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Sample records for rb1 rna levels

  1. Zebrafish usp39 mutation leads to rb1 mRNA splicing defect and pituitary lineage expansion.

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    Yesenia Ríos

    2011-01-01

    Full Text Available Loss of retinoblastoma (Rb tumor suppressor function is associated with human malignancies. Molecular and genetic mechanisms responsible for tumorigenic Rb downregulation are not fully defined. Through a forward genetic screen and positional cloning, we identified and characterized a zebrafish ubiquitin specific peptidase 39 (usp39 mutation, the yeast and human homolog of which encodes a component of RNA splicing machinery. Zebrafish usp39 mutants exhibit microcephaly and adenohypophyseal cell lineage expansion without apparent changes in major hypothalamic hormonal and regulatory signals. Gene expression profiling of usp39 mutants revealed decreased rb1 and increased e2f4, rbl2 (p130, and cdkn1a (p21 expression. Rb1 mRNA overexpression, or antisense morpholino knockdown of e2f4, partially reversed embryonic pituitary expansion in usp39 mutants. Analysis of pre-mRNA splicing status of critical cell cycle regulators showed misspliced Rb1 pre-mRNA resulting in a premature stop codon. These studies unravel a novel mechanism for rb1 regulation by a neuronal mRNA splicing factor, usp39. Zebrafish usp39 regulates embryonic pituitary homeostasis by targeting rb1 and e2f4 expression, respectively, contributing to increased adenohypophyseal sensitivity to these altered cell cycle regulators. These results provide a mechanism for dysregulated rb1 and e2f4 pathways that may result in pituitary tumorigenesis.

  2. Pumilio and nanos RNA-binding proteins counterbalance the transcriptional consequences of RB1 inactivation.

    Science.gov (United States)

    Miles, Wayne O; Dyson, Nicholas J

    2014-01-01

    The ability of the retinoblastoma protein (RB) tumor suppressor to repress transcription stimulated by the E2 promoter binding factors (E2F) is integral to its biological functions. Our recent report described a conserved feedback mechanism mediated by the RNA-binding proteins Pumilio and Nanos that increases in importance following RB loss and helps cells to tolerate deregulated E2F.

  3. Rb1 loss modifies but does not initiate alveolar rhabdomyosarcoma

    Science.gov (United States)

    2013-01-01

    Background Alveolar rhabdomyosarcoma (aRMS) is a myogenic childhood sarcoma frequently associated with a translocation-mediated fusion gene, Pax3:Foxo1a. Methods We investigated the complementary role of Rb1 loss in aRMS tumor initiation and progression using conditional mouse models. Results Rb1 loss was not a necessary and sufficient mutational event for rhabdomyosarcomagenesis, nor a strong cooperative initiating mutation. Instead, Rb1 loss was a modifier of progression and increased anaplasia and pleomorphism. Whereas Pax3:Foxo1a expression was unaltered, biomarkers of aRMS versus embryonal rhabdomyosarcoma were both increased, questioning whether these diagnostic markers are reliable in the context of Rb1 loss. Genome-wide gene expression in Pax3:Foxo1a,Rb1 tumors more closely approximated aRMS than embryonal rhabdomyosarcoma. Intrinsic loss of pRb function in aRMS was evidenced by insensitivity to a Cdk4/6 inhibitor regardless of whether Rb1 was intact or null. This loss of function could be attributed to low baseline Rb1, pRb and phospho-pRb expression in aRMS tumors for which the Rb1 locus was intact. Pax3:Foxo1a RNA interference did not increase pRb or improve Cdk inhibitor sensitivity. Human aRMS shared the feature of low and/or heterogeneous tumor cell pRb expression. Conclusions Rb1 loss from an already low pRb baseline is a significant disease modifier, raising the possibility that some cases of pleomorphic rhabdomyosarcoma may in fact be Pax3:Foxo1a-expressing aRMS with Rb1 or pRb loss of function. PMID:24274149

  4. Splicing aberrations caused by constitutional RB1 gene mutations in ...

    Indian Academy of Sciences (India)

    in this family revealed skipping of exon 22 in three members of this family. In one proband, a ... This study reveals novel effects of RB1 mutations on splicing and suggests the utility of RNA analysis as an ... of life) and presence of multiple tumors (multifocal). The ..... spliced RNA have been linked to parent of origin as well as.

  5. Ginsenoside Rb1 improves postoperative fatigue syndrome by reducing skeletal muscle oxidative stress through activation of the PI3K/Akt/Nrf2 pathway in aged rats.

    Science.gov (United States)

    Zhuang, Cheng-Le; Mao, Xiang-Yu; Liu, Shu; Chen, Wei-Zhe; Huang, Dong-Dong; Zhang, Chang-Jing; Chen, Bi-Cheng; Shen, Xian; Yu, Zhen

    2014-10-05

    Ginsenoside Rb1 is reported to possess anti-fatigue activity, but the mechanisms remain unknown. The aim of this study was to investigate the molecular mechanisms responsible for the anti-fatigue effect of ginsenoside Rb1 on postoperative fatigue syndrome induced by major small intestinal resection (MSIR) in aged rat. Aged rats with MSIR were administrated with ginsenoside Rb1 (15 mg/kg) once a day from 3 days before surgery to the day of sacrifice, or with saline as corresponding controls. Rats without MSIR but going through the same surgery procedure were administrated with saline as blank controls. Anti-fatigue effect was assessed by an open field test; superoxide dismutase, reactive oxygen species and malondialdehyde in skeletal muscle were determined. The mRNA levels of Akt2 and Nrf2 in skeletal muscle were measured by real-time quantitative PCR. The activation of Akt and Nrf2 was examined by western blot and immunohistofluorescence. Our results revealed that ginsenoside Rb1 significantly increased the journey and the rearing frequency, decreased the time of rest in aged rats with MSIR. In addition, ginsenoside Rb1 significantly reduced reactive oxygen species and malondialdehyde release and increased the superoxide dismutase activity of skeletal muscle in aged rats with MSIR. Ginsenoside Rb1 also increased the expression of Akt2 and Nrf2 mRNA, up-regulated Akt phosphorylation and Nrf2 nuclear translocation. These findings indicate that ginsenoside Rb1 has an anti-fatigue effect on postoperative fatigue syndrome in aged rat, and the mechanism possibly involves activation of the PI3K/Akt pathway with subsequent Nrf2 nuclear translocation and induction of antioxidant enzymes. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. Impact of miR-208 and its Target Gene Nemo-Like Kinase on the Protective Effect of Ginsenoside Rb1 in Hypoxia/Ischemia Injuried Cardiomyocytes

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    Xu Yan

    2016-09-01

    Full Text Available Background/Aims: Ginsenoside Rb1 (GS-Rb1 is one of the most important active pharmacological extracts of the Traditional Chinese Medicine ginseng, with extensive evidence of its cardioprotective properties. Mir-208 has been shown to act as a biomarker of acute myocardial infarction in vivo studies including man. However the impact of miR-208 on the protective effect of GS-Rb1 in hypoxia/ischemia injured cardiomyocytes remains unclear. The current study aims to investigate the target gene of miR-208 and the impact on the protective effect of GS-Rb1 in hypoxia/ischemia (H/I injuried cardiomyocytes. Materials and Methods: Primary cultures of neonatal rat cardiomyocytes (NRCMs was subjected to the H/I conditions with or without GS-Rb1. Cell viability was calculated by MTT assay and confirmed by flow cytometry analysis. Mir-208 was then detected by qRT-PCR. Luciferase reporter assay was carried out to detect the target gene of Mir-208. Then the NRCMs were transfected with miR-208 mimics and inhibitors to evaluate the impact on cardioprotective properties of Rb1. Results: The miR-208 expression level was clearly upregulated in the H/I treated NRCMs accompanied by the percentage of the apoptotic cells which could be reversed by GS-Rb1 pretreatment. The nemo-like kinase (NLK mRNA and protein expression levels were decreased in H/I group measured by RT-PCR and western blotting. Luciferase activity assay was then carried out to identify that NLK may be a direct target of mir-208. MTT assay showed that miR-208 inhibitor slightly decreased the protective effect of Rb1 on the H/I impaired NRCMs. However, results showed no statistical difference. Conclusions: These findings proved that NLK was a direct target of mir-208 and miR-208 act indirectly during Rb1 protecting H/I impaired NRCMs and further researches were needed to explore the relationship that microRNAs and other signal pathways in the protective effect of GS-Rb1 on the hypoxia/ischemia injuries in

  7. Frequent disruption of the RB1 pathway in diffuse large B cell lymphoma

    DEFF Research Database (Denmark)

    Møller, Michael Boe; Kania, P W; Ino, Y

    2000-01-01

    In the present study, we analysed 34 de novo diffuse large B cell lymphoma (DLCL) from a population-based lymphoma registry for alterations of the RB1 pathway at the genetic (RB1 and CDK4) and protein (pRb, cyclin D1, cyclin D3, CDK4, and E2F-1) level. The results were correlated with the data fr...

  8. RB1CC1 Protein Suppresses Type II Collagen Synthesis in Chondrocytes and Causes Dwarfism*

    Science.gov (United States)

    Nishimura, Ichiro; Chano, Tokuhiro; Kita, Hiroko; Matsusue, Yoshitaka; Okabe, Hidetoshi

    2011-01-01

    RB1-inducible coiled-coil 1 (RB1CC1) functions in various processes, such as cell growth, differentiation, senescence, apoptosis, and autophagy. The conditional transgenic mice with cartilage-specific RB1CC1 excess that were used in the present study were made for the first time by the Cre-loxP system. Cartilage-specific RB1CC1 excess caused dwarfism in mice without causing obvious abnormalities in endochondral ossification and subsequent skeletal development from embryo to adult. In vitro and in vivo analysis revealed that the dwarf phenotype in cartilaginous RB1CC1 excess was induced by reductions in the total amount of cartilage and the number of cartilaginous cells, following suppressions of type II collagen synthesis and Erk1/2 signals. In addition, we have demonstrated that two kinds of SNPs (T-547C and C-468T) in the human RB1CC1 promoter have significant influence on the self-transcriptional level. Accordingly, human genotypic variants of RB1CC1 that either stimulate or inhibit RB1CC1 transcription in vivo may cause body size variations. PMID:22049074

  9. The origin of the RB1 imprint.

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    Deniz Kanber

    Full Text Available The human RB1 gene is imprinted due to a differentially methylated CpG island in intron 2. This CpG island is part of PPP1R26P1, a truncated retrocopy of PPP1R26, and serves as a promoter for an alternative RB1 transcript. We show here by in silico analyses that the parental PPP1R26 gene is present in the analysed members of Haplorrhini, which comprise Catarrhini (Old World Monkeys, Small apes, Great Apes and Human, Platyrrhini (New World Monkeys and tarsier, and Strepsirrhini (galago. Interestingly, we detected the retrocopy, PPP1R26P1, in all Anthropoidea (Catarrhini and Platyrrhini that we studied but not in tarsier or galago. Additional retrocopies are present in human and chimpanzee on chromosome 22, but their distinct composition indicates that they are the result of independent retrotransposition events. Chimpanzee and marmoset have further retrocopies on chromosome 8 and chromosome 4, respectively. To examine the origin of the RB1 imprint, we compared the methylation patterns of the parental PPP1R26 gene and its retrocopies in different primates (human, chimpanzee, orangutan, rhesus macaque, marmoset and galago. Methylation analysis by deep bisulfite sequencing showed that PPP1R26 is methylated whereas the retrocopy in RB1 intron 2 is differentially methylated in all primates studied. All other retrocopies are fully methylated, except for the additional retrocopy on marmoset chromosome 4, which is also differentially methylated. Using an informative SNP for the methylation analysis in marmoset, we could show that the differential methylation pattern of the retrocopy on chromosome 4 is allele-specific. We conclude that the epigenetic fate of a PPP1R26 retrocopy after integration depends on the DNA sequence and selective forces at the integration site.

  10. RB1CC1 activates RB1 pathway and inhibits proliferation and cologenic survival in human cancer.

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    Tokuhiro Chano

    2010-06-01

    Full Text Available RB1-inducible coiled-coil 1 (RB1CC1, also known as FIP200 plays a role in the enhancement of the RB1 pathway through the direct binding to a GC-rich region 201bp upstream (from the initiation ATG of the RB1 promoter. Here, we identified hSNF5 and p53 as the binding partners of RB1CC1 by immunoprecipitation and immunofluorescence assays. Interaction between these molecules and the RB1 pathway was analyzed by the assays of chromatin immunoprecipitation, luciferase-reporter, reverse transcription-polymerase chain reaction and immunoblot. The tumor growth suppression by RB1CC1 was evaluated by flow cytometry or by a cell growth assay. The nuclear RB1CC1 complex involving hSNF5 and/or p53 activated transcription of RB1, p16 and p21, and suppressed tumor cell growth. Furthermore, nuclear RB1CC1 expression significantly correlated with those of RB1 and p16 in breast cancer tissue in vivo, and the Ki-67 proliferation index was dependent on p53 as well as RB1CC1. The present study indicates that RB1CC1 together with hSNF5 and/or p53 enhances the RB1 pathway through transcriptional activation of RB1, p16 and p21. Evaluation of RB1CC1 expression combined with RB1 and p53 status is expected to provide useful information in clinical practice and future therapeutic strategies in breast cancer.

  11. Brain Transport Profiles of Ginsenoside Rb1 by Glucose Transporter 1: In Vitro and in Vivo

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    Yu-Zhu Wang

    2018-04-01

    Full Text Available Ginsenoside Rb1 (Rb1 has been demonstrated its protection for central nervous system and is apparently highly distributed to the brain. The objective of this study was to characterize Rb1 transport at the blood–brain barrier (BBB using primary cultured rat brain microvascular endothelial cells (rBMEC, an in vitro BBB model. The initial uptake velocity of Rb1 in rBMEC was temperature- and concentration-dependent, and was significantly reduced by phloretin, an inhibitor of GLUT1 transporter, but was independent of metabolic inhibitor. Furthermore, the transport of Rb1 into rBMEC was significantly diminished in the presence of natural substrate α-D-glucose, suggesting a facilitated transport of Rb1 via GLUT1 transporter. The impact of GLUT1 on the distribution of Rb1 between brain and plasma was studied experimentally in rats. Administration of phloretin (5 mg/kg, i.v. to normal rats for consecutive 1 week before Rb1 (10 mg/kg, i.v. at 0.5, 2, and 6 h did not alter Rb1 concentrations in plasma, but resulted in significant decreased brain concentrations of Rb1 compared to in the phloretin-untreated normal rats (489.6 ± 58.3 versus 105.1 ± 15.1 ng/g, 193.8 ± 11.1 versus 84.8 ± 4.1 ng/g, and 114.2 ± 24.0 versus 39.9 ± 4.9 ng/g, respectively. The expression of GLUT1 in the phloretin-treated group by western blotting analysis in vitro and in vivo experiments was significantly decreased, indicating that the decreased transport of Rb1 in brain was well related to the down-regulated function and level of GLUT1. Therefore, our in vitro and in vivo results indicate that the transport of Rb1 at the BBB is at least partly mediated by GLUT1 transporter.

  12. Ginsenosides Rb1 and Rg1 Stimulate Melanogenesis in Human Epidermal Melanocytes via PKA/CREB/MITF Signaling

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    Mao Lin

    2014-01-01

    Full Text Available Reduced or defective melanin skin pigmentation may cause many hypopigmentation disorders and increase the risk of damage to the skin triggered by UV irradiation. Ginsenosides Rb1 and Rg1 have many molecular targets including the cAMP-response element-binding protein (CREB, which is involved in melanogenesis. This study aimed to investigate the effects of ginsenosides Rb1 and Rg1 on melanogenesis in human melanocytes and their related mechanisms. The effects of Rb1 and Rg1 on cell viability, tyrosinase activity, cellular melanin content and protein levels of tyrosinase, microphthalmia-associated transcription factor (MITF, and activation of CREB in melanocytes were assessed. Results showed that Rb1 or Rg1 significantly increased cellular melanin content and tyrosinase activity in a dose-dependent manner. By contrast, the cell viability of melanocytes remained unchanged. After exposure to Rb1 or Rg1, the protein levels of tyrosinase, MITF, and phosphorylated CREB were significantly increased. Furthermore, pretreatment with the selective PKA inhibitor H-89 significantly blocked the Rb1- or Rg1-induced increase of melanin content. These findings indicated that Rb1 and Rg1 increased melanogenesis and tyrosinase activity in human melanocytes, which was associated with activation of PKA/CREB/MITF signaling. The effects and mechanisms of Rb1 or Rg1 on skin pigmentation deserve further study.

  13. Microbial conversion of major ginsenoside Rb1 to minor ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-12-15

    Dec 15, 2009 ... minor ginsenoside Rd by Indian fermented food bacteria. Kalaiselvi ... South Korea. Accepted 17 April, 2009. Ginsenoside Rb1 is the predominant secondary metabolite (saponin) in Panax ginseng. Hydrolysis of the sugar .... culture and 200 µl of major ginsenoside Rb1. The reaction mixture was incubated ...

  14. Analysis list: RB1 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available RB1 Prostate,Uterus + hg19 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/target/R...B1.1.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/target/RB1.5.tsv http://dbarchive.biosciencedbc.jp/...kyushu-u/hg19/target/RB1.10.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/colo/RB1.Prostate.tsv,http://dbarchive.bioscience...dbc.jp/kyushu-u/hg19/colo/RB1.Uterus.tsv http://dbarchive.bioscience...dbc.jp/kyushu-u/hg19/colo/Prostate.gml,http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/colo/Uterus.gml ...

  15. Rapid detection of RB1 recurrent mutations in retinoblastoma by ...

    Indian Academy of Sciences (India)

    In about half of the patients, one mutation is inherited via the germinal cells, while in the .... mutational hot spots in the RB1 gene, making genetic testing complex and challenging ... by direct sequencing. High normal background in sequenc-.

  16. Ginsenoside Rb1 Reduces Nitric Oxide Production via Inhibition of ...

    African Journals Online (AJOL)

    Inhibition of Nuclear Factor-κB Activation in Interleukin-1β- ... 20, 40, 80 µM ginsenoside Rb1. NO concentration was assessed by the Griess reaction. ... International Pharmaceutical Abstract, Chemical Abstracts, Embase, Index Copernicus, ...

  17. Inoculation of Pichia kudriavzevii RB1 degrades the organic acids present in raw compost material and accelerates composting.

    Science.gov (United States)

    Nakasaki, Kiyohiko; Araya, Shogo; Mimoto, Hiroshi

    2013-09-01

    In this study, the yeast strain Pichia kudriavzevii RB1 was used as an inoculum to accelerate organic matter degradation of rabbit food with added organic acids, which was used as a model food waste for composting. The RB1 strain rapidly degraded the organic acids present in the raw compost material, leading to an increase in pH beyond the neutral level, within 2 days. Both mesophilic and thermophilic bacteria proliferated faster in the compost with RB1 inoculation than in that without inoculation. Although the yeast died with the increase in compost temperature, it affected the early stages of composting prior to the thermophilic stage and accelerated the composting process by 2 days by eliminating the initial lag phase seen in the growth of other microorganisms. Moreover, populations of Bacillus thermoamylovorans, Bacillus foraminis, and Bacillus coagulans became dominant during the thermophilic stages of both composting with and without RB1 inoculation. Copyright © 2013 Elsevier Ltd. All rights reserved.

  18. RB1 mutations and second primary malignancies after hereditary retinoblastoma

    NARCIS (Netherlands)

    Dommering, Charlotte J.; Marees, Tamara; van der Hout, Annemarie H.; Imhof, Saskia M.; Meijers-Heijboer, Hanne; Ringens, Peter J.; van Leeuwen, Flora E.; Moll, Annette C.

    2012-01-01

    Survivors of hereditary retinoblastoma have a high risk of second primary malignancies, but it has not been investigated whether specific RB1 germline mutations are associated with greater risk of second primary malignancies in a large cohort. We conducted a retrospective cohort study of 199

  19. RB1 mutations and second primary malignancies after hereditary retinoblastoma

    NARCIS (Netherlands)

    Dommering, Charlotte J.; Marees, Tamara; van der Hout, Annemarie H.; Imhof, Saskia M.; Meijers-Heijboer, Hanne; Ringens, Peter J.; van Leeuwen, Flora E.; Moll, Annette C.

    Survivors of hereditary retinoblastoma have a high risk of second primary malignancies, but it has not been investigated whether specific RB1 germline mutations are associated with greater risk of second primary malignancies in a large cohort. We conducted a retrospective cohort study of 199

  20. Neuroprotective effects of ginsenoside Rb1 on high glucose-induced neurotoxicity in primary cultured rat hippocampal neurons.

    Science.gov (United States)

    Liu, Di; Zhang, Hong; Gu, Wenjuan; Liu, Yuqin; Zhang, Mengren

    2013-01-01

    Ginsenoside Rb1 is one of the main active principles in traditional herb ginseng and has been reported to have a wide variety of neuroprotective effects. Endoplasmic reticulum (ER) stress has been implicated in neurodegenerative diseases, so the present study aimed to observe the effects of ginsenoside Rb1 on ER stress signaling pathways in high glucose-treated hippocampal neurons. The results from MTT, TUNEL labeling and Annexin V-FITC/PI/Hoechst assays showed that incubating neurons with 50 mM high glucose for 72 h decreased cell viability and increased the number of apoptotic cells whereas treating neurons with 1 μM Rb1 for 72 h protected the neurons against high glucose-induced cell damage. Further molecular mechanism study demonstrated that Rb1 suppressed the activation of ER stress-associated proteins including protein kinase RNA (PKR)-like ER kinase (PERK) and C/EBP homology protein (CHOP) and downregulation of Bcl-2 induced by high glucose. Moreover, Rb1 inhibited both the elevation of intracellular reactive oxygen species (ROS) and the disruption of mitochondrial membrane potential induced by high glucose. In addition, the high glucose-induced cell apoptosis, activation of ER stress, ROS accumulation and mitochondrial dysfunction can also be attenuated by the inhibitor of ER stress 4-phenylbutyric acid (4-PBA) and anti-oxidant N-acetylcysteine(NAC). In conclusion, these results suggest that Rb1 may protect neurons against high glucose-induced cell injury through inhibiting CHOP signaling pathway as well as oxidative stress and mitochondrial dysfunction.

  1. Thermodynamics, dielectric permittivity and phase diagrams of the Rb1-x(NH4xH2PO4 type proton glasses

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    S.I. Sorokov

    2010-01-01

    Full Text Available The cluster pseudospin model of proton glasses, which takes into account the energy levels of protons around the PO4 group, the long-range interactions between the hydrogen bonds, and an internal random deformational field is used to investigate thermodynamical characteristics, longitudinal and transverse dielectric permittivities of Rb1-x(ND4xD2PO4 and Rb1-x(NH4xH2AsO4 compounds. A review of experimental and theoretical works on the Rb1-x(NH4xH2PO4 type crystals is presented.

  2. RB1 mutation spectrum in a comprehensive nationwide cohort of retinoblastoma patients

    NARCIS (Netherlands)

    Dommering, Charlotte J.; Mol, Berber M.; Moll, Annette C.; Burton, Margaret; Cloos, Jacqueline; Dorsman, Josephine C.; Meijers-Heijboer, Hanne; van der Hout, Annemarie H.

    Background Retinoblastoma (Rb) is a childhood cancer of the retina, commonly initiated by biallelic inactivation of the RB1 gene. Knowledge of the presence of a heritable RB1 mutation can help in risk management and reproductive decision making. We report here on RB1 mutation scanning in a unique

  3. Mitotic defects lead to pervasive aneuploidy and accompany loss of RB1 activity in mouse LmnaDhe dermal fibroblasts.

    Directory of Open Access Journals (Sweden)

    C Herbert Pratt

    2011-03-01

    Full Text Available Lamin A (LMNA is a component of the nuclear lamina and is mutated in several human diseases, including Emery-Dreifuss muscular dystrophy (EDMD; OMIM ID# 181350 and the premature aging syndrome Hutchinson-Gilford progeria syndrome (HGPS; OMIM ID# 176670. Cells from progeria patients exhibit cell cycle defects in both interphase and mitosis. Mouse models with loss of LMNA function have reduced Retinoblastoma protein (RB1 activity, leading to aberrant cell cycle control in interphase, but how mitosis is affected by LMNA is not well understood.We examined the cell cycle and structural phenotypes of cells from mice with the Lmna allele, Disheveled hair and ears (Lmna(Dhe. We found that dermal fibroblasts from heterozygous Lmna(Dhe (Lmna(Dhe/+ mice exhibit many phenotypes of human laminopathy cells. These include severe perturbations to the nuclear shape and lamina, increased DNA damage, and slow growth rates due to mitotic delay. Interestingly, Lmna(Dhe/+ fibroblasts also had reduced levels of hypophosphorylated RB1 and the non-SMC condensin II-subunit D3 (NCAP-D3, a mitosis specific centromere condensin subunit that depends on RB1 activity. Mitotic check point control by mitotic arrest deficient-like 1 (MAD2L1 also was perturbed in Lmna(Dhe/+ cells. Lmna(Dhe/+ fibroblasts were consistently aneuploid and had higher levels of micronuclei and anaphase bridges than normal fibroblasts, consistent with chromosome segregation defects.These data indicate that RB1 may be a key regulator of cellular phenotype in laminopathy-related cells, and suggest that the effects of LMNA on RB1 include both interphase and mitotic cell cycle control.

  4. Mitotic Defects Lead to Pervasive Aneuploidy and Accompany Loss of RB1 Activity in Mouse LmnaDhe Dermal Fibroblasts

    Science.gov (United States)

    Pratt, C. Herbert; Curtain, Michelle; Donahue, Leah Rae; Shopland, Lindsay S.

    2011-01-01

    Background Lamin A (LMNA) is a component of the nuclear lamina and is mutated in several human diseases, including Emery-Dreifuss muscular dystrophy (EDMD; OMIM ID# 181350) and the premature aging syndrome Hutchinson-Gilford progeria syndrome (HGPS; OMIM ID# 176670). Cells from progeria patients exhibit cell cycle defects in both interphase and mitosis. Mouse models with loss of LMNA function have reduced Retinoblastoma protein (RB1) activity, leading to aberrant cell cycle control in interphase, but how mitosis is affected by LMNA is not well understood. Results We examined the cell cycle and structural phenotypes of cells from mice with the Lmna allele, Disheveled hair and ears (LmnaDhe). We found that dermal fibroblasts from heterozygous LmnaDhe (LmnaDhe/+) mice exhibit many phenotypes of human laminopathy cells. These include severe perturbations to the nuclear shape and lamina, increased DNA damage, and slow growth rates due to mitotic delay. Interestingly, LmnaDhe/+ fibroblasts also had reduced levels of hypophosphorylated RB1 and the non-SMC condensin II-subunit D3 (NCAP-D3), a mitosis specific centromere condensin subunit that depends on RB1 activity. Mitotic check point control by mitotic arrest deficient-like 1 (MAD2L1) also was perturbed in LmnaDhe /+ cells. LmnaDhe /+ fibroblasts were consistently aneuploid and had higher levels of micronuclei and anaphase bridges than normal fibroblasts, consistent with chromosome segregation defects. Conclusions These data indicate that RB1 may be a key regulator of cellular phenotype in laminopathy-related cells, and suggest that the effects of LMNA on RB1 include both interphase and mitotic cell cycle control. PMID:21464947

  5. Effects of Ginsenoside Rb1 on Skin Changes

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    Yoshiyuki Kimura

    2012-01-01

    Full Text Available Ginseng roots (Panax ginseng CA Meyer have been used traditionally for the treatment, especially prevention, of various diseases in China, Korea, and Japan. Both experimental and clinical studies suggest ginseng roots to have pharmacological effects in patients with life-style-related diseases such as non-insulin-dependent diabetic mellitus, atherosclerosis, hyperlipidemia, and hypertension. The topical use of ginseng roots to treat skin complaints including atopic suppurative dermatitis, wounds, and inflammation is also described in ancient Chinese texts; however, there have been relatively few studies in this area. In the present paper, we describe introduce the biological and pharmacological effects of ginsenoside Rb1 isolated from Red ginseng roots on skin damage caused by burn-wounds using male Balb/c mice (in vivo and by ultraviolet B irradiation using male C57BL/6J and albino hairless (HR-1 mice (in vivo. Furthermore, to clarify the mechanisms behind these pharmacological actions, human primary keratinocytes and the human keratinocyte cell line HaCaT were used in experiments in vitro.

  6. Purification and Characterization of a Ginsenoside Rb1-Hydrolyzing β-Glucosidase from Aspergillus niger KCCM 11239

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    Kyung Hoon Chang

    2012-09-01

    Full Text Available Rb1-hydrolyzing β-glucosidase from Aspergillus niger KCCM 11239 was studied to develop a bioconversion process for minor ginsenosides. The specific activity of the purified enzyme was 46.5 times greater than that of the crude enzyme. The molecular weight of the native enzyme was estimated to be approximately 123 kDa. The optimal pH of the purified enzyme was pH 4.0, and the enzyme proved highly stable over a pH range of 5.0–10.0. The optimal temperature was 70 °C, and the enzyme became unstable at temperatures above 60 °C. The enzyme was inhibited by Cu2+, Mg2+, Co2+, and acetic acid (10 mM. In the specificity tests, the enzyme was found to be active against ginsenoside Rb1, but showed very low levels of activity against Rb2, Rc, Rd, Re, and Rg1. The enzyme hydrolyzed the 20-C,β-(1→6-glucoside of ginsenoside Rb1 to generate ginsenoside Rd and Rg3, and hydrolyzed 3-C,β-(1→2-glucoside to generate F2. The properties of the enzyme indicate that it could be a useful tool in biotransformation applications in the ginseng industry, as well as in the development of novel drug compounds.

  7. PDGFRα depletion attenuates glioblastoma stem cells features by modulation of STAT3, RB1 and multiple oncogenic signals.

    Science.gov (United States)

    Cenciarelli, Carlo; Marei, Hany E; Felsani, Armando; Casalbore, Patrizia; Sica, Gigliola; Puglisi, Maria Ausiliatrice; Cameron, Angus J M; Olivi, Alessandro; Mangiola, Annunziato

    2016-08-16

    Platelet derived growth factor receptors (PDGFRs) play an important role in tumor pathogenesis, and they are frequently overexpressed in glioblastoma (GBM). Earlier we have shown a higher protein expression of PDGFR isoforms (α and β) in peritumoral-tissue derived cancer stem cells (p-CSC) than in tumor core (c-CSC) of several GBM affected patients. In the current study, in order to assess the activity of PDGFRα/PDGF-AA signaling axis, we performed time course experiments to monitor the effects of exogenous PDGF-AA on the expression of downstream target genes in c-CSC vs p-CSC. Interestingly, in p-CSC we detected the upregulation of Y705-phosphorylated Stat3, concurrent with a decrement of Rb1 protein in its active state, within minutes of PDGF-AA addition. This finding prompted us to elucidate the role of PDGFRα in self-renewal, invasion and differentiation in p-CSC by using short hairpin RNA depletion of PDGFRα expression. Notably, in PDGFRα-depleted cells, protein analysis revealed attenuation of stemness-related and glial markers expression, alongside early activation of the neuronal marker MAP2a/b that correlated with the induction of tumor suppressor Rb1. The in vitro reduction of the invasive capacity of PDGFRα-depleted CSC as compared to parental cells correlated with the downmodulation of markers of epithelial-mesenchymal transition phenotype and angiogenesis. Surprisingly, we observed the induction of anti-apoptotic proteins and compensatory oncogenic signals such as EDN1, EDNRB, PRKCB1, PDGF-C and PDGF-D. To conclude, we hypothesize that the newly discovered PDGFRα/Stat3/Rb1 regulatory axis might represent a potential therapeutic target for GBM treatment.

  8. ATM/RB1 mutations predict shorter overall survival in urothelial cancer.

    Science.gov (United States)

    Yin, Ming; Grivas, Petros; Emamekhoo, Hamid; Mendiratta, Prateek; Ali, Siraj; Hsu, JoAnn; Vasekar, Monali; Drabick, Joseph J; Pal, Sumanta; Joshi, Monika

    2018-03-30

    Mutations of DNA repair genes, e.g. ATM/RB1 , are frequently found in urothelial cancer (UC) and have been associated with better response to cisplatin-based chemotherapy. Further external validation of the prognostic value of ATM/RB1 mutations in UC can inform clinical decision making and trial designs. In the discovery dataset, ATM/RB1 mutations were present in 24% of patients and were associated with shorter OS (adjusted HR 2.67, 95% CI, 1.45-4.92, p = 0.002). There was a higher mutation load in patients carrying ATM/RB1 mutations (median mutation load: 6.7 versus 5.5 per Mb, p = 0.072). In the validation dataset, ATM/RB1 mutations were present in 22.2% of patients and were non-significantly associated with shorter OS (adjusted HR 1.87, 95% CI, 0.97-3.59, p = 0.06) and higher mutation load (median mutation load: 8.1 versus 7.2 per Mb, p = 0.126). Exome sequencing data of 130 bladder UC patients from The Cancer Genome Atlas (TCGA) dataset were analyzed as a discovery cohort to determine the prognostic value of ATM/RB1 mutations. Results were validated in an independent cohort of 81 advanced UC patients. Cox proportional hazard regression analysis was performed to calculate the hazard ratio (HR) and 95% confidence interval (CI) to compare overall survival (OS). ATM/RB1 mutations may be a biomarker of poor prognosis in unselected UC patients and may correlate with higher mutational load. Further studies are required to determine factors that can further stratify prognosis and evaluate predictive role of ATM/RB1 mutation status to immunotherapy and platinum-based chemotherapy.

  9. Ginsenoside Rb1 for Myocardial Ischemia/Reperfusion Injury: Preclinical Evidence and Possible Mechanisms

    Directory of Open Access Journals (Sweden)

    Qun Zheng

    2017-01-01

    Full Text Available Ginseng is an important herbal drug that has been used worldwide for many years. Ginsenoside Rb1 (G-Rb1, the major pharmacological extract from ginseng, possesses a variety of biological activities in the cardiovascular systems. Here, we conducted a preclinical systematic review to investigate the efficacy of G-Rb1 for animal models of myocardial ischemia/reperfusion injury and its possible mechanisms. Ten studies involving 211 animals were identified by searching 6 databases from inception to May 2017. The methodological quality was assessed by using the CAMARADES 10-item checklist. All the data were analyzed using RevMan 5.3 software. As a result, the score of study quality ranged from 3 to 7 points. Meta-analyses showed that G-Rb1 can significantly decrease the myocardial infarct size and cardiac enzymes (including lactate dehydrogenase, creatine kinase, and creatine kinase-MB when compared with control group (P<0.01. Significant decrease in cardiac troponin T and improvement in the degree of ST-segment depression were reported in one study (P<0.05. Additionally, the possible mechanisms of G-Rb1 for myocardial infarction are antioxidant, anti-inflammatory, antiapoptosis, promoting angiogenesis and improving the circulation. Thus, G-Rb1 is a potential cardioprotective candidate for further clinical trials of myocardial infarction.

  10. Ginsenoside Rb1 Protects Rat Neural Progenitor Cells against Oxidative Injury

    Directory of Open Access Journals (Sweden)

    Na Ni

    2014-03-01

    Full Text Available Ginseng, the root of Panax ginseng C.A. Meyer, has been used as a tonic to enhance bodily functions against various ailments for hundreds of years in Far Eastern countries without apparent adverse effects. Ginsenoside Rb1, one of the most active ingredients of ginseng, has been shown to possess various pharmacological activities. Here we report that Rb1 exhibits potent neuroprotective effects against oxidative injury induced by tert-butylhydroperoxide (t-BHP. Lactate dehydrogenase (LDH assay demonstrated that incubation with 300 µm t-BHP for 2.5 h led to a significant cell loss of cultured rat embryonic cortex-derived neural progenitor cells (NPCs and the cell viability was pronouncedly increased by 24 h pretreatment of 10 µm Rb1. TUNEL staining further confirmed that pretreatment of Rb1 significantly reduced the cell apoptosis in t-BHP-induced oxidative injury. Real time PCR revealed that pretreatment with Rb1 activated Nrf2 pathway in cultured NPCs and led to an elevated expression of HO-1. The results of the present study demonstrate that Rb1 shows a potent anti-oxidative effect on cultured NPCs by activating Nrf2 pathway.

  11. Microbial transformation of ginsenoside Rb1 to compound K by Lactobacillus paralimentarius.

    Science.gov (United States)

    Quan, Lin-Hu; Kim, Yeon-Ju; Li, Guan Hao; Choi, Kwang-Tea; Yang, Deok-Chun

    2013-06-01

    In this study, the major ginsenoside Rb1 was transformed into the more pharmacologically active minor compound K by food grade Lactobacillus paralimentarius LH4, which was isolated from kimchi, a traditional Korean fermented food. The enzymatic reaction was analyzed by TLC, HPLC, and NMR. Using the cell-free enzyme of Lactobacillus paralimentarius LH4 at optimal conditions for 30 °C at pH 6.0, 1.0 mg ml(-1) ginsenoside Rb1 was transformed into 0.52 mg ml(-1) compound K within 72 h, with a corresponding molar conversion yield of 88 %. The cell-free enzyme hydrolyzed the two glucose moieties attached to the C-3 position and the outer glucose moiety attached to the C-20 position of the ginsenoside Rb1. The cell-free enzyme hydrolyzed the ginsenoside Rb1 along the following pathway: ginsenoside Rb1 → gypenoside XVII and ginsenoside Rd → ginsenoside F2 → compound K. Our results indicate that Lactobacillus paralimentarius LH4 has the potential to be applied for the preparation of compound K in the food industry.

  12. Knockdown of hypoxia-inducible factor-1 alpha reduces proliferation, induces apoptosis and attenuates the aggressive phenotype of retinoblastoma WERI-Rb-1 cells under hypoxic conditions.

    Science.gov (United States)

    Xia, Tian; Cheng, Hao; Zhu, Yu

    2014-01-01

    Hypoxia-inducible factor-1 alpha (HIF-1α) plays a critical role in tumor cell adaption to hypoxia by inducing the transcription of numerous genes. The role of HIF-1α in malignant retinoblastoma remains unclear. We analyzed the role of HIF-1α in WERI-Rb-1 retinoblastoma cells under hypoxic conditions. CoCl2 (125 mmol/L) was added to the culture media to mimic hypoxia. HIF-1α was silenced using siRNA. Gene and protein expression were measured by semi-quantitative RT-PCR and Western blotting. Cell cycle and apoptosis were analyzed by flow cytometry. Cell proliferation, adhesion and invasion were assayed using MTT, Transwell invasion, and cell adhesion assays respectively. Hypoxia significantly upregulated HIF-1α protein expression and the HIF-1α target genes VEGF, GLUT1, and Survivin mRNA. HIF-1α mRNA expression was not affected by hypoxia. Transfection of the siRNA expression plasmid pRNAT-CMV3.2/Neo-HIF-1α silenced HIF-1α by approximately 80% in hypoxic WERI-Rb-1 cells. The knockdown of HIF-1α under hypoxic conditions downregulated VEGF, GLUT1, and Survivin mRNA. It also inhibited proliferation, promoted apoptosis, induced the G0/G1 phase cell cycle arrest, and reduced the adhesion and invasion of WERI-Rb-1 cells. HIF-1α plays a major role in the survival and aggressive phenotype of retinoblastoma cells under hypoxic conditions. Targeting HIF-1α may be a promising therapeutic strategy for human malignant retinoblastoma.

  13. Effect of enzyme on extraction of ginsenoside Rb1 and Rg3 from Panax notoginseng roots

    Science.gov (United States)

    Phuong, Nguyen Tran Xuan; Thy, Lu Thi Mong; Khang, Nguyen Luu Vinh; My, Huynh Thi Kieu; Tam, Nguyen Le Phuong; Hieu, Nguyen Huu

    2018-04-01

    Panax notoginseng is distributed throughout the north and northwest of Vietnam, especially Ha Giang, Lao Cai, and Cao Bang provinces. The root of this plant contains ginsenosides (Rb1, Rb2, Rd, Rg3), flavonoids, polyacetylene, polysaccharides, amino acids, fatty acids, and peptides. In this study, the ratios of enzyme (Viscozyme, Termamyl, Cellulase), solvent of components, and time extraction were investigated. The results showed that the highest contents of Rb1 and Rg3 were achieved in the sample extracted with the ratio of enzymes V:C:T = 1:0:0, ethanol:water (60:40, v/v) as extracting solvent in 45 minutes. Then, conditions of high performance liquid chromatography with diode array detector method to determine the content of ginsenosides Rb1 and Rg3 in the roots of Panax notoginseng were studied, including wavelength, mobile phase, and flow rate. The separation was subjected on a reversed-phase C18 column using acetonitrile (A) and water (B) as mobile phase. The gradient elution was set as follow: 0-10 min, 15-25% A; 10-20 min, 25-30% A; 20-40 min, 30-60% A; 40-60 min, 60-80% A; and 60-65 min back to 15% A before the next injection, at a flow rate of 0.5 mL/min, and the wavelength was set at 202 nm. The linear range was from 298.59 to 696.72 µg/mL for Rb1 and from 8.19 to 19.10 µg/L for Rg3. The limits of detection for Rb1 and Rg3 obtained were 0.31 µg/mL and 0.33 µg/mL, respectively. The limits of quantification were 0.95 µg/mL and 1.01 µg/mL for Rb1 and Rg3, respectively. Consequently, the high performance liquid chromatography demonstrated the highly sensitive and accurate method for determination of Rb1 and Rg3 in Panax notoginseng.

  14. Correlation between alanine aminotransferase level, HCV-RNA titer ...

    African Journals Online (AJOL)

    Reham Al Swaff

    2012-04-04

    Apr 4, 2012 ... RNA titer and/or serum ALT level in patients with chronic hepatitis C (genotype 4) infection. .... Other liver diseases such as alcoholic liver disease, non- .... Insulin resistance, obesity, and steatosis are associated with a.

  15. Changes in rRNA levels during stress invalidates results from mRNA blotting: Fluorescence in situ rRNA hybridization permits renormalization for estimation of cellular mRNA levels

    DEFF Research Database (Denmark)

    Hansen, M.C.; Nielsen, A.K.; Molin, Søren

    2001-01-01

    obtained by these techniques are compared between experiments in which differences in growth rates, strains, or stress treatments occur, the normalization procedure may have a significant impact on the results. In this report we present a solution to the normalization problem in RNA slot blotting...... the relative level of rRNA per cell, and slot blotting to rRNA probes, which estimates the level of rRNA per extracted total RNA, the amount of RNA per cell was calculated in a series of heat shock experiments with the gram-positive bacterium Lactococcus lactis. It was found that the level of rRNA per cell...... decreased to 30% in the course of the heat shock. This lowered ribosome level led to a decrease in the total RNA content, resulting in a gradually increasing overestimation of the mRNA levels throughout the experiment. Using renormalized cellular mRNA levels, the HrcA-mediated regulation of the genes...

  16. RB1 is the crucial target of the Merkel cell polyomavirus Large T antigen in Merkel cell carcinoma cells.

    Science.gov (United States)

    Hesbacher, Sonja; Pfitzer, Lisa; Wiedorfer, Katharina; Angermeyer, Sabrina; Borst, Andreas; Haferkamp, Sebastian; Scholz, Claus-Jürgen; Wobser, Marion; Schrama, David; Houben, Roland

    2016-05-31

    The pocket protein (PP) family consists of the three members RB1, p107 and p130 all possessing tumor suppressive properties. Indeed, the PPs jointly control the G1/S transition mainly by inhibiting E2F transcription factors. Notably, several viral oncoproteins are capable of binding and inhibiting PPs. Merkel cell polyomavirus (MCPyV) is considered as etiological factor for Merkel cell carcinoma (MCC) with expression of the viral Large T antigen (LT) harboring an intact PP binding domain being required for proliferation of most MCC cells. Therefore, we analyzed the interaction of MCPyV-LT with the PPs. Co-IP experiments indicate that MCPyV-LT binds potently only to RB1. Moreover, MCPyV-LT knockdown-induced growth arrest in MCC cells can be rescued by knockdown of RB1, but not by p107 or p130 knockdown. Accordingly, cell cycle arrest and E2F target gene repression mediated by the single PPs can only in the case of RB1 be significantly reverted by MCPyV-LT expression. Moreover, data from an MCC patient indicate that loss of RB1 rendered the MCPyV-positive MCC cells LT independent. Thus, our results suggest that RB1 is the dominant tumor suppressor PP in MCC, and that inactivation of RB1 by MCPyV-LT is largely sufficient for its growth supporting function in established MCPyV-positive MCC cells.

  17. Autochthonous tumors driven by Rb1 loss have an ongoing requirement for the RBP2 histone demethylase.

    Science.gov (United States)

    McBrayer, Samuel K; Olenchock, Benjamin A; DiNatale, Gabriel J; Shi, Diana D; Khanal, Januka; Jennings, Rebecca B; Novak, Jesse S; Oser, Matthew G; Robbins, Alissa K; Modiste, Rebecca; Bonal, Dennis; Moslehi, Javid; Bronson, Roderick T; Neuberg, Donna; Nguyen, Quang-De; Signoretti, Sabina; Losman, Julie-Aurore; Kaelin, William G

    2018-04-17

    Inactivation of the retinoblastoma gene ( RB1 ) product, pRB, is common in many human cancers. Targeting downstream effectors of pRB that are central to tumorigenesis is a promising strategy to block the growth of tumors harboring loss-of-function RB1 mutations. One such effector is retinoblastoma-binding protein 2 (RBP2, also called JARID1A or KDM5A), which encodes an H3K4 demethylase. Binding of pRB to RBP2 has been linked to the ability of pRB to promote senescence and differentiation. Importantly, genetic ablation of RBP2 is sufficient to phenocopy pRB's ability to induce these cellular changes in cell culture experiments. Moreover, germline Rbp2 deletion significantly impedes tumorigenesis in Rb1 +/- mice. The value of RBP2 as a therapeutic target in cancer, however, hinges on whether loss of RBP2 could block the growth of established tumors as opposed to simply delaying their onset. Here we show that conditional, systemic ablation of RBP2 in tumor-bearing Rb1 +/- mice is sufficient to slow tumor growth and significantly extend survival without causing obvious toxicity to the host. These findings show that established Rb1 -null tumors require RBP2 for growth and further credential RBP2 as a therapeutic target in human cancers driven by RB1 inactivation.

  18. Optimization of lipase-catalyzed synthesis of ginsenoside Rb1 esters using response surface methodology.

    Science.gov (United States)

    Hu, Jiang-Ning; Lee, Jeung-Hee; Zhu, Xue-Mei; Shin, Jung-Ah; Adhikari, Prakash; Kim, Jae-Kyung; Lee, Ki-Teak

    2008-11-26

    In the lipase (Novozyme 435)-catalyzed synthesis of ginsenoside Rb1 esters, different acyl donors were found to affect not only the degree of conversion but also the regioselectivity. The reaction of acyl donors with short carbon chain was more effective, showing higher conversion than those with long carbon chain. Among the three solvent systems, the reaction in tert-amyl alcohol showed the highest conversion rate, while the reaction in the mixed solvent of t-BuOH and pyridine (1:1) had the lowest conversion rate. To allow the increase of GRb1 lipophilicity, we decided to further study the optimal condition of synthesis of GRb1 with vinyl decanoate with 10 carbon chain fatty acids in tert-amyl alcohol. Response surface methodology (RSM) was employed to optimize the synthesis condition. From the ridge analysis with maximum responses, the maximum GRb1 conversion was predicted to be 61.51% in a combination of factors (40.2 h, 52.95 degrees C, substrate mole ratio 275.57, and enzyme amount 39.81 mg/mL). Further, the adequacy of the predicted model was examined by additional independent experiments at the predicted maximum synthesis conditions. Results showed that the RSM was effective to optimize a combination of factors for lipase-catalyzed synthesis of ginsenoside Rb1 with vinyl decanoate.

  19. The effect of tRNA levels on decoding times of mRNA codons.

    Science.gov (United States)

    Dana, Alexandra; Tuller, Tamir

    2014-08-01

    The possible effect of transfer ribonucleic acid (tRNA) concentrations on codons decoding time is a fundamental biomedical research question; however, due to a large number of variables affecting this process and the non-direct relation between them, a conclusive answer to this question has eluded so far researchers in the field. In this study, we perform a novel analysis of the ribosome profiling data of four organisms which enables ranking the decoding times of different codons while filtering translational phenomena such as experimental biases, extreme ribosomal pauses and ribosome traffic jams. Based on this filtering, we show for the first time that there is a significant correlation between tRNA concentrations and the codons estimated decoding time both in prokaryotes and in eukaryotes in natural conditions (-0.38 to -0.66, all P values decoding times are not correlated with aminoacyl-tRNA levels. The reported results support the conjecture that translation efficiency is directly influenced by the tRNA levels in the cell. Thus, they should help to understand the evolution of synonymous aspects of coding sequences via the adaptation of their codons to the tRNA pool. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  20. Collagen mRNA levels changes during colorectal cancer carcinogenesis

    DEFF Research Database (Denmark)

    Skovbjerg, Hanne; Anthonsen, Dorit; Lothe, Inger M B

    2009-01-01

    BACKGROUND: Invasive growth of epithelial cancers is a complex multi-step process which involves dissolution of the basement membrane. Type IV collagen is a major component in most basement membranes. Type VII collagen is related to anchoring fibrils and is found primarily in the basement membrane...... zone of stratified epithelia. Immunohistochemical studies have previously reported changes in steady-state levels of different alpha(IV) chains in several epithelial cancer types. In the present study we aimed to quantitatively determine the mRNA levels of type IV collagen (alpha1/alpha 4/alpha 6......) and type VII collagen (alpha1) during colorectal cancer carcinogenesis. METHODS: Using quantitative RT-PCR, we have determined the mRNA levels for alpha1(IV), alpha 4(IV), alpha 6(IV), and alpha1(VII) in colorectal cancer tissue (n = 33), adenomas (n = 29) and in normal tissue from the same individuals...

  1. Influence of B-Complex Vitamins on the Pharmacokinetics of Ginsenosides Rg1, Rb1, and Ro After Oral Administration.

    Science.gov (United States)

    Zheng, Peihe; Chen, Yinbin; Fu, Yangyang; Wang, Hecheng; Wang, Jia; Zheng, Siwen; Xiao, Shengyuan; Wang, Yingping

    2017-11-01

    After cultivation of ginseng, ginsenosides, which are the major active ingredients of gingeng, were approved for use by the food industry, and began to be used as added functional ingredients to try to improve the quality and price of functional foods. However, the interaction between different types of ginsenosides and nutrients needs further study. We investigated the effect of B-complex vitamins (which are essential nutrients) on the pharmacokinetics of the ginsenosides protopanaxatriol-type saponin Rg 1 , protopanaxadiol-type saponin Rb 1 , and oleanolic acid-type saponin Ro after oral administration. Ginsenosides Rg 1 , Rb 1 , and Ro, with or without B-complex vitamins, respectively, were administered orally to rats to evaluate their pharmacokinetics. The concentration of ginsenosides in plasma was determined by liquid chromatography/tandem mass spectrometry. Pharmacokinetic parameters were fitted using WinNonlin v6.2. After oral coadministration with B-complex vitamins, the area under the concentration-time curve from zero to infinity (AUC 0-∞ ) of ginsenoside Rg 1 was reduced by 70%, that of ginsenoside Rb 1 was reduced by 43%, and that of ginsenoside Ro was reduced by 34%. The AUC 0-∞ of ginsenosides Rg 1 and Rb 1 showed significant differences between different treatments, but the AUC 0-∞ of ginsenoside Ro did not. These results suggest significant ginsenoside-nutrient interactions between ginsenosides Rg 1 , Rb 1 , and B-complex vitamins.

  2. Non-functional genes repaired at the RNA level.

    Science.gov (United States)

    Burger, Gertraud

    2016-01-01

    Genomes and genes continuously evolve. Gene sequences undergo substitutions, deletions or nucleotide insertions; mobile genetic elements invade genomes and interleave in genes; chromosomes break, even within genes, and pieces reseal in reshuffled order. To maintain functional gene products and assure an organism's survival, two principal strategies are used - either repair of the gene itself or of its product. I will introduce common types of gene aberrations and how gene function is restored secondarily, and then focus on systematically fragmented genes found in a poorly studied protist group, the diplonemids. Expression of their broken genes involves restitching of pieces at the RNA-level, and substantial RNA editing, to compensate for point mutations. I will conclude with thoughts on how such a grotesquely unorthodox system may have evolved, and why this group of organisms persists and thrives since tens of millions of years. Copyright © 2016 Académie des sciences. Published by Elsevier SAS. All rights reserved.

  3. 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.

  4. p53 inhibits autophagy by interacting with the human ortholog of yeast Atg17, RB1CC1/FIP200.

    Science.gov (United States)

    Morselli, Eugenia; Shen, Shensi; Ruckenstuhl, Christoph; Bauer, Maria Anna; Mariño, Guillermo; Galluzzi, Lorenzo; Criollo, Alfredo; Michaud, Mickael; Maiuri, Maria Chiara; Chano, Tokuhiro; Madeo, Frank; Kroemer, Guido

    2011-08-15

    The tumor suppressor protein p53 tonically suppresses autophagy when it is present in the cytoplasm. This effect is phylogenetically conserved from mammals to nematodes, and human p53 can inhibit autophagy in yeast, as we show here. Bioinformatic investigations of the p53 interactome in relationship to the autophagy-relevant protein network underscored the possible relevance of a direct molecular interaction between p53 and the mammalian ortholog of the essential yeast autophagy protein Atg17, namely RB1-inducible coiled-coil protein 1 (RB1CC1), also called FAK family kinase-interacting protein of 200 KDa (FIP200). Mutational analyses revealed that a single point mutation in p53 (K382R) abolished its capacity to inhibit autophagy upon transfection into p53-deficient human colon cancer or yeast cells. In conditions in which wild-type p53 co-immunoprecipitated with RB1CC1/FIP200, p53 (K382R) failed to do so, underscoring the importance of the physical interaction between these proteins for the control of autophagy. In conclusion, p53 regulates autophagy through a direct molecular interaction with RB1CC1/FIP200, a protein that is essential for the very apical step of autophagy initiation.

  5. Analysis of the RB1 gene in children with retinoblastoma having residential connections to West Cumbria, England

    Energy Technology Data Exchange (ETDEWEB)

    Cowell, J K [Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, NY 14263 (United States); Morris, J A [Department of Pathology, Royal Lancaster Infirmary, Lancaster LA1 4RP (United Kingdom); Tawn, E J [Westlakes Research Institute, Moor Row, Cumbria CA24 3JY (United Kingdom)

    2005-03-01

    Six of eight cases of retinoblastoma previously identified as having a residential association with West Cumbria, England, in which the Sellafield nuclear installation is situated, were examined for the presence of a constitutional RB1 mutation. No mutations were detected, thus providing strong evidence against an environmental or occupational genotoxic effect causing germline mutations in the parents of these children. (note)

  6. Whole-Cell Biocatalysis for Producing Ginsenoside Rd from Rb1 Using Lactobacillus rhamnosus GG.

    Science.gov (United States)

    Ku, Seockmo; You, Hyun Ju; Park, Myeong Soo; Ji, Geun Eog

    2016-07-28

    Ginsenosides are the major active ingredients in ginseng used for human therapeutic plant medicines. One of the most well-known probiotic bacteria among the various strains on the functional food market is Lactobacillus rhamnosus GG. Biocatalytic methods using probiotic enzymes for producing deglycosylated ginsenosides such as Rd have a growing significance in the functional food industry. The addition of 2% cellobiose (w/v) to glucose-free de Man-Rogosa-Sharpe broths notably induced β-glucosidase production from L. rhamnosus GG. Enzyme production and activity were optimized at a pH, temperature, and cellobiose concentration of 6.0, 40°C, and 2% (w/v), respectively. Under these controlled conditions, β-glucosidase production in L. rhamnosus GG was enhanced by 25-fold. Additionally, whole-cell homogenates showed the highest β-glucosidase activity when compared with disrupted cell suspensions; the cell disruption step significantly decreased the β-glucosidase activity. Based on the optimized enzyme conditions, whole-cell L. rhamnosus GG was successfully used to convert ginsenoside Rb1 into Rd.

  7. Heritable factors for radiation-induced osteosarcoma and the role of Rb1 in telomere maintenance in mesenchymal stem cells

    International Nuclear Information System (INIS)

    Rosemann, M.; Gonzalez-Vasconcellos, I.; Atkinson, M. J.

    2013-01-01

    Full text: Secondary tumors following a pediatric radiotherapy become an ever growing concern, a side effect of the improved therapeutic success leading to extended life span of the patients. Osteosarcoma (OS) and other soft-tissue sarcomas arise over proportionally frequent in the radiation field of former radiooncology patients. In an effort to map and identify inherited factors that govern individual susceptibility for these secondary, therapy associated tumors, we developed a mouse model that after injection of 227 thorium develops high numbers of OS and facilitates whole genome mapping of genetic variants that modify risk. We could identify genetic risk factors on 5 different chromosomes, that by independent segregation in the germline can interact in an additive manner and in combination alter the individual risk more than 3 fold. Using additional in-vitro studies and mouse knockout technology we could confirm that the responsible gene on the principal susceptibility locus is Rb1. Mice with a bone-specific Rb1+/- status exhibits increased bone tumor risk following irradiation. We have shown recently that the Rb1 tumor suppressor gene does not only regulates the cell cycle kinetics of mammalian cells, but that in normal osteoblasts is also crucial to protect telomeres from extensive erosion. An Rb1+/- deficiency therefore exhibit accelerated telomeric loss and, following ionising irradiation an excess of chromosomal defects. In the majority of secondary RT associated human OS, Rb1 was affected by allelic loss, whereas spontaneous human OS with no known radiation etiology show only 15%-20% Rb1 losses. It is assumed that the target cells for malignant transformation leading to OS are not the differentiating osteoblasts, but rather the long-term repopulating and pluripotent mesenchymal stem cells (MSC). These normal tissue stem cells are assumed to maintain a high degree of genome stability throughout the entire life span of an organism. One of the key factors

  8. Possible role of ginsenoside Rb1 in skin wound healing via regulating senescent skin dermal fibroblast.

    Science.gov (United States)

    Hou, Jingang; Kim, Sunchang

    2018-05-05

    Cellular senescence suppresses cancer by inducing irreversible cell growth arrest. Nevertheless, senescent cells is proposed as causal link with aging and aging-related pathologies. The physiological beneficial functions of senescent cells are still of paucity. Here we show that senescent human dermal fibroblast accelerates keratinocytes scratch wound healing and stimulates differentiation of fibroblast. Using oxidative stress (100 μM H 2 O 2 exposure for 1 h) induction, we successfully triggered fibroblast senescence and developed senescence associated secretory phenotype (SASP). The induction of SASP was regulated by p38MAPK/MSK2/NF-κB pathway. Interestingly, inhibition of p38MAPK activation only partially suppressed SASP. However, SASP was significantly inhibited by SB747651A, a specific MSK inhibitor. Additionally, we demonstrate that SASP stimulates migration of keratinocytes and myofibroblast transition of fibroblast, through fold-increased secretion of growth factors, platelet-derived growth factor AA (PDGF-AA) and AB (PDGF-AB), transforming growth factor beta 1 (TGF-β1) and beta 2 (TGF-β2), vascular endothelial growth factor A (VEGF-A) and D (VEGF-D), vascular endothelial growth factor receptor 2 (VEGFR2) and 3 (VEGFR3). Importantly, we also confirmed ginsenoside Rb1 promoted SASP-mediated healing process via p38MAPK/MSK2/NF-κB pathway. The results pointed to senescent fibroblast as a potential mechanism of wound healing control in human skin. Further, it provided a candidate targeted for wound therapy. Copyright © 2018 Elsevier Inc. All rights reserved.

  9. Study of ammonium molecular ion impurity modes in Rb1-x(NH4)xI mixed crystals by inelastic incoherent neutron scattering

    International Nuclear Information System (INIS)

    Smirnov, L.S.; Natkaniec, I.; Ollivier, J.; Dianoux, J.A.; Martinez Sarrion, M.L.; Mestres, L.

    2010-01-01

    The study of ammonium dynamics in Rb 1-x (NH 4 ) x I mixed crystals was carried out by inelastic incoherent neutron scattering in the concentration region of orientationally disordered α-phase, 0.0< x<0.40, at the temperature range from 2 to 150 K. The observed resonance modes correspond to three energy regions: 0.19-0.481 (I), 0.56-3.0 (II) and 4.0-10.0 (III) meV. The modes of region I could be described by rotational tunneling energies of the multipole moments of ammonium ions. The modes within energy region II correspond to the calculated rotational tunneling energies between splitted levels of the ground librational level of ammonium ion. The modes of region III can be described as local gap modes of ammonium ion because they are located between acoustic and optic branches of RbI phonon density of states

  10. Comparison of norovirus RNA levels in outbreak-related oysters with background environmental levels.

    Science.gov (United States)

    Lowther, James A; Gustar, Nicole E; Hartnell, Rachel E; Lees, David N

    2012-02-01

    Norovirus is the principal agent of bivalve shellfish-associated gastroenteric illness worldwide. Numerous studies using PCR have demonstrated norovirus contamination in a significant proportion of both oyster and other bivalve shellfish production areas and ready-to-eat products. By comparison, the number of epidemiologically confirmed shellfish-associated outbreaks is relatively low. This suggests that factors other than the simple presence or absence of virus RNA are important contributors to the amount of illness reported. This study compares norovirus RNA levels in oyster samples strongly linked to norovirus or norovirus-type illness with the levels typically found in commercial production areas (non-outbreak-related samples). A statistically significant difference between norovirus levels in the two sets of samples was observed. The geometric mean of the levels in outbreak samples (1,048 copies per g) was almost one order of magnitude higher than for positive non-outbreak-related samples (121 copies per g). Further, while none of the outbreak-related samples contained fewer than 152 copies per g, the majority of positive results for non-outbreak-related samples was below this level. These observations support the concept of a dose-response for norovirus RNA levels in shellfish and could help inform the establishment of threshold criteria for risk management.

  11. Genomic portrait of resectable hepatocellular carcinomas: implications of RB1 and FGF19 aberrations for patient stratification.

    Science.gov (United States)

    Ahn, Sung-Min; Jang, Se Jin; Shim, Ju Hyun; Kim, Deokhoon; Hong, Seung-Mo; Sung, Chang Ohk; Baek, Daehyun; Haq, Farhan; Ansari, Adnan Ahmad; Lee, Sun Young; Chun, Sung-Min; Choi, Seongmin; Choi, Hyun-Jeung; Kim, Jongkyu; Kim, Sukjun; Hwang, Shin; Lee, Young-Joo; Lee, Jong-Eun; Jung, Wang-Rim; Jang, Hye Yoon; Yang, Eunho; Sung, Wing-Kin; Lee, Nikki P; Mao, Mao; Lee, Charles; Zucman-Rossi, Jessica; Yu, Eunsil; Lee, Han Chu; Kong, Gu

    2014-12-01

    Hepatic resection is the most curative treatment option for early-stage hepatocellular carcinoma, but is associated with a high recurrence rate, which exceeds 50% at 5 years after surgery. Understanding the genetic basis of hepatocellular carcinoma at surgically curable stages may enable the identification of new molecular biomarkers that accurately identify patients in need of additional early therapeutic interventions. Whole exome sequencing and copy number analysis was performed on 231 hepatocellular carcinomas (72% with hepatitis B viral infection) that were classified as early-stage hepatocellular carcinomas, candidates for surgical resection. Recurrent mutations were validated by Sanger sequencing. Unsupervised genomic analyses identified an association between specific genetic aberrations and postoperative clinical outcomes. Recurrent somatic mutations were identified in nine genes, including TP53, CTNNB1, AXIN1, RPS6KA3, and RB1. Recurrent homozygous deletions in FAM123A, RB1, and CDKN2A, and high-copy amplifications in MYC, RSPO2, CCND1, and FGF19 were detected. Pathway analyses of these genes revealed aberrations in the p53, Wnt, PIK3/Ras, cell cycle, and chromatin remodeling pathways. RB1 mutations were significantly associated with cancer-specific and recurrence-free survival after resection (multivariate P = 0.038 and P = 0.012, respectively). FGF19 amplifications, known to activate Wnt signaling, were mutually exclusive with CTNNB1 and AXIN1 mutations, and significantly associated with cirrhosis (P = 0.017). RB1 mutations can be used as a prognostic molecular biomarker for resectable hepatocellular carcinoma. Further study is required to investigate the potential role of FGF19 amplification in driving hepatocarcinogenesis in patients with liver cirrhosis and to investigate the potential of anti-FGF19 treatment in these patients. © 2014 by the American Association for the Study of Liver Diseases.

  12. Next generation sequencing of Cytokeratin 20-negative Merkel cell carcinoma reveals ultraviolet-signature mutations and recurrent TP53 and RB1 inactivation.

    Science.gov (United States)

    Harms, Paul W; Collie, Angela M B; Hovelson, Daniel H; Cani, Andi K; Verhaegen, Monique E; Patel, Rajiv M; Fullen, Douglas R; Omata, Kei; Dlugosz, Andrzej A; Tomlins, Scott A; Billings, Steven D

    2016-03-01

    Merkel cell carcinoma is a rare but highly aggressive cutaneous neuroendocrine carcinoma. Cytokeratin 20 (CK20) is expressed in ~95% of Merkel cell carcinomas and is useful for distinction from morphologically similar entities including metastatic small-cell lung carcinoma. Lack of CK20 expression may make diagnosis of Merkel cell carcinoma more challenging, and has unknown biological significance. Approximately 80% of CK20-positive Merkel cell carcinomas are associated with the oncogenic Merkel cell polyomavirus. Merkel cell carcinomas lacking Merkel cell polyomavirus display distinct genetic changes from Merkel cell polyomavirus-positive Merkel cell carcinoma, including RB1 inactivating mutations. Unlike CK20-positive Merkel cell carcinoma, the majority of CK20-negative Merkel cell carcinomas are Merkel cell polyomavirus-negative, suggesting CK20-negative Merkel cell carcinomas predominantly arise through virus-independent pathway(s) and may harbor additional genetic differences from conventional Merkel cell carcinoma. Hence, we analyzed 15 CK20-negative Merkel cell carcinoma tumors (10 Merkel cell polyomavirus-negative, four Merkel cell polyomavirus-positive, and one undetermined) using the Ion Ampliseq Comprehensive Cancer Panel, which assesses copy number alterations and mutations in 409 cancer-relevant genes. Twelve tumors displayed prioritized high-level chromosomal gains or losses (average 1.9 per tumor). Non-synonymous high-confidence somatic mutations were detected in 14 tumors (average 11.9 per tumor). Assessing all somatic coding mutations, an ultraviolet-signature mutational profile was present, and more prevalent in Merkel cell polyomavirus-negative tumors. Recurrent deleterious tumor suppressor mutations affected TP53 (9/15, 60%), RB1 (3/15, 20%), and BAP1 (2/15, 13%). Oncogenic activating mutations included PIK3CA (3/15, 20%), AKT1 (1/15, 7%) and EZH2 (1/15, 7%). In conclusion, CK20-negative Merkel cell carcinoma display overlapping genetic changes

  13. Next Generation Sequencing of Cytokeratin 20-Negative Merkel Cell Carcinoma Reveals Ultraviolet Signature Mutations and Recurrent TP53 and RB1 Inactivation

    Science.gov (United States)

    Harms, Paul W.; Collie, Angela M. B.; Hovelson, Daniel H.; Cani, Andi K.; Verhaegen, Monique E.; Patel, Rajiv M.; Fullen, Douglas R.; Omata, Kei; Dlugosz, Andrzej A.; Tomlins, Scott A.; Billings, Steven D.

    2016-01-01

    Merkel cell carcinoma is a rare but highly aggressive cutaneous neuroendocrine carcinoma. Cytokeratin-20 (CK20) is expressed in approximately 95% of Merkel cell carcinomas and is useful for distinction from morphologically similar entities including metastatic small cell lung carcinoma. Lack of CK20 expression may make diagnosis of Merkel cell carcinoma more challenging, and has unknown biological significance. Approximately 80% of CK20-positive Merkel cell carcinomas are associated with the oncogenic Merkel cell polyomavirus. Merkel cell carcinomas lacking Merkel cell polyomavirus display distinct genetic changes from Merkel cell polyomavirus-positive Merkel cell carcinoma, including RB1 inactivating mutations. Unlike CK20-positive Merkel cell carcinoma, the majority of CK20-negative Merkel cell carcinomas are Merkel cell polyomavirus-negative, suggesting CK20-negative Merkel cell carcinomas predominantly arise through virus-independent pathway(s) and may harbor additional genetic differences from conventional Merkel cell carcinoma. Hence, we analyzed 15 CK20-negative Merkel cell carcinoma tumors (ten Merkel cell polyomavirus-negative, four Merkel cell polyomavirus-positive, and one undetermined) using the Ion Ampliseq Comprehensive Cancer Panel, which assesses copy number alterations and mutations in 409 cancer-relevant genes. Twelve tumors displayed prioritized high-level chromosomal gains or losses (average 1.9 per tumor). Non-synonymous high confidence somatic mutations were detected in 14 tumors (average 11.9 per tumor). Assessing all somatic coding mutations, an ultraviolet-signature mutational profile was present, and more prevalent in Merkel cell polyomavirus-negative tumors. Recurrent deleterious tumor suppressor mutations affected TP53 (9/15, 60%), RB1 (3/15, 20%), and BAP1 (2/15, 13%). Oncogenic activating mutations included PIK3CA (3/15, 20%), AKT1 (1/15, 7%)) and EZH2 (1/15, 7%). In conclusion, CK20-negative Merkel cell carcinoma display overlapping

  14. Silencing of RB1 and RB2/P130 during adipogenesis of bone marrow stromal cells results in dysregulated differentiation.

    Science.gov (United States)

    Capasso, Stefania; Alessio, Nicola; Di Bernardo, Giovanni; Cipollaro, Marilena; Melone, Mariarosa Ab; Peluso, Gianfranco; Giordano, Antonio; Galderisi, Umberto

    2014-01-01

    Bone marrow adipose tissue (BMAT) is different from fat found elsewhere in the body, and only recently have some of its functions been investigated. BMAT may regulate bone marrow stem cell niche and plays a role in energy storage and thermogenesis. BMAT may be involved also in obesity and osteoporosis onset. Given the paramount functions of BMAT, we decided to better clarify the human bone marrow adipogenesis by analyzing the role of the retinoblastoma gene family, which are key players in cell cycle regulation. Our data provide evidence that the inactivation of RB1 or RB2/P130 in uncommitted bone marrow stromal cells (BMSC) facilitates the first steps of adipogenesis. In cultures with silenced RB1 or RB2/P130, we observed an increase of clones with adipogenic potential and a higher percentage of cells accumulating lipid droplets. Nevertheless, the absence of RB1 or RB2/P130 impaired the terminal adipocyte differentiation and gave rise to dysregulated adipose cells, with alteration in lipid uptake and release. For the first time, we evidenced that RB2/P130 plays a role in bone marrow adipogenesis. Our data suggest that while the inactivation of retinoblastoma proteins may delay the onset of last cell division and allow more BMSC to be committed to adipocyte, it did not allow a permanent cell cycle exit, which is a prerequisite for adipocyte terminal maturation.

  15. Highly Selective Bioconversion of Ginsenoside Rb1 to Compound K by the Mycelium of Cordyceps sinensis under Optimized Conditions

    Directory of Open Access Journals (Sweden)

    Wei-Nan Wang

    2015-10-01

    Full Text Available Compound K (CK, a highly active and bioavailable derivative obtained from protopanaxadiol ginsenosides, displays a wide variety of pharmacological properties, especially antitumor activity. However, the inadequacy of natural sources limits its application in the pharmaceutical industry. In this study, we firstly discovered that Cordyceps sinensis was a potent biocatalyst for the biotransformation of ginsenoside Rb1 into CK. After a series of investigations on the biotransformation parameters, an optimal composition of the biotransformation culture was found to be lactose, soybean powder and MgSO4 without controlling the pH. Also, an optimum temperature of 30 °C for the biotransformation process was suggested in a range of 25 °C–50 °C. Then, a biotransformation pathway of Rb1 → Rd → F2 → CK was established using high performance liquid chromatography/quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF-MS. Our results demonstrated that the molar bioconversion rate of Rb1 to CK was more than 82% and the purity of CK produced by C. sinensis under the optimized conditions was more than 91%. In conclusion, the combination of C. sinensis and the optimized conditions is applicable for the industrial preparation of CK for medicinal purposes.

  16. Impact of MicroRNA Levels, Target-Site Complementarity, and Cooperativity on Competing Endogenous RNA-Regulated Gene Expression.

    Science.gov (United States)

    Denzler, Rémy; McGeary, Sean E; Title, Alexandra C; Agarwal, Vikram; Bartel, David P; Stoffel, Markus

    2016-11-03

    Expression changes of competing endogenous RNAs (ceRNAs) have been proposed to influence microRNA (miRNA) activity and thereby regulate other transcripts containing miRNA-binding sites. Here, we find that although miRNA levels define the extent of repression, they have little effect on the magnitude of the ceRNA expression change required to observe derepression. Canonical 6-nt sites, which typically mediate modest repression, can nonetheless compete for miRNA binding, with potency ∼20% of that observed for canonical 8-nt sites. In aggregate, low-affinity/background sites also contribute to competition. Sites with extensive additional complementarity can appear as more potent, but only because they induce miRNA degradation. Cooperative binding of proximal sites for the same or different miRNAs does increase potency. These results provide quantitative insights into the stoichiometric relationship between miRNAs and target abundance, target-site spacing, and affinity requirements for ceRNA-mediated gene regulation, and the unusual circumstances in which ceRNA-mediated gene regulation might be observed. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  17. RNA.

    Science.gov (United States)

    Darnell, James E., Jr.

    1985-01-01

    Ribonucleic acid (RNA) converts genetic information into protein and usually must be processed to serve its function. RNA types, chemical structure, protein synthesis, translation, manufacture, and processing are discussed. Concludes that the first genes might have been spliced RNA and that humans might be closer than bacteria to primitive…

  18. [Correlation between RNA Expression Level and Early PMI in Human Brain Tissue].

    Science.gov (United States)

    Lü, Y H; Ma, K J; Li, Z H; Gu, J; Bao, J Y; Yang, Z F; Gao, J; Zeng, Y; Tao, L; Chen, L

    2016-08-01

    To explore the correlation between the expression levels of several RNA markers in human brain tissue and early postmortem interval (PMI). Twelve individuals with known PMI (range from 4.3 to 22.5 h) were selected and total RNA was extracted from brain tissue. Eight commonly used RNA markers were chosen including β -actin, GAPDH, RPS29, 18S rRNA, 5S rRNA, U6 snRNA, miRNA-9 and miRNA-125b, and the expression levels were detected in brain tissue by real-time fluorescent quantitative PCR. The internal reference markers with stable expression in early PMI were screened using geNorm software and the relationship between its expression level and some relevant factors such as age, gender and cause of death were analyzed. RNA markers normalized by internal reference were inserted into the mathematic model established by previous research for PMI estimation using R software. Model quality was judged by the error rate calculated with estimated PMI. 5S rRNA, miRNA-9 and miRNA-125b showed quite stable expression and their expression levels had no relation with age, gender and cause of death. The error rate of estimated PMI using β -actin was 24.6%, while GAPDH was 41.0%. 5S rRNA, miRNA-9 and miRNA-125b are suitable as internal reference markers of human brain tissue owing to their stable expression in early PMI. The expression level of β -actin correlates well with PMI, which can be used as an additional index for early PMI estimation. Copyright© by the Editorial Department of Journal of Forensic Medicine

  19. Ginsenoside Rb1 Attenuates Agonist-Induced Contractile Response via Inhibition of Store-Operated Calcium Entry in Pulmonary Arteries of Normal and Pulmonary Hypertensive Rats

    Directory of Open Access Journals (Sweden)

    Rui-Xing Wang

    2015-03-01

    Full Text Available Background: Pulmonary hypertension (PH is characterized by sustained vasoconstriction, enhanced vasoreactivity and vascular remodeling, which leads to right heart failure and death. Despite several treatments are available, many forms of PH are still incurable. Ginsenoside Rb1, a principle active ingredient of Panax ginseng, exhibits multiple pharmacological effects on cardiovascular system, and suppresses monocrotaline (MCT-induced right heart hypertrophy. However, its effect on the pulmonary vascular functions related to PH is unknown. Methods: We examined the vasorelaxing effects of ginsenoside Rb1 on endothelin-1 (ET-1 induced contraction of pulmonary arteries (PAs and store-operated Ca2+ entry (SOCE in pulmonary arterial smooth muscle cells (PASMCs from chronic hypoxia (CH and MCT-induced PH. Results: Ginsenoside Rb1 elicited concentration-dependent relaxation of ET-1-induced PA contraction. The vasorelaxing effect was unaffected by nifedipine, but abolished by the SOCE blocker Gd3+. Ginsenoside Rb1 suppressed cyclopiazonic acid (CPA-induced PA contraction, and CPA-activated cation entry and Ca2+ transient in PASMCs. ET-1 and CPA-induced contraction, and CPA-activated cation entry and Ca2+ transients were enhanced in PA and PASMCs of CH and MCT-treated rats; the enhanced responses were abolished by ginsenoside Rb1. Conclusion: Ginsenoside Rb1 attenuates ET-1-induced contractile response via inhibition of SOCE, and it can effectively antagonize the enhanced pulmonary vasoreactivity in PH.

  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

    of the ERα mRNA level, but only significantly for prochloraz, dieldrin, and tolchlofos-methyl. Alone no pesticides affected the ERβ mRNA level significantly, but chlorpyrifos increased the mRNA level weakly. Co-exposure with E2 elicited a significant increased ERβ mRNA level by prochloraz, fenarimol...

  1. Inhibition of Autophagy via Activation of PI3K/Akt Pathway Contributes to the Protection of Ginsenoside Rb1 against Neuronal Death Caused by Ischemic Insults

    Directory of Open Access Journals (Sweden)

    Tianfei Luo

    2014-09-01

    Full Text Available Lethal autophagy is a pathway leading to neuronal death caused by transient global ischemia. In this study, we examined the effect of Ginsenoside Rb1 (GRb1 on ischemia/reperfusion-induced autophagic neuronal death and investigated the role of PI3K/Akt. Ischemic neuronal death in vitro was induced by using oxygen glucose deprivation (OGD in SH-SY5Y cells, and transient global ischemia was produced by using two vessels occlusion in rats. Cellular viability of SH-SY5Y cells was assessed by MTT assay, and CA1 neuronal death was evaluated by Hematoxylin-eosin staining. Autophagic vacuoles were detected by using both fluorescent microscopy in combination with acridine orange (AO and Monodansylcadaverine (MDC staining and transmission electronic microscopy. Protein levels of LC3II, Beclin1, total Akt and phosphor-Akt at Ser473 were examined by western blotting analysis. GRb1 inhibited both OGD and transient ischemia-induced neuronal death and mitigated OGD-induced autophagic vacuoles in SH-SY5Y cells. By contrast, PI3K inhibitor LY294002 counteracted the protection of GRb1 against neuronal death caused by either OGD or transient ischemia. LY294002 not only mitigated the up-regulated protein level of phosphor Akt at Ser473 caused by GRb1, but also reversed the inhibitory effect of GRb1 on OGD and transient ischemia-induced elevation in protein levels of LC3II and Beclin1.

  2. Influence of concentration and temperature on tunneling and rotational dynamics of ammonium in Rb1-x (NH4)x I mixed crystals

    International Nuclear Information System (INIS)

    Natkaniec, I.; Dianoux, A.J.; Martinez-Sarrion, M.L.; Mestres, L.; Herraiz, M.; Smirnov, L.S.; Shuvalov, L.A.

    2001-01-01

    The Rb 1x (NH 4 ) x I mixed crystals are studied by inelastic incoherent neutron scattering using time-of-flight spectrometers in the concentration region of the x-T phase diagram 0.01≤x≤0.66 at 5≤T≤150K, where dynamic and static orientational disorder phases are generally found. It is shown that at 5 K rotational tunneling levels for ammonium concentrations x=0.01, 0.02 and 0.06 are similar. Additional tunneling levels are observed for x=0.16 which can be explained as the result of T-states splitting for account of NH 4 -NH 4 interaction. Tunneling levels are not observed for x=0.40 as the result of forming of orientational glass state. The elastic incoherent structure factors for concentrations 0.01≤x≤0.16 (dynamic orientational disordered α-phase), x=0.40 (orientational glass state) and 0.50≤x≤0.66 (orientational ordered state) have different temperature dependences

  3. Inflammation-related microRNA expression level in the bovine milk is affected by mastitis.

    Science.gov (United States)

    Lai, Yu-Chang; Fujikawa, Takuro; Maemura, Tadashi; Ando, Takaaki; Kitahara, Go; Endo, Yasuyuki; Yamato, Osamu; Koiwa, Masateru; Kubota, Chikara; Miura, Naoki

    2017-01-01

    MicroRNA (miRNA) in tissue and liquid samples have been shown to be associated with many diseases including inflammation. We aimed to identify inflammation-related miRNA expression level in the bovine mastitis milk. Expression level of inflammation-related miRNA in milk from mastitis-affected and normal cows was analyzed using qPCR. We found that expression level of miR-21, miR-146a, miR-155, miR-222, and miR-383 was significantly upregulated in California mastitis test positive (CMT+) milk. We further analyzed these miRNA using a chip-based QuantStudio Digital PCR System. The digital PCR results correlated with those of qPCR, demonstrating upregulation of miR-21, miR-146a, miR-155, miR-222, and miR-383 in CMT+ milk. In conclusion, we identified miRNA that are upregulated in CMT+ milk. These miRNA exhibited sensitivity and specificity greater than 80% for differentiating between CMT+ milk and normal milk. Our findings suggest that inflammation-related miRNA expression level in the bovine milk was affected by mastitis, and miRNA in milk have potential for use as biomarkers of bovine mastitis.

  4. Levels and patterns of HIV RNA viral load in untreated pregnant women

    DEFF Research Database (Denmark)

    NN, NN; Patel, Deven; Thorne, Claire

    2008-01-01

    OBJECTIVE: To assess pregnancy levels and patterns of HIV RNA in the absence of antiretroviral therapy, while appropriately adjusting for potential confounders, including maternal immune status and race. METHODS: Data on > or = 1 antenatal HIV RNA measurements were available for 333 untreated HIV......-infected pregnant women enrolled in the European Collaborative Study. CD4 counts and HIV RNA measurements were routinely collected from 1992 and 1998, respectively. Linear mixed effects models based on 246 women for whom complete data were available examined changes in HIV RNA levels over pregnancy, with a nested...... random effects term accounting for measurement variability within women and period of sample collection. RESULTS: The change in HIV RNA over pregnancy varied significantly by race (p=0.005): from the second trimester until delivery, HIV RNA decreased significantly by an estimated 0.019 log(10) copies...

  5. Cytokine RNA levels in transiliac bone biopsies from healthy early postmenopausal women

    DEFF Research Database (Denmark)

    Abrahamsen, Bo; Shalhoub, V; Larson, E K

    2000-01-01

    RNA was measured by competitive reverse transcriptase polymerase chain reaction. The IL-1ra/IL-1beta mRNA slope for the slow-loss group was steeper (deltaF = 23.3, p levels relative to IL-1beta....... During HRT, the IL-1beta mRNA level was inversely correlated with serum estradiol (log r2 = 0.77, p levels identical to the control group. In contrast, IL-1ra mRNA was independent of serum estradiol. Histomorphometric...... for IL-6 mRNA. The findings support the hypothesis that IL-1beta production within bone increases with declining estrogen levels, and that an increase in II-1ra protects against accelerated bone loss....

  6. Pharmacokinetics and efficiency of brain targeting of ginsenosides Rg1 and Rb1 given as Nao-Qing microemulsion.

    Science.gov (United States)

    Li, Tao; Shu, Ya-Jun; Cheng, Jia-Yin; Liang, Run-Cheng; Dian, Shao-Na; Lv, Xiao-Xun; Yang, Meng-Qi; Huang, Shu-Ling; Chen, Gang; Yang, Fan

    2015-02-01

    Nao-Qing solution has been shown to be clinically effective in the treatment of acute ischemic stroke (AIS). The purpose of this study was to improve the pharmacokinetics and brain uptake of Nao-Qing, administered as an oil-in-water microemulsion. Sprague-Dawley (SD) rats were given Nao-Qing microemulsion by intranasal or intragastric routes. Samples of blood, brain, heart, liver, lung and kidney were collected at pre-determined time intervals, and the contents of ginsenosides Rg1 and Rb1 (active ingredients of the Nao-Qing microemulsion) were analyzed by high-performance liquid chromatography (HPLC). The results showed that contents of ginsenosides Rg1 and Rb1 in Nao-Qing microemulsion was 8475.13 ± 54.61 μg/ml and 6633.42 ± 527.27 μg/ml, respectively, and that the particle size, pH and viscosity of the microemulsion were 19.9 ± 5.07 nm, 6.1 and 3.056 × 10(-3 )Pas, respectively. Absorption of ginsenoside Rg1 was higher than that of ginsenoside Rb1, which was barely detectable after intragastric administration; furthermore, the concentration of ginsenoside Rg1 in blood and other tissues at each time point was lower for intragastric than for intranasal administration. Compared with intragastric administration, intranasal administration resulted in a shorter tmax (0.08 versus 1 h), a higher Cmax (16.65 versus 11.29 μg/ml), and a higher area under the concentration-time curve (AUC) (592.91 versus 101.70 μgċh/ml) in the brain. The relative rates of uptake (Re) and the ratio of peak concentration (Ce) in the brain were 126.31% and 147.48% for ginsenoside Rg1, respectively. These data illustrate that intranasal administration can promote the absorption of drugs in Nao-Qing microemulsion and achieve fast effect.

  7. Mutation spectrum of RB1 mutations in retinoblastoma cases from Singapore with implications for genetic management and counselling.

    Directory of Open Access Journals (Sweden)

    Swati Tomar

    Full Text Available Retinoblastoma (RB is a rare childhood malignant disorder caused by the biallelic inactivation of RB1 gene. Early diagnosis and identification of carriers of heritable RB1 mutations can improve disease outcome and management. In this study, mutational analysis was conducted on fifty-nine matched tumor and peripheral blood samples from 18 bilateral and 41 unilateral unrelated RB cases by a combinatorial approach of Multiplex Ligation-dependent Probe Amplification (MLPA assay, deletion screening, direct sequencing, copy number gene dosage analysis and methylation assays. Screening of both blood and tumor samples yielded a mutation detection rate of 94.9% (56/59 while only 42.4% (25/59 of mutations were detected if blood samples alone were analyzed. Biallelic mutations were observed in 43/59 (72.9% of tumors screened. There were 3 cases (5.1% in which no mutations could be detected and germline mutations were detected in 19.5% (8/41 of unilateral cases. A total of 61 point mutations were identified, of which 10 were novel. There was a high incidence of previously reported recurrent mutations, occurring at 38.98% (23/59 of all cases. Of interest were three cases of mosaic RB1 mutations detected in the blood from patients with unilateral retinoblastoma. Additionally, two germline mutations previously reported to be associated with low-penetrance phenotypes: missense-c.1981C>T and splice variant-c.607+1G>T, were observed in a bilateral and a unilateral proband, respectively. These findings have implications for genetic counselling and risk prediction for the affected families. This is the first published report on the spectrum of mutations in RB patients from Singapore and shows that further improved mutation screening strategies are required in order to provide a definitive molecular diagnosis for every case of RB. Our findings also underscore the importance of genetic testing in supporting individualized disease management plans for patients and

  8. Integrative analysis of RNA, translation, and protein levels reveals distinct regulatory variation across humans.

    Science.gov (United States)

    Cenik, Can; Cenik, Elif Sarinay; Byeon, Gun W; Grubert, Fabian; Candille, Sophie I; Spacek, Damek; Alsallakh, Bilal; Tilgner, Hagen; Araya, Carlos L; Tang, Hua; Ricci, Emiliano; Snyder, Michael P

    2015-11-01

    Elucidating the consequences of genetic differences between humans is essential for understanding phenotypic diversity and personalized medicine. Although variation in RNA levels, transcription factor binding, and chromatin have been explored, little is known about global variation in translation and its genetic determinants. We used ribosome profiling, RNA sequencing, and mass spectrometry to perform an integrated analysis in lymphoblastoid cell lines from a diverse group of individuals. We find significant differences in RNA, translation, and protein levels suggesting diverse mechanisms of personalized gene expression control. Combined analysis of RNA expression and ribosome occupancy improves the identification of individual protein level differences. Finally, we identify genetic differences that specifically modulate ribosome occupancy--many of these differences lie close to start codons and upstream ORFs. Our results reveal a new level of gene expression variation among humans and indicate that genetic variants can cause changes in protein levels through effects on translation. © 2015 Cenik et al.; Published by Cold Spring Harbor Laboratory Press.

  9. Correlation between alanine aminotransferase level, HCV-RNA titer ...

    African Journals Online (AJOL)

    In contrast, an insignificant correlation was found between ALT level and grade of necroinflammation. In conclusion neither ALT level nor HCV viremia can reflect the histological liver change accurately. As a result, liver biopsy or other noninvasive procedures that measure liver stiffness (i.e., Fibroscan) remain essential for ...

  10. The skin protective effects of compound K, a metabolite of ginsenoside Rb1 from Panax ginseng

    Directory of Open Access Journals (Sweden)

    Eunji Kim

    2018-04-01

    Full Text Available Background: Compound K (CK is a ginsenoside, a metabolite of Panax ginseng. There is interest both in increasing skin health and antiaging using natural skin care products. In this study, we explored the possibility of using CK as a cosmetic ingredient. Methods: To assess the antiaging effect of CK, RT-PCR was performed, and expression levels of matrix metalloproteinase-1, cyclooxygenase-2, and type I collagen were measured under UVB irradiation conditions. The skin hydrating effect of CK was tested by RT-PCR, and its regulation was explored through immunoblotting. Melanin content, melanin secretion, and tyrosinase activity assays were performed. Results: CK treatment reduced the production of matrix metalloproteinase-1 and cyclooxygenase-2 in UVB irradiated NIH3T3 cells and recovered type I collagen expression level. Expression of skin hydrating factors—filaggrin, transglutaminase, and hyaluronic acid synthases-1 and -2—were augmented by CK and were modulated through the inhibitor of κBα, c-Jun N-terminal kinase, or extracellular signal-regulated kinases pathway. In the melanogenic response, CK did not regulate tyrosinase activity and melanin secretion, but increased melanin content in B16F10 cells was observed. Conclusion: Our data showed that CK has antiaging and hydrating effects. We suggest that CK could be used in cosmetic products to protect the skin from UVB rays and increase skin moisture level. Keywords: compound K, melanogenesis, Panax ginseng, skin protection, UVB irradiation

  11. The skin protective effects of compound K, a metabolite of ginsenoside Rb1 from Panax ginseng.

    Science.gov (United States)

    Kim, Eunji; Kim, Donghyun; Yoo, Sulgi; Hong, Yo Han; Han, Sang Yun; Jeong, Seonggu; Jeong, Deok; Kim, Jong-Hoon; Cho, Jae Youl; Park, Junseong

    2018-04-01

    Compound K (CK) is a ginsenoside, a metabolite of Panax ginseng . There is interest both in increasing skin health and antiaging using natural skin care products. In this study, we explored the possibility of using CK as a cosmetic ingredient. To assess the antiaging effect of CK, RT-PCR was performed, and expression levels of matrix metalloproteinase-1, cyclooxygenase-2, and type I collagen were measured under UVB irradiation conditions. The skin hydrating effect of CK was tested by RT-PCR, and its regulation was explored through immunoblotting. Melanin content, melanin secretion, and tyrosinase activity assays were performed. CK treatment reduced the production of matrix metalloproteinase-1 and cyclooxygenase-2 in UVB irradiated NIH3T3 cells and recovered type I collagen expression level. Expression of skin hydrating factors-filaggrin, transglutaminase, and hyaluronic acid synthases-1 and -2-were augmented by CK and were modulated through the inhibitor of κBα, c-Jun N-terminal kinase, or extracellular signal-regulated kinases pathway. In the melanogenic response, CK did not regulate tyrosinase activity and melanin secretion, but increased melanin content in B16F10 cells was observed. Our data showed that CK has antiaging and hydrating effects. We suggest that CK could be used in cosmetic products to protect the skin from UVB rays and increase skin moisture level.

  12. Low-level lasers alter mRNA levels from traditional reference genes used in breast cancer cells

    Science.gov (United States)

    Teixeira, A. F.; Canuto, K. S.; Rodrigues, J. A.; Fonseca, A. S.; Mencalha, A. L.

    2017-07-01

    Cancer is among the leading causes of mortality worldwide, increasing the importance of treatment development. Low-level lasers are used in several diseases, but some concerns remains on cancers. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is a technique used to understand cellular behavior through quantification of mRNA levels. Output data from target genes are commonly relative to a reference that cannot vary according to treatment. This study evaluated reference genes levels from MDA-MB-231 cells exposed to red or infrared lasers at different fluences. Cultures were exposed to red and infrared lasers, incubated (4 h, 37 °C), total RNA was extracted and cDNA synthesis was performed to evaluate mRNA levels from ACTB, GUSB and TRFC genes by RT-qPCR. Specific amplification was verified by melting curves and agarose gel electrophoresis. RefFinder enabled data analysis by geNorm, NormFinder and BestKeeper. Specific amplifications were obtained and, although mRNA levels from ACTB, GUSB or TRFC genes presented no significant variation through traditional statistical analysis, Excel-based tools revealed that the use of these reference genes are dependent of laser characteristics. Our data showed that exposure to low-level red and infrared lasers at different fluences alter the mRNA levels from ACTB, GUSB and TRFC in MDA-MB-231 cells.

  13. Ginsenoside Rb1 Protects Neonatal Rat Cardiomyocytes from Hypoxia/Ischemia Induced Apoptosis and Inhibits Activation of the Mitochondrial Apoptotic Pathway

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    Xu Yan

    2014-01-01

    Full Text Available Aim. To investigate the effect of Ginsenoside Rb1 (GS-Rb1 on hypoxia/ischemia (H/I injury in cardiomyocytes in vitro and the mitochondrial apoptotic pathway mediated mechanism. Methods. Neonatal rat cardiomyocytes (NRCMs for the H/I groups were kept in DMEM without glucose and serum, and were placed into a hypoxic jar for 24 h. GS-Rb1 at concentrations from 2.5 to 40 µM was given during hypoxic period for 24 h. NRCMs injury was determined by MTT and lactate dehydrogenase (LDH leakage assay. Cell apoptosis, ROS accumulation, and mitochondrial membrane potential (MMP were assessed by flow cytometry. Cytosolic translocation of mitochondrial cytochrome c and Bcl-2 family proteins were determined by Western blot. Caspase-3 and caspase-9 activities were determined by the assay kit. Results. GS-Rb1 significantly reduced cell death and LDH leakage induced by H/I. It also reduced H/I induced NRCMs apoptosis induced by H/I, in accordance with a minimal reactive oxygen species (ROS burst. Moreover, GS-Rb1 markedly decreased the translocation of cytochrome c from the mitochondria to the cytosol, increased the Bcl-2/ Bax ratio, and preserved mitochondrial transmembrane potential (ΔΨm. Its administration also inhibited activities of caspase-9 and caspase-3. Conclusion. Administration of GS-Rb1 during H/I in vitro is involved in cardioprotection by inhibiting apoptosis, which may be due to inhibition of the mitochondrial apoptotic pathway.

  14. Dissecting miRNA gene repression on single cell level with an advanced fluorescent reporter system

    Science.gov (United States)

    Lemus-Diaz, Nicolas; Böker, Kai O.; Rodriguez-Polo, Ignacio; Mitter, Michael; Preis, Jasmin; Arlt, Maximilian; Gruber, Jens

    2017-01-01

    Despite major advances on miRNA profiling and target predictions, functional readouts for endogenous miRNAs are limited and frequently lead to contradicting conclusions. Numerous approaches including functional high-throughput and miRISC complex evaluations suggest that the functional miRNAome differs from the predictions based on quantitative sRNA profiling. To resolve the apparent contradiction of expression versus function, we generated and applied a fluorescence reporter gene assay enabling single cell analysis. This approach integrates and adapts a mathematical model for miRNA-driven gene repression. This model predicts three distinct miRNA-groups with unique repression activities (low, mid and high) governed not just by expression levels but also by miRNA/target-binding capability. Here, we demonstrate the feasibility of the system by applying controlled concentrations of synthetic siRNAs and in parallel, altering target-binding capability on corresponding reporter-constructs. Furthermore, we compared miRNA-profiles with the modeled predictions of 29 individual candidates. We demonstrate that expression levels only partially reflect the miRNA function, fitting to the model-projected groups of different activities. Furthermore, we demonstrate that subcellular localization of miRNAs impacts functionality. Our results imply that miRNA profiling alone cannot define their repression activity. The gene regulatory function is a dynamic and complex process beyond a minimalistic conception of “highly expressed equals high repression”. PMID:28338079

  15. Gene-specific correlation of RNA and protein levels in human cells and tissues

    DEFF Research Database (Denmark)

    Edfors, Fredrik; Danielsson, Frida; Hallström, Björn M.

    2016-01-01

    An important issue for molecular biology is to establish whether transcript levels of a given gene can be used as proxies for the corresponding protein levels. Here, we have developed a targeted proteomics approach for a set of human non-secreted proteins based on parallel reaction monitoring...... to measure, at steady-state conditions, absolute protein copy numbers across human tissues and cell lines and compared these levels with the corresponding mRNA levels using transcriptomics. The study shows that the transcript and protein levels do not correlate well unless a gene-specific RNA-to-protein (RTP...

  16. Elevated levels of circulating microRNA-200 family members correlate with serous epithelial ovarian cancer

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    Kan Casina WS

    2012-12-01

    Full Text Available Abstract Background There is a critical need for improved diagnostic markers for high grade serous epithelial ovarian cancer (SEOC. MicroRNAs are stable in the circulation and may have utility as biomarkers of malignancy. We investigated whether levels of serum microRNA could discriminate women with high-grade SEOC from age matched healthy volunteers. Methods To identify microRNA of interest, microRNA expression profiling was performed on 4 SEOC cell lines and normal human ovarian surface epithelial cells. Total RNA was extracted from 500 μL aliquots of serum collected from patients with SEOC (n = 28 and age-matched healthy donors (n = 28. Serum microRNA levels were assessed by quantitative RT-PCR following preamplification. Results microRNA (miR-182, miR-200a, miR-200b and miR-200c were highly overexpressed in the SEOC cell lines relative to normal human ovarian surface epithelial cells and were assessed in RNA extracted from serum as candidate biomarkers. miR-103, miR-92a and miR -638 had relatively invariant expression across all ovarian cell lines, and with small-nucleolar C/D box 48 (RNU48 were assessed in RNA extracted from serum as candidate endogenous normalizers. No correlation between serum levels and age were observed (age range 30-79 years for any of these microRNA or RNU48. Individually, miR-200a, miR-200b and miR-200c normalized to serum volume and miR-103 were significantly higher in serum of the SEOC cohort (P  Conclusions We identified serum microRNAs able to discriminate patients with high grade SEOC from age-matched healthy controls. The addition of these microRNAs to current testing regimes may improve diagnosis for women with SEOC.

  17. Ginsenoside Rb1 protects against 6-hydroxydopamine-induced oxidative stress by increasing heme oxygenase-1 expression through an estrogen receptor-related PI3K/Akt/Nrf2-dependent pathway in human dopaminergic cells

    International Nuclear Information System (INIS)

    Hwang, Yong Pil; Jeong, Hye Gwang

    2010-01-01

    Phytoestrogens are polyphenolic non-steroidal plant compounds with estrogen-like biological activity. Ginseng, the root of Panax ginseng C.A. Meyer (Araliaceae), is a popular traditional herbal medicine. Ginsenoside Rb1 (Rb1), an active component commonly found in ginseng root, is a phytoestrogen that exerts estrogen-like activity. In this study, we demonstrate that the phytoestrogen Rb1 inhibits 6-hydroxydopamine (6-OHDA)-induced oxidative injury via an ER-dependent Gβ1/PI3K/Akt and heme oxygenase-1 (HO-1) pathway. Pretreatment of SH-SY5Y cells with Rb1 significantly reduced 6-OHDA-induced caspase-3 activation and subsequent cell death. Rb1 also up-regulated HO-1 expression, which conferred cytoprotection against 6-OHDA-induced oxidative injury. Moreover, Rb1 induced both Nrf2 nuclear translocation, which is upstream of HO-1 expression and PI3K activation, a pathway that is involved in induced Nrf2 nuclear translocation, HO-1 expression and cytoprotection. Also, Rb1-mediated increases in PI3K activation and HO-1 induction were reversed by co-treatment with ICI 182,780 and pertussis toxin. Taken together, these results suggest that Rb1 augments the cellular antioxidant defenses through ER-dependent HO-1 induction via the Gβ1/PI3K/Akt-Nrf2 signaling pathway, thereby protecting cells from oxidative stress. Thus our study indicates that Rb1 has a partial cytoprotective role in dopaminergic cell culture systems.

  18. Evaluation of glucosidases of Aspergillus niger strain comparing with other glucosidases in transformation of ginsenoside Rb1 to ginsenosides Rg3

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    Kyung Hoon Chang

    2014-01-01

    Full Text Available The transformation of ginsenoside Rb1 into a specific minor ginsenoside using Aspergillus niger KCCM 11239, as well as the identification of the transformed products and the pathway via thin layer chromatography and high performance liquid chromatography were evaluated to develop a new biologically active material. The conversion of ginsenoside Rb1 generated Rd, Rg3, Rh2, and compound K although the reaction rates were low due to the low concentration. In enzymatic conversion, all of the ginsenoside Rb1 was converted to ginsenoside Rd and ginsenoside Rg3 after 24 h of incubation. The crude enzyme (β-glucosidase from A. niger KCCM 11239 hydrolyzed the β-(1→6-glucosidic linkage at the C-20 of ginsenoside Rb1 to generate ginsenoside Rd and ginsenoside Rg3. Our experimental demonstration showing that A. niger KCCM 11239 produces the ginsenoside-hydrolyzing β-glucosidase reflects the feasibility of developing a specific bioconversion process to obtain active minor ginsenosides.

  19. Study by EPR and Dielectric Constant of Proton-Glass behavior in the system Rb1-X(NH4)XH2PO4:As

    International Nuclear Information System (INIS)

    Almanza, O.; Diaz J, M.; Diaz S

    1996-01-01

    From dielectric constant and EPR measurements of the system Rb1-X(NH4)XH2PO4:As we obtained the phase-diagram Tc Vs x. EPR measurements suggest a proton-glass behavior for 0.3= =0.8. In the doping-range 0.4=< x<=1 the system shows a splitting in the low field line

  20. Regulation of retinoschisin secretion in Weri-Rb1 cells by the F-actin and microtubule cytoskeleton.

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    Eiko Kitamura

    Full Text Available Retinoschisin is encoded by the gene responsible for X-linked retinoschisis (XLRS, an early onset macular degeneration that results in a splitting of the inner layers of the retina and severe loss in vision. Retinoschisin is predominantly expressed and secreted from photoreceptor cells as a homo-oligomer protein; it then associates with the surface of retinal cells and maintains the retina cellular architecture. Many missense mutations in the XLRS1 gene are known to cause intracellular retention of retinoschisin, indicating that the secretion process of the protein is a critical step for its normal function in the retina. However, the molecular mechanisms underlying retinoschisin's secretion remain to be fully elucidated. In this study, we investigated the role of the F-actin cytoskeleton in the secretion of retinoschisin by treating Weri-Rb1 cells, which are known to secrete retinoschisin, with cytochalasin D, jasplakinolide, Y-27632, and dibutyryl cGMP. Our results show that cytochalasin D and jasplakinolide inhibit retinoschisin secretion, whereas Y-27632 and dibutyryl cGMP enhance secretion causing F-actin alterations. We also demonstrate that high concentrations of taxol, which hyperpolymerizes microtubules, inhibit retinoschisin secretion. Our data suggest that retinoschisin secretion is regulated by the F-actin cytoskeleton, that cGMP or inhibition of ROCK alters F-actin structure enhancing the secretion, and that the microtubule cytoskeleton is also involved in this process.

  1. Regulation of Retinoschisin Secretion in Weri-Rb1 Cells by the F-Actin and Microtubule Cytoskeleton

    Science.gov (United States)

    Kitamura, Eiko; Gribanova, Yekaterina E.; Farber, Debora B.

    2011-01-01

    Retinoschisin is encoded by the gene responsible for X-linked retinoschisis (XLRS), an early onset macular degeneration that results in a splitting of the inner layers of the retina and severe loss in vision. Retinoschisin is predominantly expressed and secreted from photoreceptor cells as a homo-oligomer protein; it then associates with the surface of retinal cells and maintains the retina cellular architecture. Many missense mutations in the XLRS1 gene are known to cause intracellular retention of retinoschisin, indicating that the secretion process of the protein is a critical step for its normal function in the retina. However, the molecular mechanisms underlying retinoschisin's secretion remain to be fully elucidated. In this study, we investigated the role of the F-actin cytoskeleton in the secretion of retinoschisin by treating Weri-Rb1 cells, which are known to secrete retinoschisin, with cytochalasin D, jasplakinolide, Y-27632, and dibutyryl cGMP. Our results show that cytochalasin D and jasplakinolide inhibit retinoschisin secretion, whereas Y-27632 and dibutyryl cGMP enhance secretion causing F-actin alterations. We also demonstrate that high concentrations of taxol, which hyperpolymerizes microtubules, inhibit retinoschisin secretion. Our data suggest that retinoschisin secretion is regulated by the F-actin cytoskeleton, that cGMP or inhibition of ROCK alters F-actin structure enhancing the secretion, and that the microtubule cytoskeleton is also involved in this process. PMID:21738583

  2. Regulation of elastin synthesis in developing sheep nuchal ligament by elastin mRNA levels

    International Nuclear Information System (INIS)

    Davidson, J.M.; Smith, K.; Shibahara, S.; Tolstoshev, P.; Crystal, R.G.

    1982-01-01

    Levels of elastin production in explant culture of fetal sheep nuchal ligament and corresponding levels of translatable elastin mRNA were determined in parallel studies during a period of rapid growth of the embryo. The identity of the explant culture and cell-free proucts was confirmed by peptide mapping, immunoprecipitation, and the characteristic lack of histidine and methionine. Elastin production was quantitated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and radioimmune precipitation. The translation products could be labeled with methionine only when NH 2 -terminally donated as f-Met-tRNA/sup Met//sub f/. Explant cultures showed a large rise in elastin production from 70 days after conception to 150 days after conception. Cell free translation of RNA demonstrated a parallel in elastin mRNA levels and in elastin mRNA per cell. It appears, therefore, that the marked emphasis the differentiating muchal ligament places on elastin production is modulated, at least in part, by the quantities of available elastin in mRNA

  3. Growth-Rate Dependent Regulation of tRNA Level and Charging in Bacillus licheniformis.

    Science.gov (United States)

    Ferro, Iolanda; Liebeton, Klaus; Ignatova, Zoya

    2017-10-13

    Cellular growth crucially depends on protein synthesis and the abundance of translational components. Among them, aminoacyl-tRNAs play a central role in biosynthesis and shape the kinetics of mRNA translation, thus influencing protein production. Here, we used microarray-based approaches to determine the charging levels and tRNA abundance of Bacillus licheniformis. We observed an interesting cross-talk among tRNA expression, charging pattern, and growth rate. For a large subset of tRNAs, we found a co-regulated and augmented expression at high growth rate. Their tRNA aminoacylation level is kept relatively constant through riboswitch-regulated expression of the cognate aminoacyl-tRNA-synthetase (AARS). We show that AARSs with putative riboswitch-controlled expression are those charging tRNAs with amino acids which disfavor cell growth when individually added to the nutrient medium. Our results suggest that the riboswitch-regulated AARS expression in B. licheniformis is a powerful mechanism not only to maintain a constant ratio of aminoacyl-tRNA independent of the growth rate but concomitantly to control the intracellular level of free amino acids. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Genetic Determinants of RNA Editing Levels of ADAR Targets in Drosophila melanogaster

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    Yerbol Z. Kurmangaliyev

    2016-02-01

    Full Text Available RNA editing usually affects only a fraction of expressed transcripts and there is a vast amount of variation in editing levels of ADAR (adenosine deaminase, RNA-specific targets. Here we explore natural genetic variation affecting editing levels of particular sites in 81 natural strains of Drosophila melanogaster. The analysis of associations between editing levels and single-nucleotide polymorphisms allows us to map putative cis-regulatory regions affecting editing of 16 A-to-I editing sites (cis-RNA editing quantitative trait loci or cis-edQTLs, P < 10−8. The observed changes in editing levels are validated by independent molecular technique. All identified regulatory variants are located in close proximity of modulated editing sites. Moreover, colocalized editing sites are often regulated by same loci. Similar to expression and splicing QTL studies, the characterization of edQTLs will greatly expand our understanding of cis-regulatory evolution of gene expression.

  5. Abnormal levels of expression of plasma microRNA-33 in patients with psoriasis.

    Science.gov (United States)

    García-Rodríguez, S; Arias-Santiago, S; Orgaz-Molina, J; Magro-Checa, C; Valenzuela, I; Navarro, P; Naranjo-Sintes, R; Sancho, J; Zubiaur, M

    2014-06-01

    Circulating microRNAs (miRNA) are involved in the posttranscriptional regulation of genes associated with lipid metabolism (miRNA-33) and vascular function and angiogenesis (miRNA-126). The objective of this exploratory study was to measure plasma levels of miRNA-33 and miRNA-126 in patients with plaque psoriasis and evaluate their association with clinical parameters. We studied 11 patients with plaque psoriasis. The median Psoriasis Area Severity Index (PASI) was 13 (interquartile range [IQR], 9-14) and body surface area involvement was 12 (IQR, 11-15). Eleven healthy controls matched for age and sex were also included. We analyzed cardiovascular risk factors and subclinical carotid atheromatosis. Plasma miRNAs were evaluated using quantitative real-time polymerase chain reaction. Carotid intima-media thickness was greater in patients (0.57mm; IQR, 0.54-0.61; n=11) than in controls (0.50mm; IQR, 0.48-0.54; data available for 9 controls) (P=.0055, Mann-Whitney). Expression of miRNA-33 in patients (5.34; IQR, 3.12-7.96; n=11) was significantly higher than in controls (2.33; IQR, 1.71-2.84; only detected in 7 of 11 controls) (P=.0049, Wilcoxon signed rank). No differences in miRNA-126 levels were observed between patients and controls. In patients (n=11), we observed a positive correlation between miRNA-33 and insulin levels (r=0.7289, P=.0109) and a negative correlation between miRNA-126 and carotid intima-media thickness (r=-0.6181, P=.0426). In psoriasis patients plasma levels of lipid and glucose metabolism-related miRNA-33 are increased and correlated with insulin. The study of circulating miRNA-33 in psoriasis may provide new insights about the associated systemic inflammatory abnormalities. Copyright © 2013 Elsevier España, S.L. and AEDV. All rights reserved.

  6. Evaluation of the miRNA-146a and miRNA-155 Expression Levels in Patients with Oral Lichen Planus.

    Science.gov (United States)

    Ahmadi-Motamayel, Fatemeh; Bayat, Zeynab; Hajilooi, Mehrdad; Shahryar-Hesami, Soroosh; Mahdavinezhad, Ali; Samie, Lida; Solgi, Ghasem

    2017-12-01

    Oral Lichen Planus (OLP) is a chronic autoimmune disease that could be considered as a potential premalignant status. To evaluate the miRNA-146a and miRNA-155 expression levels in patients with oral Lichen planus lesions compared to healthy subjects with normal oral mucosa. Forty patients with oral lichen planus and 18 healthy age and gender-matched controls were recruited in this case-control study. Oral lichen planus was diagnosed clinically and pathologically. The expression levels of two miRNAs in peripheral blood samples were determined using commercial TaqMan MicroRNA Assays. Relative quantification of gene expression was calculated by the 2-ΔΔct method. The expression levels of miRNA-146a and miRNA-155 in patients with oral Lichen planus were significantly higher than those of healthy controls. Also, a direct but insignificant correlation was found between miRNA-155 and miRNA-146a expression levels among the patient group. Our findings indicate that miRNA-146a and miRNA-155 could be potential biomarkers for the immunopathogenesis of oral lichen planus.

  7. Reduced genetic distance and high replication levels increase the RNA recombination rate of hepatitis delta virus.

    Science.gov (United States)

    Lin, Chia-Chi; Yang, Zhi-Wei; Iang, Shan-Bei; Chao, Mei

    2015-01-02

    Hepatitis delta virus (HDV) replication is carried out by host RNA polymerases. Since homologous inter-genotypic RNA recombination is known to occur in HDV, possibly via a replication-dependent process, we hypothesized that the degree of sequence homology and the replication level should be related to the recombination frequency in cells co-expressing two HDV sequences. To confirm this, we separately co-transfected cells with three different pairs of HDV genomic RNAs and analyzed the obtained recombinants by RT-PCR followed by restriction fragment length polymorphism and sequencing analyses. The sequence divergence between the clones ranged from 24% to less than 0.1%, and the difference in replication levels was as high as 100-fold. As expected, significant differences were observed in the recombination frequencies, which ranged from 0.5% to 47.5%. Furthermore, varying the relative amounts of parental RNA altered the dominant recombinant species produced, suggesting that template switching occurs frequently during the synthesis of genomic HDV RNA. Taken together, these data suggest that during the host RNA polymerase-driven RNA recombination of HDV, both inter- and intra-genotypic recombination events are important in shaping the genetic diversity of HDV. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. High-Level Accumulation of Exogenous Small RNAs Not Affecting Endogenous Small RNA Biogenesis and Function in Plants

    Institute of Scientific and Technical Information of China (English)

    SHEN Wan-xia; Neil A Smith; ZHOU Chang-yong; WANG Ming-bo

    2014-01-01

    RNA silencing is a fundamental plant defence and gene control mechanism in plants that are directed by 20-24 nucleotide (nt) small interfering RNA (siRNA) and microRNA (miRNA). Infection of plants with viral pathogens or transformation of plants with RNA interference (RNAi) constructs is usually associated with high levels of exogenous siRNAs, but it is unclear if these siRNAs interfere with endogenous small RNA pathways and hence affect plant development. Here we provide evidence that viral satellite RNA (satRNA) infection does not affect siRNA and miRNA biogenesis or plant growth despite the extremely high level of satRNA-derived siRNAs. We generated transgenic Nicotiana benthamiana plants that no longer develop the speciifc yellowing symptoms generally associated with infection by Cucumber mosaic virus (CMV) Y-satellite RNA (Y-Sat). We then used these plants to show that CMV Y-Sat infection did not cause any visible phenotypic changes in comparison to uninfected plants, despite the presence of high-level Y-Sat siRNAs. Furthermore, we showed that the accumulation of hairpin RNA (hpRNA)-derived siRNAs or miRNAs, and the level of siRNA-directed transgene silencing, are not signiifcantly affected by CMV Y-Sat infection. Taken together, our results suggest that the high levels of exogenous siRNAs associated with viral infection or RNAi-inducing transgenes do not saturate the endogenous RNA silencing machineries and have no signiifcant impact on normal plant development.

  9. Photobiomodulation effects on mRNA levels from genomic and chromosome stabilization genes in injured muscle.

    Science.gov (United States)

    da Silva Neto Trajano, Larissa Alexsandra; Trajano, Eduardo Tavares Lima; da Silva Sergio, Luiz Philippe; Teixeira, Adilson Fonseca; Mencalha, Andre Luiz; Stumbo, Ana Carolina; de Souza da Fonseca, Adenilson

    2018-04-26

    Muscle injuries are the most prevalent type of injury in sports. A great number of athletes have relapsed in muscle injuries not being treated properly. Photobiomodulation therapy is an inexpensive and safe technique with many benefits in muscle injury treatment. However, little has been explored about the infrared laser effects on DNA and telomeres in muscle injuries. Thus, the aim of this study was to evaluate photobiomodulation effects on mRNA relative levels from genes related to telomere and genomic stabilization in injured muscle. Wistar male rats were randomly divided into six groups: control, laser 25 mW, laser 75 mW, injury, injury laser 25 mW, and injury laser 75 mW. Photobiomodulation was performed with 904 nm, 3 J/cm 2 at 25 or 75 mW. Cryoinjury was induced by two applications of a metal probe cooled in liquid nitrogen directly on the tibialis anterior muscle. After euthanasia, skeletal muscle samples were withdrawn and total RNA extracted for evaluation of mRNA levels from genomic (ATM and p53) and chromosome stabilization (TRF1 and TRF2) genes by real-time quantitative polymerization chain reaction. Data show that photobiomodulation reduces the mRNA levels from ATM and p53, as well reduces mRNA levels from TRF1 and TRF2 at 25 and 75 mW in injured skeletal muscle. In conclusion, photobiomodulation alters mRNA relative levels from genes related to genomic and telomere stabilization in injured skeletal muscle.

  10. Association between human papillomavirus and Epstein - Barr virus DNA and gene promoter methylation of RB1 and CDH1 in the cervical lesions: a transversal study.

    Science.gov (United States)

    McCormick, Thaís M; Canedo, Nathalie H S; Furtado, Yara L; Silveira, Filomena A; de Lima, Roberto J; Rosman, Andréa D F; Almeida Filho, Gutemberg L; Carvalho, Maria da Glória da C

    2015-06-02

    Human papillomavirus (HPV) inactivates the retinoblastoma 1 (RB1) gene by promoter methylation and reduces cellular E-cadherin expression by overexpression of DNA methyltransferase 1 (DNMT1). The Epstein-Barr virus (EBV) is an oncogenic virus that may be related to cervical carcinogenesis. In gastric cancer, it has been demonstrated that E-cadherin gene (CDH1) hypermethylation is associated with DNMT1 overexpression by EBV infection. Our aim was to analyze the gene promoter methylation frequency of RB1 and CDH1 and verify the association between that methylation frequency and HPV and EBV infection in cervical lesions. Sixty-five samples were obtained from cervical specimens: 15 normal cervices, 17 low-grade squamous intraepithelial lesions (LSIL), 15 high-grade squamous intraepithelial lesions (HSIL), and 18 cervical cancers. HPV and EBV DNA testing was performed by PCR, and the methylation status was verified by MSP. HPV frequency was associated with cervical cancer cases (p = 0.005) but not EBV frequency (p = 0.732). Viral co-infection showed a statistically significant correlation with cancer (p = 0.027). No viral infection was detected in 33.3% (5/15) of controls. RB1 methylated status was associated with cancer (p = 0.009) and HPV infection (p = 0.042). CDH1 methylation was not associated with cancer (p = 0.181). Controls and LSIL samples did not show simultaneous methylation, while both genes were methylated in 27.8% (5/18) of cancer samples. In the presence of EBV, CDH1 methylation was present in 27.8% (5/18) of cancer samples. Only cancer cases presented RB1 promoter methylation in the presence of HPV and EBV (33.3%). The methylation status of both genes increased with disease progression. With EBV, RB1 methylation was a tumor-associated event because only the cancer group presented methylated RB1 with HPV infection. HPV infection was shown to be significantly correlated with cancer conditions. The global methylation frequency was

  11. ErbB3 mRNA leukocyte levels as a biomarker for major depressive disorder

    Directory of Open Access Journals (Sweden)

    Milanesi Elena

    2012-09-01

    Full Text Available Abstract Background In recent years, the identification of peripheral biomarkers that are associated with psychiatric diseases, such as Major Depressive Disorder (MDD, has become relevant because these biomarkers may improve the efficiency of the differential diagnosis process and indicate targets for new antidepressant drugs. Two recent candidate genes, ErbB3 and Fgfr1, are growth factors whose mRNA levels have been found to be altered in the leukocytes of patients that are affected by bipolar disorder in a depressive state. On this basis, the aim of the study was to determine if ErbB3 and Fgfr1 mRNA levels could be a biomarkers of MDD. Methods We measured by Real Time PCR ErbB3 and Fgfr1 mRNA expression levels in leukocytes of MDD patients compared with controls. Successively, to assess whether ErbB3 mRNA levels were influenced by previous antidepressant treatment we stratified our patients sample in two cohorts, comparing drug-naive versus drug-free patients. Moreover, we evaluated the levels of the transcript in MDD patients after 12 weeks of antidepressant treatment, and in prefrontal cortex of rats stressed and treated with an antidepressant drug of the same class. Results These results showed that ErbB3 but not Fgfr1 mRNA levels were reduced in leukocytes of MDD patients compared to healthy subjects. Furthermore, ErbB3 levels were not affected by antidepressant treatment in either human or animal models Conclusions Our data suggest that ErbB3 might be considered as a biomarker for MDD and that its deficit may underlie the pathopsysiology of the disease and is not a consequence of treatment. Moreover the study supports the usefulness of leukocytes as a peripheral system for identifying biomarkers in psychiatric diseases.

  12. Calling genotypes from public RNA-sequencing data enables identification of genetic variants that affect gene-expression levels

    NARCIS (Netherlands)

    Deelen, Patrick; Zhernakova, Daria V.; de Haan, Mark; van der Sijde, Marijke; Bonder, Marc Jan; Karjalainen, Juha; van der Velde, K. Joeri; Abbott, Kristin M.; Fu, Jingyuan; Wijmenga, Cisca; Sinke, Richard J.; Swertz, Morris A.; Franke, Lude

    2015-01-01

    Background: RNA-sequencing (RNA-seq) is a powerful technique for the identification of genetic variants that affect gene-expression levels, either through expression quantitative trait locus (eQTL) mapping or through allele-specific expression (ASE) analysis. Given increasing numbers of RNA-seq

  13. Plasma processing conditions substantially influence circulating microRNA biomarker levels.

    Science.gov (United States)

    Cheng, Heather H; Yi, Hye Son; Kim, Yeonju; Kroh, Evan M; Chien, Jason W; Eaton, Keith D; Goodman, Marc T; Tait, Jonathan F; Tewari, Muneesh; Pritchard, Colin C

    2013-01-01

    Circulating, cell-free microRNAs (miRNAs) are promising candidate biomarkers, but optimal conditions for processing blood specimens for miRNA measurement remain to be established. Our previous work showed that the majority of plasma miRNAs are likely blood cell-derived. In the course of profiling lung cancer cases versus healthy controls, we observed a broad increase in circulating miRNA levels in cases compared to controls and that higher miRNA expression correlated with higher platelet and particle counts. We therefore hypothesized that the quantity of residual platelets and microparticles remaining after plasma processing might impact miRNA measurements. To systematically investigate this, we subjected matched plasma from healthy individuals to stepwise processing with differential centrifugation and 0.22 µm filtration and performed miRNA profiling. We found a major effect on circulating miRNAs, with the majority (72%) of detectable miRNAs substantially affected by processing alone. Specifically, 10% of miRNAs showed 4-30x variation, 46% showed 30-1,000x variation, and 15% showed >1,000x variation in expression solely from processing. This was predominantly due to platelet contamination, which persisted despite using standard laboratory protocols. Importantly, we show that platelet contamination in archived samples could largely be eliminated by additional centrifugation, even in frozen samples stored for six years. To minimize confounding effects in microRNA biomarker studies, additional steps to limit platelet contamination for circulating miRNA biomarker studies are necessary. We provide specific practical recommendations to help minimize confounding variation attributable to plasma processing and platelet contamination.

  14. Loss of heterozygosity of CDKN2A (p16INK4a) and RB1 tumor suppressor genes in testicular germ cell tumors

    International Nuclear Information System (INIS)

    Vladusic, Tomislav; Hrascan, Reno; Pecina-Slaus, Nives; Vrhovac, Ivana; Gamulin, Marija; Franekic, Jasna; Kruslin, Bozo

    2010-01-01

    Testicular germ cell tumors (TGCTs) are the most frequent malignances in young adult men. The two main histological forms, seminomas and nonseminomas, differ biologically and clinically. pRB protein and its immediate upstream regulator p16INK4a are involved in the RB pathway which is deregulated in most TGCTs. The objective of this study was to evaluate the occurrence of loss of heterozygosity (LOH) of the CDKN2A (p16INK4a) and RB1 tumor suppressor genes in TGCTs. Forty TGCTs (18 seminomas and 22 nonseminomas) were analyzed by polymerase chain reaction using the restriction fragment length polymorphism or the nucleotide repeat polymorphism method. LOH of the CDKN2A was found in two (6%) out of 34 (85%) informative cases of our total TGCT sample. The observed changes were assigned to two (11%) nonseminomas out of 18 (82%) informative samples. Furthermore, LOH of the RB1 was detected in two (6%) out of 34 (85%) informative cases of our total TGCT sample. Once again, the observed changes were assigned to two (10.5%) nonseminomas out of 19 (86%) informative samples. Both LOHs of the CDKN2A were found in nonseminomas with a yolk sac tumor component, and both LOHs of the RB1 were found in nonseminomas with an embryonal carcinoma component. The higher incidence of observed LOH in nonseminomas may provide a clue to their invasive behavior

  15. Correlation of microRNA levels during hypoxia with predicted target mRNAs through genome-wide microarray analysis

    Directory of Open Access Journals (Sweden)

    Page Grier P

    2009-03-01

    Full Text Available Abstract Background Low levels of oxygen in tissues, seen in situations such as chronic lung disease, necrotic tumors, and high altitude exposures, initiate a signaling pathway that results in active transcription of genes possessing a hypoxia response element (HRE. The aim of this study was to investigate whether a change in miRNA expression following hypoxia could account for changes in the cellular transcriptome based on currently available miRNA target prediction tools. Methods To identify changes induced by hypoxia, we conducted mRNA- and miRNA-array-based experiments in HT29 cells, and performed comparative analysis of the resulting data sets based on multiple target prediction algorithms. To date, few studies have investigated an environmental perturbation for effects on genome-wide miRNA levels, or their consequent influence on mRNA output. Results Comparison of miRNAs with predicted mRNA targets indicated a lower level of concordance than expected. We did, however, find preliminary evidence of combinatorial regulation of mRNA expression by miRNA. Conclusion Target prediction programs and expression profiling techniques do not yet adequately represent the complexity of miRNA-mediated gene repression, and new methods may be required to better elucidate these pathways. Our data suggest the physiologic impact of miRNAs on cellular transcription results from a multifaceted network of miRNA and mRNA relationships, working together in an interconnected system and in context of hundreds of RNA species. The methods described here for comparative analysis of cellular miRNA and mRNA will be useful for understanding genome wide regulatory responsiveness and refining miRNA predictive algorithms.

  16. Mucin gene mRNA levels in broilers challenged with eimeria and/or Clostridium perfringens.

    Science.gov (United States)

    Kitessa, Soressa M; Nattrass, Gregory S; Forder, Rebecca E A; McGrice, Hayley A; Wu, Shu-Biao; Hughes, Robert J

    2014-09-01

    The effects of Eimeria (EM) and Clostridium perfringens (CP) challenges on the mRNA levels of genes involved in mucin (Muc) synthesis (Muc2, Muc5ac, Muc13, and trefoil family factor-2 [TFF2]), inflammation (tumor necrosis factor alpha [TNF-alpha] and interleukin-18 [IL-18]), and metabolic processes (cluster of differentiation [CD]36) in the jejunum of broilers were investigated. Two parallel experiments involving 1) EM challenge and 2) EM and CP challenges were conducted. The first experiment was a 2 X 2 study with 12 birds per treatment (N = 48) involving fishmeal substitution (25%) in the diet (FM) and EM challenge. The treatments were: Control (FM-, EM-), Fishmeal (FM+, EM-), EM challenge (FM-, EM+), and fishmeal substitution and EM challenge (FM+, EM+). The second experiment was a 2 X 2 X 2 experiment with six birds per treatment (N = 48) involving fishmeal (FM-, FM+), Eimeria (EM-, EM+), and C perfringens (CP-, CP+). In both arms of the study, male broilers were given a starter diet for the whole period of 16 days, except those assigned to FM+, where 25% of the starter ration was replaced with fishmeal from days 8 to 14. EM inoculation was performed on day 9 and CP inoculation on days 14 and 15. The EM challenge birds were euthanatized for sampling on day 13; postmortem examination and sampling for the Eimeria plus C perfringens challenge arm of the study were on day 16. In the Eimeria challenge arm of the study, fishmeal supplementation significantly suppressed the mRNA levels of TNF-alpha, TFF2, and IL-18 pre-CP inoculation but simultaneously increased the levels of Muc13 and CD36 mRNAs. Birds challenged with Eimeria exhibited increased mRNA levels of Muc13, Muc5ac, TNF-alpha, and IL-18. In the Eimeria and C. perfringens challenge arm, birds exposed to EM challenge exhibited significantly lower mRNA levels of Muc2 and CD36. The mRNA levels of CD36 were also significantly suppressed by CP challenge. Our results showed that the transcription of mucin synthesis

  17. Analysis of circulating hem-endothelial marker RNA levels in preterm infants

    Directory of Open Access Journals (Sweden)

    Kuint Jacob

    2009-06-01

    Full Text Available Abstract Background Circulating endothelial cells may serve as novel markers of angiogenesis. These include a subset of hem-endothelial progenitor cells that play a vital role in vascular growth and repair. The presence and clinical implications of circulating RNA levels as an expression for hematopoietic and endothelial-specific markers have not been previously evaluated in preterm infants. This study aims to determine circulating RNA levels of hem-endothelial marker genes in peripheral blood of preterm infants and begin to correlate these findings with prenatal complications. Methods Peripheral blood samples from seventeen preterm neonates were analyzed at three consecutive post-delivery time points (day 3–5, 10–15 and 30. Using quantitative reverse transcription-polymerase chain reaction we studied the expression patterns of previously established hem-endothelial-specific progenitor-associated genes (AC133, Tie-2, Flk-1 (VEGFR2 and Scl/Tal1 in association with characteristics of prematurity and preterm morbidity. Results Circulating Tie-2 and SCL/Tal1 RNA levels displayed an inverse correlation to gestational age (GA. We observed significantly elevated Tie-2 levels in preterm infants born to mothers with amnionitis, and in infants with sustained brain echogenicity on brain sonography. Other markers showed similar expression patterns yet we could not demonstrate statistically significant correlations. Conclusion These preliminary findings suggest that circulating RNA levels especially Tie2 and SCL decline with maturation and might relate to some preterm complication. Further prospective follow up of larger cohorts are required to establish this association.

  18. Genetic Determinants of RNA Editing Levels of ADAR Targets in Drosophila melanogaster.

    Science.gov (United States)

    Kurmangaliyev, Yerbol Z; Ali, Sammi; Nuzhdin, Sergey V

    2015-12-12

    RNA editing usually affects only a fraction of expressed transcripts and there is a vast amount of variation in editing levels of ADAR (adenosine deaminase, RNA-specific) targets. Here we explore natural genetic variation affecting editing levels of particular sites in 81 natural strains of Drosophila melanogaster. The analysis of associations between editing levels and single-nucleotide polymorphisms allows us to map putative cis-regulatory regions affecting editing of 16 A-to-I editing sites (cis-RNA editing quantitative trait loci or cis-edQTLs, P < 10(-8)). The observed changes in editing levels are validated by independent molecular technique. All identified regulatory variants are located in close proximity of modulated editing sites. Moreover, colocalized editing sites are often regulated by same loci. Similar to expression and splicing QTL studies, the characterization of edQTLs will greatly expand our understanding of cis-regulatory evolution of gene expression. Copyright © 2016 Kurmangaliyev et al.

  19. Assessment of flhDC mRNA levels in Serratia liquefaciens swarm cells

    DEFF Research Database (Denmark)

    Tolker-Nielsen, Tim; Christensen, Allan Beck; Holmstrøm, K.

    2000-01-01

    We reported previously that artificial overexpression of the flhDC operon in liquid-grown Serratia liquefaciens resulted in the formation of filamentous, multinucleated, and hyperflagellated cells that were indistinguishable from surface-induced swarm cells (L. Eberl, G. Christiansen, S. Molin, a......, vegetative cells. This suggests that surface-induced S. liquefaciens swarm cell differentiation, although dependent on flhDC gene expression, does not occur through elevated flhDC mRNA levels....

  20. Effect of physical training on glucose transporter protein and mRNA levels in rat adipocytes

    DEFF Research Database (Denmark)

    Stallknecht, B; Andersen, P H; Vinten, J

    1993-01-01

    Physical training increases insulin-stimulated glucose transport and the number of glucose transporters in adipocytes measured by cytochalasin B binding. In the present study we used immunoblotting to measure the abundance of two glucose transporters (GLUT-4, GLUT-1) in white adipocytes from....../or intrinsic activity). GLUT-1 protein and mRNA levels/adipocyte volume did not change with age or training....

  1. Lin28a uses distinct mechanisms of binding to RNA and affects miRNA levels positively and negatively.

    Science.gov (United States)

    Nowak, Jakub Stanislaw; Hobor, Fruzsina; Downie Ruiz Velasco, Angela; Choudhury, Nila Roy; Heikel, Gregory; Kerr, Alastair; Ramos, Andres; Michlewski, Gracjan

    2017-03-01

    Lin28a inhibits the biogenesis of let-7 miRNAs by triggering the polyuridylation and degradation of their precursors by terminal uridylyltransferases TUT4/7 and 3'-5' exoribonuclease Dis3l2, respectively. Previously, we showed that Lin28a also controls the production of neuro-specific miRNA-9 via a polyuridylation-independent mechanism. Here we reveal that the sequences and structural characteristics of pre-let-7 and pre-miRNA-9 are eliciting two distinct modes of binding to Lin28a. We present evidence that Dis3l2 controls miRNA-9 production. Finally, we show that the constitutive expression of untagged Lin28a during neuronal differentiation in vitro positively and negatively affects numerous other miRNAs. Our findings shed light on the role of Lin28a in differentiating cells and on the ways in which one RNA-binding protein can perform multiple roles in the regulation of RNA processing. © 2017 Nowak et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  2. Active Center Control of Termination by RNA Polymerase III and tRNA Gene Transcription Levels In Vivo.

    Directory of Open Access Journals (Sweden)

    Keshab Rijal

    2016-08-01

    Full Text Available The ability of RNA polymerase (RNAP III to efficiently recycle from termination to reinitiation is critical for abundant tRNA production during cellular proliferation, development and cancer. Yet understanding of the unique termination mechanisms used by RNAP III is incomplete, as is its link to high transcription output. We used two tRNA-mediated suppression systems to screen for Rpc1 mutants with gain- and loss- of termination phenotypes in S. pombe. 122 point mutation mutants were mapped to a recently solved 3.9 Å structure of yeast RNAP III elongation complex (EC; they cluster in the active center bridge helix and trigger loop, as well as the pore and funnel, the latter of which indicate involvement of the RNA cleavage domain of the C11 subunit in termination. Purified RNAP III from a readthrough (RT mutant exhibits increased elongation rate. The data strongly support a kinetic coupling model in which elongation rate is inversely related to termination efficiency. The mutants exhibit good correlations of terminator RT in vitro and in vivo, and surprisingly, amounts of transcription in vivo. Because assessing in vivo transcription can be confounded by various parameters, we used a tRNA reporter with a processing defect and a strong terminator. By ruling out differences in RNA decay rates, the data indicate that mutants with the RT phenotype synthesize more RNA than wild type cells, and than can be accounted for by their increased elongation rate. Finally, increased activity by the mutants appears unrelated to the RNAP III repressor, Maf1. The results show that the mobile elements of the RNAP III active center, including C11, are key determinants of termination, and that some of the mutations activate RNAP III for overall transcription. Similar mutations in spontaneous cancer suggest this as an unforeseen mechanism of RNAP III activation in disease.

  3. RNA-Seq and molecular docking reveal multi-level pesticide resistance in the bed bug

    Directory of Open Access Journals (Sweden)

    Mamidala Praveen

    2012-01-01

    Full Text Available Abstract Background Bed bugs (Cimex lectularius are hematophagous nocturnal parasites of humans that have attained high impact status due to their worldwide resurgence. The sudden and rampant resurgence of C. lectularius has been attributed to numerous factors including frequent international travel, narrower pest management practices, and insecticide resistance. Results We performed a next-generation RNA sequencing (RNA-Seq experiment to find differentially expressed genes between pesticide-resistant (PR and pesticide-susceptible (PS strains of C. lectularius. A reference transcriptome database of 51,492 expressed sequence tags (ESTs was created by combining the databases derived from de novo assembled mRNA-Seq tags (30,404 ESTs and our previous 454 pyrosequenced database (21,088 ESTs. The two-way GLMseq analysis revealed ~15,000 highly significant differentially expressed ESTs between the PR and PS strains. Among the top 5,000 differentially expressed ESTs, 109 putative defense genes (cuticular proteins, cytochrome P450s, antioxidant genes, ABC transporters, glutathione S-transferases, carboxylesterases and acetyl cholinesterase involved in penetration resistance and metabolic resistance were identified. Tissue and development-specific expression of P450 CYP3 clan members showed high mRNA levels in the cuticle, Malpighian tubules, and midgut; and in early instar nymphs, respectively. Lastly, molecular modeling and docking of a candidate cytochrome P450 (CYP397A1V2 revealed the flexibility of the deduced protein to metabolize a broad range of insecticide substrates including DDT, deltamethrin, permethrin, and imidacloprid. Conclusions We developed significant molecular resources for C. lectularius putatively involved in metabolic resistance as well as those participating in other modes of insecticide resistance. RNA-Seq profiles of PR strains combined with tissue-specific profiles and molecular docking revealed multi-level insecticide

  4. Neutron scattering investigations of the properties of the x - T phase diagram of Rb1-x(NH4)xI mixed crystals

    International Nuclear Information System (INIS)

    Smirnov, L.S.; Natkaniec, I.; Savenko, B.N.

    2002-01-01

    The x - T phase diagram of Rb 1-x (NH 4 ) x I is studied using samples with the ammonium concentration 0.01< x<0.77 over a wide temperature region of 15 to 300 K by neutron powder diffraction and inelastic incoherent neutron scattering. The results of powder diffraction studies show that at low temperatures a phase transition from α-phase to β-phase is observed at ammonium concentrations x = 0.50 and x = 0.66. Inelastic incoherent neutron scattering detects a region of the orientional glass state at ammonium concentrations 0.29< x,0.40

  5. Let-7 and MicroRNA-148 Regulate Parathyroid Hormone Levels in Secondary Hyperparathyroidism.

    Science.gov (United States)

    Shilo, Vitali; Mor-Yosef Levi, Irit; Abel, Roy; Mihailović, Aleksandra; Wasserman, Gilad; Naveh-Many, Tally; Ben-Dov, Iddo Z

    2017-08-01

    Secondary hyperparathyroidism commonly complicates CKD and associates with morbidity and mortality. We profiled microRNA (miRNA) in parathyroid glands from experimental hyperparathyroidism models and patients receiving dialysis and studied the function of specific miRNAs. miRNA deep-sequencing showed that human and rodent parathyroids share similar profiles. Parathyroids from uremic and normal rats segregated on the basis of their miRNA expression profiles, and a similar finding was observed in humans. We identified parathyroid miRNAs that were dysregulated in experimental hyperparathyroidism, including miR-29, miR-21, miR-148, miR-30, and miR-141 (upregulated); and miR-10, miR-125, and miR-25 (downregulated). Inhibition of the abundant let-7 family increased parathyroid hormone (PTH) secretion in normal and uremic rats, as well as in mouse parathyroid organ cultures. Conversely, inhibition of the upregulated miR-148 family prevented the increase in serum PTH level in uremic rats and decreased levels of secreted PTH in parathyroid cultures. The evolutionary conservation of abundant miRNAs in normal parathyroid glands and the regulation of these miRNAs in secondary hyperparathyroidism indicates their importance for parathyroid function and the development of hyperparathyroidism. Specifically, let-7 and miR-148 antagonism modified PTH secretion in vivo and in vitro , implying roles for these specific miRNAs. These findings may be utilized for therapeutic interventions aimed at altering PTH expression in diseases such as osteoporosis and secondary hyperparathyroidism. Copyright © 2017 by the American Society of Nephrology.

  6. Lin28a uses distinct mechanisms of binding to RNA and affects miRNA levels positively and negatively

    OpenAIRE

    Nowak, Jakub Stanislaw; Hobor, Fruzsina; Downie Ruiz Velasco, Angela; Choudhury, Nila Roy; Heikel, Gregory; Kerr, Alastair; Ramos, Andres; Michlewski, Gracjan

    2017-01-01

    Lin28a inhibits the biogenesis of let-7 miRNAs by triggering the polyuridylation and degradation of their precursors by terminal uridylyltransferases TUT4/7 and 3’-5’ exoribonuclease Dis3l2, respectively. Previously, we showed that Lin28a also controls the production of neuro-specific miRNA-9 via a polyuridylation-independent mechanism. Here we reveal that the sequences and structural characteristics of pre-let-7 and pre-miRNA-9 are eliciting two distinct modes of binding to Lin28a. We presen...

  7. Association of microRNA-200c expression levels with clinicopathological factors and prognosis in endometrioid endometrial cancer.

    Science.gov (United States)

    Wilczynski, Milosz; Danielska, Justyna; Domanska-Senderowska, Daria; Dzieniecka, Monika; Szymanska, Bozena; Malinowski, Andrzej

    2018-05-01

    MicroRNAs (miRNAs) are regulators of gene expression, which play an important role in many critical cellular processes including apoptosis, proliferation and cell differentiation. Aberrant miRNA expression has been reported in a variety of human malignancies. Therefore, miRNAs may be potentially used as cancer biomarkers. miRNA-200c, which is a member of the miRNA-200 family, might play an essential role in tumor progression. The purpose of this study was to evaluate the prognostic and clinical significance of miRNA-200c in women with endometrioid endometrial cancer. Total RNA extraction from 90 archival formalin-fixed paraffin-embedded tissue samples of endometri-oid endometrial cancer and 10 normal endometrium samples was performed. After cDNA synthesis, real-time polymerase chain reaction was conducted and relative expression of miRNA-200c was assessed. Then, miRNA-200c expression levels were evaluated with regard to clinicopathological characteristics. The expression levels of miRNA-200c were significantly increased in endometrioid endometrial cancer samples. Expression of miRNA-200c maintained at significantly higher levels in the early stage endometrioid endometrial cancer compared with more advanced stages. In the Kaplan-Meier analysis, lower levels of miRNA-200c expression were associated with inferior survival. Expression levels of miRNA-200c might be associated with clinicopathological factors and survival in endometrioid endometrial cancer. © 2018 Nordic Federation of Societies of Obstetrics and Gynecology.

  8. Why weight? Modelling sample and observational level variability improves power in RNA-seq analyses.

    Science.gov (United States)

    Liu, Ruijie; Holik, Aliaksei Z; Su, Shian; Jansz, Natasha; Chen, Kelan; Leong, Huei San; Blewitt, Marnie E; Asselin-Labat, Marie-Liesse; Smyth, Gordon K; Ritchie, Matthew E

    2015-09-03

    Variations in sample quality are frequently encountered in small RNA-sequencing experiments, and pose a major challenge in a differential expression analysis. Removal of high variation samples reduces noise, but at a cost of reducing power, thus limiting our ability to detect biologically meaningful changes. Similarly, retaining these samples in the analysis may not reveal any statistically significant changes due to the higher noise level. A compromise is to use all available data, but to down-weight the observations from more variable samples. We describe a statistical approach that facilitates this by modelling heterogeneity at both the sample and observational levels as part of the differential expression analysis. At the sample level this is achieved by fitting a log-linear variance model that includes common sample-specific or group-specific parameters that are shared between genes. The estimated sample variance factors are then converted to weights and combined with observational level weights obtained from the mean-variance relationship of the log-counts-per-million using 'voom'. A comprehensive analysis involving both simulations and experimental RNA-sequencing data demonstrates that this strategy leads to a universally more powerful analysis and fewer false discoveries when compared to conventional approaches. This methodology has wide application and is implemented in the open-source 'limma' package. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  9. Serum microRNA-1 and microRNA-133a levels reflect myocardial steatosis in uncomplicated type 2 diabetes

    NARCIS (Netherlands)

    Gonzalo-Calvo, D. de; Meer, R.W. van der; Rijzewijk, L.J.; Smit, J.W.A.; Revuelta-Lopez, E.; Nasarre, L.; Escola-Gil, J.C.; Lamb, H.J.; Llorente-Cortes, V.

    2017-01-01

    Using in vitro, in vivo and patient-based approaches, we investigated the potential of circulating microRNAs (miRNAs) as surrogate biomarkers of myocardial steatosis, a hallmark of diabetic cardiomyopathy. We analysed the cardiomyocyte-enriched miRNA signature in serum from patients with

  10. Myb-binding protein 1a (Mybbp1a) regulates levels and processing of pre-ribosomal RNA.

    Science.gov (United States)

    Hochstatter, Julia; Hölzel, Michael; Rohrmoser, Michaela; Schermelleh, Lothar; Leonhardt, Heinrich; Keough, Rebecca; Gonda, Thomas J; Imhof, Axel; Eick, Dirk; Längst, Gernot; Németh, Attila

    2012-07-13

    Ribosomal RNA gene transcription, co-transcriptional processing, and ribosome biogenesis are highly coordinated processes that are tightly regulated during cell growth. In this study we discovered that Mybbp1a is associated with both the RNA polymerase I complex and the ribosome biogenesis machinery. Using a reporter assay that uncouples transcription and RNA processing, we show that Mybbp1a represses rRNA gene transcription. In addition, overexpression of the protein reduces RNA polymerase I loading on endogenous rRNA genes as revealed by chromatin immunoprecipitation experiments. Accordingly, depletion of Mybbp1a results in an accumulation of the rRNA precursor in vivo but surprisingly also causes growth arrest of the cells. This effect can be explained by the observation that the modulation of Mybbp1a protein levels results in defects in pre-rRNA processing within the cell. Therefore, the protein may play a dual role in the rRNA metabolism, potentially linking and coordinating ribosomal DNA transcription and pre-rRNA processing to allow for the efficient synthesis of ribosomes.

  11. Hamp1 mRNA and plasma hepcidin levels are influenced by sex and strain but do not predict tissue iron levels in inbred mice.

    Science.gov (United States)

    McLachlan, Stela; Page, Kathryn E; Lee, Seung-Min; Loguinov, Alex; Valore, Erika; Hui, Simon T; Jung, Grace; Zhou, Jie; Lusis, Aldons J; Fuqua, Brie; Ganz, Tomas; Nemeth, Elizabeta; Vulpe, Chris D

    2017-11-01

    Iron homeostasis is tightly regulated, and the peptide hormone hepcidin is considered to be a principal regulator of iron metabolism. Previous studies in a limited number of mouse strains found equivocal sex- and strain-dependent differences in mRNA and serum levels of hepcidin and reported conflicting data on the relationship between hepcidin ( Hamp1 ) mRNA levels and iron status. Our aim was to clarify the relationships between strain, sex, and hepcidin expression by examining multiple tissues and the effects of different dietary conditions in multiple inbred strains. Two studies were done: first, Hamp1 mRNA, liver iron, and plasma diferric transferrin levels were measured in 14 inbred strains on a control diet; and second, Hamp1 mRNA and plasma hepcidin levels in both sexes and iron levels in the heart, kidneys, liver, pancreas, and spleen in males were measured in nine inbred/recombinant inbred strains raised on an iron-sufficient or high-iron diet. Both sex and strain have a significant effect on both hepcidin mRNA (primarily a sex effect) and plasma hepcidin levels (primarily a strain effect). However, liver iron and diferric transferrin levels are not predictors of Hamp1 mRNA levels in mice fed iron-sufficient or high-iron diets, nor are the Hamp1 mRNA and plasma hepcidin levels good predictors of tissue iron levels, at least in males. We also measured plasma erythroferrone, performed RNA-sequencing analysis of liver samples from six inbred strains fed the iron-sufficient, low-iron, or high-iron diets, and explored differences in gene expression between the strains with the highest and lowest hepcidin levels. NEW & NOTEWORTHY Both sex and strain have a significant effect on both hepcidin mRNA (primarily a sex effect) and plasma hepcidin levels (primarily a strain effect). Liver iron and diferric transferrin levels are not predictors of Hamp1 mRNA levels in mice, nor are the Hamp1 mRNA and plasma hepcidin levels good predictors of tissue iron levels, at least

  12. Alternative Polyadenylation and Nonsense-Mediated Decay Coordinately Regulate the Human HFE mRNA Levels

    Science.gov (United States)

    Martins, Rute; Proença, Daniela; Silva, Bruno; Barbosa, Cristina; Silva, Ana Luísa; Faustino, Paula; Romão, Luísa

    2012-01-01

    Nonsense-mediated decay (NMD) is an mRNA surveillance pathway that selectively recognizes and degrades defective mRNAs carrying premature translation-termination codons. However, several studies have shown that NMD also targets physiological transcripts that encode full-length proteins, modulating their expression. Indeed, some features of physiological mRNAs can render them NMD-sensitive. Human HFE is a MHC class I protein mainly expressed in the liver that, when mutated, can cause hereditary hemochromatosis, a common genetic disorder of iron metabolism. The HFE gene structure comprises seven exons; although the sixth exon is 1056 base pairs (bp) long, only the first 41 bp encode for amino acids. Thus, the remaining downstream 1015 bp sequence corresponds to the HFE 3′ untranslated region (UTR), along with exon seven. Therefore, this 3′ UTR encompasses an exon/exon junction, a feature that can make the corresponding physiological transcript NMD-sensitive. Here, we demonstrate that in UPF1-depleted or in cycloheximide-treated HeLa and HepG2 cells the HFE transcripts are clearly upregulated, meaning that the physiological HFE mRNA is in fact an NMD-target. This role of NMD in controlling the HFE expression levels was further confirmed in HeLa cells transiently expressing the HFE human gene. Besides, we show, by 3′-RACE analysis in several human tissues that HFE mRNA expression results from alternative cleavage and polyadenylation at four different sites – two were previously described and two are novel polyadenylation sites: one located at exon six, which confers NMD-resistance to the corresponding transcripts, and another located at exon seven. In addition, we show that the amount of HFE mRNA isoforms resulting from cleavage and polyadenylation at exon seven, although present in both cell lines, is higher in HepG2 cells. These results reveal that NMD and alternative polyadenylation may act coordinately to control HFE mRNA levels, possibly varying its

  13. Interpretation of the CABRI-RAFT RB1 and RB2 tests through detailed data evaluation and PAPAS-2S code analysis

    International Nuclear Information System (INIS)

    Fukano, Yoshitaka; Sato, Ikken

    2001-08-01

    The CABRI-RAFT RB1 and RB2 tests were aiming at a study on impact of fuel pin failure under an overpower condition leading to fuel melting. Using a special technique, combination of through-cladding failure and fuel melting was realized. In the RB1 test, fuel ejection was prevented under a limited fuel melting condition. On the other hand, significant fuel melting was applied in the RB2 test so as to get the fuel ejection, thereby obtaining information on the fuel ejection behavior. Interpretation for these tests through the detailed experimental data evaluation and the PAPAS-2S code analysis is performed in this study. Through this study, it is indicated that molten fuel ejection can be prevented with the low smear density fuel as far as the fuel melting is not large for a slit-type cladding defect. Fuel ejection becomes possible in the case of significant fuel melting with a very thin solid fuel shell surrounding the molten fuel cavity. However, the rapidness of the fuel ejection with the low smear density fuel is less pronounced compared with that of the high smear density fuel. It is also confirmed that there is considerable DN-precursor release into the coolant flow already before fuel ejection. The result is very useful for evaluation of anomaly detection with DN signal observation. (author)

  14. Expression of Versican 3′-Untranslated Region Modulates Endogenous MicroRNA Functions

    Science.gov (United States)

    Lee, Daniel Y.; Jeyapalan, Zina; Fang, Ling; Yang, Jennifer; Zhang, Yaou; Yee, Albert Y.; Li, Minhui; Du, William W.; Shatseva, Tatiana; Yang, Burton B.

    2010-01-01

    Background Mature microRNAs (miRNAs) are single-stranded RNAs that regulate post-transcriptional gene expression. In our previous study, we have shown that versican 3′UTR, a fragment of non-coding transcript, has the ability to antagonize miR-199a-3p function thereby regulating expression of the matrix proteins versican and fibronectin, and thus resulting in enhanced cell-cell adhesion and organ adhesion. However, the impact of this non-coding fragment on tumorigenesis is yet to be determined. Methods and Findings Using computational prediction confirmed with in vitro and in vivo experiments, we report that the expression of versican 3′UTR not only antagonizes miR-199a-3p but can also lower its steady state expression. We found that expression of versican 3′UTR in a mouse breast carcinoma cell line, 4T1, decreased miR-199a-3p levels. The decrease in miRNA activity consequently translated into differences in tumor growth. Computational analysis indicated that both miR-199a-3p and miR-144 targeted a cell cycle regulator, Rb1. In addition, miR-144 and miR-136, which have also been shown to interact with versican 3′UTR, was found to target PTEN. Expression of Rb1 and PTEN were up-regulated synergistically in vitro and in vivo, suggesting that the 3′UTR binds and modulates miRNA activities, freeing Rb1 and PTEN mRNAs for translation. In tumor formation assays, cells transfected with the 3′UTR formed smaller tumors compared with cells transfected with a control vector. Conclusion Our results demonstrated that a 3′UTR fragment can be used to modulate miRNA functions. Our study also suggests that miRNAs in the cancer cells are more susceptible to degradation, due to its interaction with a non-coding 3′UTR. This non-coding component of mRNA may be used retrospectively to modulate miRNA activities. PMID:21049042

  15. Expression of versican 3'-untranslated region modulates endogenous microRNA functions.

    Science.gov (United States)

    Lee, Daniel Y; Jeyapalan, Zina; Fang, Ling; Yang, Jennifer; Zhang, Yaou; Yee, Albert Y; Li, Minhui; Du, William W; Shatseva, Tatiana; Yang, Burton B

    2010-10-25

    Mature microRNAs (miRNAs) are single-stranded RNAs that regulate post-transcriptional gene expression. In our previous study, we have shown that versican 3'UTR, a fragment of non-coding transcript, has the ability to antagonize miR-199a-3p function thereby regulating expression of the matrix proteins versican and fibronectin, and thus resulting in enhanced cell-cell adhesion and organ adhesion. However, the impact of this non-coding fragment on tumorigenesis is yet to be determined. Using computational prediction confirmed with in vitro and in vivo experiments, we report that the expression of versican 3'UTR not only antagonizes miR-199a-3p but can also lower its steady state expression. We found that expression of versican 3'UTR in a mouse breast carcinoma cell line, 4T1, decreased miR-199a-3p levels. The decrease in miRNA activity consequently translated into differences in tumor growth. Computational analysis indicated that both miR-199a-3p and miR-144 targeted a cell cycle regulator, Rb1. In addition, miR-144 and miR-136, which have also been shown to interact with versican 3'UTR, was found to target PTEN. Expression of Rb1 and PTEN were up-regulated synergistically in vitro and in vivo, suggesting that the 3'UTR binds and modulates miRNA activities, freeing Rb1 and PTEN mRNAs for translation. In tumor formation assays, cells transfected with the 3'UTR formed smaller tumors compared with cells transfected with a control vector. Our results demonstrated that a 3'UTR fragment can be used to modulate miRNA functions. Our study also suggests that miRNAs in the cancer cells are more susceptible to degradation, due to its interaction with a non-coding 3'UTR. This non-coding component of mRNA may be used retrospectively to modulate miRNA activities.

  16. Expression of versican 3'-untranslated region modulates endogenous microRNA functions.

    Directory of Open Access Journals (Sweden)

    Daniel Y Lee

    Full Text Available BACKGROUND: Mature microRNAs (miRNAs are single-stranded RNAs that regulate post-transcriptional gene expression. In our previous study, we have shown that versican 3'UTR, a fragment of non-coding transcript, has the ability to antagonize miR-199a-3p function thereby regulating expression of the matrix proteins versican and fibronectin, and thus resulting in enhanced cell-cell adhesion and organ adhesion. However, the impact of this non-coding fragment on tumorigenesis is yet to be determined. METHODS AND FINDINGS: Using computational prediction confirmed with in vitro and in vivo experiments, we report that the expression of versican 3'UTR not only antagonizes miR-199a-3p but can also lower its steady state expression. We found that expression of versican 3'UTR in a mouse breast carcinoma cell line, 4T1, decreased miR-199a-3p levels. The decrease in miRNA activity consequently translated into differences in tumor growth. Computational analysis indicated that both miR-199a-3p and miR-144 targeted a cell cycle regulator, Rb1. In addition, miR-144 and miR-136, which have also been shown to interact with versican 3'UTR, was found to target PTEN. Expression of Rb1 and PTEN were up-regulated synergistically in vitro and in vivo, suggesting that the 3'UTR binds and modulates miRNA activities, freeing Rb1 and PTEN mRNAs for translation. In tumor formation assays, cells transfected with the 3'UTR formed smaller tumors compared with cells transfected with a control vector. CONCLUSION: Our results demonstrated that a 3'UTR fragment can be used to modulate miRNA functions. Our study also suggests that miRNAs in the cancer cells are more susceptible to degradation, due to its interaction with a non-coding 3'UTR. This non-coding component of mRNA may be used retrospectively to modulate miRNA activities.

  17. Dengue viral RNA levels in peripheral blood mononuclear cells are associated with disease severity and preexisting dengue immune status.

    Directory of Open Access Journals (Sweden)

    Anon Srikiatkhachorn

    Full Text Available Infection with dengue viruses (DENV causes a wide range of manifestations from asymptomatic infection to a febrile illness called dengue fever (DF, to dengue hemorrhagic fever (DHF. The in vivo targets of DENV and the relation between the viral burden in these cells and disease severity are not known.The levels of positive and negative strand viral RNA in peripheral blood monocytes, T/NK cells, and B cells and in plasma of DF and DHF cases were measured by quantitative RT-PCR.Positive strand viral RNA was detected in monocytes, T/NK cells and B cells with the highest amounts found in B cells. Viral RNA levels in CD14+ cells and plasma were significantly higher in DHF compared to DF, and in cases with a secondary infection compared to those undergoing a primary infection. The distribution of viral RNA among cell subpopulations was similar in DF and DHF cases. Small amounts of negative strand RNA were found in a few cases only. The severity of plasma leakage correlated with viral RNA levels in plasma and in CD14+ cells.B cells were the principal cells containing DENV RNA in peripheral blood, but overall there was little active DENV RNA replication detectable in peripheral blood mononuclear cells (PBMC. Secondary infection and DHF were associated with higher viral burden in PBMC populations, especially CD14+ monocytes, suggesting that viral infection of these cells may be involved in disease pathogenesis.

  18. Chronic periodontitis can affect the levels of potential oral cancer salivary mRNA biomarkers.

    Science.gov (United States)

    Cheng, Y-S L; Jordan, L; Chen, H-S; Kang, D; Oxford, L; Plemons, J; Parks, H; Rees, T

    2017-06-01

    More than 100 salivary constituents have been found to show levels significantly different in patients with oral squamous cell carcinoma (OSCC) from those found in healthy controls, and therefore have been suggested to be potential salivary biomarkers for OSCC detection. However, many of these potential OSCC salivary biomarkers are also involved in chronic inflammation, and whether the levels of these biomarkers could be affected by the presence of chronic periodontitis was not known. The objective of this pilot study was therefore to measure the levels of seven previously reported potential OSCC salivary mRNA biomarkers in patients with chronic periodontitis and compare them to levels found in patients with OSCC and healthy controls. The seven salivary mRNAs were interleukin (IL)-8, IL-1β, dual specificity phosphatase 1, H3 histone family 3A, ornithine decarboxylase antizyme 1, S100 calcium-binding protein P (S100P) and spermidine/spermine N1-acetyltransferase 1. Unstimulated whole saliva samples were collected from a total of 105 human subjects from the following four study groups: OSCC; CPNS (chronic periodontitis, moderate to severe degree, non-smokers); CPS (chronic periodontitis, moderate to severe degree, smokers); and healthy controls. Levels of each mRNA in patient groups (OSCC or chronic periodontitis) relative to the healthy controls were determined by a pre-amplification reverse transcription-quantitative polymerase chain reaction approach with nested gene-specific primers. Results were recorded and analyzed by the Bio-Rad CFX96 Real-Time System. Mean fold changes between each pair of patient vs. control groups were analyzed by the Mann-Whitney U-test with Bonferroni corrections. Only S100P showed significantly higher levels in patients with OSCC compared to both patients with CPNS (p = 0.003) and CPS (p = 0.007). The difference in S100P levels between patients with OSCC and healthy controls was also marginally significant (p = 0.009). There was no

  19. HLA-G allelic variants are associated with differences in the HLA-G mRNA isoform profile and HLA-G mRNA levels

    DEFF Research Database (Denmark)

    Hviid, Thomas Vauvert F; Hylenius, Sine; Rørbye, Christina

    2003-01-01

    between mother and fetus in several ways. Finally, the expression of membrane-bound HLA-G and soluble HLA-G has been proposed to influence the outcome of pregnancy, and an aberrant HLA-G expression in pre-eclamptic placentas and spontaneous abortions has been reported. Here, an association between certain...... HLA-G polymorphisms and the mRNA levels of the different alternatively spliced HLA-G isoforms in first trimester trophoblast cell populations is reported. Several alternatively spliced HLA-G mRNA isoforms, including a 14-bp polymorphism in the 3'UTR end (exon 8) of the HLA-G gene, are expressed...

  20. Gestational Protein Restriction Increases Cardiac Connexin 43 mRNA levels in male adult rat offspring

    Science.gov (United States)

    Rossini, Kamila Fernanda; de Oliveira, Camila Andrea; Rebelato, Hércules Jonas; Esquisatto, Marcelo Augusto Marreto; Catisti, Rosana

    2017-01-01

    Background The dietary limitation during pregnancy influences the growth and development of the fetus and offspring and their health into adult life. The mechanisms underlying the adverse effects of gestational protein restriction (GPR) in the development of the offspring hearts are not well understood. Objectives The aim of this study was to evaluate the effects of GPR on cardiac structure in male rat offspring at day 60 after birth (d60). Methods Pregnant Wistar rats were fed a normal-protein (NP, 17% casein) or low-protein (LP, 6% casein) diet. Blood pressure (BP) values from 60-day-old male offspring were measured by an indirect tail-cuff method using an electro sphygmomanometer. Hearts (d60) were collected for assessment of connexin 43 (Cx43) mRNA expression and morphological and morphometric analysis. Results LP offspring showed no difference in body weight, although they were born lighter than NP offspring. BP levels were significantly higher in the LP group. We observed a significant increase in the area occupied by collagen fibers, a decrease in the number of cardiomyocytes by 104 µm2, and an increase in cardiomyocyte area associated with an increased Cx43 expression. Conclusion GPR changes myocardial levels of Cx43 mRNA in male young adult rats, suggesting that this mechanism aims to compensate the fibrotic process by the accumulation of collagen fibers in the heart interstitium. PMID:28678925

  1. Transcript levels, alternative splicing and proteolytic cleavage of TFIIIA control 5S rRNA accumulation during Arabidopsis thaliana development.

    Science.gov (United States)

    Layat, Elodie; Cotterell, Sylviane; Vaillant, Isabelle; Yukawa, Yasushi; Tutois, Sylvie; Tourmente, Sylvette

    2012-07-01

    Ribosome biogenesis is critical for eukaryotic cells and requires coordinated synthesis of the protein and rRNA moieties of the ribosome, which are therefore highly regulated. 5S ribosomal RNA, an essential component of the large ribosomal subunit, is transcribed by RNA polymerase III and specifically requires transcription factor IIIA (TFIIIA). To obtain insight into the regulation of 5S rRNA transcription, we have investigated the expression of 5S rRNA and the exon-skipped (ES) and exon-including (EI) TFIIIA transcripts, two transcript isoforms that result from alternative splicing of the TFIIIA gene, and TFIIIA protein amounts with respect to requirements for 5S rRNA during development. We show that 5S rRNA quantities are regulated through distinct but complementary mechanisms operating through transcriptional and post-transcriptional control of TFIIIA transcripts as well as at the post-translational level through proteolytic cleavage of the TFIIIA protein. During the reproductive phase, high expression of the TFIIIA gene together with low proteolytic cleavage contributes to accumulation of functional, full-length TFIIIA protein, and results in 5S rRNA accumulation in the seed. In contrast, just after germination, the levels of TFIIIA-encoding transcripts are low and stable. Full-length TFIIIA protein is undetectable, and the level of 5S rRNA stored in the embryo progressively decreases. After day 4, in correlation with the reorganization of 5S rDNA chromatin to a mature state, full-length TFIIIA protein with transcriptional activity accumulates and permits de novo transcription of 5S rRNA. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.

  2. Visualization and measurement of ATP levels in living cells replicating hepatitis C virus genome RNA.

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    Tomomi Ando

    Full Text Available Adenosine 5'-triphosphate (ATP is the primary energy currency of all living organisms and participates in a variety of cellular processes. Although ATP requirements during viral lifecycles have been examined in a number of studies, a method by which ATP production can be monitored in real-time, and by which ATP can be quantified in individual cells and subcellular compartments, is lacking, thereby hindering studies aimed at elucidating the precise mechanisms by which viral replication energized by ATP is controlled. In this study, we investigated the fluctuation and distribution of ATP in cells during RNA replication of the hepatitis C virus (HCV, a member of the Flaviviridae family. We demonstrated that cells involved in viral RNA replication actively consumed ATP, thereby reducing cytoplasmic ATP levels. Subsequently, a method to measure ATP levels at putative subcellular sites of HCV RNA replication in living cells was developed by introducing a recently-established Förster resonance energy transfer (FRET-based ATP indicator, called ATeam, into the NS5A coding region of the HCV replicon. Using this method, we were able to observe the formation of ATP-enriched dot-like structures, which co-localize with non-structural viral proteins, within the cytoplasm of HCV-replicating cells but not in non-replicating cells. The obtained FRET signals allowed us to estimate ATP concentrations within HCV replicating cells as ∼5 mM at possible replicating sites and ∼1 mM at peripheral sites that did not appear to be involved in HCV replication. In contrast, cytoplasmic ATP levels in non-replicating Huh-7 cells were estimated as ∼2 mM. To our knowledge, this is the first study to demonstrate changes in ATP concentration within cells during replication of the HCV genome and increased ATP levels at distinct sites within replicating cells. ATeam may be a powerful tool for the study of energy metabolism during replication of the viral genome.

  3. Quantitative correlation between promoter methylation and messenger RNA levels of the reduced folate carrier

    Directory of Open Access Journals (Sweden)

    Kheradpour Albert

    2008-05-01

    Full Text Available Abstract Background Methotrexate (MTX uptake is mediated by the reduced folate carrier (RFC. Defective drug uptake in association with decreased RFC expression is a common mechanism of MTX resistance in many tumor types. Heavy promoter methylation was previously identified as a basis for the complete silencing of RFC in MDA-MB-231 breast cancer cells, its role and prevalence in RFC transcription regulation are, however, not widely studied. Methods In the current study, RFC promoter methylation was assessed using methylation specific PCR in a panel of malignant cell lines (n = 8, including MDA-MB-231, and M805, a MTX resistant cell line directly established from the specimen of a patient with malignant fibrohistocytoma, whom received multiple doses of MTX. A quantitative approach of real-time PCR for measuring the extent of RFC promoter methylation was developed, and was validated by direct bisulfite genomic sequencing. RFC mRNA levels were determined by quantitative real-time RT-PCR and were related to the extent of promoter methylation in these cell lines. Results A partial promoter methylation and RFC mRNA down-regulation were observed in M805. Using the quantitative approach, a reverse correlation (correlation coefficient = -0.59, p Conclusion This study further suggests that promoter methylation is a potential basis for MTX resistance. The quantitative correlation identified in this study implies that promoter methylation is possibly a mechanism involved in the fine regulation of RFC transcription.

  4. Ginsenoside Rb1 Attenuates Oxygen-Glucose Deprivation-Induced Apoptosis in SH-SY5Y Cells via Protection of Mitochondria and Inhibition of AIF and Cytochrome c Release

    Directory of Open Access Journals (Sweden)

    Pengfei Ge

    2013-10-01

    Full Text Available To investigate the role of mitochondria in the protective effects of ginsenoside Rb1 on cellular apoptosis caused by oxygen-glucose deprivation, in this study, MTT assay, TUNEL staining, flow cytometry, immunocytochemistry and western blotting were used to examine the cellular viability, apoptosis, ROS level, mitochondrial membrane potential, and the distribution of apoptosis inducing factor, cytochrome c, Bax and Bcl-2 in nucleus, mitochondria and cytoplasm. We found that pretreatment with GRb1 improved the cellular viability damaged by OGD. Moreover, GRb1 inhibited apoptosis in SH-SY5Y cells induced by OGD. Further studies showed that the elevation of cellular reactive oxygen species levels and the reduction of mitochondrial membrane potential caused by OGD were both counteracted by GRb1. Additionally, GRb1 not only suppressed the translocation of apoptosis inducing factor into nucleus and cytochrome c into cytoplasm, but also inhibited the increase of Bax within mitochondria and alleviated the decrease of mitochondrial Bcl-2. Our study indicates that the protection of GRb1 on OGD-induced apoptosis in SH-SY5Y cells is associated with its protection on mitochondrial function and inhibition of release of AIF and cytochrome c.

  5. Repeat testing of low-level HIV-1 RNA: assay performance and implementation in clinical trials.

    Science.gov (United States)

    White, Kirsten; Garner, Will; Wei, Lilian; Eron, Joseph J; Zhong, Lijie; Miller, Michael D; Martin, Hal; Plummer, Andrew; Tran-Muchowski, Cecilia; Lindstrom, Kim; Porter, James; Piontkowsky, David; Light, Angela; Reiske, Heinz; Quirk, Erin

    2018-05-15

    Assess the performance of HIV-1 RNA repeat testing of stored samples in cases of low-level viremia during clinical trials. Prospective and retrospective analysis of randomized clinical trial samples and reference standards. To evaluate assay variability of the Cobas AmpliPrep/Cobas TaqMan HIV-1 Test, v2.0, three separate sources of samples were utilized: the World Health Organization (WHO) HIV reference standard (assayed using 50 independent measurements at six viral loads <200 copies/ml), retrospective analysis of four to six aliquots of plasma samples from four clinical trial participants, and prospective repeat testing of 120 samples from participants in randomized trials with low-level viremia. The TaqMan assay on the WHO HIV-1 RNA standards at viral loads <200 copies/ml performed within the expected variability according to assay specifications. However, standards with low viral loads of 36 and 18 copies/ml reported values of ≥ 50 copies/ml in 66 and 18% of tests, respectively. In participants treated with antiretrovirals who had unexpected viremia of 50-200 copies/ml after achieving <50 copies/ml, retesting of multiple aliquots of stored plasma found <50 copies/ml in nearly all cases upon retesting (14/15; 93%). Repeat testing was prospectively implemented in four clinical trials for all samples with virologic rebound of 50-200 copies/ml (n = 120 samples from 92 participants) from which 42% (50/120) had a retest result of less than 50 copies/ml and 58% (70/120) retested ≥ 50 copies/ml. The TaqMan HIV-1 RNA assay shows variability around 50 copies/ml that affects clinical trial results and may impact clinical practice. In participants with a history of viral load suppression, unexpected low-level viremia may be because of assay variability rather than low drug adherence or true virologic failure. Retesting a stored aliquot of the same sample may differentiate between assay variability and virologic failure as the source of viremia

  6. In C. elegans, high levels of dsRNA allow RNAi in the absence of RDE-4.

    Science.gov (United States)

    Habig, Jeffrey W; Aruscavage, P Joseph; Bass, Brenda L

    2008-01-01

    C. elegans Dicer requires an accessory double-stranded RNA binding protein, RDE-4, to enact the first step of RNA interference, the cleavage of dsRNA to produce siRNA. While RDE-4 is typically essential for RNAi, we report that in the presence of high concentrations of trigger dsRNA, rde-4 deficient animals are capable of silencing a transgene. By multiple criteria the silencing occurs by the canonical RNAi pathway. For example, silencing is RDE-1 dependent and exhibits a decrease in the targeted mRNA in response to an increase in siRNA. We also find that high concentrations of dsRNA trigger lead to increased accumulation of primary siRNAs, consistent with the existence of a rate-limiting step during the conversion of primary to secondary siRNAs. Our studies also revealed that transgene silencing occurs at low levels in the soma, even in the presence of ADARs, and that at least some siRNAs accumulate in a temperature-dependent manner. We conclude that an RNAi response varies with different conditions, and this may allow an organism to tailor a response to specific environmental signals.

  7. In C. elegans, high levels of dsRNA allow RNAi in the absence of RDE-4.

    Directory of Open Access Journals (Sweden)

    Jeffrey W Habig

    Full Text Available C. elegans Dicer requires an accessory double-stranded RNA binding protein, RDE-4, to enact the first step of RNA interference, the cleavage of dsRNA to produce siRNA. While RDE-4 is typically essential for RNAi, we report that in the presence of high concentrations of trigger dsRNA, rde-4 deficient animals are capable of silencing a transgene. By multiple criteria the silencing occurs by the canonical RNAi pathway. For example, silencing is RDE-1 dependent and exhibits a decrease in the targeted mRNA in response to an increase in siRNA. We also find that high concentrations of dsRNA trigger lead to increased accumulation of primary siRNAs, consistent with the existence of a rate-limiting step during the conversion of primary to secondary siRNAs. Our studies also revealed that transgene silencing occurs at low levels in the soma, even in the presence of ADARs, and that at least some siRNAs accumulate in a temperature-dependent manner. We conclude that an RNAi response varies with different conditions, and this may allow an organism to tailor a response to specific environmental signals.

  8. TP53 and ATM mRNA expression in skin and skeletal muscle after low-level laser exposure.

    Science.gov (United States)

    Guedes de Almeida, Luciana; Sergio, Luiz Philippe da Silva; de Paoli, Flavia; Mencalha, Andre Luiz; da Fonseca, Adenilson de Souza

    2017-08-01

    Low-level lasers are widespread in regenerative medicine, but the molecular mechanisms involved in their biological effects are not fully understood, particularly those on DNA stability. Therefore, this study aimed to investigate mRNA expression of genes related to DNA genomic stability in skin and skeletal muscle tissue from Wistar rats exposed to low-level red and infrared lasers. For this, TP53 (Tumor Protein 53) and ATM (Ataxia Telangiectasia Mutated gene) mRNA expressions were evaluated by real-time quantitative PCR (RT-qPCR) technique 24 hours after low-level red and infrared laser exposure. Our data showed that relative TP53 mRNA expression was not significantly altered in both tissues exposed to lasers. For ATM, relative mRNA expression in skin tissue was not significantly altered, but in muscle tissue, laser exposure increased relative ATM mRNA expression. Low-level red and infrared laser radiations alter ATM mRNA expression related to DNA stability in skeletal muscle tissue.

  9. Increased level of miRNA 30b-3p in patients with prostatic hyperplasia and testosterone with high-level of prostate-specific antigen

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    Wasnaa Jumaa Mohammad

    2018-01-01

    Full Text Available Background: Prostate cancer (PCa is the most common causing cancer-related in death in men and lack of reliable diagnostic tool. MicroRNAs are small molecules single-stranded RNA that affecting protein expression at the level of translation and dysregulation can dramatically affect cell metabolism. However, the using of circulating miRNAs as diagnostic biomarkers for diagnosis of PCa is still unknown. Methods: Ten patients with prostatic hyperplasia with high-level of PSA and 10 healthy controls were conducted in this study. The reverse transcription of miRNA based on quantitative polymerase chain reaction (qPCR were used for evaluating the dysregulation of miRNA 30b-3p and using of ELISA to evaluate the level of prostate-specific antigen (PSA and testosterone hormone. Results: Circulating miRNA 30b-3p level was increased in patients with prostatic hyperplasia with higher level of PSA as compared with healthy controls. Also, the testosterone hormone was increased in those patients as compared with normal level of testosterone in healthy individuals. Conclusion: The serum miRNA 30b-3p level increased in patients with hyperplasia in prostate and may be one of potential biomarker for diagnosis of PCa.

  10. Sputum and serum microRNA-144 levels in patients with tuberculosis before and after treatment.

    Science.gov (United States)

    Lv, Yan; Guo, Shuai; Li, Xue-Gang; Chi, Jing-Yu; Qu, Yi-Qing; Zhong, Hai-Lai

    2016-02-01

    To measure the expression levels of sputum and serum microRNA-144 (miR-144) before and after the treatment of patients with tuberculosis (TB). Details of the cases of a total of 124 TB patients were collected at Qilu Hospital of Shandong University between April 2014 and April 2015. Fifty-three of these patients had sputum positive for bacteria and a cavity on imaging (group A), 20 patients had sputum negative for bacteria and a cavity on imaging (group B), and 51 patients had sputum negative for bacteria and no cavity on imaging (group C). One hundred seventeen healthy people who attended the hospital for a physical examination were recruited as controls. Quantitative real-time PCR (qRT-PCR) was used to measure the levels of sputum and serum miR-144 before anti-TB treatment and at 1 month after treatment. Before treatment, sputum and serum miR-144 expression levels in the TB patients were both higher than those of the controls (both p<0.05). After treatment, sputum and serum miR-144 levels in the TB patients were significantly lower than those measured before treatment (both p<0.05). The levels of sputum and serum miR-144 in the improved TB patients decreased significantly after treatment compared to those measured before treatment (both p<0.001). Significant differences were found in sputum and serum miR-144 levels in the TB patients, with or without improvement, compared with the healthy controls (all p<0.05). Sputum and serum miR-144 levels were significantly upregulated in the TB patients, but were found to decrease significantly after anti-TB treatment. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  11. Dose-Response of High-Intensity Training (HIT) on Atheroprotective miRNA-126 Levels

    Science.gov (United States)

    Schmitz, Boris; Schelleckes, Katrin; Nedele, Johanna; Thorwesten, Lothar; Klose, Andreas; Lenders, Malte; Krüger, Michael; Brand, Eva; Brand, Stefan-Martin

    2017-01-01

    Aim: MicroRNA-126 (miR-126) exerts beneficial effects on vascular integrity, angiogenesis, and atherosclerotic plaque stability. The purpose of this investigation was to analyze the dose-response relationship of high-intensity interval training (HIIT) on miR-126-3p and -5p levels. Methods: Sixty-one moderately trained individuals (females = 31 [50.8%]; 22.0 ± 1.84 years) were consecutively recruited and allocated into three matched groups using exercise capacity. During a 4-week intervention a HIIT group performed three exercise sessions/week of 4 × 30 s at maximum speed (all-out), a progressive HIIT (proHIIT) group performed three exercise sessions/week of 4 × 30 s at maximum speed (all-out) with one extra session every week (up to 7 × 30 s) and a low-intensity training (LIT) control group performed three exercise sessions/week for 25 min HIIT groups (after 4 min of high-intensity running). After the intervention, the LIT group presented an increase in miR-126-3p, while in the HIIT group, miR-126-3p levels were still reduced (all p HIIT (−1.05 ± 2.6 units). Conclusions: LIT and proHIIT may be performed to increase individual miR-126 levels. HIIT without progression was less effective in increasing miR-126. PMID:28611681

  12. Integrated mRNA and microRNA analysis identifies genes and small miRNA molecules associated with transcriptional and post-transcriptional-level responses to both drought stress and re-watering treatment in tobacco.

    Science.gov (United States)

    Chen, Qiansi; Li, Meng; Zhang, Zhongchun; Tie, Weiwei; Chen, Xia; Jin, Lifeng; Zhai, Niu; Zheng, Qingxia; Zhang, Jianfeng; Wang, Ran; Xu, Guoyun; Zhang, Hui; Liu, Pingping; Zhou, Huina

    2017-01-10

    Drought stress is one of the most severe problem limited agricultural productivity worldwide. It has been reported that plants response to drought-stress by sophisticated mechanisms at both transcriptional and post-transcriptional levels. However, the precise molecular mechanisms governing the responses of tobacco leaves to drought stress and water status are not well understood. To identify genes and miRNAs involved in drought-stress responses in tobacco, we performed both mRNA and small RNA sequencing on tobacco leaf samples from the following three treatments: untreated-control (CL), drought stress (DL), and re-watering (WL). In total, we identified 798 differentially expressed genes (DEGs) between the DL and CL (DL vs. CL) treatments and identified 571 DEGs between the WL and DL (WL vs. DL) treatments. Further analysis revealed 443 overlapping DEGs between the DL vs. CL and WL vs. DL comparisons, and, strikingly, all of these genes exhibited opposing expression trends between these two comparisons, strongly suggesting that these overlapping DEGs are somehow involved in the responses of tobacco leaves to drought stress. Functional annotation analysis showed significant up-regulation of genes annotated to be involved in responses to stimulus and stress, (e.g., late embryogenesis abundant proteins and heat-shock proteins) antioxidant defense (e.g., peroxidases and glutathione S-transferases), down regulation of genes related to the cell cycle pathway, and photosynthesis processes. We also found 69 and 56 transcription factors (TFs) among the DEGs in, respectively, the DL vs. CL and the WL vs. DL comparisons. In addition, small RNA sequencing revealed 63 known microRNAs (miRNA) from 32 families and 368 novel miRNA candidates in tobacco. We also found that five known miRNA families (miR398, miR390, miR162, miR166, and miR168) showed differential regulation under drought conditions. Analysis to identify negative correlations between the differentially expressed mi

  13. Cervical Shedding of HIV-1 RNA Among Women With Low Levels of Viremia While Receiving Highly Active Antiretroviral Therapy

    Science.gov (United States)

    Neely, Michael N.; Benning, Lorie; Xu, Jiaao; Strickler, Howard D.; Greenblatt, Ruth M.; Minkoff, Howard; Young, Mary; Bremer, James; Levine, Alexandra M.; Kovacs, Andrea

    2011-01-01

    Background Among women with low o r undetectable quantities of HIV-1 RNA in plasma, factors associated with genital HIV-1 RNA shedding, including choice of treatment regimen, are poorly characterized. Methods We measured HIV-1 RNA in cervical swab specimens obtained from participants in the Women’s Interagency HIV Study who had concurrent plasma viral RNA levels <500 copies/mL, and we assessed factors associated with genital HIV shedding. The study was powered to determine the relative effects of antiretroviral protease inhibitors (PIs) versus nonnucleoside reverse transcriptase inhibitors (NNRTIs) on viral RNA shedding. Results Overall, 44 (15%) of 290 women had detectable HIV-1 RNA in cervical specimens. In the final multivariate model, shedding was independently associated with NNRTI (vs. PI) use (odds ratio [OR], 95% confidence interval [CI]: 2.24, 1.13 to 4.45) and illicit drug use (OR, 95% CI: 2.41, 0.96 to 5.69). Conclusions This is the largest study to define risks for genital HIV-1 RNA shedding in women with low/undetectable plasma virus. Shedding in this population was common, and NNRTI-based highly active antiretroviral therapy (HAART) (vs. PI-based HAART) was associated with genital HIV shedding. Further study is required to determine the impact of these findings on transmission of HIV from mother to child or to sexual partners. PMID:17106279

  14. Accurate microRNA target prediction correlates with protein repression levels

    Directory of Open Access Journals (Sweden)

    Simossis Victor A

    2009-09-01

    Full Text Available Abstract Background MicroRNAs are small endogenously expressed non-coding RNA molecules that regulate target gene expression through translation repression or messenger RNA degradation. MicroRNA regulation is performed through pairing of the microRNA to sites in the messenger RNA of protein coding genes. Since experimental identification of miRNA target genes poses difficulties, computational microRNA target prediction is one of the key means in deciphering the role of microRNAs in development and disease. Results DIANA-microT 3.0 is an algorithm for microRNA target prediction which is based on several parameters calculated individually for each microRNA and combines conserved and non-conserved microRNA recognition elements into a final prediction score, which correlates with protein production fold change. Specifically, for each predicted interaction the program reports a signal to noise ratio and a precision score which can be used as an indication of the false positive rate of the prediction. Conclusion Recently, several computational target prediction programs were benchmarked based on a set of microRNA target genes identified by the pSILAC method. In this assessment DIANA-microT 3.0 was found to achieve the highest precision among the most widely used microRNA target prediction programs reaching approximately 66%. The DIANA-microT 3.0 prediction results are available online in a user friendly web server at http://www.microrna.gr/microT

  15. mRNA levels of enzymes and receptors implicated in arachidonic acid metabolism in gliomas.

    Science.gov (United States)

    De Armas, Rafael; Durand, Karine; Guillaudeau, Angélique; Weinbreck, Nicolas; Robert, Sandrine; Moreau, Jean-Jacques; Caire, François; Acosta, Gisela; Pebet, Matias; Chaunavel, Alain; Marin, Benoît; Labrousse, François; Denizot, Yves

    2010-07-01

    Gliomas are tumors of the central nervous system derived from glial cells. They show cellular heterogeneity and lack specific diagnostic markers. Although a possible role for the eicosanoid cascade has been suggested in glioma tumorigenesis, the relationship between enzymes and receptors implicated in arachidonic acid metabolism, with histological tumor type has not yet been determined. Quantitative real-time reverse transcription-polymerase chain reaction was performed to measure and compare transcript levels of enzymes and receptors implicated in both lipoxygenase and cyclooxygenase pathways between oligodendrogliomas, astrocytomas, glioblastomas and mixed oligoastrocytomas. Arachidonic acid metabolism-related enzymes and receptor transcripts (i) were underexpressed in classical oligodendrogliomas compared to astrocytomas and/or glioblastomas, (ii) differed between astrocytomas and glioblastomas and (iii) had an intermediate expression in mixed oligoastrocytomas. mRNA levels of enzymes and receptors implicated both in lipoxygenase and cyclooxygenase pathways differed significantly in gliomas according to the histological type. Copyright 2010 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  16. Physiologically-induced changes in proopiomelanocortin mRNA levels in the pituitary gland of the amphibian Xenopus laevis.

    Science.gov (United States)

    Martens, G J; Weterings, K A; van Zoest, I D; Jenks, B G

    1987-03-13

    In the pars intermedia of the pituitary gland of the amphibian Xenopus laevis the level of mRNA encoding proopiomelanocortin (POMC), the precursor protein for alpha-melanophore-stimulating hormone (alpha-MSH), is shown to be dependent on physiological parameters. POMC mRNA levels in the pars intermedia of black-background-adapted Xenopus are much higher than those of white-adapted animals. These physiological changes in POMC mRNA levels are tissue-specific because they were not found in the pars distalis of the pituitary gland. Background transfer experiments revealed that modulation of POMC gene activity is much slower than changes in the secretion of alpha-MSH.

  17. GnRH mRNA levels in male three-spined sticklebacks, Gasterosteus aculeatus, under different reproductive conditions.

    Science.gov (United States)

    Shao, Yi Ta; Tseng, Yung Che; Chang, Chia-Hao; Yan, Hong Young; Hwang, Pung Pung; Borg, Bertil

    2015-02-01

    In vertebrates, reproduction is regulated by the brain-pituitary-gonad (BPG) axis, where the gonadotropin-releasing hormone (GnRH) is one of the key components. However, very little is known about the possible role of GnRH in the environmental and feedback control of fish reproduction. To investigate this, full-length gnrh2 (chicken GnRH II) and gnrh3 (salmon GnRH) sequences of male three-spined sticklebacks (Gasterosteus aculeatus), which are clustered with the taxa of the same GnRH type as other Euteleostei, were cloned and annotated. gnrh1 is absent in this species. The mRNA levels of gnrh2 and gnrh3 in the sticklebacks' brain were measured under breeding and post-breeding conditions as well as in castrated and sham-operated breeding fish and castrated/sham-operated fish kept under long-day (LD 16:8) and short-day (LD 8:16) conditions. Fully breeding males had considerably higher mRNA levels of gnrh2 and gnrh3 in the thalamus (Th) and in the telencephalon and preoptic area (T+POA), respectively, than post-breeding males. Sham-operated breeding males have higher gnrh3 mRNA levels than the corresponding castrated males. Moreover, higher gnrh2 mRNA levels in the Th and higher gnrh3 mRNA levels in the T+POA and hypothalamus (HypTh) were also found in long-day sham-operated males than in sham-operated fish kept under an inhibitory short day photoperiod. Nevertheless, gnrh2 and gnrh3 mRNA levels were not up-regulated in castrated males kept under long-day photoperiod, which suggests that positive feedbacks on the brain-pituitary-gonad axis are necessary for this response. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. Dose-Response of High-Intensity Training (HIT on Atheroprotective miRNA-126 Levels

    Directory of Open Access Journals (Sweden)

    Boris Schmitz

    2017-05-01

    Full Text Available Aim: MicroRNA-126 (miR-126 exerts beneficial effects on vascular integrity, angiogenesis, and atherosclerotic plaque stability. The purpose of this investigation was to analyze the dose-response relationship of high-intensity interval training (HIIT on miR-126-3p and -5p levels.Methods: Sixty-one moderately trained individuals (females = 31 [50.8%]; 22.0 ± 1.84 years were consecutively recruited and allocated into three matched groups using exercise capacity. During a 4-week intervention a HIIT group performed three exercise sessions/week of 4 × 30 s at maximum speed (all-out, a progressive HIIT (proHIIT group performed three exercise sessions/week of 4 × 30 s at maximum speed (all-out with one extra session every week (up to 7 × 30 s and a low-intensity training (LIT control group performed three exercise sessions/week for 25 min <75% of maximum heart rate. Exercise miR-126-3p/-5p plasma levels were determined using capillary blood from earlobes.Results: No exercise-induced increase in miR-126 levels was detected at baseline, neither in the LIT (after 25 min low-intensity running nor the HIIT groups (after 4 min of high-intensity running. After the intervention, the LIT group presented an increase in miR-126-3p, while in the HIIT group, miR-126-3p levels were still reduced (all p < 0.05. An increase for both, miR-126-3p and -5p levels (all p < 0.05, pre- vs. during and post-exercise was detected in the proHIIT group. Between group analysis revealed that miR-126-3p levels after LIT and proHIIT increased by 2.12 ± 2.55 and 1.24 ± 2.46 units (all p < 0.01, respectively, compared to HIIT (−1.05 ± 2.6 units.Conclusions: LIT and proHIIT may be performed to increase individual miR-126 levels. HIIT without progression was less effective in increasing miR-126.

  19. Creatine kinase and alpha-actin mRNA levels decrease in diabetic rat hearts

    International Nuclear Information System (INIS)

    Popovich, B.; Barrieux, A.; Dillmann, W.H.

    1987-01-01

    Diabetic cardiomyopathy is associated with cardiac atrophy and isoenzyme redistribution. To determine if tissue specific changes occur in mRNAs coding for α-actin and creatine kinase (CK), they performed RNA blot analysis. Total ventricular RNA from control (C) and 4 wk old diabetic (D) rats were hybridized with 32 P cDNA probes for α-actin and CK. A tissue independent cDNA probe, CHOA was also used. Signal intensity was quantified by photodensitometry. D CK mRNA was 47 +/- 16% lower in D vs C. Insulin increases CK mRNA by 20% at 1.5 hs, and completely reverses the deficit after 4 wks. D α-actin mRNA is 66 +/- 18% lower in D vs C. Insulin normalized α-actin mRNA by 5 hs. CHOA mRNA is unchanged in D vs C, but D + insulin CHOA mRNA is 30 +/- 2% lower than C. In rats with diabetic cardiomyopathy, muscle specific CK and α-actin mRNAs are decreased. Insulin treatment reverses these changes

  20. TaxCollector: Modifying Current 16S rRNA Databases for the Rapid Classification at Six Taxonomic Levels

    Directory of Open Access Journals (Sweden)

    Eric W. Triplett

    2010-07-01

    Full Text Available The high level of conservation of 16S ribosomal RNA gene (16S rRNA in all Prokaryotes makes this gene an ideal tool for the rapid identification and classification of these microorganisms. Databases such as the Ribosomal Database Project II (RDP-II and the Greengenes Project offer access to sets of ribosomal RNA sequence databases useful in identification of microbes in a culture-independent analysis of microbial communities. However, these databases do not contain all of the taxonomic levels attached to the published names of the bacterial and archaeal sequences. TaxCollector is a set of scripts developed in Python language that attaches taxonomic information to all 16S rRNA sequences in the RDP-II and Greengenes databases. These modified databases are referred to as TaxCollector databases, which when used in conjunction with BLAST allow for rapid classification of sequences from any environmental or clinical source at six different taxonomic levels, from domain to species. The TaxCollector database prepared from the RDP-II database is an important component of a new 16S rRNA pipeline called PANGEA. The usefulness of TaxCollector databases is demonstrated with two very different datasets obtained using samples from a clinical setting and an agricultural soil. The six TaxCollector scripts are freely available on http://taxcollector.sourceforge.net and on http://www.microgator.org.

  1. Pulsed low-level infrared laser alters mRNA levels from muscle repair genes dependent on power output in Wistar rats

    Science.gov (United States)

    Trajano, L. A. S. N.; Trajano, E. T. L.; Thomé, A. M. C.; Sergio, L. P. S.; Mencalha, A. L.; Stumbo, A. C.; Fonseca, A. S.

    2017-10-01

    Satellite cells are present in skeletal muscle functioning in the repair and regeneration of muscle injury. Activation of these cells depends on the expression of myogenic factor 5 (Myf5), myogenic determination factor 1(MyoD), myogenic regulatory factor 4 (MRF4), myogenin (MyoG), paired box transcription factors 3 (Pax3), and 7 (Pax7). Low-level laser irradiation accelerates the repair of muscle injuries. However, data from the expression of myogenic factors have been controversial. Furthermore, the effects of different laser beam powers on the repair of muscle injuries have been not evaluated. The aim of this study was to evaluate the effects of low-level infrared laser at different powers and in pulsed emission mode on the expression of myogenic regulatory factors and on Pax3 and Pax7 in injured skeletal muscle from Wistar rats. Animals that underwent cryoinjury were divided into three groups: injury, injury laser 25 Mw, and injury laser 75 mW. Low-level infrared laser irradiation (904 nm, 3 J cm-2, 5 kHz) was carried out at 25 and 75 mW. After euthanasia, skeletal muscle samples were withdrawn and the total RNA was extracted for the evaluation of mRNA levels from the MyoD, MyoG, MRF4, Myf5, Pax3, and Pax7 gene. Pax 7 mRNA levels did not alter, but Pax3 mRNA levels increased in the injured and laser-irradiated group at 25 mW. MyoD, MyoG, and MYf5 mRNA levels increased in the injured and laser-irradiated animals at both powers, and MRF4 mRNA levels decreased in the injured and laser-irradiated group at 75 mW. In conclusion, exposure to pulsed low-level infrared laser, by power-dependent effect, could accelerate the muscle repair process altering mRNA levels from paired box transcription factors and myogenic regulatory factors.

  2. Carbon nanotubes as carriers of Panax ginseng metabolites and enhancers of ginsenosides Rb1 and Rg1 anti-cancer activity

    Science.gov (United States)

    Lahiani, Mohamed H.; Eassa, Souzan; Parnell, Charlette; Nima, Zeid; Ghosh, Anindya; Biris, Alexandru S.; Khodakovskaya, Mariya V.

    2017-01-01

    A major benefit to nanomaterial based-medicine is the ability to provide nanosized vehicles for sporadic metabolites. Here, we describe how the conjugation of valuable ginseng secondary metabolites (ginsenoside Rb1 or Rg1) with carbon nanotubes (CNT) can enhance their anti-proliferative and anti-cancer effects. Ginsenoside-CNT conjugate (Rb-CNT or Rg-CNT) permitted the ginsenosides to be used at a low dose, yet achieve a higher incidence of cancer killing. We were able to demonstrate that the ginsenoside-CNT conjugate can decrease cell viability up to 62% in breast cancer cells (MCF-7) and enhance antiproliferation of drug-resistant pancreatic cancer cells (PANC-1) by 61%. The interaction of the ginsenoside-CNT conjugate with breast cancer cells was studied using Raman Spectroscopy mapping. Total transcriptome profiling (Affymetrix platform) of MCF-7 cells treated with the ginsenoside-CNT conjugate shows that a number of cellular, apoptotic and response to stimulus processes were affected. Therefore, our data confirmed the potential use of CNT as a drug delivery system.

  3. Molecular screening of compounds to the predicted Protein-Protein Interaction site of Rb1-E7 with p53- E6 in HPV

    Science.gov (United States)

    Shaikh, Faraz; Sanehi, Parvish; Rawal, Rakesh

    2012-01-01

    Cervical cancer is malignant neoplasm of the cervix uteri or cervical area. Human Papillomaviruses (HPVs) which are heterogeneous groups of small double stranded DNA viruses are considered as the primary cause of cervical cancer, involved in 90% of all Cervical Cancers. Two early HPV genes, E6 and E7, are known to play crucial role in tumor formation. E6 binds with p53 and prevents its translocation and thereby inhibit the ability of p53 to activate or repress target genes. E7 binds to hypophosphorylated Rb and thereby induces cells to enter into premature S-phase by disrupting Rb-E2F complexes. The strategy of the research work was to target the site of interaction of Rb1 -E7 & p53-E6. A total of 88 compounds were selected for molecular screening, based on comprehensive literature survey for natural compounds with anti-cancer activity. Molecular docking analysis was carried out with Molegro Virtual Docker, to screen the 88 chosen compounds and rank them according to their binding affinity towards the site of interaction of the viral oncoproteins and human tumor suppressor proteins. The docking result revealed that Nicandrenone a member of Withanolides family of chemical compounds as the most likely molecule that can be used as a candidate drug against HPV induced cervical cancer. Abbreviations HPV - Human Papiloma Virus, HTSP - Human Tumor Suppressor Proteins, VOP - Viral oncoproteins. PMID:22829740

  4. A hairpin within YAP mRNA 3′UTR functions in regulation at post-transcription level

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Yuen; Wang, Yuan; Feng, Jinyan; Feng, Guoxing; Zheng, Minying; Yang, Zhe; Xiao, Zelin; Lu, Zhanping [State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin 300071 (China); Ye, Lihong [State Key Laboratory of Medicinal Chemical Biology, Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin 300071 (China); Zhang, Xiaodong, E-mail: zhangxd@nankai.edu.cn [State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin 300071 (China)

    2015-04-03

    The central dogma of gene expression is that DNA is transcribed into messenger RNAs, which in turn serve as the template for protein synthesis. Recently, it has been reported that mRNAs display regulatory roles that rely on their ability to compete for microRNA binding, independent of their protein-coding function. However, the regulatory mechanism of mRNAs remains poorly understood. Here, we report that a hairpin within YAP mRNA 3′untranslated region (3′UTR) functions in regulation at post-transcription level through generating endogenous siRNAs (esiRNAs). Bioinformatics analysis for secondary structure showed that YAP mRNA displayed a hairpin structure (termed standard hairpin, S-hairpin) within its 3′UTR. Surprisingly, we observed that the overexpression of S-hairpin derived from YAP 3′UTR (YAP-sh) increased the luciferase reporter activities of transcriptional factor NF-κB and AP-1 in 293T cells. Moreover, we identified that a fragment from YAP-sh, an esiRNA, was able to target mRNA 3′UTR of NF2 (a member of Hippo-signaling pathway) and YAP mRNA 3′UTR itself in hepatoma cells. Thus, we conclude that the YAP-sh within YAP mRNA 3′UTR may serve as a novel regulatory element, which functions in regulation at post-transcription level. Our finding provides new insights into the mechanism of mRNAs in regulatory function. - Highlights: • An S-hairpin within YAP mRNA 3′UTR possesses regulatory function. • YAP-sh acts as a regulatory element for YAP at post-transcription level. • YAP-sh-3p20, an esiRNA derived from YAP-sh, targets mRNAs of YAP and NF2. • YAP-sh-3p20 depresses the proliferation of HepG2 cells in vitro.

  5. A hairpin within YAP mRNA 3′UTR functions in regulation at post-transcription level

    International Nuclear Information System (INIS)

    Gao, Yuen; Wang, Yuan; Feng, Jinyan; Feng, Guoxing; Zheng, Minying; Yang, Zhe; Xiao, Zelin; Lu, Zhanping; Ye, Lihong; Zhang, Xiaodong

    2015-01-01

    The central dogma of gene expression is that DNA is transcribed into messenger RNAs, which in turn serve as the template for protein synthesis. Recently, it has been reported that mRNAs display regulatory roles that rely on their ability to compete for microRNA binding, independent of their protein-coding function. However, the regulatory mechanism of mRNAs remains poorly understood. Here, we report that a hairpin within YAP mRNA 3′untranslated region (3′UTR) functions in regulation at post-transcription level through generating endogenous siRNAs (esiRNAs). Bioinformatics analysis for secondary structure showed that YAP mRNA displayed a hairpin structure (termed standard hairpin, S-hairpin) within its 3′UTR. Surprisingly, we observed that the overexpression of S-hairpin derived from YAP 3′UTR (YAP-sh) increased the luciferase reporter activities of transcriptional factor NF-κB and AP-1 in 293T cells. Moreover, we identified that a fragment from YAP-sh, an esiRNA, was able to target mRNA 3′UTR of NF2 (a member of Hippo-signaling pathway) and YAP mRNA 3′UTR itself in hepatoma cells. Thus, we conclude that the YAP-sh within YAP mRNA 3′UTR may serve as a novel regulatory element, which functions in regulation at post-transcription level. Our finding provides new insights into the mechanism of mRNAs in regulatory function. - Highlights: • An S-hairpin within YAP mRNA 3′UTR possesses regulatory function. • YAP-sh acts as a regulatory element for YAP at post-transcription level. • YAP-sh-3p20, an esiRNA derived from YAP-sh, targets mRNAs of YAP and NF2. • YAP-sh-3p20 depresses the proliferation of HepG2 cells in vitro

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

    DEFF Research Database (Denmark)

    Christiansen, Mette; Kveiborg, M.; Kassem, M.

    2000-01-01

    In order to understand the reasons for age-related impairment of the function of bone forming osteoblasts, we have examined the steady-state mRNA levels of the transcription factor CBFA1 and topoisomerase I during cellular aging of normal human trabecular osteoblasts, by the use of semiquantitati...

  7. Using Poisson mixed-effects model to quantify transcript-level gene expression in RNA-Seq.

    Science.gov (United States)

    Hu, Ming; Zhu, Yu; Taylor, Jeremy M G; Liu, Jun S; Qin, Zhaohui S

    2012-01-01

    RNA sequencing (RNA-Seq) is a powerful new technology for mapping and quantifying transcriptomes using ultra high-throughput next-generation sequencing technologies. Using deep sequencing, gene expression levels of all transcripts including novel ones can be quantified digitally. Although extremely promising, the massive amounts of data generated by RNA-Seq, substantial biases and uncertainty in short read alignment pose challenges for data analysis. In particular, large base-specific variation and between-base dependence make simple approaches, such as those that use averaging to normalize RNA-Seq data and quantify gene expressions, ineffective. In this study, we propose a Poisson mixed-effects (POME) model to characterize base-level read coverage within each transcript. The underlying expression level is included as a key parameter in this model. Since the proposed model is capable of incorporating base-specific variation as well as between-base dependence that affect read coverage profile throughout the transcript, it can lead to improved quantification of the true underlying expression level. POME can be freely downloaded at http://www.stat.purdue.edu/~yuzhu/pome.html. yuzhu@purdue.edu; zhaohui.qin@emory.edu Supplementary data are available at Bioinformatics online.

  8. MYC Targeted Long Noncoding RNA DANCR Promotes Cancer in Part by Reducing p21 Levels.

    Science.gov (United States)

    Lu, Yunqi; Hu, Zhongyi; Mangala, Lingegowda S; Stine, Zachary E; Hu, Xiaowen; Jiang, Dahai; Xiang, Yan; Zhang, Youyou; Pradeep, Sunila; Rodriguez-Aguayo, Cristian; Lopez-Berestein, Gabriel; DeMarzo, Angelo M; Sood, Anil K; Zhang, Lin; Dang, Chi V

    2018-01-01

    The MYC oncogene broadly promotes transcription mediated by all nuclear RNA polymerases, thereby acting as a positive modifier of global gene expression. Here, we report that MYC stimulates the transcription of DANCR, a long noncoding RNA (lncRNA) that is widely overexpressed in human cancer. We identified DANCR through its overexpression in a transgenic model of MYC-induced lymphoma, but found that it was broadly upregulated in many human cancer cell lines and cancers, including most notably in prostate and ovarian cancers. Mechanistic investigations indicated that DANCR limited the expression of cell-cycle inhibitor p21 (CDKN1A) and that the inhibitory effects of DANCR loss on cell proliferation could be partially rescued by p21 silencing. In a xenograft model of human ovarian cancer, a nanoparticle-mediated siRNA strategy to target DANCR in vivo was sufficient to strongly inhibit tumor growth. Our observations expand knowledge of how MYC drives cancer cell proliferation by identifying DANCR as a critical lncRNA widely overexpressed in human cancers. Significance: These findings expand knowledge of how MYC drives cancer cell proliferation by identifying an oncogenic long noncoding RNA that is widely overexpressed in human cancers. Cancer Res; 78(1); 64-74. ©2017 AACR . ©2017 American Association for Cancer Research.

  9. Gestational Protein Restriction Increases Cardiac Connexin 43 mRNA levels in male adult rat offspring.

    Science.gov (United States)

    Rossini, Kamila Fernanda; Oliveira, Camila Andrea de; Rebelato, Hércules Jonas; Esquisatto, Marcelo Augusto Marreto; Catisti, Rosana

    2017-07-01

    The dietary limitation during pregnancy influences the growth and development of the fetus and offspring and their health into adult life. The mechanisms underlying the adverse effects of gestational protein restriction (GPR) in the development of the offspring hearts are not well understood. The aim of this study was to evaluate the effects of GPR on cardiac structure in male rat offspring at day 60 after birth (d60). Pregnant Wistar rats were fed a normal-protein (NP, 17% casein) or low-protein (LP, 6% casein) diet. Blood pressure (BP) values from 60-day-old male offspring were measured by an indirect tail-cuff method using an electro sphygmomanometer. Hearts (d60) were collected for assessment of connexin 43 (Cx43) mRNA expression and morphological and morphometric analysis. LP offspring showed no difference in body weight, although they were born lighter than NP offspring. BP levels were significantly higher in the LP group. We observed a significant increase in the area occupied by collagen fibers, a decrease in the number of cardiomyocytes by 104 µm2, and an increase in cardiomyocyte area associated with an increased Cx43 expression. GPR changes myocardial levels of Cx43 mRNA in male young adult rats, suggesting that this mechanism aims to compensate the fibrotic process by the accumulation of collagen fibers in the heart interstitium. A limitação dietética durante a gravidez influencia o crescimento e desenvolvimento do feto e da prole e sua saúde na vida adulta. Os mecanismos subjacentes dos efeitos adversos da restrição proteica gestacional (RPG) no desenvolvimento dos corações da prole não são bem compreendidos. Avaliar os efeitos da RPG sobre a estrutura cardíaca em filhotes machos de ratas aos 60 dias após o nascimento (d60). Ratos fêmeas Wistar grávidas foram alimentadas com uma dieta de proteína normal (PN, 17% caseína) ou de baixa proteína (BP, caseína 6%). Os valores de pressão arterial (PA) de descendentes do sexo masculino de

  10. Full Viral Suppression, Low-Level Viremia, and Quantifiable Plasma HIV-RNA at the End of Pregnancy in HIV-Infected Women on Antiretroviral Treatment

    OpenAIRE

    Baroncelli, Silvia; Pirillo, Maria F.; Tamburrini, Enrica; Guaraldi, Giovanni; Pinnetti, Carmela; Antoni, Anna Degli; Galluzzo, Clementina M.; Stentarelli, Chiara; Amici, Roberta; Floridia, Marco

    2015-01-01

    There is limited information on full viral suppression and low-level HIV-RNA viremia in HIV-infected women at the end of pregnancy. We investigated HIV-RNA levels close to delivery in women on antiretroviral treatment in order to define rates of complete suppression, low-level viremia, and quantifiable HIV-RNA, exploring as potential determinants some clinical and viroimmunological variables. Plasma samples from a national study in Italy, collected between 2003 and 2012, were used. According ...

  11. Full Viral Suppression, Low-Level Viremia, and Quantifiable Plasma HIV-RNA at the End of Pregnancy in HIV-Infected Women on Antiretroviral Treatment.

    Science.gov (United States)

    Baroncelli, Silvia; Pirillo, Maria F; Tamburrini, Enrica; Guaraldi, Giovanni; Pinnetti, Carmela; Degli Antoni, Anna; Galluzzo, Clementina M; Stentarelli, Chiara; Amici, Roberta; Floridia, Marco

    2015-07-01

    There is limited information on full viral suppression and low-level HIV-RNA viremia in HIV-infected women at the end of pregnancy. We investigated HIV-RNA levels close to delivery in women on antiretroviral treatment in order to define rates of complete suppression, low-level viremia, and quantifiable HIV-RNA, exploring as potential determinants some clinical and viroimmunological variables. Plasma samples from a national study in Italy, collected between 2003 and 2012, were used. According to plasma HIV-RNA levels, three groups were defined: full suppression (target not detected), low-level viremia (target detected but HIV-RNA (≥37 copies/ml). Multivariable logistic regression was used to define determinants of full viral suppression and of quantifiable HIV-RNA. Among 107 women evaluated at a median gestational age of 35 weeks, 90 (84.1%) had HIV-RNA HIV-RNA was 109 copies/ml (IQR 46-251), with only one case showing resistance (mutation M184V; rate: 9.1%). In multivariable analyses, women with higher baseline HIV-RNA levels and with hepatitis C virus (HCV) coinfection were significantly more likely to have quantifiable HIV-RNA in late pregnancy. Full viral suppression was significantly more likely with nonnucleoside reverse transcriptase inhibitor (NNRTI)-based regimens and significantly less likely with higher HIV-RNA in early pregnancy. No cases of HIV transmission occurred. In conclusion, HIV-infected pregnant women showed a high rate of viral suppression and a low resistance rate before delivery. In most cases no target HIV-RNA was detected in plasma, suggesting a low risk of subsequent virological rebound and development of resistance. Women with high levels of HIV-RNA in early pregnancy and those who have concomitant HCV infection should be considered at higher risk of having quantifiable HIV-RNA at the end of pregnancy.

  12. RNA-SEQ reveals transcriptional level changes of poplar roots in different forms of nitrogen treatments

    Directory of Open Access Journals (Sweden)

    Chunpu eQu

    2016-02-01

    Full Text Available Poplar has emerged as a model plant for understanding molecular mechanisms of tree growth, development and response to environment. Long-term application of different forms of nitrogen (such as NO3--N and NH4+-N may cause morphological changes of poplar roots; however, the molecular level changes are still not well known. In this study, we analyzed the expression profiling of poplar roots treated by three forms of nitrogen: S1 (NH4+, S2 (NH4NO3 and S3 (NO3- by using RNA-SEQ technique. We found 463 genes significantly differentially expressed in roots by different N treatments, of which a total of 116 genes were found to differentially express between S1 and S2, 173 genes between S2 and S3, and 327 genes between S1 and S3. A cluster analysis shows significant difference in many transcription factor families and functional genes family under different N forms. Through an analysis of Mapman metabolic pathway, we found that the significantly differentially expressed genes are associated with fermentation, glycolysis and tricarboxylic acid cycle (TCA, secondary metabolism, hormone metabolism, and transport processing. Interestingly, we did not find significantly differentially expressed genes in N metabolism pathway, mitochondrial electron transport / ATP synthesis and mineral nutrition. We also found abundant candidate genes (20 transcription factors and 30 functional genes regulating morphology changes of poplar roots under the three N forms. The results obtained are beneficial to a better understanding of the potential molecular and cellular mechanisms regulating root morphology changes under different N treatments.

  13. Enoxacin elevates microRNA levels in rat frontal cortex and prevents learned helplessness

    Directory of Open Access Journals (Sweden)

    Neil R Smalheiser

    2014-02-01

    Full Text Available Major depressive disorder (MDD is a major public health concern. Despite tremendous advancement, the pathogenic mechanisms associated with MDD are still unclear. Moreover, a significant number of MDD subjects do not respond to the currently available medication. MicroRNAs (miRNAs are a class of small non-coding RNAs that control gene expression by modulating translation, mRNA degradation or stability of mRNA targets. The role of miRNAs in disease pathophysiology is emerging rapidly. Recently, we reported that miRNA expression is down-regulated in frontal cortex of depressed suicide subjects, and that rats exposed to repeated inescapable shock show differential miRNA changes depending on whether they exhibited normal adaptive responses or learned helpless behavior. Enoxacin, a fluoroquinolone used clinically as an antibacterial compound, enhances the production of miRNAs in vitro and in peripheral tissues in vivo, but has not yet been tested as an experimental tool to study the relation of miRNA expression to neural functions or behavior. Treatment of rats with 10 or 25 mg/kg enoxacin for one week increased the expression of miRNAs in frontal cortex and decreased the proportion of rats exhibiting learned helpless behavior following inescapable shock. Further studies are warranted to learn whether enoxacin may ameliorate depressive behavior in other rodent paradigms and in human clinical situations, and if so whether its mechanism is due to upregulation of miRNAs.

  14. Circulating Plasma Levels of MicroRNA-21 and MicroRNA-221 Are Potential Diagnostic Markers for Primary Intrahepatic Cholangiocarcinoma

    Science.gov (United States)

    Kemeny, Nancy; Kingham, T. Peter; Allen, Peter J.; D’Angelica, Michael I.; DeMatteo, Ronald P.; Betel, Doron; Klimstra, David; Jarnagin, William R.; Ventura, Andrea

    2016-01-01

    Background MicroRNAs (miRNAs) are potential biomarkers in various malignancies. We aim to characterize miRNA expression in intrahepatic cholangiocarcinoma (ICC) and identify circulating plasma miRNAs with potential diagnostic and prognostic utility. Methods Using deep-sequencing techniques, miRNA expression between tumor samples and non-neoplastic liver parenchyma were compared. Overexpressed miRNAs were measured in plasma from an independent cohort of patients with cholangiocarcinoma using RT-qPCR and compared with that healthy volunteers. The discriminatory ability of the evaluated plasma miRNAs between patients and controls was evaluated with receiving operating characteristic (ROC) curves. Results Small RNAs from 12 ICC and 11 tumor-free liver samples were evaluated. Unsupervised hierarchical clustering using the miRNA expression data showed clear grouping of ICC vs. non-neoplastic liver parenchyma. We identified 134 down-regulated and 128 upregulated miRNAs. Based on overexpression and high fold-change, miR21, miR200b, miR221, and miR34c were measured in plasma from an independent cohort of patients with ICC (n = 25) and healthy controls (n = 7). Significant overexpression of miR-21 and miR-221 was found in plasma from ICC patients. Furthermore, circulating miR-21 demonstrated a high discriminatory ability between patients with ICC and healthy controls (AUC: 0.94). Conclusion Among the differentially expressed miRNAs in ICC, miR-21 and miR-221 are overexpressed and detectable in the circulation. Plasma expression levels of these miRNAs, particularly miR-21, accurately differentiates patients with ICC from healthy controls and could potentially serve as adjuncts in diagnosis. Prospective validation and comparison with other hepatobiliary malignancies is required to establish their potential role as diagnostic and prognostic biomarkers. PMID:27685844

  15. Circulating MicroRNA Expression Levels Associated With Internet Gaming Disorder

    Directory of Open Access Journals (Sweden)

    Minho Lee

    2018-03-01

    Full Text Available BackgroundAddictive use of the Internet and online games is a potential psychiatric disorder termed Internet gaming disorder (IGD. Altered microRNA (miRNA expression profiles have been reported in blood and brain tissue of patients with certain psychiatric disorders and suggested as biomarkers. However, there have been no reports on blood miRNA profiles in IGD.MethodsTo discover IGD-associated miRNAs, we analyzed the miRNA expression profiles of 51 samples (25 IGD and 26 controls using the TaqMan Low Density miRNA Array. For validation, we performed quantitative reverse transcription PCR with 36 independent samples (20 IGD and 16 controls.ResultsThrough discovery and independent validation, we identified three miRNAs (hsa-miR-200c-3p, hsa-miR-26b-5p, hsa-miR-652-3p that were significantly downregulated in the IGD group. Individuals with all three miRNA alterations had a much higher risk of IGD than those with no alteration [odds ratio (OR 22, 95% CI 2.29–211.11], and the ORs increased dose dependently with number of altered miRNAs. The predicted target genes of the three miRNAs were associated with neural pathways. We explored the protein expression of the three downstream target genes by western blot and confirmed that expression of GABRB2 and DPYSL2 was significantly higher in the IGD group.ConclusionWe observed that expressions of hsa-miR-200c-3p, hsa-miR-26b-5p, and hsa-miR-652-3p were downregulated in the IGD patients. Our results will be helpful to understand the pathophysiology of IGD.

  16. Dietary fatty acids regulate hepatic low density lipoprotein (LDL) transport by altering LDL receptor protein and mRNA levels.

    Science.gov (United States)

    Horton, J D; Cuthbert, J A; Spady, D K

    1993-01-01

    The concentration of LDL in plasma is strongly influenced by the amount and the type of lipid in the diet. Recent studies in the hamster have shown that dietary fatty acids differentially affect circulating LDL levels primarily by altering receptor-dependent LDL uptake in the liver. To investigate the mechanistic basis of this effect, rates of receptor-dependent LDL transport in the liver were correlated with LDL receptor protein and mRNA levels in hamsters fed safflower oil or coconut oil and varying amounts of cholesterol. Hepatic LDL receptor activity was significantly lower in animals fed coconut oil than in animals fed safflower oil at all levels of cholesterol intake (26, 53, and 61% lower at cholesterol intakes of 0, 0.06, and 0.12%, respectively). These fatty acid-induced changes in hepatic LDL receptor activity were accompanied by parallel changes in hepatic LDL receptor protein and mRNA levels, suggesting that dietary fatty acids regulate the LDL receptor pathway largely at the mRNA level. Images PMID:8349814

  17. Relationship between depressive symptoms and miRNA expression level in monocytes of patients with depression before and after antidepressant treatment

    Directory of Open Access Journals (Sweden)

    Qiao-li ZHANG

    2015-04-01

    Full Text Available Objective To explore the correlation of depressive symptoms to the microRNA (miRNA expression level in monocytes of patients with depression before and after antidepressant treatment. Methods Eighty-one patients with depression, admitted to the 102 Hospital of PLA from Aug. 2012 to Oct. 2013, having not received antidepressants treatment and meeting the criteria as listed in Diagnostic and Statistical Manual 4th edition (DSM-IV, were selected as case group. Eighty-one normal individuals served as control group. With Affymetrix Expression Array, 26 miRNAs were identified from 3 individuals from each group as candidate miRNA, and among them 9 miRNAs (miR-146b, miR-1972, miR-26b, miR-29b, miR-338, miR-4485, miR-4498, miR-4743 and miR-874 in monocytes were selected for quantitative real-time reverse transcription polymerase chain reaction (RTPCR assessment. Twenty patients from the case group were selected for the assessment of miRNA expression levels, and the clinical symptoms and treatment effect were evaluated using Hamilton Depression Scale (HAMD and Clinical Global Impression (CGI, before and 6 weeks after antidepressant (venlafaxine, sertraline, mirtazapine, etc. treatment. Results Compared with the control group, the expression levels of miRNA-26b, miRNA-4743, miRNA-4498, miRNA-4485 and miRNA-1972 of the case group were significantly up-regulated (P<0.05. The variance of expression level of miRNA-4743, miRNA-4498, miRNA-4485 and miRNA-1972 was respectively positively correlated with improvement in retardation factors (P<0.05, meanwhile the variance of expression level of miRNA-26b was negatively correlated with the improvement of day and night change factors (P<0.05. Logistic regression analysis demonstrated that the alteration of miRNA-4485 expression may account 28.8% of retardation variance (P<0.05. Conclusion  The miRNA-4743, miRNA-4498, miRNA-4485, miRNA-1972 and miRNA-26b in monocytes may serve as the biomarkers for the

  18. Seminal Plasma HIV-1 RNA Concentration Is Strongly Associated with Altered Levels of Seminal Plasma Interferon-γ, Interleukin-17, and Interleukin-5

    Science.gov (United States)

    Hoffman, Jennifer C.; Anton, Peter A.; Baldwin, Gayle Cocita; Elliott, Julie; Anisman-Posner, Deborah; Tanner, Karen; Grogan, Tristan; Elashoff, David; Sugar, Catherine; Yang, Otto O.

    2014-01-01

    Abstract Seminal plasma HIV-1 RNA level is an important determinant of the risk of HIV-1 sexual transmission. We investigated potential associations between seminal plasma cytokine levels and viral concentration in the seminal plasma of HIV-1-infected men. This was a prospective, observational study of paired blood and semen samples from 18 HIV-1 chronically infected men off antiretroviral therapy. HIV-1 RNA levels and cytokine levels in seminal plasma and blood plasma were measured and analyzed using simple linear regressions to screen for associations between cytokines and seminal plasma HIV-1 levels. Forward stepwise regression was performed to construct the final multivariate model. The median HIV-1 RNA concentrations were 4.42 log10 copies/ml (IQR 2.98, 4.70) and 2.96 log10 copies/ml (IQR 2, 4.18) in blood and seminal plasma, respectively. In stepwise multivariate linear regression analysis, blood HIV-1 RNA level (pplasma HIV-1 RNA level. After controlling for blood HIV-1 RNA level, seminal plasma HIV-1 RNA level was positively associated with interferon (IFN)-γ (p=0.03) and interleukin (IL)-17 (p=0.03) and negatively associated with IL-5 (p=0.0007) in seminal plasma. In addition to blood HIV-1 RNA level, cytokine profiles in the male genital tract are associated with HIV-1 RNA levels in semen. The Th1 and Th17 cytokines IFN-γ and IL-17 are associated with increased seminal plasma HIV-1 RNA, while the Th2 cytokine IL-5 is associated with decreased seminal plasma HIV-1 RNA. These results support the importance of genital tract immunomodulation in HIV-1 transmission. PMID:25209674

  19. Effect of dietary betaine supplementation on mRNA level of ...

    African Journals Online (AJOL)

    Yomi

    2012-03-22

    Mar 22, 2012 ... of China. Accepted 3 January, 2012. Our aims are to determine the ... percentage of abdominal fat, ratio of liver to body weight, mRNA ... four diet groups with supplemented betaine of 0, 0.04, 0.06 and 0.08%, respectively.

  20. How to switch the motor on: RNA polymerase initiation steps at the single-molecule level

    NARCIS (Netherlands)

    Marchetti, M.; Malinowska, A.; Heller, I.; Wuite, G. J. L.

    RNA polymerase (RNAP) is the central motor of gene expression since it governs the process of transcription. In prokaryotes, this holoenzyme is formed by the RNAP core and a sigma factor. After approaching and binding the specific promoter site on the DNA, the holoenzyme-promoter complex undergoes

  1. RNA-Sequencing of Primary Retinoblastoma Tumors Provides New Insights and Challenges Into Tumor Development

    Directory of Open Access Journals (Sweden)

    Sailaja V. Elchuri

    2018-05-01

    Full Text Available Retinoblastoma is rare tumor of the retina caused by the homozygous loss of the Retinoblastoma 1 tumor suppressor gene (RB1. Loss of the RB1 protein, pRB, results in de-regulated activity of the E2F transcription factors, chromatin changes and developmental defects leading to tumor development. Extensive microarray profiles of these tumors have enabled the identification of genes sensitive to pRB disruption, however, this technology has a number of limitations in the RNA profiles that they generate. The advent of RNA-sequencing has enabled the global profiling of all of the RNA within the cell including both coding and non-coding features and the detection of aberrant RNA processing events. In this perspective, we focus on discussing how RNA-sequencing of rare Retinoblastoma tumors will build on existing data and open up new area’s to improve our understanding of the biology of these tumors. In particular, we discuss how the RB-research field may be to use this data to determine how RB1 loss results in the expression of; non-coding RNAs, causes aberrant RNA processing events and how a deeper analysis of metabolic RNA changes can be utilized to model tumor specific shifts in metabolism. Each section discusses new opportunities and challenges associated with these types of analyses and aims to provide an honest assessment of how understanding these different processes may contribute to the treatment of Retinoblastoma.

  2. Pre-storage centrifugation conditions have significant impact on measured microRNA levels in biobanked EDTA plasma samples

    DEFF Research Database (Denmark)

    Binderup, Helle Glud; Houlind, Kim; Madsen, Jonna Skov

    2016-01-01

    in platelets this may partly be explained by residual platelets in the plasma samples used. When collecting fresh plasma samples, it is possible to produce cell-free/platelet-poor plasma by centrifugation. In this study, we systematically investigated whether biobanked EDTA plasma samples could be processed...... to be suitable for miRNA analysis. Materials and methods: Blood samples were collected from ten healthy volunteers and centrifuged to produce platelet-poor-plasma (PPP) and standard biobank plasma. After one week at -80 °C the biobanked EDTA plasma was re-centrifuged by different steps to remove residual...... platelets. Using RT-qPCR the levels of 14 miRNAs in the different plasma preparations were compared to that of PPP. Results: We were able to remove residual platelets from biobanked EDTA plasma by re-centrifugation of the thawed samples. Nevertheless, for most of the investigated miRNAs, the miRNA level...

  3. A structured sparse regression method for estimating isoform expression level from multi-sample RNA-seq data.

    Science.gov (United States)

    Zhang, L; Liu, X J

    2016-06-03

    With the rapid development of next-generation high-throughput sequencing technology, RNA-seq has become a standard and important technique for transcriptome analysis. For multi-sample RNA-seq data, the existing expression estimation methods usually deal with each single-RNA-seq sample, and ignore that the read distributions are consistent across multiple samples. In the current study, we propose a structured sparse regression method, SSRSeq, to estimate isoform expression using multi-sample RNA-seq data. SSRSeq uses a non-parameter model to capture the general tendency of non-uniformity read distribution for all genes across multiple samples. Additionally, our method adds a structured sparse regularization, which not only incorporates the sparse specificity between a gene and its corresponding isoform expression levels, but also reduces the effects of noisy reads, especially for lowly expressed genes and isoforms. Four real datasets were used to evaluate our method on isoform expression estimation. Compared with other popular methods, SSRSeq reduced the variance between multiple samples, and produced more accurate isoform expression estimations, and thus more meaningful biological interpretations.

  4. Elevated levels of urinary markers of oxidatively generated DNA and RNA damage in bipolar disorder

    DEFF Research Database (Denmark)

    Munkholm, Klaus; Poulsen, Henrik Enghusen; Kessing, Lars Vedel

    2015-01-01

    OBJECTIVES: The pathophysiological mechanisms underlying bipolar disorder and its multi-system nature are unclear. Oxidatively generated damage to nucleosides has been demonstrated in metabolic disorders; however, the extent to which this occurs in bipolar disorder in vivo is unknown. We...... investigated oxidatively generated damage to DNA and RNA in patients with bipolar disorder and its relationship with the affective phase compared with healthy control subjects. METHODS: Urinary excretion of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydroguanosine (8-oxoGuo), markers...... of oxidatively generated DNA and RNA damage, respectively, was measured in 37 rapid cycling patients with bipolar disorder and in 40 age- and gender-matched healthy control subjects. Employing a longitudinal design, repeated measurements of both markers were evaluated in various affective phases in patients...

  5. Chronic Stress Reduces Nectin-1 mRNA Levels and Disrupts Dendritic Spine Plasticity in the Adult Mouse Perirhinal Cortex

    Directory of Open Access Journals (Sweden)

    Qian Gong

    2018-03-01

    Full Text Available In adulthood, chronic exposure to stressful experiences disrupts synaptic plasticity and cognitive function. Previous studies have shown that perirhinal cortex-dependent object recognition memory is impaired by chronic stress. However, the stress effects on molecular expression and structural plasticity in the perirhinal cortex remain unclear. In this study, we applied the chronic social defeat stress (CSDS paradigm and measured the mRNA levels of nectin-1, nectin-3 and neurexin-1, three synaptic cell adhesion molecules (CAMs implicated in the adverse stress effects, in the perirhinal cortex of wild-type (WT and conditional forebrain corticotropin-releasing hormone receptor 1 conditional knockout (CRHR1-CKO mice. Chronic stress reduced perirhinal nectin-1 mRNA levels in WT but not CRHR1-CKO mice. In conditional forebrain corticotropin-releasing hormone conditional overexpression (CRH-COE mice, perirhinal nectin-1 mRNA levels were also reduced, indicating that chronic stress modulates nectin-1 expression through the CRH-CRHR1 system. Moreover, chronic stress altered dendritic spine morphology in the main apical dendrites and reduced spine density in the oblique apical dendrites of perirhinal layer V pyramidal neurons. Our data suggest that chronic stress disrupts cell adhesion and dendritic spine plasticity in perirhinal neurons, which may contribute to stress-induced impairments of perirhinal cortex-dependent memory.

  6. Complex mutual regulation of facilitates chromatin transcription (FACT) subunits on both mRNA and protein levels in human cells.

    Science.gov (United States)

    Safina, Alfiya; Garcia, Henry; Commane, Mairead; Guryanova, Olga; Degan, Seamus; Kolesnikova, Kateryna; Gurova, Katerina V

    2013-08-01

    Facilitates chromatin transcription (FACT) is a chromatin remodeling complex with two subunits: SSRP1 and SPT16. Mechanisms controlling FACT levels are of interest, since the complex is not expressed in most differentiated cells, but is frequently upregulated in cancer, particularly in poorly differentiated, aggressive tumors. Moreover, inhibition of FACT expression or function in tumor cells interferes with their survival. Here we demonstrate that SSRP1 and SPT16 protein levels decline upon induction of cellular differentiation or senescence in vitro and that similar declines in protein levels for both SSRP1 and SPT16 occur upon RNAi-mediated knockdown of either SSRP1 or SPT16. The interdependence of SSRP1 and SPT16 protein levels was found to be due to their association with SSRP1 and SPT16 mRNAs, which stabilizes the proteins. In particular, presence of SSRP1 mRNA is critical for SPT16 protein stability. In addition, binding of SSRP1 and SPT16 mRNAs to the FACT complex increases the stability and efficiency of translation of the mRNAs. These data support a model in which the FACT complex is stable when SSRP1 mRNA is present, but quickly degrades when SSRP1 mRNA levels drop. In the absence of FACT complex, SSRP1 and SPT16 mRNAs are unstable and inefficiently translated, making reactivation of FACT function unlikely in normal cells. Thus, we have described a complex and unusual mode of regulation controlling cellular FACT levels that results in amplified and stringent control of FACT activity. The FACT dependence of tumor cells suggests that mechanisms controlling FACT levels could be targeted for anticancer therapy.

  7. Stability of hepatitis C virus (HCV) RNA levels among interferon-naïve HIV/HCV-coinfected individuals treated with combination antiretroviral therapy

    DEFF Research Database (Denmark)

    Grint, D; Peters, L; Reekie, J

    2013-01-01

    Infection with hepatitis C virus (HCV) is a major cause of chronic liver disease. High HCV RNA levels have been associated with poor treatment response. This study aimed to examine the natural history of HCV RNA in chronically HCV/HIV-coinfected individuals.......Infection with hepatitis C virus (HCV) is a major cause of chronic liver disease. High HCV RNA levels have been associated with poor treatment response. This study aimed to examine the natural history of HCV RNA in chronically HCV/HIV-coinfected individuals....

  8. Comparison of mRNA levels of three ethylene receptors in senescing flowers of carnation (Dianthus caryophyllus L.).

    Science.gov (United States)

    Shibuya, Kenichi; Nagata, Masayasu; Tanikawa, Natsu; Yoshioka, Toshihito; Hashiba, Teruyoshi; Satoh, Shigeru

    2002-03-01

    Three ethylene receptor genes, DC-ERS1, DC-ERS2 and DC-ETR1, were previously identified in carnation (Dianthus caryophyllus L.). Here, the presence of mRNAs for respective genes in flower tissues and their changes during flower senescence are investigated by Northern blot analysis. DC-ERS2 and DC-ETR1 mRNAs were present in considerable amounts in petals, ovaries and styles of the flower at the full-opening stage. In the petals the level of DC-ERS2 mRNA showed a decreasing trend toward the late stage of flower senescence, whereas it increased slightly in ovaries and was unchanged in styles throughout the senescence period. However, DC-ETR1 mRNA showed no or little changes in any of the tissues during senescence. Exogenously applied ethylene did not affect the levels of DC-ERS2 and DC-ETR1 mRNAs in petals. Ethylene production in the flowers was blocked by treatment with 1,1-dimethyl-4-(phenylsulphonyl)semicarbazide (DPSS), but the mRNA levels for DC-ERS2 and DC-ETR1 decreased in the petals. DC-ERS1 mRNA was not detected in any cases. These results indicate that DC-ERS2 and DC-ETR1 are ethylene receptor genes responsible for ethylene perception and that their expression is regulated in a tissue-specific manner and independently of ethylene in carnation flowers during senescence.

  9. Analysis of correlation between cerebrospinal fluid and plasma HIV-1 RNA levels in patients with neurological opportunistic diseases

    Directory of Open Access Journals (Sweden)

    Paulo Pereira Christo

    2011-08-01

    Full Text Available The question of whether HIV-1 RNA in cerebrospinal fluid (CSF is derived from viral replication in the central nervous system or simply reflects the transit of infected lymphocytes from the blood compartment has long been a matter of debate. Some studies found no correlation between CSF and plasma viral load, whereas others did. The lack of a correlation between the two compartments suggests that the presence of HIV-1 RNA is not simply due to the passive passage of the virus from blood to CSF but rather due to intrathecal replication. To evaluate the correlation between plasma and CSF HIV-1 RNA levels and to identify situations in which there is no correlation between the two compartments, seventy patients were prospectively studied. The association between CSF and plasma viral load was evaluated in the total population and in subgroups of patients with similar characteristics. A correlation between the CSF and plasma compartments was observed for patients undergoing highly active antiretroviral therapy (HAART, those with a CD4 T lymphocyte count lower than 200 cells/mm³, and those with increased CSF protein content. On the other hand, no correlation was observed for patients without adequate virological control, who had a CD4 count higher than 200 cells/mm³ and who did not use HAART. The correlation between the two compartments observed in some patients suggests that CSF HIV-1 RNA levels may reflect plasma levels in these subjects. In contrast, the lack of a correlation between the two compartments in patients who were not on HAART and who had normal CSF proteins and a poor virological control possibly indicates compartmentalization of the virus in CSF and, consequently, plasma-independent intrathecal viral replication.

  10. Resveratrol, by modulating RNA processing factor levels, can influence the alternative splicing of pre-mRNAs.

    Directory of Open Access Journals (Sweden)

    M Andrea Markus

    Full Text Available Alternative pre-mRNA splicing defects can contribute to, or result from, various diseases, including cancer. Aberrant mRNAs, splicing factors and other RNA processing factors have therefore become targets for new therapeutic interventions. Here we report that the natural polyphenol resveratrol can modulate alternative splicing in a target-specific manner. We transfected minigenes of several alternatively spliceable primary mRNAs into HEK293 cells in the presence or absence of 1, 5, 20 and 50 µM resveratrol and measured exon levels by semi-quantitative PCR after separation by agarose gel electrophoresis. We found that 20 µg/ml and 50 µg/ml of resveratrol affected exon inclusion of SRp20 and SMN2 pre-mRNAs, but not CD44v5 or tau pre-mRNAs. By Western blotting and immunofluorescence we showed that this effect may be due to the ability of resveratrol to change the protein level but not the localization of several RNA processing factors. The processing factors that increased significantly were ASF/SF2, hnRNPA1 and HuR, but resveratrol did not change the levels of RBM4, PTBP1 and U2AF35. By means of siRNA-mediated knockdown we depleted cells of SIRT1, regarded as a major target of resveratrol, and showed that the effect on splicing was not dependent on SIRT1. Our results suggest that resveratrol might be an attractive small molecule to treat diseases in which aberrant splicing has been implicated, and justify more extensive research on the effects of resveratrol on the splicing machinery.

  11. Resveratrol, by modulating RNA processing factor levels, can influence the alternative splicing of pre-mRNAs.

    Science.gov (United States)

    Markus, M Andrea; Marques, Francine Z; Morris, Brian J

    2011-01-01

    Alternative pre-mRNA splicing defects can contribute to, or result from, various diseases, including cancer. Aberrant mRNAs, splicing factors and other RNA processing factors have therefore become targets for new therapeutic interventions. Here we report that the natural polyphenol resveratrol can modulate alternative splicing in a target-specific manner. We transfected minigenes of several alternatively spliceable primary mRNAs into HEK293 cells in the presence or absence of 1, 5, 20 and 50 µM resveratrol and measured exon levels by semi-quantitative PCR after separation by agarose gel electrophoresis. We found that 20 µg/ml and 50 µg/ml of resveratrol affected exon inclusion of SRp20 and SMN2 pre-mRNAs, but not CD44v5 or tau pre-mRNAs. By Western blotting and immunofluorescence we showed that this effect may be due to the ability of resveratrol to change the protein level but not the localization of several RNA processing factors. The processing factors that increased significantly were ASF/SF2, hnRNPA1 and HuR, but resveratrol did not change the levels of RBM4, PTBP1 and U2AF35. By means of siRNA-mediated knockdown we depleted cells of SIRT1, regarded as a major target of resveratrol, and showed that the effect on splicing was not dependent on SIRT1. Our results suggest that resveratrol might be an attractive small molecule to treat diseases in which aberrant splicing has been implicated, and justify more extensive research on the effects of resveratrol on the splicing machinery.

  12. Next-Generation Sequencing to Detect Deletion of RB1 and ERBB4 Genes in Chromophobe Renal Cell Carcinoma: A Potential Role in Distinguishing Chromophobe Renal Cell Carcinoma from Renal Oncocytoma.

    Science.gov (United States)

    Liu, Qingqing; Cornejo, Kristine M; Cheng, Liang; Hutchinson, Lloyd; Wang, Mingsheng; Zhang, Shaobo; Tomaszewicz, Keith; Cosar, Ediz F; Woda, Bruce A; Jiang, Zhong

    2018-04-01

    Overlapping morphologic, immunohistochemical, and ultrastructural features make it difficult to diagnose chromophobe renal cell carcinoma (ChRCC) and renal oncocytoma (RO). Because ChRCC is a malignant tumor, whereas RO is a tumor with benign behavior, it is important to distinguish these two entities. We aimed to identify genetic markers that distinguish ChRCC from RO by using next-generation sequencing (NGS). NGS for hotspot mutations or gene copy number changes was performed on 12 renal neoplasms, including seven ChRCC and five RO cases. Matched normal tissues from the same patients were used to exclude germline variants. Rare hotspot mutations were found in cancer-critical genes (TP53 and PIK3CA) in ChRCC but not RO. The NGS gene copy number analysis revealed multiple abnormalities. The two most common deletions were tumor-suppressor genes RB1 and ERBB4 in ChRCC but not RO. Fluorescence in situ hybridization was performed on 65 cases (ChRCC, n = 33; RO, n = 32) to verify hemizygous deletion of RB1 (17/33, 52%) or ERBB4 (11/33, 33%) in ChRCC, but not in RO (0/32, 0%). In total, ChRCCs (23/33, 70%) carry either a hemizygous deletion of RB1 or ERBB4. The combined use of RB1 and ERBB4 fluorescence in situ hybridization to detect deletion of these genes may offer a highly sensitive and specific assay to distinguish ChRCC from RO. Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  13. Metabolic activity and mRNA levels of human cardiac CYP450s involved in drug metabolism.

    Directory of Open Access Journals (Sweden)

    Veronique Michaud

    2010-12-01

    Full Text Available Tissue-specific expression of CYP450s can regulate the intracellular concentration of drugs and explain inter-subject variability in drug action. The overall objective of our study was to determine in a large cohort of samples, mRNA levels and CYP450 activity expressed in the human heart.CYP450 mRNA levels were determined by RTPCR in left ventricular samples (n = 68 of explanted hearts from patients with end-stage heart failure. Samples were obtained from ischemic and non-ischemic hearts. In some instances (n = 7, samples were available from both the left and right ventricles. A technique for the preparation of microsomes from human heart tissue was developed and CYP450-dependent activity was determined using verapamil enantiomers as probe-drug substrates.Our results show that CYP2J2 mRNA was the most abundant isoform in all human heart left ventricular samples tested. Other CYP450 mRNAs of importance were CYP4A11, CYP2E1, CYP1A1 and CYP2C8 mRNAs while CYP2B6 and CYP2C9 mRNAs were present at low levels in only some of the hearts analyzed. CYP450 mRNAs did not differ between ischemic and non-ischemic hearts and appeared to be present at similar levels in the left and right ventricles. Incubation of verapamil with heart microsomes led to the formation of nine CYP450-dependent metabolites: a major finding was the observation that stereoselectivity was reversed compared to human liver microsomes, in which the R-enantiomer is metabolized to a greater extent.This study determined cardiac mRNA levels of various CYP450 isozymes involved in drug metabolism and demonstrated the prevalent expression of CYP2J2 mRNA. It revealed that cardiomyocytes can efficiently metabolize drugs and that cardiac CYP450s are highly relevant with regard to clearance of drugs in the heart. Our results support the claim that drug metabolism in the vicinity of a drug effector site can modulate drug effects.

  14. Analysis of hepatic deiodinase 2 mRNA levels in natural fish lake populations exposed to different levels of putative thyroid disrupters

    International Nuclear Information System (INIS)

    Jarque, Sergio; Bosch, Carme; Casado, Marta; Grimalt, Joan O.; Raldúa, Demetrio; Piña, Benjamin

    2014-01-01

    Hepatic mRNA levels of the dio2 gene (deiodinase 2), implicated in thyroid hormone homeostasis, were analyzed in trout from six remote lakes in the Pyrenees (Spain) and the Tatra Mountains (Slovakia). Highest levels corresponded to fish from the two coldest lakes in Pyrenees, whereas relatively low levels were found in the Tatra lakes. These values correlated with the presence of highly-brominated polybrominated diphenyl ethers (PBDE) congeners in the muscle of the same animals, reflecting the distribution of these compounds across European mountain ranges. In contrast, cyp1a expression levels, diagnostic for the presence of dioxin-like pollutants, mirrored the distribution of semi-volatile organochlorine compounds, indicating the specificity of the two types of biological responses. Exposure to PDBEs is known to increase transcription of dio2 and other thyroid-related genes in laboratory experiments; we propose that our data reflects the same phenomenon in natural populations, driven by anthropogenic pollutants at the environmental concentrations. - Highlights: • Hepatic deiodinase 2 (dio2) mRNA levels vary among mountain lake trout populations. • High dio2 expression correlated with elevated levels of PBDE 153 and 154 in muscle. • Expression patterns of dio2 and cyp1a diverge among the same fish populations. • Elevated biological responses associated to high loads of specific pollutants. • These data indicate that thyroid disruption may occur in remote ecosystems. - Deionidase dio2 expression as a marker for exposure to putative thyroid disruptors in mountain lake trout

  15. Decreased Rhes mRNA levels in the brain of patients with Parkinson's disease and MPTP-treated macaques.

    Directory of Open Access Journals (Sweden)

    Francesco Napolitano

    Full Text Available In rodent and human brains, the small GTP-binding protein Rhes is highly expressed in virtually all dopaminoceptive striatal GABAergic medium spiny neurons, as well as in large aspiny cholinergic interneurons, where it is thought to modulate dopamine-dependent signaling. Consistent with this knowledge, and considering that dopaminergic neurotransmission is altered in neurological and psychiatric disorders, here we sought to investigate whether Rhes mRNA expression is altered in brain regions of patients with Parkinson's disease (PD, Schizophrenia (SCZ, and Bipolar Disorder (BD, when compared to healthy controls (about 200 post-mortem samples. Moreover, we performed the same analysis in the putamen of non-human primate Macaca Mulatta, lesioned with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP. Overall, our data indicated comparable Rhes mRNA levels in the brain of patients with SCZ and BD, and their respective healthy controls. In sharp contrast, the putamen of patients suffering from PD showed a significant 35% reduction of this transcript, compared to healthy subjects. Interestingly, in line with observations obtained in humans, we found 27% decrease in Rhes mRNA levels in the putamen of MPTP-treated primates. Based on the established inhibitory influence of Rhes on dopamine-related responses, we hypothesize that its striatal downregulation in PD patients and animal models of PD might represent an adaptive event of the dopaminergic system to functionally counteract the reduced nigrostriatal innervation.

  16. Regulation of mRNA Levels by Decay-Promoting Introns that Recruit the Exosome Specificity Factor Mmi1

    Directory of Open Access Journals (Sweden)

    Cornelia Kilchert

    2015-12-01

    Full Text Available In eukaryotic cells, inefficient splicing is surprisingly common and leads to the degradation of transcripts with retained introns. How pre-mRNAs are committed to nuclear decay is unknown. Here, we uncover a mechanism by which specific intron-containing transcripts are targeted for nuclear degradation in fission yeast. Sequence elements within these “decay-promoting” introns co-transcriptionally recruit the exosome specificity factor Mmi1, which induces degradation of the unspliced precursor and leads to a reduction in the levels of the spliced mRNA. This mechanism negatively regulates levels of the RNA helicase DDX5/Dbp2 to promote cell survival in response to stress. In contrast, fast removal of decay-promoting introns by co-transcriptional splicing precludes Mmi1 recruitment and relieves negative expression regulation. We propose that decay-promoting introns facilitate the regulation of gene expression. Based on the identification of multiple additional Mmi1 targets, including mRNAs, long non-coding RNAs, and sn/snoRNAs, we suggest a general role in RNA regulation for Mmi1 through transcript degradation.

  17. Long-term treatment with haloperidol affects neuropeptide S and NPSR mRNA levels in the rat brain.

    Science.gov (United States)

    Palasz, Artur; Rojczyk, Ewa; Golyszny, Milosz; Filipczyk, Lukasz; Worthington, John J; Wiaderkiewicz, Ryszard

    2016-04-01

    The brainstem-derived neuropeptide S (NPS) has a multidirectional regulatory activity, especially as a potent anxiolytic factor. Accumulating data suggests that neuroleptics affect peptidergic signalling in various brain structures. However, there is no information regarding the influence of haloperidol on NPS and NPS receptor (NPSR) expression. We assessed NPS and NPSR mRNA levels in brains of rats treated with haloperidol using quantitative real-time polymerase chain reaction. Chronic haloperidol treatment (4 weeks) led to a striking upregulation of NPS and NPSR expression in the rat brainstem. Conversely, the NPSR mRNA expression was decreased in the hippocampus and striatum. This stark increase of NPS in response to haloperidol treatment supports the hypothesis that this neuropeptide is involved in the dopamine-dependent anxiolytic actions of neuroleptics and possibly also in the pathophysiology of mental disorders. Furthermore, our findings underline the complex nature of potential interactions between dopamine receptors and brain peptidergic pathways, which has potential clinical applications.

  18. DNA Methylation of MMP9 Is Associated with High Levels of MMP-9 Messenger RNA in Periapical Inflammatory Lesions.

    Science.gov (United States)

    Campos, Kelma; Gomes, Carolina Cavalieri; Farias, Lucyana Conceição; Silva, Renato Menezes; Letra, Ariadne; Gomez, Ricardo Santiago

    2016-01-01

    Matrix metalloproteinases (MMPs) are the major class of enzymes responsible for degradation of extracellular matrix components and participate in the pathogenesis of periapical inflammatory lesions. MMP expression may be regulated by DNA methylation. The purpose of the present investigation was to analyze the expression of MMP2 and MMP9 in periapical granulomas and radicular cysts and to test the hypothesis that, in these lesions, their transcription may be modulated by DNA methylation. Methylation-specific polymerase chain reaction was used to evaluate the DNA methylation pattern of the MMP2 gene in 13 fresh periapical granuloma samples and 10 fresh radicular cyst samples. Restriction enzyme digestion was used to assess methylation of the MMP9 gene in 12 fresh periapical granuloma samples and 10 fresh radicular cyst samples. MMP2 and MMP9 messenger RNA transcript levels were measured by quantitative real-time polymerase chain reaction. All periapical lesions and healthy mucosa samples showed partial methylation of the MMP2 gene; however, periapical granulomas showed higher MMP2 mRNA expression levels than healthy mucosa (P = .014). A higher unmethylated profile of the MMP9 gene was found in periapical granulomas and radicular cysts compared with healthy mucosa. In addition, higher MMP9 mRNA expression was observed in the periapical lesions compared with healthy tissues. The present study suggests that the unmethylated status of the MMP9 gene in periapical lesions may explain the observed up-regulation of messenger RNA transcription in these lesions. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  19. Comparative modeling and docking studies of p16ink4/Cyclin D1/Rb pathway genes in lung cancer revealed functionally interactive residue of RB1 and its functional partner E2F1

    Directory of Open Access Journals (Sweden)

    e Zahra Syeda Naqsh

    2013-01-01

    Full Text Available Abstract Background Lung cancer is the major cause of mortality worldwide. Major signalling pathways that could play significant role in lung cancer therapy include (1 Growth promoting pathways (Epidermal Growth Factor Receptor/Ras/ PhosphatidylInositol 3-Kinase (2 Growth inhibitory pathways (p53/Rb/P14ARF, STK11 (3 Apoptotic pathways (Bcl-2/Bax/Fas/FasL. Insilico strategy was implemented to solve the mystery behind selected lung cancer pathway by applying comparative modeling and molecular docking studies. Results YASARA [v 12.4.1] was utilized to predict structural models of P16-INK4 and RB1 genes using template 4ELJ-A and 1MX6-B respectively. WHAT CHECK evaluation tool demonstrated overall quality of predicted P16-INK4 and RB1 with Z-score of −0.132 and −0.007 respectively which showed a strong indication of reliable structure prediction. Protein-protein interactions were explored by utilizing STRING server, illustrated that CDK4 and E2F1 showed strong interaction with P16-INK4 and RB1 based on confidence score of 0.999 and 0.999 respectively. In order to facilitate a comprehensive understanding of the complex interactions between candidate genes with their functional interactors, GRAMM-X server was used. Protein-protein docking investigation of P16-INK4 revealed four ionic bonds illustrating Arg47, Arg80,Cys72 and Met1 residues as actively participating in interactions with CDK4 while docking results of RB1 showed four hydrogen bonds involving Glu864, Ser567, Asp36 and Arg861 residues which interact strongly with its respective functional interactor E2F1. Conclusion This research may provide a basis for understanding biological insights of P16-INK4 and RB1 proteins which will be helpful in future to design a suitable drug to inhibit the disease pathogenesis as we have determined the interacting amino acids which can be targeted in order to design a ligand in-vitro to propose a drug for clinical trials. Protein -protein docking of

  20. RNA deep sequencing reveals novel candidate genes and polymorphisms in boar testis and liver tissues with divergent androstenone levels.

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    Asep Gunawan

    Full Text Available Boar taint is an unpleasant smell and taste of pork meat derived from some entire male pigs. The main causes of boar taint are the two compounds androstenone (5α-androst-16-en-3-one and skatole (3-methylindole. It is crucial to understand the genetic mechanism of boar taint to select pigs for lower androstenone levels and thus reduce boar taint. The aim of the present study was to investigate transcriptome differences in boar testis and liver tissues with divergent androstenone levels using RNA deep sequencing (RNA-Seq. The total number of reads produced for each testis and liver sample ranged from 13,221,550 to 33,206,723 and 12,755,487 to 46,050,468, respectively. In testis samples 46 genes were differentially regulated whereas 25 genes showed differential expression in the liver. The fold change values ranged from -4.68 to 2.90 in testis samples and -2.86 to 3.89 in liver samples. Differentially regulated genes in high androstenone testis and liver samples were enriched in metabolic processes such as lipid metabolism, small molecule biochemistry and molecular transport. This study provides evidence for transcriptome profile and gene polymorphisms of boars with divergent androstenone level using RNA-Seq technology. Digital gene expression analysis identified candidate genes in flavin monooxygenease family, cytochrome P450 family and hydroxysteroid dehydrogenase family. Moreover, polymorphism and association analysis revealed mutation in IRG6, MX1, IFIT2, CYP7A1, FMO5 and KRT18 genes could be potential candidate markers for androstenone levels in boars. Further studies are required for proving the role of candidate genes to be used in genomic selection against boar taint in pig breeding programs.

  1. Gibberellin (GA3) enhances cell wall invertase activity and mRNA levels in elongating dwarf pea (Pisum sativum) shoots

    Science.gov (United States)

    Wu, L. L.; Mitchell, J. P.; Cohn, N. S.; Kaufman, P. B.

    1993-01-01

    The invertase (EC 3.2.1.26) purified from cell walls of dwarf pea stems to homogeneity has a molecular mass of 64 kilodaltons (kD). Poly(A)+RNA was isolated from shoots of dwarf pea plants, and a cDNA library was constructed using lambda gt11 as an expression vector. The expression cDNA library was screened with polyclonal antibodies against pea cell wall invertase. One invertase cDNA clone was characterized as a full-length cDNA with 1,863 base pairs. Compared with other known invertases, one homologous region in the amino acid sequence was found. The conserved motif, Asn-Asp-Pro-Asn-Gly, is located near the N-terminal end of invertase. Northern blot analysis showed that the amount of invertase mRNA (1.86 kb) was rapidly induced to a maximal level 4 h after GA3 treatment, then gradually decreased to the control level. The mRNA level at 4 h in GA3-treated peas was fivefold higher than that of the control group. The maximal increase in activity of pea cell wall invertase elicited by GA3 occcured at 8 h after GA3 treatment. This invertase isoform was shown immunocytochemically to be localized in the cell walls, where a 10-fold higher accumulation occurred in GA3-treated tissue compared with control tissue. This study indicates that the expression of the pea shoot cell-wall invertase gene could be regulated by GA3 at transcriptional and/or translational levels.

  2. Global survey of mRNA levels and decay rates of Chlamydia trachomatis trachoma and lymphogranuloma venereum biovars

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    Rita Ferreira

    2017-07-01

    Full Text Available Interpreting the intricate bacterial transcriptomics implies understanding the dynamic relationship established between de novo transcription and the degradation of transcripts. Here, we performed a comparative overview of gene expression levels and mRNA decay rates for different-biovar (trachoma and lymphogranuloma venereum strains of the obligate intracellular bacterium Chlamydia trachomatis. By using RNA-sequencing to measure gene expression levels at mid developmental stage and mRNA decay rates upon rifampicin-based transcription blockage, we observed that: i 60–70% of the top-50 expressed genes encode proteins with unknown function and proteins involved in “Translation, ribosomal structure and biogenesis” for all strains; ii the expression ranking by genes' functional categories was in general concordant among different-biovar strains; iii the median of the half-life time (t1/2 values of transcripts were 15–17 min, indicating that the degree of transcripts’ stability seems to correlate with the bacterial intracellular life-style, as these values are considerably higher than the ones observed in other studies for facultative intracellular and free-living bacteria; iv transcript decay rates were highly heterogeneous within each C. trachomatis strain and did not correlate with steady-state expression levels; v only at very few instances (essentially at gene functional category level was possible to unveil dissimilarities potentially underlying phenotypic differences between biovars. In summary, the unveiled transcriptomic scenario, marked by a general lack of correlation between transcript production and degradation and a huge inter-transcript heterogeneity in decay rates, likely reflects the challenges underlying the unique biphasic developmental cycle of C. trachomatis and its intricate interactions with the human host, which probably exacerbate the complexity of the bacterial transcription regulation.

  3. Global survey of mRNA levels and decay rates of Chlamydia trachomatis trachoma and lymphogranuloma venereum biovars.

    Science.gov (United States)

    Ferreira, Rita; Borges, Vítor; Borrego, Maria José; Gomes, João Paulo

    2017-07-01

    Interpreting the intricate bacterial transcriptomics implies understanding the dynamic relationship established between de novo transcription and the degradation of transcripts. Here, we performed a comparative overview of gene expression levels and mRNA decay rates for different-biovar (trachoma and lymphogranuloma venereum) strains of the obligate intracellular bacterium Chlamydia trachomatis . By using RNA-sequencing to measure gene expression levels at mid developmental stage and mRNA decay rates upon rifampicin-based transcription blockage, we observed that: i ) 60-70% of the top-50 expressed genes encode proteins with unknown function and proteins involved in "Translation, ribosomal structure and biogenesis" for all strains; ii ) the expression ranking by genes' functional categories was in general concordant among different-biovar strains; iii ) the median of the half-life time (t 1/2 ) values of transcripts were 15-17 min, indicating that the degree of transcripts' stability seems to correlate with the bacterial intracellular life-style, as these values are considerably higher than the ones observed in other studies for facultative intracellular and free-living bacteria; iv ) transcript decay rates were highly heterogeneous within each C. trachomatis strain and did not correlate with steady-state expression levels; v ) only at very few instances (essentially at gene functional category level) was possible to unveil dissimilarities potentially underlying phenotypic differences between biovars. In summary, the unveiled transcriptomic scenario, marked by a general lack of correlation between transcript production and degradation and a huge inter-transcript heterogeneity in decay rates, likely reflects the challenges underlying the unique biphasic developmental cycle of C. trachomatis and its intricate interactions with the human host, which probably exacerbate the complexity of the bacterial transcription regulation.

  4. Relationship between expression of leptin receptors mRNA in breast tissue, plasma leptin level in breast cancer patients with obesity and clinical pathologic data

    International Nuclear Information System (INIS)

    Li Chunrui; Liu Wenli; Sun Hanying; Zhou Jianfeng

    2007-01-01

    In order to investigate the expression of leptin receptors mRNA in breast tissue and plasma leptin levels in breast cancer patients with obesity and their relationship with clinical pathologic data, 124 subjects who were either obesity or had suffered from breast benign disease with obesity, or breast cancer with obesity were entered into this study. The levels of plasma leptin in all subjects were determined and leptin receptors mRNA expression levels were measured by RT-PCR in breast tissue of breast cancer patients with obesity and breast benign disease with obesity. The results showed that plasma leptin levels in breast cancer patients with obesity were significantly higher than those in breast benign disease with obesity and obesity patients alone (P<0.05). The expression of the leptin receptor long form [-Lep-R(L)-] mRNA and the leptin receptor short form [-Lep-R(S)-] mRNA in breast tissue of breast cancer patients with obesity were significantly higher than that in breast tissue of breast benign disease patients with obesity (P<0.05). The plasma leptin level had remarkable positive correlation with the expressions of the Lep-R(L) mRNA and the Lep-R(S) mRNA. The plasma leptin level and leptin receptors mRNA expression levels in patients were not correlated with the axillary node metastasis, menopause, the TNM stage or pathological type. Therefore, leptin may have a promoting effect on the carcinogenesis of breast cancer. (authors)

  5. Genome-wide siRNA Screening at Biosafety Level 4 Reveals a Crucial Role for Fibrillarin in Henipavirus Infection.

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    Celine Deffrasnes

    2016-03-01

    Full Text Available Hendra and Nipah viruses (genus Henipavirus, family Paramyxoviridae are highly pathogenic bat-borne viruses. The need for high biocontainment when studying henipaviruses has hindered the development of therapeutics and knowledge of the viral infection cycle. We have performed a genome-wide siRNA screen at biosafety level 4 that identified 585 human proteins required for henipavirus infection. The host protein with the largest impact was fibrillarin, a nucleolar methyltransferase that was also required by measles, mumps and respiratory syncytial viruses for infection. While not required for cell entry, henipavirus RNA and protein syntheses were greatly impaired in cells lacking fibrillarin, indicating a crucial role in the RNA replication phase of infection. During infection, the Hendra virus matrix protein co-localized with fibrillarin in cell nucleoli, and co-associated as a complex in pulldown studies, while its nuclear import was unaffected in fibrillarin-depleted cells. Mutagenesis studies showed that the methyltransferase activity of fibrillarin was required for henipavirus infection, suggesting that this enzyme could be targeted therapeutically to combat henipavirus infections.

  6. Circulating microRNA levels predict residual beta cell function and glycaemic control in children with type 1 diabetes mellitus

    DEFF Research Database (Denmark)

    Samandari, Nasim; Mirza, Aashiq H; Nielsen, Lotte B

    2017-01-01

    AIMS/HYPOTHESIS: We aimed to identify circulating microRNA (miRNA) that predicts clinical progression in a cohort of 123 children with new-onset type 1 diabetes mellitus. METHODS: Plasma samples were prospectively obtained at 1, 3, 6, 12 and 60 months after diagnosis from a subset of 40 children......RNAs revealed significant enrichment for pathways related to gonadotropin-releasing hormone receptor and angiogenesis pathways. CONCLUSIONS/INTERPRETATION: The miRNA hsa-miR-197-3p at 3 months was the strongest predictor of residual beta cell function 1 year after diagnosis in children with type 1 diabetes...... from the Danish Remission Phase Cohort, and profiled for miRNAs. At the same time points, meal-stimulated C-peptide and HbA1c levels were measured and insulin-dose adjusted HbA1c (IDAA1c) calculated. miRNAs that at 3 months after diagnosis predicted residual beta cell function and glycaemic control...

  7. Direct quantification of human cytomegalovirus immediate-early and late mRNA levels in blood of lung transplant recipients by competitive nucleic acid sequence-based amplification

    NARCIS (Netherlands)

    Greijer, AE; Verschuuren, EAM; Harmsen, MC; Dekkers, CAJ; Adriaanse, HMA; The, TH; Middeldorp, JM

    The dynamics of active human cytomegalovirus (HCMV) infection was monitored by competitive nucleic acid sequence-based amplification (NASBA) assays for quantification of IE1 (UL123) and pp67 (UL65) mRNA expression levels In the blood of patients after lung transplantation. RNA was isolated from 339

  8. hnRNP A2/B1 interacts with influenza A viral protein NS1 and inhibits virus replication potentially through suppressing NS1 RNA/protein levels and NS1 mRNA nuclear export

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yimeng; Zhou, Jianhong; Du, Yuchun, E-mail: ydu@uark.edu

    2014-01-20

    The NS1 protein of influenza viruses is a major virulence factor and exerts its function through interacting with viral/cellular RNAs and proteins. In this study, we identified heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) as an interacting partner of NS1 proteins by a proteomic method. Knockdown of hnRNP A2/B1 by small interfering RNA (siRNA) resulted in higher levels of NS vRNA, NS1 mRNA, and NS1 protein in the virus-infected cells. In addition, we demonstrated that hnRNP A2/B1 proteins are associated with NS1 and NS2 mRNAs and that knockdown of hnRNP A2/B1 promotes transport of NS1 mRNA from the nucleus to the cytoplasm in the infected cells. Lastly, we showed that knockdown of hnRNP A2/B1 leads to enhanced virus replication. Our results suggest that hnRNP A2/B1 plays an inhibitory role in the replication of influenza A virus in host cells potentially through suppressing NS1 RNA/protein levels and NS1 mRNA nucleocytoplasmic translocation. - Highlights: • Cellular protein hnRNP A2/B1 interacts with influenza viral protein NS1. • hnRNP A2/B1 suppresses the levels of NS1 protein, vRNA and mRNA in infected cells. • hnRNP A2/B1 protein is associated with NS1 and NS2 mRNAs. • hnRNP A2/B1 inhibits the nuclear export of NS1 mRNAs. • hnRNP A2/B1 inhibits influenza virus replication.

  9. Interleukin-6 and interleukin-10 plasma levels and mRNA expression in polytrauma patients

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    Heber B. Sapan

    2017-12-01

    Conclusion: The pattern of cytokine levels in inflammation response has great impact on the outcome of polytrauma patients. Further study at the genetic level is needed to investigate inflammation process which may influence patient's outcome.

  10. Prolonged treatment with imatinib mesylate in patients with advanced chronic myeloid leukemia causes a reduction of bcr/abl mRNA levels independent of cytogenetic response.

    Science.gov (United States)

    Cariani, E; Capucci, M; Micheletti, M; Spalenza, F; Zanella, I; Albertini, A; Rossi, G

    2003-06-01

    Bcr/abl mRNA levels were monitored in 13 patients with chronic myeloid leukemia receiving imatinib mesylate over a period of 78 weeks. During treatment median bcr/abl mRNA levels progressively declined from 77.2 normalized dose (nD) at baseline to 11.28 nD after 13 weeks ( P<0.05) and to 1.28 nD after 78 weeks ( P<0.05). After 13 weeks, bcr/abl mRNA levels were significantly lower in cytogenetic responders compared to nonresponders ( P<0.05), but subsequent decrease in the transcript levels caused the loss of any correlation to the cytogenetic status. These results suggest that bcr/abl mRNA levels may reflect cytogenetic response only during the early phases of imatinib therapy.

  11. The classification of mRNA expression levels by the phosphorylation state of RNAPII CTD based on a combined genome-wide approach

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    Tachibana Taro

    2011-10-01

    Full Text Available Abstract Background Cellular function is regulated by the balance of stringently regulated amounts of mRNA. Previous reports revealed that RNA polymerase II (RNAPII, which transcribes mRNA, can be classified into the pausing state and the active transcription state according to the phosphorylation state of RPB1, the catalytic subunit of RNAPII. However, genome-wide association between mRNA expression level and the phosphorylation state of RNAPII is unclear. While the functional importance of pausing genes is clear, such as in mouse Embryonic Stem cells for differentiation, understanding this association is critical for distinguishing pausing genes from active transcribing genes in expression profiling data, such as microarrays and RNAseq. Therefore, we examined the correlation between the phosphorylation of RNAPII and mRNA expression levels using a combined analysis by ChIPseq and RNAseq. Results We first performed a precise quantitative measurement of mRNA by performing an optimized calculation in RNAseq. We then visualized the recruitment of various phosphorylated RNAPIIs, such as Ser2P and Ser5P. A combined analysis using optimized RNAseq and ChIPseq for phosphorylated RNAPII revealed that mRNA levels correlate with the various phosphorylation states of RNAPII. Conclusions We demonstrated that the amount of mRNA is precisely reflected by the phased phosphorylation of Ser2 and Ser5. In particular, even the most "pausing" genes, for which only Ser5 is phosphorylated, were detectable at a certain level of mRNA. Our analysis indicated that the complexity of quantitative regulation of mRNA levels could be classified into three categories according to the phosphorylation state of RNAPII.

  12. Low levels of HIV-1 RNA detected in the cerebrospinal fluid after up to 10 years of suppressive therapy are associated with local immune activation.

    Science.gov (United States)

    Dahl, Viktor; Peterson, Julia; Fuchs, Dietmar; Gisslen, Magnus; Palmer, Sarah; Price, Richard W

    2014-09-24

    Though combination antiretroviral therapy reduces the concentration of HIV-1 RNA in both plasma and cerebrospinal fluid (CSF) below the detection limit of clinical assays, low levels of HIV-1 RNA are frequently detectable in plasma using more sensitive assays. We examined the frequency and magnitude of persistent low-level HIV-1 RNA in CSF and its relation to the central nervous system (CNS) immune activation. CSF and plasma HIV-1 RNA were measured using the single-copy assay with a detection limit of 0.3 copies/ml in 70 CSF and 68 plasma samples from 45 treated HIV-1-infected patients with less than 40 copies/ml of HIV-1 RNA in both fluids by standard clinical assays. We also measured CSF neopterin to assess intrathecal immune activation. Theoretical drug exposure was estimated using the CNS penetration-efficacy score of treatment regimens. CSF HIV-1 RNA was detected in 12 of the 70 CSF samples (17%) taken after up to 10 years of suppressive therapy, compared to 39 of the 68 plasma samples (57%) with a median concentration of less than 0.3 copies/ml in CSF compared to 0.3 copies/ml in plasma (P < 0.0001). CSF samples with detectable HIV-1 RNA had higher CSF neopterin levels (mean 8.2 compared to 5.7 nmol/l; P = 0.0085). Patients with detectable HIV-1 RNA in CSF did not differ in pretreatment plasma HIV-1 RNA levels, nadir CD4 cell count or CNS penetration-efficacy score. Low-level CSF HIV-1 RNA and its association with elevated CSF neopterin highlight the potential for the CNS to serve as a viral reservoir and for persistent infection to cause subclinical CNS injury.

  13. 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, S. G.

    2011-01-01

    significantly with the expression of AMHRII, but did not correlate with any of the hormones in the follicular fluid. 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 follicular......Human small antral follicles (diameter 3-9 mm) were obtained from ovaries surgically removed for fertility preservation. From the individual aspirated follicles, granulosa cells and the corresponding follicular fluid were isolated in 64 follicles, of which 55 were available for mRNA analysis (24...... and to the follicular fluid concentrations of AMH, inhibin-B, progesterone and estradiol. AR mRNA expression in granulosa cells and the follicular fluid content of androgens both showed a highly significant positive association with the expression of FSHR mRNA in granulosa cells. AR mRNA expression also correlated...

  14. Significance of the BRAF mRNA Expression Level in Papillary Thyroid Carcinoma: An Analysis of The Cancer Genome Atlas Data.

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    Young Jun Chai

    Full Text Available BRAFV600E is the most common mutation in papillary thyroid carcinoma (PTC, and it is associated with high-risk prognostic factors. However, the significance of the BRAF mRNA level in PTC remains unknown. We evaluated the significance of BRAF mRNA expression level by analyzing PTC data from The Cancer Genome Atlas (TCGA database.Data from 499 patients were downloaded from the TCGA database. After excluding other PTC variants, we selected 353 cases of classic PTC, including 193 cases with BRAFV600E and 160 cases with the wild-type BRAF. mRNA abundances were measured using RNA-Seq with the Expectation Maximization algorithm.The mean BRAF mRNA level was significantly higher in BRAFV600E patients than in patients with wild-type BRAF (197.6 vs. 179.3, p = 0.031. In wild-type BRAF patients, the mean BRAF mRNA level was higher in cases with a tumor > 2 cm than those with a tumor ≤ 2.0 cm (189.4 vs. 163.8, p = 0.046, and was also higher in cases with lymph node metastasis than in those without lymph node metastasis (188.5 vs. 157.9, p = 0.040. Within BRAFV600E patients, higher BRAF mRNA expression was associated with extrathyroidal extension (186.4 vs. 216.4, p = 0.001 and higher T stage (188.1 vs. 210.2, p = 0.016.A higher BRAF mRNA expression level was associated with tumor aggressiveness in classic PTC regardless of BRAF mutational status. Evaluation of BRAF mRNA level may be helpful in prognostic risk stratification of PTC.

  15. Insulin elevates leptin secretion and mRNA levels via cyclic AMP in 3T3-L1 adipocytes deprived of glucose

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    Tomomi Tsubai

    2016-11-01

    Conclusion: Insulin alone stimulates leptin secretion and elevates leptin mRNA levels via cAMP under the lack of glucose metabolism, while glucose is a significant and ambivalent effector on the insulin effects of leptin.

  16. Missense mutation in GRN gene affecting RNA splicing and plasma progranulin level in a family affected by frontotemporal lobar degeneration.

    Science.gov (United States)

    Luzzi, Simona; Colleoni, Lara; Corbetta, Paola; Baldinelli, Sara; Fiori, Chiara; Girelli, Francesca; Silvestrini, Mauro; Caroppo, Paola; Giaccone, Giorgio; Tagliavini, Fabrizio; Rossi, Giacomina

    2017-06-01

    Gene coding for progranulin, GRN, is a major gene linked to frontotemporal lobar degeneration. While most of pathogenic GRN mutations are null mutations leading to haploinsufficiency, GRN missense mutations do not have an obvious pathogenicity, and only a few have been revealed to act through different pathogenetic mechanisms, such as cytoplasmic missorting, protein degradation, and abnormal cleavage by elastase. The aim of this study was to disclose the pathogenetic mechanisms of the GRN A199V missense mutation, which was previously reported not to alter physiological progranulin features but was associated with a reduced plasma progranulin level. After investigating the family pedigree, we performed genetic and biochemical analysis on its members and performed RNA expression studies. We found that the mutation segregates with the disease and discovered that its pathogenic feature is the alteration of GRN mRNA splicing, actually leading to haploinsufficiency. Thus, when facing with a missense GRN mutation, its pathogenetic effects should be investigated, especially if associated with low plasma progranulin levels, to determine its nature of either benign polymorphism or pathogenic mutation. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Low levels of PRB3 mRNA are associated with dopamine-agonist resistance and tumor recurrence in prolactinomas.

    Science.gov (United States)

    Wang, Fei; Gao, Hua; Li, Chuzhong; Bai, Jiwei; Lu, Runchun; Cao, Lei; Wu, Yongtu; Hong, Lichuan; Wu, Yonggang; Lan, Xiaolei; Zhang, Yazhuo

    2014-01-01

    Prolactinomas, or prolactin-secreting adenomas, constitute the most common type of hyperfunctioning pituitary adenoma. Dopamine agonists are used as first-line medication for prolactinomas, but the tumors are resistant to the therapy in 5-18 % of patients. To explore potential mechanisms of resistance to bromocriptine (a dopamine agonist), we analyzed six responsive prolactinomas and six resistant prolactinomas by whole-exome sequencing. We identified ten genes with sequence variants that were differentially found in the two groups of tumors. The expression of these genes was then quantified by real-time reverse-transcription PCR (RT-qPCR) in the 12 prolactinomas and in six normal pituitary glands. The mRNA levels of one of the genes, PRB3, were about fourfold lower in resistant prolactinomas than in the responsive tumors (p = 0.02). Furthermore, low PRB3 expression was also associated with tumor recurrence. Our results suggest that low levels of PRB3 mRNA may have a role in dopamine-agonist resistance and tumor recurrence of prolactinomas.

  18. Extractions of High Quality RNA from the Seeds of Jerusalem Artichoke and Other Plant Species with High Levels of Starch and Lipid.

    Science.gov (United States)

    Mornkham, Tanupat; Wangsomnuk, Preeya Puangsomlee; Fu, Yong-Bi; Wangsomnuk, Pinich; Jogloy, Sanun; Patanothai, Aran

    2013-04-29

    Jerusalem artichoke (Helianthus tuberosus L.) is an important tuber crop. However, Jerusalem artichoke seeds contain high levels of starch and lipid, making the extraction of high-quality RNA extremely difficult and the gene expression analysis challenging. This study was aimed to improve existing methods for extracting total RNA from Jerusalem artichoke dry seeds and to assess the applicability of the improved method in other plant species. Five RNA extraction methods were evaluated on Jerusalem artichoke seeds and two were modified. One modified method with the significant improvement was applied to assay seeds of diverse Jerusalem artichoke accessions, sunflower, rice, maize, peanut and marigold. The effectiveness of the improved method to extract total RNA from seeds was assessed using qPCR analysis of four selected genes. The improved method of Ma and Yang (2011) yielded a maximum RNA solubility and removed most interfering substances. The improved protocol generated 29 to 41 µg RNA/30 mg fresh weight. An A260/A280 ratio of 1.79 to 2.22 showed their RNA purity. Extracted RNA was effective for downstream applications such as first-stranded cDNA synthesis, cDNA cloning and qPCR. The improved method was also effective to extract total RNA from seeds of sunflower, rice, maize and peanut that are rich in polyphenols, lipids and polysaccharides.

  19. Extractions of High Quality RNA from the Seeds of Jerusalem Artichoke and Other Plant Species with High Levels of Starch and Lipid

    Directory of Open Access Journals (Sweden)

    Tanupat Mornkham

    2013-04-01

    Full Text Available Jerusalem artichoke (Helianthus tuberosus L. is an important tuber crop. However, Jerusalem artichoke seeds contain high levels of starch and lipid, making the extraction of high-quality RNA extremely difficult and the gene expression analysis challenging. This study was aimed to improve existing methods for extracting total RNA from Jerusalem artichoke dry seeds and to assess the applicability of the improved method in other plant species. Five RNA extraction methods were evaluated on Jerusalem artichoke seeds and two were modified. One modified method with the significant improvement was applied to assay seeds of diverse Jerusalem artichoke accessions, sunflower, rice, maize, peanut and marigold. The effectiveness of the improved method to extract total RNA from seeds was assessed using qPCR analysis of four selected genes. The improved method of Ma and Yang (2011 yielded a maximum RNA solubility and removed most interfering substances. The improved protocol generated 29 to 41 µg RNA/30 mg fresh weight. An A260/A280 ratio of 1.79 to 2.22 showed their RNA purity. Extracted RNA was effective for downstream applications such as first-stranded cDNA synthesis, cDNA cloning and qPCR. The improved method was also effective to extract total RNA from seeds of sunflower, rice, maize and peanut that are rich in polyphenols, lipids and polysaccharides.

  20. Sequences within the 5' untranslated region regulate the levels of a kinetoplast DNA topoisomerase mRNA during the cell cycle.

    Science.gov (United States)

    Pasion, S G; Hines, J C; Ou, X; Mahmood, R; Ray, D S

    1996-12-01

    Gene expression in trypanosomatids appears to be regulated largely at the posttranscriptional level and involves maturation of mRNA precursors by trans splicing of a 39-nucleotide miniexon sequence to the 5' end of the mRNA and cleavage and polyadenylation at the 3' end of the mRNA. To initiate the identification of sequences involved in the periodic expression of DNA replication genes in trypanosomatids, we have mapped splice acceptor sites in the 5' flanking region of the TOP2 gene, which encodes the kinetoplast DNA topoisomerase, and have carried out deletion analysis of this region on a plasmid-encoded TOP2 gene. Block deletions within the 5' untranslated region (UTR) identified two regions (-608 to -388 and -387 to -186) responsible for periodic accumulation of the mRNA. Deletion of one or the other of these sequences had no effect on periodic expression of the mRNA, while deletion of both regions resulted in constitutive expression of the mRNA throughout the cell cycle. Subcloning of these sequences into the 5' UTR of a construct lacking both regions of the TOP2 5' UTR has shown that an octamer consensus sequence present in the 5' UTR of the TOP2, RPA1, and DHFR-TS mRNAs is required for normal cycling of the TOP2 mRNA. Mutation of the consensus octamer sequence in the TOP2 5' UTR in a plasmid construct containing only a single consensus octamer and that shows normal cycling of the plasmid-encoded TOP2 mRNA resulted in substantial reduction of the cycling of the mRNA level. These results imply a negative regulation of TOP2 mRNA during the cell cycle by a mechanism involving redundant elements containing one or more copies of a conserved octamer sequence within the 5' UTR of TOP2 mRNA.

  1. MicroRNA array and microarray evaluation of endometrial receptivity in patients with high serum progesterone levels on the day of hCG administration

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    Liu Ping

    2011-03-01

    Full Text Available Abstract Background To determine the effect of higher progesterone (P level on endometrial receptivity. Methods This was a prospective analysis conducted in the Reproductive Medical Center of Peking University Third Hospital. All patients received IVF treatment and canceled embryo transfer in the same cycle and were divided into group 1 (normal P; 7 patients and group 2 (elevated P; 12 patients. Endometrial biopsies were performed 6 days after oocyte retrieval. The global miRNA and mRNA gene expressions in endometrial biopsies were investigated with a V4.0 miRNA probe and 22 K Human Genome Array. Fold ratios were derived to compare gene regulation between the groups. Spp1 and Ang gene expression was selected to verify the array results by RT-PCR and the protein expression of osteopontin and VEGF was determined using an immunohistochemical method. Results There were 4 miRNA (all down-regulated and 22 mRNA (13 up-regulated and 9 down-regulated exhibiting differential expression between the groups on the microRNA and microarray chips. miRNA-451, Spp1, and Ang expression in RT-PCR verified the array results. Osteopontin and VEGF were also shown to have positive expression in the endometrium. Conclusions Data from microRNA and microarray analysis suggests dissimilar endometrial receptivity in patients with high P levels on the day of hCG, and elevated osteopontin and decreased VEGF had poor pregnancy rates.

  2. Target gene expression levels and competition between transfected and endogenous microRNAs are strong confounding factors in microRNA high-throughput experiments

    Science.gov (United States)

    2012-01-01

    Background MicroRNA (miRNA) target genes tend to have relatively long and conserved 3' untranslated regions (UTRs), but to what degree these characteristics contribute to miRNA targeting is poorly understood. Different high-throughput experiments have, for example, shown that miRNAs preferentially regulate genes with both short and long 3' UTRs and that target site conservation is both important and irrelevant for miRNA targeting. Results We have analyzed several gene context-dependent features, including 3' UTR length, 3' UTR conservation, and messenger RNA (mRNA) expression levels, reported to have conflicting influence on miRNA regulation. By taking into account confounding factors such as technology-dependent experimental bias and competition between transfected and endogenous miRNAs, we show that two factors - target gene expression and competition - could explain most of the previously reported experimental differences. Moreover, we find that these and other target site-independent features explain about the same amount of variation in target gene expression as the target site-dependent features included in the TargetScan model. Conclusions Our results show that it is important to consider confounding factors when interpreting miRNA high throughput experiments and urge special caution when using microarray data to compare average regulatory effects between groups of genes that have different average gene expression levels. PMID:22325809

  3. CRISPR/Cas9-mediated gene knockout is insensitive to target copy number but is dependent on guide RNA potency and Cas9/sgRNA threshold expression level.

    Science.gov (United States)

    Yuen, Garmen; Khan, Fehad J; Gao, Shaojian; Stommel, Jayne M; Batchelor, Eric; Wu, Xiaolin; Luo, Ji

    2017-11-16

    CRISPR/Cas9 is a powerful gene editing tool for gene knockout studies and functional genomic screens. Successful implementation of CRISPR often requires Cas9 to elicit efficient target knockout in a population of cells. In this study, we investigated the role of several key factors, including variation in target copy number, inherent potency of sgRNA guides, and expression level of Cas9 and sgRNA, in determining CRISPR knockout efficiency. Using isogenic, clonal cell lines with variable copy numbers of an EGFP transgene, we discovered that CRISPR knockout is relatively insensitive to target copy number, but is highly dependent on the potency of the sgRNA guide sequence. Kinetic analysis revealed that most target mutation occurs between 5 and 10 days following Cas9/sgRNA transduction, while sgRNAs with different potencies differ by their knockout time course and by their terminal-phase knockout efficiency. We showed that prolonged, low level expression of Cas9 and sgRNA often fails to elicit target mutation, particularly if the potency of the sgRNA is also low. Our findings provide new insights into the behavior of CRISPR/Cas9 in mammalian cells that could be used for future improvement of this platform. Published by Oxford University Press on behalf of Nucleic Acids Research 2017.

  4. Cytochrome P450 1B1 mRNA levels in peripheral blood cells and exposure to polycyclic aromatic hydrocarbons in Chinese coke oven workers

    Energy Technology Data Exchange (ETDEWEB)

    Hanaoka, Tomoyuki; Tsugane, Shoichiro [Epidemiology and Biostatistics Division, National Cancer Center Research Institute East, 6-5-1 Kashiwanoha, Kashiwa-shi, 277-8577 Chiba (Japan); Yamano, Yuko; Kagawa, Jun [Tokyo Womens' Medical University, 8-1 Kawadacho, Shinjuku-ku, 162-8666 Tokyo (Japan); Pan, Guowei; Zhang, Shujuan [Liaoning Provincial Center for Disease Prevention and Control, 42-1 Jixian Street, 110005 Shenyang (China); Hara, Kunio [Institute for Science of Labour, 2-8-14 Miyamae-ku, 216-8501 Kawasaki (Japan); Ichiba, Masayoshi; Zhang, Jiusong [Saga Medical School, 5-1 Nabeshima, Saga-shi, 849-8501 Saga (Japan); Liu, Tiefu; Li, Landi [Angang Public Health and Anti-epidemic Station Lishan District, 23 Shengoushi Yutian Street, 114034 Anshan (China); Takahashi, Ken [University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, 807-8555 Kitakyushu (Japan)

    2002-09-16

    Cytochrome P450 1B1 (CYP1B1) is induced through the Ah receptor and is involved in the activation of polycyclic aromatic hydrocarbons (PAHs). To determine the validity of a quantitative analysis of CYP1B1 mRNA in peripheral human blood cells for the estimation of PAH exposure, a real-time quantitative polymerase chain reaction method was used to measure the relative levels of CYP1B1 mRNA in 37 Chinese coke oven workers and 13 control workers. A large inter-individual difference in the levels was observed. The average level of the CYP1B1 mRNA in workers at the top work site, where the PAH exposure level from the coke ovens was highest, was significantly higher than in workers at the middle site (P<0.01) or the controls (P=0.02). A non-significant positive correlation was found between the CYP1B1 mRNA levels and urinary 1-hydroxypyrene (R=0.22, P=0.13), and a significant correlation between these mRNA levels and urinary cotinine (R=0.33, P=0.02). It was interesting that a significant positive correlation between CYP1B1 mRNA and 1-hydroxypyrene was observed in subjects with the Leu/Leu type of CYP1B1 Leu432Val polymorphism (R=0.33, P=0.02, n=38) and a non-significant correlation in subjects with the Leu/Val and Val/Val types (R=-0.36, P=0.25, n=12), although the number of subjects in this strata analysis was small. Our preliminary study suggests that PAH exposure in coke ovens and smoking maybe associated with CYP1B1 mRNA levels in peripheral blood cells although mRNA is generally unstable and could be expressed following exposure to other agents.

  5. Family- and genus-level 16S rRNA-targeted oligonucleotide probes for ecological studies of methanotrophic bacteria.

    Science.gov (United States)

    Gulledge, J; Ahmad, A; Steudler, P A; Pomerantz, W J; Cavanaugh, C M

    2001-10-01

    Methanotrophic bacteria play a major role in the global carbon cycle, degrade xenobiotic pollutants, and have the potential for a variety of biotechnological applications. To facilitate ecological studies of these important organisms, we developed a suite of oligonucleotide probes for quantitative analysis of methanotroph-specific 16S rRNA from environmental samples. Two probes target methanotrophs in the family Methylocystaceae (type II methanotrophs) as a group. No oligonucleotide signatures that distinguish between the two genera in this family, Methylocystis and Methylosinus, were identified. Two other probes target, as a single group, a majority of the known methanotrophs belonging to the family Methylococcaceae (type I/X methanotrophs). The remaining probes target members of individual genera of the Methylococcaceae, including Methylobacter, Methylomonas, Methylomicrobium, Methylococcus, and Methylocaldum. One of the family-level probes also covers all methanotrophic endosymbionts of marine mollusks for which 16S rRNA sequences have been published. The two known species of the newly described genus Methylosarcina gen. nov. are covered by a probe that otherwise targets only members of the closely related genus Methylomicrobium. None of the probes covers strains of the newly proposed genera Methylocella and "Methylothermus," which are polyphyletic with respect to the recognized methanotrophic families. Empirically determined midpoint dissociation temperatures were 49 to 57 degrees C for all probes. In dot blot screening against RNA from positive- and negative-control strains, the probes were specific to their intended targets. The broad coverage and high degree of specificity of this new suite of probes will provide more detailed, quantitative information about the community structure of methanotrophs in environmental samples than was previously available.

  6. Lower glutamic acid decarboxylase 65-kDa isoform messenger RNA and protein levels in the prefrontal cortex in schizoaffective disorder but not schizophrenia.

    Science.gov (United States)

    Glausier, Jill R; Kimoto, Sohei; Fish, Kenneth N; Lewis, David A

    2015-01-15

    Altered gamma-aminobutyric acid (GABA) signaling in the prefrontal cortex (PFC) has been associated with cognitive dysfunction in patients with schizophrenia and schizoaffective disorder. Levels of the GABA-synthesizing enzyme glutamic acid decarboxylase 67-kDa isoform (GAD67) in the PFC have been consistently reported to be lower in patients with these disorders, but the status of the second GABA-synthesizing enzyme, glutamic acid decarboxylase 65-kDa isoform (GAD65), remains unclear. GAD65 messenger RNA (mRNA) levels were quantified in PFC area 9 by quantitative polymerase chain reaction from 62 subjects with schizophrenia or schizoaffective disorder and 62 matched healthy comparison subjects. In a subset of subject pairs, GAD65 relative protein levels were quantified by confocal immunofluorescence microscopy. Mean GAD65 mRNA levels were 13.6% lower in subjects with schizoaffective disorder but did not differ in subjects with schizophrenia relative to their matched healthy comparison subjects. In the subjects with schizoaffective disorder, mean GAD65 protein levels were 19.4% lower and were correlated with GAD65 mRNA levels. Lower GAD65 mRNA and protein levels within subjects with schizoaffective disorder were not attributable to factors commonly comorbid with the diagnosis. In concert with previous studies, these findings suggest that schizoaffective disorder is associated with lower levels of both GAD65 and GAD67 mRNA and protein in the PFC, whereas subjects with schizophrenia have lower mean levels of only GAD67 mRNA and protein. Because cognitive function is generally better preserved in patients with schizoaffective disorder relative to patients with schizophrenia, these findings may support an interpretation that GAD65 downregulation provides a homeostatic response complementary to GAD67 downregulation that serves to reduce inhibition in the face of lower PFC network activity. Copyright © 2015 Society of Biological Psychiatry. Published by Elsevier Inc

  7. Chorionic gonadotropin regulates the transcript level of VHL, p53, and HIF-2alpha in human granulosa lutein cells.

    Science.gov (United States)

    Herr, D; Keck, C; Tempfer, C; Pietrowski, Detlef

    2004-12-01

    The ovarian corpus luteum plays a critical role in reproduction being the primary source of circulating progesterone. After ovulation the corpus luteum is build by avascular granulosa lutein cells through rapid vascularization regulated by gonadotropic hormones. The present study was performed to investigate whether this process might be influenced by the human chorionic gonadotropin (hCG)-dependent expression of different tumor suppressor genes and hypoxia dependent transcription factors. RNA was isolated from cultured granulosa lutein cells, transcribed into cDNA, and the transcript level of following genes were determined: RB-1, VHL, NF-1, NF-2, Wt-1, p53, APC, and hypoxia inducible factor-1 (HIF-1), -2, and -3alpha. Additionally, the influence of hCG on the expression of VHL, p53, and HIf2alpha were investigated. We demonstrate that in human granulosa lutein cells the tumor suppressor genes RB-1, VHL, NF-1, NF-2, Wt-1, p53, and APC and the hypoxia dependent transcription factors HIF-1alpha, -2alpha, and -3alpha are expressed. In addition, we showed that hCG regulates the expression of p53, VHL, and HIF-2alpha. Our results indicate that hCG may determine the growth and development of the corpus luteum by mediating hypoxic and apoptotic pathways in human granulosa lutein cells. Copyright 2004 Wiley-Liss, Inc.

  8. Multi-level gene/MiRNA feature selection using deep belief nets and active learning.

    Science.gov (United States)

    Ibrahim, Rania; Yousri, Noha A; Ismail, Mohamed A; El-Makky, Nagwa M

    2014-01-01

    Selecting the most discriminative genes/miRNAs has been raised as an important task in bioinformatics to enhance disease classifiers and to mitigate the dimensionality curse problem. Original feature selection methods choose genes/miRNAs based on their individual features regardless of how they perform together. Considering group features instead of individual ones provides a better view for selecting the most informative genes/miRNAs. Recently, deep learning has proven its ability in representing the data in multiple levels of abstraction, allowing for better discrimination between different classes. However, the idea of using deep learning for feature selection is not widely used in the bioinformatics field yet. In this paper, a novel multi-level feature selection approach named MLFS is proposed for selecting genes/miRNAs based on expression profiles. The approach is based on both deep and active learning. Moreover, an extension to use the technique for miRNAs is presented by considering the biological relation between miRNAs and genes. Experimental results show that the approach was able to outperform classical feature selection methods in hepatocellular carcinoma (HCC) by 9%, lung cancer by 6% and breast cancer by around 10% in F1-measure. Results also show the enhancement in F1-measure of our approach over recently related work in [1] and [2].

  9. Can balneotherapy modify microRNA expression levels in osteoarthritis? A comparative study in patients with knee osteoarthritis

    Science.gov (United States)

    Giannitti, C.; De Palma, A.; Pascarelli, N. A.; Cheleschi, S.; Giordano, N.; Galeazzi, M.; Fioravanti, Antonella

    2017-12-01

    The aim of this study was to evaluate the whole-blood levels of miR-155, miR-223, miR-181a, miR-146a, and miR-let-7e in patients with bilateral knee osteoarthritis (OA) after a cycle of mud-bath therapy (MBT). Thirty-two patients with knee OA defined by the ACR criteria were included. Twenty-one patients (MBT group) were daily treated with a combination of local mud-packs at 42 °C and baths in mineral water, at 37 °C for 15 min, for 12 applications over a period of 2 weeks, in addition to standard therapy; 11 patients (control group) continued their conventional treatment alone. Global pain score evaluated by visual analog scale (VAS), WOMAC subscores, and microRNA expression were evaluated at baseline and after 2 weeks. Peripheral whole blood was collected into PAXgene™ Blood RNA tubes, stored at - 80 °C, and total RNA was extracted. The expression of miR-155, miR-223, miR-181a, miR-146a, and miR-let-7e was determined by qRT-PCR. After MBT, we observed a statistically significant improvement of clinical parameters and a significant decrease of miR-155, miR-181a, miR-146a ( p < 0.001), and miR-223 ( p < 0.01) expression levels. No clinical and biochemical modifications were detected in the control group. No significant variations of miR-let-7e were shown in both groups after 2 weeks. In conclusion, MBT can modify the expression of miR-155, miR-181a, miR-146a, and miR-223, which are upregulated in OA. It could be due to the heat stress and the hydrostatic pressure, since some miRNAs were found to be temperature- and mechano-responsive. Further studies are needed to better explain the mechanism of action of MBT and the role of miRNAs in OA.

  10. Inhibition of Poliovirus-Induced Cleavage of Cellular Protein PCBP2 Reduces the Levels of Viral RNA Replication

    Science.gov (United States)

    Chase, Amanda J.; Daijogo, Sarah

    2014-01-01

    ABSTRACT Due to their small genome size, picornaviruses must utilize host proteins to mediate cap-independent translation and viral RNA replication. The host RNA-binding protein poly(rC) binding protein 2 (PCBP2) is involved in both processes in poliovirus infected cells. It has been shown that the viral proteinase 3CD cleaves PCBP2 and contributes to viral translation inhibition. However, cleaved PCBP2 remains active in viral RNA replication. This would suggest that both cleaved and intact forms of PCBP2 have a role in the viral RNA replication cycle. The picornavirus genome must act as a template for both translation and RNA replication. However, a template that is actively being translated cannot function as a template for RNA replication, suggesting that there is a switch in template usage from translation to RNA replication. We demonstrate that the cleavage of PCBP2 by the poliovirus 3CD proteinase is a necessary step for efficient viral RNA replication and, as such, may be important for mediating a switch in template usage from translation to RNA replication. IMPORTANCE Poliovirus, like all positive-strand RNA viruses that replicate in the cytoplasm of eukaryotic cells, uses its genomic RNA as a template for both viral protein synthesis and RNA replication. Given that these processes cannot occur simultaneously on the same template, poliovirus has evolved a mechanism(s) to facilitate the switch from using templates for translation to using them for RNA synthesis. This study explores one possible scenario for how the virus alters the functions of a host cell RNA binding protein to mediate, in part, this important transition. PMID:24371074

  11. Cerebrospinal fluid HIV-1 RNA levels in asymptomatic patients with early stage chronic HIV-1 infection: support for the hypothesis of local virus replication.

    Science.gov (United States)

    García, F; Niebla, G; Romeu, J; Vidal, C; Plana, M; Ortega, M; Ruiz, L; Gallart, T; Clotet, B; Miró, J M; Pumarola, T; Gatell, J M

    1999-08-20

    To assess HIV-1 RNA levels in cerebrospinal fluid (CSF) and their potential correlation with plasma viral load and central nervous system (CNS) HIV-1 infection markers in stable asymptomatic patients with a CD4 T cell count >500x10(6) cells/l. Consecutive patients screened for two trials were eligible for lumbar puncture assessment. At day 0, simultaneous samples of CSF and plasma were obtained and levels of total proteins, albumin, IgG, antibodies against HIV-1 p24 antigen, HIV-1 RNA (using the polymerase chain technique) and white cells were measured. The integrity of the blood-brain barrier was preserved (albumin index > or =7) in 59 out of 70 patients (84%). Intrathecal production of antibodies against HIV-1 p24 antigen was demonstrated in 55 out of 70 individuals (78%). Viral load in CSF was significantly lower than plasma values (3.13+/-0.95 versus 4.53+/-0.53, P = 0.0001). HIV-1 RNA was not detected in CSF in only three of the 70 patients (4%). Overall, there was a significant correlation between plasma and CSF HIV-1 RNA levels (r = 0.43, P = 0.0001); however, in 29 patients (41%) there were significant differences (>1.5 log10 copies/ml) between the viral loads in plasma and CSF. In the multivariate analysis, a high level of protein and white cells in CSF, but not the HIV-1 RNA plasma level, were factors independently associated with a higher level of HIV-1 RNA in CSF (P = 0.0001). HIV-1 RNA can be detected almost always in CSF of asymptomatic patients in early stages of HIV-1 infection including those with a preserved integrity of the blood-brain barrier. The important discrepancies between plasma and CSF viral load, and the independent association between CSF abnormalities and CSF viral load, support the hypothesis of local production of HIV-1.

  12. Regulation of RNA polymerase III transcription during transformation of human IMR90 fibroblasts with defined genetic elements.

    Science.gov (United States)

    Durrieu-Gaillard, Stéphanie; Dumay-Odelot, Hélène; Boldina, Galina; Tourasse, Nicolas J; Allard, Delphine; André, Fabrice; Macari, Françoise; Choquet, Armelle; Lagarde, Pauline; Drutel, Guillaume; Leste-Lasserre, Thierry; Petitet, Marion; Lesluyes, Tom; Lartigue-Faustin, Lydia; Dupuy, Jean-William; Chibon, Frédéric; Roeder, Robert G; Joubert, Dominique; Vagner, Stéphan; Teichmann, Martin

    2018-01-01

    RNA polymerase (Pol) III transcribes small untranslated RNAs that are essential for cellular homeostasis and growth. Its activity is regulated by inactivation of tumor suppressor proteins and overexpression of the oncogene c-MYC, but the concerted action of these tumor-promoting factors on Pol III transcription has not yet been assessed. In order to comprehensively analyse the regulation of Pol III transcription during tumorigenesis we employ a model system that relies on the expression of five genetic elements to achieve cellular transformation. Expression of these elements in six distinct transformation intermediate cell lines leads to the inactivation of TP53, RB1, and protein phosphatase 2A, as well as the activation of RAS and the protection of telomeres by TERT, thereby conducting to full tumoral transformation of IMR90 fibroblasts. Transformation is accompanied by moderately enhanced levels of a subset of Pol III-transcribed RNAs (7SK; MRP; H1). In addition, mRNA and/or protein levels of several Pol III subunits and transcription factors are upregulated, including increased protein levels of TFIIIB and TFIIIC subunits, of SNAPC1 and of Pol III subunits. Strikingly, the expression of POLR3G and of SNAPC1 is strongly enhanced during transformation in this cellular transformation model. Collectively, our data indicate that increased expression of several components of the Pol III transcription system accompanied by a 2-fold increase in steady state levels of a subset of Pol III RNAs is sufficient for sustaining tumor formation.

  13. Minimal alteration in the ratio of circulatory fetal DNA to fetal corticotropin-releasing hormone mRNA level in preeclampsia.

    Science.gov (United States)

    Zhong, Xiao Yan; Holzgreve, Wolfgang; Gebhardt, Stefan; Hillermann, Renate; Tofa, Kashefa Carelse; Gupta, Anurag Kumar; Huppertz, Berthold; Hahn, Sinuhe

    2006-01-01

    We have recently observed that fetal DNA and fetal corticotropin-releasing hormone (CRH) mRNA are associated with in vitro generated syncytiotrophoblast-derived microparticles, and that the ratio of fetal DNA to mRNA (CRH) varied according to whether the particles were derived by predominantly apoptotic, apo-necrotic or necrotic pathways. Hence, we examined whether these ratios varied in maternal plasma samples taken from normotensive and preeclamptic pregnancies in vivo. Maternal plasma samples were collected from 18 cases with preeclampsia and 29 normotensive term controls. Circulatory fetal CRH mRNA and DNA levels were quantified by real-time PCR and RT-PCR. Circulatory fetal mRNA and fetal DNA levels were significantly elevated in the preeclampsia study group when compared to normotensive controls. Alterations in the fetal mRNA to DNA ratio between the study and control groups were minimal, even when stratified into early (34 weeks of gestation) onset preeclampsia. Our data suggest that although circulatory fetal DNA and mRNA levels are significantly elevated in preeclampsia, the ratios in maternal plasma are not dramatically altered. Copyright 2006 S. Karger AG, Basel.

  14. Predicted RNA Binding Proteins Pes4 and Mip6 Regulate mRNA Levels, Translation, and Localization during Sporulation in Budding Yeast.

    Science.gov (United States)

    Jin, Liang; Zhang, Kai; Sternglanz, Rolf; Neiman, Aaron M

    2017-05-01

    In response to starvation, diploid cells of Saccharomyces cerevisiae undergo meiosis and form haploid spores, a process collectively referred to as sporulation. The differentiation into spores requires extensive changes in gene expression. The transcriptional activator Ndt80 is a central regulator of this process, which controls many genes essential for sporulation. Ndt80 induces ∼300 genes coordinately during meiotic prophase, but different mRNAs within the NDT80 regulon are translated at different times during sporulation. The protein kinase Ime2 and RNA binding protein Rim4 are general regulators of meiotic translational delay, but how differential timing of individual transcripts is achieved was not known. This report describes the characterization of two related NDT80 -induced genes, PES4 and MIP6 , encoding predicted RNA binding proteins. These genes are necessary to regulate the steady-state expression, translational timing, and localization of a set of mRNAs that are transcribed by NDT80 but not translated until the end of meiosis II. Mutations in the predicted RNA binding domains within PES4 alter the stability of target mRNAs. PES4 and MIP6 affect only a small portion of the NDT80 regulon, indicating that they act as modulators of the general Ime2/Rim4 pathway for specific transcripts. Copyright © 2017 American Society for Microbiology.

  15. Regulation of c-myc and c-fos mRNA levels by polyomavirus: distinct roles for the capsid protein VP1 and the viral early proteins

    International Nuclear Information System (INIS)

    Zullo, J.; Stiles, C.D.; Garcea, R.L.

    1987-01-01

    The levels of c-myc, c-fos, and JE mRNAs accumulate in a biphasic pattern following infection of quiescent BALB/c 3T3 mouse cells with polyomavirus. Maximal levels of c-myc and c-fos mRNAs were seen within 1 hr and were nearly undetectable at 6 hr after infection. At 12 hr after infection mRNA levels were again maximal and remained elevated thereafter. Empty virions (capsids) and recombinant VP 1 protein, purified from Escherichia coli, induced the early but not the late phase of mRNA accumulation. Virions, capsids, and recombinant VP 1 protein stimulated [ 3 H]thymidine nuclear labeling and c-myc mRNA accumulation in a dose-responsive manner paralleling their affinity for the cell receptor for polyoma. The second phase of mRNA accumulation is regulated by the viral early gene products, as shown by polyomavirus early gene mutants and by a transfected cell line (336a) expressing middle tumor antigen upon glucocorticoid addition. These results suggest that polyomavirus interacts with the cell membrane at the onset of infection to increase the levels of mRNA for the cellular genes associated with cell competence for DNA replication, and subsequently these levels are maintained by the action of the early viral proteins

  16. Neurotrophins and their receptors in the rat pituitary gland: regulation of BDNF and trkB mRNA levels by adrenal hormones.

    Science.gov (United States)

    Kononen, J; Soinila, S; Persson, H; Honkaniemi, J; Hökfelt, T; Pelto-Huikko, M

    1994-12-01

    We studied the expression of messenger ribonucleic acids (mRNAs) for neurotrophins and neurotrophin receptors in the rat pituitary gland and examined the influence of adrenal hormones on their mRNA levels, using in situ hybridization and Northern blot analysis. The only neurotrophin present at detectable levels in the pituitary was brain-derived neurotrophic factor (BDNF), which was observed in the anterior and intermediate lobes. Several transcripts of the putative receptor for BDNF, trkB, were present in the anterior and posterior lobes of the pituitary. A low amount of trkC mRNA was found in both the anterior and the intermediate lobe. Dexamethasone treatment decreased both BDNF and trkB mRNA levels in the anterior lobe of the pituitary. Adrenalectomy had no effect on trkB expression, but it decreased BDNF mRNA levels in comparison to the control animals. This effect could not be reversed by dexamethasone substitution, suggesting that BDNF, mRNA levels may be regulated not only by glucocorticoids but also by other adrenal hormones. These results demonstrate that BDNF, trkB and trkC are expressed in the pituitary gland and that glucocorticoids and possibly other adrenal hormones may modulate pituitary functions by regulating the expression of neurotrophic factors and their receptors. Whether BDNF acts as a secreted hormone, a trophic factor, or has autocrine/paracrine functions within the pituitary through its receptor, trkB, remains to be studied.

  17. Lower glutamic acid decarboxylase 65kD mRNA and protein levels in the prefrontal cortex in schizoaffective disorder but not schizophrenia

    Science.gov (United States)

    Glausier, JR; Kimoto, S; Fish, KN; Lewis, DA

    2014-01-01

    Background Altered GABA signaling in the prefrontal cortex (PFC) has been associated with cognitive dysfunction in schizophrenia and schizoaffective disorder. PFC levels of the GABA-synthesizing enzyme glutamic acid decarboxylase 67kD (GAD67) has been consistently reported to be lower in these disorders, but the status of the second GABA-synthesizing enzyme, GAD65, remains unclear. Methods GAD65 mRNA levels were quantified in PFC area 9 by quantitative polymerase chain reaction from 62 subjects with schizophrenia or schizoaffective disorder and 62 matched healthy comparison subjects. GAD65 relative protein levels were quantified in a subset of subject pairs by confocal immunofluorescence microscopy. Results Mean GAD65 mRNA levels were 13.6% lower in schizoaffective disorder subjects, but did not differ in schizophrenia subjects, relative to their matched healthy comparison subjects. In the subjects with schizoaffective disorder, mean GAD65 protein levels were 19.4% lower and were correlated with GAD65 mRNA levels. Lower GAD65 mRNA and protein measures within schizoaffective disorder subjects was not attributable to factors commonly comorbid with the diagnosis. Conclusions In concert with previous studies, these findings suggest that schizoaffective disorder is associated with lower levels of both GAD65 and GAD67 mRNA and protein in the PFC, whereas subjects with schizophrenia have lower mean levels of only GAD67 mRNA and protein. Because cognitive function is generally better preserved in subjects with schizoaffective disorder relative to subjects with schizophrenia, these findings may support an interpretation that GAD65 down-regulation provides a homeostatic response complementary to GAD67 down-regulation expression that serves to reduce inhibition in the face of lower PFC network activity. PMID:24993056

  18. Plasma cytokines do not reflect expression of pro- and anti-inflammatory cytokine mRNA at organ level after cardiopulmonary bypass in neonatal pigs

    DEFF Research Database (Denmark)

    Brix-Christensen, V.; Vestergaard, C.; Chew, M.

    2003-01-01

    Background: Plasma concentrations of inflammatory markers are increased in response to the trauma of cardiac surgery and cardiopulmonary bypass (CPB). It is, however, unknown whether the plasma cytokine levels and cytokine mRNA expression at organ level reflect each other. Methods: Twenty...

  19. Variation of transaminases, HCV-RNA levels and Th1/Th2 cytokine production during the post-partum period in pregnant women with chronic hepatitis C.

    Directory of Open Access Journals (Sweden)

    Angeles Ruiz-Extremera

    Full Text Available This study analyses the evolution of liver disease in women with chronic hepatitis C during the third trimester of pregnancy and the post-partum period, as a natural model of immune modulation and reconstitution. Of the 122 mothers recruited to this study, 89 were HCV-RNA+ve/HIV-ve and 33 were HCV-RNA-ve/HIV-ve/HCVantibody+ve and all were tested during the third trimester of pregnancy, at delivery and post-delivery. The HCV-RNA+ve mothers were categorized as either Type-A (66%, with an increase in ALT levels in the post-partum period (>40 U/L; P<0.001 or as Type-B (34%, with no variation in ALT values. The Type-A mothers also presented a significant decrease in serum HCV-RNA levels in the post-delivery period (P<0.001 and this event was concomitant with an increase in Th1 cytokine levels (INFγ, P = 0.04; IL12, P = 0.01 and IL2, P = 0.01. On the other hand, the Type-B mothers and the HCV-RNA-ve women presented no variations in either of these parameters. However, they did present higher Th1 cytokine levels in the partum period (INFγ and IL2, P<0.05 than both the Type-A and the HCV-RNA-ve women. Cytokine levels at the moment of delivery do not constitute a risk factor associated with HCV vertical transmission. It is concluded that differences in the ALT and HCV-RNA values observed in HCV-RNA+ve women in the postpartum period might be due to different ratios of Th1 cytokine production. In the Type-B women, the high partum levels of Th1 cytokines and the absence of post-partum variation in ALT and HCV-RNA levels may be related to permanent Th1 cytokine stimulation.

  20. Expression levels of the microRNA maturing microprocessor complex component DGCR8 and the RNA-induced silencing complex (RISC) components argonaute-1, argonaute-2, PACT, TARBP1, and TARBP2 in epithelial skin cancer.

    Science.gov (United States)

    Sand, Michael; Skrygan, Marina; Georgas, Dimitrios; Arenz, Christoph; Gambichler, Thilo; Sand, Daniel; Altmeyer, Peter; Bechara, Falk G

    2012-11-01

    The microprocessor complex mediates intranuclear biogenesis of precursor microRNAs from the primary microRNA transcript. Extranuclear, mature microRNAs are incorporated into the RNA-induced silencing complex (RISC) before interaction with complementary target mRNA leads to transcriptional repression or cleavage. In this study, we investigated the expression profiles of the microprocessor complex subunit DiGeorge syndrome critical region gene 8 (DGCR8) and the RISC components argonaute-1 (AGO1), argonaute-2 (AGO2), as well as double-stranded RNA-binding proteins PACT, TARBP1, and TARBP2 in epithelial skin cancer and its premalignant stage. Patients with premalignant actinic keratoses (AK, n = 6), basal cell carcinomas (BCC, n = 15), and squamous cell carcinomas (SCC, n = 7) were included in the study. Punch biopsies were harvested from the center of the tumors (lesional), from healthy skin sites (intraindividual controls), and from healthy skin sites in a healthy control group (n = 16; interindividual control). The DGCR8, AGO1, AGO2, PACT, TARBP1, and TARBP2 mRNA expression levels were detected by quantitative real-time reverse transcriptase polymerase chain reaction. The DGCR8, AGO1, AGO2, PACT, and TARBP1 expression levels were significantly higher in the AK, BCC, and SCC groups than the healthy controls (P  0.05). This study indicates that major components of the miRNA pathway, such as the microprocessor complex and RISC, are dysregulated in epithelial skin cancer. Copyright © 2011 Wiley Periodicals, Inc.

  1. Elevated microRNA-126 is associated with high vascular endothelial growth factor receptor 2 expression levels and high microvessel density in colorectal cancer

    DEFF Research Database (Denmark)

    Hansen, Torben Frøstrup; Andersen, Claus Lindbjerg; Nielsen, Boye Schnack

    2011-01-01

    was to analyse the possible relationship between miRNA-126, VEGFR-2 and angiogenesis in tumour tissue from patients with colorectal cancer (CRC). Tumour tissue was obtained from 81 patients. The miRNA-126 and VEGFR-2 gene expression levels were analysed by PCR and the protein concentrations of VEGFR-2 were...... the median as the cut-off. The median gene expression levels of VEGFR-2 were significantly lower in the tumours expressing low levels of miRNA-126, 0.30 (95% CI, 0.24‑0.36), compared to those expressing high levels of miRNA-126, 0.48 (95% CI, 0.28-0.60), p=0.02. A positive association was observed with VEGFR...... analysed by ELISA. Angiogenesis, visualised by the endothelial cell marker CD105 combined with caldesmon, was assessed by immunohistochemistry and the microvessel density (MVD) technique. In situ hybridisation was performed for miRNA-126. Tumours were classified as low or high miRNA‑126-expressing using...

  2. Urinary Hepcidin Levels in Iron-Deficient and Iron-Supplemented Piglets Correlate with Hepcidin Hepatic mRNA and Serum Levels and with Body Iron Status.

    Directory of Open Access Journals (Sweden)

    Robert Staroń

    Full Text Available Among livestock, domestic pig (Sus scrofa is a species, in which iron metabolism has been most intensively examined during last decade. The obvious reason for studying the regulation of iron homeostasis especially in young pigs is neonatal iron deficiency anemia commonly occurring in these animals. Moreover, supplementation of essentially all commercially reared piglets with iron entails a need for monitoring the efficacy of this routine practice followed in the swine industry for several decades. Since the discovery of hepcidin many studies confirmed its role as key regulator of iron metabolism and pointed out the assessment of its concentrations in biological fluids as diagnostic tool for iron-related disorder. Here we demonstrate that urine hepcidin-25 levels measured by a combination of weak cation exchange chromatography and time-of-flight mass spectrometry (WCX-TOF MS are highly correlated with mRNA hepcidin expression in the liver and plasma hepcidin-25 concentrations in anemic and iron-supplemented 28-day old piglets. We also found a high correlation between urine hepcidin level and hepatic non-heme iron content. Our results show that similarly to previously described transgenic mouse models of iron disorders, young pigs constitute a convenient animal model to explore accuracy and relationship between indicators for assessing systemic iron status.

  3. Study on the plasma leptin level and leptin mRNA expression in cancerous breast tissue in patients with breast carcinoma complicated with obesity

    International Nuclear Information System (INIS)

    Li Chunrui; Liu Wenli; Sun Hanying; Zhou Jianfeng

    2006-01-01

    Objective: To study the plasma leptin level and leptin mRNA expression in cancerous breast tissue in patients with breast cancer complicated with obesity. Methods: Plasma leptin levels were measured with RIA in 48 breast cancer patients with obesity, 36 patients with various benign breast disorders and obesity and 40 controls (with simple obesity only). The leptin mRNA expression in the surgical specimens from the 84 patients with breast disease was also examined with RT-PCR, Results: The plasma leptin levels in the breast cancer patients (12.02 ± 1.23 μg/L) were significantly higher than those in patients with benign breast disorders (9.84 ± 0.98 μg/L) and controls (9.79 ± 1.16 μg/L) (both P<0.05). The expression levels of leptin mRNA in specimens from malignant breast disease (0.71 ± 0.32), were significantly higher than those in specimens from benign breast diseases (0.41 ± 0.26) (P<0.05), The plasma leptin levels and the tissue leptin mRNA expression levels were mutually positively correlated (r=0.4220 ,P 0.0180). These levels were not correlated with the presence of axillary metastasis, TMN stage, menstrual status, pathological classification and other parameters. Conclusion: Leptin might be a promotive factor in the development of breast cancer. (authors)

  4. Growth arrest-specific transcript 5 associated snoRNA levels are related to p53 expression and DNA damage in colorectal cancer.

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    Jonathan Krell

    Full Text Available The growth arrest-specific transcript 5 gene (GAS5 encodes a long noncoding RNA (lncRNA and hosts a number of small nucleolar RNAs (snoRNAs that have recently been implicated in multiple cellular processes and cancer. Here, we investigate the relationship between DNA damage, p53, and the GAS5 snoRNAs to gain further insight into the potential role of this locus in cell survival and oncogenesis both in vivo and in vitro.We used quantitative techniques to analyse the effect of DNA damage on GAS5 snoRNA expression and to assess the relationship between p53 and the GAS5 snoRNAs in cancer cell lines and in normal, pre-malignant, and malignant human colorectal tissue and used biological techniques to suggest potential roles for these snoRNAs in the DNA damage response.GAS5-derived snoRNA expression was induced by DNA damage in a p53-dependent manner in colorectal cancer cell lines and their levels were not affected by DICER. Furthermore, p53 levels strongly correlated with GAS5-derived snoRNA expression in colorectal tissue.In aggregate, these data suggest that the GAS5-derived snoRNAs are under control of p53 and that they have an important role in mediating the p53 response to DNA damage, which may not relate to their function in the ribosome. We suggest that these snoRNAs are not processed by DICER to form smaller snoRNA-derived RNAs with microRNA (miRNA-like functions, but their precise role requires further evaluation. Furthermore, since GAS5 host snoRNAs are often used as endogenous controls in qPCR quantifications we show that their use as housekeeping genes in DNA damage experiments can lead to inaccurate results.

  5. Induction of vitellogenin synthesis by estrogen in avian liver: relationship between level of vitellogenin mRNA and vitellogenin synthesis.

    Science.gov (United States)

    Mullinix, K P; Wetekam, W; Deeley, R G; Gordon, J I; Meyers, M; Kent, K A; Goldberger, R F

    1976-01-01

    We have investigated the estrogen-mediated induction of vitellogenin synthesis in rooster liver. We compared the concentrations of vitellogenin messenger RNA (mRNA) in the liver with the concentrations of vitellogenin in the sera of roosters that had recieved various treatments with estrogen. We found no vitellogenin mRNA in the livers of the unstimulated roosters. An initial injection of estrogen was attended by de novo synthesis of vitellogenin mRNA in the liver and accumulation of vitellogenin in the serum. When vitellogenin was no longer present in the serum or liver (the "post-estrogen-serum-negative" state), the liver was found to contain appreciable amounts of vitellogenin mRNA. This mRNA was of the same size as that found in the liver of the rooster actively synthesizing vitellogenin in response to estrogen. Whereas vitellogenin mRNA was in large polysomes in the livers of the roosters actively synthesizing vitellogenin, the vitellogenin mRNA in the liver of the post-estrogen-serum-negative rooster was not associated with polysomes. The possible relevance of these findings to the fact that the rooster responds differently to a primary stimulation with estrogen than to subsequent stimulations is discussed. PMID:1064017

  6. Skeletal changes in osteoprotegerin and receptor activator of nuclear factor-κb ligand mRNA levels in primary hyperparathyroidism

    DEFF Research Database (Denmark)

    Stilgren, L.S.; Rettmer, E.; Eriksen, E. F.

    2004-01-01

    , and treatment of PHPT were included. A transiliac bone biopsy was done before (n = 24) and 12 months after parathyroidectomy (PTX) (n = 21). Biopsies were frozen in liquid nitrogen and RNA extracted using Trizol. A competitive RT-PCR assay for RANKL and OPG mRNA using artificial cDNA standards was developed...

  7. Oxygen regulation of uricase and sucrose synthase synthesis in soybean callus tissue is exerted at the mRNA level

    DEFF Research Database (Denmark)

    Xue, Z T; Larsen, K; Jochimsen, B U

    1991-01-01

    The effect of lowering oxygen concentration on the expression of nodulin genes in soybean callus tissue devoid of the microsymbiont has been examined. Poly(A)+ RNA was isolated from tissue cultivated in 4% oxygen and in normal atmosphere. Quantitative mRNA hybridization experiments using nodule...

  8. The effects of running exercise on oxidative capacity and PGC-1α mRNA levels in the soleus muscle of rats with metabolic syndrome.

    Science.gov (United States)

    Nagatomo, Fumiko; Fujino, Hidemi; Kondo, Hiroyo; Kouzaki, Motoki; Gu, Ning; Takeda, Isao; Tsuda, Kinsuke; Ishihara, Akihiko

    2012-03-01

    Skeletal muscles in animals with metabolic syndrome exhibit reduced oxidative capacity. We investigated the effects of running exercise on fiber characteristics, oxidative capacity, and mRNA levels in the soleus muscles of rats with metabolic syndrome [SHR/NDmcr-cp (cp/cp); CP]. We divided 5-week-old CP rats into non-exercise (CP) and exercise (CP-Ex) groups. Wistar-Kyoto rats (WKY) were used as the control group. CP-Ex rats were permitted voluntary exercise on running wheels for 10 weeks. Triglyceride levels were higher and adiponectin levels lower in the CP and CP-Ex groups than in the WKY group. However, triglyceride levels were lower and adiponectin levels higher in the CP-Ex group than in the CP group. The soleus muscles in CP-Ex rats contained only high-oxidative type I fibers, whereas those in WKY and CP rats contained type I, IIA, and IIC fibers. Muscle succinate dehydrogenase (SDH) activity was higher in the CP-Ex group than in the CP group; there was no difference in SDH activity between the WKY and CP-Ex groups. Muscle proliferator-activated receptor γ coactivator-1α (PGC-1α) mRNA levels were higher in the CP-Ex group than in the CP group; there was no difference in PGC-1α mRNA levels between the WKY and CP-Ex groups. In CP-Ex rats, longer running distance was associated with increased muscle SDH activity and PGC-1α mRNA levels. We concluded that running exercise restored decreased muscle oxidative capacity and PGC-1α mRNA levels and improved hypertriglyceridemia in rats with metabolic syndrome.

  9. Increased mRNA Levels of Sphingosine Kinases and S1P Lyase and Reduced Levels of S1P Were Observed in Hepatocellular Carcinoma in Association with Poorer Differentiation and Earlier Recurrence.

    Science.gov (United States)

    Uranbileg, Baasanjav; Ikeda, Hitoshi; Kurano, Makoto; Enooku, Kenichiro; Sato, Masaya; Saigusa, Daisuke; Aoki, Junken; Ishizawa, Takeaki; Hasegawa, Kiyoshi; Kokudo, Norihiro; Yatomi, Yutaka

    2016-01-01

    Although sphingosine 1-phosphate (S1P) has been reported to play an important role in cancer pathophysiology, little is known about S1P and hepatocellular carcinoma (HCC). To clarify the relationship between S1P and HCC, 77 patients with HCC who underwent surgical treatment were consecutively enrolled in this study. In addition, S1P and its metabolites were quantitated by LC-MS/MS. The mRNA levels of sphingosine kinases (SKs), which phosphorylate sphingosine to generate S1P, were increased in HCC tissues compared with adjacent non-HCC tissues. Higher mRNA levels of SKs in HCC were associated with poorer differentiation and microvascular invasion, whereas a higher level of SK2 mRNA was a risk factor for intra- and extra-hepatic recurrence. S1P levels, however, were unexpectedly reduced in HCC compared with non-HCC tissues, and increased mRNA levels of S1P lyase (SPL), which degrades S1P, were observed in HCC compared with non-HCC tissues. Higher SPL mRNA levels in HCC were associated with poorer differentiation. Finally, in HCC cell lines, inhibition of the expression of SKs or SPL by siRNA led to reduced proliferation, invasion and migration, whereas overexpression of SKs or SPL enhanced proliferation. In conclusion, increased SK and SPL mRNA expression along with reduced S1P levels were more commonly observed in HCC tissues compared with adjacent non-HCC tissues and were associated with poor differentiation and early recurrence. SPL as well as SKs may be therapeutic targets for HCC treatment.

  10. Green tea increases anti-inflammatory tristetraprolin and decreases pro-inflammatory tumor necrosis factor mRNA levels in rats

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    Roussel Anne M

    2007-01-01

    Full Text Available Abstract Background Tristetraprolin (TTP/ZFP36 family proteins have anti-inflammatory activity by binding to and destabilizing pro-inflammatory mRNAs such as Tnf mRNA, and represent a potential therapeutic target for inflammation-related diseases. Tea has anti-inflammatory properties but the molecular mechanisms have not been completely elucidated. We hypothesized that TTP and/or its homologues might contribute to the beneficial effects of tea as an anti-inflammatory product. Methods Quantitative real-time PCR was used to investigate the effects of green tea (0, 1, and 2 g solid extract/kg diet on the expression of Ttp family genes (Ttp/Tis11/Zfp36, Zfp36l1/Tis11b, Zfp36l2/Tis11d, Zfp36l3, pro-inflammatory genes (Tnf, Csf2/Gm-csf, Ptgs2/Cox2, and Elavl1/Hua/Hur and Vegf genes in liver and muscle of rats fed a high-fructose diet known to induce insulin resistance, oxidative stress, inflammation, and TNF-alpha levels. Results Ttp and Zfp36l1 mRNAs were the major forms in both liver and skeletal muscle. Ttp, Zfp36l1, and Zfp36l2 mRNA levels were more abundant in the liver than those in the muscle. Csf2/Gm-csf and Zfp36l3 mRNAs were undetectable in both tissues. Tea (1 g solid extract/kg diet increased Ttp mRNA levels by 50–140% but Tnf mRNA levels decreased by 30% in both tissues, and Ptgs2/Cox2 mRNA levels decreased by 40% in the muscle. Tea (2 g solid extract/kg diet increased Elavl1/Hua/Hur mRNA levels by 40% in the liver but did not affect any of the other mRNA levels in liver or muscle. Conclusion These results show that tea can modulate Ttp mRNA levels in animals and suggest that a post-transcriptional mechanism through TTP could partially account for tea's anti-inflammatory properties. The results also suggest that drinking adequate amounts of green tea may play a role in the prevention of inflammation-related diseases.

  11. Hydrostatic Pressure Regulates MicroRNA Expression Levels in Osteoarthritic Chondrocyte Cultures via the Wnt/β-Catenin Pathway

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    Sara Cheleschi

    2017-01-01

    Full Text Available Mechanical loading and hydrostatic pressure (HP regulate chondrocytes’ metabolism; however, how mechanical stimulation acts remain unclear. MicroRNAs (miRNAs play an important role in cartilage homeostasis, mechanotransduction, and in the pathogenesis of osteoarthritis (OA. This study investigated the effects of a cyclic HP (1–5 MPa, in both normal and OA human chondrocytes, on the expression of miR-27a/b, miR-140, miR-146a/b, and miR-365, and of their target genes (MMP-13, ADAMTS-5, IGFBP-5, and HDAC-4. Furthermore, we assessed the possible involvement of Wnt/β-catenin pathway in response to HP. Chondrocytes were exposed to HP for 3h and the evaluations were performed immediately after pressurization, and following 12, 24, and 48 h. Total RNA was extracted and used for real-time PCR. β-catenin was detected by Western blotting analysis and immunofluorescence. In OA chondrocytes, HP induced a significant increase (p < 0.01 of the expression levels of miR-27a/b, miR-140, and miR-146a, and a significant reduction (p < 0.01 of miR-365 at all analyzed time points. MMP-13, ADAMTS-5, and HDAC-4 were significantly downregulated following HP, while no significant modification was found for IGFBP-5. β-catenin levels were significantly increased (p < 0.001 in OA chondrocytes at basal conditions and significantly reduced (p < 0.01 by HP. Pressurization did not cause any significant modification in normal cells. In conclusion, in OA chondrocytes, HP restores the expression levels of some miRNAs, downregulates MMP-13, ADAMTS-5, and HDAC-4, and modulates the Wnt/β-catenin pathway activation.

  12. High-level mRNA quantification of proliferation marker pKi-67 is correlated with favorable prognosis in colorectal carcinoma.

    Science.gov (United States)

    Ihmann, Thomas; Liu, Jian; Schwabe, Wolfgang; Häusler, Peter; Behnke, Detlev; Bruch, Hans-Peter; Broll, Rainer; Windhövel, Ute; Duchrow, Michael

    2004-12-01

    The present study retrospectively examines the expression of pKi-67 mRNA and protein in colorectal carcinoma and their correlation to the outcome of patients. Immunohistochemistry and quantitative RT-PCR were used to analyze the expression of pKi-67 in 43 archival specimens of patients with curatively resected primary colorectal carcinoma, who were not treated with neo-adjuvant therapy. We determined a median pKi-67 (MIB-1) labeling index of 31.3% (range 10.3-66.4%), and a mean mRNA level of 0.1769 (DeltaC(T): range 0.01-0.69); indices and levels did not correlate. High pKi-67 mRNA DeltaC(T) values were associated with a significantly favorable prognosis, while pKi-67 labeling indices were not correlated to prognostic outcome. A multivariate analysis of clinical and biological factors indicated that tumor stage (UICC) and pKi-67 mRNA expression level were independent prognostic factors. Quantitatively determined pKi-67 mRNA can be a good and new prognostic indicator for primary resected colorectal carcinoma.

  13. The mRNA level of MLH1 in peripheral blood is a biomarker for the diagnosis of hereditary nonpolyposis colorectal cancer.

    Science.gov (United States)

    Yu, Hong; Li, Hui; Cui, Yongan; Xiao, Wei; Dai, Guihong; Huang, Junxing; Wang, Chaofu

    2016-01-01

    Hereditary nonpolyposis colorectal cancer (HNPCC) is caused by functional defects in mismatch repair (MMR) genes, including mutL homolog 1 (MLH1) and mutS homolog 2 (MSH2). This study aimed to assess whether the mRNA expression of MLH1 in peripheral blood could be used as a biomarkers for the diagnosis of HNPCC. The mRNA level of MLH1 was determined in 19 HNPCC families (46 members) using real-time quantitative polymerase chain reaction (qPCR). The mRNA levels of MLH1 in HNPCC were significantly lower than controls (P MLH1 for the diagnosis of HNPCC with the area under curve of 0.858. At an optimal cut-off value (0.511), the mRNA level of MLH1 had a sensitivity of 81.3% and a specificity of 86.7% for distinguishing HNPCC from controls. In conclusion, the mRNA expression of MLH1 in peripheral blood may serve as a biomarker for the diagnosis of HNPCC.

  14. Chitinase mRNA levels by quantitative PCR using the single standard DNA: acidic mammalian chitinase is a major transcript in the mouse stomach.

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    Misa Ohno

    Full Text Available Chitinases hydrolyze the β-1-4 glycosidic bonds of chitin, a major structural component of fungi, crustaceans and insects. Although mammals do not produce chitin or its synthase, they express two active chitinases, chitotriosidase (Chit1 and acidic mammalian chitinase (AMCase. These mammalian chitinases have attracted considerable attention due to their increased expression in individuals with a number of pathological conditions, including Gaucher disease, Alzheimer's disease and asthma. However, the contribution of these enzymes to the pathophysiology of these diseases remains to be determined. The quantification of the Chit1 and AMCase mRNA levels and the comparison of those levels with the levels of well-known reference genes can generate useful and biomedically relevant information. In the beginning, we established a quantitative real-time PCR system that uses standard DNA produced by ligating the cDNA fragments of the target genes. This system enabled us to quantify and compare the expression levels of the chitinases and the reference genes on the same scale. We found that AMCase mRNA is synthesized at extraordinarily high levels in the mouse stomach. The level of this mRNA in the mouse stomach was 7- to 10-fold higher than the levels of the housekeeping genes and was comparable to that the level of the mRNA for pepsinogen C (progastricsin, a major component of the gastric mucosa. Thus, AMCase mRNA is a major transcript in mouse stomach, suggesting that AMCase functions as a digestive enzyme that breaks down polymeric chitin and as part of the host defense against chitin-containing pathogens in the gastric contents. Our methodology is applicable to the quantification of mRNAs for multiple genes across multiple specimens using the same scale.

  15. Changes in angiotensin AT1 receptor mRNA levels in the rat brain after immobilization stress and inhibition of central nitric oxide synthase.

    Science.gov (United States)

    Kiss, A; Jurkovicova, D; Jezova, D; Krizanova, O

    2001-06-01

    To study functional interactions between angiotensin II AT1 receptors and nitric oxide (NO) activity in different brain areas in rats exposed to immobilization stress. Central inhibition of nitric oxide synthase (NOS) was provided by intracerebroventricular (i.c.v.) administration of (N-omega-nitro-L-arginine-methylester) L-NAME and analysis of AT1 receptor mRNA was performed using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) technique. The immobilization in prone position lasted 2 hrs and the rats were sacrificed 24 hr later. The hypothalamus, hippocampus, thalamus, and cortex were isolated from fresh brains. In the cortex, gene expression of AT1 receptors was unaffected either by L-NAME treatment, or by a single exposure to immobilization stress for 2 hours followed by 24 hours of rest. In the hippocampus, the repeated treatment with L-NAME increased mRNA levels of AT1 receptors approximately 9-times compared to those in the control (untreated) group. Immobilization also increased AT1 receptor mRNA levels in the hippocampus which was similar to that induced by the L-NAME. The increase of AT1 receptor mRNA levels in the hippocampus of immobilized rats was not further altered when the animals were pretreated with L-NAME. In control rats, exposure to immobilization resulted in a significant rise in mRNA levels coding for AT1 receptors in the hypothalamus, but not in the thalamus. L-NAME treatment showed a tendency of increase in AT1 receptor mRNA levels in the hypothalamus. Moreover, when animals treated with L-NAME were subjected to immobilization, a further increase in AT1 receptor mRNA levels was observed in the hypothalamus in comparison with corresponding controls. The present data indicate that a single immobilization stress results in increased gene expression of AT1 receptors in the hypothalamus and hippocampus. The rise in AT1 mRNA levels in the same brain structures after repeated treatment with L-NAME allow to suggest an

  16. Glypican-3 mRNA expression level in Wilms tumor: correlation with histological type, stage, and outcome.

    Science.gov (United States)

    Wari, Md Nahidul; Vallonthaiel, Archana George; Ahmed, Aijaz; Saxena, Deepali; Iyer, Venkateswaran K; Mathur, Sandeep R; Agarwala, Sandeep; Bakhshi, Sameer; Srinivas, V; Chattopadhyaya, P; Sharma, Arundhati; Gupta, S Datta; Dinda, Amit

    2017-06-01

    To correlate expression of Glypican-3 in Wilms tumor with histopathology, stage, and outcome. Glypican-3 mRNA expression by real-time PCR on tumor and normal germline samples from 75 fresh nephrectomies for Wilms tumor with fold change after normalization against GAPDH was compared. Survival analysis for event-free and overall survival (EFS, OS) with 2-year follow-up for Glypican-3 overexpression (>1.5 times) and clinicopathological parameters was performed. Glypican-3 was overexpressed in 37/75 (49.3%). It was overexpressed in 77% (10/13) cases with blastema predominance or anaplastic histology, as compared to 44% of other histologies (27/62) (p = 0.03). OS was 73 and 93%, respectively (p = 0.016), for those with and without GPC-3 overexpression. EFS was not significantly different with Glypican-3 overexpression (p = 0.11). All 5 deaths among blastema predominant tumors and 4/5 deaths among triphasic tumors had overexpressed Glypican-3. Most deaths in Stage IV, Stage III, and Stage I + II (5/7, 3/3, 1/1) had GPC-3 overexpression. On multivariate analysis, only histology and stage were found to have independent prognostic value. Glypican-3 overexpression in Wilms tumor correlates with poor OS on univariate analysis. However, only histology and stage have independent prognostic value. Glypican-3 levels may help to stratify intermediate outcome histology (triphasic) and Stage III Wilms tumors.

  17. Integration, viral charge and E2 mRNA levels in the progresion of intraepitelial cervical lesions

    Directory of Open Access Journals (Sweden)

    Esperanza Trujillo

    2018-01-01

    Full Text Available It is important to distinguish among squamous intraepithelial lesions (SIL those associated with increased risk cervical cancer. Our aim was to evaluate if the expression level of gen E2 in women with SIL and evidence of viral integration is associated to the grade of lesion. Cervical scrapes HPV16 positive from 19 women with normal histology, 45 women with low-grade SIL (LSIL and 45 women with high-grade SIL (HSIL were analyzed. Real-time PCR was used to quantify the mRNA of E2 and E2 and E6 genes to calculate viral load (E6 and the ratio E2/E6 to assess viral integration. Similar frequencies of E2 expression were found in LSIL and HSIL15/45 (33 %, the frequency in women without SIL was lower 3/19 (15.8 %, and all cases with E2 gene expression had mixed episomal and integrated viral DNA. The viral load increased significantly with the grade of SIL (p= 0.049, while E2/E6 ratio decreased (p=0.049. The ROC analysis showed low capacity of the three viral parameters analyzed to distinguish between low and high grade SIL. In conclusion although SIL with mixed and integrated viral DNA with E2 expression could be at lower risk of progression, and viral load and integration were associated with higher severity of the lesion, its clinical value as biomarkers of HSIL is limited.

  18. TR{alpha}- and TSH-mRNA levels after temporal exposition with methimazole in zebrafish, Danio rerio

    Energy Technology Data Exchange (ETDEWEB)

    Schulz, A.E.I.; Stocker, A.; Hollosi, L.; Schramm, K.W. [Inst. of Ecological Chemistry, GSF - National Research Center for Environment and Health (Germany)

    2004-09-15

    The group of dioxin and dioxin-like substances are highly persistent in the environment. There are evidences from present investigations that a variety of substances are capable of disrupting the endocrine system in the aquatic environment. These substances are called endocrine disruptors. Dioxin and related compounds can act as endocrine disruptors. Aquatic animals like amphibian and fish are especially affected of the impact of these compounds. Investigations concerned so far in particular the domain of reproduction biology and the thyroid axis especially. Recent investigations showed that the TR{alpha}-mRNA level change after a short temporal expression with T3, methimazole and amiodarone. The objective of the project is to identify effects of thyroid endocrine disruptors on the regulation of gene expression of the thyroid receptors TR{alpha}a, TR{beta} and thyroid stimulating hormone TSH and associated effects on other system. In preliminary studies the effects of the drug methimazole as model substance on gene expression of TR{alpha} and TSH were investigated. Methimazole is an inhibitor of the thyroid peroxidase so that the formation of thyroid hormones is disrupted.

  19. Regulation of the glucocorticoid receptor mRNA levels in the gills of Atlantic salmon (Salmo salar during smoltification

    Directory of Open Access Journals (Sweden)

    MAZURAIS D.

    1998-07-01

    Full Text Available The regulation of the Glucocorticoid Receptor (GR transcript was investigated in the gills of Atlantic salmon (Salmo salar during the parr-smolt transformation. Sampling of parr and smolt fish was performed between December and July and in particular during the smoltification period occurring in spring. Quantification of GR transcripts revealed differences between the two groups in March and at the beginning of April. During these dates, the amounts of GR mRNA in parr gills were respectively three and two fold lower than those measured in smolts. In order to determine which factors are responsible for these differences, we studied the long-term effects of prolactin and Cortisol treatments on GR transcript in the gills of presmolt fish. The plasma levels of these two hormones respectively drop and rise during smoltification. Contrary to Cortisol long-term treatment which did not modify the amount of gill GR transcript, short-term treatment induced a significant decrease within 12 hours. Prolactin long-term treatment caused a significant increase of GR transcript abundance after 13 days of implant treatment. This result is unexpected with regard to those obtained in the smoltification analysis but is in agreement with previous studies performed in mammary gland revealing a positive control of PRL on GR in epithelial cells. Our data suggest that the regulation of the GR transcript during the parr-smolt transformation probably involves several hormonal factors.

  20. Thyroid hormone coordinately regulates Na sup + -K sup + -ATPase. alpha. - and. beta. -subunit mRNA levels in kidney

    Energy Technology Data Exchange (ETDEWEB)

    McDonough, A.A.; Brown, T.A.; Horowitz, B.; Chiu, R.; Schlotterbeck, J.; Bowen, J.; Schmitt, C.A. (Univ. of Southern California School of Medicine, Los Angeles (USA))

    1988-02-01

    Synthesis of the sodium pump, Na{sup +}-K{sup +}-ATPase, is regulated by thyroid hormone in responsive tissues. The purpose of this study was to determine if triiodothyronine (T{sub 3}) regulates the concentration of the mRNAs coding for the two enzyme subunits, {alpha} and {beta}, and the time course of the response. A single dose of T{sub 3} was administered to hypothyroid rats that were killed at various times after injection. In the kidney cortexes of the T{sub 3}-injected animals, as well as hypothyroid and euthyroid rats, {alpha}- and {beta}-mRNA concentrations were measured by dot blot using cDNAs corresponding to the two mRNAs; {alpha}-subunit abundance was measured by Western blot using antibodies to the enzyme, and Na{sup +}-K{sup +}-ATPase activity was measured enzymatically. {alpha}- and {beta}-mRNAs increased coordinately to 1.6-fold over hypothyroid levels by 12 h after T{sub 3}. The authors conclude that T{sub 3} regulates Na{sup +}-K{sup +}-ATPase synthesis and activity by coordinately increasing the mRNAs of both the {alpha}- and {beta}-subunits of the enzyme.

  1. Viral RNA levels and env variants in semen and tissues of mature male rhesus macaques infected with SIV by penile inoculation.

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    Francis Fieni

    Full Text Available HIV is shed in semen but the anatomic site of virus entry into the genital secretions is unknown. We determined viral RNA (vRNA levels and the envelope gene sequence in the SIVmac 251 viral populations in the genital tract and semen of 5 adult male rhesus monkeys (Macaca mulatta that were infected after experimental penile SIV infection. Paired blood and semen samples were collected from 1-9 weeks after infection and the monkeys were necropsied eleven weeks after infection. The axillary lymph nodes, testes, epididymis, prostate, and seminal vesicles were collected and vRNA levels and single-genome analysis of the SIVmac251 env variants was performed. At the time of semen collection, blood vRNA levels were between 3.09 and 7.85 log10 vRNA copies/ml plasma. SIV RNA was found in the axillary lymph nodes of all five monkeys and in 3 of 5 monkeys, all tissues examined were vRNA positive. In these 3 monkeys, vRNA levels (log10 SIVgag copies/ug of total tissue RNA in the axillary lymph node (6.48 ± 0.50 were significantly higher than in the genital tract tissues: testis (3.67 ± 2.16; p<0.05, epididymis (3.08 ± 1.19; p<0.0001, prostate (3.36 ± 1.30; p<0.01, and seminal vesicle (2.67 ± 1.50; p<0.0001. Comparison of the SIVmac251 env viral populations in blood plasma, systemic lymph node, and genital tract tissues was performed in two of the macaques. Visual inspection of the Neighbor-Joining phylograms revealed that in both animals, all the sequences were generally distributed evenly among all tissue compartments. Importantly, viral populations in the genital tissues were not distinct from those in the systemic tissues. Our findings demonstrate striking similarity in the viral populations in the blood and male genital tract tissues within 3 months of penile SIV transmission.

  2. QuickRNASeq lifts large-scale RNA-seq data analyses to the next level of automation and interactive visualization.

    Science.gov (United States)

    Zhao, Shanrong; Xi, Li; Quan, Jie; Xi, Hualin; Zhang, Ying; von Schack, David; Vincent, Michael; Zhang, Baohong

    2016-01-08

    of automation and interactivity in QuickRNASeq leads to a substantial reduction in the time and effort required prior to further downstream analyses and interpretation of the analyses findings. QuickRNASeq advances primary RNA-seq data analyses to the next level of automation, and is mature for public release and adoption.

  3. Effects of growth state and 3H labeling level on RNA turnover in WI-38 fibroblasts and HeLa cells

    International Nuclear Information System (INIS)

    Sameshima, M.; Schlessinger, D.

    1981-01-01

    The rate of turnover of prelabeled RNA in WI-38 human diploid fibroblasts varied with the level of 3 H incorporated, the cell density of cultures, and the arrest of growth by senescence. The half-life of RNA in sparse cultures of growing WI-38 diploid fibroblasts depended on the level of [ 3 H]uridine incorporated; extrapolated to zero levels of incorporation, the half-life was 15 to 20 days. At any level of incorporated [ 3 H]uridine, however, RNA half-life decreased to 4 to 5 days in superconfluent cultures as the culture growth slowed. A similar shortening of half-life was observed when growth was stopped by 3 H irradiation or clonal senescence. However, the rate of turnover was not simply dependent on whether cells were growing; for example, turnover did not increase when growth was arrested by incubating cells in conditioned medium. HeLa and L cells also showed an RNA half-life of about 14 to 20 days with an increase in turnover rate of crowded cultures. However, this increase occurred at higher cell densities than with the diploid fibroblasts. Also, the growth rate and rate of RNA turnover of HeLa and L cells were much less affected by incorporated 3 H. The differential responses to confluence and 3 H label can explain the higher turnover rate of RNA in normal human fibroblasts compared to SV40-transformed cells [S.A. Liebhaber, S. Wolf, and D. Schlessinger, Cell 13, 121-127 (1978)

  4. High Intensity High Volume Interval Training Improves Endurance Performance and Induces a Nearly Complete Slow-to-Fast Fiber Transformation on the mRNA Level

    Directory of Open Access Journals (Sweden)

    Julian Eigendorf

    2018-05-01

    Full Text Available We present here a longitudinal study determining the effects of two 3 week-periods of high intensity high volume interval training (HIHVT (90 intervals of 6 s cycling at 250% maximum power, Pmax/24 s on a cycle ergometer. HIHVT was evaluated by comparing performance tests before and after the entire training (baseline, BSL, and endpoint, END and between the two training sets (intermediate, INT. The mRNA expression levels of myosin heavy chain (MHC isoforms and markers of energy metabolism were analyzed in M. vastus lateralis biopsies by quantitative real-time PCR. In incremental tests peak power (Ppeak was increased, whereas V˙O2peak was unaltered. Prolonged time-to-exhaustion was found in endurance tests with 65 and 80% Pmax at INT and END. No changes in blood levels of lipid metabolites were detected. Training-induced decreases of hematocrit indicate hypervolemia. A shift from slow MHCI/β to fast MHCIIa mRNA expression occurred after the first and second training set. The mRNA expression of peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α, a master regulator of oxidative energy metabolism, decreased after the second training set. In agreement, a significant decrease was also found for citrate synthase mRNA after the second training set, indicating reduced oxidative capacity. However, mRNA expression levels of glycolytic marker enzyme glyceraldehyde-3-phosphate dehydrogenase did not change after the first and second training set. HIHVT induced a nearly complete slow-to-fast fiber type transformation on the mRNA level, which, however, cannot account for the improvements of performance parameters. The latter might be explained by the well-known effects of hypervolemia on exercise performance.

  5. Maternal plasma levels of cell-free β-HCG mRNA as a prenatal diagnostic indicator of placenta accrete.

    Science.gov (United States)

    Zhou, J; Li, J; Yan, P; Ye, Y H; Peng, W; Wang, S; Wang, X Tong

    2014-09-01

    Several biomarkers, including maternal serum creatinine kinase and α-fetoprotein, have been described as potential tools for the diagnosis of placental abnormalities. This study aimed to determine whether maternal plasma mRNA levels of the β subunit of human chorionic gonadotropin (β-HCG) could predict placenta accreta prenatally. Sixty-eight singleton pregnant women with prior cesarean deliveries (CDs) were classified into three groups: normal placentation (35 women, control group); placenta previa alone (21 women, placenta previa group); and both placenta previa and placenta accreta (12 women, placenta previa/accreta group). Maternal plasma concentrations of cell-free β-HCG mRNA were measured by real-time reverse-transcription polymerase chain reaction and were expressed as multiples of the median (MoM). Cell-free β-HCG mRNA concentrations (MoM, range) were significantly higher in women with placenta accreta (3.65, 2.78-7.19) than in women with placenta previa (0.94, 0.00-2.97) or normal placentation (1.00, 0.00-2.69) (Steel-Dwass test, P accreta group, the concentration of cell-free β-HCG mRNA was significantly higher among women who underwent CDs with hysterectomy (4.41, 3.49-7.19) than among women whose CDs did not result in hysterectomy (3.20, 2.78-3.70) (Mann-Whitney U test, P = 0.012). An increased level of cell-free β-HCG mRNA in the maternal plasma of women with placenta accreta may arise from direct uteroplacental transfer of cell-free placental mRNA molecules. The concentration of cell-free β-HCG mRNA in maternal plasma may be applicable to the prenatal diagnosis of placenta accreta, especially to identify women with placenta accreta likely to require hysterectomy. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. Inter-individual variation, seasonal variation and close correlation of OGG1 and ERCC1 mRNA levels in full blood from healthy volunteers

    DEFF Research Database (Denmark)

    Vogel, Ulla Birgitte; Møller, Peter; Dragsted, Lars

    2002-01-01

    The mRNA levels of the nucleotide excision DNA repair gene ERCC1 and the base excision DNA repair gene OGG1 were quantified in 43 healthy volunteers in a dietary intervention trial as markers for the DNA repair capacity. Nine samples were collected from each subject over a period of 52 days. Samp...

  7. PGC-1α mRNA Level and Oxidative Capacity of the Plantaris Muscle in Rats with Metabolic Syndrome, Hypertension, and Type 2 Diabetes

    International Nuclear Information System (INIS)

    Nagatomo, Fumiko; Fujino, Hidemi; Kondo, Hiroyo; Gu, Ning; Takeda, Isao; Ishioka, Noriaki; Tsuda, Kinsuke; Ishihara, Akihiko

    2011-01-01

    We examined the fiber profiles and the mRNA levels of peroxisome proliferator-activated receptors (PPARα and PPARδ/β) and of the PPARγ coactivator-1α (PGC-1α) in the plantaris muscles of 15-week-old control (WR), metabolic syndrome (CP), hypertensive (SHR), and type 2 diabetic (GK) rats. The deep regions in the muscles of SHR and GK rats exhibited lower percentages of high-oxidative type I and IIA fibers and higher percentages of low-oxidative type IIB fibers compared with WR and CP rats. The surface regions in the muscles of CP, SHR, and GK rats exhibited lower percentages of high-oxidative type IIA fibers and higher percentages of low-oxidative type IIB fibers compared with WR rats. The muscles of SHR and GK rats had lower oxidative enzyme activity compared with WR rats. The muscles of SHR rats had the lowest PPARδ/β mRNA level. In addition, the muscles of SHR and GK rats had lower PGC-1α mRNA level compared with WR and CP rats. We concluded that the plantaris muscles of rats with hypertension and type 2 diabetes have lower oxidative capacity, which is associated with the decreased level of PGC-1α mRNA

  8. Glycogen synthase and phosphofructokinase protein and mRNA levels in skeletal muscle from insulin-resistant patients with non-insulin-dependent diabetes mellitus

    DEFF Research Database (Denmark)

    Vestergaard, H; Lund, S; Larsen, F S

    1993-01-01

    In patients with non-insulin-dependent diabetes mellitus (NIDDM) and matched control subjects we examined the interrelationships between in vivo nonoxidative glucose metabolism and glucose oxidation and the muscle activities, as well as the immunoreactive protein and mRNA levels of the rate-limit...

  9. Systematic and quantitative mRNA expression analysis of TRP channel genes at the single trigeminal and dorsal root ganglion level in mouse

    Directory of Open Access Journals (Sweden)

    Vandewauw Ine

    2013-02-01

    Full Text Available Abstract Background Somatosensory nerve fibres arising from cell bodies within the trigeminal ganglia (TG in the head and from a string of dorsal root ganglia (DRG located lateral to the spinal cord convey endogenous and environmental stimuli to the central nervous system. Although several members of the transient receptor potential (TRP superfamily of cation channels have been implicated in somatosensation, the expression levels of TRP channel genes in the individual sensory ganglia have never been systematically studied. Results Here, we used quantitative real-time PCR to analyse and compare mRNA expression of all TRP channels in TG and individual DRGs from 27 anatomically defined segments of the spinal cord of the mouse. At the mRNA level, 17 of the 28 TRP channel genes, TRPA1, TRPC1, TRPC3, TRPC4, TRPC5, TRPM2, TRPM3, TRPM4, TRPM5, TRPM6, TRPM7, TRPM8, TRPV1, TRPV2, TRPV4, TRPML1 and TRPP2, were detectable in every tested ganglion. Notably, four TRP channels, TRPC4, TRPM4, TRPM8 and TRPV1, showed statistically significant variation in mRNA levels between DRGs from different segments, suggesting ganglion-specific regulation of TRP channel gene expression. These ganglion-to-ganglion differences in TRP channel transcript levels may contribute to the variability in sensory responses in functional studies. Conclusions We developed, compared and refined techniques to quantitatively analyse the relative mRNA expression of all TRP channel genes at the single ganglion level. This study also provides for the first time a comparative mRNA distribution profile in TG and DRG along the entire vertebral column for the mammalian TRP channel family.

  10. Racial Differences in Expression Levels of miRNA Machinery-Related Genes, Dicer, Drosha, DGCR8, and AGO2, in Asian Korean Papillary Thyroid Carcinoma and Comparative Validation Using the Cancer Genome Atlas

    Directory of Open Access Journals (Sweden)

    Jaegil Kim

    2017-01-01

    Full Text Available Aberrant regulation of microRNA (miRNA machinery components is associated with various human cancers, including papillary thyroid carcinoma (PTC, which is the most common type of thyroid cancer, and a higher prevalent female malignancy. The purpose of this study is to investigate racial differences in mRNA expression levels of four miRNA machinery components, Dicer, Drosha, DGCR8, and AGO2, and their correlations with clinicopathological characteristics. Forty PTC samples from female Asian Korean PTC patients were enrolled. Using qPCR, we examined mRNA expression levels of the components and next validated our results by comparison with results of female white American in the TCGA PTC project. Interestingly, mRNA expression levels of the selected factors were altered in the TCGA PTC samples. However, only Drosha showed a significantly lower expression level in Asian Korean PTC samples. Furthermore, the mRNA expression levels of the four components showed no association with clinicopathological characteristics in both groups. On the other hand, positive correlations were observed between altered mRNA expression levels of Dicer and Drosha and DGCR8 and Drosha in TCGA PTC samples. These findings collectively revealed that altered mRNA expression levels of miRNA machinery components might be responsible for racial differences in the carcinogenesis of PTC.

  11. Analysis of MDM2 and MDM4 single nucleotide polymorphisms, mRNA splicing and protein expression in retinoblastoma.

    Directory of Open Access Journals (Sweden)

    Justina McEvoy

    Full Text Available Retinoblastoma is a childhood cancer of the developing retina that begins in utero and is diagnosed in the first years of life. Biallelic RB1 gene inactivation is the initiating genetic lesion in retinoblastoma. The p53 gene is intact in human retinoblastoma but the pathway is believed to be suppressed by increased expression of MDM4 (MDMX and MDM2. Here we quantify the expression of MDM4 and MDM2 mRNA and protein in human fetal retinae, primary retinoblastomas, retinoblastoma cell lines and several independent orthotopic retinoblastoma xenografts. We found that MDM4 is the major p53 antagonist expressed in retinoblastoma and in the developing human retina. We also discovered that MDM4 protein steady state levels are much higher in retinoblastoma than in human fetal retinae. This increase would not have been predicted based on the mRNA levels. We explored several possible post-transcriptional mechanisms that may contribute to the elevated levels of MDM4 protein. A proportion of MDM4 transcripts are alternatively spliced to produce protein products that are reported to be more stable and oncogenic. We also discovered that a microRNA predicted to target MDM4 (miR191 was downregulated in retinoblastoma relative to human fetal retinae and a subset of samples had somatic mutations that eliminated the miR-191 binding site in the MDM4 mRNA. Taken together, these data suggest that post-transcriptional mechanisms may contribute to stabilization of the MDM4 protein in retinoblastoma.

  12. Androgen receptor-beta mRNA levels in different tissues in breeding and post-breeding male and female sticklebacks, Gasterosteus aculeatus

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    Hoffmann Erik

    2012-03-01

    Full Text Available Abstract Background Androgens induce male characters by activating androgen receptors (AR. Previous quantitative studies on AR in fishes have been limited to few tissues and/or a single season/reproductive state. The aim of this investigation was to study the possible role of AR-beta expression levels in the control of male traits in the three-spined stickleback. To that end, AR-beta expression levels in major tissues in breeding and post-breeding male and female sticklebacks were examined. Methods AR-beta mRNA levels were quantified in ten tissues; eye, liver, axial muscle, heart, brain, intestine, ovary, testis, kidney and pectoral muscle in six breeding and post-breeding males and females using reverse transcription quantitative PCR. Results Breeding in contrast to post-breeding males built nests and showed secondary sexual characters (e.g. kidney hypertrophy and elevated androgen levels. Post-breeding females had lower ovarian weights and testosterone levels than breeding females. AR-beta was expressed in all studied tissues in both sexes and reproductive states with the highest expression in the gonads and in the kidneys. The kidney is an androgen target organ in sticklebacks, from which breeding males produce the protein spiggin, which is used in nest-building. There was also high AR-beta expression in the intestine, an organ that appears to take over hyperosmo-regulation in fresh water when the kidney hypertrophies in mature males and largely loses this function. The only tissue that showed effects of sex or reproductive state on AR-beta mRNA levels was the kidneys, where post-breeding males displayed higher AR-beta mRNA levels than breeding males. Conclusion The results indicate that changes in AR-beta mRNA levels play no or little role in changes in androgen dependent traits in the male stickleback.

  13. Low levels of the AhR in chronic obstructive pulmonary disease (COPD)-derived lung cells increases COX-2 protein by altering mRNA stability.

    Science.gov (United States)

    Zago, Michela; Sheridan, Jared A; Traboulsi, Hussein; Hecht, Emelia; Zhang, Yelu; Guerrina, Necola; Matthews, Jason; Nair, Parameswaran; Eidelman, David H; Hamid, Qutayba; Baglole, Carolyn J

    2017-01-01

    Heightened inflammation, including expression of COX-2, is associated with chronic obstructive pulmonary disease (COPD) pathogenesis. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is reduced in COPD-derived lung fibroblasts. The AhR also suppresses COX-2 in response to cigarette smoke, the main risk factor for COPD, by destabilizing the Cox-2 transcript by mechanisms that may involve the regulation of microRNA (miRNA). Whether reduced AhR expression is responsible for heightened COX-2 in COPD is not known. Here, we investigated the expression of COX-2 as well as the expression of miR-146a, a miRNA known to regulate COX-2 levels, in primary lung fibroblasts derived from non-smokers (Normal) and smokers (At Risk) with and without COPD. To confirm the involvement of the AhR, AhR knock-down via siRNA in Normal lung fibroblasts and MLE-12 cells was employed as were A549-AhRko cells. Basal expression of COX-2 protein was higher in COPD lung fibroblasts compared to Normal or Smoker fibroblasts but there was no difference in Cox-2 mRNA. Knockdown of AhR in lung structural cells increased COX-2 protein by stabilizing the Cox-2 transcript. There was less induction of miR-146a in COPD-derived lung fibroblasts but this was not due to the AhR. Instead, we found that RelB, an NF-κB protein, was required for transcriptional induction of both Cox-2 and miR-146a. Therefore, we conclude that the AhR controls COX-2 protein via mRNA stability by a mechanism independent of miR-146a. Low levels of the AhR may therefore contribute to the heightened inflammation common in COPD patients.

  14. Fibroblast growth factor-21 and omentin-1 hepatic mRNA expression and serum levels in morbidly obese women with non-alcoholic fatty liver disease.

    Science.gov (United States)

    Waluga, M; Kukla, M; Zorniak, M; Kajor, M; Liszka, L; Dyaczynski, M; Kowalski, G; Zadlo, D; Waluga, E; Olczyk, P; Buldak, R J; Berdowska, A; Hartleb, M

    2017-06-01

    Fibroblast growth factor-21 (FGF21) and omentin-1 have been recognized as potent antidiabetic agents with potential hepatoprotective activity. The aim of this study was to evaluate hepatic FGF21 and omentin-1 mRNA expression as well as their serum levels as predictive markers of liver injury and insulin resistance in morbidly obese women with non-alcoholic fatty liver disease (NAFLD). This study included 56 severely obese women who underwent intraoperative wedge liver biopsy during the bariatric surgery. Hepatic FGF21 and omentin-1 mRNA were assessed by quantitative real-time PCR, while their serum concentrations were measured with commercially available enzyme-linked immunosorbent assays. The FGF21 serum level was significantly higher in patients with a greater extent of steatosis (grade 2 and 3) compared to those without or with mild steatosis (grade 0 and 1) (P = 0.049). Receiver Operating Characteristic analysis, however, showed poor discriminant power for the FGF21 serum levels in differentiating between more and less extensive steatosis with an AUC = 0.666. There was a tendency towards higher levels of hepatic FGF21 mRNA in patients with lobular inflammation and fibrosis and towards lower levels in the case of hepatocyte ballooning and steatosis. There was a positive mutual correlation between hepatic FGF21 and omentin-1 mRNA levels (r = 0.78; P hepatic omentin-1 mRNA levels showed a tendency to be lower in patients with advanced steatosis and hepatocyte ballooning. In conclusion, our study, which focused on hepatic FGF21 and omentin-1 mRNA expression, confirmed marked expression of both molecules in the liver of morbidly obese patients with NAFLD. More extensive steatosis was associated with evident changes in the serum FGF21 concentration in morbidly obese women with NAFLD, but the difference did not reach statistical significance. The vast amount of fat, both visceral and subcutaneous, in severely obese patients may be the additional source and influence

  15. PAI-1 mRNA expression and plasma level in rheumatoid arthritis: relationship with 4G/5G PAI-1 polymorphism.

    Science.gov (United States)

    Muñoz-Valle, José Francisco; Ruiz-Quezada, Sandra Luz; Oregón-Romero, Edith; Navarro-Hernández, Rosa Elena; Castañeda-Saucedo, Eduardo; De la Cruz-Mosso, Ulises; Illades-Aguiar, Berenice; Leyva-Vázquez, Marco Antonio; Castro-Alarcón, Natividad; Parra-Rojas, Isela

    2012-12-01

    Rheumatoid arthritis (RA) is a chronic inflammatory disease affecting the synovial membrane, cartilage and bone. PAI-1 is a key regulator of the fibrinolytic system through which plasminogen is converted to plasmin. The plasmin activates the matrix metalloproteinase system, which is closely related with the joint damage and bone destruction in RA. The aim of this study was to investigate the relationship between 4G/5G PAI-1 polymorphism with mRNA expression and PAI-1 plasma protein levels in RA patients. 113 RA patients and 123 healthy subjects (HS) were included in the study. The 4G/5G PAI-1 polymorphism was determined by polymerase chain reaction-restriction fragment length polymorphism method; the PAI-1 mRNA expression was determined by real-time PCR; and the soluble PAI-1 (sPAI-1) levels were quantified using an ELISA kit. No significant differences in the genotype and allele frequencies of 4G/5G PAI-1 polymorphism were found between RA patients and HS. However, the 5G/5G genotype was the most frequent in both studied groups: RA (42%) and HS (44%). PAI-1 mRNA expression was slightly increased (0.67 fold) in RA patients with respect to HS (P = 0.0001). In addition, in RA patients, the 4G/4G genotype carriers showed increased PAI-1 mRNA expression (3.82 fold) versus 4G/5G and 5G/5G genotypes (P = 0.0001), whereas the sPAI-1 plasma levels did not show significant differences. Our results indicate that the 4G/5G PAI-1 polymorphism is not a marker of susceptibility in the Western Mexico. However, the 4G/4G genotype is associated with high PAI-1 mRNA expression but not with the sPAI-1 levels in RA patients.

  16. Correlation of Cyfra 21-1 levels in saliva and serum with CK19 mRNA expression in oral squamous cell carcinoma.

    Science.gov (United States)

    Malhotra, Rewa; Urs, Aadithya B; Chakravarti, Anita; Kumar, Suman; Gupta, V K; Mahajan, Bhawna

    2016-07-01

    Oral squamous cell carcinoma (OSCC) accounts for 90 % of malignant lesions of oral cavity. The study assessed the potential of Cyfra 21-1 as a tumor marker in OSCC. The study included 50 patients of OSCC to evaluate levels of Cyfra 21-1 in serum and saliva by electrochemiluminescent immunoassay (ECLIA) and CK19 messenger RNA (mRNA) expression in tissue by florescent quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR) along with healthy individuals as control. The salivary and serum Cyfra 21-1 levels in patients of OSCC were significantly higher compared to controls (p value < 0.01). There was a 2.75-fold increase in CK19 mRNA expression in OSCC cases compared to controls. A significant positive correlation was found between serum and salivary Cyfra 21-1, serum Cyfra 21-1, and CK19 mRNA expression and between salivary Cyfra 21-1 and CK19 mRNA expression. Among these, correlation between serum and salivary Cyfra 21-1 was highly significant. Salivary and serum Cyfra 21-1 showed significantly elevated levels in grade II OSCC compared to grade I histopathologically. Elevated levels of salivary Cyfra 21-1 were associated with recurrence in OSCC patients. Reverse operating curve constructed using 3 ng/ml as a cutoff for serum Cyfra 21-1 revealed the sensitivity and specificity to be 88 and 78.2 %, respectively. Using a cutoff value of 8.5 ng/ml for salivary Cyfra 21-1, the sensitivity was found to be 93.8 % and specificity 84.3 %. We advocate salivary Cyfra 21-1 as a better diagnostic marker over serum Cyfra 21-1 as well as a potential marker in the prognosis of OSCC.

  17. Thyroid hormone deiodinase type 2 mRNA levels in sea lamprey (Petromyzon marinus) are regulated during metamorphosis and in response to a thyroid challenge.

    Science.gov (United States)

    Stilborn, S Salina M; Manzon, Lori A; Schauenberg, Jennifer D; Manzon, Richard G

    2013-03-01

    Thyroid hormones (THs) are crucial for normal vertebrate development and are the one obligate morphogen that drives amphibian metamorphosis. However, contrary to other metamorphosing vertebrates, lampreys exhibit a sharp drop in serum TH early in metamorphosis, and anti-thyroid agents such as potassium perchlorate (KClO(4)) induce metamorphosis. The type 2 deiodinase (D2) enzyme is a key regulator of TH availability during amphibian metamorphosis. We set out to determine how D2 may be involved in the regulation of lamprey metamorphosis and thyroid homeostasis. We cloned a 1.8Kb Petromyzon marinus D2 cDNA that includes the entire protein coding region and a selenocysteine (Sec) codon. Northern blotting indicated that the lamprey D2 mRNA is the longest reported to date (>9Kb). Using real-time PCR, we showed that intestinal and hepatic D2 mRNA levels were elevated prior to and during the early stages of metamorphosis and then declined dramatically to low levels that were sustained for the remainder of metamorphosis. These data are consistent with previously reported changes in serum TH levels and deiodinase activity. Treatment of larvae with either TH or KClO(4) significantly affected D2 mRNA levels in the intestine and liver. These D2 mRNA levels during metamorphosis and in response to thyroid challenges suggest that D2 may function in the regulation of TH levels during lamprey metamorphosis and the maintenance of TH homeostasis. Copyright © 2013 Elsevier Inc. All rights reserved.

  18. Intake of branched-chain amino acids influences the levels of MAFbx mRNA and MuRF-1 total protein in resting and exercising human muscle.

    Science.gov (United States)

    Borgenvik, Marcus; Apró, William; Blomstrand, Eva

    2012-03-01

    Resistance exercise and amino acids are two major factors that influence muscle protein turnover. Here, we examined the effects of resistance exercise and branched-chain amino acids (BCAA), individually and in combination, on the expression of anabolic and catabolic genes in human skeletal muscle. Seven subjects performed two sessions of unilateral leg press exercise with randomized supplementation with BCAA or flavored water. Biopsies were collected from the vastus lateralis muscle of both the resting and exercising legs before and repeatedly after exercise to determine levels of mRNA, protein phosphorylation, and amino acid concentrations. Intake of BCAA reduced (P exercising legs, respectively. The level of MuRF-1 mRNA was elevated (P exercising leg two- and threefold under the placebo and BCAA conditions, respectively, whereas MuRF-1 total protein increased by 20% (P exercising muscle. In conclusion, BCAA ingestion reduced MAFbx mRNA and prevented the exercise-induced increase in MuRF-1 total protein in both resting and exercising leg. Further-more, resistance exercise differently influenced MAFbx and MuRF-1 mRNA expression, suggesting both common and divergent regulation of these two ubiquitin ligases.

  19. Photobiomodulation on Bax and Bcl-2 Proteins and SIRT1/PGC-1α Axis mRNA Expression Levels of Aging Rat Skeletal Muscle

    Directory of Open Access Journals (Sweden)

    Fang-Hui Li

    2014-01-01

    Full Text Available Objective. This study aimed to analyze the effects of low level laser irradiation (LLLI on Bax and IGF-1 and Bcl-2 protein contents and SIRT1/PGC-1α axis mRNA expression levels to prevent sarcopenia in aged rats. Material and Methods. Twenty female Sprague Dawley rats (18 months old were randomly divided into two groups (n=10 per group: control (CON and LLLI groups. The gallium-aluminum-arsenium (GaAlAs laser irradiation at 810 nm was used in the single point contact mode (3.75 J/cm2; 0.4 cm2; 125 mW/cm2; 30 s. Bax, Bcl-2, and IGF-1 proteins and SIRT1/PGC-1α axis mRNA expression were assessed 24 h after LLLI on gastrocnemius in aged rat. Results. Gastrocnemius muscle weights, gastrocnemius mass/body mass, Bcl-2/BAX ratio, Bcl-2 protein, IGF-1 protein, and the mRNA contents in SIRT1, PGC-1α, NRF1, TMF, and SOD2 were significantly (P<0.05 increased by LLLI compared to CON group without LLLI. However, levels of BAX protein and caspase 3 mRNA were significantly attenuated by LLLI compared to CON group (P<0.05. Conclusion. LLLI at 810 nm inhibits sarcopenia associated with upregulation of Bcl-2/BAX ratio and IGF-1 and SIRT1/PGC-1α axis mRNA expression in aged rats. This indicates that LLLI has potential to decrease progression of myocyte apoptosis in sarcopenic muscles.

  20. Efficacy of Omega Fatty Acid Supplementation on mRNA Expression Level of Tumor Necrosis Factor Alpha in Patients with Gastric Adenocarcinoma.

    Science.gov (United States)

    Hosseinzadeh, Asghar; Ardebili, Seyed Mojtaba Mohaddes

    2016-09-01

    Tumor necrosis factor alpha (TNF-α), a multifunctional cytokine, is involved in apoptosis, cell proliferation, cell survival, and inflammation. It plays a dual role in cancer development and progression. It has been revealed that polyunsaturated fatty acids (PUFAs) modulate the production and activity of TNF family cytokines. The objective of the present study was to evaluate the effect of PUFAs on messenger RNA expression levels of TNF-α in patients with gastric adenocarcinoma. Thirty-four chemotherapy-naive patients diagnosed with gastric adenocarcinoma were randomly divided into two groups. The first group (17 individuals) received cisplatin without supplements and the second group (17 individuals) received cisplatin plus orally administered PUFA supplements for 3 weeks, based on treatment strategies. The gastric biopsy samples were obtained from all participants before and after treatment, and TNF-α mRNA expression levels were evaluated by quantitative real-time PCR procedure. Our findings revealed that TNF-α mRNA expression is downregulated in group II, after receiving cisplatin and omega fatty acid supplement for 3 weeks. However, this difference is not statistically significant (p > 0.05). TNF-α mRNA expression did not show significant alteration in group I, after receiving cisplatin alone. Taken together, we concluded that omega fatty acids reduce TNF-α expression at the mRNA level in patients with gastric adenocarcinoma. These data suggest that TNF-α may act as a potential target for the therapy of human gastric adenocarcinoma.

  1. Gene expression change in human dental pulp cells exposed to a low-level toxic concentration of triethylene glycol dimethacrylate: an RNA-seq analysis.

    Science.gov (United States)

    Cho, Sung-Geun; Lee, Jin-Woo; Heo, Jung Sun; Kim, Sun-Young

    2014-09-01

    Dental composite resin restoration for defective tooth may lead unpolymerized resin monomers to be leached into dental pulp tissue. The aim of this study was to investigate the early gene expression change over time of human dental pulp cells (HDPCs) treated with a low-level toxic concentration of Triethylene Glycol Dimethacrylate (TEGDMA), a common dental resin monomer, by adopting the novel high-throughput transcriptome analysis of RNA-seq. The low-level toxic concentration of TEGDMA was determined through MTT assays with serially diluted concentrations. After the HDPCs were exposed to TEGDMA for 6, 12, 24 or 48 hr, the total RNA of the samples was prepared for RNA-seq. qRT-PCR for several genes was performed for validation of RNA-seq results. In the treated group, 1280 genes were differentially expressed compared with the control group. Five patterns of time-series gene expression profiles were identified through k-means clustering analysis. Angiogenesis, cell adhesion and migration, extracellular matrix organization, response to extracellular stimulus, inflammatory response and mineralization-related process were major gene ontology terms in functional annotation clustering. HMOX1, OSGIN1, SMN2, SRXN1 AKR1C1, SPP1 and TOMM40L were highly up-regulated genes, and WRAP53 and CCL2 were highly down-regulated genes over time. qRT-PCR for several genes exhibited a high level of agreement with RNA-seq. TEGDMA induced the HDPCs to show massive and dynamic gene expression changes over time. The previously suggested toxic mechanism of TEGDMA was not only verified, but new genes whose functions have yet to be determined were also found. © 2014 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).

  2. Association of Cocaine- and Amphetamine-Regulated Transcript (CART) Messenger RNA Level, Food Intake, and Growth in Channel Catfish

    Science.gov (United States)

    Cocaine-and Amphetamine-Regulated Transcript (CART) is a potent hypothalamic anorectic peptide in mammals and fish. We hypothesized that increased food intake is associated with changes in expression of CART mRNA within the brain of channel catfish. Objectives were to clone the CART gene, examine ...

  3. The relative prognostic value of plasma HIV RNA levels and CD4 lymphocyte counts in advanced HIV infection

    DEFF Research Database (Denmark)

    Cozzi-Lepri, A; Katzenstein, T L; Ullum, H

    1998-01-01

    . RESULTS: The plasma HIV RNA (median 101410 copies/ml; range (range 200-7200000) and the CD4 lymphocyte count (median 250 cells x 10(6)/l; range 1-1247) were negatively correlated (Pearson r = -0.53; P

  4. Evaluation of IL-12RB1, IL-12B, CXCR-3 and IL-17a expression in cases affected by a non-healing form of cutaneous leishmaniasis: an observational study design.

    Science.gov (United States)

    Moafi, Mohammad; Rezvan, Hossein; Sherkat, Roya; Taleban, Roya; Asilian, Ali; Zarkesh Esfahani, Seyed Hamid; Nilforoushzadeh, Mohammad Ali; Jaffary, Fariba; Feizi, Awat

    2017-01-27

    Seldom cutaneous leishmaniasis (CL) may present as a lasting and active lesion(s), known as a non-healing form of CL (NHCL). Non-functional type 1 T helper (Th1) cells are assumed the most important factor in the outcome of the disease. The present study aims to assess some molecular defects that potentially contribute to Th1 impairment in NHCL. This prospective observational study will be implemented among five groups. The first and second groups comprise patients afflicted with non-healing and healing forms of CL, respectively. The third group consists of those recovered participants who have scars as a result of CL. Those participants who have never lived or travelled to endemic areas of leishmaniasis will comprise the fourth group. The fifth group comprises participants living in hyperendemic areas for leishmaniasis, although none of them have been afflicted by CL. The aim is to recruit 10 NHCL cases and 30 participants in each of the other groups. A leishmanin skin test (LST) will be performed to assess in vivo immunity against the Leishmania infection. The cytokine profile (interleukin (IL)-12p70, interferon (IFN)-γ, C-X-C motif chemokine ligand (CXCL)-11 and IL-17a) of the isolated peripheral blood mononuclear cells (PBMCs) will be evaluated through ELISA. Real-time PCR will determine the C-X-C motif chemokine receptor (CXCR)-3 and IL-17a gene expression and expression of IL-12Rβ1 will be assessed by flow cytometry. Furthermore, IL-12B and IL-12RB1 mutation analysis will be performed. It is anticipated that the outcome of the current study will identify IL-12B and IL-12RB1 mutations, which lead to persistent lesions of CL. Furthermore, our expected results will reveal an association between NHCL and pro-inflammatory cytokines (IL-12p70, IFN-γ IL-17a and CXCL-11), as well as CXCR-3 expression. This study has been approved by a local ethical committee. The final results will be disseminated through peer-reviewed journals and scientific conferences

  5. Adipocyte resistin mRNA levels are down-regulated by laparoscopic ovarian electrocautery in both obese and lean women with polycystic ovary syndrome.

    Science.gov (United States)

    Seow, Kok-Min; Juan, Chi-Chang; Ho, Low-Tone; Hsu, Yung-Pei; Lin, Yu-Hung; Huang, Lee-Wen; Hwang, Jiann-Loung

    2007-04-01

    The aim of this study was to investigate serum and adipocyte mRNA expression of resistin in lean and obese women with polycystic ovary syndrome (PCOS) before and 3 months after laparoscopic ovarian electrocauterization (LOE). Adipose tissue obtained from 12 women with PCOS (six obese and six lean, body mass index > 27 kg m(-1) as threshold point) before and after LOE was analysed. Gene expression of resistin was measured by semi-quantitative RT-PCR. Ten lean, age-matched healthy women served as controls. Both lean and obese women with PCOS had significantly higher fasting and 2 h insulin and homeostasis model insulin resistance index (HOMA(IR)) values and lower fasting glucose-to-insulin ratios (G(0)/I(0)) than did the controls. The serum levels of glucose and insulin and HOMA(IR) were significantly decreased, and the G(0)/I(0) ratio was significantly increased 3 months after LOE. No difference was found in serum resistin levels between controls and either obese or lean women with PCOS before LOE, nor between PCOS patients before and after LOE. However, resistin mRNA expression levels in both lean and obese women with PCOS before LOE were significantly higher than that in controls and were decreased significantly after LOE back to control levels. Local resistin activity may be actively involved in the pathogenesis of PCOS. LOE reduces insulin resistance and down-regulates resistin mRNA expression in lean and obese women with PCOS.

  6. Chitinase mRNA Levels Determined by QPCR in Crab-Eating Monkey (Macaca fascicularis) Tissues: Species-Specific Expression of Acidic Mammalian Chitinase and Chitotriosidase.

    Science.gov (United States)

    Uehara, Maiko; Tabata, Eri; Ishii, Kazuhiro; Sawa, Akira; Ohno, Misa; Sakaguchi, Masayoshi; Matoska, Vaclav; Bauer, Peter O; Oyama, Fumitaka

    2018-05-09

    Mice and humans express two active chitinases: acidic mammalian chitinase (AMCase) and chitotriosidase (CHIT1). Both chitinases are thought to play important roles in specific pathophysiological conditions. The crab-eating monkey ( Macaca fascicularis ) is one of the most frequently used nonhuman primate models in basic and applied biomedical research. Here, we performed gene expression analysis of two chitinases in normal crab-eating monkey tissues by way of quantitative real-time polymerase chain reaction (qPCR) using a single standard DNA molecule. Levels of AMCase and CHIT1 messenger RNAs (mRNAs) were highest in the stomach and the lung, respectively, when compared to other tissues. Comparative gene expression analysis of mouse, monkey, and human using monkey⁻mouse⁻human hybrid standard DNA showed that the AMCase mRNA levels were exceptionally high in mouse and monkey stomachs while very low in the human stomach. As for the CHIT1 mRNA, we detected higher levels in the monkey lung when compared with those of mouse and human. The differences of mRNA expression between the species in the stomach tissues were basically reflecting the levels of the chitinolytic activities. These results indicate that gene expression of AMCase and CHIT1 differs between mammalian species and requiring special attention in handling data in chitinase-related studies in particular organisms.

  7. Foot-and-mouth disease virus leader proteinase inhibits dsRNA-induced type I interferon transcription by decreasing interferon regulatory factor 3/7 in protein levels

    International Nuclear Information System (INIS)

    Wang, Dang; Fang, Liurong; Luo, Rui; Ye, Rui; Fang, Ying; Xie, Lilan; Chen, Huanchun; Xiao, Shaobo

    2010-01-01

    Research highlights: → FMDV L pro inhibits poly(I:C)-induced IFN-α1/β mRNA expression. → L pro inhibits MDA5-mediated activation of the IFN-α1/β promoter. → L pro significantly reduced the transcription of multiple IRF-responsive genes. → L pro inhibits IFN-α1/β promoter activation by decreasing IRF-3/7 in protein levels. → The ability to process eIF-4G of L pro is not necessary to inhibit IFN-α1/β activation. -- Abstract: The leader proteinase (L pro ) of foot-and-mouth disease virus (FMDV) has been identified as an interferon-β (IFN-β) antagonist that disrupts the integrity of transcription factor nuclear factor κB (NF-κB). In this study, we showed that the reduction of double stranded RNA (dsRNA)-induced IFN-α1/β expression caused by L pro was also associated with a decrease of interferon regulatory factor 3/7 (IRF-3/7) in protein levels, two critical transcription factors for activation of IFN-α/β. Furthermore, overexpression of L pro significantly reduced the transcription of multiple IRF-responsive genes including 2',5'-OAS, ISG54, IP-10, and RANTES. Screening L pro mutants indicated that the ability to process eIF-4G of L pro is not required for suppressing dsRNA-induced activation of the IFN-α1/β promoter and decreasing IRF-3/7 expression. Taken together, our results demonstrate that, in addition to disrupting NF-κB, L pro also decreases IRF-3/7 expression to suppress dsRNA-induced type I IFN production, suggesting multiple strategies used by FMDV to counteract the immune response to viral infection.

  8. Foot-and-mouth disease virus leader proteinase inhibits dsRNA-induced type I interferon transcription by decreasing interferon regulatory factor 3/7 in protein levels

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Dang; Fang, Liurong; Luo, Rui; Ye, Rui; Fang, Ying; Xie, Lilan; Chen, Huanchun [Division of Animal Infectious Diseases, State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070 (China); Xiao, Shaobo, E-mail: shaoboxiao@yahoo.com [Division of Animal Infectious Diseases, State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070 (China)

    2010-08-13

    Research highlights: {yields} FMDV L{sup pro} inhibits poly(I:C)-induced IFN-{alpha}1/{beta} mRNA expression. {yields} L{sup pro} inhibits MDA5-mediated activation of the IFN-{alpha}1/{beta} promoter. {yields} L{sup pro} significantly reduced the transcription of multiple IRF-responsive genes. {yields} L{sup pro} inhibits IFN-{alpha}1/{beta} promoter activation by decreasing IRF-3/7 in protein levels. {yields} The ability to process eIF-4G of L{sup pro} is not necessary to inhibit IFN-{alpha}1/{beta} activation. -- Abstract: The leader proteinase (L{sup pro}) of foot-and-mouth disease virus (FMDV) has been identified as an interferon-{beta} (IFN-{beta}) antagonist that disrupts the integrity of transcription factor nuclear factor {kappa}B (NF-{kappa}B). In this study, we showed that the reduction of double stranded RNA (dsRNA)-induced IFN-{alpha}1/{beta} expression caused by L{sup pro} was also associated with a decrease of interferon regulatory factor 3/7 (IRF-3/7) in protein levels, two critical transcription factors for activation of IFN-{alpha}/{beta}. Furthermore, overexpression of L{sup pro} significantly reduced the transcription of multiple IRF-responsive genes including 2',5'-OAS, ISG54, IP-10, and RANTES. Screening L{sup pro} mutants indicated that the ability to process eIF-4G of L{sup pro} is not required for suppressing dsRNA-induced activation of the IFN-{alpha}1/{beta} promoter and decreasing IRF-3/7 expression. Taken together, our results demonstrate that, in addition to disrupting NF-{kappa}B, L{sup pro} also decreases IRF-3/7 expression to suppress dsRNA-induced type I IFN production, suggesting multiple strategies used by FMDV to counteract the immune response to viral infection.

  9. Let-7 microRNA and HMGA2 levels of expression are not inversely linked in adipocytic tumors: analysis of 56 lipomas and liposarcomas with molecular cytogenetic data.

    Science.gov (United States)

    Bianchini, Laurence; Saâda, Esma; Gjernes, Elisabet; Marty, Marion; Haudebourg, Juliette; Birtwisle-Peyrottes, Isabelle; Keslair, Frédérique; Chignon-Sicard, Bérangère; Chamorey, Emmanuel; Pedeutour, Florence

    2011-06-01

    The aim of our study was first to assess the role of HMGA2 expression in the pathogenesis of adipocytic tumors (AT) and, second, to seek a potential correlation between overexpression of HMGA2 and let-7 expression inhibition by analyzing a series of 56 benign and malignant AT with molecular cytogenetic data. We measured the levels of expression of HMGA2 mRNA and of eight members of the let-7 microRNA family using quantitative RT-PCR and expression of HMGA2 protein using immunohistochemistry. HMGA2 was highly overexpressed in 100% of well-differentiated/dedifferentiated liposarcomas (WDLPS/DDLPS), all with HMGA2 amplification, and 100% of lipomas with HMGA2 rearrangement. Overexpression of HMGA2 mRNA was detected in 76% of lipomas without HMGA2 rearrangement. HMGA2 protein expression was detected in 100% of lipomas with HMGA2 rearrangement and 48% of lipomas without HMGA2 rearrangement. We detected decreased expression levels of some let-7 members in a significant proportion of AT. Notably, let-7b and let-7g were inhibited in 61% of WDLPS/DDLPS. In lipomas, each type of let-7 was inhibited in approximately one-third of the cases. Although overexpression of both HMGA2 mRNA and protein in a majority of ordinary lipomas without HMGA2 structural rearrangement may have suggested a potential role for let-7 microRNAs, we did not observe a significant link with let-7 inhibition in such cases. Our results indicate that inhibition of let-7 microRNA expression may participate in the deregulation of HMGA2 in AT but that this inhibition is neither a prominent stimulator for HMGA2 overexpression nor a surrogate to genomic HMGA2 rearrangements. Copyright © 2011 Wiley-Liss, Inc.

  10. Elevated mRNA-levels of gonadotropin-releasing hormone and its receptor in plaque-bearing Alzheimer's disease transgenic mice.

    Directory of Open Access Journals (Sweden)

    Syed Nuruddin

    Full Text Available Research on Alzheimer's disease (AD has indicated an association between hormones of the hypothalamic-pituitary-gonadal (HPG axis and cognitive senescence, indicating that post meno-/andropausal changes in HPG axis hormones are implicated in the neuropathology of AD. Studies of transgenic mice with AD pathologies have led to improved understanding of the pathophysiological processes underlying AD. The aims of this study were to explore whether mRNA-levels of gonadotropin-releasing hormone (Gnrh and its receptor (Gnrhr were changed in plaque-bearing Alzheimer's disease transgenic mice and to investigate whether these levels and amyloid plaque deposition were downregulated by treatment with a gonadotropin-releasing hormone analog (Gnrh-a; Leuprorelin acetate. The study was performed on mice carrying the Arctic and Swedish amyloid-β precursor protein (AβPP mutations (tgArcSwe. At 12 months of age, female tgArcSwe mice showed a twofold higher level of Gnrh mRNA and more than 1.5 higher level of Gnrhr mRNA than age matched controls. Male tgArcSwe mice showed the same pattern of changes, albeit more pronounced. In both sexes, Gnrh-a treatment caused significant down-regulation of Gnrh and Gnrhr mRNA expression. Immunohistochemistry combined with quantitative image analysis revealed no significant changes in the plaque load after Gnrh-a treatment in hippocampus and thalamus. However, plaque load in the cerebral cortex of treated females tended to be lower than in female vehicle-treated mice. The present study points to the involvement of hormonal changes in AD mice models and demonstrates that these changes can be effectively counteracted by pharmacological treatment. Although known to increase in normal aging, our study shows that Gnrh/Gnrhr mRNA expression increases much more dramatically in tgArcSwe mice. Treatment with Leuprorelin acetate successfully abolished the transgene specific effects on Gnrh/Gnrhr mRNA expression. The present experimental

  11. Modulation of DNA repair capacity and mRNA expression levels of XRCC1, hOGG1 and XPC genes in styrene-exposed workers

    International Nuclear Information System (INIS)

    Hanova, Monika; Stetina, Rudolf; Vodickova, Ludmila; Vaclavikova, Radka; Hlavac, Pavel; Smerhovsky, Zdenek; Naccarati, Alessio; Polakova, Veronika; Soucek, Pavel; Kuricova, Miroslava; Manini, Paola; Kumar, Rajiv; Hemminki, Kari; Vodicka, Pavel

    2010-01-01

    Decreased levels of single-strand breaks in DNA (SSBs), reflecting DNA damage, have previously been observed with increased styrene exposure in contrast to a dose-dependent increase in the base-excision repair capacity. To clarify further the above aspects, we have investigated the associations between SSBs, micronuclei, DNA repair capacity and mRNA expression in XRCC1, hOGG1 and XPC genes on 71 styrene-exposed and 51 control individuals. Styrene concentrations at workplace and in blood characterized occupational exposure. The workers were divided into low (below 50 mg/m 3 ) and high (above 50 mg/m 3 ) styrene exposure groups. DNA damage and DNA repair capacity were analyzed in peripheral blood lymphocytes by Comet assay. The mRNA expression levels were determined by qPCR. A significant negative correlation was observed between SSBs and styrene concentration at workplace (R = - 0.38, p = 0.001); SSBs were also significantly higher in men (p = 0.001). The capacity to repair irradiation-induced DNA damage was the highest in the low exposure group (1.34 ± 1.00 SSB/10 9 Da), followed by high exposure group (0.72 ± 0.81 SSB/10 9 Da) and controls (0.65 ± 0.82 SSB/10 9 Da). The mRNA expression levels of XRCC1, hOGG1 and XPC negatively correlated with styrene concentrations in blood and at workplace (p < 0.001) and positively with SSBs (p < 0.001). Micronuclei were not affected by styrene exposure, but were higher in older persons and in women (p < 0.001). In this study, we did not confirm previous findings on an increased DNA repair response to styrene-induced genotoxicity. However, negative correlations of SSBs and mRNA expression levels of XRCC1, hOGG1 and XPC with styrene exposure warrant further highly-targeted study.

  12. MicroRNA miR-9 modifies motor neuron columns by a tuning regulation of FoxP1 levels in developing spinal cords

    OpenAIRE

    Otaegi, Gaizka; Pollock, Andrew; Hong, Janet; Sun, Tao

    2011-01-01

    The precise organization of motor neuron subtypes in a columnar pattern in developing spinal cords is controlled by cross-interactions of multiple transcription factors and segmental expressions of Hox genes and their accessory proteins. Accurate expression levels and domains of these regulators are essential for organizing spinal motor neuron columns and axonal projections to target muscles. Here, we show that microRNA miR-9 is transiently expressed in a motor neuron subtype and displays ove...

  13. Tris(2-butoxyethyl)phosphate and triethyl phosphate alter embryonic development, hepatic mRNA expression, thyroid hormone levels, and circulating bile acid concentrations in chicken embryos

    Energy Technology Data Exchange (ETDEWEB)

    Egloff, Caroline [National Wildlife Research Centre, Environment Canada, Ottawa, ON K1A 0H3 (Canada); Crump, Doug, E-mail: doug.crump@ec.gc.ca [National Wildlife Research Centre, Environment Canada, Ottawa, ON K1A 0H3 (Canada); Porter, Emily; Williams, Kim L.; Letcher, Robert J.; Gauthier, Lewis T. [National Wildlife Research Centre, Environment Canada, Ottawa, ON K1A 0H3 (Canada); Kennedy, Sean W. [National Wildlife Research Centre, Environment Canada, Ottawa, ON K1A 0H3 (Canada); Department of Biology, University of Ottawa, Ottawa, ON K1N 6N5 (Canada)

    2014-09-15

    The organophosphate flame retardants tris(2-butoxyethyl) phosphate (TBOEP) and triethyl phosphate (TEP) are used in a wide range of applications to suppress or delay the ignition and spread of fire. Both compounds have been detected in the environment and TBOEP was recently measured in free-living avian species. In this study, TBOEP and TEP were injected into the air cell of chicken embryos at concentrations ranging from 0 to 45,400 ng/g and 0 to 241,500 ng/g egg, respectively. Pipping success, development, hepatic mRNA expression of 9 target genes, thyroid hormone levels, and circulating bile acid concentrations were determined. Exposure to the highest doses of TBOEP and TEP resulted in negligible detection of the parent compounds in embryonic contents at pipping indicating their complete metabolic degradation. TBOEP exposure had limited effects on chicken embryos, with the exception of hepatic CYP3A37 mRNA induction. TEP exposure decreased pipping success to 68%, altered growth, increased liver somatic index (LSI) and plasma bile acids, and modulated genes associated with xenobiotic and lipid metabolism and the thyroid hormone pathway. Plasma thyroxine levels were decreased at all TEP doses, including an environmentally-relevant concentration (8 ng/g), and gallbladder hypotrophy was evident at ≥ 43,200 ng/g. Tarsus length and circulating thyroxine concentration emerged as potential phenotypic anchors for the modulation of transthyretin mRNA. The increase in plasma bile acids and LSI, gallbladder hypotrophy, and discoloration of liver tissue represented potential phenotypic outcomes associated with modulation of hepatic genes involved with xenobiotic and lipid metabolism. - Highlights: • TBOEP is not embryolethal to chicken embryos. • TEP affected embryonic viability, morphometric endpoints, and thyroid hormone levels. • TEP altered mRNA levels of xenobiotic and lipid metabolism genes. • TEP increased plasma bile acids and caused gallbladder hypotrophy

  14. [Selection and construction of cell line stably expressing survivin gene in lower level through eukaryotic plasmid vector of shRNA].

    Science.gov (United States)

    Wang, Wen-Xia; Sun, Shan-Zhen; Song, Ying

    2008-06-01

    To construct a short hairpin RNA(shRNA) interference expression plasmid vector of survivin gene, transfect tongue squamous cell carcinoma line Tca8113 which expressed survivin gene in a high level, and choose the cells whose survivin gene were suppressed significantly. Two pairs of oligonucleotide sequences specific for survivin gene were designed and synthesized, and cloned into pSilencer-2.1U6-neo plasmid. The recombinant plasmids (named PS1 and PS2) were amplified in Ecoli. DH5alpha was identified by restriction digestion, PCR and sequencing. The vectors were transfected into Tca8113 cells with lipofectamine 2000. After selection with G418, the stable cell clones were attained. Survivn expression was assayed with real-time quantitative PCR and Western blotting. SAS8.0 software package was used for Student t test. Two vectors were constructed successfully and stable cell clones with PS1 or PS2 plasmid were obtained. As compared with those of control, survivin expression of transfected cell with PS1 or PS2 in mRNA level was significantly suppressed (P<0.05). In protein level, only those of transfected cell with PS2 was significantly suppressed (P<0.01). The shRNA interference expression plasmid vectors of survivin gene are successfully constructed, and Tca8113 cells which express survivin gene in a stable lower level are attained, which enable us to carry out further research on gene therapy of oral squamous cell carcinoma. Supported by National Natural Science Foundation of China (Grant No.30572056).

  15. Serotonin 2A receptor mRNA levels in the neonatal dopamine-depleted rat striatum remain upregulated following suppression of serotonin hyperinnervation.

    Science.gov (United States)

    Basura, G J; Walker, P D

    1999-08-05

    Sixty days after bilateral dopamine (DA) depletion (>98%) with 6-hydroxydopamine (6-OHDA) in neonatal rats, serotonin (5-HT) content doubled and 5-HT(2A) receptor mRNA expression rose 54% within the rostral striatum. To determine if striatal 5-HT(2A) receptor mRNA upregulation is dependent on increased 5-HT levels following DA depletion, neonatal rats received dual injections of 6-OHDA and 5,7-dihydroxytryptamine (5,7-DHT) which suppressed 5-HT content by approximately 90%. In these 6-OHDA/5,7-DHT-treated rats, striatal 5-HT(2A) receptor mRNA expression was still elevated (87% above vehicle controls). Comparative analysis of 5-HT(2C) receptor mRNA expression yielded no significant changes in any experimental group. These results demonstrate that upregulated 5-HT(2A) receptor biosynthesis in the DA-depleted rat is not dependent on subsequent 5-HT hyperinnervation. Copyright 1999 Elsevier Science B.V.

  16. Assemble: an interactive graphical tool to analyze and build RNA architectures at the 2D and 3D levels.

    Science.gov (United States)

    Jossinet, Fabrice; Ludwig, Thomas E; Westhof, Eric

    2010-08-15

    Assemble is an intuitive graphical interface to analyze, manipulate and build complex 3D RNA architectures. It provides several advanced and unique features within the framework of a semi-automated modeling process that can be performed by homology and ab initio with or without electron density maps. Those include the interactive editing of a secondary structure and a searchable, embedded library of annotated tertiary structures. Assemble helps users with performing recurrent and otherwise tedious tasks in structural RNA research. Assemble is released under an open-source license (MIT license) and is freely available at http://bioinformatics.org/assemble. It is implemented in the Java language and runs on MacOSX, Linux and Windows operating systems.

  17. Kinetic proofreading at single molecular level: aminoacylation of tRNA(Ile and the role of water as an editor.

    Directory of Open Access Journals (Sweden)

    Mantu Santra

    Full Text Available Proofreading/editing in protein synthesis is essential for accurate translation of information from the genetic code. In this article we present a theoretical investigation of efficiency of a kinetic proofreading mechanism that employs hydrolysis of the wrong substrate as the discriminatory step in enzyme catalytic reactions. We consider aminoacylation of tRNA(Ile which is a crucial step in protein synthesis and for which experimental results are now available. We present an augmented kinetic scheme and then employ methods of stochastic simulation algorithm to obtain time dependent concentrations of different substances involved in the reaction and their rates of formation. We obtain the rates of product formation and ATP hydrolysis for both correct and wrong substrates (isoleucine and valine in our case, respectively, in single molecular enzyme as well as ensemble enzyme kinetics. The present theoretical scheme correctly reproduces (i the amplitude of the discrimination factor in the overall rates between isoleucine and valine which is obtained as (1.8×10(2.(4.33×10(2 = 7.8×10(4, (ii the rates of ATP hydrolysis for both Ile and Val at different substrate concentrations in the aminoacylation of tRNA(Ile. The present study shows a non-michaelis type dependence of rate of reaction on tRNA(Ile concentration in case of valine. The overall editing in steady state is found to be independent of amino acid concentration. Interestingly, the computed ATP hydrolysis rate for valine at high substrate concentration is same as the rate of formation of Ile-tRNA(Ile whereas at intermediate substrate concentration the ATP hydrolysis rate is relatively low. We find that the presence of additional editing domain in class I editing enzyme makes the kinetic proofreading more efficient through enhanced hydrolysis of wrong product at the editing CP1 domain.

  18. tRNA Is the Source of Low-Level trans-Zeatin Production in Methylobacterium spp.†‡

    Science.gov (United States)

    Koenig, Robbin L.; Morris, Roy O.; Polacco, Joe C.

    2002-01-01

    Pink-pigmented facultatively methylotrophic bacteria (PPFMs), classified as Methylobacterium spp., are persistent colonizers of plant leaf surfaces. Reports of PPFM-plant dialogue led us to examine cytokinin production by PPFMs. Using immunoaffinity and high-performance liquid chromatography (HPLC) purification, we obtained 22 to 111 ng of trans-zeatin per liter from culture filtrates of four PPFM leaf isolates (from Arabidopsis, barley, maize, and soybean) and of a Methylobacterium extorquens type culture originally recovered as a soil isolate. We identified the zeatin isolated as the trans isomer by HPLC and by a radioimmunoassay in which monoclonal antibodies specific for trans-hydroxylated cytokinins were used. Smaller and variable amounts of trans-zeatin riboside were also recovered. trans-Zeatin was recovered from tRNA hydrolysates in addition to the culture filtrates, suggesting that secreted trans-zeatin resulted from tRNA turnover rather than from de novo synthesis. The product of the miaA gene is responsible for isopentenylation of a specific adenine in some tRNAs. To confirm that the secreted zeatin originated from tRNA, we mutated the miaA gene of M. extorquens by single exchange of an internal miaA fragment into the chromosomal gene. Mutant exconjugants, confirmed by PCR, did not contain zeatin in their tRNAs and did not secrete zeatin into the medium, findings which are consistent with the hypothesis that all zeatin is tRNA derived rather than synthesized de novo. In germination studies performed with heat-treated soybean seeds, cytokinin-null (miaA) mutants stimulated germination as well as wild-type bacteria. While cytokinin production may play a role in the plant-PPFM interaction, it is not responsible for stimulation of germination by PPFMs. PMID:11889088

  19. Detection of Ebola Virus RNA through Aerosol Sampling of Animal Biosafety Level 4 Rooms Housing Challenged Nonhuman Primates

    Science.gov (United States)

    2016-08-02

    Rooms Housing Challenged Nonhuman Primates 10 11 12 13 14 15 David E. Harbourt1*, Sara C. Johnston1, James Pettitt2, Travis K. Warren1 and...Sampling of ABSL-4 Rooms Housing Challenged Nonhuman 10 Primates for publication in an edition of The Journal of Infectious Disease. This 11 manuscript...embedded in the texts. This is the first report demonstrating detection of Ebola virus 17 RNA from animal rooms housing infected nonhuman primates and

  20. Influence of developmental stage and genotype on liver mRNA levels among wild, domesticated, and hybrid rainbow trout (Oncorhynchus mykiss).

    Science.gov (United States)

    White, Samantha L; Sakhrani, Dionne; Danzmann, Roy G; Devlin, Robert H

    2013-10-02

    Release of domesticated strains of fish into nature may pose a threat to wild populations with respect to their evolved genetic structure and fitness. Understanding alterations that have occurred in both physiology and genetics as a consequence of domestication can assist in evaluating the risks posed by introgression of domesticated genomes into wild genetic backgrounds, however the molecular causes of these consequences are currently poorly defined. The present study has examined levels of mRNA in fast-growing pure domesticated (D), slow-growing age-matched pure wild (Wa), slow-growing size-matched pure wild (Ws), and first generation hybrid cross (W/D) rainbow trout (Oncorhynchus mykiss) to investigate the influence of genotype (domesticated vs. wild, and their interactions in hybrids) and developmental stage (age- or size-matched animals) on genetic responses (i.e. dominant vs. recessive) and specific physiological pathways. Highly significant differences in mRNA levels were found between domesticated and wild-type rainbow trout genotypes (321 mRNAs), with many mRNAs in the wild-domesticated hybrid progeny showing intermediate levels. Differences were also found between age-matched and size-matched wild-type trout groups (64 mRNAs), with unique mRNA differences for each of the wild-type groups when compared to domesticated trout (Wa: 114 mRNAs, Ws: 88 mRNAs), illustrating an influence of fish developmental stage affecting findings when used as comparator groups to other genotypes. Analysis of differentially expressed mRNAs (found for both wild-type trout to domesticated comparisons) among the genotypes indicates that 34.8% are regulated consistent with an additive genetic model, whereas 39.1% and 26.1% show a recessive or dominant mode of regulation, respectively. These molecular data are largely consistent with phenotypic data (growth and behavioural assessments) assessed in domesticated and wild trout strains. The present molecular data are concordant with

  1. Alterations in Circulating miRNA Levels following Early-Stage Estrogen Receptor-Positive Breast Cancer Resection in Post-Menopausal Women

    DEFF Research Database (Denmark)

    Kodahl, Annette R; Zeuthen, Pernille; Binder, Harald

    2014-01-01

    INTRODUCTION: Circulating microRNAs (miRNAs) exhibit remarkable stability and may serve as biomarkers in several clinical cancer settings. The aim of this study was to investigate changes in the levels of specific circulating miRNA following breast cancer surgery and evaluate whether these altera...... and could potentially be used to monitor whether all cancer cells have been removed at surgery and/or, subsequently, whether the patients develop recurrence.......INTRODUCTION: Circulating microRNAs (miRNAs) exhibit remarkable stability and may serve as biomarkers in several clinical cancer settings. The aim of this study was to investigate changes in the levels of specific circulating miRNA following breast cancer surgery and evaluate whether...... these alterations were also observed in an independent data set. METHODS: Global miRNA analysis was performed on prospectively collected serum samples from 24 post-menopausal women with estrogen receptor-positive early-stage breast cancer before surgery and 3 weeks after tumor resection using global LNA...

  2. Correlation of mRNA and protein levels: Cell type-specific gene expression of cluster designation antigens in the prostate

    Directory of Open Access Journals (Sweden)

    Deutsch Eric W

    2008-05-01

    Full Text Available Abstract Background: Expression levels of mRNA and protein by cell types exhibit a range of correlations for different genes. In this study, we compared levels of mRNA abundance for several cluster designation (CD genes determined by gene arrays using magnetic sorted and laser-capture microdissected human prostate cells with levels of expression of the respective CD proteins determined by immunohistochemical staining in the major cell types of the prostate – basal epithelial, luminal epithelial, stromal fibromuscular, and endothelial – and for prostate precursor/stem cells and prostate carcinoma cells. Immunohistochemical stains of prostate tissues from more than 50 patients were scored for informative CD antigen expression and compared with cell-type specific transcriptomes. Results: Concordance between gene and protein expression findings based on 'present' vs. 'absent' calls ranged from 46 to 68%. Correlation of expression levels was poor to moderate (Pearson correlations ranged from 0 to 0.63. Divergence between the two data types was most frequently seen for genes whose array signals exceeded background (> 50 but lacked immunoreactivity by immunostaining. This could be due to multiple factors, e.g. low levels of protein expression, technological sensitivities, sample processing, probe set definition or anatomical origin of tissue and actual biological differences between transcript and protein abundance. Conclusion: Agreement between these two very different methodologies has great implications for their respective use in both molecular studies and clinical trials employing molecular biomarkers.

  3. Switching off small RNA regulation with trap-mRNA

    DEFF Research Database (Denmark)

    Overgaard, Martin; Johansen, Jesper; Møller-Jensen, Jakob

    2009-01-01

    to operate at the level of transcription initiation. By employing a highly sensitive genetic screen we uncovered a novel RNA-based regulatory principle in which induction of a trap-mRNA leads to selective degradation of a small regulatory RNA molecule, thereby abolishing the sRNA-based silencing of its...

  4. Two distinct genes for ADP/ATP translocase are expressed at the mRNA level in adult human liver

    International Nuclear Information System (INIS)

    Houldsworth, J.; Attardi, G.

    1988-01-01

    Several clones hybridizing with a bovine ADP/ATP translocase cDNA were isolated from an adult human liver cDNA library in the vector pEX1. DNA sequence analysis revealed that these clones encode two distinct forms of translocase. In particular, two clones specifying the COOH-end-proximal five-sixths of the protein exhibit a 9% amino acid sequence divergence and totally dissimilar 3' untranslated regions. One of these cDNAs is nearly identical in sequence to an ADP/ATP translocase clone (hp2F1) recently isolated from a human fibroblast cDNA library with three amino acid changes and a few differences in the 3' untranslated region. Another clone isolated from the pEX1 library contains a reading frame encoding the remaining, NH 2 -end-proximal, 37 amino acids of the translocase. This sequence differs significantly (14% amino acid sequence divergence) from the corresponding segment of hp2F1, and the 5' untranslated regions of the two clones are totally dissimilar. RNA transfer hybridization experiments utilizing the clones isolated from the pEX1 library revealed the presence in HeLa cells of three distinct mRNA species. The pattern of hybridization and the sizes of these mRNAs suggest a greater complexity of organization and expression of the ADP/ATP translocase genes in human cells than indicated by the analysis of the cDNA clones

  5. Amelioration of ultraviolet-B-induced down-regulation of mRNA levels for chloroplast proteins, by high irradiance, is mediated by photosynthesis

    International Nuclear Information System (INIS)

    Mackerness, S.A. H.; Butt, P.J.; Jordan, B.R.; Thomas, B.

    1996-01-01

    The mechanism by which increasing photosynthetically active radiation (PAR) reduces the sensitivity of RNA transcripts to UV-B radiation was studied in pea (Pisum sativum L.). mRNA transcript levels for rbc S, rbc L, cab and psb A were measured over an 8 d experimental period in pea, plants supplemented with UV-B radiation under a range of conditions. Under low light (150 mu-mol m -2 s -1 ), UV-B resulted in a significant decline in the levels of transcripts for all four genes which was prevented by increasing the background irradiance to 350 mu-mol m -2 s -1 (high light) with white light from fluorescent lamps. Increasing CO 2 levels to give photosynthesis rates equivalent to the high light treatment partially protected rbc S and cab transcripts and fully protected rbc L transcripts but did not prevent visible injury. Increasing light with low pressure sodium lamps, which increase photosynthesis but are not effective for activation of the DNA repair enzyme, photolyase, gave results which were not significantly different from white fluorescent high light treatments. Protection by high light was lost in the presence of the photosynthesis inhibitors CCCP and DCMU. The UV-B induced increase in the expression of chalcone synthase (chs) genes was delayed by the treatments which increased photosynthesis rates and conferred protection. The results indicate that photosynthesis plays a key role in the amelioration of UV-B induced decline in mRNA levels for proteins. The minimal role of DNA repair by photolyase indicates that reduction in photosynthesis gene transcripts in response to UV-B represents a specific regulation rather than being a consequence of DNA damage. (author)

  6. ERCC1 and BRCA1 mRNA expression levels in metastatic malignant effusions is associated with chemosensitivity to cisplatin and/or docetaxel

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    Wang Tingting

    2008-04-01

    Full Text Available Abstract Background One of the major challenges in currently chemotherapeutic theme is lacking effective biomarkers for drug response and sensitivity. Our current study focus on two promising biomarkers, ERCC1 (excision repair cross-complementing group 1 and BRCA1 (breast cancer susceptibility gene 1. To investigate their potential role in serving as biomarkers for drug sensitivity in cancer patients with metastases, we statistically measure the mRNA expression level of ERCC1 and BRCA1 in tumor cells isolated from malignant effusions and correlate them with cisplatin and/or docetaxel chemosensitivity. Methods Real-time quantitative PCR is used to analysis related genes expression in forty-six malignant effusions prospectively collected from non-small cell lung cancer (NSCLC, gastric and gynecology cancer patients. Viable tumor cells obtained from malignant effusions are tested for their sensitivity to cisplatin and docetaxel using ATP-TCA assay. Results ERCC1 expression level is negatively correlated with the sensitivity to cisplatin in NSCLC patients (P = 0.001. In NSCLC and gastric group, BRCA1 expression level is negatively correlated with the sensitivity to cisplatin (NSCLC: P = 0.014; gastric: P = 0.002 while positively correlated with sensitivity to docetaxel (NSCLC: P = 0.008; gastric: P = 0.032. A significant interaction is found between ERCC1 and BRCA1 mRNA expressions on sensitivity to cisplatin (P = 0.010, n = 45. Conclusion Our results demonstrate that ERCC1 and BRCA1 mRNA expression levels are correlated with in vitro chemosensitivity to cisplatin and/or docetaxel in malignant effusions of NSCLC and gastric cancer patients. And combination of ERCC1 and BRCA1 may have a better role on predicting the sensitivity to cisplatin than the single one is considered.

  7. Waterborne gemfibrozil challenges the hepatic antioxidant defense system and down-regulates peroxisome proliferator-activated receptor beta (PPARβ) mRNA levels in male goldfish (Carassius auratus)

    International Nuclear Information System (INIS)

    Mimeault, C.; Trudeau, V.L.; Moon, T.W.

    2006-01-01

    The lipid regulator gemfibrozil (GEM) is one of many human pharmaceuticals found in the aquatic environment. We previously demonstrated that GEM bioconcentrates in blood and reduces plasma testosterone levels in goldfish (Carassius auratus). In this study, we address the potential of an environmentally relevant waterborne concentration of GEM (1.5 μg/l) to induce oxidative stress in goldfish liver and whether this may be linked to GEM acting as a peroxisome proliferator (PP). We also investigate the autoregulation of the peroxisome proliferator-activated receptors (PPARs) as a potential index of exposure. The three PPAR subtypes (α, β, and γ) were amplified from goldfish liver cDNA. Goldfish exposed to a concentration higher (1500 μg/l) than environmentally relevant for 14 and 28 days significantly reduce hepatic PPARβ mRNA levels (p < 0.001). Levels of CYP1A1 mRNA were unchanged. GEM exposure significantly induced the antioxidant defense enzymes catalase (p < 0.001), glutathione peroxidase (p < 0.001) and glutathione-S-transferase (p = 0.006) but not acyl-CoA oxidase or glutathione reductase. As GEM exposure failed to increase levels of thiobarbituric reactive substances (TBARS), we conclude that a sub-chronic exposure to GEM upregulates the antioxidant defense status of the goldfish as an adaptive response to this human pharmaceutical

  8. Low BMI is correlated with increased TGF-β and IL-10 mRNA levels in the peripheral blood of breast cancer patients.

    Science.gov (United States)

    Liu, Chao; Wang, Qian; Sun, Bing; Meng, Xiangying; Li, Lan; Yang, Liuchun; Cong, Yang; Liu, Jiannan; Xuan, Liang; Huang, Yan; Wu, Shikai

    2018-03-01

    Transforming growth factor-β (TGF-β), interleukin-10 (IL-10), and forkhead box P3 (Foxp3) have important roles in breast cancer development. Previous studies confirmed a correlation between these immune molecules and tumor characteristics, but their association with nutritional status in breast cancer is largely unknown. We aimed to investigate the association between body mass index (BMI), hemoglobin, total protein, albumin, globulin (GLB), albumin/GLB ratio (AGR), pre-albumin, prognostic nutritional index, and TGF-β, IL-10, and Foxp3 mRNA expression in patients with breast cancer. Quantitative real-time PCR was used to detect the mRNA expression of TGF-β, IL-10, and Foxp3 in the peripheral blood of 107 patients with breast cancer and 21 healthy controls. We found that TGF-β mRNA levels were 2.6-fold, 3.2-fold, and 2.3-fold higher in patients with low BMI (BMI (BMI BMI ≥ 25), respectively (P BMI, may strongly affect systematic immune function in patients with breast cancer. © 2018 IUBMB Life, 70(3):237-245, 2018. © 2018 International Union of Biochemistry and Molecular Biology.

  9. Canine adiponectin: cDNA structure, mRNA expression in adipose tissues and reduced plasma levels in obesity.

    Science.gov (United States)

    Ishioka, K; Omachi, A; Sagawa, M; Shibata, H; Honjoh, T; Kimura, K; Saito, M

    2006-04-01

    Adiponectin is a protein synthesized and secreted by adipocytes. Decreased adiponectin is responsible for insulin resistance and atherosclerosis associated with human obesity. We obtained a cDNA clone corresponding to canine adiponectin, whose nucleotide and deduced amino acid sequences were highly identical to those of other species. Adiponectin mRNA was detected in adipose tissues, but not in other tissues, of dogs. When 22 adult beagles were given a high-energy diet for 14 weeks, they became obese, showing heavier body weights, higher plasma leptin concentrations, but lower plasma adiponectin concentrations. The adiponectin concentrations of plasma samples collected from 71 dogs visiting veterinary practices were negatively correlated to plasma leptin concentrations, being lower in obese than non-obese dogs. These results are compatible with those reported in other species, and suggest that adiponectin is an index of adiposity and a target molecule for studies on diseases associated with obesity in dogs.

  10. Antagomirs Targeting MicroRNA-134 Increase Limk1 Levels After Experimental Seizures in Vitro and in Vivo

    Directory of Open Access Journals (Sweden)

    Jiahang Sun

    2017-09-01

    Full Text Available Background: MiR-134 is enriched in dendrites of hippocampal neurons and plays crucial roles in the progress of epilepsy. The present study aims to investigate the effects of antagomirs targeting miroRNA-134 (Ant-134 on limk1 expression and the binding of miR-134 and limk1 in experimental seizure. Methods: Status epilepticus (SE rat model was established by lithium chloride-pilocarpine injection and was treated with Ant-134 by intracerebroventricular injection. Low Mg2+-exposed primary neurons were used as an in vitro model of SE. The expression of miR-134 was determined using real-time PCR. Protein expressions of limk1 and cofilin were determined by Western blotting. Luciferase reporter assay was used to examine the binding between miR-134 and limk1 3’-untranslated region. Results: The expression of miR-134 was markedly enhanced in hippocampus of the SE rats and low Mg2+-exposed neurons. Ant-134 increased the expression of limk1 and reduced the expression of cofilin in the SE hippocampus and Low Mg2+-exposed neurons. In addition, luciferase reporter assay confirmed that miR-134 bound limk1 3’-UTR. MiR-134 overexpression inhibited limk1 mRNA and protein expressions in neurons. Conclusion: Blockage of miR-134 upregulates limk1 expression and downregulated cofilin expression in hippocampus of the SE rats. This mechanism may contribute to the neuroprotective effects of Ant-134.

  11. Loss of Pnn expression attenuates expression levels of SR family splicing factors and modulates alternative pre-mRNA splicing in vivo

    International Nuclear Information System (INIS)

    Chiu Yali; Ouyang Pin

    2006-01-01

    SR and SR-related proteins have been implicated as trans-acting factors that play an important role in splice selection and are involved at specific stages of spliceosome formation. A well-established property of SR protein splicing factors is their ability to influence selection of alternative splice sites in a concentration-dependent manner. Identification of molecules that regulate SR family protein expression is therefore of vital importance in RNA biology. Here we report that depletion of Pnn expression, a SR-related protein with functions involved in pre-mRNA splicing and mRNA export, induces reduced expression of a subset of cellular proteins, especially that of SR family proteins, including SC35, SRm300, SRp55, and SRp40, but not that of other nuclear proteins, such as p53, Mdm2, and ki67. Knocking down Pnn expression was achieved in vitro by siRNA transfection. Expression levels of SR and SR-related proteins in Pnn-depleted cells as compared to those in control cells were evaluated by immunofluorescent staining and Western blot with specific antibodies. In addition, we also demonstrate that loss of Pnn expression could modulate splice site selection of model reporter gene in vivo. Our finding is significant in terms of regulation of SR protein cellular concentration because it reveals that Pnn may play a general role in the control of the cellular amount of family SR proteins through down-regulation of its own expression, thereby providing us with a better understanding of the cellular mechanism by which Pnn fulfills its biological function

  12. Immediate-early gene response to repeated immobilization: Fos protein and arc mRNA levels appear to be less sensitive than c-fos mRNA to adaptation.

    Science.gov (United States)

    Ons, Sheila; Rotllant, David; Marín-Blasco, Ignacio J; Armario, Antonio

    2010-06-01

    Stress exposure resulted in brain induction of immediate-early genes (IEGs), considered as markers of neuronal activation. Upon repeated exposure to the same stressor, reduction of IEG response (adaptation) has been often observed, but there are important discrepancies in literature that may be in part related to the particular IEG and methodology used. We studied the differential pattern of adaptation of the IEGs c-fos and arc (activity-regulated cytoskeleton-associated protein) after repeated exposure to a severe stressor: immobilization on wooden boards (IMO). Rats repeatedly exposed to IMO showed reduced c-fos mRNA levels in response to acute IMO in most brain areas studied: the medial prefrontal cortex (mPFC), lateral septum (LS), medial amygdala (MeA), paraventricular nucleus of the hypothalamus (PVN) and locus coeruleus. In contrast, the number of neurons showing Fos-like immunoreactivity was only reduced in the MeA and the various subregions of the PVN. IMO-induced increases in arc gene expression were restricted to telencephalic regions and reduced by repeated IMO only in the mPFC. Double-labelling in the LS of IMO-exposed rats revealed that arc was expressed in only one-third of Fos+ neurons, suggesting two populations of Fos+ neurons. These data suggest that c-fos mRNA levels are more affected by repeated IMO than corresponding protein, and that arc gene expression does not reflect adaptation in most brain regions, which may be related to its constitutive expression. Therefore, the choice of a particular IEG and the method of measurement are important for proper interpretation of the impact of chronic repeated stress on brain activation.

  13. Evaluation of mRNA expression levels and electrophysiological function of neuron-like cells derived from canine bone marrow stromal cells.

    Science.gov (United States)

    Nakano, Rei; Edamura, Kazuya; Sugiya, Hiroshi; Narita, Takanori; Okabayashi, Ken; Moritomo, Tadaaki; Teshima, Kenji; Asano, Kazushi; Nakayama, Tomohiro

    2013-10-01

    To investigate the in vitro differentiation of canine bone marrow stromal cells (BMSCs) into functional, mature neurons. Bone marrow from 6 adult dogs. BMSCs were isolated from bone marrow and chemically induced to develop into neurons. The morphology of the BMSCs during neuronal induction was monitored, and immunocytochemical analyses for neuron markers were performed after the induction. Real-time PCR methods were used to evaluate the mRNA expression levels of markers for neural stem or progenitor cells, neurons, and ion channels, and western blotting was used to assess the expression of neuronal proteins before and after neuronal induction. The electrophysiological properties of the neuron-like cells induced from canine BMSCs were evaluated with fluorescent dye to monitor Ca(2)+ influx. Canine BMSCs developed a neuron-like morphology after neuronal induction. Immunocytochemical analysis revealed that these neuron-like cells were positive for neuron markers. After induction, the cells' mRNA expression levels of almost all neuron and ion channel markers increased, and the protein expression levels of nestin and neurofilament-L increased significantly. However, the neuron-like cells derived from canine BMSCs did not have the Ca(2)+ influx characteristic of spiking neurons. Although canine BMSCs had neuron-like morphological and biochemical properties after induction, they did not develop the electrophysiological characteristics of neurons. Thus, these results have suggested that canine BMSCs could have the capacity to differentiate into a neuronal lineage, but the differentiation protocol used may have been insufficient to induce development into functional neurons.

  14. RNA Crystallization

    Science.gov (United States)

    Golden, Barbara L.; Kundrot, Craig E.

    2003-01-01

    RNA molecules may be crystallized using variations of the methods developed for protein crystallography. As the technology has become available to syntheisize and purify RNA molecules in the quantities and with the quality that is required for crystallography, the field of RNA structure has exploded. The first consideration when crystallizing an RNA is the sequence, which may be varied in a rational way to enhance crystallizability or prevent formation of alternate structures. Once a sequence has been designed, the RNA may be synthesized chemically by solid-state synthesis, or it may be produced enzymatically using RNA polymerase and an appropriate DNA template. Purification of milligram quantities of RNA can be accomplished by HPLC or gel electrophoresis. As with proteins, crystallization of RNA is usually accomplished by vapor diffusion techniques. There are several considerations that are either unique to RNA crystallization or more important for RNA crystallization. Techniques for design, synthesis, purification, and crystallization of RNAs will be reviewed here.

  15. Elevated mRNA-levels of distinct mitochondrial and plasma membrane Ca2+ transporters in individual hypoglossal motor neurons of endstage SOD1 transgenic mice.

    Directory of Open Access Journals (Sweden)

    Tobias eMühling

    2014-11-01

    Full Text Available Disturbances in Ca2+ homeostasis and mitochondrial dysfunction have emerged as major pathogenic features in familial and sporadic forms of Amyotrophic Lateral Sclerosis (ALS, a fatal degenerative motor neuron disease. However, the distinct molecular ALS-pathology remains unclear. Recently, an activity-dependent Ca2+ homeostasis deficit, selectively in highly vulnerable cholinergic motor neurons in the hypoglossal nucleus (hMNs from a common ALS mouse model, endstage superoxide dismutase SOD1G93A transgenic mice, was described. This functional deficit was defined by a reduced hMN mitochondrial Ca2+ uptake capacity and elevated Ca2+ extrusion across the plasma membrane. To address the underlying molecular mechanisms, here we quantified mRNA-levels of respective potential mitochondrial and plasma membrane Ca2+ transporters in individual, choline-acetyltransferase (ChAT positive hMNs from wildtype (WT and endstage SOD1G93A mice, by combining UV laser microdissection with RT-qPCR techniques, and specific data normalization. As ChAT cDNA levels as well as cDNA and genomic DNA levels of the mitochondrially encoded NADH dehydrogenase ND1 were not different between hMNs from WT and endstage SOD1G93A mice, these genes were used to normalize hMN-specific mRNA-levels of plasma membrane and mitochondrial Ca2+ transporters, respectively. We detected about 2-fold higher levels of the mitochondrial Ca2+ transporters MCU/MICU1, Letm1 and UCP2 in remaining hMNs from endstage SOD1G93A mice. These higher expression-levels of mitochondrial Ca2+ transporters in individual hMNs were not associated with a respective increase in number of mitochondrial genomes, as evident from hMN specific ND1 DNA quantification. Normalized mRNA-levels for the plasma membrane Na2+/Ca2+exchanger NCX1 was also about 2-fold higher in hMNs from SOD1G93A mice. Thus, pharmacological stimulation of Ca2+ transporters in highly vulnerable hMNs might offer a novel neuroprotective strategy for ALS.

  16. An image-based, dual fluorescence reporter assay to evaluate the efficacy of shRNA for gene silencing at the single-cell level [v1; ref status: indexed, http://f1000r.es/2tt

    Directory of Open Access Journals (Sweden)

    Shin-ichiro Kojima

    2014-02-01

    Full Text Available RNA interference (RNAi is widely used to suppress gene expression in a specific manner. The efficacy of RNAi is mainly dependent on the sequence of small interfering RNA (siRNA in relation to the target mRNA. Although several algorithms have been developed for the design of siRNA, it is still difficult to choose a really effective siRNA from among multiple candidates. In this article, we report the development of an image-based, quantitative, ratiometric fluorescence reporter assay to evaluate the efficacy of RNAi at the single-cell level. Two fluorescence reporter constructs are used. One expresses the candidate small hairpin RNA (shRNA together with an enhanced green fluorescent protein (EGFP; the other expresses a 19-nt target sequence inserted into a cassette expressing a red fluorescent protein (either DsRed or mCherry. Effectiveness of the candidate shRNA is evaluated as the extent to which it knocks down expression of the red fluorescent protein. Thus, the red-to-green fluorescence intensity ratio (appropriately normalized to controls is used as the read-out for quantifying the siRNA efficacy at the individual cell level. We tested this dual fluorescence assay and compared predictions to actual endogenous knockdown levels for three different genes (vimentin, lamin A/C and Arp3 and twenty different shRNAs. For each of the genes, our assay successfully predicted the target sequences for effective RNAi. To further facilitate testing of RNAi efficacy, we developed a negative selection marker (ccdB method for construction of shRNA and red fluorescent reporter plasmids that allowed us to purify these plasmids directly from transformed bacteria without the need for colony selection and DNA sequencing verification.

  17. An image-based, dual fluorescence reporter assay to evaluate the efficacy of shRNA for gene silencing at the single-cell level [v2; ref status: indexed, http://f1000r.es/39j

    Directory of Open Access Journals (Sweden)

    Shin-ichiro Kojima

    2014-05-01

    Full Text Available RNA interference (RNAi is widely used to suppress gene expression in a specific manner. The efficacy of RNAi is mainly dependent on the sequence of small interfering RNA (siRNA in relation to the target mRNA. Although several algorithms have been developed for the design of siRNA, it is still difficult to choose a really effective siRNA from among multiple candidates. In this article, we report the development of an image-based, quantitative, ratiometric fluorescence reporter assay to evaluate the efficacy of RNAi at the single-cell level. Two fluorescence reporter constructs are used. One expresses the candidate small hairpin RNA (shRNA together with an enhanced green fluorescent protein (EGFP; the other expresses a 19-nt target sequence inserted into a cassette expressing a red fluorescent protein (either DsRed or mCherry. Effectiveness of the candidate shRNA is evaluated as the extent to which it knocks down expression of the red fluorescent protein. Thus, the red-to-green fluorescence intensity ratio (appropriately normalized to controls is used as the read-out for quantifying the siRNA efficacy at the individual cell level. We tested this dual fluorescence assay and compared predictions to actual endogenous knockdown levels for three different genes (vimentin, lamin A/C and Arp3 and twenty different shRNAs. For each of the genes, our assay successfully predicted the target sequences for effective RNAi. To further facilitate testing of RNAi efficacy, we developed a negative selection marker (ccdB method for construction of shRNA and red fluorescent reporter plasmids that allowed us to purify these plasmids directly from transformed bacteria without the need for colony selection and DNA sequencing verification.

  18. Modifying a standard method allows simultaneous extraction of RNA and protein, enabling detection of enzymes in the rat retina with low expressions and protein levels.

    Science.gov (United States)

    Agardh, Elisabet; Gustavsson, Carin; Hagert, Per; Nilsson, Marie; Agardh, Carl-David

    2006-02-01

    The aim of the study was to evaluate messenger RNA and protein expression in limited amounts of tissue with low protein content. The Chomczynski method was used for simultaneous extraction of RNA, and protein was modified in the protein isolation step. Template mass and cycling time for the complementary DNA synthesis step of real-time reverse transcription-polymerase chain reaction (RT-PCR) for analysis of catalase, copper/zinc superoxide dismutase, manganese superoxide dismutase, the catalytic subunit of glutamylcysteine ligase, glutathione peroxidase 1, and the endogenous control cyclophilin B (CypB) were optimized before PCR. Polymerase chain reaction accuracy and efficacy were demonstrated by calculating the regression (R2) values of the separate amplification curves. Appropriate antibodies, blocking buffers, and running conditions were established for Western blot, and protein detection and multiplex assays with CypB were performed for each target. During the extraction procedure, the protein phase was dissolved in a modified washing buffer containing 0.1% sodium dodecyl sulfate, followed by ultrafiltration. Enzyme expression on real-time RT-PCR was accomplished with high reliability and reproducibility (R2, 0.990-0.999), and all enzymes except for glutathione peroxidase 1 were detectable in individual retinas on Western blot. Western blot multiplexing with CypB was possible for all targets. In conclusion, connecting gene expression directly to protein levels in the individual rat retina was possible by simultaneous extraction of RNA and protein. Real-time RT-PCR and Western blot allowed accurate detection of retinal protein expressions and levels.

  19. RNA Origami

    DEFF Research Database (Denmark)

    Sparvath, Steffen Lynge

    introducerede vores gruppe den enkeltstrengede RNA-origami metode, der giver mulighed for cotranscriptional foldning af veldefinerede nanostrukturer, og er en central del af arbejdet præsenteret heri. Denne ph.d.-afhandling udforsker potentielle anvendelser af RNA-origami nanostrukturer, som nanomedicin eller...... biosensorer. Afhandlingen består af en introduktion til RNA-nanoteknologi feltet, en introduktion af enkeltstrenget RNA-origami design, og fire studier, der beskriver design, produktion og karakterisering af både strukturelle og funktionelle RNA-origamier. Flere RNA-origami designs er blevet undersøgt, og...... projekterne, der indgår i denne afhandling, inkluderer de nyeste fremskridt indenfor strukturel RNA-nanoteknologi og udvikling af funktionelle RNA-baserede enheder. Det første studie beskriver konstruktion og karakterisering af en enkeltstrenget 6-helix RNA-origami stuktur, som er den første demonstration af...

  20. The effects of dietary Myrtle (Myrtus communis) on skin mucus immune parameters and mRNA levels of growth, antioxidant and immune related genes in zebrafish (Danio rerio).

    Science.gov (United States)

    Safari, Roghieh; Hoseinifar, Seyed Hossein; Van Doan, Hien; Dadar, Maryam

    2017-07-01

    Myrtle (Myrtus communis L., Myrtaceae) is a significant plant which naturally distributed around the globe. Although numerous studies have demonstrated the benefits of myrtle in different species, studies using the oral route are rare in the literature. In the present study, we evaluated the effect of myrtle intake on the antioxidant, immune, appetite and growth related genes as well as mucosal immune responses in zebrafish (Danio rerio) model. Zebrafish were fed control or myrtle (5, 10 and 20 g kg -1 myrtle) supplemented diets for sixty days. The results showed that, oral administration of Myrtle significantly improved mucosal immune responses (the activity of lysozyme, total Ig and protease). Furthermore, fish fed 20 g kg -1 showed remarkably higher antioxidant (sod and cat) enzymes gene expression compared other treatment. There were significant difference between myrtle fed fish and control group regarding tnf-alpha and lyz expression. Also, evaluation of growth (gh and igf1) related genes revealed remarkable upregulation in 20 g kg -1 myrtle treatment compared other myrtle treatments and control group. Similar results was observed regarding the mRNA levels of appetite related genes (ghrl) in zebrafish fed 20 g kg -1 myrtle. The present results indicated that dietary administration of myrtle improved mucosal immune parameters and altered mRNA levels of selected genes. These results on zebrafish model also highlights the potential use of Myrtle supplements as additive in human diets. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. In vitro Effects of Four Native Brazilian Medicinal Plants in CYP3A4 mRNA Gene Expression, Glutathione Levels, and P-Glycoprotein Activity.

    Science.gov (United States)

    Mazzari, Andre L D A; Milton, Flora; Frangos, Samantha; Carvalho, Ana C B; Silveira, Dâmaris; de Assis Rocha Neves, Francisco; Prieto, Jose M

    2016-01-01

    Erythrina mulungu Benth. (Fabaceae), Cordia verbenacea A. DC. (Boraginaceae), Solanum paniculatum L. (Solanaceae) and Lippia sidoides Cham. (Verbenaceae) are medicinal plant species native to Brazil shortlisted by the Brazilian National Health System for future clinical use. However, nothing is known about their effects in metabolic and transporter proteins, which could potentially lead to herb-drug interactions (HDI). In this work, we assess non-toxic concentrations (100 μg/mL) of the plant infusions for their in vitro ability to modulate CYP3A4 mRNA gene expression and intracellular glutathione levels in HepG2 cells, as well as P-glycoprotein (P-gp) activity in vincristine-resistant Caco-2 cells (Caco-2 VCR). Their mechanisms of action were further studied by measuring the activation of human pregnane X receptor (hPXR) in transiently co-transfected HeLa cells and the inhibition of γ-glutamyl transferase (GGT) in HepG2 cells. Our results show that P-gp activity was not affected in any case and that only Solanum paniculatum was able to significantly change CYP3A4 mRNA gene expression (twofold decrease, p effect upon hPXR (EC50 = 0.38 mg/mL). Total intracellular glutathione levels were significantly depleted by exposure to Solanum paniculatum (-44%, p Cordia verbenacea (-47%, p activity (-48%, p active pharmacovigilance is recommended for the other three species, especially in the case of Solanum paniculatum.

  2. ERBB2 in Cat Mammary Neoplasias Disclosed a Positive Correlation between RNA and Protein Low Expression Levels: A Model for erbB-2 Negative Human Breast Cancer

    Science.gov (United States)

    Abreu, Rui M. V.; Bastos, Estela; Amorim, Irina; Gut, Ivo G.; Gärtner, Fátima; Chaves, Raquel

    2013-01-01

    Human ERBB2 is a proto-oncogene that codes for the erbB-2 epithelial growth factor receptor. In human breast cancer (HBC), erbB-2 protein overexpression has been repeatedly correlated with poor prognosis. In more recent works, underexpression of this gene has been described in HBC. Moreover, it is also recognised that oncogenes that are commonly amplified or deleted encompass point mutations, and some of these are associated with HBC. In cat mammary lesions (CMLs), the overexpression of ERBB2 (27%–59.6%) has also been described, mostly at the protein level and although cat mammary neoplasias are considered to be a natural model of HBC, molecular information is still scarce. In the present work, a cat ERBB2 fragment, comprising exons 10 to 15 (ERBB2_10–15) was achieved for the first time. Allelic variants and genomic haplotype analyses were also performed, and differences between normal and CML populations were observed. Three amino acid changes, corresponding to 3 non-synonymous genomic sequence variants that were only detected in CMLs, were proposed to damage the 3D structure of the protein. We analysed the cat ERBB2 gene at the DNA (copy number determination), mRNA (expression levels assessment) and protein levels (in extra- and intra protein domains) in CML samples and correlated the last two evaluations with clinicopathological features. We found a positive correlation between the expression levels of the ERBB2 RNA and erbB-2 protein, corresponding to the intracellular region. Additionally, we detected a positive correlation between higher mRNA expression and better clinical outcome. Our results suggest that the ERBB2 gene is post-transcriptionally regulated and that proteins with truncations and single point mutations are present in cat mammary neoplastic lesions. We would like to emphasise that the recurrent occurrence of low erbB-2 expression levels in cat mammary tumours, suggests the cat mammary neoplasias as a valuable model for erbB-2 negative HBC

  3. Annotation Of Novel And Conserved MicroRNA Genes In The Build 10 Sus scrofa Reference Genome And Determination Of Their Expression Levels In Ten Different Tissues

    DEFF Research Database (Denmark)

    Thomsen, Bo; Nielsen, Mathilde; Hedegaard, Jakob

    The DNA template used in the pig genome sequencing project was provided by a Duroc pig named TJ Tabasco. In an effort to annotate microRNA (miRNA) genes in the reference genome we have conducted deep sequencing to determine the miRNA transcriptomes in ten different tissues isolated from Pinky......, a genetically identical clone of TJ Tabasco. The purpose was to generate miRNA sequences that are highly homologous to the reference genome sequence, which along with computational prediction will improve confidence in the genomic annotation of miRNA genes. Based on homology searches of the sequence data...... against miRBase, we identified more than 600 conserved known miRNA/miRNA*, which is a significant increase relative to the 211 porcine miRNA/miRNA* deposited in the current version of miRBase. Furthermore, the genome-wide transcript profiles provided important information on the relative abundance...

  4. Relationship between Sustained Reductions in Plasma Lipid and Lipoprotein Concentrations with Apheresis and Plasma Levels and mRNA Expression of PTX3 and Plasma Levels of hsCRP in Patients with HyperLp(a)lipoproteinemia

    Science.gov (United States)

    Stefanutti, Claudia; Mazza, Fabio; Steiner, Michael; Watts, Gerald F.; De Nève, Joel; Pasqualetti, Daniela; Paal, Juergen

    2016-01-01

    The effect of lipoprotein apheresis (Direct Adsorption of Lipids, DALI) (LA) on plasma levels of pentraxin 3 (PTX3), an inflammatory marker that reflects coronary plaque vulnerability, and expression of PTX3 mRNA was evaluated in patients with hyperLp(a)lipoproteinemia and angiographically defined atherosclerosis/coronary artery disease. Eleven patients, aged 55 ± 9.3 years (mean ± SD), were enrolled in the study. PTX3 soluble protein levels in plasma were unchanged by 2 sessions of LA; however, a downregulation of mRNA expression for PTX3 was observed, starting with the first session of LA (p < 0.001). The observed reduction was progressively increased in the interval between the first and second LA sessions to achieve a maximum decrease by the end of the second session. A statistically significantly greater treatment-effect correlation was observed in patients undergoing weekly treatments, compared with those undergoing treatment every 15 days. A progressive reduction in plasma levels of C-reactive protein was also seen from the first session of LA, with a statistically significant linear correlation for treatment-effect in the change in plasma levels of this established inflammatory marker (R 2 = 0.99; p < 0.001). Our findings suggest that LA has anti-inflammatory and endothelium protective effects beyond its well-established efficacy in lowering apoB100-containing lipoproteins. PMID:26903710

  5. Relationship between Sustained Reductions in Plasma Lipid and Lipoprotein Concentrations with Apheresis and Plasma Levels and mRNA Expression of PTX3 and Plasma Levels of hsCRP in Patients with HyperLp(alipoproteinemia

    Directory of Open Access Journals (Sweden)

    Claudia Stefanutti

    2016-01-01

    Full Text Available The effect of lipoprotein apheresis (Direct Adsorption of Lipids, DALI (LA on plasma levels of pentraxin 3 (PTX3, an inflammatory marker that reflects coronary plaque vulnerability, and expression of PTX3 mRNA was evaluated in patients with hyperLp(alipoproteinemia and angiographically defined atherosclerosis/coronary artery disease. Eleven patients, aged 55±9.3 years (mean ± SD, were enrolled in the study. PTX3 soluble protein levels in plasma were unchanged by 2 sessions of LA; however, a downregulation of mRNA expression for PTX3 was observed, starting with the first session of LA (p<0.001. The observed reduction was progressively increased in the interval between the first and second LA sessions to achieve a maximum decrease by the end of the second session. A statistically significantly greater treatment-effect correlation was observed in patients undergoing weekly treatments, compared with those undergoing treatment every 15 days. A progressive reduction in plasma levels of C-reactive protein was also seen from the first session of LA, with a statistically significant linear correlation for treatment-effect in the change in plasma levels of this established inflammatory marker (R2=0.99; p<0.001. Our findings suggest that LA has anti-inflammatory and endothelium protective effects beyond its well-established efficacy in lowering apoB100-containing lipoproteins.

  6. Alterations of type IV collagen alpha chains in patients with chronic acquired glomerulopathies: mRNA levels, protein expression and urinary loss.

    Science.gov (United States)

    Sanna-Cherchi, Simone; Carnevali, Maria Luisa; Martorana, Davide; Cravedi, Paolo; Maggiore, Umberto; Alinovi, Rossella; Bovino, Achiropita; Mattei, Silvia; Orlandini, Guido; Gatti, Rita; Savi, Mario; Sado, Yoshikazu; Neri, Tauro M; Allegri, Landino

    2007-01-01

    Type IV collagen is a major structural component of the normal kidney glomerulus. However, its role in chronic acquired glomerulopathies has been only partially elucidated. Urinary levels of col(IV)alpha1, col(IV)alpha3 and col(IV)alpha5 collagen chains were analyzed in 107 patients with chronic acquired glomerulopathies. In a subgroup of 33 patients, tissue mRNA levels, protein expression and urinary excretion were evaluated for all col(IV)alpha chains, from col(IV)alpha1 to col(IV)alpha5. The renal specimens were examined to get a semiquantitative score of the acute and chronic activity of the histological lesions. Urines obtained from 13 healthy subjects and 10 normal renal tissue samples were used as controls. Urinary levels of col(IV)alpha1, col(IV)alpha3, col(IV)alpha5 chains were significantly higher in patients than in controls [p < 0.01 for all], while only col(IV)alpha1 and col(IV)alpha3 urinary excretion correlated with the degree of chronic histological damage [col(IV)alpha1 R = 0.44, p < 0.001; col(IV)alpha3: R = 0.47, p < 0.001]. Compared with controls, patients showed a renal expression of mRNA for col(IV)alpha5 chain significantly higher [p = 0.001], while having a significantly lower protein expression of col(IV)alpha3, col(IV)alpha4 and col(IV)alpha5 chains [p < 0.01 for all]. Patients with chronic acquired glomerulopathies show important alterations in the col(IV)alpha chain network mimicking some molecular features of the X-linked Alport's syndrome. Further studies are needed to show whether urinary levels of the col(IV)alpha chains may be used as markers for monitoring renal injury. Copyright 2007 S. Karger AG, Basel.

  7. Quantifying Temporal Autocorrelations for the Expression of Geobacter species mRNA Gene Transcripts at Variable Ammonium Levels during in situ U(VI) Bioremediation

    Science.gov (United States)

    Mouser, P. J.

    2010-12-01

    In order to develop decision-making tools for the prediction and optimization of subsurface bioremediation strategies, we must be able to link the molecular-scale activity of microorganisms involved in remediation processes with biogeochemical processes observed at the field-scale. This requires the ability to quantify changes in the in situ metabolic condition of dominant microbes and associate these changes to fluctuations in nutrient levels throughout the bioremediation process. It also necessitates a need to understand the spatiotemporal variability of the molecular-scale information to develop meaningful parameters and constraint ranges in complex bio-physio-chemical models. The expression of three Geobacter species genes (ammonium transporter (amtB), nitrogen fixation (nifD), and a housekeeping gene (recA)) were tracked at two monitoring locations that differed significantly in ammonium (NH4+) concentrations during a field-scale experiment where acetate was injected into the subsurface to simulate Geobacteraceae in a uranium-contaminated aquifer. Analysis of amtB and nifD mRNA transcript levels indicated that NH4+ was the primary form of fixed nitrogen during bioremediation. Overall expression levels of amtB were on average 8-fold higher at NH4+ concentrations of 300 μM or more than at lower NH4+ levels (average 60 μM). The degree of temporal correlation in Geobacter species mRNA expression levels was calculated at both locations using autocorrelation methods that describe the relationship between sample semi-variance and time lag. At the monitoring location with lower NH4+, a temporal correlation lag of 8 days was observed for both amtB and nifD transcript patterns. At the location where higher NH4+ levels were observed, no discernable temporal correlation lag above the sampling frequency (approximately every 2 days) was observed for amtB or nifD transcript fluctuations. Autocorrelation trends in recA expression levels at both locations indicated that

  8. Constitutive mRNA expression and protein activity levels of nine ABC efflux transporters in seven permanent cell lines derived from different tissues of rainbow trout (Oncorhynchus mykiss).

    Science.gov (United States)

    Fischer, Stephan; Loncar, Jovica; Zaja, Roko; Schnell, Sabine; Schirmer, Kristin; Smital, Tvrtko; Luckenbach, Till

    2011-01-25

    Permanent fish cell lines have become common model systems for determining ecotoxicological effects of pollutants. For these cell lines little is known on the cellular active transport mechanisms that control the amount of a compound entering the cell, such as the MXR (multixenobiotic resistance) system mediated by ATP binding cassette (ABC) transport proteins. Therefore, for toxic evaluation of chemicals with those cells information on MXR is important. We here present data on constitutive mRNA expression and protein activity levels of a series of ABC efflux transporters in seven permanent cell lines derived from liver (RTL-W1; R1) and liver hepatoma (RTH-149), gill (RTgill-W1), gonad (RTG-2), gut (RTgutGC) and brain (RTbrain) of rainbow trout (Oncorhynchus mykiss). In addition to known transporters abcb1 (designated here abcb1a), abcb11, abcc1-3, abcc5 and abcg2, we quantified expression levels of a newly identified abcb1 isoform (abcb1b) and abcc4, previously unknown in trout. Quantitative real time PCR (qPCR) indicated that mRNA of the examined ABC transporters was constitutively expressed in all cell lines. Transporter mRNA expression patterns were similar in all cell lines, with expression levels of abcc transporters being 80 to over 1000 fold higher than for abcg2, abcb1a/b and abcb11 (abcc1-5>abcg2>abcb1a/b, 11). Transporter activity in the cell lines was determined by measuring uptake of transporter type specific fluorescent substrates in the presence of activity inhibitors. The combination of the ABCB1 and ABCC transporter substrate calcein-AM with inhibitors cyclosporine A, PSC833 and MK571 resulted in a concentration-dependent fluorescence increase of up to 3-fold, whereas reversin 205 caused a slight, but not concentration-dependent fluorescence increase. Accumulation of the dyes Hoechst 33342 and 2',7'-dichlorodihydrofluorescein diacetate was basically unchanged in the presence of Ko134 and taurocholate, respectively, indicating low Abcg2 and Abcb11

  9. MicroRNA-15a finetunes the level of Delta-like 1 homologue (DLK1) in proliferating 3T3-L1 preadipocytes

    DEFF Research Database (Denmark)

    Andersen, Ditte C; Jensen, Charlotte H; Schneider, Mikael

    2010-01-01

    Delta like 1 homologue (Dlk1) exists in both transmembrane and soluble molecular forms, and is implicated in cellular growth and plays multiple roles in development, tissue regeneration, and cancer. Thus, DLK1 levels are critical for cell function, and abnormal DLK1 expression can be lethal...... increases with cell density, and peaks at the same stage where membrane DLK1(M) and soluble DLK1(S) are found at maximum levels. Remarkably, miR-15a represses the amount of all Dlk1 variants at the mRNA level but also the level of DLK1(M) protein while it increases the amount of DLK1(S) supporting a direct...... while increasing cell numbers, scenarios that were completely rescued by addition of purified DLK1(S). Our data thus imply that miR-15a regulates cell size and proliferation by fine-tuning Dlk1 among others, and further emphasize miR-15a and DLK1 levels to play important roles in growth signaling...

  10. A standardized seabuckthorn leaf extract (SBL-1) counters radiation induced renal histopathology, oxidative stress as well as changes in mRNA levels

    International Nuclear Information System (INIS)

    Saini, Manu; Madhu Bala; Prasad, Jagdish; Farooqi, Humaira; Abdin, M.Z.

    2014-01-01

    Hippophae rhamnoides L., (common name Seabuckthorn; Family: Elaeagnaceae) is a plant growing naturally as well as cultivated in North-West Himalayas at 7000-15,000 feet. It is known for antioxidant and medicinal properties. Our earlier studies showed that administration of SBL-1 (30 mg/kg body weight), 30 minutes before 60 Co-γ-radiation (10 Gy, lethal dose) rendered > 90% survival in mice population. The present study was planned to investigate the effects of radioprotective dose (30 mg/kg body weight) dose of SBL-1 treatment in kidneys of irradiated and control animals. Strain 'A' male mice (weighing 28±2 g) were irradiated without or 30 minutes after administration of SBL-1. Animals were sacrificed at different days (1, 2, 3, 5, 7 and 15) after the treatment. Histopathology and biochemical assays were performed using standardized procedures. Gene expression study was performed by PCR of mRNA. The 60 Co γ-irradiated animals showed a significant reduction in total thiol (T-SH) content till day 5 (p< 0.05), activity of catalase, superoxide dismutase (SOD) (p<0.01) and glutathione-s-transferase (GST) (p<0.05) and significant increase in LPx, ALP and free iron content (p<0.05) on all study days. Histopathology showed Glomeruli shrinkage, nuclear degeneration, tubular dilations, widening of tubular lumen and collapsing of cellular architecture which increased from day 2 till day 7. Significant alterations in mRNA levels of some of the key genes involved in acute renal failure were observed. In animals treated with SBL-1 before irradiation the T-SH increased significantly at day 2, 3 and 5, activity of catalase, SOD and GST decreased significantly (p<0.05) only at day 2 and 3 respectively. Significant increase in (p<0.05) LPx was observed till day 3, ALP levels only at day 3 while free iron content till day 5. Only mild changes in the tissues histology were observed at day 2, 5 and 7. By day 10 no significant difference was observed in comparison to

  11. Circadian transitions in radiation dose-dependent augmentation of mRNA levels for DNA damage-induced genes elicited by accurate real-time RT-PCR quantification

    International Nuclear Information System (INIS)

    Ishihara, Hiroshi; Tanaka, Izumi; Yakumaru, Haruko

    2010-01-01

    Molecular mechanisms of intracellular response after DNA-damage by exposure to ionizing radiation have been studied. In the case of cells isolated from living body of human and experimental animals, alteration of the responsiveness by physiological oscillation such as circadian rhythm must be considered. To examine the circadian variation in the response of p53-responsible genes p21, mdm2, bax, and puma, we established a method to quantitate their mRNA levels with high reproducibility and accuracy based on real-time reverse transcription polymerase chain reaction (RT-PCR) and compared the levels of responsiveness in mouse hemocytes after diurnal irradiation to that after nocturnal irradiation. Augmentations of p21 and mdm2 mRNA levels with growth-arrest and of puma mRNA before apoptosis were confirmed by time-course experiment in RAW264.7, and dose-dependent increases in the peak levels of all the RNA were shown. Similarly, the relative RNA levels of p21, mdm2, bax, and puma per glyceraldehyde-3-phosphate dehydrogenase (GAPDH) also increased dose-dependently in peripheral blood and bone marrow cells isolated from whole-body-irradiated mice. Induction levels of all messages reduced by half after nighttime irradiation as compared with daytime irradiation in blood cells. In marrow cells, nighttime irradiation enhanced the p21 and mdm2 mRNA levels than daytime irradiation. No significant difference in bax or puma mRNA levels was observed between nighttime and daytime irradiation in marrow cells. This suggests that early-stage cellular responsiveness in DNA damage-induced genes is modulated between diurnal and nocturnal irradiation. (author)

  12. Comparison of mRNA, Protein, and Urinary Nucleic Acid Levels of S100A8 and S100A9 between Prostate Cancer and BPH.

    Science.gov (United States)

    Yun, Seok Joong; Yan, Chunri; Jeong, Pildu; Kang, Ho Won; Kim, Ye-Hwan; Kim, Eun-Ah; Lee, Ok-Jun; Kim, Won Tae; Moon, Sung-Kwon; Kim, Isaac Yi; Choi, Yung-Hyun; Kim, Wun-Jae

    2015-07-01

    Infections and inflammation in the prostate play a critical role in carcinogenesis, and S100A8 and S100A9 are key mediators in acute and chronic inflammation. Therefore, we investigated the differences of S100A8/A9 expression between prostate cancer (CaP) and benign prostatic hyperplasia (BPH) tissues, and we evaluated the possibilities of urinary nucleic acids of S100A8/A9 as diagnostic and prognostic markers. Tissues from 132 CaP patients who underwent prostatectomy or transurethral resection and 90 BPH patients who underwent transurethral prostatectomy were assessed.sd In addition, S100A8 and S100A9 nucleic acid levels were measured in the urine of 283 CaP patients and 363 BPH controls. S100A8 and S100A9 mRNA levels were lower in CaP than BPH tissues (P BPH tissues stained more strongly for both S100A8 and S100A9 than CaP tissues (P BPH (P = 0.001 and BPH. Both were more highly expressed in patients with aggressive disease and shorter biochemical recurrence-free time. S100A8/A9 urinary cell-free nucleic acid levels correlated positively with expression levels obtained from tissue staining. Therefore, S100A8/A9 measurement in tissues and urine may have diagnostic and prognostic value in CaP.

  13. Molecular profiling of short-term and long-term surviving patients identifies CD34 mRNA level as prognostic for glioblastoma survival

    DEFF Research Database (Denmark)

    Michaelsen, Signe Regner; Urup, Thomas; Olsen, Lars Rønn

    2018-01-01

    Despite extensive treatment, overall survival (OS) for glioblastoma (GBM) remains poor. A small proportion of patients present long survival over 3 years, but the underlying molecular background separating these long-term survivors (LTS) from short-term survivors (STS) are insufficiently understood....... Accordingly, study aim was to identify independent prognostic biomarkers for survival. Study cohort consisted of 93 primary GBM patients treated with radiation-, chemo- and bevacizumab therapy, among which 14 STS (OS ≤ 12 months) and 6 LTS (OS ≥ 36 months) were identified, all confirmed being IDH wild......-type. RNA expression levels in diagnostic tumor specimen for 792 genes were analyzed by NanoString technology. While no differences were found with regard to GBM subtype between LTS versus STS, comparative analysis of individual genes identified 14 significantly differently expressed candidate genes...

  14. Molecular response to imatinib & its correlation with mRNA expression levels of imatinib influx & efflux transporters in patients with chronic myeloid leukaemia in chronic phase

    Directory of Open Access Journals (Sweden)

    Hemant Malhotra

    2015-01-01

    Full Text Available Background & objectives: Imatinib is the standard first-line treatment for chronic myeloid leukaemia (CML patients. About 20 to 30 per cent patients develop resistance to imatinib and fail imatinib treatment. One of the mechanisms proposed is varying expression levels of the drug transporters. This study was aimed to determine the expression levels of imatinib transporter genes (OCT1, ABCB1, ABCG2 in CML patients and to correlate these levels with molecular response. Methods: Sixty three CML chronic phase patients who were on 400 mg/day imatinib for more than two years were considered for gene expression analysis study for OCT1, ABCB1 and ABCG2 genes. These were divided into responders and non-responders. The relative transcript expression levels of the three genes were compared between these two categories. The association between the expression values of these three genes was also determined. Results: No significant difference in the expression levels of OCT1, ABCB1 and ABCG2 was found between the two categories. The median transcript expression levels of OCT1, ABCB1 and ABCG2 genes in responders were 26.54, 10.78 and 0.64 versus 33.48, 7.09 and 0.53 in non-responders, respectively. A positive association was observed between the expression of the ABCB1 and ABCG2 transporter genes (r=0.407, P<0.05 while no association was observed between the expression of either of the ABC transporter genes with the OCT1 gene. Interpretation & conclusions: Our findings demonstrated that the mRNA expression levels of imatinib transporter genes were not correlated with molecular response in CML patients. Further studies need to be done on a large sample of CML patients to confirm these findings.

  15. Effect of low levels of dietary available phosphorus on phosphorus utilization, bone mineralization, phosphorus transporter mRNA expression and performance in growing pigs.

    Science.gov (United States)

    Pokharel, Bishwo B; Regassa, Alemu; Nyachoti, Charles M; Kim, Woo K

    2017-06-03

    A study was conducted to examine the effects of different dietary levels of available phosphorus (aP) on P excretion, bone mineralization, performance and the mRNA expression of sodium-dependent P transporters in growing pigs. Sixty-day old growing pigs (n = 54) with an average initial BW of 19.50 ± 1.11 kg were randomly allocated to a control diet (C) containing 0.23% available phosphorus (aP), T1 containing 0.17% aP and T2 containing 0.11% aP. There were 6 pens per treatment with 3 pigs per pen. Body weight and feed intake were measured weekly. At the end of each week, one pig from each pen was housed in a metabolic crate for 24 h to collect fecal and urine samples and then sacrificed to obtain third metacarpal (MC3) bones and jejunal and kidney samples. Bones were scanned by Dual Energy X-ray Absorptiometry (DEXA). Fecal and urine samples were sub-sampled and analyzed for P content. The expression of P transporter mRNA in jejunum and kidney samples was measured using quantitative real-time polymerase chain reaction (qRT-PCR). Data were analyzed using GLM procedure of the Statistical Analysis System (SAS Institute version 9.2). Pigs fed the T2 diet had reduced (P reduced (P reduced (P reduced ADG, bone mineralization and urinary P level, but moderate reduction in dietary P up to 0.17% aP in the diet has the potential to reduce environmental pollution by reducing P concentration in swine manure and without compromising performance.

  16. In vitro effects of four native Brazilian medicinal plants in CYP3A4 mRNA gene expression, glutathione levels and P-glycoprotein activity.

    Directory of Open Access Journals (Sweden)

    Andre Luis Dias Araujo Mazzari

    2016-08-01

    Full Text Available Erythrina mulungu Benth. (Fabaceae, Cordia verbenacea A. DC. (Boraginaceae, Solanum paniculatum L. (Solanaceae and Lippia sidoides Cham. (Verbenaceae are medicinal plants species native to Brazil shortlisted by the Brazilian National Health System for future clinical use. However, nothing is known about their effects in metabolic and transporter proteins, which could potentially lead to herb-drug interactions (HDI. In this work we assess non-toxic concentrations (100μg/mL of their infusions for their in vitro ability to modulate CYP3A4 mRNA gene expression and intracellular glutathione levels in HepG2 cells, as well as P-glycoprotein (P-gp activity in vincristine-resistant Caco-2 cells (Caco-2 VCR. Their mechanisms of action were further studied by measuring the activation of human pregnane X receptor (hPXR in transiently co-transfected HeLa cells and the inhibition of γ-glutamyl transferase (GGT in HepG2 cells. Our results show that P-gp activity was not affected in any case and that only Solanum paniculatum was able to significantly change CYP3A4 mRNA gene expression (two-fold decrease, p<0.05, this being correlated with an antagonist effect upon hPXR (EC50 = 0.38mg/mL. Total intracellular glutathione levels were significantly depleted by exposure to Solanum paniculatum (-44%, p<0.001, Lippia sidoides (-12%, p<0.05 and Cordia verbenacea (-47%, p<0.001. The later plant extract was able to decrease GGT activity (-48%, p<0.01. In conclusion, this preclinical study shows that the administration of some of these herbal medicines may be able to cause disturbances to metabolic mechanisms in vitro. Although Erythrina mulungu appears safe in our tests, active pharmacovigilance is recommended for the other three species, especially in the case of Solanum paniculatum.

  17. A predominance of R5-like HIV genotypes in vaginal secretions is associated with elevated plasma HIV-1 RNA levels and the absence of anti-retroviral therapy

    Directory of Open Access Journals (Sweden)

    Lacour Nedra

    2008-07-01

    Full Text Available Abstract HIV expressed in genital secretions provides the inoculum from which transmitting variants are selected, both in sexual transmission and mother-to-infant transmission during partuition. Characterization of HIV levels and genotypes found in vaginal secretions and the impact of anti-retroviral therapy (ART on this virus can provide valuable insight for the prevention of HIV transmission. Vaginal HIV was evaluated in a cohort of 43 women attending a New Orleans HIV outpatient clinic. Predominant vaginal genotypes were characterized as R5- or X4-like by heteroduplex tracking analyses of the envelope V3 region. Most women (67.4% shed R5-like genotypes in vaginal secretions which was associated with elevated plasma HIV levels (≥ 10,000 copies HIV-RNA/mL and absence of ART. Because R5-like genotypes are more frequently associated with transmission, these observations suggest that the majority of women shedding HIV in genital secretions present a transmission risk. The levels of vaginal virus were similar between both groups, but shedding of X4-like genotypes was associated with lower plasma viral loads and the use of ART, suggesting that ART use may impact the genotypes of virus found in the female genital compartment.

  18. Associations of Haplotypes Upstream of IRS1 with Insulin Resistance, Type 2 Diabetes, Dyslipidemia, Preclinical Atherosclerosis, and Skeletal Muscle LOC646736 mRNA Levels

    Directory of Open Access Journals (Sweden)

    Selma M. Soyal

    2015-01-01

    Full Text Available The genomic region ~500 kb upstream of IRS1 has been implicated in insulin resistance, type 2 diabetes, adverse lipid profile, and cardiovascular risk. To gain further insight into this chromosomal region, we typed four SNPs in a cross-sectional cohort and subjects with type 2 diabetes recruited from the same geographic region. From 16 possible haplotypes, 6 haplotypes with frequencies >0.01 were observed. We identified one haplotype that was protective against insulin resistance (determined by HOMA-IR and fasting plasma insulin levels, type 2 diabetes, an adverse lipid profile, increased C-reactive protein, and asymptomatic atherosclerotic disease (assessed by intima media thickness of the common carotid arteries. BMI and total adipose tissue mass as well as visceral and subcutaneous adipose tissue mass did not differ between the reference and protective haplotypes. In 92 subjects, we observed an association of the protective haplotype with higher skeletal muscle mRNA levels of LOC646736, which is located in the same haplotype block as the informative SNPs and is mainly expressed in skeletal muscle, but only at very low levels in liver or adipose tissues. These data suggest a role for LOC646736 in human insulin resistance and warrant further studies on the functional effects of this locus.

  19. Charging Levels of Four tRNA Species in Escherichia coli Rel+ and REL- Strains during Amino Acid Starvation: A Simple Model for the Effect of ppGpp on Translational Accuracy

    DEFF Research Database (Denmark)

    Sørensen, M.A.

    2001-01-01

    Escherichia coli strains mutated in the relA gene lack the ability to produce ppGpp during amino acid starvation. One consequence of this deficiency is a tenfold increase in misincorporation at starved codons compared to the wild-type. Previous work had shown that the charging levels of tRNAs were...... the same in Rel+ and Rel- strains and reduced, at most, two- to fivefold in both strains during starvation. The present reinvestigation of the charging levels of tRNA2Arg, tRNA1Thr, tRNA1Leu and tRNAHis during starvation of isogenic Rel+ and Rel- strains showed that starvation reduced charging levels...... tenfold to 40-fold. This reduction corresponds much better with the decreased rate of protein synthesis during starvation than that reported earlier. The determination of the charging levels of tRNA2Arg and tRNA1Thr during starvation were accurate enough to demonstrate that charging levels were at least...

  20. p38 mitogen-activated protein kinase (p38MAPK) upregulates catalase levels in response to low dose H2O2 treatment through enhancement of mRNA stability.

    Science.gov (United States)

    Sen, Prosenjit; Chakraborty, Prabir Kumar; Raha, Sanghamitra

    2005-08-15

    V79 fibroblasts were repetitively stressed through multiple exposures to a low dose (30 microM) H2O2 in culture for 4 weeks. Catalase activity, protein levels and mRNA levels increased markedly (5-6-fold) during this time and these augmentations were inhibited by the simultaneous presence of SB203580, an inhibitor of p38 mitogen-activated protein kinase (p38MAPK). p38MAPK became dually phosphorylated and ATF-2, a p38MAPK substrate also became increasingly phosphorylated over the repetitive stress period. Short interfering RNA that induced effective silencing of p38MAPK, was used to silence p38MAPK in V79 fibroblasts. Silencing of p38MAPK drastically hindered the elevation in catalase (protein and mRNA) levels observed after a single low dose (50 microM) of H2O. The rise in catalase mRNA levels induced by low concentration (single and multiple dose) H2O2 treatment was established to be unconnected with transcriptional upregulation but was brought forth primarily by an enhancement in catalase mRNA stability through the action of p38MAPK. Therefore, our data strongly indicate that activation of p38MAPK is a key controlling step in the upregulation of catalase levels by low dose H2O2 treatment.

  1. Protein arginine methyltransferase 7-mediated microRNA-221 repression maintains Oct4, Nanog, and Sox2 levels in mouse embryonic stem cells.

    Science.gov (United States)

    Chen, Tsai-Yu; Lee, Sung-Hun; Dhar, Shilpa S; Lee, Min Gyu

    2018-03-16

    The stemness maintenance of embryonic stem cells (ESCs) requires pluripotency transcription factors, including Oct4, Nanog, and Sox2. We have previously reported that protein arginine methyltransferase 7 (PRMT7), an epigenetic modifier, is an essential pluripotency factor that maintains the stemness of mouse ESCs, at least in part, by down-regulating the expression of the anti-stemness microRNA (miRNA) miR-24-2. To gain greater insight into the molecular basis underlying PRMT7-mediated maintenance of mouse ESC stemness, we searched for new PRMT7-down-regulated anti-stemness miRNAs. Here, we show that miR-221 gene-encoded miR-221-3p and miR-221-5p are anti-stemness miRNAs whose expression levels in mouse ESCs are directly repressed by PRMT7. Notably, both miR-221-3p and miR-221-5p targeted the 3' untranslated regions of mRNA transcripts of the major pluripotency factors Oct4, Nanog, and Sox2 to antagonize mouse ESC stemness. Moreover, miR-221-5p silenced also the expression of its own transcriptional repressor PRMT7. Transfection of miR-221-3p and miR-221-5p mimics induced spontaneous differentiation of mouse ESCs. CRISPR-mediated deletion of the miR-221 gene, as well as specific antisense inhibitors of miR-221-3p and miR-221-5p, inhibited the spontaneous differentiation of PRMT7-depleted mouse ESCs. Taken together, these findings reveal that the PRMT7-mediated repression of miR-221-3p and miR-221-5p expression plays a critical role in maintaining mouse ESC stemness. Our results also establish miR-221-3p and miR-221-5p as anti-stemness miRNAs that target Oct4 , Nanog , and Sox2 mRNAs in mouse ESCs. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Phosphatase Rtr1 Regulates Global Levels of Serine 5 RNA Polymerase II C-Terminal Domain Phosphorylation and Cotranscriptional Histone Methylation.

    Science.gov (United States)

    Hunter, Gerald O; Fox, Melanie J; Smith-Kinnaman, Whitney R; Gogol, Madelaine; Fleharty, Brian; Mosley, Amber L

    2016-09-01

    In eukaryotes, the C-terminal domain (CTD) of Rpb1 contains a heptapeptide repeat sequence of (Y1S2P3T4S5P6S7)n that undergoes reversible phosphorylation through the opposing action of kinases and phosphatases. Rtr1 is a conserved protein that colocalizes with RNA polymerase II (RNAPII) and has been shown to be important for the transition from elongation to termination during transcription by removing RNAPII CTD serine 5 phosphorylation (Ser5-P) at a selection of target genes. In this study, we show that Rtr1 is a global regulator of the CTD code with deletion of RTR1 causing genome-wide changes in Ser5-P CTD phosphorylation and cotranscriptional histone H3 lysine 36 trimethylation (H3K36me3). Using chromatin immunoprecipitation and high-resolution microarrays, we show that RTR1 deletion results in global changes in RNAPII Ser5-P levels on genes with different lengths and transcription rates consistent with its role as a CTD phosphatase. Although Ser5-P levels increase, the overall occupancy of RNAPII either decreases or stays the same in the absence of RTR1 Additionally, the loss of Rtr1 in vivo leads to increases in H3K36me3 levels genome-wide, while total histone H3 levels remain relatively constant within coding regions. Overall, these findings suggest that Rtr1 regulates H3K36me3 levels through changes in the number of binding sites for the histone methyltransferase Set2, thereby influencing both the CTD and histone codes. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  3. Detection of high levels of European bat lyssavirus type-1 viral RNA in the thyroid gland of experimentally-infected Eptesicus fuscus bats.

    Science.gov (United States)

    Fooks, A R; Johnson, N; Müller, T; Vos, A; Mansfield, K; Hicks, D; Nunez, A; Freuling, C; Neubert, L; Kaipf, I; Denzinger, A; Franka, R; Rupprecht, C E

    2009-08-01

    Two common bat lyssavirus species have been identified in many European countries: European bat lyssavirus type-1 and -2 (EBLV-1 and EBLV-2). Only limited knowledge on the susceptibility of the natural EBLV-hosts, insectivorous bats, to lyssavirus infection is available. Our study was undertaken to evaluate the susceptibility and pathology associated with an EBLV-1 infection in Eptesicus fuscus following different routes of virus inoculation including intracranial (n = 6), intramuscular (n = 14), oral (n = 7) and intranasal (n = 7). Blood and saliva samples were collected from all bats on a monthly basis. Four bats inoculated intracranially developed rabies with a mean of 11 days to death, whilst seven bats inoculated intramuscularly developed rabies, with an extended incubation period prior to death. We did not observe any mortality in the oral (p.o.) or intranasal (i.n.) groups and both groups had detectable levels of virus neutralizing antibodies (data not shown). Virus shedding was demonstrated in the saliva by virus isolation and the detection of viral RNA in ill bats, particularly immediately prior to the development of disease. In addition, the presence of virus and viral RNA was detected in the thyroid gland in bats challenged experimentally with EBLV-1, which exceeded that detected in all other extra-neural tissue. The significance of detecting EBLV-1 in the thyroid gland of rabid bats is not well understood. We speculate that the infection of the thyroid gland may cause subacute thyroiditis, a transient form of thyroiditis causing hyperthyroidism, resulting in changes in adrenocortical activity that could lead to hormonal dysfunction, thereby distinguishing the clinical presentation of rabies in the rabid host.

  4. In vitro Effects of Four Native Brazilian Medicinal Plants in CYP3A4 mRNA Gene Expression, Glutathione Levels, and P-Glycoprotein Activity

    Science.gov (United States)

    Mazzari, Andre L. D. A.; Milton, Flora; Frangos, Samantha; Carvalho, Ana C. B.; Silveira, Dâmaris; de Assis Rocha Neves, Francisco; Prieto, Jose M.

    2016-01-01

    Erythrina mulungu Benth. (Fabaceae), Cordia verbenacea A. DC. (Boraginaceae), Solanum paniculatum L. (Solanaceae) and Lippia sidoides Cham. (Verbenaceae) are medicinal plant species native to Brazil shortlisted by the Brazilian National Health System for future clinical use. However, nothing is known about their effects in metabolic and transporter proteins, which could potentially lead to herb-drug interactions (HDI). In this work, we assess non-toxic concentrations (100 μg/mL) of the plant infusions for their in vitro ability to modulate CYP3A4 mRNA gene expression and intracellular glutathione levels in HepG2 cells, as well as P-glycoprotein (P-gp) activity in vincristine-resistant Caco-2 cells (Caco-2 VCR). Their mechanisms of action were further studied by measuring the activation of human pregnane X receptor (hPXR) in transiently co-transfected HeLa cells and the inhibition of γ-glutamyl transferase (GGT) in HepG2 cells. Our results show that P-gp activity was not affected in any case and that only Solanum paniculatum was able to significantly change CYP3A4 mRNA gene expression (twofold decrease, p Cordia verbenacea (-47%, p < 0.001). The latter plant extract was able to decrease GGT activity (-48%, p < 0.01). In conclusion, this preclinical study shows that the administration of some of these herbal medicines may be able to cause disturbances to metabolic mechanisms in vitro. Although Erythrina mulungu appears safe in our tests, active pharmacovigilance is recommended for the other three species, especially in the case of Solanum paniculatum. PMID:27594838

  5. Expression of insulin-like growth factor I receptors at mRNA and protein levels during metamorphosis of Japanese flounder (Paralichthys olivaceus).

    Science.gov (United States)

    Zhang, Junling; Shi, Zhiyi; Cheng, Qi; Chen, Xiaowu

    2011-08-01

    Insulin-like growth factor I (IGF-I) is an important regulator of fish growth and development, and its biological actions are initiated by binding to IGF-I receptor (IGF-IR). Our previous study has revealed that IGF-I could play an important role during metamorphosis of Japanese flounder, Paralichthys olivaceus. The analysis of IGF-IR expression thus helps further elucidate the IGF-I regulation of metamorphic processes. In this study, the spatial-temporal expression of two distinct IGF-IR mRNAs was investigated by real-time RT-PCR. The spatial distribution of two IGF-IR mRNAs in adult tissues is largely overlapped, but they exhibit distinct temporal expression patterns during larval development. A remarkable decrease in IGF-IR-2 mRNA was detected during metamorphosis. In contrast, a significant increase in IGF-IR-1 mRNA was determined from pre-metamorphosis to metamorphic completion. These indicate that they may play different function roles during the flounder metamorphosis. The levels and localization of IGF-IR proteins during larval development were further studied by Western blotting and immunohistochemistry. Immunoreactive IGF-IRs were detected throughout larval development, and the IGF-IR proteins displayed a relatively abundant expression during metamorphosis. Moreover, the IGF-IR proteins appeared in key tissues, such as thickened skin beneath the migrating eye, developing intestine, gills and kidney during metamorphosis. These results further suggest that the IGF-I system may be involved in metamorphic development of Japanese flounder. Copyright © 2011 Elsevier Inc. All rights reserved.

  6. A step-by-step workflow for low-level analysis of single-cell RNA-seq data with Bioconductor [version 2; referees: 1 approved, 4 approved with reservations

    Directory of Open Access Journals (Sweden)

    Aaron T.L. Lun

    2016-10-01

    Full Text Available Single-cell RNA sequencing (scRNA-seq is widely used to profile the transcriptome of individual cells. This provides biological resolution that cannot be matched by bulk RNA sequencing, at the cost of increased technical noise and data complexity. The differences between scRNA-seq and bulk RNA-seq data mean that the analysis of the former cannot be performed by recycling bioinformatics pipelines for the latter. Rather, dedicated single-cell methods are required at various steps to exploit the cellular resolution while accounting for technical noise. This article describes a computational workflow for low-level analyses of scRNA-seq data, based primarily on software packages from the open-source Bioconductor project. It covers basic steps including quality control, data exploration and normalization, as well as more complex procedures such as cell cycle phase assignment, identification of highly variable and correlated genes, clustering into subpopulations and marker gene detection. Analyses were demonstrated on gene-level count data from several publicly available datasets involving haematopoietic stem cells, brain-derived cells, T-helper cells and mouse embryonic stem cells. This will provide a range of usage scenarios from which readers can construct their own analysis pipelines.

  7. Altered microRNA expression levels in mononuclear cells of patients with pulmonary and pleural tuberculosis and their relation with components of the immune response.

    Science.gov (United States)

    Spinelli, Silvana V; Diaz, Ariana; D'Attilio, Luciano; Marchesini, Marcela M; Bogue, Cristina; Bay, Maria L; Bottasso, Oscar A

    2013-03-01

    Different lines of evidence demonstrate that microRNAs (miRNAs) play an important role in host-pathogen interactions. In this study we investigated the expression patterns of several miRNAs, most of them involved in regulating inflammatory responses, in patients with tuberculosis (TB). In order to understand the events occurring at the site of infection, we employed mononuclear cells obtained from both peripheral blood (PBMC) and pleural fluids (PFMC) of patients. Interestingly, we found that the miRNA signature of each compartment is different, with a strong down-regulation in PFMCs of miR-223, miR-144* and miR-421. In addition, we observed that miR-146a expression is also down-regulated in tuberculosis patients, both in PBMCs and PFMCs while miR-424 levels are elevated only in the peripheral compartments. We also showed that systemic expression of these miRNAs changes upon specific treatment and is associated with IL-6 levels, a cytokine playing a substantial role in TB immunopathology. Present results contribute to a better knowledge of the host responses in TB pathogenesis, pointing out the role of miRNAs in this disease. Copyright © 2012 Elsevier Ltd. All rights reserved.

  8. Plant RNA Regulatory Network and RNA Granules in Virus Infection

    Directory of Open Access Journals (Sweden)

    Kristiina Mäkinen

    2017-12-01

    Full Text Available Regulation of post-transcriptional gene expression on mRNA level in eukaryotic cells includes translocation, translation, translational repression, storage, mRNA decay, RNA silencing, and nonsense-mediated decay. These processes are associated with various RNA-binding proteins and cytoplasmic ribonucleoprotein complexes many of which are conserved across eukaryotes. Microscopically visible aggregations formed by ribonucleoprotein complexes are termed RNA granules. Stress granules where the translationally inactive mRNAs are stored and processing bodies where mRNA decay may occur present the most studied RNA granule types. Diverse RNP-granules are increasingly being assigned important roles in viral infections. Although the majority of the molecular level studies on the role of RNA granules in viral translation and replication have been conducted in mammalian systems, some studies link also plant virus infection to RNA granules. An increasing body of evidence indicates that plant viruses require components of stress granules and processing bodies for their replication and translation, but how extensively the cellular mRNA regulatory network is utilized by plant viruses has remained largely enigmatic. Antiviral RNA silencing, which is an important regulator of viral RNA stability and expression in plants, is commonly counteracted by viral suppressors of RNA silencing. Some of the RNA silencing suppressors localize to cellular RNA granules and have been proposed to carry out their suppression functions there. Moreover, plant nucleotide-binding leucine-rich repeat protein-mediated virus resistance has been linked to enhanced processing body formation and translational repression of viral RNA. Many interesting questions relate to how the pathways of antiviral RNA silencing leading to viral RNA degradation and/or repression of translation, suppression of RNA silencing and viral RNA translation converge in plants and how different RNA granules and

  9. Plant RNA Regulatory Network and RNA Granules in Virus Infection.

    Science.gov (United States)

    Mäkinen, Kristiina; Lõhmus, Andres; Pollari, Maija

    2017-01-01

    Regulation of post-transcriptional gene expression on mRNA level in eukaryotic cells includes translocation, translation, translational repression, storage, mRNA decay, RNA silencing, and nonsense-mediated decay. These processes are associated with various RNA-binding proteins and cytoplasmic ribonucleoprotein complexes many of which are conserved across eukaryotes. Microscopically visible aggregations formed by ribonucleoprotein complexes are termed RNA granules. Stress granules where the translationally inactive mRNAs are stored and processing bodies where mRNA decay may occur present the most studied RNA granule types. Diverse RNP-granules are increasingly being assigned important roles in viral infections. Although the majority of the molecular level studies on the role of RNA granules in viral translation and replication have been conducted in mammalian systems, some studies link also plant virus infection to RNA granules. An increasing body of evidence indicates that plant viruses require components of stress granules and processing bodies for their replication and translation, but how extensively the cellular mRNA regulatory network is utilized by plant viruses has remained largely enigmatic. Antiviral RNA silencing, which is an important regulator of viral RNA stability and expression in plants, is commonly counteracted by viral suppressors of RNA silencing. Some of the RNA silencing suppressors localize to cellular RNA granules and have been proposed to carry out their suppression functions there. Moreover, plant nucleotide-binding leucine-rich repeat protein-mediated virus resistance has been linked to enhanced processing body formation and translational repression of viral RNA. Many interesting questions relate to how the pathways of antiviral RNA silencing leading to viral RNA degradation and/or repression of translation, suppression of RNA silencing and viral RNA translation converge in plants and how different RNA granules and their individual

  10. Association of expression of selenoprotein P in mRNA and protein levels with metabolic syndrome in subjects with cardiovascular disease: Results of the Selenegene study.

    Science.gov (United States)

    Gharipour, Mojgan; Sadeghi, Masoumeh; Salehi, Mansour; Behmanesh, Mehrdad; Khosravi, Elham; Dianatkhah, Minoo; Haghjoo Javanmard, Shaghayegh; Razavi, Rouzbeh; Gharipour, Amin

    2017-03-01

    Selenoprotein P (SeP) is involved in transporting selenium from the liver to target tissues. Because SeP confers protection against disease by reducing chronic oxidative stress, the present study aimed to assess the level of SeP in the serum of patients with metabolic syndrome (MetS) with a history of cardiovascular disease (CVD). A cross-sectional study was conducted in 63 and 71 subjects with and without MetS in the presence of documented CVD. All demographic, anthropometric and cardiometabolic variables (lipids, blood glucose, blood pressure) were assessed. Lifestyle-related factors and personal history and familial CVD risk factors were recorded. The expression of SELP in mRNA and protein levels in the serum was measured, and MetS was determined using ATPIII criteria. Binary logistic regression analysis demonstrated MetS and SeP to be dependent and independent variables, respectively. Mean of systolic and diastolic blood pressure, triglyceride, high-density lipoprotein-cholesterol, fasting blood sugar, body mass index and waist circumference were higher among subjects with MetS (p = 0.05). The mean of selenium was higher among subjects with MetS, whereas the mean of SeP was lower among subjects with MetS (p family history, smoking status and nutrition. SeP and waist circumference show a significant relationship (OR =0.995; 95% CI = 0.990-1.00) (p < 0.033). We have demonstrated a significant decrease in circulating SeP levels according to MetS status in patients with documented cardiovascular disease. Copyright © 2017 John Wiley & Sons, Ltd.

  11. MicroRNA-27a decreases the level and efficiency of the LDL receptor and contributes to the dysregulation of cholesterol homeostasis.

    Science.gov (United States)

    Alvarez, M Lucrecia; Khosroheidari, Mahdieh; Eddy, Elena; Done, Stefania C

    2015-10-01

    A strong risk factor for atherosclerosis- the leading cause of heart attacks and strokes- is the elevation of low-density lipoprotein cholesterol (LDL-C) in blood. The LDL receptor (LDLR) is the primary pathway for LDL-C removal from circulation, and their levels are increased by statins -the main treatment for high blood LDL-C. However, statins have low efficiency because they also increase PCSK9 which targets LDLR for degradation. Since microRNAs have recently emerged as key regulators of cholesterol homeostasis, our aim was to identify potential microRNA-based therapeutics to decrease blood LDL-C and prevent atherosclerosis. We over expressed and knocked down miR-27a in HepG2 cells to assess its effect on the expression of key players in the LDLR pathway using PCR Arrays, Elisas, and Western blots. We found that miR-27a decreases LDLR levels by 40% not only through a direct binding to its 3' untranslated region but also indirectly by inducing a 3-fold increase in PCSK9, which enhances LDLR degradation. Interestingly, miR-27a also directly decreases LRP6 and LDLRAP1, two other key players in the LDLR pathway that are required for efficient endocytosis of the LDLR-LDL-C complex in the liver. The inhibition of miR-27a using lock nucleic acids induced a 70% increase in LDLR levels and, therefore, it would be a more efficient treatment for hypercholesterolemia because of its desirable effects not only on LDLR but also on PCSK9. The results presented here provide evidence supporting the potential of miR-27a as a novel therapeutic target for the prevention of atherosclerosis. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  12. 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)

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

    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 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 < .01 and P < .05, respectively. We found a sensitivity of 100% and a specificity of 61.5% for the prediction of invasive 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. PMID:23744883

  13. 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

  14. mRNA Levels of Placental Iron and Zinc Transporter Genes Are Upregulated in Gambian Women with Low Iron and Zinc Status.

    Science.gov (United States)

    Jobarteh, Modou Lamin; McArdle, Harry J; Holtrop, Grietje; Sise, Ebrima A; Prentice, Andrew M; Moore, Sophie E

    2017-07-01

    Background: The role of the placenta in regulating micronutrient transport in response to maternal status is poorly understood. Objective: We investigated the effect of prenatal nutritional supplementation on the regulation of placental iron and zinc transport. Methods: In a randomized trial in rural Gambia [ENID (Early Nutrition and Immune Development)], pregnant women were allocated to 1 of 4 nutritional intervention arms: 1 ) iron and folic acid (FeFol) tablets (FeFol group); 2 ) multiple micronutrient (MMN) tablets (MMN group); 3 ) protein energy (PE) as a lipid-based nutrient supplement (LNS; PE group); and 4 ) PE and MMN (PE+MMN group) as LNS. All arms included iron (60 mg/d) and folic acid (400 μg/d). The MMN and PE+MMN arms included 30 mg supplemental Zn/d. In a subgroup of ∼300 mother-infant pairs, we measured maternal iron status, mRNA levels of genes encoding for placental iron and zinc transport proteins, and cord blood iron levels. Results: Maternal plasma iron concentration in late pregnancy was 45% and 78% lower in the PE and PE+MMN groups compared to the FeFol and MMN groups, respectively ( P Zinc supplementation in the MMN arm was associated with higher maternal plasma zinc concentrations (10% increase; P zinc-uptake proteins, in this case zrt, irt-like protein (ZIP) 4 and ZIP8, were 96-205% lower in the PE+MMN arm than in the intervention arms without added zinc ( P zinc, the placenta upregulates the gene expression of iron and zinc uptake proteins, presumably in order to meet fetal demands in the face of low maternal supply. The ENID trial was registered at www.controlled-trials.com as ISRCTN49285450.

  15. Molecular characterization of cytochrome P450 1A and 3A and the effects of perfluorooctanoic acid on their mRNA levels in rare minnow (Gobiocypris rarus) gills

    Energy Technology Data Exchange (ETDEWEB)

    Liu Yong; Wang Jianshe; Wei Yanhong; Zhang Hongxia; Liu Yang [Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Datun Road, Beijing 100101 (China); Dai Jiayin [Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Datun Road, Beijing 100101 (China)], E-mail: daijy@ioz.ac.cn

    2008-07-07

    Perfluorooctanoic acid (PFOA), a potentially toxic perfluorinated compound (PFC), has been widely disseminated in the environment. In the present study, rare minnows (Gobiocypris rarus) exposed to PFOA exhibited histopathological gill damage, including epithelial hyperplasia of the lamellae, inflammatory cell infiltration, and lamellar fusion. Cytochrome P450s (CYPs) play a central role in the metabolism and biotransformation of a wide range of endogenous substrates and foreign compounds. Thus, we studied the CYPs and the effects of waterborne PFOA on their corresponding mRNA levels in the gills of rare minnows. Two novel CYP cDNAs (CYP1A and CYP3A) were identified in rare minnow and their mRNAs were ubiquitously expressed in all tissues examined. Upregulation of CYP3A mRNA was observed in the gills of male rare minnows exposed to 30 mg/L PFOA, while no significant changes occurred in exposed females. In contrast, down regulation of CYP1A mRNA was detected in the gills of male and female minnows exposed to PFOA. However, the effect of PFOA on gill mRNA levels of their potential regulators, aryl hydrocarbon receptor (AhR) for CYP1A, and pregnane X receptor (PXR) for CYP3A, were not consistent with the observed effects of PFOA on the corresponding CYP mRNA concentrations. This suggests a different or more complex transcriptional regulation of CYP expression following PFOA exposure.

  16. Macrophage Migration Inhibitory Factor Promoter Polymorphisms (−794 CATT5–8 and −173 G>C: Relationship with mRNA Expression and Soluble MIF Levels in Young Obese Subjects

    Directory of Open Access Journals (Sweden)

    Inés Matia-García

    2015-01-01

    Full Text Available We analyzed the relationship of −794 CATT5–8 and −173 G>C MIF polymorphisms with mRNA and soluble MIF in young obese subjects. A total of 250 young subjects, 150 normal-weight and 100 obese subjects, were recruited in the study. Genotyping of −794 CATT5–8 and −173 G>C MIF polymorphisms was performed by PCR and PCR-RFLP, respectively. MIF mRNA expression was determined by real-time PCR and serum MIF levels were measured using an ELISA kit. For both MIF promoter polymorphisms, no significant differences in the genotype and allele frequencies between groups were observed. MIF mRNA expression was slightly higher in obese subjects than in normal-weight subjects (1.38-fold, while soluble MIF levels did not show differences between groups. In addition, we found an increase in MIF mRNA expression in carriers of the 6,6 and C/C genotypes and the 6G haplotype of the −794 CATT5–8 and −173 G>C MIF polymorphisms, although it was not significant. In conclusion, this study found no relationship between obesity and MIF gene promoter polymorphisms with MIF mRNA expression in young obese subjects.

  17. Effect of different levels of feed restriction and fish oil fatty acid supplementation on fat deposition by using different techniques, plasma levels and mRNA expression of several adipokines in broiler breeder hens.

    Directory of Open Access Journals (Sweden)

    Namya Mellouk

    Full Text Available Reproductive hens are subjected to a restricted diet to limit the decline in fertility associated with change in body mass. However, endocrine and tissue responses to diet restriction need to be documented.We evaluated the effect of different levels of feed restriction, with or without fish oil supplementation, on metabolic parameters and adipokine levels in plasma and metabolic tissues of reproductive hens.We designed an in vivo protocol involving 4 groups of hens; RNS: restricted (Rt unsupplemented, ANS: ad libitum (Ad, receiving an amount of feed 1.7 times greater than animals on the restricted diet unsupplemented, RS: Rt supplemented, and AS: Ad supplemented. The fish oil supplement was used at 1% of the total diet composition.Hens fed with the Rt diet had a significantly (P < 0.0001 lower growth than Ad hens, while the fish oil supplementation had no effect on these parameters. Furthermore, the bioelectrical impedance analysis (BIA and the fat ultrasonographic examinations produced similar results to the other methods that required animals to be killed (carcass analysis and weight of adipose tissue. In addition, the Rt diet significantly (P < 0.05 decreased plasma levels of triglycerides, phospholipids, glucose and ADIPOQ, and fish oil supplementation decreased plasma levels of RARRES2. We also showed a positive correlation between insulin values and ADIPOQ or NAMPT or RARRES2 values, and a negative correlation of fat percentage to RARRES2 values. Moreover, the effects of the Rt diet and fish oil supplementation on the mRNA expression depended on the factors tested and the hen age.Rt diet and fish oil supplementation are able to modulate metabolic parameters and the expression of adipokines and their receptors in metabolic tissue.

  18. A new synthetic drug 5-(2-aminopropyl)indole (5-IT) induces rewarding effects and increases dopamine D1 receptor and dopamine transporter mRNA levels.

    Science.gov (United States)

    Botanas, Chrislean Jun; Yoon, Seong Shoon; de la Peña, June Bryan; Dela Peña, Irene Joy; Kim, Mikyung; Custodio, Raly James; Woo, Taeseon; Seo, Joung-Wook; Jang, Choon-Gon; Yang, Ji Seul; Yoon, Yoon Mi; Lee, Yong Sup; Kim, Hee Jin; Cheong, Jae Hoon

    2018-04-02

    In recent years, there has been a marked increase in the use of recreational synthetic psychoactive substances, which is a cause of concern among healthcare providers and legal authorities. In particular, there have been reports on the misuse of 5-(2-aminopropyl)indole (5-API; 5-IT), a new synthetic drug, and of fatal and non-fatal intoxication. Despite these reports, little is known about its psychopharmacological effects and abuse potential. Here, we investigated the abuse potential of 5-IT by evaluating its rewarding and reinforcing effects through conditioned place preference (CPP) (1, 10, and 30 mg/kg, i.p.) in mice and self-administration test (0.1, 0.3, 1, and 3 mg/kg/inf., i.v.) in rats. We also examined whether 5-IT (1, 3, and 10 mg/kg, i.p.) induces locomotor sensitization in mice following a 7-day treatment and drug challenge. Then, we explored the effects of 5-IT (10 mg/kg, i.p.) on dopamine-related genes in the striatum, prefrontal cortex (PFC), and substantia nigra pars compacta (SNc)/ventral tegmental (VTA) of mice by quantitative real-time polymerase chain reaction. 5-IT produced CPP in mice but was not reliably self-administered by rats. 5-IT also induced locomotor sensitization following repeated administration and drug challenge. Moreover, 5-IT increased mRNA levels of dopamine D1 receptor in the striatum and PFC and dopamine transporter in the SNc/VTA of mice. These results indicate that 5-IT has psychostimulant and rewarding properties, which may be attributed to its ability to affect the dopaminergic system in the brain. These findings suggest that 5-IT poses a substantial risk for abuse and addiction in humans. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Maternal exposure to titanium dioxide nanoparticles during pregnancy and lactation alters offspring hippocampal mRNA BAX and Bcl-2 levels, induces apoptosis and decreases neurogenesis.

    Science.gov (United States)

    Ebrahimzadeh Bideskan, Alireza; Mohammadipour, Abbas; Fazel, Alireza; Haghir, Hossein; Rafatpanah, Houshang; Hosseini, Mahmoud; Rajabzadeh, Aliakbar

    2017-07-05

    The usage of Titanium dioxide nanoparticles (TiO 2 -NPs) covers a vast area in different fields ranging from cosmetics and food to the production of drugs. Maternal exposure to TiO 2 -NPs during developmental period has been associated with hippocampal injury and with a decrease in learning and memory status of the offspring. However, little is known about its injury mechanism. This paper describes the in vivo neurotoxic effects of TiO 2 -NPs on rat offspring hippocampus during developmental period. Pregnant and lactating Wistar rats received intragastric TiO 2 -NPs (100mg/kg body weight) daily from gestational day (GD) 2 to (GD) 21 and postnatal day (PD) 2 to (PD) 21 respectively. Animals in the control groups received an equal volume of distilled water via gavage. At the end of the treatment process, offspring were deeply anesthetized and sacrificed. Then brains of each group were collected and sections of the rat offspring's brains were stained using TUNEL staining (for detection of apoptotic cells) and immunostaining (for neurogenesis). Moreover, the right hippocampus (n=6 per each group) were removed from the right hemisphere for evaluating the expression of Bax and Bcl-2 level. Results of histopatological examination by TUNEL staining showed that maternal exposure to TiO 2 -NPs during pregnancy and lactation periods increased apoptotic cells significantly (P<0.01) in the offspring hippocampus. The immunolabeling of double cortin (DCX) protein as neurogenesis marker also showed that TiO 2 -NPs reduced neurogenesis in the hippocampus of the offspring (P<0.05). Moreover, in comparison with the control group, the mRNA levels of Bax and Bcl-2 in the TiO 2 -NPs group significantly increased and decreased, respectively (P<0.01). These findings provide strong evidence that maternal exposure to TiO 2 -NPs significantly impact hippocampal neurogenesis and apoptosis in the offspring. The potential impact of nanoparticle exposure for millions of pregnant mothers and

  20. A nonsense mutation causing decreased levels of insulin receptor mRNA: Detection by a simplified technique for direct sequencing of genomic DNA amplified by the polymerase chain reaction

    International Nuclear Information System (INIS)

    Kadowaki, T.; Kadowaki, H.; Taylor, S.I.

    1990-01-01

    Mutations in the insulin receptor gene can render the cell resistant to the biological action of insulin. The authors have studied a patient with leprechaunism (leprechaun/Minn-1), a genetic syndrome associated with intrauterine growth retardation and extreme insulin resistance. Genomic DNA from the patient was amplified by the polymerase chain reaction catalyzed by Thermus aquaticus (Taq) DNA polymerase, and the amplified DNA was directly sequenced. A nonsense mutations was identified at codon 897 in exon 14 in the paternal allele of the patient's insulin receptor gene. Levels of insulin receptor mRNA are decreased to <10% of normal in Epstein-Barr virus-transformed lymphoblasts and cultured skin fibroblasts from this patient. Thus, this nonsense mutation appears to cause a decrease in the levels of insulin receptor mRNA. In addition, they have obtained indirect evidence that the patient's maternal allele of the insulin receptor gene contains a cis-acting dominant mutation that also decreases the level of mRNA, but by a different mechanism. The nucleotide sequence of the entire protein-coding domain and the sequences of the intron-exon boundaries for all 22 exons of the maternal allele were normal. Presumably, the mutation in the maternal allele maps elsewhere in the insulin receptor gene. Thus, they conclude that the patient is a compound heterozygote for two cis-acting dominant mutations in the insulin receptor gene: (i) a nonsense mutation in the paternal allel that reduces the level of insulin receptor mRNA and (ii) an as yet unidentified mutation in the maternal allele that either decreases the rate of transcription or decreases the stability of the mRNA

  1. Disease-associated mutations that alter the RNA structural ensemble.

    Directory of Open Access Journals (Sweden)

    Matthew Halvorsen

    2010-08-01

    Full Text Available Genome-wide association studies (GWAS often identify disease-associated mutations in intergenic and non-coding regions of the genome. Given the high percentage of the human genome that is transcribed, we postulate that for some observed associations the disease phenotype is caused by a structural rearrangement in a regulatory region of the RNA transcript. To identify such mutations, we have performed a genome-wide analysis of all known disease-associated Single Nucleotide Polymorphisms (SNPs from the Human Gene Mutation Database (HGMD that map to the untranslated regions (UTRs of a gene. Rather than using minimum free energy approaches (e.g. mFold, we use a partition function calculation that takes into consideration the ensemble of possible RNA conformations for a given sequence. We identified in the human genome disease-associated SNPs that significantly alter the global conformation of the UTR to which they map. For six disease-states (Hyperferritinemia Cataract Syndrome, beta-Thalassemia, Cartilage-Hair Hypoplasia, Retinoblastoma, Chronic Obstructive Pulmonary Disease (COPD, and Hypertension, we identified multiple SNPs in UTRs that alter the mRNA structural ensemble of the associated genes. Using a Boltzmann sampling procedure for sub-optimal RNA structures, we are able to characterize and visualize the nature of the conformational changes induced by the disease-associated mutations in the structural ensemble. We observe in several cases (specifically the 5' UTRs of FTL and RB1 SNP-induced conformational changes analogous to those observed in bacterial regulatory Riboswitches when specific ligands bind. We propose that the UTR and SNP combinations we identify constitute a "RiboSNitch," that is a regulatory RNA in which a specific SNP has a structural consequence that results in a disease phenotype. Our SNPfold algorithm can help identify RiboSNitches by leveraging GWAS data and an analysis of the mRNA structural ensemble.

  2. RNA oxidation

    DEFF Research Database (Denmark)

    Kjaer, L. K.; Cejvanovic, V.; Henriken, T.

    2015-01-01

    .9 significant hazard ratio for death compared with the quartile with the lowest 8oxoGuo excretion when adjusted for age, sex, BMI, smoker status, s-HbA1c, urine protein excretion and s-cholesterol. We conclude that it is now established that RNA oxidation is an independent risk factor for death in type 2...

  3. Dynamic changes in HCV RNA levels and viral quasispecies in a patient with chronic hepatitis C after telaprevir-based treatment

    NARCIS (Netherlands)

    de Bruijne, Joep; Sullivan, James C.; Kieffer, Tara L.; Botfield, Martyn; Shames, Ben; Schinkel, Janke; Molenkamp, Richard; Weegink, Christine; Reesink, Henk

    2012-01-01

    Background: Telaprevir is a selective inhibitor of the hepatitis C virus NS3 center dot 4A serine protease. Treatment with telaprevir resulted in a rapid HCV-RNA decline in chronic hepatitis C genotype 1 patients. Objectives: To report the clinical and viral course of a patient treated with

  4. Unusually effective microRNA targeting within repeat-rich coding regions of mammalian mRNAs

    Science.gov (United States)

    Schnall-Levin, Michael; Rissland, Olivia S.; Johnston, Wendy K.; Perrimon, Norbert; Bartel, David P.; Berger, Bonnie

    2011-01-01

    MicroRNAs (miRNAs) regulate numerous biological processes by base-pairing with target messenger RNAs (mRNAs), primarily through sites in 3′ untranslated regions (UTRs), to direct the repression of these targets. Although miRNAs have sometimes been observed to target genes through sites in open reading frames (ORFs), large-scale studies have shown such targeting to be generally less effective than 3′ UTR targeting. Here, we show that several miRNAs each target significant groups of genes through multiple sites within their coding regions. This ORF targeting, which mediates both predictable and effective repression, arises from highly repeated sequences containing miRNA target sites. We show that such sequence repeats largely arise through evolutionary duplications and occur particularly frequently within families of paralogous C2H2 zinc-finger genes, suggesting the potential for their coordinated regulation. Examples of ORFs targeted by miR-181 include both the well-known tumor suppressor RB1 and RBAK, encoding a C2H2 zinc-finger protein and transcriptional binding partner of RB1. Our results indicate a function for repeat-rich coding sequences in mediating post-transcriptional regulation and reveal circumstances in which miRNA-mediated repression through ORF sites can be reliably predicted. PMID:21685129

  5. The 0.3-kb fragment containing the R-U5-5'leader sequence of Friend murine leukemia virus influences the level of protein expression from spliced mRNA.

    Science.gov (United States)

    Choo, Yeng Cheng; Seki, Yohei; Machinaga, Akihito; Ogita, Nobuo; Takase-Yoden, Sayaka

    2013-04-19

    A neuropathogenic variant of Friend murine leukemia virus (Fr-MLV) clone A8 induces spongiform neurodegeneration when infected into neonatal rats. Studies with chimeras constructed from the A8 virus and the non-neuropathogenic Fr-MLV clone 57 identified a 0.3-kb KpnI-AatII fragment containing a R-U5-5'leader sequence as an important determinant for inducing spongiosis, in addition to the env gene of A8 as the primary determinant. This 0.3-kb fragment contains a 17-nucleotide difference between the A8 and 57 sequences. We previously showed that the 0.3-kb fragment influences expression levels of Env protein in both cultured cells and rat brain, but the corresponding molecular mechanisms are not well understood. Studies with expression vectors constructed from the full-length proviral genome of Fr-MLV that incorporated the luciferase (luc) gene instead of the env gene found that the vector containing the A8-0.3-kb fragment yielded a larger amount of spliced luc-mRNA and showed higher expression of luciferase when compared to the vector containing the 57-0.3-kb fragment. The amount of total transcripts from the vectors, the poly (A) tail length of their mRNAs, and the nuclear-cytoplasm distribution of luc-mRNA in transfected cells were also evaluated. The 0.3-kb fragment did not influence transcription efficiency, mRNA polyadenylation or nuclear export of luc-mRNA. Mutational analyses were carried out to determine the importance of nucleotides that differ between the A8 and 57 sequences within the 0.3-kb fragment. In particular, seven nucleotides upstream of the 5'splice site (5'ss) were found to be important in regulating the level of protein expression from spliced messages. Interestingly, these nucleotides reside within the stem-loop structure that has been speculated to limit the recognition of 5'ss. The 0.3-kb fragment containing the R-U5-5'leader sequence of Fr-MLV influences the level of protein expression from the spliced-mRNA by regulating the splicing

  6. High BMI levels associate with reduced mRNA expression of IL10 and increased mRNA expression of iNOS (NOS2) in human frontal cortex

    DEFF Research Database (Denmark)

    Lauridsen, J K; Olesen, R H; Vendelbo, J

    2017-01-01

    unknown. Therefore we aim to examine the relationship between BMI and gene expression of central inflammatory markers in the human frontal cortex. Microarray data of 141 neurologically and psychiatrically healthy individuals were obtained through the BrainCloud database. A simple linear regression...... correlated (Plinear regression analyses with BMI, age, sex and race as variables were performed in order to identify potential confounders. In conclusion, increasing BMI could affect the IL10-mediated anti...... analysis was performed with BMI as variable on data on IL10, IL1β, IL6, PTGS2 (COX2) and NOS2 (iNOS). Increasing BMI is associated with a decrease in the mRNA expression of IL10 (P=0.014) and an increase in the expression of NOS2 (iNOS; P=0.040). Expressions of IL10 and NOS2 (iNOS) were negatively...

  7. High BMI levels associate with reduced mRNA expression of IL10 and increased mRNA expression of iNOS (NOS2) in human frontal cortex

    DEFF Research Database (Denmark)

    Lauridsen, J K; Olesen, R H; Vendelbo, J

    2017-01-01

    analysis was performed with BMI as variable on data on IL10, IL1β, IL6, PTGS2 (COX2) and NOS2 (iNOS). Increasing BMI is associated with a decrease in the mRNA expression of IL10 (P=0.014) and an increase in the expression of NOS2 (iNOS; P=0.040). Expressions of IL10 and NOS2 (iNOS) were negatively...... correlated (PIL10 was mostly affected by individuals with BMI ⩾40. Multiple linear regression analyses with BMI, age, sex and race as variables were performed in order to identify potential confounders. In conclusion, increasing BMI could affect the IL10-mediated anti...

  8. Identification of microRNAs controlling hepatic mRNA levels for metabolic genes during the metabolic transition from embryonic to posthatch development in the chicken.

    Science.gov (United States)

    Hicks, Julie A; Porter, Tom E; Liu, Hsiao-Ching

    2017-09-05

    The transition from embryonic to posthatch development in the chicken represents a massive metabolic switch from primarily lipolytic to primarily lipogenic metabolism. This metabolic switch is essential for the chick to successfully transition from the metabolism of stored egg yolk to the utilization of carbohydrate-based feed. However, regulation of this metabolic switch is not well understood. We hypothesized that microRNAs (miRNAs) play an important role in the metabolic switch that is essential to efficient growth of chickens. We used high-throughput RNA sequencing to characterize expression profiles of mRNA and miRNA in liver during late embryonic and early posthatch development of the chicken. This extensive data set was used to define the contributions of microRNAs to the metabolic switch during development that is critical to growth and nutrient utilization in chickens. We found that expression of over 800 mRNAs and 30 miRNAs was altered in the embryonic liver between embryonic day 18 and posthatch day 3, and many of these differentially expressed mRNAs and miRNAs are associated with metabolic processes. We confirmed the regulation of some of these mRNAs by miRNAs expressed in a reciprocal pattern using luciferase reporter assays. Finally, through the use of yeast one-hybrid screens, we identified several proteins that likely regulate expression of one of these important miRNAs. Integration of the upstream regulatory mechanisms governing miRNA expression along with monitoring the downstream effects of this expression will ultimately allow for the construction of complete miRNA regulatory networks associated with the hepatic metabolic switch in chickens. Our findings support a key role for miRNAs in controlling the metabolic switch that occurs between embryonic and posthatch development in the chicken.

  9. Differential Regulation of rRNA and tRNA Transcription from the rRNA-tRNA Composite Operon in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Hiraku Takada

    Full Text Available Escherichia coli contains seven rRNA operons, each consisting of the genes for three rRNAs (16S, 23S and 5S rRNA in this order and one or two tRNA genes in the spacer between 16S and 23S rRNA genes and one or two tRNA genes in the 3' proximal region. All of these rRNA and tRNA genes are transcribed from two promoters, P1 and P2, into single large precursors that are afterward processed to individual rRNAs and tRNAs by a set of RNases. In the course of Genomic SELEX screening of promoters recognized by RNA polymerase (RNAP holoenzyme containing RpoD sigma, a strong binding site was identified within 16S rRNA gene in each of all seven rRNA operons. The binding in vitro of RNAP RpoD holoenzyme to an internal promoter, referred to the promoter of riRNA (an internal RNA of the rRNA operon, within each 16S rRNA gene was confirmed by gel shift assay and AFM observation. Using this riRNA promoter within the rrnD operon as a representative, transcription in vitro was detected with use of the purified RpoD holoenzyme, confirming the presence of a constitutive promoter in this region. LacZ reporter assay indicated that this riRNA promoter is functional in vivo. The location of riRNA promoter in vivo as identified using a set of reporter plasmids agrees well with that identified in vitro. Based on transcription profile in vitro and Northern blot analysis in vivo, the majority of transcript initiated from this riRNA promoter was estimated to terminate near the beginning of 23S rRNA gene, indicating that riRNA leads to produce the spacer-coded tRNA. Under starved conditions, transcription of the rRNA operon is markedly repressed to reduce the intracellular level of ribosomes, but the levels of both riRNA and its processed tRNAGlu stayed unaffected, implying that riRNA plays a role in the continued steady-state synthesis of tRNAs from the spacers of rRNA operons. We then propose that the tRNA genes organized within the spacers of rRNA-tRNA composite operons

  10. Inferior clinical outcome of the CD4+ cell count-guided antiretroviral treatment interruption strategy in the SMART study: role of CD4+ Cell counts and HIV RNA levels during follow-up

    DEFF Research Database (Denmark)

    Lundgren, Jens; Babiker, Abdel; El-Sadr, Wafaa

    2008-01-01

    BACKGROUND AND METHODS: The SMART study compared 2 strategies for using antiretroviral therapy-drug conservation (DC) and viral suppression (VS)-in 5,472 human immunodeficiency virus (HIV)-infected patients with CD4+ cell counts >350 cells/microL. Rates and predictors of opportunistic disease...... or death (OD/death) and the relative risk (RR) in DC versus VS groups according to the latest CD4+ cell count and HIV RNA level are reported. RESULTS: During a mean of 16 months of follow-up, DC patients spent more time with a latest CD4+ cell count ...%) and with a latest HIV RNA level >400 copies/mL (71% vs. 28%) and had a higher rate of OD/death (3.4 vs. 1.3/100 person-years) than VS patients. For periods of follow- up with a CD4+ cell count

  11. Effectiveness of memantine on depression-like behavior, memory deficits and brain mRNA levels of BDNF and TrkB in rats subjected to repeated unpredictable stress

    DEFF Research Database (Denmark)

    Amidfar, Meysam; Kim, Yong-Ku; Wiborg, Ove

    2018-01-01

    downregulation. Administration of memantine reversed depression-like behavior and memory impairment and significantly increased BDNF and TrkB mRNA levels in both prefrontal cortex and hippocampus of stress exposed rats. CONCLUSIONS: Our study supports the hypothesis that drugs with antagonistic properties...... administration in rats subjected to the repeated unpredictable stress (RUS) paradigm. METHODS: Rats were split into four groups at random including control + saline, control + memantine, stressed + saline and stressed + memantine. After 10 days of exposure to the RUS paradigm, rats were administered memantine...... (20 mg/kg) intraperitoneally (ip) for 14 days. Depression-like behavior and memory performance were assessed by measuring immobility time in the forced swim test and passive avoidance test, respectively. The mRNA levels of BDNF and TrkB in the prefrontal cortex and hippocampus were measured by real...

  12. mRNA and Protein Levels for GABA[subscript A][alpha]4, [alpha]5, [beta]1 and GABA[subscript B]R1 Receptors are Altered in Brains from Subjects with Autism

    Science.gov (United States)

    Fatemi, S. Hossein; Reutiman, Teri J.; Folsom, Timothy D.; Rooney, Robert J.; Patel, Diven H.; Thuras, Paul D.

    2010-01-01

    We have shown altered expression of gamma-aminobutyric acid A (GABA[subscript A]) and gamma-aminobutyric acid B (GABA[subscript B]) receptors in the brains of subjects with autism. In the current study, we sought to verify our western blotting data for GABBR1 via qRT-PCR and to expand our previous work to measure mRNA and protein levels of 3…

  13. Comparison of traditional phenotypic identification methods with partial 5' 16S rRNA gene sequencing for species-level identification of nonfermenting Gram-negative bacilli.

    Science.gov (United States)

    Cloud, Joann L; Harmsen, Dag; Iwen, Peter C; Dunn, James J; Hall, Gerri; Lasala, Paul Rocco; Hoggan, Karen; Wilson, Deborah; Woods, Gail L; Mellmann, Alexander

    2010-04-01

    Correct identification of nonfermenting Gram-negative bacilli (NFB) is crucial for patient management. We compared phenotypic identifications of 96 clinical NFB isolates with identifications obtained by 5' 16S rRNA gene sequencing. Sequencing identified 88 isolates (91.7%) with >99% similarity to a sequence from the assigned species; 61.5% of sequencing results were concordant with phenotypic results, indicating the usability of sequencing to identify NFB.

  14. Triiodothyronine increases mRNA and protein leptin levels in short time in 3T3-L1 adipocytes by PI3K pathway activation.

    Directory of Open Access Journals (Sweden)

    Miriane de Oliveira

    Full Text Available The present study aimed to examine the effects of thyroid hormone (TH, more precisely triiodothyronine (T3, on the modulation of leptin mRNA expression and the involvement of the phosphatidyl inositol 3 kinase (PI3K signaling pathway in adipocytes, 3T3-L1, cell culture. We examined the involvement of this pathway in mediating TH effects by treating 3T3-L1 adipocytes with physiological (P=10nM or supraphysiological (SI=100 nM T3 dose during one hour (short time, in the absence or the presence of PI3K inhibitor (LY294002. The absence of any treatment was considered the control group (C. RT-qPCR was used for mRNA expression analyzes. For data analyzes ANOVA complemented with Tukey's test was used at 5% significance. T3 increased leptin mRNA expression in P (2.26 ± 0.36, p 0.001. These results demonstrate that the activation of the PI3K signaling pathway has a role in TH-mediated direct and indirect leptin gene expression in 3T3-L1 adipocytes.

  15. 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, a......% of the a2 expression, and no reliable detection of a3 and a4 was possible. In conclusion, activation of additional muscle mass does not result in a higher exercise-induced increase in Na+-K+-ATPase subunit-specific mRNA.......4, ß1, ß2, and ß3 mRNA in human skeletal muscle was investigated. On two occasions, eight subjects performed one-legged knee extension exercise (L) or combined one-legged knee extension and bilateral arm cranking (AL) for 5.00, 4.25, 3.50, 2.75, and 2.00 min separated by 3 min of rest. Leg exercise...

  16. Identifying microRNA/mRNA dysregulations in ovarian cancer.

    Science.gov (United States)

    Miles, Gregory D; Seiler, Michael; Rodriguez, Lorna; Rajagopal, Gunaretnam; Bhanot, Gyan

    2012-03-27

    MicroRNAs are a class of noncoding RNA molecules that co-regulate the expression of multiple genes via mRNA transcript degradation or translation inhibition. Since they often target entire pathways, they may be better drug targets than genes or proteins. MicroRNAs are known to be dysregulated in many tumours and associated with aggressive or poor prognosis phenotypes. Since they regulate mRNA in a tissue specific manner, their functional mRNA targets are poorly understood. In previous work, we developed a method to identify direct mRNA targets of microRNA using patient matched microRNA/mRNA expression data using an anti-correlation signature. This method, applied to clear cell Renal Cell Carcinoma (ccRCC), revealed many new regulatory pathways compromised in ccRCC. In the present paper, we apply this method to identify dysregulated microRNA/mRNA mechanisms in ovarian cancer using data from The Cancer Genome Atlas (TCGA). TCGA Microarray data was normalized and samples whose class labels (tumour or normal) were ambiguous with respect to consensus ensemble K-Means clustering were removed. Significantly anti-correlated and correlated genes/microRNA differentially expressed between tumour and normal samples were identified. TargetScan was used to identify gene targets of microRNA. We identified novel microRNA/mRNA mechanisms in ovarian cancer. For example, the expression level of RAD51AP1 was found to be strongly anti-correlated with the expression of hsa-miR-140-3p, which was significantly down-regulated in the tumour samples. The anti-correlation signature was present separately in the tumour and normal samples, suggesting a direct causal dysregulation of RAD51AP1 by hsa-miR-140-3p in the ovary. Other pairs of potentially biological relevance include: hsa-miR-145/E2F3, hsa-miR-139-5p/TOP2A, and hsa-miR-133a/GCLC. We also identified sets of positively correlated microRNA/mRNA pairs that are most likely result from indirect regulatory mechanisms. Our findings identify

  17. RNase-assisted RNA chromatography

    Science.gov (United States)

    Michlewski, Gracjan; Cáceres, Javier F.

    2010-01-01

    RNA chromatography combined with mass spectrometry represents a widely used experimental approach to identify RNA-binding proteins that recognize specific RNA targets. An important drawback of most of these protocols is the high background due to direct or indirect nonspecific binding of cellular proteins to the beads. In many cases this can hamper the detection of individual proteins due to their low levels and/or comigration with contaminating proteins. Increasing the salt concentration during washing steps can reduce background, but at the cost of using less physiological salt concentrations and the likely loss of important RNA-binding proteins that are less stringently bound to a given RNA, as well as the disassembly of protein or ribonucleoprotein complexes. Here, we describe an improved RNA chromatography method that relies on the use of a cocktail of RNases in the elution step. This results in the release of proteins specifically associated with the RNA ligand and almost complete elimination of background noise, allowing a more sensitive and thorough detection of RNA-binding proteins recognizing a specific RNA transcript. PMID:20571124

  18. NGS Reveals Molecular Pathways Affected by Obesity and Weight Loss-Related Changes in miRNA Levels in Adipose Tissue

    Directory of Open Access Journals (Sweden)

    Alina Kuryłowicz

    2017-12-01

    Full Text Available Both obesity and weight loss may cause molecular changes in adipose tissue. This study aimed to characterize changes in adipose tissue miRNome in order to identify molecular pathways affected by obesity and weight changes. Next generation sequencing (NGS was applied to identify microRNAs (miRNAs differentially expressed in 47 samples of visceral (VAT and subcutaneous (SAT adipose tissues from normal-weight (N, obese (O and obese after surgery-induced weight loss (PO individuals. Subsequently miRNA expression was validated by real-time PCR in 197 adipose tissues and bioinformatics analysis performed to identify molecular pathways affected by obesity-related changes in miRNA expression. NGS identified 344 miRNAs expressed in adipose tissues with ≥5 reads per million. Using >2 and <−2 fold change as cut-offs we showed that the expression of 54 miRNAs differed significantly between VAT-O and SAT-O. Equally, between SAT-O and SAT-N, the expression of 20 miRNAs differed significantly, between SAT-PO and SAT-N the expression of 79 miRNAs differed significantly, and between SAT-PO and SAT-O, the expression of 61 miRNAs differed significantly. Ontological analyses disclosed several molecular pathways regulated by these miRNAs in adipose tissue. NGS-based miRNome analysis characterized changes of the miRNA profile of adipose tissue, which are associated with changes of weight possibly responsible for a differential regulation of molecular pathways in adipose tissue when the individual is obese and after the individual has lost weight.

  19. Effects of varying dietary iodine supplementation levels as iodide or iodate on thyroid status as well as mRNA expression and enzyme activity of antioxidative enzymes in tissues of grower/finisher pigs.

    Science.gov (United States)

    Li, Qimeng; Mair, Christiane; Schedle, Karl; Hellmayr, Isabella; Windisch, Wilhelm

    2013-02-01

    The objective of this study was to investigate the influence of high dietary iodine supply and different iodine sources on thyroid status and oxidative stress in target tissues of the thyroid hormones in fattening pigs. Eighty castrates (body weight: 33.3 ± 0.4 kg) were randomly allotted into five different treatments: The control diet contained 150 μg I/kg as KI, the other feeding groups were supplemented with 4,000 μg I/kg (as KI and KIO(3)) and 10,000 μg I/kg (as KI and KIO(3)), respectively. The mRNA expression levels of sodium/iodide symporter (NIS) and key antioxidant enzymes (Cu/Zn SOD, CAT, GPx) were analyzed in thyroid gland, liver, kidney, muscle, and adipose tissue sampled during slaughter. Furthermore, antioxidant enzyme activities and the effect on lipid peroxidation (MDA) were determined in liver and muscle. In thyroid gland, a significant downregulation of NIS and Cu/Zn SOD mRNA expression was observed in high-iodine groups. In liver, a source effect on the mRNA expression of Cu/Zn SOD between KI and KIO(3) at 4,000 μg I/kg was shown. In contrast, not SOD but GPx activity was affected by iodine source with strongest downregulation in high KIO(3) group. In muscle, GPx activity was affected by both iodine source and dose, showing stronger downregulation in KI groups. In kidney and adipose tissue, oxidative stress parameters showed no or only unsystematic changes. However, variation in iodine supply had no effect on MDA concentrations. NIS expression was significantly decreased with increased iodine supplementation, which is to ensure the thyroid gland function. However, the alleviating effect of iodine supplementation observed in antioxidant enzyme mRNA expression and activity did not reflect on the lipid peroxide level.

  20. MicroRNA 128a increases intracellular ROS level by targeting Bmi-1 and inhibits medulloblastoma cancer cell growth by promoting senescence.

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    Sujatha Venkataraman

    Full Text Available BACKGROUND: MicroRNAs (miRNAs are a class of short non-coding RNAs that regulate cell homeostasis by inhibiting translation or degrading mRNA of target genes, and thereby can act as tumor suppressor genes or oncogenes. The role of microRNAs in medulloblastoma has only recently been addressed. We hypothesized that microRNAs differentially expressed during normal CNS development might be abnormally regulated in medulloblastoma and are functionally important for medulloblastoma cell growth. METHODOLOGY AND PRINCIPAL FINDINGS: We examined the expression of microRNAs in medulloblastoma and then investigated the functional role of one specific one, miR-128a, in regulating medulloblastoma cell growth. We found that many microRNAs associated with normal neuronal differentiation are significantly down regulated in medulloblastoma. One of these, miR-128a, inhibits growth of medulloblastoma cells by targeting the Bmi-1 oncogene. In addition, miR-128a alters the intracellular redox state of the tumor cells and promotes cellular senescence. CONCLUSIONS AND SIGNIFICANCE: Here we report the novel regulation of reactive oxygen species (ROS by microRNA 128a via the specific inhibition of the Bmi-1 oncogene. We demonstrate that miR-128a has growth suppressive activity in medulloblastoma and that this activity is partially mediated by targeting Bmi-1. This data has implications for the modulation of redox states in cancer stem cells, which are thought to be resistant to therapy due to their low ROS states.

  1. Probiotics Differently Affect Gut-Associated Lymphoid Tissue Indolamine-2,3-Dioxygenase mRNA and Cerebrospinal Fluid Neopterin Levels in Antiretroviral-Treated HIV-1 Infected Patients: A Pilot Study.

    Science.gov (United States)

    Scagnolari, Carolina; Corano Scheri, Giuseppe; Selvaggi, Carla; Schietroma, Ivan; Najafi Fard, Saeid; Mastrangelo, Andrea; Giustini, Noemi; Serafino, Sara; Pinacchio, Claudia; Pavone, Paolo; Fanello, Gianfranco; Ceccarelli, Giancarlo; Vullo, Vincenzo; d'Ettorre, Gabriella

    2016-09-27

    Recently the tryptophan pathway has been considered an important determinant of HIV-1 infected patients' quality of life, due to the toxic effects of its metabolites on the central nervous system (CNS). Since the dysbiosis described in HIV-1 patients might be responsible for the microbial translocation, the chronic immune activation, and the altered utilization of tryptophan observed in these individuals, we speculated a correlation between high levels of immune activation markers in the cerebrospinal fluid (CSF) of HIV-1 infected patients and the over-expression of indolamine-2,3-dioxygenase (IDO) at the gut mucosal surface. In order to evaluate this issue, we measured the levels of neopterin in CSF, and the expression of IDO mRNA in gut-associated lymphoid tissue (GALT), in HIV-1-infected patients on effective combined antiretroviral therapy (cART), at baseline and after six months of probiotic dietary management. We found a significant reduction of neopterin and IDO mRNA levels after the supplementation with probiotic. Since the results for the use of adjunctive therapies to reduce the levels of immune activation markers in CSF have been disappointing so far, our pilot study showing the efficacy of this specific probiotic product should be followed by a larger confirmatory trial.

  2. Prenatal Stress Impairs Spatial Learning and Memory Associated with Lower mRNA Level of the CAMKII and CREB in the Adult Female Rat Hippocampus.

    Science.gov (United States)

    Sun, Hongli; Wu, Haibin; Liu, Jianping; Wen, Jun; Zhu, Zhongliang; Li, Hui

    2017-05-01

    Prenatal stress (PS) results in various behavioral and emotional alterations observed in later life. In particular, PS impairs spatial learning and memory processes but the underlying mechanism involved in this pathogenesis still remains unknown. Here, we reported that PS lowered the body weight in offspring rats, particularly in female rats, and impaired spatial learning and memory of female offspring rats in the Morris water maze. Correspondingly, the decreased CaMKII and CREB mRNA in the hippocampus were detected in prenatally stressed female offspring, which partially explained the effect of PS on the spatial learning and memory. Our findings suggested that CaMKII and CREB may be involved in spatial learning and memory processes in the prenatally stressed adult female offspring.

  3. [Satellite RNA (RNA3) of tomato black ring virus is found with one of the 2 major RNAs (RNA2) in a new capsid nucleoprotein].

    Science.gov (United States)

    Doz, B; Dunez, J; Bove, J M

    1977-12-19

    Tomato Black Ring Virus (TBRV) like other NEPOviruses posseses two nucleoproteins M and B and two major RNAs, RNA1 and RNA2 respectively distributed in B and M. A new nucleoprotein has just been discovered and comprises one molecule of RNA2 associated with one molecule of RNA3. RNA3 is a small RNA of molecular weight 500,000 d considered to be a satellite RNA. Its level appears to depend on the infection stage, local or systemic. RNA3 is able to modify the relative proportions of nucleoproteins M and B and their respective RNAs. The satellite RNA, might be part of the genome and represent a monocistronic mRNA for protein capsid synthesis. However it seems perhaps more tempting to correlate TBRV-RNA3 with satellite RNA5 of certain strains of Cucumber mosaic virus.

  4. 5-hydroxytryptamine1C receptor density and mRNA levels in choroid plexus epithelial cells after treatment with mianserin and (-)-1-(4-bromo-2,5-dimethoxyphenyl)-2-aminopropane.

    Science.gov (United States)

    Barker, E L; Sanders-Bush, E

    1993-10-01

    5-Hydroxytryptamine (5HT)1C and 5HT2 receptors display paradoxical down-regulation when exposed to receptor antagonists in vivo, a property that is unique to these two subtypes of serotonin (5HT) receptors. Because of the absence of cell culture model systems, the mechanisms involved in this paradoxical down-regulation have been difficult to explore. The present study focuses on the regulation of 5HT1C receptors in primary cultures of rat choroid plexus epithelial cells. Exposure of the epithelial cell cultures to 100 nM mianserin, a receptor antagonist, or (-)-1-(4-bromo-2,5-dimethoxyphenyl)-2-aminopropane, an agonist, for 72 hr caused a loss of 5HT1C receptor binding sites, as determined by [3H]mesulergine binding to crude membrane preparations. No significant changes in Kd values were observed. Neither the agonist nor antagonist caused a significant change in binding sites after 24 hr. A solution hybridization assay was used to determine whether the down-regulation by mianserin or (-)-1-(4-bromo-2,5-dimethoxyphenyl)-2-aminopropane was accompanied by a decrease in the steady state level of 5HT1C receptor mRNA. These studies showed that neither treatment caused an alteration in the levels of 5HT1C receptor mRNA. Thus, it is possible to reproduce the in vivo regulatory effects of drugs on 5HT1C receptors in choroid plexus epithelial cells in culture, including the atypical down-regulation by receptor antagonists. Using this cell culture model system, indirect transynaptic effects and decreases in receptor mRNA levels have been ruled out as mechanisms accounting for the down-regulation.

  5. Targeted CRISPR disruption reveals a role for RNase MRP RNA in human preribosomal RNA processing.

    Science.gov (United States)

    Goldfarb, Katherine C; Cech, Thomas R

    2017-01-01

    MRP RNA is an abundant, essential noncoding RNA whose functions have been proposed in yeast but are incompletely understood in humans. Mutations in the genomic locus for MRP RNA cause pleiotropic human diseases, including cartilage hair hypoplasia (CHH). Here we applied CRISPR-Cas9 genome editing to disrupt the endogenous human MRP RNA locus, thereby attaining what has eluded RNAi and RNase H experiments: elimination of MRP RNA in the majority of cells. The resulting accumulation of ribosomal RNA (rRNA) precursor-analyzed by RNA fluorescent in situ hybridization (FISH), Northern blots, and RNA sequencing-implicates MRP RNA in pre-rRNA processing. Amelioration of pre-rRNA imbalance is achieved through rescue of MRP RNA levels by ectopic expression. Furthermore, affinity-purified MRP ribonucleoprotein (RNP) from HeLa cells cleaves the human pre-rRNA in vitro at at least one site used in cells, while RNP isolated from cells with CRISPR-edited MRP loci loses this activity, and ectopic MRP RNA expression restores cleavage activity. Thus, a role for RNase MRP in human pre-rRNA processing is established. As demonstrated here, targeted CRISPR disruption is a valuable tool for functional studies of essential noncoding RNAs that are resistant to RNAi and RNase H-based degradation. © 2017 Goldfarb and Cech; Published by Cold Spring Harbor Laboratory Press.

  6. High levels of microRNA-21 in the stroma of colorectal cancers predict short disease-free survival in stage II colon cancer patients

    DEFF Research Database (Denmark)

    Nielsen, Boye Schnack; Jørgensen, Stine; Fog, Jacob Ulrik

    2011-01-01

    Approximately 25% of all patients with stage II colorectal cancer will experience recurrent disease and subsequently die within 5 years. MicroRNA-21 (miR-21) is upregulated in several cancer types and has been associated with survival in colon cancer. In the present study we developed a robust...... in situ hybridization assay using high-affinity Locked Nucleic Acid (LNA) probes that specifically detect miR-21 in formalin-fixed paraffin embedded (FFPE) tissue samples. The expression of miR-21 was analyzed by in situ hybridization on 130 stage II colon and 67 stage II rectal cancer specimens. The mi...... relative to the nuclear density (TBR) obtained using a red nuclear stain. High TBR (and TB) estimates of miR-21 expression correlated significantly with shorter disease-free survival (p = 0.004, HR = 1.28, 95% CI: 1.06-1.55) in the stage II colon cancer patient group, whereas no significant correlation...

  7. Putative pacemakers in the eyestalk and brain of the crayfish Procambarus clarkii show circadian oscillations in levels of mRNA for crustacean hyperglycemic hormone.

    Directory of Open Access Journals (Sweden)

    Janikua Nelson-Mora

    Full Text Available Crustacean hyperglycemic hormone (CHH synthesizing cells in the optic lobe, one of the pacemakers of the circadian system, have been shown to be present in crayfish. However, the presence of CHH in the central brain, another putative pacemaker of the multi-oscillatory circadian system, of this decapod and its circadian transcription in the optic lobe and brain have yet to be explored. Therefore, using qualitative and quantitative PCR, we isolated and cloned a CHH mRNA fragment from two putative pacemakers of the multi-oscillatory circadian system of Procambarus clarkii, the optic lobe and the central brain. This CHH transcript synchronized to daily light-dark cycles and oscillated under dark, constant conditions demonstrating statistically significant daily and circadian rhythms in both structures. Furthermore, to investigate the presence of the peptide in the central brain of this decapod, we used immunohistochemical methods. Confocal microscopy revealed the presence of CHH-IR in fibers and cells of the protocerebral and tritocerebal clusters and neuropiles, particularly in some neurons located in clusters 6, 14, 15 and 17. The presence of CHH positive neurons in structures of P. clarkii where clock proteins have been reported suggests a relationship between the circadian clockwork and CHH. This work provides new insights into the circadian regulation of CHH, a pleiotropic hormone that regulates many physiological processes such as glucose metabolism and osmoregulatory responses to stress.

  8. Increases in [3H]muscimol and [3H]flumazenil binding in the dorsolateral prefrontal cortex in schizophrenia are linked to α4 and γ2S mRNA levels respectively.

    Directory of Open Access Journals (Sweden)

    Mathieu Verdurand

    Full Text Available GABA(A receptors (GABA(AR are composed of several subunits that determine sensitivity to drugs, synaptic localisation and function. Recent studies suggest that agonists targeting selective GABA(AR subunits may have therapeutic value against the cognitive impairments observed in schizophrenia. In this study, we determined whether GABA(AR binding deficits exist in the dorsolateral prefrontal cortex (DLPFC of people with schizophrenia and tested if changes in GABA(AR binding are related to the changes in subunit mRNAs. The GABA orthosteric and the benzodiazepine allosteric binding sites were assessed autoradiographically using [(3H]Muscimol and [(3H]Flumazenil, respectively, in a large cohort of individuals with schizophrenia (n = 37 and their matched controls (n = 37. We measured, using qPCR, mRNA of β (β1, β2, β3, γ (γ1, γ2, γ2S for short and γ2L for long isoform, γ3 and δ subunits and used our previous measurements of GABA(AR α subunit mRNAs in order to relate mRNAs and binding through correlation and regression analysis.Significant increases in both [(3H]Muscimol (p = 0.016 and [(3H]Flumazenil (p = 0.012 binding were found in the DLPFC of schizophrenia patients. Expression levels of mRNA subunits measured did not show any significant difference in schizophrenia compared to controls. Regression analysis revealed that in schizophrenia, the [(3H]Muscimol binding variance was most related to α4 mRNA levels and the [(3H]Flumazenil binding variance was most related to γ2S subunit mRNA levels. [(3H]Muscimol and [(3H]Flumazenil binding were not affected by the lifetime anti-psychotics dose (chlorpromazine equivalent.We report parallel increases in orthosteric and allosteric GABA(AR binding sites in the DLPFC in schizophrenia that may be related to a "shift" in subunit composition towards α4 and γ2S respectively, which may compromise normal GABAergic modulation and function. Our results may have implications for the

  9. Ethnic variations of a retinoblastoma susceptibility gene (RB1 ...

    Indian Academy of Sciences (India)

    Unknown

    1Department of Paediatrics, National University of Singapore, Lower Kent Ridge Road, Singapore 119074. 2National Neuroscience Institute, Singapore 308433. 3Department of Paediatrics, University Diponegoro, Indonesia 50241. 4Department of Human Genetics, University of Pittsburgh, Pittsburgh, PA 15261, USA.

  10. In vitro Rb-1 gene transfer to retinoblastoma cell lines

    International Nuclear Information System (INIS)

    Choi, Sang Wook; Ham, Yong Hoh; Kim, Mee Heui

    1994-04-01

    After transfection of Rb-vector to packaging cell line (CRIP) by Ca-P precipitation method, we could select nineteen colonies of G-418 resistant clone by ring cloning. Each colony was transduced to NIH3T3 cells to select the one which produces high titer virus. After NIH3T3 cells transduction, we could get 28 colony counts for the high, 127 for the middle, and 6 for the low viral titer. With the supernatant of the high viral titer colony (CRIPRb 2-5). We transduct retinoblastoma cell lines. 5 figs, 11 refs. (Author)

  11. Extracellular RNA Communication (ExRNA)

    Data.gov (United States)

    Federal Laboratory Consortium — Until recently, scientists believed RNA worked mostly inside the cell that produced it. Some types of RNA help translate genes into proteins that are necessary for...

  12. Reduced basal and novelty-induced levels of activity-regulated cytoskeleton associated protein (Arc) and c-Fos mRNA in the cerebral cortex and hippocampus of APPswe/PS1ΔE9 transgenic mice

    DEFF Research Database (Denmark)

    Christensen, Ditte Z; Thomsen, Morten Skøtt; Mikkelsen, Jens D

    2013-01-01

    to a novel open field environment was compromised in different neocortical areas and the hippocampal formation in APP/PS1ΔE9 transgenic mice characterized by pronounced accumulation and deposition of beta amyloid (Aβ). Notably, the basal level of Arc and c-fos mRNA in the neocortex was significantly lower...... in APP/PS1ΔE9 compared to wild-type mice. Novelty exposure induced an increase in Arc and c-Fos mRNA in the medial prefrontal cortex (mPFC), parietal cortex, and hippocampal formation in both APP/PS1ΔE9 transgenic and wild-type mice. However, novelty-induced IEG expression did not reach the same levels...... in a transgenic mouse model of Alzheimer's disease, which is most pronounced in cortical regions, indicating that a decreased functional response in IEG expression could be partly responsible for the cognitive deficits observed in patients with Alzheimer's disease....

  13. Effect of phenylurea herbicides on soil microbial communities estimated by analysis of 16S rRNA gene fingerprints and community-level physiological profiles.

    Science.gov (United States)

    el Fantroussi, S; Verschuere, L; Verstraete, W; Top, E M

    1999-03-01

    The effect of three phenyl urea herbicides (diuron, linuron, and chlorotoluron) on soil microbial communities was studied by using soil samples with a 10-year history of treatment. Denaturing gradient gel electrophoresis (DGGE) was used for the analysis of 16S rRNA genes (16S rDNA). The degree of similarity between the 16S rDNA profiles of the communities was quantified by numerically analysing the DGGE band patterns. Similarity dendrograms showed that the microbial community structures of the herbicide-treated and nontreated soils were significantly different. Moreover, the bacterial diversity seemed to decrease in soils treated with urea herbicides, and sequence determination of several DGGE fragments showed that the most affected species in the soils treated with diuron and linuron belonged to an uncultivated bacterial group. As well as the 16S rDNA fingerprints, the substrate utilization patterns of the microbial communities were compared. Principal-component analysis performed on BIOLOG data showed that the functional abilities of the soil microbial communities were altered by the application of the herbicides. In addition, enrichment cultures of the different soils in medium with the urea herbicides as the sole carbon and nitrogen source showed that there was no difference between treated and nontreated soil in the rate of transformation of diuron and chlorotoluron but that there was a strong difference in the case of linuron. In the enrichment cultures with linuron-treated soil, linuron disappeared completely after 1 week whereas no significant transformation was observed in cultures inoculated with nontreated soil even after 4 weeks. In conclusion, this study showed that both the structure and metabolic potential of soil microbial communities were clearly affected by a long-term application of urea herbicides.

  14. Lowering the quantification limit of the QubitTM RNA HS assay using RNA spike-in.

    Science.gov (United States)

    Li, Xin; Ben-Dov, Iddo Z; Mauro, Maurizio; Williams, Zev

    2015-05-06

    RNA quantification is often a prerequisite for most RNA analyses such as RNA sequencing. However, the relatively low sensitivity and large sample consumption of traditional RNA quantification methods such as UV spectrophotometry and even the much more sensitive fluorescence-based RNA quantification assays, such as the Qubit™ RNA HS Assay, are often inadequate for measuring minute levels of RNA isolated from limited cell and tissue samples and biofluids. Thus, there is a pressing need for a more sensitive method to reliably and robustly detect trace levels of RNA without interference from DNA. To improve the quantification limit of the Qubit™ RNA HS Assay, we spiked-in a known quantity of RNA to achieve the minimum reading required by the assay. Samples containing trace amounts of RNA were then added to the spike-in and measured as a reading increase over RNA spike-in baseline. We determined the accuracy and precision of reading increases between 1 and 20 pg/μL as well as RNA-specificity in this range, and compared to those of RiboGreen(®), another sensitive fluorescence-based RNA quantification assay. We then applied Qubit™ Assay with RNA spike-in to quantify plasma RNA samples. RNA spike-in improved the quantification limit of the Qubit™ RNA HS Assay 5-fold, from 25 pg/μL down to 5 pg/μL while maintaining high specificity to RNA. This enabled quantification of RNA with original concentration as low as 55.6 pg/μL compared to 250 pg/μL for the standard assay and decreased sample consumption from 5 to 1 ng. Plasma RNA samples that were not measurable by the Qubit™ RNA HS Assay were measurable by our modified method. The Qubit™ RNA HS Assay with RNA spike-in is able to quantify RNA with high specificity at 5-fold lower concentration and uses 5-fold less sample quantity than the standard Qubit™ Assay.

  15. Time- and dose-dependent differential regulation of copper-zinc superoxide dismutase and manganese superoxide dismutase enzymatic activity and mRNA level by vitamin E in rat blood cells.

    Science.gov (United States)

    Hajiani, Maliheh; Razi, Farideh; Golestani, Aboualfazl; Frouzandeh, Mehdi; Owji, Ali Akbar; Khaghani, Shahnaz; Ghannadian, Naghmeh; Shariftabrizi, Ahmad; Pasalar, Parvin

    2012-01-01

    Vitamin E is the most important lipid-soluble antioxidant. Recently, it has been proposed as a gene regulator, and its gene modulation effects have been observed at different levels of gene expression and cell signaling. This study was performed to investigate the effects of vitamin E on the activity and expression of the most important endogenous antioxidant enzyme, superoxide dismutase (SOD), in rat plasma. Twenty-eight male Sprauge-Dawley rats were divided into four groups: control group and three dosing groups. The control group received the vehicle (liquid paraffin), and the dosing groups received twice-weekly intraperitoneal injections of 10, 30, and 100 mg/kg of vitamin E ((±)-α-Tocopherol) for 6 weeks. Quantitative real-time reverse transcription-polymerase chain reaction and enzyme assays were used to assess the levels of Cu/Zn-SOD and Mn-SOD mRNA and enzyme activity levels in blood cells at 0, 2, 4, and 6 weeks following vitamin E administration. Catalase enzyme activity and total antioxidant capacity were also assessed in plasma at the same time intervals. Mn-SOD activity was significantly increased in the 100 and 30 mg/kg dosing groups after 4 and 6 weeks, with corresponding significant increase in their mRNA levels. Cu/Zn-SOD activity was not significantly changed in response to vitamin E administration at any time points, whereas Cu/Zn-SOD mRNA levels were significantly increased after longer time points with high doses (30 and 100 mg/kg) of vitamin E. Catalase enzyme activity was transiently but significantly increased after 4 weeks of vitamin E treatment in 30 and 100 mg/kg dosing groups. Total antioxidant status was significantly increased after 4 and 6 weeks in the 100 mg/kg dosing group. Only the chronic administration of higher doses of alpha-tocopherol is associated with the increased activity and expression of Mn-SOD in rats. Cu/Zn-SOD activity and expression does not dramatically change in response to vitamin E.

  16. RNA-Binding Proteins Revisited – The Emerging Arabidopsis mRNA Interactome

    KAUST Repository

    Kö ster, Tino; Marondedze, Claudius; Meyer, Katja; Staiger, Dorothee

    2017-01-01

    RNA–protein interaction is an important checkpoint to tune gene expression at the RNA level. Global identification of proteins binding in vivo to mRNA has been possible through interactome capture – where proteins are fixed to target RNAs by UV crosslinking and purified through affinity capture of polyadenylated RNA. In Arabidopsis over 500 RNA-binding proteins (RBPs) enriched in UV-crosslinked samples have been identified. As in mammals and yeast, the mRNA interactomes came with a few surprises. For example, a plethora of the proteins caught on RNA had not previously been linked to RNA-mediated processes, for example proteins of intermediary metabolism. Thus, the studies provide unprecedented insights into the composition of the mRNA interactome, highlighting the complexity of RNA-mediated processes.

  17. RNA-Binding Proteins Revisited – The Emerging Arabidopsis mRNA Interactome

    KAUST Repository

    Köster, Tino

    2017-04-13

    RNA–protein interaction is an important checkpoint to tune gene expression at the RNA level. Global identification of proteins binding in vivo to mRNA has been possible through interactome capture – where proteins are fixed to target RNAs by UV crosslinking and purified through affinity capture of polyadenylated RNA. In Arabidopsis over 500 RNA-binding proteins (RBPs) enriched in UV-crosslinked samples have been identified. As in mammals and yeast, the mRNA interactomes came with a few surprises. For example, a plethora of the proteins caught on RNA had not previously been linked to RNA-mediated processes, for example proteins of intermediary metabolism. Thus, the studies provide unprecedented insights into the composition of the mRNA interactome, highlighting the complexity of RNA-mediated processes.

  18. Alterations in messenger RNA and small nuclear RNA metabolism resulting from fluorouracil incorporation

    International Nuclear Information System (INIS)

    Armstrong, R.D.; Cadman, E.C.

    1985-01-01

    Studies were completed to examine the effect of 5-fluorouracil (FUra) incorporation on messenger RNA (mRNA) and small molecular weight nuclear RNA (SnRNA) metabolism. Studies of mRNA were completed using cDNA-mRNA hybridization methods to specifically examine dihydrofolate reductase (DHFR) mRNA. C 3 -L5178Y murine leukemia cells which are gene-amplified for DHFR, were exposed to FUra for 6, 12 or 24 hr, and the nuclear and cytoplasmic levels of DHFR-mRNA determined by hybridization with 32 P-DHFR-cDNA. FUra produced a dose-dependent increase in nuclear DHFR-mRNA levels, while total cytoplasmic DHFR-mRNA levels appeared to be unchanged. To examine only mRNA synthesized during FUra exposure, cells were also treated concurrently with [ 3 H] cytidine, and the [ 3 H]mRNA-cDNA hybrids measured following S 1 -nuclease treatment. FUra produced a concentration-dependent increase in nascent nuclear DHFR-mRNA levels, and a decrease in nascent cytoplasmic DHFR-mRNAs levels. These results suggest that FUra produces either an inhibition of mRNA processing, or an inhibition of nuclear-cytoplasmic transport. Preliminary experiments to examine ATP-dependent mRNA transport were completed with isolated nuclei from cells treated with FUra for 1 or 24 hr and then pulse-labeled for 1 hr with [ 3 H] cytidine. The results demonstrate a FUra-concentration and time-dependent inhibition of ATP-mediated mRNA efflux

  19. A universal protocol to generate consensus level genome sequences for foot-and-mouth disease virus and other positive-sense polyadenylated RNA viruses using the Illumina MiSeq.

    Science.gov (United States)

    Logan, Grace; Freimanis, Graham L; King, David J; Valdazo-González, Begoña; Bachanek-Bankowska, Katarzyna; Sanderson, Nicholas D; Knowles, Nick J; King, Donald P; Cottam, Eleanor M

    2014-09-30

    Next-Generation Sequencing (NGS) is revolutionizing molecular epidemiology by providing new approaches to undertake whole genome sequencing (WGS) in diagnostic settings for a variety of human and veterinary pathogens. Previous sequencing protocols have been subject to biases such as those encountered during PCR amplification and cell culture, or are restricted by the need for large quantities of starting material. We describe here a simple and robust methodology for the generation of whole genome sequences on the Illumina MiSeq. This protocol is specific for foot-and-mouth disease virus (FMDV) or other polyadenylated RNA viruses and circumvents both the use of PCR and the requirement for large amounts of initial template. The protocol was successfully validated using five FMDV positive clinical samples from the 2001 epidemic in the United Kingdom, as well as a panel of representative viruses from all seven serotypes. In addition, this protocol was successfully used to recover 94% of an FMDV genome that had previously been identified as cell culture negative. Genome sequences from three other non-FMDV polyadenylated RNA viruses (EMCV, ERAV, VESV) were also obtained with minor protocol amendments. We calculated that a minimum coverage depth of 22 reads was required to produce an accurate consensus sequence for FMDV O. This was achieved in 5 FMDV/O/UKG isolates and the type O FMDV from the serotype panel with the exception of the 5' genomic termini and area immediately flanking the poly(C) region. We have developed a universal WGS method for FMDV and other polyadenylated RNA viruses. This method works successfully from a limited quantity of starting material and eliminates the requirement for genome-specific PCR amplification. This protocol has the potential to generate consensus-level sequences within a routine high-throughput diagnostic environment.

  20. Cisplatin Targeting of Bacterial Ribosomal RNA Hairpins

    Directory of Open Access Journals (Sweden)

    Gayani N. P. Dedduwa-Mudalige

    2015-09-01

    Full Text Available Cisplatin is a clinically important chemotherapeutic agent known to target purine bases in nucleic acids. In addition to major deoxyribonucleic acid (DNA intrastrand cross-links, cisplatin also forms stable adducts with many types of ribonucleic acid (RNA including siRNA, spliceosomal RNAs, tRNA, and rRNA. All of these RNAs play vital roles in the cell, such as catalysis of protein synthesis by rRNA, and therefore serve as potential drug targets. This work focused on platination of two highly conserved RNA hairpins from E. coli ribosomes, namely pseudouridine-modified helix 69 from 23S rRNA and the 790 loop of helix 24 from 16S rRNA. RNase T1 probing, MALDI mass spectrometry, and dimethyl sulfate mapping revealed platination at GpG sites. Chemical probing results also showed platination-induced RNA structural changes. These findings reveal solvent and structural accessibility of sites within bacterial RNA secondary structures that are functionally significant and therefore viable targets for cisplatin as well as other classes of small molecules. Identifying target preferences at the nucleotide level, as well as determining cisplatin-induced RNA conformational changes, is important for the design of more potent drug molecules. Furthermore, the knowledge gained through studies of RNA-targeting by cisplatin is applicable to a broad range of organisms from bacteria to human.

  1. Effects of Ethanol on the Expression Level of Various BDNF mRNA Isoforms and Their Encoded Protein in the Hippocampus of Adult and Embryonic Rats

    Directory of Open Access Journals (Sweden)

    Shahla Shojaei

    2015-12-01

    Full Text Available We aimed to compare the effects of oral ethanol (Eth alone or combined with the phytoestrogen resveratrol (Rsv on the expression of various brain-derived neurotrophic factor (BDNF transcripts and the encoded protein pro-BDNF in the hippocampus of pregnant and embryonic rats. A low (0.25 g/kg body weight (BW/day dose of Eth produced an increase in the expression of BDNF exons I, III and IV and a decrease in that of the exon IX in embryos, but failed to affect BDNF transcript and pro-BDNF protein expression in adults. However, co-administration of Eth 0.25 g/kg·BW/day and Rsv led to increased expression of BDNF exons I, III and IV and to a small but significant increase in the level of pro-BDNF protein in maternal rats. A high (2.5 g/kg·BW/day dose of Eth increased the expression of BDNF exons III and IV in embryos, but it decreased the expression of exon IX containing BDNF mRNAs in the maternal rats. While the high dose of Eth alone reduced the level of pro-BDNF in adults, it failed to change the levels of pro-BDNF in embryos. Eth differentially affects the expression pattern of BDNF transcripts and levels of pro-BDNF in the hippocampus of both adult and embryonic rats.

  2. Levels of microRNA miR-16 and miR-155 are altered in serum of patients with tuberculosis and associate with responses to therapy.

    Science.gov (United States)

    Wagh, Vishal; Urhekar, Anant; Modi, Deepak

    2017-01-01

    Identification of blood biomarkers that can be useful for predicting Mycobacterium tuberculosis (M.TB) infection, effect of therapy and Multi Drug Resistant (MDR) TB infected individuals is clinically useful for combating tuberculosis epidemic. In this study, we have evaluated the levels of selected miRNAs in serum of TB and MDR TB patients. In addition, we have studied their levels in serum of patients post-therapy. The levels of 4-miRNAs (miR-16, miR-29a, miR-125b and miR-155) were measured in 30 newly diagnosed TB patients, 19 Multi Drug Resistant (MDR) TB patients, 10 patients who completed TB therapy and were TB negative. 30 healthy individuals were recruited as controls. The levels of the miRNAs were estimated by qRT-PCR. Of the four miRNAs studied, the levels of miR-16 were significantly elevated and miR-155 were significantly reduced in serum of TB patients as compared to uninfected controls. The Receiver Operating Characteristic (ROC) curve of miR-16 and miR-155 exhibited a significant distinguishing efficiency with an AUC value of 1 (95% CI, 1 to 1) and 0.967 (95% CI, 0.92-1.04) respectively. Following the therapy, the levels of miR-16 and miR-155 returned to those observed in healthy subjects. In patients with MDR TB, miR-155 was lower as compared to healthy controls and TB treated group but higher as compared to TB naïve patients. miR-16 levels were lowest in serum of MDR TB patients compared to TB naïve, TB treated group and healthy controls. In conclusion, miR-16 and miR-155 in serum may act as surrogate biomarker for studying TB infection, progression of therapy and MDR TB. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Role of RNase MRP in viral RNA degradation and RNA recombination.

    Science.gov (United States)

    Jaag, Hannah M; Lu, Qiasheng; Schmitt, Mark E; Nagy, Peter D

    2011-01-01

    RNA degradation, together with RNA synthesis, controls the steady-state level of viral RNAs in infected cells. The endoribonucleolytic cleavage of viral RNA is important not only for viral RNA degradation but for RNA recombination as well, due to the participation of some RNA degradation products in the RNA recombination process. To identify host endoribonucleases involved in degradation of Tomato bushy stunt virus (TBSV) in a Saccharomyces cerevisiae model host, we tested eight known endoribonucleases. Here we report that downregulation of SNM1, encoding a component of the RNase MRP, and a temperature-sensitive mutation in the NME1 gene, coding for the RNA component of RNase MRP, lead to reduced production of the endoribonucleolytically cleaved TBSV RNA in yeast. We also show that the highly purified yeast RNase MRP cleaves the TBSV RNA in vitro, resulting in TBSV RNA degradation products similar in size to those observed in yeast cells. Knocking down the NME1 homolog in Nicotiana benthamiana also led to decreased production of the cleaved TBSV RNA, suggesting that in plants, RNase MRP is involved in TBSV RNA degradation. Altogether, this work suggests a role for the host endoribonuclease RNase MRP in viral RNA degradation and recombination.

  4. A link between expression level of long-non-coding RNA ZFAS1 in breast tissue of healthy women and obesity.

    Science.gov (United States)

    Mansoori, Yaser; Tabei, Mohammad Bagher; Askari, Alireza; Izadi, Pantea; Daraei, Abdolreza; Naghizadeh, Mohammad Mehdi; Zendehbad, Zahra; Bastami, Milad; Nariman-Saleh-Fam, Ziba; Mansoori, Hosein; Tavakkoly-Bazzaz, Javad

    2018-04-01

    Epidemiological and experimental literature indicates that the risk of breast cancer incidence is strongly linked to hormone-dependent factors, including reproductive history and obesity. However, the molecular mechanisms underlying the association between these factors and breast cancer risk are poorly understood. The aim of this study, therefore, was to determine whether obesity and reproductive history are associated with expression levels of two breast cancer-related long non-coding RNAs (lncRNAs), namely ZFAS1 and SRA1 in cancer-free breast tissues of women. In the current research, 145 healthy women were recruited, and the quantitative expression levels of the two lncRNAs were determined through qPCR assay after gathering the mammoplasty breast tissue samples. It was found that women with body mass index (BMI)≥30 kg/m 2 and BMI 25-29 kg/m 2 show a low expression of ZFAS1 compared to the BMI<25 kg/m 2 ( P=0.031 and P=0.027, respectively). Then, the correlation analysis disclosed a negative correlation of ZFAS1 low expression with increasing BMI (r=-0.194, P=0.019). Interestingly, this analysis demonstrated a negative correlation between low expression of the ZFAS1 and high BMI in women with menarche age below 14 (r=-221; P=0.028). Lastly, it was also revealed that there was a negative association of the low expression level of ZFAS1 with increasing BMI in women through regression models (B=-0.048, P=0.019). These findings suggest interesting clues about the links between high BMI and the expression levels of ZFAS1 in non-diseased breasts that may help us better understand the underlying mechanisms through which obesity contributes to breast carcinogenesis. However, such results need more validations in future research.

  5. Nucleolin Mediates MicroRNA-directed CSF-1 mRNA Deadenylation but Increases Translation of CSF-1 mRNA*

    Science.gov (United States)

    Woo, Ho-Hyung; Baker, Terri; Laszlo, Csaba; Chambers, Setsuko K.

    2013-01-01

    CSF-1 mRNA 3′UTR contains multiple unique motifs, including a common microRNA (miRNA) target in close proximity to a noncanonical G-quadruplex and AU-rich elements (AREs). Using a luciferase reporter system fused to CSF-1 mRNA 3′UTR, disruption of the miRNA target region, G-quadruplex, and AREs together dramatically increased reporter RNA levels, suggesting important roles for these cis-acting regulatory elements in the down-regulation of CSF-1 mRNA. We find that nucleolin, which binds both G-quadruplex and AREs, enhances deadenylation of CSF-1 mRNA, promoting CSF-1 mRNA decay, while having the capacity to increase translation of CSF-1 mRNA. Through interaction with the CSF-1 3′UTR miRNA common target, we find that miR-130a and miR-301a inhibit CSF-1 expression by enhancing mRNA decay. Silencing of nucleolin prevents the miRNA-directed mRNA decay, indicating a requirement for nucleolin in miRNA activity on CSF-1 mRNA. Downstream effects followed by miR-130a and miR-301a inhibition of directed cellular motility of ovarian cancer cells were found to be dependent on nucleolin. The paradoxical effects of nucleolin on miRNA-directed CSF-1 mRNA deadenylation and on translational activation were explored further. The nucleolin protein contains four acidic stretches, four RNA recognition motifs (RRMs), and nine RGG repeats. All three domains in nucleolin regulate CSF-1 mRNA and protein levels. RRMs increase CSF-1 mRNA, whereas the acidic and RGG domains decrease CSF-1 protein levels. This suggests that nucleolin has the capacity to differentially regulate both CSF-1 RNA and protein levels. Our finding that nucleolin interacts with Ago2 indirectly via RNA and with poly(A)-binding protein C (PABPC) directly suggests a nucleolin-Ago2-PABPC complex formation on mRNA. This complex is in keeping with our suggestion that nucleolin may work with PABPC as a double-edged sword on both mRNA deadenylation and translational activation. Our findings underscore the complexity of

  6. Up-regulated EMMPRIN/CD147 protein expression might play a role in colorectal carcinogenesis and its subsequent progression without an alteration of its glycosylation and mRNA level.

    Science.gov (United States)

    Zheng, Hua-chuan; Wang, Wei; Xu, Xiao-yan; Xia, Pu; Yu, Miao; Sugiyama, Toshiro; Takano, Yasuo

    2011-04-01

    Extracellular matrix metalloproteinase inducer (EMMPRIN) was reported to involve in the invasion and metastasis of malignancies by regulating the expression of vascular endothelial growth factor (VEGF) in stromal and cancer cells. The study aimed to clarify the role of EMMPRIN expression in tumorigenesis and progression of colorectal carcinomas (CRC). EMMPRIN expression was examined on tissue microarray containing colorectal carcinomas, adenoma and non-neoplastic mucosa (NNM) by immunohistochemistry and in situ hybridization (ISH). Colorectal carcinoma cell lines (DLD-1, HCT-15, SW480 and WiDr) and tissues were studied for EMMPRIN expression by Western blot or RT-PCR, followed by sequencing. All carcinoma cell lines showed EMMPRIN expression at both mRNA and protein levels. Two synonymous mutations were found in carcinoma cell lines at codon109 (GCT → GCC: Ala) or 179 (GAT → GAC: Asp). Frozen CRC tissues displayed higher EMMPRIN expression than paired NNM (P EMMPRIN expression was immunohistochemically stronger in colorectal high-grade adenoma, adenocarcinoma and metastatic carcinoma than non-neoplastic superficial epithelium and low-grade adenoma (P 0.05). Immunohistochemically, EMMPRIN expression was positively correlated with tumor size, depth of invasion, vascular or lymphatic invasion, grade of infiltration (INF), ki-67 and VEGF expression of CRCs (P EMMPRIN expression in CRCs (P EMMPRIN protein expression might contribute to colorectal carcinogenesis without the alteration of its glycosylation and mRNA level. Aberrant EMMPRIN protein expression might promote growth or invasion of CRCs possibly through increased ki-67 expression and inducible angiogenesis via up-regulating VEGF expression.

  7. Protein and mRNA levels support the notion that a genetic regulatory circuit controls growth phases in E. coli populations

    Directory of Open Access Journals (Sweden)

    Agustino Martinez-Antonio

    2015-09-01

    Full Text Available Bacterial populations transition between growing and non-growing phases, based on nutrient availability and stresses conditions. The hallmark of a growing state is anabolism, including DNA replication and cell division. In contrast, bacteria in a growth-arrested state acquire a resistant physiology and diminished metabolism. However, there is little knowledge on how this transition occurs at the molecular level. Here, we provide new evidence that a multi-element genetic regulatory circuit might work to maintain genetic control among growth-phase transitions in Escherichia coli. This work contributes to the discovering of design principles behind the performance of biological functions, which could be of relevance on the new disciplines of biological engineering and synthetic biology.

  8. Combinatorics of RNA-RNA interaction

    DEFF Research Database (Denmark)

    Li, Thomas J X; Reidys, Christian

    2012-01-01

    RNA-RNA binding is an important phenomenon observed for many classes of non-coding RNAs and plays a crucial role in a number of regulatory processes. Recently several MFE folding algorithms for predicting the joint structure of two interacting RNA molecules have been proposed. Here joint structure...... means that in a diagram representation the intramolecular bonds of each partner are pseudoknot-free, that the intermolecular binding pairs are noncrossing, and that there is no so-called "zigzag" configuration. This paper presents the combinatorics of RNA interaction structures including...

  9. siRNA and innate immunity.

    Science.gov (United States)

    Robbins, Marjorie; Judge, Adam; MacLachlan, Ian

    2009-06-01

    Canonical small interfering RNA (siRNA) duplexes are potent activators of the mammalian innate immune system. The induction of innate immunity by siRNA is dependent on siRNA structure and sequence, method of delivery, and cell type. Synthetic siRNA in delivery vehicles that facilitate cellular uptake can induce high levels of inflammatory cytokines and interferons after systemic administration in mammals and in primary human blood cell cultures. This activation is predominantly mediated by immune cells, normally via a Toll-like receptor (TLR) pathway. The siRNA sequence dependency of these pathways varies with the type and location of the TLR involved. Alternatively nonimmune cell activation may also occur, typically resulting from siRNA interaction with cytoplasmic RNA sensors such as RIG1. As immune activation by siRNA-based drugs represents an undesirable side effect due to the considerable toxicities associated with excessive cytokine release in humans, understanding and abrogating this activity will be a critical component in the development of safe and effective therapeutics. This review describes the intracellular mechanisms of innate immune activation by siRNA, the design of appropriate sequences and chemical modification approaches, and suitable experimental methods for studying their effects, with a view toward reducing siRNA-mediated off-target effects.

  10. Correlation of immune activation with HIV-1 RNA levels assayed by real-time RT-PCR in HIV-1 Subtype C infected patients in Northern India

    Science.gov (United States)

    Agarwal, Atima; Sankaran, Sumathi; Vajpayee, Madhu; Sreenivas, V; Seth, Pradeep; Dandekar, Satya

    2014-01-01

    Background Assays with specificity and cost effectiveness are needed for the measurement of HIV-1 burden to monitor disease progression or response to anti-retroviral therapy (ART) in HIV-1 subtype C infected patients. Objectives The objective of this study was to develop and validate an affordable; one step Real-Time RT-PCR assay with high specificity and sensitivity to measure plasma HIV-1 loads in HIV-1 subtype C infected patients. Results We developed an RT-PCR assay to detect and quantitate plasma HIV-1 levels in HIV-1 subtype C infected patients. An inverse correlation between plasma viral loads (PVL) and CD4+ T-cell numbers was detected at all CDC stages. Significant correlations were found between CD8+ T-cell activation and PVL, as well as with the clinical and immunological status of the patients. Conclusions The RT-PCR assay provides a sensitive method to measure PVL in HIV-1 subtype C infected patients. Viral loads correlated with immune activation and can be used to monitor HIV care in India. PMID:17962068

  11. Effect of antiretroviral drugs on maternal CD4 lymphocyte counts, HIV-1 RNA levels, and anthropometric parameters of their neonates Efeito das drogas anti-retrovirais sobre os valores dos linfócitos TCD4, RNA do HIV-1 e parâmetros antropométricos de neonatos de gestantes portadoras do HIV-1

    Directory of Open Access Journals (Sweden)

    Patrícia El Beitune

    2005-06-01

    Full Text Available PURPOSE: To study the effect of antiretroviral drugs administered during pregnancy on CD4 lymphocyte counts and HIV-1 RNA levels of pregnant women and on the anthropometric parameters of their neonates. METHODS: A prospective study was conducted on 57 pregnant women and their neonates divided into 3 groups: ZDV Group, HIV-infected mothers taking zidovudine (n = 20; triple therapy (TT Group, mothers taking zidovudine + lamivudine + nelfinavir (n = 25, and Control Group, normal women (n = 12. CD4 lymphocyte counts and HIV-1 RNA levels of pregnant women were analyzed during two periods of pregnancy. The perinatal prognosis took into account preterm rates, birth weight, intrauterine growth restriction, perinatal death, and vertical transmission of HIV-1. Data were analyzed statistically using the nonparametric chi-square, Mann-Whitney, Friedman, Kruskal-Wallis, and Wilcoxon matched pairs tests, with the level of significance set at P OBJETIVOS: Estudar o efeito das drogas anti-retrovirais sobre a quantificação dos linfócitos TCD4 e RNA do HIV-1 de gestantes portadoras do HIV-1 e parâmetros antropométricos de seus neonatos. MÉTODOS: Estudo prospectivo avaliando 57 gestantes e seus neonatos em três grupos: Grupo AZT, gestantes portadoras do HIV utilizando zidovudina (n=20; Grupo TT, mães utilizando zidovudina+lamivudina+nelfinavir (n=25, e Grupo Controle, mulheres saudáveis (n=12. A quantificação dos linfócitos TCD4 e RNA do HIV-1 de gestantes portadoras do HIV foi analisada em dois períodos durante a gestação. O prognóstico perinatal levou em consideração as taxas de pré-termos, restrição de crescimento intra-útero, mortalidade perinatal e transmissão vertical do HIV-1. Os dados foram analisados utilizando-se testes não paramétricos de qui-quadrado, Mann-Whitney, Friedman, Kruskal-Wallys e Wilcoxon para amostras pareadas, considerando-se significativos valores associados a p<0,05. RESULTADOS: Observou-se homogeneidade entre

  12. RNA-Seq transcriptomic analysis of the Morus alba L. leaves exposed to high-level UVB with or without dark treatment.

    Science.gov (United States)

    Guan, Qijie; Yu, Jiaojiao; Zhu, Wei; Yang, Bingxian; Li, Yaohan; Zhang, Lin; Tian, Jingkui

    2018-03-01

    Ultraviolet-B (UVB) irradiation induces oxidative stress in plant cells due to the generation of excessive reactive oxygen species. Morus alba L. (M. abla) is an important medicinal plant used for the treatment of human diseases. Also, its leaves are widely used as food for silkworms. In our previous research, we found that a high level of UVB irradiation with dark incubation led to the accumulation of secondary metabolites in M. abla leaf. The aim of the present study was to describe and compare M. alba leaf transcriptomics with different treatments (control, UVB, UVB+dark). Leaf transcripts from M. alba were sequenced using an Illumina Hiseq 2000 system, which produced 14.27Gb of data including 153,204,462 paired-end reads among the three libraries. We de novo assembled 133,002 transcripts with an average length of 1270bp and filtered 69,728 non-redundant unigenes. A similarity search was performed against the non-redundant National Center of Biotechnology Information (NCBI) protein database, which returned 41.08% hits. Among the 20,040 unigenes annotated in UniProtKB/SwissProt database, 16,683 unigenes were assigned 102,232 gene ontology terms and 6667 unigenes were identified in 287 known metabolic pathways. Results of differential gene expression analysis together with real-time quantitative PCR tests indicated that UVB irradiation with dark incubation enhanced the flavonoid biosynthesis in M. alba leaf. Our findings provided a valuable proof for a better understanding of the metabolic mechanism under abiotic stresses in M. alba leaf. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Molecular Characterization of Aquaporin 1 and Aquaporin 3 from the Gills of the African Lungfish, Protopterus annectens, and Changes in Their Branchial mRNA Expression Levels and Protein Abundance during Three Phases of Aestivation.

    Science.gov (United States)

    Chng, You R; Ong, Jasmine L Y; Ching, Biyun; Chen, Xiu L; Hiong, Kum C; Wong, Wai P; Chew, Shit F; Lam, Siew H; Ip, Yuen K

    2016-01-01

    African lungfishes can undergo long periods of aestivation on land during drought. During aestivation, lungfishes are confronted with desiccation and dehydration, and their gills become non-functional and covered with a thick layer of dried mucus. Aquaporins (Aqps) are a superfamily of integral membrane proteins which generally facilitate the permeation of water through plasma membranes. This study aimed to obtain the complete cDNA coding sequences of aqp1 and aqp3 from the gills of Protopterus annectens , and to determine their branchial mRNA and protein expression levels during the induction, maintenance and arousal phases of aestivation. Dendrogramic analyses of the deduced Aqp1 and Aqp3 amino acid sequences of P. annectens revealed their close relationships with those of Latimeria chalumnae and tetrapods. During the induction phase, there were significant decreases in the transcript levels of aqp1 and aqp3 in the gills of P. annectens , but the branchial Aqp1 and Aqp3 protein abundance remained unchanged. As changes in transcription might precede changes in translation, this could be regarded as an adaptive response to decrease the protein abundance of Aqp1 and Aqp3 in the subsequent maintenance phase of aestivation. As expected, the branchial transcript levels and protein abundance of aqp1 /Aqp1 and aqp3 /Aqp3 were significantly down-regulated during the maintenance phase, probably attributable to the shutdown of branchial functions and the cessation of volume regulation of branchial epithelial cells. Additionally, these changes could reduce the loss of water through branchial epithelial surfaces, supplementing the anti-desiccating property of the dried mucus. Upon arousal, it was essential for the lungfish to restore branchial functions. Indeed, the protein abundance of Aqp1 recovered partially, with complete recovery of mRNA expression level and protein abundance of Aqp3, in the gills of P. annectens after 3 days of arousal. These results provide insights

  14. RNA Thermodynamic Structural Entropy.

    Science.gov (United States)

    Garcia-Martin, Juan Antonio; Clote, Peter

    2015-01-01

    Conformational entropy for atomic-level, three dimensional biomolecules is known experimentally to play an important role in protein-ligand discrimination, yet reliable computation of entropy remains a difficult problem. Here we describe the first two accurate and efficient algorithms to compute the conformational entropy for RNA secondary structures, with respect to the Turner energy model, where free energy parameters are determined from UV absorption experiments. An algorithm to compute the derivational entropy for RNA secondary structures had previously been introduced, using stochastic context free grammars (SCFGs). However, the numerical value of derivational entropy depends heavily on the chosen context free grammar and on the training set used to estimate rule probabilities. Using data from the Rfam database, we determine that both of our thermodynamic methods, which agree in numerical value, are substantially faster than the SCFG method. Thermodynamic structural entropy is much smaller than derivational entropy, and the correlation between length-normalized thermodynamic entropy and derivational entropy is moderately weak to poor. In applications, we plot the structural entropy as a function of temperature for known thermoswitches, such as the repression of heat shock gene expression (ROSE) element, we determine that the correlation between hammerhead ribozyme cleavage activity and total free energy is improved by including an additional free energy term arising from conformational entropy, and we plot the structural entropy of windows of the HIV-1 genome. Our software RNAentropy can compute structural entropy for any user-specified temperature, and supports both the Turner'99 and Turner'04 energy parameters. It follows that RNAentropy is state-of-the-art software to compute RNA secondary structure conformational entropy. Source code is available at https://github.com/clotelab/RNAentropy/; a full web server is available at http

  15. RNA Thermodynamic Structural Entropy.

    Directory of Open Access Journals (Sweden)

    Juan Antonio Garcia-Martin

    Full Text Available Conformational entropy for atomic-level, three dimensional biomolecules is known experimentally to play an important role in protein-ligand discrimination, yet reliable computation of entropy remains a difficult problem. Here we describe the first two accurate and efficient algorithms to compute the conformational entropy for RNA secondary structures, with respect to the Turner energy model, where free energy parameters are determined from UV absorption experiments. An algorithm to compute the derivational entropy for RNA secondary structures had previously been introduced, using stochastic context free grammars (SCFGs. However, the numerical value of derivational entropy depends heavily on the chosen context free grammar and on the training set used to estimate rule probabilities. Using data from the Rfam database, we determine that both of our thermodynamic methods, which agree in numerical value, are substantially faster than the SCFG method. Thermodynamic structural entropy is much smaller than derivational entropy, and the correlation between length-normalized thermodynamic entropy and derivational entropy is moderately weak to poor. In applications, we plot the structural entropy as a function of temperature for known thermoswitches, such as the repression of heat shock gene expression (ROSE element, we determine that the correlation between hammerhead ribozyme cleavage activity and total free energy is improved by including an additional free energy term arising from conformational entropy, and we plot the structural entropy of windows of the HIV-1 genome. Our software RNAentropy can compute structural entropy for any user-specified temperature, and supports both the Turner'99 and Turner'04 energy parameters. It follows that RNAentropy is state-of-the-art software to compute RNA secondary structure conformational entropy. Source code is available at https://github.com/clotelab/RNAentropy/; a full web server is available at http

  16. Molecular characterization of three Rhesus glycoproteins from the gills of the African lungfish, Protopterus annectens, and effects of aestivation on their mRNA expression levels and protein abundance.

    Directory of Open Access Journals (Sweden)

    You R Chng

    Full Text Available African lungfishes are ammonotelic in water. They can aestivate for long periods on land during drought. During aestivation, the gills are covered with dried mucus and ammonia excretion ceases. In fishes, ammonia excretion through the gills involves Rhesus glycoproteins (RhGP/Rhgp. This study aimed to obtain the complete cDNA coding sequences of rhgp from the gills of Protopterus annectens, and to determine their branchial mRNA and protein expression levels during the induction, maintenance and arousal phases of aestivation. Three isoforms of rhgp (rhag, rhbg and rhcg were obtained in the gills of P. annectens. Their complete cDNA coding sequences ranged between 1311 and 1398 bp, coding for 436 to 465 amino acids with estimated molecular masses between 46.8 and 50.9 kDa. Dendrogramic analyses indicated that Rhag was grouped closer to fishes, while Rhbg and Rhcg were grouped closer to tetrapods. During the induction phase, the protein abundance of Rhag, but not its transcript level, was down-regulated in the gills, suggesting that there could be a decrease in the release of ammonia from the erythrocytes to the plasma. Furthermore, the branchial transcript levels of rhbg and rhcg decreased significantly, in preparation for the subsequent shutdown of gill functions. During the maintenance phase, the branchial expression levels of rhag/Rhag, rhbg/Rhbg and rhcg/Rhcg decreased significantly, indicating that their transcription and translation were down-regulated. This could be part of an overall mechanism to shut down branchial functions and save metabolic energy used for transcription and translation. It could also be regarded as an adaptive response to stop ammonia excretion. During the arousal phase, it is essential for the lungfish to regain the ability to excrete ammonia. Indeed, the protein abundance of Rhag, Rhbg and Rhcg recovered to the corresponding control levels after 1 day or 3 days of recovery from 6 months of aestivation.

  17. Shielding the messenger (RNA): microRNA-based anticancer therapies

    Science.gov (United States)

    Sotillo, Elena; Thomas-Tikhonenko, Andrei

    2011-01-01

    It has been a decade since scientists realized that microRNAs (miRNAs) are not an oddity invented by worms to regulate gene expression at post-transcriptional levels. Rather, many of these 21–22-nucleotide-short RNAs exist in invertebrates and vertebrates alike and some of them are in fact highly conserved. miRNAs are now recognized as an important class of non-coding small RNAs that inhibit gene expression by targeting mRNA stability and translation. In the last ten years, our knowledge of the miRNAs world was expanding at vertiginous speed, propelled by the development of computational engines for miRNA identification and target prediction, biochemical tools and techniques to modulate miRNA activity, and last but not least, the emergence of miRNA-centric animal models. One important conclusion that has emerged from this effort is that many microRNAs and their cognate targets are strongly implicated in cancer, either as oncogenes or tumor and metastasis suppressors. In this review we will discuss the diverse role that miRNAs play in cancer initiation and progression and also the tools with which miRNA expression could be corrected in vivo. While the idea of targeting microRNAs towards therapeutic ends is getting considerable traction, basic, translational, and clinical research done in the next few years will tell whether this promise is well-founded. PMID:21514318

  18. RNA modifications by oxidation

    DEFF Research Database (Denmark)

    Poulsen, Henrik E; Specht, Elisabeth; Broedbaek, Kasper

    2012-01-01

    to encompass various classes of novel regulatory RNAs, including, e.g., microRNAs. It is well known that DNA is constantly oxidized and repaired by complex genome maintenance mechanisms. Analogously, RNA also undergoes significant oxidation, and there are now convincing data suggesting that oxidation......The past decade has provided exciting insights into a novel class of central (small) RNA molecules intimately involved in gene regulation. Only a small percentage of our DNA is translated into proteins by mRNA, yet 80% or more of the DNA is transcribed into RNA, and this RNA has been found......, and the consequent loss of integrity of RNA, is a mechanism for disease development. Oxidized RNA is found in a large variety of diseases, and interest has been especially devoted to degenerative brain diseases such as Alzheimer disease, in which up to 50-70% of specific mRNA molecules are reported oxidized, whereas...

  19. MicroRNA mimicry blocks pulmonary fibrosis

    Science.gov (United States)

    Montgomery, Rusty L; Yu, Guoying; Latimer, Paul A; Stack, Christianna; Robinson, Kathryn; Dalby, Christina M; Kaminski, Naftali; van Rooij, Eva

    2014-01-01

    Over the last decade, great enthusiasm has evolved for microRNA (miRNA) therapeutics. Part of the excitement stems from the fact that a miRNA often regulates numerous related mRNAs. As such, modulation of a single miRNA allows for parallel regulation of multiple genes involved in a particular disease. While many studies have shown therapeutic efficacy using miRNA inhibitors, efforts to restore or increase the function of a miRNA have been lagging behind. The miR-29 family has gained a lot of attention for its clear function in tissue fibrosis. This fibroblast-enriched miRNA family is downregulated in fibrotic diseases which induces a coordinate increase of many extracellular matrix genes. Here, we show that intravenous injection of synthetic RNA duplexes can increase miR-29 levels in vivo for several days. Moreover, therapeutic delivery of these miR-29 mimics during bleomycin-induced pulmonary fibrosis restores endogenous miR-29 function whereby decreasing collagen expression and blocking and reversing pulmonary fibrosis. Our data support the feasibility of using miRNA mimics to therapeutically increase miRNAs and indicate miR-29 to be a potent therapeutic miRNA for treating pulmonary fibrosis. PMID:25239947

  20. Comparison of protocols and RNA carriers for plasma miRNA isolation. Unraveling RNA carrier influence on miRNA isolation

    Science.gov (United States)

    Martos, Laura; Fernández-Pardo, Álvaro; Oto, Julia; Medina, Pilar; España, Francisco; Navarro, Silvia

    2017-01-01

    microRNAs are promising biomarkers in biological fluids in several diseases. Different plasma RNA isolation protocols and carriers are available, but their efficiencies have been scarcely compared. Plasma microRNAs were isolated using a phenol and column-based procedure and a column-based procedure, in the presence or absence of two RNA carriers (yeast RNA and MS2 RNA). We evaluated the presence of PCR inhibitors and the relative abundance of certain microRNAs by qRT-PCR. Furthermore, we analyzed the association between different isolation protocols, the relative abundance of the miRNAs in the sample, the GC content and the free energy of microRNAs. In all microRNAs analyzed, the addition of yeast RNA as a carrier in the different isolation protocols used gave lower raw Cq values, indicating higher microRNA recovery. Moreover, this increase in microRNAs recovery was dependent on their own relative abundance in the sample, their GC content and the free-energy of their own most stable secondary structure. Furthermore, the normalization of microRNA levels by an endogenous microRNA is more reliable than the normalization by plasma volume, as it reduced the difference in microRNA fold abundance between the different isolation protocols evaluated. Our thorough study indicates that a standardization of pre- and analytical conditions is necessary to obtain reproducible inter-laboratory results in plasma microRNA studies. PMID:29077772

  1. Effects of simultaneously elevated temperature and CO2 levels on Nicotiana benthamiana and its infection by different positive-sense RNA viruses are cumulative and virus type-specific.

    Science.gov (United States)

    Del Toro, Francisco J; Rakhshandehroo, Farshad; Larruy, Beatriz; Aguilar, Emmanuel; Tenllado, Francisco; Canto, Tomás

    2017-11-01

    We have studied how simultaneously elevated temperature and CO 2 levels [climate change-related conditions (CCC) of 30°C, 970 parts-per-million (ppm) of CO 2 vs. standard conditions (SC) of 25°C, ~ 405ppm CO 2 ] affect physiochemical properties of Nicotiana benthamiana leaves, and also its infection by several positive-sense RNA viruses. In previous works we had studied effects of elevated temperature, CO 2 levels separately. Under CCC, leaves of healthy plants almost doubled their area relative to SC but contained less protein/unit-of-area, similarly to what we had found under conditions of elevated CO 2 alone. CCC also affected the sizes/numbers of different foliar cell types differently. Under CCC, infection outcomes in titers and symptoms were virus type-specific, broadly similar to those observed under elevated temperature alone. Under either condition, infections did not significantly alter the protein content of leaf discs. Therefore, effects of elevated temperature and CO 2 combined on properties of the pathosystems studied were overall cumulative. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Species-independent MicroRNA Gene Discovery

    KAUST Repository

    Kamanu, Timothy K.

    2012-12-01

    genes are not only species-specific but may also be DNA strand-specific and chromosome-specific; the genomic distribution of miRNA genes is conserved at the chromosomal level across species; miRNA are conserved; More than one miRNA with different regulatory targets can be excised from one miRNA gene; Repeat-related miRNA and miRNA genes with palindromic sequences may be the largest subclass of miRNA class that have eluded detection by most computational and experimental methods.

  3. RNA-Binding Proteins in Plant Immunity

    Directory of Open Access Journals (Sweden)

    Virginia Woloshen

    2011-01-01

    Full Text Available Plant defence responses against pathogen infection are crucial to plant survival. The high degree of regulation of plant immunity occurs both transcriptionally and posttranscriptionally. Once transcribed, target gene RNA must be processed prior to translation. This includes polyadenylation, 5′capping, editing, splicing, and mRNA export. RNA-binding proteins (RBPs have been implicated at each level of RNA processing. Previous research has primarily focused on structural RNA-binding proteins of yeast and mammals; however, more recent work has characterized a number of plant RBPs and revealed their roles in plant immune responses. This paper provides an update on the known functions of RBPs in plant immune response regulation. Future in-depth analysis of RBPs and other related players will unveil the sophisticated regulatory mechanisms of RNA processing during plant immune responses.

  4. Semi-Nested Real-Time Reverse Transcription Polymerase Chain Reaction Methods for the Successful Quantitation of Cytokeratin mRNA Expression Levels for the Subtyping of Non-Small-Cell Lung Carcinoma Using Paraffin-Embedded and Microdissected Lung Biopsy Specimens

    International Nuclear Information System (INIS)

    Nakanishi, Yoko; Shimizu, Tetsuo; Tsujino, Ichiro; Obana, Yukari; Seki, Toshimi; Fuchinoue, Fumi; Ohni, Sumie; Oinuma, Toshinori; Kusumi, Yoshiaki; Yamada, Tsutomu; Takahashi, Noriaki; Hashimoto, Shu; Nemoto, Norimichi

    2013-01-01

    In patients with inoperable advanced non-small cell lung carcinomas (NSCLCs), histological subtyping using small-mount biopsy specimens was often required to decide the indications for drug treatment. The aim of this study was to assess the utility of highly sensitive mRNA quantitation for the subtyping of advanced NSCLC using small formalin fixing and paraffin embedding (FFPE) biopsy samples. Cytokeratin (CK) 6, CK7, CK14, CK18, and thyroid transcription factor (TTF)-1 mRNA expression levels were measured using semi-nested real-time quantitative (snq) reverse-transcribed polymerase chain reaction (RT-PCR) in microdissected tumor cells collected from 52 lung biopsies. Our results using the present snqRT-PCR method showed an improvement in mRNA quantitation from small FFPE samples, and the mRNA expression level using snqRT-PCR was correlated with the immunohistochemical protein expression level. CK7, CK18, and TTF-1 mRNA were expressed at significantly higher levels (P<0.05) in adenocarcinoma (AD) than in squamous cell carcinoma (SQ), while CK6 and CK14 mRNA expression was significantly higher (P<0.05) in SQ than in AD. Each histology-specific CK, particularly CK18 in AD and CK6 in SQ, were shown to be correlated with a poor prognosis (P=0.02, 0.02, respectively). Our results demonstrated that a quantitative CK subtype mRNA analysis from lung biopsy samples can be useful for predicting the histology subtype and prognosis of advanced NSCLC

  5. Working with RNA

    DEFF Research Database (Denmark)

    Nielsen, Henrik

    2011-01-01

    Working with RNA is not a special discipline in molecular biology. However, RNA is chemically and structurally different from DNA and a few simple work rules have to be implemented to maintain the integrity of the RNA. Alkaline pH, high temperatures, and heavy metal ions should be avoided when po...

  6. Inhibition of carrageenan-induced expression of tissue and plasma prekallikreins mRNA by low level laser therapy in a rat paw edema model Inibição da expressão de RNA mensageiro de pré-calicreínas tecidual e plasmática pela laserterapia em modelo de edema de pata induzido pela carragenina em ratos

    Directory of Open Access Journals (Sweden)

    Moisés P. Silva

    2011-02-01

    Full Text Available BACKGROND: Low level laser therapy (LLLT has been used clinically in order to treat inflammation, where tissue and plasma prekallikrein have crucial importance. Plasma prekallikrein (PPK is synthesized by the hepatocytes and secreted into the bloodstream, where it participates in the surface-dependent activation of blood coagulation, fibrinolysis, kinin generation and inflammation. Tissue prekallikrein is associated with important disease states (including cancer, inflammation, and neurodegeneration and has been utilized or proposed as clinically important biomarker or therapeutic target of interest. OBJECTIVE: To evaluate if LLLT modulates tissue and plasma prekallikreins mRNA expression in the carrageenan-induced rat paw edema. METHODS: Experimental groups were assigned as followed: A1 (Control-saline, A2 (Carrageenan-only, A3 (laser 660nm only and A4 (Carrageenan + laser 660nm. Edema was measured by a plethysmometer. Subplantar tissue was collected for the quantification of prekallikreins mRNA by Real time-Polymerase Chain Reaction. RESULTS: A significantly decrease in the edema was observed after laser irradiation. Expression of prekallikreins increased after carrageenan injection. Tissue and plasma prekallikrein mRNA expression significantly decreased after LLLT's 660nm wavelength. CONCLUSION: These results suggest that expression of tissue and plasma prekallikreins is modulated by LLLT, which can be used in clinical practice due to its anti-inflammatory effects.CONTEXTUALIZAÇÃO: A laserterapia de baixa potência tem sido usada para o tratamento de processos inflamatórios diversos em que a calicreína tecidual e a plasmática possuem participação ativa. A pré-calicreína plasmática (PPK é sintetizada pelos hepatócitos e secretada na corrente sanguínea, onde participa da ativação da coagulação, fibrinólise, geração de cininas e inflamação. A pré-calicreína tecidual está associada com importantes doenças (incluindo c

  7. Effect of different levels of feed restriction and fish oil fatty acid supplementation on fat deposition by using different techniques, plasma levels and mRNA expression of several adipokines in broiler breeder hens.

    Science.gov (United States)

    Mellouk, Namya; Ramé, Christelle; Marchand, Maxime; Staub, Christophe; Touzé, Jean-Luc; Venturi, Éric; Mercerand, Frédéric; Travel, Angélique; Chartrin, Pascal; Lecompte, François; Ma, Linlin; Froment, Pascal; Dupont, Joëlle

    2018-01-01

    Reproductive hens are subjected to a restricted diet to limit the decline in fertility associated with change in body mass. However, endocrine and tissue responses to diet restriction need to be documented. We evaluated the effect of different levels of feed restriction, with or without fish oil supplementation, on metabolic parameters and adipokine levels in plasma and metabolic tissues of reproductive hens. We designed an in vivo protocol involving 4 groups of hens; RNS: restricted (Rt) unsupplemented, ANS: ad libitum (Ad, receiving an amount of feed 1.7 times greater than animals on the restricted diet) unsupplemented, RS: Rt supplemented, and AS: Ad supplemented. The fish oil supplement was used at 1% of the total diet composition. Hens fed with the Rt diet had a significantly (P hens, while the fish oil supplementation had no effect on these parameters. Furthermore, the bioelectrical impedance analysis (BIA) and the fat ultrasonographic examinations produced similar results to the other methods that required animals to be killed (carcass analysis and weight of adipose tissue). In addition, the Rt diet significantly (P hen age. Rt diet and fish oil supplementation are able to modulate metabolic parameters and the expression of adipokines and their receptors in metabolic tissue.

  8. Effect of Genetic Variability in the CYP4F2, CYP4F11, and CYP4F12 Genes on Liver mRNA Levels and Warfarin Response

    Directory of Open Access Journals (Sweden)

    J. E. Zhang

    2017-05-01

    Full Text Available Genetic polymorphisms in the gene encoding cytochrome P450 (CYP 4F2, a vitamin K oxidase, affect stable warfarin dose requirements and time to therapeutic INR. CYP4F2 is part of the CYP4F gene cluster, which is highly polymorphic and exhibits a high degree of linkage disequilibrium, making it difficult to define causal variants. Our objective was to examine the effect of genetic variability in the CYP4F gene cluster on expression of the individual CYP4F genes and warfarin response. mRNA levels of the CYP4F gene cluster were quantified in human liver samples (n = 149 obtained from a well-characterized liver bank and fine mapping of the CYP4F gene cluster encompassing CYP4F2, CYP4F11, and CYP4F12 was performed. Genome-wide association study (GWAS data from a prospective cohort of warfarin-treated patients (n = 711 was also analyzed for genetic variations across the CYP4F gene cluster. In addition, SNP-gene expression in human liver tissues and interactions between CYP4F genes were explored in silico using publicly available data repositories. We found that SNPs in CYP4F2, CYP4F11, and CYP4F12 were associated with mRNA expression in the CYP4F gene cluster. In particular, CYP4F2 rs2108622 was associated with increased CYP4F2 expression while CYP4F11 rs1060467 was associated with decreased CYP4F2 expression. Interestingly, these CYP4F2 and CYP4F11 SNPs showed similar effects with warfarin stable dose where CYP4F11 rs1060467 was associated with a reduction in daily warfarin dose requirement (∼1 mg/day, Pc = 0.017, an effect opposite to that previously reported with CYP4F2 (rs2108622. However, inclusion of either or both of these SNPs in a pharmacogenetic algorithm consisting of age, body mass index (BMI, gender, baseline clotting factor II level, CYP2C9∗2 rs1799853, CYP2C9∗3 rs1057910, and VKORC1 rs9923231 improved warfarin dose variability only by 0.5–0.7% with an improvement in dose prediction accuracy of ∼1–2%. Although there is complex

  9. Methods for RNA Analysis

    DEFF Research Database (Denmark)

    Olivarius, Signe

    of the transcriptome, 5’ end capture of RNA is combined with next-generation sequencing for high-throughput quantitative assessment of transcription start sites by two different methods. The methods presented here allow for functional investigation of coding as well as noncoding RNA and contribute to future...... RNAs rely on interactions with proteins, the establishment of protein-binding profiles is essential for the characterization of RNAs. Aiming to facilitate RNA analysis, this thesis introduces proteomics- as well as transcriptomics-based methods for the functional characterization of RNA. First, RNA...

  10. Differential effects of cortisol and 11-deoxycorticosterone on ion transport protein mRNA levels in gills of two euryhaline teleosts, Mozambique tilapia (Oreochromis mossambicus) and striped bass (Morone saxatilis).

    Science.gov (United States)

    Kiilerich, Pia; Tipsmark, Christian K; Borski, Russell J; Madsen, Steffen S

    2011-04-01

    The role of cortisol as the only corticosteroid in fish osmoregulation has recently been challenged with the discovery of a mineralocorticoid-like hormone, 11-deoxycorticosterone (DOC), and necessitates new studies of the endocrinology of osmoregulation in fish. Using an in vitro gill explant incubation approach, DOC-mediated regulation of selected osmoregulatory target genes in the gill was investigated and compared with that of cortisol in two euryhaline teleosts, Mozambique tilapia (Oreochromis mossambicus) and striped bass (Morone saxatilis). The effects were tested in gills from both fresh water (FW)- and seawater (SW)-acclimated fish. Both cortisol and DOC caused an up-regulation of the Na(+),K(+)-ATPase α1 subunit in SW-acclimated tilapia but had no effect in FW-acclimated fish. Cortisol conferred an increase in Na(+),K(+),2Cl(-) cotransporter (NKCC) isoform 1a transcript levels in FW- and SW-acclimated tilapia, whereas DOC had a stimulatory effect only in SW-acclimated fish. Cortisol had no effect on NKCC isoform 1b mRNA levels at both salinities, while DOC stimulated this isoform in SW-acclimated fish. In striped bass, cortisol conferred an up-regulation of Na(+),K(+)-ATPase α1 and NKCC transcript levels in FW- and SW-acclimated fish, whereas DOC resulted in down-regulation of these transcripts in FW-acclimated fish. It was also found that both corticosteroids may rapidly (30 min) alter the mitogen-activated protein kinase signalling pathway in gill, inducing phosphorylation of extracellular signal-regulated kinase 1 (ERK1) and ERK2 in a salinity-dependent manner. The study shows a disparate organisation of corticosteroid signalling mechanisms involved in ion regulation in the two species and adds new evidence to a role of DOC as a mineralocorticoid hormone in teleosts.

  11. Cytoplasmic Z-RNA

    International Nuclear Information System (INIS)

    Zarling, D.A.; Calhoun, C.J.; Hardin, C.C.; Zarling, A.H.

    1987-01-01

    Specific immunochemical probes for Z-RNA were generated and characterized to search for possible Z-RNA-like double helices in cells. Z-RNA was detected in the cytoplasm of fixed protozoan cells by immunofluorescence microscopy using these anti-Z-RNA IgCs. In contrast, autoimmune or experimentally elicited anti-DNA antibodies, specifically reactive with B-DNA or Z-DNA, stained the nuclei. Pre-or nonimmune IgGs did not bind to the cells. RNase A or T1 digestion eliminated anti-Z-RNA IgG binding to cytoplasmic determinants; however, DNase I or mung bean nuclease had no effect. Doxorubicin and ethidium bromide prevented anti-Z-RNA antibody binding; however, actinomycin D, which does not bind double-stranded RNA, did not. Anti-Z-RNA immunofluorescence was specifically blocked in competition assays by synthetic Z-RNA but not Z-DNA, A-RNA, or single-stranded RNAs. Thus, some cytoplasmic sequences in fixed cells exist in the left-handed Z-RNA conformation

  12. Cancer-Related Triplets of mRNA-lncRNA-miRNA Revealed by Integrative Network in Uterine Corpus Endometrial Carcinoma

    Directory of Open Access Journals (Sweden)

    Chenglin Liu

    2017-01-01

    Full Text Available The regulation of transcriptome expression level is a complex process involving multiple-level interactions among molecules such as protein coding RNA (mRNA, long noncoding RNA (lncRNA, and microRNA (miRNA, which are essential for the transcriptome stability and maintenance and regulation of body homeostasis. The availability of multilevel expression data enables a comprehensive view of the regulatory network. In this study, we analyzed the coding and noncoding gene expression profiles of 301 patients with uterine corpus endometrial carcinoma (UCEC. A new method was proposed to construct a genome-wide integrative network based on variance inflation factor (VIF regression method. The cross-regulation relations of mRNA, lncRNA, and miRNA were then selected based on clique-searching algorithm from the network, when any two molecules of the three were shown as interacting according to the integrative network. Such relation, which we call the mRNA-lncRNA-miRNA triplet, demonstrated the complexity in transcriptome regulation process. Finally, six UCEC-related triplets were selected in which the mRNA participates in endometrial carcinoma pathway, such as CDH1 and TP53. The multi-type RNAs are proved to be cross-regulated as to each of the six triplets according to literature. All the triplets demonstrated the association with the initiation and progression of UCEC. Our method provides a comprehensive strategy for the investigation of transcriptome regulation mechanism.

  13. Neonatal paternal deprivation impairs social recognition and alters levels of oxytocin and estrogen receptor α mRNA expression in the MeA and NAcc, and serum oxytocin in mandarin voles.

    Science.gov (United States)

    Cao, Yan; Wu, Ruiyong; Tai, Fadao; Zhang, Xia; Yu, Peng; An, Xiaolei; Qiao, Xufeng; Hao, Ping

    2014-01-01

    Paternal care is necessary for the healthy development of social behavior in monogamous rodents and social recognition underpins social behavior in these animals. The effects of paternal care on the development of social recognition and underlying neuroendocrine mechanisms, especially the involvement of oxytocin and estrogen pathways, remain poorly understood. We investigated the effects of paternal deprivation (PD: father was removed from neonatal pups and mother alone raised the offspring) on social recognition in mandarin voles (Microtus mandarinus), a socially monogamous rodent. Paternal deprivation was found to inhibit the development of social recognition in female and male offspring according to a habituation-dishabituation paradigm. Paternal deprivation resulted in increased inactivity and reduced investigation during new encounters with other animals. Paternal deprivation reduced oxytocin receptor (OTR) and estrogen receptor α (ERα) mRNA expression in the medial amygdala and nucleus accumbens. Paternal deprivation reduced serum oxytocin (OT) concentration in females, but had no effect on males. Our results provide substantial evidence that paternal deprivation inhibits the development of social recognition in female and male mandarin voles and alters social behavior later in life. This is possibly the result of altered expression of central OTR and ERα and serum OT levels caused by paternal deprivation. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. Cloning of the DNA-binding subunit of human nuclear factor κB: The level of its mRNA is strongly regulated by phorbol ester or tumor necrosis factor α

    International Nuclear Information System (INIS)

    Meyer, R.; Hatada, E.N.; Bartsch, C.; Scheidereit, C.; Hohmann, H.P.; Haiker, M.; Roethlisberger, U.; Lahm, H.W.; Schlaeger, E.J.; van Loon, A.P.G.M.

    1991-01-01

    The DNA binding subunit of nuclear factor κB (NF-κB), a B-cell protein that interacts with the immunoglobulin κ light-chain gene enhancer, has been purified from nuclei of human HL-60 cells stimulated with tumor necrosis factor α (TNFα), and internal peptide sequences were obtained. Overlapping cDNA clones were isolated and sequenced. The encoded open reading frame of about 105 kDa contained at its N-terminal half all six tryptic peptide sequences, suggesting that the 51-kDa NF-κB protein is processed from a 105-kDa precursor. An in vitro synthesized protein containing most of the N-terminal half of the open reading frame bound specifically to an NF-κB binding site. This region also showed high homology to a domain shared by the Drosophila dorsal gene and the avian and mammalian rel (proto)oncogene products. The level of the 3.8-kilobase mRNA was strongly increased after stimulation with TNFα or phorbol ester. Thus, both factors not only activate NF-κB protein, as described previously, but also induce expression of the gene encoding the DNA-binding subunit of NF-κB

  15. Role of uncontrolled HIV RNA level and immunodeficiency in the occurrence of malignancy in HIV-infected patients during the combination antiretroviral therapy era: Agence Nationale de Recherche sur le Sida (ANRS) CO3 Aquitaine Cohort.

    Science.gov (United States)

    Bruyand, Mathias; Thiébaut, Rodolphe; Lawson-Ayayi, Sylvie; Joly, Pierre; Sasco, Annie Jeanne; Mercié, Patrick; Pellegrin, Jean Luc; Neau, Didier; Dabis, François; Morlat, Philippe; Chêne, Geneviève; Bonnet, Fabrice

    2009-10-01

    Human immunodeficiency virus (HIV)-infected patients are at higher risk of malignancies. In addition to traditional determinants, a specific deleterious effect of HIV and immunodeficiency is speculated. We aimed at studying the association between immunological and virological characteristics of HIV-infected patients in care and the risk of acquired immunodeficiency syndrome (AIDS)-defining and non-AIDS-defining malignancies. Patients consecutively enrolled in the hospital-based Agence Nationale de Recherche sur le Sida (ANRS) CO3 Aquitaine Cohort were included if the duration of follow-up was >3 months during the period 1998-2006. Multivariate modeling used an extended Cox proportional hazards model for time-dependent covariates and delayed entry. The 4194 patients included in the study developed 251 first malignancies during 22,389 person-years. A higher incidence of AIDS-defining malignancies (107 cases) was independently associated with (1) both longer and current exposures to a plasma HIV RNA level >500 copies/mL (hazard ratio [HR], 1.27 per year [P500 cells/mm(3) to prevent the occurrence of malignancy, this should be integrated to malignancy-prevention policies.

  16. Validation of reference genes for normalization of qPCR mRNA expression levels in Staphylococcus aureus exposed to osmotic and lactic acid stress conditions encountered during food production and preservation.

    Science.gov (United States)

    Sihto, Henna-Maria; Tasara, Taurai; Stephan, Roger; Johler, Sophia

    2014-07-01

    Staphylococcus aureus represents the most prevalent cause of food-borne intoxications worldwide. While being repressed by competing bacteria in most matrices, this pathogen exhibits crucial competitive advantages during growth at high salt concentrations or low pH, conditions frequently encountered in food production and preservation. We aimed to identify reference genes that could be used to normalize qPCR mRNA expression levels during growth of S. aureus in food-related osmotic (NaCl) and acidic (lactic acid) stress adaptation models. Expression stability of nine housekeeping genes was evaluated in full (LB) and nutrient-deficient (CYGP w/o glucose) medium under conditions of osmotic (4.5% NaCl) and acidic stress (lactic acid, pH 6.0) after 2-h exposure. Among the set of candidate reference genes investigated, rplD, rpoB,gyrB, and rho were most stably expressed in LB and thus represent the most suitable reference genes for normalization of qPCR data in osmotic or lactic acid stress models in a rich medium. Under nutrient-deficient conditions, expression of rho and rpoB was highly stable across all tested conditions. The presented comprehensive data on changes in expression of various S. aureus housekeeping genes under conditions of osmotic and lactic acid stress facilitate selection of reference genes for qPCR-based stress response models. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  17. Pseudogenes regulate parental gene expression via ceRNA network.

    Science.gov (United States)

    An, Yang; Furber, Kendra L; Ji, Shaoping

    2017-01-01

    The concept of competitive endogenous RNA (ceRNA) was first proposed by Salmena and colleagues. Evidence suggests that pseudogene RNAs can act as a 'sponge' through competitive binding of common miRNA, releasing or attenuating repression through sequestering miRNAs away from parental mRNA. In theory, ceRNAs refer to all transcripts such as mRNA, tRNA, rRNA, long non-coding RNA, pseudogene RNA and circular RNA, because all of them may become the targets of miRNA depending on spatiotemporal situation. As binding of miRNA to the target RNA is not 100% complementary, it is possible that one miRNA can bind to multiple target RNAs and vice versa. All RNAs crosstalk through competitively binding to miRNAvia miRNA response elements (MREs) contained within the RNA sequences, thus forming a complex regulatory network. The ratio of a subset of miRNAs to the corresponding number of MREs determines repression strength on a given mRNA translation or stability. An increase in pseudogene RNA level can sequester miRNA and release repression on the parental gene, leading to an increase in parental gene expression. A massive number of transcripts constitute a complicated network that regulates each other through this proposed mechanism, though some regulatory significance may be mild or even undetectable. It is possible that the regulation of gene and pseudogene expression occurring in this manor involves all RNAs bearing common MREs. In this review, we will primarily discuss how pseudogene transcripts regulate expression of parental genes via ceRNA network and biological significance of regulation. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  18. The Old and New RNA World

    Directory of Open Access Journals (Sweden)

    Zofia Szweykowska-Kulińska

    2014-12-01

    Full Text Available Among the numerous hypotheses offering a scenario for the origin of life on Earth, the one called “The RNA World” has gained the most attention. According to this hypothesis RNA acted as a genetic information storage material, as a catalyst of all metabolic reactions, and as a regulator of all processes in the primordial world. Various experiments show that RNA molecules could have been synthesized abiotically, with the potential to mediate a whole repertoire of metabolic reactions. Ribozymes carrying out aminoacyl-tRNA reactions have been found in SELEX (systematic evolution of ligands by exponential enrichment approaches and the development of a ribosome from a RNA-built protoribosome is easy to imagine. Transfer RNA aminoacylation, protoribosome origin, and the availability of amino acids on early Earth allowed the genetic code to evolve. Encoded proteins most likely stabilized RNA molecules and were able to create channels across membranes. In the modern cell, DNA replaced RNA as the main depositor of genetic information and proteins carry out almost all metabolic reactions. However, RNA is still playing versatile, crucial roles in the cell. Apart from its classical functions in the cell, a huge small RNA world is controlling gene expression, chromatin condensation, response to environmental cues, and protecting the cell against the invasion of various nucleic acids forms. Long non-coding RNAs act as crucial gene expression regulators. Riboswitches act at the level of transcription, splicing or translation and mediate feedback regulation on biosynthesis and transport of the ligand they sense. Alternative splicing generates genetic variability and increases the protein repertoire in response to developmental or environmental changes. All these regulatory functions are essential in shaping cell plasticity in the changing milieu. Recent discoveries of new, unexpected and important functions of RNA molecules support the hypothesis that we

  19. Adaptive capability as indicated by behavioral and physiological responses, plasma HSP70 level, and PBMC HSP70 mRNA expression in Osmanabadi goats subjected to combined (heat and nutritional) stressors

    Science.gov (United States)

    Shilja, Shaji; Sejian, V.; Bagath, M.; Mech, A.; David, C. G.; Kurien, E. K.; Varma, Girish; Bhatta, Raghavendra

    2016-09-01

    A study was conducted to assess the impact of heat and nutritional stress simultaneously on the adaptive capability as indicated by behavioral and physiological responses, plasma heat shock protein 70 (HSP70) level, and peripheral blood mononuclear cells (PBMC) HSP70 gene expression in goats. Twenty-four adult Osmanabadi bucks (average body weight (BW) 16.0 kg) were used in the present study. The bucks were divided into four groups viz., C ( n = 6; control), HS ( n = 6; heat stress), NS ( n = 6; nutritional stress), and CS ( n = 6; combined stress). The study was conducted for a period of 45 days. C and HS bucks had ad libitum access to their feed while NS and CS bucks were under restricted feed (30 % intake of C bucks) to induce nutritional stress. The HS and CS bucks were exposed to solar radiation for 6 h a day between 10:00 a.m. and 4:00 p.m. to induce heat stress. The data was analyzed using repeated measures analysis of variance. The standing time differed significantly ( P < 0.01) between ad libitum fed groups (C and HS) and restricted feeding groups (NS and CS). The highest ( P < 0.01) lying time was recorded in the CS group while the lowest in the C and HS groups. The highest ( P < 0.01) drinking frequency was also recorded in the CS group. Water intake recorded was significantly ( P < 0.01) higher in both the HS and CS groups. The highest respiration rate (RR), pulse rate (PR), and rectal temperature (RT) during the afternoon were also recorded in the CS group. Further, skin temperature of the head, flank, and scrotum during the afternoon was also higher ( P < 0.01) in the CS group. In addition, both plasma HSP70 concentration and PBMC HSP70 messenger RNA (mRNA) transcript expression were also significantly ( P < 0.01) higher in the CS group. It can be concluded from this study that when two stressors occur simultaneously, they may have severe impact on adaptive capabilities of Osmanabadi bucks as compared to that would occur individually. Further, the

  20. RNA interference analyses suggest a transcript-specific regulatory role for mitochondrial RNA-binding proteins MRP1 and MRP2 in RNA editing and other RNA processing in Trypanosoma brucei

    NARCIS (Netherlands)

    Vondrusková, Eva; van den Burg, Janny; Zíková, Alena; Ernst, Nancy Lewis; Stuart, Kenneth; Benne, Rob; Lukes, Julius

    2005-01-01

    Mitochondrial RNA-binding proteins MRP1 and MRP2 occur in a heteromeric complex that appears to play a role in U-insertion/deletion editing in trypanosomes. Reduction in the levels of MRP1 (gBP21) and/or MRP2 (gBP25) mRNA by RNA interference in procyclic Trypanosoma brucei resulted in severe growth

  1. Interactions between the HIV-1 Unspliced mRNA and Host mRNA Decay Machineries

    Directory of Open Access Journals (Sweden)

    Daniela Toro-Ascuy

    2016-11-01

    Full Text Available The human immunodeficiency virus type-1 (HIV-1 unspliced transcript is used both as mRNA for the synthesis of structural proteins and as the packaged genome. Given the presence of retained introns and instability AU-rich sequences, this viral transcript is normally retained and degraded in the nucleus of host cells unless the viral protein REV is present. As such, the stability of the HIV-1 unspliced mRNA must be particularly controlled in the nucleus and the cytoplasm in order to ensure proper levels of this viral mRNA for translation and viral particle formation. During its journey, the HIV-1 unspliced mRNA assembles into highly specific messenger ribonucleoproteins (mRNPs containing many different host proteins, amongst which are well-known regulators of cytoplasmic mRNA decay pathways such as up-frameshift suppressor 1 homolog (UPF1, Staufen double-stranded RNA binding protein 1/2 (STAU1/2, or components of miRNA-induced silencing complex (miRISC and processing bodies (PBs. More recently, the HIV-1 unspliced mRNA was shown to contain N6-methyladenosine (m6A, allowing the recruitment of YTH N6-methyladenosine RNA binding protein 2 (YTHDF2, an m6A reader host protein involved in mRNA decay. Interestingly, these host proteins involved in mRNA decay were shown to play positive roles in viral gene expression and viral particle assembly, suggesting that HIV-1 interacts with mRNA decay components to successfully accomplish viral replication. This review summarizes the state of the art in terms of the interactions between HIV-1 unspliced mRNA and components of different host mRNA decay machineries.

  2. Presence of albumin mRNA precursors in nuclei of analbuminemic rat liver lacking cytoplasmic albumin mRNA.

    OpenAIRE

    Esumi, H; Takahashi, Y; Sekiya, T; Sato, S; Nagase, S; Sugimura, T

    1982-01-01

    Analbuminemic rats, which lack serum albumin, were previously found to have no albumin mRNA in the cytoplasm of the liver. In the present study, the existence of nuclear albumin mRNA precursors in the liver of analbuminemic rats was examined by RNA X cDNA hybridization kinetics. Albumin mRNA precursors were present in the nuclei of analbuminemic rat liver at almost normal levels, despite the absence of albumin mRNA from the cytoplasm. Nuclear RNA of analbuminemic rat liver was subjected to el...

  3. The Conserved RNA Exonuclease Rexo5 Is Required for 3′ End Maturation of 28S rRNA, 5S rRNA, and snoRNAs

    Directory of Open Access Journals (Sweden)

    Stefanie Gerstberger

    2017-10-01

    Full Text Available Non-coding RNA biogenesis in higher eukaryotes has not been fully characterized. Here, we studied the Drosophila melanogaster Rexo5 (CG8368 protein, a metazoan-specific member of the DEDDh 3′-5′ single-stranded RNA exonucleases, by genetic, biochemical, and RNA-sequencing approaches. Rexo5 is required for small nucleolar RNA (snoRNA and rRNA biogenesis and is essential in D. melanogaster. Loss-of-function mutants accumulate improperly 3′ end-trimmed 28S rRNA, 5S rRNA, and snoRNA precursors in vivo. Rexo5 is ubiquitously expressed at low levels in somatic metazoan cells but extremely elevated in male and female germ cells. Loss of Rexo5 leads to increased nucleolar size, genomic instability, defective ribosome subunit export, and larval death. Loss of germline expression compromises gonadal growth and meiotic entry during germline development.

  4. The microRNA and messengerRNA profile of the RNA-induced silencing complex in human primary astrocyte and astrocytoma cells.

    Science.gov (United States)

    Moser, Joanna J; Fritzler, Marvin J

    2010-10-18

    GW/P bodies are cytoplasmic ribonucleoprotein-rich foci involved in microRNA (miRNA)-mediated messenger RNA (mRNA) silencing and degradation. The mRNA regulatory functions within GW/P bodies are mediated by GW182 and its binding partner hAgo2 that bind miRNA in the RNA-induced silencing complex (RISC). To date there are no published reports of the profile of miRNA and mRNA targeted to the RISC or a comparison of the RISC-specific miRNA/mRNA profile differences in malignant and non-malignant cells. RISC mRNA and miRNA components were profiled by microarray analysis of malignant human U-87 astrocytoma cells and its non-malignant counterpart, primary human astrocytes. Total cell RNA as well as RNA from immunoprecipitated RISC was analyzed. The novel findings were fourfold: (1) miRNAs were highly enriched in astrocyte RISC compared to U-87 astrocytoma RISC, (2) astrocytoma and primary astrocyte cells each contained unique RISC miRNA profiles as compared to their respective cellular miRNA profiles, (3) miR-195, 10b, 29b, 19b, 34a and 455-3p levels were increased and the miR-181b level was decreased in U-87 astrocytoma RISC as compared to astrocyte RISC, and (4) the RISC contained decreased levels of mRNAs in primary astrocyte and U-87 astrocytoma cells. The observation that miR-34a and miR-195 levels were increased in the RISC of U-87 astrocytoma cells suggests an oncogenic role for these miRNAs. Differential regulation of mRNAs by specific miRNAs is evidenced by the observation that three miR34a-targeted mRNAs and two miR-195-targeted mRNAs were downregulated while one miR-195-targeted mRNA was upregulated. Biological pathway analysis of RISC mRNA components suggests that the RISC plays a pivotal role in malignancy and other conditions. This study points to the importance of the RISC and ultimately GW/P body composition and function in miRNA and mRNA deregulation in astrocytoma cells and possibly in other malignancies.

  5. The microRNA and messengerRNA profile of the RNA-induced silencing complex in human primary astrocyte and astrocytoma cells.

    Directory of Open Access Journals (Sweden)

    Joanna J Moser

    2010-10-01

    Full Text Available GW/P bodies are cytoplasmic ribonucleoprotein-rich foci involved in microRNA (miRNA-mediated messenger RNA (mRNA silencing and degradation. The mRNA regulatory functions within GW/P bodies are mediated by GW182 and its binding partner hAgo2 that bind miRNA in the RNA-induced silencing complex (RISC. To date there are no published reports of the profile of miRNA and mRNA targeted to the RISC or a comparison of the RISC-specific miRNA/mRNA profile differences in malignant and non-malignant cells.RISC mRNA and miRNA components were profiled by microarray analysis of malignant human U-87 astrocytoma cells and its non-malignant counterpart, primary human astrocytes. Total cell RNA as well as RNA from immunoprecipitated RISC was analyzed. The novel findings were fourfold: (1 miRNAs were highly enriched in astrocyte RISC compared to U-87 astrocytoma RISC, (2 astrocytoma and primary astrocyte cells each contained unique RISC miRNA profiles as compared to their respective cellular miRNA profiles, (3 miR-195, 10b, 29b, 19b, 34a and 455-3p levels were increased and the miR-181b level was decreased in U-87 astrocytoma RISC as compared to astrocyte RISC, and (4 the RISC contained decreased levels of mRNAs in primary astrocyte and U-87 astrocytoma cells.The observation that miR-34a and miR-195 levels were increased in the RISC of U-87 astrocytoma cells suggests an oncogenic role for these miRNAs. Differential regulation of mRNAs by specific miRNAs is evidenced by the observation that three miR34a-targeted mRNAs and two miR-195-targeted mRNAs were downregulated while one miR-195-targeted mRNA was upregulated. Biological pathway analysis of RISC mRNA components suggests that the RISC plays a pivotal role in malignancy and other conditions. This study points to the importance of the RISC and ultimately GW/P body composition and function in miRNA and mRNA deregulation in astrocytoma cells and possibly in other malignancies.

  6. HIV-infected individuals with the CCR delta32/CCR5 genotype have lower HIV RNA levels and higher CD4 cell counts in the early years of the infection than do patients with the wild type. Copenhagen AIDS Cohort Study Group

    DEFF Research Database (Denmark)

    Katzenstein, T L; Eugen-Olsen, J; Hofmann, B

    1997-01-01

    The relations among serum HIV RNA levels, CD4 cell counts, presence of the mutant CCR5-allele in heterozygous form, and clinical outcome was analyzed in 96 patients from the Copenhagen AIDS Cohort. In the early years of the infection, patients with the CCR5 delta32/CCR5 genotype had significantly...

  7. Pathways of cellular internalisation of liposomes delivered siRNA and effects on siRNA engagement with target mRNA and silencing in cancer cells.

    Science.gov (United States)

    Alshehri, Abdullah; Grabowska, Anna; Stolnik, Snow

    2018-02-28

    Design of an efficient delivery system is a generally recognised bottleneck in translation of siRNA technology into clinic. Despite research efforts, cellular processes that determine efficiency of siRNA silencing achieved by different delivery formulations remain unclear. Here, we investigated the mechanism(s) of cellular internalisation of a model siRNA-loaded liposome system in a correlation to the engagement of delivered siRNA with its target and consequent silencing by adopting siRNA molecular beacon technology. Probing of cellular internalisation pathways by a panel of pharmacological inhibitors indicated that clathrin-mediated (dynamin-dependent) endocytosis, macropinocytosis (dynamine independent), and cell membrane cholesterol dependent process(es) (clathrin and caveolea-independent) all play a role in the siRNA-liposomes internalization. The inhibition of either of these entry routes was, in general, mirrored by a reduction in the level of siRNA engagement with its target mRNA, as well as in a reduction of the target gene silencing. A dramatic increase in siRNA engagement with its target RNA was observed on disruption of endosomal membrane (by chloroquine), accompanied with an increased silencing. The work thus illustrates that employing molecular beacon siRNA technology one can start to assess the target RNA engagement - a stage between initial cellular internalization and final gene silencing of siRNA delivery systems.

  8. RNA decay by messenger RNA interferases

    DEFF Research Database (Denmark)

    Christensen-Dalsgaard, Mikkel; Overgaard, Martin; Winther, Kristoffer Skovbo

    2008-01-01

    Two abundant toxin-antitoxin (TA) gene families, relBE and mazEF, encode mRNA cleaving enzymes whose ectopic overexpression abruptly inhibits translation and thereby induces a bacteriostatic condition. Here we describe and discuss protocols for the overproduction, purification, and analysis of mR...... cleaving enzymes such as RelE of Escherichia coli and the corresponding antitoxin RelB. In particular, we describe a set of plasmid vectors useful for the detailed analysis of cleavage sites in model mRNAs.......Two abundant toxin-antitoxin (TA) gene families, relBE and mazEF, encode mRNA cleaving enzymes whose ectopic overexpression abruptly inhibits translation and thereby induces a bacteriostatic condition. Here we describe and discuss protocols for the overproduction, purification, and analysis of mRNA...

  9. Regulatory effects of cotranscriptional RNA structure formation and transitions.

    Science.gov (United States)

    Liu, Sheng-Rui; Hu, Chun-Gen; Zhang, Jin-Zhi

    2016-09-01

    RNAs, which play significant roles in many fundamental biological processes of life, fold into sophisticated and precise structures. RNA folding is a dynamic and intricate process, which conformation transition of coding and noncoding RNAs form the primary elements of genetic regulation. The cellular environment contains various intrinsic and extrinsic factors that potentially affect RNA folding in vivo, and experimental and theoretical evidence increasingly indicates that the highly flexible features of the RNA structure are affected by these factors, which include the flanking sequence context, physiochemical conditions, cis RNA-RNA interactions, and RNA interactions with other molecules. Furthermore, distinct RNA structures have been identified that govern almost all steps of biological processes in cells, including transcriptional activation and termination, transcriptional mutagenesis, 5'-capping, splicing, 3'-polyadenylation, mRNA export and localization, and translation. Here, we briefly summarize the dynamic and complex features of RNA folding along with a wide variety of intrinsic and extrinsic factors that affect RNA folding. We then provide several examples to elaborate RNA structure-mediated regulation at the transcriptional and posttranscriptional levels. Finally, we illustrate the regulatory roles of RNA structure and discuss advances pertaining to RNA structure in plants. WIREs RNA 2016, 7:562-574. doi: 10.1002/wrna.1350 For further resources related to this article, please visit the WIREs website. © 2016 Wiley Periodicals, Inc.

  10. Hydration dependent dynamics in RNA

    International Nuclear Information System (INIS)

    Olsen, Greg L.; Bardaro, Michael F.; Echodu, Dorothy C.; Drobny, Gary P.; Varani, Gabriele

    2009-01-01

    The essential role played by local and collective motions in RNA function has led to a growing interest in the characterization of RNA dynamics. Recent investigations have revealed that even relatively simple RNAs experience complex motions over multiple time scales covering the entire ms-ps motional range. In this work, we use deuterium solid-state NMR to systematically investigate motions in HIV-1 TAR RNA as a function of hydration. We probe dynamics at three uridine residues in different structural environments ranging from helical to completely unrestrained. We observe distinct and substantial changes in 2 H solid-state relaxation times and lineshapes at each site as hydration levels increase. By comparing solid-state and solution state 13 C relaxation measurements, we establish that ns-μs motions that may be indicative of collective dynamics suddenly arise in the RNA as hydration reaches a critical point coincident with the onset of bulk hydration. Beyond that point, we observe smaller changes in relaxation rates and lineshapes in these highly hydrated solid samples, compared to the dramatic activation of motion occurring at moderate hydration

  11. Mutations in the RNA-binding domains of tombusvirus replicase proteins affect RNA recombination in vivo

    International Nuclear Information System (INIS)

    Panaviene, Zivile; Nagy, Peter D.

    2003-01-01

    RNA recombination, which is thought to occur due to replicase errors during viral replication, is one of the major driving forces of virus evolution. In this article, we show evidence that the replicase proteins of Cucumber necrosis virus, a tombusvirus, are directly involved in RNA recombination in vivo. Mutations within the RNA-binding domains of the replicase proteins affected the frequency of recombination observed with a prototypical defective-interfering (DI) RNA, a model template for recombination studies. Five of the 17 replicase mutants tested showed delay in the formation of recombinants when compared to the wild-type helper virus. Interestingly, two replicase mutants accelerated recombinant formation and, in addition, these mutants also increased the level of subgenomic RNA synthesis (Virology 308 (2003), 191-205). A trans-complementation system was used to demonstrate that mutation in the p33 replicase protein resulted in altered recombination rate. Isolated recombinants were mostly imprecise (nonhomologous), with the recombination sites clustered around a replication enhancer region and a putative cis-acting element, respectively. These RNA elements might facilitate the proposed template switching events by the tombusvirus replicase. Together with data in the article cited above, results presented here firmly establish that the conserved RNA-binding motif of the replicase proteins is involved in RNA replication, subgenomic RNA synthesis, and RNA recombination

  12. Lysosomal putative RNA transporter SIDT2 mediates direct uptake of RNA by lysosomes.

    Science.gov (United States)

    Aizawa, Shu; Fujiwara, Yuuki; Contu, Viorica Raluca; Hase, Katsunori; Takahashi, Masayuki; Kikuchi, Hisae; Kabuta, Chihana; Wada, Keiji; Kabuta, Tomohiro

    2016-01-01

    Lysosomes are thought to be the major intracellular compartment for the degradation of macromolecules. We recently identified a novel type of autophagy, RNautophagy, where RNA is directly taken up by lysosomes in an ATP-dependent manner and degraded. However, the mechanism of RNA translocation across the lysosomal membrane and the physiological role of RNautophagy remain unclear. In the present study, we performed gain- and loss-of-function studies with isolated lysosomes, and found that SIDT2 (SID1 transmembrane family, member 2), an ortholog of the Caenorhabditis elegans putative RNA transporter SID-1 (systemic RNA interference deficient-1), mediates RNA translocation during RNautophagy. We also observed that SIDT2 is a transmembrane protein, which predominantly localizes to lysosomes. Strikingly, knockdown of Sidt2 inhibited up to ˜50% of total RNA degradation at the cellular level, independently of macroautophagy. Moreover, we showed that this impairment is mainly due to inhibition of lysosomal RNA degradation, strongly suggesting that RNautophagy plays a significant role in constitutive cellular RNA degradation. Our results provide a novel insight into the mechanisms of RNA metabolism, intracellular RNA transport, and atypical types of autophagy.

  13. Topology of RNA-RNA interaction structures

    DEFF Research Database (Denmark)

    Andersen, Jørgen Ellegaard; Huang, Fenix Wenda; Penner, Robert

    2012-01-01

    Abstract The topological filtration of interacting RNA complexes is studied, and the role is analyzed of certain diagrams called irreducible shadows, which form suitable building blocks for more general structures. We prove that, for two interacting RNAs, called interaction structures, there exist...

  14. RNA Localization in Astrocytes

    DEFF Research Database (Denmark)

    Thomsen, Rune

    2012-01-01

    , regulation of the blood brain barrier and glial scar tissue formation. Despite the involvement in various CNS functions only a limited number of studies have addressed mRNA localization in astrocytes. This PhD project was initially focused on developing and implementing methods that could be used to asses mRNA......Messenger RNA (mRNA) localization is a mechanism by which polarized cells can regulate protein synthesis to specific subcellular compartments in a spatial and temporal manner, and plays a pivotal role in multiple physiological processes from embryonic development to cell differentiation...... localization in astrocyte protrusions, and following look into the subcellular localization pattern of specific mRNA species of both primary astrocytes isolated from cortical hemispheres of newborn mice, and the mouse astrocyte cell line, C8S. The Boyden chamber cell fractionation assay was optimized, in a way...

  15. Phage T4 SegB protein is a homing endonuclease required for the preferred inheritance of T4 tRNA gene region occurring in co-infection with a related phage.

    Science.gov (United States)

    Brok-Volchanskaya, Vera S; Kadyrov, Farid A; Sivogrivov, Dmitry E; Kolosov, Peter M; Sokolov, Andrey S; Shlyapnikov, Michael G; Kryukov, Valentine M; Granovsky, Igor E

    2008-04-01

    Homing endonucleases initiate nonreciprocal transfer of DNA segments containing their own genes and the flanking sequences by cleaving the recipient DNA. Bacteriophage T4 segB gene, which is located in a cluster of tRNA genes, encodes a protein of unknown function, homologous to homing endonucleases of the GIY-YIG family. We demonstrate that SegB protein is a site-specific endonuclease, which produces mostly 3' 2-nt protruding ends at its DNA cleavage site. Analysis of SegB cleavage sites suggests that SegB recognizes a 27-bp sequence. It contains 11-bp conserved sequence, which corresponds to a conserved motif of tRNA TpsiC stem-loop, whereas the remainder of the recognition site is rather degenerate. T4-related phages T2L, RB1 and RB3 contain tRNA gene regions that are homologous to that of phage T4 but lack segB gene and several tRNA genes. In co-infections of phages T4 and T2L, segB gene is inherited with nearly 100% of efficiency. The preferred inheritance depends absolutely on the segB gene integrity and is accompanied by the loss of the T2L tRNA gene region markers. We suggest that SegB is a homing endonuclease that functions to ensure spreading of its own gene and the surrounding tRNA genes among T4-related phages.

  16. Size, Shape, and Sequence-Dependent Immunogenicity of RNA Nanoparticles.

    Science.gov (United States)

    Guo, Sijin; Li, Hui; Ma, Mengshi; Fu, Jian; Dong, Yizhou; Guo, Peixuan

    2017-12-15

    RNA molecules have emerged as promising therapeutics. Like all other drugs, the safety profile and immune response are important criteria for drug evaluation. However, the literature on RNA immunogenicity has been controversial. Here, we used the approach of RNA nanotechnology to demonstrate that the immune response of RNA nanoparticles is size, shape, and sequence dependent. RNA triangle, square, pentagon, and tetrahedron with same shape but different sizes, or same size but different shapes were used as models to investigate the immune response. The levels of pro-inflammatory cytokines induced by these RNA nanoarchitectures were assessed in macrophage-like cells and animals. It was found that RNA polygons without extension at the vertexes were immune inert. However, when single-stranded RNA with a specific sequence was extended from the vertexes of RNA polygons, strong immune responses were detected. These immunostimulations are sequence specific, because some other extended sequences induced little or no immune response. Additionally, larger-size RNA square induced stronger cytokine secretion. 3D RNA tetrahedron showed stronger immunostimulation than planar RNA triangle. These results suggest that the immunogenicity of RNA nanoparticles is tunable to produce either a minimal immune response that can serve as safe therapeutic vectors, or a strong immune response for cancer immunotherapy or vaccine adjuvants. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  17. Size, Shape, and Sequence-Dependent Immunogenicity of RNA Nanoparticles

    Directory of Open Access Journals (Sweden)

    Sijin Guo

    2017-12-01

    Full Text Available RNA molecules have emerged as promising therapeutics. Like all other drugs, the safety profile and immune response are important criteria for drug evaluation. However, the literature on RNA immunogenicity has been controversial. Here, we used the approach of RNA nanotechnology to demonstrate that the immune response of RNA nanoparticles is size, shape, and sequence dependent. RNA triangle, square, pentagon, and tetrahedron with same shape but different sizes, or same size but different shapes were used as models to investigate the immune response. The levels of pro-inflammatory cytokines induced by these RNA nanoarchitectures were assessed in macrophage-like cells and animals. It was found that RNA polygons without extension at the vertexes were immune inert. However, when single-stranded RNA with a specific sequence was extended from the vertexes of RNA polygons, strong immune responses were detected. These immunostimulations are sequence specific, because some other extended sequences induced little or no immune response. Additionally, larger-size RNA square induced stronger cytokine secretion. 3D RNA tetrahedron showed stronger immunostimulation than planar RNA triangle. These results suggest that the immunogenicity of RNA nanoparticles is tunable to produce either a minimal immune response that can serve as safe therapeutic vectors, or a strong immune response for cancer immunotherapy or vaccine adjuvants.

  18. Assembling RNA Nanoparticles.

    Science.gov (United States)

    Xiao, Shou-Jun

    2017-01-01

    RNA nanoparticles are designed and self-assembled according to noncanonical interactions of naturally conserved RNA motifs and/or canonical Watson-Crick base-pairing interactions, which have potential applications in gene therapy and nanomedicine. These artificially engineered nanoparticles are mainly synthesized from in vitro transcribed RNAs, purified by denaturing and native polyacrylamide gel electrophoresis (PAGE), and characterized with native PAGE, AFM, and TEM technologies. The protocols of in vitro transcription, denaturing and native PAGE, and RNA nanoparticle self-assembly are described in detail.

  19. Increased 5S rRNA oxidation in Alzheimer's disease.

    Science.gov (United States)

    Ding, Qunxing; Zhu, Haiyan; Zhang, Bing; Soriano, Augusto; Burns, Roxanne; Markesbery, William R

    2012-01-01

    It is widely accepted that oxidative stress is involved in neurodegenerative disorders such as Alzheimer's disease (AD). Ribosomal RNA (rRNA) is one of the most abundant molecules in most cells and is affected by oxidative stress in the human brain. Previous data have indicated that total rRNA levels were decreased in the brains of subjects with AD and mild cognitive impairment concomitant with an increase in rRNA oxidation. In addition, level of 5S rRNA, one of the essential components of the ribosome complex, was significantly lower in the inferior parietal lobule (IP) brain area of subjects with AD compared with control subjects. To further evaluate the alteration of 5S rRNA in neurodegenerative human brains, multiple brain regions from both AD and age-matched control subjects were used in this study, including IP, superior and middle temporal gyro, temporal pole, and cerebellum. Different molecular pools including 5S rRNA integrated into ribosome complexes, free 5S rRNA, cytoplasmic 5S rRNA, and nuclear 5S rRNA were studied. Free 5S rRNA levels were significantly decreased in the temporal pole region of AD subjects and the oxidation of ribosome-integrated and free 5S rRNA was significantly increased in multiple brain regions in AD subjects compared with controls. Moreover, a greater amount of oxidized 5S rRNA was detected in the cytoplasm and nucleus of AD subjects compared with controls. These results suggest that the increased oxidation of 5S rRNA, especially the oxidation of free 5S rRNA, may be involved in the neurodegeneration observed in AD.

  20. RNA precursor pool metabolism and RNA synthesis in X-irradiated Tetrahymena

    International Nuclear Information System (INIS)

    Stephens, R.E.; Paul, I.J.; Zimmerman, A.M.

    1976-01-01

    The incorporation of a radioactive RNA precursor ( 3 H-uridine) has been used in many studies as an index for measuring the synthesis of RNA, yet there is a distinct possibility that the results so obtained were significantly influenced by radiation-induced effects on the metabolism of this precursor into UTP (the primary immediate precursor of RNA) before its incorporation into RNA. A direct examination was therefore undertaken of the effects of X-irradiation on the metabolism of 3 H-uridine and its relationship to RNA synthesis as determined by incorporation. X-irradiation of logarithmically growing Tetrahymena pyriformis caused a dose-dependent depression of total cellular RNA synthesis. Ribosomal RNA (which comprises about 80 per cent of total cellular RNA) synthesis was also depressed by X-irradiation in a dose-dependent manner. Measurements of the levels of radioactivity present in the UTP precursor pool of both irradiated and unirradiated cells were obtained by means of DEAE-cellulose column chromatography of the extracted free nucleotides. Metabolism of 3 H-uridine into UMP, UDP and UTP was depressed by 40%, 26% and 27% respectively, whereas incorporation of 3 H-uridine into RNA was depressed by 77%. The results show that about one-third of the observed (apparent) depression in RNA synthesis was due to radiation-induced effects on the precursor pool, and the remaining two-thirds due to some definite effect of radiation at the transcription level leading to depressed synthesis of RNA. (U.K.)