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Sample records for ercc1 mrna levels

  1. Association between ERCC1 and TS mRNA levels and disease free survival in colorectal cancer patients receiving oxaliplatin and fluorouracil (5-FU) adjuvant chemotherapy.

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    Li, Sheng; Zhu, Liangjun; Yao, Li; Xia, Lei; Pan, Liangxi

    2014-08-29

    Aim was to explore the association of ERCC1 and TS mRNA levels with the disease free survival (DFS) in Chinese colorectal cancer (CRC) patients receiving oxaliplatin and 5-FU based adjuvant chemotherapy. Total 112 Chinese stage II-III CRC patients were respectively treated by four different chemotherapy regimens after curative operation. The TS and ERCC1 mRNA levels in primary tumor were measured by real-time RT-PCR. Kaplan-Meier curves and log-rank tests were used for DFS analysis. The Cox proportional hazards model was used for prognostic analysis. In univariate analysis, the hazard ratio (HR) for the mRNA expression levels of TS and ERCC1 (logTS: HR = 0.820, 95% CI = 0.600 - 1.117, P = 0.210; logERCC1: HR = 1.054, 95% CI = 0.852 - 1.304, P = 0.638) indicated no significant association of DFS with the TS and ERCC1 mRNA levels. In multivariate analyses, tumor stage (IIIc: reference, P = 0.083; IIb: HR = 0.240, 95% CI = 0.080 - 0.724, P = 0.011; IIc: HR TS and ERCC1 mRNA levels were not significantly associated with the DFS (TS: P = 0.264; ERCC1: P = 0.484). The mRNA expression of ERCC1 and TS were not applicable to predict the DFS of Chinese stage II-III CRC patients receiving 5-FU and oxaliplatin based adjuvant chemotherapy.

  2. 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.

  3. XPG mRNA expression levels modulate prognosis in resected non-small-cell lung cancer in conjunction with BRCA1 and ERCC1 expression.

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    Bartolucci, Roberta; Wei, Jia; Sanchez, Jose Javier; Perez-Roca, Laia; Chaib, Imane; Puma, Francesco; Farabi, Raffaele; Mendez, Pedro; Roila, Fausto; Okamoto, Tatsuro; Taron, Miquel; Rosell, Rafael

    2009-01-01

    Molecular markers can help identify patients with early-stage non-small-cell lung cancer (NSCLC) with a high risk of relapse. Excision repair cross-complementing 1 (ERCC1), Xeroderma pigmentosum group G (XPG), and breast cancer 1 (BRCA1) are involved in DNA damage repair, whereas ribonucleotide reductase M1 (RRM1) is implicated in DNA synthesis. Expression levels of these molecules might therefore have a prognostic role in lung cancer. We examined ERCC1, RRM1, XPG, and BRCA1 mRNA levels by real-time quantitative polymerase chain reaction in 54 patients with stage IB-IIB resected NSCLC. A strong correlation was observed between the 4 genes. For patients with low BRCA1, regardless of XPG mRNA expression levels, disease-free survival (DFS) was not reached. For patients with intermediate/high BRCA1 and high XPG, DFS was 50.7 months. However, for patients with intermediate/high BRCA1 and low/intermediate XPG, DFS decreased to 16.3 months (P = .002). Similar differences were observed in overall survival, with median survival not reached for patients with low BRCA1, regardless of XPG levels, or for patients with intermediate/high BRCA1 and high XPG. Conversely, for patients with intermediate/high BRCA1 levels and low/intermediate XPG levels, median survival dropped to 25.5 months (P = .007). BRCA1 and XPG were identified as independent prognostic factors for both median survival and DFS. High BRCA1 mRNA expression confers poor prognosis in early NSCLC, and the combination of high BRCA1 and low XPG expression still further increases the risk of shorter survival. These findings can help optimize the customization of adjuvant chemotherapy.

  4. Correlation between expressions of ERCC1/TS mRNA and effects of gastric cancer to chemotherapy in the short term.

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    Chen, Liqi; Li, Guoli; Li, Jieshou; Fan, Chaogang; Xu, Jian; Wu, Bo; Liu, Kun; Zhang, Caihua

    2013-04-01

    To study the correlation between expression levels of ERCC1/TS mRNA and the susceptibility of preoperative chemotherapy for patients with gastric cancer. A total of forty cases with advanced gastric cancer of T3-4N1-2M0 were treated with preoperative chemotherapy according to FLEEOX regimen based on endarterial-intravenous coadministration. Sufficient, fresh gastric tissue specimens were obtained with the help of gastroscope, and the expression levels of ERCC1/TS mRNA were detected by qRT-PCR before chemotherapy. The chemotherapeutic response was evaluated with Choi Criteria after chemotherapy, and pathologic remission extent was observed after surgery. The correlation between the expression levels of ERCC1/TS mRNA before chemotherapy and the chemotherapeutic effect based on imageology and pathology was analyzed. The response rate of Chemotherapy in this cohort was 80.0 % based on imageology and 51.43 % based on pathology. The expression levels of ERCC1/TS mRNA were significantly associated with imageology remission extent (P = 0.033, P = 0.025) and pathologic remission extent (P = 0.044, P = 0.016), respectively. The chemotherapeutic effect on patients with low-expression levels of ERCC1/TS mRNA was better. From the perspective of pathology and imageology evaluating the preoperative chemotherapeutic response for patients with gastric cancer, ERCC1 and TS were used as the molecular predictors and provided prognostic information in this study.

  5. TS and ERCC-1 mRNA expressions and clinical outcome in patients with metastatic colon cancer in CONFIRM-1 and -2 clinical trials.

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    Grimminger, P P; Shi, M; Barrett, C; Lebwohl, D; Danenberg, K D; Brabender, J; Vigen, C L P; Danenberg, P V; Winder, T; Lenz, H-J

    2012-10-01

    To validate established cutoff levels of thymidylate synthase (TS) and excision repair cross-complementing (ERCC-1) intratumoral mRNA expressions in tumor samples from metastatic colorectal cancer (mCRC) patients treated with PTK787/ZK222584 (PTK/ZK). From 122 samples of patients with mCRC enrolled in CONFIRM-1 (Colorectal Oral Novel Therapy for the Inhibition of Angiogenesis and Retarding of Metastases) or CONFIRM-2, mRNA was isolated of microdissected formalin-fixed paraffin-embedded samples and quantitated using TaqMan-based technology. Existing TS and ERCC-1 cutoff levels were tested for their prognostic value in first-line and second-line therapy. TS expression was associated with overall survival (OS) in first-line, but not second-line therapy. ERCC-1 was associated with OS in patients treated with first-line and second-line FOLFOX4. In first-line FOLFOX4, combination of high TS and/or high ERCC-1 was associated with shorter OS. A correlation was observed between ERCC-1 expression and benefit from PTK/ZK+FOLFOX4 treatment. TS and ERCC-1 expression is associated with clinical outcome in mCRC. Baseline TS and ERCC-1 levels may allow the selection of patients who benefit from FOLFOX4 chemotherapy.

  6. [Effect of TUBB3, TS and ERCC1 mRNA expression on chemoresponse and clinical outcome of advanced gastric cancer by multiplex branched-DNA liquid chip technology].

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    Huang, Jin; Hu, Huabin; Xie, Yangchun; Tang, Youhong; Liu, Wei; Zhong, Meizuo

    2013-06-01

    To analyze the impact of β-tubulin-III (TUBB3), thymidylate synthase (TS) and excision repair cross complementation group 1 (ERCC1) mRNA expression on chemoresponse and clinical outcome of patients with advanced gastric cancer treated with TXT/CDDP/FU (DCF) regimen chemotherapy. The study population consisted of 48 patients with advanced gastric cancer. All patients were treated with DCF regimen palliative chemotherapy. The mRNA expressions of TUBB3, TS and ERCC1 of primary tumors were examined by multiplex branched-DNA liquid chip technology. The patients with low TUBB3 mRNA expression had higher response rate to chemotherapy than patients with high TUBB3 expression (P=0.011). There were no significant differences between response rate and TS or ERCC1 expression pattern. Median overall survival (OS) and median time to progression (TTP) were significantly longer in patients with low TUBB3 mRNA expression (P=0.002, PTS or ERCC1 expression was not correlated with TTP and OS. In the combined analysis including TUBB3, TS and ERCC1, the patients with 0 or 1 high expression gene had better response rate, TTP and OS than the remaining patients (all PTS and ERCC1 expression can promote the individual treatment in advanced gastric cancer.

  7. Prediction of response to chemotherapy by ERCC1 immunohistochemistry and ERCC1 polymorphism in ovarian cancer

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    Dahl Steffensen, Karina; Waldstrøm, Marianne; Jeppesen, Ulla

    Background: The response of tumor cells to platinum-based drugs involves DNA repair mechanisms. Platinum-DNA adducts are repaired by nucleotide excision repair (NER) enzymes that recognize the DNA damage and excise the platinum-DNA adducts from the injured DNA strand. Excision repair cross......-complementation group 1 (ERCC1) is one of the genes that encode the proteins of the NER complex and several studies have linked ERCC1 to platinum resistance in cell lines and in human cancers. Cells with a high repair capacity, e.g. high level of ERCC1 expression may therefore be resistant to platinum......-based chemotherapy, and conversely, polymorphisms within encoding DNA repair enzymes or low repair capacity may confer sensitivity. A common single nucleotide polymorphism (SNP) of ERCC1 at codon 118 have been proposed to impair ERCC1 translation and reduce ERCC1 protein expression and consequently influence...

  8. Low ERCC1 Expression Correlates with Prolonged Survival after Cisplatin plus Gemcitabine Chemotherapy in Non-Small Cell Lung Cancer

    National Research Council Canada - National Science Library

    Reginald V. N. Lord; Jan Brabender; David Gandara; Vicente Alberola; Carlos Camps; Manuel Domine; Felip Cardenal; José M. Sánchez; Paul H. Gumerlock; Miquel Tarón; José J. Sánchez; Kathleen D. Danenberg; Peter V. Danenberg; Rafael Rosell

    2002-01-01

    .... We investigated whether ERCC1 mRNA expression levels were associated with clinical outcomes after treatment with a combination Gem/CDDP regimen for patients with advanced stage non-small cell lung cancer (NSCLC...

  9. Prediction of response to chemotherapy by ERCC1 immunohistochemistry and ERCC1 polymorphism in ovarian cancer

    DEFF Research Database (Denmark)

    Dahl Steffensen, Karina; Waldstrøm, M.; Jeppesen, Ulla

    2007-01-01

    The response of tumor cells to platinum-based chemotherapy involves DNA repair mechanisms. Excision repair cross-complementation group 1 (ercc1) is one of the leading genes involved in DNA repair, and several studies have linked ercc1 to platinum resistance in cell lines and in human cancers....... A common single nucleotide polymorphism (SNP) of ercc1 at codon 118 has been proposed to impair ercc1 translation and reduce ERCC1 protein expression and consequently influence the response to platinum-based chemotherapy. The primary aim of the present study was to evaluate ERCC1 expression and ercc1 codon...... 118 polymorphism in epithelial ovarian cancer (EOC) and their possible predictive value in patients treated with platinum-based chemotherapy. Formalin-fixed, paraffin-embedded tissue sections from 159 patients with advanced EOC were used for immunohistochemistry. Ercc1 codon 118 SNP genotyping...

  10. Differential expression and prognostic value of ERCC1 and thymidylate synthase in resected gastric adenocarcinoma.

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    Squires, Malcolm H; Fisher, Sarah B; Fisher, Kevin E; Patel, Sameer H; Kooby, David A; El-Rayes, Bassel F; Staley, Charles A; Farris, Alton B; Maithel, Shishir K

    2013-09-01

    Excision repair cross-complementing gene-1 (ERCC1) and thymidylate synthase (TS) are key regulatory enzymes whose expression patterns are associated with overall survival (OS) in several malignancies. Their expression patterns and prognostic value in resected gastric adenocarcinoma (GAC) are not known. In total, 109 patients who underwent resection for GAC between January 2000 and June 2011 had tissue available for analysis. The primary objective was to assess for the differential expression of ERCC1 and TS using immunohistochemistry. The secondary objective was to assess for the association between OS and the expression of ERCC1 and TS. The median follow-up was 21.2 months, and the median OS was 28.8 months. Resected GAC exhibited differential expression of ERCC1 (high expression, 23%; n = 25) and TS (high expression, 43%; n = 47). ERCC1 and TS expression were not associated with OS. In a subset analysis of patients who received chemotherapy (n = 73), high ERCC1 expression was associated with decreased OS (16.7 months vs 53.8 months; P = 0.03). After controlling for known adverse pathologic features, high ERCC1 expression persisted as a negative prognostic factor in multivariate Cox regression analysis (hazard ratio, 2.5; 95% confidence interval, 1.03-6.0; P = .04). Conversely, in patients who underwent resection only (n = 35), high ERCC1 expression demonstrated a trend toward improved OS (40.4 months vs 12.7 months; P = .10); a positive prognostic influence also was present on multivariate analysis (hazard ratio, 0.20; 95% confidence interval, 0.04-0.86; P = .03). Resected GAC exhibited differential expression of TS and ERCC1. Among all patients, ERCC1 and TS expression levels were not associated with OS. High ERCC1 tumor expression was associated with decreased OS in the patients who received chemotherapy but was associated with increased OS in those who underwent surgery alone. ERCC1 expression had prognostic value in resected

  11. ERCC1 and ERCC2 haplotype modulates induced BPDE-DNA adducts in primary cultured lymphocytes.

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    Xiaobo Lu

    Full Text Available BACKGROUND: Benzo[a]pyrene(B[a]P, and its ultimate metabolite Benzo[a]pyrene 7,8-diol 9,10-epoxide (BPDE, are classic DNA damaging carcinogens. DNA damage caused by BPDE is normally repaired by Nucleotide Excision Repair (NER, of which ERCC1 and ERCC2/XPD exert an indispensable role. Genetic variations in ERCC1 and ERCC2 have been related to DNA repair efficiency. In this study we used lymphocytes from healthy individuals to show that polymorphisms in ERCC1 and ERCC2 are directly associated with decreased DNA repair efficiency. METHODS: ERCC1 (rs3212986 and rs11615 and ERCC2 (rs13181, rs1799793 and rs238406 were genotyped in 818 healthy Han individuals from the northeast of China. BPDE induced DNA adducts in lymphocytes were assessed by high performance liquid chromatography (HPLC in 282 randomly selected participants. The effect of ERCC1 rs3212986 and ERCC2 rs238406 on DNA damage caused by B[a]P was assessed with a modified comet assay. RESULTS: We found that the variant genotypes of ERCC1 rs3212986 and ERCC2 rs238406 were associated with the high levels of BPDE-DNA adducts. Especially ERCC1 rs3212986 A-allele variant was significantly associated with the high BPDE-DNA adducts. Haplotype analysis showed that the ERCC1 haplotype AC (OR = 2.36, 95% CI = 1.84-2.97, ERCC2 haplotype AGA (OR = 1.51, 95% CI = 1.06-2.15 and haplotype block AGAAC (OR = 5.28, 95% CI = 2.95-9.43, AGCAC (OR = 1.35 95% CI = 1.13-1.60 were linked with high BPDE-DNA adducts. In addition, we found that the combined minor alleles of ERCC1 rs3212986 and ERCC2 rs238406 were associated with a reduced DNA repair capacity. CONCLUSIONS: Our results suggest that the variant genotypes of ERCC1 rs3212986 and ERCC2 rs238406 are associated with decreased repair efficiency of BPDE induced DNA damage, and may be predictive for an individual's DNA repair capacity in response to environmental carcinogens.

  12. Using protein microarray technology to screen anti-ERCC1 monoclonal antibodies for specificity and applications in pathology

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    Ma Donghui

    2012-11-01

    Full Text Available Abstract Background An antibody with cross-reactivity can create unexpected side effects or false diagnostic reports if used for clinical purposes. ERCC1 is being explored as a predictive diagnostic biomarker for cisplatin-based chemotherapy. High ERCC1 expression is linked to drug resistance on cisplatin-based chemotherapy. 8F1 is one of the most commonly used monoclonal antibodies for evaluating ERCC1 expression levels in lung cancer patient tissues, but it has been noted that this antibody cross-reacts with an unknown protein. Results By using a high density protein microarray chip technology, we discovered that 8F1 not only reacts with its authentic target, ERCC1, but also cross-reacts with an unrelated nuclear membrane protein, PCYT1A. The cross-reactivity is due to a common epitope presented on these two unrelated proteins. Similar to the subcellular localization of ERCC1, IHC tests demonstrated that PCYT1A is localized mainly on nuclear membrane. In this study, we also discovered that the PCYT1A gene expression level is significantly higher than the ERCC1 gene expression level in a certain population of lung cancer patient tissue samples. To develop the best monoclonal antibody for ERCC1 IHC analysis, 18 monoclonal antibodies were generated and 6 of them were screened against our protein microarray chip. Two clones showed high mono-specificity on the protein microarray chip test and both worked for the IHC application. Conclusion In summary, the 8F1 clone is not suitable for ERCC1 IHC assay due to its cross-reactivity with PCYT1A protein. Two newly generated monoclonal antibodies, 4F9 and 2E12, demonstrated ultra-specificity against ERCC1 protein and superior performance for IHC analyses.

  13. Synaptic proteome changes in a DNA repair deficient Ercc1 mouse model of accelerated aging

    NARCIS (Netherlands)

    M.J. Végh (Marlene); M.C. de Waard (Monique); I. van der Pluijm (Ingrid); Y. Ridwan (Yanto); M.J.M. Sassen (Marion J.); P. van Nierop (Pim); R.C. van der Schors (Roel); K.W. Li (Ka Wan); J.H.J. Hoeijmakers (Jan); A.B. Smit (August); R.E. van Kesteren (Ronald)

    2012-01-01

    textabstractCognitive decline is one of the earliest hallmarks of both normal and pathological brain aging. Here we used Ercc1 mutant mice, which are impaired in multiple DNA repair systems and consequently show accelerated aging and progressive memory deficits, to identify changes in the levels of

  14. Synaptic proteome changes in a DNA repair deficient ercc1 mouse model of accelerated aging

    NARCIS (Netherlands)

    Vegh, M.J.; de Waard, M.C.; van der Pluijm, I.; Ridwan, Y; Sassen, M.J.M.; van Nierop, P.; van der Schors, R.C.; Li, K.W.; Hoeijmakers, J.H.J.; Smit, A.B.; van Kesteren, R.E.

    2012-01-01

    Cognitive decline is one of the earliest hallmarks of both normal and pathological brain aging. Here we used Ercc1 mutant mice, which are impaired in multiple DNA repair systems and consequently show accelerated aging and progressive memory deficits, to identify changes in the levels of hippocampal

  15. ERCC1 and TS Expression as Prognostic and Predictive Biomarkers in Metastatic Colon Cancer.

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    Choueiri, Michel B; Shen, John Paul; Gross, Andrew M; Huang, Justin K; Ideker, Trey; Fanta, Paul

    2015-01-01

    In patients with metastatic colon cancer, response to first line chemotherapy is a strong predictor of overall survival (OS). Currently, oncologists lack diagnostic tests to determine which chemotherapy regimen offers the greatest chance for response in an individual patient. Here we present the results of gene expression analysis for two genes, ERCC1 and TS, measured with the commercially available ResponseDX: Colon assay (Response Genetics, Los Angeles, CA) in 41 patients with de novo metastatic colon cancer diagnosed between July 2008 and August 2013 at the University of California, San Diego. In addition ERCC1 and TS expression levels as determined by RNAseq and survival data for patients in TCGA were downloaded from the TCGA data portal. We found that patients with low expression of ERCC1 (n = 33) had significantly longer median OS (36.0 vs. 10.1 mo, HR 0.29, 95% CI .095 to .84, log-rank p = 9.0x10-6) and median time to treatment to failure (TTF) following first line chemotherapy (14.1 vs. 2.4 mo, HR 0.17, 95% CI 0.048 to 0.58, log-rank p = 5.3x10-4) relative to those with high expression (n = 4). After accounting for the covariates age, sex, tumor grade and ECOG performance status in a Cox proportional hazard model the association of low ERCC1 with longer OS (HR 0.18, 95% CI 0.14 to 0.26, p = 0.0448) and TTF (HR 0.16, 95% CI 0.14 to 0.21, p = 0.0053) remained significant. Patients with low TS expression (n = 29) had significantly longer median OS (36.0 vs. 14.8 mo, HR 0.25, 95% CI 0.074 to 0.82, log-rank p = 0.022) relative to those with high expression (n = 12). The combined low expression of ERCC1/TS was predictive of response in patients treated with FOLFOX (40% vs. 91%, RR 2.3, Fisher's exact test p = 0.03, n = 27), but not with FOLFIRI (71% vs. 71%, RR 1.0, Fisher's exact test p = 1, n = 14). Overall, these findings suggest that measurement of ERCC1 and TS expression has potential clinical utility in managing patients with metastatic colorectal cancer.

  16. Accelerated loss of hearing and vision in the DNA-repair deficient Ercc1(δ/-) mouse.

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    Spoor, Marcella; Nagtegaal, A Paul; Ridwan, Yanto; Borgesius, Nils Z; van Alphen, Bart; van der Pluijm, Ingrid; Hoeijmakers, Jan H J; Frens, Maarten A; Borst, J Gerard G

    2012-01-01

    Age-related loss of hearing and vision are two very common disabling conditions, but the underlying mechanisms are still poorly understood. Damage by reactive oxygen species and other reactive cellular metabolites, which in turn may damage macromolecules such as DNA, has been implicated in both processes. To investigate whether DNA damage can contribute to age-related hearing and vision loss, we investigated hearing and vision in Ercc1(δ/-) mutant mice, which are deficient in DNA repair of helix-distorting DNA lesions and interstrand DNA crosslinks. Ercc1(δ/-) mice showed a progressive, accelerated increase of hearing level thresholds over time, most likely arising from deteriorating cochlear function. Ercc1(δ/-) mutants also displayed a progressive decrease in contrast sensitivity followed by thinning of the outer nuclear layer of the eyeball. The strong parallels with normal ageing suggest that unrepaired DNA damage can induce age-related decline of the auditory and visual system. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  17. Virtual screening and biological evaluation of inhibitors targeting the XPA-ERCC1 interaction.

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    Khaled H Barakat

    Full Text Available BACKGROUND: Nucleotide excision repair (NER removes many types of DNA lesions including those induced by UV radiation and platinum-based therapy. Resistance to platinum-based therapy correlates with high expression of ERCC1, a major element of the NER machinery. The interaction between ERCC1 and XPA is essential for a successful NER function. Therefore, one way to regulate NER is by inhibiting the activity of ERCC1 and XPA. METHODOLOGY/PRINCIPAL FINDINGS: Here we continued our earlier efforts aimed at the identification and characterization of novel inhibitors of the ERCC1-XPA interaction. We used a refined virtual screening approach combined with a biochemical and biological evaluation of the compounds for their ability to interact with ERCC1 and to sensitize cells to UV radiation. Our findings reveal a new validated ERCC1-XPA inhibitor that significantly sensitized colon cancer cells to UV radiation indicating a strong inhibition of the ERCC1-XPA interaction. CONCLUSIONS: NER is a major factor in acquiring resistance to platinum-based therapy. Regulating the NER pathway has the potential of improving the efficacy of platinum treatments. One approach that we followed is to inhibit the essential interaction between the two NER elements, ERCC1 and XPA. Here, we performed virtual screening against the ERCC1-XPA interaction and identified novel inhibitors that block the XPA-ERCC1 binding. The identified inhibitors significantly sensitized colon cancer cells to UV radiation indicating a strong inhibition of the ERCC1-XPA interaction.

  18. Predictive value of BRCA1, ERCC1, ATP7B, PKM2, TOPOI, TOPΟ-IIA, TOPOIIB and C-MYC genes in patients with small cell lung cancer (SCLC who received first line therapy with cisplatin and etoposide.

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    Niki Karachaliou

    Full Text Available BACKGROUND: The aim of the study was to evaluate the predictive value of genes involved in the action of cisplatin-etoposide in Small Cell Lung Cancer (SCLC. METHODS: 184 SCLC patients' primary tumour samples were analyzed for ERCCI, BRCA1, ATP7B, PKM2 TOPOI, TOPOIIA, TOPOIIB and C-MYC mRNA expression. All patients were treated with cisplatin-etoposide. RESULTS: The patients' median age was 63 years and 120 (65% had extended stage, 75 (41% had increased LDH serum levels and 131 (71% an ECOG performance status was 0-1. Patients with limited stage, whose tumours expressed high ERCC1 (p=0.028, PKM2 (p=0.046, TOPOI (p=0.008, TOPOIIA (p=0.002 and TOPOIIB (p<0.001 mRNA had a shorter Progression Free Survival (PFS. In limited stage patients, high expression of ERCC1 (p=0.014, PKM2 (p=0.026, TOPOIIA (p=0.021 and TOPOIIB (p=0.019 was correlated with decreased median overall survival (mOS while in patients with extended stage, only high TOPOIIB expression had a negative impact on Os (p=0.035. The favorable expression signature expression signature (low expression of ERCC1, PKM2, TOPOIIA and TOPOIIB was correlated with significantly better PFS and Os in both LS-SCLC (p<0.001 and p=0.007, respectively and ES-SCLC (p=0.007 and (p=0.011, respectively group. The unfavorable expression signature was an independent predictor for poor PFS (HR: 3.18; p=0.002 and HR: 3.14; p=0.021 and Os (HR: 4.35; p=0.001and HR: 3.32; p=0.019 in both limited and extended stage, respectively. CONCLUSIONS: Single gene's expression analysis as well as the integrated analysis of ERCC1, PKM2, TOPOIIA and TOPOIIB may predict treatment outcome in patients with SCLC. These findings should be further validated in a prospective study.

  19. The ERCC1 and ERCC4 (XPF) genes and gene products.

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    Manandhar, Mandira; Boulware, Karen S; Wood, Richard D

    2015-09-15

    The ERCC1 and ERCC4 genes encode the two subunits of the ERCC1-XPF nuclease. This enzyme plays an important role in repair of DNA damage and in maintaining genomic stability. ERCC1-XPF nuclease nicks DNA specifically at junctions between double-stranded and single-stranded DNA, when the single-strand is oriented 5' to 3' away from a junction. ERCC1-XPF is a core component of nucleotide excision repair and also plays a role in interstrand crosslink repair, some pathways of double-strand break repair by homologous recombination and end-joining, as a backup enzyme in base excision repair, and in telomere length regulation. In many of these activities, ERCC1-XPF complex cleaves the 3' tails of DNA intermediates in preparation for further processing. ERCC1-XPF interacts with other proteins including XPA, RPA, SLX4 and TRF2 to perform its functions. Disruption of these interactions or direct targeting of ERCC1-XPF to decrease its DNA repair function might be a useful strategy to increase the sensitivity of cancer cells to some DNA damaging agents. Complete deletion of either ERCC1 or ERCC4 is not compatible with viability in mice or humans. However, mutations in the ERCC1 or ERCC4 genes cause a remarkable array of rare inherited human disorders. These include specific forms of xeroderma pigmentosum, Cockayne syndrome, Fanconi anemia, XFE progeria and cerebro-oculo-facio-skeletal syndrome. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. ERCC1 and histopathology in advanced NSCLC patients randomized in a large multicenter phase III trial

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    Vilmar, Adam Christian; Santoni-Rugiu, E; Sørensen, J B

    2010-01-01

    Customized chemotherapy is likely to improve outcome in patients with advanced non-small-cell lung cancer (NSCLC). Excision repair cross-complementation group 1 (ERCC1) is a promising biomarker; however, current evidence is inadequate. Impact of ERCC1 status was evaluated among patients participa......Customized chemotherapy is likely to improve outcome in patients with advanced non-small-cell lung cancer (NSCLC). Excision repair cross-complementation group 1 (ERCC1) is a promising biomarker; however, current evidence is inadequate. Impact of ERCC1 status was evaluated among patients...

  1. RRM1, ERCC1 and TS1 Immunofluorescence Expression in Leiomyosarcoma: A Tissue Microarray Study with Clinical Outcome Correlation Analysis.

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    Zheng, Sam D; Bui, Katherine; Chiappori, Alberto; Bepler, Gerold; Bui, Marilyn M

    2016-07-01

    ERCC1, RRM1 and TS1 are reportedly linked to chemotherapy resistance in lung and other cancers. However, there are currently no studies reporting the relationship between these genes and clinical parameters in leiomyosarcomas. This study investigated the expression pattern of ERCC1, RRM1 and TS1 in forty-four leiomyosarcoma samples by the use of tissue microarray (TMA), immunofluorescence and AQUA methods. The results were then analyzed for expression level and correlations were made with clinical outcome to determine their potential prognostic value in leiomyosarcoma. In the forty-four samples studied, the expression level of these three proteins can be well quantified in the AQUA system and reflected by the AQUA score. RRM1 and ERCC1 expression levels did not show any relationship with overall survival. However, a correlation was found between TS1 expression in the cytoplasm and overall survival. The high expression group had a shorter overall survival time (log-rank p = 0.0498). This trend was confirmed by the Cox proportional hazards model. The poor overall survival of leiomyosarcoma is linked to TS1 cytoplasm expression which may be useful in predicting prognoses of this tumor, methods targeting expression of TS1 may lead to improved overall survival in leiomyosarcoma, though more detailed information regarding treatment information and a larger sample size is needed to confirm this phenomenon.

  2. ERCC1 and XRCC1 as biomarkers for lung and head and neck cancer

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    Vaezi A

    2011-07-01

    Full Text Available Alec Vaezi1,2, Chelsea H Feldman2, Laura J Niedernhofer2,31Department of Otolaryngology and Head and Neck Surgery, University of Pittsburgh School of Medicine, 2University of Pittsburgh Cancer Institute, 3Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine and Cancer Institute, Pittsburgh, PA, USAAbstract: Advanced stage non-small cell lung cancer and head and neck squamous cell carcinoma are both treated with DNA damaging agents including platinum-based compounds and radiation therapy. However, at least one quarter of all tumors are resistant or refractory to these genotoxic agents. Yet the agents are extremely toxic, leading to undesirable side effects with potentially no benefit. Alternative therapies exist, but currently there are no tools to predict whether the first-line genotoxic agents will work in any given patient. To maximize therapeutic success and limit unnecessary toxicity, emerging clinical trials aim to inform personalized treatments tailored to the biology of individual tumors. Worldwide, significant resources have been invested in identifying biomarkers for guiding the treatment of lung and head and neck cancer. DNA repair proteins of the nucleotide excision repair pathway (ERCC1 and of the base excision repair pathway (XRCC1, which are instrumental in clearing DNA damage caused by platinum drugs and radiation, have been extensively studied as potential biomarkers of clinical outcomes in lung and head and neck cancers. The results are complex and contradictory. Here we summarize the current status of single nucleotide polymorphisms, mRNA, and protein expression of ERCC1 and XRCC1 in relation to cancer risk and patient outcomes.Keywords: nucleotide excision repair, base excision repair, DNA damage, DNA repair, chemotherapy, NSCLC, HNSCC, single nucleotide polymorphism

  3. ERCC1 and histopathology in advanced NSCLC patients randomized in a large multicenter phase III trial

    DEFF Research Database (Denmark)

    Vilmar, Adam Christian; Santoni-Rugiu, E; Sørensen, J B

    2010-01-01

    Customized chemotherapy is likely to improve outcome in patients with advanced non-small-cell lung cancer (NSCLC). Excision repair cross-complementation group 1 (ERCC1) is a promising biomarker; however, current evidence is inadequate. Impact of ERCC1 status was evaluated among patients participa...

  4. The Cerebro-oculo-facio-skeletal Syndrome Point Mutation F231L in the ERCC1 DNA Repair Protein Causes Dissociation of the ERCC1-XPF Complex.

    Science.gov (United States)

    Faridounnia, Maryam; Wienk, Hans; Kovačič, Lidija; Folkers, Gert E; Jaspers, Nicolaas G J; Kaptein, Robert; Hoeijmakers, Jan H J; Boelens, Rolf

    2015-08-14

    The ERCC1-XPF heterodimer, a structure-specific DNA endonuclease, is best known for its function in the nucleotide excision repair (NER) pathway. The ERCC1 point mutation F231L, located at the hydrophobic interaction interface of ERCC1 (excision repair cross-complementation group 1) and XPF (xeroderma pigmentosum complementation group F), leads to severe NER pathway deficiencies. Here, we analyze biophysical properties and report the NMR structure of the complex of the C-terminal tandem helix-hairpin-helix domains of ERCC1-XPF that contains this mutation. The structures of wild type and the F231L mutant are very similar. The F231L mutation results in only a small disturbance of the ERCC1-XPF interface, where, in contrast to Phe(231), Leu(231) lacks interactions stabilizing the ERCC1-XPF complex. One of the two anchor points is severely distorted, and this results in a more dynamic complex, causing reduced stability and an increased dissociation rate of the mutant complex as compared with wild type. These data provide a biophysical explanation for the severe NER deficiencies caused by this mutation. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. RPA activates the XPF‐ERCC1 endonuclease to initiate processing of DNA interstrand crosslinks

    KAUST Repository

    Abdullah, Ummi B

    2017-06-13

    During replication‐coupled DNA interstrand crosslink (ICL) repair, the XPF‐ERCC1 endonuclease is required for the incisions that release, or “unhook”, ICLs, but the mechanism of ICL unhooking remains largely unknown. Incisions are triggered when the nascent leading strand of a replication fork strikes the ICL. Here, we report that while purified XPF‐ERCC1 incises simple ICL‐containing model replication fork structures, the presence of a nascent leading strand, modelling the effects of replication arrest, inhibits this activity. Strikingly, the addition of the single‐stranded DNA (ssDNA)‐binding replication protein A (RPA) selectively restores XPF‐ERCC1 endonuclease activity on this structure. The 5′–3′ exonuclease SNM1A can load from the XPF‐ERCC1‐RPA‐induced incisions and digest past the crosslink to quantitatively complete the unhooking reaction. We postulate that these collaborative activities of XPF‐ERCC1, RPA and SNM1A might explain how ICL unhooking is achieved in vivo.

  6. The relationship of platinum resistance and ERCC1 protein expression in epithelial ovarian cancer.

    Science.gov (United States)

    Steffensen, Karina Dahl; Waldstrøm, Marianne; Jakobsen, Anders

    2009-07-01

    Although platinum-based chemotherapy remains the cornerstone for treatment of ovarian cancer, some patients are resistant to the treatment and will therefore not benefit from the standard platinum-based chemotherapy. Preclinical and clinical data have suggested a potential use of excision repair cross-complementation group 1 enzyme (ERCC1) as a molecular predictor of clinical resistance to platinum-based chemotherapy. Excision repair cross-complementation group 1 enzyme is a key enzyme in the nucleotide excision repair pathway which is involved in the DNA repair mechanisms in tumor cells damaged by treatment with platinum agents. The primary aim of the present study was to investigate if immunohistochemical expression of ERCC1 protein was associated with resistance to standard combination carboplatin and paclitaxel chemotherapy in newly diagnosed ovarian cancer patients. Formalin-fixed, paraffin-embedded tissue sections from 101 patients with newly diagnosed ovarian cancer were used for immunohistochemical staining for the ERCC1 protein. All patients received carboplatin-paclitaxel combination chemotherapy. Excision repair cross-complementation group 1 enzyme protein overexpression was found in 13.9% of the tumors. Platinum resistance was found in 75% of the tumors with positive ERCC1 protein expression compared with 27% among the patients with negative tumor staining for ERCC1 (P = 0.0013). These findings translated into a significant difference in progression-free survival in both univariate (P = 0.0012) and in multivariate analysis (P = 0.006). The data presented suggest a positive association between positive ERCC1 protein expression and clinical resistance to platinum-based chemotherapy.

  7. The relationship of platinum resistance and ERCC1 protein expression in epithelial ovarian cancer

    DEFF Research Database (Denmark)

    Steffensen, Karina Dahl; Waldstrøm, Marianne; Jakobsen, Anders

    2009-01-01

    cells damaged by treatment with platinum agents. The primary aim of the present study was to investigate if immunohistochemical expression of ERCC1 protein was associated with resistance to standard combination carboplatin and paclitaxel chemotherapy in newly diagnosed ovarian cancer patients. METHODS......: Formalin-fixed, paraffin-embedded tissue sections from 101 patients with newly diagnosed ovarian cancer were used for immunohistochemical staining for the ERCC1 protein. All patients received carboplatin-paclitaxel combination chemotherapy. RESULTS: Excision repair cross-complementation group 1 enzyme...

  8. Association of ERCC1 protein expression to platinum resistance in epithelial ovarian cancer

    DEFF Research Database (Denmark)

    Dahl Steffensen, Karina; Waldstrøm, Marianne; Jakobsen, Anders

    was to investigate if immunohistochemical expression of ERCC1 protein was associated with resistance to standard combination carboplatin and paclitaxel chemotherapy in newly diagnosed ovarian cancer patients. Methods: Formalin-fixed, paraffin-embedded tissue sections from 101 patients with newly diagnosed ovarian...... by multiplying the staining intensity (0-3) with the percentage score. The tumor was considered positive when the H-score was > 1.0. All patients received carboplatin-paclitaxel combination chemotherapy. Results: ERCC1 protein overexpression was found in 13.9 % of the tumors. Platinum resistance were found in 75...

  9. Enhancement of damage-specific DNA binding of XPA by interaction with the ERCC1 DNA repair protein

    NARCIS (Netherlands)

    A. Nagai; M. Saijo (Masafumi); I. Kuraoka; T. Matsuda (Toshiro); N. Kodo (Naohiko); Y. Nakatsu (Yoshimichi); T. Mimaki; M. Mino; M. Biggerstaff (Maureen); R.D. Wood (Richard); A.M. Sijbers (Anneke); J.H.J. Hoeijmakers (Jan); K. Tanaka (Kiyoji)

    1995-01-01

    textabstractThe human XPA and ERCC1 proteins, which are involved in early steps of nucleotide excision repair of DNA, specifically interacted in an in vitro binding assay and a yeast two-hybrid assay. A stretch of consecutive glutamic acid residues in XPA was needed for binding to ERCC1. Binding of

  10. Disruption of mouse ERCC1 results in a novel repair syndrome with growth failure, nuclear abnormalities and senescence.

    NARCIS (Netherlands)

    G. Weeda (Geert); I. Donker (Ingrid); J. de Wit (Jan); H. Morreau (Hans); R. Janssens (Rick); C.J. Vissers; A. Nigg; H. van Steeg (Harry); D. Bootsma (Dirk); J.H.J. Hoeijmakers (Jan)

    1997-01-01

    textabstractBACKGROUND: The structure-specific ERCC1/XPF endonuclease complex that contains the ERCC1 and XPF subunits is implicated in the repair of two distinct types of lesions in DNA: nucleotide excision repair (NER) for ultraviolet-induced lesions and bulky chemical adducts; and recombination

  11. Correlations between microsatellite instability, ERCC1/XRCC1 polymorphism and clinical characteristics, and FOLFOX adjuvant chemotherapy effect of colorectal cancer patients.

    Science.gov (United States)

    Zhang, Liping; Zhao, Jiangman; Yu, Bin; Song, Xinjiang; Sun, Guogang; Han, Lijiang; Wang, Lu; Dong, Shu

    2017-12-01

    Patients with MSI colorectal tumor have good prognosis and cannot benefit from 5-fluorouracil (5-Fu)-based chemotherapy reported by previous studies. While, single nucleotide polymorphisms (SNP) of ERCC1 and XRCC1 have be proved to influence clinical outcome of colorectal cancer patients treated with oxaliplatin-based chemotherapy. We aim to study the correlation between molecular status and clinical- pathological features, and their effect on CRC patients' clinical outcome treated with mFOLFOX6 adjuvant chemotherapy. In this study, MSI status was determined in tumors and paired normal tissues from 101 colorectal cancer (CRC) patients. We also examined SNP of ERCC1 (c. C354T) and XRCC1 (c.G1196A) in 83 and 85 patients' blood. MSI was found to be significantly correlated with well/moderate histopathological differentiation (p = 0.038) and CRC family history (p = 0.003). CEA and CA19-9 levels was positive correlated significantly by Spearman correlation analysis (Pearson's r = 0.823, p history (p = 0.003). The MSI status, ERCC1 and XRCC1 polymorphisms may influence the clinical outcome of colorectal cancer patients treated with mFOLFOX6 adjuvant chemotherapy. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  12. Mutational analysis of the human nucleotide excision repair gene ERCC1.

    NARCIS (Netherlands)

    A.M. Sijbers (Anneke); P.J. van der Spek (Peter); H. Odijk (Hanny); J.H. van den Berg (Jan); M. van Duin (Mark); A. Westerveld (Andries); N.G.J. Jaspers (Nicolaas); D. Bootsma (Dirk); J.H.J. Hoeijmakers (Jan)

    1996-01-01

    textabstractThe human DNA repair protein ERCC1 resides in a complex together with the ERCC4, ERCC11 and XP-F correcting activities, thought to perform the 5' strand incision during nucleotide excision repair (NER). Its yeast counterpart, RAD1-RAD10, has an additional engagement in a mitotic

  13. An explorative analysis of ERCC1-19q13 copy number aberrations in a chemonaive stage III colorectal cancer cohort

    DEFF Research Database (Denmark)

    Smith, David Hersi; Christensen, Ib Jarle; Jensen, Niels Frank

    2013-01-01

    line panel and in tumor sections from 152 stage III CRC chemonaive patients. Relationships between biomarker status and clinical endpoints (overall survival, time to recurrence, and local recurrence in rectal cancer) were analyzed by survival statistics. Results: ERCC1-19q13 copy number alterations...... investigation is to determine the presence, frequency and prognostic impact of ERCC1 or ERCC4 gene copy number alterations in colorectal cancer (CRC). Methods: Fluorescent in situ hybridization probes directed at ERCC1 and ERCC4 with relevant reference probes were constructed. Probes were tested in a CRC cell...... were observed in a single cell line metaphase (HT29). In patient material, ERCC1-19q13 copy number gains (ERCC1-19q13/CEN-2 ≥ 1.5) were detected in 27.0% of specimens, whereas ERCC1-19q13 deletions (ERCC1-19q13/CEN-2

  14. DNA repair gene expression level in peripheral blood and tumour tissue from non-small cell lung cancer and head and neck squamous cell cancer patients.

    Science.gov (United States)

    Schena, Marina; Guarrera, Simonetta; Buffoni, Lucio; Salvadori, Angelica; Voglino, Floriana; Allione, Alessandra; Pecorari, Giancarlo; Ruffini, Enrico; Garzino-Demo, Paolo; Bustreo, Sara; Consito, Lorena; Bironzo, Paolo; Matullo, Giuseppe

    2012-04-01

    The nucleotide excision repair pathway is crucial for cellular DNA integrity and the ERCC1 helicase is also potentially involved in resistance to platinum-based chemotherapy, and high levels of ERCC1 mRNA in tumours have been associated with cisplatin resistance in different human cancers. The aim of this work was to investigate the correlation between DNA repair gene expression levels in tumour tissue, normal tissue and peripheral blood samples from patients with two common human cancers, non-small cell lung cancer (NSCLC) and squamous cell carcinoma of the head and neck (HNSCC), to test if blood gene expression could be a proxy for tumour tissue gene expression to predict response to platinum-based chemotherapy. Using RT-qPCR we determined ERCC1, ERCC2, ERCC4, XPA, XPC, XRCC1, XRCC3, APEX, OGG1, MGMT mRNA levels in fresh NSCLC, normal lung and HNSCC tissue, as well as blood, from NSCLC and HNSCC patients who were treated surgically. Target gene expression in NSCLC and HNSCC tissue was higher than in blood. A statistically significant correlation (pAPEX, ERCC1, ERCC2, ERCC4, XRCC1 and XRCC3 in HNSCC. The existence of a significant correlation between blood and tumour tissue expression of some genes of clinical interest, such as ERCC1 in NSCLC and HNSCC, could allow the introduction in clinical practice of a simple test that would measure mRNA levels of DNA repair genes in peripheral blood samples instead of tissue samples to determine prognostic and predictive factors in NSCLC and HNSCC patients. Copyright © 2012 Elsevier B.V. All rights reserved.

  15. Evaluation of correlation between CT image features and ERCC1 protein expression in assessing lung cancer prognosis

    Science.gov (United States)

    Tan, Maxine; Emaminejad, Nastaran; Qian, Wei; Sun, Shenshen; Kang, Yan; Guan, Yubao; Lure, Fleming; Zheng, Bin

    2014-03-01

    Stage I non-small-cell lung cancers (NSCLC) usually have favorable prognosis. However, high percentage of NSCLC patients have cancer relapse after surgery. Accurately predicting cancer prognosis is important to optimally treat and manage the patients to minimize the risk of cancer relapse. Studies have shown that an excision repair crosscomplementing 1 (ERCC1) gene was a potentially useful genetic biomarker to predict prognosis of NSCLC patients. Meanwhile, studies also found that chronic obstructive pulmonary disease (COPD) was highly associated with lung cancer prognosis. In this study, we investigated and evaluated the correlations between COPD image features and ERCC1 gene expression. A database involving 106 NSCLC patients was used. Each patient had a thoracic CT examination and ERCC1 genetic test. We applied a computer-aided detection scheme to segment and quantify COPD image features. A logistic regression method and a multilayer perceptron network were applied to analyze the correlation between the computed COPD image features and ERCC1 protein expression. A multilayer perceptron network (MPN) was also developed to test performance of using COPD-related image features to predict ERCC1 protein expression. A nine feature based logistic regression analysis showed the average COPD feature values in the low and high ERCC1 protein expression groups are significantly different (p < 0.01). Using a five-fold cross validation method, the MPN yielded an area under ROC curve (AUC = 0.669±0.053) in classifying between the low and high ERCC1 expression cases. The study indicates that CT phenotype features are associated with the genetic tests, which may provide supplementary information to help improve accuracy in assessing prognosis of NSCLC patients.

  16. ERCC1 and XRCC1 but not XPA single nucleotide polymorphisms correlate with response to chemotherapy in endometrial carcinoma

    Directory of Open Access Journals (Sweden)

    Chen L

    2016-11-01

    Full Text Available Liang Chen,1 Mei-Mei Liu,1 Hui Liu,1 Dan Lu,2 Xiao-Dan Zhao,3 Xue-Jing Yang4 1Department of Gynecology and Obstetrics, 2Department of Oncology, 3Department of Clinical Laboratory, The 2nd Affiliated Hospital, Harbin Medical University, 4Nursing Department, Harbin Chest Hospital, Harbin, People’s Republic of China Abstract: Our study aimed to investigate the correlation between single nucleotide polymorphisms of ERCC1/XRCC1/XPA genes and postoperative chemotherapy efficacy and prognosis of endometrial carcinoma. Our study included 108 patients with endometrial carcinoma and 100 healthy participants. ERCC1 rs11615/XRCC1 rs25487/XPA rs1800975 gene polymorphisms were detected by polymerase chain reaction–restriction fragment length polymorphism. Then the chemotherapy efficacy and toxic effects of the patients were assessed. The genotype and allele frequency of ERCC1 rs11615/XRCC1 rs25487 in the case group were significantly different from that in the control group (all P<0.05. The patients with AA + GA in ERCC1 rs11615 had an increased risk of endometrial carcinoma than those with GG, and the risk of endometrial carcinoma for patients with AA + GA was also higher in comparison with patients with GG genotype in XRCC1 rs25487 (all P<0.05. GG on both ERCC1 rs11615/XRCC1 rs25487 had a higher effective rate of chemotherapy than GA + AA (all P<0.05. ERCC1 rs11615/XRCC1 rs25487 gene polymorphisms were linked with toxic effects in liver, kidney, and nervous system. ERCC1 rs11615/XRCC1 rs25487, muscular invasion, and tumor stage were independent risk factors for the prognosis of endometrial carcinoma (all P<0.05. However, no significant associations were observed between XPA rs1800975 polymorphism and chemotherapy efficacy and prognosis of endometrial carcinoma (all P>0.05. These results indicated that ERCC1 and XRCC1 but not XPA polymorphisms correlate with response to chemotherapy in endometrial carcinoma. Keywords: ERCC1, XRCC1, XPA, single nucleotide

  17. Role of ERCC1 variants in response to chemotherapy and clinical outcome of advanced non-small cell lung cancer.

    Science.gov (United States)

    Huang, Shao-jun; Wang, Yu-fei; Jin, Zhi-yong; Sun, Jia-yang; Guo, Zhan-lin

    2014-05-01

    Excision repair cross-complementation group 1 (ERCC1) and xeroderma pigmentosum-F (XPF) in the nucleotide excision repair pathway have been effectively repairing DNA damage induced by chemotherapeutic agents. We conducted a cohort study to assess the associations of ERCC1 and XPF polymorphisms with response to platinum-based chemotherapy and clinical outcome of non-small-cell lung cancer (NSCLC). One hundred eighty-seven NSCLC cases treated with platinum-based chemotherapy were prospectively analyzed. The predictive value of four SNPs in ERCC1 and two SNPs in XPF in patient's response and survival related to platinum-based chemotherapy were analyzed using χ(2) tests, Kaplan-Meier method, log-rank test, and Cox proportional hazards regression. The overall chemotherapy response rate for treatment was 51.18%. One hundred eighty-seven patients were followed up, and the median survival time is 17.6 months (ranged from 1 to 50 months). A total of 106 patients (56.68%) died from NSCLC during the follow-up period. Carriers of the rs3212986 AA and A allele had a borderline significantly lower response rate to the chemotherapy. In the Cox proportional hazards model, patients carrying the ERCC1 rs3212986 AA genotype were significantly associated with increased risk of death from NSCLC when compared with those with CC genotype as a reference variable. This study reported that variants in ERCC1 can be used as a prognostic maker to platinum-based chemotherapy in NSCLC patients.

  18. High pretherapeutic thymidylate synthetase and MRP-1 protein levels are associated with nonresponse to neoadjuvant chemotherapy in oesophageal adenocarcinoma patients.

    Science.gov (United States)

    Langer, Rupert; Ott, Katja; Feith, Marcus; Lordick, Florian; Specht, Katja; Becker, Karen; Hofler, Heinz

    2010-10-01

    The aim of this study was to determine whether pretherapeutic protein expression levels of the excision repair cross-complementing (ERCC1) enzyme, thymidylate synthetase (TS), multidrug-resistance protein 1 (MRP-1) and P-glycoprotein (P-gp) are associated with tumour response to cisplatin and fluorouracil (5-FU)-based neoadjuvant chemotherapy in oesophageal adenocarcinomas. The expression levels of ERCC1, TS, MDR-1 and P-gp were determined immunohistochemically in pretherapeutic tumour biopsies from 40 oesophageal adenocarcinoma patients and were correlated with histopathological tumour regression and with patient survival. Protein expression was compared to mRNA data, which was previously published for ERCC1, TS and MRP-1 and newly determined for the purpose of this study for MDR-1/P-gp. High-TS and -MRP-1 protein expression was correlated with tumour non-response to chemotherapy (P = 0.001 and P = 0.036, respectively). For ERCC-1 and P-gp, no association between pretherapeutic protein expression and response was found. There was no correlation between mRNA levels and protein expression for all investigated markers. Survival analysis revealed a trend towards increased survival for low-ERCC-1 expression (P = 0.079). The pattern of pretherapeutic expression of TS and MRP-1 is related to chemotherapy response in oesophageal adenocarcinoma patients. Immunohistochemical assessment of these markers may be helpful for response prediction. J. Surg. Oncol. 2010;102:503-508. © 2010 Wiley-Liss, Inc.

  19. Pre-treatment tumor expression of ERCC1 in women with advanced stage epithelial ovarian cancer is not predictive of clinical outcomes: a Gynecologic Oncology Group study.

    Science.gov (United States)

    Rubatt, Jennifer M; Darcy, Kathleen M; Tian, Chunqiao; Muggia, Franco; Dhir, Rajiv; Armstrong, Deborah K; Bookman, Michael A; Niedernhofer, Laura J; Deloia, Julie; Birrer, Michael; Krivak, Thomas Carl

    2012-05-01

    Excision repair cross-complementation group 1 (ERCC1) is required for the repair of platinum-induced DNA damage. This study sought to assess the prognostic value of ERCC1 expression, measured by immunohistochemistry (IHC) using a highly specific antibody, in advanced epithelial ovarian cancer (EOC) patients treated with platinum-based chemotherapy. Formalin-fixed, paraffin-embedded tumors were collected from two GOG phase III trials (GOG-172 and GOG-182) of patients with stage III/IV EOC treated with platinum-based chemotherapy. ERCC1 was detected by (IHC) using FL297 polyclonal antibody and tumors were categorized as negative or positive, based on nuclear staining of tumor cells. ERCC1 genotyping was performed as previously reported. Associations between ERCC1 expression and clinical characteristics, platinum responsiveness, progression-free survival (PFS) or overall survival (OS) were evaluated. Of 408 eligible patients, 27% had tumors that were ERCC1 positive. ERCC1 expression was not associated with clinical characteristics or platinum-responsiveness. Women with ERCC1-positive versus -negative tumors had similar median PFS (17.9 months versus 17.5 months, respectively, p=0.59), median OS (52.0 months versus 47.0 months, respectively, p=0.30), risk of disease progression (adjusted hazard ratio [HR]=0.90, 95% confidence interval (CI): 0.71-1.15, p=0.41), and risk of death (adjusted HR=0.81, 95% CI: 0.61-1.07, p=0.14). ERCC1 expression, as measured by IHC, was not associated with single nucleotide polymorphisms (SNPs), in codon 118 and C8092A, of the ERCC1 gene. ERCC1 expression, measured by IHC in pre-treatment tumor specimens, using a highly specific antibody, has limited clinical value in patients with advanced EOC treated with platinum and taxane based chemotherapy. Published by Elsevier Inc.

  20. Mislocalization of XPF-ERCC1 nuclease contributes to reduced DNA repair in XP-F patients.

    Directory of Open Access Journals (Sweden)

    Anwaar Ahmad

    2010-03-01

    Full Text Available Xeroderma pigmentosum (XP is caused by defects in the nucleotide excision repair (NER pathway. NER removes helix-distorting DNA lesions, such as UV-induced photodimers, from the genome. Patients suffering from XP exhibit exquisite sun sensitivity, high incidence of skin cancer, and in some cases neurodegeneration. The severity of XP varies tremendously depending upon which NER gene is mutated and how severely the mutation affects DNA repair capacity. XPF-ERCC1 is a structure-specific endonuclease essential for incising the damaged strand of DNA in NER. Missense mutations in XPF can result not only in XP, but also XPF-ERCC1 (XFE progeroid syndrome, a disease of accelerated aging. In an attempt to determine how mutations in XPF can lead to such diverse symptoms, the effects of a progeria-causing mutation (XPF(R153P were compared to an XP-causing mutation (XPF(R799W in vitro and in vivo. Recombinant XPF harboring either mutation was purified in a complex with ERCC1 and tested for its ability to incise a stem-loop structure in vitro. Both mutant complexes nicked the substrate indicating that neither mutation obviates catalytic activity of the nuclease. Surprisingly, differential immunostaining and fractionation of cells from an XFE progeroid patient revealed that XPF-ERCC1 is abundant in the cytoplasm. This was confirmed by fluorescent detection of XPF(R153P-YFP expressed in Xpf mutant cells. In addition, microinjection of XPF(R153P-ERCC1 into the nucleus of XPF-deficient human cells restored nucleotide excision repair of UV-induced DNA damage. Intriguingly, in all XPF mutant cell lines examined, XPF-ERCC1 was detected in the cytoplasm of a fraction of cells. This demonstrates that at least part of the DNA repair defect and symptoms associated with mutations in XPF are due to mislocalization of XPF-ERCC1 into the cytoplasm of cells, likely due to protein misfolding. Analysis of these patient cells therefore reveals a novel mechanism to potentially

  1. Association studies of ERCC1 polymorphisms with lung cancer susceptibility: a systematic review and meta-analysis.

    Directory of Open Access Journals (Sweden)

    Jinhong Zhu

    Full Text Available BACKGROUND: Excision repair cross-complimentary group 1 (ERCC1 is an essential component of the nucleotide excision repair system that is responsible for repairing damaged DNA. Functional genetic variations in the ERCC1 gene may alter DNA repair capacity and modulate cancer risk. The putative roles of ERCC1 gene polymorphisms in lung cancer susceptibility have been widely investigated. However, the results remain controversial. OBJECTIVES: An updated meta-analysis was conducted to explore whether lung cancer risk could be attributed to the following ERCC1 polymorphisms: rs11615 (T>C, rs3212986 (C>A, rs3212961 (A>C, rs3212948 (G>C, rs2298881 (C>A. METHODS: Several major databases (MEDLINE, EMBASE and Scopus and the Chinese Biomedical database were searched for eligible studies. Crude odds ratios (ORs with 95% confidence intervals (CIs were used to measure the strength of associations. RESULTS: Sixteen studies with 10,106 cases and 13,238 controls were included in this meta-analysis. Pooled ORs from 11 eligible studies (8,215 cases vs. 11,402 controls suggested a significant association of ERCC1 rs11615 with increased risk for lung cancer (homozygous: CC versus TT, OR = 1.24, 95% CI: 1.04-1.48, P = 0.02. However, such an association was disproportionately driven by a single study. Removal of that study led to null association. Moreover, initial analyses suggested that ERCC1 rs11615 exerts a more profound effect on the susceptibility of non-smokers to lung cancer than that of smokers. Moreover, no statistically significant association was found between remaining ERCC1 polymorphisms of interest and lung cancer risk, except for rs3212948 variation (heterozygous: CG vs.GG, OR = 0.78, 95% CI: 0.67-0.90, P = 0.001; dominant: CG/CC vs.GG, OR = 0.79, 95% CI: 0.69-0.91, P = 0.001. CONCLUSION: Overall, this meta-analysis suggests that ERCC1 rs3212948 G>C, but not others, is a lung cancer risk-associated polymorphism. Carefully

  2. Collagen mRNA levels changes during colorectal cancer carcinogenesis

    DEFF Research Database (Denmark)

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

    2009-01-01

    . In addition, corresponding tissue was examined from healthy volunteers (n = 20). mRNA levels were normalized to beta-actin. Immunohistochemical analysis of the distributions of type IV and type VII collagens were performed on normal and affected tissues from colorectal cancer patients. RESULTS: The alpha1(IV......). The level of alpha 6(IV) was 5-fold lower in colorectal cancer tissue as compared to healthy individuals (p alpha 6(IV) mRNA coincides...... 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...

  3. [The expression of thymidylate synthase (TS) and excision repair complementing-1 (ERCC-1) protein in patients with unresectable colorectal cancer treated with mFOLFOX6 therapy].

    Science.gov (United States)

    Ishibashi, Keiichiro; Okada, Norimichi; Ishiguro, Toru; Kuwabara, Kouki; Ohsawa, Tomonori; Yokoyama, Masaru; Kumamoto, Kensuke; Haga, Norihiro; Mori, Takashi; Yamada, Hirofumi; Miura, Ichiro; Tamaru, Junichi; Itoyama, Shinji; Ishida, Hideyuki

    2010-11-01

    Thymidylate synthase (TS) and excision repair complementing-1 (ERCC-1) were known to be important biomarkers to predict a tumor response to 5-fluorouracil (5-FU) and oxaliplatin, but the relationship between these expressions and tumor response were still unclear. The aim of this study was to determine whether the expression of TS and ERCC-1 protein predict a tumor response in patients with unresectable colorectal cancer treated with mFOLFOX6 therapy as first-line treatment. Fifty patients with unresectable colorectal cancer treated with mFOLFOX6 therapy were enrolled in this study. The expression of TS and ERCC-1 protein in primary cancer cells were examined using immunohistochemistry. There were no significant differences between response rate and the expression of TS or ERCC-1 protein (TS: p>0.99, ERCC-1: p= 0.50). There were no significant differences between progression-free survival time and the expression of TS or ERCC-1 protein (TS: p=0.60, ERCC-1: p=0.60). In this study, the expression TS and ERCC-1 protein may not be useful for the prediction of tumor response in patients with unresectable colorectal cancer treated with mFOLFOX6 therapy.

  4. Recruitment and positioning determine the specific role of the XPF-ERCC1 endonuclease in interstrand crosslink repair

    NARCIS (Netherlands)

    Klein Douwel, Daisy; Hoogenboom, Wouter S; Boonen, Rick Acm; Knipscheer, Puck

    2017-01-01

    XPF-ERCC1 is a structure-specific endonuclease pivotal for several DNA repair pathways and, when mutated, can cause multiple diseases. Although the disease-specific mutations are thought to affect different DNA repair pathways, the molecular basis for this is unknown. Here we examine the function of

  5. The structure-specific endonuclease Ercc1-Xpf is required for targeted gene replacement in embryonic stem cells

    NARCIS (Netherlands)

    L.J. Niedernhofer (Laura); J. Essers (Jeroen); G. Weeda (Geert); H.B. Beverloo (Berna); J. de Wit (Jan); M. Muijtjens (Manja); H. Odijk (Hanny); J.H.J. Hoeijmakers (Jan); R. Kanaar (Roland)

    2001-01-01

    textabstractThe Ercc1-Xpf heterodimer, a highly conserved structure-specific endonuclease, functions in multiple DNA repair pathways that are pivotal for maintaining genome stability, including nucleotide excision repair, interstrand crosslink repair and homologous recombination. Erccl-Xpf incises

  6. Low ERCC1 expression in malignant pleural mesotheliomas treated with cisplatin and vinorelbine predicts prolonged progression-free survival

    DEFF Research Database (Denmark)

    Zimling, Zarah Glad; Sørensen, Jens Benn; Gerds, Thomas Alexander

    2012-01-01

    The relationship between excision repair cross-complementation group 1 (ERCC1) expression and outcome, in patients with malignant pleural mesothelioma (MPM), treated with cisplatin/vinorelbine combination-therapy, was retrospectively evaluated in a patient population from a previously published...

  7. Hfq affects mRNA levels independently of degradation

    Directory of Open Access Journals (Sweden)

    Hajnsdorf Eliane

    2010-02-01

    Full Text Available Abstract Background The bacterial Lsm protein, Hfq, is an RNA chaperone involved in many reactions related to RNA metabolism, such as replication and stability, control of small RNA activity and polyadenylation. Despite this wide spectrum of known functions, the global role of Hfq is almost certainly undervalued; its capacity to bind DNA and to interact with many other proteins are only now beginning to be taken into account. Results The role of Hfq in the maturation and degradation of the rpsO mRNA of E. coli was investigated in vivo. The data revealed a decrease in rpsO mRNA abundance concomitant to an increase in its stability when Hfq is absent. This indicates that the change in mRNA levels in hfq mutants does not result from its modification of RNA stability. Moreover, a series of independent experiments have revealed that the decrease in mRNA level is not a consequence of a reduction of translation efficiency and that Hfq is not directly implicated in translational control of rpsO expression. Reduced steady-state mRNA levels in the absence of Hfq were also shown for rpsT, rpsB and rpsB-tsf, but not for lpp, pnp or tRNA transcripts. The abundance of chimeric transcripts rpsO-lacZ and rpsB-lacZ, whose expression was driven by rpsO and rpsB promoters, respectively, was also lower in the hfq null-mutants, while the β-galactosidase yield remained about the same as in the parent wild-type strain. Conclusions The data obtained suggest that alteration of rpsO, rpsT and rpsB-tsf transcript levels observed under conditions of Hfq deficiency is not caused by the post-transcriptional events, such as mRNA destabilization or changes in translation control, and may rather result from changes in transcriptional activity. So far, how Hfq affects transcription remains unclear. We propose that one of the likely mechanisms of Hfq-mediated modulation of transcription might operate early in the elongation step, when interaction of Hfq with a nascent transcript

  8. First reported patient with human ERCC1 deficiency has cerebro-oculo-facio-skeletal syndrome with a mild defect in nucleotide excision repair and severe developmental failure.

    Science.gov (United States)

    Jaspers, Nicolaas G J; Raams, Anja; Silengo, Margherita Cirillo; Wijgers, Nils; Niedernhofer, Laura J; Robinson, Andria Rasile; Giglia-Mari, Giuseppina; Hoogstraten, Deborah; Kleijer, Wim J; Hoeijmakers, Jan H J; Vermeulen, Wim

    2007-03-01

    Nucleotide excision repair (NER) is a genome caretaker mechanism responsible for removing helix-distorting DNA lesions, most notably ultraviolet photodimers. Inherited defects in NER result in profound photosensitivity and the cancer-prone syndrome xeroderma pigmentosum (XP) or two progeroid syndromes: Cockayne and trichothiodystrophy syndromes. The heterodimer ERCC1-XPF is one of two endonucleases required for NER. Mutations in XPF are associated with mild XP and rarely with progeria. Mutations in ERCC1 have not been reported. Here, we describe the first case of human inherited ERCC1 deficiency. Patient cells showed moderate hypersensitivity to ultraviolet rays and mitomycin C, yet the clinical features were very severe and, unexpectedly, were compatible with a diagnosis of cerebro-oculo-facio-skeletal syndrome. This discovery represents a novel complementation group of patients with defective NER. Further, the clinical severity, coupled with a relatively mild repair defect, suggests novel functions for ERCC1.

  9. Association between the ERCC1 polymorphism and platinum-based chemotherapy effectiveness in ovarian cancer: a meta-analysis.

    Science.gov (United States)

    Tang, Ning; Lyu, Dan; Zhang, Yan; Liu, Haiping

    2017-06-17

    Ovarian cancer is a prominent public health problem which affects people all around the world. Platinum-based chemotherapy is a common treatment for ovarian cancer, however, the effectiveness of chemotherapy varies from patient to patient. The excision repair cross complementation group 1 (ERCC1) protein may mediate chemotherapy resistance. A meta-analysis was conducted to explore whether platinum-based chemotherapy effectiveness could be attributed to the ERCC1 C19007T polymorphisms. Seven major databases (EMBASE, Web of Science, Pubmed, Springer Link, Chinese National Knowledge Infrastructure (CNKI), EBSCO and Science Direct databases) were searched for eligible studies. Crude odds ratios (ORs) with 95% confidence intervals (CIs) were calculated to evaluate the results. In this meta-analysis, 1169 subjects (425 non-responders and 744 responders) from 8 studies were included. The overall OR (C vs. T alleles) using random model was 1.07 (95% CI 0.75-1.52, P = 0.7), which was not statistically significant. Moreover, there was no significant difference in the analysis by race. There is no association between the ERCC1 C19007T polymorphism and platinum-based chemotherapy effectiveness in ovarian cancer. The polymorphism did not have a significant impact on platinum-based chemotherapy in non-responders and responders.

  10. Integrin beta 4 MRNA expression levels in bronchial asthma patients ...

    African Journals Online (AJOL)

    Serum total IgE was measured by ELISA and mRNA expression of ITGβ4 was assessed by reverse transcriptase PCR (RT-PCR) using real time PCR.. ITGβ4 mRNA expression was significantly down regulated with increased serum total IgE in patients with asthma compared to controls. Moreover, ITGβ4 expression was ...

  11. High ERCC1 expression predicts cisplatin-based chemotherapy resistance and poor outcome in unresectable squamous cell carcinoma of head and neck in a betel-chewing area

    Directory of Open Access Journals (Sweden)

    Chien Chih-Yen

    2011-03-01

    Full Text Available Abstract Background This study was to evaluate the effect of excision repair cross-complementation group 1(ERCC1 expression on response to cisplatin-based induction chemotherapy (IC followed by concurrent chemoradiation (CCRT in locally advanced unresectable head and neck squamous cell carcinoma (HNSCC patients. Methods Fifty-seven patients with locally advanced unresectable HNSCC who received cisplatin-based IC followed by CCRT from January 1, 2006 through January 1, 2008. Eligibility criteria included presence of biopsy-proven HNSCC without a prior history of chemotherapy or radiotherapy. Immunohistochemistry was used to assess ERCC1 expression in pretreatment biopsy specimens from paraffin blocks. Clinical parameters, including smoking, alcohol consumption and betel nuts chewing, were obtained from the medical records. Results The 12-month progression-free survival (PFS and 2-year overall survival (OS rates of fifty-seven patients were 61.1% and 61.0%, respectively. Among these patients, thirty-one patients had low ERCC1 expression and forty-one patients responded to IC followed by CCRT. Univariate analyses showed that patients with low expression of ERCC1 had a significantly higher 12-month PFS rates (73.3% vs. 42.3%, p Conclusions Our study suggest that a high expression of ERCC1 predict a poor response and survival to cisplatin-based IC followed by CCRT in patients with locally advanced unresectable HNSCC in betel nut chewing area.

  12. Expression and clinical implication of Beclin1, HMGB1, p62, survivin, BRCA1 and ERCC1 in epithelial ovarian tumor tissues.

    Science.gov (United States)

    Ju, L-L; Zhao, C Y; Ye, K-F; Yang, H; Zhang, J

    2016-05-01

    The aim of the present study is to investigate the differential expression of Beclin1, HMGB1, p62, survivin, ERCC1 and BRCA1 protein in epithelial ovarian cancer (EOC) and to evaluate the relationship between autophagy and platinum resistance of EOC patients during platinum-based chemotherapy with the protein expression. Expression of Beclin1, HMGB1, p62, survivin, ERCC1 and BRCA1 were detected with immunohistochemistry in 60 patients, including 39 with epithelial ovarian cancer (EOC), 13 benign epithelial ovarian tumor tissue (BET) and 8 borderline ovarian tumor tissue. Beclin, p62 and ERCC1 expression was significantly higher in the EOC than the BET (p0.05). BRCA1 expression was lower in EOC than BET (pepithelial ovarian cancer.

  13. ERCC1 and Ki67 in Small Cell Lung Carcinoma and Other Neuroendocrine Tumors of the Lung Distribution and Impact on Survival

    DEFF Research Database (Denmark)

    Skov, Birgit Guldhammer; Holm, B.; Erreboe, A.

    2010-01-01

    ), typical carcinoid (TC), atypical carcinoid (AC), and large cell neuroendocrine carcinoma (LCNEC) were determined. Materials and Methods: We included a consecutive series of 186 patients with SCLC treated with platinum-based chemotherapy and surgically treated patients with TC (n = 48), AC (n = 15......) and LCNEC (n = 27). ERCC1 and Ki 67 were measured by immunohistochemistry and scored using published criteria. Results: The expression of ERCC1 was different among the different tumor types (p disease as well as extensive disease SCLC, no association of ERCC1 expression.......001). The difference between TC and AC was significant (p = 0.02), as was the difference between low grade (TC + AC) and high grade NE (LCNEC + SCLC) (p treated...

  14. Supplementation with Lactobacillus plantarum WCFS1 prevents Decline of Mucus Barrier in Colon of Accelerated Aging Ercc1-/Δ7 Mice

    Directory of Open Access Journals (Sweden)

    Adriaan A Van Beek

    2016-10-01

    Full Text Available Although it is clear that probiotics improve intestinal barrier function, little is known about the effects of probiotics on the aging intestine. We investigated effects of 10-wk bacterial supplementation of Lactobacillus plantarum WCFS1, Lactobacillus casei BL23, or Bifidobacterium breve DSM20213 on gut barrier and immunity in 16-week-old accelerated aging Ercc1-/Δ7 mice, which have a median lifespan of ~20wk, and their wild-type littermates. The colonic barrier in Ercc1-/Δ7 mice was characterized by a thin (<10µm mucus layer. L. plantarum prevented this decline in mucus integrity in Ercc1-/Δ7 mice, whereas B. breve exacerbated it. Bacterial supplementations affected the expression of immune-related genes, including Toll-like receptor 4. Regulatory T cell frequencies were increased in the mesenteric lymph nodes of L. plantarum- and L. casei-treated Ercc1-/Δ7 mice. L. plantarum- and L. casei-treated Ercc1-/Δ7 mice showed increased specific antibody production in a T cell-dependent immune response in vivo. By contrast, the effects of bacterial supplementation on wild-type control mice were negligible. Thus, supplementation with L. plantarum – but not with L. casei and B. breve – prevented the decline in the mucus barrier in Ercc1-/Δ7 mice. Our data indicate that age is an important factor influencing beneficial or detrimental effects of candidate probiotics. These findings also highlight the need for caution in translating beneficial effects of probiotics observed in young animals or humans to the elderly.

  15. Low-level lasers and mRNA levels of reference genes used in Escherichia coli

    Science.gov (United States)

    Teixeira, A. F.; Machado, Y. L. R. C.; Fonseca, A. S.; Mencalha, A. L.

    2016-11-01

    Low-level lasers are widely used for the treatment of diseases and antimicrobial photodynamic therapy. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is widely used to evaluate mRNA levels and output data from a target gene are commonly relative to a reference mRNA that cannot vary according to treatment. In this study, the level of reference genes from Escherichia coli exposed to red or infrared lasers at different fluences was evaluated. E. coli AB1157 cultures were exposed to red (660 nm) and infrared (808 nm) lasers, incubated (20 min, 37 °C), the total RNA was extracted, and cDNA synthesis was performed to evaluate mRNA levels from arcA, gyrA and rpoA genes by RT-qPCR. Melting curves and agarose gel electrophoresis were carried out to evaluate specific amplification. Data were analyzed by geNorm, NormFinder and BestKeeper. The melting curve and agarose gel electrophoresis showed specific amplification. Although mRNA levels from arcA, gyrA or rpoA genes presented no significant variations trough a traditional statistical analysis, Excel-based tools revealed that these reference genes are not suitable for E. coli cultures exposed to lasers. Our data showed that exposure to low-level red and infrared lasers at different fluences alter the mRNA levels from arcA, gyrA and rpoA in E. coli cells.

  16. Molecular characterization of the human excision repair gene ERCC-1: cDNA cloning and aminoacid homology with the yeast DNA repair gene RAD10.

    NARCIS (Netherlands)

    M. van Duin (Mark); J. de Wit (Jan); H. Odijk (Hanny); A. Westerveld (Andries); A. Yasui (Akira); M.H.M. Koken (Marcel); J.H.J. Hoeijmakers (Jan); D. Bootsma (Dirk)

    1986-01-01

    textabstractThe human excision repair gene ERCC-7 was cloned after DNA mediated gene transfer to the CHO mutant 43-38, which is sensitive to ultraviolet light and mitomycin-C. We describe the cloning and sequence analysis of the ERCC-7 cDNA and partial characterization of the gene. ERCC.1 has a size

  17. Dietary restriction but not angiotensin II type 1 receptor blockade improves DNA damage-related vasodilator dysfunction in rapidly aging Ercc1Δ/- mice.

    NARCIS (Netherlands)

    Wu, Haiyan; van Thiel, Bibi S; Bautista-Niño, Paula K; Reiling, Erwin; Durik, Matej; Leijten, Frank P J; Ridwan, Yanto; Brandt, Renata M C; van Steeg, Harry; Dollé, Martijn E T; Vermeij, Wilbert P; Hoeijmakers, Jan H J; Essers, Jeroen; van der Pluijm, Ingrid; Danser, A H Jan; Roks, Anton J M

    2017-01-01

    DNA damage is an important contributor to endothelial dysfunction and age-related vascular disease. Recently, we demonstrated in a DNA repair-deficient, prematurely aging mouse model (Ercc1Δ/- mice) that dietary restriction (DR) strongly increases life- and health span, including ameliorating

  18. Frontline Science: Tryptophan restriction arrests B cell development and enhances microbial diversity in WT and prematurely aging Ercc1-/Δ7 mice.

    Science.gov (United States)

    van Beek, Adriaan A; Hugenholtz, Floor; Meijer, Ben; Sovran, Bruno; Perdijk, Olaf; Vermeij, Wilbert P; Brandt, Renata M C; Barnhoorn, Sander; Hoeijmakers, Jan H J; de Vos, Paul; Leenen, Pieter J M; Hendriks, Rudi W; Savelkoul, Huub F J

    2017-04-01

    With aging, tryptophan metabolism is affected. Tryptophan has a crucial role in the induction of immune tolerance and the maintenance of gut microbiota. We, therefore, studied the effect of dietary tryptophan restriction in young wild-type (WT) mice (118-wk life span) and in DNA-repair deficient, premature-aged (Ercc1-/Δ7 ) mice (20-wk life span). First, we found that the effect of aging on the distribution of B and T cells in bone marrow (BM) and in the periphery of 16-wk-old Ercc1-/Δ7 mice was comparable to that in 18-mo-old WT mice. Dietary tryptophan restriction caused an arrest of B cell development in the BM, accompanied by diminished B cell frequencies in the periphery. In general, old Ercc1-/Δ7 mice showed similar responses to tryptophan restriction compared with young WT mice, indicative of age-independent effects. Dietary tryptophan restriction increased microbial diversity and made the gut microbiota composition of old Ercc1-/Δ7 mice more similar to that of young WT mice. The decreased abundances of Alistipes and Akkermansia spp. after dietary tryptophan restriction correlated significantly with decreased B cell precursor numbers. In conclusion, we report that dietary tryptophan restriction arrests B cell development and concomitantly changes gut microbiota composition. Our study suggests a beneficial interplay between dietary tryptophan, B cell development, and gut microbial composition on several aspects of age-induced changes. © Society for Leukocyte Biology.

  19. TS mRNA levels can predict pemetrexed and raltitrexed sensitivity in colorectal cancer.

    Science.gov (United States)

    Zhang, Qun; Shen, Jie; Wang, Hao; Hu, Jing; Yu, Lixia; Xie, Li; Wei, Jia; Liu, Baorui; Guan, Wenxian; Qian, Xiaoping

    2014-02-01

    The purpose of the study is to analyze the relationship between tumor thymidylate synthase (TS) mRNA expression levels and raltitrexed/pemetrexed/5-FU sensitivity. We collected freshly removed colorectal tumor specimens from 50 patients. Chemosensitivities to anticancer drugs were evaluated by histoculture drug response assay. We adopted quantitative reverse transcription polymerase chain reaction for TS mRNA detection and immunohistochemical staining for assessing TS expression in tumor tissues. There is a significant relationship between TS mRNA expression levels and in vitro chemosensitivity of freshly removed colorectal tumor specimens to pemetrexed (P TS mRNA expression levels can predict pemetrexed and raltitrexed sensitivity in colorectal cancer.

  20. Malfunction of nuclease ERCC1-XPF results in diverse clinical manifestations and causes Cockayne syndrome, xeroderma pigmentosum, and Fanconi anemia.

    Science.gov (United States)

    Kashiyama, Kazuya; Nakazawa, Yuka; Pilz, Daniela T; Guo, Chaowan; Shimada, Mayuko; Sasaki, Kensaku; Fawcett, Heather; Wing, Jonathan F; Lewin, Susan O; Carr, Lucinda; Li, Tao-Sheng; Yoshiura, Koh-ichiro; Utani, Atsushi; Hirano, Akiyoshi; Yamashita, Shunichi; Greenblatt, Danielle; Nardo, Tiziana; Stefanini, Miria; McGibbon, David; Sarkany, Robert; Fassihi, Hiva; Takahashi, Yoshito; Nagayama, Yuji; Mitsutake, Norisato; Lehmann, Alan R; Ogi, Tomoo

    2013-05-02

    Cockayne syndrome (CS) is a genetic disorder characterized by developmental abnormalities and photodermatosis resulting from the lack of transcription-coupled nucleotide excision repair, which is responsible for the removal of photodamage from actively transcribed genes. To date, all identified causative mutations for CS have been in the two known CS-associated genes, ERCC8 (CSA) and ERCC6 (CSB). For the rare combined xeroderma pigmentosum (XP) and CS phenotype, all identified mutations are in three of the XP-associated genes, ERCC3 (XPB), ERCC2 (XPD), and ERCC5 (XPG). In a previous report, we identified several CS cases who did not have mutations in any of these genes. In this paper, we describe three CS individuals deficient in ERCC1 or ERCC4 (XPF). Remarkably, one of these individuals with XP complementation group F (XP-F) had clinical features of three different DNA-repair disorders--CS, XP, and Fanconi anemia (FA). Our results, together with those from Bogliolo et al., who describe XPF alterations resulting in FA alone, indicate a multifunctional role for XPF. Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

  1. RT-PCR versus immunohistochemistry for correlation and quantification of ERCC1, BRCA1, TUBB3 and RRM1 in NSCLC

    DEFF Research Database (Denmark)

    Vilmar, Adam Christian; Garcia-Foncillas, J; Huarriz, M

    2012-01-01

    Customized chemotherapy is increasingly used in the management of patients with advanced non-small cell lung cancer (NSCLC). However, the most reliable methodology to determine biomarker status is neither fully elucidated nor agreed upon. Accordingly, we evaluated the predictive efficiency of q......RT-PCR and immunohistochemical analysis (IHC) on excision cross complementation group 1 (ERCC1), breast cancer susceptibility gene 1 (BRCA1), ribonucleotide reductase subunit M1 (RRM1) and class III ß-tubulin (TUBB3)....

  2. First Reported Patient with Human ERCC1 Deficiency Has Cerebro-Oculo-Facio-Skeletal Syndrome with a Mild Defect in Nucleotide Excision Repair and Severe Developmental Failure

    OpenAIRE

    Jaspers, Nicolaas G.J.; Raams, Anja; Silengo, Margherita Cirillo; Wijgers, Nils; Niedernhofer, Laura J; Robinson, Andria Rasile; Giglia-Mari, Giuseppina; Hoogstraten, Deborah; Kleijer, Wim J.; Hoeijmakers, Jan H.J.; Vermeulen, Wim

    2007-01-01

    Nucleotide excision repair (NER) is a genome caretaker mechanism responsible for removing helix-distorting DNA lesions, most notably ultraviolet photodimers. Inherited defects in NER result in profound photosensitivity and the cancer-prone syndrome xeroderma pigmentosum (XP) or two progeroid syndromes: Cockayne and trichothiodystrophy syndromes. The heterodimer ERCC1-XPF is one of two endonucleases required for NER. Mutations in XPF are associated with mild XP and rarely with progeria. Mutati...

  3. Maternal mRNA expression levels of H19 are inversely associated with risk of macrosomia.

    Science.gov (United States)

    Jiang, Hua; Yu, Yang; Xun, Pengcheng; Zhang, Jun; Luo, Guanghua; Wang, Qiuwei

    2014-06-29

    To investigate the associations between the mRNA levels of H19 in term placenta and risk of macrosomia. Term placentas were collected from 37 macrosomia and 37 matched neonates with normal birth weight (controls) born in Changzhou Women and Children Health Hospital, Jiangsu province, P. R. China from March 1 to June 30, 2008. The mRNA levels of H19 in those placentas were measured by real-time polymerase chain reaction (PCR). Simple and multiple logistic regression models were used to explore the risk factors in the development of macrosomia. All analyses were performed using Stata 10.0 (StataCorp, College Station, Texas, USA). The average H19 mRNA level of the macrosomia group was 1.450 ±0.456 while in the control group it was 2.080 ±1.296. Based on the result of Student's t test, there was a significant difference in H19 mRNA level between the macrosomia group and the control group (p = 0.008). After controlling for potential confounders, the multivariable adjusted odds ratio (OR) of macrosomia for those in the highest tertile of H19 mRNA level was 0.12 (95% CI: 0.02-0.59) when compared to those in the lowest tertile (p for linear trend = 0.009). The term placental H19 mRNA levels were inversely related to the occurrence of macrosomia. Our findings suggest that the low expression of H19 mRNA may contribute to the development of macrosomia.

  4. Thyroid hormones regulate levels of thyrotropin-releasing-hormone mRNA in the paraventricular nucleus

    Energy Technology Data Exchange (ETDEWEB)

    Koller, K.J.; Wolff, R.S.; Warden, M.K.; Zoeller, R.T.

    1987-10-01

    Cellular levels of messenger RNA encoding thyrotropin-releasing hormone (TRH) were measured in the paraventricular nucleus of the hypothalamus and the reticular nucleus of the thalamus in male rats after chemical thyroidectomy and thyroid hormone, replacement. TRH mRNA levels were measured by quantitative in situ hybridization histochemistry using a /sup 35/S-labeled synthetic 48-base oligodeoxynucleotide probe and quantitative autoradiography. Chemical thyroidectomy, produced by the administration of 6-(n-propyl)-2-thiouracil (PrSur), reduced plasma thyroxine below detection limits and significantly increased TRH mRNA in the paraventricular nucleus. Treatments with exogenous L-triiodothyronine (T/sub 3/) reduced TRH mRNA to the same level in both hypothyroid and euthyroid animals. Neither PrSur treatment nor T/sub 3/ replacement influenced TRH mRNA levels in the reticular nucleus of the thalamus. Blot hybridization analysis of electrophoretically fractionated total RNA from pituitaries of these animals indicated that thyrotropin-..beta.. mRNA levels were elevated after thyroidectomy and reduced by T/sub 3/ treatment, showing that the pituitary-thyroid axis was indeed stimulated by PrSur treatment. These results suggest that thyroid hormones are involved, either directly or indirectly, in regulating the biosynthesis of TRH in the thyrotropic center of the hypothalamus.

  5. 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.

  6. Effects of codon optimization on the mRNA levels of heterologous genes in filamentous fungi.

    Science.gov (United States)

    Tanaka, Mizuki; Tokuoka, Masafumi; Gomi, Katsuya

    2014-05-01

    Filamentous fungi, particularly Aspergillus species, have recently attracted attention as host organisms for recombinant protein production. Because the secretory yields of heterologous proteins are generally low compared with those of homologous proteins or proteins from closely related fungal species, several strategies to produce substantial amounts of recombinant proteins have been conducted. Codon optimization is a powerful tool for improving the production levels of heterologous proteins. Although codon optimization is generally believed to improve the translation efficiency of heterologous genes without affecting their mRNA levels, several studies have indicated that codon optimization causes an increase in the steady-state mRNA levels of heterologous genes in filamentous fungi. However, the mechanism that determines the low mRNA levels when native heterologous genes are expressed was poorly understood. We recently showed that the transcripts of heterologous genes are polyadenylated prematurely within the coding region and that the heterologous gene transcripts can be stabilized significantly by codon optimization, which is probably attributable to the prevention of premature polyadenylation in Aspergillus oryzae. In this review, we describe the detailed mechanism of premature polyadenylation and the rapid degradation of mRNA transcripts derived from heterologous genes in filamentous fungi.

  7. 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.

  8. Exome-Wide Meta-Analysis Identifies Rare 3′-UTR Variant in ERCC1/CD3EAP Associated with Symptoms of Sleep Apnea

    Directory of Open Access Journals (Sweden)

    Ashley van der Spek

    2017-10-01

    Full Text Available Obstructive sleep apnea (OSA is a common sleep breathing disorder associated with an increased risk of cardiovascular and cerebrovascular diseases and mortality. Although OSA is fairly heritable (~40%, there have been only few studies looking into the genetics of OSA. In the present study, we aimed to identify genetic variants associated with symptoms of sleep apnea by performing a whole-exome sequence meta-analysis of symptoms of sleep apnea in 1,475 individuals of European descent. We identified 17 rare genetic variants with at least suggestive evidence of significance. Replication in an independent dataset confirmed the association of a rare genetic variant (rs2229918; minor allele frequency = 0.3% with symptoms of sleep apnea (p-valuemeta = 6.98 × 10−9, βmeta = 0.99. Rs2229918 overlaps with the 3′ untranslated regions of ERCC1 and CD3EAP genes on chromosome 19q13. Both genes are expressed in tissues in the neck area, such as the tongue, muscles, cartilage and the trachea. Further, CD3EAP is localized in the nucleus and mitochondria and involved in the tumor necrosis factor-alpha/nuclear factor kappa B signaling pathway. Our results and biological functions of CD3EAP/ERCC1 genes suggest that the 19q13 locus is interesting for further OSA research.

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

    Directory of Open Access Journals (Sweden)

    Johann Edge

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

  10. Digestive enzyme activity and mRNA level of trypsin in embryonic redclaw crayfish, Cherax quadricarnatus

    Science.gov (United States)

    Luo, Wen; Zhao, Yunlong; Zhou, Zhongliang; An, Chuanguang; Ma, Qiang

    2008-02-01

    The digestive enzyme activity and mRNA level of trypsin during the embryonic development of Cherax quadricarinatus were analyzed using biochemical and Fluorogenic Quantitative PCR (FQ—PCR) methods. The results show that the activities of trypsin and chymotrypsin had two different change patterns. Trypsin specific activity increased rapidly in the early stages of development and still remained high in preparation for the hatch stage. However, chymotrypsin activity peaked in stage 4 of embryonic development and decreased significantly in the last stage. The mRNA level of trypsin was elevated in all stages and two peak values were observed in stages 2 and 5 respectively. The results indicate that trypsin is very important for the utilization of the yolk during embryonic development and for the assimilation of dietary protein for larvae. The gene of trypsin is probably regulated at transcriptional level. The mRNA levels of trypsin can reflect not only trypsin activity, but also the regulatory mechanism for expression of trypsin gene to a certain degree.

  11. 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

    Regulation of gene expression can be analyzed by a number of different techniques. Some techniques monitor the level of specific mRNA directly, and others monitor indirectly by determining the level of enzymes encoded by the mRNA. Each method has its own inherent way of normalization. When result...

  12. 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

    trained rats. Furthermore, the abundance of the mRNAs for these proteins and glucose transport was measured. Rats were swim-trained for 10 wk, and adipocytes were isolated from epididymal fat pads. The amount of GLUT-4/adipocyte volume unit was significantly higher in trained animals compared with both...... age- and cell size-matched animals. The amount of GLUT-4 mRNA was also increased by training and it decreased with increasing age. Furthermore, young age as well as training was accompanied by relatively low GLUT-4 protein/mRNA and relatively high overall GLUT-4 efficiency (recruitability and....../or intrinsic activity). GLUT-1 protein and mRNA levels/adipocyte volume did not change with age or training....

  13. Correlation between TS, MTHFR, and ERCC1 gene polymorphisms and the efficacy of platinum in combination with pemetrexed first-line chemotherapy in mesothelioma patients.

    Science.gov (United States)

    Powrózek, Tomasz; Kowalski, Dariusz M; Krawczyk, Paweł; Ramlau, Rodryg; Kucharczyk, Tomasz; Kalinka-Warzocha, Ewa; Knetki-Wróblewska, Magdalena; Winiarczyk, Kinga; Dyszkiewicz, Wojciech; Krzakowski, Maciej; Milanowski, Janusz

    2014-11-01

    The combination of pemetrexed and platinum compound represents the standard regimen for first-line chemotherapy in malignant pleural mesothelioma patients. Pemetrexed is a multitarget antifolate agent that inhibits folate-dependent enzymes (eg, thymidylate synthase [TS]) and thus synthesis of nucleotides and DNA. Expression of TS and folate availability, regulated by gene polymorphisms, have implications for effectiveness of chemotherapy and the outcome of mesothelioma patients. The aim of this retrospective multicenter study was to assess the correlation between TS, 5,10-methylenetetrahydrofolate reductase (MTHFR) and excision repair cross-complementing group 1 (ERCC1) gene polymorphisms and the efficacy of pemetrexed-based first-line chemotherapy of mesothelioma patients. Fifty-nine mesothelioma patients (31 men with a median age of 62 years) treated in first-line chemotherapy with platinum in combination with pemetrexed or pemetrexed monotherapy were enrolled. Genomic DNA was isolated from peripheral blood. Using polymerase chain reaction and high resolution melt methods, the variable number of tandem repeat, the G>C single nucleotide polymorphism (SNP) in these repeats, and 6-base pair (bp) insertion/deletion polymorphism of the TS gene, the SNP of 677C>T in MTHFR, and 19007C>T in the ERCC1 gene were analyzed and correlated with disease control rate, progression-free survival (PFS), and overall survival (OS) of mesothelioma patients. Greater risk of early disease progression (PD), and shortening of PFS and OS were associated with several clinical factors (eg, anemia for early PD and OS), weight loss (for PFS and OS), and previous surgical treatment (for early PD, PFS, and OS). Insertion of 6-bp in both alleles of the TS gene (1494del6) was the only genetic factor that increased the incidence of early progression (P = .028) and shortening of median PFS (P = .06) in patients treated with pemetrexed-based chemotherapy. In multivariate analysis, the 1494del6 in the

  14. Polymorphisms in TS, MTHFR and ERCC1 genes as predictive markers in first-line platinum and pemetrexed therapy in NSCLC patients.

    Science.gov (United States)

    Krawczyk, Paweł; Kucharczyk, Tomasz; Kowalski, Dariusz M; Powrózek, Tomasz; Ramlau, Rodryg; Kalinka-Warzocha, Ewa; Winiarczyk, Kinga; Knetki-Wróblewska, Magdalena; Wojas-Krawczyk, Kamila; Kałakucka, Katarzyna; Dyszkiewicz, Wojciech; Krzakowski, Maciej; Milanowski, Janusz

    2014-12-01

    We presented retrospective analysis of up to five polymorphisms in TS, MTHFR and ERCC1 genes as molecular predictive markers for homogeneous Caucasian, non-squamous NSCLC patients treated with pemetrexed and platinum front-line chemotherapy. The following polymorphisms in DNA isolated from 115 patients were analyzed: various number of 28-bp tandem repeats in 5'-UTR region of TS gene, single nucleotide polymorphism (SNP) within the second tandem repeat of TS gene (G>C); 6-bp deletion in 3'-UTR region of the TS (1494del6); 677C>T SNP in MTHFR; 19007C>T SNP in ERCC1. Molecular examinations' results were correlated with disease control rate, progression-free survival (PFS) and overall survival. Polymorphic tandem repeat sequence (2R, 3R) in the enhancer region of TS gene and G>C SNP within the second repeat of 3R allele seem to be important for the effectiveness of platinum and pemetrexed in first-line chemotherapy. The insignificant shortening of PFS in 3R/3R homozygotes as compared to 2R/2R and 2R/3R genotypes were observed, while it was significantly shorter in patients carrying synchronous 3R allele and G nucleotide. The combined analysis of TS VNTR and MTHFR 677C>T SNP revealed shortening of PFS in synchronous carriers of 3R allele in TS and two C alleles in MTHFR. The strongest factors increased the risk of progression were poor PS, weight loss, anemia and synchronous presence of 3R allele and G nucleotide in the second repeat of 3R allele in TS. Moreover, lack of application of second-line chemotherapy, weight loss and poor performance status and above-mentioned genotype of TS gene increased risk of early mortality. The examined polymorphisms should be accounted as molecular predictor factors for pemetrexed- and platinum-based front-line chemotherapy in non-squamous NSCLC patients.

  15. The structure of the XPF-ssDNA complex underscores the distinct roles of the XPF and ERCC1 helix- hairpin-helix domains in ss/ds DNA recognition.

    Science.gov (United States)

    Das, Devashish; Folkers, Gert E; van Dijk, Marc; Jaspers, Nicolaas G J; Hoeijmakers, Jan H J; Kaptein, Robert; Boelens, Rolf

    2012-04-04

    Human XPF/ERCC1 is a structure-specific DNA endonuclease that nicks the damaged DNA strand at the 5' end during nucleotide excision repair. We determined the structure of the complex of the C-terminal domain of XPF with 10 nt ssDNA. A positively charged region within the second helix of the first HhH motif contacts the ssDNA phosphate backbone. One guanine base is flipped out of register and positioned in a pocket contacting residues from both HhH motifs of XPF. Comparison to other HhH-containing proteins indicates a one-residue deletion in the second HhH motif of XPF that has altered the hairpin conformation, thereby permitting ssDNA interactions. Previous nuclear magnetic resonance studies showed that ERCC1 in the XPF-ERCC1 heterodimer can bind dsDNA. Combining the two observations gives a model that underscores the asymmetry of the human XPF/ERCC1 heterodimer in binding at an ss/ds DNA junction. Copyright © 2012 Elsevier Ltd. All rights reserved.

  16. The structure of the XPF-ssDNA complex underscores the distinct roles of the XPF and ERCC1 helix- hairpin-helix domains in ss/ds DNA recognition

    NARCIS (Netherlands)

    Das, Devashish; Folkers, Gert; van Dijk, Marc; Jaspers, Nicolaas G.J.; Hoeijmakers, Jan H.J.; Kaptein, Robert; Boelens, Rolf

    2012-01-01

    Human XPF/ERCC1 is a structure-specific DNA endonuclease that nicks the damaged DNA strand at the 5' end during nucleotide excision repair. We determined the structure of the complex of the C-terminal domain of XPF with 10 nt ssDNA. A positively charged region within the second helix of the first

  17. Intestinal PTGS2 mRNA Levels, PTGS2 Gene Polymorphisms, and Colorectal Carcinogenesis

    DEFF Research Database (Denmark)

    Vogel, Lotte K.; Saebo, Mona; Hoyer, Helle

    2014-01-01

    Background & Aims: Inflammation is a major risk factor for development of colorectal cancer (CRC). Prostaglandin synthase cyclooxygenase-2 (COX-2) encoded by the PTGS2 gene is the rate limiting enzyme in prostaglandin synthesis and therefore plays a distinct role as regulator of inflammation....... Methods: PTGS2 mRNA levels were determined in intestinal tissues from 85 intestinal adenoma cases, 115 CRC cases, and 17 healthy controls. The functional PTGS2 polymorphisms A-1195G (rs689466), G-765C (rs20417), T8473C (rs5275) were assessed in 200 CRC cases, 991 adenoma cases and 399 controls from...

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

    DEFF Research Database (Denmark)

    Christiansen, M; Kveiborg, Marie; Kassem, M

    2000-01-01

    -transformed human lung fibroblast cell line MRC5V2 have 20 to 40% higher levels of CBFA1 mRNA. Similar levels of CBFA1 mRNA are detectable in normal human skin fibroblasts, and these cells also exhibit an age-related decline to the same extent. In addition, the expression of topoisomerase I is reduced by 40......% in senescent osteoblasts, and the mRNA levels are significantly higher (40-70%) in transformed osteoblasts and fibroblasts. These changes in gene expression may be among the causes of impaired osteoblast functions, resulting in reduced bone formation during aging....

  19. Analysis of xanthine dehydrogenase mRNA levels in mutants affecting the expression of the rosy locus.

    OpenAIRE

    Covington, M; Fleenor, D; Devlin, R B

    1984-01-01

    Xanthine dehydrogenase (XDH) mRNA levels were measured in a number of mutants and natural variants affecting XDH gene expression. Two variants, ry+4 and ry+10, contain cis-acting elements which map to a region flanking the 5' end of the XDH gene. Ry+4, which has 2-3 times more XDH protein than a wild type strain, has 3.2 times more XDH mRNA. Ry+10 has 50% of the wild type XDH level and 54% of the wild type XDH mRNA level. Three rosy mutants which map within the structural gene were also exami...

  20. 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.......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......, and M. Givskov, J. Bacteriol. 178:554-559, 1996). In the present report we show by means of reporter gene measurements, Northern analysis, and in situ reverse transcription-PCR that the amount of flhDC mRNA in surface-grown swarm cells does not exceed the maximum level found in nondifferentiated...

  1. Mitotic regulator Nlp interacts with XPA/ERCC1 complexes and regulates nucleotide excision repair (NER) in response to UV radiation.

    Science.gov (United States)

    Ma, Xiao-Juan; Shang, Li; Zhang, Wei-Min; Wang, Ming-Rong; Zhan, Qi-Min

    2016-04-10

    Cellular response to DNA damage, including ionizing radiation (IR) and UV radiation, is critical for the maintenance of genomic fidelity. Defects of DNA repair often result in genomic instability and malignant cell transformation. Centrosomal protein Nlp (ninein-like protein) has been characterized as an important cell cycle regulator that is required for proper mitotic progression. In this study, we demonstrate that Nlp is able to improve nucleotide excision repair (NER) activity and protects cells against UV radiation. Upon exposure of cells to UVC, Nlp is translocated into the nucleus. The C-terminus (1030-1382) of Nlp is necessary and sufficient for its nuclear import. Upon UVC radiation, Nlp interacts with XPA and ERCC1, and enhances their association. Interestingly, down-regulated expression of Nlp is found to be associated with human skin cancers, indicating that dysregulated Nlp might be related to the development of human skin cancers. Taken together, this study identifies mitotic protein Nlp as a new and important member of NER pathway and thus provides novel insights into understanding of regulatory machinery involved in NER. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  2. Study of association between pre-senile cataracts and rs11615 of ERCC1, rs13181 of ERCC2, and rs25487 of XRCC1 polymorphisms in a Spanish population.

    Science.gov (United States)

    López-Valverde, Gloria; Garcia-Martin, Elena; Fernández-Mateos, Javier; Cruz-González, Fernando; Larrosa-Povés, José M; Polo-Llorens, Vicente; Pablo-Júlvez, Luis E; González-Sarmiento, Rogelio

    2017-01-01

    The purpose of this study was to attempt to determine if the presence of certain polymorphisms in the DNA repair genes (ERCC1, ERCC2, and XRCC1) is associated with pre-senile cataract development. We performed a retrospective study over three groups of patients. The first group with pre-senile cataract was formed by 72 patients younger than 55 years with cataract surgery. The second group with senile cataract was formed by 101 patients older than 55 years with cataract surgery. And the third group, without cataract, was formed by 42 subjects older than 55 years without lens opacities. We analyzed the presence of SNP rs11615 from ERCC1, rs13181 from ERCC2, and rs25487 from XRCC1 and the relationship between risk factors such as smoking, alcohol intake, hypertension, and diabetes. The comparison of the genotype distribution in ERCC1 and ERCC2 did not show any statistically significant association in any of our analyses (p > 0.05). The comparison of the genotype distribution in XRCC1 within the different groups did not show any statistically significant associations (p > 0.05), except for the comparison between the pre-senile cataract group and the group without cataract, where an increased risk of developing pre-senile cataract for the genotype Gln/Gln (p = 0.029; OR = 1.02-40.67) in recessive inheritance models was observed when adjusting for risk factors. Allelic variants in ERCC1 and ERCC2 are not associated with an increased risk of developing pre-senile cataract. The presence of Gln/Gln in XRCC1 in the pre-senile cataract group with regard to the group without cataract is associated with a major risk of developing pre-senile cataract.

  3. Chronic social stress alters levels of corticotropin-releasing factor and arginine vasopressin mRNA in rat brain.

    Science.gov (United States)

    Albeck, D S; McKittrick, C R; Blanchard, D C; Blanchard, R J; Nikulina, J; McEwen, B S; Sakai, R R

    1997-06-15

    In the visible burrow system model of chronic social stress, male rats housed in mixed-sex groups quickly form a dominance hierarchy in which the subordinates appear to be severely stressed. A subgroup of subordinates have an impaired corticosterone response after presentation of a novel restraint stressor, leading to their designation as nonresponsive subordinates. To examine the mechanism underlying the blunted corticosterone response in these animals, in situ hybridization histochemistry was used to quantify corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) mRNA expression in the brain. In two separate visible burrow system experiments, the nonresponsive subordinates expressed a significantly lower average number of CRF mRNA grains per cell in the paraventricular hypothalamic nucleus compared with stress-responsive subordinates, dominants (DOM), or cage-housed control (CON) rats. The number of CRF mRNA labeled cells was also significantly lower in nonresponders than in responsive subordinates or DOM. In the central amygdala, CRF mRNA levels were increased in both groups of subordinates compared with CON rats, whereas responsive subordinates exhibited higher levels than the DOM rats as well. AVP mRNA levels did not vary with behavioral rank in any subdivision of the paraventricular hypothalamic nucleus. In the medial amygdala, the number of cells expressing AVP mRNA was significantly greater in CON rats compared with both groups of subordinates, although the average number of AVP mRNA grains per cell did not vary with rank. In addition, the number of AVP-positive cells significantly correlated with plasma testosterone level.

  4. 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

    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......During pregnancy, the human extra-villous trophoblast in the contact zone between maternal and fetal tissue in the placenta does not express the classical MHC class I and II molecules. Instead, HLA-G and -C, and possibly HLA-E, are expressed. HLA-G may modulate the immunological relationship...... 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...

  5. Vaspin mRNA levels in the liver of morbidly obese women with nonalcoholic fatty liver disease.

    Science.gov (United States)

    Waluga, Marek; Kukla, Michał; Żorniak, Michał; Grabiec, Marta; Kajor, Maciej; Dyaczyński, Michał; Kowalski, Grzegorz; Żądło, Dominika; Berdowska, Agnieszka; Kotulski, Rafał; Bułdak, Rafał J; Sawczyn, Tomasz; Waluga, Ewa; Olczyk, Paweł; Hartleb, Marek

    The aim of this study was to evaluate hepatic vaspin mRNA in morbidly obese women with nonalcoholic fatty liver disease (NAFLD) and to look for its relationships with metabolic and histopathological features. The study included 56 severely obese women who underwent intraoperative wedge liver biopsy during bariatric surgery. Hepatic vaspin mRNA was assessed by quantitative real-time PCR. Vaspin mRNA found in all included patients was markedly higher in patients with body mass index (BMI) ≥ 40 kg/m2 (4.59 ±3.09 vs. 0.44 ±0.33; p = 0.05). An evident but statistically insignificant difference in vaspin mRNA levels was observed between patients with and without hepatocyte ballooning (4.77 ±4.23 vs. 0.45 ±0.29, respectively), with and without steatosis (4.80 ±4.20 vs. 0.41 ±0.29, respectively), without and with fibrosis (0.25 ±0.80 vs. 6.23 ±7.2, respectively), and those without and with lobular inflammation (0.27 ±1.0 vs. 5.55 ±10.1, respectively). There was marked difference in vaspin mRNA between patients with simple steatosis/borderline nonalcoholic steatohepatitis (NASH) compared to those with definite NASH (0.24 ±0.96 vs. 10.5 ±10.4). Adiposity is an undoubted confounding factor influencing vaspin levels. Hepatic vaspin mRNA seems to be markedly elevated in morbidly obese patients with more advanced NAFLD and when hallmarks of NASH were observed. Pointing to non-linear mRNA levels within the NAFLD spectrum and an evident increase in patients with fibrosis and definite NASH, the detrimental action of vaspin cannot be excluded.

  6. Increased interferon alpha receptor 2 mRNA levels is associated with renal cell carcinoma metastasis

    Directory of Open Access Journals (Sweden)

    Yamanishi Tomonori

    2007-08-01

    Full Text Available Abstract Background Interferon-α (IFN-α is one of the central agents in immunotherapy for renal cell carcinoma (RCC and binds to the IFN-α receptor (IFNAR. We investigated the role of IFNAR in RCC. Methods We quantified IFNAR mRNA expression in paired tumor and non-tumor samples from the surgical specimens of 103 consecutive patients with RCC using a real-time reverse transcription polymerase chain reaction (RT-PCR, and IFNAR2 protein using Western blotting. Results The absolute level of IFNAR1 and IFNAR2 mRNAs in tumor and non-tumor tissues did not correlate with the malignant and metastatic profiles. The relative yields of the PCR product from the tumor tissue to that from the corresponding non-tumor tissue (T/N for the expression of IFNAR mRNAs were calculated. While the T/N ratio of IFNAR1 did not correlate with any factor, a high T/N ratio of IFNAR2 correlated with poor differentiation (P P P P P Conclusion IFNAR2 is associated with the progression of RCC.

  7. 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

  8. Intrahepatic mRNA levels of type I interferon receptor and interferon-stimulated genes in genotype 1b chronic hepatitis C. Association between IFNAR1 mRNA level and sustained response to interferon therapy.

    Science.gov (United States)

    Taniguchi, Hideaki; Iwasaki, Yoshiaki; Takahashi, Akira; Shimomura, Hiroyuki; Moriya, Akio; Yu, Piao Cheng; Umeoka, Fumi; Fujioka, Shin-ichi; Koide, Norio; Shiratori, Yasushi

    2007-01-01

    The aim of this study was to determine the association between pretreatment intrahepatic mRNA levels of interferon receptor and interferon-stimulated genes and response to interferon therapy for genotype 1b chronic hepatitis C. Forty-four patients with genotype 1b chronic hepatitis C who underwent liver biopsy and then received interferon therapy participated in this study. Pretreatment intrahepatic mRNA levels of interferon receptor genes (IFNAR1, IFNAR2b, and IFNAR2c) and interferon-stimulated genes (OAS1 and PKR) were quantified by competitive polymerase chain reaction. In the genes examined, only IFNAR1 mRNA level was significantly higher in patients with sustained virological and biochemical response to interferon therapy versus those with nonsustained response (p IFNAR1 to IFNAR2 were also significantly higher in patients with sustained virological and biochemical response to IFN therapy (p IFNAR1 and mRNA ratio of IFNAR1 to IFNAR2 before treatment may be associated with a favorable response to interferon therapy. Copyright 2007 S. Karger AG, Basel.

  9. Higher LPA2 and LPA6 mRNA Levels in Hepatocellular Carcinoma Are Associated with Poorer Differentiation, Microvascular Invasion and Earlier Recurrence with Higher Serum Autotaxin Levels.

    Directory of Open Access Journals (Sweden)

    Kenichiro Enooku

    Full Text Available Hepatocellular carcinoma (HCC commonly develops in patients with liver fibrosis; in these patients, the blood levels of lysophosphatidic acid (LPA and its generating enzyme autotaxin (ATX increase with the liver fibrosis stage. We aimed to examine the potential relevance of ATX and LPA in HCC. Fifty-eight HCC patients who underwent surgical treatment were consecutively enrolled in the study. Among the LPA receptors in HCC, higher LPA2 mRNA levels correlated with poorer differentiation, and higher LPA6 mRNA levels correlated with microvascular invasion, which suggested a higher malignant potential of HCC with increased LPA2 and LPA6 expression. In patients with primary HCC, neither LPA2 nor LPA6 mRNA levels were associated with recurrence. However, when serum ATX levels were combined for analysis as a surrogate for plasma LPA levels, the cumulative intra-hepatic recurrence rate was higher in patients in whom both serum ATX levels and LPA2 or LPA6 mRNA levels were higher than the median. However, the mRNA level of phosphatidic acid-selective phospholipase A1ɑ, another LPA-generating enzyme, in HCC patients was not associated with pathological findings or recurrence, even in combination with the expression of LPA receptors. Higher LPA2 mRNA levels were associated with poorer differentiation, and higher LPA6 levels were associated with microvascular invasion in HCC; both became a risk factor for recurrence after surgical treatment when combined with increased serum ATX levels. ATX and LPA receptors merit consideration as therapeutic targets of HCC.

  10. Increased interferon alpha receptor 2 mRNA levels is associated with renal cell carcinoma metastasis.

    Science.gov (United States)

    Kamai, Takao; Yanai, Yoshiaki; Arai, Kyoko; Abe, Hideyuki; Yamanishi, Tomonori; Kurimoto, Masashi; Yoshida, Ken-Ichiro

    2007-08-15

    Interferon-alpha (IFN-alpha) is one of the central agents in immunotherapy for renal cell carcinoma (RCC) and binds to the IFN-alpha receptor (IFNAR). We investigated the role of IFNAR in RCC. We quantified IFNAR mRNA expression in paired tumor and non-tumor samples from the surgical specimens of 103 consecutive patients with RCC using a real-time reverse transcription polymerase chain reaction (RT-PCR), and IFNAR2 protein using Western blotting. The absolute level of IFNAR1 and IFNAR2 mRNAs in tumor and non-tumor tissues did not correlate with the malignant and metastatic profiles. The relative yields of the PCR product from the tumor tissue to that from the corresponding non-tumor tissue (T/N) for the expression of IFNAR mRNAs were calculated. While the T/N ratio of IFNAR1 did not correlate with any factor, a high T/N ratio of IFNAR2 correlated with poor differentiation (P < 0.05), local invasion (P < 0.001), and metastasis (P < 0.0001). By multivariate analysis, a high T/N ratio of IFNAR2 predicted a shortened overall survival in all cases (P < 0.05) and a shorter disease-free survival in those without metastasis (M0; 68 cases, P < 0.05). Impressively, patients with a poorer response to IFN-alpha treatment had a higher IFNAR2 T/N ratio than those who had a good response (P < 0.05). IFNAR2c protein expression was higher in the primary tumors in patients with metastases (M1; 35 cases) compared to those without ( P < 0.0001). IFNAR2 is associated with the progression of RCC.

  11. Validation of two reference genes for mRNA level studies of murine disease models in neurobiology

    DEFF Research Database (Denmark)

    Meldgaard, Michael; Fenger, Christina; Lambertsen, Kate Lykke

    2006-01-01

    Reverse transcription of extracted cellular RNA combined with real-time PCR is now an established method for sensitive detection and quantification of specific mRNA level changes in experimental models of neurological diseases. To neutralize the impact of experimental error and make quantificatio...

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

    DEFF Research Database (Denmark)

    Theander Grünfeld, Heidi; Bonefeld-Jørgensen, Eva Cecilie

    2004-01-01

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

  13. High levels of DNA polymerase β mRNA corresponding with the high activity in Graves' thyroid tissue.

    Science.gov (United States)

    Hayakawa, N; Sato, Y; Nagasaka, A; Mano, Y; Nagasaka, T; Nakai, A; Iwase, K; Yoshida, S

    2017-04-01

    High DNA polymerase β activity has been observed in the thyroid tissue of patients with Graves' disease (Nagasaka et al. in Metabolism 37:1051-1054, 1988). This fact aroused our interest in whether the alteration of DNA polymerase β activity depends on DNA polymerase β (DNA poly β) mRNA levels, which may be modulated by thyroid-stimulating hormone (TSH) or thyroid-stimulating substances, i.e. TSH receptor antibody (TRAb). Addition of TSH or TRAb to primary cultures of Graves' disease thyroid cells for 4 h led to no increase in DNA poly β mRNA levels. In contrast, thyroid hormone synthesizing enzyme, peroxidase, mRNA levels increased fivefold after coculture with TSH and TRAb, even though DNA poly β activity and mRNA levels are already significantly higher in Graves' disease thyroid tissues, compared with normal thyroid tissue. These results indicate that DNA poly β expression in Graves' disease thyroid cells may be maximally activated or plateau in response to thyroid-stimulating immunoglobulins, or that the activation of to poly β expression may occur via pathways other than the G protein and cyclic AMP system.

  14. 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.

  15. Elevated levels of transferrin receptor 2 mRNA, not transferrin receptor 1 mRNA, are associated with increased survival in acute myeloid leukaemia.

    Science.gov (United States)

    Nakamaki, Tsuyoshi; Kawabata, Hiroshi; Saito, Bungo; Matsunawa, Manabu; Suzuki, Junko; Adachi, Daisuke; Tomoyasu, Shigeru; Phillip Koeffler, H

    2004-04-01

    Transferrin receptor 1 (TfR1) is a type II membrane protein that mediates cellular iron uptake. Transferrin receptor 2(TfR2), another receptor for transferrin (Tf), has recently been cloned. We examined expression levels of TfR1, TfR2-alpha (membrane form) and TfR2-beta (non-membrane form) transcripts in cells from 67 patients with de novo acute myeloid leukaemia (AML) using reverse transcription-polymerase chain reaction (RT-PCR), and correlated the results with a variety of clinical features and disease outcomes of these patients. Significant correlations were noted between the levels of both TfR1 and TfR2-alpha (r = 0.771, P TfR2-beta (r = 0.534, P TfR2-alpha (r = -0.486, P TfR2-beta (r = -0.435, P = 0.0003). Only TfR2 expression was significantly associated with either serum iron (r = -0.270, P = 0.045) or serum ferritin (r = -0.364, P = 0.008). Multivariate analyses using Cox's proportional hazard model showed that elevated TfR2-alpha, but not TfR1 or TfR2-beta mRNA levels significantly contributed to a better prognosis for AML patients. Furthermore, a group with high expression levels of both TfR2-alpha and TfR2-beta survived significantly longer than a group without high expression of both of them (P TfR2 (especially the alpha form) is a novel prognostic factor for patients with AML.

  16. DETERMINATION OF LEVEL EXPRESSION OF mRNA SPLICING VARIANTS FOR DR3 IN BLOOD CELLS IN INFECTIOUS MONONUCLEOSIS

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    V. D. Cvetkova

    2016-01-01

    Full Text Available The DR3 «death receptor» plays an important role in the initiation of apoptosis, proliferation, or inflammation. This receptor is shown to be involved in various diseases, including infectious conditions. Different variants of mRNA DR3 are formed as a result of alternative splicing. These variant transcripts encode membrane and soluble forms of the receptor which have different functions. Features of their expression and contribution of individual DR3 variants to the immune pathogenesis of infectious mononucleosis (IM are poorely understood.The purpose of this work was to develop, validate and test the techniques of DR3 gene expression assays, as well as to evaluate the DR3 mRNA splice variants by means of real-time RT-PCR and RT-PCR in the IM patients.The original version of real-time RT-PCR allowed to determine relative amounts of DR3 mRNA, DR3 membrane variants (LARD1a + LARD8, and ratios of mRNAs encoding membrane and soluble forms of the receptor. The technique proved to be specific and sensitive (a semi-quantitative detection limit = 34-35 cycles when tested in healthy volunteers and patients with acute infectious mononucleosis (AIM. Lower expression levels were shown for two alternative membrane variants of DR3 mRNA (LARD1b and DR3beta thus regarding these isoforms as minor fractions. The relative levels of total DR3 mRNA expression were decreased in patients with AIM, as compared to healthy volunteers, whereas mRNA expression of membrane receptor variants did not differ between IM and controls.To determine a qualitative contribution of either LARD1a and LARD8 variants into the expression of membrane forms of DR3, a two-step «nested» version of RT-PCR has been developed. It was shown that, in majority of control and IM samples, both main LARD1a, and alternative LARD8 membrane forms are contributing to mRNA expression of membrane DR3 variants.The presented methods for evaluation of expression and occurrence of DR3 mRNA variants allow

  17. Acquisition of a novel behavior induces higher levels of Arc mRNA than does overtrained performance.

    Science.gov (United States)

    Kelly, M P; Deadwyler, S A

    2002-01-01

    Arc (also termed activity-regulated cytoskeleton-associated protein or Arg3.1), is an effector immediate early gene whose upregulation has been demonstrated during events of synaptic plasticity. In the present study, the possibility that Arc would be specifically upregulated in rats during the acquisition of a quickly learned behavioral task but not in overtrained animals was investigated. Three groups of rats, pseudotrained, newly trained and overtrained, were examined with respect to Arc expression following training on a simple operant lever-pressing task. Newly trained animals were killed 30 min following the session in which they demonstrated acquisition of the task, and overtrained animals were trained on the same task for 13-14 days and then killed. Relative to base level measures taken 6 h following the session, all three groups demonstrated significant levels of induction of Arc mRNA; however, newly trained animals exhibited heightened induction of Arc mRNA relative to both pseudotrained and overtrained animals. The increased levels of Arc mRNA in newly trained animals were located in the CA1 and CA3 fields of hippocampus, the subiculum, and the anterior cingulate, piriform, infra/prelimbic, perirhinal and entorhinal cortical areas. Additionally, Arc mRNA was expressed differentially across the above anatomic structures in a relative pattern that was the same in all three groups. Finally, levels of Arc mRNA in specific brain regions of newly trained animals correlated negatively with the rate of task acquisition, such that slow learners exhibited higher levels of Arc mRNA than fast learners. From these results we suggest that Arc is upregulated in an experience-dependent manner, with higher levels of induction occurring during the initial stage of learning. Furthermore, the finding of increased Arc levels in slow versus fast learners indicates that Arc expression may be associated with the length of time required to: (1) form new associations or (2

  18. Effects of extremely low frequency electromagnetic field and cisplatin on mRNA levels of some DNA repair genes.

    Science.gov (United States)

    Sanie-Jahromi, Fatemeh; Saadat, Iraj; Saadat, Mostafa

    2016-12-01

    It has been shown that exposure to extremely-low frequency (˂300Hz) oscillating electromagnetic field (EMF) can affect gene expression. The effects of different exposure patterns of 50-Hz EMF and co-treatment of EMF plus cisplatin (CDDP) on mRNA levels of seven genes involved in DNA repair pathways (GADD45A, XRCC1, XRCC4, Ku70, Ku80, DNA-PKcs and LIG4) were evaluated. Two 50-Hz EMF intensities (0.25 and 0.50mT), three exposure patterns (5min field-on/5min field-off, 15min field-on/15min field-off, 30min field-on continuously) and two cell lines (MCF-7 and SH-SY5Y) were used. The mRNA levels were measured using quantitative real-time PCR. The examined genes had tendency to be down-regulated in MCF-7 cells treated with EMF. In the pattern of 15min field-on/15min field-off of the 0.50mT EMF, no increase in mRNA levels were observed, but the mRNA levels of GADD45A, XRCC1, XRCC4, Ku80, Ku70, and LIG4 were down-regulated. A significant elevation in IC50 of CDDP was observed when MCF-7 and SH-SY5Y cells were co-treated with CDDP+EMF in comparison with the cells treated with CDDP alone. GADD45A mRNA levels in MCF-7 and SH-SY5Y cells co-treated with CDDP+EMF were increased and at the same time the mRNA levels of XRCC4, Ku80, Ku70 and DNA-PKcs were down-regulated. Present study provides evidence that co-treatment of CDDP+EMF can enhance down-regulation of the genes involved in non-homologous end-joining pathway. It might be suggested that co-treatment of CDDP+EMF could be more promising for sensitizing cancer cells to DNA double strand breaks. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Effects of polymorphisms in ERCC1, ASE-1 and RAI on the risk of colorectal carcinomas and adenomas: a case control study

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    Wallin Håkan

    2006-07-01

    Full Text Available Abstract Background The risk of sporadic colorectal cancer is mainly associated with lifestyle factors and may be modulated by several genetic factors of low penetrance. Genetic variants represented by single nucleotide polymorphisms in genes encoding key players in the adenoma carcinoma sequence may contribute to variation in susceptibility to colorectal cancer. In this study, we aimed to evaluate whether the recently identified haplotype encompassing genes of DNA repair and apoptosis, is associated with increased risk of colorectal adenomas and carcinomas. Methods We used a case-control study design (156 carcinomas, 981 adenomas and 399 controls to test the association between polymorphisms in the chromosomal region 19q13.2-3, encompassing the genes ERCC1, ASE-1 and RAI, and risk of colorectal adenomas and carcinomas in a Norwegian cohort. Odds ratio (OR and 95% confidence interval (CI were estimated by binary logistic regression model adjusting for age and gender. Results The ASE-1 polymorphism was associated with an increased risk of adenomas, OR of 1.39 (95% CI 1.06–1.81, which upon stratification was apparent among women only, OR of 1.66 (95% CI 1.15–2.39. The RAI polymorphism showed a trend towards risk reduction for both adenomas (OR of 0.70, 95% CI 0.49–1.01 and carcinomas (OR of 0.49, 95% CI 0.21–1.13 among women, although not significant. Women who were homozygous carriers of the high risk haplotype had an increased risk of colorectal cancer, OR of 2.19 (95% CI 0.95–5.04 compared to all non-carriers although the estimate was not statistically significant. Conclusion We found no evidence that the studied polymorphisms were associated with risk of adenomas or colorectal cancer among men, but we found weak indications that the chromosomal region may influence risk of colorectal cancer and adenoma development in women.

  20. Stimulation of glyceraldehyde-3-phosphate dehydrogenase mRNA levels by endogenous nitric oxide in cytokine-activated endothelium.

    Science.gov (United States)

    Bereta, J; Bereta, M

    1995-12-05

    Previous studies have shown that endogenous nitric oxide (NO) potentiates glycolysis in the cytokine-activated murine microvascular endothelial cells (MME). In the present study we investigate the influence of NO on the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an enzyme of the glycolytic pathway. Activation of MME with TNF-alpha and IFN-gamma resulted in a strong elevation of GAPDH mRNA levels. This effect was impaired in the presence of L-NMMA, the inhibitor of NO synthesis. We discuss the possibility that NO-mediated elevation of GAPDH mRNA levels may compensate for NO-mediated inhibition of GAPDH enzymatic activity, representing another adaptive mechanism which protects cells producing large amounts of NO against its cytotoxic effects.

  1. ESTRADIOL IN FEMALES MAY NEGATE SKELETAL MUSCLE MYOSTATIN MRNA EXPRESSION AND SERUM MYOSTATIN PROPEPTIDE LEVELS AFTER ECCENTRIC MUSCLE CONTRACTIONS

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    Darryn S. Willoughby

    2006-12-01

    Full Text Available Eccentric contractions produce a significant degree of inflammation and muscle injury that may increase the expression of myostatin. Due to its anti- oxidant and anti-flammatory effects, circulating 17-β estradiol (E2 may attenuate myostatin expression. Eight males and eight females performed 7 sets of 10 reps of eccentric contractions of the knee extensors at 150% 1-RM. Each female performed the eccentric exercise bout on a day that fell within her mid-luteal phase (d 21-23 of her 28-d cycle. Blood and muscle samples were obtained before and 6 and 24 h after exercise, while additional blood samples were obtained at 48 and 72 h after exercise. Serum E2 and myostatin LAP/propeptide (LAP/pro levels were determined with ELISA, and myostatin mRNA expression determined using RT-PCR. Data were analyzed with two-way ANOVA and bivariate correlations (p 0.05. Compared to pre-exercise, males had significant increases (p < 0.05 in LAP/propetide and mRNA of 78% and 28%, respectively, at 24 h post-exercise, whereas females underwent respective decreases of 10% and 21%. E2 and LAP/propeptide were correlated at 6 h (r = -0.804, p = 0.016 and 24 h post- exercise (r = -0.841, p = 0.009 in males, whereas in females E2 levels were correlated to myostatin mRNA at 6 h (r =0.739, p = 0.036 and 24 h (r = 0.813, p = 0.014 post-exercise and LAP/propeptide at 6 h (r = 0.713, p = 0.047 and 24 h (r = 0.735, p = 0.038. In females, myostatin mRNA expression and serum LAP/propeptide levels do not appear to be significantly up-regulated following eccentric exercise, and may be due to higher levels of circulating E2

  2. Size and Levels of mRNA for Acid Invertase in Ripe Tomato Fruit

    OpenAIRE

    Masakazu, Endo; Hiroki, Nakagawa; Nagao, OGURA; Takahide, Sato; Department of Agricultural Chemistry, Faculty of Horticulture, Chiba University

    1990-01-01

    Poly(A)^+RNA was isolated from ripe tomato fruit and translated in a wheat germ cell-free translation system. A 74-kDa polypeptide was detected as a putative precursor of acid invertase by immunoprecipitation with antiserum raised against SDS-treated acid invertase (denatured form) from tomato fruit. The molecular mass of the mRNA for acid invertase was estimated to be about 8 × 10^5 Da (2.4 k nucleotides) by sucrose density gradient centrifugation. Mature, green tomato fruit contained very l...

  3. IGF-I mRNA levels in bovine satellite cell cultures: effects of fusion and anabolic steroid treatment.

    Science.gov (United States)

    Kamanga-Sollo, E; Pampusch, M S; Xi, G; White, M E; Hathaway, M R; Dayton, W R

    2004-11-01

    Androgenic and estrogenic steroids enhance muscle growth in a number of species; however, the mechanism by which anabolic steroids enhance muscle growth is not known. Castrated male cattle (steers) provide a particularly good model system in which to study the effects of anabolic steroids on muscle growth because they respond dramatically to treatment with both estrogens and androgens. The goal of this study was to determine if treatment of bovine satellite cell (BSC) cultures with 17beta-estradiol (E(2)) or trenbolone (a synthetic androgen) directly affects proliferation rate or level of mRNA for estrogen receptor (ER)-alpha, androgen receptor, and growth factors that have been shown to affect muscle growth (insulin-like growth factor (IGF)-I, IGF binding protein (IGFBP)-3, and myostatin). BSC cultures were established from the semimembranosus muscles of steers and then treated for 48 h with various concentrations of E(2) or trenbolone ranging from 0.001 to 10 nM. IGF-I mRNA levels in proliferating BSC cultures were significantly increased at 0.01 (1.9-times control values, P steroids. Although, levels of IGF-I mRNA were 10-times greater (P steroids have direct anabolic effects on cells present in the BSC culture. Copyright 2004 Wiley-Liss, Inc.

  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. Ovarian carcinoma cells in serous effusions show altered MMP-2 and TIMP-2 mRNA levels.

    Science.gov (United States)

    Davidson, B; Reich, R; Berner, A; Givant-Horwitz, V; Goldberg, I; Risberg, B; Kristensen, G B; Trope, C G; Bryne, M; Kopolovic, J; Nesland, J M

    2001-11-01

    The expression of matrix metalloproteinases (MMP) and their inhibitor TIMP-2 in serous effusions from patients with ovarian carcinoma and its association with clinico-pathological parameters were analysed. The findings in carcinoma cells in effusions were compared with corresponding primary and metastatic lesions. Sixty-six effusions and 96 tissue sections were stained for MMP-1, MMP-2 and MMP-9 applying immunohistochemistry (IHC) and analysed for MMP-2, MMP-9 and TIMP-2 expression using mRNA in situ hybridisation (ISH). MMP-2 and MMP-9 mRNA levels in 30 effusions were subsequently analysed using reverse transcription- polymerase chain reaction (RT-PCR). MMP and TIMP expression was detected in both carcinoma and mesothelial cells in effusions. The levels were consistently higher in malignant cells, significantly so for MMP-1 (P=0.016) and MMP-2 (P=0.036) proteins, as well as for TIMP-2 mRNA (P=0.008). In tissue sections, MMP-1, MMP-2 and MMP-9 protein expression was mostly localised to tumour cells, while MMP-2, MMP-9 and TIMP-2 mRNA were predominantly detected in stromal cells. Adenocarcinoma cells in effusions showed a significant upregulation of MMP-2 expression compared with primary tumours, with a concomitant downregulation of TIMP-2. RT-PCR demonstrated the presence of MMP-2 and MMP-9 in 28/30 and 0/30 specimens, respectively. MMP and TIMP are thus mainly synthesised by cancer cells in effusions, while stromal cells have a similar role in solid tumours. MMP-1 and MMP-2 production predominates over that of MMP-9 in effusions. Increased MMP-2 and reduced TIMP-2 levels are seen in ovarian carcinoma cells in effusions, possibly marking the acquisition of a metastatic phenotype.

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

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    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.

  7. Adenosine methylation in Arabidopsis mRNA is associated with the 3’ end and reduced levels cause developmental defects

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    Zsuzsanna eBodi

    2012-03-01

    Full Text Available We previously showed that the N6-methyladenosine (m6A mRNA methylase is essential during Arabidopsis thaliana embryonic development. We also demonstrated that this modification is present at varying levels in all mature tissues. However, the requirement for the m6A in the mature plant was not tested. Here we show that a 95% reduction in m6A levels during later growth stages gives rise to plants with altered growth patterns and reduced apical dominance. The flowers of these plants commonly show defects in their floral organ number, size and identity. The global analysis of gene expression from reduced m6A plants show that a significant number of down regulated genes are involved in transport, or targeted transport, and most of the upregulated genes are involved in stress and stimulus response processes. An analysis of m6A distribution in fragmented mRNA suggests that the m6A is predominantly positioned towards the 3’ end of transcripts in a region 100-150 bp before the poly(A tail. In addition to the analysis of the phenotypic changes in the low methylation Arabidopsis plants we will review the latest advances in the field of mRNA internal methylation

  8. Association of a PIT1 gene polymorphism with growth hormone mRNA levels in pig pituitary glands

    Directory of Open Access Journals (Sweden)

    Maurício Machaim Franco

    2005-03-01

    Full Text Available Fourty-six non-castrated, halothane-free, male Landrace pigs were genotyped by PCR-RFLP for the Rsa I polymorphism in the PIT1 gene and classified into AA and AB genotypes. Total RNA was extracted from the pituitaries and the relative quantities of growth hormone (GH mRNA were determined by semi-quantitative RT-PCR. Pigs with the AB genotype had higher levels of GH mRNA than those with the AA genotype (p = 0.034; Kruskal-Wallis test. This result suggests that the Rsa I polymorphism may be involved in Pit-1 protein expression or function, which in turn may influence GH transcription and expression. Thus, the Rsa I PIT1 gene polymorphism in this pig line may be used as a molecular marker to identify higher GH expression and possibly select for carcass and performance traits affected by GH.

  9. 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.

  10. Excision repair cross-complementation group 1 (ERCC1) in platinum-based treatment of non-small cell lung cancer with special emphasis on carboplatin: a review of current literature

    DEFF Research Database (Denmark)

    Vilmar, A.; Sorensen, J.B.

    2009-01-01

    BACKGROUND: Patients diagnosed with advanced non-small cell lung cancer have a dismal prognosis and are often relative resistant to chemotherapy. A need for markers has emerged based on tumour biology in order to predict which patients will respond to treatment. Excision repair cross-complementat......BACKGROUND: Patients diagnosed with advanced non-small cell lung cancer have a dismal prognosis and are often relative resistant to chemotherapy. A need for markers has emerged based on tumour biology in order to predict which patients will respond to treatment. Excision repair cross......-complementation group 1 (ERCC1) has shown potential as a predictive marker in patients with NSCLC treated with cisplatin-based chemotherapy. Carboplatin has gained widespread use in the treatment of advanced NSCLC and its mechanisms of action are likely similar to that of cisplatin. MATERIALS AND METHODS: A literature...... articles and 1 clinical abstract were identified. Laboratory methods were mainly RT-PCR (reverse transcriptase polymerase chain reaction) or immunohistochemistry (IHC) for expression of ERCC1. Preclinical studies pointed towards similar mechanisms of chemotherapy-resistance among platinum compounds...

  11. Effects of metoclopramide on mRNA levels of steroid 5α-reductase isozymes in prostate of adult rats.

    Science.gov (United States)

    Sánchez, Pilar; Torres, Jesús M; Castro, Beatriz; Frías, José F; Ortega, Esperanza

    2013-03-01

    The rising incidence of prostate cancer and benign prostatic hypertrophy in the Western world is a cause of increasing public health concern. The most active androgen in the prostate is 5α-dihydrotestosterone obtained from testosterone (T) by the enzyme 5α-reductase (5α-R), expressed in the prostate as two isozymes, 5α-R1 and 5α-R2. These isozymes are involved in the growth and development of normal prostate and in the onset and progression of prostate disease. Besides androgens, prolactin (PRL) may also play a role, although it is not clear whether its effects on the prostate are in synergism with or independent of those of androgens. We previously demonstrated that sulpiride, an inductor of hyperprolactinemia, increased mRNA levels of 5α-R isozymes in prostate of adult rat. We hypothesized a possible interrelationship between PRL levels and 5α-R, although the effects of sulpiride per se cannot be ruled out. In the present study, one-step quantitative reverse transcription polymerase chain reaction coupled with laser-induced fluorescence capillary electrophoresis was used to quantify mRNA levels of both 5α-R isozymes in prostate of adult rat after administration of metoclopramide (MTC), another inductor of PRL secretion. With the administration regimens studied, MTC produced an increase in prostate weight and mRNA levels of 5α-R1 and 5α-R2 in adult rats. Given our finding that MTC per se or MTC-induced hyperprolactinemia modifies prostate disease-related parameters in animals with reduced plasma T levels, further investigation is warranted into the possibility that MTC use by aging males may increase their risk of prostate disease.

  12. Ethylmalonic acid modulates Na+, K(+)-ATPase activity and mRNA levels in rat cerebral cortex.

    Science.gov (United States)

    Schuck, Patrícia Fernanda; De Assis, Dênis Reis; Viegas, Carolina Maso; Pereira, Talita Carneiro Brandão; Machado, Jéssica Luca; Furlanetto, Camila Brulezi; Bogo, Mauricio Reis; Streck, Emilio Luiz; Ferreira, Gustavo Costa

    2013-03-01

    Ethylmalonic acid (EMA) accumulates in tissues of patients affected by short-chain acyl-CoA dehydrogenase deficiency and ethylmalonic encephalopathy, illnesses characterized by variable neurological symptoms. In this work, we investigated the in vitro and in vivo EMA effects on Na(+), K(+)-ATPase (NAK) activity and mRNA levels in cerebral cortex from 30-day-old rats. For in vitro studies, cerebral cortex homogenates were incubated in the presence of EMA at 0.5, 1, or 2.5 mM concentrations for 1 h. For in vivo experiments, animals received three subcutaneous EMA injections (6 μmol g(-1); 90-min interval) and were killed 60 min after the last injection. After that, NAK activity and its mRNA expression were measured. We observed that EMA did not affect this enzyme activity in vitro. In contrast, EMA administration significantly increased NAK activity and decreased mRNA NAK expression as assessed by semiquantitative reverse transcriptase polymerase chain reaction when compared with control group. Considering the high score of residues prone to phosphorylation on NAK, this profile can be associated with a possible regulation by specific phosphorylation sites of the enzyme. Altogether, the present results suggest that NAK alterations may be involved in the pathophysiology of brain damage found in patients in which EMA accumulates. Copyright © 2012 Wiley Periodicals, Inc.

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

    Directory of Open Access Journals (Sweden)

    John Walmsley

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

  14. Transforming growth factor β1 protein and mRNA levels in inflammatory bowel diseases: towards solving the contradictions by longitudinal assessment of the protein and mRNA amounts.

    Science.gov (United States)

    Liberek, Anna; Kmieć, Zbigniew; Wierzbicki, Piotr M; Jakóbkiewicz-Banecka, Joanna; Liberek, Tomasz; Łuczak, Grażyna; Plata-Nazar, Katarzyna; Słomińska-Frączek, Magdalena; Kaszubowska, Lucyna; Gabig-Cimińska, Magdalena; Węgrzyn, Alicja

    2013-01-01

    Previously published studies on levels of the transforming growth factor-β1 (TGF-β1) protein and mRNA of the corresponding gene in patients suffering from inflammatory bowel diseases (IBD) gave varying results, leading to contradictory conclusions. To solve the contradictions, we aimed to assess longitudinally TGF-β1 protein and mRNA levels at different stages of the disease in children suffering from IBD. The study group consisted of 19 pediatric patients with IBD at the age between 3.5 and 18.4 years. The control group consisted of 42 children aged between 2.0 and 18.0 years. The plasma TGF-β1 concentration was measured with ELISA. mRNA levels of the TGF-β1 gene isolated from samples of the intestinal tissue were assessed by reverse transcription and real-time PCR. Levels of TGF-β1 protein in plasma and corresponding mRNA in intestinal tissue were significantly higher in IBD patients than in controls. TGF-β1 and corresponding transcripts were also more abundant in plasma and intestinal tissue, respectively, in patients at the active stage of the disease than during remission. In every single IBD patient, plasma TGF-β1 level and mRNA level in intestinal tissue was higher at the active stage of the disease than during remission. Levels of TGF-β1 and corresponding mRNA are elevated during the active stage of IBD but not during the remission. Longitudinal assessment of this cytokine in a single patient may help to monitor the clinical course of IBD.

  15. The peptidyl-prolyl isomerase Pin1 determines parathyroid hormone mRNA levels and stability in rat models of secondary hyperparathyroidism.

    Science.gov (United States)

    Nechama, Morris; Uchida, Takafumi; Mor Yosef-Levi, Irit; Silver, Justin; Naveh-Many, Tally

    2009-10-01

    Secondary hyperparathyroidism is a major complication of chronic kidney disease (CKD). In experimental models of secondary hyperparathyroidism induced by hypocalcemia or CKD, parathyroid hormone (PTH) mRNA levels increase due to increased PTH mRNA stability. K-homology splicing regulator protein (KSRP) decreases the stability of PTH mRNA upon binding a cis-acting element in the PTH mRNA 3' UTR region. As the peptidyl-prolyl isomerase (PPIase) Pin1 has recently been shown to regulate the turnover of multiple cytokine mRNAs, we investigated the role of Pin1 in regulating PTH mRNA stability in rat parathyroids and transfected cells. The data generated were consistent with Pin1 being a PTH mRNA destabilizing protein. Initial analysis indicated that Pin1 activity was decreased in parathyroid protein extracts from both hypocalcemic and CKD rats and that pharmacologic inhibition of Pin1 increased PTH mRNA levels posttranscriptionally in rat parathyroid and in transfected cells. Pin1 mediated its effects via interaction with KSRP, which led to KSRP dephosphorylation and activation. In the rat parathyroid, Pin1 inhibition decreased KSRP-PTH mRNA interactions, increasing PTH mRNA levels. Furthermore, Pin1-/- mice displayed increased serum PTH and PTH mRNA levels, suggesting that Pin1 determines basal PTH expression in vivo. These results demonstrate that Pin1 is a key mediator of PTH mRNA stability and indicate a role for Pin1 in the pathogenesis of secondary hyperparathyroidism in individuals with CKD.

  16. Arsenic Trioxide Attenuates NF-κB and Cytokine mRNA Levels in the Livers of Cocks.

    Science.gov (United States)

    Zhang, Kexin; Zhao, Panpan; Guo, Guangyang; Guo, Ying; Tian, Li; Sun, Xiao; Li, Siwen; He, Ying; Sun, Ying; Chai, Hongliang; Zhang, Wen; Xing, Mingwei

    2016-04-01

    Arsenic (As) is a trace element widely found in nature. It exists in several forms, including organic arsenic, inorganic arsenic, and trivalent arsenic, the most toxic. Arsenic trioxide (As2O3) is widespread in nature. This form tends to accumulate in animals and humans and therefore has a potential harm for them. Cytokines play essential roles in the immune response and inflammation. Although the importance of cytokines in the responses to arsenic exposure has been demonstrated in many types of mammals, the function of these in poultry, especially in chickens, remains unclear. The purpose of the present study was to examine the effect of As2O3 exposure on cytokines in cock livers. In this study, 72 1-day-old male Hy-line cocks were randomly divided into four groups including the control group, low-As group, middle-As group, and high-As group. The livers were collected on days 30, 60, and 90 of the experiment. The levels of nuclear factor-kappa B (NF-κB), tumor necrosis factor-alpha (TNF-α), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-12 beta (IL-12β), and interleukin-1 beta (IL-1β) mRNA in the livers of the cocks were measured using real-time PCR. The results showed that the expression levels of IL-6, IL-8, TNF-α, and NF-κB which seemed to be a critical mediator in the inflammatory response tended to increase in the birds chronically treated with As2O3. However, the mRNA expression levels of IL-4, IL-12β, and IL-1β were decreased in the experiment. The information regarding the effects of As2O3 on cytokine mRNA expression generated in this study will be important information for arsenic toxicology evaluation.

  17. Rapid, Transient Fluconazole Resistance in Candida albicans Is Associated with Increased mRNA Levels of CDR

    Science.gov (United States)

    Marr, Kieren A.; Lyons, Christopher N.; Rustad, Tiger; Bowden, Raleigh A.; White, Theodore C.

    1998-01-01

    Fluconazole-resistant Candida albicans, a cause of recurrent oropharyngeal candidiasis in patients with human immunodeficiency virus infection, has recently emerged as a cause of candidiasis in patients receiving cancer chemotherapy and marrow transplantation (MT). In this study, we performed detailed molecular analyses of a series of C. albicans isolates from an MT patient who developed disseminated candidiasis caused by an azole-resistant strain 2 weeks after initiation of fluconazole prophylaxis (K. A. Marr, T. C. White, J. A. H. vanBurik, and R. A. Bowden, Clin. Infect. Dis. 25:908–910, 1997). DNA sequence analysis of the gene (ERG11) for the azole target enzyme, lanosterol demethylase, revealed no difference between sensitive and resistant isolates. A sterol biosynthesis assay revealed no difference in sterol intermediates between the sensitive and resistant isolates. Northern blotting, performed to quantify mRNA levels of genes encoding enzymes in the ergosterol biosynthesis pathway (ERG7, ERG9, and ERG11) and genes encoding efflux pumps (MDR1, ABC1, YCF, and CDR), revealed that azole resistance in this series is associated with increased mRNA levels for members of the ATP binding cassette (ABC) transporter superfamily, CDR genes. Serial growth of resistant isolates in azole-free media resulted in an increased susceptibility to azole drugs and corresponding decreased mRNA levels for the CDR genes. These results suggest that C. albicans can become transiently resistant to azole drugs rapidly after exposure to fluconazole, in association with increased expression of ABC transporter efflux pumps. PMID:9756759

  18. EGF receptor inhibitors increase ErbB3 mRNA and protein levels in breast cancer cells

    DEFF Research Database (Denmark)

    Grøvdal, Lene Melsæther; Kim, Jiyoung; Holst, Mikkel Roland

    2012-01-01

    gene expression of FAS which is involved in apoptotic signaling. Together, our data strongly suggest that resistance to EGFR inhibitors may result from the compensation of other family members and that combinations of anti-cancer drugs are required to increase the sensitivity of these treatments....... induced by gefitinib treatment on mRNA levels of the most common genes known to be involved in breast cancer. As expected, we found that gefitinib downregulated genes whose functions were linked to cellular proliferation, such as Ki-67, topoisomerase II alpha and cyclins, and surprisingly downregulated...

  19. α2A -Adrenergic receptor polymorphisms and mRNA expression levels are associated with delay discounting in cocaine users.

    Science.gov (United States)

    Havranek, Michael M; Hulka, Lea M; Tasiudi, Eve; Eisenegger, Christoph; Vonmoos, Matthias; Preller, Katrin H; Mössner, Rainald; Baumgartner, Markus R; Seifritz, Erich; Grünblatt, Edna; Quednow, Boris B

    2017-03-01

    Cocaine users characteristically display preferences for smaller immediate rewards over larger delayed rewards, and this delay discounting (DD) has been proposed as an endophenotype of cocaine addiction. Recent evidence suggests that the norepinephrine system and more specifically the α2A -adrenergic receptor (ADRA2A) are impacted by chronic cocaine use while also being potentially involved in the neural mechanisms underlying DD. Hence, we investigated the effects of ADRA2A polymorphisms and ADRA2A mRNA expression levels on DD of cocaine users and stimulant-naïve controls. Two hundred and twenty-three participants (129 cocaine users and 94 stimulant-naïve healthy controls) completed a computerized DD paradigm and were genotyped for three single nucleotide polymorphisms (SNPs; rs1800544, rs521674 and rs602618) in the ADRA2A gene, while their peripheral ADRA2A mRNA expression was quantified in whole blood samples. The three SNPs were in near-perfect linkage disequilibrium. Accordingly, significant group*genotype interactions were found for all three ADRA2A variants revealing steeper DD in cocaine users (but not in controls) carrying the G-allele of SNP rs1800544, the T-allele of rs521674 and the C-allele of rs602618. Similarly, high ADRA2A mRNA expression levels were significantly associated with a reduced tendency to choose smaller more immediate rewards (over larger delayed rewards) in cocaine users but not in controls. As the relationship between DD and cocaine use was moderated by ADRA2A SNPs and by peripheral ADRA2A gene expression, we propose that the norepinephrine system is involved in DD deficits observed in cocaine using individuals. Consequently, pharmacological compounds targeting ADRA2As might be considered for the symptom-specific treatment of delay aversion in stimulant addiction. © 2015 Society for the Study of Addiction.

  20. Exogenous glucagon-like peptide-2 (GLP-2) augments GLP-2 receptor mRNA and maintains proglucagon mRNA levels in resected rats

    DEFF Research Database (Denmark)

    Koopmann, Matthew C; Nelson, David W; Murali, Sangita G

    2008-01-01

    ), crypt-villus height, and crypt cell proliferation (by bromodeoxyuridine staining) were determined. Plasma bioactive GLP-2 (by radioimmunoassay), proglucagon and GLP-2 receptor mRNA expression (by Northern blot and real-time reverse transcriptase quantitative polymerase chain reaction) were measured. GLP...... activity. Plasma bioactive GLP-2 concentration increased 70% upon resection, with an additional 54% increase upon GLP-2 infusion in resected rats (P ... GLP-2 receptor expression 3-fold in resected animals and was colocalized to vasoactive intestinal peptide-positive and endothelial nitric oxide synthase-expressing enteric neurons and serotonin-containing enteroendocrine cells in the jejunum and ileum of resected rats. CONCLUSIONS: Exogenous GLP-2...

  1. Effects of sulpiride on mRNA levels of steroid 5alpha-reductase isozymes in prostate of adult rats.

    Science.gov (United States)

    Sánchez, Pilar; Torres, Jesús M; Vílchez, Pablo; Del Moral, Raimundo G; Ortega, Esperanza

    2008-01-01

    Prolactin (PRL) is implicated in prostate growth and in the development and regulation of benign prostatic hypertrophy (BPH) and prostate cancer (PCa). PRL may exert its effects on prostate in synergism with androgens. The most active androgen in the prostate is the 5alpha-dihydrotestosterone (DHT) obtained from testosterone by the 5alpha-reductase (5alpha-R) enzyme, which is expressed in the prostate as two isozymes, 5alpha-R1 and 5alpha-R2. In this study, sulpiride, a prolactin-secretion inductor, was administered to male rats. mRNA levels of 5alpha-R1 and 5alpha-R2 were measured in prostate of controls and sulpiride-treated rats, using one-step quantitative RT-PCR coupled with laser-induced fluorescence capillary electrophoresis (LIF-CE). Results demonstrated that sulpiride-induced hyperprolactinemia is associated with an increase in mRNA levels of both 5alpha-R1 and 5alpha-R2 in prostate of adult rats. Although a direct effect of sulpiride on prostate gland cannot be ruled out, hyperprolactinemia may be a factor to be considered in aging males, in whom prostatic diseases such as BPH and PCa are more frequent.

  2. Vesicular monoamine transporter 2 mRNA levels are reduced in platelets from patients with Parkinson's disease.

    Science.gov (United States)

    Sala, Gessica; Brighina, Laura; Saracchi, Enrico; Fermi, Silvia; Riva, Chiara; Carrozza, Veronica; Pirovano, Marta; Ferrarese, Carlo

    2010-09-01

    Despite advances in neuroimaging, the diagnosis of idiopathic Parkinson's disease (PD) remains clinical. The identification of biological markers for an early diagnosis is of great interest to start a neuroprotective therapy aimed at slowing, blocking or reversing the disease progression. Vesicular monoamine transporter 2 (VMAT2) sequesters cytoplasmic dopamine into synaptic vesicles for storage and release. Thus, VMAT2 impairment can regulate intra- and extracellular dopamine levels, influencing oxidative stress and neuronal death. Because in vivo imaging studies have demonstrated a VMAT2 reduction in PD patients greater than would be explained by neuronal loss alone, as an exploratory study we assessed VMAT2 mRNA and protein levels in platelets from 39 PD patients, 39 healthy subjects and 10 patients with vascular parkinsonism (VP) to identify a possible peripheral biomarker for PD. A significant reduction (p platelets. Although further studies in a greater number of cases are needed to confirm our data, the reduction in VMAT2 mRNA in platelets from PD patients suggests the existence of a systemic impairment of this transporter possibly contributing to PD pathology.

  3. Salt Stress Increases the Level of Translatable mRNA for Phosphoenolpyruvate Carboxylase in Mesembryanthemum crystallinum1

    Science.gov (United States)

    Ostrem, James A.; Olson, Steve W.; Schmitt, Jürgen M.; Bohnert, Hans J.

    1987-01-01

    Mesembryanthemum crystallinum responds to salt stress by switching from C3 photosynthesis to Crassulacean acid metabolism (CAM). During this transition the activity of phosphoenolpyruvate carboxylase (PEPCase) increases in soluble protein extracts from leaf tissue. We monitored CAM induction in plants irrigated with 0.5 molar NaCl for 5 days during the fourth, fifth, and sixth week after germination. Our results indicate that the age of the plant influenced the response to salt stress. There was no increase in PEPCase protein or PEPCase enzyme activity when plants were irrigated with 0.5 molar NaCl during the fourth and fifth week after germination. However, PEPCase activity increased within 2 to 3 days when plants were salt stressed during the sixth week after germination. Immunoblot analysis with anti-PEPCase antibodies showed that PEPCase synthesis was induced in both expanded leaves and in newly developing axillary shoot tissue. The increase in PEPCase protein was paralleled by an increase in PEPCase mRNA as assayed by immunoprecipitation of PEPCase from the in vitro translation products of RNA from salt-stressed plants. These results demonstrate that salinity increased the level of PEPCase in leaf and shoot tissue via a stress-induced increase in the steady-state level of translatable mRNA for this enzyme. Images Fig. 2 Fig. 3 Fig. 4 PMID:16665596

  4. Abnormal mRNA Expression Levels of Telomere-Binding Proteins Represent Biomarkers in Myelodysplastic Syndromes: A Case-Control Study

    Directory of Open Access Journals (Sweden)

    Baoshan Liu

    2017-09-01

    Full Text Available Objective: As evidence was shown that abnormal shortening of telomeres begins to accumulate in myelodysplastic syndrome (MDS patients, this study was conducted to determine the relationship between the mRNA expression levels of telomere-binding proteins (TRF1/TRF2/TIN2/TPP1/POT1/RAP1 and the risk level in MDS. Materials and Methods: There were 40 patients with MDS and 40 normal controls in this study. Methods including telomere content assays and quantitative reverse transcription-polymerase chain reaction were used to examine the mRNA levels of TRF1/TRF2/TIN2/ TPP1/POT1/RAP1 in patients with MDS. Results: Compared to the normal group used as a control, the mRNA expression levels of RAP1/POT1/TPP1 of the patients with MDS were decreased, whereas their levels of TRF1/TRF2 and TIN2 were increased. A positive correlation was found between the TRF1, TRF2, and TIN2 mRNA expression levels and the risk level of the International Prognostic Scoring System (IPSS and the World Health Organization Prognostic Scoring System (WPSS criteria; however, a negative correlation was found between RAP1/POT1/TPP1 mRNA expression levels and the risk levels of IPSS and WPSS criteria. Conclusion: Because the reduction of TRF1/TRF2/TIN2 mRNA expression and the increase of RAP1/POT1/TPP1 mRNA expression are closely related to the risk levels of the IPSS and WPSS criteria in MDS, it is thought that these telomere-binding proteins could lead to abnormal telomere length and function, which cause chromosomal abnormalities in MDS. With this evidence, we suggest that those proteins’ mRNA expressions could be used as biomarkers for the assessment of the risk degree of MDS patients.

  5. Miconia sp. Increases mRNA Levels of PPAR Gamma and Inhibits Alpha Amylase and Alpha Glucosidase

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    David Mizael Ortíz-Martinez

    2016-01-01

    Full Text Available Diabetes mellitus is a public health problem worldwide. For this reason, ethanolic extract of Miconia sp. from Oaxaca, Mexico, was selected in search of an alternative against this disease. The effect of Miconia sp. on mRNA expression of PPARγ on cell line 3T3-L1, its effect on alpha amylase and alpha glucosidase, lipid accumulation during adipogenesis, and cell viability on VERO cells were evaluated. The mRNA levels of PPARγ increased on 1.393±0.008 folds, lipid accumulation was increased by 29.55% with Miconia sp. extract and 34.57% with rosiglitazone, and α-amylase and α-glycosidase were inhibited with IC50 values from 28.23±2.15 μg/mL and 1.95±0.15 μg/mL, respectively; the IC50 on antiproliferative activity on VERO cells was 314.54±45.40 μg/mL. In case of α-amylase and α-glycosidase assays, IC50 (inhibitory concentration 50 refers to necessary extract amounts to inhibit 50% of enzymatic activity. On the other hand, on antiproliferative activity, IC50 (inhibitory concentration 50 refers to necessary extract amounts to inhibit 50% of cell proliferation. It was concluded that the compounds present in Miconia sp. ethanolic extract increase mRNA expression of PPARγ, inhibit α-amylase and α-glucosidase, and increase lipid accumulation. It constitutes an alternative as adjuvant in diabetes mellitus treatment; therefore, we recommend continuing identifying the compounds responsible for its promising in vivo antidiabetic activity.

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

    Directory of Open Access Journals (Sweden)

    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.

  7. 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.

  8. 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.

  9. A haplotype of polymorphisms in ASE-1, RAI and ERCC1 and the effects of tobacco smoking and alcohol consumption on risk of colorectal cancer: a Danish prospective case-cohort study

    DEFF Research Database (Denmark)

    Hansen, Rikke D; Sørensen, Mette; Tjønneland, Anne

    2008-01-01

    cancer, and investigated gene-environment associations between the polymorphisms and the haplotype and smoking status at enrolment, smoking duration, average smoking intensity and alcohol consumption, respectively, in relation to risk of colorectal cancer. METHODS: Associations between the three...... of colorectal cancer were found. No statistically significant interactions between the genotypes and the lifestyle exposures smoking or alcohol consumption were observed. CONCLUSION: Our results suggest that the ASE-1 G-21A, RAI IVS1 A4364G and ERCC1 Asn118Asn polymorphisms and the previously identified...... haplotype are not associated with risk of colorectal cancer. We found no evidence of gene-environment interaction between the three polymorphisms and the haplotype and smoking intensity and alcohol consumption, respectively, in relation to the risk of colorectal cancer. Udgivelsesdato: 2008-null...

  10. Correlation of thyroid stimulating hormone receptor mRNA expression levels in peripheral blood with undesirable clinicopathological features in papillary thyroid carcinoma patients

    Science.gov (United States)

    Liu, Riming; Hao, Shaolong; Zhang, Hua; Ma, Jihong; Liu, Xincheng; Xu, Jie; Liu, Xin; Ning, Jinyao; Sun, Yan; Jiang, Lixin; Li, Guojun; Song, Xicheng; Zheng, Haitao

    2017-01-01

    To determine the extent to which thyroid stimulating hormone receptor (TSHR) mRNA in peripheral blood (PB) has diagnostic value for papillary thyroid carcinoma (PTC). We obtained pre- and postoperative PB samples from 104 thyroid disease patients and collected 11 healthy volunteers’ PB samples twice apiece at different times. We used reverse transcription polymerase chain reaction (RT-PCR) to quantify TSHR mRNA expression levels in the samples. T-test and chi-square test were used to compare quantitative data and rates. The mean preoperative PB TSHR mRNA expression level of the PTC patients was significantly higher than that of the healthy volunteers. However, on the postoperative day 1, PB TSHR mRNA level of PTC patients significantly decreased but not for healthy controls. Preoperative PB TSHR mRNA expression levels were significantly associated with patient age, capsular invasion status, lymph node metastasis status, and BRAFV600E mutation status (P cancer foci, or Hashimoto thyroiditis status. Preoperative assessment of the PB TSHR mRNA expression level combined with ultrasonography of the thyroid had better accuracy in the diagnosis of PTC than either method alone did. Moreover, TSHR mRNA expression significantly affected recurrence of PTC patients. Our findings suggest that PB TSHR mRNA expression level is a promising novel biomarker for the early detection, diagnosis, and treatment of PTC. It may serve as a noninvasive means of PTC detection and a prognostic biomarker of residual tumor and help guide further treatment. PMID:29088773

  11. 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.

  12. TREM-1 SNP rs2234246 regulates TREM-1 protein and mRNA levels and is associated with plasma levels of L-selectin.

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    Alex-Ander Aldasoro Arguinano

    Full Text Available High levels of TREM-1 are associated with cardiovascular and inflammatory diseases risks and the most recent studies have showed that TREM-1 deletion or blockade is associated with up to 60% reduction of the development of atherosclerosis. So far, it is unknown whether the levels of TREM-1 protein are genetically regulated. Moreover, TREM family receptors have been suggested to regulate the cellular adhesion process. The goal of this study was to investigate whether polymorphisms within TREM-1 are regulating the variants of serum TREM-1 levels and the expression levels of their mRNA. Furthermore, we aimed to point out associations between polymorphisms on TREM-1 and blood levels of selectins. Among the 10 SNPs studied, the minor allele T of rs2234246, was associated with increased sTREM-1 in the discovery population (p-value = 0.003, explaining 33% of its variance, and with increased levels of mRNA (p-value = 0.007. The same allele was associated with increased soluble L-selectin levels (p-value = 0.011. The higher levels of sTREM-1 and L-selectin were confirmed in the replication population (p-value = 0.0007 and p-value = 0.018 respectively. We demonstrated for the first time one SNP on TREM-1, affecting its expression levels. These novel results, support the hypothesis that TREM-1 affects monocytes extravasation and accumulation processes leading to atherogenesis and atherosclerotic plaque progression, possibly through increased inflammation and subsequent higher expression of sL-selectin.

  13. Effects of the pesticides prochloraz and methiocarb on human estrogen receptor alpha and beta mRNA levels analyzed by on-line RT-PCR.

    Science.gov (United States)

    Hofmeister, M V; Bonefeld-Jørgensen, E C

    2004-08-01

    Exposure to endocrine disrupters such as dioxins, PCBs and certain pesticides are suspected to affect human reproductive health. We have analyzed the effect of the currently used pesticides prochloraz and methiocarb on the estrogen receptor (ER)alpha and beta mRNA levels in parallel with the natural ligand, 17beta-estradiol (E2). Using the highly sensitive on-line RT-PCR technique we were able to quantify the ERalpha and ERbeta mRNA levels in the human breast cancer cell line, MCF7-BUS. Upon exposure with E2 or prochloraz a down regulation of ERalpha and ERbeta mRNAs was observed after 48 h of treatment. Co-treatment with the ER antagonist ICI 182,780 abolished these mRNA down regulations. Western blot analyses elicited a decreased ER protein level after 3 h of exposure with prochloraz but after 24 h the ERalpha protein level had recovered to basal level. Methiocarb exposure had no effect on the ERalpha mRNA level, whereas an increase in the ERbeta mRNA level was observed after 3 h of exposure. Our study demonstrates that like E2, prochloraz had the potential to down regulate the expression of ERalpha and ERbeta mRNAs as well as the ERalpha protein level in MCF7-BUS cells.

  14. Blood glutathione peroxidase-1 mRNA levels can be used as molecular biomarkers to determine dietary selenium requirements in rats.

    Science.gov (United States)

    Sunde, Roger A; Thompson, Kevin M; Evenson, Jacqueline K; Thompson, Britta M

    2009-11-01

    Transcript (mRNA) levels are increasingly being used in medicine as molecular biomarkers for disease and disease risk, including use of whole blood as a target tissue for analysis. Development of blood molecular biomarkers for nutritional status, too, has potential application that parallels opportunities in medicine, including providing solid data for individualized nutrition. We previously reported that blood glutathione peroxidase-1 (Gpx1) mRNA was expressed at levels comparable to major tissues in rats and humans. To determine the efficacy of using blood Gpx1 mRNA to assess selenium (Se) status and requirements, we fed graded levels of Se (0-0.3 microg Se/g as selenite) to weanling male rats. Se status was determined by liver Se concentration and selenoenzyme activity, and selenoprotein mRNA abundance in liver and blood was determined by ribonuclease protection analysis. Liver Se and plasma glutathione peroxidase-3 and liver Gpx1 activities indicated that minimal Se requirements were at 0.08 microg Se/g diet. When total RNA was isolated from whole blood, Gpx1 mRNA in Se-deficient rats decreased to 10% of levels in Se-adequate (0.2 microg Se/g diet) rats. With Se supplementation, blood Gpx1 mRNA levels increased sigmoidally to a plateau with a minimum Se requirement of 0.08 microg Se/g diet, whereas glutathione peroxidase-4 mRNA levels were unaffected. Similarly, Gpx1 mRNA in RNA isolated from fractionated red blood cells decreased in Se-deficient rats to 23% of Se-adequate levels, with a minimum Se requirement of 0.09 microg Se/g diet. Additional studies showed that the preponderance of whole blood Gpx1 mRNA arises from erythroid cells, most likely reticulocytes and young erythrocytes. In summary, whole blood selenoprotein mRNA levels can be used as molecular biomarkers for assessing Se requirements, illustrating that whole blood has potential as a target tissue in development of molecular biomarkers for use in nutrition as well as in medicine.

  15. Quantification of Chitinase mRNA Levels in Human and Mouse Tissues by Real-Time PCR: Species-Specific Expression of Acidic Mammalian Chitinase in Stomach Tissues.

    Science.gov (United States)

    Ohno, Misa; Togashi, Yuto; Tsuda, Kyoko; Okawa, Kazuaki; Kamaya, Minori; Sakaguchi, Masayoshi; Sugahara, Yasusato; Oyama, Fumitaka

    2013-01-01

    Chitinase hydrolyzes chitin, which is an N-acetyl-D-glucosamine polymer that is present in a wide range of organisms, including insects, parasites and fungi. Although mammals do not contain any endogenous chitin, humans and mice express two active chitinases, chitotriosidase (Chit1) and acidic mammalian chitinase (AMCase). Because the level of expression of these chitinases is increased in many inflammatory conditions, including Gaucher disease and mouse models of asthma, both chitinases may play important roles in the pathophysiologies of these and other diseases. We recently established a quantitative PCR system using a single standard DNA and showed that AMCase mRNA is synthesized at extraordinarily high levels in mouse stomach tissues. In this study, we applied this methodology to the quantification of chitinase mRNAs in human tissues and found that both chitinase mRNAs were widely expressed in normal human tissues. Chit1 mRNA was highly expressed in the human lung, whereas AMCase mRNA was not overexpressed in normal human stomach tissues. The levels of these mRNAs in human tissues were significantly lower than the levels of housekeeping genes. Because the AMCase expression levels were quite different between the human and mouse stomach tissues, we developed a quantitative PCR system to compare the mRNA levels between human and mouse tissues using a human-mouse hybrid standard DNA. Our analysis showed that Chit1 mRNA is expressed at similar levels in normal human and mouse lung. In contrast, the AMCase expression level in human stomach was significantly lower than that expression level observed in mouse stomach. These mRNA differences between human and mouse stomach tissues were reflecting differences in the chitinolytic activities and levels of protein expression. Thus, the expression level of the AMCase in the stomach is species-specific.

  16. Acid tolerance and gad mRNA levels of Escherichia coli O157:H7 grown in foods.

    Science.gov (United States)

    Yokoigawa, Kumio; Takikawa, Akiko; Okubo, Yoko; Umesako, Seiichi

    2003-05-15

    We examined the acid tolerance and gad mRNA levels of Escherichia coli O157:H7 (three strains) and nonpathogenic E. coli (strains K12, W1485, and B) grown in foods. The E. coli cells (approximately 30,000 cells) were inoculated on the surface of 10 g of solid food samples (asparagus, broccoli, carrot, celery, cucumber, eggplant, ginger, green pepper, onion, potato, radish, tomato and beef) and in 10 ml of cow's milk, cultured statically at 10-25 degrees C for 1-14 days, and subjected to an acid challenge at 37 degrees C for 1 h in LB medium (pH 3.0). When grown at 20 and 25 degrees C in all foods, except for tomato and ginger, the strains showed a stationary-phase specific acid tolerance. The acid tolerance of the O157 strains changed depending on the types of foods (3-10% survival), but was clearly lower than that of the cells grown in EC medium (more than 90% survival). Tomato and ginger induced relatively high acid tolerances (10-30% survival) in the O157 strains irrespective of the growth phase, probably because of their acidity. No remarkable difference was observed in the acid tolerance between the O157 and nonpathogenic strains grown in all foods. When grown at 10 and 15 degrees C in the foods and EC medium, none of the strains showed the stationary-phase specific acid tolerance. In beef, broccoli, celery, potato and radish, the acid tolerance showed a tendency to decrease with the prolonged cultivation time. In other foods, the acid tolerance was almost constant (about 0.1% survival) irrespective of the growth stage. The mRNA level of glutamate decarboxylase genes (gadA and gadB) correlated to the acid tolerance level when the E. coli cells were grown at 25 degrees C, but was very low even in the stationary phase when the E. coli cells were grown at 15 degrees C or below.

  17. Clinical Usefulness of Monitoring Expression Levels of CCL24 (Eotaxin-2) mRNA on the Ocular Surface in Patients with Vernal Keratoconjunctivitis and Atopic Keratoconjunctivitis.

    Science.gov (United States)

    Shiraki, Yukiko; Shoji, Jun; Inada, Noriko

    2016-01-01

    Purpose. This study aimed to evaluate the clinical efficacy of using expression levels of CCL24 (eotaxin-2) mRNA on the ocular surface as a biomarker in patients with vernal keratoconjunctivitis (VKC) and atopic keratoconjunctivitis (AKC). Methods. Eighteen patients with VKC or AKC (VKC/AKC group) and 12 control subjects (control group) were enrolled in this study. The VKC/AKC clinical score was determined by objective findings in patients by using the 5-5-5 exacerbation grading scale. All subjects underwent modified impression cytology and specimens were obtained from the upper tarsal conjunctiva. Expression levels of CCL24 (eotaxin-2) mRNA on the ocular surface were determined using real-time reverse transcription polymerase chain reaction. Results. The VKC group was divided into two subgroups, depending on the clinical score: the active stage subgroup with 100 points or more of clinical scores and the stable stage subgroup with 100 points or less. CCL24 (eotaxin-2) mRNA expression levels in the active VKC/AKC stage subgroup were significantly higher than those in the stable VKC/AKC subgroup and the control group. Clinical scores correlated significantly with CCL24 (eotaxin-2) mRNA expression levels in the VKC group. Conclusions. CCL24 (eotaxin-2) mRNA expression levels on the ocular surface are a useful biomarker for clinical severity of VKC/AKC.

  18. Expression of NK1 receptor at the protein and mRNA level in the porcine female reproductive system.

    Science.gov (United States)

    Bukowski, R

    2014-01-01

    The presence and distribution of substance P (SP) receptor NK1 was studied in the ovary, the oviduct and the uterus (uterine horn and cervix) of the domestic pig using the methods of molecular biology (RT-PCR and immunoblot) and immunohistochemistry. The expression of NK1 receptor at mRNA level was confirmed with RT-PCR in all the studied parts of the porcine female reproductive system by the presence of 525 bp PCR product and at the protein level by the detection of 46 kDa protein band in immunoblot. Immunohistochemical staining revealed the cellular distribution of NK1 receptor protein. In the ovary NKI receptor was present in the wall of arterial blood vessels, as well as in ovarian follicles of different stages of development. In the tubular organs the NK1 receptor immunohistochemical stainings were observed in the wall of the arterial blood vessels, in the muscular membrane, as well as in the mucosal epithelium. The study confirmed the presence of NK1 receptor in the tissues of the porcine female reproductive tract which clearly points to the possibility that SP can influence porcine ovary, oviduct and uterus.

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

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    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.

  20. 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

  1. Increased mRNA Levels of TCF7L2 and MYC of the Wnt Pathway in Tg-ArcSwe Mice and Alzheimer's Disease Brain

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    Elin S. Blom

    2011-01-01

    Full Text Available Several components in the Wnt pathway, including β-catenin and glycogen synthase kinase 3 beta, have been implied in AD pathogenesis. Here, mRNA brain levels from five-month-old tg-ArcSwe and nontransgenic mice were compared using Affymetrix microarray analysis. With surprisingly small overall changes, Wnt signaling was the most affected pathway with altered expression of nine genes in tg-ArcSwe mice. When analyzing mRNA levels of these genes in human brain, transcription factor 7-like 2 (TCF7L2 and v-myc myelocytomatosis viral oncogene homolog (MYC, were increased in Alzheimer's disease (AD (P<.05. Furthermore, no clear differences in TCF7L2 and MYC mRNA were found in brains with frontotemporal lobar degeneration, suggesting that altered regulation of these Wnt-related genes could be specific to AD. Finally, mRNA levels of three neurogenesis markers were analyzed. Increased mRNA levels of dihydropyrimidinase-like 3 were observed in AD brain, suggesting that altered Wnt pathway regulation may signify synaptic rearrangement or neurogenesis.

  2. Green tea polyphenols improve cardiac muscle mRNA, and protein levels of signal pathways related to insulin and lipid metabolism and inflammation in insulin-resistant rats

    Science.gov (United States)

    Epidemiologic studies indicate that the consumption of green tea polyphenols (GTP) may reduce the risk of coronary artery disease. To explore the underlying mechanisms of action at the molecular level, we examined the effects of GTP on cardiac mRNA and protein levels of genes involved in insulin an...

  3. Positive correlation between patency and mRNA levels for cyclooxygenase-2 and prostaglandin E synthase in the uterine cervix of bitches with pyometra.

    Science.gov (United States)

    Tamada, Hiromichi; Adachi, Nahoko; Kawate, Noritoshi; Inaba, Toshio; Hatoya, Shingo; Sawada, Tsutomu

    2016-03-01

    Factors involved in patency of uterine cervices in the bitch with pyometra remain to be clarified. This study examined relationship between patency and mRNA levels for inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-1, COX-2 and prostaglandin E synthase (PGES) in the uterine cervix of bitches with pyometra. Cervical patency was measured by inserting the stainless steel rods with different diameter into cervical canals. Levels of mRNA expression were determined by semi-quantitative reverse transcription-polymerase chain reaction. The cervical patency was positively correlated with mRNA levels for COX-2 and PGES, but not those for iNOS and COX-1. The results suggest that gene expression of COX-2 and PGES may be involved in the regulation of patency in the uterine cervix of bitches with pyometra.

  4. 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...... increase in OI and increased plasma IL-8 and IL-10 concentrations in the CPB-pigs compared with the sham-pigs. Conclusion: The cytokine mRNA expression pattern was very different for the pigs killed already 0.5 h after the CPB procedure compared with the pigs killed 4 h post-CPB. The plasma cytokine levels...... poorly reflected mRNA expression of the pro- and anti-inflammatory cytokines....

  5. AFP mRNA level in enriched circulating tumor cells from hepatocellular carcinoma patient blood samples is a pivotal predictive marker for metastasis.

    Science.gov (United States)

    Jin, Junhua; Niu, Xiaojuan; Zou, Lihui; Li, Lin; Li, Shugang; Han, Jingli; Zhang, Peiying; Song, Jinghai; Xiao, Fei

    2016-08-01

    Circulating tumor cells (CTCs) quantification may be helpful for evaluating cancer dissemination, predicting prognosis and assessing therapeutic effectiveness and safety. In the present study, CTCs from blood samples of 72 patients with hepatocellular carcinoma (HCC) were enriched with anti-EpCAM nanoparticles. AFP mRNA level was detected by nested polymerase chain reaction (PCR) after enrichment of CTCs from HCC blood samples at 0, 3, 6, 9 and 12 months after hepatectomy, respectively. AFP mRNA expression in CTCs was positive in 43 patients (59.7%) and negative in 29 patients (40.3%) before hepatectomy. Among 43 patients with positive AFP mRNA expression in CTCs before hepatectomy, 10 and 11 were diagnosed as intrahepatic/extrahepatic metastasis before and after hepatectomy, respectively. In addition, these 21 patients with metastasis had persisting positive AFP mRNA of CTCs during the whole tested year. Specifically, 3 patients with AFP mRNA negative in CTCs before hepatectomy changed to be positive at 6 and 9 months, and 2 of them were diagnosed as metastasis 12 months after hepatectomy. We conclude that the positive AFP mRNA of CTCs can be a pivotal predictor for HCC metastasis before and after hepatectomy. The release of AFP expression from hepatocellular carcinoma cells into circulation must be a major source of HCC metastasis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  6. Developmental changes in the hypothalamic mRNA levels of prepro-orexin and orexin receptors and their sensitivity to fasting in male and female rats.

    Science.gov (United States)

    Iwasa, Takeshi; Matsuzaki, Toshiya; Munkhzaya, Munkhsaikhan; Tungalagsuvd, Altankhuu; Kuwahara, Akira; Yasui, Toshiyuki; Irahara, Minoru

    2015-11-01

    Orexin, which is also called as hypocretin (Hcrt), a product of the prepro-orexin (pp-orexin//Hcrt) gene, affects various physiological and behavioral functions, such as the sleep-wake cycle and appetite. The developmental changes in the hypothalamic mRNA levels of pp-prexin and the orexin receptors OX1R and OX2R and their sensitivity to fasting were evaluated in both male and female rats. During development, hypothalamic pp-orexin/Hcrt mRNA expression increased in both male and female rats, whereas hypothalamic OX1R mRNA expression decreased in both sexes. In addition, hypothalamic OX2R mRNA expression increased in male rats, but did not change in female rats. Fasting did not affect hypothalamic pp-orexin/Hcrt mRNA expression in either sex. Hypothalamic OX1R mRNA expression was increased by fasting in the prepubertal period (postnatal days 20 and 30) in female rats, but was not affected by fasting in males. In male rats, hypothalamic OX2R mRNA expression was decreased by fasting during the neonatal period (postnatal day 10), but not the prepubertal period (postnatal days 20 and 30). In females, hypothalamic OX2R mRNA expression was also decreased by fasting; however, the fasting-induced downregulation of hypothalamic OX2R expression persisted until postnatal day 20. These results indicate that the developmental patterns of components of the orexin system and their sensitivity to fasting during the neonatal and prepubertal periods only differ slightly between the sexes. These differences might be involved in the development of some physiological and behavioral functions. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

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    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.

  8. Trypsinogen-like cDNAs and quantitative analysis of mRNA levels from the Indianmeal moth, Plodia interpunctella.

    Science.gov (United States)

    Zhu, Y C; Kramer, K J; Dowdy, A K; Baker, J E

    2000-11-01

    -like specificity to the enzymes. Quantitative RT-PCR analyses showed that, in fourth instar larvae, RC688s had 1.6-fold higher PiT2a trypsinogen-like mRNA than did HD198r. Expression of PiT2b mRNA was 3.4-fold higher in HD198r than in RC688s. Expression of PiT2c mRNA was 2.8-fold higher in RC688s than in HD198r. Mean accumulation levels of mRNAs for all three trypsinogen-like proteins were slightly higher in RC688s than in HD198r based on total RNA, and 1.3-fold higher in RC688s than in HD198r based on wet weight of larval body tissues.

  9. 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.

  10. Prognostic Impact of mRNA Expression Levels of HER1–4 (ERBB1–4 in Patients with Locally Advanced Rectal Cancer

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    Melanie Kripp

    2016-01-01

    Full Text Available Background. No predictive or prognostic biomarker is available for patients with locally advanced rectal cancer (LARC undergoing perioperative chemoradiotherapy (CRT. Members of the human epidermal growth factor receptor (HER family of receptor tyrosine kinases EGFR (HER1, ERBB1, HER2 (ERBB2, HER3 (ERBB3, and HER4 (ERBB4 are therapeutic targets in several cancers. The analysis was performed to assess expression levels and study the potential prognostic impact for disease-free and overall survival in patients with LARC. Patients and Methods. ERBB1–4 mRNA expression and tumor proliferation using Ki-67 (MKI67 mRNA were evaluated using RT-quantitative PCR in paraffin-embedded tumor samples from 86 patients (median age: 63 treated with capecitabine or 5-fluorouracil-based CRT within a phase 3 clinical trial. Results. A positive correlation of HER4 and HER2, HER3 and HER2, and HER4 and HER3 with each other was observed. Patients with high mRNA expression of ERBB1 (EGFR, HER1 had significantly increased risk for recurrence and death. Patients with high mRNA expression of MKI67 had reduced risk for relapse. Conclusion. This analysis suggests a prognostic impact of both ERBB1 and MKi67 mRNA expression in LARC patients treated with capecitabine or fluorouracil-based chemoradiotherapy.

  11. Biofilm Formation by Bacillus subtilis Requires an Endoribonuclease-containing Multisubunit Complex that Controls mRNA Levels for the Matrix Gene Repressor SinR

    Science.gov (United States)

    DeLoughery, Aaron; Dengler, Vanina; Chai, Yunrong; Losick, Richard

    2016-01-01

    Biofilm formation by Bacillus subtilis is largely governed by a circuit in which the response regulator Spo0A turns on the gene for the anti-repressor SinI. SinI, in turn, binds to and inactivates SinR, a dedicated repressor of genes for matrix production. Mutants of the genes ylbF, ymcA, and yaaT are blocked in biofilm formation, but the mechanism by which they act has been mysterious. A recent report attributed their role in biofilm formation to stimulating Spo0A activity. However, we detect no measurable effect on the transcription of sinI. Instead, we find that the block in biofilm formation is caused by an increase in the levels of SinR and of its mRNA. Evidence is presented that YlbF, YmcA and YaaT interact with, and control the activity of, RNase Y, which is known to destabilize sinR mRNA. We show that the processing of another target of RNase Y, cggR-gapA mRNA, similarly depends on YlbF and YmcA. Our work suggests that sinR mRNA stability is an additional posttranscriptional control mechanism governing the switch to multicellularity and raises the possibility that YlbF, YmcA, and YaaT broadly regulate mRNA stability as part of an RNase Y-containing, multi-subunit complex. PMID:26434553

  12. [Effect of processed Polygonum multiflorum on mRNA expression level of five subtypes of CYP450 enzymes in rat liver].

    Science.gov (United States)

    Huang, Chun-Lian; Fan, Xue-Mei; Li, Qian; Wang, Yi-Ming; Wang, Shu-Mei; Gong, Meng-Juan; Luo, Guo-An

    2017-01-01

    To observe the effect of processed Polygonum multiflorum on mRNA expression levels of five subtypes of CYP450 enzymes in rat liver. SD rats were randomly divided into the normal control group, processed P. multiflorum high dose and low dose groups (5.40 g•kg⁻¹ and 1.08 g•kg⁻¹). The rats in administration groups were continuously given with processed P. mutiflorum for 7 days by ig administration, and the rats in normal control group were given with the same volume of distilled water. After successive administration of 7 days, the serum biochemical indications were detected, and Real-time quantitative PCR technology was used to detect the mRNA expression levels of five subtypes of CYP450 enzymes in rat liver. Experimental results showed that AST was decreased significantly in both low and high dose groups. ALT was significantly decreased in low dose group and significantly increased in high dose group. The mRNA expression levels of five subtypes of CYP450 enzymes in rat liver were decreased in high dose and low dose groups in a dose-dependent manner. Especially the high dose processed P. multiflorum could significantly inhibit CYP1A2 and CYP2E1 mRNA expression levels in rats. The study showed that high dose P. multiflorum water extract had hepatotoxicity, and the degree of liver damage was increased with the increase of dose. It shall be noted that 5.40 g•kg⁻¹ water extract of P. multiflorum could significantly inhibit CYP1A2 and CYP2E1 mRNA expression levels in the liver of rats. Copyright© by the Chinese Pharmaceutical Association.

  13. siRNA down-regulation of FGA mRNA in HepG2 cells demonstrated that heterozygous abnormality of the Aα-chain gene does not affect the plasma fibrinogen level.

    Science.gov (United States)

    Takezawa, Yuka; Matsuda, Kazuyuki; Terasawa, Fumiko; Sugano, Mitsutoshi; Honda, Takayuki; Okumura, Nobuo

    2013-04-01

    We encountered two afibrinogenemia patients with homozygous and compound heterozygous FGA mutation. Of interest, the patients' parents, who are heterozygous, had normal levels of plasma fibrinogen; thus, we hypothesized that liver FGA mRNA levels were higher than those of FGB and/or FGG mRNA. To test the hypothesis, we quantitated mRNA levels of a normal liver and a human hepatocyte cell line, HepG2 cells, and performed siRNA-mediated down-regulation of the fibrinogen gene in HepG2 cells. mRNA levels were determined using real-time quantitative RT- PCR for three normal livers and HepG2 cells. Down-regulation of FGA, FGB, or FGG in HepG2 cells was performed by the addition of siRNA corresponding to each of the three genes, and the mRNA levels determined in the cells and the secreted fibrinogen concentration in media. The mRNA level of normal human liver was FGA=FGB>FGG and the FGG mRNA level was about 2-fold lower than the others, that of HepG2 cells was FGA>FGG>FGB and FGA mRNA was approximately 2- or 4-fold higher than FGG mRNA and FGB mRNA. When FGA, FGB, or FGG mRNA expression levels were down-regulated by nearby 50%, fibrinogen concentrations in media were 78%, 49%, or 57% of the control, respectively. Our results suggest that FGG mRNA levels limit fibrinogen expression in normal liver and HepG2 cells and that 50% reduction of FGA mRNA levels would not limit fibrinogen expression in normal liver and HepG2 cells. Copyright © 2013 Elsevier Ltd. All rights reserved.

  14. 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.

  15. Marked increase of CYP24A1 mRNA level in hepatocellular carcinoma cell lines following vitamin D administration.

    Science.gov (United States)

    Horvath, Evelin; Lakatos, Péter; Balla, Bernadett; Kósa, János Pál; Tóbiás, Bálint; Jozilan, Hasan; Borka, Katalin; Horváth, Henrik Csaba; Kovalszky, Ilona; Szalay, Ferenc

    2012-11-01

    1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) inhibits cell growth and induces apoptosis in numerous types of tumors. We aimed to examine the mRNA and protein expression of 1,25(OH)(2)D(3)-inactivating CYP24A1 and mRNA expression of the activating CYP27B1 enzymes, as well as that of vitamin D receptor (VDR), in hepatocellular carcinoma (HCC) cell cultures in response to 1,25(OH)(2)D(3) administration. Increasing amounts of 1,25(OH)(2)D(3) (0.256-10 nM) were added to cultures of HepG2, Huh-Neo, Hep3B, Huh5-15 human HCC cell lines and cells then incubated for various time periods (30 min-28 h). The mRNA expression was analyzed by real time reverse transcription-polymerase chain reaction (RT-PCR). CYP24A1 protein in HepG2 cells was detected by immuncytochemistry. CYP24A1 mRNA expression significantly (pVDR gene expression in any cell cultures. Immuncytochemistry in HepG2 cells proved that gene activation was followed by CYP24A1 protein synthesis. Our novel data indicate that administration of 1,25(OH)(2)D(3) results in a marked increase of CYP24A1 mRNA expression in some, but not all, human HCC lines in vitro. These differences could be dependent upon the origin of the tumor cells.

  16. Castration decreases single cell levels of mRNA encoding glutamic acid decarboxylase in the diagonal band of broca and the sexually dimorphic nucleus of the preoptic area.

    Science.gov (United States)

    Sagrillo, C A; Selmanoff, M

    1997-09-01

    Using quantitative in situ hybridization histochemistry (ISHH), we determined the effect of castration on single cell levels of glutamic acid decarboxylase (GAD) mRNA in discrete hypothalamic regions of the male rat brain associated with the control of gonadotropin secretion. A 48-base oligodeoxynucleotide probe was used to detect with equal affinity the two isoforms of GAD message, GAD65 and GAD67. GAD message also was quantitated in a number of selected areas of the brain to contrast GAD gene expression amongst several populations of GABAergic neurons. Comparison of 11 brain regions demonstrated a 9.3-fold range in the quantity of single cell GAD mRNA with levels being highest in the amygdala and the diagonal band of Broca, moderate in the piriform cortex, caudate nucleus, substantia innominata, globus pallidus, cingulate cortex and medial septal nucleus, and lowest in the lateral septal nucleus and the medial preoptic nucleus (MPN). Castration markedly reduced single cell GAD mRNA levels in the DBB and the MPN, two discrete hypothalamic structures known to contain dendritic fields, cell bodies, and axons of GnRH neurons projecting to the median eminence. A striking finding was a dense core of steroid-sensitive GABAergic neurons within the MPN comprising the sexually dimorphic nucleus of the preoptic area (SDN-POA). Similar to the MPN as a whole, the amount of GAD mRNA expressed by cells in the SDN-POA of sham operated control rats was greater than in castrated animals. GAD mRNA levels were inversely related to serum LH titers, suggesting a role for these neurons in the mechanism controlling gonadal steroid negative feedback on LH secretion. This report provides the basis for future work to determine if GAD65, GAD67 or whether both isoforms are affected by gonadal steroid input.

  17. Developmental changes in the hypothalamic mRNA expression levels of PACAP and its receptor PAC1 and their sensitivity to fasting in male and female rats.

    Science.gov (United States)

    Iwasa, Takeshi; Matsuzaki, Toshiya; Tungalagsuvd, Altankhuu; Munkhzaya, Munkhsaikhan; Yiliyasi, Maira; Kato, Takeshi; Kuwahara, Akira; Irahara, Minoru

    2016-08-01

    The actions and responses of hypothalamic appetite regulatory and factors change markedly during the neonatal to pre-pubertal period. Pituitary adenylate cyclase-activating polypeptide (PACAP) has been found to play pivotal roles in the regulation of metabolic and nutritional status through its specific receptor PAC1. PACAP/PAC1 have anorectic roles, and their functions are regulated by leptin in adulthood. In the present study, we showed that hypothalamic PACAP mRNA expression decreases during the neonatal to pre-pubertal period (from postnatal day 10-30) in both male and female rats. During this period, hypothalamic PACAP mRNA expression was not affected by 24h fasting in either sex, while the serum leptin levels (leptin is a positive regulator of hypothalamic PACAP expression in adulthood) of both sexes were decreased by fasting. On the other hand, hypothalamic PAC1 mRNA expression did not change during the neonatal to pre-pubertal period in either sex; however, its levels were consistently higher in males than in females. Hypothalamic PAC1 mRNA expression was decreased by 24h fasting in males, but no such changes were observed in females. These results indicate while hypothalamic PACAP expression is sensitive to a negative energy state and the serum leptin level in adulthood, no such relationships are seen in the pre-pubertal period. In addition, we speculate that differences in the gonadal steroidal milieu might induce sexual dimorphism in the basal hypothalamic PAC1 mRNA level and its response to fasting. The mechanisms responsible for and the physiological effects of such changes in hypothalamic PACAP and PAC1 expression during the developmental period remain to be clarified. Copyright © 2016 ISDN. Published by Elsevier Ltd. All rights reserved.

  18. 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.

  19. CD40L and IL-4 stimulation of acute lymphoblastic leukemia cells results in upregulation of mRNA level of FLICE--an important component of apoptosis.

    Directory of Open Access Journals (Sweden)

    Radosław Jaworowski

    2007-03-01

    Full Text Available The use of cancer vaccines based on dendritic cells (DC presenting tumor antigens can be a promising tool in the treatment of leukemia. The functional characteristics of leukemia derived DC is still to be elucidated. CD40 promotes survival, proliferation and differentiation of normal B cells. CD40 triggering was used to enhance the poor antigen-presenting capacity of leukemic B-cells. Since it is still unclear whether CD40 ligation drives neoplastic B-cells to apoptosis or not, we assessed the mRNA expression of FLICE, FAS, FADD and TRADD - important components of apoptosis machinery, using real-time PCR in acute lymphoblastic leukemia cells before and after CD40 and IL-4 stimulation. ALL cells stimulated with CD40L/IL-4 expressed dendritic cell phenotype at mRNA and protein levels (upregulation of main costimulatory and adhesion molecules noted in real-time RT PCR and flow cytometry; they also expressed higher amounts of mRNA for FLICE, TRADD and FADD after CD40L/IL-4 stimulation. However differences statistically significant comparing cells cultured with CD40L/IL-4 and medium alone regarded only FLICE. Concluding, we showed upregulation of important elements of apoptosis at mRNA level in ALL cells after CD40 ligation.

  20. 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.

  1. Molecular analysis of methylmalonic acidemia: Identification of novel mutations in the methylmalonyl-CoA mutase gene with decreased level of mutant mRNA

    Energy Technology Data Exchange (ETDEWEB)

    Ogasawara, M.; Matsubara, Y.; Mikami, H.; Narisawa, K. [Tohoku Univ. School of Medicine, Sendai (Japan)

    1994-09-01

    Deficiency of methylmalonyl-CoA mutase (MCM) results in methylmalonic acidemia, which is inherited as an autosomal recessive trait and clinically characterized by metabolic ketoacidosis. Previous studies of Caucasian and African American patients identified seven MCM mutations, and we also detected four missense substitutions (Ala197Thr, Val368Asp, Arg369His and Val669Glu). However, mutations with decreased level of MCM mRNA, which accounts for at least 25% of mutations among Caucasian patients, have not been reported. Our study on eight Japanese patients indicated that 13 of 16 mutant alleles (81%) showed decreased level of MCM mRNA, suggesting that these {open_quotes}low message{close_quotes} alleles are likely to be common contributors to MCM deficiency. Reverse transcription/polymerase chain reaction (RT-PCR) of MCM mRNA followed by analysis on a fluorescent fragment analyzer indicated that the level of these mutant mRNAs was less than 1% controls. We were able to amplify such mutant mRNAs by nested PCR and directly determine the primary structure. Sequence analysis revealed three novel mutations: a G-to-T substitution at nucleotide position 425, a 2 bp deletion at nt 769 and 770, and a G-to-T substitution at nt 326. The first mutation (G425T) resulted in the substitution of a termination codon for glutamic acid at amino acid position 117. The analysis of 17 Japanese patients revealed the presence of G425T in 7 alleles (21%), suggesting a relatively high incidence of the mutation among Japanese patients. This observation is in sharp contrast to previous reports describing diverse heterogeneity of MCM mutations among Caucasians. Our report is the first to identify MCM mutations that decrease the stability of MCM mRNA. Amplification of trace amount of mRNA followed by sequencing analysis may provide useful tool for identifying such mutations.

  2. Differential dopamine-induced prolactin mRNA levels in various prolactin-secreting cell (sub)populations.

    Science.gov (United States)

    Kazemzadeh, M; Velkeniers, B; Herregodts, P; Collumbien, R; Finné, E; Derde, M P; Vanhaelst, L; Hooghe-Peters, E L

    1992-03-01

    We have examined the effects of dopamine on prolactin gene expression using quantitative in-situ hybridization histochemistry in different pituitary cell (sub)populations separated according to their density on a discontinuous Percoll gradient. Administration of dopamine resulted in a drastic reduction in hybridization of 35S-labelled DNA probe complementary to prolactin mRNA in total pituitary cells and in lactotrophs with low density. In contrast, dopamine significantly stimulated mRNA accumulation in prolactin-secreting cells with high density compared with other cell layers. The combined use of Percoll gradient and quantitative in-situ hybridization is a valuable and sensitive method with which to examine prolactin-secreting cell response to a given stimulation. Prolactin-secreting cells with high and low density clearly show functional heterogeneity in their response to dopamine.

  3. 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

    power output was the same in AL and L, but heart rate at the end of each exercise interval was higher in AL compared with L. One minute after exercise, arm venous blood lactate was higher in AL than in L. A higher level of blood epinephrine and norepinephrine was evident 3 min after exercise in AL...... compared with L. Nevertheless, none of the exercise-induced increases in a1, a2, ß1, and ß3 mRNA expression levels were higher in AL compared with L. The most abundant Na+-K+-ATPase subunit at the mRNA level was ß1, which was expressed 3.4 times than a2. Expression of a1, ß2, and ß3 was less than 5...

  4. Sublethal effects of chlorantraniliprole on juvenile hormone levels and mRNA expression of JHAMT and FPPS genes in the rice stem borer, Chilo suppressalis.

    Science.gov (United States)

    Xu, Beibei; Qian, Kun; Zhang, Nan; Miao, Lijun; Cai, Jingxuan; Lu, Mingxing; Du, Yuzhou; Wang, Jianjun

    2017-10-01

    Juvenile hormone (JH) regulates the development and reproduction of insects. The sublethal effects of chlorantraniliprole on JH levels and mRNA expression of JH acid methyltransferase gene (CsJHAMT) and farnesyl diphosphate synthase genes (CsFPPS1 and CsFPPS2) in Chilo suppressalis (Walker) were investigated. Exposure of sublethal concentrations of chlorantraniliprole (LC10 and LC30 ) to the third instar larvae of C. suppressalis significantly increased the JH levels in all developmental stages investigated including larvae 72 h after treatment, the first, third and fifth day of female pupae, as well as newly emerged, 12-h-old and 24-h-old female adults. A general trend of increased mRNA expression levels of CsJHAMT, CsFPPS1and CsFPPS2 was also observed in LC10 and LC30 treatment groups. Notably, the mRNA expression level of CsJHAMT significantly increased by 7.46-fold in the larvae 72 h after LC30 treatment. A significant increase of the mRNA expression levels of CsFPPS2 was also observed in the fifth day female pupae of LC10 and LC30 treatment groups (2.60-fold and 2.62-fold, respectively) as well as in 12-h-old female adults of the LC30 treatment group (3.45-fold). Sublethal concentrations of chlorantraniliprole might upregulate the expression of JH biosynthesis genes and in turn result in an increase of JH level in C. suppressalis. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  5. Cytokine mRNA expressions after racing at a high altitude and at sea level in horses with exercise-induced pulmonary hemorrhage.

    Science.gov (United States)

    Saulez, Montague N; Godfroid, Jacques; Bosman, Anamarie; Stiltner, Jackie L; Breathnach, Cormac C; Horohov, David W

    2010-04-01

    To determine concentrations of cytokine mRNA in horses with exercise-induced pulmonary hemorrhage (EIPH) after racing. 97 Thoroughbreds. Following tracheobronchoscopy, the severity of EIPH was graded (scale of 0 to 4), and venous blood samples were collected from 10 horses in each grade. After RNA isolation and cDNA synthesis, real-time PCR assay was conducted to detect cytokinespecific mRNA for interleukin (IL)-1, IL-6, and IL-10; interferon (INF)-gamma; and tumor necrosis factor (TNF)-alpha. Neither location nor grade of EIPH affected the expression of IL-1 and INF-gamma. There was significantly greater overall expression of IL-6 mRNA at sea level, with significantly more IL-6 expressed in horses with grade 4 EIPH than in horses with grade 0, 1, or 2 EIPH. At a high altitude, no difference was detected for IL-6 expression among the various EIPH grades. There was significantly greater overall expression of TNF-alpha mRNA at a high altitude; however, there was no difference within the various grades of EIPH. Expression of IL-10 was significantly affected by grade of EIPH because horses with grade 3 EIPH expressed significantly more IL-10 mRNA than did horses with grade 0 or 2 EIPH; this expression was not affected by location. At sea level, increased IL-6 expression was associated with more severe EIPH, and altitude may affect gene expressions of the proinflammatory cytokine TNF-alpha and anti-inflammatory cytokine IL-6. Studies on protein concentrations of cytokine expression are needed. The pathophysiologic importance of these findings remains to be explained.

  6. 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.

  7. The Co-Induced Effects of Molybdenum and Cadmium on the Trace Elements and the mRNA Expression Levels of CP and MT in Duck Testicles.

    Science.gov (United States)

    Xia, Bing; Chen, Hua; Hu, Guoliang; Wang, Liqi; Cao, Huabin; Zhang, Caiying

    2016-02-01

    To investigate the chronic toxicity of molybdenum (Mo) and cadmium (Cd) on the trace elements and the mRNA expression levels of ceruloplasmin (CP) and metallothionein (MT) in duck testicles, 120 healthy 11-day-old male ducks were randomly divided into six groups with 20 ducks in each group. Ducks were treated with the diet containing different dosages of Mo or Cd. The source of Mo and Cd was hexaammonium molybdate ([(NH4)6Mo7O24·4H2O]) and cadmium sulfate (3CdSO4·8H2O), respectively, in this study. After being treated for 60 and 120 days, ten male birds in each group were randomly selected and euthanized and then testicles were aseptically collected for determining the mRNA expression levels of MT and CP, antioxidant indexes, and contents of trace elements in the testicle. In addition, testicle tissues at 120 days were subjected to histopathological analysis with the optical microscope. The results showed that co-exposure to Mo and Cd resulted in an increase in malondialdehyde (MDA) level while decrease in xanthine oxidase (XOD) and catalase (CAT) activities. The mRNA expression level of MT gene was upregulated while CP was decreased in combination groups. Contents of Mo, copper (Cu), iron (Fe), and zinc (Zn) decreased in combined groups while Cd increased in Cd and combined groups at 120 days. Furthermore, severe congestion, low sperm count, and malformation were observed in low dietary of Mo combined with Cd group and high dietary of Mo combined with Cd group. Our results suggested that Mo and Cd might aggravate testicular degeneration synergistically through altering the mRNA expression levels of MT and CP, increasing lipid peroxidation through inhibiting related enzyme activities and disturbing homeostasis of trace elements in testicles. Interaction of Mo and Cd may have a synergistic effect on the testicular toxicity.

  8. High levels of MMP-2, MMP-9, MT1-MMP and TIMP-2 mRNA correlate with poor survival in ovarian carcinoma.

    Science.gov (United States)

    Davidson, B; Goldberg, I; Gotlieb, W H; Kopolovic, J; Ben-Baruch, G; Nesland, J M; Berner, A; Bryne, M; Reich, R

    1999-01-01

    The object of this study was to analyze the potential association between the expression of MMP-2, MMP-9, MT1-MMP and TIMP-2, and disease outcome in advanced-stage ovarian carcinomas. Sections from 70 paraffin-embedded blocks (36 primary ovarian carcinomas and 34 metastatic lesions) from 45 patients diagnosed with advanced stage ovarian carcinomas (FIGO stages III-IV) were studied using mRNA in situ hybridization (ISH) technique. Patients were divided retrospectively in two groups based on disease outcome. Long-term survivors (21 patients) and short-term survivors (24 patients) were defined using a double cut-off of 36 months for disease-free survival (DFS) and 60 months for overall survival (OS). Mean follow-up period for patients that were diagnosed with advanced-stage carcinoma was 70 months. The mean values for DFS and OS were 109 and 125 months for long-term survivors, as compared to 3 and 21 months for short-term survivors, respectively. Intense mRNA signals were detected more frequently in tumor cells of short-term survivors with use of all four probes. Comparable findings were observed in peritumoral stromal cells with ISH for MMP-2, MMP-9 and TIMP-2 mRNA. Notably, primary tumors with intense mRNA signal for TIMP-2 (No = 14) were uniformly associated with a fatal outcome. In univariate analysis of primary tumors, mRNA levels of TIMP-2 in stromal cells (P = 0.0002), as well as for MMP-9 (P = 0.012) and TIMP-2 (P = 0.02) in tumor cells, correlated with poor outcome. In univariate analysis of metastatic lesions, mRNA levels of TIMP-2 in stromal cells (P = 0.031), as well as for MMP-2 (P = 0.027) and MT1-MMP (P = 0.008) in tumor cells, correlated with poor outcome. Interestingly, the presence of MT1-MMP in stromal cells correlated with longer survival (P = 0.025). In a multivariate analysis of ISH results for primary tumors, TIMP-2 levels in stromal cells (P = 0.006) and MMP-9 levels in tumor cells (P = 0.011) retained their predictive value. We conclude that

  9. Demonstration of elevated type I and type III procollagen mRNA levels in cutaneous wounds treated with helium-neon laser. Proposed mechanism for enhanced wound healing.

    Science.gov (United States)

    Saperia, D; Glassberg, E; Lyons, R F; Abergel, R P; Baneux, P; Castel, J C; Dwyer, R M; Uitto, J

    1986-08-14

    To assess laser modulation of wound healing, full-thickness cutaneous wounds were produced in the backs of pigs, and subjected to treatment with helium-neon laser. For comparison, some wounds were treated with non-laser energy source (a tungsten light) or left untreated as controls. Type I and type III procollagen mRNA levels were determined in the wounds by molecular hybridization with cDNA probes. The results indicated that type I and type III mRNA levels were markedly increased at days 17 and 28 of the healing in wounds treated with He-Ne laser, when compared to control or tungsten light-treated wounds. The results suggest that helium-neon laser stimulates wound healing by enhancing procollagen gene expression. These observations may have relevance to previous clinical studies suggesting that helium-neon laser stimulates wound healing.

  10. Effects of sodium saccharin and linoleic acid on mRNA levels of Her2/neu and p53 in a human breast epithelial cell line.

    Science.gov (United States)

    Ogretmen, B; Ratajczak, H; Gendel, S M; Stark, B C

    1996-04-19

    The effects of two food-related chemicals (sodium saccharin and linoleic acid) on the levels of Her2/neu and p53 mRNA in a non-cancerous human breast epithelial cell line (HBL-100) were tested in comparison with the effects of the known tumor promoter phorbol 12-myristate 13-acetate (TPA). Treatments were made both with and without prior treatment with two well-known tumor initiators, N-nitroso-N-methylurea (NMU) or 7,12-dimethylbenz[a]anthracene (DMBA). The effects in general were small, the greatest being increases of 46-67% in Her2/neu mRNA levels in response to treatments with TPA or sodium saccharin following NMU treatments. These results demonstrate that sodium saccharin following NMU treatments might be involved in transcriptional regulation of Her2/neu in HBL-100 cells and suggest that its effects may not be limited to urinary bladder.

  11. 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

  12. The Allergic Airway Inflammation Repository - a user-friendly, curated resource of mRNA expression levels in studies of allergic airways

    OpenAIRE

    Gawel, Danuta; James, A. Rani; Benson, Mikael; Liljenstrom, R.; Muraro, A.; Nestor, Colm; Zhang, Huan; Gustafsson, Mika

    2014-01-01

    Public microarray databases allow analysis of expression levels of candidate genes in different contexts. However, finding relevant microarray data is complicated by the large number of available studies. We have compiled a user-friendly, open-access database of mRNA microarray experiments relevant to allergic airway inflammation, the Allergic Airway Inflammation Repository (AAIR, http://aair.cimed.ike.liu.se/). The aim is to allow allergy researchers to determine the expression profile of th...

  13. Effects of bamboo vinegar powder on growth performance and mRNA expression levels of interleukin-10, interleukin-22, and interleukin-25 in immune organs of weaned piglets

    Directory of Open Access Journals (Sweden)

    Yongjiu Huo

    2016-06-01

    Full Text Available The aim of this study was to explore the effects of bamboo vinegar powder on growth performance, diarrhea situation and mRNA expression levels of cytokines i.e., interleukin-10 (IL-10, interleukin-22 (IL-22, and interleukin-25 (IL-25 in immune organs of weaned piglets, and to accumulate theoretical data for the application of bamboo vinegar powder in weaned piglet production. Forty-five crossbred (Duroc × Landrace × Yorkshire, all male weaned piglets with similar body weight (6.74 ± 0.17 kg at 31 days of age were randomly assigned to 5 treatments with 3 replicates per treatment and 3 piglets in each replicate. The five treatments were as follows: CON (a basal diet, ANT (the basal diet + 0.12% antibiotics, BV1 (the basal diet + 0.1% bamboo vinegar powder, BV5 (the basal diet + 0.5% bamboo vinegar powder, BV10 (the basal diet + 1.0% bamboo vinegar powder. This experiment lasted 35 days. The growth performance and diarrhea situation were recorded. The relative mRNA expression levels of IL-10, IL-22 and IL-25 in liver, spleen, duodenum and mesenteric lymph nodes were detected by real-time PCR. Feed: gain of BV5 was significantly lower than that of CON (P < 0.05. In comparison with CON, diarrhea rate and diarrhea index of BV1 and BV5 all tended to decrease (P < 0.1. Compared with CON, mRNA expression level of IL-10 in liver of ANT tended to be lower (P < 0.1 and these of BV1, BV5 and BV10 were significantly reduced (P < 0.05. The mRNA expression levels of IL-10 in duodenum of ANT, BV1, BV5 and BV10 were all lower than those of CON, of which BV10 had significantly decreased IL-10 mRNA expression in duodenum (P < 0.05. The mRNA expression levels of IL-22 in duodenum of ANT, BV1, BV5 and BV10 all tended to be inhibited compared with CON (P < 0.1. With the increase of bamboo vinegar powder dosage, mRNA expression levels of IL-25 in spleen and mesenteric lymph nodes of BV1, BV5 and BV10 tended to be up-regulated. Overall

  14. 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-s...... was about 5-fold at 4% oxygen. No expression at atmospheric oxygen or in response to low oxygen was observed when using cDNA probes for other nodulin genes such as leghemoglobin c3, nodulin-22 and nodulin-44. Udgivelsesdato: 1991-May...

  15. Developmental changes in the hypothalamic mRNA expression levels of brain-derived neurotrophic factor and serum leptin levels: Their responses to fasting in male and female rats.

    Science.gov (United States)

    Iwasa, Takeshi; Matsuzaki, Toshiya; Yano, Kiyohito; Munkhzaya, Munkhsaikhan; Tungalagsuvd, Altankhuu; Yiliyasi, Maira; Kuwahara, Akira; Irahara, Minoru

    2016-11-01

    The actions and responses of hypothalamic appetite regulatory factors change markedly during the neonatal to pre-pubertal period in order to maintain appropriate metabolic and nutritional conditions. In this study, we examined the developmental changes in the hypothalamic mRNA levels of brain-derived neurotrophic factor (BDNF), which is a potent anorectic factor and the changes in the sensitivity of the hypothalamic expression of this factor to fasting during the neonatal to pre-pubertal period. Under fed conditions, hypothalamic BDNF mRNA expression decreased during development in both male and female rats. Similarly, the serum levels of leptin, which is a positive regulator of hypothalamic BDNF expression, also tended to fall during the developmental period. The serum leptin level and the hypothalamic BDNF mRNA level were found to be positively correlated in both sexes under the fed conditions. Hypothalamic BDNF mRNA expression was decreased by 24h fasting (separating the rats from their mothers) in the early neonatal period (postnatal day 10) in both males and females, but no such changes were seen at postnatal day 20. Twenty-four hours' fasting (food deprivation) did not affect hypothalamic BDNF mRNA expression in the pre-pubertal period (postnatal day 30). On the other hand, the rats' serum leptin levels were decreased by 24h fasting (separating the rats from their mothers at postnatal day 10 and 20, and food deprivation at postnatal day 30) throughout the early neonatal to pre-pubertal period. The correlation between serum leptin and hypothalamic BDNF mRNA levels was not significant under the fasted conditions. It can be speculated that leptin partially regulates hypothalamic BDNF mRNA levels, but only in fed conditions. Such changes in hypothalamic BDNF expression might play a role in maintaining appropriate metabolic and nutritional conditions and promoting normal physical development. In addition, because maternal separation induces a negative energy

  16. Effects of ischemic preconditioning on myocardium Caspase-3, SOCS-1, SOCS-3, TNF-α and IL-6 mRNA expression levels in myocardium IR rats.

    Science.gov (United States)

    Ma, Jiangwei; Qiao, Zengyong; Xu, Biao

    2013-10-01

    The aim of this study was to characterise the effects of ischemic preconditioning (IP) on heart function parameters (ΔST and ΔT), activities of serum creatine kinase (CK), lactate dehydrogenase (LDH), and levels of serum nitric oxide (NO), malondialdehyde (MDA), and myocardium Caspase-3 mRNA, SOCS-1, SOCS-3, tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) expression levels and Apoptosis index in myocardium IR rats. Results showed that ΔST and ΔST values in IP group were markedly lower than those in IR group. Compared with IR group, IP significantly (p myocardium Caspase-3 mRNA (0.303 ± 0.021 vs 0.515 ± 0.022) gene expression (p myocardium SOCS-1 (0.241 ± 0.031 vs 0.596 ± 0.036), SOCS-3 (0.258 ± 0.031 vs 0.713 ± 0.057), TNF-α (0.137 ± 0.011 vs 0.427 ± 0.035) and IL-6 (0.314 ± 0.021 vs 0.719 ± 0.064) mRNA gene expression (p < 0.01) compared to IR model group. We conclude that IP is effective in the therapy of heart disease. These findings may have implications for the clinical development of preconditioning-based therapies for ischemic heart disease.

  17. Hepatic mRNA expression and plasma levels of insulin-like growth factor-I (IGF-I in broiler chickens selected for different growth rates

    Directory of Open Access Journals (Sweden)

    Poliana Fernanda Giachetto

    2004-01-01

    Full Text Available The hepatic expression and plasma concentrations of IGF-I were investigated in three broiler chicken strains selected for different growth rates (HP-Hubbard-Pettersen, a fast growing strain; NN-Naked-neck, a strain with an intermediate growth rate and a heterozygous genotype, and C-Caipira, a slow growing crossbred strain. The chickens were studied at 1, 21 and 42 days of age and had free access to food throughout the study. Hepatic IGF-I mRNA expression was assessed by dot blot analysis using a randomly labeled chicken IGF-I cDNA as the probe and plasma IGF-I concentrations were assayed by radioimmunoassay. The hepatic levels of IGF-I mRNA increased from 1 to 21 days of age in all strains, with NN chickens showing a higher (p < 0.05 IGF-I expression than the other strains. Plasma IGF-I concentrations increased (p < 0.05 with broiler chicken age, but there were no significant differences among the strains. These results indicate that despite differences in the growth rates among the strains, the changes in the expression of IGF-I mRNA in liver and in the plasma levels of IGF-I were independent of broiler chicken strain, but varied with chicken age.

  18. Characterization of a chitin synthase cDNA and its increased mRNA level associated with decreased chitin synthesis in Anopheles quadrimaculatus exposed to diflubenzuron.

    Science.gov (United States)

    Zhang, Jianzhen; Zhu, Kun Yan

    2006-09-01

    Chitin synthase (EC 2.4.1.16) is a crucial enzyme responsible for chitin biosynthesis in all chitin-containing organisms. This paper reports a complete cDNA encoding chitin synthase 1 (AqCHS1), change of AqCHS1 mRNA level in response to diflubenzuron exposure, and concentration-dependent effect of diflubenzuron on chitin synthesis in the common malaria mosquito (Anopheles quadrimaculatus). The cDNA consists of 5723 nucleotides, including an open reading frame (ORF) of 4734 nucleotides that encode 1578 amino acid residues and a non-translated region of 989 nucleotides. The deduced amino acid sequence contains all the chitin synthase signature motifs (EDR, QRRRW and SWGTR) and shows 97% identity to that of An. gambiae (AgCHS1, XM_321337). Northern blot and real-time quantitative PCR analyses revealed a significant increase of AqCHS1 mRNA level in the larvae exposed to diflubenzuron at 100 and 500 microg/L. As confirmed by real-time quantitative PCR, AqCHS1 mRNA level was enhanced by 2-fold in the larvae exposed to diflubenzuron at 500 microg/L for 24 h. In contrast, exposures of the larvae to diflubenzuron at 4.0, 20, 100 and 500 microg/L for 48 h resulted in decreases of chitin content by 9.0%, 43%, 58% and 76%, respectively. Significantly increased AqCHS1 mRNA level associated with decreased chitin synthesis may imply possible inhibition of chitin synthase, or abnormal chitin synthase translocation or chitin microfibril assembly conferred by diflubenzuron. Increased AqCHS1 expression due to increased transcription and/or increased mRNA stability may serve as a feedback mechanism to compensate such an effect in the mosquitoes. Further studies are necessary to elucidate the relationship between reduced chitin synthesis and increased expression of AqCHS1 in order to shed new light on trafficking and regulation of chitin biosynthesis in the mosquito affected by diflubenzuron.

  19. 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.

  20. Low-level light-emitting diode therapy increases mRNA expressions of IL-10 and type I and III collagens on Achilles tendinitis in rats.

    Science.gov (United States)

    Xavier, Murilo; de Souza, Renato Aparecido; Pires, Viviane Araújo; Santos, Ana Paula; Aimbire, Flávio; Silva, José Antônio; Albertini, Regiane; Villaverde, Antonio Balbin

    2014-01-01

    The present study investigated the effects of low-level light-emitting diode (LED) therapy (880 ± 10 nm) on interleukin (IL)-10 and type I and III collagen in an experimental model of Achilles tendinitis. Thirty male Wistar rats were separated into six groups (n = 5), three groups in the experimental period of 7 days, control group, tendinitis-induced group, and LED therapy group, and three groups in the experimental period of 14 days, tendinitis group, LED therapy group, and LED group with the therapy starting at the 7th day after tendinitis induction (LEDT delay). Tendinitis was induced in the right Achilles tendon using an intratendinous injection of 100 μL of collagenase. The LED parameters were: optical power of 22 mW, spot area size of 0.5 cm(2), and irradiation time of 170 s, corresponding to 7.5 J/cm(2) of energy density. The therapy was initiated 12 h after the tendinitis induction, with a 48-h interval between irradiations. The IL-10 and type I and III collagen mRNA expression were evaluated by real-time polymerase chain reaction at the 7th and 14th days after tendinitis induction. The results showed that LED irradiation increased IL-10 (p < 0.001) in treated group on 7-day experimental period and increased type I and III collagen mRNA expression in both treated groups of 7- and 14-day experimental periods (p < 0.05), except by type I collagen mRNA expression in LEDT delay group. LED (880 nm) was effective in increasing mRNA expression of IL-10 and type I and III collagen. Therefore, LED therapy may have potentially therapeutic effects on Achilles tendon injuries.

  1. Interleukin-8 and vascular endothelial growth factor mRNA and protein levels are down-regulated in ovarian carcinoma cells in serous effusions.

    Science.gov (United States)

    Davidson, Ben; Reich, Reuven; Kopolovic, Juri; Berner, Aasmund; Nesland, Jahn M; Kristensen, Gunnar B; Tropé, Claes G; Bryne, Magne; Risberg, Bjørn; van de Putte, Gregg; Goldberg, Iris

    2002-01-01

    Angiogenic factors are involved in tumor growth and spread. The aim of this study was to evaluate the expression of angiogenesis-related genes in malignant serous effusions of patients with advanced-stage (FIGO stage III and IV) ovarian carcinoma. In addition, to compare the results for carcinoma cells in effusions with corresponding primary tumors and metastatic lesions, and analyze their prognostic role. Sections from 66 effusions and 90 primary and metastatic lesions from 62 ovarian and primary peritoneal carcinoma patients, were evaluated for expression of basic fibroblast factor (bFGF), interleukin-8 (IL-8), and vascular endothelial growth factor (VEGF) using mRNA in situ hybridization (ISH). Protein expression was evaluated in a subset of specimens using immunohistochemistry (IHC). ISH results were correlated with clinical parameters. In both effusions and solid tumors, bFGF mRNA was the most commonly expressed factor (93% of effusions and 95% of solid tumors) followed by IL-8, while VEGF was expressed in a minority of the specimens (P 0.05). Peritoneal and pleural effusions showed similar expression patterns. In conclusion, bFGF is the major angiogenic factor expressed in ovarian carcinoma at the mRNA level. It is highly expressed in both solid tumors and serous effusions, while IL-8 and VEGF are down regulated in carcinoma cells in effusions, possibly due to the lack of interaction with stromal cells. mRNA expression of VEGF, bFGF, and IL-8 does not appear to be a predictor of disease outcome in advanced-stage ovarian carcinoma. Carcinoma cells in pleural and peritoneal effusions show a similar metastatic expression profile, in agreement with our previous findings, supporting the true metastatic nature of ovarian carcinoma cells in ascites.

  2. The relative expression levels of insulin-like growth factor 1 and myostatin mRNA in the asynchronous development of skeletal muscle in ducks during early development.

    Science.gov (United States)

    Hu, Yan; Liu, Hongxiang; Shan, Yanju; Ji, Gaige; Xu, Wenjuan; Shu, Jingting; Li, Huifang

    2015-08-10

    Genetic selection is a powerful tool for modifying poultry muscle yield. Insulin-like growth factor I (IGF-I) and myostatin (MSTN) are important regulators of muscle growth, especially in the myogenesis stage. This study compared the developmental pattern of the pectoralis major (PM) and lateral gastrocnemius (LM) muscles, mRNA expression characterization of IGF-I and MSTN-A and their correlation between 14 days in ovo and 1 week post-hatch in two Chinese local duck breeds. During early development, the growth of duck PM and LM followed an asynchronous pattern. Variations in PM growth rate observed with development followed the relative variations of MSTN and IGF-I expression; however, the same behavior was not observed in LM. Moreover, the profile of IGF-I expression in duck skeletal muscles indicated that genetic selection for high meat-yield poultry has altered the temporal expression of IGF-I and affected cellular characteristics and mass by hatch in a PM-specific manner. The MSTN-A expression profile showed synchronization with the growth of skeletal muscle and peaks of myofiber proliferation. The expression patterns of IGF-I and MSTN suggest that duck pectoralis fibers are prioritized for proliferation in embryogenesis. The IGF-1/MSTN-A mRNA ratios in PM and LM presented very similar trends in the changes of myofiber characteristics, and differences in the IGF-1/MSTN-A mRNA ratio in PM between the two breeds corresponded to the timing of differences in PM mass between the varieties. Our results support the hypothesis that relative levels of IGF-I and MSTN mRNA may participate in ordering muscle growth rates with selected development. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Gender differences in tryptophan hydroxylase-2 mRNA, serotonin, and 5-hydroxytryptophan levels in the brain of catfish, Clarias gariepinus, during sex differentiation.

    Science.gov (United States)

    Raghuveer, K; Sudhakumari, C C; Senthilkumaran, B; Kagawa, H; Dutta-Gupta, A; Nagahama, Y

    2011-03-01

    Tryptophan hydroxylase (tph) is the key regulator in serotonin (5-HT) biosynthesis that stimulates the release of GnRH and gonadotropins by acting at the level of hypothalamo-hypophyseal axis. In brain, 5-HT is expressed predominantly in preoptic area-hypothalamus (POA-HYP) region in teleosts. Therefore, in the present study we isolated tph2 from catfish brain to evaluate its expression pattern in male and female brains during early development. Tph2 cloned from catfish brain is 2.768 Kb in length which encodes predicted protein of 488 amino acid residues. The characterization of recombinant tph2 was done by transient transfection in CHO cells. Tissue distribution of tph2 revealed ubiquitous expression except ovary. Real time PCR analysis in discrete regions of adult male brain revealed that tph2 mRNA was abundant in the POA-HYP and optic tectum+cerebellum+thalamus (OCT) regions. Differential expression of tph2 was observed at mRNA and protein levels in the POA-HYP and OCT regions of male and female brains during development that further correlate with the 5-hydroxytryptophan (5-HTP) and 5-HT levels measured using HPLC method in these regions of male and female brains. Tph2 immunoreactive neurons were observed in different regions of brain at 50 days post hatch using catfish specific tph2 antibody. Changes in tph2 mRNA expression, 5-HTP, and 5-HT levels in the POA-HYP+OCT region of brains of methyltestosterone and para-chlorophenylalanine treated fishes during development further endorse our results. Based on our results, we propose that the serotonergic system is involved in brain sex differentiation in teleosts. Copyright © 2010 Elsevier Inc. All rights reserved.

  4. Anguillicola crassus infection significantly affects the silvering related modifications in steady state mRNA levels in gas gland tissue of the European eel

    Directory of Open Access Journals (Sweden)

    Bernd ePelster

    2016-05-01

    Full Text Available Using Illumina sequencing, transcriptional changes occurring during silvering in swimbladder tissue of the European eel have been analyzed by comparison of yellow eel and silver eel tissue samples. Functional annotation analysis based on GO terms revealed significant expression changes in a number of genes related to the extracellular matrix, important for the control of gas permeability of the swimbladder, and to ROS (reactive oxygen species defense, important to cope with reactive oxygen species generated under hyperbaric oxygen partial pressures. Focusing on swimbladder tissue metabolism, levels of several mRNA species encoding glucose transport proteins were several-fold higher in silver eels, while enzymes of the glycolytic pathway were not affected. The significantly higher steady state level of a transcript encoding for membrane bound carbonic anhydrase, however, suggested that CO2 production in the pentose phosphate shunt and diffusion of CO2 was of particular importance in silver eel swimbladder. In addition, the mRNA level of a large number of genes related to immune response and to sexual maturation was significantly modified in the silver eel swimbladder. The modification of several processes related to protein metabolism and transport, cell cycle, and apoptosis suggested that these changes in swimbladder metabolism and permeability were achieved by increasing cell turn-over. The impact of an infection of the swimbladder with the nematode Anguillicola crassus has been assessed by comparing these expression changes with expression changes observed between uninfected yellow eel swimbladder tissue and infected silver eel swimbladder tissue. In contrast to uninfected silver eel swimbladder tissue, in infected tissue the mRNA level of several glycolytic enzymes was significantly elevated, and with respect to extracellular matrix, several mucin genes were many-fold higher in their mRNA level. Modification of many immune related genes and

  5. A novel long non-coding RNA in the rheumatoid arthritis risk locus TRAF1-C5 influences C5 mRNA levels.

    Science.gov (United States)

    Messemaker, T C; Frank-Bertoncelj, M; Marques, R B; Adriaans, A; Bakker, A M; Daha, N; Gay, S; Huizinga, T W; Toes, R E M; Mikkers, H M M; Kurreeman, F

    2016-03-01

    Long non-coding RNAs (lncRNAs) can regulate the transcript levels of genes in the same genomic region. These locally acting lncRNAs have been found deregulated in human disease and some have been shown to harbour quantitative trait loci (eQTLs) in autoimmune diseases. However, lncRNAs linked to the transcription of candidate risk genes in loci associated to rheumatoid arthritis (RA) have not yet been identified. The TRAF1 and C5 risk locus shows evidence of multiple eQTLs and transcription of intergenic non-coding sequences. Here, we identified a non-coding transcript (C5T1lncRNA) starting in the 3' untranslated region (UTR) of C5. RA-relevant cell types express C5T1lncRNA and RNA levels are further enhanced by specific immune stimuli. C5T1lncRNA is expressed predominantly in the nucleus and its expression correlates positively with C5 mRNA in various tissues (P=0.001) and in peripheral blood mononuclear cells (P=0.02) indicating transcriptional co-regulation. Knockdown results in a concurrent decrease in C5 mRNA levels but not of other neighbouring genes. Overall, our data show the identification of a novel lncRNA C5T1lncRNA that is fully located in the associated region and influences transcript levels of C5, a gene previously linked to RA pathogenesis.

  6. Clinical Usefulness of Monitoring Expression Levels of CCL24 (Eotaxin-2) mRNA on the Ocular Surface in Patients with Vernal Keratoconjunctivitis and Atopic Keratoconjunctivitis

    OpenAIRE

    Yukiko Shiraki; Jun Shoji; Noriko Inada

    2016-01-01

    Purpose. This study aimed to evaluate the clinical efficacy of using expression levels of CCL24 (eotaxin-2) mRNA on the ocular surface as a biomarker in patients with vernal keratoconjunctivitis (VKC) and atopic keratoconjunctivitis (AKC). Methods. Eighteen patients with VKC or AKC (VKC/AKC group) and 12 control subjects (control group) were enrolled in this study. The VKC/AKC clinical score was determined by objective findings in patients by using the 5-5-5 exacerbation grading scale. All su...

  7. Elevated alpha-synuclein mRNA levels in individual UV-laser-microdissected dopaminergic substantia nigra neurons in idiopathic Parkinson's disease.

    Science.gov (United States)

    Gründemann, Jan; Schlaudraff, Falk; Haeckel, Olga; Liss, Birgit

    2008-04-01

    The presynaptic protein alpha-synuclein is involved in several neurodegenerative diseases, including Parkinson's disease (PD). In rare familial forms of PD, causal mutations (PARK1) as well as multiplications (PARK4) of the alpha-synuclein gene have been identified. In sporadic, idiopathic PD, abnormal accumulation and deposition of alpha-synuclein might also cause degeneration of dopaminergic midbrain neurons, the clinically most relevant neuronal population in PD. Thus, cell-specific quantification of alpha-synuclein expression-levels in dopaminergic neurons from idiopathic PD patients in comparison to controls would provide essential information about contributions of alpha-synuclein to the etiology of PD. However, a number of previous studies addressing this question at the tissue-level yielded varying results regarding alpha-synuclein expression. To increase specificity, we developed a cell-specific approach for mRNA quantification that also took into account the important issue of variable RNA integrities of the individual human postmortem brain samples. We demonstrate that PCR -amplicon size can confound quantitative gene-expression analysis, in particular of partly degraded RNA. By combining optimized UV-laser microdissection- and quantitative RT-PCR-techniques with suitable PCR assays, we detected significantly elevated alpha-synuclein mRNA levels in individual, surviving neuromelanin- and tyrosine hydroxylase-positive substantia nigra dopaminergic neurons from idiopathic PD brains compared to controls. These results strengthen the pathophysiologic role of transcriptional dysregulation of the alpha-synuclein gene in sporadic PD.

  8. Elevated α-synuclein mRNA levels in individual UV-laser-microdissected dopaminergic substantia nigra neurons in idiopathic Parkinson's disease

    Science.gov (United States)

    Gründemann, Jan; Schlaudraff, Falk; Haeckel, Olga; Liss, Birgit

    2008-01-01

    The presynaptic protein α-synuclein is involved in several neurodegenerative diseases, including Parkinson's disease (PD). In rare familial forms of PD, causal mutations (PARK1) as well as multiplications (PARK4) of the α-synuclein gene have been identified. In sporadic, idiopathic PD, abnormal accumulation and deposition of α-synuclein might also cause degeneration of dopaminergic midbrain neurons, the clinically most relevant neuronal population in PD. Thus, cell-specific quantification of α-synuclein expression-levels in dopaminergic neurons from idiopathic PD patients in comparison to controls would provide essential information about contributions of α-synuclein to the etiology of PD. However, a number of previous studies addressing this question at the tissue-level yielded varying results regarding α-synuclein expression. To increase specificity, we developed a cell-specific approach for mRNA quantification that also took into account the important issue of variable RNA integrities of the individual human postmortem brain samples. We demonstrate that PCR –amplicon size can confound quantitative gene-expression analysis, in particular of partly degraded RNA. By combining optimized UV-laser microdissection- and quantitative RT–PCR-techniques with suitable PCR assays, we detected significantly elevated α-synuclein mRNA levels in individual, surviving neuromelanin- and tyrosine hydroxylase-positive substantia nigra dopaminergic neurons from idiopathic PD brains compared to controls. These results strengthen the pathophysiologic role of transcriptional dysregulation of the α-synuclein gene in sporadic PD. PMID:18332041

  9. 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.

  10. Irbesartan inhibits advanced glycation end product (AGE)-induced up-regulation of vascular cell adhesion molecule-1 (VCAM-1) mRNA levels in glomerular endothelial cells.

    Science.gov (United States)

    Matsui, Takanori; Nishino, Yuri; Maeda, Sayaka; Takeuchi, Masayoshi; Yamagishi, Sho-ichi

    2011-05-01

    Renin-angiotensin system (RAS) plays a central role in the development and progression of diabetic nephropathy. There is a growing body of evidence that advanced glycation end products (AGE) and inflammation contribute to diabetic nephropathy as well. However, the pathophysiological crosstalk between the RAS and AGE in inflammatory reactions in glomerular endothelial cells (ECs) remains unknown. In this study, we examined whether and how irbesartan, an angiotensin II type 1 receptor blocker (ARB), inhibited the AGE-induced vascular cell adhesion molecule-1 (VCAM-1) gene expression in cultured human glomerular ECs. Irbesartan or an anti-oxidant N-acetylcysteine inhibited the AGE-induced increase in reactive oxygen species (ROS) generation and subsequently blocked up-regulation of VCAM-1 mRNA levels in glomerular ECs. AGE significantly stimulated angiotensin II production by glomerular ECs. Furthermore, irbesartan completely suppressed up-regulation of VCAM-1 mRNA levels in AGE plus angiotensin II-exposed glomerular ECs. Our present data suggest that there exists a crosstalk between the RAS and AGE in inflammatory reactions in glomerular ECs. Irbesartan may play a protective role against diabetic nephropathy by blocking the deleterious effects of AGE-elicited angiotensin II and ROS. Copyright © 2011 Elsevier Inc. All rights reserved.

  11. Nerve Growth Factor Increases mRNA Levels for the Prion Protein and the β -amyloid Protein Precursor in Developing Hamster Brain

    Science.gov (United States)

    Mobley, William C.; Neve, Rachael L.; Prusiner, Stanley B.; McKinley, Michael P.

    1988-12-01

    Deposition of amyloid filaments serves as a pathologic hallmark for some neurodegenerative disorders. The prion protein (PrP) is found in amyloid of animals with scrapie and humans with Creutzfeldt-Jakob disease; the β protein is present in amyloid deposits in Alzheimer disease and Down syndrome patients. These two proteins are derived from precursors that in the brain are expressed primarily in neurons and are membrane bound. We found that gene expression for PrP and the β -protein precursor (β -PP) is regulated in developing hamster brain. Specific brain regions showed distinct patterns of ontogenesis for PrP and β -PP mRNAs. The increases in PrP and β -PP mRNAs in developing basal forebrain coincided with an increase in choline acetyltransferase activity, raising the possibility that these markers might be coordinately controlled in cholinergic neurons and regulated by nerve growth factor (NGF). Injections of NGF into the brains of neonatal hamsters increased both PrP and β -PP mRNA levels. Increased PrP and β -PP mRNA levels induced by NGF were confined to regions that contain NGF-responsive cholinergic neurons and were accompanied by elevations in choline acetyltransferase. It remains to be established whether or not exogenous NGF acts to increase PrP and β -PP gene expression selectively in forebrain cholinergic neurons in the developing hamster and endogenous NGF regulates expression of these genes.

  12. Alterations in mRNA and protein levels of metalloproteinases-2, -9, and -14 and tissue inhibitor of metalloproteinase-2 responses to traumatic skeletal muscle injury.

    Science.gov (United States)

    Barnes, Brian R; Szelenyi, Eric R; Warren, Gordon L; Urso, Maria L

    2009-12-01

    This study characterizes the temporal relationship of membrane type-1 matrix metalloproteinase (MT1-MMP) and tissue inhibitor of metalloproteinase-2 (TIMP-2) expression in skeletal muscle following injury. Tibialis anterior (TA) muscles from 60 mice were exposed and injured by applying a cold steel probe (-79 degrees C) to the muscle for 10 s. Thereafter, TA muscles from uninjured and injured legs were collected at 3, 10, 24, 48, and 72 h postinjury for analysis of local MT1-MMP, TIMP-2, and matrix metalloproteinases-2 and -9 (MMP-2 and MMP-9) mRNA and protein content via quantitative RT-PCR, immunoblotting, zymography, and immunofluorescence. All data are expressed as fold change of injured leg vs. uninjured leg. MT1-MMP mRNA levels were decreased significantly at 48 and 72 h postinjury by approximately 9- and 21-fold, respectively (P muscle injury initiates a sequence of events in the MT1-MMP proteolytic cascade resulting in elevated levels of the soluble (50 kDa) fragment of MT1-MMP, which could enhance pericellular extracellular matrix remodeling.

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

    Directory of Open Access Journals (Sweden)

    Rossie Sandra

    2004-06-01

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

  14. Amiodarone decreases gene expression of low-density lipoprotein receptor at both the mRNA and the protein level

    NARCIS (Netherlands)

    Hudig, F.; Bakker, O.; Wiersinga, W. M.

    1998-01-01

    Amiodarone, a potent antiarrhythmic drug, decreases plasma and tissue triiodothyronine (T3) and increases plasma cholesterol levels, resembling changes seen during hypothyroidism. The increase of serum cholesterol during amiodarone medication is associated with a decreased expression of the hepatic

  15. Evaluation of immune and stress status in harbour porpoises (Phocoena phocoena): can hormones and mRNA expression levels serve as indicators to assess stress?

    Science.gov (United States)

    Müller, Sabine; Lehnert, Kristina; Seibel, Henrike; Driver, Jörg; Ronnenberg, Katrin; Teilmann, Jonas; van Elk, Cornelius; Kristensen, Jakob; Everaarts, Eligius; Siebert, Ursula

    2013-07-17

    The harbour porpoise is exposed to increasing pressure caused by anthropogenic activities in its marine environment. Numerous offshore wind farms are planned or under construction in the North and Baltic Seas, which will increase underwater noise during both construction and operation. A better understanding of how anthropogenic impacts affect the behaviour, health, endocrinology, immunology and physiology of the animals is thus needed. The present study compares levels of stress hormones and mRNA expression of cytokines and acute-phase proteins in blood samples of harbour porpoises exposed to different levels of stress during handling, in rehabilitation or permanent human care.Free-ranging harbour porpoises, incidentally caught in pound nets in Denmark, were compared to harbour porpoises in rehabilitation at SOS Dolfijn in Harderwijk, the Netherlands, and individuals permanently kept in human care in the Dolfinarium Harderwijk and Fjord & Belt Kerteminde, Denmark. Blood samples were investigated for catecholamines, adrenaline, noradrenaline and dopamine, as well as for adrenocorticotropic hormone (ACTH), cortisol, metanephrine and normetanephrine. mRNA expression levels of relevant cell mediators (cytokines IL-10 and TNFα, acute-phase proteins haptoglobin and C-reactive protein and the heat shock protein HSP70) were measured using real-time PCR. Biomarker expression levels varied between free-ranging animals and porpoises in human care. Hormone and cytokine ranges showed correlations to each other and to the health status of investigated harbour porpoises. Hormone concentrations were higher in free-ranging harbour porpoises than in animals in human care. Adrenaline can be used as a parameter for the initial reaction to acute stress situations; noradrenaline, dopamine, ACTH and cortisol are more likely indicators for the following minutes of acute stress. There is evidence for different correlations between production of normetanephrine, metanephrine, cortisol and

  16. Predictive value of PD-L1 based on mRNA level in the treatment of stage IV melanoma with ipilimumab.

    Science.gov (United States)

    Brüggemann, C; Kirchberger, M C; Goldinger, S M; Weide, B; Konrad, A; Erdmann, M; Schadendorf, D; Croner, R S; Krähenbühl, L; Kähler, K C; Hafner, C; Leisgang, W; Kiesewetter, F; Dummer, R; Schuler, G; Stürzl, M; Heinzerling, L

    2017-10-01

    PD-L1 is established as a predictive marker for therapy of non-small cell lung cancer with pembrolizumab. Furthermore, PD-L1 positive melanoma has shown more favorable outcomes when treated with anti-PD1 antibodies and dacarbazine compared to PD-L1 negative melanoma. However, the role of PD-L1 expression with regard to response to checkpoint inhibition with anti-CTLA-4 is not clear, yet. In addition, the lack of standardization in the immunohistochemical assessment of PD-L1 makes the comparison of results difficult. In this study, we investigated the PD-L1 gene expression with a new fully automated technique via RT-PCR and correlated the findings with the response to the anti-CTLA-4 antibody ipilimumab. Within a retrospective multi-center trial, PD-L1 gene expression was evaluated in 78 melanoma patients in a total of 111 pre-treatment tumor samples from 6 skin cancer centers and analyzed with regard to response to ipilimumab. For meaningful statistical analysis, the cohort was enriched for responders with 30 responders and 48 non-responders. Gene expression was assessed by quantitative RT-PCR after extracting mRNA from formalin-fixed paraffin embedded tumor tissue and correlated with results from immunohistochemical (IHC) stainings. The evaluation of PD-L1 expression based on mRNA level is feasible. Correlation between PD-L1 expression as assessed by IHC and RT-PCR showed varying levels of concordance depending on the antibody employed. RT-PCR should be further investigated to measure PD-L1 expression, since it is a semi-quantitative method with observer-independent evaluation. With this approach, there was no statistical significant difference in the PD-L1 expression between responders and non-responders to the therapy with ipilimumab. The evaluation of PD-L1 expression based on mRNA level is feasible. Correlation between PD-L1 expression as assessed by IHC and RT-PCR showed varying levels of concordance depending on the antibody employed. RT-PCR should be

  17. Placental leptin mRNA expression and serum leptin levels in pre-eclampsia associated with HIV infection.

    Science.gov (United States)

    Haffejee, Firoza; Naicker, Thajasvarie; Singh, Moganavelli; Kharsany, Ayesha B M; Adhikari, Miriam; Singh, Ravesh; Maharaj, Niren; Moodley, Jagidesa

    2017-01-01

    Leptin, primarily produced by adipocytes, is implicated in the development of pre-eclampsia. This study examines placental leptin production and serum leptin levels in HIV infected and uninfected normotensive and pre-eclamptic pregnancies. Placental leptin production was analysed by RT-PCR and serum leptin levels by ELISA in normotensive (n = 90) and pre-eclamptic (n = 90) pregnancies which were further stratified by HIV status. Placental leptin production was higher in pre-eclampsia compared to normotensive pregnancies irrespective of HIV status (p = .04). Serum leptin was non-significantly raised in HIV uninfected (p = .42) but lower in HIV-infected (p = .03) pre-eclampsia. The latter had lower BMI (p = .007) and triceps skin-fold thickness (p leptin and triceps skin-fold thickness (p leptin. Thus, serum leptin levels are not indicative of increased placental production when pre-eclampsia is associated with HIV infection.

  18. Hind limb unloading of mice modulates gene expression at the protein and mRNA level in mesenchymal bone cells

    Directory of Open Access Journals (Sweden)

    Palmieri Daniela

    2010-07-01

    Full Text Available Abstract Background We investigated the extent, modalities and reversibility of changes at cellular level in the expression of genes and proteins occurring upon Hind limb unloading (HU in the tibiae of young C57BL/6J male mice. We focused on the effects of HU in chondrogenic, osteogenic, and marrow mesenchymal cells. Methods We analyzed for expression of genes and proteins at two time points after HU (7 and 14 days, and at 14 days after recovery from HU. Levels of mRNAs were tested by in situ hybridization. Protein levels were tested by immunohistochemistry. We studied genes involved in osteogenesis (alkaline phosphatase (AP, osteocalcin (OC, bonesialoprotein (BSP, membrane type1 matrix metalloproteinase (MT1-MMP, in extracellular matrix (ECM formation (procollagenases (BMP1, procollagenase enhancer proteins (PCOLCE and remodeling (metalloproteinase-9 (MMP9, RECK, and in bone homeostasis (Stro-1, CXCL12, CXCR4, CD146. Results We report the following patterns and timing of changes in gene expression induced by HU: 1 transient or stable down modulations of differentiation-associated genes (AP, OC, genes of matrix formation, maturation and remodelling, (BMP1, PCOLCEs MMP9 in osteogenic, chondrogenic and bone marrow cells; 2 up modulation of MT1-MMP in these same cells, and uncoupling of its expression from that of AP; 3 transient down modulation of the osteoblast specific expression of BSP; 4 for genes involved in bone homeostasis, up modulation in bone marrow cells at distal epiphysis for CXCR4, down modulation of CXCL12, and transient increases in osteoblasts and marrow cells for Stro1. 14 days after limb reloading expression returned to control levels for most genes and proteins in most cell types, except AP in all cells, and CXCL12, only in bone marrow. Conclusions HU induces the coordinated modulation of gene expression in different mesenchymal cell types and microenvironments of tibia. HU also induces specific patterns of expression for

  19. Changes in var gene mRNA levels during erythrocytic development in two phenotypically distinct Plasmodium falciparum parasites

    DEFF Research Database (Denmark)

    Dahlbäck, Madeleine; Lavstsen, Thomas; Salanti, Ali

    2007-01-01

    points along the 48 hours intra-erythrocytic cycle for extraction of RNA and for analysis of expression of variant surface antigens by flow cytometry. Total RNA from each parasite sample was extracted and cDNA synthesized. Quantitative real-time PCR was performed using gene-specific primers for all var...... genes. Samples for flow cytometry were labelled with rabbit IgG targeting DBL5epsilon of VAR2CSA and serum IgG from malaria-exposed men and pregnant women. RESULTS: var transcripts were detected at all time points of the intra-erythrocytic cycle by quantitative real-time PCR, although transcription......BACKGROUND: The var multigene family encodes PfEMP1, which are expressed on the surface of infected erythrocytes and bind to various host endothelial receptors. Antigenic variation of PfEMP1 plays a key role in malaria pathogenesis, a process partially controlled at the level of var gene...

  20. Expression of human amyloid precursor protein in Drosophila melanogaster nerve cells causes a decrease in presynaptic gene mRNA levels.

    Science.gov (United States)

    Rodin, D I; Schwarzman, A L; Sarantseva, S V

    2015-08-10

    Amyloid precursor protein (APP) is a key player in Alzheimer's disease. The proteolytic cleavage of APP results in various short peptide fragments including the toxic amyloid-beta peptide, which is a main component of senile plaques. However, the functions of APP and its processed fragments are not yet well understood. Here, using real-time polymerase chain reaction, we demonstrate that exogenous expression of APP, its mutant form APP-Swedish, or two truncated forms in Drosophila melanogaster causes a significant (P ≤ 0.05) drop in the mRNA levels of the presynaptic proteins synaptotagmin-1 and neuronal synaptobrevin. The results obtained from this study suggest a potential role of APP or its fragments in the regulation of synaptic gene transcription.

  1. Dataset of mRNA levels for dopaminergic receptors, adrenoceptors and tyrosine hydroxylase in lymphocytes from subjects with clinically isolated syndromes

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    Marco Cosentino

    2016-12-01

    Full Text Available This data article presents a dataset of mRNA levels for dopaminergic receptors, adrenoceptors and for tyrosine hydoxylase, the rate-limiting enzyme in the synthesis of catecholamines, in peripheral blood mononuclear cells as well as in CD4+ T effector and regulatory cells from subjects with clinically isolated syndromes (CIS, which is a first episode of neurological disturbance(s suggestive of multiple sclerosis. CIS subjects are divided into two groups according to their eventual progression, after 12 months from CIS, to clinically established multiple sclerosis. The data reported are related to the article entitled "Dopaminergic receptors and adrenoceptors in circulating lymphocytes as putative biomarkers for the early onset and progression of multiple sclerosis" (M. Cosentino, M. Zaffaroni, M. Legnaro, R. Bombelli, L. Schembri, D. Baroncini, A. Bianchi, R. Clerici, M. Guidotti, P. Banfi, G. Bono, F. Marino, 2016 [1].

  2. The Allergic Airway Inflammation Repository--a user-friendly, curated resource of mRNA expression levels in studies of allergic airways.

    Science.gov (United States)

    Gawel, D R; Rani James, A; Benson, M; Liljenström, R; Muraro, A; Nestor, C E; Zhang, H; Gustafsson, M

    2014-08-01

    Public microarray databases allow analysis of expression levels of candidate genes in different contexts. However, finding relevant microarray data is complicated by the large number of available studies. We have compiled a user-friendly, open-access database of mRNA microarray experiments relevant to allergic airway inflammation, the Allergic Airway Inflammation Repository (AAIR, http://aair.cimed.ike.liu.se/). The aim is to allow allergy researchers to determine the expression profile of their genes of interest in multiple clinical data sets and several experimental systems quickly and intuitively. AAIR also provides quick links to other relevant information such as experimental protocols, related literature and raw data files. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  3. Levels of estrogen receptor B splice variant (ERBΔ5 mRNA correlates with progesterone receptor in breast carcinomas

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    Mandušić Vesna

    2010-01-01

    Full Text Available It is well known that breast tumors which are estrogen positive ER(+ are more likely to respond to hormone therapy. However, a certain percentage of ER(+/PR(+ tumors do not respond to this therapy. Identification of the second estrogen receptor, named estrogen receptor beta (ERβ, as well as the existence of numerous isoforms/splice variants of both ERα and ERβ, suggests that a complex regulation of estrogen action exists. In this study, we analyzed the expression ratio of ERβ1 isoform and ERβΔ5 splice variant mRNAs, and its correlation with ER/PR status by quantitative RT-PCR and clinical and histopathological parameters. We found that the relative proportion of ERβΔ5 in the total ERβ1 transcript 'pool' inversely correlates with the PR level (p = -0,359, p< 0,003, Spearman. It may be that the ERβΔ5 variant modulates the ERα activity of downstream targets. In addition, we suggest that the determination of the expression profiles of ERα and ERβ isoforms and splice variants in the defined groups of patients are necessary for elucidating their involvement in endocrine resistance.

  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 < 0.05), this being correlated with an antagonist effect upon hPXR (EC50 = 0.38 mg/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 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. 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.

  6. Assessment of placental and maternal stress responses in patients with pregnancy related complications via monitoring of heat shock protein mRNA levels.

    Science.gov (United States)

    Hromadnikova, Ilona; Dvorakova, Lenka; Kotlabova, Katerina; Kestlerova, Andrea; Hympanova, Lucie; Novotna, Veronika; Doucha, Jindrich; Krofta, Ladislav

    2015-03-01

    The study describes the stress response in the central cotyledon zone of placental tissue and in maternal whole peripheral blood to pregnancy related complications including gestational hypertension (n = 31), preeclampsia w or w/o fetal growth restriction (n = 95), and fetal growth restriction (n = 39) using real-time RT-PCR and genes encoding Hsp27, Hsp60, Hsp70, Hsp90 and HspBP1 proteins. The placental tissue does not respond to pregnancy induced hypertension, fetal growth restriction and short-term severe preeclampsia that requires immediate termination of gestation. Upregulation of Hsp27, Hsp90 and HspBP1 appears just in case of long-term deteriorated conditions (usually in mild preeclampsia, that enable further continuation of gestation, when properly treated). On the other hand, maternal circulation is able to reflect both maternal and fetal pathologic conditions. While pregnancy related complications always induce upregulation of Hsp70 and downregulation of Hsp90 in maternal whole peripheral blood, the increase of Hsp60 mRNA levels occurs entirely in patients with preeclampsia and/or fetal growth restriction. Hsp60, Hsp70 and Hsp90 are dysregulated in maternal circulation irrespective of the severity of the disease (in both mild and severe preeclampsia) and the requirements for the delivery (before and after 34th week of gestation). Nevertheless, the highest Hsp60 mRNA levels may be observed in pregnancies with signs of the centralization of the fetal circulation associated with fetal hypoxia.

  7.  The impact of IL18 gene polymorphisms on mRNA levels and interleukin-18 release by peripheral blood mononuclear cells

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    Violetta Dziedziejko

    2012-06-01

    Full Text Available  Introduction:Interleukin-18 (IL-18 is a pleiotropic cytokine playing an important role as a modulator of immune responses, found to play a role in pathogenesis of numerous inflammatory-associated disorders. In the present study a potential association between 7 common single-nucleotide polymorphisms (SNPs spanning the whole IL18 gene, gene expression and the release of IL-18 from the stimulated peripheral blood mononuclear cells (PBMCs was investigated.Materials/Methods:PBMCs were isolated from peripheral blood of 29 healthy volunteers, genotyped for the presence of IL18 SNPs: rs1946518: A>C, rs187238: G>C, rs360718: A>C, rs360722: C>T, rs360721: C>G, rs549908: T>G, and rs5744292: A>G. IL-18 concentration and IL18 mRNA levels were investigated after incubation of cells for 48 h with different stimulants (PHA, LPS, and anti-CD3/CD28 antibodies.Results:After treatment with LPS and antibodies IL-18 concentrations were significantly lower in rs1946518AA homozygotes than in C allele carriers. When differences in IL18 mRNA levels between non-stimulated and stimulated cells were analyzed, significantly decreased gene expression was noted in rs1946518 AA homozygotes (as compared with C allele carriers in samples treated with PHA and LPS. Similar trends were observed in the case of rs187238 SNP; however, the differences reached statistical significance only after PHA treatment.Conclusions:Our study supports the role of rs1946518 (-607A>C and rs187238 (-137G>C SNPs as genetic determinants of the observed variability in IL18 expression.

  8. MicroRNA-145 ModulatesN6-Methyladenosine Levels by Targeting the 3'-Untranslated mRNA Region of theN6-Methyladenosine Binding YTH Domain Family 2 Protein.

    Science.gov (United States)

    Yang, Zhe; Li, Jiong; Feng, Guoxing; Gao, Shan; Wang, Yuan; Zhang, Shuqin; Liu, Yunxia; Ye, Lihong; Li, Yueguo; Zhang, Xiaodong

    2017-03-03

    N 6 -Methyladenosine (m 6 A) is a prevalent modification present in the mRNAs of higher eukaryotes. YTH domain family 2 (YTHDF2), an m 6 A "reader" protein, can recognize mRNA m 6 A sites to mediate mRNA degradation. However, the regulatory mechanism of YTHDF2 is poorly understood. To this end, we investigated the post-transcriptional regulation of YTHDF2. Bioinformatics analysis suggested that the microRNA miR-145 might target the 3'-untranslated region (3'-UTR) of YTHDF2 mRNA. The levels of miR-145 were negatively correlated with those of YTHDF2 mRNA in clinical hepatocellular carcinoma (HCC) tissues, and immunohistochemical staining revealed that YTHDF2 was closely associated with malignancy of HCC. Interestingly, miR-145 decreased the luciferase activities of 3'-UTR of YTHDF2 mRNA. Mutation of predicted miR-145 binding sites in the 3'-UTR of YTHDF2 mRNA abolished the miR-145-induced decrease in luciferase activity. Overexpression of miR-145 dose-dependently down-regulated YTHDF2 expression in HCC cells at the levels of both mRNA and protein. Conversely, inhibition of miR-145 resulted in the up-regulation of YTHDF2 in the cells. Dot blot analysis and immunofluorescence staining revealed that the overexpression of miR-145 strongly increased m 6 A levels relative to those in control HCC cells, and this increase could be blocked by YTHDF2 overexpression. Moreover, miR-145 inhibition strongly decreased m 6 A levels, which were rescued by treatment with a small interfering RNA-based YTHDF2 knockdown. Thus, we conclude that miR-145 modulates m 6 A levels by targeting the 3'-UTR of YTHDF2 mRNA in HCC cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. 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 < 0.05) average daily gain (ADG) and gain to feed (G:F) compared to those fed the C diet during week 2. Overall, ADG and G:F were also reduced (P < 0.05) in pigs fed the T2 diet compared to those fed the C and T1 diets. Bone mineral density (BMD) and bone mineral content (BMC) were reduced (P < 0.05) in pigs fed the T2 diet compared to those fed the C diet throughout the experiment. At week 1, jejunal mRNA expression of Na (+)-dependent phosphate transporter 2 (SLC34A2) was increased (P < 0.01) in pigs fed the T2 diet compared to C diet. Renal mRNA expression of Na(+)-dependent phosphate transporter 1 (SLC34A1) and SLC34A3 were increased (P < 0.05) in pigs fed the T2 diet compared to those fed the C diet at week 2 and was accompanied by lower (P < 0.05) urinary P in pigs fed the T2 diet during week 2

  10. Associations of Haplotypes Upstream of IRS1 with Insulin Resistance, Type 2 Diabetes, Dyslipidemia, Preclinical Atherosclerosis, and Skeletal Muscle LOC646736 mRNA Levels

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    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.

  11. [Effect of dangua recipe on glycolipid metabolism and VCAM-1 and its mRNA expression level in Apo E(-/-) mice with diabetes mellitus].

    Science.gov (United States)

    Heng, Xian-Pei; Li, Liang; Huang, Su-Ping; Chen, Yan; Lin, Miao-Xian; Zhuang, Huai-Shan; Yan, Qun-Fang; Yang, Liu-Qing; Chen, Ling; Lin, Qing; Cheng, Xin-Ling; Chen, Min-Ling; Chen, Yi-Chu; Lan, Yuan-Long; Wang, Zhi-Ta; Yao, Shu-Hong; Zhang, Zhi-San

    2014-09-01

    To study the effect of Dangua Recipe (DGR) on glycolipid metabolism, vascular cell adhesion molecule-1 (VCAM-1) and its mRNA expression level of transgenic Apo E(-/-) mouse with spontaneous atherosclerosis, thus revealing its partial mechanism for curing diabetes mellitus (DM) with angiopathy. Diabetic model was prepared by peritoneally injecting streptozotocin (STZ) to Apo E(-/-) mouse. Totally 32 modeled mice were stratified by body weight, and then divided into 4 groups referring to blood glucose levels from low to high by random digit table, i.e., the model group (MOD, fed with sterile water, at the daily dose of 15 mL/kg), the DGR group (fed with DGR at the daily dose of 15 mL/kg), the combination group (COM, fed with DGR at the daily dose of 15 mL/kg and pioglitazone at the daily dose of 4.3 mg/kg), and the pioglitazone group (PIO, at the daily dose of 4.3 mg/kg), 8 in each group. Another 8 normal glucose C57 mouse of the same age and strain were recruited as the control group. All interventions lasted for 12 weeks by gastrogavage. The fasting blood glucose (FBG), body weight, food intake, water intake, skin temperature, the length of tail, and the degree of fatty liver were monitored. The hemoglobin A1c (HbA1c), total cholesterol (TC), and LDL-C were determined. Endothelin-1 (ET-1) was determined by radioimmunoassay. Nitrogen monoxidum (NO) was determined by nitrate reductase. The kidney tissue VCAM-1 level was analyzed with ELISA. The expression of VCAM-1 mRNA in the kidney tissue was detected with real time quantitative PCR. Compared with the control group, the body weight and food intake decreased, water intake increased in all the other model groups (P food intake and water intake increased more and the tail length was longer in the DRG group (P < 0.01). There was no statistical difference in the level of NO among groups. The degree of fatty liver in the model group was significantly severer than that in the control group (P < 0.05). It was obviously

  12. Galanin-like peptide (GALP) neurone-specific phosphoinositide 3-kinase signalling regulates GALP mRNA levels in the hypothalamus of males and luteinising hormone levels in both sexes.

    Science.gov (United States)

    Aziz, R; Beymer, M; Negrón, A L; Newshan, A; Yu, G; Rosati, B; McKinnon, D; Fukuda, M; Lin, R Z; Mayer, C; Boehm, U; Acosta-Martínez, M

    2014-07-01

    Galanin-like peptide (GALP) neurones participate in the metabolic control of reproduction and are targets of insulin and leptin regulation. Phosphoinositide 3-kinase (PI3K) is common to the signalling pathways utilised by both insulin and leptin. Therefore, we investigated whether PI3K signalling in neurones expressing GALP plays a role in the transcriptional regulation of the GALP gene and in the metabolic control of luteinising hormone (LH) release. Accordingly, we deleted PI3K catalytic subunits p110α and p110β via conditional gene targeting (cKO) in mice (GALP-p110α/β cKO). To monitor PI3K signalling in GALP neurones, these animals were also crossed with Cre-dependent FoxO1GFP reporter mice. Compared to insulin-infused control animals, the PI3K-Akt-dependent FoxO1GFP nuclear exclusion in GALP neurones was abolished in GALP-p110α/β cKO mice. We next used food deprivation to investigate whether the GALP-neurone specific ablation of PI3K activity affected the susceptibility of the gonadotrophic axis to negative energy balance. Treatment did not affect LH levels in either sex. However, a significant genotype effect on LH levels was observed in females. By contrast, no genotype effect on LH levels was observed in males. A sex-specific genotype effect on hypothalamic GALP mRNA was observed, with fed and fasted GALP-p110α/β cKO males having lower GALP mRNA expression compared to wild-type fed males. Finally, the effects of gonadectomy and steroid hormone replacement on GALP mRNA levels were investigated. Compared to vehicle-treated mice, steroid hormone replacement reduced mediobasal hypothalamus GALP expression in wild-type and GALP-p110α/β cKO animals. In addition, within the castrated and vehicle-treated group and compared to wild-type mice, LH levels were lower in GALP-p110α/β cKO males. Double immunofluorescence using GALP-Cre/R26-YFP mice showed androgen and oestrogen receptor co-localisation within GALP neurones. Our data demonstrate that GALP

  13. 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.

  14. Analysis of SLC40A1 gene at the mRNA level reveals rapidly the causative mutations in patients with hereditary hemochromatosis type IV.

    Science.gov (United States)

    Speletas, Matthaios; Kioumi, Anna; Loules, Gedeon; Hytiroglou, Prodromos; Tsitouridis, John; Christakis, John; Germenis, Anastasios E

    2008-01-01

    Mutations in the SLC40A1 gene result in a dominant genetic disorder [ferroportin disease; hereditary hemochromatosis type (HH) IV], characterized by iron overload with two different clinical manifestations, normal transferrin saturation with macrophage iron accumulation (the most prevalent type) or high transferrin saturation with hepatocyte iron accumulation (classical hemochromatosis phenotype). In previous studies, the mutational analysis of SLC40A1 gene has been performed at the genomic DNA level by PCR amplification and direct sequencing of all coding regions and flanking intron-exon boundaries (usually in 9 PCR reactions). In this study, we analyzed the SLC40A1 gene at the mRNA level, in two RT-PCR reactions, followed by direct sequencing and/or NIRCA (non-isotopic RNase cleavage assay). This protocol turned out to be rapid, sensitive and reliable, facilitating the detection of the SLC40A1 gene mutations in two patients with hyperferritinemia, normal transferrin saturation and iron accumulation predominantly in macrophages and Kupffer cells. The first one displayed the well-described alteration V162 Delta and the second a novel mutation (R178G) that was further detected in two relatives in a pedigree analysis. The proposed procedure would facilitate the wide-range molecular analysis of the SLC40A1 gene, contributing to better understanding the pathogenesis of the ferroportin disease.

  15. CORRELATION BETWEEN PROTEIN-WITH-MOLECULAR-WEIGHT-53 (P53, BURKIT CELL LYMPHOMA 2 (BCL2, AND FAS LIGAND (FASL AND VASCULAR-CELL-ADHESION-MOLECULE-1 (VCAM-1 MRNA EXPRESSION LEVELS IN A PATHOGENESIS STUDY OF PREECLAMPSIA

    Directory of Open Access Journals (Sweden)

    Mintareja Teguh

    2014-06-01

    Full Text Available Objective: To determine the role of protein-with-molecular-weight-53 (p53, burkit cell lymphoma 2 (Bcl2, Fas ligand (FasL mRNA, and vascular cell adhesion molecule 1 (VCAM-1, known as the apoptosis-related molecular pathway, in preeclamptic patients. Methods: Observation on the correlation between the mRNA levels of p53, Bcl2 and FasL and VCAM-1 in 31 subjects at 28-42 weeks gestational age was performed in this study using the real time reverse transcriptase-polymerase chain reaction (RT-PCR. Results: The results showed that p53 mRNA increased (>1.2350 ng/μL in the preeclampsia group compared to the normal pregnancy group (p=0.010, Bcl2 mRNA was lower (≤0.9271 ng/μL in the preeclampsia group than the control group (p=0.041. There was also a tendency of increased FasL mRNA expression (>0.5509 ng/μL in the preeclampsia group compared to the normal pregnancy group (p=0.300. The level of VCAM-1 elevated (>890.08 ng/mL in the preeclampsia group compared to the normal pregnancy group (p=0.001. In preeclampsia, the correlation between the Bcl2/p53 ratio and VCAM-1 was r=0.541 (p=0.002, whereas the correlation in normal pregnancy was r=0.099 (p=0.595. Conclusions: There are correlations between the mRNA expression levels of p53 and Bcl2 as an intrinsic pathway of apoptosis along with the VCAM-1 levels in the incidence of preeclampsia. However, no correlation is found between FasL mRNA expression and the incidence of preeclampsia.

  16. Production of a Locus- and Allele-Specific Monoclonal Antibody for the Characterization of SLA-1*0401 mRNA and Protein Expression Levels in MHC-Defined Microminipigs.

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    Yoshie Kametani

    Full Text Available The class I major histocompatibility complex (MHC presents self-developed peptides to specific T cells to induce cytotoxity against infection. The MHC proteins are encoded by multiple loci that express numerous alleles to preserve the variability of the antigen-presenting ability in each species. The mechanism regulating MHC mRNA and protein expression at each locus is difficult to analyze because of the structural and sequence similarities between alleles. In this study, we examined the correlation between the mRNA and surface protein expression of swine leukocyte antigen (SLA-1*0401 after the stimulation of peripheral blood mononuclear cells (PBMCs by Staphylococcus aureus superantigen toxic shock syndrome toxin-1 (TSST-1. We prepared a monoclonal antibody (mAb against a domain composed of Y102, L103 and L109 in the α2 domain. The Hp-16.0 haplotype swine possess only SLA-1*0401, which has the mAb epitope, while other haplotypes possess 0 to 3 SLA classical class I loci with the mAb epitopes. When PBMCs from SLA-1*0401 homozygous pigs were stimulated, the SLA-1*0401 mRNA expression level increased until 24 hrs and decreased at 48 hrs. The kinetics of the interferon regulatory transcription factor-1 (IRF-1 mRNA level were similar to those of the SLA-1*0401 mRNA. However, the surface protein expression level continued to increase until 72 hrs. Similar results were observed in the Hp-10.0 pigs with three mAb epitopes. These results suggest that TSST-1 stimulation induced both mRNA and surface protein expression of class I SLA in the swine PBMCs differentially and that the surface protein level was sustained independently of mRNA regulation.

  17. 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...

  18. High protein and mRNA expression levels of TUBB3 (class III ß-tubulin) are associated with aggressive tumor features in esophageal adenocarcinomas.

    Science.gov (United States)

    Loeser, Heike; Schallenberg, Simon; von Winterfeld, Moritz; Tharun, Lars; Alakus, Hakan; Hölscher, Arnulf; Bollschweiler, Elfriede; Buettner, Reinhard; Zander, Thomas; Quaas, Alexander

    2017-12-29

    Esophageal adenocarcinomas show an increasing incidence in the Western world and their overall survival remains low. Microtubules are multifunctional cytoskeletal proteins involved in crucial cellular roles, including maintenance of cell shape, intracellular transport, meiosis, and mitosis. Microtubulus-TUBB3 was found overexpressed in several carcinomas suggesting a significant role in cancer development. High levels of TUBB3 expression were also described to be associated with poor clinical outcome in various cancers. It was shown that overexpression of TUBB3 could be related to reduced efficiency of taxane-based targeting anticancer drugs in several cancer types. There is a statistically significant association between high TUBB3 protein and TUBB3 mRNA expression and shortened survival (pimportance of TUBB3 in esophageal adenocarcinoma. TUBB3 serves as an independent prognostic marker and may be a valuable biomarker for routine application in esophageal adenocarcinoma especially to address the need for adjuvant treatment in individuals following neoadjuvant therapy and surgery. Future prospective studies are needed which include the results of TUBB3 in preoperative biopsy material to proof the prognostic impact of TUBB3. 280 esophageal adenocarcinomas that underwent primary surgical resection or resection after neoadjuvant therapy were analyzed by mRNA-in-situ-hybridization (RNAscope ® ) and by immunohistochemistry (TUBB3 rabbit monoclonal antibody; Epitomics).

  19. 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 iron uptake protein transferrin receptor 1 were 30-49% higher in the PE and PE+MMN arms than in the FeFol arm (P iron and in the absence of supplemental 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.

  20. Effect of Salvia miltiorrhiza root extract on brain acetylcholinesterase and butyrylcholinesterase activities, their mRNA levels and memory evaluation in rats.

    Science.gov (United States)

    Ozarowski, Marcin; Mikolajczak, Przemyslaw L; Piasecka, Anna; Kujawski, Radoslaw; Bartkowiak-Wieczorek, Joanna; Bogacz, Anna; Szulc, Michal; Kaminska, Ewa; Kujawska, Malgorzata; Gryszczynska, Agnieszka; Kachlicki, Piotr; Buchwald, Waldemar; Klejewski, Andrzej; Seremak-Mrozikiewicz, Agnieszka

    2017-05-01

    Salvia miltiorrhiza (Lamiaceae), one of the most important and popular plants of traditional medicine of Asia, is used for the prevention and treatment of cardiovascular diseases and in central nervous system disturbances. The main aim of this study was to assess the influence of subchronic (28-fold) administration of Salvia miltiorrhiza root extract (SE, 200mg/kg, p.o.) on behavioural activity and memory of rats and to evaluate the activities of cholinesterases (AChE and BuChE) and gene expression levels of AChE and BuChE as well as of beta-secretase (BACE1) in the hippocampus and frontal cortex in vivo. Huperzine A (HU, 0.5mg/kg b.w., p.o.) served as a positive control substance, whereas scopolamine (0.5mg/kg, i.p.) injection was used as a well-known model of memory impairment. The results showed that subchronic administration of SE led to an improvement of long-term memory of rats. Strong inhibition of AChE and BuChE mRNA transcription in the frontal cortex of rats treated with SE or HU was observed. The BACE1 transcript level was significantly decreased. AChE activity was statistically significantly inhibited in the frontal cortex and the hippocampus by SE (47% and 55%, respectively). Similar effects were observed in the case of HU. In summary, activity of SE provides evidence that the plant can be a source of drugs used in the treatment of Alzheimer disease. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Variable levels of 37-kDa/67-kDa laminin receptor (RPSA) mRNA in ovine tissues: potential contribution to the regulatory processes of PrPSc propagation?

    Science.gov (United States)

    Qiao, Jun-Wen; Su, Xiao-Ou; Li, Yu-Xing; Yang, Jian-Min; Wang, Yi-Qin; Kouadir, Mohammed; Zhou, Xiang-Mei; Yang, Li-Feng; Yin, Xiao-Min; Zhao, De-Ming

    2009-01-01

    The 37-kDa laminin receptor precursor/67-kDa laminin receptor (LRP/LR, also known as ribosomal protein SA, RPSA) has been reported to be involved in cancer development and prion internalization. Previous studies have shown that the LRP/LR is expressed in a wide variety of tissues. In particular, expression of LRP/LR mRNA may be closely related to the degree of PrP(Sc) propagation. This study presents a detailed investigation of the LRP/LR mRNA expression levels in eleven normal ovine tissues. Using real-time quantitative PCR, the highest LRP/LR expression was found in neocortex (p < 0.05). Slightly lower levels were found in the heart and obex. Intermediate levels were seen in hippocampus, cerebellum, spleen, thalamus, mesenteric lymph node, and the lowest levels were present in liver, kidney, and lung. In general, the LRP/LR mRNA levels were much higher in neuronal tissues than in peripheral tissues. The observation that differences in LRP/LR mRNA expression levels are consistent with the corresponding variation in PrP(Sc) accumulation suggests that the 37-kDa/67-kDa laminin receptor may be involved in the regulation of PrP(Sc) propagation.

  2. 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.

  3. 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

    Science.gov (United States)

    Matia-García, Inés; Salgado-Goytia, Lorenzo; Muñoz-Valle, José F.; García-Arellano, Samuel; Hernández-Bello, Jorge; Salgado-Bernabé, Aralia B.; Parra-Rojas, Isela

    2015-01-01

    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. PMID:25972622

  4. 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.

  5. Innate differences in neuropeptide Y (NPY) mRNA expression in discrete brain regions between alcohol-preferring (P) and -nonpreferring (NP) rats: a significantly low level of NPY mRNA in dentate gyrus of the hippocampus and absence of NPY mRNA in the medial habenular nucleus of P rats.

    Science.gov (United States)

    Hwang, Bang H; Suzuki, Ryoji; Lumeng, Lawrence; Li, T-K; McBride, William J

    2004-12-01

    The neuropeptide Y (NPY) gene in rat chromosome 4 has been shown to play an important role in alcohol-seeking behavior. NPY knockout mice drink more alcohol than wild-type mice, implicating a link between NPY deficiency and high alcohol intake. This is supported by recent studies showing that intracerebroventricular injections of NPY reduce alcohol intake in both alcohol-preferring (P) and high alcohol-drinking rats. However, it is unknown which anatomical NPY systems are involved in alcohol preference. This study was designed to investigate whether there are innate differences in NPY mRNA in cerebral cortical areas, dentate gyrus (DG) of the hippocampus and medial habenular nucleus (MHb) between P and alcohol-nonpreferring (NP) rats, as these discrete brain regions are rich in NPY mRNA. [(33)P]-labeled 28-mer oligodeoxynucleotide probe was applied for the in situ hybridization study to detect the NPY mRNA, measured using quantitative autoradiography. This study revealed an absence of NPY mRNA in the MHb of P rats. We found that NPY mRNA was significantly lower in the DG of P rats than NP rats. This innate difference of NPY mRNA expression in the DG between P and NP rats is region specific. For example, in most of the cerebral cortical areas examined, an innate difference was not seen. Our study suggests that lower NPY gene expression in the DG and MHb of P rats may be factors contributing to some of the phenotypic differences observed between the P and NP lines of rats.

  6. Expression levels of mRNA for insulin-like growth factors 1 and 2, IGF receptors and IGF binding proteins in in vivo and in vitro grown bovine follicles.

    Science.gov (United States)

    Rebouças, Emanuela L; Costa, José J N; Passos, Maria J; Silva, Anderson W B; Rossi, Rodrigo O D S; van den Hurk, Robert; Silva, José R V

    2014-11-01

    This study investigated mRNA levels for insulin-like growth factors (IGFs) IGF1 (IGF-I) and IGF2 (IGF-II), IGF receptors (IGF1R and IGF2R), and binding proteins (IGFBP-1, IGFBP-2. IGFBP-3, IGFBP-4, IGFBP-5 and IGFBP-6) in bovine follicles of 0.2, 0.5 or 1.0 mm in diameter. mRNA expression levels in in vitro cultured follicles that reached approximately 0.5 mm were compared with that of in vivo grown follicles. IGF1R and IGF2R expression levels in 0.5 mm in vivo follicles were higher than in 1.0 or 0.2 mm follicles, respectively. IGFBP-1, IGFBP-2. IGFBP-3, IGFBP-4, IGFBP-5 and IGFBP-6 showed variable expression in the follicular size classes analyzed. In vitro grown follicles had significantly reduced expression levels for IGF1, IGF1R, IGFBP-3, IGFBP-5 and IGFBP-6 mRNA when compared with 0.2 mm follicles, but, when compared with in vivo grown follicles (0.5 mm), only IGFBP-1, IGFBP-2, IGFBP-3 and IGFBP-6 showed a reduction in their expression. In conclusion, IGFs, their receptors and IGFBPs showed variable expression of mRNA levels in the follicular size classes analyzed.

  7. Transcript copy number of genes for DNA repair and translesion synthesis in yeast: contribution of transcription rate and mRNA stability to the steady-state level of each mRNA along with growth in glucose-fermentative medium.

    Science.gov (United States)

    Michán, Carmen; Monje-Casas, Fernando; Pueyo, Carmen

    2005-04-04

    We quantitated the copy number of mRNAs (NTG1, NTG2, OGG1, APN1, APN2, MSH2, MSH6, REV3, RAD30) encoding different DNA repair enzymes and translesion-synthesis polymerases in yeast. Quantitations reported examine how the steady-state number of each transcript is modulated in association with the growth in glucose-fermentative medium, and evaluate the respective contribution of the rate of mRNA degradation and transcription initiation to the specific mRNA level profile of each gene. Each transcript displayed a unique growth-related profile, therefore altering the relative abundance of mRNAs coding for proteins with similar functions, as cells proceed from exponential to stationary phase. Nonetheless, as general trend, they exhibited maximal levels when cells proliferate rapidly and minimal values when cells cease proliferation. We found that previous calculations on the stability of the investigated mRNAs might be biased, in particular regarding those that respond to heat shock stress. Overall, the mRNAs experienced drastic increments in their stabilities in response to gradual depletion of essential nutrients in the culture. However, differences among the mRNA stability profiles suggest a dynamic modulation rather than a passive process. As general rule, the investigated genes were much more frequently transcribed during the fermentative growth than later during the diauxic arrest and the stationary phase, this finding conciliating low steady-state levels with increased mRNA stabilities. Interestingly, while the rate at which each gene is transcribed appeared as the only determinant of the number of mRNA copies at the exponential growth, later, when cell growth is arrested, the rate of mRNA degradation becomes also a key factor for gene expression. In short, our results raise the question of how important the respective contribution of transcription and mRNA stability mechanisms is for the steady-state profile of a given transcript, and how this contribution may

  8. Effect of Gu Tong Xian capsule on expression level of type I, II collagen and BMP-2 mRNA in rabbits with fracture during long-distance running

    Directory of Open Access Journals (Sweden)

    Liang Li

    2017-05-01

    Full Text Available The study aims to analyze and investigate the effects of Gu Tong Xian Capsule on the expression level of type I, II collagen and BMP-2 mRNA in rabbits with fracture during long-distance running. 60 adult healthy rabbits were selected as research objects, and then randomly divided into three groups including model group, positive control group and treatment group, each containing 20 rabbits. The three groups were treated with saline gastric lavage, powder for fracture and trauma, and Gu Tong Xian capsule, respectively. The rabbits of the three groups were respectively sacrificed at 1st week, 2nd weeks and 4th week after operation for sample collection. After that, the expression levels of bone collagen type I, II and BMP-2 of three groups were measured and compared with each other. At all stages, the transcriptional level of type I collagen mRNA in the treatment group were significantly higher than that in the positive control group and model group (p < 0.05; Transcriptional level of type II collagen mRNA in the treatment group increased significantly in the first week, then gradually declined in the 2nd and 4th week, with significantly difference to the model group and the positive control group (p < 0.05. In addition, the transcriptional level of bone morphogenetic protein BMP-2 mRNA at fracture site of the treatment group was higher than that of model group and positive control group (p < 0.05. Gu Tong Xian Capsule can significantly promote fracture healing of experiment rabbits and reduce fracture healing time. Moreover, it can well regulate the expression levels of type I, II collagen and transcriptional level ofBMP-2 mRNA in experiment rabbits with fracture.

  9. Coordinate Changes in Histone Modifications, mRNA Levels, and Metabolite Profiles in Clonal INS-1 832/13 β-Cells Accompany Functional Adaptations to Lipotoxicity*

    Science.gov (United States)

    Malmgren, Siri; Spégel, Peter; Danielsson, Anders P.H.; Nagorny, Cecilia L.; Andersson, Lotta E.; Nitert, Marloes Dekker; Ridderstråle, Martin; Mulder, Hindrik; Ling, Charlotte

    2013-01-01

    Lipotoxicity is a presumed pathogenetic process whereby elevated circulating and stored lipids in type 2 diabetes cause pancreatic β-cell failure. To resolve the underlying molecular mechanisms, we exposed clonal INS-1 832/13 β-cells to palmitate for 48 h. We observed elevated basal insulin secretion but impaired glucose-stimulated insulin secretion in palmitate-exposed cells. Glucose utilization was unchanged, palmitate oxidation was increased, and oxygen consumption was impaired. Halting exposure of the clonal INS-1 832/13 β-cells to palmitate largely recovered all of the lipid-induced functional changes. Metabolite profiling revealed profound but reversible increases in cellular lipids. Glucose-induced increases in tricarboxylic acid cycle intermediates were attenuated by exposure to palmitate. Analysis of gene expression by microarray showed increased expression of 982 genes and decreased expression of 1032 genes after exposure to palmitate. Increases were seen in pathways for steroid biosynthesis, cell cycle, fatty acid metabolism, DNA replication, and biosynthesis of unsaturated fatty acids; decreases occurred in the aminoacyl-tRNA synthesis pathway. The activity of histone-modifying enzymes and histone modifications of differentially expressed genes were reversibly altered upon exposure to palmitate. Thus, Insig1, Lss, Peci, Idi1, Hmgcs1, and Casr were subject to epigenetic regulation. Our analyses demonstrate that coordinate changes in histone modifications, mRNA levels, and metabolite profiles accompanied functional adaptations of clonal β-cells to lipotoxicity. It is highly likely that these changes are pathogenetic, accounting for loss of glucose responsiveness and perturbed insulin secretion. PMID:23476019

  10. CDH1 (E-cadherin) in testicular germ cell neoplasia: suppressed translation of mRNA in pre-invasive carcinoma in situ but increased protein levels in advanced tumours

    DEFF Research Database (Denmark)

    Sonne, Si Brask; Hoei-Hansen, Christina E; Nielsen, John E

    2006-01-01

    E-cadherin (CDH1) is a transmembrane glycoprotein involved in cellular adhesion. In our recent microarray studies of testicular germ cell tumours (TGCTs) and the common precursor carcinoma in situ (CIS), CDH1 mRNA was highly expressed in CIS and embryonal carcinoma. It has previously been reported...... that the CDH1 protein is not expressed in CIS. To resolve the discrepancy, we performed a detailed analysis of the expression of CDH1 mRNA and protein in a series of normal and neoplastic testes. High expression of CDH1 mRNA in CIS was confirmed by real-time PCR and in situ hybridisation. At the protein level...... higher levels in patients with advanced disease (stage II/III) when compared to healthy individuals and patients with stage I TGCT. In conclusion, despite high mRNA levels, the CDH1 protein is not expressed in CIS, suggesting translational suppression of CDH1 protein expression. CDH1 serum levels may...

  11. Long-term in-vitro treatment of human growth hormone (GH)-secreting pituitary adenoma cells with octreotide causes accumulation of intracellular GH and GH mRNA levels.

    Science.gov (United States)

    Hofland, L J; Velkeniers, B; vd Lely, A J; van Koetsveld, P M; Kazemzadeh, M; Waaijers, M; Hooghe-Peters, E L; Lamberts, S W

    1992-09-01

    We studied the effects of long-term in-vitro exposure of human GH secreting pituitary adenoma cells to octreotide on GH release, intracellular GH concentrations and GH messenger ribonucleic acid (mRNA) levels. Human GH-secreting pituitary adenoma cells were cultured for periods from 4 days up to 3 weeks without or with octreotide (10 nM) and/or bromocriptine (10 nM). The effects of these drugs were measured on GH release, intracellular GH concentrations and intracellular GH mRNA levels. Thirteen patients with GH-secreting pituitary adenomas were studied. Twelve patients were untreated, one had been pretreated with octreotide (12 weeks, 3 x 100 micrograms daily). GH, PRL, alpha-subunit and IGF-I concentrations in plasma, media and cell extracts were determined by immunoradiometric or radioimmuno-assays. GH mRNA levels were determined by automatic quantification of grain numbers in individual adenoma cells. Incubation of the adenoma cells for 4 days with 10 nM octreotide induced a dose-dependent inhibition of GH release and a parallel increase (increase varying between 124 and 617% of control) in the intracellular GH levels was observed in six of seven adenomas. In addition, bromocriptine, when effective in inhibiting GH release by the adenomas, also induced an increase in intracellular GH levels. Even after 3 weeks of exposure to 10 nM octreotide in vitro there was a statistically significant increase in intracellular GH levels (between 191 and 923% of control). Withdrawal of octreotide after 6 days of incubation resulted in a lowering of intracellular GH levels to control values, showing that the octreotide-induced increase in intracellular GH is reversible. In a 96-hour incubation with 10 nM octreotide, GH mRNA levels were increased in two, and slightly decreased in one of the three adenomas tested. This effect was time dependent in that there was no significant effect of 10 nM octreotide on GH mRNA levels in a 24-hour incubation. (1) Long-term in-vitro exposure

  12. Effect of Bifidobacterium on the mRNA expression levels of TRAF6, GSK-3β, and microRNA-146a in LPS-stimulated rat intestinal epithelial cells.

    Science.gov (United States)

    Zhou, W; Yuan, Y; Li, J; Yuan, W M; Huang, L G; Zheng, S W

    2015-08-21

    We investigated the effect of inactivated Bifidobacterium on the mRNA expression of TRAF6, GSK-3β, and microRNA-146a in lipopolysaccharide (LPS)-stimulated rat small intestinal epithelial cells (IEC-6s). IEC-6s were randomly divided into an LPS group, a culture supernatant group, and an inactivated bacteria group. After stimulation with LPS for 5 h, the three groups were treated as follows: the LPS group was cultured for 24 h with sterile saline; the culture supernatant group was cultured with Bifidobacterium (infantis strain) culture supernatant for 24 h; and the inactivated bacteria group was cultured with inactivated infantis Bifidobacterium for 24 h. Reverse transcription polymerase chain reaction was used to determine mRNA expression levels. The mRNA expression levels of TRAF-6 and GSK-3β in the culture supernatant group were lower, and microRNA-146a expression was higher, compared with the LPS group (t = 5.278, P = 0.000; t = 6.316, P = 0.000; t = 13.218, P = 0.000, respectively). GSK-3β mRNA expression in the inactivated bacteria group was lower than in the LPS group (t = 4.837, P = 0.000). There was no difference in the mRNA expression levels of TRAF-6 and microRNA-146a between the two groups (t = 0.732, P = 0.472 and t = 1.463, P = 0.164). Both the culture supernatant and the inactivated Bifidobacterium had a protective effect on LPS-stimulated IEC-6s. The protective effect of Bifidobacterium may be achieved through increased microRNA-146a by reducing levels of TRAF6 and GSK-3β; the protective effect of inactivated Bifidobacterium may be achieved by reducing levels of GSK-3β.

  13. Increase in IL-8, IL-10, IL-13, and RANTES mRNA levels (in situ hybridization) in the nasal mucosa after nasal allergen provocation

    NARCIS (Netherlands)

    Kleinjan, A.; Dijkstra, M. D.; Boks, S. S.; Severijnen, L. A.; Mulder, P. G.; Fokkens, W. J.

    1999-01-01

    BACKGROUND: Allergic inflammation is regulated by the local production and release of several cytokines. OBJECTIVES: This study was designed to assess the changes in mRNA cytokine-positive cells after allergen provocation and to compare these cytokines with tissue eosinophilia as a marker of

  14. The effects of progesterone on oxytocin mRNA levels in the paraventricular nucleus of the female rat can be altered by the administration of diazepam or RU486.

    Science.gov (United States)

    Thomas, A; Shughrue, P J; Merchenthaler, I; Amico, J A

    1999-02-01

    Oxytocin (OT) facilitates the onset of maternal behaviour in the late pregnant rat, enhances uterine contractility at parturition, and elicits milk ejection during lactation. If the rising estradiol (E2 and declining progesterone (P) of late pregnancy is reproduced in a virgin ovariectomized rat by implanting E2- and P-filled capsules for 2 weeks followed by removal of P-containing implants 36-48 h prior to death, OT messenger ribonucleic acid (mRNA) levels increase in the paraventricular and supraoptic nuclei (PVN and SON) of the rat. Both E2 administration and P withdrawal are necessary to increase OT mRNA, but the mechanisms of these effects are not understood. P may work within the PVN although P receptors are reported to be sparse or non-existent in the PVN or outside the PVN on PR-containing neurones that project to OT-containing neurones or via membrane bound receptors that are known to bind neurosteroids and gamma aminobutyric acid (GABA). To determine the mechanism through which P may inhibit or P withdrawal may increase OT mRNA levels, virgin ovariectomized (OVX) rats received sequential E2 and P via Silastic implants for 14 days. On day 13, prior to removal of P capsules on day 14, the rats were given the benzodiazepine agonist, diazepam, or saline injections subcutaneously (s.c.) twice daily until death on day 16. OT mRNA levels were increased in the steroid-treated group that received saline but not diazepam. In experiment 2, P capsules were removed on day 14 or pharmacological P withdrawal was induced by injecting RU486 injections s.c. twice daily until death 48 h later. OT mRNA levels were increased in the steroid-treated group that received RU486. Subsequent studies demonstrated the expression of PR mRNA within the rat PVN. The data suggest that gonadal steroids may influence PVN OT mRNA levels by modulating the GABA(A) receptor or by directly altering gene transcription via the PR.

  15. 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

    Energy Technology Data Exchange (ETDEWEB)

    Kadowaki, T.; Kadowaki, H.; Taylor, S.I. (National Institutes of Health, Bethesda, MD (USA))

    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.

  16. EZH2 promotes malignant behaviors via cell cycle dysregulation and its mRNA level associates with prognosis of patient with non-small cell lung cancer.

    Directory of Open Access Journals (Sweden)

    Wei Cao

    Full Text Available Epigenetic silencing is a common mechanism to inactivate tumor suppressor genes during carcinogenesis. Enhancer of Zeste 2 (EZH2 is the histone methyltransferase subunit in polycomb repressive complex 2 which mediates transcriptional repression through histone methylation. EZH2 overexpression has been linked to aggressive phenotypes of certain cancers. However, the mechanism that EZH2 played in promoting malignancy in non-small cell lung cancer (NSCLC remains unclear. In addition, the correlation of EZH2 overexpression and the prognosis of NSCLC patients in non-Asian cohort need to be determined.Up-regulation of EZH2 was found in NSCLC cells compared with normal human bronchial epithelial cells by western blot assay. Upon EZH2 knockdown using small interfering RNA (siRNA, the proliferation, anchorage-independent growth and invasion of NSCLC cells were remarkably suppressed with profound induction of G1 arrest. Furthermore, the expression of cyclin D1 was notably reduced whereas p15(INK4B, p21(Waf1/Cip1 and p27(Kip1 were increased in NSCLC cells after EZH2-siRNA delivery. To determine whether EZH2 expression contributes to disease progression in patients with NSCLC, Taqman quantitative real-time RT-PCR was used to measure the expression of EZH2 in paired tumor and normal samples. Univariate analysis revealed that patients with NSCLC whose tumors had a higher EZH2 expression had significantly inferior overall, disease-specific, and disease-free survivals compared to those whose tumors expressed lower EZH2 (P = 0.005, P = 0.001 and P = 0.003, respectively. In multivariate analysis, EZH2 expression was an independent predictor of disease-free survival (hazard ratio = 0.450, 95% CI: 0.270 to 0.750, P = 0.002.Our results demonstrate that EZH2 overexpression is critical for NSCLC progression. EZH2 mRNA levels may serve as a prognostic predictor for patients with NSCLC.

  17. [Transcription factors NF-kB, HIF-1, HIF-2, growth factor VEGF, VEGFR2 and carboanhydrase IX mRNA and protein level in the development of kidney cancer metastasis].

    Science.gov (United States)

    Spirina, L V; Usynin, Y A; Yurmazov, Z A; Slonimskaya, E M; Kolegova, E S; Kondakova, I V

    2017-01-01

    Here, we have investigated the participation of nuclear factors NF-kB, HIF-1 and HIF-2, VEGF, VEGFR2, and carboanhydrase IX in clear-cell renal cancer. We have determined the expression and protein level of transcription factors, VEGF, VEGFR2, and carboanhydrase IX in tumor and normal tissues of 30 patients with kidney cancer. The Real-Time PCR and ELISA were used in the study. The low levels of HIF-1 mRNA expression associated with high levels of HIF-1 protein were also associated with metastasis. The expression levels of VEGF, VEGFR2, and their protein levels are increased in primary tumors of patients with disseminated kidney cancer compared to nonmetastatic cancer. No correlation was revealed between the content of mRNA and encoded proteins in the kidney cancer tissues. The changes in the ratios of mRNA levels and the respective proteins (HIF-1α, HIF-2, NF-kB, VEGF, VEGFR2, and carboanhydrase IX) may contribute to kidney-cancer metastasis.

  18. Effects of environmental stress on mRNA expression levels of seven genes related to oxidative stress and growth in Atlantic salmon Salmo salar L. of farmed, hybrid and wild origin

    Science.gov (United States)

    2012-01-01

    Background Ten generations of domestication selection has caused farmed Atlantic salmon Salmo salar L. to deviate from wild salmon in a range of traits. Each year hundreds of thousands of farmed salmon escape into the wild. Thus, interbreeding between farmed escapees and wild conspecifics represents a significant threat to the genetic integrity of wild salmon populations. In a previous study we demonstrated how domestication has inadvertently selected for reduced responsiveness to stress in farmed salmon. To complement that study, we have evaluated the expression of seven stress-related genes in head kidney of salmon of farmed, hybrid and wild origin exposed to environmentally induced stress. Results In general, the crowding stressor used to induce environmental stress did not have a strong impact on mRNA expression levels of the seven genes, except for insulin-like growth factor-1 (IGF-1) that was downregulated in the stress treatment relative to the control treatment. mRNA expression levels of glutathione reductase (GR), Cu/Zn superoxide dismutase (Cu/Zn SOD), Mn superoxide dismutase (Mn SOD), glutathione peroxidase (GP) and IGF-1 were affected by genetic origin, thus expressed significantly different between the salmon of farmed, hybrid or wild origin. A positive relationship was detected between body size of wild salmon and mRNA expression level of the IGF-1 gene, in both environments. No such relationship was observed for the hybrid or farmed salmon. Conclusion Farmed salmon in this study displayed significantly elevated mRNA levels of the IGF-1 gene relative to the wild salmon, in both treatments, while hybrids displayed a non additive pattern of inheritance. As IGF-1 mRNA levels are positively correlated to growth rate, the observed positive relationship between body size and IGF-1 mRNA levels detected in the wild but neither in the farmed nor the hybrid salmon, could indicate that growth selection has increased IGF-1 levels in farmed salmon to the extent

  19. Brain region specific alterations in the protein and mRNA levels of protein kinase A subunits in the post-mortem brain of teenage suicide victims.

    Science.gov (United States)

    Pandey, Ghanshyam N; Dwivedi, Yogesh; Ren, Xinguo; Rizavi, Hooriyah S; Mondal, Amal C; Shukla, Pradeep K; Conley, Robert R

    2005-08-01

    Protein kinase A (PKA), a critical component of the adenylyl cyclase signaling system, phosphorylates crucial proteins and has been implicated in the pathophysiology of depression and suicide. The objective of the study was to examine if changes in PKA activity or in the protein and messenger RNA (mRNA) expression of any of its subunits are related to the pathophysiology of teenage suicide. We determined PKA activity and the protein and mRNA expression of different subunits of PKA in cytosol and membrane fractions obtained from the prefrontal cortex, (PFC) hippocampus, and nucleus accumbens (NA) of post-mortem brain from 17 teenage suicide victims and 17 nonpsychiatric control subjects. PKA activity was significantly decreased in the PFC but not the hippocampus of teenage suicide victims as compared with controls. However, the protein and mRNA expression of only two PKA subunits, that is, PKA RIalpha and PKA RIbeta, but not any other subunits were significantly decreased in both membrane and cytosol fractions of the PFC and protein expression of RIalpha and RIbeta in the NA of teenage suicide victims as compared to controls. A decrease in protein and mRNA expression of two specific PKA subunits may be associated with the pathogenesis of teenage suicide, and this decrease may be brain region specific, which may be related to the specific behavioral functions associated with these brain areas. Whether these changes in PKA subunits are related to suicidal behavior or are a result of suicide or are specific to suicide is not clear at this point.

  20. 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.

  1. Evaluation of alpha 1-antitrypsin and the levels of mRNA expression of matrix metalloproteinase 7, urokinase type plasminogen activator receptor and COX-2 for the diagnosis of colorectal cancer.

    Directory of Open Access Journals (Sweden)

    Luis Bujanda

    Full Text Available BACKGROUND: Colorectal cancer (CRC is the second most common cause of death from cancer in both men and women in the majority of developed countries. Molecular tests of blood could potentially provide this ideal screening tool. AIM: Our objective was to assess the usefulness of serum markers and mRNA expression levels in the diagnosis of CRC. METHODS: In a prospective study, we measured mRNA expression levels of 13 markers (carbonic anhydrase, guanylyl cyclase C, plasminogen activator inhibitor, matrix metalloproteinase 7 (MMP7, urokinase-type plasminogen activator receptor (uPAR, urokinase-type plasminogen activator, survivin, tetranectin, vascular endothelial growth factor (VEGF, cytokeratin 20, thymidylate synthase, cyclooxygenase 2 (COX-2, and CD44 and three proteins in serum (alpha 1 antitrypsin, carcinoembryonic antigen (CEA and activated C3 in 42 patients with CRC and 33 with normal colonoscopy results. RESULTS: Alpha 1-antitrypsin was the serum marker that was most useful for CRC diagnosis (1.79 ± 0.25 in the CRC group vs 1.27 ± 0.25 in the control group, P<0.0005. The area under the ROC curve for alpha 1-antitrypsin was 0.88 (0.79-0.96. The mRNA expression levels of five markers were statistically different between CRC cases and controls: those for which the ROC area was over 75% were MMP7 (0.81 and tetranectin (0.80, COX-2 (0.78, uPAR (0.78 and carbonic anhydrase (0.77. The markers which identified early stage CRC (Stages I and II were alpha 1-antitrypsin, uPAR, COX-2 and MMP7. CONCLUSIONS: Serum alpha 1-antitrypsin and the levels of mRNA expression of MMP7, COX-2 and uPAR have good diagnostic accuracy for CRC, even in the early stages.

  2. 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…

  3. 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

    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...

  4. Epithelial remodeling and claudin mRNA abundance in the gill and kidney of puffer fish (Tetraodon biocellatus) acclimated to altered environmental ion levels.

    Science.gov (United States)

    Duffy, Nicole M; Bui, Phuong; Bagherie-Lachidan, Mazdak; Kelly, Scott P

    2011-02-01

    In water of varying ion content, the gills and kidney of fishes contribute significantly to the maintenance of salt and water balance. However, little is known about the molecular architecture of the tight junction (TJ) complex and the regulation of paracellular permeability characteristics in these tissues. In the current studies, puffer fish (Tetraodon biocellatus) were acclimated to freshwater (FW), seawater (SW) or ion-poor freshwater (IPW) conditions. Following acclimation, alterations in systemic endpoints of hydromineral status were examined in conjunction with changes in gill and kidney epithelia morphology/morphometrics, as well as claudin TJ protein mRNA abundance. T. biocellatus were able to maintain endpoints of hydromineral status within relatively tight limits across the broad range of water ion content examined. Both gill and kidney tissue exhibited substantial alterations in morphology as well as claudin TJ protein mRNA abundance. These responses were particularly pronounced when comparing fish acclimated to SW versus those acclimated to IPW. TEM observations of IPW-acclimated fish gills revealed the presence of cells that exhibited the typical characteristics of gill mitochondria-rich cells (e.g. voluminous, Na(+)-K(+)-ATPase-immunoreactive, exposed to the external environment at the apical surface), but were not mitochondria-rich. To our knowledge, this type of cell has not previously been described in hyperosmoregulating fish gills. Furthermore, modifications in the morphometrics and claudin mRNA abundance of kidney tissue support the notion that spatial alterations in claudin TJ proteins along the nephron of fishes will likely play an important role in the regulation of salt and water balance in these organisms.

  5. The effect of 1,25 dihydroxyvitamin D3 treatment on the mRNA levels of β catenin target genes in mice with colonic inactivation of both APC alleles.

    Science.gov (United States)

    DeWitt, Marsha; Johnson, Robert L; Snyder, Paul; Fleet, James C

    2015-04-01

    In colon cancer, adenomatous polyposis coli (APC) inactivating gene mutations increase nuclear β-catenin levels and stimulate proliferation. In vitro, 1,25 dihydroxyvitamin D (1,25(OH)2D), suppresses β-catenin-mediated gene transcription by inducing vitamin D receptor (VDR)-β-catenin interactions. We examined whether acute treatment with 1,25(OH)2D could suppress β-catenin-mediated gene transcription in the hyperplastic colonic lesions of mice with colon-specific deletion of both APC gene alleles (CAC; APC(Δ580/Δ580)). At four weeks of age, CAC; APC(Δ580/Δ580) and control mice were injected with vehicle or 1,25(OH)2D (1μg/kg body weight) once a day for three days and then killed six hours after the last injection. mRNA levels of β-catenin target genes were elevated in the colon of CAC; APC(Δ580/Δ580) mice. 1,25(OH)2D increased 25 hydroxyvitamin D-24 hydroxylase mRNA levels in the colon of CAC; APC(Δ580/Δ580) and control mice indicating the treatments activated the VDR. However, 1,25(OH)2D had no effect on either β-catenin target gene mRNA levels or the proliferation index in CAC; APC(Δ580/Δ580) or control mice. VDR mRNA and protein levels were lower (-65% and -90%) in the colon of CAC; APC(Δ580/Δ580) mice compared to control mice, suggesting loss of colon responsiveness to vitamin D. Consistent with this, vitamin D-induced expression of transient receptor potential cation channel, subfamily V, member 6 mRNA was reduced in the colon of CAC; APC(Δ580/Δ580) mice. Our data show that short term exposure to 1,25(OH)2D does not suppress colonic β-catenin signaling in vivo. This article is part of a special issue entitled '17th Vitamin D Workshop'. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Analysis of TLR4 (Asp299Gly and Thr399Ile) gene polymorphisms and mRNA level in patients with dengue infection: A case-control study.

    Science.gov (United States)

    Sharma, Swati; Singh, Satyendra K; Kakkar, Kavita; Nyari, Nikky; Dhole, Tapan N; Kashyap, Rajesh; Hasan, Saba

    2016-09-01

    Dengue is a systemic viral infection that spreads to humans by the bite of infected Aedes mosquitoes. The secreted NS1 protein of dengue virus activates macrophages and human PBMCs via TLR4 and induce the release of pro-inflammatory cytokines which is responsible for the pathogenesis of disease. Mutations in TLR4 gene have been associated with the increased susceptibility to many viral, bacterial and parasitic diseases. To study the impact of TLR4 Asp299Gly (rs4986790) and Thr399Ile (rs4986791) gene polymorphisms with susceptibility to dengue infection. A total of 120 dengue infected (57; DHF/DSS and 63; DF) and 200 healthy controls were included in the study. TLR4 Asp299Gly and Thr399Ile gene polymorphisms was studied by PCR-RFLP. Expression of TLR4 mRNA was evaluated by rRT-PCR. Individuals with heterozygous genotype for TLR4 Asp299Gly and Thr399Ile polymorphisms had increased susceptibility to dengue infection (OR-1.70, 95% CI=1.01-2.86 P=0.042 and OR-2.17, 95% CI=1.10-4.28, P=0.024, respectively). The frequency of Gly and Ile alleles were higher in dengue patients as compared to controls (OR-1.67, 95% CI=1.05-2.64, P=0.029 and OR-2.20, 95% CI=1.19-4.07, P=0.011, respectively). IIe/Gly haplotype was associated with the risk of the disease when compared with controls (OR=3.15, 95% CI=1.09-9.09, P=0.035). The mRNA expression was higher in DF when compared with DHF/DSS and controls (P=0.040 and 0.009, respectively). A higher expression of TLR4 mRNA was associated with DF. The TLR4 Asp299Gly and Thr399Ile gene polymorphisms were associated with the susceptibility of dengue infection probably by altering the immune response. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. 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.

  8. 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.

  9. Comparison of transcript levels and mRNA half-lives for the subunits of the branched-chain {alpha}-keto acid dehydrogenase (BCKD) complex in two human cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Bowers, B.A.; Danner, D.J. [Emory Univ., Atlanta, GA (United States)

    1994-09-01

    BCKD is a mitochondrial multienzyme complex that catalyzes the committed step in catabolism of the keto acid derivatives of leucine, isoleucine and valine. Three subunits, El{alpha}, E1{beta} and E2 are specific to the complex. The subunits are nuclearly encoded from genes located on separate chromosomes, and it is not yet understood how gene expression of the components is regulated to maintain proper stoichiometry of the complex. The focus of the present study is to establish mRNA half-lives for the BCKD subunits in two human cell lines and to examine whether expression of transcripts for the subunits is similar in different cell types. HepG2 cells, a hepatocarcinoma cell line, and DG75 cells, a Burkitt`s lymphoma cell line, express comparable levels of BCKD complex based on total enzyme activity. Half-lives of the mRNAs for each subunit have been determined in HepG2 cells and are presently being defined in DG75 cells. mRNA half-lives were calculated by quantifying message levels over a 24 hour period following an actinomycin D block. Transcripts for the BCKD subunits are relatively stable in HepG2 cells with mRNA half-lives for the E1{alpha} of 11 hours, E1{beta}, 24 hours and E2, 22 hours. Steady-state message levels have been analyzed in both cell lines by RNase protection and quantified as a percentage of total RNA. mRNA levels for all three subunits are higher in DG75 cells than in HepG2 cells (E1{alpha}, 4-fold; E1{beta}, 1.9-fold; E2, 1.8-fold). Preliminary data indicates that the half-life of the E1{alpha} transcript in DG75 cells is approximately 29 hours, and it is possible that differences in steady-state levels of the mRNAs are achieved through different half-lives of the transcripts. The relationship between transcript levels and protein levels for the three subunits is being examined in both cell types.

  10. Identification of a novel P450 gene belonging to the CYP4 family in the clam Ruditapes philippinarum, and analysis of basal- and benzo(a)pyrene-induced mRNA expression levels in selected tissues.

    Science.gov (United States)

    Pan, Luqing; Liu, Na; Xu, Chaoqun; Miao, Jingjing

    2011-11-01

    A novel full-length cDNA encoding a CYP4 protein was initially cloned from the clam, Ruditapes philippinarum. The nucleotide sequence contained an open reading frame coding for 442 amino acids and the deduced amino acid sequence showed 42.6-49.1% identity with other species CYP4s. The phylogenetic analysis demonstrated that the clam CYP4 was clustered within the CYP4s branch. The clam CYP4 mRNA expression was detected in gill, digestive gland, adductor muscle and mantle, and highest transcription level was observed in digestive gland compared to other tissues. Quantitative real-time RT-PCR analysis revealed that there was no notable change in CYP4 mRNA expression in gill of R. philippinarum exposure to benzo(a)pyrene (BaP), while the mRNA expression was induced significantly in the digestive gland of the clam by 0.2 ppb (μgL(-1)) BaP (pclam may serve as a useful biomarker of marine environmental PAH pollution. Copyright © 2011 Elsevier B.V. All rights reserved.

  11. Determination of mRNA, and protein levels of p53, MDM2 and protein kinase CK2 subunits in F9 cells after treatment with the apoptosis-inducing drugs cisplatin and carboplatin

    DEFF Research Database (Denmark)

    Siemer, S; Ornskov, D; Guerra, B

    1999-01-01

    cisplatin and carboplatin on the mRNA and protein levels of p53, MDM2 and CK2 in a murine teratocarcinoma cell line F9. Northern and Western blot analyses were performed and the CK2 activity was determined. The degree of apoptosis after drug treatment was assessed using the TUNEL test. Six hours after...... cisplatin and carboplatin treatment, the RNA level of p53 dropped by 59% +/- 9% and 86% +/- 8% respectively, whereas the observed level of p53 protein rose to 7 and 10 times over the untreated control, respectively. Treatment with 33 microM cisplatin prompted apoptosis as early as 4 h after drug treatment....... More than 50% apoptotic cells were seen after 6 h. We conclude that cisplatin and its second generation drug carboplatin act similarly i.e. both drugs cause a concomitant decrease in p53 mRNA and an increase in p53 protein level. After 4 h treatment with either of the two drugs, p53 levels reach...

  12. Are we missing a mineralocorticoid in teleost fish? Effects of cortisol, deoxycorticosterone and aldosterone on osmoregulation, gill Na+,K+-ATPase activity and isoform mRNA levels in Atlantic salmon

    Science.gov (United States)

    McCormick, S.D.; Regish, A.; O'Dea, M. F.; Shrimpton, J.M.

    2008-01-01

    It has long been held that cortisol, acting through a single receptor, carries out both glucocorticoid and mineralocorticoid actions in teleost fish. The recent finding that fish express a gene with high sequence similarity to the mammalian mineralocorticoid receptor (MR) suggests the possibility that a hormone other than cortisol carries out some mineralocorticoid functions in fish. To test for this possibility, we examined the effect of in vivo cortisol, 11-deoxycorticosterone (DOC) and aldosterone on salinity tolerance, gill Na+,K+-ATPase (NKA) activity and mRNA levels of NKA α1a and α1b in Atlantic salmon. Cortisol treatment for 6–14 days resulted in increased, physiological levels of cortisol, increased gill NKA activity and improved salinity tolerance (lower plasma chloride after a 24 h seawater challenge), whereas DOC and aldosterone had no effect on either NKA activity or salinity tolerance. NKA α1a and α1b mRNA levels, which increase in response to fresh water and seawater acclimation, respectively, were both upregulated by cortisol, whereas DOC and aldosterone were without effect. Cortisol, DOC and aldosterone had no effect on gill glucocorticoid receptor GR1, GR2 and MR mRNA levels, although there was some indication of possible upregulation of GR1 by cortisol (p = 0.07). The putative GR blocker RU486 inhibited cortisol-induced increases in salinity tolerance, NKA activity and NKA α1a and α1b transcription, whereas the putative MR blocker spironolactone had no effect. The results provide support that cortisol, and not DOC or aldosterone, is involved in regulating the mineralocorticoid functions of ion uptake and salt secretion in teleost fish.

  13. 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.

  14. Active immunization with recombinant GnRH fusion protein in boars reduces both testicular development and mRNA expression levels of GnRH receptor in pituitary.

    Science.gov (United States)

    Fang, Fugui; Li, Haidong; Liu, Ya; Zhang, Yunhai; Tao, Yong; Li, Yunsheng; Cao, Hongguo; Wang, Suolu; Wang, Lin; Zhang, Xiaorong

    2010-06-01

    Immunization using recombinant maltose binding protein-gonadotropin releasing hormone (MBP-GnRH6) altered both testicular development and transcription of the pituitary GnRH receptor (GnRHR) gene in boars. Scrotal measurement and blood samples were taken at 4-week interval after immunization at 9 weeks of age. The concentrations of testosterone and anti-GnRH antibodies in serum were determined by radioimmunoassay and enzyme-linked immunosorbent assay, respectively. The results showed that active immunization with MBP-GnRH6 increased the serum concentration of anti-GnRH antibodies (Pimmunized animals as compared with MBP immunized boars. MBP-GnRH6 immunized pigs exhibited mounting behavior 4 weeks later than MBP immunized boars. No mature spermatozoa were observed from MBP-GnRH6 immunized animals. By real-time quantitative PCR analysis, the amount of GnRHR mRNA in the pituitary tissue was found to be significantly lower in MBP-GnRH6 immunized animals than in controls (P<0.05). These data demonstrate that recombinant MBP-GnRH6 was effective in immunological castration in boars. Copyright (c) 2010 Elsevier B.V. All rights reserved.

  15. The effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on corticotrophin-releasing hormone, arginine vasopressin, and pro-opiomelanocortin mRNA levels in the hypothalamus of the cynomolgus monkey.

    Science.gov (United States)

    Shridhar, S; Farley, A; Reid, R L; Foster, W G; Van Vugt, D A

    2001-10-01

    2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a widespread environmental contaminant that has profound deleterious effects on development and reproduction. TCDD may act at one or more levels to alter the hypothalamic-pituitary-adrenal (HPA) and hypothalamic-pituitary-gonadal (HPG) axes. The objective of this study was to investigate whether TCDD modulates neuroendocrine systems by altering gene expression of arginine vasopressin (AVP), corticotrophin-releasing hormone (CRH), or pro-opiomelanocortin (POMC), which are important neuroregulators of the HPA and HPG axes. Four groups of female cynomolgus monkeys (Macaca fascicularis) were administered daily oral doses of gelatin capsule containing TCDD (0, 1, 5, or 25 ng/kg body weight) mixed with glucose 5 days a week for 1 year. At the end of the dosing period, animals were euthanized and brains were harvested. CRH, AVP, and POMC mRNA levels were semiquantified by in situ hybridization histochemistry on 30-microm coronal sections of the brain. Blood collected on the day of euthanasia was assayed for cortisol and progesterone. CRH mRNA levels in the paraventricular nucleus (PVN) were significantly increased by the 2 higher TCDD doses (5 and 25 ng/kg/day) compared to controls (p HPA axis by a central effect involving CRH, but had no effect on the HPG axis at the doses tested.

  16. 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

  17. Lycopene-rich tomato oleoresin modulates plasma adiponectin concentration and mRNA levels of adiponectin, SIRT1, and FoxO1 in adipose tissue of obese rats.

    Science.gov (United States)

    Luvizotto, R A M; Nascimento, A F; Miranda, N C M; Wang, X-D; Ferreira, A L A

    2015-06-01

    To investigate whether lycopene can modulate adiponectin levels and SIRT1 and FoxO1 gene expression in the adipose tissue of diet-induced obese rats. Male Wistar rats were first fed with hypercaloric diet (HD, n = 12) for 6 weeks, and afterward, these rats were randomly assigned to receive HD (n = 6) or HD with lycopene-rich tomato oleoresin (equivalent to lycopene 10 mg/kg body weight (BW)/day, HD + L, n = 6) by gavage for additional 6 weeks. Plasma lycopene and adiponectin levels were analyzed by high-performance liquid chromatography and immunoassay, respectively. The messenger RNA (mRNA) expressions of adiponectin, Sirtuin 1 (SIRT1), Forkhead box O 1 (FoxO1), fatty acid translocase/cluster of differentiation 36 (FAT/CD36), and PPARγ in adipose tissues were determined by quantitative polymerase chain reaction. Lycopene was detected in the plasma of rats in HD + L group but not in the HD group. Although both BW and adiposity were not different between the two groups, there was a significant increase in both plasma concentration and mRNA expression of adiponectin in the adipose tissue of the HD + L group. In addition, the lycopene supplementation upregulated mRNA expressions of SIRT1, FoxO1, and FAT/CD36 but downregulated PPARγ in adipose tissue of obese rats. These data suggest that lycopene, in the concentration used, is not toxic and also its health benefits in adipose tissue may play a role against obesity-related complications. © The Author(s) 2014.

  18. Insulin immuno-neutralization decreases food intake in chickens without altering hypothalamic mRNA levels for genes involved in regulation of food intake and metabolism

    Science.gov (United States)

    Chickens are characterized by rather unique glucose homeostasis, with relatively high blood glucose levels, reduced glucose sensitivity of pancreatic cells, and large resistance to exogenous insulin. In mammals, insulin regulates blood glucose level but also plays a key role in appetite regulation ...

  19. Low-level laser therapy (LLLT; 780 nm) acts differently on mRNA expression of anti- and pro-inflammatory mediators in an experimental model of collagenase-induced tendinitis in rat.

    Science.gov (United States)

    Pires, Débora; Xavier, Murilo; Araújo, Tiago; Silva, José Antônio; Aimbire, Flavio; Albertini, Regiane

    2011-01-01

    Low-level laser therapy (LLLT) has been found to produce anti-inflammatory effects in a variety of disorders. Tendinopathies are directly related to unbalance in expression of pro- and anti-inflammatory cytokines which are responsible by degeneration process of tendinocytes. In the current study, we decided to investigate if LLLT could reduce mRNA expression for TNF-α, IL-1β, IL-6, TGF-β cytokines, and COX-2 enzyme. Forty-two male Wistar rats were divided randomly in seven groups, and tendinitis was induced with a collagenase intratendinea injection. The mRNA expression was evaluated by real-time PCR in 7th and 14th days after tendinitis. LLLT irradiation with wavelength of 780 nm required for 75 s with a dose of 7.7 J/cm(2) was administered in distinct moments: 12 h and 7 days post tendinitis. At the 12 h after tendinitis, the animals were irradiated once in intercalate days until the 7th or 14th day in and them the animals were killed, respectively. In other series, 7 days after tendinitis, the animals were irradiated once in intercalated days until the 14th day and then the animals were killed. LLLT in both acute and chronic phases decreased IL-6, COX-2, and TGF-β expression after tendinitis, respectively, when compared to tendinitis groups: IL-6, COX-2, and TGF-β. The LLLT not altered IL-1β expression in any time, but reduced the TNF-α expression; however, only at chronic phase. We conclude that LLLT administered with this protocol reduces one of features of tendinopathies that is mRNA expression for pro-inflammatory mediators.

  20. Molecular identification of an androgen receptor and its changes in mRNA levels during 17α-methyltestosterone-induced sex reversal in the orange-spotted grouper Epinephelus coioides.

    Science.gov (United States)

    Shi, Yu; Liu, Xiaochun; Zhang, Haifa; Zhang, Yong; Lu, Danqi; Lin, Haoran

    2012-09-01

    Androgens play a crucial role in sex differentiation, sexual maturation, and spermatogenesis in vertebrates. The action of androgens is mediated via androgen receptors (ARs). The present study reports the cloning of the cDNA sequence of the ar in the orange-spotted grouper, with high expression in testis and relatively low in subdivision of brain areas. The cDNA sequence of ar was 2358 bp, encoding a protein of 759 amino acids (aa). Phylogenetic analysis showed that the ar cDNA sequence was closely related to that of threespot wrasse (Halichoeres trimaculatus) and medaka (Oryzias latipes) arβ. As deduced from the phylogenetic tree and the high amino acid identity with the ARβ subtype of other teleosts, grouper ar seems to be more closely related to the beta than the alpha subtype cloned to date. In the first week after 17α-methyltestosterone (MT) implantation, the transcript levels of ar in the hypothalamus declined significantly, and consistently stayed at low level expression to the second week, but increased back to the control levels in the third and fourth week. In the gonad, the mRNA expression of ar was not changed in the first week compared with the control, but increased significantly in the second week, consistently reached the highest level in the third week, dropped slightly but still higher than that of the control in the fourth week. The expression pattern of ar in hypothalamus and gonad during MT-induced sex reversal suggests the involvement of ar in regulating this process in the orange-spotted grouper. The present study provides the data of the changes in the mRNA levels of ar during MT-induced sex reversal in detail to help understand the complicated signals under sex reversal. Copyright © 2012 Elsevier Inc. All rights reserved.

  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. The TNF-alpha system in heart failure and after heart transplantation : plasma protein levels, mRNA expression, soluble receptors and plasma buffer capacity

    NARCIS (Netherlands)

    van Riemsdijk-van Overbeeke, I C; Baan, C C; Niesters, H G; Hesse, C J; Loonen, E H; Balk, A H; Maat, A P; Weimar, W

    BACKGROUND: The two soluble tumour necrosis factor (TNF) receptors (sTNF-R1, sTNF-R2) can bind TNF-alpha, which is a cytokine with cardiodepressant properties. In heart failure and after heart transplantation, the TNF-alpha system is unbalanced, due to elevated levels of sTNF receptors. AIM: To

  3. Tumor BRCA1, RRM1 and RRM2 mRNA expression levels and clinical response to first-line gemcitabine plus docetaxel in non-small-cell lung cancer patients.

    Directory of Open Access Journals (Sweden)

    Ioannis Boukovinas

    Full Text Available BACKGROUND: Overexpression of RRM1 and RRM2 has been associated with gemcitabine resistance. BRCA1 overexpression increases sensitivity to paclitaxel and docetaxel. We have retrospectively examined the effect of RRM1, RRM2 and BRCA1 expression on outcome to gemcitabine plus docetaxel in advanced non-small-cell lung cancer (NSCLC patients. METHODOLOGY AND PRINCIPAL FINDINGS: Tumor samples were collected from 102 chemotherapy-naïve advanced NSCLC patients treated with gemcitabine plus docetaxel as part of a randomized trial. RRM1, RRM2 and BRCA1 mRNA levels were assessed by quantitative PCR and correlated with response, time to progression and survival. As BRCA1 levels increased, the probability of response increased (Odds Ratio [OR], 1.09: p = 0.01 and the risk of progression decreased (hazard ratio [HR], 0.99; p = 0.36. As RRM1 and RRM2 levels increased, the probability of response decreased (RRM1: OR, 0.97; p = 0.82; RRM2: OR, 0.94; p<0.0001 and the risk of progression increased (RRM1: HR, 1.02; p = 0.001; RRM2: HR, 1.005; p = 0.01. An interaction observed between BRCA1 and RRM1 allowed patients to be classified in three risk groups according to combinations of gene expression levels, with times to progression of 10.13, 4.17 and 2.30 months (p = 0.001. Low BRCA1 expression was the only factor significantly associated with longer time to progression in 31 patients receiving cisplatin-based second-line therapy. CONCLUSIONS: The mRNA expression of BRCA1, RRM1 and RRM2 is potentially a useful tool for selecting NSCLC patients for individualized chemotherapy and warrants further investigation in prospective studies.

  4. Association between fertilin beta, protamines 1 and 2 and spermatid-specific linker histone H1-like protein mRNA levels, fertilization ability of human spermatozoa, and quality of preimplantation embryos.

    Directory of Open Access Journals (Sweden)

    Bartosz Kempisty

    2008-04-01

    Full Text Available Fertilization involves a series of cellular interactions culminating in the fusion of gamete membranes, creating a zygote and then an embryo. During the process of human fertilization in vivo or in conventional in vitro fertilization (IVF, sperm must be capable of undergoing the acrosome reaction, binding to the zona pellucida (ZP, and penetrating the ZP to fuse with the oolema. The key role in this process is played by fertilin beta. Protamines and histones are the proteins that bind to sperm chromatin and contribute in chromatin remodeling during early spermiogenesis. It has been suggested that these proteins may also participate in successful fertilization and embryo development. Using reverse transcription and real-time quantitative PCR reaction (QR-PCR methods and zygote and embryo scoring, we compared fertilin beta, protamine 1 (PRM1, protamine 2 (PRM2, spermatid-specific linker histone 1 (HILS1 mRNAs levels, in vitro fertilization ability of mature spermatozoa, and quality of embryos obtained from in vitro fertilization (IVF. We found significantly lower contents of fertilin beta transcript in spermatozoa from patients in which IVF fertilization failed (p<0.001. We also noticed a correlation between high levels of fertilin beta and increased quality of embryos (p<0.05. We observed an increase in PRM1 and PRM2 mRNA levels in spermatozoa obtained from patients with successful in vitro fertilization versus compared to the number of these transcripts isolated from spermatozoa of patients in which in vitro fertilization failed (P<0.001, (P<0.001, respectively. We found direct correlation between PRM1 and PRM2 mRNA levels to the quality of embryos (r=0.31, P=0.012, (r=0.31, P=0.011, respectively. The differences in HILS1 mRNA contents between these two groups were not statistically significant (P>0,05. We did not observe an association between HILS1 transcript contents and quality of embryos (r=0.22, P=0.076. We suggest that fertilin beta and

  5. mRNA levels of related Abcb genes change opposite to each other upon histone deacetylase inhibition in drug-resistant rat hepatoma cells.

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    Adám Sike

    Full Text Available The multidrug-resistant phenotype of tumor cells is acquired via an increased capability of drug efflux by ABC transporters and causes serious problems in cancer treatment. With the aim to uncover whether changes induced by epigenetic mechanisms in the expression level of drug transporter genes correlates with changes in the drug resistance phenotypes of resistant cells, we studied the expression of drug transporters in rat hepatoma cell lines. We found that of the three major rat ABC transporter genes Abcb1a, Abcb1b and Abcc1 the activity of only Abcb1b increased significantly in colchicine-selected, drug-resistant cells. Increased transporter expression in drug-resistant cells results primarily from transcriptional activation. A change in histone modification at the regulatory regions of the chromosomally adjacent Abcb1a and Abcb1b genes differentially affects the levels of corresponding mRNAs. Transcriptional up- and down-regulation accompany an increase in acetylation levels of histone H3 lysine 9 at the promoter regions of Abcb1b and Abcb1a, respectively. Drug efflux activity, however, does not follow tightly the transcriptional activity of drug transporter genes in hepatoma cells. Our results point out the need for careful analysis of cause-and-effect relationships between changes in histone modification, drug transporter expression and drug resistance phenotypes.

  6. KRAS mutation is a predictor of oxaliplatin sensitivity in colon cancer cells.

    Directory of Open Access Journals (Sweden)

    Yu-Lin Lin

    Full Text Available Molecular biomarkers to determine the effectiveness of targeted therapies in cancer treatment have been widely adopted in colorectal cancer (CRC, but those to predict chemotherapy sensitivity remain poorly defined. We tested our hypothesis that KRAS mutation may be a predictor of oxaliplatin sensitivity in CRC. KRAS was knocked-down in KRAS-mutant CRC cells (DLD-1(G13D and SW480(G12V by small interfering RNAs (siRNA and overexpressed in KRAS-wild-type CRC cells (COLO320DM by KRAS-mutant vectors to generate paired CRC cells. These paired CRC cells were tested by oxaliplatin, irinotecan and 5FU to determine the change in drug sensitivity by MTT assay and flow cytometry. Reasons for sensitivity alteration were further determined by western blot and real-time quantitative reverse transcriptase polymerase chain reaction (qRT -PCR. In KRAS-wild-type CRC cells (COLO320DM, KRAS overexpression by mutant vectors caused excision repair cross-complementation group 1 (ERCC1 downregulation in protein and mRNA levels, and enhanced oxaliplatin sensitivity. In contrast, in KRAS-mutant CRC cells (DLD-1(G13D and SW480(G12V, KRAS knocked-down by KRAS-siRNA led to ERCC1 upregulation and increased oxaliplatin resistance. The sensitivity of irinotecan and 5FU had not changed in the paired CRC cells. To validate ERCC1 as a predictor of sensitivity for oxaliplatin, ERCC1 was knocked-down by siRNA in KRAS-wild-type CRC cells, which restored oxaliplatin sensitivity. In contrast, ERCC1 was overexpressed by ERCC1-expressing vectors in KRAS-mutant CRC cells, and caused oxaliplatin resistance. Overall, our findings suggest that KRAS mutation is a predictor of oxaliplatin sensitivity in colon cancer cells by the mechanism of ERCC1 downregulation.

  7. Measuring ERCC1 protein expression in cancer specimens

    DEFF Research Database (Denmark)

    Smith, David Hersi; Fiehn, Anne-Marie Kanstrup; Fogh, Louise

    2014-01-01

    Platinum chemotherapy remains part of standard therapies in the management of a variety of cancers. Severe side effects and a high degree of resistance to platinum drugs have led numerous researchers to search for predictive biomarkers, which could aid in identifying patients that are the most li...

  8. Structure and Stability of ERCC1-XPF DNA Repair Complexes

    NARCIS (Netherlands)

    Faridounnia, M.

    2015-01-01

    Understanding DNA repair pathways such as Nucleotide Excision Repair, Double Strand Break repair and Interstrand Cross-Link repair is of basic interest for understanding fundamental cellular processes. It also forms the basis for understanding molecular details of diseases when defects occur in

  9. Nucleotide excision repair: ERCC1 and TFIIH complexes

    NARCIS (Netherlands)

    A.J. van Vuuren (Hanneke)

    1995-01-01

    textabstractDNA is the carrier of genetic information in living organisms. The information stored in the nucleotide sequence of DNA is transmitted to the offspring by generating identical copies of the parental DNA molecules. Damage in DNA can cause loss of genetic information. Nevertheless, the DNA

  10. Expression of Thyroid Hormone Receptors in Villous Trophoblasts and Decidual Tissue at Protein and mRNA Levels Is Downregulated in Spontaneous and Recurrent Miscarriages.

    Science.gov (United States)

    Ziegelmüller, Brigitte; Vattai, Aurelia; Kost, Bernd; Kuhn, Christina; Hofmann, Simone; Bayer, Birgit; Toth, Bettina; Jeschke, Udo; Ditsch, Nina

    2015-07-01

    Thyroid hormones are essential for the maintenance of pregnancy, and a deficiency in maternal thyroid hormones has been associated with early pregnancy losses. The expression of THRα1, THRβ1 and THRα2 increases with gestational age. The aim of this study was the investigation of the protein and mRNA-levels of THR isoforms THRα1, THRα2, THRβ1 and THRβ2 in normal, spontaneous and recurrent miscarriages. The identification of THR-expressing cells in the decidua was done with double immunofluorescence. The nuclear expression of THRα1, THRα2, THRβ1 and THRβ2 is downregulated at protein level in spontaneous and recurrent miscarriages in villous trophoblast tissue. In decidual tissue, we found a significant downregulation only for THRα1 in spontaneous miscarriages. For recurrent miscarriages, THRα1 and THRβ1 were both significantly downregulated in decidual tissue. By applying HLA-G as a trophoblast marker, we found a significant co-expression only for THRβ2. The results of our study show that thyroid hormone receptors THRα1, THRα2, THRβ1 and THRβ2 are downregulated in spontaneous and recurrent miscarriages. The majority of cells expressing the thyroid hormone receptors in the decidua are decidual stromal cells. © The Author(s) 2015.

  11. Three mutations in Zea mays affecting zein accumulation: a comparison of zein polypeptides, in vitro synthesis and processing, mRNA levels, and genomic organization

    Energy Technology Data Exchange (ETDEWEB)

    Burr, F.A.; Burr, B.

    1982-07-01

    Researchers studied three mutations, opaque-2 (o2), opaque-7 (o7), and floury-2(fl2), each of which causes a depression in zein synthesis. Researchers examined the processing efficiencies of the rough endoplasmic reticulum membranes in vitro, the levels of RNA transcription using cloned zein probes, and the genomic organization of the zein sequences as possible sites for the genetic defects. The results obtained indicate that the steps in prezein translation and processing occurring on the protein body membranes are not accountable for the lowered zein content in any ofl the mutations. The o2 mutation that typically shows a paucity of 22.5-kdalton zein polypeptides was found to have a concomitant reduction in a particular subgroup of mRNAs coding for this size class. Southern analyses suggest that the 02 mutation is not the result of a large deletion of tandem-linked zein genes.

  12. Protein and mRNA levels support the notion that a genetic regulatory circuit controls growth phases in E. coli populations

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    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.

  13. Expression profile of peripheral tissue antigen genes in medullary thymic epithelial cells (mTECs) is dependent on mRNA levels of autoimmune regulator (Aire).

    Science.gov (United States)

    Oliveira, Ernna H; Macedo, Claudia; Donate, Paula B; Almeida, Renata S; Pezzi, Nicole; Nguyen, Catherine; Rossi, Marcos A; Sakamoto-Hojo, Elza T; Donadi, Eduardo A; Passos, Geraldo A

    2013-01-01

    In the thymus of non-obese diabetic (NOD) mice, the expression of the autoimmune regulator (Aire) gene varies with age, and its down-regulation in young mice precedes the later emergence of type 1 diabetes mellitus (T1D). In addition, the insulin (Ins2) peripheral tissue antigen (PTA) gene, which is Aire-dependent, is also deregulated in these mice. Based in these findings, we hypothesized that the imbalance in PTA gene expression in the thymus can be associated with slight variations in Aire transcript levels. To test this, we used siRNA to knockdown Aire by in vivo electro-transfection of the thymus of BALB/c mice. The efficiency of the electro-transfection was monitored by assessing the presence of irrelevant Cy3-labeled siRNA in the thymic stroma. Importantly, Aire-siRNA reached medullary thymic epithelial cells (mTECs) down-regulating Aire. As expected, the in vivo Aire knockdown was partial and transient; the maximum 59% inhibition occurred in 48 h. The Aire knockdown was sufficient to down-regulate PTA genes; however, surprisingly, several others, including Ins2, were up-regulated. The modulation of these genes after in vivo Aire knockdown was comparable to that observed in NOD mice before the emergence of T1D. The in vitro transfections of 3.10 mTEC cells with Aire siRNA resulted in samples featuring partial (69%) and complete (100%) Aire knockdown. In these Aire siRNA-transfected 3.10 mTECs, the expression of PTA genes, including Ins2, was down-regulated. This suggests that the expression profile of PTA genes in mTECs is affected by fine changes in the transcription level of Aire. Copyright © 2012 Elsevier GmbH. All rights reserved.

  14. Digital multiplexed mRNA analysis of functionally important genes in single human oocytes and correlation of changes in transcript levels with oocyte protein expression☆

    Science.gov (United States)

    Riris, Solon; Webster, Philippa; Homer, Hayden

    2014-01-01

    Objective To investigate functionally important transcripts in single human oocytes with the use of NanoString technology and determine whether observed differences are biologically meaningful. Design Analysis of human oocytes with the use of NanoString and immunoblotting. Setting University-affiliated reproductive medicine unit. Patients Women undergoing in vitro fertilization. Intervention Human oocytes were analyzed with the use of NanoString or immunoblotting. Main Outcome Measures The abundance of transcripts for ten functionally important genes—AURKA, AURKC, BUB1, BUB1B (encoding BubR1), CDK1, CHEK1, FYN, MOS, MAP2K1, and WEE2—and six functionally dispensable genes were analyzed with the use of NanoString. BubR1 protein levels in oocytes from younger and older women were compared with the use of immunoblotting. Result(s) All ten functional genes but none of the six dispensable genes were detectable with the use of NanoString in single oocytes. There was 3- to 5-fold variation in BUB1, BUB1B, and CDK1 transcript abundance among individual oocytes from a single patient. Transcripts for these three genes—all players within the spindle assembly checkpoint surveillance mechanism for preventing aneuploidy—were reduced in the same oocyte from an older patient. Mean BUB1B transcripts were reduced by 1.5-fold with aging and associated with marked reductions in BubR1 protein levels. Conclusion(s) The abundance of functionally important transcripts exhibit marked oocyte-to-oocyte heterogeneity to a degree that is sufficient to affect protein expression. Observed variations in transcript abundance are therefore likely to be biologically meaningful, especially if multiple genes within the same pathway are simultaneously affected. PMID:24444598

  15. Digital multiplexed mRNA analysis of functionally important genes in single human oocytes and correlation of changes in transcript levels with oocyte protein expression.

    Science.gov (United States)

    Riris, Solon; Webster, Philippa; Homer, Hayden

    2014-03-01

    To investigate functionally important transcripts in single human oocytes with the use of NanoString technology and determine whether observed differences are biologically meaningful. Analysis of human oocytes with the use of NanoString and immunoblotting. University-affiliated reproductive medicine unit. Women undergoing in vitro fertilization. Human oocytes were analyzed with the use of NanoString or immunoblotting. The abundance of transcripts for ten functionally important genes-AURKA, AURKC, BUB1, BUB1B (encoding BubR1), CDK1, CHEK1, FYN, MOS, MAP2K1, and WEE2-and six functionally dispensable genes were analyzed with the use of NanoString. BubR1 protein levels in oocytes from younger and older women were compared with the use of immunoblotting. All ten functional genes but none of the six dispensable genes were detectable with the use of NanoString in single oocytes. There was 3- to 5-fold variation in BUB1, BUB1B, and CDK1 transcript abundance among individual oocytes from a single patient. Transcripts for these three genes-all players within the spindle assembly checkpoint surveillance mechanism for preventing aneuploidy-were reduced in the same oocyte from an older patient. Mean BUB1B transcripts were reduced by 1.5-fold with aging and associated with marked reductions in BubR1 protein levels. The abundance of functionally important transcripts exhibit marked oocyte-to-oocyte heterogeneity to a degree that is sufficient to affect protein expression. Observed variations in transcript abundance are therefore likely to be biologically meaningful, especially if multiple genes within the same pathway are simultaneously affected. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

    Michaelsen, Signe Regner; Urup, Thomas; Olsen, Lars Rønn; Broholm, Helle; Lassen, Ulrik; Poulsen, Hans Skovgaard

    2018-01-05

    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. Univariate analysis in the whole patient cohort found that 12 of these were significantly associated with OS, of which increased IFNG, CXCL9, LGALS4, CD34 and decreased MGMT levels remained significant associated with prolonged OS in multivariate analysis correcting for known prognostic variables. Validation analyses in an independent dataset from the AVAglio study confirmed CD34 as significant in comparative analysis between STS and LTS patients and as an independent prognostic factor. Analysis of this dataset further supported CD34 expression to be associated with improved bevacizumab efficacy, while CD34 immunohistochemistry indicated variation in CD34 expression to result primarily from varying tumor vascularization. Collectively, CD34 expression candidates as a prognostic biomarker in GBM able to identify survival outliers and could also be predictive for efficacy of bevacizumab.

  17. Matrix metalloprotease-3 and tissue inhibitor of metalloprotease-1 mRNA and protein levels are altered in response to traumatic skeletal muscle injury.

    Science.gov (United States)

    Urso, Maria L; Szelenyi, Eric R; Warren, Gordon L; Barnes, Brian R

    2010-07-01

    The purpose of this study was to characterize the time course of matrix metalloprotease-3 (MMP-3) and tissue inhibitor of metalloprotease-1 (TIMP-1) expression in mouse tibialis anterior (TA) muscle post-injury. Mice were anesthetized, the TA muscle exposed, and injury induced by applying a cold steel probe (-79 degrees C) to the muscle for 10 s. Muscle was collected from uninjured and injured legs at 3, 10, 24, 48, and 72 h post-injury. qRT-PCR, immunoblotting, and immunohistochemistry were used to quantify/localize MMP-3 and TIMP-1. MMP-3 transcripts increased 19- and 12-fold, 10 and 24 h post-injury (p injury (p = 0.01), respectively, with a subsequent decrease 72 h post-injury (p injury and remained elevated (p injury (p injury, returning to baseline by 72 h post-injury. In response to injury, injured skeletal muscle preferentially produces increased levels of the latent form of the MMP-3 protein with a concomitant decrease in the active form, and a significant decrease in TIMP-1 expression. The altered pattern of MMP-3/TIMP-1 expression may be due to alterations in post-transcriptional mechanisms that are responsible for specific regulation of the MMP-3/TIMP-1 system. These data suggest that there is a disproportionate regulation of the MMP-3/TIMP-1 system following traumatic injury and this response may contribute to impaired extracellular matrix remodeling.

  18. cDNA sequences and mRNA levels of two hexamerin storage proteins PinSP1 and PinSP2 from the Indianmeal moth, Plodia interpunctella.

    Science.gov (United States)

    Zhu, Yu Cheng; Muthukrishnan, Subbaratnam; Kramer, Karl J

    2002-05-01

    In insects, storage proteins or hexamerins accumulate apparently to serve as sources of amino acids during metamorphosis and reproduction. Two storage protein-like cDNAs obtained from a cDNA library prepared from fourth instar larvae of the Indianmeal moth (Plodia interpunctella) were cloned and sequenced. The first clone, PinSP1, contained 2431 nucleotides with a 2295 nucleotide open reading frame (ORF) encoding a protein with 765 amino acid residues. The second cDNA, PinSP2, consisted of 2336 nucleotides with a 2250-nucleotide ORF encoding a protein with 750 amino acid residues. PinSP1 and PinSP2 shared 59% nucleotide sequence identity and 44% deduced amino acid sequence identity. A 17-amino acid signal peptide and a molecular mass of 90.4 kDa were predicted for the PinSP1 protein, whereas a 15-amino acid signal peptide and a mass of 88 kDa were predicted for PinSP2. Both proteins contained conserved insect larval storage protein signature sequence patterns and were 60-70% identical to other lepidopteran larval storage proteins. Expression of mRNA for both larval storage proteins was determined using the quantitative reverse transcription polymerase chain reaction method. Only very low levels were present in the second instar, but both mRNAs dramatically increased during the third instar, peaked in the fourth instar, decreased dramatically late in the same instar and pupal stages, and were undetectable during the adult stage. Males and females exhibited similar mRNA expression levels for both storage proteins during the pupal and adult stages. The results support the hypothesis that P. interpunctella, a species that does not feed after the larval stage, accumulates these two storage proteins as reserves during larval development for subsequent use in the pupal and adult stages.

  19. Diversities in hepatic HIF-1, IGF-I/IGFBP-1, LDH/ICD, and their mRNA expressions induced by CoCl(2) in Qinghai-Tibetan plateau mammals and sea level mice.

    Science.gov (United States)

    Chen, Xue-Qun; Wang, Shi-Jun; Du, Ji-Zeng; Chen, Xiao-Cheng

    2007-01-01

    Ochotona curzoniae and Microtus oeconomus are the native mammals living on the Qinghai-Tibetan-Plateau of China. The molecular mechanisms of their acclimatization to the Plateau-hypoxia remain unclear. Expressions of hepatic hypoxia-inducible factor (HIF)-1alpha, insulin-like growth factor-I (IGF-I)/IGF binding protein (BP)-1(IGFBP-1; including genes), and key metabolic enzymatic genes [lactate dehydrogenase (LDH)-A/isocitrate dehydrogenase (ICD)] are compared in Qinghai-Tibetan-Plateau mammals and sea-level mice after injection of CoCl(2) (20, 40, or 60 mg/kg) and normobaric hypoxia (16.0% O(2), 10.8% O(2), and 8.0% O(2)) for 6 h, tested by histochemistry, Western blot analysis, ELISA, and RT-PCR. Major results are CoCl(2) markedly increased 1) HIF-1alpha only in mice, 2) hepatic and circulatory IGF-I in M. oeconomus, 3) hepatic IGFBP-1 in mice and O. curzoniae, and 4) LDH-A but reduced ICD mRNA in mice (CoCl(2) 20 mg/kg) but were unchanged in the Tibetan mammals. Normobaric hypoxia markedly 1) increased HIF-1alpha and LDH-A mRNA in mice and M. oeconomus (8.0% O(2)) not in O. curzoniae, and 2) reduced ICD mRNA in mice and M. oeconomus (8.0% O(2)) not in O. curzoniae. Results suggest that 1) HIF-1alpha responsiveness to hypoxia is distinct in lowland mice and plateau mammals, reflecting a diverse tolerance of the three species to hypoxia; 2) CoCl(2) induces diversities in HIF-1, IGF-I/IGFBP-1 protein or genes in mice, M. oeconomus, and O. curzoniae. In contrast, HIF-1 mediates IGFBP-1 transcription only in mice and in M. oeconomus (subjected to severe hypoxia); 3) differences in IGF-I/IGFBP-1 expressions induced by CoCl(2) reflect significant diversities in hormone regulation and cell protection from damage; and 4) activation of anaerobic glycolysis and reduction of Krebs cycle represents strategies of lowland-animals vs. the stable metabolic homeostasis of plateau-acclimatized mammals.

  20. A 3'UTR polymorphism marks differential KLRG1 mRNA levels through disruption of a miR-584-5p binding site and associates with pemphigus foliaceus susceptibility.

    Science.gov (United States)

    Cipolla, Gabriel A; Park, Jong Kook; de Oliveira, Liana A; Lobo-Alves, Sara Cristina; de Almeida, Rodrigo C; Farias, Ticiana D J; Lemos, Débora de S; Malheiros, Danielle; Lavker, Robert M; Petzl-Erler, Maria Luiza

    2016-10-01

    Genetic variations mapping to 3' untranslated regions (3'UTRs) may overlap with microRNA (miRNA) binding sites, therefore potentially interfering with translation inhibition or messenger RNA (mRNA) degradation. The aim of this study was to investigate whether single nucleotide polymorphisms (SNPs) located within the 3'UTRs of six candidate genes and predicted to interfere with miRNA ligation could account for disease-relevant differential mRNA levels. Focusing on pemphigus foliaceus (PF) - an autoimmune blistering skin condition with unique endemic patterns - we investigated whether nine 3'UTR SNPs from the CD1D, CTLA4, KLRD1, KLRG1, NKG7, and TNFSF13B genes differentially expressed in PF were disease-associated. The heterozygous genotype of the KLRG1 rs1805672 polymorphism was associated with increased predisposition to PF (A/G vs. A/A: P=0.038; OR=1.60), and a trend for augmented susceptibility was observed for carriers of the G allele (P=0.094; OR=1.44). In silico analyses suggested that rs1805672 G allele could disrupt binding of miR-584-5p, and indicated rs1805672 as an expression Quantitative Trait Locus (eQTL), with an effect on KLRG1 gene expression. Dual-luciferase assay showed that miR-584-5p mediated approximately 50% downregulation of the reporter gene's activity through the 3'UTR of KLRG1 harboring rs1805672 A allele (vs. miRNA-negative condition, P=0.006). This silencing relationship was lost after site-directed mutation to G allele (vs. miRNA-negative condition, P=0.391; vs. rs1805672 A allele, P=0.005). Collectively, these results suggest that a disease-associated SNP located within the 3'UTR of KLRG1 directly interferes with miR-584-5p binding, allowing for KLRG1 mRNA differential accumulation, which in turn may contribute to pathogenesis of autoimmune diseases, such as pemphigus. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. A 3’UTR polymorphism marks differential KLRG1 mRNA levels through disruption of a miR-584-5p binding site and associates with pemphigus foliaceus susceptibility

    Science.gov (United States)

    Cipolla, Gabriel A.; Park, Jong K.; de Oliveira, Liana A.; Lobo-Alves, Sara Cristina; de Almeida, Rodrigo C.; Farias, Ticiana D. J.; Lemos, Débora de S.; Malheiros, Danielle; Lavker, Robert M.; Petzl-Erler, Maria Luiza

    2016-01-01

    Genetic variations mapping to 3’ untranslated regions (3’UTRs) may overlap with microRNA (miRNA) binding sites, therefore potentially interfering with translation inhibition or messenger RNA (mRNA) degradation. The aim of this study was to investigate whether single nucleotide polymorphisms (SNPs) located within the 3’UTRs of six candidate genes and predicted to interfere with miRNA ligation could account for disease-relevant differential mRNA levels. Focusing on pemphigus foliaceus (PF) – an autoimmune blistering skin condition with unique endemic patterns – we investigated if nine 3’UTR SNPs from the CD1D, CTLA4, KLRD1, KLRG1, NKG7, and TNFSF13B genes differentially expressed in PF were disease-associated. The heterozygous genotype of the KLRG1 rs1805672 polymorphism was associated with increased predisposition to PF (A/G vs. A/A: P=0.038; OR=1.60), and a trend for augmented susceptibility was observed for carriers of the G allele (P=0.094; OR=1.44). In silico analyses suggested that rs1805672 G allele could disrupt binding of miR-584-5p, and indicated rs1805672 as an expression Quantitative Trait Locus (eQTL), with an effect on KLRG1 gene expression. Dual-luciferase assay showed that miR-584-5p mediated approximately 50% downregulation of the reporter gene’s activity through the 3’UTR of KLRG1 harboring rs1805672 A allele (vs. miRNA-negative condition, P=0.006). This silencing relationship was lost after site-directed mutation to G allele (vs. miRNA-negative condition, P=0.391; vs. rs1805672 A allele, P=0.005). Collectively, these results suggest that a disease-associated SNP located within the 3’UTR of KLRG1 directly interferes with miR-584-5p binding, allowing for KLRG1 mRNA differential accumulation, which in turn may contribute to pathogenesis of autoimmune diseases, such as pemphigus. PMID:27424220

  2. Hypolipidemic effects of crude extract of adlay seed (Coix lachrymajobi var. mayuen) in obesity rat fed high fat diet: relations of TNF-alpha and leptin mRNA expressions and serum lipid levels.

    Science.gov (United States)

    Kim, Sung Ok; Yun, Su-Jin; Jung, Bomi; Lee, Eunjoo H; Hahm, Dae-Hyun; Shim, Insop; Lee, Hye-Jung

    2004-07-30

    To find out whether the expressions of these adipocyte markers are influenced by oriental medicine, obesity rats induced by high fat diet (HFD) for 8 weeks were injected with 50 mg/100 g body weight adlay seed crude extract (ACE), daily for 4 weeks. The results are summarized as follows: HFD + ACE group significantly reduced food intakes and body weights. Weights of epididymal and peritoneal fat were dramatically increased in HFD groups compared with those of normal diet (ND) group but significantly decreased more in HFD + ACE group than those of HFD + saline group (sham). Those of brown adipocytes were increased in HFD + ACE group compared to ND and sham groups but there was no significant difference. The sizes in white adipose tissue (WAT) by microscope were markedly larger in HFD groups than ND group but considerably reduced in HFD + ACE group compared with sham group. The levels of triglyceride, total-cholesterol and leptin in blood serum were significantly decreased in HFD + ACE group compared to those of sham group. Leptin and TNF-alpha mRNA expressions in WAT of rats were remarkably increased more in sham group than in those of ND group. Those of HFD + ACE group were significantly decreased compared with those of sham group, especially. TNF-alpha mRNA expression in HFD + ACE group was declined more than that of ND group. In conclusion, treatments of ACE modulated expressions of leptin and TNF-alpha and reduced body weights, food intake, fat size, adipose tissue mass and serum hyperlipidemia in obesity rat fed HFD. Accordingly, the oriental medicine extract, adlay seed crude extract, can be considered for obesity therapies controlling.

  3. Lipopolysaccharide-Induced CXCL10 mRNA Level and Six Stimulant-mRNA Combinations in Whole Blood: Novel Biomarkers for Bortezomib Responses Obtained from a Prospective Multicenter Trial for Patients with Multiple Myeloma.

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    Takashi Watanabe

    Full Text Available To identify predictive biomarkers for clinical responses to bortezomib treatment, 0.06 mL of each whole blood without any cell separation procedures was stimulated ex vivo using five agents, and eight mRNAs were quantified. In six centers, heparinized peripheral blood was prospectively obtained from 80 previously treated or untreated, symptomatic multiple myeloma (MM patients with measurable levels of M-proteins. The blood sample was procured prior to treatment as well as 2-3 days and 1-3 weeks after the first dose of bortezomib, which was intravenously administered biweekly or weekly, during the first cycle. Six stimulant-mRNA combinations; that is, lipopolysaccharide (LPS-granulocyte-macrophage colony-stimulating factor (GM-CSF, LPS-CXCL chemokine 10 (CXCL10, LPS-CCL chemokine 4 (CCL4, phytohemagglutinin-CCL4, zymosan A (ZA-GMCSF and ZA-CCL4 showed significantly higher induction in the complete and very good partial response group than in the stable and progressive disease group, as determined by both parametric (t-test and non-parametric (unpaired Mann-Whitney test tests. Moreover, LPS-induced CXCL10 mRNA expression was significantly suppressed 2-3 days after the first dose of bortezomib in all patients, as determined by both parametric (t-test and non-parametric (paired Wilcoxon test tests, whereas the complete and very good partial response group showed sustained suppression 1-3 weeks after the first dose. Thus, pretreatment LPS-CXCL10 mRNA and/or the six combinations may serve as potential biomarkers for the response to bortezomib treatment in MM patients.

  4. 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

    Directory of Open Access Journals (Sweden)

    You R. Chng

    2016-11-01

    Full Text Available 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 may 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. These results provide insights into how P

  5. The Presence of the Y-Chromosome, Not the Absence of the Second X-Chromosome, Alters the mRNA Levels Stored in the Fully Grown XY Mouse Oocyte

    Science.gov (United States)

    Xu, Baozeng; Obata, Yayoi; Cao, Feng; Taketo, Teruko

    2012-01-01

    The oocytes of B6.YTIR sex-reversed female mouse mature in culture but fail to develop after fertilization because of their cytoplasmic defects. To identify the defective components, we compared the gene expression profiles between the fully-grown oocytes of B6.YTIR (XY) females and those of their XX littermates by cDNA microarray. 173 genes were found to be higher and 485 genes were lower in XY oocytes than in XX oocytes by at least 2-fold. We compared the transcript levels of selected genes by RT-PCR in XY and XX oocytes, as well as in XO oocytes missing paternal X-chromosomes. All genes tested showed comparable transcript levels between XX and XO oocytes, indicating that mRNA accumulation is well adjusted in XO oocytes. By contrast, in addition to Y-encoded genes, many genes showed significantly different transcript levels in XY oocytes. We speculate that the presence of the Y-chromosome, rather than the absence of the second X-chromosome, caused dramatic changes in the gene expression profile in the XY fully-grown oocyte. PMID:22792347

  6. MicroRNA-92 expression may be associated with reduced estrogen receptor β1 mRNA levels in cervical portion of uterosacral ligaments in women with pelvic organ prolapse.

    Science.gov (United States)

    He, Ke; Niu, Gang; Gao, Jun; Liu, Jun-Xiu; Qu, Hu

    2016-03-01

    This study examined microRNA-92 (miR-92) expression level in relation to the mRNA level of its potential target gene, estrogen receptor β1 (ERβ1), in female patients diagnosed with pelvic organ prolapse (POP). Between July 2012 and September 2014, a total of 104 patients were recruited at the First Affiliated Hospital of Sun Yat-sen University, which included 56 POP patients and 48 non-POP control subjects. Based on POP-Q score, the POP patients were further categorized into POP II and POP III groups. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to quantify miR-92 expression level. ERβ1 tissue expression was measured by western blot and immunohistochemistry (IHC) methods. SPSS 19.0 software was used for statistical analysis. No remarkable differences were observed between the POP group and non-POP group, and between the POP II and POP III groups, with respect to age, body mass index (BMI), parity, menopause status, and family history of POP. The expression level of miR-92 in the POP group was dramatically higher than the non-POP group (Pligament tissue showed inverse correlation between miR-92 and ERβ1 expression levels in POP patients (Pligaments of women diagnosed with POP, compared to non-POP subjects POP III patients exhibited more severe changes than POP II patients. Further, ERβ1expression is inversely correlated to miR-92 expression. Taken together, our results suggest that miR-92 and ERβ1 expression levels may be used as reliable diagnostic markers for assessing the severity of POP. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  7. 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.

  8. Effects of pectin pentaoligosaccharide from Hawthorn ( Crataegus pinnatifida Bunge. var. Major) on the activity and mRNA levels of enzymes involved in fatty acid oxidation in the liver of mice fed a high-fat diet.

    Science.gov (United States)

    Li, Tuo-Ping; Zhu, Ru-Gang; Dong, Yin-Ping; Liu, Yong-Hui; Li, Su-Hong; Chen, Gang

    2013-08-07

    The regulatory effects of haw pectin pentaoligosaccharide (HPPS) on fatty acid oxidation-related enzyme activities and mRNA levels were investigated in the liver of high fat diet induced hyperlipidemic mice. Results showed that HPPS (150 mg/kg for 10 weeks) significantly suppresses weight gain (32.3 ± 0.26 and 21.1 ± 0.14 g for high-fat diet and HPPS groups, respectively), decreases serum triacylglycerol levels (1.64 ± 0.09 and 0.91 ± 0.02 mmol/L, respectively), and increases lipid excretion in feces (55.7 ± 0.38 and 106.4 ± 0.57 mg/g for total lipid, respectively), compared to high-fat diet as control. HPPS significantly increased the hepatic fatty acid oxidation-related enzyme activities of acyl-CoA oxidase, carnitine palmitoyltransferase I, 3-ketoacyl-CoA thiolase, and 2,4-dienoyl-CoA reductase by 53.8, 74.2, 47.1, and 24.2%, respectively. Meanwhile, the corresponding mRNAs were up-regulated by 89.6, 85.8, 82.9, and 30.9%, respectively. Moreover, HPPS was able to up-regulate the gene and protein expressions of peroxisome proliferator-activated receptor α. Results suggest that continuous HPPS ingestion may be used as dietary therapy to prevent obesity and cardiovascular diseases.

  9. 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

  10. Methods for quantification of in situ hybridization signals obtained by film autoradiography and phosphorimaging applied for estimation of regional levels of calmodulin mRNA classes in the rat brain.

    Science.gov (United States)

    Vizi, S; Palfi, A; Hatvani, L; Gulya, K

    2001-08-01

    Comparative analysis of the regional abundances of the various mRNAs in neural tissues requires the quantitation of target nucleic acid sequences while their tissue distribution is preserved. A quantitative in situ hybridization protocol is presented for the assessment of regional levels of calmodulin (CaM) I, II and III mRNAs in the rat brain. Coronal brain cryostat sections were hybridized with multiple CaM [35S]cRNA probes and co-exposed to an autoradiographic film or storage phosphor screen, together with a membrane-based radioactive standard scale. The membrane scale was calibrated against a brain paste standard scale. Regression analyses of the sensitometric graphs of standard scales corresponding to the autoradiographic film and to the storage phosphor screen were performed by means of exponential (ROD=p(1)(1-exp[-p(2)x])) and linear (LI=ax) functions, respectively (ROD is relative optical density, LI is labeling intensity, and x is radioactivity). The ROD/LI values for the hybridized brain regions were converted into cRNA probe copy numbers (estimations of mRNA copy numbers) through use of the above standard scales. This method was applied to compare the regional abundances of multiple CaM mRNAs in the brains of control, dehydrated, chronic ethanol-treated and ethanol withdrawal-treated animals.

  11. 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.

  12. The involvement of miR-100 in bladder urothelial carcinogenesis changing the expression levels of mRNA and proteins of genes related to cell proliferation, survival, apoptosis and chromosomal stability.

    Science.gov (United States)

    Morais, Denis R; Reis, Sabrina T; Viana, Nayara; Piantino, Camila Berfort; Massoco, Cristina; Moura, Caio; Dip, Nelson; Silva, Iran A; Srougi, Miguel; Leite, Katia Rm

    2014-01-01

    MicroRNAs (miRNA) are small non-coding RNAs that play an important role in the control of gene expression by inhibiting protein translation or promoting messenger RNA degradation. Today, miRNAs have been shown to be involved in various physiological and pathological cellular processes, including cancer, where they can act as oncogenes or tumor suppressor genes. Recently, lowered expression of miR-100, resulting in upregulation of FGFR3, has been correlated with low-grade, non-invasive bladder urothelial cancer, as an alternative oncogenesis pathway to the typical FGFR3 gene mutation. Our aim is to analyze the role of miR-100 in bladder cancer cell lines in controlling the expression of some of its possible target genes, including FGFR3 and its relationship with proliferation, apoptosis and DNA ploidy. The bladder cancer cell lines RT4 and T24 were transfected with pre-miR 100, anti-miR 100 and their respective controls using a lipid-based formulation. After transfection mRNA and protein levels of its supposed target genes THAP2, BAZ2A, mTOR, SMARCA5 and FGFR3 were analyzed by quantitative real time polymerase chain reaction (qRT-PCR) and western blotting. Cell proliferation, apoptosis and DNA ploidy were analyzed by flow cytometry. For statistical analysis, a t-test was applied, p RT4, mTOR (p = 0.023) and SMARCA5 (p = 0.015) in T24. There was a reduction in the expression of all proteins, variable from 22.5% to 57.1% in both cell lines. In T24 miR-100 promoted an increase in cell proliferation and anti-miR 100 promoted apoptosis characterizing miR-100 as an oncomiR in this cell line representative of a high-grade urothelial carcinoma. miR-100 transfection reduces expression of BAZ2A, mTOR and SMARCA5 mRNA and protein in BC cell lines. miR-100 would be classified as an oncomiR in T24 cells representative of high grade urothelial carcinoma promoting increase in cell proliferation and reduction in apoptosis. The knowledge of miRNA role in tumors will allow their use

  13. Deregulated expression of hnRNP A/B proteins in human non-small cell lung cancer: parallel assessment of protein and mRNA levels in paired tumour/non-tumour tissues

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    Boukakis Georgios

    2010-08-01

    Full Text Available Abstract Background Heterogeneous nuclear ribonucleoproteins (hnRNPs of the A/B type (hnRNP A1, A2/B1, A3 are highly related multifunctional proteins participating in alternative splicing by antagonising other splicing factors, notably ASF/SF2. The altered expression pattern of hnRNP A2/B1 and/or splicing variant B1 alone in human lung cancer and their potential to serve as molecular markers for early diagnosis remain issues of intense investigation. The main objective of the present study was to use paired tumour/non-tumour biopsies from patients with non-small cell lung cancer (NSCLC to investigate the expression profiles of hnRNP A1, A2/B1 and A3 in conjunction with ASF/SF2. Methods We combined western blotting of tissue homogenates with immunohistochemical examination of fixed tissue sections and quantification of mRNA expression levels in tumour versus adjacent normal-looking areas of the lung in the same patient. Results Our study, in addition to clear evidence of mostly uncoupled deregulation of hnRNPs A/B, has revealed hnRNP A1 to be the most deregulated protein with a high frequency of over-expression (76%, followed by A3 (52% and A2/B1 (43%. Moreover, direct comparison of protein/mRNA levels showed a lack of correlation in the case of hnRNP A1 (as well as of ASF/SF2, but not of A2/B1, suggesting that different mechanisms underlie their deregulation. Conclusion Our results provide strong evidence for the up-regulation of hnRNP A/B in NSCLC, and they support the existence of distinct mechanisms responsible for their deregulated expression.

  14. Increased IL-10 mRNA and IL-23 mRNA expression in multiple sclerosis

    DEFF Research Database (Denmark)

    Krakauer, Martin; Sorensen, P; Khademi, M

    2008-01-01

    volunteers served to confirm initial findings. mRNA was analyzed by real-time reverse transcriptase polymerase chain reaction (PCR). RESULTS: We found elevated expression of interleukin (IL)-23 and IL-10 in untreated MS patients. IFN-beta therapy increased IL-10 and decreased IL-23 expression independently...... of the regulatory cytokine IL-10. The elevated IL-23 mRNA levels in MS patients are noteworthy in view of the newly discovered IL-23-driven Th17 T-cell subset, which is crucial in animal models of MS. Since IFN-beta therapy resulted in decreased IL-23 mRNA levels, the Th17 axis could be another target of IFN...

  15. The siRNA-mediated knockdown of GluN3A in 46C-derived neural stem cells affects mRNA expression levels of neural genes, including known iGluR interactors

    Science.gov (United States)

    Eilebrecht, Elke; Schöneborn, Hendrik; Neumann, Sebastian; Benecke, Arndt G.; Hollmann, Michael

    2018-01-01

    For years, GluN3A was solely considered to be a dominant-negative modulator of NMDARs, since its incorporation into receptors alters hallmark features of conventional NMDARs composed of GluN1/GluN2 subunits. Only recently, increasing evidence has accumulated that GluN3A plays a more diversified role. It is considered to be critically involved in the maturation of glutamatergic synapses, and it might act as a molecular brake to prevent premature synaptic strengthening. Its expression pattern supports a putative role during neural development, since GluN3A is predominantly expressed in early pre- and postnatal stages. In this study, we used RNA interference to efficiently knock down GluN3A in 46C-derived neural stem cells (NSCs) both at the mRNA and at the protein level. Global gene expression profiling upon GluN3A knockdown revealed significantly altered expression of a multitude of neural genes, including genes encoding small GTPases, retinal proteins, and cytoskeletal proteins, some of which have been previously shown to interact with GluN3A or other iGluR subunits. Canonical pathway enrichment studies point at important roles of GluN3A affecting key cellular pathways involved in cell growth, proliferation, motility, and survival, such as the mTOR pathway. This study for the first time provides insights into transcriptome changes upon the specific knockdown of an NMDAR subunit in NSCs, which may help to identify additional functions and downstream pathways of GluN3A and GluN3A-containing NMDARs. PMID:29438442

  16. Quantification of rainbow trout (Oncorhynchus mykiss) zona radiata and vitellogenin mRNA levels using real-time PCR after in vivo treatment with estradiol-17 beta or alpha-zearalenol.

    Science.gov (United States)

    Celius, T; Matthews, J B; Giesy, J P; Zacharewski, T R

    2000-12-15

    Estrogen receptor-mediated induction of zona radiata (ZR) and vitellogenin (VTG) mRNA and protein in rainbow trout (Oncorhynchus mykiss) was compared to assess their utility as biomarkers for exposure to estrogenic compounds. Partial sequences of rainbow trout ZR and beta-actin were cloned by reverse transcriptase polymerase chain reaction (RT-PCR) using degenerate primers based on conserved regions across a number of species. A 549 bp fragment of the rainbow trout ZR-gene showed a high degree of amino acid sequence identity to that of salmon (77%), winter flounder (64%), carp ZP2 (63%) and medaka (61%) ZR-proteins. The 1020 bp beta-actin fragment was approximately 100% identical to sequences from several species. Real-time PCR was used to quantify the induction of ZR-gene and VTG in rainbow trout liver after in vivo exposure to estradiol-17 beta (E(2)) (0.01, 0.1, 1.0 or 10 mg/kg body weight (bw) fish) or alpha-zearalenol (alpha-ZEA) (0.1, 1.0 or 10 mg/kg bw). Real-time PCR and indirect enzyme-linked immunosorbent assay (ELISA) showed that ZR and VTG were induced in both the liver and the plasma after a single injection of E(2) or alpha-ZEA. ZR was more responsive to low levels of E(2) and alpha-ZEA than VTG, and real-time PCR was shown to be more sensitive than the ELISA. Rainbow trout ZR-gene and proteins provide a sensitive biomarker for assessing estrogenic activity.

  17. The tight junction protein transcript abundance changes and oxidative damage by tryptophan deficiency or excess are related to the modulation of the signalling molecules, NF-κB p65, TOR, caspase-(3,8,9) and Nrf2 mRNA levels, in the gill of young grass carp (Ctenopharyngodon idellus).

    Science.gov (United States)

    Jiang, Wei-Dan; Wen, Hai-Lang; Liu, Yang; Jiang, Jun; Kuang, Sheng-Yao; Wu, Pei; Zhao, Juan; Tang, Ling; Tang, Wu-Neng; Zhang, Yong-An; Zhou, Xiao-Qiu; Feng, Lin

    2015-10-01

    This study is for the first time to explore the possible effects of dietary tryptophan (Trp) on structural integrity and the related signalling factor gene expression in the gill of young grass carp (Ctenopharyngodon idella). Fish were fed with six different experimental diets containing graded levels of Trp at 0.7 (control), 1.7, 3.1, 4.0, 5.2 and 6.1 g kg(-1) diet for 8 weeks. The results firstly demonstrated that Trp deficiency or excess caused increases in reactive oxygen species (ROS) contents, and severe oxidative damage (lipid peroxidation and protein oxidation) in the gill of fish, and those negative effects could be reversed by optimal Trp levels. Secondly, compared with the optimal Trp levels, Trp deficiency could cause decreases in the mRNA levels of the barrier functional proteins (occludin, zonula occludens-1, claudin-c, and -3) and increases in the mRNA levels of the pore-formation proteins (claudin-12 and -15) mRNA levels in the gill of fish, and those were reversed by the optimal levels of Trp. The negative effects of Trp deficiency on those tight junction protein gene expression might be partly related to the increases in the mRNA levels of pro-inflammatory cytokines and related signalling factors (tumor necrosis factor α, interleukin 8, interleukin 1β and transcription factor-κB) and decreases in the mRNA levels of anti-inflammatory cytokines and related signalling factors [interleukin 10, transforming growth factor-β1, nuclear inhibitor factor κBα (iκBα), target of rapamyc and ribosome protein S6 kinase 1 (S6K1)] in the gill of fish. In addition, optimal dietary Trp protected the gill of fish against its deficiency-caused increases in the mRNA levels of the apoptosis signalling (caspase-3, caspase-8, caspase-9) and decreases in anti-superoxide radicals capacity, anti-hydroxyl radical capacity, glutathione contents and the activities of Cu/Zn superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase

  18. 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

  19. Stress granules: sites of mRNA triage that regulate mRNA stability and translatability.

    Science.gov (United States)

    Kedersha, N; Anderson, P

    2002-11-01

    Mammalian stress granules (SGs) are cytoplasmic domains into which mRNAs are sorted dynamically in response to phosphorylation of eukaryotic initiation factor (eIF) 2alpha, a key regulatory step in translational initiation. The activation of one or more of the eIF2alpha kinases leads to SG assembly by decreasing the levels of eIF2-GTP-tRNA(Met), the ternary complex that is normally required for loading the initiator methionine onto the 48 S preinitiation complex to begin translation. This stress-induced scarcity of eIF2-GTP-tRNA(Met) allows the RNA-binding proteins TIA-1 (T-cell internal antigen-1) and TIAR (TIA-1-related protein) to bind the 48 S complex in lieu of the ternary complex, thereby promoting polysome disassembly and the concurrent routing of the mRNA into a SG. The actual formation of SGs occurs upon auto-aggregation of the prion-like C-termini of TIA-1 proteins; this aggregation is reversed in vivo by overexpression of the heat-shock protein (HSP) chaperone HSP70. Remarkably, HSP70 mRNA is excluded from SGs and is preferentially translated during stress, indicating that the RNA composition of the SG is selective. Moreover, the effects of HSP70 on TIA aggregation suggest a feedback loop whereby HSP70 synthesis is auto-regulated. Proteins that promote mRNA stability [e.g. HuR (Hu protein R)] and destabilize mRNA [i.e. tristetraprolin (TTP)] are also recruited to SGs, suggesting that SGs effect a process of mRNA triage, by promoting polysome disassembly and routing mRNAs to cytoplasmic domains enriched for HuR and TTP. This model reveals connections between the eIF2alpha kinase system, mRNA stability and cellular chaperone levels.

  20. Effect of repeated L-DOPA, bromocriptine, or lisuride administration on preproenkephalin-A and preproenkephalin-B mRNA levels in the striatum of the 6-hydroxydopamine-lesioned rat.

    Science.gov (United States)

    Henry, B; Crossman, A R; Brotchie, J M

    1999-02-01

    dorsally, while PPE-B expression was reduced in the striatum at all rostrocaudal levels. Repeated l-DOPA administration was accompanied by elevations in striatal PPE-B mRNA levels and a further elevation, above lesion-induced levels, in PPE-A expression. This further elevation was restricted to the dorsolateral striatum. However, following repeated bromocriptine or lisuride administration no increase in PPE-B expression was observed and the lesion-induced increase in PPE-A expression was normalized to prelesion levels. Increased PPE-A and PPE-B levels may, through decreasing GABA and glutamate release, respectively, in output nuclei of the basal ganglia, play a role in the development of L-DOPA- and dopamine-agonist induced dyskinesia in Parkinson's disease. These studies suggest that anti-parkinsonian treatments which are not associated with an elevation in PPE-B and/or normalize elevated PPE-A precursor expression, such as NMDA-receptor antagonists or long-acting dopamine D2 receptor agonists, e.g., cabergoline or ropinirole, may reduce dyskinesia in Parkinson's disease. Copyright 1999 Academic Press.

  1. Benefits of Hormone Therapy Estrogens Depend on Estrogen Type: 17β-Estradiol and Conjugated Equine Estrogens Have Differential Effects on Cognitive, Anxiety-Like, and Depressive-Like Behaviors and Increase Tryptophan Hydroxylase-2 mRNA Levels in Dorsal Raphe Nucleus Subregions.

    Science.gov (United States)

    Hiroi, Ryoko; Weyrich, Giulia; Koebele, Stephanie V; Mennenga, Sarah E; Talboom, Joshua S; Hewitt, Lauren T; Lavery, Courtney N; Mendoza, Perla; Jordan, Ambra; Bimonte-Nelson, Heather A

    2016-01-01

    Decreased serotonin (5-HT) function is associated with numerous cognitive and affective disorders. Women are more vulnerable to these disorders and have a lower rate of 5-HT synthesis than men. Serotonergic neurons in the dorsal raphe nucleus (DRN) are a major source of 5-HT in the forebrain and play a critical role in regulation of stress-related disorders. In particular, polymorphisms of tryptophan hydroxylase-2 (TpH2, the brain-specific, rate-limiting enzyme for 5-HT biosynthesis) are implicated in cognitive and affective disorders. Administration of 17β-estradiol (E2), the most potent naturally circulating estrogen in women and rats, can have beneficial effects on cognitive, anxiety-like, and depressive-like behaviors. Moreover, E2 increases TpH2 mRNA in specific subregions of the DRN. Although conjugated equine estrogens (CEE) are a commonly prescribed estrogen component of hormone therapy in menopausal women, there is a marked gap in knowledge regarding how CEE affects these behaviors and the brain 5-HT system. Therefore, we compared the effects of CEE and E2 treatments on behavior and TpH2 mRNA. Female Sprague-Dawley rats were ovariectomized, administered either vehicle, CEE, or E2 and tested on a battery of cognitive, anxiety-like, and depressive-like behaviors. The brains of these animals were subsequently analyzed for TpH2 mRNA. Both CEE and E2 exerted beneficial behavioral effects, although efficacy depended on the distinct behavior and for cognition, on the task difficulty. Compared to CEE, E2 generally had more robust anxiolytic and antidepressant effects. E2 increased TpH2 mRNA in the caudal and mid DRN, corroborating previous findings. However, CEE increased TpH2 mRNA in the caudal and rostral, but not the mid, DRN, suggesting that distinct estrogens can have subregion-specific effects on TpH2 gene expression. We also found differential correlations between the level of TpH2 mRNA in specific DRN subregions and behavior, depending on the type of

  2. Benefits of Hormone Therapy Estrogens Depend on Estrogen Type: 17β-Estradiol and Conjugated Equine Estrogens Have Differential Effects on Cognitive, Anxiety-Like, and Depressive-Like Behaviors and Increase Tryptophan Hydroxylase-2 mRNA Levels in Dorsal Raphe Nucleus Subregions

    Science.gov (United States)

    Hiroi, Ryoko; Weyrich, Giulia; Koebele, Stephanie V.; Mennenga, Sarah E.; Talboom, Joshua S.; Hewitt, Lauren T.; Lavery, Courtney N.; Mendoza, Perla; Jordan, Ambra; Bimonte-Nelson, Heather A.

    2016-01-01

    Decreased serotonin (5-HT) function is associated with numerous cognitive and affective disorders. Women are more vulnerable to these disorders and have a lower rate of 5-HT synthesis than men. Serotonergic neurons in the dorsal raphe nucleus (DRN) are a major source of 5-HT in the forebrain and play a critical role in regulation of stress-related disorders. In particular, polymorphisms of tryptophan hydroxylase-2 (TpH2, the brain-specific, rate-limiting enzyme for 5-HT biosynthesis) are implicated in cognitive and affective disorders. Administration of 17β-estradiol (E2), the most potent naturally circulating estrogen in women and rats, can have beneficial effects on cognitive, anxiety-like, and depressive-like behaviors. Moreover, E2 increases TpH2 mRNA in specific subregions of the DRN. Although conjugated equine estrogens (CEE) are a commonly prescribed estrogen component of hormone therapy in menopausal women, there is a marked gap in knowledge regarding how CEE affects these behaviors and the brain 5-HT system. Therefore, we compared the effects of CEE and E2 treatments on behavior and TpH2 mRNA. Female Sprague-Dawley rats were ovariectomized, administered either vehicle, CEE, or E2 and tested on a battery of cognitive, anxiety-like, and depressive-like behaviors. The brains of these animals were subsequently analyzed for TpH2 mRNA. Both CEE and E2 exerted beneficial behavioral effects, although efficacy depended on the distinct behavior and for cognition, on the task difficulty. Compared to CEE, E2 generally had more robust anxiolytic and antidepressant effects. E2 increased TpH2 mRNA in the caudal and mid DRN, corroborating previous findings. However, CEE increased TpH2 mRNA in the caudal and rostral, but not the mid, DRN, suggesting that distinct estrogens can have subregion-specific effects on TpH2 gene expression. We also found differential correlations between the level of TpH2 mRNA in specific DRN subregions and behavior, depending on the type of

  3. Carboxylesterase 1 gene duplication and mRNA expression in adipose tissue are linked to obesity and metabolic function

    DEFF Research Database (Denmark)

    Friedrichsen, Martin; Poulsen, Pernille; Wojtaszewski, Jørgen

    2013-01-01

    involved in the control of mRNA expression. Here, we investigated mRNA expression level in adipose tissue and its association with measures of adiposity and metabolic function in a population of elderly twins. Furthermore, the heritability of mRNA expression level in adipose tissue and the effect of gene...

  4. Sequence of the human glycogen-associated regulatory subunit of type 1 protein phosphatase and analysis of its coding region and mRNA level in muscle from patients with NIDDM

    DEFF Research Database (Denmark)

    Chen, Y H; Hansen, L; Chen, Min

    1994-01-01

    of protein phosphatase 1 (PP1 G-subunit) plays a key role in the insulin stimulation of glycogen synthesis and the activity of PP1 is decreased in insulin-resistant subjects, we have now cloned the human G-subunit cDNA to search for abnormalities in the corresponding gene (designated PPP1R3 in the human...... genome nomenclature) in patients with NIDDM. The human cDNA was isolated from a skeletal muscle cDNA library and was found to encode a 126-kDa protein, which shows 73% amino acid identity to the rabbit PP1 G-subunit. The human G-subunit cDNA from 30 insulin-resistant NIDDM patients was analyzed...... for genetic variations in the G-subunit by using single-stranded conformation polymorphism (SSCP) scanning of reversely transcribed mRNA. One variant SSCP profile was detected in the region encoding the COOH-terminal part of the PP1 G-subunit in only one NIDDM patient, and subsequent nucleotide sequencing...

  5. Increased IL-10 mRNA and IL-23 mRNA expression in multiple sclerosis: interferon-beta treatment increases IL-10 mRNA expression while reducing IL-23 mRNA expression

    DEFF Research Database (Denmark)

    Krakauer, M.; Sorensen, P.; Khademi, M.

    2008-01-01

    volunteers served to confirm initial findings. mRNA was analyzed by real-time reverse transcriptase polymerase chain reaction (PCR). RESULTS: We found elevated expression of interleukin (IL)-23 and IL-10 in untreated MS patients. IFN-beta therapy increased IL-10 and decreased IL-23 expression independently...... of the regulatory cytokine IL-10. The elevated IL-23 mRNA levels in MS patients are noteworthy in view of the newly discovered IL-23-driven Th17 T-cell subset, which is crucial in animal models of MS. Since IFN-beta therapy resulted in decreased IL-23 mRNA levels, the Th17 axis could be another target of IFN...

  6. Impact of age, BMI and HbA1c levels on the genome-wide DNA methylation and mRNA expression patterns in human adipose tissue and identification of epigenetic biomarkers in blood

    DEFF Research Database (Denmark)

    Rönn, Tina; Volkov, Petr; Gillberg, Linn

    2015-01-01

    Increased age, BMI and HbA1c levels are risk factors for several non-communicable diseases. However, the impact of these factors on the genome-wide DNA methylation pattern in human adipose tissue remains unknown. We analyzed the DNA methylation of ∼480 000 sites in human adipose tissue from 96 ma...

  7. Voltage-Gated Na+ Channel Isoforms and Their mRNA Expression Levels and Protein Abundance in Three Electric Organs and the Skeletal Muscle of the Electric Eel Electrophorus electricus.

    Directory of Open Access Journals (Sweden)

    Biyun Ching

    Full Text Available This study aimed to obtain the coding cDNA sequences of voltage-gated Na+ channel (scn α-subunit (scna and β-subunit (scnb isoforms from, and to quantify their transcript levels in, the main electric organ (EO, Hunter's EO, Sach's EO and the skeletal muscle (SM of the electric eel, Electrophorus electricus, which can generate both high and low voltage electric organ discharges (EODs. The full coding sequences of two scna (scn4aa and scn4ab and three scnb (scn1b, scn2b and scn4b were identified for the first time (except scn4aa in E. electricus. In adult fish, the scn4aa transcript level was the highest in the main EO and the lowest in the Sach's EO, indicating that it might play an important role in generating high voltage EODs. For scn4ab/Scn4ab, the transcript and protein levels were unexpectedly high in the EOs, with expression levels in the main EO and the Hunter's EO comparable to those of scn4aa. As the key domains affecting the properties of the channel were mostly conserved between Scn4aa and Scn4ab, Scn4ab might play a role in electrogenesis. Concerning scnb, the transcript level of scn4b was much higher than those of scn1b and scn2b in the EOs and the SM. While the transcript level of scn4b was the highest in the main EO, protein abundance of Scn4b was the highest in the SM. Taken together, it is unlikely that Scna could function independently to generate EODs in the EOs as previously suggested. It is probable that different combinations of Scn4aa/Scn4ab and various Scnb isoforms in the three EOs account for the differences in EODs produced in E. electricus. In general, the transcript levels of various scn isoforms in the EOs and the SM were much higher in adult than in juvenile, and the three EOs of the juvenile fish could be functionally indistinct.

  8. CD36 mRNA and Protein Expression Levels Are Significantly Increased in the Heart and Testis of apoE Deficient Mice in Comparison to Wild Type (C57BL/6

    Directory of Open Access Journals (Sweden)

    Kazem Zibara

    2002-01-01

    Full Text Available CD36, an 88kd-adhesion molecule, plays a major role as a scavenging receptor implicated in cellular lipid metabolism. Secretory mammary epithelium, microvasculature endothelium, adipocytes, smooth muscle cells, and platelets express CD36. In addition, CD36 expression is significantly enhanced in macrophages differentiating into foam cells. The effect of pathological levels of cholesterol, as observed in apoE−/−, on vascular CD36 expression is, at this stage, not known. In this study, a quantitative analysis of CD36 transcription and protein expression levels, present in tissues of male C57BL/6 and apolipoprotein-E (apoE deficient mice was carried out by Northern and Western blots. Four-week-old animals were fed a chow diet over different periods of time (0, 6, 16, or 20 weeks. Immunohistochemistry was used to localize CD36 protein expression in the heart and testis. Results indicate that CD36 transcription is increased in hearts of apoE deficient animals (100% higher at 6 weeks, and 30% higher at 16 and 20 weeks in comparison to wild type. This was confirmed at the protein level, which showed an increase of at least 100% at 6 weeks, and between 40% to 50% increase at 16 and 20 weeks of apoE−/− mice compared to controls. In addition, CD36 transcription levels were significantly increased in testis of apoE animals (at least 100% at 6, 16, and 20 weeks compared to C57BL/6 wild type. Such an increase was also confirmed at the protein level (65% increase at 16 weeks in apoE mice compared to control. Finally, localization of CD36 protein expression by immunohistochemistry showed that it was expressed in the capillaries of heart and testis endothelial cells and also at the head of spermatozoid during spermatogenesis. These results indicate that high circulating cholesterol levels, in apoE deficient mice, significantly enhance the expression of CD36 in the heart and testis. Such enhanced CD36 expression might lead to organ remodeling and

  9. Translational repression of mRNA for eucaryotic elongation factors in Friend erythroleukemia cells.

    Science.gov (United States)

    Slobin, L I; Jordan, P

    1984-11-15

    Poly(A)-containing mRNA was prepared from polyribosomes and postpolyribosomal messenger ribonucleoprotein particles (mRNP) from Friend erythroleukemic cells. Both mRNA types were translated in vitro and the 35S-labeled translation products examined by two-dimensional gel electrophoresis. Among the most abundant untranslated mRNA species was the mRNA coding for eucaryotic elongation factor Tu (eEF-Tu). In addition, the mRNA for eucaryotic elongation factor Ts was also present in Friend cells in untranslated form. Calculations based on translation assays indicate that eEF-Tu represents about 15% of the translation products of RNP mRNA and that approximately 40% of the eEF-Tu synthesized in vitro is encoded by translationally repressed mRNA. This repressed mRNA can be activated by addition of cycloheximide to cell cultures. At the level of 0.1 micrograms/ml, cycloheximide was found to inhibit cellular protein synthesis by about 50% while augmenting the relative rate of eEF-Tu synthesis 1.6-fold. This result suggested that eEF-Tu mRNA might initiate poorly. However, addition of supersaturating levels of mRNA to a reticulocyte lysate augmented eEF-Tu synthesis about twofold, while generally depressing the synthesis of other proteins by about 40%. Thus the storage of large amounts of eEF-Tu mRNA in vivo is unlikely to be due directly to the ineffectiveness of the mRNA in competing for the initiation machinery of the cell. The results presented in this report suggest that the supply of active eEF-Tu in erythroleukemic cells is controlled, at least in part, by a translational mechanism.

  10. Comparison of protein and mRNA expression evolution in humans and chimpanzees.

    Directory of Open Access Journals (Sweden)

    Ning Fu

    Full Text Available Even though mRNA expression levels are commonly used as a proxy for estimating functional differences that occur at the protein level, the relation between mRNA and protein expression is not well established. Further, no study to date has tested whether the evolutionary differences in mRNA expression observed between species reflect those observed in protein expression. Since a large proportion of mRNA expression differences observed between mammalian species appears to have no functional consequences for the phenotype, it is conceivable that many or most mRNA expression differences are not reflected at the protein level. If this is true, then differences in protein expression may largely reflect functional adaptations observed in species phenotypes. In this paper, we present the first direct comparison of mRNA and protein expression differences seen between humans and chimpanzees. We reproducibly find a significant positive correlation between mRNA expression and protein expression differences. This correlation is comparable in magnitude to that found between mRNA and protein expression changes at different developmental stages or in different physiological conditions within one species. Noticeably, this correlation is mainly due to genes with large expression differences between species. Our study opens the door to a new level of understanding of regulatory evolution and poses many new questions that remain to be answered.

  11. Analysis of mRNA recognition by human thymidylate synthase.

    Science.gov (United States)

    Brunn, Nicholas D; Dibrov, Sergey M; Kao, Melody B; Ghassemian, Majid; Hermann, Thomas

    2014-12-23

    Expression of hTS (human thymidylate synthase), a key enzyme in thymidine biosynthesis, is regulated on the translational level through a feedback mechanism that is rarely found in eukaryotes. At low substrate concentrations, the ligand-free enzyme binds to its own mRNA and stabilizes a hairpin structure that sequesters the start codon. When in complex with dUMP (2'-deoxyuridine-5'-monophosphate) and a THF (tetrahydrofolate) cofactor, the enzyme adopts a conformation that is unable to bind and repress expression of mRNA. Here, we have used a combination of X-ray crystallography, RNA mutagenesis and site-specific cross-linking studies to investigate the molecular recognition of TS mRNA by the hTS enzyme. The interacting mRNA region was narrowed to the start codon and immediately flanking sequences. In the hTS enzyme, a helix-loop-helix domain on the protein surface was identified as the putative RNA-binding site.

  12. Regulation of mRNA Trafficking by Nuclear Pore Complexes

    Directory of Open Access Journals (Sweden)

    Amandine Bonnet

    2014-09-01

    Full Text Available Over the last two decades, multiple studies have explored the mechanisms governing mRNA export out of the nucleus, a crucial step in eukaryotic gene expression. During transcription and processing, mRNAs are assembled into messenger ribonucleoparticles (mRNPs. mRNPs are then exported through nuclear pore complexes (NPCs, which are large multiprotein assemblies made of several copies of a limited number of nucleoporins. A considerable effort has been put into the dissection of mRNA export through NPCs at both cellular and molecular levels, revealing the conserved contributions of a subset of nucleoporins in this process, from yeast to vertebrates. Several reports have also demonstrated the ability of NPCs to sort out properly-processed mRNPs for entry into the nuclear export pathway. Importantly, changes in mRNA export have been associated with post-translational modifications of nucleoporins or changes in NPC composition, depending on cell cycle progression, development or exposure to stress. How NPC modifications also impact on cellular mRNA export in disease situations, notably upon viral infection, is discussed.

  13. Different profiles of the mRNA levels of DNA repair genes in MCF-7 and SH-SY5Y cells after treatment with combination of cisplatin, 50-Hz electromagnetic field and bleomycin.

    Science.gov (United States)

    Sanie-Jahromi, Fatemeh; Saadat, Mostafa

    2017-10-01

    Neurotoxicity is known to be a major dose-limiting adverse effect of cisplatin (CDDP), alone or in combination with other chemicals. DNA repair capacity serve as a neuroprotective factor against CDDP. The purpose of this study was to evaluate the effect of 50-Hz electromagnetic field (EMF) in combination with CDDP and bleomycin (Bleo) on expression of some of DNA repair genes (GADD45A, XRCC1, XRCC4, Ku70, Ku80, DNA-PKcs and LIG4) in MCF-7 (breast cancer) and SH-SY5Y (neuroblastoma) cell lines. MCF-7 and SH-SY5Y cells were pre-treated with CDDP in the presence or absence of EMF and then exposed to different concentration of Bleo. EMF (0.50mT intensity) was used in the intermittenet pattern of "15min field on/15min field off" with 30min total exposure. Cell viability assay was done and then the transcript levels of the examined genes were measured using quantitative real-time PCR in "CDDP+Bleo" and "CDDP+EMF+Bleo" treatments. Our results indicated that MCF-7 cells treated with "CDDP+EMF+Bleo" showed more susceptibility compared with "CDDP+Bleo" treated ones, while SH-SY5Y susceptibility was not changed between the two treatments. The represented data indicated that MCF-7 and SH-SY5Y cells showed non-random disagreement in DNA repair gene expression in 11 conditions (out of 14 conditions) with each other (χ2=4.52, df=1, P=0.033). This finding can be promising for sensitizing breast cancer cells while protecting against CDDP induced neuropathy in cancer patients. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  14. Digoxin up-regulates multidrug resistance transporter (MDR1) mRNA and simultaneously down-regulates steroid xenobiotic receptor mRNA.

    Science.gov (United States)

    Takara, Kohji; Takagi, Kentaro; Tsujimoto, Masayuki; Ohnishi, Noriaki; Yokoyama, Teruyoshi

    2003-06-20

    A steroid xenobiotic receptor (SXR) is involved in the induction of MDR1/P-glycoprotein. MDR1 up-regulation by digoxin was previously demonstrated in human colon adenocarcinoma Caco-2 cells, but the participation of SXR remains unclear. Herein, the participation of SXR in MDR1 up-regulation was examined using reverse transcription-polymerase chain reaction in Caco-2 cells, and digoxin-tolerant cells (Caco/DX) as well as human colon carcinoma LS180 cells, which expressed SXR. MDR1 mRNA expression in Caco-2 or LS180 cells was increased by exposure to 1 microM digoxin for 24h, in a concentration-dependent manner, but SXR mRNA decreased concentration-dependently and was undetectable or significantly lower at 1 microM digoxin, indicating antithetical changes in MDR1 and SXR mRNA expression. Moreover, the MDR1 mRNA level was higher in Caco/DX cells than Caco-2 cells, whereas the SXR mRNA level was lower in Caco/DX cells. Consequently, digoxin was demonstrated to up-regulate MDR1 mRNA and simultaneously down-regulate SXR mRNA expression.

  15. Xplore mRNA assays for the quantification of IL-1 beta and TNF-alpha mRNA in lipopolysaccharide-induced mouse macrophages.

    Science.gov (United States)

    Van Arsdell, S W; Murphy, K P; Pazmany, C; Erickson, D; Burns, C; Moody, M D

    2000-06-01

    Because the accurate measurement of a number of cytokine mRNA transcripts provides valuable knowledge about cytokine gene regulation, we have developed the Xplore assay for the quantification of cytokine mRNA. This microplate-based assay is rapid (under four hours), quantitative over three orders of magnitude and carries no risk of false-positive values from contamination with amplified target. Here, we describe the use of Xplore assays to measure the steady-state mRNA levels of TNF-alpha and IL-1 beta produced by mouse WEHI and J774 macrophage-like cell lines.

  16. Polysome Fractionation to Analyze mRNA Distribution Profiles.

    Science.gov (United States)

    Panda, Amaresh C; Martindale, Jennifer L; Gorospe, Myriam

    2017-02-05

    Eukaryotic cells adapt to changes in external or internal signals by precisely modulating the expression of specific gene products. The expression of protein-coding genes is controlled at the transcriptional and post-transcriptional levels. Among the latter steps, the regulation of translation is particularly important in cellular processes that require rapid changes in protein expression patterns. The translational efficiency of mRNAs is altered by RNA-binding proteins (RBPs) and noncoding (nc)RNAs such as microRNAs (Panda et al., 2014a and 2014b; Abdelmohsen et al., 2014). The impact of factors that regulate selective mRNA translation is a critical question in RNA biology. Polyribosome (polysome) fractionation analysis is a powerful method to assess the association of ribosomes with a given mRNA. It provides valuable information about the translational status of that mRNA, depending on the number of ribosomes with which they are associated, and identifies mRNAs that are not translated (Panda et al., 2016). mRNAs associated with many ribosomes form large polysomes that are predicted to be actively translated, while mRNAs associated with few or no ribosomes are expected to be translated poorly if at all. In sum, polysome fractionation analysis allows the direct determination of translation efficiencies at the level of the whole transcriptome as well as individual mRNAs.

  17. Self-amplifying mRNA vaccines.

    Science.gov (United States)

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

    2015-01-01

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

  18. Coordinated Regulations of mRNA Synthesis and Decay during Cold Acclimation in Arabidopsis Cells.

    KAUST Repository

    Arae, Toshihiro

    2017-04-18

    Plants possess a cold acclimation system to acquire freezing tolerance through pre-exposure to non-freezing low temperatures. The transcriptional cascade of C-repeat binding factors (CBFs)/dehydration response element-binding factors (DREBs) is considered a major transcriptional regulatory pathway during cold acclimation. However, little is known regarding the functional significance of mRNA stability regulation in the response of gene expression to cold stress. The actual level of individual mRNAs is determined by a balance between mRNA synthesis and degradation. Therefore, it is important to assess the regulatory steps to increase our understanding of gene regulation. Here, we analyzed temporal changes in mRNA amounts and half-lives in response to cold stress in Arabidopsis cell cultures based on genome-wide analysis. In this mRNA decay array method, mRNA half-life measurements and microarray analyses were combined. In addition, temporal changes in the integrated value of transcription rates were estimated from the above two parameters using a mathematical approach. Our results showed that several cold-responsive genes, including Cold-regulated 15a, were relatively destabilized, whereas the mRNA amounts were increased during cold treatment by accelerating the transcription rate to overcome the destabilization. Considering the kinetics of mRNA synthesis and degradation, this apparently contradictory result supports that mRNA destabilization is advantageous for the swift increase in CBF-responsive genes in response to cold stress.

  19. Synergistic anticancer effects of polyphyllin I and evodiamine on freshly-removed human gastric tumors.

    Directory of Open Access Journals (Sweden)

    Guofeng Yue

    Full Text Available The present study was designed to examine the anticancer effect of Traditional Chinese Medicine of polyphyllin I (PPI and evodiamine (EVO on freshly-removed gastric tumor tissues.Sixty freshly-removed gastric tumor tissues were collected. Their sensitivity to PPI, EVO, platinum (Pt, 5-FU, irinotecan (CPT-11 were determined by histoculture drug response assay (HDRA. Those samples were also formalin-fixed and paraffin-embedded, which were used to examine the mRNA expression levels of aprataxin(APTX, excision repair cross-complementing 1(ERCC1, thymidylate synthase(TS and topoisomerase I(TOPO1 by quantitative RT-PCR. The association of the gene expression levels and in vitro sensitivity were analyzed.PPI, EVO, Pt, 5-FU and CPT-11 had anticancer effects on the freshly-removed gastric tumor tissues with average inhibition rates of 20.64%±14.25% for PPI, 21.14%±13.43% for EVO, 50.57%±22.37% for Pt, 53.54%±22.03% for 5-FU, and 39.33%±24.79% for CPT-11, respectively. Combination of PPI and Pt, EVO and Pt, EVO and 5-FU had higher inhibition rates than any single drug of them (P<0.001, P = 0.028, P = 0.017, respectively. The mRNA expression levels of ERCC1 were correlated with Pt sensitivity (rho = -0.645, P<0.001; the mRNA expression levels of TS were correlated with 5-FU sensitivity (rho = -0.803, P<0.001. There were also weak but significant correlations between APTX mRNA expression levels and CPT-11 sensitivity (rho = -0.376, P = 0.017 or EVO sensitivity (rho = -0.322, P = 0.036. ERCC1 mRNA expression levels was markedly suppressed by the presentation of PPI (P = 0.001 and slightly suppressed by the presentation of EVO (P = 0.04; whereas, TS mRNA expression levels was markedly decreased by the presentation of EVO (P = 0.017 and slightly decreased by the presentation of PPI (P = 0.047.PPI and EVO both could inhibit the activity of freshly-removed gastric tumor, and they could enhance the anticancer

  20. Making the cut : How XPF-ERCC1 unhooks DNA interstrand crosslinks

    NARCIS (Netherlands)

    Klein Douwel, Daisy

    2017-01-01

    DNA interstrand crosslinks (ICLs) are highly toxic lesions that bind both strands of the DNA helix together, which prevents the DNA from unwinding. This blocks important cellular processes such as DNA replication and transcription. ICL inducing agents were among the first chemotherapeutic drugs,

  1. Transcriptional profiling reveals progeroid Ercc1-/Δ mice as a model system for glomerular aging

    NARCIS (Netherlands)

    B. Schumacher (Björn); V. Bartels (Valerie); P. Frommolt (Peter); B. Habermann (Bianca); F. Braun (Fabian); J.L. Schultze (Joachim); M. Roodbergen (Marianne); J.H.J. Hoeijmakers (Jan); P. Nürnberg (Peter); M.E.T. Dollé (Martijn); T. Benzing (Thomas); R.-U. Müller (Roman-Ulrich); C.E. Kurschat (Christine)

    2013-01-01

    textabstractBackground: Aging-related kidney diseases are a major health concern. Currently, models to study renal aging are lacking. Due to a reduced life-span progeroid models hold the promise to facilitate aging studies and allow examination of tissue-specific changes. Defects in genome

  2. Cloning and characterization of the human DNA-excision repair gene ERCC-1

    NARCIS (Netherlands)

    M. van Duin (Michel)

    1988-01-01

    textabstractIt is the aim of the work described in this thesis to isolate and characterize human genes involved DNA excision repair. This will facilitate the understanding of the mechanism of this repair process whereas it also provides an important step to better understand the relationship

  3. Cup regulates oskar mRNA stability during oogenesis.

    Science.gov (United States)

    Broyer, Risa M; Monfort, Elena; Wilhelm, James E

    2017-01-01

    The proper regulation of the localization, translation, and stability of maternally deposited transcripts is essential for embryonic development in many organisms. These different forms of regulation are mediated by the various protein subunits of the ribonucleoprotein (RNP) complexes that assemble on maternal mRNAs. However, while many of the subunits that regulate the localization and translation of maternal transcripts have been identified, relatively little is known about how maternal mRNAs are stockpiled and stored in a stable form to support early development. One of the best characterized regulators of maternal transcripts is Cup - a broadly conserved component of the maternal RNP complex that in Drosophila acts as a translational repressor of the localized message oskar. In this study, we have found that loss of cup disrupts the localization of both the oskar mRNA and its associated proteins to the posterior pole of the developing oocyte. This defect is not due to a failure to specify the oocyte or to disruption of RNP transport. Rather, the localization defects are due to a drop in oskar mRNA levels in cup mutant egg chambers. Thus, in addition to its role in regulating oskar mRNA translation, Cup also plays a critical role in controlling the stability of the oskar transcript. This suggests that Cup is ideally positioned to coordinate the translational control function of the maternal RNP complex with its role in storing maternal transcripts in a stable form. Published by Elsevier Inc.

  4. CD133 mRNA expression and microsatellite instability in colorectal carcinoma.

    Science.gov (United States)

    Huh, Jung Wook; Park, Yeon Sun; Lee, Jae Hyuk; Kim, Hyeong Rok; Shin, Myung Geun; Kim, Young Jin

    2010-12-01

    The present study was performed to examine the CD133 expression in colorectal cancer and to analyze its relationship with microsatellite instability (MSI) and the clinicopathological factors, including patient survival. The CD133 mRNA levels in 61 primary colorectal adenocarcinomas were analyzed by reverse transcriptase-polymerase chain reaction, with normalization relative to GAPDH. Five microsatellite markers were analyzed to evaluate MSI. A CD133 mRNA expression was significantly associated with the depth of invasion (P = 0.017), lymph node involvement (P = 0.012), and lymphovascular invasion (P = 0.019). A CD133 expression was significantly correlated with the MSI status (P = 0.035). With a median follow-up period of 45 months, the 5-year disease-free survival rate of patients with a low CD133 mRNA expression was significantly higher than that of those patients with high levels of CD133 mRNA expression (82.9% and 59.0%, respectively; P = 0.027). However, on the multivariate analysis, a CD133 mRNA expression was not an independent predictor of disease-free survival. Elevated CD133 mRNA levels may represent more aggressive tumor biology and poorer survival in patients with colorectal cancer, correlating with a high level of MSI status. Larger prospective studies are required to confirm these findings. © 2010 Wiley-Liss, Inc.

  5. Na+/K+-ATPase α-subunit (nkaα isoforms and their mRNA expression levels, overall Nkaα protein abundance, and kinetic properties of Nka in the skeletal muscle and three electric organs of the electric eel, Electrophorus electricus.

    Directory of Open Access Journals (Sweden)

    Biyun Ching

    Full Text Available This study aimed to obtain the coding cDNA sequences of Na+/K+-ATPase α (nkaα isoforms from, and to quantify their mRNA expression in, the skeletal muscle (SM, the main electric organ (EO, the Hunter's EO and the Sach's EO of the electric eel, Electrophorus electricus. Four nkaα isoforms (nkaα1c1, nkaα1c2, nkaα2 and nkaα3 were obtained from the SM and the EOs of E. electricus. Based on mRNA expression levels, the major nkaα expressed in the SM and the three EOs of juvenile and adult E. electricus were nkaα1c1 and nkaα2, respectively. Molecular characterization of the deduced Nkaα1c1 and Nkaα2 sequences indicates that they probably have different affinities to Na+ and K+. Western blotting demonstrated that the protein abundance of Nkaα was barely detectable in the SM, but strongly detected in the main and Hunter's EOs and weakly in the Sach's EO of juvenile and adult E. electricus. These results corroborate the fact that the main EO and Hunter's EO have high densities of Na+ channels and produce high voltage discharges while the Sach's EO produces low voltage discharges. More importantly, there were significant differences in kinetic properties of Nka among the three EOs of juvenile E. electricus. The highest and lowest Vmax of Nka were detected in the main EO and the Sach's EO, respectively, with the Hunter's EO having a Vmax value intermediate between the two, indicating that the metabolic costs of EO discharge could be the highest in the main EO. Furthermore, the Nka from the main EO had the lowest Km (or highest affinity for Na+ and K+ among the three EOs, suggesting that the Nka of the main EO was more effective than those of the other two EOs in maintaining intracellular Na+ and K+ homeostasis and in clearing extracellular K+ after EO discharge.

  6. Technical validation of an RT-qPCR in vitro diagnostic test system for the determination of breast cancer molecular subtypes by quantification of ERBB2, ESR1, PGR and MKI67 mRNA levels from formalin-fixed paraffin-embedded breast tumor specimens

    National Research Council Canada - National Science Library

    Laible, Mark; Schlombs, Kornelia; Kaiser, Katharina; Veltrup, Elke; Herlein, Stefanie; Lakis, Sotiris; Stöhr, Robert; Eidt, Sebastian; Hartmann, Arndt; Wirtz, Ralph M; Sahin, Ugur

    2016-01-01

    ... (HER2 positive or negative), HER2 positive (non-luminal) and Triple negative (ductal) according to the mRNA expression of ERBB2, ESR1, PGR and MKI67 and the St Gallen consensus surrogate clinical definition...

  7. 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

  8. Association of chemerin mRNA expression in human epicardial adipose tissue with coronary atherosclerosis

    Directory of Open Access Journals (Sweden)

    Wang Linjie

    2011-10-01

    Full Text Available Abstract Background Growing evidence suggests that epicardial adipose tissue (EAT may play a key role in the pathogenesis and development of coronary artery disease (CAD by producing several inflammatory adipokines. Chemerin, a novel adipokine, has been reported to be involved in regulating immune responses and glucolipid metabolism. Given these properties, chemerin may provide an interesting link between obesity, inflammation and atherosclerosis. In this study, we sought to determine the relationship of chemerin expression in EAT and the severity of coronary atherosclerosis in Han Chinese patients. Methods Serums and adipose tissue biopsies (epicardial and thoracic subcutaneous were obtained from CAD (n = 37 and NCAD (n = 16 patients undergoing elective cardiac surgery. Gensini score was used to assess the severity of CAD. Serum levels of chemerin, adiponectin and insulin were measured by ELISA. Chemerin protein expression in adipose tissue was detected by immunohistochemistry. The mRNA levels of chemerin, chemR23, adiponectin and TNF-alpha in adipose tissue were detected by RT-PCR. Results We found that EAT of CAD group showed significantly higher levels of chemerin and TNF-alpha mRNA, and significantly lower level of adiponectin mRNA than that of NCAD patients. In CAD group, significantly higher levels of chemerin mRNA and protein were observed in EAT than in paired subcutaneous adipose tissue (SAT, whereas such significant difference was not found in NCAD group. Chemerin mRNA expression in EAT was positively correlated with Gensini score (r = 0.365, P P P P P P P > 0.05. Conclusions The expressions of chemerin mRNA and protein are significantly higher in EAT from patients with CAD in Han Chinese patients. Furthermore, the severity of coronary atherosclerosis is positive correlated with the level of chemerin mRNA in EAT rather than its circulating level.

  9. Heat shock response in yeast involves changes in both transcription rates and mRNA stabilities.

    Directory of Open Access Journals (Sweden)

    Laia Castells-Roca

    Full Text Available We have analyzed the heat stress response in the yeast Saccharomyces cerevisiae by determining mRNA levels and transcription rates for the whole transcriptome after a shift from 25 °C to 37 °C. Using an established mathematical algorithm, theoretical mRNA decay rates have also been calculated from the experimental data. We have verified the mathematical predictions for selected genes by determining their mRNA decay rates at different times during heat stress response using the regulatable tetO promoter. This study indicates that the yeast response to heat shock is not only due to changes in transcription rates, but also to changes in the mRNA stabilities. mRNA stability is affected in 62% of the yeast genes and it is particularly important in shaping the mRNA profile of the genes belonging to the environmental stress response. In most cases, changes in transcription rates and mRNA stabilities are homodirectional for both parameters, although some interesting cases of antagonist behavior are found. The statistical analysis of gene targets and sequence motifs within the clusters of genes with similar behaviors shows that both transcriptional and post-transcriptional regulons apparently contribute to the general heat stress response by means of transcriptional factors and RNA binding proteins.

  10. Analysis of mRNA transcripts in chronic myeloid leukemia patients

    Directory of Open Access Journals (Sweden)

    Meissner Rosely de V.

    1999-01-01

    Full Text Available The nature of BCR/ABL hybrid mRNA was analyzed by RT-PCR in cells from 33 patients (22 males, 11 females with chronic myeloid leukemia (CML. b3a2 mRNA was found in 14 cases, whereas 13 patients had b2a2 mRNA and six had both kinds of mRNA, with a predominance of the b3a2 type. The type of mRNA present showed no significant correlation with age, hemoglobin level, number of leukocytes and platelets, percentage of blasts or basophils or the presence of splenomegaly at diagnosis. There was also no correlation with sex or duration of the chronic phase. When these results were combined with those reported by other groups, a significant association (P = 0.029 was observed for mRNA type vs. sex, with a predominance of men in the groups expressing b2a2 (2.68:1 and b3a2 (1.33:1. We conclude that the classification of patients according to mRNA type does not homogenize the clinical and hematological data within groups, where variance is large, nor does it allow a differentiation between groups.

  11. Prefrontal cortical–striatal dopamine receptor mRNA expression predicts distinct forms of impulsivity

    Science.gov (United States)

    Simon, Nicholas W.; Beas, Blanca S.; Montgomery, Karienn S.; Haberman, Rebecca P.; Bizon, Jennifer L.; Setlow, Barry

    2014-01-01

    Variation in dopamine receptor levels has been associated with different facets of impulsivity. To further delineate the neural substrates underlying impulsive action (inability to withhold a prepotent motor response) and impulsive choice (delay aversion), we characterised rats in the Differential Reinforcement of Low Rates of Responding task and a delay discounting task. We also measured performance on an effort-based discounting task. We then assessed D1 and D2 dopamine receptor mRNA expression in subregions of the prefrontal cortex and nucleus accumbens using in situ hybridisation, and compared these data with behavioral performance. Expression of D1 and D2 receptor mRNA in distinct brain regions was predictive of impulsive action. A dissociation within the nucleus accumbens was observed between subregions and receptor subtypes; higher D1 mRNA expression in the shell predicted greater impulsive action, whereas lower D2 mRNA expression in the core predicted greater impulsive action. We also observed a negative correlation between impulsive action and D2 mRNA expression in the prelimbic cortex. Interestingly, a similar relationship was present between impulsive choice and prelimbic cortex D2 mRNA, despite the fact that behavioral indices of impulsive action and impulsive choice were uncorrelated. Finally, we found that both high D1 mRNA expression in the insular cortex and low D2 mRNA expression in the infralimbic cortex were associated with willingness to exert effort for rewards. Notably, dopamine receptor mRNA in these regions was not associated with either facet of impulsivity. The data presented here provide novel molecular and neuroanatomical distinctions between different forms of impulsivity, as well as effort-based decision-making. PMID:23510331

  12. Impairment of FOS mRNA stabilization following translation arrest in granulocytes from myelodysplastic syndrome patients.

    Science.gov (United States)

    Feng, Xiaomin; Shikama, Yayoi; Shichishima, Tsutomu; Noji, Hideyoshi; Ikeda, Kazuhiko; Ogawa, Kazuei; Kimura, Hideo; Takeishi, Yasuchika; Kimura, Junko

    2013-01-01

    Although quantitative and qualitative granulocyte defects have been described in myelodysplastic syndromes (MDS), the underlying molecular basis of granulocyte dysfunction in MDS is largely unknown. We recently found that FOS mRNA elevation under translation-inhibiting stimuli was significantly smaller in granulocytes from MDS patients than in healthy individuals. The aim of this study is to clarify the cause of the impaired FOS induction in MDS. We first examined the mechanisms of FOS mRNA elevation using granulocytes from healthy donors cultured with the translation inhibitor emetine. Emetine increased both transcription and mRNA stability of FOS. p38 MAPK inhibition abolished the emetine-induced increase of FOS transcription but did not affect FOS mRNA stabilization. The binding of an AU-rich element (ARE)-binding protein HuR to FOS mRNA containing an ARE in 3'UTR was increased by emetine, and the knockdown of HuR reduced the FOS mRNA stabilizing effect of emetine. We next compared the emetine-induced transcription and mRNA stabilization of FOS between MDS patients and healthy controls. Increased rates of FOS transcription by emetine were similar in MDS and controls. In the absence of emetine, FOS mRNA decayed to nearly 17% of initial levels in 45 min in both groups. In the presence of emetine, however, 76.7±19.8% of FOS mRNA remained after 45 min in healthy controls, versus 37.9±25.5% in MDS (Pgranulocytes.

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

    DEFF Research Database (Denmark)

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

    2006-01-01

    Visfatin [pre-beta-cell colony-enhancing factor (PBEF)] is a novel adipokine that is produced by adipose tissue, skeletal muscle, and liver and has insulin-mimetic actions. Regular exercise enhances insulin sensitivity. In the present study, we therefore examined visfatin mRNA expression...... by elevated levels of plasma visfatin. Recombinant human IL-6 infusion to mimic the exercise-induced IL-6 response (n = 6) had no effect on visfatin mRNA expression in adipose tissue compared with the effect of placebo infusion (n = 6). The finding that exercise enhances subcutaneous adipose tissue visfatin mRNA...... in abdominal subcutaneous adipose tissue and skeletal muscle biopsies obtained from healthy young men at time points 0, 3, 4.5, 6, 9, and 24 h in relation to either 3 h of ergometer cycle exercise at 60% of Vo(2 max) or rest. Adipose tissue visfatin mRNA expression increased threefold at the time points 3, 4...

  14. UCP2 mRNA expression is dependent on glucose metabolism in pancreatic islets

    Energy Technology Data Exchange (ETDEWEB)

    Dalgaard, Louise T., E-mail: ltd@ruc.dk [Division of Endocrinology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA (United States); Department of Science, Systems and Models, Roskilde University (Denmark)

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer UCP2 mRNA levels are decreased in islets of Langerhans from glucokinase deficient mice. Black-Right-Pointing-Pointer UCP2 mRNA up-regulation by glucose is dependent on glucokinase. Black-Right-Pointing-Pointer Absence of UCP2 increases GSIS of glucokinase heterozygous pancreatic islets. Black-Right-Pointing-Pointer This may protect glucokinase deficient mice from hyperglycemic damages. -- Abstract: Uncoupling Protein 2 (UCP2) is expressed in the pancreatic {beta}-cell, where it partially uncouples the mitochondrial proton gradient, decreasing both ATP-production and glucose-stimulated insulin secretion (GSIS). Increased glucose levels up-regulate UCP2 mRNA and protein levels, but the mechanism for UCP2 up-regulation in response to increased glucose is unknown. The aim was to examine the effects of glucokinase (GK) deficiency on UCP2 mRNA levels and to characterize the interaction between UCP2 and GK with regard to glucose-stimulated insulin secretion in pancreatic islets. UCP2 mRNA expression was reduced in GK+/- islets and GK heterozygosity prevented glucose-induced up-regulation of islet UCP2 mRNA. In contrast to UCP2 protein function UCP2 mRNA regulation was not dependent on superoxide generation, but rather on products of glucose metabolism, because MnTBAP, a superoxide dismutase mimetic, did not prevent the glucose-induced up-regulation of UCP2. Glucose-stimulated insulin secretion was increased in UCP2-/- and GK+/- islets compared with GK+/- islets and UCP2 deficiency improved glucose tolerance of GK+/- mice. Accordingly, UCP2 deficiency increased ATP-levels of GK+/- mice. Thus, the compensatory down-regulation of UCP2 is involved in preserving the insulin secretory capacity of GK mutant mice and might also be implicated in limiting disease progression in MODY2 patients.

  15. Conserved noncoding sequences are associated with rates of mRNA decay in Arabidopsis

    Directory of Open Access Journals (Sweden)

    Jacob B Spangler

    2013-05-01

    Full Text Available Steady-state mRNA levels are tightly regulated through a combination of transcriptional and post-transcriptional control mechanisms. The discovery of cis-acting DNA elements that encode these control mechanisms is of high importance. We have investigated the influence of conserved non-coding sequences (CNSs, DNA patterns retained after an ancient whole genome duplication event, on the breadth of gene expression and the rates of mRNA decay in Arabidopsis thaliana. The absence of CNSs near α duplicate genes was associated with a decrease in breadth of gene expression and slower mRNA decay rates while the presence CNSs near α duplicates was associated with an increase in breadth of gene expression and faster mRNA decay rates. The observed difference in mRNA decay rate was fastest in genes with CNSs in both nontranscribed and transcribed regions, albeit through an unknown mechanism. This study supports the notion that some Arabidopsis CNSs regulate the steady-state mRNA levels through post-transcriptional control mechanisms and that CNSs also play a role in controlling the breadth of gene expression.

  16. Tau mRNA 3'UTR-to-CDS ratio is increased in Alzheimer disease.

    Science.gov (United States)

    García-Escudero, Vega; Gargini, Ricardo; Martín-Maestro, Patricia; García, Esther; García-Escudero, Ramón; Avila, Jesús

    2017-08-10

    Neurons frequently show an imbalance in expression of the 3' untranslated region (3'UTR) relative to the coding DNA sequence (CDS) region of mature messenger RNAs (mRNA). The ratio varies among different cells or parts of the brain. The Map2 protein levels per cell depend on the 3'UTR-to-CDS ratio rather than the total mRNA amount, which suggests powerful regulation of protein expression by 3'UTR sequences. Here we found that MAPT (the microtubule-associated protein tau gene) 3'UTR levels are particularly high with respect to other genes; indeed, the 3'UTR-to-CDS ratio of MAPT is balanced in healthy brain in mouse and human. The tau protein accumulates in Alzheimer diseased brain. We nonetheless observed that the levels of RNA encoding MAPT/tau were diminished in these patients' brains. To explain this apparently contradictory result, we studied MAPT mRNA stoichiometry in coding and non-coding regions, and found that the 3'UTR-to-CDS ratio was higher in the hippocampus of Alzheimer disease patients, with higher tau protein but lower total mRNA levels. Our data indicate that changes in the 3'UTR-to-CDS ratio have a regulatory role in the disease. Future research should thus consider not only mRNA levels, but also the ratios between coding and non-coding regions. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Association of chemerin mRNA expression in human epicardial adipose tissue with coronary atherosclerosis.

    Science.gov (United States)

    Gao, Xiuying; Mi, Shuhua; Zhang, Fuzhuang; Gong, Fengying; Lai, Yongqiang; Gao, Feng; Zhang, Xiaoxia; Wang, Linjie; Tao, Hong

    2011-10-07

    Growing evidence suggests that epicardial adipose tissue (EAT) may play a key role in the pathogenesis and development of coronary artery disease (CAD) by producing several inflammatory adipokines. Chemerin, a novel adipokine, has been reported to be involved in regulating immune responses and glucolipid metabolism. Given these properties, chemerin may provide an interesting link between obesity, inflammation and atherosclerosis. In this study, we sought to determine the relationship of chemerin expression in EAT and the severity of coronary atherosclerosis in Han Chinese patients. Serums and adipose tissue biopsies (epicardial and thoracic subcutaneous) were obtained from CAD (n = 37) and NCAD (n = 16) patients undergoing elective cardiac surgery. Gensini score was used to assess the severity of CAD. Serum levels of chemerin, adiponectin and insulin were measured by ELISA. Chemerin protein expression in adipose tissue was detected by immunohistochemistry. The mRNA levels of chemerin, chemR23, adiponectin and TNF-alpha in adipose tissue were detected by RT-PCR. We found that EAT of CAD group showed significantly higher levels of chemerin and TNF-alpha mRNA, and significantly lower level of adiponectin mRNA than that of NCAD patients. In CAD group, significantly higher levels of chemerin mRNA and protein were observed in EAT than in paired subcutaneous adipose tissue (SAT), whereas such significant difference was not found in NCAD group. Chemerin mRNA expression in EAT was positively correlated with Gensini score (r = 0.365, P correlation remained statistically significant (r = 0.357, P age, gender, BMI and waist circumference. Chemerin mRNA expression in EAT was also positively correlated with BMI (r = 0.305, P correlated with adiponectin mRNA expression in EAT (r = -0.322, P chemerin or adiponectin between the two groups. Likewise, neither serum chemerin nor serum adiponectin was associated with Gensini score (P > 0.05). The expressions of chemerin mRNA and

  18. mRNA quality control pathways in Saccharomyces cerevisiae

    Indian Academy of Sciences (India)

    2013-07-10

    Jul 10, 2013 ... mRNPs in the nucleus very often leads to the formation of aberrant and faulty messages along with their functional .... Figure 1. mRNA life-cycle in eukaryotic cell: Schematic view of the nuclear and cytoplasmic phases of mRNA life cycle, namely ..... structure is characteristic and critical feature of an mRNA.

  19. Connecting protein and mRNA burst distributions for stochastic models of gene expression

    CERN Document Server

    Elgart, Vlad; Fenley, Andrew T; Kulkarni, Rahul V

    2011-01-01

    The intrinsic stochasticity of gene expression can lead to large variability in protein levels for genetically identical cells. Such variability in protein levels can arise from infrequent synthesis of mRNAs which in turn give rise to bursts of protein expression. Protein expression occurring in bursts has indeed been observed experimentally and recent studies have also found evidence for transcriptional bursting, i.e. production of mRNAs in bursts. Given that there are distinct experimental techniques for quantifying the noise at different stages of gene expression, it is of interest to derive analytical results connecting experimental observations at different levels. In this work, we consider stochastic models of gene expression for which mRNA and protein production occurs in independent bursts. For such models, we derive analytical expressions connecting protein and mRNA burst distributions which show how the functional form of the mRNA burst distribution can be inferred from the protein burst distributio...

  20. Increased thymidylate synthase mRNA concentration in blood leukocytes following an experimental stressor

    DEFF Research Database (Denmark)

    Ehrnrooth, Eva; Zacharia, Robert; Svendsen, Gunner

    2002-01-01

    of an experimental stressor on mRNA levels in peripheral blood leukocytes of thymidylate synthase (TS), a gene necessary for cell division, while investigating possible individual differences in stress reactivity. METHODS: Fifteen healthy subjects were investigated under three experimental conditions: (1) exposure...... to a computerized mental stressor; (2) relaxation, and (3) control. Measurements included TS mRNA levels, total leukocyte number, leukocyte subtypes, and serum cortisol before (baseline), immediately after, and 1 h after each experimental condition. RESULTS: While no significant differences were found between...... in percentage of neutrophil cells after stress. CONCLUSION: The results suggest that TS mRNA levels in peripheral leukocytes may be sensitive to mental stress and confirm previous findings indicating that subjects scoring high on the personality trait of absorption exhibit greater physiological stress...

  1. Viscum album-mediated COX-2 inhibition implicates destabilization of COX-2 mRNA.

    Science.gov (United States)

    Saha, Chaitrali; Hegde, Pushpa; Friboulet, Alain; Bayry, Jagadeesh; Kaveri, Srinivas V

    2015-01-01

    Extensive use of Viscum album (VA) preparations in the complementary therapy of cancer and in several other human pathologies has led to an increasing number of cellular and molecular approaches to explore the mechanisms of action of VA. We have recently demonstrated that, VA preparations exert a potent anti-inflammatory effect by selectively down-regulating the COX-2-mediated cytokine-induced secretion of prostaglandin E2 (PGE2), one of the important molecular signatures of inflammatory reactions. In this study, we observed a significant down-regulation of COX-2 protein expression in VA-treated A549 cells however COX-2 mRNA levels were unaltered. Therefore, we hypothesized that VA induces destabilisation of COX-2 mRNA, thereby depleting the available functional COX-2 mRNA for the protein synthesis and for the subsequent secretion of PGE2. To address this question, we analyzed the molecular degradation of COX-2 protein and its corresponding mRNA in A549 cell line. Using cyclohexamide pulse chase experiment, we demonstrate that, COX-2 protein degradation is not affected by the treatment with VA whereas experiments on transcriptional blockade with actinomycin D, revealed a marked reduction in the half life of COX-2 mRNA due to its rapid degradation in the cells treated with VA compared to that in IL-1β-stimulated cells. These results thus demonstrate that VA-mediated inhibition of PGE2 implicates destabilization of COX-2 mRNA.

  2. Viscum album-mediated COX-2 inhibition implicates destabilization of COX-2 mRNA.

    Directory of Open Access Journals (Sweden)

    Chaitrali Saha

    Full Text Available Extensive use of Viscum album (VA preparations in the complementary therapy of cancer and in several other human pathologies has led to an increasing number of cellular and molecular approaches to explore the mechanisms of action of VA. We have recently demonstrated that, VA preparations exert a potent anti-inflammatory effect by selectively down-regulating the COX-2-mediated cytokine-induced secretion of prostaglandin E2 (PGE2, one of the important molecular signatures of inflammatory reactions. In this study, we observed a significant down-regulation of COX-2 protein expression in VA-treated A549 cells however COX-2 mRNA levels were unaltered. Therefore, we hypothesized that VA induces destabilisation of COX-2 mRNA, thereby depleting the available functional COX-2 mRNA for the protein synthesis and for the subsequent secretion of PGE2. To address this question, we analyzed the molecular degradation of COX-2 protein and its corresponding mRNA in A549 cell line. Using cyclohexamide pulse chase experiment, we demonstrate that, COX-2 protein degradation is not affected by the treatment with VA whereas experiments on transcriptional blockade with actinomycin D, revealed a marked reduction in the half life of COX-2 mRNA due to its rapid degradation in the cells treated with VA compared to that in IL-1β-stimulated cells. These results thus demonstrate that VA-mediated inhibition of PGE2 implicates destabilization of COX-2 mRNA.

  3. Elevated proto-oncogene and collagen mRNA expression in PVR retinas.

    Science.gov (United States)

    Hollborn, Margrit; Faude, Frank; Wiedemann, Peter; Kohen, Leon

    2003-05-01

    Retinal detachment is often accompanied by proliferation and migration of retinal cells and by increased synthesis of structural proteins, known as proliferative vitreoretinopathy (PVR). Herein we investigate the messenger RNA (mRNA) expression of proto-oncogenes responsible for cell proliferation and of structural proteins that have a role in membrane formation. Retinal samples were obtained from patients undergoing vitreoretinal surgery for the treatment of retinal detachment complicated by PVR. Normal human control retinas were obtained from cornea donors. The mRNA expression of the proto-oncogenes c- myc, c- fos and the proliferation marker Ki67, as well as of collagen type III and type IV, were investigated using the ribonuclease protection assay. Ki67 mRNA expression was not detectable in either sample type, but c- fos and c- myc mRNA expression was found in normal and PVR retinas. Whereas the expression of c- myc showed a marginal increase, the up-regulation in c- fos expression was strongly significant (5.07-fold). The mRNA of collagen type III was detectable at widely varying levels in all the PVR retinas but was found in only 2 of the 16 analysed normal samples. Collagen type IV mRNA was expressed in both PVR and control samples but was higher (2.21-fold) in the PVR retinas. These results indicate that an up-regulation of the proto-oncogene c- fos occurs in human PVR retinas. An increase in mRNA expression of collagen types III and IV takes place simultaneously. These changes in mRNA expression appear to be mainly connected to the initiation of cell proliferation, dedifferentiation and formation of tractional membranes.

  4. mRNA transcripts as molecular biomarkers in medicine and nutrition

    Science.gov (United States)

    Sunde, Roger A.

    2010-01-01

    In medicine, mRNA transcripts are being developed as molecular biomarkers for the diagnosis and treatment of a number of diseases. These biomarkers offer early and more accurate prediction and diagnosis of disease and disease progression, and ability to identify individuals at risk. Use of microarrays also offers opportunity to identify orthogonal (uncorrelated) biomarkers not known to be linked with conventional biomarkers. Investigators are increasingly using blood as a surrogate tissue for biopsy and analysis; total RNA isolated from whole blood is predominantly from erythroid cells, and whole blood mRNA share more than 80% of the transcriptome with major tissues. Thus blood mRNA biomarkers for individualized disease prediction and diagnosis are an exciting area in medicine; mRNA biomarkers in nutrition have potential application that parallel these opportunities. Assessment of selenium (Se) status and requirements is one area where tissue mRNA levels have been used successfully. Selenoprotein-H and selenoprotein-W as well as glutathione peroxidase-1 (Gpx1) mRNAs are highly down-regulated in Se deficiency in rat liver, and the minimum dietary Se requirement is 0.06–0.07 μg Se/g based on these biomarkers, similar to requirements determined using conventional biomarkers. Blood Gpx1 mRNA can also be used to determine Se requirements in rats, showing that blood mRNA has potential for assessment of nutrient status. Future research is needed to develop mRNA biomarker panels for all nutrients that will discriminate between deficient, marginal, adequate, and supernutritional individuals and populations, and differentiate between individuals that will benefit versus be adversely affected by nutrient supplementation. PMID:20303730

  5. Matrin 3 binds and stabilizes mRNA.

    Directory of Open Access Journals (Sweden)

    Maayan Salton

    Full Text Available Matrin 3 (MATR3 is a highly conserved, inner nuclear matrix protein with two zinc finger domains and two RNA recognition motifs (RRM, whose function is largely unknown. Recently we found MATR3 to be phosphorylated by the protein kinase ATM, which activates the cellular response to double strand breaks in the DNA. Here, we show that MATR3 interacts in an RNA-dependent manner with several proteins with established roles in RNA processing, and maintains its interaction with RNA via its RRM2 domain. Deep sequencing of the bound RNA (RIP-seq identified several small noncoding RNA species. Using microarray analysis to explore MATR3's role in transcription, we identified 77 transcripts whose amounts depended on the presence of MATR3. We validated this finding with nine transcripts which were also bound to the MATR3 complex. Finally, we demonstrated the importance of MATR3 for maintaining the stability of several of these mRNA species and conclude that it has a role in mRNA stabilization. The data suggest that the cellular level of MATR3, known to be highly regulated, modulates the stability of a group of gene transcripts.

  6. Regulation of the growth hormone (GH) receptor and GH-binding protein mRNA

    Energy Technology Data Exchange (ETDEWEB)

    Kaji, Hidesuke; Ohashi, Shin-Ichirou; Abe, Hiromi; Chihara, Kazuo [Kobe Univ. School of Medicine, Kobe (Japan)

    1994-12-31

    In fasting rats, a transient increase in growth hormone-binding protein (GHBP) mRNA levels was observed after 1 day, in muscle, heart, and liver, but not in fat tissues. The liver GH receptor (GHR) mRNA level was significantly increased after 1 day (but not after 5 days) of bovine GH (bGH) treatment in fed rats. Both the liver GHR mRNA level and the net increment of plasma IGF-I markedly decreased after 5 days of bGH administration in fasting rats. These findings suggest that GHR and GHBP mRNAs in the liver are expressed in a different way and that the expression of GHBP mRNA is regulated differently between tissues, at least in rats. The results also suggest that refractoriness to GH in a sustained fasting state might be beneficial in preventing anabolic effects of GH. In humans, GHR mRNA in lymphocytes, from subjects with either GH-deficiency or acromegaly, could be detected by the reverse transcription-polymerase chain reaction method. In one patient with partial GH insensitivity, a heterozygous missense mutation (P561T) was identified in the cytoplasmic domain of GHR. 15 refs., 4 figs.

  7. Messenger RNA (mRNA) nanoparticle tumour vaccination

    Science.gov (United States)

    Phua, Kyle K. L.; Nair, Smita K.; Leong, Kam W.

    2014-06-01

    Use of mRNA-based vaccines for tumour immunotherapy has gained increasing attention in recent years. A growing number of studies applying nanomedicine concepts to mRNA tumour vaccination show that the mRNA delivered in nanoparticle format can generate a more robust immune response. Advances in the past decade have deepened our understanding of gene delivery barriers, mRNA's biological stability and immunological properties, and support the notion for engineering innovations tailored towards a more efficient mRNA nanoparticle vaccine delivery system. In this review we will first examine the suitability of mRNA for engineering manipulations, followed by discussion of a model framework that highlights the barriers to a robust anti-tumour immunity mediated by mRNA encapsulated in nanoparticles. Finally, by consolidating existing literature on mRNA nanoparticle tumour vaccination within the context of this framework, we aim to identify bottlenecks that can be addressed by future nanoengineering research.

  8. Stabilization of circulating thyroglobulin mRNA transcripts in patients treated for differentiated thyroid carcinoma

    DEFF Research Database (Denmark)

    Feddersen, Søren; Bastholt, Lars; Pedersen, Susanne Møller

    2017-01-01

    BACKGROUND: The clinical utility of serum thyroglobulin (TG) in the follow-up of patients with differentiated thyroid carcinoma (DTC) may be compromised by the presence of endogenous anti-thyroglobulin antibodies (TGAb). To prevent interference by TGAb several groups have developed real-time PCR...... (RT-PCR) based assays for quantification of blood TG mRNA levels. For accurate quantification of TG mRNA in blood preanalytical factors must be recognized and controlled. In this study we evaluate the effect of different blood RNA stabilizing systems - the Tempus(T) (M) Blood RNA system...... hours, and RNA yield, integrity and purity was determined. TG, GAPDH and ACTB mRNA levels were quantified by semi-quantitative RT-PCR. RESULTS: The RNA yield was significantly higher for blood collected in Tempus tubes compared to PAXgene tubes following storage for 72 hours at room temperature (p = 0...

  9. Tuning protein expression using synonymous codon libraries targeted to the 5' mRNA coding region

    DEFF Research Database (Denmark)

    Goltermann, Lise; Borch Jensen, Martin; Bentin, Thomas

    2011-01-01

    sequence allowed tuning of protein expression over ~300-fold with preservation of amino acid identity. This approach is simple and should be generally applicable in bacteria. The data support that features in the 5' mRNA coding region near the AUG start codon are key in determining translation output......In bacteria, the 5' mRNA coding region plays an important role in determining translation output. Here, we report synthetic sequences that when placed in the 5'-mRNA coding region, leading to recombinant proteins containing short N-terminal extensions, virtually abolish, enhance or produce...... intermediate expression levels of green fluorescent protein in Escherichia coli. At least in one case, no apparent effect on protein stability was observed, pointing to RNA level effects as the principal reason for the observed expression differences. Targeting a synonymous codon library to the 5' coding...

  10. Changes in mRNA for metabotropic glutamate receptors after transient cerebral ischaemia

    DEFF Research Database (Denmark)

    Rosdahl, D; Seitzberg, D A; Christensen, Thomas

    1994-01-01

    Using a rat 4-vessel occlusion model of cerebral ischaemia we studied the changes in the mRNA level for the metabotropic receptor subtypes mGluR1 alpha, mGluR1 beta, mGluR2, mGluR3, mGluR4, and mGluR5 by means of in situ hybridization with oligonucleotides. After 24 hours of reperfusion the mRNA...... levels were significantly increased for mGluR2 and mGluR4 while it was significantly decreased for mGluR5. These results suggest that vulnerable neurones react to an increased extracellular glutamate concentration by differential regulation of the mRNA for metabotropic glutamate receptor subtypes which...... perhaps reflects the different pre- or postsynaptic location and different involvement in ischaemic neurodegeneration....

  11. Conserved CPEs in the p53 3' untranslated region influence mRNA stability and protein synthesis

    DEFF Research Database (Denmark)

    Rosenstierne, Maiken W; Vinther, Jeppe; Mittler, Gerhard

    2008-01-01

    -type p53 3'UTR reduced mRNA steady state levels of the reporter gene and point mutations in the CPEs rescued the mRNA steady state levels in the MCF-7 cells, but not in the HaCaT cells. In both cell lines, the CPEs had a significant effect on translation of the reporter and influenced the effect of UV...

  12. EXPRESSION OF AID-SPECIFIC mRNA IN PERIPHERAL BLOOD LYMPHOCYTES IN BRONCHIAL ASTHMA

    Directory of Open Access Journals (Sweden)

    V. N. Mineev

    2015-01-01

    Full Text Available The aim of this study was to evaluate a possible role of AID (AICDA – activation-induced (cytidine deaminase in the bronchial asthma (BA pathogenesis. Materials and methods. We have examined twelve healthy control persons, forty-two patients with allergic bronchial asthma (ABA and twenty-seven patients with non-allergic bronchial asthma (NABA. The AID mRNA expression was evaluated by means of RT-PCR. Results: AID mRNA was more significantly expressed in BA, than in healthy controls. Meanwhile, no significant differences were revealed between ABA and NABA groups. Correlation analysis of AID mRNA expression levels in peripheral blood lymphocytes has shown that CHε mRNA and total serum IgE revealed significant negative correlation, in the NABA group only. The levels of AID mRNA expression in peripheral blood lymphocytes exhibited positive and significant correlations with clinical characteristics, reflecting severity and stage of the disease, in BA patients. Moreover, a significant positive correlation was revealed with eosinophil contents in sputum. Conclusion. It was concluded that normal regulation of IgE class switching normally based on feedback regulation, was impaired in ABA but not affected in NABA. 

  13. Peripheral Mononuclear Cell Resistin mRNA Expression Is Increased in Type 2 Diabetic Women

    Directory of Open Access Journals (Sweden)

    Panayoula C. Tsiotra

    2008-01-01

    Full Text Available Resistin has been shown to cause insulin resistance and to impair glucose tolerance in rodents, but in humans its physiological role still remains elusive. The aim of this study was to examine whether resistin mRNA expression in human peripheral mononuclear cells (PBMCs and its corresponding plasma levels are altered in type 2 diabetes. Resistin mRNA levels were easily detectable in human PBMC, and found to be higher in DM2 compared to healthy women (P=.05. Similarly, mononuclear mRNA levels of the proinflammatory cytokines IL-1β, TNF-α, and IL-6 were all significantly higher in DM2 compared to control women (P<.001. The corresponding plasma resistin levels were slightly, but not significantly, increased in DM2 women (P=.051, and overall, they correlated significantly with BMI (r=0.406, P=.010 and waist circumference (r=0.516, P=.003, but not with fasting insulin levels or HOMA-IR. Resistin mRNA expression is increased in PBMC from DM2 women, together with increased expression of the inflammatory cytokines IL-1β, TNF-α, and IL-6, independent of obesity. These results suggest that resistin and cytokines might contribute to the low-grade inflammation and the increased atherogenic risk observed in these patients.

  14. Reduced m6A mRNA methylation is correlated with the progression of human cervical cancer.

    Science.gov (United States)

    Wang, Xiuli; Li, Zenghui; Kong, Beihua; Song, Chen; Cong, Jianglin; Hou, Jianqing; Wang, Shaoguang

    2017-11-17

    The m6A mRNA methylation involves in mRNA splicing, degradation and translation. Recent studies have revealed that reduced m6A mRNA methylation might promote cancer development. However, the role of m6A mRNA methylation in cervical cancer development remains unknown. Therefore, we investigated the role of m6A methylation in cervical cancer in the current study. We first evaluated the m6A mRNA methylation level in 286 pairs of cervical cancer samples and their adjacent normal tissues by dot blot assay. Then the role of m6A on patient survival rates and cervical cancer progression were assessed. The m6A level was significantly reduced in the cervical cancer when comparing with the adjacent normal tissue. The m6A level reduction was significantly correlated with the FIGO stage, tumor size, differentiation, lymph invasion and cancer recurrence. It was also shown to be an independent prognostic indicator of disease-free survival and overall survival for patients with cervical cancer. Reducing m6A level via manipulating the m6A regulators expression promoted cervical cancer cell proliferation. And increasing m6A level significantly suppressed tumor development both in vitro and in vivo. Our results showed that the reduced m6A level is tightly associated with cervical cancer development and m6A mRNA methylation might be a potential therapeutic target in cervical cancer.

  15. Conventional and unconventional mechanisms for capping viral mRNA.

    Science.gov (United States)

    Decroly, Etienne; Ferron, François; Lescar, Julien; Canard, Bruno

    2011-12-05

    In the eukaryotic cell, capping of mRNA 5' ends is an essential structural modification that allows efficient mRNA translation, directs pre-mRNA splicing and mRNA export from the nucleus, limits mRNA degradation by cellular 5'-3' exonucleases and allows recognition of foreign RNAs (including viral transcripts) as 'non-self'. However, viruses have evolved mechanisms to protect their RNA 5' ends with either a covalently attached peptide or a cap moiety (7-methyl-Gppp, in which p is a phosphate group) that is indistinguishable from cellular mRNA cap structures. Viral RNA caps can be stolen from cellular mRNAs or synthesized using either a host- or virus-encoded capping apparatus, and these capping assemblies exhibit a wide diversity in organization, structure and mechanism. Here, we review the strategies used by viruses of eukaryotic cells to produce functional mRNA 5'-caps and escape innate immunity.

  16. Analysis of novel NEFL mRNA targeting microRNAs in amyotrophic lateral sclerosis.

    Directory of Open Access Journals (Sweden)

    Muhammad Ishtiaq

    Full Text Available Amyotrophic lateral sclerosis (ALS is a fatal disease characterized by progressive motor neuron degeneration and neurofilament aggregate formation. Spinal motor neurons in ALS also show a selective suppression in the levels of low molecular weight neurofilament (NEFL mRNA. We have been interested in investigating the role of microRNAs (miRNAs in NEFL transcript stability. MiRNAs are small, 20-25 nucleotide, non-coding RNAs that act as post-transcriptional gene regulators by targeting the 3' untranslated region (3'UTR of mRNA resulting in mRNA decay or translational silencing. In this study, we characterized putative novel miRNAs from a small RNA library derived from control and sporadic ALS (sALS spinal cords. We detected 80 putative novel miRNAs, 24 of which have miRNA response elements (MREs within the NEFL mRNA 3'UTR. From this group, we determined by real-time PCR that 10 miRNAs were differentially expressed in sALS compared to controls. Functional analysis by reporter gene assay and relative quantitative RT-PCR showed that two novel miRNAs, miR-b1336 and miR-b2403, were downregulated in ALS spinal cord and that both stabilize NEFL mRNA. We confirmed the direct effect of these latter miRNAs using anit-miR-b1336 and anti-miR-b2403. These results demonstrate that the expression of two miRNAs (miRNAs miR-b1336 and miR-b2403 whose effect is to stabilize NEFL mRNA are down regulated in ALS, the net effect of which is predicted to contribute directly to the loss of NEFL steady state mRNA which is pathognomic of spinal motor neurons in ALS.

  17. mRNA vaccines - a new era in vaccinology.

    Science.gov (United States)

    Pardi, Norbert; Hogan, Michael J; Porter, Frederick W; Weissman, Drew

    2018-01-12

    mRNA vaccines represent a promising alternative to conventional vaccine approaches because of their high potency, capacity for rapid development and potential for low-cost manufacture and safe administration. However, their application has until recently been restricted by the instability and inefficient in vivo delivery of mRNA. Recent technological advances have now largely overcome these issues, and multiple mRNA vaccine platforms against infectious diseases and several types of cancer have demonstrated encouraging results in both animal models and humans. This Review provides a detailed overview of mRNA vaccines and considers future directions and challenges in advancing this promising vaccine platform to widespread therapeutic use.

  18. The potential lipolysis function of musclin and its mRNA expression ...

    African Journals Online (AJOL)

    Musclin is a newly discovered factor and its functions remain to be defined. This study investigated the tissue expression pattern of musclin gene and its potential effect on lipid metabolism. Musclin mRNA levels in adipose, muscle tissues and primary adipocytes were examined by quantitative PCR. The musclin gene ...

  19. Dynamics of Translation of Single mRNA Molecules in Vivo

    NARCIS (Netherlands)

    Yan, Xiaowei; Hoek, Tim A.; Vale, Ronald D.; Tanenbaum, Marvin E.

    2016-01-01

    Regulation of mRNA translation, the process by which ribosomes decode mRNAs into polypeptides, is used to tune cellular protein levels. Currently, methods for observing the complete process of translation from single mRNAs in vivo are unavailable. Here, we report the long-term (>1 hr) imaging of

  20. Human Cytomegalovirus Strategies to Maintain and Promote mRNA Translation.

    Science.gov (United States)

    Vincent, Heather A; Ziehr, Benjamin; Moorman, Nathaniel J

    2016-04-13

    mRNA translation requires the ordered assembly of translation initiation factors and ribosomal subunits on a transcript. Host signaling pathways regulate each step in this process to match levels of protein synthesis to environmental cues. In response to infection, cells activate multiple defenses that limit viral protein synthesis, which viruses must counteract to successfully replicate. Human cytomegalovirus (HCMV) inhibits host defenses that limit viral protein expression and manipulates host signaling pathways to promote the expression of both host and viral proteins necessary for virus replication. Here we review key regulatory steps in mRNA translation, and the strategies used by HCMV to maintain protein synthesis in infected cells.

  1. Hepatic chemerin mRNA in morbidly obese patients with nonalcoholic fatty liver disease.

    Science.gov (United States)

    Kajor, Maciej; Kukla, Michał; Waluga, Marek; Liszka, Łukasz; Dyaczyński, Michał; Kowalski, Grzegorz; Żądło, Dominika; Berdowska, Agnieszka; Chapuła, Mateusz; Kostrząb-Zdebel, Anna; Bułdak, Rafał J; Sawczyn, Tomasz; Hartleb, Marek

    The aim of this study was to investigate hepatic chemerin mRNA, serum chemerin concentration, and immunohistochemical staining for chemerin and and chemokine receptor-like 1 (CMKLR1) in hepatic tissue in 56 morbidly obese women with nonalcoholic fatty liver disease (NAFLD) and to search for a relationship with metabolic and histopathological features. Chemerin mRNA was assessed by quantitative real-time PCR, chemerin, and CMKLR1 immunohistochemical expression with specific antibodies, while serum chemerin concentration was assessed with commercially available enzyme-linked immunosorbent assays. Serum chemerin concentration reached 874.1 ±234.6 ng/ml. There was no difference in serum chemerin levels between patients with BMI Liver chemerin mRNA was observed in all included patients and was markedly, but insignificantly, higher in those with BMI ≥ 40 kg/m2, hepatocyte ballooning, greater extent of steatosis, and definite NASH. Hepatic chemerin mRNA might be a predictor of hepatic steatosis, hepatocyte ballooning, and NAFLD activity score (NAS) but seemed not to be a primary driver regulating liver necroinflammatory activity and fibrosis. The lack of association between serum chemerin and hepatic chemerin mRNA may suggest that adipose tissue but not the liver is the main source of chemerin in morbidly obese women.

  2. Modulation of IFNAR1 mRNA expression in multiple sclerosis patients.

    Science.gov (United States)

    Serana, Federico; Sottini, Alessandra; Ghidini, Claudia; Zanotti, Cinzia; Capra, Ruggero; Cordioli, Cinzia; Caimi, Luigi; Imberti, Luisa

    2008-06-15

    Interferon-beta receptor (IFNAR) is composed of 2 subunits, IFNAR1 and IFNAR2, the latter of which is expressed as functional (IFNAR2.2), non-functional (IFNAR2.1) and soluble (IFNAR2.3) isoform. Real-Time PCR analysis of mRNA for all IFNAR components in multiple sclerosis patients naïve for therapy and undergoing long-term treatment with interferon-beta shows that IFNAR1 mRNA level is lower than in healthy controls. If long-term treated patients are divided according to the production of mRNA for Myxovirus protein-A, a marker of interferon-beta bioactivity, IFNAR1 mRNA reaches the values observed in controls only in Myxovirus protein-A-induced patients. Since chronic cell stimulation by interferon-beta induces IFNAR protein down-regulation, we suggest that the increase of IFNAR1 mRNA might serve as a mechanism for counterbalancing the loss of protein receptor, enhancing, at least in this sub-group of patients, cell responsiveness to interferon-beta.

  3. Localization of BDNF mRNA with the Huntington's disease protein in rat brain

    Directory of Open Access Journals (Sweden)

    Chao Moses V

    2010-05-01

    Full Text Available Abstract Background Studies have implicated reduced levels of brain-derived neurotrophic factor (BDNF in the pathogenesis of Huntington's disease. Mutant huntingtin (Htt protein was previously reported to decrease BDNF gene transcription and axonal transport of BDNF. We recently showed that wild-type Htt is associated with the Argonaute 2 microRNA-processing enzyme involved in gene silencing. In dendrites, Htt co-localizes with components of neuronal granules and mRNAs, indicating that it might play a role in post-transcriptional processing/transport of dendritic mRNAs. Results We conducted imaging experiments in cultured cortical neurons to demonstrate the co-localization of endogenous Htt and BDNF mRNA in fixed cells, and co-trafficking of BDNF 3'UTR mRNA with endogenous and fluorescently tagged Htt in live neurons. We used an enhanced technique that combines FISH and immunofluorescent staining to co-localize BDNF mRNA with Htt, Ago2, CPEB and dynein in thick vibratome sections of the rat cortex. Conclusions In cultured neurons and sections of the rat cortex, we found BDNF mRNA associated with Htt and components of neuronal RNA granules, which are centers for regulating RNA transport and local translation. Htt may play a role in post-transcriptional transport/targeting of mRNA for BDNF, thus contributing to neurotrophic support and neuron survival.

  4. Technical validation of an RT-qPCR in vitro diagnostic test system for the determination of breast cancer molecular subtypes by quantification of ERBB2, ESR1, PGR and MKI67 mRNA levels from formalin-fixed paraffin-embedded breast tumor specimens.

    Science.gov (United States)

    Laible, Mark; Schlombs, Kornelia; Kaiser, Katharina; Veltrup, Elke; Herlein, Stefanie; Lakis, Sotiris; Stöhr, Robert; Eidt, Sebastian; Hartmann, Arndt; Wirtz, Ralph M; Sahin, Ugur

    2016-07-07

    MammaTyper is a novel CE-marked in vitro diagnostic RT-qPCR assay which assigns routinely processed breast cancer specimens into the molecular subtypes Luminal A-like, Luminal B-like (HER2 positive or negative), HER2 positive (non-luminal) and Triple negative (ductal) according to the mRNA expression of ERBB2, ESR1, PGR and MKI67 and the St Gallen consensus surrogate clinical definition. Until now and regarding formalin-fixed, paraffin-embedded material (FFPE), this has been a task mostly accomplished by immunohistochemistry (IHC). However the discrepancy rates of IHC for the four breast cancer biomarkers are frequently under debate, especially for Ki-67 which carries the highest degree of inter- and even intra-observer variability. Herein we describe a series of studies in FFPE specimens which aim to fully validate the analytical performance of the MammaTyper assay, including the site to site reproducibility of the individual marker measurements. Tumor RNA was extracted with the novel RNXtract RNA extraction kit. Synthetic RNA was used to assess the sensitivity of the RNXtract kit. DNA and RNA specific qPCR assays were used so as to determine analyte specificity of RNXtract. For the assessment of limit of blank, limit of detection, analytical measurement range and PCR efficiency of the MammaTyper kit serial dilutions of samples were used. Analytical precision studies of MammaTyper were built around two different real time PCR platforms and involved breast tumor samples belonging to different subtypes analyzed across multiple sites and under various stipulated conditions. The MammaTyper assay robustness was tested against RNA input variations, alternative extraction methods and tumor cell content. Individual assays were linear up to at least 32.33 and 33.56 Cqs (quantification cycles) for the two qPCR platforms tested. PCR efficiency ranged from 99 to 109 %. In qPCR platform 1, estimates for assay specific inter-site standard deviations (SD) were between 0.14 and

  5. 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.

  6. Importance of a 5′ Stem-Loop for Longevity of papA mRNA in Escherichia coli

    OpenAIRE

    Bricker, Angela L.; Belasco, Joel G.

    1999-01-01

    High-level expression of the major pilus subunit (PapA) of uropathogenic strains of Escherichia coli results in part from the unusually long lifetime of the mRNA that encodes this protein. Here we report that the longevity of papA mRNA derives in large measure from the protection afforded by its 5′ untranslated region. This papA RNA segment can prolong the lifetime of an otherwise short-lived mRNA to which it is fused. In vivo alkylation studies indicate that, in its natural milieu, the papA ...

  7. Novel RNA-binding activity of NQO1 promotes SERPINA1 mRNA translation.

    Science.gov (United States)

    Di Francesco, Andrea; Di Germanio, Clara; Panda, Amaresh C; Huynh, Phu; Peaden, Robert; Navas-Enamorado, Ignacio; Bastian, Paul; Lehrmann, Elin; Diaz-Ruiz, Alberto; Ross, David; Siegel, David; Martindale, Jennifer L; Bernier, Michel; Gorospe, Myriam; Abdelmohsen, Kotb; de Cabo, Rafael

    2016-10-01

    NAD(P)H: quinone oxidoreductase (NQO1) is essential for cell defense against reactive oxidative species, cancer, and metabolic stress. Recently, NQO1 was found in ribonucleoprotein (RNP) complexes, but NQO1-interacting mRNAs and the functional impact of such interactions are not known. Here, we used ribonucleoprotein immunoprecipitation (RIP) and microarray analysis to identify comprehensively the subset of NQO1 target mRNAs in human hepatoma HepG2 cells. One of its main targets, SERPINA1 mRNA, encodes the serine protease inhibitor α-1-antitrypsin, A1AT, which is associated with disorders including obesity-related metabolic inflammation, chronic obstructive pulmonary disease (COPD), liver cirrhosis and hepatocellular carcinoma. Biotin pulldown analysis indicated that NQO1 can bind the 3' untranslated region (UTR) and the coding region (CR) of SERPINA1 mRNA. NQO1 did not affect SERPINA1 mRNA levels; instead, it enhanced the translation of SERPINA1 mRNA, as NQO1 silencing decreased the size of polysomes forming on SERPINA1 mRNA and lowered the abundance of A1AT. Luciferase reporter analysis further indicated that NQO1 regulates SERPINA1 mRNA translation through the SERPINA1 3'UTR. Accordingly, NQO1-KO mice had reduced hepatic and serum levels of A1AT and increased activity of neutrophil elastase (NE), one of the main targets of A1AT. We propose that this novel mechanism of action of NQO1 as an RNA-binding protein may help to explain its pleiotropic biological effects. Published by Elsevier Inc.

  8. Extracellular tumor-related mRNA in plasma of lymphoma patients and survival implications.

    Directory of Open Access Journals (Sweden)

    Vanesa Garcia

    Full Text Available BACKGROUND: We studied anomalous extracellular mRNAs in plasma from patients with diffuse large B-cell lymphoma (DLBCL and their survival implications. mRNAs studied have been reported in the literature as markers of poor (BCL2, CCND2, MYC and favorable outcome (LMO2, BCL6, FN1 in tumors. These markers were also analyzed in lymphoma tissues to test possible associations with their presence in plasma. METHODOLOGY/PRINCIPAL FINDINGS: mRNA from 42 plasma samples and 12 tumors from patients with DLBCL was analyzed by real-time PCR. Samples post-treatment were studied. The immunohistochemistry of BCL2 and BCL6 was defined. Presence of circulating tumor cells was determined by analyzing the clonality of the immunoglobulin heavy-chain genes by PCR. In DLBCL, MYC mRNA was associated with short overall survival. mRNA targets with unfavorable outcome in tumors were associated with characteristics indicative of poor prognosis, with partial treatment response and with short progression-free survival in patients with complete response. In patients with low IPI score, unfavorable mRNA targets were related to shorter overall survival, partial response, high LDH levels and death. mRNA disappeared in post-treatment samples of patients with complete response, and persisted in those with partial response or death. No associations were found between circulating tumor cells and plasma mRNA. Absence of BCL6 protein in tumors was associated with presence of unfavorable plasma mRNA. CONCLUSIONS/SIGNIFICANCE: Through a non-invasive procedure, tumor-derived mRNAs can be obtained in plasma. mRNA detected in plasma did not proceed from circulating tumor cells. In our study, unfavorable targets in plasma were associated with poor prognosis in B-cell lymphomas, mainly MYC mRNA. Moreover, the unfavorable targets in plasma could help us to classify patients with poor outcome within the good prognosis group according to IPI.

  9. Phencyclidine rapidly decreases neuronal mRNA of brain-derived neurotrophic factor.

    Science.gov (United States)

    Katanuma, Yusuke; Numakawa, Tadahiro; Adachi, Naoki; Yamamoto, Noriko; Ooshima, Yoshiko; Odaka, Haruki; Inoue, Takafumi; Kunugi, Hiroshi

    2014-06-01

    Downregulation of brain-derived neurotrophic factor (BDNF), a member of neurotrophin family, has been implicated in psychiatric diseases including schizophrenia. However, detailed mechanisms of its reduction in patients with schizophrenia remain unclear. Here, using cultured cortical neurons, we monitored BDNF mRNA levels following acute application of phencyclidine [PCP; an N-methyl-d-aspartate (NMDA) receptor blocker], which is known to produce schizophrenia-like symptoms. We found that PCP rapidly caused a reduction in total amount of BDNF transcripts without effect on cell viability, while mRNA levels of nerve growth factor was intact. Actinomycin-D (ActD), an RNA synthesis inhibitor, decreased total BDNF mRNA levels similar to PCP, and coapplication of ActD with PCP did not show further reduction in BDNF mRNA compared with solo application of each drug. Among BDNF exons I, IV, and VI, the exon IV, which is positively regulated by neuronal activity, was highly sensitive to PCP. Furthermore, PCP inactivated cAMP response element-binding protein (CREB; a regulator of transcriptional activity of exon IV). The inactivation of CREB was also achieved by an inhibitor for Ca(2+) /calmodulin kinase II (CaMKII), although coapplication with PCP induced no further inhibition on the CREB activity. It is possible that PCP decreases BDNF transcription via blocking the NMDA receptor/CaMKII/CREB signaling. Copyright © 2014 Wiley Periodicals, Inc.

  10. Allelic Imbalance of mRNA Associated with α2-HS Glycoprotein (Fetuin-A) Polymorphism.

    Science.gov (United States)

    Inaoka, Yoshihiko; Osawa, Motoki; Mukasa, Nahoko; Miyashita, Keiko; Satoh, Fumiko; Kakimoto, Yu

    2015-01-01

    Alpha 2-HS glycoprotein (AHSG), also designated as fetuin-A, exhibits polymorphism in population genetics consisting of two major alleles of AHSG(∗) 1 and AHSG(∗) 2. The serum level in the AHSG(∗) 1 homozygote is significantly higher than that of the AHSG(∗) 2 homozygote. This study examined the molecular mechanism for the cis-regulatory expression. To quantitate allele-specific mRNA in intra-assays of the heterozygote, RT-PCR method employing primers that were incorporated to the two closely located SNPs was developed. The respective magnitudes of AHSG(∗) 1 to AHSG(∗) 2 in the liver tissues and hepatic culture cells of PLC/PRF/5 were determined quantitatively as 2.5-fold and 6.2-fold. The mRNA expressional difference of two major alleles was observed, which is consistent with that in the serum level. The culture cells carried heterozygous genotypes in rs4917 and rs4918, but homozygous one in rs2248690. It was unlikely that the imbalance was derived from the SNP located in the promotor site. Furthermore, to investigate the effect of mRNA degradation, RNA synthesis in the cell culture was inhibited potently by the addition of actinomycin-D. No marked change was apparent between the two alleles. The results indicated that the cis-regulatory expressional difference is expected to occur at the level of transcription or splicing of mRNA.

  11. Allelic Imbalance of mRNA Associated with α2-HS Glycoprotein (Fetuin-A Polymorphism

    Directory of Open Access Journals (Sweden)

    Yoshihiko Inaoka

    2015-01-01

    Full Text Available Alpha 2-HS glycoprotein (AHSG, also designated as fetuin-A, exhibits polymorphism in population genetics consisting of two major alleles of AHSG∗1 and AHSG∗2. The serum level in the AHSG∗1 homozygote is significantly higher than that of the AHSG∗2 homozygote. This study examined the molecular mechanism for the cis-regulatory expression. To quantitate allele-specific mRNA in intra-assays of the heterozygote, RT-PCR method employing primers that were incorporated to the two closely located SNPs was developed. The respective magnitudes of AHSG∗1 to AHSG∗2 in the liver tissues and hepatic culture cells of PLC/PRF/5 were determined quantitatively as 2.5-fold and 6.2-fold. The mRNA expressional difference of two major alleles was observed, which is consistent with that in the serum level. The culture cells carried heterozygous genotypes in rs4917 and rs4918, but homozygous one in rs2248690. It was unlikely that the imbalance was derived from the SNP located in the promotor site. Furthermore, to investigate the effect of mRNA degradation, RNA synthesis in the cell culture was inhibited potently by the addition of actinomycin-D. No marked change was apparent between the two alleles. The results indicated that the cis-regulatory expressional difference is expected to occur at the level of transcription or splicing of mRNA.

  12. Impact of fasting followed by short-term exposure to interleukin-6 on cytochrome P450 mRNA in mice.

    Science.gov (United States)

    Rasmussen, Martin Krøyer; Bertholdt, Lærke; Gudiksen, Anders; Pilegaard, Henriette; Knudsen, Jakob G

    2018-01-05

    The gene expression of the cytochrome P450 (CYP) enzyme family is regulated by numerous factors. Fasting has been shown to induce increased hepatic CYP mRNA in both humans and animals. However, the coordinated regulation of CYP, CYP-regulating transcription factors, and transcriptional co-factors in the liver linking energy metabolism to detoxification has never been investigated. Interleukin-6 (IL-6) has been suggested to be released during fasting and has been shown to regulate CYP expression. The present study investigated the hepatic mRNA content of selected CYP, AhR, CAR, PXR and PPARα in mice fasted for 18h and subsequently exposed to IL-6. Furthermore, the impact of fasting on PGC-1α, HNF-4α, SIRT1 and SIRT3 mRNA was examined. Fasting induced a marked increase in Cyp2b10, Cyp2e1 and Cyp4a10 mRNA, while CYP1a1, Cyp1a2, Cyp2a4 and Cyp3a11 mRNA levels remained unchanged. In accordance, the mRNA levels of CAR and PPARα were also increased with fasting. The PGC-1α, SIRT1 and SIRT3 mRNA levels were also increased after fasting, while the HNF-4α mRNA levels remained unchanged. In mice subjected to IL-6 injection, the fasting-induced PXR, PPARα and PGC-1α mRNA responses were lower than after saline injection. In conclusion, fasting was demonstrated to be a strong inducer of hepatic CYP mRNA as well as selected transcription factors controlling the expression of the investigated CYP. Moreover, the mRNA levels of transcriptional co-factors acting as energy sensors and co-factors for CYP regulation was also increased in the liver, suggesting crosstalk at the molecular level between regulation of energy metabolism and detoxification. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Regulation of Egr-1, VIP, and Shh mRNA and Egr-1 protein in the mouse retina by light and image quality.

    Science.gov (United States)

    Brand, Christine; Burkhardt, Eva; Schaeffel, Frank; Choi, Jeong Won; Feldkaemper, Marita Pauline

    2005-04-28

    To analyze mRNA expression changes of Egr-1, VIP, and Shh under different light and treatment conditions in mice. The mRNA expression levels of the three genes and additionally the Egr-1 protein expression were compared in form deprived eyes and eyes with normal vision. Moreover, the influence of dark to light and light to dark transitions and of changes in retinal illumination on mRNA levels was investigated. Form deprivation of mice was induced by fitting frosted diffusers over one eye and an attentuation matched neutral density (ND) filter over the other eye. To measure the effects of retinal illumination changes on mRNA expression, animals were bilaterally fitted with different ND filters. Semiquantitative real-time RT-PCR was used to measure the mRNA levels and immunohistochemistry was applied to localize and detect Egr-1 protein. The expression levels of both Egr-1 mRNA and protein were reduced in form deprived eyes compared to their fellow eyes after 30 min and 1 h, respectively. Egr-1 mRNA was strikingly upregulated both after dark to light and light to dark transitions, whereas minor changes in retinal illumination by covering the eyes with neutral density filters did not alter Egr-1 mRNA expression. In mice, the mRNA levels of VIP and Shh were not affected by form deprivation, but they were found to be regulated depending on the time of day. Both Egr-1 mRNA and protein expression levels were strongly regulated by light, especially by transitions between light and darkness. Image contrast may exert an additional influence on mRNA and protein expression of Egr-1, particularly in the cells in the ganglion cell layer and in bipolar cells.

  14. NPY mRNA Expression in the Prefrontal Cortex: Selective Reduction in the Superficial White Matter of Subjects with Schizoaffective Disorder

    Science.gov (United States)

    Morris, Harvey M.; Stopczynski, Rachelle E.; Lewis, David A.

    2009-01-01

    Background Alterations in the inhibitory circuitry of the dorsolateral prefrontal cortex (DLPFC) in schizophrenia include reduced expression of the messenger RNA (mRNA) for somatostatin (SST), a neuropeptide present in a subpopulation of γ-aminobutyric acid (GABA) neurons. Neuropeptide Y (NPY) is expressed in a subset of SST-containing interneurons and lower levels of NPY mRNA have also been reported in schizophrenia spectrum disorders. However, whether the alterations in these two transcripts identify the same, particularly vulnerable, subset of GABA neurons has not been examined. Methods We used in situ hybridization to quantify NPY mRNA levels in DLPFC gray and white matter from 23 pairs of subjects with schizophrenia or schizoaffective disorder and matched normal control subjects; results were compared to those from a previous study of SST mRNA expression in the same subjects. Results In contrast to SST mRNA, NPY mRNA levels were not significantly lower in the gray matter of subjects with schizophrenia or schizoaffective disorder. However, NPY, but not SST, mRNA expression was significantly lower in the superficial white matter of subjects with schizoaffective disorder. Conclusion These findings suggest that the alterations in SST-containing interneurons in schizophrenia and schizoaffective disorder are selective for the subset that do not express NPY mRNA, and that lower NPY mRNA expression in the superficial white matter may distinguish subjects with schizoaffective disorder from those with schizophrenia. PMID:19804960

  15. Functional Integration of mRNA Translational Control Programs

    Directory of Open Access Journals (Sweden)

    Melanie C. MacNicol

    2015-07-01

    Full Text Available Regulated mRNA translation plays a key role in control of cell cycle progression in a variety of physiological and pathological processes, including in the self-renewal and survival of stem cells and cancer stem cells. While targeting mRNA translation presents an attractive strategy for control of aberrant cell cycle progression, mRNA translation is an underdeveloped therapeutic target. Regulated mRNAs are typically controlled through interaction with multiple RNA binding proteins (RBPs but the mechanisms by which the functions of distinct RBPs bound to a common target mRNA are coordinated are poorly understood. The challenge now is to gain insight into these mechanisms of coordination and to identify the molecular mediators that integrate multiple, often conflicting, inputs. A first step includes the identification of altered mRNA ribonucleoprotein complex components that assemble on mRNAs bound by multiple, distinct RBPs compared to those recruited by individual RBPs. This review builds upon our knowledge of combinatorial control of mRNA translation during the maturation of oocytes from Xenopus laevis, to address molecular strategies that may mediate RBP diplomacy and conflict resolution for coordinated control of mRNA translational output. Continued study of regulated ribonucleoprotein complex dynamics promises valuable new insights into mRNA translational control and may suggest novel therapeutic strategies for the treatment of disease.

  16. Phytochrome B mRNA expression enhances biomass yield and ...

    African Journals Online (AJOL)

    The present study shows successful transformation and mRNA expression in Phytochrome B transformed CIM 482 cotton plants. Transgenic cotton plants expressing Phytochrome B mRNA have showed more than two times increase in relative leaf growth rate (RLGR) and photosynthetic rate, more than one time increase in ...

  17. mRNA pseudoknot structures can act as ribosomal roadblocks

    DEFF Research Database (Denmark)

    Hansen, Jesper Tholstrup; Oddershede, Lene Broeng; Sørensen, Michael Askvad

    2012-01-01

    Several viruses utilize programmed ribosomal frameshifting mediated by mRNA pseudoknots in combination with a slippery sequence to produce a well defined stochiometric ratio of the upstream encoded to the downstream-encoded protein. A correlation between the mechanical strength of mRNA pseudoknots...

  18. Testosterone Regulates NUCB2 mRNA Expression in Male Mouse Hypothalamus and Pituitary Gland.

    Science.gov (United States)

    Seon, Sojeong; Jeon, Daun; Kim, Heejeong; Chung, Yiwa; Choi, Narae; Yang, Hyunwon

    2017-03-01

    Nesfatin-1/NUCB2 is known to take part in the control of the appetite and energy metabolism. Recently, many reports have shown nesfatin-1/NUCB2 expression and function in various organs. We previously demonstrated that nesfatin-1/NUCB2 expression level is higher in the pituitary gland compared to other organs and its expression is regulated by 17β-estradiol and progesterone secreted from the ovary. However, currently no data exist on the expression of nesfatin-1/NUCB2 and its regulation mechanism in the pituitary of male mouse. Therefore, we examined whether nesfatin-1/NUCB2 is expressed in the male mouse pituitary and if its expression is regulated by testosterone. As a result of PCR and western blotting, we found that a large amount of nesfatin-1/NUCB2 was expressed in the pituitary and hypothalamus. The NUCB2 mRNA expression level in the pituitary was decreased after castration, but not in the hypothalamus. In addition, its mRNA expression level in the pituitary was increased after testosterone treatment in the castrated mice, whereas, the expression level in the hypothalamus was significantly decreased after the treatment with testosterone. The in vitro experiment to elucidate the direct effect of testosterone on NUCB2 mRNA expression showed that NUCB2 mRNA expression was significantly decreased with testosterone in cultured hypothalamus tissue, but increased with testosterone in cultured pituitary gland. The present study demonstrated that nesfatin-1/NUCB2 was highly expressed in the male mouse pituitary and was regulated by testosterone. This data suggests that reproductive-endocrine regulation through hypothalamus-pituitary-testis axis may contribute to NUCB2 mRNA expression in the mouse hypothalamus and pituitary gland.

  19. Regulation and dysregulation of vitellogenin mRNA accumulation in daphnids (Daphnia magna)

    Energy Technology Data Exchange (ETDEWEB)

    Hannas, Bethany R.; Wang, Ying H.; Thomson, Susanne; Kwon, Gwijun; Li Hong [Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, NC 27695-7633 (United States); LeBlanc, Gerald A., E-mail: Gerald_LeBlanc@ncsu.edu [Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, NC 27695-7633 (United States)

    2011-01-25

    The induction of vitellogenin in oviparous vertebrates has become the gold standard biomarker of exposure to estrogenic chemicals in the environment. This biomarker of estrogen exposure also has been used in arthropods, however, little is known of the factors that regulate the expression of vitellogenin in these organisms. We investigated changes in accumulation of mRNA products of the vitellogenin gene Vtg2 in daphnids (Daphnia magna) exposed to a diverse array of chemicals. We further evaluated the involvement of hormonal factors in the regulation of vitellogenin expression that may be targets of xenobiotic chemicals. Expression of the Vtg2 gene was highly responsive to exposure to various chemicals with an expression range spanning approximately four orders of magnitude. Chemicals causing the greatest induction were piperonyl butoxide, chlordane, 4-nonylphenol, cadmium, and chloroform. Among these, only 4-nonylphenol is recognized to be estrogenic. Exposure to several chemicals also suppressed Vtg2 mRNA levels, as much as 100-fold. Suppressive chemicals included cyproterone acetate, acetone, triclosan, and atrazine. Exposure to the estrogens diethylstilbestrol and bisphenol A had little effect on vitellogenin mRNA levels further substantiating that these genes are not induced by estrogen exposure. Exposure to the potent ecdysteroids 20-hydroxyecdysone and ponasterone A revealed that Vtg2 was subject to strong suppressive control by these hormones. Vtg2 mRNA levels were not significantly affected from exposure to several juvenoid hormones. Results indicate that ecdysteroids are suppressors of vitellogenin gene expression and that vitellogenin mRNA levels can be elevated or suppressed in daphnids by xenobiotics that elicit antiecdysteroidal or ecdysteroidal activity, respectively. Importantly, daphnid Vtg2 is not elevated in response to estrogenic activity.

  20. Effects of the β2 -agonist clenbuterol on testicular steroidogenic acute regulatory protein mRNA expression in adult rats.

    Science.gov (United States)

    Ma, J-K; Zhu, W-J

    2010-12-01

    This study was carried out to investigate the effects of clenbuterol (CLB) on the testicular (steroidogenic acute regulatory, StAR) protein mRNA expression in rats. Thirty adult male rats were administered CLB by gavage daily at the doses of 0.4, 2.0 and 18.5 mg/kg bw for 14 days in the subacute experiment, whereas 20 rats received a single treatment with CLB at the doses of 20 and 40 mg/kg bw in the acute experiment and 20 rats were treated with 0.9% NaCl solution as vehicle groups. Testicular tissues were collected and snap-frozen in liquid nitrogen and stored at -70 °C until use. The levels of StAR mRNA were detected by RT-PCR. The levels of StAR mRNA were markedly increased (P  0.05) in the expression levels of StAR mRNA, and the mRNA levels were recovered to near normal level in the groups treated with CLB at dosages of 0.4 and 2.0 mg/kg bw/day following a 7-day withdrawal period, compared with the control animals. The mRNA levels of StAR showed a significant decrease in the groups treated with CLB at the dosage of 18.5 mg/kg bw/day (P < 0.05) after a 1- or 7-day withdrawal period with respect to the control animals. These results demonstrated transient stimulative effects of CLB on testicular StAR mRNA levels and inhibitory effects after treatment with CLB for 14 consecutive days. © 2010 Blackwell Publishing Ltd.

  1. Sequence-engineered mRNA Without Chemical Nucleoside Modifications Enables an Effective Protein Therapy in Large Animals

    Science.gov (United States)

    Thess, Andreas; Grund, Stefanie; Mui, Barbara L; Hope, Michael J; Baumhof, Patrick; Fotin-Mleczek, Mariola; Schlake, Thomas

    2015-01-01

    Being a transient carrier of genetic information, mRNA could be a versatile, flexible, and safe means for protein therapies. While recent findings highlight the enormous therapeutic potential of mRNA, evidence that mRNA-based protein therapies are feasible beyond small animals such as mice is still lacking. Previous studies imply that mRNA therapeutics require chemical nucleoside modifications to obtain sufficient protein expression and avoid activation of the innate immune system. Here we show that chemically unmodified mRNA can achieve those goals as well by applying sequence-engineered molecules. Using erythropoietin (EPO) driven production of red blood cells as the biological model, engineered Epo mRNA elicited meaningful physiological responses from mice to nonhuman primates. Even in pigs of about 20 kg in weight, a single adequate dose of engineered mRNA encapsulated in lipid nanoparticles (LNPs) induced high systemic Epo levels and strong physiological effects. Our results demonstrate that sequence-engineered mRNA has the potential to revolutionize human protein therapies. PMID:26050989

  2. Inhibition of pyrimidine synthesis reverses viral virulence factor-mediated block of mRNA nuclear export.

    Science.gov (United States)

    Zhang, Liang; Das, Priyabrata; Schmolke, Mirco; Manicassamy, Balaji; Wang, Yaming; Deng, Xiaoyi; Cai, Ling; Tu, Benjamin P; Forst, Christian V; Roth, Michael G; Levy, David E; García-Sastre, Adolfo; de Brabander, Jef; Phillips, Margaret A; Fontoura, Beatriz M A

    2012-02-06

    The NS1 protein of influenza virus is a major virulence factor essential for virus replication, as it redirects the host cell to promote viral protein expression. NS1 inhibits cellular messenger ribonucleic acid (mRNA) processing and export, down-regulating host gene expression and enhancing viral gene expression. We report in this paper the identification of a nontoxic quinoline carboxylic acid that reverts the inhibition of mRNA nuclear export by NS1, in the absence or presence of the virus. This quinoline carboxylic acid directly inhibited dihydroorotate dehydrogenase (DHODH), a host enzyme required for de novo pyrimidine biosynthesis, and partially reduced pyrimidine levels. This effect induced NXF1 expression, which promoted mRNA nuclear export in the presence of NS1. The release of NS1-mediated mRNA export block by DHODH inhibition also occurred in the presence of vesicular stomatitis virus M (matrix) protein, another viral inhibitor of mRNA export. This reversal of mRNA export block allowed expression of antiviral factors. Thus, pyrimidines play a necessary role in the inhibition of mRNA nuclear export by virulence factors. © 2012 Zhang et al.

  3. Developmental changes in hypothalamic oxytocin and oxytocin receptor mRNA expression and their sensitivity to fasting in male and female rats.

    Science.gov (United States)

    Matsuzaki, Toshiya; Iwasa, Takeshi; Munkhzaya, Munkhsaikhan; Tungalagsuvd, Altankhuu; Kawami, Takako; Murakami, Masahiro; Yamasaki, Mikio; Yamamoto, Yuri; Kato, Takeshi; Kuwahara, Akira; Yasui, Toshiyuki; Irahara, Minoru

    2015-04-01

    Oxytocin (OT) affects the central nervous system and is involved in a variety of social and non-social behaviors. Recently, the role played by OT in energy metabolism and its organizational effects on estrogen receptor alpha (ER-α) during the neonatal period have gained attention. In this study, the developmental changes in the hypothalamic mRNA levels of OT, the OT receptor (OTR), and ER-α were evaluated in male and female rats. In addition, the fasting-induced changes in the hypothalamic mRNA levels of OT and the OTR were evaluated. Hypothalamic explants were taken from postnatal day (PND) 10, 20, and 30 rats, and the mRNA level of each molecule was measured. Hypothalamic OT mRNA expression increased throughout the developmental period in both sexes. The rats' hypothalamic OTR mRNA levels were highest on PND 10 and decreased throughout the developmental period. In the male rats, the hypothalamic mRNA levels of ER-α were higher on PND 30 than on PND 10. On the other hand, no significant differences in hypothalamic ER-α mRNA expression were detected among the examined time points in the female rats, although hypothalamic ER-α mRNA expression tended to be higher on PND 30 than on PND 10. Significant positive correlations were detected between hypothalamic OT and ER-α mRNA expression in both the male and female rats. Hypothalamic OT mRNA expression was not affected by fasting at any of the examined time points in either sex. These results indicate that hypothalamic OT expression is not sensitive to fasting during the developmental period. In addition, as a positive correlation was detected between hypothalamic OT and ER-α mRNA expression, these two molecules might interact with each other to induce appropriate neuronal development. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. Pol II-directed short RNAs suppress the nuclear export of mRNA.

    Science.gov (United States)

    Komarova, Tatiana V; Schwartz, Anton M; Frolova, Olga Y; Zvereva, Anna S; Gleba, Yuri Y; Citovsky, Vitaly; Dorokhov, Yuri L

    2010-12-01

    The synthesis and subsequent nuclear export of non-coding RNA (ncRNA) directed by RNA polymerase (Pol) II is very sensitive to abiotic and biotic external stimuli including pathogen challenges. To assess whether stress-induced ncRNAs may suppress the nuclear export of mRNA, we exploited the ability of Agrobacterium tumefaciens to co-deliver Pol I, II and III promoter-based vectors for the transcription of short (s) ncRNAs, GFP mRNA or genomic RNA of plant viruses (Tobacco mosaic virus, TMV; or Potato virus X, PVX) into the nucleus of Nicotiana benthamiana cells. We showed that, in contrast to Pol I- and Pol III-derived sncRNAs, all tested Pol II-derived sncRNAs (U6 RNA, tRNA or artificial RNAs) resulted in decreased expression of GFP and host mRNA. The level of this inhibitory effect depended on the non-coding transcript length and promoter strength. Short coding RNA (scRNA) can also compete with mRNA for nuclear export. We showed that scRNA, an artificial 117-nt short sequence encoding Elastin-Like peptide element tandems with FLAG sequence (ELF) and the 318-nt N. benthamiana antimicrobial peptide thionin (defensin) gene efficiently decreased GFP expression. The stress-induced export of Pol II-derived sncRNA and scRNA into the cytoplasm via the mRNA export pathway may block nucleocytoplasmic traffic including the export of mRNA responsible for antivirus protection. Consistent with this model, we observed that Pol II-derived sncRNAs as well as scRNA, thionin and ELF strongly enhanced the cytoplasmic reproduction of TMV and PVX RNA.

  5. An investigation of nutrient-dependent mRNA translation in Drosophila larvae

    Directory of Open Access Journals (Sweden)

    Sabarish Nagarajan

    2014-10-01

    Full Text Available The larval period of the Drosophila life cycle is characterized by immense growth. In nutrient rich conditions, larvae increase in mass approximately two hundred-fold in five days. However, upon nutrient deprivation, growth is arrested. The prevailing view is that dietary amino acids drive this larval growth by activating the conserved insulin/PI3 kinase and Target of rapamycin (TOR pathways and promoting anabolic metabolism. One key anabolic process is protein synthesis. However, few studies have attempted to measure mRNA translation during larval development or examine the signaling requirements for nutrient-dependent regulation. Our work addresses this issue. Using polysome analyses, we observed that starvation rapidly (within thirty minutes decreased larval mRNA translation, with a maximal decrease at 6–18 hours. By analyzing individual genes, we observed that nutrient-deprivation led to a general reduction in mRNA translation, regardless of any starvation-mediated changes (increase or decrease in total transcript levels. Although sugars and amino acids are key regulators of translation in animal cells and are the major macronutrients in the larval diet, we found that they alone were not sufficient to maintain mRNA translation in larvae. The insulin/PI3 kinase and TOR pathways are widely proposed as the main link between nutrients and mRNA translation in animal cells. However, we found that genetic activation of PI3K and TOR signaling, or regulation of two effectors – 4EBP and S6K – could not prevent the starvation-mediated translation inhibition. Similarly, we showed that the nutrient stress-activated eIF2α kinases, GCN2 and PERK, were not required for starvation-induced inhibition of translation in larvae. These findings indicate that nutrient control of mRNA translation in larvae is more complex than simply amino acid activation of insulin and TOR signaling.

  6. Functional characterization of the HuR:CD83 mRNA interaction.

    Directory of Open Access Journals (Sweden)

    Dorothea Pieper

    Full Text Available Maturation of dendritic cells (DC is characterized by expression of CD83, a surface protein that appears to be necessary for the effective activation of naïve T-cells and T-helper cells by DC. Lately it was shown that CD83 expression is regulated on the posttranscriptional level by interaction of the shuttle protein HuR with a novel posttranscriptional regulatory RNA element (PRE, which is located in the coding region of the CD83 transcript. Interestingly, this interaction commits the CD83 mRNA to efficient nuclear export via the CRM1 pathway. To date, however, the structural basis of this interaction, which potentially involves three distinct RNA recognition motifs (RRM1-3 in HuR and a complex three-pronged RNA stem-loop element in CD83 mRNA, has not been investigated in detail. In the present work we analyzed this interaction in vitro and in vivo using various HuR- and CD83 mRNA mutants. We are able to demonstrate that both, RRM1 and RRM2 are crucial for binding, whereas RRM3 as well as the HuR hinge region contributed only marginally to this protein:RNA interaction. Furthermore, mutation of uridine rich patches within the PRE did not disturb HuR:CD83 mRNA complex formation while, in contrast, the deletion of specific PRE subfragments from the CD83 mRNA prevented HuR binding in vitro and in vivo. Interestingly, the observed inhibition of HuR binding to CD83 mRNA does not lead to a nuclear trapping of the transcript but rather redirected this transcript from the CRM1- towards the NXF1/TAP-specific nuclear export pathway. Thus, the presence of a functional PRE permits nucleocytoplasmic trafficking of the CD83 transcript via the CRM1 pathway.

  7. Changes in the responsiveness of hypothalamic prokineticin 2 mRNA expression to food deprivation in developing female rats.

    Science.gov (United States)

    Iwasa, Takeshi; Matsuzaki, Toshiya; Munkhzaya, Munkhsaikhan; Tungalagsuvd, Altankhuu; Kawami, Takako; Murakami, Masahiro; Yamasaki, Mikio; Kato, Takeshi; Kuwahara, Akira; Yasui, Toshiyuki; Irahara, Minoru

    2014-05-01

    Prokineticin 2 (PK2) is highly expressed in several regions of the central nervous system, including the hypothalamus. Recently, it has been suggested that PK2 plays a role in appetite regulation. In adult male rodents, the administration of PK2 decreased food intake, and PK2 mRNA expression was reduced by food deprivation. Usually, the changes in the expression levels of appetite-regulating factors induced in response to fasting are not fully established during the neonatal period. Thus, we investigated the developmental changes in hypothalamic PK2 mRNA expression and the alterations in hypothalamic PK2 mRNA expression induced by fasting during the pre-pubertal period in female rats. The changes in hypothalamic neuropeptide Y (NPY) mRNA expression were also examined because NPY is a potent appetite regulatory factor. Hypothalamic PK2 mRNA expression was extremely high during the early neonatal period (postnatal day (PND) 5) compared with that observed during subsequent periods (PND15, 25, and 42), while hypothalamic NPY mRNA expression did not differ among any of the examined periods. A fasting-induced reduction in hypothalamic PK2 mRNA expression was observed on PND5, but no fasting-induced increase in hypothalamic NPY mRNA expression was seen during the same period. In addition, the fasting-induced reduction in hypothalamic PK2 mRNA expression observed on PND5 was more marked than that seen on PND25. These results suggest that the sensitivity of hypothalamic PK2 expression to undernutrition develops during the early neonatal period, when the responses of other appetite regulatory factors to such pressures remain immature. Copyright © 2014 ISDN. Published by Elsevier Ltd. All rights reserved.

  8. The silence of MUC2 mRNA induced by promoter hypermethylation associated with HBV in Hepatocellular Carcinoma

    Directory of Open Access Journals (Sweden)

    Ling Yang

    2013-01-01

    Full Text Available Abstract Background To evaluate the promoter methylation status of MUC2 gene and mRNA expression in patients with hepatocellular carcinoma. Methods We analyzed MUC2 methylation by MSP, and MUC2 mRNA by real-time PCR in 74 HCC. Results MUC2 mRNA were lower in HCC tissues (Mean -ΔCt = −4.70 than that in Non-HCC tissues (Mean -ΔCt = −2.98. Expression of MUC2 was elevated in only 23 (31.08% of the 74 HCC patients. MUC2 promoter was hypermethylated in 62.2% (46/74 of HCCs, and in only 18.9% (14/74 of non-tumor samples. MUC2 mRNA were lower in HCC patients with hypermethylation (Mean -ΔΔCt = −2.25 than those with demethylation (Mean -ΔΔCt = −0.22, and there is a decreased tendency for MUC2 mRNA in HCC patients with promoter hypermethylation (p = 0.011. There was a significantly correlation found between MUC2 mRNA and HBV and AFP in HCC. The loss of MUC2 mRNA and hypermethylation could be poor prognostic factors. After treated by 5-Aza-CdR and TSA, we found that MUC2 mRNA induced significantly in 7721, Huh7 and HepG2 cells. Conclusion The results suggested that MUC2 mRNA silenced by promoter hypermethylation is associated with high levels HBV in HCC.

  9. Distinct gene expression profiles of proximal and distal colorectal cancer: implications for cytotoxic and targeted therapy.

    Science.gov (United States)

    Maus, M K H; Hanna, D L; Stephens, C L; Astrow, S H; Yang, D; Grimminger, P P; Loupakis, F; Hsiang, J H; Zeger, G; Wakatsuki, T; Barzi, A; Lenz, H-J

    2015-08-01

    Colorectal cancer (CRC) is a heterogeneous disease with genetic profiles and clinical outcomes dependent on the anatomic location of the primary tumor. How location has an impact on the molecular makeup of a tumor and how prognostic and predictive biomarkers differ between proximal versus distal colon cancers is not well established. We investigated the associations between tumor location, KRAS and BRAF mutation status, and the messenger RNA (mRNA) expression of proteins involved in major signaling pathways, including tumor growth (epidermal growth factor receptor (EGFR)), angiogenesis (vascular endothelial growth factor receptor 2 (VEGFR2)), DNA repair (excision repair cross complement group 1 (ERCC1)) and fluoropyrimidine metabolism (thymidylate synthase (TS)). Formalin-fixed paraffin-embedded tumor specimens from 431 advanced CRC patients were analyzed. The presence of seven different KRAS base substitutions and the BRAF V600E mutation was determined. ERCC1, TS, EGFR and VEGFR2 mRNA expression levels were detected by reverse transcriptase-PCR. BRAF mutations were significantly more common in the proximal colon (Pexpression in multivariate analysis. In a subgroup analysis, this association remained significant for all genes in the proximal colon and for VEGFR2 expression in rectal cancers. The mRNA expression patterns of predictive and prognostic biomarkers, as well as associations with KRAS and BRAF mutation status depend on primary tumor location. Prospective studies are warranted to confirm these findings and determine the underlying mechanisms.

  10. Cellular retinoic-acid-binding-protein and retinol-binding-protein mRNA expression in the cells of the rat seminiferous tubules and their regulation by retinoids.

    Science.gov (United States)

    Faraonio, R; Galdieri, M; Colantuoni, V

    1993-02-01

    The levels of the mRNA corresponding to the intracellular binding proteins for retinoic acid and retinol (CRABP1 and CRBP1, respectively) were studied in primary cultures of somatic and germ cells of the rat seminiferous tubules. We show that the CRABP1 mRNA is expressed in Sertoli and germ cells and a single molecular species of mRNA is detected. CRBP1 mRNA is detected in Sertoli and peritubular cells. The regulation of the expression of both genes by retinoids was studied in Sertoli cells. CRABP1 mRNA levels are not affected by either retinoic acid or retinol, whereas both compounds positively regulate CRBP1 mRNA synthesis in a dose-dependent manner. A fivefold increase in CRBP1 mRNA levels was observed 32-48 h after addition of either agent. These results demonstrate that in Sertoli cells the expression of CRABP1 is not affected by retinoids, similar to the situation observed in vivo and in other in-vitro cultures. CRBP1-gene expression is, instead, induced and the variations in CRBP1-mRNA levels may regulate the intracellular concentrations of retinoids, as a response to changes in the vitamin-A nutritional status.

  11. Increased shelterin mRNA expression in peripheral blood mononuclear cells and skeletal muscle following an ultra-long-distance running event

    DEFF Research Database (Denmark)

    Laye, Matthew J; Solomon, Thomas; Karstoft, Kristian

    2012-01-01

    telomere length, telomerase activity, hTert mRNA, or hterc mRNAs found in PBMCs. Higher protein concentrations of TRF2 were found in skeletal muscle vs. PBMCs at rest. Mean telomere length in skeletal muscle did not change and did not contain detectable levels of htert mRNA or telomerase activity...

  12. The coupled and uncoupled mechanisms by which trans-acting factors regulate mRNA stability and translation.

    Science.gov (United States)

    Fukao, Akira; Fujiwara, Toshinobu

    2017-04-01

    In mammals, spatiotemporal control of protein synthesis plays a key role in the post-transcriptional regulation of gene expression during cell proliferation, development and differentiation and RNA-binding proteins (RBPs) and microRNAs (miRNAs) are required for this phenomenon. RBPs and miRNAs control the levels of mRNA protein products by regulating mRNA stability and translation. Recent studies have shown that RBPs and miRNAs simultaneously regulate mRNA stability and translation, and that the differential functions of RBPs and miRNAs are dependent on their interaction partners. Here, we summarize the coupled- and uncoupled mechanisms by which trans-acting factors regulate mRNA stability and translation. © The Authors 2016. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  13. Changes of rat kidney AQP2 and Na,K-ATPase mRNA expression in lithium-induced nephrogenic diabetes insipidus.

    Science.gov (United States)

    Laursen, Ulla Helt; Pihakaski-Maunsbach, Kaarina; Kwon, Tae-Hwan; Østergaard Jensen, Erik; Nielsen, Søren; Maunsbach, Arvid B

    2004-01-01

    In a rat model, lithium treatment is associated with polyuria and severe downregulation of aquaporin-2 (AQP2) protein in the inner medulla (IM) or in the whole kidney. However, it is not known (1) to what extent this downregulation occurs at the mRNA level; (2) whether the main sodium transporter of the nephron, Na,K-ATPase, is regulated in parallel at the mRNA level, and (3) whether lithium treatment induces zonal or segmental differences in AQP2 and Na,K-ATPase mRNA levels. We examined the changes in mRNA expression levels for AQP2 and Na,K-ATPase in kidney cortex, inner stripe of the outer medulla (ISOM), and IM of rats treated with lithium orally using semiquantitative Northern blot analyses and in situ hybridization at the light and electron microscopic levels. The AQP2 mRNA levels decreased significantly (p dabetes insipidus and moderately decreased Na,K-ATPase mRNA levels in the ISOM and in the IM. The results suggest that decreased mRNA expressions of AQP2 and Na,K-ATPase contribute to the development of lithium-induced nephrogenic diabetes insipidus. Copyright 2004 S. Karger AG, Basel

  14. Role of mRNA Methylation in Prostate Cancer

    Science.gov (United States)

    2015-02-01

    regulation of mRNA. Recent technological advances have made it possible to detect mRNA methylation . The m6A was found near regulatory regions and...TERMS mRNA methylation , FTO, MeRIP-seq, RNA-seq, m6A 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF PAGES 19a. NAME OF...10 9. Appendices……………………………………………………………10 1. INTRODUCTION: Methylation at the N6 position of adenosine ( m6A ) is a post-transcriptional modification of

  15. Arsenic Induces Polyadenylation of Canonical Histone mRNA by Down-regulating Stem-Loop-binding Protein Gene Expression*

    Science.gov (United States)

    Brocato, Jason; Fang, Lei; Chervona, Yana; Chen, Danqi; Kiok, Kathrin; Sun, Hong; Tseng, Hsiang-Chi; Xu, Dazhong; Shamy, Magdy; Jin, Chunyuan; Costa, Max

    2014-01-01

    The replication-dependent histone genes are the only metazoan genes whose messenger RNA (mRNA) does not terminate with a poly(A) tail at the 3′-end. Instead, the histone mRNAs display a stem-loop structure at their 3′-end. Stem-loop-binding protein (SLBP) binds the stem-loop and regulates canonical histone mRNA metabolism. Here we report that exposure to arsenic, a carcinogenic metal, decreased cellular levels of SLBP by inducing its proteasomal degradation and inhibiting SLBP transcription via epigenetic mechanisms. Notably, arsenic exposure dramatically increased polyadenylation of canonical histone H3.1 mRNA possibly through down-regulation of SLBP expression. The polyadenylated H3.1 mRNA induced by arsenic was not susceptible to normal degradation that occurs at the end of S phase, resulting in continued presence into mitosis, increased total H3.1 mRNA, and increased H3 protein levels. Excess expression of canonical histones have been shown to increase sensitivity to DNA damage as well as increase the frequency of missing chromosomes and induce genomic instability. Thus, polyadenylation of canonical histone mRNA following arsenic exposure may contribute to arsenic-induced carcinogenesis. PMID:25266719

  16. mRNA overexpression of BAALC: A novel prognostic factor for pediatric acute lymphoblastic leukemia

    Science.gov (United States)

    AZIZI, ZAHRA; RAHGOZAR, SOHEILA; MOAFI, ALIREZA; DABAGHI, MOHAMMAD; NADIMI, MOTAHAREH

    2015-01-01

    BAALC is a novel molecular marker in leukemia that is highly expressed in patients with acute leukemia. Increased expression levels of BAALC are known as poor prognostic factors in adult acute myeloid and lymphoid leukemia. The purpose of the present study was to evaluate the prognostic significance of the BAALC gene expression levels in pediatric acute lymphoblastic leukemia (ALL) and its association with MDR1. Using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), the mRNA expression levels of BAALC and MRD1 were measured in bone marrow samples of 28 new diagnosed childhood ALL patients and 13 children without cancer. Minimal residual disease (MRD) was measured one year after the initiation of the chemotherapy using the RT-qPCR method. The high level expression of BAALC had a significant association with the pre-B-ALL subtype, leukocytosis and positive MRD after one year of treatment in leukemic patients. In addition, a positive correlation between BAALC and MDR1 mRNA expression was shown in this group. In conclusion, to the best of our knowledge, the increase of BAALC expression as a poor prognostic factor for childhood ALL is shown for the first time. Additionally, the correlation between BAALC and MDR1 in mRNA expression levels can aid for an improved understanding of the mechanism through which BAALC may function in ALL and multidrug resistance. PMID:26137238

  17. Diet-induced changes in hypothalamic pro-opio-melanocortin mRNA in the rat hypothalamus.

    Science.gov (United States)

    Torri, Carla; Pedrazzi, Patrizia; Leo, Giuseppina; Müller, Eugenio E; Cocchi, Daniela; Agnati, Luigi F; Zoli, Michele

    2002-06-01

    Hypothalamic mRNA and peptide levels of pro-opio-melanocortin (POMC) and other neuropeptides were studied in rats that either develop obesity (diet-induced obese, DIO), when fed a palatable and hypercaloric diet (cafeteria diet, caf) or do not develop obesity (diet resistant, DR), when fed the same diet. cafDIO rats showed a significant increase in POMC, but not in melanin concentrating hormone, mRNA levels as determined by semiquantitative in situ hybridization. cafDR and cafDIO rats showed no change in POMC-derived peptide levels, whereas neuropeptide Y immunoreactivity was significantly increased in cafDR rats. POMC mRNA levels were also studied in high-fat diet-fed rats but no significant change was observed. Altered hypothalamic transmission by POMC-derived peptides may contribute to the susceptibility of cafDIO rats to the weight promoting action of caf diet.

  18. Effect of vitrification on the mRNA transcriptome of bovine oocytes.

    Science.gov (United States)

    Wang, N; Li, C-Y; Zhu, H-B; Hao, H-S; Wang, H-Y; Yan, C-L; Zhao, S-J; Du, W-H; Wang, D; Liu, Y; Pang, Y-W; Zhao, X-M

    2017-08-01

    Vitrification has been shown to decrease the developmental capacity of mammalian oocytes, and this is closely associated with the abnormal mRNA expressions of vitrified oocytes. However, the effect of vitrification on transcriptional machinery of oocytes examined by RNA sequencing (RNA-seq) has yet to be defined. In the present study, the mRNA transcriptomes of fresh and vitrified bovine oocytes were analysed by Smart-seq2 with the differently expressed genes determined by DEseq2 (an adjusted p-value of .05 and a minimum fold change of 2). The differentially expressed mRNAs were then searched against the Gene Ontology (GO) and Genomes (KEGG) database. Finally, the mRNA expressions of 10 candidate genes were validated using quantitative real-time PCR (qRT-PCR). Approximately 12,000 genes were detected in each sample of fresh or vitrified oocytes. Of these, the expression levels of 102 genes differed significantly in vitrified groups: 12 genes mainly involved in cell cycle, fertilization and glucose metabolism were upregulated, and 90 genes mainly involved in mitochondria, ribosomal protein, cytoskeleton, transmembrane protein, cell cycle and calcium ions were downregulated. GO analysis showed that these genes were mainly enriched in terms of membrane-bounded organelles, macromolecular complex, and intracellular part. The mRNA expression levels of 10 candidate genes selected randomly were in agreement with the results of the RNA-seq. In conclusion, our results showed that vitrification affected the mRNA transcriptome of bovine oocytes by downregulating genes, which contributed to the decreased developmental capacity of vitrified oocytes. Our findings will be useful in determining approaches to improve the efficiency of vitrified oocytes. © 2017 Blackwell Verlag GmbH.

  19. Differential regulation of renal cyclooxygenase mRNA by dietary salt intake

    DEFF Research Database (Denmark)

    Jensen, B L; Kurtz, A

    1997-01-01

    Experiments were done to investigate the influence of dietary salt intake on renal cyclooxygenase (COX) I and II mRNA levels. To this end rats were fed either a low NaCl diet (LS; 0.02% NaCl wt/wt) or a high NaCl diet (HS diet; 4% NaCl wt/wt) for 5, 10 and 20 days. After 10 days Na excretion...... differed 760-fold, plasma renin activity and renin mRNA were increased eight- and threefold in LS compared to HS animals. Total renal COX I mRNA decreased 50% following the LS diet and did not change after the HS diet. Conversely, COX II mRNA declined after HS intake and transiently increased after salt...... depletion. COX I and II mRNAs were unevenly distributed along the cortical-medullary axis with ratios of the cortex:outer medulla:papilla of 1:3:23 and 1:1:2, respectively. Cortical COX mRNAs were inversely regulated by salt intake with eightfold changes in COX II. Conversely, in medullary zones, COX I m...

  20. Structure of an RNA dimer of a regulatory element from human thymidylate synthase mRNA

    Energy Technology Data Exchange (ETDEWEB)

    Dibrov, Sergey; McLean, Jaime; Hermann, Thomas (UCSD)

    2011-09-27

    A sequence around the start codon of the mRNA of human thymidylate synthase (TS) folds into a secondary-structure motif in which the initiation site is sequestered in a metastable hairpin. Binding of the protein to its own mRNA at the hairpin prevents the production of TS through a translation-repression feedback mechanism. Stabilization of the mRNA hairpin by other ligands has been proposed as a strategy to reduce TS levels in anticancer therapy. Rapidly proliferating cells require high TS activity to maintain the production of thymidine as a building block for DNA synthesis. The crystal structure of a model oligonucleotide (TS1) that represents the TS-binding site of the mRNA has been determined. While fluorescence studies showed that the TS1 RNA preferentially adopts a hairpin structure in solution, even at high RNA concentrations, an asymmetric dimer of two hybridized TS1 strands was obtained in the crystal. The TS1 dimer contains an unusual S-turn motif that also occurs in the 'off' state of the human ribosomal decoding site RNA.

  1. Cellular volume is a global controller of mRNA abundance

    Science.gov (United States)

    Padovan-Merhar, Olivia; Raj, Arjun

    2013-03-01

    Many researchers have observed large variability in the numbers of RNA and protein molecules from cell to cell, a phenomenon thought to result from random bursts of transcription. These findings hold even for genes involved in core cellular processes, raising questions as to how cells can function in the presence of such molecular noise. However, biochemical processes typically depend on concentrations of cellular constituents rather than absolute numbers, so we use RNA fluorescence in situ hybridization to measure mRNA counts and cellular volume in single cells. We find that while both mRNA numbers and volume vary widely between cells, mRNA density does not. Thus, for many genes, mRNA abundance is precisely controlled to match the volume of the cell, as though the genes know how big the cell is. We measure transcription on a global and single-gene scale, and find that transcriptional activity scales with volume, suggesting that density is regulated at a transcriptional level. We present a mathematical model explaining which transcriptional bursting parameters account for the presence or lack of density conservation. Our findings suggest that global properties of RNA dynamics require a reassessment of our understanding of cellular heterogeneity and stochastic gene expression.

  2. A small RNA activates CFA synthase by isoform-specific mRNA stabilization.

    Science.gov (United States)

    Fröhlich, Kathrin Sophie; Papenfort, Kai; Fekete, Agnes; Vogel, Jörg

    2013-11-13

    Small RNAs use a diversity of well-characterized mechanisms to repress mRNAs, but how they activate gene expression at the mRNA level remains not well understood. The predominant activation mechanism of Hfq-associated small RNAs has been translational control whereby base pairing with the target prevents the formation of an intrinsic inhibitory structure in the mRNA and promotes translation initiation. Here, we report a translation-independent mechanism whereby the small RNA RydC selectively activates the longer of two isoforms of cfa mRNA (encoding cyclopropane fatty acid synthase) in Salmonella enterica. Target activation is achieved through seed pairing of the pseudoknot-exposed, conserved 5' end of RydC to an upstream region of the cfa mRNA. The seed pairing stabilizes the messenger, likely by interfering directly with RNase E-mediated decay in the 5' untranslated region. Intriguingly, this mechanism is generic such that the activation is equally achieved by seed pairing of unrelated small RNAs, suggesting that this mechanism may be utilized in the design of RNA-controlled synthetic circuits. Physiologically, RydC is the first small RNA known to regulate membrane stability.

  3. A small RNA activates CFA synthase by isoform-specific mRNA stabilization

    Science.gov (United States)

    Fröhlich, Kathrin Sophie; Papenfort, Kai; Fekete, Agnes; Vogel, Jörg

    2013-01-01

    Small RNAs use a diversity of well-characterized mechanisms to repress mRNAs, but how they activate gene expression at the mRNA level remains not well understood. The predominant activation mechanism of Hfq-associated small RNAs has been translational control whereby base pairing with the target prevents the formation of an intrinsic inhibitory structure in the mRNA and promotes translation initiation. Here, we report a translation-independent mechanism whereby the small RNA RydC selectively activates the longer of two isoforms of cfa mRNA (encoding cyclopropane fatty acid synthase) in Salmonella enterica. Target activation is achieved through seed pairing of the pseudoknot-exposed, conserved 5′ end of RydC to an upstream region of the cfa mRNA. The seed pairing stabilizes the messenger, likely by interfering directly with RNase E-mediated decay in the 5′ untranslated region. Intriguingly, this mechanism is generic such that the activation is equally achieved by seed pairing of unrelated small RNAs, suggesting that this mechanism may be utilized in the design of RNA-controlled synthetic circuits. Physiologically, RydC is the first small RNA known to regulate membrane stability. PMID:24141880

  4. mRNA cap methylation influences pathogenesis of vesicular stomatitis virus in vivo.

    Science.gov (United States)

    Ma, Yuanmei; Wei, Yongwei; Zhang, Xiaodong; Zhang, Yu; Cai, Hui; Zhu, Yang; Shilo, Konstantin; Oglesbee, Michael; Krakowka, Steven; Whelan, Sean P J; Li, Jianrong

    2014-03-01

    One role of mRNA cap guanine-N-7 (G-N-7) methylation is to facilitate the efficient translation of mRNA. The role of mRNA cap ribose 2'-O methylation is enigmatic, although recent work has implicated this as a signature to avoid detection of RNA by the innate immune system (S. Daffis, K. J. Szretter, J. Schriewer, J. Q. Li, S. Youn, J. Errett, T. Y. Lin, S. Schneller, R. Zust, H. P. Dong, V. Thiel, G. C. Sen, V. Fensterl, W. B. Klimstra, T. C. Pierson, R. M. Buller, M. Gale, P. Y. Shi, M. S. Diamond, Nature 468:452-456, 2010, doi:10.1038/nature09489). Working with vesicular stomatitis virus (VSV), we previously showed that a panel of recombinant VSVs carrying mutations at a predicted methyltransferase catalytic site (rVSV-K1651A, -D1762A, and -E1833Q) or S-adenosylmethionine (SAM) binding site (rVSV-G1670A, -G1672A, and -G4A) were defective in cap methylation and were also attenuated for growth in cell culture. Here, we analyzed the virulence of these recombinants in mice. We found that rVSV-K1651A, -D1762A, and -E1833Q, which are defective in both G-N-7 and 2'-O methylation, were highly attenuated in mice. All three viruses elicited a high level of neutralizing antibody and provided full protection against challenge with the virulent VSV. In contrast, mice inoculated with rVSV-G1670A and -G1672A, which are defective only in G-N-7 methylation, were attenuated in vivo yet retained a low level of virulence. rVSV-G4A, which is completely defective in both G-N-7 and 2'-O methylation, also exhibited low virulence in mice despite the fact that productive viral replication was not detected in lung and brain. Taken together, our results suggest that abrogation of viral mRNA cap methylation can serve as an approach to attenuate VSV, and perhaps other nonsegmented negative-strand RNA viruses, for potential application as vaccines and viral vectors. Nonsegmented negative-sense (NNS) RNA viruses include a wide range of significant human, animal, and plant pathogens. For many

  5. Progressive APOBEC3B mRNA expression in distant breast cancer metastases.

    Directory of Open Access Journals (Sweden)

    Anieta M Sieuwerts

    Full Text Available APOBEC3B was recently identified as a gain-of-function enzymatic source of mutagenesis, which may offer novel therapeutic options with molecules that specifically target this enzyme. In primary breast cancer, APOBEC3B mRNA is deregulated in a substantial proportion of cases and its expression is associated with poor prognosis. However, its expression in breast cancer metastases, which are the main causes of breast cancer-related death, remained to be elucidated.RNA was isolated from 55 primary breast cancers and paired metastases, including regional lymph node (N = 20 and distant metastases (N = 35. APOBEC3B mRNA levels were measured by RT-qPCR. Expression levels of the primary tumors and corresponding metastases were compared, including subgroup analysis by estrogen receptor (ER/ESR1 status.Overall, APOBEC3B mRNA levels of distant metastases were significantly higher as compared to the corresponding primary breast tumor (P = 0.0015, an effect that was not seen for loco-regional lymph node metastases (P = 0.23. Subgroup analysis by ER-status showed that increased APOBEC3B levels in distant metastases were restricted to metastases arising from ER-positive primary breast cancers (P = 0.002. However, regarding ER-negative primary tumors, only loco-regional lymph node metastases showed increased APOBEC3B expression when compared to the corresponding primary tumor (P = 0.028.APOBEC3B mRNA levels are significantly higher in breast cancer metastases as compared to the corresponding ER-positive primary tumors. This suggests a potential role for APOBEC3B in luminal breast cancer progression, and consequently, a promising role for anti-APOBEC3B therapies in advanced stages of this frequent form of breast cancer.

  6. [Influence of Heat-reinforcing Needling on Expression of Plasma Atp 5 O mRNA and Atp 6 V 1 B 2 mRNA in Patients with Rheumatoid Arthritis of Wind-cold-damp Retention Type].

    Science.gov (United States)

    Du, Xiao-Zheng; Wang, Jin-Hai; Zhang, Xing-Hua; Tian, Jie-Xiang; Qin, Xiao-Guang; Fang, Xiao-Li; Tian, Liang; Yuan, Bo

    2016-08-25

    To observe influences of heat-reinforcing needling (HRN) on scores of traditional Chinese medicine (TCM) symptoms and expression of plasma ATP synthase subunit O (Atp 5 O) mRNA and lysosomal V 1 subunit B 2 (Atp 6 V 1 B 2) mRNA in patients with wind-cold-damp retention type rheumatoid arthritis (RA), so as to investigate its biological mechanisms in "heat production". Sixty wind-cold-damp retention type RA patients were randomly allocated to HRN group (n=30) and control group (n=30). Guanyuan (CV 4), Qihai (CV 6), bilateral Zusanli (ST 36), and local acupoints near the knee-joint were selected for needling stimulation. Patients of the HRN group were treated by manipulating the acupuncture needle with HRN, and those of the control group treated by manipulating the needle with uniform reinforcing-reducing method. The treatments were performed once daily, 5 days a week, and two weeks altogether. The other 30 healthy volunteers were recruited as the normal control group. The TCM symptom scoring system (0-31 points, 11 items as the severities of pain, swelling and tenderness of the knee-joint) was used to evaluate the status of RA, and quantitative real-time PCR (RT-PCR) was used to detect the expression of plasma Atp 5 O mRNA and Atp 6 V 1 B 2 mRNA following removal of red blood cells. After the treatment, the TCM scores of both the HRN and control groups were significantly decreased (PB 2 mRNA in RA patients were significantly lower than those of the normal group (PB 2 mRNA were significantly increased in both HRN and control groups compared to pre-treatment in the same one group (PB 2 mRNA levels were remarkably higher in the control group than in the HRN group (PB 2 mRNA.

  7. Demonstration that a mRNA Binding Protein is Responsible for GADD45 mRNA Destabilization

    National Research Council Canada - National Science Library

    Abcouwer, Steve

    2003-01-01

    ...) Using regions of the GADD45 mRNA 3'-untranslated region (UTR) in KNA gel shift assays, we have observed that glutamine causes distinct changes in RBP activities in cytoplasmic and nuclear protein extracts...

  8. Molecular basis of maple syrup urine disease: Novel mutations at the E1[alpha] locus that impair E1([alpha][sub 2][beta][sub 2]) assembly or decrease steady-state E1[alpha] mRNA levels of branched-chain [alpha]-keto acid dehydrogenase complex

    Energy Technology Data Exchange (ETDEWEB)

    Chuang, J.L.; Fisher, C.R.; Chuang, D.T.; Cox, R.P. (Univ. of Texas Southwestern Medical Center, Dallas, TX (United States))

    1994-08-01

    The authors report the occurrence of three novel mutations in the E1[alpha] (BCKDHA) locus of the branched-chain [alpha]-keto acid dehydrogenase (BCKAD) complex that cause maple syrup urine disease (MSUD). An 8-bp deletion in exon 7 is present in one allele of a compound-heterozygous patient (GM-649). A single C nucleotide insertion in exon 2 occurs in one allele of an intermediate-MSUD patient (Lo). The second allele of patient Lo carries an A-to-G transition in exon 9 of the E1[alpha] gene. This missense mutation changes Tyr-368 to Cys (Y368C) in the E1[alpha] subunit. Both the 8-bp deletion and the single C insertion generate a downstream nonsense codon. Both mutations appear to be associated with a low abundance of the mutant E1[alpha] mRNA, as determined by allele-specific oligonucleotide probing. Transfection studies strongly suggest that the Y368C substitution in the E1[alpha] subunit impairs its proper assembly with the normal E1[beta]. Unassembled as well as misassembled E1[alpha] and E1[beta] subunits are degraded in the cell. 32 refs., 8 figs.

  9. [Expression of PEPT2 mRNA in lung tissue of rats with pulmonary fibrosis].

    Science.gov (United States)

    Li, Li; Wang, Dianhua; Zhang, Xuan; Song, Xin; Ma, Xiaobiao; Hu, Zaoxiu

    2013-10-20

    Pulmonary fibrosis is a common pathological phenomenon in lung cancer patients after chemotherapy or radiotherapy. It is also a key hindrance to the transport of drugs to lung tissue. Peptide transporters have become a target of the rational design of peptides and peptide drugs. The aim of this study is to investigates the expression of peptide transporter 2 (PEPT2) mRNA in the lungs of rats with bleomycin (BLM)-induced pulmonary fibrosis. Fifty healthy adult Sprague-Dawley rats were randomly divided into five groups. One group was untreated (control), the second group was injected with normal saline solution (NS), and the three remaining groups were treated with a single dose of bleomycin to induce pulmonary fibrosis (BLM). Rats from the NS group were killed by exsanguination on day 14. Rats from the BLM group were killed by exsanguination on days 7, 14, and 28. The lung samples were observed under light microscopy and the hydroxyproline concentration was determined. The expression levels of PEPT2 mRNA were measured by RT-PCR. The morphological study showed that collagenous fiber proliferated in the lungs of rats injected with BLM, indicating pulmonary fibrosis. This proliferation was apparent at 14 d post-injection and especially at 28 d post-injection. Hydroxyproline levels increased seven days post-injection compared with the control group and NS group, but there was no significant statistical difference (P>0.05). Hydroxyproline levels significantly increased (Ppulmonary PEPT2 mRNA expression levels among the different groups (P>0.05). PEPT2 is a potential peptide drug target in the treatment of pulmonary fibrosis, although there were no significant changes of PEPT2 mRNA expression in the lungs of rats with bleomycin-induced pulmonary fibrosis.

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

    Science.gov (United States)

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

    2017-07-22

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

  11. Downregulation of TIM-3 mRNA expression in peripheral blood mononuclear cells from patients with systemic lupus erythematosus

    Energy Technology Data Exchange (ETDEWEB)

    Cai, X.Z. [Central Laboratory, First Affiliated Hospital, China Medical University, Shenyang (China); Department of Immunology, College of Basic Medical Sciences, China Medical University, Shenyang (China); Huang, W.Y.; Qiao, Y.; Chen, Y.; Du, S.Y.; Chen, D.; Yu, S. [Central Laboratory, First Affiliated Hospital, China Medical University, Shenyang (China); Liu, N. [Department of Nephrology, First Affiliated Hospital, China Medical University, Shenyang (China); Dou, L.Y. [Central Laboratory, First Affiliated Hospital, China Medical University, Shenyang (China); Jiang, Y. [Central Laboratory, First Affiliated Hospital, China Medical University, Shenyang (China); Department of Immunology, College of Basic Medical Sciences, China Medical University, Shenyang (China); Department of Dermatology, First Affiliated Hospital, China Medical University, Shenyang (China)

    2014-10-17

    The T-cell immunoglobulin and mucin domain (TIM) family is associated with autoimmune diseases, but its expression level in the immune cells of systemic lupus erythematosus (SLE) patients is not known. The aim of this study was to investigate whether the expression of TIM-3 mRNA is associated with pathogenesis of SLE. Quantitative real-time reverse transcription-polymerase chain reaction analysis (qRT-PCR) was used to determine TIM-1, TIM-3, and TIM-4 mRNA expression in peripheral blood mononuclear cells (PBMCs) from 132 patients with SLE and 62 healthy controls. The PBMC surface protein expression of TIMs in PBMCs from 20 SLE patients and 15 healthy controls was assayed by flow cytometry. Only TIM-3 mRNA expression decreased significantly in SLE patients compared with healthy controls (P<0.001). No significant differences in TIM family protein expression were observed in leukocytes from SLE patients and healthy controls (P>0.05). SLE patients with lupus nephritis (LN) had a significantly lower expression of TIM-3 mRNA than those without LN (P=0.001). There was no significant difference in the expression of TIM-3 mRNA within different classes of LN (P>0.05). Correlation of TIM-3 mRNA expression with serum IgA was highly significant (r=0.425, P=0.004), but was weakly correlated with total serum protein (r{sub s}=0.283, P=0.049) and serum albumin (r{sub s}=0.297, P=0.047). TIM-3 mRNA expression was weakly correlated with the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI; r{sub s}=-0.272, P=0.032). Our results suggest that below-normal expression of TIM-3 mRNA in PBMC may be involved in the pathogenesis of SLE.

  12. Light differentially regulates cell division and the mRNA abundance of pea nucleolin during de-etiolation

    Science.gov (United States)

    Reichler, S. A.; Balk, J.; Brown, M. E.; Woodruff, K.; Clark, G. B.; Roux, S. J.

    2001-01-01

    The abundance of plant nucleolin mRNA is regulated during de-etiolation by phytochrome. A close correlation between the mRNA abundance of nucleolin and mitosis has also been previously reported. These results raised the question of whether the effects of light on nucleolin mRNA expression were a consequence of light effects on mitosis. To test this we compared the kinetics of light-mediated increases in cell proliferation with that of light-mediated changes in the abundance of nucleolin mRNA using plumules of dark-grown pea (Pisum sativum) seedlings. These experiments show that S-phase increases 9 h after a red light pulse, followed by M-phase increases in the plumule leaves at 12 h post-irradiation, a time course consistent with separately measured kinetics of red light-induced increases in the expression of cell cycle-regulated genes. These increases in cell cycle-regulated genes are photoreversible, implying that the light-induced increases in cell proliferation are, like nucleolin mRNA expression, regulated via phytochrome. Red light stimulates increases in the mRNA for nucleolin at 6 h post-irradiation, prior to any cell proliferation changes and concurrent with the reported timing of phytochrome-mediated increases of rRNA abundance. After a green light pulse, nucleolin mRNA levels increase without increasing S-phase or M-phase. Studies in animals and yeast indicate that nucleolin plays a significant role in ribosome biosynthesis. Consistent with this function, pea nucleolin can rescue nucleolin deletion mutants of yeast that are defective in rRNA synthesis. Our data show that during de-etiolation, the increased expression of nucleolin mRNA is more directly regulated by light than by mitosis.

  13. Effect of pioglitazone, quercetin, and hydroxy citric acid on vascular endothelial growth factor messenger RNA (VEGF mRNA) expression in experimentally induced nonalcoholic steatohepatitis (NASH).

    Science.gov (United States)

    Surapaneni, Krishna Mohan; Vishnu Priya, Veeraraghavan; Mallika, Jainu

    2015-01-01

    Vascular endothelial growth factor (VEGF) is associated with various ischemic and inflammatory diseases, and plays an important role in the development of liver fibrosis and hepatocarcinogenesis in nonalcoholic steatohepatitis (NASH). In this study, the comparative effect of pioglitazone, quercetin, and hydroxy citric acid on VEGF mRNA in experimentally induced NASH was investigated. The experimental protocol consisted of five groups: control, NASH, NASH + pioglitazone, NASH + quercetin, and NASH + hydroxy citric acid. The VEGF mRNA expression was evaluated by reverse transcription polymerase chain reaction (RT- PCR) analysis for all experimental groups, and the levels of VEGF mRNA were quantitatively measured by densitometry. A higher expression of VEGF mRNA was found in the hepatic cells of rats with experimentally induced NASH compared to the control group. A very mild increase in VEGF mRNA expression was observed in the rats treated with quercetin. In contrast, a mild increase in the expression of VEGF mRNA was observed in the rats treated with pioglitazone and hydroxy citric acid. Quercetin exhibited an effective inhibition of VEGF mRNA expression, while a lower inhibition of the VEGF mRNA level was observed in the hydroxy citric acid- and the pioglitazone-treated rats.

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

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    Taylor John M

    2001-03-01

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

  15. Differential histone deacetylase mRNA expression patterns in amyotrophic lateral sclerosis.

    Science.gov (United States)

    Janssen, Claas; Schmalbach, Sonja; Boeselt, Sebastian; Sarlette, Alexander; Dengler, Reinhard; Petri, Susanne

    2010-06-01

    Histone deacetylases (HDACs) are important regulators of gene expression and cell differentiation. The HDAC inhibitors have recently been considered as potential novel neuroprotective drugs for the treatment of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). A major limitation, however, lies in the broad spectrum of action of currently available HDAC inhibitors that may cause a variety of toxic side effects. The mRNA expression levels of the HDAC isoforms HDACs 1 to 11 have previously been characterized in rat brain but have not been studied in human tissue. Using in situ hybridization histochemistry and immunohistochemistry we assessed the distribution and expression levels of HDACs 1to 11 in postmortem ALS and control brain and spinal cord specimens (n = 6 cases each) to determine alterations in the mRNA expression pattern that could provide a basis for disease-specific therapies. We found a reduction of HDAC 11 mRNA and increased HDAC 2 levels in ALS brain and spinal cord compared with controls. A more precise knowledge of the disease-related expression pattern could lead to the development of more specific pharmacotherapeutic approaches.

  16. Basal keratin expression in breast cancer by quantification of mRNA and by immunohistochemistry

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    Pluciennik Elzbieta

    2010-04-01

    Full Text Available Abstract Definitions of basal-like breast cancer phenotype vary, and microarray-based expression profiling analysis remains the gold standard for the identification of these tumors. Immunohistochemical identification of basal-like carcinomas is hindered with a fact, that on microarray level not all of them express basal-type cytokeratin 5/6, 14 and 17. We compared expression of cytokeratin 5, 14 and 17 in 115 patients with operable breast cancer estimated by real-time RT-PCR and immunohistochemistry. Despite the method of dichotomization and statistical analysis, there were cases with discordant results comparing immunohistochemistry and RT-PCR analysis. For dichotomisation based on quartiles and ROC, 14% of cases were negative on immunohistochemical examination for CK5/6, but presented high CK5 mRNA levels. There were also 48-55% cases, which were CK5/6-immunopositive, but were negative by mRNA examination. Similar discordances were observed for CK14 and CK17. Basal keratin mRNAs did not correlate with ER mRNA levels, while immunohistochemistry produced significant relationship with ER status. Our observation suggest that both method may produce different results in a small proportion of cases. Discordance between immunohistochemistry and RT-PCR may confound attempts to establish a simple methods for identification of basal-like tumors.

  17. Ferroportin mRNA is down-regulated in granulosa and cervical cells from infertile women.

    Science.gov (United States)

    Moreno-Navarrete, José Maria; López-Navarro, Eva; Candenas, Luz; Pinto, Francisco; Ortega, Francisco J; Sabater-Masdeu, Mònica; Fernández-Sánchez, Manuel; Blasco, Victor; Romero-Ruiz, Antonio; Fontán, Marina; Ricart, Wifredo; Tena-Sempere, Manuel; Fernández-Real, José M

    2017-01-01

    To explore the relationship between iron and infertility by investigating iron-related gene expression in granulosa and uterine cervical cells. Case-control study. Two tertiary hospitals. Two independent cohorts of fertile (n = 18 and n = 17) and infertile (n = 31 and n = 35) women. In vitro fertilization. Gene expression levels of ferritin light chain (FTL), ferritin heavy chain (FTH), transferrin receptor (TFRC), and ferroportin (SLC40A1) mRNA were analyzed in granulosa and cervical cells. In the first cohort, fertile and infertile women were similar in body mass index. Ferroportin mRNA levels were decreased in granulosa cells from infertile women in parallel with increased serum hepcidin levels. A positive association between ferroportin and TFRC mRNA, a gene associated with intracellular iron deficiency, was observed only in granulosa cells from fertile women. The major findings were replicated in a second independent cohort. Ferroportin mRNAs and circulating hepcidin identify a subset of infertile women and may constitute a target for therapy. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  18. Expression of clusterin (testosterone-repressed prostate message-2) mRNA during growth and regeneration of rat liver

    Energy Technology Data Exchange (ETDEWEB)

    Bursch, W. [Inst. fuer Tumorbiologie-Krebsforschung, Univ. Wien (Austria); Gleeson, T. [Dept. of Biochemistry, Univ. of Ottawa, ON (Canada); Kleine, L. [Dept. of Biochemistry, Univ. of Ottawa, ON (Canada); Tenniswood, M. [Dept. of Biochemistry, Univ. of Ottawa, ON (Canada)

    1995-02-01

    In the present study, we have looked at the expression of clusterin during the growth and regression of rat liver induced by short term administration of the hepatomitogen, cyproterone acetate. The steady state level of clusterin mRNA, as measured by Northern and slot blot analysis, is low in control hepatocytes. Following administration of cyproterone acetate, the clusterin mRNA level is increased during both liver growth and regression. In situ hybridization reveals that clusterin is expressed in all hepatocytes, indicating that it is not confined to cell death by apoptosis. These results suggest that the gene product may be involved in maintaining membrane integrity, which is necessary during both mitosis and apoptosis. To determine whether clusterin mRNA is induced by membrane remodelling independent of either mitosis or apoptosis, we examined the expression of clusterin mRNA in the liver after a necrogenic dose of carbon tetrachloride. During the first 24-48 h of this time period, necrosis is the predominant form of cell death and liver regeneration starts after approximately 24 h. Elevated levels of clusterin mRNA are found as early as 12 h after carbon tetrachloride administration and persist for at least 72 h. Clusterin expression in tissues such as the prostate and mammary gland appears to be confined to the apoptotic pathway; however, our results suggest that in hepatocytes, the expression of the gene is induced in processes other than apoptosis, including mitosis and necrosis. (orig.)

  19. Detection of siRNA induced mRNA silencing by RT-qPCR: considerations for experimental design

    Directory of Open Access Journals (Sweden)

    Chapman Elinor A

    2010-03-01

    Full Text Available Abstract Background RNA interference (RNAi has been one of the most rapidly expanding areas of biological research in the past decade, revolutionizing the ability to analyze gene function. Thorough validation of siRNA duplexes is required prior to use in experimental systems, ideally by western blotting to show a reduction in protein levels. However, in many cases good antibodies are not available, and researchers must rely on RT-qPCR to detect knockdown of the mRNA species. Findings We have observed a phenomenon that gives a disparity between analyzing small interfering RNA (siRNA efficacy by western blotting of the protein levels and real-time quantitative PCR (RT-qPCR measurement of mRNA levels. Detection of this phenomenon was dependent upon the location of the target amplicon for PCR primers within the mRNA. Conclusions Our data suggests that for certain mRNAs, degradation of the 3' mRNA fragment resulting from siRNA mediated cleavage is blocked, leaving an mRNA fragment that can act as a template for cDNA synthesis, giving rise to false negative results and the rejection of a valid siRNA duplex. We show that this phenomenon may be avoided by the careful design of RT-qPCR primers for each individual siRNA experiment.

  20. Interaction between thymidylate synthase and its cognate mRNA in zebrafish embryos.

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    Yuyan Zhang

    Full Text Available Thymidylate synthase (TS, which catalyzes the de novo synthesis of dUMP, is an important target for cancer therapy. In this report, the effects of 5-fluorouracil (5-FU and ZD1694 on the regulation of TS gene expression were evaluated in zebrafish embryos. Our results revealed that the expression of TS was increased by about six-fold when embryos were treated with 1.0 microM 5-FU and there was a greater than 10-fold increase in the TS protein level after treatment with 0.4 microM ZD1694. Northern blot analysis confirmed that expression of TS mRNA was identical in treated or untreated embryos. Gel shift and immunoprecipitation assays revealed that zebrafish TS was specifically bound with its cognate mRNA in vitro and in vivo. We identified a 20 nt RNA sequence, TS:N20, localized to the 5'-UTR of TS mRNA, which corresponded to nt 13-32; TS:N20 bound to the TS protein with an affinity similar to that of the full-length TS mRNA. The MFold program predicted that TS:N20 formed a stable stem-loop structure similar to that of the cis-acting element found in human TS mRNA. Variant RNAs with either a deletion or mutation in the core motif of TS:N20 were unable to bind to the TS protein. In vitro translation experiments, using the rabbit lysate system, confirmed that zebrafish TS mRNA translation was significantly repressed when an excess amount of TS protein was included in the system. Additionally, a TS stability experiment confirmed that treatment of zebrafish embryos with 5-FU could increase the TS stability significantly, and the half life of TS protein was about 2.7 times longer than in untreated embryos. Our study revealed a structural requirement for the interaction of TS RNA with TS protein. These findings also demonstrated that the increase in TS protein induced by 5-FU occurs at the post-transcriptional level and that increased stability and translation efficiency both contributed to the increase in TS protein levels induced by TS inhibitors.

  1. Orosomucoid serum concentrations and fat depot-specific mRNA and protein expression in humans.

    Science.gov (United States)

    Alfadda, Assim A; Fatma, Sumbul; Chishti, M Azhar; Al-Naami, Mohammed Y; Elawad, Ruba; Mendoza, Carmen Deanna O; Jo, Hyunsun; Lee, Yun Sok

    2012-01-01

    Obesity is associated with chronic low-grade inflammation, which contributes to systemic metabolic irregularities and obesity-linked metabolic disorders. Orosomucoid (ORM), an acute phase reactant protein, was shown to be produced in response to metabolic and inflammatory signals in the adipose tissue of obese mice, which protects them from severe inflammation and subsequent metabolic dysfunction. In this study, we examined whether there are site-specific differences between visceral and subcutaneous adipose tissue (VAT and SAT, respectively) ORM gene and protein expression from individuals with a wide range of obesity and the relationship between expressed and circulating ORM levels and measures of adiposity, insulin resistance, and pro- and anti-inflammatory markers and adipokines. The level of circulating ORM correlated positively with BMI, body fat mass, and serum leptin. It also correlated with fasting insulin, HOMA-IR values and C-reactive protein in men. There were no site-specific differences in ORM mRNA and protein expression between VAT and SAT, nor did we find a relationship between circulating ORM levels and its mRNA expression in either fat depot. We found that ORM mRNA expression correlated with mRNA expression of TNF-α, IL-6, and adiponectin in VAT, and with TNF-α and adiponectin in SAT. These observations are the first description linking adipose tissue ORM and pro- and anti-inflammatory molecules in humans. The close links of ORM and measures of adiposity, insulin resistance, and adipose tissue inflammation in humans reinforce previous experimental data and warrant further studies to explore a possible role of ORM in the pathogenesis of obesity-associated metabolic derangements.

  2. Nebulisation of IVT mRNA Complexes for Intrapulmonary Administration.

    Directory of Open Access Journals (Sweden)

    Sarah M Johler

    Full Text Available During the last years the potential role of in vitro transcribed (IVT mRNA as a vehicle to deliver genetic information has come into focus. IVT mRNA could be used for anti-cancer therapies, vaccination purposes, generation of pluripotent stem cells and also for genome engineering or protein replacement. However, the administration of IVT mRNA into the target organ is still challenging. The lung with its large surface area is not only of interest for delivery of genetic information for treatment of e.g. for cystic fibrosis or alpha-1-antitrypsin deficiency, but also for vaccination purposes. Administration of IVT mRNA to the lung can be performed by direct intratracheal instillation or by aerosol inhalation/nebulisation. The latter approach shows a non-invasive tool, although it is not known, if IVT mRNA is resistant during the process of nebulisation. Therefore, we investigated the transfection efficiency of non-nebulised and nebulised IVT mRNA polyplexes and lipoplexes in human bronchial epithelial cells (16HBE. A slight reduction in transfection efficiency was observed for lipoplexes (Lipofectamine 2000 in the nebulised part compared to the non-nebulised which can be overcome by increasing the amount of Lipofectamine. However, Lipofectamine was more than three times more efficient in transfecting 16HBE than DMRIE and linear PEI performed almost 10 times better than its branched derivative. By contrast, the nebulisation process did not affect the cationic polymer complexes. Furthermore, aerosolisation of IVT mRNA complexes did neither affect the protein duration nor the toxicity of the cationic complexes. Taken together, these data show that aerosolisation of cationic IVT mRNA complexes constitute a potentially powerful means to transfect cells in the lung with the purpose of protein replacement for genetic diseases such as cystic fibrosis or alpha-1-antitrypsin deficiency or for infectious disease vaccines, while bringing along the advantages

  3. Protein targeting to subcellular organelles via MRNA localization.

    Science.gov (United States)

    Weis, Benjamin L; Schleiff, Enrico; Zerges, William

    2013-02-01

    Cells have complex membranous organelles for the compartmentalization and the regulation of most intracellular processes. Organelle biogenesis and maintenance requires newly synthesized proteins, each of which needs to go from the ribosome translating its mRNA to the correct membrane for insertion or transclocation to an a organellar subcompartment. Decades of research have revealed how proteins are targeted to the correct organelle and translocated across one or more organelle membranes ro the compartment where they function. The paradigm examples involve interactions between a peptide sequence in the protein, localization factors, and various membrane embedded translocation machineries. Membrane translocation is either cotranslational or posttranslational depending on the protein and target organelle. Meanwhile research in embryos, neurons and yeast revealed an alternative targeting mechanism in which the mRNA is localized and only then translated to synthesize the protein in the correct location. In these cases, the targeting information is coded by the cis-acting sequences in the mRNA ("Zipcodes") that interact with localization factors and, in many cases, are transported by the molecular motors on the cytoskeletal filaments. Recently, evidence has been found for this "mRNA based" mechanism in organelle protein targeting to endoplasmic reticulum, mitochondria, and the photosynthetic membranes within chloroplasts. Here we review known and potential roles of mRNA localization in protein targeting to and within organelles. This article is part of a Special Issue entitled: Protein Import and Quality Control in Mitochondria and Plastids.

  4. The translation initiation factor DAP5 promotes IRES-driven translation of p53 mRNA.

    Science.gov (United States)

    Weingarten-Gabbay, S; Khan, D; Liberman, N; Yoffe, Y; Bialik, S; Das, S; Oren, M; Kimchi, A

    2014-01-30

    Translational regulation of the p53 mRNA can determine the ratio between p53 and its N-terminal truncated isoforms and therefore has a significant role in determining p53-regulated signaling pathways. Although its importance in cell fate decisions has been demonstrated repeatedly, little is known about the regulatory mechanisms that determine this ratio. Two internal ribosome entry sites (IRESs) residing within the 5'UTR and the coding sequence of p53 mRNA drive the translation of full-length p53 and Δ40p53 isoform, respectively. Here, we report that DAP5, a translation initiation factor shown to positively regulate the translation of various IRES containing mRNAs, promotes IRES-driven translation of p53 mRNA. Upon DAP5 depletion, p53 and Δ40p53 protein levels were decreased, with a greater effect on the N-terminal truncated isoform. Functional analysis using bicistronic vectors driving the expression of a reporter gene from each of these two IRESs indicated that DAP5 preferentially promotes translation from the second IRES residing in the coding sequence. Furthermore, p53 mRNA expressed from a plasmid carrying this second IRES was selectively shifted to lighter polysomes upon DAP5 knockdown. Consequently, Δ40p53 protein levels and the subsequent transcriptional activation of the 14-3-3σ gene, a known target of Δ40p53, were strongly reduced. In addition, we show here that DAP5 interacts with p53 IRES elements in in vitro and in vivo binding studies, proving for the first time that DAP5 directly binds a target mRNA. Thus, through its ability to regulate IRES-dependent translation of the p53 mRNA, DAP5 may control the ratio between different p53 isoforms encoded by a single mRNA.

  5. The p53 target Wig-1 regulates p53 mRNA stability through an AU-rich element

    DEFF Research Database (Denmark)

    Vilborg, Anna; Glahder, Jacob-Andreas Harald; Wilhelm, Margareta T

    2009-01-01

    The p53 target gene Wig-1 encodes a double-stranded-RNA-binding zinc finger protein. We show here that Wig-1 binds to p53 mRNA and stabilizes it through an AU-rich element (ARE) in the 3' UTR of the p53 mRNA. This effect is mirrored by enhanced p53 protein levels in both unstressed cells and cells...... exposed to p53-activating stress agents. Thus, the p53 target Wig-1 is a previously undescribed ARE-regulating protein that acts as a positive feedback regulator of p53, with implications both for the steady-state levels of p53 and for the p53 stress response. Our data reveal a previously undescribed link...... between the tumor suppressor p53 and posttranscriptional gene regulation via AREs in mRNA....

  6. Recognition of nonsense mRNA: towards a unified model.

    Science.gov (United States)

    Mühlemann, Oliver

    2008-06-01

    Among the different cellular surveillance mechanisms that ensure accurate gene expression, nonsense-mediated mRNA decay rapidly degrades mRNAs harbouring PTCs (premature translation-termination codons) and thereby prevents the accumulation of potentially deleterious proteins with C-terminal truncations. In the present article, I review recent data from yeast, fluitflies, nematode worms and human cells and endeavour to merge these results into a unified model for recognition of nonsense mRNA. According to this model, the distinction between translation termination at PTCs and at 'normal' termination codons relies on the physical distance between the terminating ribosome and PABP [poly(A)-binding protein]. Correct translation termination is promoted by a PABP-mediated signal to the terminating ribosome, whereas the absence of this signal leads to the assembly of an mRNA decay-promoting protein complex including the conserved NMD factors UPF (up-frameshift) 1-3.

  7. Post-transcriptional gene regulation by mRNA modifications

    Science.gov (United States)

    Zhao, Boxuan Simen; Roundtree, Ian A.; He, Chuan

    2016-01-01

    The recent discovery of reversible mRNA methylation has opened a new realm of post-transcriptional gene regulation in eukaryotes. The identification and functional characterization of proteins that specifically recognize RNA N6-methyladenosine (m6A) unveiled it as a modification that cells utilize to accelerate mRNA metabolism and translation. N6-adenosine methylation directs mRNAs to distinct fates by grouping them for differential processing, translation and decay in processes such as cell differentiation, embryonic development and stress responses. Other mRNA modifications, including N1-methyladenosine (m1A), 5-methylcytosine (m5C) and pseudouridine, together with m6A form the epitranscriptome and collectively code a new layer of information that controls protein synthesis. PMID:27808276

  8. Effect of atrophy and contractions on myogenin mRNA concentration in chick and rat myoblast omega muscle cells

    Science.gov (United States)

    Krebs, J. M.; Denney, R. M.

    1997-01-01

    The skeletal rat myoblast omega (RMo) cell line forms myotubes that exhibit spontaneous contractions under appropriate conditions in culture. We examined if the RMo cells would provide a model for studying atrophy and muscle contraction. To better understand how to obtain contractile cultures, we examined levels of contraction under different growing conditions. The proliferation medium and density of plating affected the subsequent proportion of spontaneously contracting myotubes. Using a ribonuclease protection assay, we found that exponentially growing RMo myoblasts contained no detectable myogenin or herculin mRNA, while differentiating myoblasts contained high levels of myogenin mRNA but no herculin mRNA. There was no increase in myogenin mRNA concentration in either primary chick or RMo myotubes whose contractions were inhibited by depolarizing concentrations of potassium (K+). Thus, altered myogenin mRNA concentrations are not involved in atrophy of chick myotubes. Depolarizing concentrations of potassium inhibited spontaneous contractions in both RMo cultures and primary chick myotube cultures. However, we found that the myosin concentration of 6-d-old contracting RMo cells fed medium plus AraC was 11 +/- 3 micrograms myosin/microgram DNA, not significantly different from 12 +/- 4 micrograms myosin/microgram DNA (n = 3), the myosin concentration of noncontracting RMo cells (treated with 12 mM K+ for 6 d). Resolving how RMo cells maintained their myosin content when contraction is inhibited may be important for understanding atrophy.

  9. Screening of Pleural Mesotheliomas for DNA-damage Repair Players by Digital Gene Expression Analysis Can Enhance Clinical Management of Patients Receiving Platin-Based Chemotherapy.

    Science.gov (United States)

    Walter, Robert Fred Henry; Vollbrecht, Claudia; Werner, Robert; Mairinger, Thomas; Schmeller, Jan; Flom, Elena; Wohlschlaeger, Jeremias; Barbetakis, Nikolaos; Paliouras, Dimitrios; Chatzinikolaou, Fotios; Adamidis, Vasilis; Tsakiridis, Kosmas; Zarogoulidis, Paul; Trakada, Georgia; Christoph, Daniel Christian; Schmid, Kurt Werner; Mairinger, Fabian Dominik

    2016-01-01

    Malignant pleural mesothelioma (MPM) is a rare, predominantly asbestos-related and biologically highly aggressive tumour leading to a dismal prognosis. Multimodality therapy consisting of platinum-based chemotherapy is the treatment of choice. The reasons for the rather poor efficacy of platinum compounds remain largely unknown. For this exploratory mRNA study, 24 FFPE tumour specimens were screened by digital gene expression analysis. Based on data from preliminary experiments and recent literature, a total of 366 mRNAs were investigated using a Custom CodeSet from NanoString. All statistical analyses were calculated with the R i386 statistical programming environment. CDC25A and PARP1 gene expression were correlated with lymph node spread, BRCA1 and TP73 expression levels with higher IMIG stage. NTHL1 and XRCC3 expression was associated with TNM stage. CHECK1 as well as XRCC2 expression levels were correlated with tumour progression in the overall cohort of patients. CDKN2A and MLH1 gene expression influenced overall survival in this collective. In the adjuvant treated cohort only, CDKN2A , CHEK1 as well as ERCC1 were significantly associated with overall survival. Furthermore, TP73 expression was associated with progression in this subgroup. DNA-damage response plays a crucial role in response to platin-based chemotherapeutic regimes. In particular, CHEK1 , XRCC2 and TP73 are strongly associated with tumour progression. ERCC1, MLH1 , CDKN2A and most promising CHEK1 are prognostic markers for OS in MPM. TP73 , CDKN2A , CHEK1 and ERCC1 seem to be also predictive markers in adjuvant treated MPMs. After a prospective validation, these markers may improve clinical and pathological practice, finally leading to a patients' benefit by an enhanced clinical management.

  10. Simple and inexpensive ribosome profiling analysis of mRNA translation.

    Science.gov (United States)

    Reid, David W; Shenolikar, Shirish; Nicchitta, Christopher V

    2015-12-01

    The development and application of ribosome profiling has markedly advanced our understanding of ribosomes and mRNA translation. The experimental approach, which relies on deep sequencing of ribosome-protected mRNA fragments generated by treatment of polyribosomes with exogenous nucleases, provides a transcriptome-wide assessment of translation. The broad application of ribosome profiling has been slowed by the complexity and expense of the protocol. Here, we provide a simplified ribosome profiling method that uses micrococcal nuclease to generate ribosome footprints in crude cellular extracts, which are then purified simply by size selection via polyacrylamide gel electrophoresis. This simplification removes the laborious or expensive purification of ribosomes that has typically been used. This direct extraction method generates gene-level ribosome profiling data that are similar to a method that includes ribosome purification. This protocol should significantly ease the barrier to entry for research groups interested in employing ribosome profiling. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. High interleukin-6 mRNA expression is a predictor of relapse in colon cancer

    DEFF Research Database (Denmark)

    Olsen, Jesper; Kirkeby, Lene T.; Olsen, Jørgen

    2015-01-01

    Aim: To investigate the expression of interleukin-6 (IL6) in colon cancer tissue, and to examine if the risk of relapse is influenced by IL6 expression. Materials and Methods: Fresh-frozen biopsies from tumor and normal adjacent tissues were taken from patients with colon cancer during surgery...... and stored at −80°C. mRNA expression for interleukin-6 was evaluated with reverse transcription real time quantitative polymerase chain reaction. Survival analyses were carried-out using a Cox competing risk regression model. Results: IL6 mRNA was significantly more highly expressed in tumor tissue compared...... for clinicopathological characteristics (Hazard Ratio=2.16, 95% CI=1.07-4.40; pInterleukin-6 is up-regulated in colon cancer tissue at the transcriptional level and is significantly associated with increased risk of relapse....

  12. High interleukin-6 mRNA expression is a predictor of relapse in colon cancer

    DEFF Research Database (Denmark)

    Olsen, Jesper; Kirkeby, Lene T; Olsen, Jørgen

    2015-01-01

    AIM: To investigate the expression of interleukin-6 (IL6) in colon cancer tissue, and to examine if the risk of relapse is influenced by IL6 expression. MATERIALS AND METHODS: Fresh-frozen biopsies from tumor and normal adjacent tissues were taken from patients with colon cancer during surgery...... and stored at -80 °C. mRNA expression for interleukin-6 was evaluated with reverse transcription real time quantitative polymerase chain reaction. Survival analyses were carried-out using a Cox competing risk regression model. RESULTS: IL6 mRNA was significantly more highly expressed in tumor tissue compared...... for clinicopathological characteristics (Hazard Ratio=2.16, 95% CI=1.07-4.40; pInterleukin-6 is up-regulated in colon cancer tissue at the transcriptional level and is significantly associated with increased risk of relapse....

  13. Long Open Reading Frame Transcripts Escape Nonsense-Mediated mRNA Decay in Yeast

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    Laurence Decourty

    2014-02-01

    Full Text Available Nonsense-mediated mRNA decay (NMD destabilizes eukaryotic transcripts with long 3′ UTRs. To investigate whether other transcript features affect NMD, we generated yeast strains expressing chromosomal-derived mRNAs with 979 different promoter and open reading frame (ORF regions and with the same long, destabilizing 3′ UTR. We developed a barcode-based DNA microarray strategy to compare the levels of each reporter mRNA in strains with or without active NMD. The size of the coding region had a significant negative effect on NMD efficiency. This effect was not specific to the tested 3′ UTR because two other different NMD reporters became less sensitive to NMD when ORF length was increased. Inefficient NMD was not due to a lack of association of Upf1 to long ORF transcripts. In conclusion, in addition to a long 3′ UTR, short translation length is an important feature of NMD substrates in yeast.

  14. Two tandem RNase III cleavage sites determine betT mRNA stability in response to osmotic stress in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Minji Sim

    Full Text Available While identifying genes regulated by ribonuclease III (RNase III in Escherichia coli, we observed that steady-state levels of betT mRNA, which encodes a transporter mediating the influx of choline, are dependent on cellular concentrations of RNase III. In the present study, we also observed that steady-state levels of betT mRNA are dependent on RNase III activity upon exposure to osmotic stress, indicating the presence of cis-acting elements controlled by RNase III in betT mRNA. Primer extension analyses of betT mRNA revealed two tandem RNase III cleavage sites in its stem-loop region, which were biochemically confirmed via in vitro cleavage assays. Analyses of cleavage sites suggested the stochastic selection of cleavage sites by RNase III, and mutational analyses indicated that RNase III cleavage at either site individually is insufficient for efficient betT mRNA degradation. In addition, both the half-life and abundance of betT mRNA were significantly increased in association with decreased RNase III activity under hyper-osmotic stress conditions. Our findings demonstrate that betT mRNA stability is controlled by RNase III at the post-transcriptional level under conditions of osmotic stress.

  15. Adaptive effects of the beta2-agonist clenbuterol on expression of beta2-adrenoceptor mRNA in rat fast-twitch fiber-rich muscles.

    Science.gov (United States)

    Sato, Shogo; Nomura, Sachiko; Kawano, Fuuun; Tanihata, Jun; Tachiyashiki, Kaoru; Imaizumi, Kazuhiko

    2010-03-01

    Administration of the beta(2)-agonist clenbuterol has been shown to reduce the expression of beta(2)-adrenoceptor (AR) mRNA in fast-twitch fiber-rich (extensor digitorum longus, EDL) muscle without changing that in slow-twitch fiber-rich (soleus, SOL) muscle in rats. However, the regulatory mechanism for muscle fiber type-dependent down-regulation of the expression of beta(2)-AR mRNA induced by clenbuterol is still unclear. Therefore, mRNA expression of transcriptional and post-transcriptional regulatory factors for beta(2)-AR mRNA levels in fast-twitch fiber-rich (EDL and plantaris, PLA) and slow-twitch fiber-rich (SOL) muscles in clenbuterol-administered (1.0 mg/kg body weight/day for 10 days, subcutaneous) rats was studied by real-time reverse transcription-polymerase chain reaction. Administration of clenbuterol significantly reduced expression of beta(2)-AR mRNA in EDL and PLA muscles without changing that in SOL muscle. Administration of clenbuterol also significantly reduced the mRNA expression of transcriptional regulatory factor (glucocorticoid receptor) and mRNA stabilizing factor (Hu antigen R) in EDL and PLA muscles without changing those in SOL muscle. These results suggest that muscle fiber type-dependent effects of clenbuterol on expression of beta(2)-AR mRNA are closely related to the down-regulation of mRNA expression of transcriptional and post-transcriptional regulatory factors for beta(2)-AR mRNA levels.

  16. Complexity on Acute Myeloid Leukemia mRNA Transcript Variant

    Directory of Open Access Journals (Sweden)

    Carlo Cattani

    2011-01-01

    Full Text Available This paper deals with the sequence analysis of acute myeloid leukemia mRNA. Six transcript variants of mlf1 mRNA, with more than 2000 bps, are analyzed by focusing on the autocorrelation of each distribution. Through the correlation matrix, some patches and similarities are singled out and commented, with respect to similar distributions. The comparison of Kolmogorov fractal dimension will be also given in order to classify the six variants. The existence of a fractal shape, patterns, and symmetries are discussed as well.

  17. Effect of glutamine on the mRNA level of key enzymes of malate-aspartate shuttle in the rat intestine subjected to ischemia reperfusion Efeito da glutamina sobre o nível de RNA Mensageiro das enzimas-chave do ciclo malato-aspartato no intestino de ratos submetidos à isquemia e reperfusão

    Directory of Open Access Journals (Sweden)

    Paulo Roberto Cavalcante de Vasconcelos

    2011-01-01

    Full Text Available PURPOSE: To determine the effects of oral L-glutamine (L-Gln and the dipeptide l-alanyl-glutamine (L-Ala-Gln upon the activity of the malate-aspartate shuttle in the rat distal small intestine following ischemia and reperfusion. METHODS: Seventy-two Wistar rats (350-400g, were randomized in 2 groups (n = 36: group S (Sham and Group T (Treatment and divided into 12 subgroups (n = 6: A-A6, and B1-B6. The subgroups A1-A3 were subjected to sham procedures at 30 and 60 minutes. Thirty minutes before the study, rats were treated with calcium caseinate, 0.5g/Kg (subgroups A1, A4, B1, B4, L-Gln, 0.5g / kg (subgroups A2, A5, B2 and B5 or L-Ala-Gln, 0.75g/Kg (subgroups A3, A6, B3, B6, administered by gavage. Ischemia was achieved by clamping the mesenteric vessels, delimiting a segment of bowel 5 cm long and 5 cm apart from the ileocecal valve. Samples were collected 30 and 60 minutes after start of the study for real-time PCR assay of malate dehydrogenases (MDH1-2 and aspartate-aminotransferases (GOT1-2 enzymes. RESULTS: Tissue MDH and GOT mRNA expression in intestinal samples from rats preconditioned with either L-Gln or L-Ala-Gln showed no significant differences both during ischemia and early reperfusion. CONCLUSION: Activation of the malate-aspartate shuttle system appears not to be the mechanism of glutamine-mediated elevation of glucose oxidation in rat intestine during ischemia/reperfusion injury.OBJETIVO: Determinar os efeitos da administração oral de L-glutamina (L-Gln e do dipeptídeo L-alanil-glutamina (L-Ala-Gln sobre a atividade do ciclo malato-aspartato no intestino delgado distal de ratos após isquemia/reperfusão. MÉTODOS: Setenta e dois ratos Wistar (350-400g foram randomizados em 2 grupos (n = 36: T grupo S (Sham e grupo (Tratamento e distribuídos em 12 subgrupos (n = 6: A-A6, e B1-B6. Os subgrupos A1-A3 foram submetidos a procedimentos "sham" aos 30 e 60 minutos. Trinta minutos antes do estudo, os ratos foram tratados com

  18. Reduced expression of androgen receptor and myosin heavy chain mRNA in cremaster muscle of boys with nonsyndromic cryptorchidism.

    Science.gov (United States)

    Barthold, Julia Spencer; Wang, Yanping; Reilly, Anita; Robbins, Alan; Figueroa, T Ernesto; Banihani, Ahmad; Hagerty, Jennifer; Akins, Robert E

    2012-10-01

    To better define the developmental mechanisms of nonsyndromic cryptorchidism, we measured the expression of hormone receptor and muscle type specific mRNAs in target tissues of boys with and those without nonsyndromic cryptorchidism. Prospectively collected cremaster muscle and/or hernia sac tissues from boys with congenital (79) or acquired (66) nonsyndromic cryptorchidism and hernia/hydrocele (controls, 84) were analyzed for hormone receptor (RXFP2, AR, ESR1, ESR2) and myosin heavy chain specific (MYH1, MYH2, MYH7) mRNA expression using real-time reverse transcriptase polymerase chain reaction. Log transformed mRNA, phenotype and feeding history data were statistically analyzed using Pearson's correlation, ANOVA and 2-sample t tests. AR mRNA expression was higher in cremaster muscle than in sac tissue, and significantly lower in congenital and acquired nonsyndromic cryptorchidism cases vs controls (p MYH7 mRNA expression was also significantly reduced in both nonsyndromic cryptorchidism groups (p ≤ 0.002), while a reduction in type 2 (fast) MYH2 expression was more modest and significant only for the congenital cryptorchidism group (p MYH7 and AR levels were strongly correlated (r(2) = 0.751, p MYH7 and ESR1 mRNA levels were higher and lower, respectively, in boys with nonsyndromic cryptorchidism who were fed soy formula. Expression of other genes was not measurable. Our data suggest that boys with congenital and acquired nonsyndromic cryptorchidism differentially express AR and slow twitch specific MYH7 mRNA in the cremaster muscle, and that MYH7 expression is correlated with AR levels and soy formula use. These differences in gene expression may reflect aberrant hormonal signaling and/or innervation during development with the potential for secondary functional effects and failed testicular descent. Copyright © 2012 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    David G Hendrickson

    2009-11-01

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

  20. Xplore mRNA assays for the quantification of IL-1 beta and TNF-alpha mRNA in lipopolysaccharide-induced mouse macrophages

    National Research Council Canada - National Science Library

    Van Arsdell, S W; Murphy, K P; Pazmany, C; Erickson, D; Burns, C; Moody, M D

    2000-01-01

    Because the accurate measurement of a number of cytokine mRNA transcripts provides valuable knowledge about cytokine gene regulation, we have developed the Xplore assay for the quantification of cytokine mRNA...

  1. Association between VDAC1 mRNA expression and intracellular ...

    African Journals Online (AJOL)

    One way in which xenobiotics induce apoptotic cell death is to alter the selective permeability of the intracellular voltage-dependent anion channel (VDAC1) in the mitochondrial membrane. In this study, we explored the association between VDAC1 mRNA expression and mitochondrial function during hexavalent chromium ...

  2. Cytokine mRNA expression during experimental corneal allograft rejection

    NARCIS (Netherlands)

    Torres, P. F.; de Vos, A. F.; van der Gaag, R.; Martins, B.; Kijlstra, A.

    1996-01-01

    Allograft rejection is the main cause of corneal graft failure. T lymphocytes and macrophages have been implied to be involved in corneal rejection, but little is known about the molecular mechanism in this process. In this study, cytokine mRNA expression in the cornea was analysed during

  3. Human mRNA response to exercise and temperature.

    Science.gov (United States)

    Slivka, D R; Dumke, C L; Tucker, T J; Cuddy, J S; Ruby, B

    2012-02-01

    The purpose of this research was to determine the mRNA response to exercise in different environmental temperatures. 9 recreationally active males (27±1 years, 77.4±2.7  kg, 13.5±1.5% fat, 4.49±0.15  L · min (-1) VO2 max) completed 3 trials consisting of 1 h cycling exercise at 60% Wmax followed by a 3 h recovery in the cold (7°C), room temperature (20°C), and hot (33°C) environments. Muscle biopsies were obtained pre, post, and 3 h post exercise for the analysis of glycogen and mRNA. Expired gases were collected to calculate substrate use. PGC-1α increased to a greater degree in the cold trial than in the room temperature trial (p=0.036) and the hot trial (p=0.006). PGC1-α mRNA was also higher after the room temperature trial than the hot trial (p=0.050). UCP3 and MFN2 mRNA increased with exercise (pcold than exercise in the heat. However, VO2 was higher during recovery in the cold trial than in the room temperature and hot trials (p<0.05). This study presents evidence of PGC-1α temperature sensitivity in human skeletal muscle. © Georg Thieme Verlag KG Stuttgart · New York.

  4. Cloning and mRNA expression pattern analysis under low ...

    African Journals Online (AJOL)

    This research cloned endochitinase-antifreeze protein precursor (EAPP) gene of Dong-mu 70 rye (Secale cereale) by designing special primers according to Genbank's EAPP gene sequence, and analyzing the influence of low temperature stress on the expression of mRNA with RT-PCR. The results indicated that the ...

  5. Cloning and mRNA expression pattern analysis under low ...

    African Journals Online (AJOL)

    Jane

    2011-07-13

    Jul 13, 2011 ... This research cloned endochitinase-antifreeze protein precursor (EAPP) gene of Dong-mu 70 rye. (Secale cereale) by designing special primers according to Genbank's EAPP gene sequence, and analyzing the influence of low temperature stress on the expression of mRNA with RT-PCR. The results.

  6. MRNA-based skin identification for forensic applications

    NARCIS (Netherlands)

    M. Visser (Mijke); D. Zubakov (Dmitry); K. Ballantyne (Kaye); M.H. Kayser (Manfred)

    2011-01-01

    textabstractAlthough the identification of human skin cells is of important relevance in many forensic cases, there is currently no reliable method available. Here, we present a highly specific and sensitive messenger RNA (mRNA) approach for skin identification, meeting the key requirements in

  7. The mRNA Expression Status of Dopamine Receptor D2, Dopamine Receptor D3 and DARPP-32 in T Lymphocytes of Patients with Early Psychosis

    Directory of Open Access Journals (Sweden)

    Yin Cui

    2015-11-01

    Full Text Available Peripheral blood lymphocytes are an attractive tool because there is accumulating evidence indicating that lymphocytes may be utilized as a biomarker in the field of psychiatric study as they could reveal the condition of cells distributed in the brain. Here, we measured the mRNA expression status of dopamine receptor D2 (DRD2, DRD3, and dopamine and cyclic adenosine 3′,5′-monophosphate regulated phosphoprotein-32 (DARPP-32 in T lymphocytes of patients with early psychosis by quantitative real-time polymerase chain reaction (q-PCR and explored the relationships between their mRNA levels and the psychopathological status of patients. The present study demonstrated that the mRNA expression levels of DRD3 in T lymphocytes were significantly different among controls, and in patients with psychotic disorder not otherwise specified (NOS and schizophrenia/schizophreniform disorder. However, no significant differences in mRNA expression levels of DRD2 and DARPP-32 were found among the three groups. We found a significant positive correlation between the DRD2 mRNA level and the score of the excited factor of the Positive and Negative Syndrome Scale (PANSS in patients with schizophrenia/schizophreniform disorder. These findings suggest that DRD3 mRNA levels may serve as a potential diagnostic biomarker differentiating patients with early psychosis from controls.

  8. Decreases in mRNA by an siRNA Complex Do not Alter Blood Pressure in Mice

    Directory of Open Access Journals (Sweden)

    Su-Min Ji

    2012-03-01

    Full Text Available Recent genomewide association studies of large samples have identified genes that are associated with blood pressure. The Global Blood Pressure Genetics (Global BPgen and Cohorts for Heart and Aging Research in Genome Epidemiology (CHARGE consortiums identified 14 loci that govern blood pressure on a genomewide significance level, one of which is CASZ1 confirmed in both Europeans and Asians. CASZ1 is a zinc finger transcription factor that controls apoptosis and cell fate and suppresses neuroblastoma tumor growth by reprogramming gene expression, like a tumor suppressor. To validate the function of CASZ1 in blood pressure, we decreased Casz1 mRNA levels in mice by siRNA. Casz1 siRNA reduced mRNA levels by 59% in a mouse cell line. A polyethylenimine-mixed siRNA complex was injected into mouse tail veins, reducing Casz1 mRNA expression to 45% in the kidney. However, blood pressure in the treated mice was unaffected, despite a 55% reduction in Casz1 mRNA levels in the kidney on multiple siRNA injections daily. Even though Casz1 siRNA-treated mice did not experience any significant change in blood pressure, our study demonstrates the value of in vivo siRNA injection in analyzing the function of candidate genes identified by genomewide association studies.

  9. Regulation of adeno-associated virus gene expression in 293 cells: control of mRNA abundance and translation

    Energy Technology Data Exchange (ETDEWEB)

    Trempe, J.P.; Carter, B.J.

    1988-01-01

    The authors studied the effects of the adeno-associated virus (AAV) rep gene on the control of gene expression from the AAV p/sub 40/ promoter in 293 cells in the absence of an adenovirus coinfection. AAV vectors containing the chloramphenicol acetyltransferase (cat) gene were used to measure the levels of cat expression and steady-state mRNA from p/sub 40/. When the rep gene was present in cis or in trans, cat expression from p/sub 40/ was decreased 3- to 10-fold, but there was a 2- to 10-fold increase in the level of p/sub 40/ mRNA. Conversely, cat expression increased and the p/sub 40/ mRNA level decreased in the absence of the rep gene. Both wild-type and carboxyl-terminal truncated Rep proteins were capable of eliciting both effects. These data suggest two roles for the pleiotropic AAV rep gene: as a translational inhibitor and as a positive regulator of p/sub 40/ mRNA levels. They also provide additional evidence for a cis-acting negative regulatory region which decreases RNA from the AAV p/sub 5/ promoter in a fashion independent of rep.

  10. Suppression of sterol 27-hydroxylase mRNA and transcriptional activity by bile acids in cultured rat hepatocytes

    NARCIS (Netherlands)

    Twisk, J.; Wit, E.C.M. de; Princen, H.M.G.

    1995-01-01

    In previous work we have demonstrated suppression of cholesterol 7α-hydroxylase by bile acids at the level of mRNA and transcription, resulting in a similar decline in bile acid synthesis in cultured rat hepatocytes. In view of the substantial contribution of the 'alternative' or '27-hydroxylase'