tissues, showing that the mRNA expression level of NPC tissues was significantly down-regulated (P0.05), but was closely related to lymph node metastasis (P<0.05). Conclusion:Parkin gene mRNA expression, serving as a cancer suppressor gene in the occurrence and development of NPC, is inactivated and regulated by methylation, which also has a negative correlation with lymph node metastasis and could be considered as the judgment of predictive index of clinical prognosis of NPC.

  10. mRNA levels of TLR4 and TLR5 are independent of H pylori

    Institute of Scientific and Technical Information of China (English)

    Elvira Garza-González; Virgilio Bocanegra-García; Francisco Javier Bosques-Padilla; Juan Pablo Flores-Gutiérrez; Francisco Moreno; Guillermo Ignacio Perez-Perez

    2008-01-01

    AIM:To determine if the presence Hpylori or its virulence affect toll-like receptor 4 (TLR4) and TLR5 mRNA expression levels.METHODS:For the in vivo assays,gastric biopsies were obtained from 40 patients and H pylori status was determined.For the in vitro assays,human gastric adenocarcinoma mucosal cells (AGS) were cultured in the presence or absence of twelve selected H pylori strains.H pylori strains isolated from culture-positive patients and selected strains were genotyped for cagA and vacA.The cDNA was obtained from mRNA extracted from biopsies and from infected AGS cells.TLR4 and TLR5 mRNA levels were examined by real-time PCR.RESULTS:The presence of Hpylori did not affect the mRNA levels of TLR4 or TLR5 in gastric biopsies.The mRNA levels of both receptors were not influenced by the vacA status (P>0.05 for both receptors) and there were no differences in TLR4 or TLR5 mRNA levels among the different clinical presentations/histological findings (P>0.05).In the in vitro assay,the mRNA levels of TLR4 or TLR5 in AGS cells were not influenced by the vacAsl status or the clinical condition associated with the strains (P>0.05 for both TLR4 and TLR5).CONCLUSION:The results of this study show that the mRNA levels of TLR4 and TLR5 in gastric cells,both in vivo and in vitro,are independent of H pylori colonization and suggest that vacA may not be a significant player in the first step of innate immune recognition mediated by TLR4 or TLR5.

  11. Packaging of the recombinant lentivirus delivering microRNA-383sequences and its positive effects on Foxp3 mRNA in human PBMCs%携带microRNA-383重组慢病毒的包装及其上调PBMCs中Foxp3mRNA的表达

    Institute of Scientific and Technical Information of China (English)

    周峰; 张庆; 张海清; 刘晓梅; 史震; 郑葵阳

    2011-01-01

    目的 包装携带microRNA-383(miR-383)的重组慢病毒,并观察其对正常人外周血单个核细胞(peripheral blood mononuclear ce11s,PBMCs)中Foxp3 mRNA转录的影响.方法 将4个串联的miRNA-383前体序列亚克隆人慢病毒转移载体pHAGE-CMV-MCS-IZsGreen,构建重组载体,命名为pHAGE-miR-383.将pHAGE-383、包装质粒psPAX2与包膜质粒pMD2.G共转染入HEK293T细胞进行重组慢病毒包装.进而分别采用定量PCR和RT-PCR检测慢病毒miR-383和Mock感染48 h后细胞中的miR-383的表达和转录因子Foxp3 mRNA的转录水平.结果 定量PCR证实携带miR-383的重组慢病毒包装成功,滴度为1×10(7)TU/ml.以MOI为5的慢病毒感染HEK293T细胞后miR-383表达明显增多.RT-PCR结果显示,miR-383能够上调PBMCs中Foxp3 mRNA的表达.结论 成功包装出含有miR-383前体序列的重组慢病毒,而且慢病毒miR-383能够上调PBMCs中Foxp3 mRNA的转录水平,为进一步研究其对调节性T细胞的调控作用奠定基础.%MicroRNAs (miRNAs)-mediated RNA interference is a vital regulatory pathway for many biological processes, such as development, differentiation, and homeostasis. As potent immune regulators, regulatory T cells (Tregs) are strictly regulated by Foxp3, a transcription factor. To produce the recombinant lentivirus containing pre-microRNA-383 (miR-383) sequences and to determinate the effect miR-383 on F0XP3 mRNA level in human peripheral blood mononuclear cells (PBMCs), four pre-miR-383 sequences in series were subcloned into the lentiviral transfected plasmid pHAGE-CMV-MCS-IZsGreen (named as pHAGE-miR-383). And then the pHAGE-miR-383, packaged plasmid psPAX2, and envelope plasmid pMD2.G were co-transfected into the HEK 293T cells with lipofectamine reagent. The viral titer was checked by observing the expression of green fluorescent protein (GFP). Moreover, real-time PCR assay was performed to measure miR-383 level and RT-PCR to test Foxp3 mRNA level in cells infected by lentiviral mi

  12. Use of human stem cells in Huntington disease modeling and translational research.

    Science.gov (United States)

    Golas, Monika M; Sander, Bjoern

    2016-04-01

    Huntington disease (HD) is a devastating neurological disorder caused by an extended CAG repeat in exon 1 of the gene that encodes the huntingtin (HTT) protein. HD pathology involves a loss of striatal medium spiny neurons (MSNs) and progressive neurodegeneration affects the striatum and other brain regions. Because HTT is involved in multiple cellular processes, the molecular mechanisms of HD pathogenesis should be investigated on multiple levels. On the cellular level, in vitro stem cell models, such as induced pluripotent stem cells (iPSCs) derived from HD patients and HD embryonic stem cells (ESCs), have yielded progress. Approaches to differentiate functional MSNs from ESCs, iPSCs, and neural stem/progenitor cells (NSCs/NPCs) have been established, enabling MSN differentiation to be studied and disease phenotypes to be recapitulated. Isolation of target stem cells and precursor cells may also provide a resource for grafting. In animal models, transplantation of striatal precursors differentiated in vitro to the striatum has been reported to improve disease phenotype. Initial clinical trials examining intrastriatal transplantation of fetal neural tissue suggest a more favorable clinical course in a subset of HD patients, though shortcomings persist. Here, we review recent advances in the development of cellular HD models and approaches aimed at cell regeneration with human stem cells. We also describe how genome editing tools could be used to correct the HTT mutation in patient-specific stem cells. Finally, we discuss the potential and the remaining challenges of stem cell-based approaches in HD research and therapy development.

  13. (Online & Print): 2305-7432. htt

    African Journals Online (AJOL)

    Peter Berglez

    Factors affecting the growth of the Malawian film industry. Mufunanji Magalasi .... impact on Ghanaians reports on the role played by the British colonialists not only in .... Mpakati in the lobby of the Sheraton Hotel in Harare. Then the ..... Malawian vulnerable girl child, in the hands of the middle class, places the inspiration.

  14. 14-3-3 protein binds to the low molecular weight neurofilament (NFL) mRNA 3' UTR.

    Science.gov (United States)

    Ge, Wei-Wen; Volkening, Kathryn; Leystra-Lantz, Cheryl; Jaffe, Howard; Strong, Michael J

    2007-01-01

    We have previously reported that altered stability of low molecular weight neurofilament (NFL) mRNA in lumbar spinal cord homogenates in amyotrophic lateral sclerosis (ALS) is associated with altered expression of trans-acting 3' UTR mRNA binding proteins. We have identified two hexanucleotide motifs as the main cis elements and, using LC/MS/MS of peptide digests of NFL 3' UTR interacting proteins from human spinal cord, observed that 14-3-3 proteins interact with these motifs. 14-3-3 beta, zeta, tau, gamma, and eta isoforms were found to be expressed in human spinal cord. Each isoform was expressed in vitro and shown to interact with NFL 3' UTR mRNA. Mutation of one or both motifs resulted in decreased 14-3-3 interaction, changes in predicted mRNA structure or alteration in stability of the mRNA. These data show a novel interaction for 14-3-3 with NFL mRNA, and suggests that 14-3-3 may play a role in regulating NFL mRNA stability.

  15. Messenger RNA (mRNA) nanoparticle tumour vaccination

    Science.gov (United States)

    Phua, Kyle K. L.; Nair, Smita K.; Leong, Kam W.

    2014-06-01

    Use of mRNA-based vaccines for tumour immunotherapy has gained increasing attention in recent years. A growing number of studies applying nanomedicine concepts to mRNA tumour vaccination show that the mRNA delivered in nanoparticle format can generate a more robust immune response. Advances in the past decade have deepened our understanding of gene delivery barriers, mRNA's biological stability and immunological properties, and support the notion for engineering innovations tailored towards a more efficient mRNA nanoparticle vaccine delivery system. In this review we will first examine the suitability of mRNA for engineering manipulations, followed by discussion of a model framework that highlights the barriers to a robust anti-tumour immunity mediated by mRNA encapsulated in nanoparticles. Finally, by consolidating existing literature on mRNA nanoparticle tumour vaccination within the context of this framework, we aim to identify bottlenecks that can be addressed by future nanoengineering research.

  16. mRNA fragments in in vitro culture media are associated with bovine preimplantation embryonic development.

    Science.gov (United States)

    Kropp, Jenna; Khatib, Hasan

    2015-01-01

    In vitro production (IVP) systems have been used to bypass problems of fertilization and early embryonic development. However, embryos produced by IVP are commonly selected for implantation based on morphological assessment, which is not a strong indicator of establishment and maintenance of pregnancy. Thus, there is a need to identify additional indicators of embryonic developmental potential. Previous studies have identified microRNA expression in in vitro culture media to be indicative of embryo quality in both bovine and human embryos. Like microRNAs, mRNAs have been shown to be secreted from cells into the extracellular environment, but it is unknown whether or not these RNAs are secreted by embryos. Thus, the objective of the present study was to determine whether mRNAs are secreted into in vitro culture media and if their expression in the media is indicative of embryo quality. In vitro culture medium was generated and collected from both blastocyst and degenerate (those which fail to develop from the morula to blastocyst stage) embryos. Small-RNA sequencing revealed that many mRNA fragments were present in the culture media. A total of 17 mRNA fragments were differentially expressed between blastocyst and degenerate conditioned media. Differential expression was confirmed by quantitative real-time PCR for fragments of mRNA POSTN and VSNL-1, in four additional biological replicates of media. To better understand the mechanisms of mRNA secretion into the media, the expression of a predicted RNA binding protein of POSTN, PUM2, was knocked down using an antisense oligonucleotide gapmer. Supplementation of a PUM2 gapmer significantly reduced blastocyst development and decreased secretion of POSTN mRNA into the media. Overall, differential mRNA expression in the media was repeatable and sets the framework for future study of mRNA biomarkers in in vitro culture media to improve predictability of reproductive performance.

  17. Expression of SART-1 mRNA in canine squamous cell carcinomas.

    Science.gov (United States)

    Takaishi, Yumi; Yoshida, Yukari; Nakagaki, Kazuhide; Fujita, Michio; Taniguchi, Akiko; Orima, Hiromitsu

    2008-12-01

    SART-1, a squamous cell carcinoma (SCC) antigen recognized by cytotoxic T lymphocytes, has been useful in human cancer therapy. The SART-1(259) peptide is a potential candidate for vaccine. The present study examined an orthologue of the mRNA coding this peptide in canine SCCs. Specimens were obtained from seven canine patients with SCC, and the mRNA was isolated from the samples. The SART-1 and beta-actin genes were amplified by reverse-transcription polymerase chain reaction, using the isolated mRNA as a template. Canine SART-1 was amplified in six of the seven specimens, while beta-actin was detected in all the samples. In dogs, carcinomas expressing SART-1 could be a target for cytotoxic T lymphocyte mediated immunotherapy.

  18. A chicken mRNA similar to heterogeneous nuclear ribonucleoprotein H1.

    Science.gov (United States)

    Mozdziak, Paul E; Giamario, Carol; Dibner, Julia J; McCoy, Darell W

    2004-01-01

    Heterogeneous nuclear ribonucleoproteins are predominantly nuclear RNA-binding proteins that function in a variety of cellular activities. The objective of these experiments was to clone a cDNA for a chicken protein similar to other previously reported heterogeneous ribonucleoproteins for other species. The 5' and 3' ends of the chicken mRNA were cloned using Rapid Amplification of cDNA Ends (RACE). Subsequently, the expression of the mRNA sequence was confirmed via Northern analysis. The deduced amino acid sequence was approximately 86% identical to corresponding regions of human, mouse, or zebrafish proteins similar to heterogeneous nuclear ribonucleoprotein H1. The expression data confirmed the size of the predicted mRNA sequence. The newly identified sequence may be employed in future studies aimed at understanding the role of heterogeneous nuclear ribonucleoproteins in avian species.

  19. Interventional effect of triiodothyronineon thyroid hormone receptor mRNA expression during the differentiation of human embryonic brain-derived neural stem cells%三碘甲状腺原氨酸对人神经干细胞分化过程中甲状腺激素受体表达的干预效应

    Institute of Scientific and Technical Information of China (English)

    刘春蓉; 李兰英; 刘奔; 臧晓怡; 陈祖培

    2007-01-01

    甲状腺原氨酸诱导后表达量逐渐下降,至2周时达最低,此后表达量有所回升,但仍低于神经干细胞状态(F=32.49,P=0.008).甲状腺激素受体α2mRNA表达变化趋势与甲状腺激素受体α1相同.甲状腺激素受体β1mRNA在神经干细胞状态时表达量最低,三碘甲状腺原氨酸诱导后表达量逐渐升高,2周时达最高,且超过同时间点甲状腺激素受体α1的表达(t=15.64,P=0.001),至诱导3周时表达水平降至最低.甲状腺激素受体α3mRNA在三碘甲状腺原氨酸诱导后呈下降趋势,2周时接近干细胞状态(F=51.94,P=0.378),此后又降至较低水平.结论:三碘甲状腺原氨酸能诱导神经干细胞分化为神经元、少突胶质细胞和星形胶质细胞,且分化过程中甲状腺激素受体mRNA存在不同时间顺序的表达.%BACKGROUND: Triiodothyronine (T3) is an important regulation factor at the critical period of brain development. It maybe control the successive differentiation during the development of central nervous system (CNS).OBJECTIVE: To monitor the differentiation of neural stem cells (NSCs) induced by T3 and the thyroid hormone receptor (TR) mRNA expression changes.DESIGN: Open experiment.SETTING: Department of Pathology, Tianjin Medical College of Chinese People's Armed Police Force; Institute of Endocrinology of Tianjin Medical University.MATERIALS: This study was carried out in the Tianjin Medical University between January 2003 and March 2005.Ten-to-twelve-week-old aborted fetuses were obtained from the General Hospital of Tianjin Medical University with the approval of the local ethical committee. Informed consents were obtained from the mothers and their relatives.METHODS: ①Under the aseptic condition, the bilateral cortex of human fetal brain was removed and dissociated by brief mechanical trituration in D-Hanks. Then, 20 μg/L bFGF and 30 nmol/L T3 were used to induce the proliferation of NSCs and inoculated to poly-L-lysine-coated 24-well plate

  20. Alternative polyadenylation of mRNA precursors

    Science.gov (United States)

    Tian, Bin; Manley, James L.

    2017-01-01

    Alternative polyadenylation (APA) is an RNA-processing mechanism that generates distinct 3′ termini on mRNAs and other RNA polymerase II transcripts. It is widespread across all eukaryotic species and is recognized as a major mechanism of gene regulation. APA exhibits tissue specificity and is important for cell proliferation and differentiation. In this Review, we discuss the roles of APA in diverse cellular processes, including mRNA metabolism, protein diversification and protein localization, and more generally in gene regulation. We also discuss the molecular mechanisms underlying APA, such as variation in the concentration of core processing factors and RNA-binding proteins, as well as transcription-based regulation. PMID:27677860

  1. Recombinant human erythropoietin promotes expression of HO-1 mRNA after renal ischemia reperfusion injury in mice%重组人促红细胞生成素促进小鼠缺血再灌注损伤肾脏HO-1表达

    Institute of Scientific and Technical Information of China (English)

    张顺; 李炎; 张明; 张建军

    2011-01-01

    Objective To investigate the effects of recombinant human erythropoietin (rhEPO) on the expression of heme oxygenase-l(HO-1) mRNA after renal ischemia/reperfusion (IR) in mice. Methods Ninety male C57BL/6 mice were randomly divided into three groups, namely, the sham operation group (n= 30), renal IR group (n = 30), and rhEPO treatment group (n=30). Mice were sacrificed at 1, 2, 3, 6, 24, and 48 h after renal reperfusion, and the renal function was evaluated by determining blood creatinine. Histological damages were observed using a semi-quantitative histomorphological scoring system from 0 to 4. Cell apoptosis was analyzed by TUNEL staining in each group. HO-1 and IL-6 mRNA expression was examined by real-time PCR. Results Compared with renal IR group, the expression of HO-1 mRNA was significantly higher in rhEPO treatment group at 3, 6, and 24 h after reperfusion(P<0. O5). The expression of IL-6 mRNA was significantly lower in the rhEPO treatment group at 66, 24, and 48 h after reperfusion(P<0.05). Serum creatinine level in the rhEPO treatment group was significantly lower than that in the renal IR group at 24 h after reperfusion(P<0.05). Compared with the renal IR group,renal histology injury was greatly attenuated by rhEPO in rhEPO treatment group. TUNEL staining analysis indicated that the apoptotic cells in the IR group were significantly more than those in the sham operation group(P<0. 05), and those in the rhEPO treatment group was significantly less than those in the IR group(P<0.05). Conclusion rhEPO can attenuate renal ischemia/reperfusion injury in mice, probably through promoting the renal expression of HO-1 mRNA.%目的 观察重组人促红细胞生成素(rhEPO)对小鼠缺血再灌注损伤(IR)肾脏血红素加氧酶-1(HO-1) mRNA表达的影响,探讨rhEPO对IR的保护作用.方法 90只雄性C57BL/6小鼠随机分为3组:假手术组(n=30)、IR组(n=30)及rhEPO干预组(n=30),分别于再灌注1、2、3、6、24、48 h处死小鼠取

  2. Regulation of the growth hormone (GH) receptor and GH-binding protein mRNA

    Energy Technology Data Exchange (ETDEWEB)

    Kaji, Hidesuke; Ohashi, Shin-Ichirou; Abe, Hiromi; Chihara, Kazuo [Kobe Univ. School of Medicine, Kobe (Japan)

    1994-12-31

    In fasting rats, a transient increase in growth hormone-binding protein (GHBP) mRNA levels was observed after 1 day, in muscle, heart, and liver, but not in fat tissues. The liver GH receptor (GHR) mRNA level was significantly increased after 1 day (but not after 5 days) of bovine GH (bGH) treatment in fed rats. Both the liver GHR mRNA level and the net increment of plasma IGF-I markedly decreased after 5 days of bGH administration in fasting rats. These findings suggest that GHR and GHBP mRNAs in the liver are expressed in a different way and that the expression of GHBP mRNA is regulated differently between tissues, at least in rats. The results also suggest that refractoriness to GH in a sustained fasting state might be beneficial in preventing anabolic effects of GH. In humans, GHR mRNA in lymphocytes, from subjects with either GH-deficiency or acromegaly, could be detected by the reverse transcription-polymerase chain reaction method. In one patient with partial GH insensitivity, a heterozygous missense mutation (P561T) was identified in the cytoplasmic domain of GHR. 15 refs., 4 figs.

  3. MOS11: a new component in the mRNA export pathway.

    Directory of Open Access Journals (Sweden)

    Hugo Germain

    2010-12-01

    Full Text Available Nucleocytoplasmic trafficking is emerging as an important aspect of plant immunity. The three related pathways affecting plant immunity include Nuclear Localization Signal (NLS-mediated nuclear protein import, Nuclear Export Signal (NES-dependent nuclear protein export, and mRNA export relying on MOS3, a nucleoporin belonging to the Nup107-160 complex. Here we report the characterization, identification, and detailed analysis of Arabidopsis modifier of snc1, 11 (mos11. Mutations in MOS11 can partially suppress the dwarfism and enhanced disease resistance phenotypes of snc1, which carries a gain-of-function mutation in a TIR-NB-LRR type Resistance gene. MOS11 encodes a conserved eukaryotic protein with homology to the human RNA binding protein CIP29. Further functional analysis shows that MOS11 localizes to the nucleus and that the mos11 mutants accumulate more poly(A mRNAs in the nucleus, likely resulting from reduced mRNA export activity. Epistasis analysis between mos3-1 and mos11-1 revealed that MOS11 probably functions in the same mRNA export pathway as MOS3, in a partially overlapping fashion, before the mRNA molecules pass through the nuclear pores. Taken together, MOS11 is identified as a new protein contributing to the transfer of mature mRNA from the nucleus to the cytosol.

  4. Quantitation of HDAC1 mRNA expression in invasive carcinoma of the breast

    Institute of Scientific and Technical Information of China (English)

    Zhenhuan Zhang; Hirotaka Iwase; Hiroko Yamashita; Tatsuya Toyama; Hiroshi Sugiura; Yoshiaki Ando; Keiko Mita; Maho Hamaguchi; Yasuo Hara; Shunzo Kobayashi

    2006-01-01

    Estrogen is well-established as a mitogenic factor implicated in the tumorigenesis and progression of breast cancer via its binding to the estrogen receptor a(ERα). Recent data indicate that chromatin inactivation mediated by histone deacetylation(HDAC) and DNA methylation is a critical component of ERα silencing in human breast cancer cells. The aim of this study was to determine the expression of the HDAC1 gene in malignant human breast tissue and to correlate our observations with available clinical information. In the present study, the level of expression of HDAC1 mRNA was assessed by LightCycler-based quantitative real-time reverse transcriptase (RT)-PCR analvsis in 162 cases of invasive carcinoma of the breast. Associations between HDAC1 mRNA expression and different clinicopathological factors were sought. It was found that HDAC1 mRNA was expressed at significantly higher levels in tumors from patients over 50 years of age and in those tumors without axillary lymph node involvement, that are less than 2 cm, that are of a non-high histological grade, that are HER2 negative and that are ERα/PgR positive. Patients with tumors displaying high levels of HDAC1 mRNA expression tended to have a better prognosis in terms of both disease-free and overall survival. However, univariate and multivariate analysis did not show HDAC1 mRNA expression level to be an independent prognostic factor for either disease-free or overall survival. These results imply that HDAC1 mRNA expression could have potential as an endocrine response marker and may have prognostic implications for breast cancer progression.

  5. Nuclear accumulation of CDH1 mRNA in hepatocellular carcinoma cells

    Science.gov (United States)

    Ghafoory, S; Mehrabi, A; Hafezi, M; Cheng, X; Breitkopf-Heinlein, K; Hick, M; Huichalaf, M; Herbel, V; Saffari, A; Wölfl, S

    2015-01-01

    Expression of E-cadherin has a central role in maintaining epithelial morphology. In solid tumors, reduction of E-cadherin results in disruption of intercellular contacts. Consequently, cells lose adhesive properties and gain more invasive mesenchymal properties. Nevertheless, the mechanism of E-cadherin regulation is not completely elucidated. Here we analyzed the distribution of E-cadherin expression at the cell level in human hepatocellular carcinoma, in which human liver paraffin blocks from 25 hepatocellular carcinoma patients were prepared from cancerous (CA) and noncancerous areas (NCA). In situ hybridization (ISH) was performed to detect E-cadherin and hypoxia-induced factor-1α (HIF1α) mRNAs and immunohistochemistry to stain E-cadherin protein. In parallel, RNA was extracted from CA and NCA, and E-cadherin and HIF1α were quantified by quantitative reverse transcription PCR. ISH revealed abundant E-cadherin mRNA in nuclei of hepatocellular carcinoma cells (HCCs), whereas immunohistochemistry showed depletion of E-cadherin protein from these areas. In sections of NCA, E-cadherin mRNA was also found in the cytosol, and E-cadherin protein was detected on the membrane of cells. Experiments in cell lines confirmed E-cadherin mRNA in nuclei of cells negative for E-cadherin protein. HIF1α expression is elevated in CAs, which is associated with a clear cytosolic staining for this mRNA. Our results demonstrate that E-caderhin mRNA is selectively retained in nuclei of HCCs, whereas other mRNAs are still exported, suggesting that translocation of E-cadherin mRNA from nuclei to cytoplasm has a role in regulating E-cadherin protein levels during epithelial mesenchymal transition. PMID:26029826

  6. [Determination of the species specificity of interferons in the translation of the their mRNA from various cell cultures].

    Science.gov (United States)

    Nosik, D N; Novokhatskiĭ, A S; Liakh, L A; Khil'ko, S N; Aspetov, R D

    1983-01-01

    Interferons obtained on induction of human lymphocytes with Newcastle viruses and staphylococcal enterotoxin A and diploid fibroblast cells of human embryos with poly (I).poly (C), as well as translation products of interferon mRNA obtained from these cells were analysed serologically. It was shown that the main type of interferon produced by the cells depended on the cell culture and inductor nature. It was defined at the level of the respective gene depression. Effective translation of mRNA of the interferons of the 3 types makes possible production of cDNA and creation of bacterial plasmids coding the genetic information for the synthesis of human interferon.

  7. A pathogenic mechanism in Huntington's disease involves small CAG-repeated RNAs with neurotoxic activity.

    Science.gov (United States)

    Bañez-Coronel, Mónica; Porta, Silvia; Kagerbauer, Birgit; Mateu-Huertas, Elisabet; Pantano, Lorena; Ferrer, Isidre; Guzmán, Manuel; Estivill, Xavier; Martí, Eulàlia

    2012-01-01

    Huntington's disease (HD) is an autosomal dominantly inherited disorder caused by the expansion of CAG repeats in the Huntingtin (HTT) gene. The abnormally extended polyglutamine in the HTT protein encoded by the CAG repeats has toxic effects. Here, we provide evidence to support that the mutant HTT CAG repeats interfere with cell viability at the RNA level. In human neuronal cells, expanded HTT exon-1 mRNA with CAG repeat lengths above the threshold for complete penetrance (40 or greater) induced cell death and increased levels of small CAG-repeated RNAs (sCAGs), of ≈21 nucleotides in a Dicer-dependent manner. The severity of the toxic effect of HTT mRNA and sCAG generation correlated with CAG expansion length. Small RNAs obtained from cells expressing mutant HTT and from HD human brains significantly decreased neuronal viability, in an Ago2-dependent mechanism. In both cases, the use of anti-miRs specific for sCAGs efficiently blocked the toxic effect, supporting a key role of sCAGs in HTT-mediated toxicity. Luciferase-reporter assays showed that expanded HTT silences the expression of CTG-containing genes that are down-regulated in HD. These results suggest a possible link between HD and sCAG expression with an aberrant activation of the siRNA/miRNA gene silencing machinery, which may trigger a detrimental response. The identification of the specific cellular processes affected by sCAGs may provide insights into the pathogenic mechanisms underlying HD, offering opportunities to develop new therapeutic approaches.

  8. CDKN2A (p16) mRNA decreased expression is a marker of poor prognosis in malignant high-grade glioma.

    Science.gov (United States)

    Sibin, M K; Bhat, Dhananjaya I; Narasingarao, K V L; Lavanya, Ch; Chetan, G K

    2015-09-01

    Human high-grade glioma is heterogeneous in nature based on pathological and genetic profiling. Various tumour suppressor gene alterations are considered as prognostic markers in high-grade glioma. Gene expression of CDKN2A (p16) is used in various cancers as a prognostic biomarker along with methylation and deletion status of this gene. Expression levels of p16 mRNA were not studied as a biomarker in gliomas before. In this study, we have performed mRNA quantification analysis on 48 high-grade glioma tissues and checked for a possible prognostic role. The decreased expression of p16 mRNA in majority of the tumour tissues (57.1 %) was observed when compared to control tissues (P = 0.02). mRNA expression level was correlated with clinical variables also. p16 deletion status and BMI1 mRNA expression were also considered for comparison. p16 mRNA was negatively correlated with the BMI1 mRNA (P = p16 deletion. p16 mRNA expression, midline shift in MRI and tumour type were able to predict patient survival in overall survival (OS) and progression-free survival (PFS). p16 mRNA could independently predict prognosis of OS (P = 0.0146) and PFS (P = 0.0305) in multivariate analysis. We have shown that p16 mRNA expression can act as an independent prognostic biomarker in high-grade glioma.

  9. Influenza polymerase encoding mRNAs utilize atypical mRNA nuclear export.

    Science.gov (United States)

    Larsen, Sean; Bui, Steven; Perez, Veronica; Mohammad, Adeba; Medina-Ramirez, Hilario; Newcomb, Laura L

    2014-08-28

    Influenza is a segmented negative strand RNA virus. Each RNA segment is encapsulated by influenza nucleoprotein and bound by the viral RNA dependent RNA polymerase (RdRP) to form viral ribonucleoproteins responsible for RNA synthesis in the nucleus of the host cell. Influenza transcription results in spliced mRNAs (M2 and NS2), intron-containing mRNAs (M1 and NS1), and intron-less mRNAs (HA, NA, NP, PB1, PB2, and PA), all of which undergo nuclear export into the cytoplasm for translation. Most cellular mRNA nuclear export is Nxf1-mediated, while select mRNAs utilize Crm1. Here we inhibited Nxf1 and Crm1 nuclear export prior to infection with influenza A/Udorn/307/1972(H3N2) virus and analyzed influenza intron-less mRNAs using cellular fractionation and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). We examined direct interaction between Nxf1 and influenza intron-less mRNAs using immuno purification of Nxf1 and RT-PCR of associated RNA. Inhibition of Nxf1 resulted in less influenza intron-less mRNA export into the cytoplasm for HA and NA influenza mRNAs in both human embryonic kidney cell line (293 T) and human lung adenocarcinoma epithelial cell line (A549). However, in 293 T cells no change was observed for mRNAs encoding the components of the viral ribonucleoproteins; NP, PA, PB1, and PB2, while in A549 cells, only PA, PB1, and PB2 mRNAs, encoding the RdRP, remained unaffected; NP mRNA was reduced in the cytoplasm. In A549 cells NP, NA, HA, mRNAs were found associated with Nxf1 but PA, PB1, and PB2 mRNAs were not. Crm1 inhibition also resulted in no significant difference in PA, PB1, and PB2 mRNA nuclear export. These results further confirm Nxf1-mediated nuclear export is functional during the influenza life cycle and hijacked for select influenza mRNA nuclear export. We reveal a cell type difference for Nxf1-mediated nuclear export of influenza NP mRNA, a reminder that cell type can influence molecular mechanisms. Importantly, we

  10. Derivation of Huntington Disease affected Genea046 human embryonic stem cell line

    Directory of Open Access Journals (Sweden)

    Biljana Dumevska

    2016-03-01

    Full Text Available The Genea046 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying HTT gene CAG expansion of 45 repeats, indicative of Huntington Disease. Following ICM outgrowth on inactivated human feeders, karyotype was confirmed as 46, XX by CGH and STR analysis demonstrated a female Allele pattern. The hESC line had pluripotent cell morphology, 85% of cells expressed Nanog, 92% Oct4, 75% Tra1–60 and 99% SSEA4 and demonstrated Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and visible contamination.

  11. Translational control of maskin mRNA by its 3' untranslated region

    Science.gov (United States)

    Meijer, Hedda A.; Radford, Helois E.; Wilson, Lolita S.; Lissenden, Sarah; de Moor, Cornelia H.

    2007-01-01

    Background information. Maskin is a member of the acidic transforming coiled-coil (TACC) domain proteins found in Xenopus leavis oocytes and embryos. It is implicated in the coordination of the spindle and has been reported to mediate translational repression of cyclin B1 mRNA. Results We report here that maskin mRNA is translationally repressed at the level of initiation in stage 4 oocytes and becomes activated in stage 6 oocytes. The translational repression of maskin mRNA correlates with the presence of a short poly(A) tail on this mRNA in stage 4 oocytes. The 3' UTR of maskin can confer the translational regulation to a reporter mRNA, and so can the 3' UTR of human TACC3. A conserved GUCU repeat element was found to repress translation in both stage 4 and stage 6 oocytes, but deletion of this element did not abrogate repression in stage 4 oocytes. UV crosslinking experiments indicated that overlapping sets of proteins bind efficiently to both the maskin and the cyclin B1 3' UTRs. As previously reported, CPEB binds to the cyclin B1 3' UTR, but its binding to the maskin 3' UTR is minimal. By RNA affinity chromatography and mass spectrometry, we identified the embryonic deadenylation element binding protein (EDEN-BP) as one of the proteins binding to both the maskin and the cyclin B1 3' UTRs. Conclusion Maskin mRNA is translationally regulated by at least two repressor elements and an activation element. One of the repessor elements is the evolutionarily conserved GUCU repeat. EDEN-BP binds to both the maskin and cyclin B1 3' UTRs, indicating it may be involved in the deadenylation of these mRNAs. PMID:17241108

  12. AGEs and Glucose Levels Modulate Type I and III Procollagen mRNA Synthesis in Dermal Fibroblasts Cells Culture

    Directory of Open Access Journals (Sweden)

    Serban Iren Andreea

    2008-01-01

    Full Text Available In the dermis, fibroblasts play an important role in the turnover of the dermal extracellular matrix. Collagen I and III, the most important dermal proteins of the extracellular matrix, are progressively altered during ageing and diabetes. For mimicking diabetic conditions, the cultured human dermal fibroblasts were incubated with increasing amounts of AGE-modified BSA and D-glucose for 24 hours. The expression of procollagen α2(I and procollagen α1(III mRNA was analyzed by quantitative real-time PCR. Our data revealed that the treatment of fibroblasts with AGE-modified BSA upregulated the expression of procollagen α2(I and procollagen α1(III mRNA in a dose-dependent manner. High glucose levels mildly induced a profibrogenic pattern, increasing the procollagen α2(I mRNA expression whereas there was a downregulation tendency of procollagen α1(III mRNA.

  13. Coordinate Regulation of mRNA Decay Networks by GU-rich Elements and CELF1

    Science.gov (United States)

    Louis, Irina Vlasova-St.; Bohjanen, Paul R.

    2011-01-01

    The GU-rich element (GRE) was identified as a conserved sequence enriched in the 3′ UTR of human transcripts that exhibited rapid mRNA turnover. In mammalian cells, binding to GREs by the protein CELF1 coordinates mRNA decay of networks of transcripts involved in cell growth, migration, and apoptosis. Depending on the context, GREs and CELF1 also regulate pre-mRNA splicing and translation. GREs are highly conserved throughout evolution and play important roles in development of organisms ranging from worms to man. In humans, abnormal GRE-mediated regulation contributes to disease states and cancer. Thus, GREs and CELF proteins serve critical functions in gene expression regulation and define an important evolutionarily conserved posttranscriptional regulatory network. PMID:21497082

  14. Triple Therapy with Prednisolone, Pegylated Interferon and Sodium Valproate Improves Clinical Outcome and Reduces Human T-Cell Leukemia Virus Type 1 (HTLV-1) Proviral Load, Tax and HBZ mRNA Expression in Patients with HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis

    National Research Council Canada - National Science Library

    Boostani, Reza; Vakili, Rosita; Hosseiny, Samane Sadat; Shoeibi, Ali; Fazeli, Bahare; Etemadi, Mohammad Mehdi; Sabet, Faeze; Valizade, Narges; Rezaee, Seyed Abdolrahim

    2015-01-01

    .... HTLV-1 proviral load (PVL), and HBZ and Tax mRNA expression levels were measured in peripheral blood mononuclear cells of 13 patients with HTLV-1-associated myelopathy/tropical spastic paraparesis before and after treatment with 180 μ...

  15. High-Risk Human Papillomavirus (hrHPV) E6/E7 mRNA Testing by PreTect HPV-Proofer for Detection of Cervical High-Grade Intraepithelial Neoplasia and Cancer among hrHPV DNA-Positive Women with Normal Cytology

    Science.gov (United States)

    Rijkaart, D. C.; Heideman, D. A. M.; Coupe, V. M. H.; Brink, A. A. T. P.; Verheijen, R. H. M.; Skomedal, H.; Karlsen, F.; Morland, E.; Snijders, P. J. F.

    2012-01-01

    Our aim was to investigate whether high-risk HPV (hrHPV) mRNA detection by PreTect HPV-Proofer can be used to stratify hrHPV DNA-positive women of different cytology classes for risk of high-grade cervical intraepithelial neoplasia or worse (cervical precancer or cancer, i.e., cervical intraepithelial neoplasia grade 2 or higher [≥CIN2]). A total of 375 women participating in population-based screening, with a GP5+/6+-PCR hrHPV DNA-positive cervical scrape with normal cytology (n = 202), borderline or mild dyskaryosis (BMD) (n = 88), or moderate dyskaryosis or worse (>BMD) (n = 85), were enrolled. Cervical scrapes were additionally subjected to HPV16/18/31/33/45 E6/E7 mRNA analysis by PreTect HPV-Proofer (mRNA test). Referral and follow-up policies were based on cytology, hrHPV DNA, and mRNA testing. The primary study endpoint was the number of ≥CIN2 detected within 3 years of follow-up. The mRNA positivity increased with the severity of cytological abnormality, ranging from 32% (64/202) in hrHPV DNA-positive women with normal cytology to 47% (41/88) in BMD and 68% (58/85) in >BMD groups (P cytology, i.e., 0.55 (95% confidence interval [95% CI], 0.34 to 0.76) in mRNA-positive versus 0.20 (95% CI, 0.07 to 0.33) in mRNA-negative women. In hrHPV DNA-positive women with BMD or >BMD, the result of the mRNA test did not influence the ≥CIN2 risk. In conclusion, mRNA testing by PreTect HPV-Proofer might be of value to select hrHPV DNA-positive women with normal cytology in need of immediate referral for colposcopy. PMID:22553244

  16. mRNA pseudoknot structures can act as ribosomal roadblocks

    DEFF Research Database (Denmark)

    Hansen, Jesper Tholstrup; Oddershede, Lene Broeng; Sørensen, Michael Askvad

    2012-01-01

    Several viruses utilize programmed ribosomal frameshifting mediated by mRNA pseudoknots in combination with a slippery sequence to produce a well defined stochiometric ratio of the upstream encoded to the downstream-encoded protein. A correlation between the mechanical strength of mRNA pseudoknot...

  17. Functional Integration of mRNA Translational Control Programs

    Directory of Open Access Journals (Sweden)

    Melanie C. MacNicol

    2015-07-01

    Full Text Available Regulated mRNA translation plays a key role in control of cell cycle progression in a variety of physiological and pathological processes, including in the self-renewal and survival of stem cells and cancer stem cells. While targeting mRNA translation presents an attractive strategy for control of aberrant cell cycle progression, mRNA translation is an underdeveloped therapeutic target. Regulated mRNAs are typically controlled through interaction with multiple RNA binding proteins (RBPs but the mechanisms by which the functions of distinct RBPs bound to a common target mRNA are coordinated are poorly understood. The challenge now is to gain insight into these mechanisms of coordination and to identify the molecular mediators that integrate multiple, often conflicting, inputs. A first step includes the identification of altered mRNA ribonucleoprotein complex components that assemble on mRNAs bound by multiple, distinct RBPs compared to those recruited by individual RBPs. This review builds upon our knowledge of combinatorial control of mRNA translation during the maturation of oocytes from Xenopus laevis, to address molecular strategies that may mediate RBP diplomacy and conflict resolution for coordinated control of mRNA translational output. Continued study of regulated ribonucleoprotein complex dynamics promises valuable new insights into mRNA translational control and may suggest novel therapeutic strategies for the treatment of disease.

  18. Probing dimensionality beyond the linear sequence of mRNA.

    Science.gov (United States)

    Del Campo, Cristian; Ignatova, Zoya

    2016-05-01

    mRNA is a nexus entity between DNA and translating ribosomes. Recent developments in deep sequencing technologies coupled with structural probing have revealed new insights beyond the classic role of mRNA and place it more centrally as a direct effector of a variety of processes, including translation, cellular localization, and mRNA degradation. Here, we highlight emerging approaches to probe mRNA secondary structure on a global transcriptome-wide level and compare their potential and resolution. Combined approaches deliver a richer and more complex picture. While our understanding on the effect of secondary structure for various cellular processes is quite advanced, the next challenge is to unravel more complex mRNA architectures and tertiary interactions.

  19. Herpes Simplex Virus 1 Infection Alters the mRNA Translation Processing in L-02 Cells

    Institute of Scientific and Technical Information of China (English)

    Min HONG; Yan-chun CHE; Gui-zhen TANG; Wei CUN; Xue-mei ZHANG; Long-ding LIU; Qi-han LI

    2008-01-01

    HSV-1 infection-mediated regulation of mRNA translation in host cells is a systematic and complicated process. Investigation of the details of this mechanism will facilitate understanding of biological variations in the viral replication process and host cells. In this study, a comparative proteomics technology platform was applied by two-dimension electrophoresis of HSV-1 infected normal human L-02 cell and control cell lysates. The observed protein spots were analyzed qualitatively and quantitatively by the PDQuest software package. A number of the different observed protein spots closely associated with cellular protein synthesis were identified by matrix-assisted laser-desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS). The expression levels of the RPLP1 protein, which is required for mRNA translation, and KHSRP protein, which is involved in rapid decay of mRNA, were up-regulated, whereas the expression level of RNP H2, which is involved in positive regulation on the mRNA splicing process, was down-regulated. All of these results suggest that HSV-1 infection can influence cellular protein synthesis via modulation of cellular regulatory proteins involved in RNA splicing, translation and decay, resulting in optimisation of viral protein synthesis when cellular protein synthesis is shut off. Although there is need for further investigations regarding the detailed mechanisms of cellular protein control, our studies provide new insight into the targeting of varied virus signaling pathways involved in host cellular protein synthesis.

  20. Cell cycle-dependent regulation of Aurora kinase B mRNA by the Microprocessor complex.

    Science.gov (United States)

    Jung, Eunsun; Seong, Youngmo; Seo, Jae Hong; Kwon, Young-Soo; Song, Hoseok

    2014-03-28

    Aurora kinase B regulates the segregation of chromosomes and the spindle checkpoint during mitosis. In this study, we showed that the Microprocessor complex, which is responsible for the processing of the primary transcripts during the generation of microRNAs, destabilizes the mRNA of Aurora kinase B in human cells. The Microprocessor-mediated cleavage kept Aurora kinase B at a low level and prevented premature entrance into mitosis. The cleavage was reduced during mitosis leading to the accumulation of Aurora kinase B mRNA and protein. In addition to Aurora kinase B mRNA, the processing of other primary transcripts of miRNAs were also decreased during mitosis. We found that the cleavage was dependent on an RNA helicase, DDX5, and the association of DDX5 and DDX17 with the Microprocessor was reduced during mitosis. Thus, we propose a novel mechanism by which the Microprocessor complex regulates stability of Aurora kinase B mRNA and cell cycle progression.

  1. Quantification of mRNA by RT-competitive-PCR and high performance liquid chromatography

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The use of RT-competitive-PCR with high performance liquid chromatography (HPLC) detection to quantify the absolute number of mRNA copies in mammalian cells is reported. As an example, the glutathione transferase (GST)-α mRNA in human hepatoma Hep G2 cells has been estimated. A PCR-generated internal standard was used as a competitor, co-amplified with the GST-α target sequence. The RT-competitive-PCR method was improved by designing target and competitor molecules which differed in only 30 base pairs. This allowed the two sequences to be co-amplified with the same efficiency. This improvement also facilitated a wider ratio to be used than previous methods (target:competitor ratio between 0.2 and 5). Products were baseline separated by HPLC using an ion-exchange column readily quantified at 260 nm. To validate the improved methodology, the effect of a known GST-α inducer, the anticancer drug oltipraz, was shown to induce GST-α mRNA up to 3-fold in Hep G2 cells. The RT-competitive PCR-HPLC method provides a reliable and sensitive way to quantify the amount of specific mRNA with 0.1 ng of total RNA.

  2. c-Myc deregulation induces mRNA capping enzyme dependency

    Science.gov (United States)

    Lombardi, Olivia; Varshney, Dhaval; Phillips, Nicola M.; Cowling, Victoria H.

    2016-01-01

    c-Myc is a potent driver of many human cancers. Since strategies for directly targeting c-Myc protein have had limited success, upstream regulators and downstream effectors of c-Myc are being investigated as alternatives for therapeutic intervention. c-Myc regulates transcription and formation of the mRNA cap, which is important for transcript maturation and translation. However, the direct mechanism by which c-Myc upregulates mRNA capping is unclear. mRNA cap formation initiates with the linkage of inverted guanosine via a triphosphate bridge to the first transcribed nucleotide, catalysed by mRNA capping enzyme (CE/RNGTT). Here we report that c-Myc increases the recruitment of catalytically active CE to RNA polymerase II and to its target genes. c-Myc-induced target gene expression, cell proliferation and cell transformation is highly dependent on CE, but only when c-Myc is deregulated. Cells retaining normal control of c-Myc expression are insensitive to repression of CE. c-Myc expression is also dependent on CE. Therefore, inhibiting CE provides an attractive route for selective therapeutic targeting of cancer cells which have acquired deregulated c-Myc. PMID:27756891

  3. c-Myc deregulation induces mRNA capping enzyme dependency.

    Science.gov (United States)

    Lombardi, Olivia; Varshney, Dhaval; Phillips, Nicola M; Cowling, Victoria H

    2016-12-13

    c-Myc is a potent driver of many human cancers. Since strategies for directly targeting c-Myc protein have had limited success, upstream regulators and downstream effectors of c-Myc are being investigated as alternatives for therapeutic intervention. c-Myc regulates transcription and formation of the mRNA cap, which is important for transcript maturation and translation. However, the direct mechanism by which c-Myc upregulates mRNA capping is unclear. mRNA cap formation initiates with the linkage of inverted guanosine via a triphosphate bridge to the first transcribed nucleotide, catalysed by mRNA capping enzyme (CE/RNGTT). Here we report that c-Myc increases the recruitment of catalytically active CE to RNA polymerase II and to its target genes. c-Myc-induced target gene expression, cell proliferation and cell transformation is highly dependent on CE, but only when c-Myc is deregulated. Cells retaining normal control of c-Myc expression are insensitive to repression of CE. c-Myc expression is also dependent on CE. Therefore, inhibiting CE provides an attractive route for selective therapeutic targeting of cancer cells which have acquired deregulated c-Myc.

  4. Cellular restriction of retrovirus particle-mediated mRNA transfer.

    Science.gov (United States)

    Galla, Melanie; Schambach, Axel; Towers, Greg J; Baum, Christopher

    2008-03-01

    Analyzing cellular restriction mechanisms provides insight into viral replication strategies, identifies targets for antiviral drug design, and is crucial for the development of novel tools for experimental or therapeutic delivery of genetic information. We have previously shown that retroviral vector mutants that are unable to initiate reverse transcription mediate a transient expression of any sequence which replaces the gag-pol transcription unit, a process we call retrovirus particle-mediated mRNA transfer (RMT). Here, we further examined the mechanism of RMT by testing its sensitivity to cellular restriction factors and short hairpin RNAs (shRNAs). We found that both human TRIM5alpha and, to a lesser extent, Fv1 effectively restrict RMT if the RNA is delivered by a restriction-sensitive capsid. While TRIM5alpha restriction of RMT led to reduced levels of retroviral mRNA in target cells, restriction by Fv1 did not. Treatment with the proteasome inhibitor MG132 partially relieved TRIM5alpha-mediated restriction of RMT. Finally, cells expressing shRNAs specifically targeting the retroviral mRNA inhibited RMT particles, but not reverse-transcribing particles. Retroviral mRNA may thus serve as a translation template if not used as a template for reverse transcription. Our data imply that retroviral nucleic acids become accessible to host factors, including ribosomes, as a result of particle remodeling during cytoplasmic trafficking.

  5. TDP-43 suppresses tau expression via promoting its mRNA instability.

    Science.gov (United States)

    Gu, Jianlan; Wu, Feng; Xu, Wen; Shi, Jianhua; Hu, Wen; Jin, Nana; Qian, Wei; Wang, Xinglong; Iqbal, Khalid; Gong, Cheng-Xin; Liu, Fei

    2017-06-02

    In the brains of individuals with Alzheimer's disease (AD) and chronic traumatic encephalopathy, tau pathology is accompanied usually by intracellular aggregation of transactive response DNA-binding protein 43 (TDP-43). However, the role of TDP-43 in tau pathogenesis is not understood. Here, we investigated the role of TDP-43 in tau expression in vitro and in vivo. We found that TDP-43 suppressed tau expression by promoting its mRNA instability through the UG repeats of its 3΄-untranslated region (3΄-UTR). The C-terminal region of TDP-43 was required for this function. Neurodegenerative diseases-causing TDP-43 mutations affected tau mRNA instability differentially, in that some promoted and others did not significantly affect tau mRNA instability. The expression levels of tau and TDP-43 were inverse in the frontal cortex and the cerebellum. Accompanied with cytoplasmic accumulation of TDP-43, tau expression was elevated in TDP-43M337V transgenic mouse brains. The level of TDP-43, which is decreased in AD brains, was found to correlate negatively with the tau level in human brain. Our findings indicate that TDP-43 suppresses tau expression by promoting the instability of its mRNA. Down-regulation of TDP-43 may be involved in the tau pathology in AD and related neurodegenerative disorders. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  6. Dendritically targeted Bdnf mRNA is essential for energy balance and response to leptin.

    Science.gov (United States)

    Liao, Guey-Ying; An, Juan Ji; Gharami, Kusumika; Waterhouse, Emily G; Vanevski, Filip; Jones, Kevin R; Xu, Baoji

    2012-03-18

    Mutations in the Bdnf gene, which produces transcripts with either short or long 3' untranslated regions (3' UTRs), cause human obesity; however, the precise role of brain-derived neurotrophic factor (BDNF) in the regulation of energy balance is unknown. Here we show the relationship between Bdnf mRNA with a long 3' UTR (long 3' UTR Bdnf mRNA), leptin, neuronal activation and body weight. We found that long 3' UTR Bdnf mRNA was enriched in the dendrites of hypothalamic neurons and that insulin and leptin could stimulate its translation in dendrites. Furthermore, mice harboring a truncated long Bdnf 3' UTR developed severe hyperphagic obesity, which was completely reversed by viral expression of long 3' UTR Bdnf mRNA in the hypothalamus. In these mice, the ability of leptin to activate hypothalamic neurons and inhibit food intake was compromised despite normal activation of leptin receptors. These results reveal a novel mechanism linking leptin action to BDNF expression during hypothalamic-mediated regulation of body weight, while also implicating dendritic protein synthesis in this process.

  7. EXPRESSION OF E-SELECTIN AND E-SELECTIN mRNA AND THE POTENTIAL CLINICAL IMPLICATIONS IN HUMAN BLADDER TRANSITIONAL CELL CARCINOMA%膀胱移行细胞癌中E-Selectin、E-Selectin mRNA的表达结果及其临床意义的初步研究

    Institute of Scientific and Technical Information of China (English)

    曾向阳; 范本祎; 孙芳浒; 张轶庠; 祖雄兵; 黄江波; 申鹏飞

    2003-01-01

    目的:通过检测膀胱移行细胞癌(BTCC)中E-Selectin、E-Selectin mRNA的表达结果,探讨其表达与肿瘤分级、分期的关系,并初步探讨其临床意义.方法:以免疫组化SABC法及原位杂交法分别检测32例膀胱移行细胞癌中E-Selectin、E-Selectin mRNA的表达,分析其与肿瘤分期、分级的关系.结果:32例膀胱移行细胞癌组织中23例E-Selectin mRNA的表达阳性,占71.8%,9例表达阴性,占28.2%;20例E-Selectin表达阳性,占62.5%,12例表达阴性,占37.5%;其表达与肿瘤的分级无关(P>0.05),但与肿瘤的临床分期明显相关(P<0.05);11例复发的BTCC患者中,11例E-Selectin mRNA表达阳性,10例E-Selectin表达阳性,其表达与肿瘤的复发显著相关.结论:在膀胱移行细胞癌中,E-Selectin及E-Selectin mRNA的表达明显上调,可能促进了膀胱移行细胞癌的血源性转移,且其表达与肿瘤的分期及复发有明显关系,可能成为判断膀胱移行细胞癌预后的一项重要指标.%Objective:To make a preliminary study of the expression of the E-Selectin mRNA and E-Selectin in bladder transitional cell carcinoma (BTCC), and then their possible clinic significance.Methods:32 fresh surgical specimens of BTCC were examined by immunohistochemistry for E-Selectin protein expression and in situ hybridization for E-Selectin mRNA expression. We discussed the correlation between the expression of E-Selectin mRNA, E-Selectin and tumor clinical stage and tumor pathological grade.Results:23/32 tumor specimens had positively expressed E-Selectin mRNA,20/32 tumor specimens showed positive E-Selectin expression. Abnormal expression of E-Selectin mRNA and E-Selectin had a significantly association with tumor clinical stage but not with increased tumor grade. 11 recurrent tumor specimens showed positive expression of E-Selectin mRNA, of which 10 tumors showed positive expression of E-Selectin. The abnormal expression had a significant association with tumor recurrence

  8. PolyA-specific ribonuclease (PARN-1) function in stage-specific mRNA turnover in Trypanosoma brucei.

    Science.gov (United States)

    Utter, Christopher J; Garcia, Stacey A; Milone, Joseph; Bellofatto, Vivian

    2011-09-01

    Deadenylation is often the rate-limiting event in regulating the turnover of cellular mRNAs in eukaryotes. Removal of the poly(A) tail initiates mRNA degradation by one of several decay pathways, including deadenylation-dependent decapping, followed by 5' to 3' exonuclease decay or 3' to 5' exosome-mediated decay. In trypanosomatids, mRNA degradation is important in controlling the expression of differentially expressed genes. Genomic annotation studies have revealed several potential deadenylases. Poly(A)-specific RNase (PARN) is a key deadenylase involved in regulating gene expression in mammals, Xenopus oocytes, and higher plants. Trypanosomatids possess three different PARN genes, PARN-1, -2, and -3, each of which is expressed at the mRNA level in two life-cycle stages of the human parasite Trypanosoma brucei. Here we show that T. brucei PARN-1 is an active deadenylase. To determine the role of PARN-1 on mRNA stability in vivo, we overexpressed this protein and analyzed perturbations in mRNA steady-state levels as well as mRNA half-life. Interestingly, a subset of mRNAs was affected, including a family of mRNAs that encode stage-specific coat proteins. These data suggest that PARN-1 functions in stage-specific protein production.

  9. Rhythmic expression of Nocturnin mRNA in multiple tissues of the mouse

    Directory of Open Access Journals (Sweden)

    Green Carla B

    2001-05-01

    Full Text Available Abstract Background Nocturnin was originally identified by differential display as a circadian clock regulated gene with high expression at night in photoreceptors of the African clawed frog, Xenopus laevis. Although encoding a novel protein, the nocturnin cDNA had strong sequence similarity with a C-terminal domain of the yeast transcription factor CCR4, and with mouse and human ESTs. Since its original identification others have cloned mouse and human homologues of nocturnin/CCR4, and we have cloned a full-length cDNA from mouse retina, along with partial cDNAs from human, cow and chicken. The goal of this study was to determine the temporal pattern of nocturnin mRNA expression in multiple tissues of the mouse. Results cDNA sequence analysis revealed a high degree of conservation among vertebrate nocturnin/CCR4 homologues along with a possible homologue in Drosophila. Northern analysis of mRNA in C3H/He and C57/Bl6 mice revealed that the mNoc gene is expressed in a broad range of tissues, with greatest abundance in liver, kidney and testis. mNoc is also expressed in multiple brain regions including suprachiasmatic nucleus and pineal gland. Furthermore, mNoc exhibits circadian rhythmicity of mRNA abundance with peak levels at the time of light offset in the retina, spleen, heart, kidney and liver. Conclusion The widespread expression and rhythmicity of mNoc mRNA parallels the widespread expression of other circadian clock genes in mammalian tissues, and suggests that nocturnin plays an important role in clock function or as a circadian clock effector.

  10. Effects of DNA replication on mRNA noise.

    Science.gov (United States)

    Peterson, Joseph R; Cole, John A; Fei, Jingyi; Ha, Taekjip; Luthey-Schulten, Zaida A

    2015-12-29

    There are several sources of fluctuations in gene expression. Here we study the effects of time-dependent DNA replication, itself a tightly controlled process, on noise in mRNA levels. Stochastic simulations of constitutive and regulated gene expression are used to analyze the time-averaged mean and variation in each case. The simulations demonstrate that to capture mRNA distributions correctly, chromosome replication must be realistically modeled. Slow relaxation of mRNA from the low copy number steady state before gene replication to the high steady state after replication is set by the transcript's half-life and contributes significantly to the shape of the mRNA distribution. Consequently both the intrinsic kinetics and the gene location play an important role in accounting for the mRNA average and variance. Exact analytic expressions for moments of the mRNA distributions that depend on the DNA copy number, gene location, cell doubling time, and the rates of transcription and degradation are derived for the case of constitutive expression and subsequently extended to provide approximate corrections for regulated expression and RNA polymerase variability. Comparisons of the simulated models and analytical expressions to experimentally measured mRNA distributions show that they better capture the physics of the system than previous theories.

  11. Effects of APP17-mer peptide on oxidative damage and expression of MMP-1 mRNA in cultured human skin fibrobiasts irradiated with ultraviolet light%淀粉样肽前体蛋白17肽对紫外线照射后皮肤成纤维细胞内活性氧簇和基质金属蛋白酶-1 mRNA表达的作用

    Institute of Scientific and Technical Information of China (English)

    陈慧; 朱威; 连石; 王蓉; 张景燕; 姬志娟; 蔡彦宁; 刘姝

    2008-01-01

    Objective To establish an ultraviolet-irradiation damage model in cultured fibroblasts derived from human skin and to explore the potential protective effects and mechanisms of amyloid precursor protein 17-met peptide (APP17-mer peptide) on the oxidative damage and collagen metabolism in cultured fibroblasts after ultraviolet irradiation. Methods Human dermal fibroblast cultures were established by outgrowth from foreskin biopsies of a healthy donor and were irradiated by a single exposure to ultraviolet rays and cultured in a series of concentrations of APP17-mer peptide (0, 20, 40, 80 μmol/L).The activity of fibroblasts was detected by the assay of MTT. The intracellular ROS level was measured with a confocal microscope. The expression of MMP-1 mRNA was analyzed real-time quantitatively following RT-PCR. Results Primary cultures of human skin fibroblasts were established from human foreskin in DMEM supplemented with 10 % fetal bovine serum. UV irradiation depressed cellular activity and increased intracellular level of ROS (P0. 05). 40μmol/L APP17-mer peptide could depress the level of ROS in irradiated libroblasts. A single exposure of fibroblasts to UV irradiation resulted in 1.78 foldup-regulation of MMP-1 mRNA compared with unirradiated sample, 40μmol/L and 80μmol/L APP17-mer peptide decreased the expression of MMP-1 mRNA (P<0.05 and P<0.01, respectively).Conclusion APP17-mer peptide can enhance cellular activity under UV-induced oxidative stress and in-hibit collagen degradation in fibroblasts irradiated with ultraviolet rays. Inhibition of ROS production may be involved in the protective mechanism of APP17 peptide.%目的 通过建立体外培养人皮肤成纤维细胞的紫外线损伤模型,探讨淀粉样肽前体蛋白319~335肽段(APP17肽)对紫外线(ultraviolet, UV)照射后人皮肤成纤维细胞的保护作用及其可能的机制.方法 从健康青年男性的包皮组织原代培养成纤维细胞.UV照射培养的成纤维细

  12. Protor-2 interacts with tristetraprolin to regulate mRNA stability during stress

    Science.gov (United States)

    Holmes, Brent; Artinian, Nicholas; Anderson, Lauren; Martin, Jheralyn; Masri, Janine; Cloninger, Cheri; Bernath, Andrew; Bashir, Tariq; Benavides-Serrato, Angelica; Gera, Joseph

    2011-01-01

    The A/U-rich RNA binding protein tristetraprolin (TTP) is an mRNA destabilizing factor which plays a role in the regulated turnover of many transcripts encoding proteins involved in immune function and cell growth control. TTP also plays a role in stress-induced destabilization of mRNAs. Here we report the interaction of TTP with a component of the mTORC2 kinase, Protor-2 (PRR5-L, protein Q6MZQ0/FLJ14213/CAE45978). Protor-2 is structurally similar to human PRR5 and has been demonstrated to bind mTORC2 via Rictor and/or Sin1 and may signal downstream events promoting apoptosis. Protor-2 dissociates from mTORC2 upon hyperactivation of the kinase and is not required for mTORC2 integrity or activity. We identified Protor-2 in a yeast two-hybrid screen as a TTP interactor using the C-terminal mRNA decay domain of TTP as bait. The interaction of Protor-2 with TTP was also confirmed in vivo in co-immunoprecipitation experiments and Protor-2 was also detected in immunoprecipitates of rictor. Protor-2 was shown to stimulate TTP-mediated mRNA turnover of several TTP-associated mRNAs (TNF-α, GM-CSF, IL-3 and COX-2) in Jurkat cells when overexpressed while the half-lives of transcripts which do not decay via a TTP-mediated mechanism were unaffected. Knockdown of Protor-2 via RNAi inhibited TTP-mediated mRNA turnover of these TTP-associated mRNAs and inhibited association of TTP with cytoplasmic stress granules (SG) or mRNA processing bodies (P-bodies) following induction of the integrated stress response. These results suggest that Protor-2 associates with TTP to accelerate TTP-mediated mRNA turnover and functionally links the control of TTP regulated mRNA stability to mTORC2 activity. PMID:21964062

  13. mRNA trafficking and local translation: the Yin and Yang of regulating mRNA localization in neurons

    Institute of Scientific and Technical Information of China (English)

    John R. Sinnamon; Kevin Czaplinski

    2011-01-01

    Localized translation and the requisite trafficking of the mRNA template play significant roles in the nervous system including the establishment of dendrites and axons,axon path-finding,and synaptic plasticity.We provide a brief review on the regulation of localizing mRNA in mammalian neurons through critical posttranslational modifications of the factors involved.These examples highlight the relationship between mRNA trafficking and the translational regulation of trafficked mRNAs and provide insight into how extracellular signals target these events during signal transduction.

  14. The role of the DEAD-box RNA helicase DDX3 in mRNA metabolism.

    Science.gov (United States)

    Soto-Rifo, Ricardo; Ohlmann, Théophile

    2013-01-01

    DDX3 belongs to the DEAD-box proteins, a large family of ATP-dependent RNA helicases that participate in all aspects of RNA metabolism. Human DDX3 is a component of several messenger ribonucleoproteins that are found in the spliceosome, the export and the translation initiation machineries but also in different cytoplasmic mRNA granules. DDX3 has been involved in several cellular processes such as cell cycle progression, apoptosis, cancer, innate immune response, and also as a host factor for viral replication. Interestingly, not all these functions require the catalytic activities of DDX3 and thus, the precise roles of this apparently multifaceted protein remain largely obscure. The aim of this review is to provide a rapid and critical overview of the structure and functions of DDX3 with a particular emphasis on its role during mRNA metabolism.

  15. Increased IL-10 mRNA and IL-23 mRNA expression in multiple sclerosis: interferon-beta treatment increases IL-10 mRNA expression while reducing IL-23 mRNA expression

    DEFF Research Database (Denmark)

    Krakauer, M.; Sorensen, P.; Khademi, M.

    2008-01-01

    of the regulatory cytokine IL-10. The elevated IL-23 mRNA levels in MS patients are noteworthy in view of the newly discovered IL-23-driven Th17 T-cell subset, which is crucial in animal models of MS. Since IFN-beta therapy resulted in decreased IL-23 mRNA levels, the Th17 axis could be another target of IFN...

  16. Vitamin D and the RNA transcriptome: more than mRNA regulation

    Directory of Open Access Journals (Sweden)

    Moray J Campbell

    2014-05-01

    Full Text Available The GRCh37.p13 primary assembly of the human genome contains 20805 protein coding mRNA, and 37147 non-protein coding genes and pseudogenes that as a result of RNA processing and editing generate 196501 gene transcripts. Given the size and diversity of the human transcriptome, it is timely to revisit what is known of VDR function in the regulation and targeting of transcription.Early transcriptomic studies using microarray approaches focused on the protein coding mRNA that were regulated by the VDR, usually following treatment with ligand. These studies quickly established the approxamte size, and surprising diversity of the VDR transcriptome, revealing it to be highly heterogenous and cell type and time dependent. With the discovery of microRNA, investigators also considered VDR regulation of these non-protein coding RNA. Again, cell and time dependency has emerged. Attempts to integrate mRNA and miRNA regulation patterns are beginning to reveal patterns of co-regulation and interaction that allow for greater control of mRNA expression, and the capacity to govern more complex cellular events. As the awareness of the diversity of non-coding RNA increases, it is evident that VDR actions are mediated through these molecules also. Key knowledge gaps remain over the VDR transcriptome. The causes for the cell and type dependent transcriptional heterogenetiy remain enigmatic. ChIP-Seq approaches have confirmed that VDR binding choices differ very significantly by cell type, but as yet the underlying causes distilling VDR binding choices are unclear. Similarly, it is clear that many of the VDR binding sites are non-canonical in nature but again the mechanisms underlying these interactions are unclear. Finally, although alternative splicing is clearly a very significant process in cellular transcriptional control, the lack of RNA-Seq data centered on VDR function are currently limiting the global assessment of the VDR transcriptome. VDR focused research

  17. The manganese superoxide dismutase Ala16Val dimorphism modulates both mitochondrial import and mRNA stability.

    Science.gov (United States)

    Sutton, Angela; Imbert, Audrey; Igoudjil, Anissa; Descatoire, Véronique; Cazanave, Sophie; Pessayre, Dominique; Degoul, Françoise

    2005-05-01

    A genetic dimorphism incorporates either alanine (Ala) or valine (Val) in the mitochondrial targeting sequence (MTS) of manganese superoxide dismutase (MnSOD). The Ala-MTS confers a 40% higher MnSOD activity than the Val-MTS after import into isolated mitochondria in vitro. The present study aimed to characterize functional consequences in whole cells. HuH7 human hepatoma cells were transfected with vectors encoding for the human Ala- or Val-MnSOD variants fused to a Myc-His-tag. The Ala-variant resulted in four-fold higher levels of the mature exogenous protein and MnSOD activity than the Val-variant. Studies with a proteasome inhibitor indicated that precursor proteins are either imported into the mitochondria or degraded by the proteasome. Despite identical levels 8 h after transfection, mRNA levels at 36 h were two-fold higher for the Ala-encoding mRNA than the Val-mRNA. Decreasing the mitochondrial membrane potential decreased both MnSOD mitochondrial import and its mRNA levels. Much larger differences in the activity of the human Val- and Ala-MnSOD variants are observed in whole cells rather than after import experiments performed in vitro. First, the slowly imported Val-MnSOD is degraded by the proteasome in cells. Second, the slower mitochondrial import of the Val-variant may be associated with decreased mRNA stability, possibly due to impaired cotranslational import.

  18. Myometrial prostaglandin E2 synthetic enzyme mRNA expression: spatial and temporal variations with pregnancy and labour.

    Science.gov (United States)

    Sooranna, S R; Grigsby, P L; Engineer, N; Liang, Z; Sun, K; Myatt, L; Johnson, M R

    2006-10-01

    We have investigated the hypothesis that the expression of the enzymes involved in PGE(2) synthesis in the human uterus is co-ordinated. We have studied (i) the mRNA expression of the enzymes involved in PGE(2) synthesis [phospholipases (cPLA(2) and sPLA(2)), prostaglandin H synthase (PGHS)-2 and PG E synthases (PGES-1 and -2)] and their relationship to the expression of inflammatory cytokines in samples of myometrium obtained from pregnant women undergoing caesarean section (LSCS) either before or after the onset of labour at or before term; and (ii) the effect of IL-1beta, IL-6, TNF-alpha, PGE(2) and stretch on PGE(2) enzyme mRNA expression. We found that cPLA(2), sPLA(2) and PGHS-2 mRNA expression were greater in labour samples; cPLA(2), sPLA(2), PGHS-2, PGES-1 and -2 mRNA expression were greater in lower- than upper-segment samples; and there was no effect of gestational age. PGHS-2 mRNA levels correlated with those of PGES-1, cPLA(2), IL-1beta and IL-8; PGES-1 mRNA levels correlated with those of IL-1beta, IL-8 and cPLA(2). In primary cultures of uterine myocytes, cPLA(2) mRNA expression was increased by IL-1beta and IL-6; PGHS-2 mRNA expression was increased by IL-1beta, PGE(2) and stretch; and PGES-1 mRNA expression was increased by IL-1beta only. These data show that labour is associated with increased expression of the enzymes involved in PGE(2) synthesis and their expression is greater in the lower uterine segment. The presence of associations between the levels of PGE(2) enzyme mRNA expression and the effects of IL-1beta suggest that their expression is co-ordinated and that IL-1beta is the responsible factor.

  19. An essential nuclear protein in trypanosomes is a component of mRNA transcription/export pathway.

    Directory of Open Access Journals (Sweden)

    Mariana Serpeloni

    Full Text Available In eukaryotic cells, different RNA species are exported from the nucleus via specialized pathways. The mRNA export machinery is highly integrated with mRNA processing, and includes a different set of nuclear transport adaptors as well as other mRNA binding proteins, RNA helicases, and NPC-associated proteins. The protozoan parasite Trypanosoma cruzi is the causative agent of Chagas disease, a widespread and neglected human disease which is endemic to Latin America. Gene expression in Trypanosoma has unique characteristics, such as constitutive polycistronic transcription of protein-encoding genes and mRNA processing by trans-splicing. In general, post-transcriptional events are the major points for regulation of gene expression in these parasites. However, the export pathway of mRNA from the nucleus is poorly understood. The present study investigated the function of TcSub2, which is a highly conserved protein ortholog to Sub2/ UAP56, a component of the Transcription/Export (TREX multiprotein complex connecting transcription with mRNA export in yeast/human. Similar to its orthologs, TcSub2 is a nuclear protein, localized in dispersed foci all over the nuclei -except the fibrillar center of nucleolus- and at the interface between dense and non-dense chromatin areas, proposing the association of TcSub2 with transcription/processing sites. These findings were analyzed further by BrUTP incorporation assays and confirmed that TcSub2 is physically associated with active RNA polymerase II (RNA pol II, but not RNA polymerase I (RNA pol I or Spliced Leader (SL transcription, demonstrating participation particularly in nuclear mRNA metabolism in T. cruzi. The double knockout of the TcSub2 gene is lethal in T. cruzi, suggesting it has an essential function. Alternatively, RNA interference assays were performed in Trypanosoma brucei. It allowed demonstrating that besides being an essential protein, its knockdown causes mRNA accumulation in the nucleus and

  20. Regulation of metallothionein-III (GIF) mRNA in the brain of patients with Alzheimer disease is not impaired.

    Science.gov (United States)

    Amoureux, M C; Van Gool, D; Herrero, M T; Dom, R; Colpaert, F C; Pauwels, P J

    1997-01-01

    Contradictory results have been reported on the downregulation and role of the brain-specific protein metallothionein-III (MT-III, GIF) in Alzheimer disease (AD). In this article, the importance of MT-III downregulation in AD brain was re-evaluated in temporal and frontal cortex, hippocampus, and cerebellum of 11 AD patients and two groups of five and six control subjects, respectively. Reverse transcription-polymerase chain reaction (RT-PCR) was used to quantify the levels of MT-III mRNA relative to the levels of three constitutive RNAs: beta-actin, glyceraldehyde-3-phosphate dehydrogenase (G3PDH), and ribosomal RNA 18S (rRNA 18S). The distribution of MT-III was similar to that of each of the three constitutive RNAs. The relative levels of each of these RNAs was high in brain regions examined in both AD patients and control subjects. Our findings do not support a downregulation of MT-III mRNA in the frontal cortex as well as the temporal cortex and hippocampus of AD patients. However, the level of MT-III mRNA was not constant in the investigated samples, suggesting that MT-III mRNA regulation could be controlled by factors other than AD pathology. Brain-derived neurotrophic factor (BDNF) mRNA levels were hardly detectable by RT-PCR in human brain tissue; a trend for a decrease was apparent in the temporal cortex of AD patients. In conclusion, the content of MT-III mRNA in the brain of AD patients was not detectably impaired, whereas BDNF mRNA may be affected.

  1. DISTRIBUTION OF VEGF mRNA IN BREAST CANCER WITH NONRADIOACTIVE IN SITU HYBRIDIZATION AT ELECTRON MICROSCOPIC LEVELS

    Institute of Scientific and Technical Information of China (English)

    王医术; 林; 王心蕊; 李一雷; 吴珊; 张丽红

    2002-01-01

    Object: To localize the mRNA coding for VEGF at Ultrastractural level in human breast cancer by using digoxigenin-labeled cDNA probes. Methods: Nonradio- active in situ hybridization at electron microscopic level was employed to detected VEGF mRNA in breast cancer. Result: Cancer cells and endothelial cell of angiogensis show dark color in experiment sections. No dark color can be found in control sections. Positive hybridization signals showed dark dot and were locatedin various compartments of the breast cancer cell and endothelial cell in experiment section. No labeling was observed in control sections. In experiment sections, the staining appeared concentrated in cytoplasm and nucleus of the breast cancer cell and endothelial cell. Conclusion: Nonradioactive in situ hybridization at electron microscopic level is efficient for direct observation of the target site mRNA of VEGF in the cytoplasm and nucleus.

  2. Interaction between thymidylate synthase and its cognate mRNA in zebrafish embryos.

    Directory of Open Access Journals (Sweden)

    Yuyan Zhang

    Full Text Available Thymidylate synthase (TS, which catalyzes the de novo synthesis of dUMP, is an important target for cancer therapy. In this report, the effects of 5-fluorouracil (5-FU and ZD1694 on the regulation of TS gene expression were evaluated in zebrafish embryos. Our results revealed that the expression of TS was increased by about six-fold when embryos were treated with 1.0 microM 5-FU and there was a greater than 10-fold increase in the TS protein level after treatment with 0.4 microM ZD1694. Northern blot analysis confirmed that expression of TS mRNA was identical in treated or untreated embryos. Gel shift and immunoprecipitation assays revealed that zebrafish TS was specifically bound with its cognate mRNA in vitro and in vivo. We identified a 20 nt RNA sequence, TS:N20, localized to the 5'-UTR of TS mRNA, which corresponded to nt 13-32; TS:N20 bound to the TS protein with an affinity similar to that of the full-length TS mRNA. The MFold program predicted that TS:N20 formed a stable stem-loop structure similar to that of the cis-acting element found in human TS mRNA. Variant RNAs with either a deletion or mutation in the core motif of TS:N20 were unable to bind to the TS protein. In vitro translation experiments, using the rabbit lysate system, confirmed that zebrafish TS mRNA translation was significantly repressed when an excess amount of TS protein was included in the system. Additionally, a TS stability experiment confirmed that treatment of zebrafish embryos with 5-FU could increase the TS stability significantly, and the half life of TS protein was about 2.7 times longer than in untreated embryos. Our study revealed a structural requirement for the interaction of TS RNA with TS protein. These findings also demonstrated that the increase in TS protein induced by 5-FU occurs at the post-transcriptional level and that increased stability and translation efficiency both contributed to the increase in TS protein levels induced by TS inhibitors.

  3. Optimal Down Regulation of mRNA Translation

    Science.gov (United States)

    Zarai, Yoram; Margaliot, Michael; Tuller, Tamir

    2017-01-01

    Down regulation of mRNA translation is an important problem in various bio-medical domains ranging from developing effective medicines for tumors and for viral diseases to developing attenuated virus strains that can be used for vaccination. Here, we study the problem of down regulation of mRNA translation using a mathematical model called the ribosome flow model (RFM). In the RFM, the mRNA molecule is modeled as a chain of n sites. The flow of ribosomes between consecutive sites is regulated by n + 1 transition rates. Given a set of feasible transition rates, that models the outcome of all possible mutations, we consider the problem of maximally down regulating protein production by altering the rates within this set of feasible rates. Under certain conditions on the feasible set, we show that an optimal solution can be determined efficiently. We also rigorously analyze two special cases of the down regulation optimization problem. Our results suggest that one must focus on the position along the mRNA molecule where the transition rate has the strongest effect on the protein production rate. However, this rate is not necessarily the slowest transition rate along the mRNA molecule. We discuss some of the biological implications of these results.

  4. Optimal Down Regulation of mRNA Translation

    Science.gov (United States)

    Zarai, Yoram; Margaliot, Michael; Tuller, Tamir

    2017-01-01

    Down regulation of mRNA translation is an important problem in various bio-medical domains ranging from developing effective medicines for tumors and for viral diseases to developing attenuated virus strains that can be used for vaccination. Here, we study the problem of down regulation of mRNA translation using a mathematical model called the ribosome flow model (RFM). In the RFM, the mRNA molecule is modeled as a chain of n sites. The flow of ribosomes between consecutive sites is regulated by n + 1 transition rates. Given a set of feasible transition rates, that models the outcome of all possible mutations, we consider the problem of maximally down regulating protein production by altering the rates within this set of feasible rates. Under certain conditions on the feasible set, we show that an optimal solution can be determined efficiently. We also rigorously analyze two special cases of the down regulation optimization problem. Our results suggest that one must focus on the position along the mRNA molecule where the transition rate has the strongest effect on the protein production rate. However, this rate is not necessarily the slowest transition rate along the mRNA molecule. We discuss some of the biological implications of these results. PMID:28120903

  5. Detection of mRNA of the cyclin D1 breast cancer marker by a novel duplex-DNA probe.

    Science.gov (United States)

    Segal, Meirav; Yavin, Eylon; Kafri, Pinhas; Shav-Tal, Yaron; Fischer, Bilha

    2013-06-27

    Previously, we have described 5-((4-methoxy-phenyl)-trans-vinyl)-2'-deoxy-uridine, 6, as a fluorescent uridine analogue exhibiting a 3000-fold higher quantum yield (Φ 0.12) and maximum emission (478 nm) which is 170 nm red-shifted as compared to uridine. Here, we utilized 6 for preparation of labeled oligodeoxynucleotide (ODN) probes based on MS2 and cyclin D1 (a known breast cancer mRNA marker) sequences. Cyclin D1-derived labeled-ssODN showed a 9.5-fold decrease of quantum yield upon duplex formation. On the basis of this finding, we developed the ds-NIF (nucleoside with intrinsic fluorescence)-probe methodology for detection of cyclin D1 mRNA, by which the fluorescent probe is released upon recognition of target mRNA by the relatively dark NIF-duplex-probe. Indeed, we successfully detected, a ss-deoxynucleic acid (DNA) variant of cyclin D1 mRNA using a dark NIF-labeled duplex-probe, and monitoring the recognition process by fluorescence spectroscopy and gel electrophoresis. Furthermore, we successfully detected cyclin D1 mRNA in RNA extracted from cancerous human cells, using ds-NIF methodology.

  6. Nucleocytoplasmic transport of luciferase gene mRNA requires CRM1/Exportin1 and RanGTPase.

    Science.gov (United States)

    Kimura, Tominori; Hashimoto, Iwao; Nishikawa, Masao; Yamada, Hisao

    2009-06-01

    Human immunodeficiency virus type 1 Rev (regulator of the expression of the virion) protein was shown to reduce the expression level of the co-transfected luciferase reporter gene (luc+) introduced to monitor transfection efficiency. We studied the mechanism of the inhibitory Rev effect. The effect, caused by nuclear retention of luc+ mRNA, was reversed if rev had a point mutation that makes its nuclear export signal (NES) unable to associate with cellular transport factors. The Rev NES receptor CRM1 (chromosome region maintenance 1)-specific inhibitor, leptomycin B, blocked luc+ mRNA export. This finding was also supported by the overexpression of delta CAN, another specific CRM1 inhibitor that caused inhibition of luciferase gene expression. Experiments involving tsBN2 cells, which have a temperature-sensitive RCC1 (regulator of chromosome condensation 1) allele, demonstrated that luc+ expression required generation of the GTP-bound form of RanGTPase (RanGTP) by RCC1. The constitutive transport element (CTE)-mediated nuclear export of luc+ mRNA was found to also depend upon RanGTP. Nuclear export of luc+ mRNA is thus suggested to involve CRM1 and RanGTP, which Rev employs to transport viral mRNA. The Rev effect is therefore considered to involve competition between two molecules for common transport factors.

  7. Calprotectin mRNA (MRP8/MRP14 expression in neutrophils of periodontitis patients with type 2 diabetes mellitus

    Directory of Open Access Journals (Sweden)

    Ahmad Syaify

    2009-09-01

    Full Text Available Background: Calprotectin, a major cytosolic protein of leukocytes, is detected in neutrophils and monocytes/machrophages. This protein is known to be a marker for several inflammatory diseases including periodontitis. In type 2 diabetes mellitus patients, the severity of periodontitis was strongly thought to be caused by decreasing of leukocytes function such as neutrophils. Previous research found that the calprotectin level in serum of periodontitis patients with type 2 DM is higher than periodontits patients non DM. Purpose: The aim of this study was to determine calprotectin mRNA (MRP8/MRP14 expression in human neutrophils of periodontitis patients with type 2 diabetes mellitus. Methods: Neutrophils were isolated from the peripheral blood of periodontitis patients with uncontrolled type 2 DM, controlled type 2 DM, and non DM. The expression of calprotectin mRNA (MRP8 and MRP14 were detected by RTPCR. Result: The result showed that the value of mRNA calprotectin expression in DM patients were higher than non DM, and the highest expression was on the uncontrolled type 2 DM. Conclusion: The basal level of calprotectin mRNA MRP8/MRP14 expression increased in neutrophil of periodontitis patient with type 2 DM compared non diabetic subjects. It was suggested that high basal level of calprotectin mRNA has a role in the regulation of periodontitis severity with diabetes mellitus patients.

  8. MiR-506 suppresses liver cancer angiogenesis through targeting sphingosine kinase 1 (SPHK1) mRNA.

    Science.gov (United States)

    Lu, Zhanping; Zhang, Weiying; Gao, Shan; Jiang, Qiulei; Xiao, Zelin; Ye, Lihong; Zhang, Xiaodong

    MicroRNAs acting as oncogenes or tumor suppressor genes play crucial roles in human cancers. Sphingosine kinase 1 (SPHK1) and its metabolite sphingosine 1-phosphate (S1P) contribute to tumor angiogenesis. We have reported that the down-regulation of miR-506 targeting YAP mRNA results in the hepatocarcinogenesis. In the present study, we report a novel function of miR-506, which suppresses tumor angiogenesis through targeting SPHK1 mRNA in liver cancer. Bioinformatics analysis showed that miR-506 might target 3'-untranslated region (3'UTR) of SPHK1 mRNA. Then, we validated that by luciferase reporter gene assays. MiR-506 was able to reduce the expression of SPHK1 at the levels of mRNA and protein using reverse transcription-polymerase chain reaction and Western blot analysis in hepatoma HepG2 cells. Functionally, human umbilical vein endothelial cell (HUVEC) tube formation assays demonstrated that the forced miR-506 expression remarkably inhibited the production of S1P in the supernatant of hepatoma cells. The supernatant resulted in the inhibition of tumor angiogenesis. Interestingly, the supernatant with overexpression of SPHK1 could rescue the inhibition of angiogenesis of liver cancer mediated by miR-506. Anti-miR-506 increased the production of S1P in the supernatant of hepatoma cells, but the supernatant with silencing of SPHK1 abolished anti-miR-506-induced acceleration of tumor angiogenesis. Clinically, we observed that the levels of miR-506 were negatively related to those of SPHK1 mRNA in liver cancer tissues. Thus, we conclude that miR-506 depresses the angiogenesis of liver cancer through targeting 3'UTR of SPHK1 mRNA. Our finding provides new insights into the mechanism of tumor angiogenesis.

  9. Quantification of GPCR mRNA using real-time RT-PCR.

    Science.gov (United States)

    Brattelid, Trond; Levy, Finn Olav

    2011-01-01

    Characterisation of G-protein-coupled receptor (GPCR) mRNA expression under normal, different pharmacological and pathological conditions in experimental animal models and human tissue biopsies by quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) is a valuable approach to understand the regulation of GPCR expression. RT-qPCR is specific and sensitive with a broad dynamic range, which allows precise quantification of mRNA species of interest. In addition to measuring the relative levels of mRNA in a tissue or changes in expression levels between groups of genes of interest, RT-qPCR is also used to identify splice variants and single nucleotide polymorphisms (SNPs) of GPCRs. Even though RT-qPCR has become the standard method for quantification of gene expression, RT-qPCR is sensitive to RNA quality, assay design, normalisation approach and data analysis. This protocol is meant as a guide to RT-qPCR methodology with references to the best standard methods available at present.

  10. Downregulation of LH receptor mRNA in the rat uterus.

    Science.gov (United States)

    Kasahara, Yoshimitsu; Kitahara, Yoshikazu; Nakamura, Kazuto; Minegishi, Takashi

    2012-05-01

    We detected luteinizing hormone receptor (LHR) mRNA in the immature rat uterus by northern blotting and downregulation of this receptor mRNA after pregnant mare serum gonadotropin (PMSG)-human chorionic gonadotropin (hCG) treatment. After administration of hCG, the mRNA levels in the rat uterus declined to an extremely low level from Days 1 to 3 and then rebounded and reached higher than pretreatment values at Day 4. At Day 5 the levels were 3-fold higher than the control levels. The cultured uterus displayed an hCG concentration-dependent increase in cAMP production in the medium. Immunohistochemical experiments showed that these receptor proteins were expressed in the epithelial cells of the endometrium. These results suggest that functional LHRs are present in the immature rat uterus and are downregulated by signals resulting from hCG treatment. These data may support the idea that LH acts on the uterus to inhibit contraction at ovulation. Although the precise role of the LHR in the uterus remains unknown, this study may provide a model with which to investigate the regulation of LHR.

  11. Anabolic androgenic steroid nandrolone decanoate reduces hypothalamic proopiomelanocortin mRNA levels.

    Science.gov (United States)

    Lindblom, Jonas; Kindlundh, Anna M S; Nyberg, Fred; Bergström, Lena; Wikberg, Jarl E S

    2003-10-03

    Supratherapeutical doses of anabolic androgenic steroids (AASs) have dramatic effects on metabolism in humans, and also inhibit feeding and reduce the rate of body weight gain in rats. In order to test the hypothesis that the AAS metabolic syndrome is accompanied by alterations in the central melanocortin system, we evaluated body weight, food intake and hypothalamic agouti-related protein (AgRP) and proopiomelanocortin (POMC) mRNA levels following administration of different doses of the anabolic androgenic steroid nandrolone decanoate. In order to distinguish changes induced by the steroid treatment per se from those resulting from the reduced food intake and growth rate, we also compared the effect of nandrolone decanoate on AgRP and POMC mRNA expression with both normally fed, and food restricted control groups. We here report that administration of nandrolone specifically reduces arcuate nucleus POMC mRNA levels while not affecting the expression level of AgRP. The effect on POMC expression was not observed in the food restricted controls, excluding the possibility that the observed effect was a mere response to the reduced food intake and body weight. These results raise the possibility that some of the metabolic and behavioural consequences of AAS abuse may be the result of alterations in the melanocortin system.

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

    Directory of Open Access Journals (Sweden)

    Taylor John M

    2001-03-01

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

  13. Total RNA-seq to identify pharmacological effects on specific stages of mRNA synthesis.

    Science.gov (United States)

    Boswell, Sarah A; Snavely, Andrew; Landry, Heather M; Churchman, L Stirling; Gray, Jesse M; Springer, Michael

    2017-03-06

    Pharmacological perturbation is a powerful tool for understanding mRNA synthesis, but identification of the specific steps of this multi-step process that are targeted by small molecules remains challenging. Here we applied strand-specific total RNA sequencing (RNA-seq) to identify and distinguish specific pharmacological effects on transcription and pre-mRNA processing in human cells. We found unexpectedly that the natural product isoginkgetin, previously described as a splicing inhibitor, inhibits transcription elongation. Compared to well-characterized elongation inhibitors that target CDK9, isoginkgetin caused RNA polymerase accumulation within a broader promoter-proximal band, indicating that elongation inhibition by isoginkgetin occurs after release from promoter-proximal pause. RNA-seq distinguished isoginkgetin and CDK9 inhibitors from topoisomerase I inhibition, which alters elongation across gene bodies. We were able to detect these and other specific defects in mRNA synthesis at low sequencing depth using simple metagene-based metrics. These metrics now enable total-RNA-seq-based screening for high-throughput identification of pharmacological effects on individual stages of mRNA synthesis.

  14. The in vitro effect of desflurane preconditioning on endothelial adhesion molecules and mRNA expression.

    Science.gov (United States)

    Biao, Zhu; Zhanggang, Xue; Hao, Jiang; Changhong, Miao; Jing, Cang

    2005-04-01

    Lower expression of intercellular adhesion molecule-1 (ICAM-1), vascular adhesion molecule-1 (VCAM-1), and E-selectin may be responsible for attenuated ischemic-reperfusion neutrophil adhesion to vascular endothelium. Desflurane reduces ischemia-reperfusion injury. Therefore, we assessed whether desflurane affects the protein expression of ICAM-1 and E-selectin and mRNA expression of ICAM-1 and VCAM-1 of human umbilical venous endothelial cells (HUVEC) stimulated with tumor necrosis factor-alpha (TNF-alpha). HUVEC were preconditioned for 60 min with 1 minimum alveolar concentration desflurane before stimulating with TNF-alpha. Protein expression of adhesion molecules ICAM-1 and E-selectin of HUVEC were evaluated via immunocytochemical techniques combined with image cytometry. ICAM-1 and VCAM-1 mRNA expression of HUVEC were determined via reverse transcription-polymerase chain reaction. Desflurane not only reduced the protein expression of ICAM-1 and E-selectin but also ICAM-1 and VCAM-1 mRNA expression of the HUVEC. The adhesion rate of neutrophils with desflurane-treated HUVEC was slower. The decreased neutrophil adhesion on the desflurane-treated HUVEC correlated well with the decrease in adhesion molecule expression. These results show that desflurane affects the expression of adhesion molecules involved in the multistep process of neutrophil recruitment. Desflurane related ischemia-reperfusion injury reduction correlates well with expression inhibition of ICAM-1, VCAM-1, and E-selectin that mediates neutrophil rotation and firm adhesion on the vascular endothelium.

  15. PolyA_DB: a database for mammalian mRNA polyadenylation.

    Science.gov (United States)

    Zhang, Haibo; Hu, Jun; Recce, Michael; Tian, Bin

    2005-01-01

    Messenger RNA polyadenylation is one of the key post-transcriptional events in eukaryotic cells. A large number of genes in mammalian species can undergo alternative polyadenylation, which leads to mRNAs with variable 3' ends. As the 3' end of mRNAs often contains cis elements important for mRNA stability, mRNA localization and translation, the implications of the regulation of polyadenylation can be multifold. Alternative polyadenylation is controlled by cis elements and trans factors, and is believed to occur in a tissue- or disease-specific manner. Given the availability of many databases devoted to other aspects of mRNA metabolism, such as transcriptional initiation and splicing, systematic information on polyadenylation, including alternative polyadenylation and its regulation, is noticeably lacking. Here, we present a database named polyA_DB, through which we strive to provide several types of information regarding polyadenylation in mammalian species: (i) polyadenylation sites and their locations with respect to the genomic structure of genes; (ii) cis elements surrounding polyadenylation sites; (iii) comparison of polyadenylation configuration between orthologous genes; and (iv) tissue/organ information for alternative polyadenylation sites. Currently, polyA_DB contains 45,565 polyadenylation sites for 25,097 human and mouse genes, representing the most comprehensive polyadenylation database till date. The database is accessible via the website (http://polya.umdnj.edu/polyadb).

  16. Yeast Ataxin-7 links histone deubiquitination with gene gating and mRNA export.

    Science.gov (United States)

    Köhler, Alwin; Schneider, Maren; Cabal, Ghislain G; Nehrbass, Ulf; Hurt, Ed

    2008-06-01

    Targeting of a gene to the nuclear pore complexes (NPCs), known as gene gating, can affect its transcriptional state. However, the mechanism underlying gene gating is poorly understood. Here, we have identified SAGA-associated Sgf73 (ref. 10), the yeast orthologue of human Ataxin-7 (ref. 11), as a regulator of histone H2B ubiquitin levels, a modification linked to both transcription initiation and elongation. Sgf73 is a key component of a minimal histone-deubiquitinating complex. Activation of the H2B deubiquitinating protease, Ubp8, is cooperative and requires complex formation with the amino-terminal zinc-finger-containing domain of Sgf73 and Sgf11-Sus1. Through a separate domain, Sgf73 mediates recruitment of the TREX-2 mRNA export factors Sac3 and Thp1 to SAGA and their stable interaction with Sus1-Cdc31. This latter step is crucial to target TREX-2 to the NPC. Loss of Sgf73 from SAGA abrogates gene gating of GAL1 and causes a GAL1 mRNA export defect. Thus, Sgf73 provides a molecular scaffold to integrate the regulation of H2B ubiquitin levels, tethering of a gene to the NPC and export of mRNA.

  17. Nonsense-mediated mRNA decay among coagulation factor genes

    Directory of Open Access Journals (Sweden)

    Shirin Shahbazi

    2016-04-01

    Full Text Available Objective(s: Haemostasis prevents blood loss following vascular injury. It depends on the unique concert of events involving platelets and specific blood proteins, known as coagulation factors. The clotting system requires precise regulation and coordinated reactions to maintain the integrity of the vasculature. Clotting insufficiency mostly occurs due to genetically inherited coagulation factor deficiencies such as hemophilia. Materials and Methods: A relevant literature search of PubMed was performed using the keywords coagulation factors, Nonsense-mediated mRNA decay and premature translation termination codons. Search limitations included English language and human-based studies. Results: Mutations that cause premature translation termination codons probably account for one-third of genetically inherited diseases. Transcripts bearing aberrant termination codons are selectively identified and eliminated by an evolutionarily conserved posttranscriptional pathway known as nonsense-mediated mRNA decay (NMD. There are many pieces of evidence of decay among coagulation factor genes. However, the hemophilia gene (F8 does not seem to be subjected to NMD. Since the F8 gene is located on the X-chromosome, a connection between X-linked traits and mRNA decay could be assumed. Conclusion: Considering that not all genes go through decay, this review focuses on the basics of the mechanism in coagulation genes. It is interesting to determine whether this translation-coupled surveillance system represents a general rule for the genes encoding components of the same physiological cascade.

  18. Expression of matrix extracellular phosphoglycoprotein mRNA in human periodontal ligament cell osteogenic differentiation%细胞外基质磷酸化糖蛋白mRNA在人牙周韧带细胞成骨分化过程中的表达

    Institute of Scientific and Technical Information of China (English)

    吴莉萍; 韦曦; 凌均棨; 刘路

    2008-01-01

    Objective To investigate the mineralization capacity of periodontal ligament stem cells (PDLC) by determining the mRNA expressions of alkaline phesphatase (ALP), osteocalein (OCN) and matrix extracellular phosphoglycoprotein (MEPE) and to explore the potential of MEPE as a differentiation marker for PDLC, and its possible function in PDLC osteogenic differentiation. Methods PDLC were digested and cultured by a solution containing collagenase type Ⅰ and dispase. PDLC were preceded to osteogenic induction for 7, 14 and 21 days respectively, and the cells before induction served as controls.Mineralization nodules and the expression of OCN in PDLC were investigated by alizarin red and immunohistochemistry respectively. The expressions of ALP, OCN and MEPE mRNA were investigated by quantitative real-time RT-PCR analysis. Statistical analysis was performed to compare the differences of mRNA expression levels among cell samples collected at different time points. Results The mRNA expressions of ALP, OCN and MEPE in PDLC before induction were 72, 1.1 and 534 respectively, but increased time-dependently in the induction cultures. The mRNA expressions of ALP, OCN and MEPE were 78,9.56 and 629.6 on day 7;290, 133 and 638.3 on day 14;1108, 925 and 2261.1 on day 21 respectively. The relative mRNA levels of OCN,ALP on day 14 and 21, MEPE on day 21 were significantly higher than control group (P < 0.05 ). Conclusions PDLC showed analogously temporal expression of ALP, OCN and MEPE mRNA while differentiating into cementoblast/osteoblast-like cells in vitro. MEPE may play a regulatory role in PDLC osteogenie differentiation, and may be a potential osteogenic differentiation marker along with ALP and OCN.%目的 检测人牙周韧带细胞(periodontal ligament stem cell,PDLC)诱导矿化过程中碱性磷酸酶(alkaline phosphatase,ALP)、骨钙素(osteocalcin,OCN)和细胞外基质磷酸化糖蛋白(matrix extracellular phosphoglycoprotein,MEPE)mRNA的表达,探讨MEPE能否作

  19. Androgen-dependent loss of muscle BDNF mRNA in two mouse models of SBMA.

    Science.gov (United States)

    Halievski, Katherine; Henley, Casey L; Domino, Laurel; Poort, Jessica E; Fu, Martina; Katsuno, Masahisa; Adachi, Hiroaki; Sobue, Gen; Breedlove, S Marc; Jordan, Cynthia L

    2015-07-01

    Transgenic expression of neurotrophic factors in skeletal muscle has been found to protect mice from neuromuscular disease, including spinal bulbar muscular atrophy (SBMA), triggering renewed interest in neurotrophic factors as therapeutic agents for treating neuromuscular disease