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Sample records for abcb1 mrna expression

  1. Drug resistance to paclitaxel is not only associated with ABCB1 mRNA expression but also with drug accumulation in intracellular compartments in human lung cancer cell lines.

    PubMed

    Shimomura, Masanori; Yaoi, Takeshi; Itoh, Kyoko; Kato, Daishiro; Terauchi, Kunihiko; Shimada, Junichi; Fushiki, Shinji

    2012-04-01

    In order to clarify the mechanisms of resistance to paclitaxel in lung cancer, three human lung cancer cell lines which exhibit different sensitivity to paclitaxel were investigated from the following viewpoints: overexpression of ATP-binding cassette, sub-family B, member 1 (ABCB1), mutations on paclitaxel binding site of β-tubulin genes, quantity of polymerized tubulin and the intracellular localization of paclitaxel. ABCB1 expression was evaluated by real-time RT-PCR. No correlations were noted between the ABCB1 expression in the sensitive and resistant cell lines at the mRNA level. No mutations on the paclitaxel binding site of the β-tubulin genes were detected in either the resistant or sensitive cells. Live cell images obtained by confocal laser microscopy revealed that the resistant cell line, RERF-LC-KJ, had more accumulation of Oregon Green® 488 conjugated paclitaxel in the lysosomal and extra-lysosomal compartments of cytoplasm than other cell lines. The results obtained in this study indicated that the changes in the subcellular localization could contribute to the production of paclitaxel resistance in lung cancer cell lines. Further studies should be conducted to elucidate the molecular mechanisms that differentiate the intracellular localization of paclitaxel. PMID:22179563

  2. Drug resistance to paclitaxel is not only associated with ABCB1 mRNA expression but also with drug accumulation in intracellular compartments in human lung cancer cell lines

    PubMed Central

    SHIMOMURA, MASANORI; YAOI, TAKESHI; ITOH, KYOKO; KATO, DAISHIRO; TERAUCHI, KUNIHIKO; SHIMADA, JUNICHI; FUSHIKI, SHINJI

    2012-01-01

    In order to clarify the mechanisms of resistance to paclitaxel in lung cancer, three human lung cancer cell lines which exhibit different sensitivity to paclitaxel were investigated from the following viewpoints: overexpression of ATP-binding cassette, sub-family B, member 1 (ABCB1), mutations on paclitaxel binding site of β-tubulin genes, quantity of polymerized tubulin and the intracellular localization of paclitaxel. ABCB1 expression was evaluated by real-time RT-PCR. No correlations were noted between the ABCB1 expression in the sensitive and resistant cell lines at the mRNA level. No mutations on the paclitaxel binding site of the β-tubulin genes were detected in either the resistant or sensitive cells. Live cell images obtained by confocal laser microscopy revealed that the resistant cell line, RERF-LC-KJ, had more accumulation of Oregon Green® 488 conjugated paclitaxel in the lysosomal and extra-lysosomal compartments of cytoplasm than other cell lines. The results obtained in this study indicated that the changes in the subcellular localization could contribute to the production of paclitaxel resistance in lung cancer cell lines. Further studies should be conducted to elucidate the molecular mechanisms that differentiate the intracellular localization of paclitaxel. PMID:22179563

  3. Effects of rifampicin, dexamethasone, St. John's Wort and Thyroxine on maternal and foetal expression of Abcb1 and organ distribution of talinolol in pregnant rats.

    PubMed

    Saljé, Karen; Lederer, Kirstin; Oswald, Stefan; Dazert, Eike; Warzok, Rolf; Siegmund, Werner

    2012-08-01

    It is well accepted that ABCB1 plays a critical role in absorption, distribution and elimination of many xenobiotics and drugs. Only little is known about the regulation and function of ABCB1 during pregnancy. Thus, the aim of this study is to investigate maternal, placental and foetal Abcb1 expression and function in pregnant rats after induction with rifampicin, dexamethasone, St. John's wort (SJW) or thyroxine. Wistar rats were orally treated with rifampicin (250 mg/kg), SJW (1.0 g/kg), thyroxine (9 μg/kg), dexamethasone (1 mg/kg) or 0.5% methylcellulose suspension (control) for 9 days during late pregnancy (each N = 5). Afterwards, organ mRNA expression and protein content of Abcb1a were determined. Tissue concentrations of the ABCB1 probe drug talinolol were measured after repeated administration of the drug (100 mg/kg, 9 days) and after induction with oral rifampicin (250 mg/kg, 9 days, N = 5). Abcb1 expression was substantially lower in foetal than in maternal organs. Abcb1 was significantly induced by SJW in the maternal jejunum and placenta, by dexamethasone in foetal brain and liver and by thyroxine in the placenta and maternal and foetal brain. Rifampicin induced Abcb1 in all maternal and foetal organs. However, organ distribution of talinolol was not influenced by comedication of rifampicin. In conclusion, maternal and foetal Abcb1 organ expression in pregnant rats is inducible by nuclear receptor agonists. Although rifampicin regulates maternal and foetal Abcb1 expression, organ distribution of talinolol remains unchanged most likely caused by the known inhibitory effect of rifampicin on Abcb1 function.

  4. Paclitaxel sensitivity in relation to ABCB1 expression, efflux and single nucleotide polymorphisms in ovarian cancer

    PubMed Central

    Gao, Bo; Russell, Amanda; Beesley, Jonathan; Chen, Xiao Qing; Healey, Sue; Henderson, Michelle; Wong, Mark; Emmanuel, Catherine; Galletta, Laura; Johnatty, Sharon E.; Bowtell, David; Bowtell, David; Chenevix-Trench, Georgia; deFazio, Anna; Gertig, Dorota; Green, Adle; Webb, Penelope; Hung, Jillian; Moore, Sue; Traficante, Nadia; Fereday, Sian; Harrap, Karen; Sadkowsky, Troy; Pandeya, Nirmala; Stuart-Harris, Robin; Kirsten, Fred; Rutovitz, Josie; Clingan, Peter; Glasgow, Amanda; Proietto, Anthony; Braye, Stephen; Otton, Greg; Shannon, Jennifer; Bonaventura, Tony; Stewart, James; Begbie, Stephen; Friedlander, Michael; Bell, David; Baron-Hay, Sally; Ferrier, Alan; Gard, Greg; Nevell, David; Pavlakis, Nick; Valmadre, Sue; Young, Barbara; Camaris, Catherine; Crouch, Roger; Edwards, Lyndal; Hacker, Neville; Marsden, Donald; Robertson, Greg; Beale, Phillip; Beith, Jane; Carter, Jonothan; Dalrymple, Chris; Hamilton, Anne; Houghton, Roger; Russell, Peter; Links, Matthew; Grygiel, John; Hill, Jane; Brand, Alison; Byth, Karen; Jaworski, Richard; Harnett, Paul; Sharma, Raghwa; Achen, Anita; Wain, Gerard; Ward, Bruce; Papadimos, David; Crandon, Alex; Cummings, Margaret; Horwood, Ken; Obermair, Andreas; Perrin, Lew; Wyld, David; Nicklin, Jim; Davy, Margaret; Oehler, Martin K; Hall, Chris; Dodd, Tom; Healy, Tabitha; Pittman, Ken; Henderson, Doug; Miller, John; Pierdes, John; Blomfield, Penny; Challis, David; McIntosh, Robert; Parker, Andrew; Brown, Bob; Rome, Robert; Allen, David; Grant, Peter; Hyde, Simon; Laurie, Rohan; Robbie, Melissa; Healy, David; Jobling, Tom; Manolitsas, Tom; McNealage, Jane; Rogers, Peter; Susil, Beatrice; Sumithran, Eric; Simpson, Ian; Phillips, Kelly; Rischin, Danny; Fox, Stephen; Johnson, Daryl; Waring, Paul; Lade, Stephen; Loughrey, Maurice; O’Callaghan, Neil; Murray, William; Billson, Virginia; Pyman, Jan; Neesham, Debra; Quinn, Michael; Underhill, Craig; Bell, Richard; Ng, Leong-Fook; Blum, Robert; Ganju, Vinod; Hammond, Ian; Leung, Yee; McCartney, Anthony; Buck, Martin; Haviv, Izak; Purdie, David; Whiteman, David; Zeps, Nikolajs; Malt, Mary-Rose; Mellon, Anne; Robertson, Randall; Bergh, Trish Vanden; Jones, Marian; Mackenzie, Patricia; Maidens, Jane; Nattress, Kath; Chiew, Yoke-Eng; Stenlake, Annie; Sullivan, Helen; Alexander, Barbara; Ashover, Pat; Brown, Sue; Corrish, Tracy; Green, Lyn; Jackman, Leah; Ferguson, Kaltin; Martin, Karen; Martyn, Adam; Ranieri, Barbara; White, Jo; Jayde, Victoria; Bowes, Leanne; Mamers, Pamela; Galletta, Laura; Giles, Debra; Hendley, Joy; Alsop, Katherine; Schmidt, Trudy; Shirley, Helen; Ball, Colleen; Young, Cherry; Viduka, Suzanna; Tran, Hoa; Bilic, Sanela; Glavinas, Lydia; Brooks, Julia; Haber, Michelle; Norris, Murray; Harnett, Paul; Chenevix-Trench, Georgia; Balleine, Rosemary L.; deFazio, Anna

    2014-01-01

    ABCB1 (adenosine triphosphate-binding cassette transporter B1) mediates cellular elimination of many chemotherapeutic agents including paclitaxel, which is commonly used to treat ovarian cancer. A significant association between common single nucleotide polymorphisms (SNPs) in ABCB1 and progression-free survival has been reported in patients with ovarian cancer. Variable paclitaxel clearance due to genotype specific differences in ABCB1 activity in cancer cells and/or normal tissues may underlie the association. Using cell-based models, we evaluated the correlations between ABCB1 expression, polymorphisms, transporter activity and paclitaxel sensitivity in ovarian cancer (n = 10) and lymphoblastoid (n = 19) cell lines. Close associations between ABCB1 expression, transporter function and paclitaxel sensitivity were found in lymphoblastoid cell lines, although we could not demonstrate an association with common SNPs. In ovarian cancer cell lines, ABCB1 expression was low and the association between expression and function was lost. These results suggest that ABCB1 related survival difference in ovarian cancer patients is more likely to be due to differential whole body paclitaxel clearance mediated by normal cells rather than a direct effect on cancer cells. PMID:24810093

  5. Influence of a time-restricted feeding schedule on the daily rhythm of abcb1a gene expression and its function in rat intestine.

    PubMed

    Hayashi, Yohei; Ushijima, Kentarou; Ando, Hitoshi; Yanagihara, Hayato; Ishikawa, Eiko; Tsuruoka, Shu-Ichi; Sugimoto, Ko-Ichi; Fujimura, Akio

    2010-11-01

    P-glycoprotein (P-gp) is one of the ATP-binding cassette transporters and acts as an efflux pump for cytotoxic substances. P-gp mRNA expression and transporting activity show the daily rhythm and contribute to the chrono-pharmacokinetic profiles of many drugs. It is reported that the daily rhythm of abcb1a mRNA is regulated by a circadian clock-controlled output pathway. Time-restricted feeding is well known to shift the peripheral circadian phase of clock gene expression without changing the central clock function. This study was undertaken to examine the influence of a time-restricted feeding procedure during the light phase on the daily rhythms of abcb1a mRNA expression and P-gp activity. The abcb1a mRNA and P-gp activity showed a daily rhythm with a peak early in the dark phase in rat intestine under ad libitum feeding. Time-restricted feeding during the light phase shifted these rhythms to 12-h advance. The mRNA expression of clock genes (DBP and HLF, the transcript activators of abcb1a) also showed daily rhythms, and their phases were shifted by the time-restricted feeding procedure. The peak time of DBP mRNA expression was similar to that of abcb1a mRNA expression under ad libitum feeding and time-restricted feeding conditions. These results indicate that a time-restricted feeding procedure changes DBP mRNA expression, which in turn influences abcb1a mRNA expression and P-gp activity.

  6. Sex differences in cyclosporine pharmacokinetics and ABCB1 gene expression in mononuclear blood cells in African American and Caucasian renal transplant recipients.

    PubMed

    Tornatore, Kathleen M; Brazeau, Daniel; Dole, Kiran; Danison, Ryan; Wilding, Gregory; Leca, Nicolae; Gundroo, Aijaz; Gillis, Kathryn; Zack, Julia; DiFrancesco, Robin; Venuto, Rocco C

    2013-10-01

    Cyclosporine exhibits pharmacokinetic and pharmacodynamic variability in renal transplant recipients (RTR) attributed to P-glycoprotein (P-gp), an ABCB1 efflux transporter that influences bioavailability and intracellular distribution. Data on race and sex influences on P-gp in RTR are lacking. We investigated sex and race influences on cyclosporine pharmacokinetics and ABCB1 gene expression in peripheral blood mononuclear cells (PBMC). Fifty-four female and male African American and Caucasian stable RTR receiving cyclosporine and mycophenolic acid completed a 12-hour study. ABCB1 gene expression was assessed in PBMCs pre-dose and 4 hours after cyclosporine. Statistical analysis used mixed effects models on transformed, normalized ABCB1 expression and cyclosporine pharmacokinetics. Sex and race differences were observed for the dose-normalized area under the concentration curve (AUC0-12 /Dose) [P = .0004], apparent clearance [P = .0004] and clearance/body mass index (CL/BMI) [P = .027] with slowest clearance and greatest drug exposure in females. Sex and race differences were found pre-dose and 4 hours for ABCB1 [P < .0001] with females having less expression than males. ABCB1 differences were observed between pre-dose and 4 hours [P = .0009]. Female RTR had slower cyclosporine clearance and lower ABCB1 gene expression in PBMC suggesting reduced efflux activity and greater intracellular drug exposure.

  7. The Pim kinase inhibitor SGI-1776 decreases cell surface expression of P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) and drug transport by Pim-1-dependent and -independent mechanisms

    PubMed Central

    Natarajan, Karthika; Bhullar, Jasjeet; Shukla, Suneet; Burcu, Mehmet; Chen, Zhe-Sheng; Ambudkar, Suresh V.; Baer, Maria R.

    2013-01-01

    Overexpression of the ATP-binding cassette (ABC) drug efflux proteins P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) on malignant cells is associated with inferior chemotherapy outcomes. Both, ABCB1 and ABCG2, are substrates of the serine/threonine kinase Pim-1; Pim-1 knockdown decreases their cell surface expression, but SGI-1776, the first clinically tested Pim inhibitor, was shown to reverse drug resistance by directly inhibiting ABCB1-mediated transport. We sought to characterize Pim-1-dependent and -independent effects of SGI-1776 on drug resistance. SGI-1776 at the Pim-1-inhibitory and non-cytotoxic concentration of 1 μM decreased the IC50s of the ABCG2 and ABCB1 substrate drugs in cytotoxicity assays in resistant cells, with no effect on the IC50 of non-substrate drug, nor in parental cells. SGI-1776 also increased apoptosis of cells overexpressing ABCG2 or ABCB1 exposed to substrate chemotherapy drugs and decreased their colony formation in the presence of substrate, but not non-substrate, drugs, with no effect on parental cells. SGI-1776 decreased ABCB1 and ABCG2 surface expression on K562/ABCB1 and K562/ABCG2 cells, respectively, with Pim-1 overexpression, but not HL60/VCR and 8226/MR20 cells, with lower-level Pim-1 expression. Finally, SGI-1776 inhibited uptake of ABCG2 and ABCB1 substrates in a concentration-dependent manner irrespective of Pim-1 expression, inhibited ABCB1 and ABCG2 photoaffinity labeling with the transport substrate [125I]iodoarylazidoprazosin ([125I]IAAP) and stimulated ABCB1 and ABCG2 ATPase activity. Thus SGI-1776 decreases cell surface expression of ABCB1 and ABCG2 and inhibits drug transport by Pim-1-dependent and -independent mechanisms, respectively. Decrease in ABCB1 and ABCG2 cell surface expression mediated by Pim-1 inhibition represents a novel mechanism of chemosensitization. PMID:23261525

  8. Wnt pathway activation and ABCB1 expression account for attenuation of Proteasome inhibitor-mediated apoptosis in multidrug-resistant cancer cells

    PubMed Central

    Chong, Kowit Yu; Hsu, Chih-Jung; Hung, Tsai-Hsien; Hu, Han-Shu; Huang, Tsung-Teng; Wang, Tzu-Hao; Wang, Chihuei; Chen, Chuan-Mu; Choo, Kong Bung; Tseng, Ching-Ping

    2015-01-01

    Multiple drug resistance (MDR) is a major obstacle to attenuating the effectiveness of chemotherapy to many human malignancies. Proteasome inhibition induces apoptosis in a variety of cancer cells and is recognized as a novel anticancer therapy approach. Despite its success, some multiple myeloma patients are resistant or become refractory to ongoing treatment by bortezomib suggesting that chemoresistant cancer cells may have developed a novel mechanism directed against the proteasome inhibitor. The present study aimed to investigate potential mechanism(s) of attenuation in a MDR cell line, MES-SA/Dx5. We found that compared to the parental human uterus sarcoma cell line MES-SA cells, MES-SA/Dx5 cells highly expressed the ABCB1 was more resistant to MG132 and bortezomib, escaping the proteasome inhibitor-induced apoptosis pathway. The resistance was reversed by co-treatment of MG132 and the ABCB1 inhibitor verapamil. The data indicated that ABCB1 might play a role in the efflux of MG132 from the MES-SA/Dx5 cells to reduce MG132-induced apoptosis. Furthermore, the canonical Wnt pathway was found activated only in the MES-SA/Dx5 cells through active β-catenin and related transactivation activities. Western blot analysis demonstrated that Wnt-targeting genes, including c-Myc and cyclin D1, were upregulated and were relevant in inhibiting the expression of p21 in MES-SA/Dx5 cells. On the other hand, MES-SA cells expressed high levels of p21 and downregulated cyclin D1 and caused cell cycle arrest. Together, our study demonstrated the existence and participation of ABCB1 and the Wnt pathway in an MDR cell line that attenuated proteasome inhibitor-induced apoptosis. PMID:25590413

  9. Regulation of expression of ABCB1 and LRP genes by mitogen-activated protein kinase/extracellular signal-regulated kinase pathway and its role in generation of side population cells in canine lymphoma cell lines.

    PubMed

    Tomiyasu, Hirotaka; Watanabe, Manabu; Goto-Koshino, Yuko; Fujino, Yasuhito; Ohno, Koichi; Sugano, Sumio; Tsujimoto, Hajime

    2013-06-01

    The concept of the cancer stem cell (CSC) has been recognized as key for elucidation of the mechanisms that confer the multidrug resistance (MDR) phenotype to tumor cells, and the side population (SP) fraction has been shown to be enriched by cells with the CSC phenotype. The purpose of the present study was to identify the mechanism that induces a difference of phenotype between the SP and the remaining major population (MP) using two canine lymphoma cell lines. Expression levels of ABCB1 and LRP genes, which encode efflux pumps, were significantly higher in the SP than in the MP. Microarray analysis revealed up-regulation of the expression of transforming growth factor-β (TGF-β) type II receptor in SP compared with MP, and the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway was more up-regulated in the SP than in the MP. Stimulation of the MAPK/ERK pathway significantly increased the mRNA expression of both ABCB1 and LRP genes. These results indicate increased expression of the efflux pumps through the MAPK/ERK pathway in SP cells.

  10. Wallichinine reverses ABCB1-mediated cancer multidrug resistance.

    PubMed

    Lv, Min; Qiu, Jian-Ge; Zhang, Wen-Ji; Jiang, Qi-Wei; Qin, Wu-Ming; Yang, Yang; Zheng, Di-Wei; Chen, Yao; Huang, Jia-Rong; Wang, Kun; Wei, Meng-Ning; Cheng, Ke-Jun; Shi, Zhi

    2016-01-01

    Overexpression of ABCB1 in cancer cells is one of the main reasons of cancer multidrug resistance (MDR). Wallichinine is a compound isolated from piper wallichii and works as an antagonist of platelet activiating factor receptor to inhibit the gathering of blood platelet. In this study, we investigate the effect of wallichinine on cancer MDR mediated by ABCB1 transporter. Wallichinine significantly potentiates the effects of two ABCB1 substrates vincristine and doxorubicin on inhibition of growth, arrest of cell cycle and induction of apoptosis in ABCB1 overexpressing cancer cells. Furthermore, wallichinine do not alter the sensitivity of non-ABCB1 substrate cisplatin. Mechanistically, wallichinine blocks the drug-efflux activity of ABCB1 to increase the intracellular accumulation of rhodamine 123 and doxorubicin and stimulates the ATPase of ABCB1 without alteration of the expression of ABCB1. The predicted binding mode shows the hydrophobic interactions of wallichinine within the large drug binding cavity of ABCB1. At all, our study of the interaction of wallichinine with ABCB1 presented herein provides valuable clues for the development of novel MDR reversal reagents from natural products. PMID:27508017

  11. Wallichinine reverses ABCB1-mediated cancer multidrug resistance

    PubMed Central

    Lv, Min; Qiu, Jian-Ge; Zhang, Wen-Ji; Jiang, Qi-Wei; Qin, Wu-Ming; Yang, Yang; Zheng, Di-Wei; Chen, Yao; Huang, Jia-Rong; Wang, Kun; Wei, Meng-Ning; Cheng, Ke-Jun; Shi, Zhi

    2016-01-01

    Overexpression of ABCB1 in cancer cells is one of the main reasons of cancer multidrug resistance (MDR). Wallichinine is a compound isolated from piper wallichii and works as an antagonist of platelet activiating factor receptor to inhibit the gathering of blood platelet. In this study, we investigate the effect of wallichinine on cancer MDR mediated by ABCB1 transporter. Wallichinine significantly potentiates the effects of two ABCB1 substrates vincristine and doxorubicin on inhibition of growth, arrest of cell cycle and induction of apoptosis in ABCB1 overexpressing cancer cells. Furthermore, wallichinine do not alter the sensitivity of non-ABCB1 substrate cisplatin. Mechanistically, wallichinine blocks the drug-efflux activity of ABCB1 to increase the intracellular accumulation of rhodamine 123 and doxorubicin and stimulates the ATPase of ABCB1 without alteration of the expression of ABCB1. The predicted binding mode shows the hydrophobic interactions of wallichinine within the large drug binding cavity of ABCB1. At all, our study of the interaction of wallichinine with ABCB1 presented herein provides valuable clues for the development of novel MDR reversal reagents from natural products. PMID:27508017

  12. Wnt5A regulates ABCB1 expression in multidrug-resistant cancer cells through activation of the non-canonical PKA/β-catenin pathway

    PubMed Central

    Hung, Tsai-Hsien; Hsu, Sheng-Chi; Cheng, Ching-Yi; Choo, Kong-Bung; Tseng, Ching-Ping; Chen, Tse-Ching; Lan, Ying-Wei; Huang, Tsung-Teng; Lai, Hsin-Chih; Chen, Chuan-Mu; Chong, Kowit-Yu

    2014-01-01

    Multidrug resistance in cancer cells arises from altered drug permeability of the cell. We previously reported activation of the Wnt pathway in ABCB1-overexpressed human uterus sarcoma drug-resistant MES-SA/Dx5 cells through active β-catenin and associated transactivation activities, and upregulation of Wnt-targeting genes. In this study, Wnt5A was found to be significantly upregulated in MES-SA/Dx5 and MCF7/ADR2 cells, suggesting an important role for the Wnt5A signaling pathway in cancer drug resistance. Higher cAMP response elements and Tcf/Lef transcription activities were shown in the drug-resistant cancer cells. However, expression of Wnt target genes and CRE activities was downregulated in Wnt5A shRNA stably-transfected MES-SA/Dx5 cells. Cell viability of the drug-resistant cancer cells was also reduced by doxorubicin treatment and Wnt5A shRNA transfection, or by Wnt5A depletion. The in vitro data were supported by immunohistochemical analysis of 24 paired breast cancer biopsies obtained pre- and post-chemotherapeutic treatment. Wnt5A, VEGF and/or ABCB1 were significantly overexpressed after treatment, consistent with clinical chemoresistance. Taken together, the Wnt5A signaling pathway was shown to contribute to regulating the drug-resistance protein ABCB1 and β-catenin-related genes in antagonizing the toxic effects of doxorubicin in the MDR cell lines and in clinical breast cancer samples. PMID:25401518

  13. Nobiletin enhances the efficacy of chemotherapeutic agents in ABCB1 overexpression cancer cells.

    PubMed

    Ma, Wenzhe; Feng, Senling; Yao, Xiaojun; Yuan, Zhongwen; Liu, Liang; Xie, Ying

    2015-12-22

    Multidrug resistance (MDR) is the major obstacle to the successful chemotherapy treatment of many cancers. Here we found that nobiletin, a citrus methoxyflavone, significantly sensitized ABCB1 overexpressing cells A2780/T and A549/T to chemotherapeutic agents such as paclitaxel (a 433-fold reversal of MDR to PTX at 9 μM), doxorubicin (DOX), docetaxel and dounorubicin. Nobiletin profoundly inhibited ABCB1 transporter activity since it significantly increased the intracellular accumulation of DOX and Flutax-2 in A2780/T cells and decreased the efflux of ABCB1 substrates in Caco2 cells without altering the mRNA and protein expression of ABCB1. Moreover, nobiletin stimulated ATPase activity and inhibited verapamil-stimulated ATPase activity in a concentration-dependent manner, indicating a direct interaction with the transporter. Consistent with these findings, molecular docking analysis also identified favorable binding of nobiletin with the transmemberane region site 1 of homology modeled human ABCB1 transporter. Moreover, the Nrf2 protein expression and phosphorylation levels of AKT/ERK were suppressed by co-treated with nobiletin and PTX at the reversal concentrations, suggesting that inhibition of the AKT/ERK/Nrf2 pathway was associated with the sensitizing effect of nobiletin. These findings encourage further animal and clinical MDR studies with the combination therapy of nobiletin and chemotherapeutic drugs.

  14. Nobiletin enhances the efficacy of chemotherapeutic agents in ABCB1 overexpression cancer cells

    PubMed Central

    Ma, Wenzhe; Feng, Senling; Yao, Xiaojun; Yuan, Zhongwen; Liu, Liang; Xie, Ying

    2015-01-01

    Multidrug resistance (MDR) is the major obstacle to the successful chemotherapy treatment of many cancers. Here we found that nobiletin, a citrus methoxyflavone, significantly sensitized ABCB1 overexpressing cells A2780/T and A549/T to chemotherapeutic agents such as paclitaxel (a 433-fold reversal of MDR to PTX at 9 μM), doxorubicin (DOX), docetaxel and dounorubicin. Nobiletin profoundly inhibited ABCB1 transporter activity since it significantly increased the intracellular accumulation of DOX and Flutax-2 in A2780/T cells and decreased the efflux of ABCB1 substrates in Caco2 cells without altering the mRNA and protein expression of ABCB1. Moreover, nobiletin stimulated ATPase activity and inhibited verapamil-stimulated ATPase activity in a concentration-dependent manner, indicating a direct interaction with the transporter. Consistent with these findings, molecular docking analysis also identified favorable binding of nobiletin with the transmemberane region site 1 of homology modeled human ABCB1 transporter. Moreover, the Nrf2 protein expression and phosphorylation levels of AKT/ERK were suppressed by co-treated with nobiletin and PTX at the reversal concentrations, suggesting that inhibition of the AKT/ERK/Nrf2 pathway was associated with the sensitizing effect of nobiletin. These findings encourage further animal and clinical MDR studies with the combination therapy of nobiletin and chemotherapeutic drugs. PMID:26689156

  15. Trametinib modulates cancer multidrug resistance by targeting ABCB1 transporter

    PubMed Central

    Qiu, Jian-Ge; Zhang, Yao-Jun; Li, Yong; Zhao, Jin-Ming; Zhang, Wen-Ji; Jiang, Qi-Wei; Mei, Xiao-Long; Xue, You-Qiu; Qin, Wu-Ming; Yang, Yang; Zheng, Di-Wei; Chen, Yao; Wei, Meng-Ning; Shi, Zhi

    2015-01-01

    Overexpression of adenine triphosphate (ATP)-binding cassette (ABC) transporters is one of the main reasons of multidrug resistance (MDR) in cancer cells. Trametinib, a novel specific small-molecule mitogen-activated extracellular signal-regulated kinase (MEK) inhibitor, is currently used for the treatment of melanoma in clinic. In this study, we investigated the effect of trametinib on MDR mediated by ABC transporters. Trametinib significantly potentiated the effects of two ABCB1 substrates vincristine and doxorubicin on inhibition of growth, arrest of cell cycle and induction of apoptosis in cancer cells overexpressed ABCB1, but not ABCC1 and ABCG2. Furthermore, trametinib did not alter the sensitivity of non-ABCB1 substrate cisplatin. Mechanistically, trametinib potently blocked the drug-efflux activity of ABCB1 to increase the intracellular accumulation of rhodamine 123 and doxorubicin and stimulates the ATPase of ABCB1 without alteration of the expression of ABCB1. Importantly, trametinib remarkably enhanced the effect of vincristine against the xenografts of ABCB1-overexpressing cancer cells in nude mice. The predicted binding mode showed the hydrophobic interactions of trametinib within the large drug binding cavity of ABCB1. Consequently, our findings may have important implications for use of trametinib in combination therapy for cancer treatment. PMID:25915534

  16. Protein phosphatase complex PP5/PPP2R3C dephosphorylates P-glycoprotein/ABCB1 and down-regulates the expression and function.

    PubMed

    Katayama, Kazuhiro; Yamaguchi, Miho; Noguchi, Kohji; Sugimoto, Yoshikazu

    2014-04-01

    P-glycoprotein (P-gp)/ABCB1 is a key molecule of multidrug resistance in cancer. Protein phosphatase (PP) 2A, regulatory subunit B, gamma (PPP2R3C), which is a regulatory subunit of PP2A and PP5, was identified as a binding candidate to P-gp. Immunoprecipitation-western blotting revealed that PP5 and PPP2R3C were coprecipitated with P-gp, while PP2A was not. PP5/PPP2R3C dephosphorylated protein kinase A/protein kinase C-phosphorylation of P-gp. Knockdown of PP5 and/or PPP2R3C increased P-gp expression and lowered the sensitivity to vincristine and doxorubicin. Consequently, our results indicate that PP5/PPP2R3C negatively regulates P-gp expression and function.

  17. Human ABCB1 confers cells resistance to cytotoxic guanidine alkaloids from Pterogyne nitens.

    PubMed

    Satake, Kazuhiro; Tsukamoto, Megumi; Mitani, Yuji; Regasini, Luis Octavio; da Silva Bolzani, Vanderlan; Efferth, Thomas; Nakagawa, Hiroshi

    2015-01-01

    Multidrug resistance (MDR) caused by human ABCB1 (P-glycoprotein/MDR1) is one of the major obstacles in chemotherapy. To understand the mechanism of MDR by ABCB1 and circumvent the MDR, in the present study, we established human ABCB1-expressing cells (Flp-In-293/ABCB1 cells) and examined the cytotoxic effects of four guanidine alkaloids from Pterogyne nitens (galegine, nitensidine A, pterogynidine and pterogynine) using Flp-In-293/Mock and Flp-In-293/ABCB1 cells. The activity of ABCB1 in Flp-In-293/ABCB1 cells were confirmed by typical substrates for ABCB1 (taxol and vinblastine) in MTT assay. Flp-In-293/ABCB1 cells were also resistant to the four guanidine alkaloids as well as taxol and vinblastine compared to Flp-In-293/Mock cells although the four guanidine alkaloids exhibited cytotoxicity against the two Flp-In-293 cells. Furthermore, the four guanidine alkaloids were also found to stimulate the ATPase activity of ABCB1 in ATPase assays. These results suggest that ABCB1 can confer the resistance to the cytotoxic guanidine alkaloids by transporting them.

  18. Butorphanol, a synthetic opioid, sensitizes ABCB1-mediated multidrug resistance via inhibition of the efflux function of ABCB1 in leukemia cells.

    PubMed

    Wen, Jing; Zhang, Tao; Shan, Zhi-Ming; Qi, Min-Yue; Xiu, Huan-Huan; Liu, Lei; Wu, Shi-Zhe; Jia, Zhen; Xu, Kang-Qing

    2015-08-01

    Multidrug resistance (MDR) remains a formidable challenge in the use of chemotherapy and represents a powerful obstacle to the treatment of leukemia. ATP-binding cassette subfamily B member 1 (ABCB1) is a recognized factor which causes MDR and is closely related to poor outcome and relapse in leukemia. Ongoing research concerning the strategy for inhibiting the abnormally high activity of the ABCB1 transporter is critically needed. In the present study, we sought to elucidate the interaction between ABCB1 transporter and butorphanol. Our results showed that butorphanol significantly antagonized ABCB1-mediated drug efflux and increased the intracellular drug concentration by inhibiting the transport activity of ABCB1 in leukemia cells. Mechanistic investigations demonstrated that butorphanol did not alter the protein expression or localization of ABCB1 in HL60/VCR and K562/ADR cells. Furthermore, homology modeling indicated that butorphanol could fit into the large drug-binding cavity of ABCB1 and form a binding conformation. In conclusion, butorphanol reversed the ABCB1-mediated MDR in leukemia cells by directly suppressing the efflux activity of ABCB1. PMID:26062728

  19. Semi-synthetic ocotillol analogues as selective ABCB1-mediated drug resistance reversal agents

    PubMed Central

    Zhang, Guan-Nan; Wang, Yi-Jun; Kathawala, Rishil J.; Si, Rui; Patel, Bhargav A.; Xu, Jinyi; Chen, Zhe-Sheng

    2015-01-01

    Overexpression of ATP-Binding Cassette transporters leads to multidrug resistance in cancer cells and results in the failure of chemotherapy. In this in-vitro study, we investigated whether or not (20S, 24R/S)-epoxy-12β, 25-dihydroxy-dommarane-3β-amine (ORA and OSA), a pair of semi-synthetic ocotillol analogue epimers, could inhibit the ABCB1 transporter. ORA (1 μM and 3 μM) significantly reversed the resistance to paclitaxel and vincristine in ABCB1-overexpressing SW620/Ad300 and HEK/ABCB1 cells, whereas OSA had no significant effects. In addition, ORA (3 μM) significantly increased the intracellular accumulation of [3H]-paclitaxel by suppressing the efflux function of ABCB1. Meanwhile, both ORA (3 μM) and OSA (3 μM) did not significantly alter the expression level or the subcellular location of ABCB1 protein. Moreover, the ABCB1 ATPase study suggested that ORA had a stronger stimulatory effect on the ATPase activity than OSA. ORA also exhibited a higher docking score as compared with OSA inside transmembrane domain of ABCB1. Overall, we concluded that ORA reverse ABCB1-mediated MDR by competitively inhibiting the ABCB1 drug efflux function. PMID:26296969

  20. ABCB1 in children's brain tumours.

    PubMed

    Coyle, Beth; Kessler, Maya; Sabnis, Durgagauri H; Kerr, Ian D

    2015-10-01

    Tumours of the central nervous system are the most common solid tumour, accounting for a quarter of the 1500 cases of childhood cancer diagnosed each year in the U.K. They are the most common cause of cancer-related death in children. Treatment consists of surgery followed by adjuvant chemotherapy and/or radiotherapy. Survival rates have generally increased, but many survivors suffer from radiotherapy-related neurocognitive and endocrine side effects as well as an increased risk of secondary cancer. Adjuvant chemotherapy is normally given in combination to circumvent chemoresistance, but several studies have demonstrated it to be ineffective in the absence of radiotherapy. The identification of children with drug-resistant disease at the outset could allow stratification of those that are potentially curable by chemotherapy alone. Ultimately, however, what is required is a means to overcome this drug resistance and restore the effectiveness of chemotherapy. Medulloblastomas and ependymomas account for over 30% of paediatric brain tumours. Advances in neurosurgery, adjuvant radiotherapy and chemotherapy have led to improvements in 5-year overall survival rates. There remain, however, significant numbers of medulloblastoma patients that have intrinsically drug-resistant tumours and/or present with disseminated disease. Local relapse in ependymoma is also common and has an extremely poor prognosis with only 25% of children surviving first relapse. Each of these is consistent with the acquisition of drug and radiotherapy resistance. Since the majority of chemotherapy drugs currently used to treat these patients are transport substrates for ATP-binding cassette sub-family B member 1 (ABCB1) we will address the hypothesis that ABCB1 expression underlies this drug resistance. PMID:26517917

  1. Osimertinib (AZD9291), a Mutant-Selective EGFR Inhibitor, Reverses ABCB1-Mediated Drug Resistance in Cancer Cells.

    PubMed

    Zhang, Xiao-Yu; Zhang, Yun-Kai; Wang, Yi-Jun; Gupta, Pranav; Zeng, Leli; Xu, Megan; Wang, Xiu-Qi; Yang, Dong-Hua; Chen, Zhe-Sheng

    2016-01-01

    In recent years, tyrosine kinase inhibitors (TKIs) have been shown capable of inhibiting the ATP-binding cassette (ABC) transporter-mediated multidrug resistance (MDR). In this study, we determine whether osimertinib, a novel selective, irreversible EGFR (epidermal growth factor receptor) TKI, could reverse ABC transporter-mediated MDR. The results showed that, at non-toxic concentrations, osimertinib significantly sensitized both ABCB1-transfected and drug-selected cell lines to substrate anticancer drugs colchicine, paclitaxel, and vincristine. Osimertinib significantly increased the accumulation of [³H]-paclitaxel in ABCB1 overexpressing cells by blocking the efflux function of ABCB1 transporter. In contrast, no significant alteration in the expression levels and localization pattern of ABCB1 was observed when ABCB1 overexpressing cells were exposed to 0.3 µM osimertinib for 72 h. In addition, ATPase assay showed osimertinib stimulated ABCB1 ATPase activity. Molecular docking and molecular dynamic simulations showed osimertinib has strong and stable interactions at the transmembrane domain of human homology ABCB1. Taken together, our findings suggest that osimertinib, a clinically-approved third-generation EGFR TKI, can reverse ABCB1-mediated MDR, which supports the combination therapy with osimertinib and ABCB1 substrates may potentially be a novel therapeutic stategy in ABCB1-positive drug resistant cancers. PMID:27649127

  2. Epigenetic modulation of the drug resistance genes MGMT, ABCB1 and ABCG2 in glioblastoma multiforme

    PubMed Central

    2013-01-01

    Background Resistance of the highly aggressive glioblastoma multiforme (GBM) to drug therapy is a major clinical problem resulting in a poor patient’s prognosis. Beside promoter methylation of the O 6 -methylguanine-DNA-methyltransferase (MGMT) gene the efflux transporters ABCB1 and ABCG2 have been suggested as pivotal factors contributing to drug resistance, but the methylation of ABCB1 and ABCG2 has not been assessed before in GBM. Methods Therefore, we evaluated the proportion and prognostic significance of promoter methylation of MGMT, ABCB1 and ABCG2 in 64 GBM patient samples using pyrosequencing technology. Further, the single nucleotide polymorphisms MGMT C-56 T (rs16906252), ABCB1 C3435T (rs1045642) and ABCG2 C421A (rs2231142) were determined using the restriction fragment length polymorphism method (RFLP). To study a correlation between promoter methylation and gene expression, we analyzed MGMT, ABCB1 and ABCG2 expression in 20 glioblastoma and 7 non-neoplastic brain samples. Results Despite a significantly increased MGMT and ABCB1 promoter methylation in GBM tissue, multivariate regression analysis revealed no significant association between overall survival of glioblastoma patients and MGMT or ABCB1 promoter methylation. However, a significant negative correlation between promoter methylation and expression could be identified for MGMT but not for ABCB1 and ABCG2. Furthermore, MGMT promoter methylation was significantly associated with the genotypes of the MGMT C-56 T polymorphism showing a higher methylation level in the T allele bearing GBM. Conclusions In summary, the data of this study confirm the previous published relation of MGMT promoter methylation and gene expression, but argue for no pivotal role of MGMT, ABCB1 and ABCG2 promoter methylation in GBM patients’ survival. PMID:24380367

  3. Cetuximab enhanced the efficacy of chemotherapeutic agent in ABCB1/P-glycoprotein-overexpressing cancer cells.

    PubMed

    Wang, Fang; Chen, Yifan; Huang, Lihua; Liu, Tao; Huang, Yue; Zhao, Jianming; Wang, Xiaokun; Yang, Ke; Ma, Shaolin; Huang, Liyan; To, Kenneth Kin Wah; Gu, Yong; Fu, Liwu

    2015-12-01

    The overexpression of ATP-binding cassette (ABC) transporters is closely associated with the development of multidrug resistance (MDR) in certain types of cancer, which represents a formidable obstacle to the successful cancer chemotherapy. Here, we investigated that cetuximab, an EGFR monoclonal antibody, reversed the chemoresistance mediated by ABCB1, ABCG2 or ABCC1. Our results showed that cetuximab significantly enhanced the cytotoxicity of ABCB1 substrate agent in ABCB1-overexpressing MDR cells but had no effect in their parental drug sensitive cells and ABCC1, ABCG2 overexpressing cells. Furthermore, cetuximab markedly increased intracellular accumulation of doxorubicin (DOX) and rhodamine 123 (Rho 123) in ABCB1-overexpressing MDR cancer cells in a concentration-dependent manner. Cetuximab stimulated the ATPase activity but did not alter the expression level of ABCB1 or block phosphorylation of AKT and ERK. Interestingly, cetuximab decreased the cell membrane fluidity which was known to decrease the function of ABCB1. Our findings advocate further clinical investigation of combination chemotherapy of cetuximab and conventional chemotherapeutic drugs in ABCB1 overexpressing cancer patients.

  4. Cetuximab enhanced the efficacy of chemotherapeutic agent in ABCB1/P-glycoprotein-overexpressing cancer cells

    PubMed Central

    Liu, Tao; Huang, Yue; Zhao, Jianming; Wang, Xiaokun; Yang, Ke; Ma, Shaolin; Huang, Liyan; Wah To, Kenneth Kin; Gu, Yong; Fu, Liwu

    2015-01-01

    The overexpression of ATP-binding cassette (ABC) transporters is closely associated with the development of multidrug resistance (MDR) in certain types of cancer, which represents a formidable obstacle to the successful cancer chemotherapy. Here, we investigated that cetuximab, an EGFR monoclonal antibody, reversed the chemoresistance mediated by ABCB1, ABCG2 or ABCC1. Our results showed that cetuximab significantly enhanced the cytotoxicity of ABCB1 substrate agent in ABCB1-overexpressing MDR cells but had no effect in their parental drug sensitive cells and ABCC1, ABCG2 overexpressing cells. Furthermore, cetuximab markedly increased intracellular accumulation of doxorubicin (DOX) and rhodamine 123 (Rho 123) in ABCB1-overexpressing MDR cancer cells in a concentration-dependent manner. Cetuximab stimulated the ATPase activity but did not alter the expression level of ABCB1 or block phosphorylation of AKT and ERK. Interestingly, cetuximab decreased the cell membrane fluidity which was known to decrease the function of ABCB1. Our findings advocate further clinical investigation of combination chemotherapy of cetuximab and conventional chemotherapeutic drugs in ABCB1 overexpressing cancer patients. PMID:26506420

  5. Vatalanib sensitizes ABCB1 and ABCG2-overexpressing multidrug resistant colon cancer cells to chemotherapy under hypoxia.

    PubMed

    To, Kenneth K W; Poon, Daniel C; Wei, Yuming; Wang, Fang; Lin, Ge; Fu, Li-wu

    2015-09-01

    Cancer microenvironment is characterized by significantly lower oxygen concentration. This hypoxic condition is known to reduce drug responsiveness to cancer chemotherapy via multiple mechanisms, among which the upregulation of the ATP-binding cassette (ABC) efflux transporters confers resistance to a wide variety of structurally unrelated anticancer drugs. Vatalanib (PTK787/ZK22584) is a multitargeted tyrosine kinase inhibitor for all isoforms of VEGFR, PDGFR and c-Kit, which exhibit potent anticancer activity in vitro and in vivo. We investigated the potentiation effect of vatalanib on the anticancer activity of conventional cytotoxic drugs in colon cancer cell lines under both normoxic and hypoxic conditions. Mechanistically, vatalanib was found to inhibit ABCG2 and ABCB1 efflux activity, presumably by acting as a competitive inhibitor and interfering with their ATPase activity. Under hypoxic growth condition, ABCG2 and ABCB1-overexpressing cells sorted out by FACS technique as side population (SP) were found to be significantly more responsive to SN-38 (ABCG2 and ABCB1 substrate anticancer drug) in the presence of vatalanib. The anchorage independent soft agar colony formation capacity of the SP cells was remarkably reduced upon treatment with a combination of SN-38 and vatalanib, compared to SN-38 alone. However, vatalanib, at concentrations that produced the circumvention of the transporters-mediated resistance, did not appreciably alter ABCG2/ABCB1 mRNA or protein expression levels or the phosphorylation of Akt and extracellular signal-regulated kinase (ERK1/2). Our study thus advocates the further investigation of vatalanib for use in combination chemotherapy to eradicate drug-resistant cancer cells under hypoxia. PMID:26206183

  6. The function of the ATP-binding cassette (ABC) transporter ABCB1 is not susceptible to actin disruption.

    PubMed

    Meszaros, Peter; Hummel, Ina; Klappe, Karin; Draghiciu, Oana; Hoekstra, Dick; Kok, Jan W

    2013-02-01

    Previously we have shown that the activity of the multidrug transporter ABCC1 (multidrug resistance protein 1), and its localization in lipid rafts, depends on cortical actin (Hummel I, Klappe K, Ercan C, Kok JW. Mol. Pharm. 2011 79, 229-40). Here we show that the efflux activity of the ATP-binding cassette (ABC) family member ABCB1 (P-glycoprotein), did not depend on actin, neither in ABCB1 over expressing murine National Institutes of Health (NIH) 3T3 MDR1 G185 cells nor in human SK-N-FI cells, which endogenously express ABCB1. Disruption of the actin cytoskeleton, upon treatment of the cells with latrunculin B or cytochalasin D, caused severe changes in cell and membrane morphology, and concomitant changes in the subcellular distribution of ABCB1, as revealed by confocal laser scanning and electron microscopy. Nevertheless, irrespective of actin perturbation, the cell surface pool of ABCB1 remained unaltered. In NIH 3T3 MDR1 G185 cells, ABCB1 is partly localized in detergent-free lipid rafts, which partitioned in two different density gradient regions, both enriched in cholesterol and sphingolipids. Interestingly, disruption of the actin cytoskeleton did not change the density gradient distribution of ABCB1. Our data demonstrate that the functioning of ABCB1 as an efflux pump does not depend on actin, which is due to its distribution in both cell surface-localized non-raft membrane areas and lipid raft domains, which do not depend on actin stabilization.

  7. ABCB1 regulation through LRPPRC is influenced by the methylation status of the GC -100 box in its promoter

    PubMed Central

    Corrêa, Stephany; Binato, Renata; Du Rocher, Bárbara; Ferreira, Gerson; Cappelletti, Paola; Soares-Lima, Sheila; Pinto, Luis Felipe; Mencalha, André; Abdelhay, Eliana

    2014-01-01

    One of the potential mechanisms of imatinib mesylate (IM) resistance in chronic myeloid leukemia (CML) is increased level of P-glycoprotein (Pgp). Pgp is an efflux pump capable of activating the multidrug resistance (MDR) phenotype. The gene encoding Pgp (ABCB1) has several binding sites in its promoter region, along with CpG islands and GC boxes, involved in its epigenetic control. In previous work, we performed a proteomic study to identify proteins involved in IM cross-resistance in acute leukemia. Among these proteins, we identified LRPPRC as a potential regulator of ABCB1 transcription via an invMED1 binding site in ABCB1. Interestingly, this invMED1 binding site overlaps with the GC -100 box. In this work, we investigated the potential role of LRPPRC in the regulation of ABCB1 transcriptional activity in CML resistance. In addition, we evaluated the potential connection between this regulation and the methylation status of the ABCB1 promoter in its GC -100 box. Our results show that LRPPRC binds prominently to the ABCB1 promoter in Lucena cells, an IM-resistant cell line. Luciferase assays showed that ABCB1 transcription is positively regulated by LRPPRC upon its knockdown. Pyrosequencing analysis showed that the ABCB1 promoter is differentially methylated at its GC -100 box in K562 cells compared with Lucena cells, and in CML patients with different response to IM. Chromatin immunoprecipitation and Pgp expression after DNA demethylation treatment showed that LRPPRC binding is affected by the methylation status of ABCB1 GC -100 box. Taken together, our findings indicate that LRPPRC is a transcription factor related to ABCB1 expression and highlight the importance of epigenetic regulation in CML resistance. PMID:25089713

  8. ABCB1 regulation through LRPPRC is influenced by the methylation status of the GC -100 box in its promoter.

    PubMed

    Corrêa, Stephany; Binato, Renata; Du Rocher, Bárbara; Ferreira, Gerson; Cappelletti, Paola; Soares-Lima, Sheila; Pinto, Luis Felipe; Mencalha, André; Abdelhay, Eliana

    2014-08-01

    One of the potential mechanisms of imatinib mesylate (IM) resistance in chronic myeloid leukemia (CML) is increased level of P-glycoprotein (Pgp). Pgp is an efflux pump capable of activating the multidrug resistance (MDR) phenotype. The gene encoding Pgp (ABCB1) has several binding sites in its promoter region, along with CpG islands and GC boxes, involved in its epigenetic control. In previous work, we performed a proteomic study to identify proteins involved in IM cross-resistance in acute leukemia. Among these proteins, we identified LRPPRC as a potential regulator of ABCB1 transcription via an invMED1 binding site in ABCB1. Interestingly, this invMED1 binding site overlaps with the GC -100 box. In this work, we investigated the potential role of LRPPRC in the regulation of ABCB1 transcriptional activity in CML resistance. In addition, we evaluated the potential connection between this regulation and the methylation status of the ABCB1 promoter in its GC -100 box. Our results show that LRPPRC binds prominently to the ABCB1 promoter in Lucena cells, an IM-resistant cell line. Luciferase assays showed that ABCB1 transcription is positively regulated by LRPPRC upon its knockdown. Pyrosequencing analysis showed that the ABCB1 promoter is differentially methylated at its GC -100 box in K562 cells compared with Lucena cells, and in CML patients with different response to IM. Chromatin immunoprecipitation and Pgp expression after DNA demethylation treatment showed that LRPPRC binding is affected by the methylation status of ABCB1 GC -100 box. Taken together, our findings indicate that LRPPRC is a transcription factor related to ABCB1 expression and highlight the importance of epigenetic regulation in CML resistance.

  9. ABCB1 regulation through LRPPRC is influenced by the methylation status of the GC -100 box in its promoter.

    PubMed

    Corrêa, Stephany; Binato, Renata; Du Rocher, Bárbara; Ferreira, Gerson; Cappelletti, Paola; Soares-Lima, Sheila; Pinto, Luis Felipe; Mencalha, André; Abdelhay, Eliana

    2014-08-01

    One of the potential mechanisms of imatinib mesylate (IM) resistance in chronic myeloid leukemia (CML) is increased level of P-glycoprotein (Pgp). Pgp is an efflux pump capable of activating the multidrug resistance (MDR) phenotype. The gene encoding Pgp (ABCB1) has several binding sites in its promoter region, along with CpG islands and GC boxes, involved in its epigenetic control. In previous work, we performed a proteomic study to identify proteins involved in IM cross-resistance in acute leukemia. Among these proteins, we identified LRPPRC as a potential regulator of ABCB1 transcription via an invMED1 binding site in ABCB1. Interestingly, this invMED1 binding site overlaps with the GC -100 box. In this work, we investigated the potential role of LRPPRC in the regulation of ABCB1 transcriptional activity in CML resistance. In addition, we evaluated the potential connection between this regulation and the methylation status of the ABCB1 promoter in its GC -100 box. Our results show that LRPPRC binds prominently to the ABCB1 promoter in Lucena cells, an IM-resistant cell line. Luciferase assays showed that ABCB1 transcription is positively regulated by LRPPRC upon its knockdown. Pyrosequencing analysis showed that the ABCB1 promoter is differentially methylated at its GC -100 box in K562 cells compared with Lucena cells, and in CML patients with different response to IM. Chromatin immunoprecipitation and Pgp expression after DNA demethylation treatment showed that LRPPRC binding is affected by the methylation status of ABCB1 GC -100 box. Taken together, our findings indicate that LRPPRC is a transcription factor related to ABCB1 expression and highlight the importance of epigenetic regulation in CML resistance. PMID:25089713

  10. Novel and functional ABCB1 gene variant in sporadic Parkinson's disease.

    PubMed

    Li, Yuequn; Li, Yonghua; Pang, Shuchao; Huang, Wenhui; Zhang, Aimei; Hawley, Robert G; Yan, Bo

    2014-04-30

    Parkinson's disease (PD) is a common progressive neurodegenerative disease. Most cases of PD are sporadic, which is caused by interaction of genetic and environmental factors. To date, genetic causes for sporadic PD remain largely unknown. ATP-binding cassette sub-family B member 1 (ABCB1) is a membrane-associated protein that acts as an efflux transporter for many substrates, including chemotherapeutic agents, anti-epilepsy medicine, antibiotics and drugs for PD. ABCB1 gene is widely expressed in human tissues, including endothelial cells of capillary blood vessels at blood-brain barrier sites. In PD patients, decreased ABCB1 levels have been reported. We speculated that misregulation of ABCB1 gene expression, caused by DNA sequence variants (DSVs) within its regulatory regions, may be involved in PD development. In this study, we genetically and functionally analyzed the proximal promoter of the human ABCB1 gene, which is required for constitutive expression, in sporadic PD patients and healthy controls. The results showed that a novel and heterozygous DSV g.117077G>A was identified in one PD patient, but in none of the controls. This DSV significantly altered the transcriptional activity of the ABCB1 gene promoter in transiently transfected HEK-293 cells. A heterozygous DSV g.116347T>C was only found in one control. Four single-nucleotide polymorphisms, g.116154T>C (rs28746504), g.117130A>G (rs2188524), g.117356C>G (rs34976462) and g.117372T>C (rs3213619), and one heterozygous deletion DSV g.116039del were found in PD patients and controls with similar frequencies. Therefore, our findings suggest that ABCB1 gene promoter DSVs may contribute to PD development as a rare risk factor. PMID:24572589

  11. Membrane Assays to Characterize Interaction of Drugs with ABCB1.

    PubMed

    Fekete, Zsolt; Rajnai, Zsuzsanna; Nagy, Tünde; Jakab, Katalin Tauberné; Kurunczi, Anita; Gémes, Katalin; Herédi-Szabó, Krisztina; Fülöp, Ferenc; Tóth, Gábor K; Czerwinski, Maciej; Loewen, Greg; Krajcsi, Peter

    2015-12-01

    ATP-binding cassette sub-family B member 1 (ABCB1) [P-glycoprotein (P-gp), multidrug resistance protein 1 (MDR1)] can affect the pharmacokinetics, safety, and efficacy of drugs making it important to identify compounds that interact with ABCB1. The ATPase assay and vesicular transport (VT) assay are membrane based assays that can be used to measure the interaction of compounds with ABCB1 at a lower cost and higher throughput compared to cellular-based assays and therefore can be used earlier in the drug development process. To that end, we tested compounds previously identified as ABCB1 substrates and inhibitors for interaction with ABCB1 using the ATPase and VT assays. All compounds tested interacted with ABCB1 in both the ATPase and VT assays. All compounds previously identified as ABCB1 substrates activated ABCB1-mediated ATPase activity in the ATPase assay. All compounds previously identified as ABCB1 inhibitors inhibited the ABCB1-mediated transport in the VT assay. Interestingly, six of the ten compounds previously identified as ABCB1 inhibitors activated the basal ATPase activity in activation assays suggesting that the compounds are substrates of ABCB1 but can inhibit ABCB1 in inhibition assays. Importantly, for ATPase activators the EC50 of activation correlated with the IC50 values from the VT assay showing that interactions of compounds with ABCB1 can be measured with similar levels of potency in either assay. For ATPase nonactivators the IC50 values from the ATPase inhibition and VT inhibition assay showed correlation. These results demonstrate the utility of membrane assays as tools to detect and rank order drug-transporter interactions. PMID:25926125

  12. Abcb and Abcc transporter homologs are expressed and active in larvae and adults of zebra mussel and induced by chemical stress.

    PubMed

    Navarro, Anna; Weißbach, Susann; Faria, Melissa; Barata, Carlos; Piña, Benjamin; Luckenbach, Till

    2012-10-15

    Multixenobiotic resistance (MXR) of aquatic invertebrates has so far been associated with cellular efflux activity mediated by P-glycoprotein (ABCB1) and MRP (multidrug resistance protein; ABCC) type ABC (ATP binding cassette) transporters. Expression and activity of an abcb1/Abcb1 homolog has been shown in eggs and larvae of the zebra mussel Dreissena polymorpha. Here we report identification of a partial cDNA sequence of an abcc/Abcc homolog from zebra mussel that is transcribed and active as a cellular efflux transporter in embryos and gill tissue of adult mussels. Transcript expression levels were comparatively low in eggs and sharply increased after fertilization, then maintaining high expression levels in 1 and 2 dpf (days post fertilization) larvae. MK571, a known inhibitor of mammalian ABCC transporters, blocks efflux of calcein-am in larvae and gill tissue as indicated by elevated calcein fluorescence; this indicates the presence of active Abcc protein in cells of the larvae and gills. Dacthal and mercury used as chemical stressors both induced expression of abcb1 and abcc mRNAs in larvae; accordingly, assays with calcein-am and ABCB1 inhibitor reversin 205 and ABCC inhibitor MK571 indicated enhanced Abcb1 and Abcc efflux activities. Responses to chemicals were different in gills, where abcb1 transcript abundances were enhanced in dacthal and mercury treatments, whereas abcc mRNA was only increased with mercury. Abcb1 and Abcc activities did not in all cases show increases that were according to respective mRNA levels; thus, Abcc activity was significantly higher with dacthal, whereas Abcb1 activity was unchanged with mercury. Our data indicate that abcb1/Abcb1 and abcc/Abcc transporters are expressed and active in larvae and adult stages of zebra mussel. Expression of both genes is induced as cellular stress response, but regulation appears to differ in larvae and tissue of adult stages. PMID:22819804

  13. Effect of FosPeg® mediated photoactivation on P-gp/ABCB1 protein expression in human nasopharyngeal carcinoma cells.

    PubMed

    Wu, R W K; Chu, E S M; Huang, Z; Xu, C S; Ip, C W; Yow, C M N

    2015-07-01

    Multidrug resistance (MDR) refers to the ability of cancer cells to develop cross resistance to a range of anticancer drugs which are structurally and functionally unrelated. P-glycoprotein (P-gp) is the best studied MDR phenotype in photodynamic therapy (PDT) treated cells. Our pervious study demonstrated that FosPeg® mediated PDT is effective to NPC cell line models. In this in vitro study, the expression of MDR1 gene and its product P-gp in undifferentiated, poorly differentiated and well differentiated human nasopharyngeal carcinoma (NPC) cells were investigated. The influence of P-gp efflux activities on photosensitizer FosPeg® was also examined. Regardless of the differentiation status, PDT tested NPC cell lines all expressed P-gp protein. Results indicated that FosPeg® photoactivation could heighten the expression of MDR1 gene and P-gp transporter protein in a dose dependent manner. Up to 2-fold increase of P-gp protein expression were seen in NPC cells after FosPeg® mediated PDT. Interestingly, our finding demonstrated that FosPeg® mediated PDT efficiency is independent to the MDR1 gene and P-gp protein expression in NPC cells. FosPeg® itself is not the substrate of P-gp transporter protein and no efflux of FosPeg® were observed in NPC cells. Therefore, the PDT efficiency would not be affected even though FosPeg® mediated PDT could induce MDR1 gene and P-gp protein expression in NPC cells. FosPeg® mediated PDT could be a potential therapeutic approach for MDR cancer patients. PMID:25900553

  14. Human-Mouse Chimeras with Normal Expression and Function Reveal That Major Domain Swapping Is Tolerated by P-Glycoprotein (ABCB1).

    PubMed

    Pluchino, Kristen M; Hall, Matthew D; Moen, Janna K; Chufan, Eduardo E; Fetsch, Patricia A; Shukla, Suneet; Gill, Deborah R; Hyde, Stephen C; Xia, Di; Ambudkar, Suresh V; Gottesman, Michael M

    2016-02-23

    The efflux transporter P-glycoprotein (P-gp) plays a vital role in the transport of molecules across cell membranes and has been shown to interact with a panoply of functionally and structurally unrelated compounds. How human P-gp interacts with this large number of drugs has not been well understood, although structural flexibility has been implicated. To gain insight into this transporter's broad substrate specificity and to assess its ability to accommodate a variety of molecular and structural changes, we generated human-mouse P-gp chimeras by the exchange of homologous transmembrane and nucleotide-binding domains. High-level expression of these chimeras by BacMam- and baculovirus-mediated transduction in mammalian (HeLa) and insect cells, respectively, was achieved. There were no detectable differences between wild-type and chimeric P-gp in terms of cell surface expression, ability to efflux the P-gp substrates rhodamine 123, calcein-AM, and JC-1, or to be inhibited by the substrate cyclosporine A and the inhibitors tariquidar and elacridar. Additionally, expression of chimeric P-gp was able to confer a paclitaxel-resistant phenotype to HeLa cells characteristic of P-gp-mediated drug resistance. P-gp ATPase assays and photo-cross-linking with [(125)I]iodoarylazidoprazosin confirmed that transport and biochemical properties of P-gp chimeras were similar to those of wild-type P-gp, although differences in drug binding were detected when human and mouse transmembrane domains were combined. Overall, chimeras with one or two mouse P-gp domains were deemed functionally equivalent to human wild-type P-gp, demonstrating the ability of human P-gp to tolerate major structural changes.

  15. Human-Mouse Chimeras with Normal Expression and Function Reveal That Major Domain Swapping Is Tolerated by P-Glycoprotein (ABCB1).

    PubMed

    Pluchino, Kristen M; Hall, Matthew D; Moen, Janna K; Chufan, Eduardo E; Fetsch, Patricia A; Shukla, Suneet; Gill, Deborah R; Hyde, Stephen C; Xia, Di; Ambudkar, Suresh V; Gottesman, Michael M

    2016-02-23

    The efflux transporter P-glycoprotein (P-gp) plays a vital role in the transport of molecules across cell membranes and has been shown to interact with a panoply of functionally and structurally unrelated compounds. How human P-gp interacts with this large number of drugs has not been well understood, although structural flexibility has been implicated. To gain insight into this transporter's broad substrate specificity and to assess its ability to accommodate a variety of molecular and structural changes, we generated human-mouse P-gp chimeras by the exchange of homologous transmembrane and nucleotide-binding domains. High-level expression of these chimeras by BacMam- and baculovirus-mediated transduction in mammalian (HeLa) and insect cells, respectively, was achieved. There were no detectable differences between wild-type and chimeric P-gp in terms of cell surface expression, ability to efflux the P-gp substrates rhodamine 123, calcein-AM, and JC-1, or to be inhibited by the substrate cyclosporine A and the inhibitors tariquidar and elacridar. Additionally, expression of chimeric P-gp was able to confer a paclitaxel-resistant phenotype to HeLa cells characteristic of P-gp-mediated drug resistance. P-gp ATPase assays and photo-cross-linking with [(125)I]iodoarylazidoprazosin confirmed that transport and biochemical properties of P-gp chimeras were similar to those of wild-type P-gp, although differences in drug binding were detected when human and mouse transmembrane domains were combined. Overall, chimeras with one or two mouse P-gp domains were deemed functionally equivalent to human wild-type P-gp, demonstrating the ability of human P-gp to tolerate major structural changes. PMID:26820614

  16. The multidrug resistance pump ABCB1 is a substrate for the ubiquitin ligase NEDD4-1

    PubMed Central

    Akkaya, Begum G.; Zolnerciks, Joseph K.; Ritchie, Tasha K.; Bauer, Bjoern; Hartz, Anika M.S.; Sullivan, James A.; Linton, Kenneth J.

    2016-01-01

    The ATP Binding Cassette transporter ABCB1 can export the neurotoxic peptide β-amyloid from endothelial cells that line the blood-brain barrier (BBB). This has the potential to lower cerebral levels of β-amyloid, but ABCB1 expression in the BBB appears to be progressively reduced in patients with Alzheimer's disease. The surface density of many membrane proteins is regulated by ubiquitination catalysed by ubiquitin E3 ligases. In brain capillaries of mice challenged with β-amyloid ex vivo, we show that the level of the ubiquitin ligase Nedd4 increases concomitant with reduction in Abcb1. In vitro we show that human ABCB1 is a substrate for human NEDD4-1 ligase. Recombinant ABCB1 was purified from Sf21 insect cells and incubated with recombinant NEDD4-1 purified from E. coli. The treated ABCB1 had reduced mobility on SDS-PAGE, and mass spectrometry identified eight lysine residues, K271, K272, K575, K685, K877, K885, K887 and K1062 that were ubiquitinated by NEDD4-1. Molecular modelling showed that all of the residues are exposed on the surface of the intracellular domains of ABCB1. K877, K885 and K887 in particular, are located in the intracellular loop of transmembrane helix 10 (TMH10) in close proximity, in the tertiary fold, to a putative NEDD4-1 binding site in the intracellular helix extending from TMH12 (PxY motif, residues 996-998). Transient expression of NEDD4-1 in HEK293 Flp-In cells stably expressing ABCB1 was shown to reduce the surface density of the transporter. Together, the data identify this ubiquitin ligase as a potential target for intervention in the pathophysiology of Alzheimer's disease. PMID:26006083

  17. The Multidrug Resistance 1 Gene Abcb1 in Brain and Placenta: Comparative Analysis in Human and Guinea Pig

    PubMed Central

    Pappas, Jane J.; Petropoulos, Sophie; Suderman, Matthew; Iqbal, Majid; Moisiadis, Vasilis; Turecki, Gustavo; Matthews, Stephen G.; Szyf, Moshe

    2014-01-01

    The Multidrug Resistance 1 (MDR1; alternatively ABCB1) gene product P-glycoprotein (P-gp), an ATP binding cassette transporter, extrudes multiple endogenous and exogenous substrates from the cell, playing an important role in normal physiology and xenobiotic distribution and bioavailability. To date, the predominant animal models used to investigate the role of P-gp have been the mouse and rat, which have two distinct genes, Abcb1a and Abcb1b. In contrast, the human has a single gene, ABCB1, for which only a single isoform has been validated. We and others have previously shown important differences between Abcb1a and Abcb1b, limiting the extrapolation from rodent findings to the human. Since the guinea pig has a relatively long gestation, hemomonochorial placentation and neuroanatomically mature offspring, it is more similar to the human, and may provide a more comparable model for investigating the regulation of P-gp in the brain and placenta, however, to date, the Abcb1 gene in the guinea pig remains to be characterized. The placenta and fetal brain are barrier sites that express P-gp and that play a critical role of protection of the fetus and the fetal brain from maternally administered drugs and other xenobiotics. Using RNA sequencing (RNA-seq), reverse transcription-polymerase chain reaction (RT-PCR) and quantitative PCR (QPCR) to sequence the expressed isoforms of guinea pig Abcb1, we demonstrate that like the human, the guinea pig genome contains one gene for Abcb1 but that it is expressed as at least three different isoforms via alternative splicing and alternate exon usage. Further, we demonstrate that these isoforms are more closely related to human than to rat or mouse isoforms. This striking, overall similarity and evolutionary relatedness between guinea pig Abcb1 and human ABCB1 indicate that the guinea pig represents a relevant animal model for investigating the function and regulation of P-gp in the placenta and brain. PMID:25353162

  18. Inhibition of ABCB1 (MDR1) Expression by an siRNA Nanoparticulate Delivery System to Overcome Drug Resistance in Osteosarcoma

    PubMed Central

    Ryu, Keinosuke; Choy, Edwin; Hornicek, Francis J.; Mankin, Henry; Milane, Lara; Amiji, Mansoor M.; Duan, Zhenfeng

    2010-01-01

    Background The use of neo-adjuvant chemotherapy in treating osteosarcoma has improved patients' average 5 year survival rate from 20% to 70% in the past 30 years. However, for patients who progress after chemotherapy, its effectiveness diminishes due to the emergence of multi-drug resistance (MDR) after prolonged therapy. Methodology/Principal Findings In order to overcome both the dose-limiting side effects of conventional chemotherapeutic agents and the therapeutic failure resulting from MDR, we designed and evaluated a novel drug delivery system for MDR1 siRNA delivery. Novel biocompatible, lipid-modified dextran-based polymeric nanoparticles were used as the platform for MDR1 siRNA delivery; and the efficacy of combination therapy with this system was evaluated. In this study, multi-drug resistant osteosarcoma cell lines (KHOSR2 and U-2OSR2) were treated with the MDR1 siRNA nanocarriers and MDR1 protein (P-gp) expression, drug retention, and immunofluoresence were analyzed. Combination therapy of the MDR1 siRNA loaded nanocarriers with increasing concentrations of doxorubicin was also analyzed. We observed that MDR1 siRNA loaded dextran nanoparticles efficiently suppresses P-gp expression in the drug resistant osteosarcoma cell lines. The results also demonstrated that this approach may be capable of reversing drug resistance by increasing the amount of drug accumulation in MDR cell lines. Conclusions/Significance Lipid-modified dextran-based polymeric nanoparticles are a promising platform for siRNA delivery. Nanocarriers loaded with MDR1 siRNA are a potential treatment strategy for reversing MDR in osteosarcoma. PMID:20520719

  19. Bafetinib (INNO-406) reverses multidrug resistance by inhibiting the efflux function of ABCB1 and ABCG2 transporters

    PubMed Central

    Zhang, Yun-Kai; Zhang, Guan-Nan; Wang, Yi-Jun; Patel, Bhargav A.; Talele, Tanaji T.; Yang, Dong-Hua; Chen, Zhe-Sheng

    2016-01-01

    ATP-Binding Cassette transporters are involved in the efflux of xenobiotic compounds and are responsible for decreasing drug accumulation in multidrug resistant (MDR) cells. Discovered by structure-based virtual screening algorithms, bafetinib, a Bcr-Abl/Lyn tyrosine kinase inhibitor, was found to have inhibitory effects on both ABCB1- and ABCG2-mediated MDR in this in-vitro investigation. Bafetinib significantly sensitized ABCB1 and ABCG2 overexpressing MDR cells to their anticancer substrates and increased the intracellular accumulation of anticancer drugs, particularly doxorubicin and [3H]-paclitaxel in ABCB1 overexpressing cells; mitoxantrone and [3H]-mitoxantrone in ABCG2 overexpressing cells, respectively. Bafetinib stimulated ABCB1 ATPase activities while inhibited ABCG2 ATPase activities. There were no significant changes in the expression level or the subcellular distribution of ABCB1 and ABCG2 in the cells exposed to 3 μM of bafetinib. Overall, our study indicated that bafetinib reversed ABCB1- and ABCG2-mediated MDR by blocking the drug efflux function of these transporters. These findings might be useful in developing combination therapy for MDR cancer treatment. PMID:27157787

  20. The effect of ABCB1 polymorphisms on the outcome of breast cancer treatment

    PubMed Central

    Tulsyan, Sonam; Mittal, Rama Devi; Mittal, Balraj

    2016-01-01

    The ABCB1 gene encodes a permeability glycoprotein, which is one of the most extensively studied human adenosine-triphosphate (ATP)-dependent efflux transporters. Permeability glycoprotein is expressed in the apical membranes of tissues such as intestine, liver, blood–brain barrier, kidney, placenta, and testis and contributes to intracellular drug disposition. It is also highly expressed in tumor cells conferring drug resistance, which is one of the major problems in the efficacy of cancer chemotherapy treatment. ABCB1 is highly polymorphic, and three well-known single-nucleotide polymorphisms such as 1236C>T, 2677G>T/A, and 3435C>T have been found to be associated with altered messenger RNA levels, protein folding, and drug pharmacokinetics. Many association studies and meta-analyses have demonstrated the clinical impact of ABCB1 polymorphisms in breast cancer treatment outcomes with respect to therapeutic response, chemotoxicity, and overall survival. Therefore, the aim of this review was to evaluate the effects of ABCB1 polymorphisms on the outcome of breast cancer treatment which, in future, would be important for tailoring individualized anticancer therapy. PMID:27175090

  1. Complete Knockout of Endogenous Mdr1 (Abcb1) in MDCK Cells by CRISPR-Cas9.

    PubMed

    Simoff, Ivailo; Karlgren, Maria; Backlund, Maria; Lindström, Anne-Christine; Gaugaz, Fabienne Z; Matsson, Pär; Artursson, Per

    2016-02-01

    Madin-Darby canine kidney II cells transfected with one or several transport proteins are commonly used models to study drug transport. In these cells, however, endogenous transporters such as canine Mdr1/P-glycoprotein (Abcb1) complicate the interpretation of transport studies. The aim of this investigation was to establish a Madin-Darby canine kidney II cell line using CRISPR-Cas9 gene-editing technology to knock out endogenous canine Mdr1 (cMdr1) expression. CRISPR-Cas9-mediated Abcb1 homozygous disruption occurred at frequencies of around 20% and resulted in several genotypes. We selected 1 clonal cell line, cMdr1 KO Cl2, for further examination. Consistent with an on-target effect of CRISPR-Cas9 in specific regions of the endogenous canine Abcb1 gene, we obtained a cell clone with Abcb1 gene alterations and without any cMdr1 expression, as confirmed by genome sequencing and quantitative protein analysis. Functional studies of these cells, using digoxin and other prototypic MDR1 substrates, showed close to identical transport in the apical-to-basolateral and basolateral-to-apical directions, resulting in efflux ratios indistinguishable from unity.

  2. The effect of ABCB1 polymorphisms on the outcome of breast cancer treatment.

    PubMed

    Tulsyan, Sonam; Mittal, Rama Devi; Mittal, Balraj

    2016-01-01

    The ABCB1 gene encodes a permeability glycoprotein, which is one of the most extensively studied human adenosine-triphosphate (ATP)-dependent efflux transporters. Permeability glycoprotein is expressed in the apical membranes of tissues such as intestine, liver, blood-brain barrier, kidney, placenta, and testis and contributes to intracellular drug disposition. It is also highly expressed in tumor cells conferring drug resistance, which is one of the major problems in the efficacy of cancer chemotherapy treatment. ABCB1 is highly polymorphic, and three well-known single-nucleotide polymorphisms such as 1236C>T, 2677G>T/A, and 3435C>T have been found to be associated with altered messenger RNA levels, protein folding, and drug pharmacokinetics. Many association studies and meta-analyses have demonstrated the clinical impact of ABCB1 polymorphisms in breast cancer treatment outcomes with respect to therapeutic response, chemotoxicity, and overall survival. Therefore, the aim of this review was to evaluate the effects of ABCB1 polymorphisms on the outcome of breast cancer treatment which, in future, would be important for tailoring individualized anticancer therapy. PMID:27175090

  3. Nickel-induced cell death and survival pathways in cultured renal proximal tubule cells: roles of reactive oxygen species, ceramide and ABCB1.

    PubMed

    Dahdouh, Faouzi; Raane, Maximilian; Thévenod, Frank; Lee, Wing-Kee

    2014-04-01

    Nickel and nickel compounds are carcinogens that target the lungs and kidneys causing cell death or cell survival adaptation. The multidrug resistance P-glycoprotein ABCB1 protects cells against toxic metabolites and xenobiotics and is upregulated in many cancer cell types. Here, we investigated the role of ABCB1 in nickel-induced stress signaling mediated by reactive oxygen species (ROS) and ceramides. In renal proximal tubule cells, nickel chloride (0.1-0.25 mM) increased both ROS formation, detected by 5-(and-6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate, and cellular ceramides, which were determined by lipid dot blot and surface immunostaining, culminating in decreased cell viability, increased DNA fragmentation, augmented PARP-1 cleavage, and increased ABCB1 mRNA and protein. Inhibitors of the de novo ceramide synthesis pathway (fumonisin B1, L-cycloserine) and an antioxidant (α-tocopherol) attenuated nickel-induced toxicity as well as induction of ABCB1. ABCB1 protects against nickel toxicity as PSC833, an ABCB1 blocker, augmented the decrease in cell viability by nickel. Moreover, nickel toxicity was attenuated in renal MDCK cells stably overexpressing ABCB1. In agreement with previous data that demonstrated extrusion of (glucosyl)ceramides by ABCB1 (Lee et al. in Toxicol Sci 121:343, 2011), PSC833 increased total cellular ceramides by >2-fold after nickel treatment. Further, glucosylceramide synthase (GCS) mRNA is upregulated by nickel at 3 h by ~1.5-fold but declined with prolonged exposures (6-24 h). Inhibition of GCS with C9DGJ or knockdown of GCS with siRNA significantly attenuated nickel toxicity. In conclusion, nickel induces a ROS-ceramide pathway to cause apoptotic cell death as well as activate adaptive survival responses, including upregulation of ABCB1, which improves cell survival by extruding proapoptotic (glucosyl)ceramides. PMID:24448832

  4. Ferulic acid reverses ABCB1-mediated paclitaxel resistance in MDR cell lines.

    PubMed

    Muthusamy, Ganesan; Balupillai, Agilan; Ramasamy, Karthikeyan; Shanmugam, Mohana; Gunaseelan, Srithar; Mary, Beaulah; Prasad, N Rajendra

    2016-09-01

    Multidrug resistance (MDR) remains a major obstacle in cancer chemotherapy. The use of the dietary phytochemicals as chemosensitizing agents to enhance the efficacy of conventional cytostatic drugs has recently gained the attention as a plausible approach for overcoming the drug resistance. The aim of this study was to investigate whether a naturally occurring diet-based phenolic acid, ferulic acid, could sensitize paclitaxel efficacy in ABCB1 overexpressing (P-glycoprotein) colchicine selected KB Ch(R)8-5 cell line. In vitro drug efflux assays demonstrated that ferulic acid inhibits P-glycoprotein transport function in drug resistant KB Ch(R)8-5 cell lines. However, ferulic acid significantly downregulates ABCB1 expression in a concentration dependent manner. Cytotoxicity assay reveals that ferulic acid decreased paclitaxel resistance in KBCh(R)8-5 and HEK293/ABCB1 cells, which indicates its chemosensitizing potential. Clonogenic cell survival assay and apoptotic morphological staining further confirm the chemosensitizing potential of ferulic acid in drug resistant KB Ch(R)8-5 cell lines. Ferulic acid treatment enhances paclitaxel mediated cell cycle arrest and upregulates paclitaxel-induced apoptotic signaling in KB resistant cells. Hence, it has been concluded that downregulation of ABCB1 and subsequent induction of paclitaxel-mediated cell cycle arrest and apoptotic signaling may be the cause for the chemosensitizing potential of ferulic acid in P-gp overexpressing cell lines. PMID:27262378

  5. 1236 C/T and 3435 C/T polymorphisms of the ABCB1 gene in Mexican breast cancer patients.

    PubMed

    Gutierrez-Rubio, S A; Quintero-Ramos, A; Durán-Cárdenas, A; Franco-Topete, R A; Castro-Cervantes, J M; Oceguera-Villanueva, A; Jiménez-Pérez, L M; Balderas-Peña, L M A; Morgan-Villela, G; Del-Toro-Arreola, A; Daneri-Navarro, A

    2015-02-13

    MDR1, which is encoded by the ABCB1 gene, is involved in multidrug resistance (hydrophobic), as well as the elimination of xenotoxic agents. The association between ABCB1 gene polymorphisms and breast cancer risk in different populations has been described previously; however, the results have been inconclusive. In this study, we examined the association between polymorphisms 3435 C/T and 1236 C/T in the ABCB1 gene and breast cancer development in Mexican women according to their menopausal status and molecular classification. Molecular subtypes as well as allele and genotype frequencies were analyzed. A total of 248 women with initial breast cancer diagnosis and 180 ethnically matched, healthy, unrelated individuals were enrolled. Polymerase chain reaction-restriction fragment length polymorphism was performed to detect polymorphisms 3435 C/T and 1236 C/T in the ABCB1 gene. Premenopausal T allele carriers of the 3435 C/T polymorphism showed a 2-fold increased risk of breast cancer with respect to the reference and postmenopausal groups, as well as triple-negative expression regarding the luminal A/B molecular subrogated subtypes. In contrast, the CT genotype of the 1236 polymorphism was a protective factor against breast cancer. We conclude that the T allele carrier of the 3435 C/T polymorphism in the ABCB1 gene in combination with an estrogen receptor-negative status may be an important risk factor for breast cancer development in premenopausal women.

  6. ABCB1 Overexpression Is a Key Initiator of Resistance to Tyrosine Kinase Inhibitors in CML Cell Lines.

    PubMed

    Eadie, Laura N; Hughes, Timothy P; White, Deborah L

    2016-01-01

    The tyrosine kinase inhibitor (TKI) imatinib has resulted in excellent responses in the majority of Chronic Myeloid Leukaemia (CML) patients; however, resistance is observed in 20-30% of patients. More recently, resistance to the second generation TKIs, nilotinib and dasatinib, has also been observed albeit at a lower incidence. ABCB1 has previously been implicated in TKI export and its overexpression linked to TKI resistance. In this study the dynamics of nilotinib resistance was studied in CML cell lines with particular focus on ABCB1 expression levels during development of resistance. Results revealed ABCB1 overexpression is likely an important initiator of nilotinib resistance in vitro. ABCB1 overexpression was also observed in cell lines as an intermediate step during development of resistance to imatinib and dasatinib in vitro. We conclude that ABCB1 overexpression may provide an initial platform to facilitate development of additional mechanisms for resistance to TKIs. This provides a rationale for investigating this phenomenon in patients undergoing TKI therapy. PMID:27536777

  7. ABCB1 Overexpression Is a Key Initiator of Resistance to Tyrosine Kinase Inhibitors in CML Cell Lines

    PubMed Central

    Hughes, Timothy P.; White, Deborah L.

    2016-01-01

    The tyrosine kinase inhibitor (TKI) imatinib has resulted in excellent responses in the majority of Chronic Myeloid Leukaemia (CML) patients; however, resistance is observed in 20–30% of patients. More recently, resistance to the second generation TKIs, nilotinib and dasatinib, has also been observed albeit at a lower incidence. ABCB1 has previously been implicated in TKI export and its overexpression linked to TKI resistance. In this study the dynamics of nilotinib resistance was studied in CML cell lines with particular focus on ABCB1 expression levels during development of resistance. Results revealed ABCB1 overexpression is likely an important initiator of nilotinib resistance in vitro. ABCB1 overexpression was also observed in cell lines as an intermediate step during development of resistance to imatinib and dasatinib in vitro. We conclude that ABCB1 overexpression may provide an initial platform to facilitate development of additional mechanisms for resistance to TKIs. This provides a rationale for investigating this phenomenon in patients undergoing TKI therapy. PMID:27536777

  8. Impact of ABCB1 1236C > T-2677G > T-3435C > T polymorphisms on the anti-proliferative activity of imatinib, nilotinib, dasatinib and ponatinib

    PubMed Central

    Dessilly, Géraldine; Panin, Nadtha; Elens, Laure; Haufroid, Vincent; Demoulin, Jean-Baptiste

    2016-01-01

    Overexpression of ABCB1 (also called P-glycoprotein) confers resistance to multiple anticancer drugs, including tyrosine kinase inhibitors (TKIs). Several ABCB1 single nucleotide polymorphisms affect the transporter activity. The most common ABCB1 variants are 1236C > T, 2677G > T, 3435C > T and have been associated with clinical response to imatinib in chronic myelogenous leukaemia (CML) in some studies. We evaluated the impact of these polymorphisms on the anti-proliferative effect and the intracellular accumulation of TKIs (imatinib, nilotinib, dasatinib and ponatinib) in transfected HEK293 and K562 cells. ABCB1 overexpression increased the resistance of cells to doxorubicin, vinblastine and TKIs. Imatinib anti-proliferative effect and accumulation were decreased to a larger extent in cells expressing the ABCB1 wild-type protein compared with the 1236T-2677T-3435T variant relatively to control cells. By contrast, ABCB1 polymorphisms influenced the activity of nilotinib, dasatinib and ponatinib to a much lesser extent. In conclusion, our data suggest that wild-type ABCB1 exports imatinib more efficiently than the 1236T-2677T-3435T variant protein, providing a molecular basis for the reported association between ABCB1 polymorphisms and the response to imatinib in CML. Our results also point to a weaker impact of ABCB1 polymorphisms on the activity of nilotinib, dasatinib and ponatinib. PMID:27405085

  9. The FLT3 and PDGFR inhibitor crenolanib is a substrate of the multidrug resistance protein ABCB1 but does not inhibit transport function at pharmacologically relevant concentrations.

    PubMed

    Mathias, Trevor J; Natarajan, Karthika; Shukla, Suneet; Doshi, Kshama A; Singh, Zeba N; Ambudkar, Suresh V; Baer, Maria R

    2015-04-01

    Background Crenolanib (crenolanib besylate, 4-piperidinamine, 1-[2-[5-[(3-methyl-3-oxetanyl)methoxy]-1H-benzimidazol-1-yl]-8-quinolinyl]-, monobenzenesulfonate) is a potent and specific type I inhibitor of fms-like tyrosine kinase 3 (FLT3) that targets the active kinase conformation and is effective against FLT3 with internal tandem duplication (ITD) with point mutations induced by, and conferring resistance to, type II FLT3 inhibitors in acute myeloid leukemia (AML) cells. Crenolanib is also an inhibitor of platelet-derived growth factor receptor alpha and beta and is in clinical trials in both gastrointestinal stromal tumors and gliomas. Methods We tested crenolanib interactions with the multidrug resistance-associated ATP-binding cassette proteins ABCB1 (P-glycoprotein), ABCG2 (breast cancer resistance protein) and ABCC1 (multidrug resistance-associated protein 1), which are expressed on AML cells and other cancer cells and are important components of the blood-brain barrier. Results We found that crenolanib is a substrate of ABCB1, as evidenced by approximate five-fold resistance of ABCB1-overexpressing cells to crenolanib, reversal of this resistance by the ABCB1-specific inhibitor PSC-833 and stimulation of ABCB1 ATPase activity by crenolanib. In contrast, crenolanib was not a substrate of ABCG2 or ABCC1. Additionally, it did not inhibit substrate transport by ABCB1, ABCG2 or ABCC1, at pharmacologically relevant concentrations. Finally, incubation of the FLT3-ITD AML cell lines MV4-11 and MOLM-14 with crenolanib at a pharmacologically relevant concentration of 500 nM did not induce upregulation of ABCB1 cell surface expression. Conclusions Thus ABCB1 expression confers resistance to crenolanib and likely limits crenolanib penetration of the central nervous system, but crenolanib at therapeutic concentrations should not alter cellular exposure to ABC protein substrate chemotherapy drugs.

  10. Involvement of ABCB1 and ABCC1 transporters in sea urchin Echinometra lucunter fertilization.

    PubMed

    Silva-Neta, Helena Lima; Torrezan, Elis; de Araújo Leite, Jocelmo Cássio; Santi-Gadelha, Tatiane; Marques-Santos, Luis Fernando

    2012-12-01

    Fertilization is an ordered sequence of cellular interactions that promotes gamete fusion to form a new individual. Since the pioneering work of Oskar Hertwig conducted on sea urchins, echinoderms have contributed to the understanding of cellular and molecular aspects of the fertilization processes. Studies on sea urchin spermatozoa reported the involvement of a plasma membrane protein that belongs to the ABC proteins superfamily in the acrosome reaction. ABC transporters are expressed in membranes of eukaryotic and prokaryotic cells, and are associated with the transport of several compounds or ions across biomembranes. We aimed to investigate ABCB1 and ABCC1 transporter activity in sea urchin spermatozoa and their involvement in fertilization. Our results indicate that Echinometra lucunter spermatozoa exhibit a low intracellular calcein accumulation (18.5% stained cells); however, the ABC blockers reversin205, verapamil, and MK571 increased dye accumulation (93.0-96.6% stained cells). We also demonstrated that pharmacologically blocking ABCB1 and ABCC1 decreased spermatozoa fertilizing capacity (70% inhibition), and this phenotype was independent of extracellular calcium. These data suggest that functional spermatozoa ABCB1 and ABCC1 transporters are crucial for a successful fertilization. Additional studies must be performed to investigate the involvement of membrane lipid homeostasis in the fertilization process.

  11. P-glycoprotein (ABCB1) inhibited network of mitochondrion transport along microtubule and BMP signal-induced cell shape in chimpanzee left cerebrum by systems-theoretical analysis.

    PubMed

    Lin, Hong; Wang, Lin; Jiang, Minghu; Huang, Juxiang; Qi, Lianxiu

    2012-10-01

    We constructed the significant low-expression P-glycoprotein (ABCB1) inhibited transport and signal network in chimpanzee compared with high-expression (fold change ≥2) the human left cerebrum in GEO data set, by using integration of gene regulatory activated and inhibited network inference method with gene ontology (GO) analysis. Our result showed that ABCB1 transport and signal upstream network RAB2A inhibited ABCB1, and downstream ABCB1-inhibited SMAD1_2, NCK2, SLC25A46, GDF10, RASGRP1, EGFR, LRPPRC, RASSF2, RASA4, CA2, CBLB, UBR5, SLC25A16, ITGB3BP, DDIT4, PDPN, RAB2A in chimpanzee left cerebrum. We obtained that the different biological processes of ABCB1 inhibited transport and signal network repressed carbon dioxide transport, ER to Golgi vesicle-mediated transport, folic acid transport, mitochondrion transport along microtubule, water transport, BMP signaling pathway, Ras protein signal transduction, transforming growth factor beta receptor signaling pathway in chimpanzee compared with the inhibited network of the human left cerebrum, as a result of inducing inhibition of mitochondrion transport along microtubule and BMP signal-induced cell shape in chimpanzee left cerebrum. Our hypothesis was verified by the same and different biological processes of ABCB1 inhibited transport and signal network of chimpanzee compared with the corresponding activated network of chimpanzee and the human left cerebrum, respectively.

  12. Cyclic-AMP Mediated Regulation of ABCB mRNA Expression in Mussel Haemocytes

    PubMed Central

    Franzellitti, Silvia; Fabbri, Elena

    2013-01-01

    Background The multixenobiotic resistance system (MXR) allows aquatic organisms to cope with their habitat despite high pollution levels by over-expressing membrane and intracellular transporters, including the P-glycoprotein (Pgp). In mammals transcription of the ABCB1 gene encoding Pgp is under cAMP/PKA-mediated regulation; whether this is true in mollusks is not fully clarified. Methodology/Principal Findings cAMP/PKA regulation and ABCB mRNA expression were assessed in haemocytes from Mediterranean mussels (Mytilus galloprovincialis) exposed in vivo for 1 week to 0.3 ng/L fluoxetine (FX) alone or in combination with 0.3 ng/L propranolol (PROP). FX significantly decreased cAMP levels and PKA activity, and induced ABCB mRNA down-regulation. FX effects were abolished in the presence of PROP. In vitro experiments using haemocytes treated with physiological agonists (noradrenaline and serotonin) and pharmacological modulators (PROP, forskolin, dbcAMP, and H89) of the cAMP/PKA system were performed to obtain clear evidence about the involvement of the signaling pathway in the transcriptional regulation of ABCB. Serotonin (5-HT) decreased cAMP levels, PKA activity and ABCB mRNA expression but increased the mRNA levels for a putative 5-HT1 receptor. Interestingly, 5-HT1 was also over-expressed after in vivo exposures to FX. 5-HT effects were counteracted by PROP. Forskolin and dbcAMP increased PKA activity as well as ABCB mRNA expression; the latter effect was abolished in the presence of the PKA inhibitor H89. Conclusions This study provides the first direct evidence for the cAMP/PKA-mediated regulation of ABCB transcription in mussels. PMID:23593491

  13. Knockdown of c-MET induced apoptosis in ABCB1-overexpressed multidrug-resistance cancer cell lines.

    PubMed

    Hung, T-H; Li, Y-H; Tseng, C-P; Lan, Y-W; Hsu, S-C; Chen, Y-H; Huang, T-T; Lai, H-C; Chen, C-M; Choo, K-B; Chong, K-Y

    2015-05-01

    Inappropriate c-MET signaling in cancer can enhance tumor cell proliferation, survival, motility, and invasion. Inhibition of c-MET signaling induces apoptosis in a variety of cancers. It has also been recognized as a novel anticancer therapy approach. Furthermore, reports have also indicated that constitutive expression of P-glycoprotein (ABCB1) is involved in the HGF/c-MET-related pathway of multidrug resistance ABCB1-positive human hepatocellular carcinoma cell lines. We previously reported that elevated expression levels of PKCδ and AP-1 downstream genes, and HGF receptor (c-MET) and ABCB1, in the drug-resistant MES-SA/Dx5 cells. Moreover, leukemia cell lines overexpressing ABCB1 have also been shown to be more resistant to the tyrosine kinase inhibitor imatinib mesylate. These findings suggest that chemoresistant cancer cells may also develop a similar mechanism against chemotherapy agents. To circumvent clinical complications arising from drug resistance during cancer therapy, the present study was designed to investigate apoptosis induction in ABCB1-overexpressed cancer cells using c-MET-targeted RNA interference technology in vitro and in vivo. The results showed that cell viability decreased and apoptosis rate increased in c-MET shRNA-transfected HGF/c-MET pathway-positive MES-SA/Dx5 and MCF-7/ADR2 cell lines in a dose-dependent manner. In vivo reduction of tumor volume in mice harboring c-MET shRNA-knockdown MES-SA/Dx5 cells was clearly demonstrated. Our study demonstrated that downregulation of c-MET by shRNA-induced apoptosis in a multidrug resistance cell line.

  14. Genome-wide association data suggest ABCB1 and immune-related gene sets may be involved in adult antisocial behavior.

    PubMed

    Salvatore, J E; Edwards, A C; McClintick, J N; Bigdeli, T B; Adkins, A; Aliev, F; Edenberg, H J; Foroud, T; Hesselbrock, V; Kramer, J; Nurnberger, J I; Schuckit, M; Tischfield, J A; Xuei, X; Dick, D M

    2015-04-28

    Adult antisocial behavior (AAB) is moderately heritable, relatively common and has adverse consequences for individuals and society. We examined the molecular genetic basis of AAB in 1379 participants from a case-control study in which the cases met criteria for alcohol dependence. We also examined whether genes of interest were expressed in human brain. AAB was measured using a count of the number of Antisocial Personality Disorder criteria endorsed under criterion A from the Diagnostic and Statistical Manual of Mental Disorders, 4th Edition (DSM-IV). Participants were genotyped on the Illumina Human 1M BeadChip. In total, all single-nucleotide polymorphisms (SNPs) accounted for 25% of the variance in AAB, although this estimate was not significant (P=0.09). Enrichment tests indicated that more significantly associated genes were over-represented in seven gene sets, and most were immune related. Our most highly associated SNP (rs4728702, P=5.77 × 10(-7)) was located in the protein-coding adenosine triphosphate-binding cassette, sub-family B (MDR/TAP), member 1 (ABCB1). In a gene-based test, ABCB1 was genome-wide significant (q=0.03). Expression analyses indicated that ABCB1 was robustly expressed in the brain. ABCB1 has been implicated in substance use, and in post hoc tests we found that variation in ABCB1 was associated with DSM-IV alcohol and cocaine dependence criterion counts. These results suggest that ABCB1 may confer risk across externalizing behaviors, and are consistent with previous suggestions that immune pathways are associated with externalizing behaviors. The results should be tempered by the fact that we did not replicate the associations for ABCB1 or the gene sets in a less-affected independent sample.

  15. Genome-wide association data suggest ABCB1 and immune-related gene sets may be involved in adult antisocial behavior

    PubMed Central

    Salvatore, J E; Edwards, A C; McClintick, J N; Bigdeli, T B; Adkins, A; Aliev, F; Edenberg, H J; Foroud, T; Hesselbrock, V; Kramer, J; Nurnberger, J I; Schuckit, M; Tischfield, J A; Xuei, X; Dick, D M

    2015-01-01

    Adult antisocial behavior (AAB) is moderately heritable, relatively common and has adverse consequences for individuals and society. We examined the molecular genetic basis of AAB in 1379 participants from a case–control study in which the cases met criteria for alcohol dependence. We also examined whether genes of interest were expressed in human brain. AAB was measured using a count of the number of Antisocial Personality Disorder criteria endorsed under criterion A from the Diagnostic and Statistical Manual of Mental Disorders, 4th Edition (DSM-IV). Participants were genotyped on the Illumina Human 1M BeadChip. In total, all single-nucleotide polymorphisms (SNPs) accounted for 25% of the variance in AAB, although this estimate was not significant (P=0.09). Enrichment tests indicated that more significantly associated genes were over-represented in seven gene sets, and most were immune related. Our most highly associated SNP (rs4728702, P=5.77 × 10−7) was located in the protein-coding adenosine triphosphate-binding cassette, sub-family B (MDR/TAP), member 1 (ABCB1). In a gene-based test, ABCB1 was genome-wide significant (q=0.03). Expression analyses indicated that ABCB1 was robustly expressed in the brain. ABCB1 has been implicated in substance use, and in post hoc tests we found that variation in ABCB1 was associated with DSM-IV alcohol and cocaine dependence criterion counts. These results suggest that ABCB1 may confer risk across externalizing behaviors, and are consistent with previous suggestions that immune pathways are associated with externalizing behaviors. The results should be tempered by the fact that we did not replicate the associations for ABCB1 or the gene sets in a less-affected independent sample. PMID:25918995

  16. Impact of Genetic Polymorphisms of ABCB1 (MDR1, P-Glycoprotein) on Drug Disposition and Potential Clinical Implications: Update of the Literature.

    PubMed

    Wolking, Stefan; Schaeffeler, Elke; Lerche, Holger; Schwab, Matthias; Nies, Anne T

    2015-07-01

    ATP-binding cassette transporter B1 (ABCB1; P-glycoprotein; multidrug resistance protein 1) is an adenosine triphosphate (ATP)-dependent efflux transporter located in the plasma membrane of many different cell types. Numerous structurally unrelated compounds, including drugs and environmental toxins, have been identified as substrates. ABCB1 limits the absorption of xenobiotics from the gut lumen, protects sensitive tissues (e.g. the brain, fetus and testes) from xenobiotics and is involved in biliary and renal secretion of its substrates. In recent years, a large number of polymorphisms of the ABCB1 [ATP-binding cassette, sub-family B (MDR/TAP), member 1] gene have been described. The variants 1236C>T (rs1128503, p.G412G), 2677G>T/A (rs2032582, p.A893S/T) and 3435C>T (rs1045642, p.I1145I) occur at high allele frequencies and create a common haplotype; therefore, they have been most widely studied. This review provides an overview of clinical studies published between 2002 and March 2015. In summary, the effect of ABCB1 variation on P-glycoprotein expression (messenger RNA and protein expression) and/or activity in various tissues (e.g. the liver, gut and heart) appears to be small. Although polymorphisms and haplotypes of ABCB1 have been associated with alterations in drug disposition and drug response, including adverse events with various ABCB1 substrates in different ethnic populations, the results have been majorly conflicting, with limited clinical relevance. Future research activities are warranted, considering a deep-sequencing approach, as well as well-designed clinical studies with appropriate sample sizes to elucidate the impact of rare ABCB1 variants and their potential consequences for effect sizes.

  17. Fentanyl Enhances Hepatotoxicity of Paclitaxel via Inhibition of CYP3A4 and ABCB1 Transport Activity in Mice.

    PubMed

    Xie, Jing-Dun; Huang, Yang; Chen, Dong-Tai; Pan, Jia-Hao; Bi, Bing-Tian; Feng, Kun-Yao; Huang, Wan; Zeng, Wei-An

    2015-01-01

    Fentanyl, a potent opioid analgesic that is used to treat cancer pain, is commonly administered with paclitaxel in advanced tumors. However, the effect of fentanyl on the hepatotoxicity of paclitaxel and its potential mechanism of action is not well studied. The purpose of this study was to investigate the effect of fentanyl on the hepatotoxicity of paclitaxel and its potential mechanisms of action. Pharmacokinetic parameters of paclitaxel were tested using reversed phase high-performance liquid chromatography (RP-HPLC). Aspartate transaminase (AST), alanine aminotransferase (ALT), and mouse liver histopathology were examined. Moreover, the cytotoxicity of anti-carcinogens was examined using 1-(4, 5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT), and the intracellular accumulation of doxorubicin and rhodamine 123 was detected by flow cytometry. Furthermore, the expression of ABCB1 and the activity of ABCB1 ATPase and CYP3A4 were also examined. In this study, the co-administration of fentanyl and paclitaxel prolonged the half-life (t1/2) of paclitaxel from 1.455 hours to 2.344 hours and decreased the clearance (CL) from 10.997 ml/h to 7.014 ml/h in mice. Fentanyl significantly increased the levels of ALT in mice to 88.2 U/L, which is more than 2-fold higher than the level detected in the control group, and it increased the histological damage in mouse livers. Furthermore, fentanyl enhanced the cytotoxicity of anti-carcinogens that are ABCB1 substrates and increased the accumulation of doxorubicin and rhodamine 123. Additionally, fentanyl stimulated ABCB1 ATPase activity and inhibited CYP3A4 activity in the liver microsomes of mice. Our study indicates that the obvious hepatotoxicity during this co-administration was due to the inhibition of CYP3A4 activity and ABCB1 transport activity. These findings suggested that the accumulation-induced hepatotoxicity of paclitaxel when it is combined with fentanyl should be avoided.

  18. Interrogation of multidrug resistance (MDR1) P-glycoprotein (ABCB1) expression in human pancreatic carcinoma cells: correlation of 99mTc-Sestamibi uptake with western blot analysis.

    PubMed

    Harpstrite, Scott E; Gu, Hannah; Natarajan, Radhika; Sharma, Vijay

    2014-10-01

    Histopathological studies indicate that ∼63% of pancreatic tumors express multidrug resistance (MDR1) P-glycoprotein (Pgp) and its polymorphic variants. However, Pgp expression detected at the mRNA or protein level does not always correlate with functional transport activity. Because Pgp transport activity is affected by specific mutations and the phosphorylation state of the protein, altered or less active forms of Pgp may also be detected by PCR or immunohistochemistry, which do not accurately reflect the status of tumor cell resistance. To interrogate the status of the functional expression of MDR1 Pgp in MiaPaCa-2 and PANC-1 cells, cellular transport studies using Tc-Sestamibi were performed and correlated with western blot analysis. Biochemical transport assays in human pancreatic carcinoma MiaPaCa-2 and PANC-1 cells, human epidermal carcinoma drug-sensitive KB-3-1 cells, and human breast carcinoma MCF-7 cells (negative controls), and human epidermal carcinoma drug-resistant KB-8-5 cells, human breast carcinoma stably transfected with Pgp MCF-7/MDR1Pgp cells, and liver carcinoma HepG2 cells (positive controls) were performed. Protein levels were determined using a monoclonal antibody C219. Tc-Sestamibi demonstrates accumulation in human pancreatic carcinoma MiaPaCa-2 and PANC-1 cells. Uptake profiles are not affected by treatment with LY335979, a Pgp inhibitor, and correlate with western blot analysis. These cellular transport studies indicate an absence of Pgp at a functional level in MiaPaCa-2 and PANC-1 cells. Because major pancreatic tumors originate from the pancreatic duct and Tc-Sestamibi undergoes a dominant hepatobiliary mode of excretion, it would not be a sensitive probe for imaging pancreatic adenocarcinomas. Following interrogation of the functional status of Pgp in other pancreatic carcinoma cells, chemotherapeutic drugs that are also MDR1 substrates could offer alternative therapeutics for treating pancreatic adenocarcinomas.

  19. Effect of ceritinib (LDK378) on enhancement of chemotherapeutic agents in ABCB1 and ABCG2 overexpressing cells in vitro and in vivo

    PubMed Central

    Hu, Jing; Zhang, Xu; Wang, Fang; Wang, Xiaokun; Yang, Ke; Xu, Meng; To, Kenneth K.W.

    2015-01-01

    Multidrug resistance (MDR) is the leading cause of treatment failure in cancer chemotherapy. The overexpression of ATP-binding cassette (ABC) transporters, particularly ABCB1, ABCC1 and ABCG2, play a key role in mediating MDR by pumping anticancer drugs out from cancer cells. Ceritinib (LDK378) is a second-generation tyrosine kinase inhibitor of anaplastic lymphoma kinase (ALK) currently in phase III clinical trial for the treatment of non-small cell lung cancer. Here, we found that ceritinib remarkably enhanced the efficacy of chemotherapeutic drugs in ABCB1 or ABCG2 over-expressing cancer cells in vitro and in vivo. Ceritinib significantly increased the intracellular accumulation of chemotherapeutic agents such as doxorubicin (DOX) by inhibiting ABCB1 or ABCG2-mediated drug efflux in the transporters-overexpressing cells. Mechanistically, ceritinib is likely a competitive inhibitor of ABCB1 and ABCG2 because it competed with [125I]-iodoarylazidoprazosin for photo affinity labeling of the transporters. On the other hand, at the transporters-inhibiting concentrations, ceritinib did not alter the expression level of ABCB1 and ABCG2, and phosphorylation status of AKT and ERK1/2. Thus the findings advocate further clinical investigation of combination chemotherapy of ceritinib and other conventional chemotherapeutic drugs in chemo-refractory cancer patients. PMID:26556876

  20. Genomewide analysis of ABCBs with a focus on ABCB1 and ABCB19 in Malus domestica.

    PubMed

    Ma, Juan Juan; Han, Mingyu

    2016-03-01

    The B subfamily of ATP-binding cassette (ABC) proteins (ABCB) plays a vital role in auxin efflux. However, no systematic study has been done in apple. In this study, we performed genomewide identification and expression analyses of the ABCB family in Malus domestica for the first time. We identified a total of 25 apple ABCBs that were divided into three clusters based on the phylogenetic analysis. Most ABCBs within the same cluster demonstrated a similar exon-intron organization. Additionally, the digital expression profiles of ABCB genes shed light on their functional divergence. ABCB1 and ABCB19 are two well-studied auxin efflux carrier genes, and we found that their expression levels are higher in young shoots of M106 than in young shoots ofM9. Since young shoots are the main source of auxin synthesis and auxin efflux involves in tree height control. This suggests that ABCB1 and ABCB19 may also take a part in the auxin efflux and tree height control in apple. PMID:27019441

  1. A novel screening strategy to identify ABCB1 substrates and inhibitors.

    PubMed

    von Richter, Oliver; Glavinas, Hristos; Krajcsi, Peter; Liehner, Stephanie; Siewert, Beate; Zech, Karl

    2009-01-01

    We tested the hypothesis whether data on ABCB1 ATPase activity and passive permeability can be used in combination to identify ABCB1 substrates and inhibitors. We determined passive permeability using an artificial membrane permeability assay (HDM-PAMPA) and ABCB1 function, i.e., vanadate-sensitive ATPase activity for a training set (40 INN drugs) and a validation set (26 development compounds). In parallel experiments, we determined ABCB1 function, i.e., vectorial transport in a Caco-2 cell monolayer, and ABCB1 inhibition, i.e., calcein AM extrusion out of K562-MDR cells, to cross-validate the results with cellular assays. We found that compounds that did not modulate ABCB1-ATPase did also not affect calcein AM extrusion and were not actively transported by ABCB1 in Caco-2 cell monolayers. The results corroborated the effect of passive permeability as an important covariate of active transport: active transport in Caco-2 monolayer was only apparent for compounds showing low passive permeability (<5.0 cmx10(-6)/s) in the HDM-PAMPA assay whereas compounds with high passive permeability (>50 cmx10(-6)/s) were shown to inhibit calcein AM efflux with IC50 values close to their respective Km value obtained for ABCB1-ATPase. The use of HDM-PAMPA in combination with ABCB1-ATPase offers a simple, inexpensive experimental approach capable of identifying ABCB1 inhibitors as well as transported substrates.

  2. Novel understanding of ABC transporters ABCB1/MDR/P-glycoprotein, ABCC2/MRP2, and ABCG2/BCRP in colorectal pathophysiology

    PubMed Central

    Andersen, Vibeke; Svenningsen, Katrine; Knudsen, Lina Almind; Hansen, Axel Kornerup; Holmskov, Uffe; Stensballe, Allan; Vogel, Ulla

    2015-01-01

    ABCB1 expression identifies a subpopulation of pro-inflammatory Th17 cells which were resistant to treatment with glucocorticoids. The evidence for the involvement of ABCC2 and ABCG2 in colonic pathophysiology was weak. CONCLUSION: ABCB1, diet, and gut microbes mutually interact in colonic inflammation, a well-known risk factor for CRC. Further insight may be translated into preventive and treatment strategies. PMID:26557010

  3. Identification of a putatively multixenobiotic resistance related Abcb1 transporter in amphipod species endemic to the highly pristine Lake Baikal.

    PubMed

    Pavlichenko, Vasiliy V; Protopopova, Marina V; Timofeyev, Maxim; Luckenbach, Till

    2015-04-01

    The fauna of Lake Baikal in Eastern Siberia, the largest freshwater body on Earth, is characterized by high degrees of biodiversity and endemism. Amphipods, a prominent taxon within the indigenous fauna, occur in an exceptionally high number of endemic species. Considering the specific water chemistry of Lake Baikal with extremely low levels of potentially toxic natural organic compounds, it seems conceivable that certain adaptions to adverse environmental factors are missing in endemic species, such as cellular defense mechanisms mitigating toxic effects of chemicals. The degree to which the endemic fauna is affected by the recently occurring anthropogenic water pollution of Lake Baikal may depend on the existence of such cellular defense mechanisms in those species. We here show that endemic amphipods express transcripts for Abcb1, a major component of the cellular multixenobiotic resistance (MXR) defense against toxic chemicals. Based on a partial abcb1 cDNA sequence from Gammarus lacustris, an amphipod species common across Northern Eurasia but only rarely found in Lake Baikal, respective homologous sequences were cloned from five amphipods endemic to Lake Baikal, Eulimnogammarus verrucosus, E. vittatus, E. cyaneus, E. marituji, and Gmelinoides fasciatus, confirming that abcb1 is transcribed in those species. The effects of thermal (25 °C) and chemical stress (1-2 mg L(-1) phenanthrene) in short-term exposures (up to 24 h) on transcript levels of abcb1 and heat shock protein 70 (hsp70), used as a proxy for cellular stress in the experiments, were exemplarily examined in E. verrucosus, E. cyaneus, and Gammarus lacustris. Whereas increases of abcb1 transcripts upon treatments occurred only in the Baikalian species E. verrucosus and E. cyaneus but not in Gammarus lacustris, changes of hsp70 transcript levels were seen in all three species. At least for species endemic to Lake Baikal, the data thus indicate that regulation of the identified amphipod abcb1 is

  4. High-throughput flow cytometry to detect selective inhibitors of ABCB1, ABCC1, and ABCG2 transporters.

    PubMed

    Ivnitski-Steele, Irena; Larson, Richard S; Lovato, Debbie M; Khawaja, Hadya M; Winter, Stuart S; Oprea, Tudor I; Sklar, Larry A; Edwards, Bruce S

    2008-04-01

    Up-regulation of pump (transporter) expression and selection of resistant cancer cells result in cancer multidrug resistance to diverse substrates of these transporters. While more than 48 members of the ATP binding cassette (ABC) transporter superfamily have been identified, up to now only three human ABC transporters-ABCB1, ABCC1, and ABCG2-have unambiguously been shown to contribute to cancer multidrug resistance. The use of low-toxicity and high-specificity agents as a targeted transporter inhibition strategy is necessary to effectively overcome multiple drug resistance. An objective of the present studies was to develop and validate HyperCyt (IntelliCyt, Albuquerque, NM) flow cytometry high-throughput screeening assays to assess the specificity of test compounds that inhibited transporters as an integral part of the screen. Two separate duplex assays were constructed: one in which ABCB1 and ABCG2 transporters were evaluated in parallel using fluorescent J-aggregate-forming lipophilic cation 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide as substrate, and the other in which ABCB1 and ABCC1 transporters were evaluated in parallel using fluorescent calcein acetoxymethyl ester as substrate. ABCB1-expressing cells were color-coded to allow their distinction from cells expressing the alternate transporter. The assays were validated in a screen of the Prestwick Chemical Library (Illkirch, France). Three novel selective inhibitors of the ABCC1 transporter were identified in the screen, and the activity of each was confirmed in follow-up chemosensitivity shift and reversal studies. This high-throughput screening assay provides an efficient approach for identifying selective inhibitors of individual ABC transporters, promising as probes of transporter function and therapeutic tools for treating chemotherapy-resistant cancers. PMID:18205550

  5. Structural determinants of peripheral O-arylcarbamate FAAH inhibitors render them dual substrates for Abcb1 and Abcg2 and restrict their access to the brain

    PubMed Central

    Moreno-Sanz, Guillermo; Barrera, Borja; Armirotti, Andrea; Bertozzi, Sine M.; Scarpelli, Rita; Bandiera, Tiziano; Prieto, Julio G.; Duranti, Andrea; Tarzia, Giorgio; Merino, Gracia

    2014-01-01

    The blood-brain barrier (BBB) is the main entry route for chemicals into the mammalian central nervous system (CNS). Two transmembrane transporters of the ATP-binding cassette (ABC) family – Breast Cancer Resistance Protein (ABCG2 in humans, Abcg2 in rodents) and P-glycoprotein (ABCB1 in humans, Abcb1 in rodents) – play a key role in mediating this process. Pharmacological and genetic evidence suggests that Abcg2 prevents CNS access to a group of highly potent and selective O-arylcarbamate fatty-acid amidohydrolase (FAAH) inhibitors, which include the compound URB937 (cyclohexylcarbamic acid 3′-carbamoyl-6-hydroxybiphenyl-3-yl ester). To define structure-activity relationships of the interaction of these molecules with Abcg2, in the present study we tested various peripherally restricted and non-restricted O-arylcarbamate FAAH inhibitors for their ability to serve as transport substrates in monolayer cultures of Madin-Darby Canine Kidney-II (MDCKII) cells over-expressing Abcg2. Surprisingly, we found that the majority of compounds tested – even those able to enter the CNS in vivo – were substrates for Abcg2 in vitro. Additional experiments in MDCKII cells overexpressing ABCB1 revealed that only those compounds that were dual substrates for ABCB1 and Abcg2 in vitro were also peripherally restricted in vivo. The extent of such restriction seems to depend upon other physicochemical features of the compounds, in particular the polar surface area. Consistent with these in vitro results, we found that URB937 readily enters the brain in dual knockout mice lacking both Abcg2 and Abcb1, whereas it is either partially or completely excluded from the brain of mice lacking either transporter alone. The results suggest that Abcg2 and Abcb1 act together to restrict the access of URB937 to the CNS. PMID:24993496

  6. The Alterations in the Expression and Function of P-Glycoprotein in Vitamin A-Deficient Rats as well as the Effect of Drug Disposition in Vivo.

    PubMed

    Wang, Yubang; Qin, Heng; Zhang, Chengxiang; Huan, Fei; Yan, Ting; Zhang, Lulu

    2015-12-29

    This study was aimed to investigate whether vitamin A deficiency could alter P-GP expression and function in tissues of rats and whether such effects affected the drug distribution in vivo of vitamin A-deficient rats. We induced vitamin A-deficient rats by giving them a vitamin A-free diet for 12 weeks. Then, Abcb1/P-GP expression was evaluated by qRT-PCR and Western blot. qRT-PCR analysis revealed that Abcb1a mRNA levels were increased in hippocampus and liver. In kidney, it only showed an upward trend. Abcb1b mRNA levels were increased in hippocampus, but decreased in cerebral cortex, liver and kidney. Western blot results were in good accordance with the alterations of Abcb1b mRNA levels. P-GP function was investigated through tissue distribution and body fluid excretion of rhodamine 123 (Rho123), and the results proclaimed that P-GP activities were also in good accordance with P-GP expression in cerebral cortex, liver and kidney. The change of drug distribution was also investigated through the tissue distribution of vincristine, and the results showed a significantly upward trend in all indicated tissues of vitamin A-deficient rats. In conclusion, vitamin A deficiency may alter Abcb1/P-GP expression and function in rat tissues, and the alterations may increase drug activity/toxicity through the increase of tissue accumulation.

  7. P-glycoprotein (ABCB1) transports the primary active tamoxifen metabolites endoxifen and 4-hydroxytamoxifen and restricts their brain penetration.

    PubMed

    Iusuf, Dilek; Teunissen, Sebastiaan F; Wagenaar, Els; Rosing, Hilde; Beijnen, Jos H; Schinkel, Alfred H

    2011-06-01

    P-glycoprotein (P-gp, ABCB1) is a highly efficient drug efflux pump expressed in brain, liver, and small intestine, but also in tumor cells, that affects pharmacokinetics and confers therapy resistance for many anticancer drugs. The aim of this study was to investigate the impact of P-gp on tamoxifen and its primary active metabolites, 4-hydroxytamoxifen, N-desmethyltamoxifen, and endoxifen. We used in vitro transport assays and Abcb1a/1b(-/-) mice to investigate the impact of P-gp on the oral availability and brain penetration of tamoxifen and its metabolites. Systemic exposure of tamoxifen and its metabolites after oral administration of tamoxifen (50 mg/kg) was not changed in the absence of P-gp. However, brain accumulation of tamoxifen, 4-hydroxytamoxifen, and N-desmethyltamoxifen were modestly, but significantly (1.5- to 2-fold), increased. Endoxifen, however, displayed a 9-fold higher brain penetration at 4 h after administration. Endoxifen was transported by P-gp in vitro. Upon direct oral administration of endoxifen (20 mg/kg), systemic exposure was slightly decreased in Abcb1a/1b(-/-) mice, but brain accumulation of endoxifen was dramatically increased (up to 23-fold at 4 h after administration). Shortly after high-dose intravenous administration (5 or 20 mg/kg), endoxifen brain accumulation was increased only 2-fold in Abcb1a/1b(-/-) mice compared with wild-type mice, suggesting a partial saturation of P-gp at the blood-brain barrier. Endoxifen, the clinically most relevant metabolite of tamoxifen, is a P-gp substrate in vitro and in vivo, where P-gp limits its brain penetration. P-gp might thus be relevant for tamoxifen/endoxifen resistance of P-gp-positive breast cancer and tumors positioned behind a functional blood-brain barrier. PMID:21378205

  8. Sildenafil reverses ABCB1- and ABCG2-mediated chemotherapeutic drug resistance.

    PubMed

    Shi, Zhi; Tiwari, Amit K; Shukla, Suneet; Robey, Robert W; Singh, Satyakam; Kim, In-Wha; Bates, Susan E; Peng, Xingxiang; Abraham, Ioana; Ambudkar, Suresh V; Talele, Tanaji T; Fu, Li-Wu; Chen, Zhe-Sheng

    2011-04-15

    Sildenafil is a potent and selective inhibitor of the type 5 cGMP (cyclic guanosine 3',5'-monophosphate)-specific phosphodiesterase that is used clinically to treat erectile dysfunction and pulmonary arterial hypertension. Here, we report that sildenafil has differential effects on cell surface ABC transporters such as ABCB1, ABCC1, and ABCG2 that modulate intracompartmental and intracellular concentrations of chemotherapeutic drugs. In ABCB1-overexpressing cells, nontoxic doses of sildenafil inhibited resistance and increased the effective intracellular concentration of ABCB1 substrate drugs such as paclitaxel. Similarly, in ABCG2-overexpressing cells, sildenafil inhibited resistance to ABCG2 substrate anticancer drugs, for example, increasing the effective intracellular concentration of mitoxantrone or the fluorescent compound BODIPY-prazosin. Sildenafil also moderately inhibited the transport of E(2)17βG and methotrexate by the ABCG2 transporter. Mechanistic investigations revealed that sildenafil stimulated ABCB1 ATPase activity and inhibited photolabeling of ABCB1 with [(125)I]-iodoarylazidoprazosin (IAAP), whereas it only slightly stimulated ABCG2 ATPase activity and inhibited photolabeling of ABCG2 with [(125)I]-IAAP. In contrast, sildenafil did not alter the sensitivity of parental, ABCB1-, or ABCG2-overexpressing cells to non-ABCB1 and non-ABCG2 substrate drugs, nor did sildenafil affect the function of another ABC drug transporter, ABCC1. Homology modeling predicted the binding conformation of sildenafil within the large cavity of the transmembrane region of ABCB1. Overall, we found that sildenafil inhibits the transporter function of ABCB1 and ABCG2, with a stronger effect on ABCB1. Our findings suggest a possible strategy to enhance the distribution and potentially the activity of anticancer drugs by jointly using a clinically approved drug with known side effects and drug-drug interactions. PMID:21402712

  9. Fentanyl Enhances Hepatotoxicity of Paclitaxel via Inhibition of CYP3A4 and ABCB1 Transport Activity in Mice

    PubMed Central

    Pan, Jia-Hao; Bi, Bing-Tian; Feng, Kun-Yao; Huang, Wan; Zeng, Wei-An

    2015-01-01

    Fentanyl, a potent opioid analgesic that is used to treat cancer pain, is commonly administered with paclitaxel in advanced tumors. However, the effect of fentanyl on the hepatotoxicity of paclitaxel and its potential mechanism of action is not well studied. The purpose of this study was to investigate the effect of fentanyl on the hepatotoxicity of paclitaxel and its potential mechanisms of action. Pharmacokinetic parameters of paclitaxel were tested using reversed phase high-performance liquid chromatography (RP-HPLC). Aspartate transaminase (AST), alanine aminotransferase (ALT), and mouse liver histopathology were examined. Moreover, the cytotoxicity of anti-carcinogens was examined using 1-(4, 5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT), and the intracellular accumulation of doxorubicin and rhodamine 123 was detected by flow cytometry. Furthermore, the expression of ABCB1 and the activity of ABCB1 ATPase and CYP3A4 were also examined. In this study, the co-administration of fentanyl and paclitaxel prolonged the half-life (t1/2) of paclitaxel from 1.455 hours to 2.344 hours and decreased the clearance (CL) from 10.997 ml/h to 7.014 ml/h in mice. Fentanyl significantly increased the levels of ALT in mice to 88.2 U/L, which is more than 2-fold higher than the level detected in the control group, and it increased the histological damage in mouse livers. Furthermore, fentanyl enhanced the cytotoxicity of anti-carcinogens that are ABCB1 substrates and increased the accumulation of doxorubicin and rhodamine 123. Additionally, fentanyl stimulated ABCB1 ATPase activity and inhibited CYP3A4 activity in the liver microsomes of mice. Our study indicates that the obvious hepatotoxicity during this co-administration was due to the inhibition of CYP3A4 activity and ABCB1 transport activity. These findings suggested that the accumulation-induced hepatotoxicity of paclitaxel when it is combined with fentanyl should be avoided. PMID:26633878

  10. Xenobiotic Metabolizing Enzyme and Transporter Gene Expression in Primary Cultures of Human Hepatocytes Modulated by ToxCast Chemicals

    EPA Science Inventory

    ToxCast chemicals were assessed for induction or suppression of xenobiotic metabolizing enzyme and transporter gene expression using primary human hepatocytes. The mRNA levels of 14 target and 2 control genes were measured: ABCB1, ABCB11, ABCG2, SLCO1B1, CYP1A1, CYP1A2, CYP2B6, C...

  11. Modulation of Xenobiotic Metabolizing Enzyme and Transporter Gene Expression in Primary Cultures of Human Hepatocytes by ToxCast Chemicals

    EPA Science Inventory

    ToxCast chemicals were assessed for induction or suppression of xenobiotic metabolizing enzyme and transporter gene expression using primary human hepatocytes. The mRNA levels of 14 target and 2 control genes were measured: ABCB1, ABCB11, ABCG2, SLCO1B1, CYP1A1, CYP1A2, CYP2B6, C...

  12. Abcb1 in Pigs: Molecular cloning, tissues distribution, functional analysis, and its effect on pharmacokinetics of enrofloxacin.

    PubMed

    Guo, Tingting; Huang, Jinhu; Zhang, Hongyu; Dong, Lingling; Guo, Dawei; Guo, Li; He, Fang; Bhutto, Zohaib Ahmed; Wang, Liping

    2016-01-01

    P-glycoprotein (P-gp) is one of the best-known ATP-dependent efflux transporters, contributing to differences in pharmacokinetics and drug-drug interactions. Until now, studies on pig P-gp have been scarce. In our studies, the full-length porcine P-gp cDNA was cloned and expressed in a Madin-Darby Canine Kidney (MDCK) cell line. P-gp expression was then determined in tissues and its role in the pharmacokinetics of oral enrofloxacin in pigs was studied. The coding region of pig Abcb1 gene was 3,861 bp, encoding 1,286 amino acid residues (Mw = 141,966). Phylogenetic analysis indicated a close evolutionary relationship between porcine P-gp and those of cow and sheep. Pig P-gp was successfully stably overexpressed in MDCK cells and had efflux activity for rhodamine 123, a substrate of P-gp. Tissue distribution analysis indicated that P-gp was highly expressed in brain capillaries, small intestine, and liver. In MDCK-pAbcb1 cells, enrofloxacin was transported by P-gp with net efflux ratio of 2.48 and the efflux function was blocked by P-gp inhibitor verapamil. High expression of P-gp in the small intestine could modify the pharmacokinetics of orally administrated enrofloxacin by increasing the Cmax, AUC and Ka, which was demonstrated using verapamil, an inhibitor of P-gp. PMID:27572343

  13. Abcb1 in Pigs: Molecular cloning, tissues distribution, functional analysis, and its effect on pharmacokinetics of enrofloxacin

    PubMed Central

    Guo, Tingting; Huang, Jinhu; Zhang, Hongyu; Dong, Lingling; Guo, Dawei; Guo, Li; He, Fang; Bhutto, Zohaib Ahmed; Wang, Liping

    2016-01-01

    P-glycoprotein (P-gp) is one of the best-known ATP-dependent efflux transporters, contributing to differences in pharmacokinetics and drug-drug interactions. Until now, studies on pig P-gp have been scarce. In our studies, the full-length porcine P-gp cDNA was cloned and expressed in a Madin-Darby Canine Kidney (MDCK) cell line. P-gp expression was then determined in tissues and its role in the pharmacokinetics of oral enrofloxacin in pigs was studied. The coding region of pig Abcb1 gene was 3,861 bp, encoding 1,286 amino acid residues (Mw = 141,966). Phylogenetic analysis indicated a close evolutionary relationship between porcine P-gp and those of cow and sheep. Pig P-gp was successfully stably overexpressed in MDCK cells and had efflux activity for rhodamine 123, a substrate of P-gp. Tissue distribution analysis indicated that P-gp was highly expressed in brain capillaries, small intestine, and liver. In MDCK-pAbcb1 cells, enrofloxacin was transported by P-gp with net efflux ratio of 2.48 and the efflux function was blocked by P-gp inhibitor verapamil. High expression of P-gp in the small intestine could modify the pharmacokinetics of orally administrated enrofloxacin by increasing the Cmax, AUC and Ka, which was demonstrated using verapamil, an inhibitor of P-gp. PMID:27572343

  14. Substrate-specific effects of pirinixic acid derivatives on ABCB1-mediated drug transport

    PubMed Central

    Michaelis, Martin; Rothweiler, Florian; Wurglics, Mario; Aniceto, Natália; Dittrich, Michaela; Zettl, Heiko; Wiese, Michael; Wass, Mark; Ghafourian, Taravat; Schubert-Zsilavecz, Manfred; Cinatl, Jindrich

    2016-01-01

    Pirinixic acid derivatives, a new class of drug candidates for a range of diseases, interfere with targets including PPARα, PPARγ, 5-lipoxygenase (5-LO), and microsomal prostaglandin and E2 synthase-1 (mPGES1). Since 5-LO, mPGES1, PPARα, and PPARγ represent potential anti-cancer drug targets, we here investigated the effects of 39 pirinixic acid derivatives on prostate cancer (PC-3) and neuroblastoma (UKF-NB-3) cell viability and, subsequently, the effects of selected compounds on drug-resistant neuroblastoma cells. Few compounds affected cancer cell viability in low micromolar concentrations but there was no correlation between the anti-cancer effects and the effects on 5-LO, mPGES1, PPARα, or PPARγ. Most strikingly, pirinixic acid derivatives interfered with drug transport by the ATP-binding cassette (ABC) transporter ABCB1 in a drug-specific fashion. LP117, the compound that exerted the strongest effect on ABCB1, interfered in the investigated concentrations of up to 2μM with the ABCB1-mediated transport of vincristine, vinorelbine, actinomycin D, paclitaxel, and calcein-AM but not of doxorubicin, rhodamine 123, or JC-1. In silico docking studies identified differences in the interaction profiles of the investigated ABCB1 substrates with the known ABCB1 binding sites that may explain the substrate-specific effects of LP117. Thus, pirinixic acid derivatives may offer potential as drug-specific modulators of ABCB1-mediated drug transport. PMID:26887049

  15. Association of ABCB1 and ABCG2 single nucleotide polymorphisms with clinical findings and response to chemotherapy treatments in Kurdish patients with breast cancer.

    PubMed

    Ghafouri, Houshiyar; Ghaderi, Bayazid; Amini, Sabrieh; Nikkhoo, Bahram; Abdi, Mohammad; Hoseini, Abdolhakim

    2016-06-01

    The possible interaction between gene polymorphisms and risk of cancer progression is very interesting. Polymorphisms in multi-drug resistance genes have an important role in response to anti-cancer drugs. The present study was aimed to evaluate the possible effects of ABCB1 C3435T and ABCG2 C421A single nucleotide polymorphisms on clinical and pathological outcomes of Kurdish patients with breast cancer. One hundred breast cancer patients and 200 healthy controls were enrolled in this case-control study. Clinical and pathological findings of all individuals were reported, and immunohistochemistry staining was used to assess the tissue expression of specific breast cancer proteins. The ABCB1 C3435T and ABCG2 C421 genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism method (PCR-RFLP). The distribution of different genotypes between patient and control groups was only significant for ABCG2 C421A. A allele of ABCG2 C421A polymorphisms were significantly higher in patients than in controls. Patients with AA genotype of ABCG2 C421A were at higher risk of progressing breast cancer. Patients with A allele of ABCG2 had complete response to chemotherapeutic agents. There was no statistically significant association between ABCB1 C3435T and ABCG2 C421A polymorphisms and tissue expression of ER, PR, Her2/neu, and Ki67. The ABCB1 C3435T has no correlation with clinical findings and treatment with chemotherapy drugs. The A allele of ABCG2 C421A may be a risk factor for progression of breast cancer in Kurdish patients. In addition, breast cancer patients with C allele of this polymorphism have weaker response to treatments with anthracyclines and Paclitaxol.

  16. Association between ABCB1 genetic polymorphism and the effect on epilepsy following phenytoin treatment

    PubMed Central

    Sun, Fei; Cao, Bo-Qiang; Wang, Bo; Wu, Shi-Qiang; Jiang, De-Hua

    2016-01-01

    The aim of the study was to analyze the effect of ABCB1 genetic polymorphisms on the efficacy of phenytoin (PHT) treatment in epilepsy patients. In total, 200 epilepsy patients who were administered PHT were divided into the responsive and pharmaco-resistance groups depending on the clinical data of PHT treatment in epilepsy patients. The serum concentration of PHT was detected by high-performance liquid chromatography (HPLC). ABCB1 polymorphisms were analyzed by the polymerase chain reaction restriction-fragment length polymorphism method. The C1236T, C3435T and G2677T/A haplotypes were reconstructed for the ABCB1 gene using SHEsis programs. One-way analysis of variance was used for data analysis. In ABCB1 C1236T, the rate of the CC genotype in pharmaco-resistance (17.5%) was higher than that of the responsive group (2.1%), while the rate of the TT genotype in pharmaco-resistance (41.6%) was lower than that of the responsive group (55.4%) (P<0.05). In ABCB1 G2677T/A, the rate of the GG genotype in pharmaco-resistance (29.6%) was higher than that of the responsive group (9.7%), while the rate of the TT genotype in pharmaco-resistance (4.6%) was lower than that of the responsive group (30.4%) (P<0.05). The rate of the TTC haploid in pharmaco-resistance (24.1%) was higher than that of the responsive group (8.8%) (P<0.05). The PHT serum concentration had no statistical significance in the patients with different genotypes. In conclusion, there was no association between ABCB1 genetic polymorphism and PHT serum concentration, although the polymorphisms affected the efficacy of PHT treatment in patients with epilepsy. PMID:27602091

  17. Association between ABCB1 genetic polymorphism and the effect on epilepsy following phenytoin treatment

    PubMed Central

    Sun, Fei; Cao, Bo-Qiang; Wang, Bo; Wu, Shi-Qiang; Jiang, De-Hua

    2016-01-01

    The aim of the study was to analyze the effect of ABCB1 genetic polymorphisms on the efficacy of phenytoin (PHT) treatment in epilepsy patients. In total, 200 epilepsy patients who were administered PHT were divided into the responsive and pharmaco-resistance groups depending on the clinical data of PHT treatment in epilepsy patients. The serum concentration of PHT was detected by high-performance liquid chromatography (HPLC). ABCB1 polymorphisms were analyzed by the polymerase chain reaction restriction-fragment length polymorphism method. The C1236T, C3435T and G2677T/A haplotypes were reconstructed for the ABCB1 gene using SHEsis programs. One-way analysis of variance was used for data analysis. In ABCB1 C1236T, the rate of the CC genotype in pharmaco-resistance (17.5%) was higher than that of the responsive group (2.1%), while the rate of the TT genotype in pharmaco-resistance (41.6%) was lower than that of the responsive group (55.4%) (P<0.05). In ABCB1 G2677T/A, the rate of the GG genotype in pharmaco-resistance (29.6%) was higher than that of the responsive group (9.7%), while the rate of the TT genotype in pharmaco-resistance (4.6%) was lower than that of the responsive group (30.4%) (P<0.05). The rate of the TTC haploid in pharmaco-resistance (24.1%) was higher than that of the responsive group (8.8%) (P<0.05). The PHT serum concentration had no statistical significance in the patients with different genotypes. In conclusion, there was no association between ABCB1 genetic polymorphism and PHT serum concentration, although the polymorphisms affected the efficacy of PHT treatment in patients with epilepsy.

  18. P-glycoprotein in sheep liver and small intestine: gene expression and transport efflux activity.

    PubMed

    Ballent, M; Wilkens, M R; Maté, L; Muscher, A S; Virkel, G; Sallovitz, J; Schröder, B; Lanusse, C; Lifschitz, A

    2013-12-01

    The role of the transporter P-glycoprotein (P-gp) in the disposition kinetics of different drugs therapeutically used in veterinary medicine has been demonstrated. Considering the anatomo-physiological features of the ruminant species, the constitutive expression of P-gp (ABCB1) along the sheep gastrointestinal tract was studied. Additionally, the effect of repeated dexamethasone (DEX) administrations on the ABCB1 gene expression in the liver and small intestine was also assessed. The ABCB1 mRNA expression was determined by real-time quantitative PCR. P-gp activity was evaluated in diffusion chambers to determine the efflux of rhodamine 123 (Rho 123) in the ileum from experimental sheep. The constitutive ABCB1 expression was 65-fold higher in the liver than in the intestine (ileum). The highest ABCB1 mRNA expression along the small intestine was observed in the ileum (between 6- and 120-fold higher). The treatment with DEX did not elicit a significant effect on the P-gp gene expression levels in any of the investigated gastrointestinal tissues. Consistently, no significant differences were observed in the intestinal secretion of Rho 123, between untreated control (Peff S-M = 3.99 × 10(-6)  ± 2.07 × 10(-6) ) and DEX-treated animals (Peff S-M = 6.00 × 10(-6)  ± 2.5 × 10(-6) ). The understanding of the efflux transporters expression and activity along the digestive tract may help to elucidate clinical implications emerging from drug interactions in livestock.

  19. Targeting ABCB1-mediated tumor multidrug resistance by CRISPR/Cas9-based genome editing

    PubMed Central

    Yang, Yang; Qiu, Jian-Ge; Li, Yong; Di, Jin-Ming; Zhang, Wen-Ji; Jiang, Qi-Wei; Zheng, Di-Wei; Chen, Yao; Wei, Meng-Ning; Huang, Jia-Rong; Wang, Kun; Shi, Zhi

    2016-01-01

    The RNA-guided clustered regularly interspaced short palindromic (CRISPR) in combination with a CRISPR-associated nuclease 9 (Cas9) nuclease system is a new rapid and precise technology for genome editing. In the present study, we applied the CRISPR/Cas9 system to target ABCB1 (also named MDR1) gene which encodes a 170 kDa transmembrane glycoprotein (P-glycoprotein/P-gp) transporting multiple types of chemotherapeutic drugs including taxanes, epipodophyllotoxins, vinca alkaloids and anthracyclines out of cells to contribute multidrug resistance (MDR) in cancer cells. Our data showed that knockout of ABCB1 by CRISPR/Cas9 system was succesfully archieved with two target sgRNAs in two MDR cancer cells due to the alteration of genome sequences. Knockout of ABCB1 by CRISPR/Cas9 system significantly enhances the sensitivity of ABCB1 substrate chemotherapeutic agents and the intracellular accumulation of rhodamine 123 and doxorubicin in MDR cancer cells. Although now there are lots of limitations to the application of CRISPR/Cas9 for editing cancer genes in human patients, our study provides valuable clues for the use of the CRISPR/Cas9 technology in the investigation and conquest of cancer MDR. PMID:27725879

  20. Effect of ABCB1 polymorphisms and atorvastatin on sitagliptin pharmacokinetics in healthy volunteers

    PubMed Central

    Aquilante, Christina L.; Wempe, Michael F.; Sidhom, Maha S.; Kosmiski, Lisa A.; Predhomme, Julie A.

    2013-01-01

    Objectives The objectives of this study were to determine if ABCB1 polymorphisms are associated with interindividual variability in sitagliptin pharmacokinetics, and if atorvastatin alters the pharmacokinetic disposition of sitagliptin in healthy volunteers. Methods In this open-label, randomized, two-phase crossover study, healthy volunteers were prospectively stratified according to ABCB1 1236/2677/3435 diplotype (n=9, CGC/CGC; n=10, CGC/TTT; and n=10, TTT/TTT). In one phase, participants received a single 100 mg dose of sitagliptin. In the other phase, participants received 40 mg of atorvastatin for five days, with a single 100 mg dose of sitagliptin administered on day 5. A 24 hour pharmacokinetic study followed each sitagliptin dose, and the study phases were separated by a 14-day washout period. Results Sitagliptin pharmacokinetic parameters did not differ significantly between ABCB1 CGC/CGC, CGC/TTT, and TTT/TTT diplotype groups during the monotherapy phase. Atorvastatin administration did not significantly affect sitagliptin pharmacokinetics, with GMRs (90% CIs) for sitagliptin Cmax, AUC0-∞, CLR, and fe of 0.93 (0.86, 1.01), 0.96 (0.91, 1.01), 1.02 (0.93, 1.12), and 0.98 (0.90, 1.06), respectively. Conclusions ABCB1 CGC/CGC, CGC/TTT, and TTT/TTT diplotypes did not influence sitagliptin pharmacokinetics in healthy volunteers. Furthermore, atorvastatin had no effect on the pharmacokinetics of sitagliptin in the setting of ABCB1 CGC/CGC, CGC/TTT, and TTT/TTT diplotypes. PMID:23407853

  1. mRNA modifications: Dynamic regulators of gene expression?

    PubMed Central

    Hoernes, Thomas Philipp; Hüttenhofer, Alexander; Erlacher, Matthias David

    2016-01-01

    ABSTRACT The expression of a gene is a tightly regulated process and is exerted by a myriad of different mechanisms. Recently, RNA modifications located in coding sequences of mRNAs, have been identified as potential regulators of gene expression. N6-methyladenosine (m6A), 5-methylcytosine (m5C), pseudouridine (Ψ) and N1-methyladenosine (m1A) have been found within open reading frames of mRNAs. The presence of these mRNA modifications has been implicated to modulate the fate of an mRNA, ranging from maturation to its translation and even degradation. However, many aspects concerning the biological functions of mRNA modifications remain elusive. Recently, systematic in vitro studies allowed a first glimpse of the direct interplay of mRNA modifications and the efficiency and fidelity of ribosomal translation. It thereby became evident that the effects of mRNA modifications were, astonishingly versatile, depending on the type, position or sequence context. The incorporation of a single modification could either prematurely terminate protein synthesis, reduce the peptide yield or alter the amino acid sequence identity. These results implicate that mRNA modifications are a powerful mechanism to post-transcriptionally regulate gene expression. PMID:27351916

  2. Pilot PET Study to Assess the Functional Interplay Between ABCB1 and ABCG2 at the Human Blood–Brain Barrier

    PubMed Central

    Bauer, M; Römermann, K; Karch, R; Wulkersdorfer, B; Stanek, J; Philippe, C; Maier‐Salamon, A; Haslacher, H; Jungbauer, C; Wadsak, W; Jäger, W; Löscher, W; Hacker, M; Zeitlinger, M

    2016-01-01

    ABCB1 and ABCG2 work together at the blood–brain barrier (BBB) to limit brain distribution of dual ABCB1/ABCG2 substrates. In this pilot study we used positron emission tomography (PET) to assess brain distribution of two model ABCB1/ABCG2 substrates ([11C]elacridar and [11C]tariquidar) in healthy subjects without (c.421CC) or with (c.421CA) the ABCG2 single‐nucleotide polymorphism (SNP) c.421C>A. Subjects underwent PET scans under conditions when ABCB1 and ABCG2 were functional and during ABCB1 inhibition with high‐dose tariquidar. In contrast to the ABCB1‐selective substrate (R)‐[11C]verapamil, [11C]elacridar and [11C]tariquidar showed only moderate increases in brain distribution during ABCB1 inhibition. This provides evidence for a functional interplay between ABCB1 and ABCG2 at the human BBB and suggests that both ABCB1 and ABCG2 need to be inhibited to achieve substantial increases in brain distribution of dual ABCB1/ABCG2 substrates. During ABCB1 inhibition c.421CA subjects had significantly higher increases in [11C]tariquidar brain distribution than c.421CC subjects, pointing to impaired cerebral ABCG2 function. PMID:26940368

  3. MDR1/ABCB1 gene polymorphisms in patients with chronic myeloid leukemia

    PubMed Central

    Lardo, Mabel; Castro, Marcelo; Moiraghi, Beatriz; Rojas, Francisca; Borda, Natalia; Rey, Jorge A

    2015-01-01

    Background Tyrosine kinase inhibitors (TKIs) are the recommended treatment for patients with chronic myeloid leukemia (CML). The MDR1/ABCB1 gene plays a role in resistance to a wide spectrum of drugs, including TKIs. However, the association of MDR1/ABCB1 gene polymorphisms (SNPs) such as C1236T, G2677T/A, and C3435T with the clinical therapeutic evolution of CML has been poorly studied. We investigated these gene polymorphisms in CML-patients treated with imatinib, nilotinib and/or dasatinib. Methods ABCB1-SNPs were studied in 22 CML-patients in the chronic phase (CP) and 2 CML-patients in blast crisis (BC), all of whom were treated with TKIs, and compared with 25 healthy controls using nested-PCR and sequencing techniques. Results Seventeen different haplotypes were identified: 7 only in controls, 6 only in CML-patients, and the remaining 4 in both groups. The distribution ratios of homozygous TT-variants present on each exon between controls and CML-patients were 2.9 for exon 12, and 0.32 for the other 2 exons. Heterozygous T-variants were observed in all controls (100%) and 75% of CML-patients. Wt-haplotype (CC-GG-CC) was observed in 6 CML-patients (25%). In this wt-group, two were treated with nilotinib and reached a major molecular response. The remaining 4 cases had either a minimal or null molecular response, or developed bone marrow aplasia. Conclusion Our results suggest that SNPs of the MDR1/ABCB1 gene could help to characterize the prognosis and the clinical-therapeutic evolution of CML-patients treated with TKIs. Wt-haplotype could be associated with a higher risk of developing CML, and a worse clinical-therapeutic evolution. PMID:26457282

  4. Association of Extrarenal Adverse Effects of Posttransplant Immunosuppression With Sex and ABCB1 Haplotypes

    PubMed Central

    Venuto, Rocco C.; Meaney, Calvin J.; Chang, Shirley; Leca, Nicolae; Consiglio, Joseph D.; Wilding, Gregory E.; Brazeau, Daniel; Gundroo, Aijaz; Nainani, Neha; Morse, Sarah E.; Cooper, Louise M.; Tornatore, Kathleen M.

    2015-01-01

    Abstract Extrarenal adverse effects (AEs) associated with calcineurin inhibitor (CNI) and mycophenolic acid (MPA) occur frequently but are unpredictable posttransplant complications. AEs may result from intracellular CNI accumulation and low activity of P-glycoprotein, encoded by the ABCB1 gene. Since ABCB1 single nucleotide polymorphisms (SNPs) and sex influence P-glycoprotein, we investigated haplotypes and extrarenal AEs. A prospective, cross-sectional study evaluated 149 patients receiving tacrolimus and enteric coated mycophenolate sodium or cyclosporine and mycophenolate mofetil. Immunosuppressive AE assessment determined individual and composite gastrointestinal, neurologic, aesthetic, and cumulative AEs. Lipids were quantitated after 12-hour fast. ABCB1 SNPs: c.1236C>T (rs1128503), c.2677G>T/A (rs2032582), and c.3435C>T (rs1045642) were determined with haplotype associations computed using the THESIAS program, and evaluated by immunosuppression, sex and race using multivariate general linear models. Tacrolimus patients exhibited more frequent and higher gastrointestinal AE scores compared with cyclosporine with association to CTT (P = 0.018) and sex (P = 0.01). Aesthetic AE score was 3 times greater for cyclosporine with TTC haplotype (P = 0.005). Females had higher gastrointestinal (P = 0.022), aesthetic (P < 0.001), neurologic (P = 0.022), and cumulative AE ratios (P < 0.001). Total cholesterol (TCHOL), low-density lipoproteins (LDL), and triglycerides were higher with cyclosporine. The TTC haplotype had higher TCHOL (P < 0.001) and LDL (P = 0.005). Higher triglyceride (P = 0.034) and lower high-density lipoproteins (P = 0.057) were associated with TTT with sex-adjusted analysis. ABCB1 haplotypes and sex were associated with extrarenal AEs. Using haplotypes, certain female patients manifested more AEs regardless of CNI. Haplotype testing may identify patients with greater susceptibility to AEs and facilitate CNI

  5. The Central Cavity of ABCB1 Undergoes Alternating Access During ATP Hydrolysis

    PubMed Central

    McDevitt, Christopher A.; Thomson, Andrew J.; Kerr, Ian D.; MacMillan, Fraser; Callaghan, Richard

    2016-01-01

    Understanding the process that underlies multi-drug recognition and efflux by P-glycoprotein (ABCB1) remains a key biological challenge. Structural data has recently become available for the murine and C. elegans homologues of ABCB1; however all structures were obtained in the absence of nucleotide. A feature of these structures was the presence of a central cavity that is inaccessible from the extracellular face of the protein. To determine the conformational dynamics of this region several residues in transmembrane helices TM6 (331, 343 and 354) and TM12 (980) were mutated to cysteine. Based upon structural predictions these residues are proposed to line, or reside proximal to, the central cavity. The mutant isoforms were labelled with a paramagnetic probe enabling the application of electron paramagnetic resonance (EPR) spectroscopic methods. Power saturation EPR spectra were recorded in the presence of hydrophobic (O2) or hydrophilic (NiEDDA) quenching agents to study the local environment of each residue. ABCB1 was trapped in both its nucleotide bound and post-hydrolytic conformations and EPR spectra were again recorded in the presence and absence of quenching agents. The EPR line shapes provide information on the movements of these residues within TM6 and TM12 during ATP hydrolysis. Rationalisation of the data with molecular dynamic simulations indicate that the cavity is converted to a configuration open to the aqueous phase following nucleotide binding, thereby suggesting alternating access to the cavity on opposite sides of the membrane during translocation. PMID:24597976

  6. Basal and benzo[a]pyrene-induced expression profile of phase I and II enzymes and ABC transporter mRNA in the early life stage of Chinese rare minnows (Gobiocypris rarus).

    PubMed

    Yuan, Lilai; Lv, Biping; Zha, Jinmiao; Wang, Weimin; Wang, Zijian

    2014-08-01

    ATP-binding cassette (ABC) transporters together with phase I and II detoxification enzymes have been considered as included in a cellular detoxification system. Previous studies have highlighted the involvement of aryl hydrocarbon receptor (AHR) and Cyp1a in PAH-induced embryo toxicity. However, the response of other xenobiotic enzymes/transporters in PAH-mediated embryo toxicity is not fully characterized. In the present study, rare minnow embryos were exposed to 10 and 100µg/L BaP within 4h post-fertilization (hpf) up to 168 hpf. RNA was extracted at 24, 48, 96, and 168 hpf. The basal and BaP-induced expression of phase I enzyme genes (cyp1a, 1b1, and 1c1), phase II enzyme gene (gstm and ugt1a), and ABC transporter genes (abcb1, abcc1, abcc2, and abcg2) mRNA was determined using real-time PCR. Severe developmental defects (e.g., spinal deformities, pericardial and yolk-sac edema) were observed in the BaP treated groups. The basal expression showed that gstm was most strongly expressed, followed by abcb1, ugt1a, and abcc2, whereas cyp1a, 1b1, 1c1, and abcg2 showed weak expression. BaP significantly induced the mRNA expression of three CYP1s (cyp1a, 1b1, and 1c1) (p<0.05) and the ABC transporters (abcc1, abcc2, and abcg2) in a dose-dependent manner. However, the mRNA expression of Phase II enzymes (gstm, ugt1a) for the BaP treatments showed no significant difference with that of the controls. Furthermore, distinct induced patterns of these genes were observed during different exposure periods. Simultaneous up-regulation of the cyp and ABC transporter gene transcripts suggests that a possible involvement and cooperation in the detoxification process could provide protection against the BaP toxicity of rare minnows at the early life stage.

  7. Use of a combined effect model approach for discriminating between ABCB1- and ABCC1-type efflux activities in native bivalve gill tissue.

    PubMed

    Faria, Melissa; Pavlichenko, Vasiliy; Burkhardt-Medicke, Kathleen; Soares, Amadeu M V M; Altenburger, Rolf; Barata, Carlos; Luckenbach, Till

    2016-04-15

    Aquatic organisms, such as bivalves, employ ATP binding cassette (ABC) transporters for efflux of potentially toxic chemicals. Anthropogenic water contaminants can, as chemosensitizers, disrupt efflux transporter function enabling other, putatively toxic compounds to enter the organism. Applying rapid amplification of cDNA ends (RACE) PCR we identified complete cDNAs encoding ABCB1- and ABCC1-type transporter homologs from zebra mussel providing the molecular basis for expression of both transporter types in zebra mussel gills. Further, efflux activities of both transporter types in gills were indicated with dye accumulation assays where efflux of the dye calcein-am was sensitive to both ABCB1- (reversin 205, verapamil) and ABCC1- (MK571) type specific inhibitors. The assumption that different inhibitors targeted different efflux pump types was confirmed when comparing measured effects of binary inhibitor compound mixtures in dye accumulation assays with predictions from mixture effect models. Effects by the MK571/reversin 205 mixture corresponded better with independent action, whereas reversin 205/verapamil joint effects were better predicted by the concentration addition model indicating different and equal targets, respectively. The binary mixture approach was further applied to identify the efflux pump type targeted by environmentally relevant chemosensitizing compounds. Pentachlorophenol and musk ketone, which were selected after a pre-screen of twelve compounds that previously had been identified as chemosensitizers, showed mixture effects that corresponded better with concentration addition when combined with reversine 205 but with independent action predictions when combined with MK571 indicating targeting of an ABCB1-type efflux pump by these compounds. PMID:26929997

  8. Vibrational force alters mRNA expression in osteoblasts

    NASA Technical Reports Server (NTRS)

    Tjandrawinata, R. R.; Vincent, V. L.; Hughes-Fulford, M.

    1997-01-01

    Serum-deprived mouse osteoblastic (MC3T3E1) cells were subjected to a vibrational force modeled by NASA to simulate a space shuttle launch (7.83 G rms). The mRNA levels for eight genes were investigated to determine the effect of vibrational force on mRNA expression. The mRNA levels of two growth-related protooncogenes, c-fos and c-myc, were up-regulated significantly within 30 min after vibration, whereas those of osteocalcin as well as transforming growth factor-beta1 were decreased significantly within 3 h after vibration. No changes were detected in the levels of beta-actin, histone H4, or cytoplasmic phospholipase A2 after vibration. No basal levels of cyclooxygenase-2 expression were detected. In addition, the extracellular concentrations of prostaglandin E2 (PGE2), a potent autocrine/paracrine growth factor in bone, were not significantly altered after vibration most likely due to the serum deprivation state of the osteoblasts. In comparison with the gravitational launch profile, vibrational-induced changes in gene expression were greater both in magnitude and number of genes activated. Taken together, these data suggest that the changes in mRNA expression are due to a direct mechanical effect of the vibrational force on the osteoblast cells and not to changes in the local PGE2 concentrations. The finding that launch forces induce gene expression is of utmost importance since many of the biological experiments do not dampen vibrational loads on experimental samples. This lack of dampening of vibrational forces may partially explain why 1-G onboard controls sometimes do not reflect 1-G ground controls. These data may also suggest that scientists use extra ground controls that are exposed to launch forces, have these forces dampened on launched samples, or use facilities such as Biorack that provide an onboard 1-G centrufuge in order to control for space shuttle launch forces.

  9. Vibrational force alters mRNA expression in osteoblasts.

    PubMed

    Tjandrawinata, R R; Vincent, V L; Hughes-Fulford, M

    1997-05-01

    Serum-deprived mouse osteoblastic (MC3T3E1) cells were subjected to a vibrational force modeled by NASA to simulate a space shuttle launch (7.83 G rms). The mRNA levels for eight genes were investigated to determine the effect of vibrational force on mRNA expression. The mRNA levels of two growth-related protooncogenes, c-fos and c-myc, were up-regulated significantly within 30 min after vibration, whereas those of osteocalcin as well as transforming growth factor-beta1 were decreased significantly within 3 h after vibration. No changes were detected in the levels of beta-actin, histone H4, or cytoplasmic phospholipase A2 after vibration. No basal levels of cyclooxygenase-2 expression were detected. In addition, the extracellular concentrations of prostaglandin E2 (PGE2), a potent autocrine/paracrine growth factor in bone, were not significantly altered after vibration most likely due to the serum deprivation state of the osteoblasts. In comparison with the gravitational launch profile, vibrational-induced changes in gene expression were greater both in magnitude and number of genes activated. Taken together, these data suggest that the changes in mRNA expression are due to a direct mechanical effect of the vibrational force on the osteoblast cells and not to changes in the local PGE2 concentrations. The finding that launch forces induce gene expression is of utmost importance since many of the biological experiments do not dampen vibrational loads on experimental samples. This lack of dampening of vibrational forces may partially explain why 1-G onboard controls sometimes do not reflect 1-G ground controls. These data may also suggest that scientists use extra ground controls that are exposed to launch forces, have these forces dampened on launched samples, or use facilities such as Biorack that provide an onboard 1-G centrufuge in order to control for space shuttle launch forces.

  10. Leptin mRNA expresses in the bull reproductive organ.

    PubMed

    Abavisani, A; Baghbanzadeh, A; Shayan, P; Tajik, P; Dehghani, H; Mirtorabi, M

    2009-12-01

    Leptin, a 167-amino acid hormone, is secreted mainly by fat tissue. It has some powerful effects on the regulation of metabolism and reproductive function through endocrine and probably paracrine mechanisms. The contribution rate of leptin function on the male reproductive system is not still clear. Characterization of leptin expression in reproductive organs will suggest that in addition to its endocrine action, leptin has also paracrine/autocrine effects on reproduction. The expression of functional leptin receptor mRNA has been already recognized in testis of rodents, human and cattle. Thus, the aim of the present study was to investigate the presence of leptin mRNA in the bovine testis, because it will be the first step for understanding of its paracrine/autocrine effects on the male reproductive organs in cattle. The present study was the first to showed leptin mRNA expression in the testis of Holstein cattle using reverse transcription and polymerase chain reaction (RT-PCR) analysis. RT-PCR products were amplified with nested PCR using inner leptin primer pairs to emphasis the first results. Besides, bovine beta actin gene was acted as an internal positive control as well as RNA purification marker. Our findings suggest that in addition to its endocrine actions at the hypothalamic-pituitary axis, leptin can has an autocrine and/or paracrine role in bull testicular function.

  11. Association between ABCB1 polymorphisms and haplotypes and Alzheimer's disease: a meta-analysis.

    PubMed

    Zhong, Xin; Liu, Ming-Yan; Sun, Xiao-Hong; Wei, Min-Jie

    2016-01-01

    Although several epidemiological studies have investigated the association between ATP-binding cassette subfamily B member 1 (ABCB1) gene polymorphisms and Alzheimer's disease (AD) susceptibility, controversial results exist. Here, we performed a meta-analysis to assess whether ABCB1 polymorphisms 3435C > T (rs1045642), 2677G > T/A (rs2032582), 1236C > T (rs1128503) and haplotypes were associated with AD risk. Nine independent publications were included and analyzed. Crude odds ratio (OR) and 95% confidence interval (CI) were applied to investigate the strength of the association. Sensitivity analysis was conducted to measure the robustness of our analysis. A funnel plot and trim and fill method were used to test and adjust for publication bias. The results showed a significant association between the 3435C > T single nucleotide polymorphism (SNP) and AD susceptibility (CT vs. CC: OR = 1.24, 95% CI = 1.06-1.45, P = 0.01; CT + TT vs. CC: OR = 1.21, 95% CI = 1.04-1.41, P = 0.01) in the total population, as well as in Caucasian subgroup. The 2677G > T/A SNP was related to a decreased AD risk in Caucasian subgroup (TT + TA + AA vs. GT + GA + GG: OR = 0.68, 95% CI = 0.47-0.98, P = 0.04). Moreover, the ABCB1 haplotype analysis showed that the 1236T/2677T/3435C haplotype was associated with a higher risk of AD (OR = 1.99, 95% CI = 1.24-3.18, P = 0.00). Our results suggest that the ABCB1 3435C > T SNP, the 2677G > T/A SNP and 1236T/2677T/3435C haplotype are significantly associated with AD susceptibility. PMID:27600024

  12. Association between ABCB1 polymorphisms and haplotypes and Alzheimer’s disease: a meta-analysis

    PubMed Central

    Zhong, Xin; Liu, Ming-Yan; Sun, Xiao-Hong; Wei, Min-Jie

    2016-01-01

    Although several epidemiological studies have investigated the association between ATP-binding cassette subfamily B member 1 (ABCB1) gene polymorphisms and Alzheimer’s disease (AD) susceptibility, controversial results exist. Here, we performed a meta-analysis to assess whether ABCB1 polymorphisms 3435C > T (rs1045642), 2677G > T/A (rs2032582), 1236C > T (rs1128503) and haplotypes were associated with AD risk. Nine independent publications were included and analyzed. Crude odds ratio (OR) and 95% confidence interval (CI) were applied to investigate the strength of the association. Sensitivity analysis was conducted to measure the robustness of our analysis. A funnel plot and trim and fill method were used to test and adjust for publication bias. The results showed a significant association between the 3435C > T single nucleotide polymorphism (SNP) and AD susceptibility (CT vs. CC: OR = 1.24, 95% CI = 1.06–1.45, P = 0.01; CT + TT vs. CC: OR = 1.21, 95% CI = 1.04–1.41, P = 0.01) in the total population, as well as in Caucasian subgroup. The 2677G > T/A SNP was related to a decreased AD risk in Caucasian subgroup (TT + TA + AA vs. GT + GA + GG: OR = 0.68, 95% CI = 0.47–0.98, P = 0.04). Moreover, the ABCB1 haplotype analysis showed that the 1236T/2677T/3435C haplotype was associated with a higher risk of AD (OR = 1.99, 95% CI = 1.24–3.18, P = 0.00). Our results suggest that the ABCB1 3435C > T SNP, the 2677G > T/A SNP and 1236T/2677T/3435C haplotype are significantly associated with AD susceptibility. PMID:27600024

  13. An update on ABCB1 pharmacogenetics: insights from a 3D model into the location and evolutionary conservation of residues corresponding to SNPs associated with drug pharmacokinetics.

    PubMed

    Wolf, S J; Bachtiar, M; Wang, J; Sim, T S; Chong, S S; Lee, C G L

    2011-10-01

    The human ABCB1 protein, (P-glycoprotein or MDR1) is a membrane-bound glycoprotein that harnesses the energy of ATP hydrolysis to drive the unidirectional transport of substrates from the cytoplasm to the extracellular space. As a large range of therapeutic agents are known substrates of ABCB1 protein, its role in the onset of multidrug resistance has been the focus of much research. This role has been of particular interest in the field of pharmacogenomics where genetic variation within the ABCB1 gene, particularly in the form of single nucleotide polymorphisms (SNPs), is believed to contribute to inter-individual variation in ABCB1 function and drug response. In this review we provide an update on the influence of coding region SNPs within the ABCB1 gene on drug pharmacokinetics. By utilizing the crystal structure of the mouse ABCB1 homolog (Abcb1a), which is 87% homologous to the human sequence, we accompany this discussion with a graphical representation of residue location for amino acids corresponding to human ABCB1 coding region SNPs. Also, an assessment of residue conservation, which is calculated following multiple sequence alignment of 11 confirmed sequences of ABCB1 homologs, is presented and discussed. Superimposing a 'heat map' of residue homology to the Abcb1a crystal structure has permitted additional insights into both the conservation of individual residues and the conservation of their immediate surroundings. Such graphical representation of residue location and conservation supplements this update of ABCB1 pharmacogenetics to help clarify the often confounding reports on the influence of ABCB1 polymorphisms on drug pharmacokinetics and response.

  14. Targeting ABCB1 and ABCC1 with their Specific Inhibitor CBT-1® can Overcome Drug Resistance in Osteosarcoma.

    PubMed

    Fanelli, Marilù; Hattinger, Claudia Maria; Vella, Serena; Tavanti, Elisa; Michelacci, Francesca; Gudeman, Beth; Barnett, Daryl; Picci, Piero; Serra, Massimo

    2016-01-01

    Clinical treatment response achievable with conventional chemotherapy in high-grade osteosarcoma (OS) is severely limited by the presence of intrinsic or acquired drug resistance, which in previous studies has been mainly addressed for overexpression of ABCB1 (MDR1/P-glycoprotein). This study was aimed to estimate the impact on OS drug resistance of a group of ATP binding cassette (ABC) transporters, which in other human tumors have been associated with unresponsiveness to the drugs that represent the backbone of multidrug treatment regimens for OS (doxorubicin, methotrexate, cisplatin). By using a group of 6 drug-sensitive and 20 drug-resistant human OS cell lines, the most relevant transporter which proved to be associated with the degree of drug resistance in OS cells, in addition to ABCB1, was ABCC1. We therefore evaluated the in vitro activity of the orally administrable ABCB1/ABCC1 inhibitor CBT-1(®) (Tetrandrine, NSC-77037). We found that in our OS cell lines this agent was able to revert the ABCB1/ABCC1-mediated resistance against doxorubicin, as well as against the drugs used in second-line OS treatments that are substrates of these transporters (taxotere, etoposide, vinorelbine). Our findings indicated that inhibiting ABCB1 and ABCC1 with CBT-1(®), used in association with conventional chemotherapeutic drugs, may become an interesting new therapeutic option for unresponsive or relapsed OS patients. PMID:26548759

  15. Association of single nucleotide polymorphisms of ABCB1, OPRM1 and COMT with pain perception in cancer patients.

    PubMed

    Wang, Xu-shi; Song, Hai-bin; Chen, Si; Zhang, Wei; Liu, Jia-qi; Huang, Chao; Wang, Hao-ran; Chen, Yuan; Chu, Qian

    2015-10-01

    Pain perception is influenced by multiple factors. The single nucleotide polymorphisms (SNPs) of some genes were found associated with pain perception. This study aimed to examine the association of the genotypes of ABCB1 C3435T, OPRM1 A118G and COMT V108/158M (valine 108/158 methionine) with pain perception in cancer patients. We genotyped 146 cancer pain patients and 139 cancer patients without pain for ABCB1 C3435T (rs1045642), OPRM1 A118G (rs1799971) and COMT V108/158M (rs4680) by the fluorescent dye-terminator cycle sequencing method, and compared the genotype distribution between groups with different pain intensities by chi-square test and pain scores between groups with different genotypes by non-parametric test. The results showed that in these cancer patients, the frequency of variant T allele of ABCB1 C3435T was 40.5%; that of G allele of OPRM1 A118G was 38.5% and that of A allele of COMT V108/158M was 23.3%. No significant difference in the genotype distribution of ABCB1 C3435T (rs1045642) and OPRM1 A118G (rs1799971) was observed between cancer pain group and control group (P=0.364 and 0.578); however, significant difference occurred in the genotype distribution of COMT V108/158M (rs4680) between the two groups (P=0.001). And the difference could not be explained by any other confounding factors. Moreover, we found that the genotypes of COMT V108/158M and ABCB1 C3435T were associated with the intensities of pain in cancer patients. In conclusion, our results indicate that the SNPs of COMT V108/158M and ABCB1 C3435T significantly influence the pain perception in Chinese cancer patients.

  16. Incorporation of ABCB1-mediated transport into a physiologically-based pharmacokinetic model of docetaxel in mice

    PubMed Central

    Hudachek, Susan F.

    2015-01-01

    Docetaxel is one of the most widely used anticancer agents. While this taxane has proven to be an effective chemotherapeutic drug, noteworthy challenges exist in relation to docetaxel administration due to the considerable interindividual variability in efficacy and toxicity associated with the use of this compound, largely attributable to differences between individuals in their ability to metabolize and eliminate docetaxel. Regarding the latter, the ATP-binding cassette transporter B1 (ABCB1, PGP, MDR1) is primarily responsible for docetaxel elimination. To further understand the role of ABCB1 in the biodistribution of docetaxel in mice, we utilized physiologically-based pharmacokinetic (PBPK) modeling that included ABCB1-mediated transport in relevant tissues. Transporter function was evaluated by studying docetaxel pharmacokinetics in wild-type FVB and Mdr1a/b constitutive knockout (KO) mice and incorporating this concentration–time data into a PBPK model comprised of eight tissue compartments (plasma, brain, heart, lung, kidney, intestine, liver and slowly perfused tissues) and, in addition to ABCB1-mediated transport, included intravenous drug administration, specific binding to intracellular tubulin, intestinal and hepatic metabolism, glomerular filtration and tubular reabsorption. For all tissues in both the FVB and KO cohorts, the PBPK model simulations closely mirrored the observed data. Furthermore, both models predicted AUC values that were with 15 % of the observed AUC values, indicating that our model-simulated drug exposures accurately reflected the observed tissue exposures. Overall, our PBPK model furthers the understanding of the role of ABCB1 in the biodistribution of docetaxel. Additionally, this exemplary model structure can be applied to investigate the pharmacokinetics of other ABCB1 transporter substrates. PMID:23616082

  17. Sequence and expression of ferredoxin mRNA in barley

    SciTech Connect

    Zielinski, R.; Funder, P.M.; Ling, V. )

    1990-05-01

    We have isolated and structurally characterized a full-length cDNA clone encoding ferredoxin from a {lambda}gt10 cDNA library prepared from barley leaf mRNA. The ferredoxin clone (pBFD-1) was fused head-to-head with a partial-length cDNA clone encoding calmodulin, and was fortuitously isolated by screening the library with a calmodulin-specific oligonucleotide probe. The mRNA sequence from which pBFD-1 was derived is expressed exclusively in the leaf tissues of 7-d old barley seedlings. Barley pre-ferredoxin has a predicted size of 15.3 kDal, of which 4.6 kDal are accounted for by the transit peptide. The polypeptide encoded by pBFD-1 is identical to wheat ferredoxin, and shares slightly more amino acid sequence similarity with spinach ferredoxin I than with ferredoxin II. Ferredoxin mRNA levels are rapidly increased 10-fold by white light in etiolated barley leaves.

  18. Rheumatoid Arthritis Disease Activity Is Determinant for ABCB1 and ABCG2 Drug-Efflux Transporters Function

    PubMed Central

    Atisha-Fregoso, Yemil; Lima, Guadalupe; Pascual-Ramos, Virginia; Baños-Peláez, Miguel; Fragoso-Loyo, Hilda; Jakez-Ocampo, Juan; Contreras-Yáñez, Irazú; Llorente, Luis

    2016-01-01

    Objective To compare drug efflux function of ABCB1 and ABCG2 transporters in rheumatoid arthritis (RA) patients with active disease and in remission. Methods Twenty two active RA patients (DAS28 ≥3.2) and 22 patients in remission (DAS28<2.6) were selected from an early RA clinic. All patients were evaluated at study inclusion and six months later. ABCB1 and ABCG2 functional activity was measured in peripheral lymphocytes by flow cytometry. The percentage of cells able to extrude substrates for ABCB1 and ABCG2 was recorded. Results Active patients had higher ABCB1 and ABCG2 activity compared with patients in remission (median [interquartile range]): 3.9% (1.4–22.2) vs (1.3% (0.6–3.2), p = 0.003 and 3.9% (1.1–13.3) vs 0.9% (0.5–1.9) p = 0.006 respectively. Both transporters correlated with disease activity assessed by DAS28, rho = 0.45, p = 0.002 and rho = 0.47, p = 0.001 respectively. Correlation was observed between the time from the beginning of treatment and transporter activity: rho = 0.34, p = 0.025 for ABCB1 and rho = 0.35, p = 0.018 for ABCG2. The linear regression model showed that DAS28 and the time from the onset of treatment are predictors of ABCB1 and ABCG2 functional activity, even after adjustment for treatment. After six months we calculated the correlation between change in DAS28 and change in the functional activity in both transporters and found a moderate and significant correlation for ABCG2 (rho = 0.28, p = 0.04) and a non-significant correlation for ABCB1 (rho = 0.22, p = 0.11). Conclusions Patients with active RA have an increased function of ABCB1 and ABCG2, and disease activity is the main determinant of this phenomena. PMID:27442114

  19. Screening of mRNA Chemical Modification to Maximize Protein Expression with Reduced Immunogenicity

    PubMed Central

    Uchida, Satoshi; Kataoka, Kazunori; Itaka, Keiji

    2015-01-01

    Chemical modification of nucleosides in mRNA is an important technology to regulate the immunogenicity of mRNA. In this study, various previously reported mRNA formulations were evaluated by analyzing in vitro protein expression and immunogenicity in multiple cell lines. For the macrophage-derived cell line, RAW 264.7, modified mRNA tended to have reduced immunogenicity and increased protein expression compared to the unmodified mRNA. In contrast, in some cell types, such as hepatocellular carcinoma cells (HuH-7) and mouse embryonic fibroblasts (MEFs), protein expression was decreased by mRNA modification. Further analyses revealed that mRNA modifications decreased translation efficiency but increased nuclease stability. Thus, mRNA modification is likely to exert both positive and negative effects on the efficiency of protein expression in transfected cells and optimal mRNA formulation should be determined based on target cell types and transfection purposes. PMID:26213960

  20. Screening of mRNA Chemical Modification to Maximize Protein Expression with Reduced Immunogenicity.

    PubMed

    Uchida, Satoshi; Kataoka, Kazunori; Itaka, Keiji

    2015-01-01

    Chemical modification of nucleosides in mRNA is an important technology to regulate the immunogenicity of mRNA. In this study, various previously reported mRNA formulations were evaluated by analyzing in vitro protein expression and immunogenicity in multiple cell lines. For the macrophage-derived cell line, RAW 264.7, modified mRNA tended to have reduced immunogenicity and increased protein expression compared to the unmodified mRNA. In contrast, in some cell types, such as hepatocellular carcinoma cells (HuH-7) and mouse embryonic fibroblasts (MEFs), protein expression was decreased by mRNA modification. Further analyses revealed that mRNA modifications decreased translation efficiency but increased nuclease stability. Thus, mRNA modification is likely to exert both positive and negative effects on the efficiency of protein expression in transfected cells and optimal mRNA formulation should be determined based on target cell types and transfection purposes. PMID:26213960

  1. VEGF, eNOS, and ABCB1 genetic polymorphisms may increase the risk of osteonecrosis of the femoral head.

    PubMed

    Zhou, Z C; Gu, S Z; Wu, J; Liang, Q W

    2015-01-01

    We investigated the associations between vascular endothelial growth factors (VEGF), endothelial nitric oxide synthase (eNOS), and ATP-binding cassette subfamily B member 1 transporter (ABCB1) polymorphisms and the risk of osteonecrosis of the femoral head (ONFH). Published studies were reviewed and analyzed based on predefined selection criteria. The strength of the association between VEGF, eNOS, and ABCB1 polymorphisms and ONFH risk was evaluated based on the odds ratio with corresponding 95%CIs. Meta-analysis was performed using the Comprehensive Meta-analysis 2.0 software. A total of 135 relevant articles were retrieved, of which 10 studies met the selection criteria, and included a total of 1025 patients with ONFH and 1730 healthy controls. The meta-analysis study results revealed that the VEGF rs2010963 G>C polymorphism increased the risk of ONFH, while the VEGF rs2010963 G>C and ABCB1 rs1045642 C>T polymorphisms increased the risk of ONFH under the allele model. In conclusion, the VEGF, eNOS, and ABCB1 polymorphisms may contribute to ONFH, but further studies including larger sample sizes are needed to confirm the results. PMID:26535684

  2. Variants in CDA and ABCB1 are predictors of capecitabine-related adverse reactions in colorectal cancer

    PubMed Central

    García, María I.; García-Alfonso, Pilar; Robles, Luis; Grávalos, Cristina; González-Haba, Eva; Marta, Pellicer; Sanjurjo, María; López-Fernández, Luis A.

    2015-01-01

    Adverse reactions to capecitabine-based chemotherapy limit full administration of cytotoxic agents. Likewise, genetic variations associated with capecitabine-related adverse reactions are associated with controversial results and a low predictive value. Thus, more evidence on the role of these variations is needed. We evaluated the association between nine polymorphisms in MTHFR, CDA, TYMS, ABCB1, and ENOSF1 and adverse reactions, dose reductions, treatment delays, and overall toxicity in 239 colorectal cancer patients treated with capecitabine-based regimens. The ABCB1*1 haplotype was associated with a high risk of delay in administration or reduction in the dose of capecitabine, diarrhea, and overall toxicity. CDA rs2072671 A was associated with a high risk of overall toxicity. TYMS rs45445694 was associated with a high risk of delay in administration or reduction in the dose of capecitabine, HFS >1 and HFS >2. Finally, ENOSF1 rs2612091 was associated with HFS >1, but was a poorer predictor than TYMS rs45445694. A score based on ABCB1-CDA polymorphisms efficiently predicts patients at high risk of severe overall toxicity (PPV, 54%; sensitivity, 43%) in colorectal cancer patients treated with regimens containing capecitabine. Polymorphisms in ABCB1, CDA, ENOSF1,and TYMS could help to predict specific and overall severe adverse reactions to capecitabine. PMID:25691056

  3. Ensemble Rule-Based Classification of Substrates of the Human ABC-Transporter ABCB1 Using Simple Physicochemical Descriptors.

    PubMed

    Demel, Michael A; Kraemer, Oliver; Ettmayer, Peter; Haaksma, Eric; Ecker, Gerhard F

    2010-03-15

    Within the last decades, the detailed knowledge on the impact of membrane bound drug efflux transporters of the ATP binding cassette (ABC) protein family on the pharmacological profile of drugs has enormously increased. Especially, ABCB1 (P-glycoprotein, P-gp, MDR1) has attracted particular interest in medicinal chemistry, since it determines the clinical efficacy, side effects and toxicity risks of drug candidates. Based on this, the development of in silico models that provide rapid and cost-effective screening tools for the classification of substrates and nonsubstrates of ABCB1 is an urgent need in contemporary ADMET profiling. A characteristic hallmark feature of this transporter is its polyspecific ligand recognition pattern. In this study we describe a method for classifying ABCB1 ligands in terms of simple, conjunctive rules (RuleFit) based on interpretable ADMET features. The retrieved results showed that models based on large, very diverse data sets gave better classification performance than models based on smaller, more homogenous training sets. The best model achieved gave a correct classification rate of 0.90 for an external validation set. Furthermore, from the interpretation of the best performing model it could be concluded that in comparison to nonsubstrates ABCB1 substrates generally show a higher number of hydrogen-bond acceptors, are more flexible and exhibit higher logP values.

  4. Improving the stability and function of purified ABCB1 and ABCA4: the influence of membrane lipids.

    PubMed

    Pollock, Naomi L; McDevitt, Christopher A; Collins, Richard; Niesten, Petronella H M; Prince, Stephen; Kerr, Ian D; Ford, Robert C; Callaghan, Richard

    2014-01-01

    ATP Binding Cassette (ABC) transporters play prominent roles in numerous cellular processes and many have been implicated in human diseases. Unfortunately, detailed mechanistic information on the majority of ABC transporters has not yet been elucidated. The slow rate of progress of molecular and high resolution structural studies may be attributed to the difficulty in the investigation of integral membrane proteins. These difficulties include the expression of functional, non-aggregated protein in heterologous systems. Furthermore, the extraction of membrane proteins from source material remains a major bottle-neck in the process since there are relatively few guidelines for selection of an appropriate detergent to achieve optimal extraction. Whilst affinity tag strategies have simplified the purification of membrane proteins; many challenges remain. For example, the chromatographic process and associated steps can rapidly lead to functional inactivation, random aggregation, or even precipitation of the target protein. Furthermore, optimisation of high yield and purity, does not guarantee successful structure determination. Based on this series of potential issues, any investigation into structure-function of membrane proteins requires a systematic evaluation of preparation quality. In particular, the evaluation should focus on function, homogeneity and mono-dispersity. The present investigation provides a detailed assessment of the quality of purified ATP Binding Cassette (ABC) transporters; namely ABCB1 (P-gp) and ABCA4 (ABCR). A number of suggestions are provided to facilitate the production of functional, homogeneous and mono-disperse preparations using the insect cell expression system. Finally, the ABCA4 samples have been used to provide structural insights into this essential photo-receptor cell protein. PMID:24036079

  5. Endothelin-1 mRNA expression in the rat kidney.

    PubMed Central

    Nunez, D J; Taylor, E A; Oh, V M; Schofield, J P; Brown, M J

    1991-01-01

    Cultured pig and bovine endothelial cells are capable of synthesizing endothelin-1 (ET-1). Thus the observation that the kidney contains a large number of binding sites for ET distributed in close proximity to endothelial cells suggests that ET-1 may be released from the endothelium to act locally on these receptors. In support of this hypothesis, using the technique of reverse transcription with specific amplification of cDNA, we report here that ET-1 mRNA is expressed in the rat kidney. The partial sequence of the amplified rat ET-1 cDNA confirms that the mature rat peptide is identical to that of the mouse, man and pig, but with some differences in codon usage. PMID:2039460

  6. The functional influences of common ABCB1 genetic variants on the inhibition of P-glycoprotein by Antrodia cinnamomea extracts.

    PubMed

    Sheu, Ming-Jyh; Teng, Yu-Ning; Chen, Ying-Yi; Hung, Chin-Chuan

    2014-01-01

    Antrodia cinnamomea is a traditional healthy food that has been demonstrated to possess anti-inflammatory, antioxidative, and anticacer effects. The purpose of this study was to evaluate whether the ethanolic extract of A. cinnamomea (EEAC) can affect the efflux function of P-glycoprotein (P-gp) and the effect of ABCB1 genetic variants on the interaction between EEAC and P-gp. To investigate the mechanism of this interaction, Flp-In™-293 cells stably transfected with various genotypes of human P-gp were established and the expression of P-gp was confirmed by Western blot. The results of the rhodamine 123 efflux assay demonstrated that EEAC efficiently inhibited wild-type P-gp function at an IC50 concentration of 1.51 ± 0.08 µg/mL through non-competitive inhibition. The IC50 concentrations for variant-type 1236T-2677T-3435T P-gp and variant-type 1236T-2677A-3435T P-gp were 5.56 ± 0.49 µg/mL and 3.33±0.67 µg/mL, respectively. In addition, the inhibition kinetics of EEAC also changed to uncompetitive inhibition in variant-type 1236T-2677A-3435T P-gp. The ATPase assay revealed that EEAC was an ATPase stimulator and was capable of reducing verapamil-induced ATPase levels. These results indicate that EEAC may be a potent P-gp inhibitor and higher dosages may be required in subjects carrying variant-types P-gp. Further studies are required to translate this basic knowledge into clinical applications.

  7. Nilotinib reverses ABCB1/P-glycoprotein-mediated multidrug resistance but increases cardiotoxicity of doxorubicin in a MDR xenograft model.

    PubMed

    Zhou, Zhi-Yong; Wan, Li-Li; Yang, Quan-Jun; Han, Yong-Long; Li, Dan; Lu, Jin; Guo, Cheng

    2016-09-30

    The BCR-Abl tyrosine kinase inhibitor (TKI), nilotinib, was developed to surmount resistance or intolerance to imatinib in patients with Philadelphia-positive chronic myelogenous leukemia. Recent studies have shown that nilotinib induces potent sensitization to anticancer agents by blocking the functions of ABCB1/P-glycoprotein (P-gp) in multidrug resistance (MDR). However, changes in P-gp expression or function affect the cardiac disposition and prolong the presence of both doxorubicin (DOX) and doxorubicinol (DOXol) in cardiac tissue, thus, enhancing the risk of cardiotoxicity. In this study, we used a MDR xenograft model to evaluate the antitumor activity, tissue distribution and cardiotoxicity of DOX when co-administered with nilotinib. This information will provide more insight into the pharmacological role of nilotinib in MDR reversal and the risk of DOX cardiotoxicity. Our results showed that nilotinib significantly enhanced DOX cytotoxicity and increased intracellular rhodamine 123 accumulation in MG63/DOX cells in vitro and strongly enhanced DOX inhibition of growth of P-gp-overexpressing MG63/DOX cell xenografts in nude mice. Additionally, nilotinib significantly increased DOX and DOXol accumulation in serum, heart, liver and tumor tissues. Importantly, nilotinib induced a disproportionate increase in DOXol in cardiac tissue. In the co-administration group, CBR1 and AKR1A1 protein levels were significantly increased in cardiac tissue, with more severe necrosis and vacuole formation. These results indicate that nilotinib reverses P-gp- mediated MDR by blocking the efflux function and potentiates DOX-induced cardiotoxicity. These findings represent a guide for the design of future clinical trials and studies of pharmacokinetic interactions and may be useful in guiding the use of nilotinib in combination therapy of cancer in clinical practice.

  8. The prognostic value of epidermal growth factor receptor mRNA expression in primary ovarian cancer.

    PubMed Central

    Bartlett, J. M.; Langdon, S. P.; Simpson, B. J.; Stewart, M.; Katsaros, D.; Sismondi, P.; Love, S.; Scott, W. N.; Williams, A. R.; Lessells, A. M.; Macleod, K. G.; Smyth, J. F.; Miller, W. R.

    1996-01-01

    The expression of mRNA for the epidermal growth factor (EGF) receptor, EGF and transforming growth factor alpha (TGF-alpha) was determined in 76 malignant, six borderline and 15 benign primary ovarian tumours using the reverse transcriptase-polymerase chain reaction and related to clinical and pathological parameters. Of the malignant tumours, 70% (53/76) expressed EGF receptor mRNA, 31% (23/75) expressed EGF mRNA and 35% (26/75) expressed TGF-alpha mRNA. For the borderline tumours, four of six (67%) expressed EGF receptor mRNA, 1/6 (17%) expressed TGF-alpha mRNA and none expressed EGF mRNA. Finally, 33% (5/15) of the benign tumours expressed EGF receptor mRNA, whereas 40% (6/15) expressed EGF mRNA and 7% (1/15) expressed TGF-alpha mRNA. The presence of the EGF receptor in malignant tumours was associated with that of TGF-alpha (P = 0.0015) but not with EGF (P = 1.00), whereas there was no relationship between the presence of EGF and TGF-alpha (P = 1.00). EGF receptor mRNA expression was significantly and positively associated with serous histology (P = 0.006) but not with stage or grade. Neither EGF nor TGF-alpha showed any link with histological subtype or stage. The survival of patients with malignant tumours possessing EGF receptor mRNA was significantly reduced compared with that of patients whose tumours were negative (P = 0.030 for all malignant tumours; P = 0.007 for malignant epithelial tumours only). In contrast, neither the expression of TGF-alpha nor EGF was related to survival. These data suggest that the presence of EGF receptor mRNA is associated with poor prognosis in primary ovarian cancer. Images Figure 1 PMID:8562334

  9. Preproglucagon mRNA expression in adult rat submandibular glands.

    PubMed

    Egéa, J C; Hirtz, C; Deville de Périère, D

    2003-04-01

    Salivary glands of various animal species have been reported to contain and suggested to produce glucagon or glucagon-like material, but the origin and the nature of this salivary peptide are still doubtful. The present study was undertaken to ascertain whether the glucagon gene is expressed in rat submandibular glands and in an immortalized murine cell line derived from salivary glands (SCA-9 cell line). For this purpose, total RNA was isolated from submandibular glands or cultured cells and submitted to reverse transcription. The cDNAs obtained were amplified by a nested polymerase chain reaction using preproglucagon primers. The results showed that the preproglucagon mRNA was expressed in adult rat submandibular glands but not in the SCA-9 cell line. Determination of cyclic DNA (cDNA) sequence established identity with the coding regions of rat pancreatic pre-proglucagon gene. In conclusion, these results strongly support the idea that rat submandibular glands could represent a source of extrapancreatic glucagon or of its precursor's peptide.

  10. A comprehensive study of polymorphisms in the ABCB1, ABCC2, ABCG2, NR1I2 genes and lymphoma risk

    PubMed Central

    Campa, Daniele; Butterbach, Katja; Slager, Susan L; Skibola, Christine F; de Sanjosé, Silvia; Benavente, Yolanda; Becker, Nikolaus; Foretova, Lenka; Maynadie, Marc; Cocco, Pierluigi; Staines, Anthony; Kaaks, Rudolf; Boffetta, Paolo; Brennan, Paul; Conde, Lucia; Bracci, Paige M; Caporaso, Neil E; Strom, Sara S; Camp, Nicola J; Cerhan, James R; Canzian, Federico; Nieters, Alexandra

    2012-01-01

    Owing to their role in controlling the efflux of toxic compounds, transporters are central players in the process of detoxification and elimination of xenobiotics, which in turn is related to cancer risk. Among these transporters, ATP-binding cassette B1/multidrug resistance 1 (ABCB1/MDR1), ABCC2/multidrug resistance protein 2 (MRP2), and ABCG2/breast cancer resistance protein (BCRP) affect susceptibility to many hematopoietic malignancies. The maintenance of regulated expression of these transporters is governed through the activation of intracellular “xenosensors” like the nuclear receptor 1I2/pregnane X receptor (NR1I2/PXR). SNPs in genes encoding these regulators have also been implicated in the risk of several cancers. Using a tagging approach, we tested the hypothesis that common polymorphisms in the transporter genes ABCB1, ABCC2, ABCG2, and the regulator gene NR1I2 could be implicated in lymphoma risk. We selected 68 SNPs in the 4 genes, and we genotyped them in 1,481 lymphoma cases and 1,491 controls of the European cases-control study (EpiLymph) using the Illumina™ GoldenGate assay technology.Carriers of the SNP rs6857600 minor allele in ABCG2, was associated with a decrease in risk of B-cell lymphoma (B-NHL) overall (p<0.001). Furthermore, a decreased risk of chronic lymphocytic leukemia (CLL) was associated with the ABCG2 rs2231142 variant (p=0.0004), which could be replicated in an independent population. These results suggest a role for this gene in B-NHL susceptibility, especially for CLL. PMID:21918980

  11. Molecular model of the outward facing state of the human P-glycoprotein (ABCB1), and comparison to a model of the human MRP5 (ABCC5)

    PubMed Central

    Ravna, Aina W; Sylte, Ingebrigt; Sager, Georg

    2007-01-01

    Background Multidrug resistance is a particular limitation to cancer chemotherapy, antibiotic treatment and HIV medication. The ABC (ATP binding cassette) transporters human P-glycoprotein (ABCB1) and the human MRP5 (ABCC5) are involved in multidrug resistance. Results In order to elucidate structural and molecular concepts of multidrug resistance, we have constructed a molecular model of the ATP-bound outward facing conformation of the human multidrug resistance protein ABCB1 using the Sav1866 crystal structure as a template, and compared the ABCB1 model with a previous ABCC5 model. The electrostatic potential surface (EPS) of the ABCB1 substrate translocation chamber, which transports cationic amphiphilic and lipophilic substrates, was neutral with negative and weakly positive areas. In contrast, EPS of the ABCC5 substrate translocation chamber, which transports organic anions, was generally positive. Positive-negative ratios of amino acids in the TMDs of ABCB1 and ABCC5 were also analyzed, and the positive-negative ratio of charged amino acids was higher in the ABCC5 TMDs than in the ABCB1 TMDs. In the ABCB1 model residues Leu65 (transmembrane helix 1 (TMH1)), Ile306 (TMH5), Ile340 (TMH6) and Phe343 (TMH6) may form a binding site, and this is in accordance with previous site directed mutagenesis studies. Conclusion The Sav1866 X-ray structure may serve as a suitable template for the ABCB1 model, as it did with ABCC5. The EPS in the substrate translocation chambers and the positive-negative ratio of charged amino acids were in accordance with the transport of cationic amphiphilic and lipophilic substrates by ABCB1, and the transport of organic anions by ABCC5. PMID:17803828

  12. Influence of ABCB1 polymorphisms and haplotypes on tacrolimus nephrotoxicity and dosage requirements in children with liver transplant

    PubMed Central

    Hawwa, Ahmed F; McKiernan, Patrick J; Shields, Michael; Millership, Jeff S; Collier, Paul S; McElnay, James C

    2009-01-01

    AIMS The aim of this study was to investigate the influence of genetic polymorphisms in ABCB1 on the incidence of nephrotoxicity and tacrolimus dosage-requirements in paediatric patients following liver transplantation. METHODS Fifty-one paediatric liver transplant recipients receiving tacrolimus were genotyped for ABCB1 C1236>T, G2677>T and C3435>T polymorphisms. Dose-adjusted tacrolimus trough concentrations and estimated glomerular filtration rates (EGFR) indicative of renal toxicity were determined and correlated with the corresponding genotypes. RESULTS The present study revealed a higher incidence of the ABCB1 variant-alleles examined among patients with renal dysfunction (≥30% reduction in EGFR) at 6 months post-transplantation (1236T allele: 63.3% vs 37.5% in controls, P= 0.019; 2677T allele: 63.3% vs. 35.9%, p = 0.012; 3435T allele: 60% vs. 39.1%, P= 0.057). Carriers of the G2677->T variant allele also had a significant reduction (%) in EGFR at 12 months post-transplant (mean difference = 22.6%; P= 0.031). Haplotype analysis showed a significant association between T-T-T haplotypes and an increased incidence of nephrotoxicity at 6 months post-transplantation (haplotype-frequency = 52.9% in nephrotoxic patients vs 29.4% in controls; P= 0.029). Furthermore, G2677->T and C3435->T polymorphisms and T-T-T haplotypes were significantly correlated with higher tacrolimus dose-adjusted pre-dose concentrations at various time points examined long after drug initiation. CONCLUSIONS These findings suggest that ABCB1 polymorphisms in the native intestine significantly influence tacrolimus dosage-requirement in the stable phase after transplantation. In addition, ABCB1 polymorphisms in paediatric liver transplant recipients may predispose them to nephrotoxicity over the first year post-transplantation. Genotyping future transplant recipients for ABCB1 polymorphisms, therefore, could have the potential to individualize better tacrolimus immunosuppressive therapy and

  13. Multiple Transport-Active Binding Sites Are Available for a Single Substrate on Human P-Glycoprotein (ABCB1)

    PubMed Central

    Chufan, Eduardo E.; Kapoor, Khyati; Sim, Hong-May; Singh, Satyakam; Talele, Tanaji T.; Durell, Stewart R.; Ambudkar, Suresh V.

    2013-01-01

    P-glycoprotein (Pgp, ABCB1) is an ATP-Binding Cassette (ABC) transporter that is associated with the development of multidrug resistance in cancer cells. Pgp transports a variety of chemically dissimilar amphipathic compounds using the energy from ATP hydrolysis. In the present study, to elucidate the binding sites on Pgp for substrates and modulators, we employed site-directed mutagenesis, cell- and membrane-based assays, molecular modeling and docking. We generated single, double and triple mutants with substitutions of the Y307, F343, Q725, F728, F978 and V982 residues at the proposed drug-binding site with cys in a cysless Pgp, and expressed them in insect and mammalian cells using a baculovirus expression system. All the mutant proteins were expressed at the cell surface to the same extent as the cysless wild-type Pgp. With substitution of three residues of the pocket (Y307, Q725 and V982) with cysteine in a cysless Pgp, QZ59S-SSS, cyclosporine A, tariquidar, valinomycin and FSBA lose the ability to inhibit the labeling of Pgp with a transport substrate, [125I]-Iodoarylazidoprazosin, indicating these drugs cannot bind at their primary binding sites. However, the drugs can modulate the ATP hydrolysis of the mutant Pgps, demonstrating that they bind at secondary sites. In addition, the transport of six fluorescent substrates in HeLa cells expressing triple mutant (Y307C/Q725C/V982C) Pgp is also not significantly altered, showing that substrates bound at secondary sites are still transported. The homology modeling of human Pgp and substrate and modulator docking studies support the biochemical and transport data. In aggregate, our results demonstrate that a large flexible pocket in the Pgp transmembrane domains is able to bind chemically diverse compounds. When residues of the primary drug-binding site are mutated, substrates and modulators bind to secondary sites on the transporter and more than one transport-active binding site is available for each substrate

  14. Genetic association of NOS1 exon18, NOS1 exon29, ABCB1 1236C/T, and ABCB1 3435C/T polymorphisms with the risk of Parkinson's disease

    PubMed Central

    Huang, Hongbin; Peng, Cong; Liu, Yong; Liu, Xu; Chen, Qicong; Huang, Zunnan

    2016-01-01

    Abstract Background: Parkinson's disease (PD) is the second most frequent neurodegenerative disorder. Previous publications have investigated the association of NOS1 and ABCB1 polymorphisms with PD risk. However, those studies have provided some contradictory results. Methods: Literature searches were performed using PubMed, Embase, PDgene, China National Knowledge Infrastructure database, and Google Scholar. Odds ratios (ORs) with 95% confidence intervals (CIs) were applied to evaluate the strength of association. Results: The analysis results indicated that NOS1 exon18 polymorphism was associated with developing PD in 4 genetic models (allelic: OR = 1.25, 95%CI 1.09–1.44, P = 0.001; homozygous: OR = 1.79, 95%CI 1.32–2.45, P < 0.001; recessive: OR = 1.70, 95%CI 1.26–2.28, P < 0.001; dominant: OR = 1.22, 95%CI 1.02–1.46, P = 0.03), whereas exon29 polymorphism was not correlated to PD susceptibility. In addition, ABCB1 1236C/T polymorphism was related to PD in the recessive (OR = 0.80, 95%CI 0.66–0.97, P = 0.025) and overdominant (OR = 1.21, 95%CI 1.03–1.43, P = 0.02) models, which might indicate the opposite effects of 2 minor variants of this locus on Parkinson's disease. However, this associated result was not robust enough to withstand statistically significant correction. On the other hand, no association was found between ABCB1 3435C/T polymorphism and the predisposition to PD in 5 genetic models, and such an absence of relationship was further confirmed by subgroup analysis in Caucasians and Asians. Whether the polymorphisms of these 4 loci were linked to PD or not, our study provided some interesting findings that differ from the previous results with regard to their genetic susceptibility. Conclusion: The NOS1 exon18 and ABCB1 1236C/T variants might play a role in the risk of Parkinson's disease, whereas NOS1 exon29 and ABCB1 3435C/T polymorphisms might not contribute to PD susceptibility. PMID

  15. Influence of ABCB1 polymorphisms and docetaxel pharmacokinetics on pathological response to neoadjuvant chemotherapy in breast cancer patients.

    PubMed

    Lévy, Pierre; Gligorov, Joseph; Antoine, Martine; Rezai, Keyvan; Lévy, Eric; Selle, Frédéric; Saintigny, Pierre; Lokiec, François; Avenin, Danielle; Beerblock, Karine; Lotz, Jean-Pierre; Bernaudin, Jean-François; Fajac, Anne

    2013-06-01

    We have previously reported an association between ABCB1 C3435T polymorphism and docetaxel pharmacokinetics in breast cancer patients. We therefore investigated whether these parameters could account for variations in pathological response. Five ABCB1 polymorphisms including C3435T polymorphism were analyzed in breast cancer patients receiving neoadjuvant chemotherapy with doxorubicin and docetaxel (n = 101). Pathological response was assessed using the Sataloff classification. Pharmacokinetic analysis was performed for the first course of docetaxel (n = 84). No significant association was found between ABCB1 polymorphisms or docetaxel pharmacokinetics and pathological complete response. C3435T genotype was an independent predictive factor of good response in breast (response >50 %, i.e., Sataloff T-A and T-B): OR: 4.6 (95 % CI: 1.3-16.1), p = 0.015, for TT patients versus CT and CC patients. Area under the plasma concentration-time curve (AUC) of docetaxel was the only independent predictive factor of the total absence of response in breast (Sataloff T-D): OR: 14.3, (95 % CI: 1.7-118), p = 0.015, for AUC of docetaxel <3,500 μg h/L versus ≥3,500 μg h/L. These results suggest that C3435T polymorphism and docetaxel exposure are involved in the response to neoadjuvant chemotherapy in breast cancer patients and may be useful to optimize individualized therapy.

  16. Host genetic variants of ABCB1 and IL15 influence treatment outcome in paediatric acute lymphoblastic leukaemia

    PubMed Central

    Lu, Y; Kham, S K Y; Ariffin, H; Oei, A M I; Lin, H P; Tan, A M; Quah, T C; Yeoh, A E J

    2014-01-01

    Background: Host germline variations and their potential prognostic importance is an emerging area of interest in paediatric ALL. Methods: We investigated the associations between 20 germline variations and various clinical end points in 463 children with ALL. Results: After adjusting for known prognostic factors, variants in two genes were found to be independently associated with poorer EFS: ABCB1 T/T at either 2677 (rs2032582) or 3435 (rs1045642) position (P=0.003) and IL15 67276493G/G (rs17015014; P=0.022). These variants showed a strong additive effect affecting outcome (P<0.001), whereby patients with both risk genotypes had the worst EFS (P=0.001), even after adjusting for MRD levels at the end of remission induction. The adverse effect of ABCB1 T/T genotypes was most pronounced in patients with favourable cytogenetics (P=0.011) while the IL15 67276493G/G genotype mainly affected patients without common chromosomal abnormalities (P=0.022). In both cytogenetic subgroups, increasing number of such risk genotypes still predicted worsening outcome (P<0.001 and=0.009, respectively). Conclusion: These results point to the prognostic importance of host genetic variants, although the specific mechanisms remain unclarified. Inclusion of ABCB1 and IL15 variants may help improve risk assignment strategies in paediatric ALL. PMID:24434428

  17. Oxysterols decrease apical-to-basolateral transport of Aß peptides via an ABCB1-mediated process in an in vitro Blood-brain barrier model constituted of bovine brain capillary endothelial cells.

    PubMed

    Saint-Pol, Julien; Candela, Pietra; Boucau, Marie-Christine; Fenart, Laurence; Gosselet, Fabien

    2013-06-23

    It is known that activation of the liver X receptors (LXRs) by natural or synthetic agonists decreases the amyloid burden and enhances cognitive function in transgenic murine models of Alzheimer's disease (AD). Recent evidence suggests that LXR activation may affect the transport of amyloid ß (Aß) peptides across the blood-brain barrier (the BBB, which isolates the brain from the peripheral circulation). By using a well-characterized in vitro BBB model, we demonstrated that LXR agonists (24S-hydroxycholesterol, 27-hydroxycholesterol and T0901317) modulated the expression of target genes involved in cholesterol homeostasis (such as ATP-binding cassette sub-family A member 1 (ABCA1)) and promoted cellular cholesterol efflux to apolipoprotein A-I and high density lipoproteins. Interestingly, we also observed a decrease in Aß peptide influx across brain capillary endothelial cells, although ABCA1 did not appear to be directly involved in this process. By focusing on others receptors and transporters that are thought to have major roles in Aß peptide entry into the brain, we then demonstrated that LXR stimulation provoked an increase in expression of the ABCB1 transporter (also named P-glycoprotein (P-gp)). Further investigations confirmed ABCB1's involvement in the restriction of Aß peptide influx. Taken as a whole, our results not only reinforce the BBB's key role in cerebral cholesterol homeostasis but also demonstrate the importance of the LXR/ABCB1 axis in Aß peptide influx-highlighting an attractive new therapeutic approach whereby the brain could be protected from peripheral Aß peptide entry.

  18. Glucocorticoids modulate BDNF mRNA expression in the rat hippocampus after traumatic brain injury.

    PubMed

    Grundy, P L; Patel, N; Harbuz, M S; Lightman, S L; Sharples, P M

    2000-10-20

    Brain-derived neurotrophic factor (BDNF) expression in rat hippocampus is increased after experimental traumatic brain injury (TBI) and may be neuroprotective. Glucocorticoids are important regulators of brain neurotrophin levels and are often prescribed following TBI. The effect of adrenalectomy (ADX) on the expression of BDNF mRNA in the hippocampus after TBI has not been investigated to date. We used fluid percussion injury (FPI) and in situ hybridization to evaluate the expression of BDNF mRNA in the hippocampus 4 h after TBI in adrenal-intact or adrenalectomized rats (with or without corticosterone replacement). FPI and ADX independently increased expression of BDNF mRNA. In animals undergoing FPI, prior ADX caused further elevation of BDNF mRNA and this upregulation was prevented by corticosterone replacement in ADX rats. These findings suggest that glucocorticoids are involved in the modulation of the BDNF mRNA response to TBI.

  19. Calpain expression in lymphoid cells. Increased mRNA and protein levels after cell activation.

    PubMed

    Deshpande, R V; Goust, J M; Chakrabarti, A K; Barbosa, E; Hogan, E L; Banik, N L

    1995-02-10

    Although calpain is ubiquitously present in human tissues and is thought to play a role in demyelination, its activity is very low in resting normal lymphocytes. To determine the nature of calpain expression at the mRNA and protein levels in human lymphoid cells, we studied human T lymphocytic, B lymphocytic, and monocytic lines as well as peripheral blood mononuclear cells. Stimulation of cells with the phorbol ester phorbol myristate acetate and the calcium ionophore A23187 resulted in increased calpain mRNA and protein expression. Calpain mRNA expression is also increased in human T cells stimulated with anti-CD3. A dissociation between the increases of RNA and protein suggested that calpain could be released from the cells; the subsequent experiments showed its presence in the extracellular environment. 5,6-Dichloro-1b-D-ribofuranosylbenzimidazole, a reversible inhibitor of mRNA synthesis, reduced calpain mRNA levels by 50-67% and protein levels by 72-91%. Its removal resulted in resumption of both calpain mRNA and protein synthesis. Cycloheximide, a translational inhibitor, reduced calpain protein levels by 77-81% and calpain mRNA levels by 96% in activated THP-1 cells. Interferon-gamma induced calpain mRNA and protein in U-937 and THP-1 cells. Dexamethasone increased mRNA expression in THP-1 cells. Our results indicate that activation of lymphoid cells results in de novo synthesis and secretion of calpain. PMID:7852311

  20. Oscillatory kinetics of gene expression: Protein conversion and slow mRNA transport

    SciTech Connect

    Zhdanov, V. P.

    2009-06-15

    The negative feedback between mRNA and regulatory-protein production may result in oscillations in the kinetics of gene expression if the mRNA-protein interplay includes protein conversion. Using a mean-field kinetic model, we show that such oscillations can be amplified due to limitations of the mRNA transport between the nucleus and cytoplasm. This effect may be dramatic for the mRNA population in the nucleus.

  1. COX-2 mRNA expression in esophageal squamous cell carcinoma (ESCC) and effect by NSAID.

    PubMed

    Liu, X; Li, P; Zhang, S-T; You, H; Jia, J-D; Yu, Z-L

    2008-01-01

    To investigate cyclooxygenase-2 (COX-2) mRNA expression in human esophageal squamous cell carcinoma and the effect of a non-steroidal anti-inflammatory drug (NSAID) on it, in order to explore the mechanism of COX-2 in esophageal squamous cell carcinoma (ESCC) carcinogenesis and the ability of NSAID to prevent or treat ESCC. Frozen specimens of human ESCC and adjacent normal esophageal squamous epithelium pairs (n = 22) were examined for COX-2 mRNA expression by reverse-transcription polymerase chain reaction (RT-PCR). After incubation with aspirin (a non-selective COX inhibitor) or Nimesulide (a selective COX-2 inhibitor), the proliferation status of two human esophageal squamous cancer cell lines, EC-9706 and EC-109, was quantified by 3-(4,5-dimethyl-thiazol-2yl)-2,5-diphenyltetrazolium bromide assay. The expression of COX-2 mRNA in these cells was detected by RT-PCR. COX-2 mRNA was expressed in 12 of 22 (54.5%) ESCC tissue samples, but it was undetectable in all the specimens of adjacent normal esophageal squamous epithelium COX-2 mRNA expression. Both aspirin (5-20 mmol/L) and Nimesulide (0.1-0.8 mmol/L) inhibited EC-9706 cell line proliferation and suppressed its COX-2 mRNA expression dose-dependently. However, only aspirin (5-20 mmol/L) could inhibit proliferation in the EC-109 cell line and suppress COX-2 mRNA expression. Nimesulide (0.1-0.8 mmol/L) could neither inhibit EC-109 cell growth nor suppress COX-2 mRNA expression. COX-2 mRNA expression is a frequent phenomenon in human ESCC tissue samples and plays an important role in the carcinogenesis of ESCC. NSAID may be useful in the chemoprevention and therapy of human ESCC and its effects are likely to be mediated by modulating COX-2 activity.

  2. Development and characterization of P-glycoprotein 1 (Pgp1, ABCB1)-mediated doxorubicin-resistant PLHC-1 hepatoma fish cell line

    SciTech Connect

    Zaja, Roko; Caminada, Daniel; Loncar, Jovica; Fent, Karl; Smital, Tvrtko

    2008-03-01

    The development of the multidrug resistance (MDR) phenotype in mammals is often mediated by the overexpression of the P-glycoprotein1 (Pgp, ABCB1) or multidrug resistance-associated protein (MRP)-like ABC transport proteins. A similar phenomenon has also been observed and considered as an important part of the multixenobiotic resistance (MXR) defence system in aquatic organisms. We have recently demonstrated the presence of ABC transporters in the widely used in vitro fish model, the PLHC-1 hepatoma cell line. In the present study we were able to select a highly resistant PLHC-1 sub-clone (PLHC-1/dox) by culturing the wild-type cells in the presence of 1 {mu}M doxorubicin. Using quantitative PCR a 42-fold higher expression of ABCB1 gene was determined in the PLHC-1/dox cells compared to non-selected wild-type cells (PLHC-1/wt). The efflux rates of model fluorescent Pgp1 substrates rhodamine 123 and calcein-AM were 3- to 4-fold higher in the PLHC-1/dox in comparison to the PLHC-1/wt cells. PLHC-1/dox were 45-fold more resistant to doxorubicin cytotoxicity than PLHC-1/wt. Similarly to mammalian cell lines, typical cross-resistance to cytotoxicity of other chemotherapeutics such as daunorubicin, vincristine, vinblastine, etoposide and colchicine, occurred. Furthermore, cyclosporine A, verapamil and PSC833, specific inhibitors of Pgp1 transport activity, completely reversed resistance of PLHC-1/dox cells to all tested drugs, resulting in EC50 values similar to the EC50 values found for PLHC-1/wt. In contrast, MK571, a specific inhibitor of MRP type of efflux transporters, sensitized PLHC-1/dox cells, neither to doxorubicin, nor to any other of the chemotherapeutics used in the study. These data demonstrate for the first time that a specific Pgp1-mediated doxorubicin resistance mechanism is present in the PLHC-1 fish hepatoma cell line. In addition, the fact that low micromolar concentrations of specific inhibitors may completely reverse a highly expressed doxorubicin

  3. Dietary glycerol for quail: association between productive performance and COX III mRNA expression.

    PubMed

    Silva, S C C; Gasparino, E; Batista, E; Tanamati, F; Vesco, A P D; Lala, B; de Oliveira, D P

    2016-01-01

    This study was carry out to evaluate mRNA expression of mitochondrial cytochrome c oxidase III in the Pectoralis superficialis muscle of 28-day-old quails fed diets containing 0, 8, and 12% glycerol. Total RNA was extracted (N = 10) and cDNA was amplified using specifics primers for qRT-PCR. Feed efficiency and feed intake were evaluated. COX III mRNA expression in breast muscle was higher in the group fed with 12% glycerol (0.863 AU); no differences were observed in the expression of this gene between the muscle of animals fed diets without glycerol (0.357 AU) and 8% glycerol (0.415 AU). Quails that showed greater COX III mRNA expression also showed the lowest feed efficiency. These results show that there is a difference in COX III mRNA expression in breast muscle of 28-day-old quail fed diets different concentrations of glycerol. PMID:27323091

  4. Polymorphisms in the BRCA1 and ABCB1 genes modulate menopausal hormone therapy associated breast cancer risk in postmenopausal women.

    PubMed

    2010-04-01

    Menopausal hormone therapy (HT) is associated with an increased breast cancer risk among postmenopausal women. In this study, we investigated genetic effect modification of HT associated breast cancer risk in 3,149 postmenopausal breast cancer patients and 5,489 controls from the two German population-based case-control studies MARIE and GENICA. Twenty-eight polymorphisms of 14 candidate genes including two drug and hormone transporter genes (ABCB1/MDR1 and SHBG), four genes involved in cell cycle regulation (BRCA1, P21/CDKN1A, STK15/AURKA and TP53), six cytokine genes (IGFBP3, IL6, TGFB1, TNF, LTA and IGF1), and two cytokine receptor genes (EGFR and ERBB2) were genotyped using validated methods. Conditional logistic regression was used to assess multiplicative statistical interaction between polymorphisms and duration of estrogen-progestagen therapy and estrogen monotherapy use with regard to breast cancer risk assuming log-additive and co-dominant modes of inheritance. Women homozygous for the major ABCB1_rs2214102_G allele were found to be at a significantly increased breast cancer risk associated with combined estrogen-progestagen therapy [odds ratio (OR) = 1.17, 95% confidence interval (CI) = 1.12-1.23, P (interaction) = 0.022]. Additionally, risk associated with estrogen monotherapy was modified by BRCA1_rs799917. We observed a trend with increasing minor T alleles leading to the highest risk in homozygous carriers of the minor allele [OR (95% CI) = 1.17 (0.98-1.39), 1.06 (0.98-1.14), and 1.02 (0.94-1.11) for homozygous minor, heterozygous, and homozygous major allele carriers, respectively; P (interaction) = 0.032]. Our results suggest that genetic variants in ABCB1 and BRCA1 may modify the effect of HT on postmenopausal breast cancer risk.

  5. Bis-cyclopropane analog of disorazole C1 is a microtubule-destabilizing agent active in abcb1-overexpressing human colon cancer cells

    PubMed Central

    Wu, Shaoyu; Guo, Zhijian; Hopkins, Chad D.; Wei, Ning; Chu, Edward; Wipf, Peter; Schmitz, John C.

    2015-01-01

    The novel, chemically stabilized disorazole analog, (−)-CP2-disorazole C1 (1) displayed potent anti-proliferative activity against a broad-spectrum of human colorectal cancer cells. HCT15 and H630R1 cell lines expressing high basal levels of the ABCB1 protein, known to cause multi-drug resistance, were also sensitive to growth inhibition by 1 but were resistant to both vincristine and docetaxel, two commonly used microtubule inhibitors. Compound 1 exhibited strong inhibition of tubulin polymerization at a level comparable to vincristine. In addition, treatment with 1 resulted in decreased protein levels of β-tubulin but not α-tubulin. An analysis of cellular proteins known to interact with microtubules showed that 1 caused decreased expression of c-Myc, APC, Rb, and additional key cellular signaling pathways in CRC cells. Treatment with compound 1 also resulted in G2/M cell cycle arrest and induction of apoptosis, but not senescence. Furthermore, endothelial spheroid sprouting assays demonstrated that 1 suppressed angiogenesis and can, therefore, potentially prevent cancer cells from spreading and metastasizing. Taken together, these findings suggest that the microtubule disruptor 1 may be a potential drug candidate for the treatment of mCRC. PMID:26506423

  6. Alternative splicing of parathyroid hormone-related protein mRNA: expression and stability

    PubMed Central

    Sellers, R S; Luchin, A I; Richard, V; Brena, R M; Lima, D; Rosol, T J

    2011-01-01

    Parathyroid hormone-related protein (PTHrP) is a multifunctional protein that is often dysregulated in cancer. The human PTHrP gene is alternatively spliced into three isoforms, each with a unique 3′-untranslated region (3′-UTR), encoding 139, 173 and 141 amino acid proteins. The regulation of PTHrP mRNA isoform expression has not been completely elucidated, but it may be affected by transforming growth factor-β1 (TGF-β1). In this study, we examined differences in the PTHrP mRNA isoform expression in two squamous carcinoma cell lines (SCC2/88 and HARA), an immortalized keratinocyte cell line (HaCaT), and spontaneous human lung cancer with adjacent normal tissue. In addition, the effect of TGF-β1 on PTHrP mRNA isoform expression and stability was examined. Cell-type specific expression of PTHrP mRNA isoforms occurred between the various cell lines, normal human lung, and immortalized human keratinocytes (HaCaT). PTHrP isoform expression pattern was significantly altered between normal lung tissue and the adjacent lung cancer. In vitro studies revealed that TGF-β1 differentially altered the mRNA steady-state levels and mRNA stability of the PTHrP isoforms. Protein–RNA binding studies identified different proteins binding to the 3′-UTR of the PTHrP isoforms (139) and (141), which may be important in the differential mRNA stability and response to cytokines between the PTHrP isoforms. The data demonstrate that there is cell-type specific expression of PTHrP mRNA isoforms, and disruption of the normal regulation during cancer progression may in part be associated with TGF-β1-induced changes in PTHrP mRNA isoform expression and stability. PMID:15291755

  7. Aberrant Maspin mRNA Expression is Associated with Clinical Outcome in Patients with Pulmonary Adenocarcinoma

    PubMed Central

    Lu, Mingjie; Li, Jun; Huang, Zebo; Du, Yiping; Jin, Shidai; Wang, Jian

    2016-01-01

    Background The aim of this study was to investigate the expression level of maspin mRNA in pulmonary adenocarcinoma and to clarify its clinical significance in prediction of prognosis. Material/Methods RNA was extracted from formalin-fixed paraffin-embedded (FFPE) tumor tissue blocks of 30 pairs of pulmonary adenocarcinoma (AC) tissues and adjacent noncancerous tissues (ANT) and in another 81 AC tissues. Expression of maspin mRNA was tested by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and the potential relationship between maspin mRNA expression and clinic pathological features of AC patients was analyzed. Results The expression of maspin mRNA was upregulated in AC samples compared with the ANT (p<0.001). Patients at advanced clinical stage (III) and patients with lymphatic metastasis showed higher maspin mRNA expression level than those in early-stage patients (I and II) (p=0.038) or with non-lymphatic metastasis (p=0.034). The Kaplan-Meier survival curves indicated that disease-free survival (DFS) was significantly worse in high maspin mRNA expression AC patients (p=0.007). Furthermore, multivariate analysis revealed that the expression of maspin mRNA was an independent prognostic marker for AC (p=0.040). Conclusions Our study reveals that maspin mRNA was significantly up-regulated in tissues of AC patients. Maspin mRNA may be useful as a new marker of prognosis in AC. PMID:26757744

  8. ABCB1 and ABCC2 and the risk of distant metastasis in Thai breast cancer patients treated with tamoxifen

    PubMed Central

    Sensorn, Insee; Sukasem, Chonlaphat; Sirachainan, Ekaphop; Chamnanphon, Montri; Pasomsub, Ekawat; Trachu, Narumol; Supavilai, Porntip; Pinthong, Darawan; Wongwaisayawan, Sansanee

    2016-01-01

    Background Genetic polymorphisms of drug-metabolizing enzymes and transporters have been extensively studied with regard to tamoxifen treatment outcomes. However, the results are inconclusive. Analysis of organ-specific metastasis may reveal the association of these pharmacogenetic factors. The aim of this study is to investigate the impact of CYP3A5, CYP2D6, ABCB1, and ABCC2 polymorphisms on the risk of all distant and organ-specific metastases in Thai patients who received tamoxifen adjuvant therapy. Methods Genomic DNA was extracted from blood samples of 73 patients with breast cancer who received tamoxifen adjuvant therapy. CYP3A5 (6986A>G), CYP2D6 (100C>T), ABCB1 (3435C>T), and ABCC2 (−24C>T) were genotyped using allelic discrimination real-time polymerase chain reaction assays. The impacts of prognostic clinical factors and genetic variants on disease-free survival were analyzed using the Kaplan–Meier method and Cox regression analysis. Results In the univariate analysis, primary tumor size >5 cm was significantly associated with increased risk of distant metastasis (P=0.004; hazard ratio [HR] =3.05; 95% confidence interval [CI], 1.44–6.47). In the multivariate analysis, tumor size >5 cm remained predictive of distant metastasis (P<0.001; HR=5.49; 95% CI, 2.30–13.10). ABCC2 −24CC were shown to be associated with increased risk of distant metastasis (P=0.040; adjusted HR=2.34; 95% CI, 1.04–5.27). The combined genotype of ABCC2 −24CC − ABCB1 3435 CT+TT was associated with increased risk of distant and bone metastasis (P=0.020; adjusted HR=2.46; 95% CI, 1.15–5.26 and P=0.040; adjusted HR=3.70; 95% CI, 1.06–12.89, respectively). Conclusion This study indicates that polymorphisms of ABCC2 and ABCB1 are independently associated with bone metastasis. Further prospective studies with larger sample sizes are needed to verify this finding. PMID:27110128

  9. CXCL10 mRNA expression predicts response to neoadjuvant chemoradiotherapy in rectal cancer patients.

    PubMed

    Li, Cong; Wang, Zhimin; Liu, Fangqi; Zhu, Ji; Yang, Li; Cai, Guoxiang; Zhang, Zhen; Huang, Wei; Cai, Sanjun; Xu, Ye

    2014-10-01

    Chemoradiotherapy has been commonly used as neoadjuvant therapy for rectal cancer to allow for less aggressive surgical approaches and to improve quality of life. In cancer, it has been reported that CXCL10 has an anti-tumor function. However, the association between CXCL10 and chemoradiosensitivity has not been fully investigated. We performed this study to investigate the relationship between CXCL10 expression and chemoradiosensitivity in rectal cancer patients. Ninety-five patients with rectal cancer who received neoadjuvant chemoradiotherapy (NCRT) were included. Clinical parameters were compared with the outcome of NCRT and CXCL10 messenger RNA (mRNA) expression between the pathological complete response (pCR) group and non-pathological complete response (npCR) group. CXCL10 mRNA and protein expressions between groups were analyzed using the Student's t test and chi-square test. The mean mRNA level of CXCL10 in the pCR group was significantly higher than that in the npCR group (p = 0.010). In the pCR group, 73.7 % of the patients had high CXCL10 mRNA expression, and 61.4 % of the patients in the npCR group had low CXCL10 mRNA expression. Subjects with high CXCL10 mRNA expression demonstrated a higher sensitivity to NCRT (p = 0.011). The receiver operating characteristic curve showed that the diagnostic performance of CXCL10 mRNA expression had an area under the curve of 0.720 (95 % confidence interval, 0.573-0.867). There were no differences between the pCR and npCR groups in CXCL10 protein expression (p > 0.05). High CXCL10 mRNA expression is associated with a better tumor response to NCRT in rectal cancer patients and may predict the outcome of NCRT in this malignancy.

  10. Increased oral availability and brain accumulation of the ALK inhibitor crizotinib by coadministration of the P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) inhibitor elacridar.

    PubMed

    Tang, Seng Chuan; Nguyen, Luan N; Sparidans, Rolf W; Wagenaar, Els; Beijnen, Jos H; Schinkel, Alfred H

    2014-03-15

    Crizotinib is an oral tyrosine kinase inhibitor approved for treating patients with non-small cell lung cancer (NSCLC) containing an anaplastic lymphoma kinase (ALK) rearrangement. We used knockout mice to study the roles of P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) in plasma pharmacokinetics and brain accumulation of oral crizotinib, and the feasibility of improving crizotinib kinetics using coadministration of the dual ABCB1/ABCG2 inhibitor elacridar. In vitro, crizotinib was a good transport substrate of human ABCB1, but not of human ABCG2 or murine Abcg2. With low-dose oral crizotinib (5 mg/kg), Abcb1a/1b(-/-) and Abcb1a/1b;Abcg2(-/-) mice had an approximately twofold higher plasma AUC than wild-type mice, and a markedly (~40-fold) higher brain accumulation at 24 hr. Also at 4 hr, crizotinib brain concentrations were ∼25-fold, and brain-to-plasma ratios ~14-fold higher in Abcb1a/1b(-/-) and Abcb1a/1b;Abcg2(-/-) mice than in wild-type mice. High-dose oral crizotinib (50 mg/kg) resulted in comparable plasma pharmacokinetics between wild-type and Abcb1a/1b(-/-) mice, suggesting saturation of intestinal Abcb1. Nonetheless, brain accumulation at 24 hr was still ~70-fold higher in Abcb1a/1b(-/-) than in wild-type mice. Importantly, oral elacridar coadministration increased the plasma and brain concentrations and brain-to-plasma ratios of crizotinib in wild-type mice, equaling the levels in Abcb1a/1b;Abcg2(-/-) mice. Our results indicate that crizotinib oral availability and brain accumulation were primarily restricted by Abcb1 at a non-saturating dose, and that coadministration of elacridar with crizotinib could substantially increase crizotinib oral availability and delivery to the brain. This principle might be used to enhance therapeutic efficacy of crizotinib against brain metastases in NSCLC patients.

  11. Induction of cysteine-rich motor neuron 1 mRNA expression in vascular endothelial cells.

    PubMed

    Nakashima, Yukiko; Takahashi, Satoru

    2014-08-22

    Cysteine-rich motor neuron 1 (CRIM1) is expressed in vascular endothelial cells and plays a crucial role in angiogenesis. In this study, we investigated the expression of CRIM1 mRNA in human umbilical vein endothelial cells (HUVECs). CRIM1 mRNA levels were not altered in vascular endothelial growth factor (VEGF)-stimulated monolayer HUVECs or in cells in collagen gels without VEGF. In contrast, the expression of CRIM1 mRNA was elevated in VEGF-stimulated cells in collagen gels. The increase in CRIM1 mRNA expression was observed even at 2h when HUVECs did not form tubular structures in collagen gels. Extracellular signal-regulated kinase (Erk) 1/2, Akt and focal adhesion kinase (FAK) were activated by VEGF in HUVECs. The VEGF-induced expression of CRIM1 mRNA was significantly abrogated by PD98059 or PF562271, but was not affected by LY294002. These results demonstrate that CRIM1 is an early response gene in the presence of both angiogenic stimulation (VEGF) and environmental (extracellular matrix) factors, and Erk and FAK might be involved in the upregulation of CRIM1 mRNA expression in vascular endothelial cells.

  12. Population pharmacokinetic approach to evaluate the effect of CYP2D6, CYP3A, ABCB1, POR and NR1I2 genotypes on donepezil clearance

    PubMed Central

    Noetzli, Muriel; Guidi, Monia; Ebbing, Karsten; Eyer, Stephan; Wilhelm, Laurence; Michon, Agnès; Thomazic, Valérie; Stancu, Ioana; Alnawaqil, Abdel-Messieh; Bula, Christophe; Zumbach, Serge; Gaillard, Michel; Giannakopoulos, Panteleimon; von Gunten, Armin; Csajka, Chantal; Eap, Chin B

    2014-01-01

    Aims A large interindividual variability in plasma concentrations has been reported in patients treated with donepezil, the most frequently prescribed antidementia drug. We aimed to evaluate clinical and genetic factors influencing donepezil disposition in a patient population recruited from a naturalistic setting. Methods A population pharmacokinetic study was performed including data from 129 older patients treated with donepezil. The patients were genotyped for common polymorphisms in the metabolic enzymes CYP2D6 and CYP3A, in the electron transferring protein POR and the nuclear factor NR1I2 involved in CYP activity and expression, and in the drug transporter ABCB1. Results The average donepezil clearance was 7.3 l h−1 with a 30% interindividual variability. Gender markedly influenced donepezil clearance (P < 0.01). Functional alleles of CYP2D6 were identified as unique significant genetic covariate for donepezil clearance (P < 0.01), with poor metabolizers and ultrarapid metabolizers demonstrating, respectively, a 32% slower and a 67% faster donepezil elimination compared with extensive metabolizers. Conclusion The pharmacokinetic parameters of donepezil were well described by the developed population model. Functional alleles of CYP2D6 significantly contributed to the variability in donepezil disposition in the patient population and should be further investigated in the context of individual dose optimization to improve clinical outcome and tolerability of the treatment. PMID:24433464

  13. Expression of D2 dopamine receptor mRNA in the arterial chemoreceptor afferent pathway.

    PubMed

    Czyzyk-Krzeska, M F; Lawson, E E; Millhorn, D E

    1992-11-01

    Dopamine is a major neurotransmitter in the arterial chemoreceptor pathway. In the present study we wished to determine if messenger RNAs for dopamine D1 and D2 receptor are expressed in carotid body (type I cells), in sensory neurons of the petrosal ganglion which innervate the carotid body and in sympathetic neurons of the superior cervical ganglion. We failed to detect D1 receptor mRNA in any of these tissues. However, we found that D2 receptor mRNA was expressed by dopaminergic carotid body type I cells. D2 receptor mRNA was also found in petrosal ganglion neurons that innervated the carotid sinus and carotid body. In addition, a large number of sympathetic postganglionic neurons in the superior cervical ganglion expressed D2 receptor mRNA. PMID:1362730

  14. Transient resistance to DNA damaging agents is associated with expression of microRNAs-135b and -196b in human leukemia cell lines.

    PubMed

    Ho, Tsui-Ting; He, Xiaolong; Mo, Yin-Yuan; Beck, William T

    2016-01-01

    The acquisition of resistance to anticancer drugs is widely viewed as a key obstacle to successful cancer therapy. However, detailed knowledge of the initial molecular events in the response of cancer cells to these chemotherapeutic and stress responses, and how these lead to the development of chemoresistance, remains incompletely understood. Using microRNA array and washout and rechallenge experiments, we found that short term treatment of leukemia cells with etoposide led a few days later to transient resistance that was associated with a corresponding transient increase in expression of ABCB1 mRNA, as well as microRNA (miR)-135b and miR-196b. This phenomenon was associated with short-term exposure to genotoxic agents, such as etoposide, topotecan, doxorubicin and ionizing radiation, but not agents that do not directly damage DNA. Further, this appeared to be histiotype-specific, and was seen in leukemic cells, but not in cell lines derived from solid tumors. Treatment of leukemic cells with either 5-aza-deoxycytidine or tricostatin A produced similar increased expression of ABCB1, miR-135b, and miR-196b, suggesting a role for epigenetic regulation of this phenomenon. Bioinformatics analyses revealed that CACNA1E, ARHGEF2, PTK2, SIAH1, ARHGAP6, and NME4 may be involved in the initial events in the development of drug resistance following the upregulation of ABCB1, miR-135b and miR-196b. In summary, we report herein that short-term exposure of cells to DNA damaging agents leads to transient drug resistance, which is associated with elevations in ABCB1, miR-135b and miR-196b, and suggests novel components that may be involved in the development of anticancer drug resistance. PMID:27570640

  15. Transient resistance to DNA damaging agents is associated with expression of microRNAs-135b and -196b in human leukemia cell lines

    PubMed Central

    Ho, Tsui-Ting; He, Xiaolong; Mo, Yin-Yuan; Beck, William T

    2016-01-01

    The acquisition of resistance to anticancer drugs is widely viewed as a key obstacle to successful cancer therapy. However, detailed knowledge of the initial molecular events in the response of cancer cells to these chemotherapeutic and stress responses, and how these lead to the development of chemoresistance, remains incompletely understood. Using microRNA array and washout and rechallenge experiments, we found that short term treatment of leukemia cells with etoposide led a few days later to transient resistance that was associated with a corresponding transient increase in expression of ABCB1 mRNA, as well as microRNA (miR)-135b and miR-196b. This phenomenon was associated with short-term exposure to genotoxic agents, such as etoposide, topotecan, doxorubicin and ionizing radiation, but not agents that do not directly damage DNA. Further, this appeared to be histiotype-specific, and was seen in leukemic cells, but not in cell lines derived from solid tumors. Treatment of leukemic cells with either 5-aza-deoxycytidine or tricostatin A produced similar increased expression of ABCB1, miR-135b, and miR-196b, suggesting a role for epigenetic regulation of this phenomenon. Bioinformatics analyses revealed that CACNA1E, ARHGEF2, PTK2, SIAH1, ARHGAP6, and NME4 may be involved in the initial events in the development of drug resistance following the upregulation of ABCB1, miR-135b and miR-196b. In summary, we report herein that short-term exposure of cells to DNA damaging agents leads to transient drug resistance, which is associated with elevations in ABCB1, miR-135b and miR-196b, and suggests novel components that may be involved in the development of anticancer drug resistance. PMID:27570640

  16. Single-cell detection of mRNA expression using nanofountain-probe electroporated molecular beacons.

    PubMed

    Giraldo-Vela, Juan P; Kang, Wonmo; McNaughton, Rebecca L; Zhang, Xuemei; Wile, Brian M; Tsourkas, Andrew; Bao, Gang; Espinosa, Horacio D

    2015-05-01

    New techniques for single-cell analysis enable new discoveries in gene expression and systems biology. Time-dependent measurements on individual cells are necessary, yet the common single-cell analysis techniques used today require lysing the cell, suspending the cell, or long incubation times for transfection, thereby interfering with the ability to track an individual cell over time. Here a method for detecting mRNA expression in live single cells using molecular beacons that are transfected into single cells by means of nanofountain probe electroporation (NFP-E) is presented. Molecular beacons are oligonucleotides that emit fluorescence upon binding to an mRNA target, rendering them useful for spatial and temporal studies of live cells. The NFP-E is used to transfect a DNA-based beacon that detects glyceraldehyde 3-phosphate dehydrogenase and an RNA-based beacon that detects a sequence cloned in the green fluorescence protein mRNA. It is shown that imaging analysis of transfection and mRNA detection can be performed within seconds after electroporation and without disturbing adhered cells. In addition, it is shown that time-dependent detection of mRNA expression is feasible by transfecting the same single cell at different time points. This technique will be particularly useful for studies of cell differentiation, where several measurements of mRNA expression are required over time.

  17. Expression of beta 3-adrenoceptor mRNA in rat tissues.

    PubMed Central

    Evans, B. A.; Papaioannou, M.; Bonazzi, V. R.; Summers, R. J.

    1996-01-01

    1. This study examines the expression of beta 3-adrenoceptor messenger RNA (beta 3-AR mRNA) in rat tissues to allow comparison with atypical beta-adrenoceptors determined by functional and radioligand binding techniques. 2. A reverse transcription/polymerase chain reaction protocol has been developed for determining the relative amounts of beta 3-AR mRNA in rat tissues. 3. Measurement of adipsin and uncoupling protein (UCP) mRNA was used to examine all tissues for the presence of white and brown adipose tissue which may contribute beta 3-AR mRNA. 4. The beta 3-AR mRNA is expressed at high levels in brown and white adipose tissue, stomach fundus, the longitudinal/circular smooth muscle of both colon and ileum, and colon submucosa. There was substantial expression of adipsin in colon submucosa and moderate expression in fundus, suggesting that in these regions at least some of the beta 3-AR signal may be contributed by fat. Pylorus and colon mucosa showed moderate levels of beta 3-AR mRNA with lower levels of adipsin. Ileum mucosa and submucosa showed low but readily detectable levels of beta 3-AR. 5. Expression of adipsin in rat skeletal muscles coupled to very low levels of beta 3-AR mRNA indicates that the observed beta 3-AR may be due to the presence of intrinsic fat. beta 3-AR mRNA was virtually undetectable in heart, lung and liver. These results raise the possibility that the atypical beta-AR demonstrated by functional and/or binding studies in muscle and in heart is not the beta 3-AR. 6. By use of two different sets of primers for amplification of beta 3-AR cDNA, no evidence was found for differential splicing of the mRNA in any of the tissues examined. 7. The detection of beta 3-AR mRNA in the gut mucosa and submucosa suggests that in addition to its established roles in lipolysis, thermogenesis and regulation of gut motility beta 3-AR may subserve other functions in the gastrointestinal tract. The absence of beta 3-AR mRNA in rat heart or its presence with

  18. Eosinophil cationic protein mRNA expression in children with bronchial asthma.

    PubMed

    Yu, H Y; Li, X Y; Cai, Z F; Li, L; Shi, X Z; Song, H X; Liu, X J

    2015-11-13

    Studies have shown that eosinophils are closely related to pathogenesis of bronchial asthma. Eosinophils release eosinophil cationic protein (ECP), which plays an important role in infection and allergic reactions. Serum ECP mRNA expression in children with bronchial asthma has not been adequately investigated. We analyzed serum ECP mRNA expression in 63 children with bronchial asthma and 21 healthy children by using reverse-transcriptase polymerase chain reaction to understand the role of ECP in children with bronchial asthma. The children with bronchial asthma were segregated into acute-phase and stable-phase groups, based on the severity of the illness. Serum ECP mRNA expression in children with bronchial asthma (0.375 ± 0.04) was significantly higher than that in healthy controls (0.20 ± 0.02; P < 0.05). Additionally, children in the acute-phase group showed higher ECP mRNA expression level (0.44 ± 0.06) than those in the stable-phase (0.31 ± 0.03) and healthy control groups (0.20 ± 0.02; P < 0.05), while the level in the stable-phase (0.31 ± 0.03) was markedly higher than that in the healthy control group (0.20 ± 0.02; P < 0.05). Detection of serum ECP mRNA expression level has possible applications in the diagnosis and treatment of children with bronchial asthma.

  19. Expression of Melanocortin-4 Receptor mRNA in Male Rat Hypothalamus During Chronic Stress

    PubMed Central

    Karami Kheirabad, Maryam; Namavar Jahromi, Bahia; Tamadon, Amin; Ramezani, Amin; Ahmadloo, Somayeh; Sabet Sarvestan, Fatemeh; Koohi-Hosseinabadi, Omid

    2015-01-01

    The effects of chronic stress and glucocorticoids receptor antagonist (RU486) on expression of melanocortin 4 receptor (MC4R) mRNA in arcuate nucleus (ARC) of male rats were evaluated. In this study, adult male Sprague Dawley rats were placed into four groups (n=6/group); stress, RU486, stress/RU486, and control groups. In stress group, the rats were restrained, 1 h/day, for 12 days. In RU486 group, the rats were injected RU486 for 12 days. In stress/RU486 group, the rats were injected RU486 1 h before the stress process for 12 days. Relative expression of MC4R mRNA was determined using real-time PCR. Relative expression of MC4R mRNA in the stress group was higher than that of the control rats (P<0.05). Relative expressions of MC4R mRNA were not different between the stress, RU486 and stress/RU486 groups (P>0.05). Chronic restraint stress causes increase in mRNA expression of MC4R in ARC and blockade of glucocorticoid receptors has no effect on this up-regulation. PMID:26629487

  20. Cytochrome P450IA mRNA expression in feral Hudson River tomcod

    SciTech Connect

    Kreamer, G.L.; Squibb, K.; Gioeli, D.; Garte, S.J.; Wirgin, I. )

    1991-06-01

    The authors sought to determine if levels of cytochrome P450IA gene expression are environmentally induced in feral populations of Hudson River tomcod, a cancer prone fish, and whether laboratory exposure of tomcod to artificially spiked and naturally contaminated Hudson sediments can elicit a significant response. Using Northern blot analysis, they found levels of P450IA mRNA in tomcod collected from two Hudson River sites higher than those in tomcod from a river in Maine. Depuration of environmentally induced Hudson tomcod P450IA mRNA was rapid, with an initial detectable decline in P450 gene expression by 8 hr and basal levels reached by 5 days. Intraperitoneal injection of {beta}-napthoflavone in depurated Hudson tomcod resulted in a 15-fold induction of P450 gene expression within 26 hr. Exposure of depurated Hudson tomcod to natural sediment spiked with two PAHs resulted in a 7-fold induction of P450 gene expression. Exposure of depurated tomcod to sediment from a contaminated Hudson site also resulted in a 7- to 15-fold induction of P450IA mRNA expression. Northern blot analysis revealed a second polymorphic cytochrome P450IA mRNA band in some tomcod which was also detected by Southern blot analysis. Induction of cytochrome P450IA mRNA in Atlantic tomcod may provide a sensitive biomarker of environmentally relevant concentrations of some pollutants in the Hudson and other northeastern tidal rivers.

  1. Cytochrome P450IA mRNA expression in feral Hudson River tomcod.

    PubMed

    Kreamer, G L; Squibb, K; Gioeli, D; Garte, S J; Wirgin, I

    1991-06-01

    We sought to determine if levels of cytochrome P450IA gene expression are environmentally induced in feral populations of Hudson River tomcod, a cancer prone fish, and whether laboratory exposure of tomcod to artificially spiked and naturally contaminated Hudson sediments can elicit a significant response. Using Northern blot analysis, we found levels of P450IA mRNA in tomcod collected from two Hudson River sites higher than those in tomcod from a river in Maine. Depuration of environmentally induced Hudson tomcod P450IA mRNA was rapid, with an initial detectable decline in P450 gene expression by 8 hr and basal levels reached by 5 days. Intraperitoneal injection of beta-napthoflavone in depurated Hudson tomcod resulted in a 15-fold induction of P450 gene expression within 26 hr. Exposure of depurated Hudson tomcod to natural sediment spiked with two PAHs resulted in a 7-fold induction of P450 gene expression. Exposure of depurated tomcod to sediment from a contaminated Hudson site also resulted in a 7- to 15-fold induction of P450IA mRNA expression. Northern blot analysis revealed a second polymorphic cytochrome P450IA mRNA band in some tomcod which was also detected by Southern blot analysis. Induction of cytochrome P450IA mRNA in Atlantic tomcod may provide a sensitive biomarker of environmentally relevant concentrations of some pollutants in the Hudson and other northeastern tidal rivers. PMID:1855491

  2. Amygdala kindling increases fear responses and decreases glucocorticoid receptor mRNA expression in hippocampal regions.

    PubMed

    Kalynchuk, Lisa E; Meaney, Michael J

    2003-12-01

    Amygdala kindling dramatically increases fearful behavior in rats. Because kindling-induced fear increases in magnitude as rats receive more stimulations, kindling provides an excellent model for studying the nature and neural mechanisms of fear sensitization. In the present experiment, we studied whether the development of kindling-induced fear is related to changes in glucocorticoid receptor (GR) mRNA expression in various brain regions. Rats received 20, 60 or 100 amygdala kindling stimulations or 100 sham stimulations. One day after the final stimulation, their fearful behavior was assessed in an unfamiliar open field. Then, the rats were sacrificed and their brains were processed for in situ hybridization of GR mRNA expression. We found that compared with the sham-stimulated rats, the rats that received 60 or 100 kindling stimulations were significantly more fearful in the open field and also had significantly less GR mRNA expression in the dentate gyrus and CA1 subfield of the hippocampus. Importantly, the changes in fearful behavior were significantly correlated with the changes in GR mRNA expression. These results suggest that alterations in GR mRNA expression in hippocampal regions may play a role in the development of kindling-induced fear.

  3. Endoplasmic reticulum stress increases AT1R mRNA expression via TIA-1-dependent mechanism

    PubMed Central

    Backlund, Michael; Paukku, Kirsi; Kontula, Kimmo K.; Lehtonen, Jukka Y.A.

    2016-01-01

    As the formation of ribonucleoprotein complexes is a major mechanism of angiotensin II type 1 receptor (AT1R) regulation, we sought to identify novel AT1R mRNA binding proteins. By affinity purification and mass spectroscopy, we identified TIA-1. This interaction was confirmed by colocalization of AT1R mRNA and TIA-1 by FISH and immunofluorescence microscopy. In immunoprecipitates of endogenous TIA- 1, reverse transcription-PCR amplified AT1R mRNA. TIA-1 has two binding sites within AT1R 3′-UTR. The binding site proximal to the coding region is glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-dependent whereas the distal binding site is not. TIA-1 functions as a part of endoplasmic reticulum (ER) stress response leading to stress granule (SG) formation and translational silencing. We and others have shown that AT1R expression is increased by ER stress-inducing factors. In unstressed cells, TIA-1 binds to AT1R mRNA and decreases AT1R protein expression. Fluorescence microscopy shows that ER stress induced by thapsigargin leads to the transfer of TIA-1 to SGs. In FISH analysis AT1R mRNA remains in the cytoplasm and no longer colocalizes with TIA-1. Thus, release of TIA-1-mediated suppression by ER stress increases AT1R protein expression. In conclusion, AT1R mRNA is regulated by TIA-1 in a ER stress-dependent manner. PMID:26681690

  4. Drosophila glutamate receptor mRNA expression and mRNP particles.

    PubMed

    Ganesan, Subhashree; Karr, Julie E; Featherstone, David E

    2011-01-01

    The processes controlling glutamate receptor expression early in synaptogenesis are poorly understood. Here, we examine glutamate receptor (GluR) subunit mRNA expression and localization in Drosophila embryonic/larval neuromuscular junctions (NMJs). We show that postsynaptic GluR subunit gene expression is triggered by contact from the presynaptic nerve, approximately halfway through embryogenesis. After contact, GluRIIA and GluRIIB mRNA abundance rises quickly approximately 20-fold, then falls within a few hours back to very low levels. Protein abundance, however, gradually increases throughout development. At the same time that mRNA levels decrease following their initial spike, GluRIIA, GluRIIB, and GluRIIC subunit mRNA aggregates become visible in the cytoplasm of postsynaptic muscle cells. These mRNA aggregates do not colocalize with eIF4E, but nevertheless presumably represent mRNP particles of unknown function. Multiplex FISH shows that different GluR subunit mRNAs are found in different mRNPs. GluRIIC mRNPs are most common, followed by GluRIIA and then GluRIIB mRNPs. GluR mRNP density is not increased near NMJs, for any subunit; if anything, GluR mRNP density is highest away from NMJs and near nuclei. These results reveal some of the earliest events in postsynaptic development and provide a foundation for future studies of GluR mRNA biology.

  5. Expression of Npas4 mRNA in Telencephalic Areas of Adult and Postnatal Mouse Brain

    PubMed Central

    Damborsky, Joanne C.; Slaton, G. Simona; Winzer-Serhan, Ursula H.

    2015-01-01

    The transcription factor neuronal PAS domain-containing protein 4 (Npas4) is an inducible immediate early gene which regulates the formation of inhibitory synapses, and could have a significant regulatory role during cortical circuit formation. However, little is known about basal Npas4 mRNA expression during postnatal development. Here, postnatal and adult mouse brain sections were processed for isotopic in situ hybridization using an Npas4 specific cRNA antisense probe. In adults, Npas4 mRNA was found in the telencephalon with very restricted or no expression in diencephalon or mesencephalon. In most telencephalic areas, including the anterior olfactory nucleus (AON), piriform cortex, neocortex, hippocampus, dorsal caudate putamen (CPu), septum and basolateral amygdala nucleus (BLA), basal Npas4 expression was detected in scattered cells which exhibited strong hybridization signal. In embryonic and neonatal brain sections, Npas4 mRNA expression signals were very low. Starting at postnatal day 5 (P5), transcripts for Npas4 were detected in the AON, CPu and piriform cortex. At P8, additional Npas4 hybridization was found in CA1 and CA3 pyramidal layer, and in primary motor cortex. By P13, robust mRNA expression was located in layers IV and VI of all sensory cortices, frontal cortex and cingulate cortex. After onset of expression, postnatal spatial mRNA distribution was similar to that in adults, with the exception of the CPu, where Npas4 transcripts became gradually restricted to the most dorsal part. In conclusion, the spatial distribution of Npas4 mRNA is mostly restricted to telencephalic areas, and the temporal expression increases with developmental age during postnatal development, which seem to correlate with the onset of activity-driven excitatory transmission. PMID:26633966

  6. Detection of MDR1 mRNA expression with optimized gold nanoparticle beacon

    NASA Astrophysics Data System (ADS)

    Zhou, Qiumei; Qian, Zhiyu; Gu, Yueqing

    2016-03-01

    MDR1 (multidrug resistance gene) mRNA expression is a promising biomarker for the prediction of doxorubicin resistance in clinic. However, the traditional technical process in clinic is complicated and cannot perform the real-time detection mRNA in living single cells. In this study, the expression of MDR1 mRNA was analyzed based on optimized gold nanoparticle beacon in tumor cells. Firstly, gold nanoparticle (AuNP) was modified by thiol-PEG, and the MDR1 beacon sequence was screened and optimized using a BLAST bioinformatics strategy. Then, optimized MDR1 molecular beacons were characterized by transmission electron microscope, UV-vis and fluorescence spectroscopies. The cytotoxicity of MDR1 molecular beacon on L-02, K562 and K562/Adr cells were investigated by MTT assay, suggesting that MDR1 molecular beacon was low inherent cytotoxicity. Dark field microscope was used to investigate the cellular uptake of hDAuNP beacon assisted with ultrasound. Finally, laser scanning confocal microscope images showed that there was a significant difference in MDR1 mRNA expression in K562 and K562/Adr cells, which was consistent with the results of q-PCR measurement. In summary, optimized MDR1 molecular beacon designed in this study is a reliable strategy for detection MDR1 mRNA expression in living tumor cells, and will be a promising strategy for in guiding patient treatment and management in individualized medication.

  7. Estradiol-induced hypophagia is associated with the differential mRNA expression of hypothalamic neuropeptides.

    PubMed

    Silva, L E C M; Castro, M; Amaral, F C; Antunes-Rodrigues, J; Elias, L L K

    2010-08-01

    Estradiol participates in the control of energy homeostasis, as demonstrated by an increase in food intake and in body weight gain after ovariectomy in rats. In the present study, female Wistar rats (200-230 g, N = 5-15 per group), with free access to chow, were individually housed in metabolic cages. We investigated food intake, body weight, plasma leptin levels, measured by specific radioimmunoassay, and the hypothalamic mRNA expression of orexigenic and anorexigenic neuropeptides, determined by real-time PCR, in ovariectomized rats with (OVX+E) and without (OVX) estradiol cypionate treatment (10 microg/kg body weight, sc, for 8 days). Hormonal and mRNA expression were determined at pre-feeding and 4 h after food intake. OVX+E rats showed lower food intake, less body weight gain and lower plasma leptin levels. In the OVX+E group, we also observed a reduction of neuropeptide Y (NPY), agouti-related protein (AgRP) and cocaine- and amphetamine-regulated transcript (CART) mRNA expression in the arcuate nucleus and a decrease in orexin A in the lateral hypothalamic area (LHA). There was an increase in leptin receptor (LepRb), melanocortin-4 receptor (MC4-R), CART, and mainly corticotropin-releasing hormone (CRH) mRNA in the paraventricular nucleus and LepRb and CART mRNA in the LHA. These data show that hypophagia induced by estradiol treatment is associated with reduced hypothalamic expression of orexigenic peptides such as NPY, AgRP and orexin A, and increased expression of the anorexigenic mediators MC4-R, LepRb and CRH. In conclusion, estradiol decreases food intake, and this effect seems to be mediated by peripheral factors such as leptin and the differential mRNA expression of neuropeptides in the hypothalamus.

  8. Prediction of microRNAs affecting mRNA expression during retinal development

    PubMed Central

    2010-01-01

    Background MicroRNAs (miRNAs) are small RNA molecules (~22 nucleotides) which have been shown to play an important role both in development and in maintenance of adult tissue. Conditional inactivation of miRNAs in the eye causes loss of visual function and progressive retinal degeneration. In addition to inhibiting translation, miRNAs can mediate degradation of targeted mRNAs. We have previously shown that candidate miRNAs affecting transcript levels in a tissue can be deduced from mRNA microarray expression profiles. The purpose of this study was to predict miRNAs which affect mRNA levels in developing and adult retinal tissue and to confirm their expression. Results Microarray expression data from ciliary epithelial retinal stem cells (CE-RSCs), developing and adult mouse retina were generated or downloaded from public repositories. Analysis of gene expression profiles detected the effects of multiple miRNAs in CE-RSCs and retina. The expression of 20 selected miRNAs was confirmed by RT-PCR and the cellular distribution of representative candidates analyzed by in situ hybridization. The expression levels of miRNAs correlated with the significance of their predicted effects upon mRNA expression. Highly expressed miRNAs included miR-124, miR-125a, miR-125b, miR-204 and miR-9. Over-expression of three miRNAs with significant predicted effects upon global mRNA levels resulted in a decrease in mRNA expression of five out of six individual predicted target genes assayed. Conclusions This study has detected the effect of miRNAs upon mRNA expression in immature and adult retinal tissue and cells. The validity of these observations is supported by the experimental confirmation of candidate miRNA expression and the regulation of predicted target genes following miRNA over-expression. Identified miRNAs are likely to be important in retinal development and function. Misregulation of these miRNAs might contribute to retinal degeneration and disease. Conversely, manipulation

  9. MRNA expression of genes regulating lipid metabolism in ringed seals (Pusa hispida) from differently polluted areas.

    PubMed

    Castelli, Martina Galatea; Rusten, Marte; Goksøyr, Anders; Routti, Heli

    2014-01-01

    There is a growing concern about the ability of persistent organic pollutants (POPs) to influence lipid metabolism. Although POPs are found at high concentrations in some populations of marine mammals, for example in the ringed seal (Pusa hispida) from the Baltic Sea, little is known about the effects of POPs on their lipid metabolism. An optimal regulation of lipid metabolism is crucial for ringed seals during the fasting/molting season. This is a physiologically stressful period, during which they rely on the energy stored in their fat reserves. The mRNA expression levels for seven genes involved in lipid metabolism were analyzed in liver and/or blubber tissue from molting ringed seals from the polluted Baltic Sea and a less polluted reference location, Svalbard (Norway). mRNA expression of genes encoding peroxisome proliferator-activated receptors (PPAR) α and γ and their target genes acyl-coenzyme A oxidase 1 (ACOX1) and cluster of differentiation 36 (CD36) were analyzed in liver. mRNA expression level of genes encoding PPARβ, PPARγ and their target genes encoding fatty acid binding protein 4 (FABP4) and adiponectin (ADIPOQ) were measured in inner and middle blubber layers. In addition, we evaluated the influence of molting status on hepatic mRNA expression of genes encoding PPARs and their target genes in ringed seals from Svalbard. Our results show higher mRNA expression of genes encoding hepatic PPARγ and adipose PPARβ, FABP4, and ADIPOQ in the Baltic seals compared to the Svalbard seals. A positive relationship between mRNA expressions of genes encoding hepatic PPARγ, adipose FABP4, adipose ADIPOQ and ΣPOP concentrations was observed. These findings suggest that lipid metabolism may be affected by contaminant exposure in the Baltic population. mRNA expression of genes encoding PPARβ, PPARγ, FABP4 and ADIPOQ were similar between the mid and inner adipose layer. Hepatic mRNA expression of genes encoding PPARα and PPARγ was higher in the pre

  10. Peripheral blood mRNA expressions of stress biomarkers in manic episode and subsequent remission.

    PubMed

    Köse Çinar, Rugül; Sönmez, Mehmet Bülent; Görgülü, Yasemin

    2016-08-01

    Theoretical models of the neuroprogressive nature of bipolar disorder (BD) are based on the hypothesis that it is an accelerated aging disease, with the allostatic load playing a major role. Glucocorticoids, oxidative stress markers, inflammatory cytokines and neurotrophins play important roles in BD. The messenger ribonucleic acid (mRNA) expressions of brain-derived neurotrophic factor (BDNF), tissue plasminogen activator (tPA), glucocorticoid receptor (GR), heat shock protein 70 (HSP70), tumour necrosis factor-alpha (TNF-α) were examined in the peripheral blood of 20 adult male, drug-free BD patients during manic and remission periods and in 20 adult male, healthy controls. mRNA expression was measured using the quantitative real-time polymerase chain reaction (qRT-PCR). Compared to the controls, the expressions of BDNF and tPA mRNA were down-regulated in mania. In remission, BNDF and tPA mRNA levels increased, but they were still lower than those of the controls. Between mania and remission periods, only the change in mRNA levels of BDNF reached statistical significance. The results suggest that BDNF and tPA may be biomarkers of BD and that proteolytic conversion of BDNF may be important in the pathophysiology of BD. The change in BDNF levels between mania and remission could be adaptive and used to follow the progression of BD. PMID:27138695

  11. Decreased TIM-3 mRNA expression in peripheral blood mononuclear cells from nephropathy patients.

    PubMed

    Cai, X Z; Liu, N; Qiao, Y; Du, S Y; Chen, Y; Chen, D; Yu, S; Jiang, Y

    2015-01-01

    Increasing evidence shows that TIM-1 and TIM-3 in-fluence chronic autoimmune diseases, and their expression levels in immune cells from nephritic patients are still unknown. Real-time transcription-polymerase chain reaction analysis was used to deter-mine expression levels of TIM-1 and TIM-3 mRNA in peripheral blood mononuclear cells (PBMCs) from 36 patients with minimal change glo-merulopathy (MCG), 65 patients with lupus nephritis (LN), 78 patients with IgA nephropathy (IgAN), 55 patients with membranous nephropa-thy (MN), 22 patients with crescentic glomerulonephritis (CGN), 26 patients with anaphylactoid purpura nephritis (APN), and 63 healthy controls. TIM-3 mRNA expression significantly decreased in PBMCs from nephritic patients (LN, P < 0.0001; MCG, P < 0.0001; MN, P = 0.0031; CGN, P = 0.0464; IgAN, P = 0.0002; APN, P = 0.0392) com-pared with healthy controls. In contrast, there was no significant differ-ence in TIM-1 mRNA expression between the patients and the healthy controls. Our results suggest that insufficient expression of TIM-3 mRNA may be involved in the pathogenesis of nephropathy.

  12. mRNA Expression of Ovine Angiopoietin-like Protein 4 Gene in Adipose Tissues.

    PubMed

    Zhang, Jing; Jing, Jiong-Jie; Jia, Xia-Li; Qiao, Li-Ying; Liu, Jian-Hua; Liang, Chen; Liu, Wen-Zhong

    2016-05-01

    Angiopoietin-like protein 4 (ANGPTL4) is involved in a variety of functions, including lipoprotein metabolism and angiogenesis. To reveal the role of ANGPTL4 in fat metabolism of sheep, ovine ANGPTL4 mRNA expression was analyzed in seven adipose tissues from two breeds with distinct tail types. Forty-eight animals with the gender ratio of 1:1 for both Guangling Large Tailed (GLT) and Small Tailed Han (STH) sheep were slaughtered at 2, 4, 6, 8, 10, and 12 months of age, respectively. Adipose tissues were collected from greater and lesser omental, subcutaneous, retroperitoneal, perirenal, mesenteric, and tail fats. Ontogenetic mRNA expression of ANGPTL4 in these adipose tissues from GTL and STH was studied by quantitative real time polymerase chain reaction. The results showed that ANGPTL4 mRNA expressed in all adipose tissues studied with the highest in subcutaneous and the lowest in mesenteric fat depots. Months of age, tissue and breed are the main factors that significantly influence the mRNA expression. These results provide new insights into ovine ANGPTL4 gene expression and clues for its function mechanism.

  13. Soya phytoestrogens change cortical and hippocampal expression of BDNF mRNA in male rats.

    PubMed

    File, Sandra E; Hartley, David E; Alom, Nazmul; Rattray, Marcus

    2003-02-27

    Adult male hooded Lister rats were either fed a diet containing 150 microg/g soya phytoestrogens or a soya-free diet for 18 days. This concentration of phytoestrogens should have been sufficient to occupy the oestrogen-beta, but not the oestrogen-alpha, receptors. Using in situ hybridisation, significant reductions were found in brain-derived neurotrophic factor (BDNF) mRNA expression in the CA3 and CA4 region of the hippocampus and in the cerebral cortex in the rats fed the diet containing phytoestrogens, compared with those on the soya-free diet. No changes in glutamic acid decarboxylase-67 or glial fibrillary acidic protein mRNA were found. This suggests a role for oestrogen-beta receptors in regulating BDNF mRNA expression.

  14. Selecting Reliable mRNA Expression Measurements Across Platforms Improves Downstream Analysis.

    PubMed

    Tong, Pan; Diao, Lixia; Shen, Li; Li, Lerong; Heymach, John Victor; Girard, Luc; Minna, John D; Coombes, Kevin R; Byers, Lauren Averett; Wang, Jing

    2016-01-01

    With increasing use of publicly available gene expression data sets, the quality of the expression data is a critical issue for downstream analysis, gene signature development, and cross-validation of data sets. Thus, identifying reliable expression measurements by leveraging multiple mRNA expression platforms is an important analytical task. In this study, we propose a statistical framework for selecting reliable measurements between platforms by modeling the correlations of mRNA expression levels using a beta-mixture model. The model-based selection provides an effective and objective way to separate good probes from probes with low quality, thereby improving the efficiency and accuracy of the analysis. The proposed method can be used to compare two microarray technologies or microarray and RNA sequencing measurements. We tested the approach in two matched profiling data sets, using microarray gene expression measurements from the same samples profiled on both Affymetrix and Illumina platforms. We also applied the algorithm to mRNA expression data to compare Affymetrix microarray data with RNA sequencing measurements. The algorithm successfully identified probes/genes with reliable measurements. Removing the unreliable measurements resulted in significant improvements for gene signature development and functional annotations.

  15. Norepinephrine does not alter NPY and POMC mRNA expression in neonatal chicks.

    PubMed

    Katayama, Sachiko; Tomonaga, Shozo; Sato, Momoka; Yamane, Haruka; Tsuneyoshi, Yousuke; Denbow, D Michael; Furuse, Mitsuhiro

    2010-05-01

    Norepinephrine (NE), synthesized in both the central and peripheral nervous system, is involved in food intake regulation of both mammals and chickens. Neuropeptide Y (NPY), a potent orexigenic peptide, is colocalized with NE neurons in the central and peripheral nervous system, suggesting an interaction. Proopiomelanocortin (POMC) is the precursor of alpha-melanocyte stimulating hormone, a potent anorexigenic peptide synthesized in the hypothalamus. In this study, two experiments were conducted to examine the effect of intracerebroventricular (ICV) injection of NE on appetite mediators in neonatal chicks (Gallus gallus). Experiment 1 was done to confirm the effect of centrally administered NE (0, 25, 50, and 100 microg) on food intake following a 3h fast, and to determine the change in NPY mRNA expression in the central nervous system (CNS). In Experiment 2, chicks fed ad libitum were treated ICV with NE (50 microg) to determine if changes occurred in brain NPY and POMC mRNA levels. In Experiment 1, the ICV injection of NE dose-dependently reduced food intake, but there was no change in NPY mRNA expression in the CNS. In Experiment 2, there was no significant change in NPY and POMC mRNA expression between the control and NE-treated group, indicating that ICV injection of NE may not be associated with changes in NPY or POMC gene expression.

  16. EXPRESSION OF AHR AND ARNT MRNA IN CULTURED HUMAN ENDOMETRIAL EXPLANTS EXPOSED TO TCDD

    EPA Science Inventory

    Expression of AhR and ARNT mRNA in cultured human endometrial explants exposed to TCDD.

    Pitt JA, Feng L, Abbott BD, Schmid J, Batt RE, Costich TG, Koury ST, Bofinger DP.

    Curriculum in Toxicology, University of North Carolina, Chapel Hill, NC 27599, USA.

    Endom...

  17. mRNA expression and protein localization of dentin matrix protein 1 during dental root formation.

    PubMed

    Toyosawa, S; Okabayashi, K; Komori, T; Ijuhin, N

    2004-01-01

    Dentin matrix protein 1 (DMP1) is an acidic phosphoprotein. DMP1 was initially detected in dentin and later in other mineralized tissues including cementum and bone, but the DMP1 expression pattern in tooth is still controversial. To determine the precise localization of DMP1 messenger RNA (mRNA) and the protein in the tooth, we performed in situ hybridization and immunohistochemical analyses using rat molars and incisors during various stages of root formation. During root dentin formation of molars, DMP1 mRNA was detected in root odontoblasts in parallel with mineralization of the dentin. However, the level of DMP1 mRNA expression in root odontoblasts decreased near the coronal part and was absent in coronal odontoblasts. DMP1 protein was localized along dentinal tubules and their branches in mineralized root dentin, and the distribution of DMP1 shifted from the end of dentinal tubules to the base of the tubules as dentin formation progressed. During the formation of the acellular cementum, DMP1 mRNA was detected in cementoblasts lining the acellular cementum where its protein was localized. During the formation of the cellular cementum, DMP1 mRNA was detected in cementocytes embedded in the cellular cementum but not in cementoblasts, and its protein was localized in the pericellular cementum of cementocytes including their processes. During dentin formation of incisors, DMP1 mRNA was detected in odontoblasts on the cementum-related dentin, where its protein was localized along dentinal tubules near the mineralization front. The localization of DMP1 mRNA and protein in dentin and cementum was related to their mineralization, suggesting that one of the functions of DMP1 may be involved in the mineralization of dentin and cementum during root formation. PMID:14751569

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

    SciTech Connect

    Dalgaard, Louise T.

    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.

  19. Expression and regulation of Icer mRNA in the Syrian hamster pineal gland.

    PubMed

    Diaz, Elena; Garidou, Marie-Laure; Dardente, Hugues; Salingre, Anthony; Pévet, Paul; Simonneaux, Valérie

    2003-04-10

    Inducible-cAMP early repressor (ICER) is a potent inhibitor of CRE (cAMP-related element)-driven gene transcription. In the rat pineal gland, it has been proposed to be part of the mechanisms involved in the shutting down of the transcription of the gene coding for arylalkylamine N-acetyltransferase (AA-NAT, the melatonin rhythm-generating enzyme). In this study, we report that ICER is expressed in the pineal gland of the photoperiodic rodent Syrian hamster although with some difference compared to the rat. In the Syrian hamster pineal, Icer mRNA levels, low at daytime, displayed a 20-fold increase during the night. Nighttime administration of a beta-adrenergic antagonist, propranolol, significantly reduced Icer mRNA levels although daytime administration of a beta-adrenergic agonist, isoproterenol, was unable to raise the low amount of Icer mRNA. These observations indicate that Icer mRNA expression is induced by the clock-driven norepinephrine release and further suggest that this stimulation is restricted to nighttime, as already observed for Aa-nat gene transcription. Furthermore, we found that the daily profile of Icer mRNA displayed photoperiodic variation with a lengthening of the nocturnal peak in short versus long photoperiod. These data indicate that ICER may be involved in both daily and seasonal regulation of melatonin synthesis in the Syrian hamster.

  20. Influenza A viruses suppress cyclooxygenase-2 expression by affecting its mRNA stability

    PubMed Central

    Dudek, Sabine Eva; Nitzsche, Katja; Ludwig, Stephan; Ehrhardt, Christina

    2016-01-01

    Infection with influenza A viruses (IAV) provokes activation of cellular defence mechanisms contributing to the innate immune and inflammatory response. In this process the cyclooxygenase-2 (COX-2) plays an important role in the induction of prostaglandin-dependent inflammation. While it has been reported that COX-2 is induced upon IAV infection, in the present study we observed a down-regulation at later stages of infection suggesting a tight regulation of COX-2 by IAV. Our data indicate the pattern-recognition receptor RIG-I as mediator of the initial IAV-induced COX-2 synthesis. Nonetheless, during on-going IAV replication substantial suppression of COX-2 mRNA and protein synthesis could be detected, accompanied by a decrease in mRNA half-life. Interestingly, COX-2 mRNA stability was not only imbalanced by IAV replication but also by stimulation of cells with viral RNA. Our results reveal tristetraprolin (TTP), which is known to bind COX-2 mRNA and promote its rapid degradation, as regulator of COX-2 expression in IAV infection. During IAV replication and viral RNA accumulation TTP mRNA synthesis was induced, resulting in reduced COX-2 levels. Accordingly, the down-regulation of TTP resulted in increased COX-2 protein expression after IAV infection. These findings indicate a novel IAV-regulated cellular mechanism, contributing to the repression of host defence and therefore facilitating viral replication. PMID:27265729

  1. Cistanches Herba aqueous extract affecting serum BGP and TRAP and bone marrow Smad1 mRNA, Smad5 mRNA, TGF-β1 mRNA and TIEG1 mRNA expression levels in osteoporosis disease.

    PubMed

    Liang, Hai-Dong; Yu, Fang; Tong, Zhi-Hong; Zhang, Hong-Quan; Liang, Wu

    2013-02-01

    We studied molecular mechanism of Cistanches Herba aqueous extract (CHAE) in ovariectomized (OVX) rats, as an experimental model of postmenopausal osteoporosis. Female rats were either sham-operated or bilaterally OVX; and at 60 days postoperatively. The OVX group (n = 8) received an ovariectomy and treatment with normal saline for 90 days commencing from 20th post ovariectomy day. The ovariectomized +CHAE (OVX + CHAE) group (n = 8) received an ovariectomy and were treated with Cistanches Herba aqueous extract of 100 mg/kg body weight daily for 90 days commencing from 22nd post ovariectomy day. The ovariectomy +CHAE (OVX + CHAE) group (n = 8) received an ovariectomy, and were treated with the of 200 mg/kg body weight daily for 90 days commencing from 20th post ovariectomy day. Serum BGP and TRAP, E2, FSH and LH level, bone marrow Smad1, Smad5, TGF-β1 and TIEG1 mRNA expression levels were examined. Results showed that serum BGP and TRAP, FSH and LH levels were significantly increased, whereas E2, Smad1, Smad5, TGF-β1 and TIEG1 mRNA and proteins expression levels were significantly decreased in OVX rats compared to sham rats. 90 days of CHAE treatment could significantly decrease serum BGP and TRAP, FSH and LH levels, and increase E2, Smad1, Smad5, TGF-β1 and TIEG1 mRNA and proteins expression levels in OVX rats. It can be concluded that CHAE play its protective effect against OVX-induced bone degeneration partly by regulating some bone metabolism related genes, e.g. Smad1, Smad5, TGF-β1 and TIEG1.

  2. BAY11 enhances OCT4 synthetic mRNA expression in adult human skin cells

    PubMed Central

    2013-01-01

    Introduction The OCT4 transcription factor is involved in many cellular processes, including development, reprogramming, maintaining pluripotency and differentiation. Synthetic OCT4 mRNA was recently used (in conjunction with other reprogramming factors) to generate human induced pluripotent stem cells. Here, we discovered that BAY 11-7082 (BAY11), at least partially through an NF-κB-inhibition based mechanism, could significantly increase the expression of OCT4 following transfection of synthetic mRNA (synRNA) into adult human skin cells. Methods We tested various chemical and molecular small molecules on their ability to suppress the innate immune response seen upon synthetic mRNA transfection. Three molecules - B18R, BX795, and BAY11 - were used in immunocytochemical and proliferation-based assays. We also utilized global transcriptional meta-analysis coupled with quantitative PCR to identify relative gene expression downstream of OCT4. Results We found that human skin cells cultured in the presence of BAY11 resulted in reproducible increased expression of OCT4 that did not inhibit normal cell proliferation. The increased levels of OCT4 resulted in significantly increased expression of genes downstream of OCT4, including the previously identified SPP1, DUSP4 and GADD45G, suggesting the expressed OCT4 was functional. We also discovered a novel OCT4 putative downstream target gene SLC16A9 which demonstrated significantly increased expression following elevation of OCT4 levels. Conclusions For the first time we have shown that small molecule-based stabilization of synthetic mRNA expression can be achieved with use of BAY11. This small molecule-based inhibition of innate immune responses and subsequent robust expression of transfected synthetic mRNAs may have multiple applications for future cell-based research and therapeutics. PMID:23388106

  3. Beta-integrin of Anopheles gambiae: mRNA cloning and analysis of structure and expression.

    PubMed

    Mahairaki, V; Lycett, G; Blass, C; Louis, C

    2001-06-01

    We have isolated an mRNA encoding a beta integrin subunit of the malaria mosquito Anopheles gambiae. Our analysis predicts a protein that is very similar to betaPS, the fruitfly orthologue. The gene is expressed during all developmental stages and it is found in all body parts, including the midgut. Finally, the expression of the gene does not seem to be modulated during blood meals, except for a substantial increase 48 h posthaematophagy, when digestion is nearly complete. PMID:11437913

  4. Regulation of leptin mRNA and protein expression in pituitary somatotropes.

    PubMed

    McDuffie, Iris A; Akhter, Noor; Childs, Gwen V

    2004-02-01

    Leptin, the ob protein, regulates food intake and satiety and can be found in the anterior pituitary. Leptin antigens and mRNA were studied in the anterior pituitary (AP) cells of male and female rats to learn more about its regulation. Leptin antigens were found in over 40% of cells in diestrous or proestrous female rats and in male rats. Lower percentages of AP cells were seen in the estrous population (21 +/- 7%). During peak expression of antigens, co-expression of leptin and growth hormone (GH) was found in 27 +/- 4% of AP cells. Affinity cytochemistry studies detected 24 +/- 3% of AP cells with leptin proteins and growth hormone releasing hormone (GHRH) receptors. These data suggested that somatotropes were a significant source of leptin. To test regulatory factors, estrous and diestrous AP populations were treated with estrogen (100 pM) and/or GHRH (2 nM) to learn if either would increase leptin expression in GH cells. To rule out the possibility that the immunoreactive leptin was bound to receptors in somatotropes, leptin mRNA was also detected by non-radioactive in situ hybridization in this group of cells. In estrous female rats, 39 +/- 0.9% of AP cells expressed leptin mRNA, indicating that the potential for leptin production was greater than predicted from the immunolabeling. Estrogen and GHRH together (but not alone) increased percentages of cells with leptin protein (41 +/- 9%) or mRNA (57 +/- 5%). Estrogen and GHRH also increased the percentages of AP cells that co-express leptin mRNA and GH antigens from 20 +/- 2% of AP cells to 37 +/- 5%. Although the significance of leptin in GH cells is not understood, it is clearly increased after stimulation with GHRH and estrogen. Because GH cells also have leptin receptors, this AP leptin may be an autocrine or paracrine regulator of pituitary cell function.

  5. Regulation of bovine pyruvate carboxylase mRNA and promoter expression by thermal stress.

    PubMed

    White, H M; Koser, S L; Donkin, S S

    2012-09-01

    Pyruvate carboxylase (PC) catalyzes the rate-limiting step in gluconeogenesis from lactate and is a determinant of tricarboxylic acid cycle carbon flux. Bovine PC 5' untranslated region (UTR) mRNA variants are the products of a single PC gene containing 3 promoter regions (P3, P2, and P1, 5' to 3') that are responsive to physiological and nutritional stressors. The objective of this study was to determine the direct effects of thermal stress on PC mRNA and gene expression in bovine hepatocyte monolayer cultures, rat hepatoma (H4IIE) cells, and Madin-Darby bovine kidney epithelial (MDBK) cells. Hepatocytes were isolated from 3 Holstein bull calves and used to prepare monolayer cultures. Rat hepatoma cells and MDBK cells were obtained from American Type Culture Collection, Manassas, VA. Beginning 24 h after initial seeding, cells were subjected to either 37°C (control) or 42°C (thermal stress) for 24 h. Treatments were applied in triplicate in a minimum of 3 independent cell preparations. For bovine primary hepatocytes, endogenous expression of bovine PC mRNA increased (P < 0.1) with 24 h of thermal stress (1.31 vs. 2.79 ± 0.49, arbitrary units, control vs. thermal stress, respectively), but there was no change (P ≥ 0.1) in cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) mRNA expression. Similarly, exposure of MDBK cells to thermal stress increased (P < 0.1) expression of bovine PC mRNA without altering (P ≥ 0.1) PEPCK-C mRNA expression. Conversely, there was no effect (P ≥ 0.1) of thermal stress on endogenous rat PC (0.47 vs. 0.30 ± 0.08, control vs. thermal stress) or PEPCK-C (1.61 vs. 1.20 ± 0.48, arbitrary units, control vs. thermal stress, respectively) mRNA expressions in H4IIE cells. To further investigate the regulation of PC, H4IIE cells were transiently transfected with bovine promoter-luciferase constructs containing either P1, P2, or P3, and exposed to thermal stress for 23 h. Activity of P1 was suppressed (P < 0.1) 5-fold, activity of P2

  6. Decreased relative expression level of trefoil factor 3 mRNA to galectin-3 mRNA distinguishes thyroid follicular carcinoma from adenoma.

    PubMed

    Takano, Toru; Miyauchi, Akira; Yoshida, Hiroshi; Kuma, Kanji; Amino, Nobuyuki

    2005-02-28

    The expression level of trefoil factor 3 (TFF3) mRNA is a marker for distinguishing thyroid follicular adenomas from carcinomas. However, when measuring the expression level of TFF3 mRNA in fine needle aspiration biopsies, an appropriate internal control mRNA, of which expression is restricted in thyroid epithelial--derived cells, is necessary, since they are often contaminated with a considerable number of blood cells, which do not express TFF3 mRNA. In this study, we evaluated the efficiency of molecular-based diagnosis of thyroid follicular carcinoma by measuring the relative expression of TFF3 mRNA by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) using galectin-3 mRNA as an internal control. The TFF3/galectin-3 mRNA ratio (T/G ratio) was measured in 54 follicular adenomas and 29 follicular carcinomas. It was markedly decreased in 7 follicular carcinomas of widely invasive type and with evident distant metastases. When the cutoff point was set at 16.0 by a receiver operator characteristic curve, the TG ratio showed good agreement with the pathological diagnosis [kappa=0.55; 95% confidence interval (CI), 0.34-0.77]. This agreement was better when the pathologically questionable cases were excluded (kappa=0.72; 95% CI, 0.49-0.95). Quantification of the T/G ratio may be a useful tool for the distinction between follicular adenomas and carcinomas, which is the most difficult in thyroid pathology.

  7. Alpha1-adrenoreceptor in human hippocampus: binding and receptor subtype mRNA expression.

    PubMed

    Szot, Patricia; White, Sylvia S; Greenup, J Lynne; Leverenz, James B; Peskind, Elaine R; Raskind, Murray A

    2005-10-01

    Alpha1-adrenoreceptors (AR), of which three subtypes exist (alpha1A-, alpha1B- and alpha1D-AR) are G-protein-coupled receptors that mediate the actions of norepinephrine and epinephrine both peripherally and centrally. In the CNS, alpha1-ARs are found in the hippocampus where animal studies have shown the ability of alpha1-AR agents to modulate long-term potentiation and memory; however, the precise distribution of alpha1-AR expression and its subtypes in the human brain is unknown making functional comparisons difficult. In the human hippocampus, 3H-prazosin (alpha1-AR antagonist) labels only the dentate gyrus (molecular, granule and polymorphic layers) and the stratum lucidum of the CA3 homogeneously. Human alpha1A-AR mRNA in the hippocampus is observed only in the dentate gyrus granule cell layer, while alpha1D-AR mRNA expression is observed only in the pyramidal cell layers of CA1, CA2 and CA3, regions where 3H-prazosin did not bind. alpha1B-AR mRNA is not expressed at detectable levels in the human hippocampus. These results confirm a difference in hippocampal alpha1-AR localization between rat and humans and further describe a difference in the localization of the alpha1A- and alpha1D-AR mRNA subtype between rats and humans. PMID:16039007

  8. Leishmania amazonensis: Anionic currents expressed in oocytes upon microinjection of mRNA from the parasite.

    PubMed

    Lagos M, Luisa F; Moran, Oscar; Camacho, Marcela

    2007-06-01

    Transport mechanisms involved in pH homeostasis are relevant for the survival of Leishmania parasites. The presence of chloride conductive pathways in Leishmania has been anticipated since anion channel inhibitors limit the proton extrusion mediated by the H+ATPase, which is the major regulator of intracellular pH in amastigotes. In this study, we used Xenopus laevis oocytes as a heterologous expression system in which to study the expression of ion channels upon microinjection of polyA mRNA from Leishmania amazonensis. After injection of polyA mRNA into the oocytes, we measured three different types of currents. We discuss the possible origin of each, and propose that Type 3 currents could be the result of the heterologous expression of proteins from Leishmania since they show different pharmacological and biophysical properties as compared to endogenous oocyte currents. PMID:17328895

  9. Adrenocorticotropin receptors: Functional expression from rat adrenal mRNA in Xenopus laevis oocytes

    SciTech Connect

    Mertz, L.M.; Catt, K.J. )

    1991-10-01

    The adrenocorticotropin (ACTH) receptor, which binds corticotropin and stimulates adenylate cyclase and steroidogenesis in adrenocortical cells, was expressed in Xenopus laevis oocytes microinjected with rat adrenal poly(A){sup +} RNA. Expression of the ACTH receptor in individual stage 5 and 6 oocytes was monitored by radioimmunoassay of ligand-stimulated cAMP production. Injection of 5-40 ng of adrenal mRNA caused dose-dependent increases in ACTH-responsive cAMP production. Size fractionation of rat adrenal poly(A){sup +}RNA by sucrose density-gradient centrifugation revealed that mRNA encoding the ACTH receptor was present in the 1.1-to 2.0-kilobase fraction. These data indicate that ACTH receptors can be expressed from adrenal mRNA in Xenopus oocytes and are fully functional in terms of ligand specificity and signal generation. The extracellular cAMP response to ACTH is a sensitive and convenient index of receptor expression. This system should permit more complete characterization and expression cloning of the ACTH receptor.

  10. Acute stress increases neuropsin mRNA expression in the mouse hippocampus through the glucocorticoid pathway.

    PubMed

    Harada, Akiko; Shiosaka, Sadao; Ishikawa, Yasuyuki; Komai, Shoji

    2008-05-01

    Stress affects synaptic plasticity and may alter various types of behaviour, including anxiety or memory formation. In the present study, we examined the effects of acute stress (1 h restraint with or without tail-shock) on mRNA levels of a plasticity-related serine protease neuropsin (NP) in the hippocampus using semiquantitative RT-PCR and in situ hybridization. We found that NP mRNA expression was dramatically increased shortly after exposure to the acute restraint tail-shock stress and remained at high level for at least 24 h. The level of NP mRNA would be correlated to the elevated plasma concentration of the glucocorticoid corticosterone (CORT) and to the stress intensity. Application of CORT either onto primary cultured hippocampal neurons (5 nM) or in vivo to adrenalectomized (ADX) mice (10 mg/kg B.W., s.c.) mimicked the effect of stress and significantly elevated NP mRNA. These results suggest that the upregulation of NP mRNA after stress is CORT-dependent and point to a role for neuropsin in stress-induced neuronal plasticity.

  11. ABCB1 C3435T and G2677T/A polymorphism decreased the risk for steroid-induced osteonecrosis of the femoral head after kidney transplantation.

    PubMed

    Asano, Takeshi; Takahashi, Kenji A; Fujioka, Mikihiro; Inoue, Shigehiro; Okamoto, Masahiko; Sugioka, Nobuyuki; Nishino, Hoyoku; Tanaka, Takashi; Hirota, Yoshio; Kubo, Toshikazu

    2003-11-01

    Advances in transplantation technology have brought about great benefits to patients suffering from organ failure, but the problem still remains of complications induced by steroids used for post-transplant immunosuppression. Among the side-effects caused by steroids, non-traumatic osteonecrosis of the femoral head (ONF) constitutes a serious problem. The same protocol for steroid administration induces ONF in some patients, but not in others, indicating the presence of individual difference in steroid sensitivity. We hypothesized that this difference might be mediated by the drug-transport protein, P-glycoprotein (P-gp), and investigated the relationship between single nucleotide polymorphisms in the multidrug resistance gene 1 (ABCB1, MDR1) encoding P-gp and ONF. Subjects comprised 136 patients receiving kidney transplantation. Thirty patients developed post-transplant ONF. Genomic DNA was extracted from peripheral blood, and genotypes of ABCB1 C3435T (exon 26) and G2677T/A (exon 21) were determined by direct sequencing. Multivariate analyses based on clinical information were performed to determine the relationship between ABCB1 genotypes and ONF. The dose/concentration (D/C) ratios of tacrolimus were also determined to estimate the activity of P-gp in patients with different genotypes of ABCB1 C3435T (CC, CT, TT), and in those who did and did not develop ONF. The ABCB1 3435TT genotype showed a significantly lower incidence of ONF (adjusted odds ratio = 0.10, P = 0.034). The D/C ratio in the 3435TT genotype was significantly higher than that in the 3435CC genotype. The D/C ratio in patients developing ONF was significantly higher than in those patients who did not develop ONF. The results suggest increased activity of P-gp in patients with the 3435TT genotype and in those who did not develop ONF. The ABCB1 2677 homozygous variant type also showed a lower incidence of ONF (adjusted odds ratio = 0.26, P = 0.056). The 3435T and 3435C alleles were in linkage

  12. Genetic association analysis of ATP binding cassette protein family reveals a novel association of ABCB1 genetic variants with epilepsy risk, but not with drug-resistance.

    PubMed

    Balan, Shabeesh; Bharathan, Sumitha Prameela; Vellichiramal, Neetha Nanoth; Sathyan, Sanish; Joseph, Vijai; Radhakrishnan, Kurupath; Banerjee, Moinak

    2014-01-01

    Epilepsy constitutes a heterogeneous group of disorders that is characterized by recurrent unprovoked seizures due to widely different etiologies. Multidrug resistance remains a major issue in clinical epileptology, where one third of patients with epilepsy continue to have seizures. Role of efflux transporters in multidrug resistant epilepsy has been attributed to drug-resistant epilepsy although, with discrepant observation in genetic studies. These discrepancies could be attributed to variety of factors such as variable definition of the anti-epileptic drug (AED)-resistance, variable epilepsy phenotypes and ethnicities among the studies. In the present study we inquired the role of multidrug transporters ABCB1 and ABCG2 variants in determining AED-resistance and susceptibility to epilepsy in three well-characterized cohorts comprising of mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE-HS) (prototype for AED-resistant epilepsy); juvenile myoclonic epilepsy (JME) (prototype for AED-responsive epilepsy); and healthy non-epileptic controls, in 738 subjects of Malayalam speaking south Indian ancestry. ABCB1 and ABCG2 variants were not found to be associated with drug resistance when AED-resistant and AED-responsive cohorts were compared. However, a significant association was observed between ABCB1 (C3435T) rs1045642 and risk of having epilepsy (MTLE-HS and JME pooled cohort; genotypic p-value = 0.0002; allelic p-value = 0.004). This association was seen persistent with MTLE-HS (genotypic p-value = 0.0008; allelic p-value = 0.004) and also with JME (genotypic p-value = 0.01; allelic p-value = 0.05) cohort individually. In-silico functional prediction indicated that ABCB1 rs1045642 has a deleterious impact on protein coding function and in splicing regulation. We conclude that the ABCB1 and ABCG2 variants do not confer to AED-resistance in the study population. However, ABCB1 rs1045642 increases vulnerability to epilepsy with greater tendency for MTLE

  13. Expression of survivin mRNA in gallbladder cancer: a diagnostic and prognostic marker?

    PubMed

    Nigam, Jaya; Chandra, Abhijit; Kazmi, Hasan Raza; Parmar, Devendra; Singh, Devendra; Gupta, Vishal; M, Noushif

    2014-09-01

    Survivin, an inhibitor of apoptosis, has been shown to be expressed in various malignancies. However, its role in gallbladder cancer (GBC) has not been evaluated yet. We investigated its expression in peripheral blood of patients with gallbladder diseases (gallstone disease (GSD), n = 30; GBC, n = 39) and compared with healthy controls (n = 25). Survivin expression was correlated with clinicopathological parameters, diagnosis, and prognosis of patients with GBC. Expression of survivin messenger RNA (mRNA) in blood was evaluated by real-time PCR. Significantly higher (P < 0.0001) expression of survivin mRNA was observed in GBC (2.2-fold) and GSD (1.52-fold) as compared to control. In GBC, increased survivin expression was significantly associated with higher tumor stage (stage III vs. stage II; P < 0.0001) and tumor differentiation (poor and moderate vs. well differentiated; P < 0.0001). No significant correlation was observed with any of the other clinicopathological parameters (age, gender, and presence or absence of gallstones) studied. Cutoff value of survivin mRNA relative quantification (RQ) was 1.08, with a sensitivity of 98.55 % and specificity of 100 % for the diseased group (GSD or GBC). RQ value of 1.71 differentiated GBC from GSD with a sensitivity of 89.74 % and specificity of 100 %. Increased expression of survivin was associated with a shorter median overall survival (12 vs. 18 months) in GBC patients. Differential expression of survivin in GBC suggests its possible role and association with poor prognosis. Expression of survivin in peripheral blood could be useful both in the diagnosis and prognosis of GBC.

  14. Differential expression of melanopsin mRNA and protein in Brown Norwegian rats.

    PubMed

    Hannibal, Jens; Georg, Birgitte; Fahrenkrug, Jan

    2013-01-01

    Melanopsin is expressed in a subpopulation of retinal ganglion cells rendering these cells intrinsically photosensitive (ipRGCs). The ipRGCs are the primary RGCs mediating light entrainment of the circadian clock and control of the pupillary light reflex, light regulated melatonin secretion and negative masking behaviour. Previous studies have demonstrated that melanopsin expression in albino rats is regulated by light and darkness. The present study was undertaken to study the influence of light and darkness during the circadian day and after extended periods of constant light and darkness on melanopsin expression in the pigmented retina of the Brown Norwegian rat (Rattus norvegicus). The diurnal and circadian expressions were examined in retinal extracts from rats euthanized every 4 h during a 24 h light/dark (LD) and a 24 h dark cycle (DD) using quantitative real-time PCR and Western blotting. To study whether light regulates melanopsin expression, rats were sacrificed after being placed in either constant light (LL) or darkness for 3 or 21 d. Flat mount retinas from animals kept during either LL or DD were also examined by immunohistochemistry. Melanopsin mRNA expression displayed a significant rhythmic change during the LD cycle with peak expression around dusk and nadir at dawn. Melanopsin protein also changed over the LD cycle with peak expression at the end of the night and nadir at dusk. Rhythmic expression of melanopsin mRNA but not melanopsin protein was found in constant darkness. After 3 or 21 d in either LL or DD melanopsin mRNA expression was unaltered. Melanopsin protein was at the same high level after 3 and 21 d in DD, whereas a significant decrease was found after prolonging the light period for 3 or 21 d. The change in melanopsin protein was primarily due to change in immunoreactivity in the dendritic processes. In conclusion we found that light and darkness are important for regulation of melanopsin protein expression whereas input from a

  15. Constitutive and allergen-induced expression of eotaxin mRNA in the guinea pig lung

    PubMed Central

    1995-01-01

    Eotaxin is a member of the C-C family of chemokines and is related during antigen challenge in a guinea pig model of allergic airway inflammation (asthma). Consistent with its putative role in eosinophilic inflammation, eotaxin induces the selective infiltration of eosinophils when injected into the lung and skin. Using a guinea pig lung cDNA library, we have cloned full-length eotaxin cDNA. The cDNA encodes a protein of 96 amino acids, including a putative 23-amino acid hydrophobic leader sequence, followed by 73 amino acids composing the mature active eotaxin protein. The protein-coding region of this cDNA is 73, 71, 50, and 48% identical in nucleic acid sequence to those of human macrophage chemoattractant protein (MCP) 3, MCP-1, macrophage inflammatory protein (MIP) 1 alpha, and RANTES, respectively. Analysis of genomic DNA suggested that there is a single eotaxin gene in guinea pig which is apparently conserved in mice. High constitutive levels of eotaxin mRNA expression were observed in the lung, while the intestines, stomach, spleen, liver, heart, thymus, testes, and kidney expressed lower levels. To determine if eotaxin mRNA levels are elevated during allergen-induced eosinophilic airway inflammation, ovalbumin (OVA)-sensitized guinea pigs were challenged with aerosolized antigen. Compared with the lungs from saline-challenged animals, eotaxin mRNA levels increased sixfold within 3 h and returned to baseline by 6 h. Thus, eotaxin mRNA levels are increased in response to allergen challenge during the late phase response. The identification of constitutive eotaxin mRNA expression in multiple tissues suggests that in addition to regulating airway eosinophilia, eotaxin is likely to be involved in eosinophil recruitment into other tissues as well as in baseline tissue homing. PMID:7869037

  16. Baseline MxA mRNA Expression Predicts Interferon Beta Response in Multiple Sclerosis Patients

    PubMed Central

    Matas, Elisabet; Bau, Laura; Martínez-Iniesta, María; Romero-Pinel, Lucía; Mañé, M. Alba; Cobo-Calvo, Álvaro; Martínez-Yélamos, Sergio

    2014-01-01

    Background Myxovirus resistance protein A (MxA) is a molecule induced after interferon-beta injection, mostly used to evaluate its bioactivity. There is little available data on clinical utility of baseline MxA mRNA status. The objective of the study is to investigate whether baseline MxA mRNA expression can predict relapse and disease progression in multiple sclerosis patients treated with interferon-beta. Methods Baseline blood samples were obtained before the first interferon-beta dose was administered to evaluate MxA mRNA expression using real-time polymerase chain reaction (PCR). Demographic and clinical variables were prospectively recorded to define treatment responder and non responder groups. Results 104 patients were included in the study. Baseline MxA mRNA expression was significantly lower in the group of patients who met the definition of responders (1.07 vs 1.95, Student t test, p<0.0001). A threshold of 1.096 was established using Receiver Operating Characteristic analysis to differentiate between responders and non-responders (sensitivity 73.9%, specificity 69.0%). Survival analysis using this threshold showed that time to next relapse (p<0.0001) and to EDSS progression (p = 0.01) were significantly higher in patients with lower MxA titers. Conclusion The results suggest that baseline MxA mRNA levels may be useful for predicting whether multiple sclerosis patients will respond or not to interferon-beta treatment. PMID:25396411

  17. Unmasking Upstream Gene Expression Regulators with miRNA-corrected mRNA Data.

    PubMed

    Bollmann, Stephanie; Bu, Dengpan; Wang, Jiaqi; Bionaz, Massimo

    2015-01-01

    Expressed micro-RNA (miRNA) affects messenger RNA (mRNA) abundance, hindering the accuracy of upstream regulator analysis. Our objective was to provide an algorithm to correct such bias. Large mRNA and miRNA analyses were performed on RNA extracted from bovine liver and mammary tissue. Using four levels of target scores from TargetScan (all miRNA:mRNA target gene pairs or only the top 25%, 50%, or 75%). Using four levels of target scores from TargetScan (all miRNA:mRNA target gene pairs or only the top 25%, 50%, or 75%) and four levels of the magnitude of miRNA effect (ME) on mRNA expression (30%, 50%, 75%, and 83% mRNA reduction), we generated 17 different datasets (including the original dataset). For each dataset, we performed upstream regulator analysis using two bioinformatics tools. We detected an increased effect on the upstream regulator analysis with larger miRNA:mRNA pair bins and higher ME. The miRNA correction allowed identification of several upstream regulators not present in the analysis of the original dataset. Thus, the proposed algorithm improved the prediction of upstream regulators.

  18. Unmasking Upstream Gene Expression Regulators with miRNA-corrected mRNA Data

    PubMed Central

    Bollmann, Stephanie; Bu, Dengpan; Wang, Jiaqi; Bionaz, Massimo

    2015-01-01

    Expressed micro-RNA (miRNA) affects messenger RNA (mRNA) abundance, hindering the accuracy of upstream regulator analysis. Our objective was to provide an algorithm to correct such bias. Large mRNA and miRNA analyses were performed on RNA extracted from bovine liver and mammary tissue. Using four levels of target scores from TargetScan (all miRNA:mRNA target gene pairs or only the top 25%, 50%, or 75%). Using four levels of target scores from TargetScan (all miRNA:mRNA target gene pairs or only the top 25%, 50%, or 75%) and four levels of the magnitude of miRNA effect (ME) on mRNA expression (30%, 50%, 75%, and 83% mRNA reduction), we generated 17 different datasets (including the original dataset). For each dataset, we performed upstream regulator analysis using two bioinformatics tools. We detected an increased effect on the upstream regulator analysis with larger miRNA:mRNA pair bins and higher ME. The miRNA correction allowed identification of several upstream regulators not present in the analysis of the original dataset. Thus, the proposed algorithm improved the prediction of upstream regulators. PMID:27279737

  19. Combining miRNA and mRNA Expression Profiles in Wilms Tumor Subtypes

    PubMed Central

    Ludwig, Nicole; Werner, Tamara V.; Backes, Christina; Trampert, Patrick; Gessler, Manfred; Keller, Andreas; Lenhof, Hans-Peter; Graf, Norbert; Meese, Eckart

    2016-01-01

    Wilms tumor (WT) is the most common childhood renal cancer. Recent findings of mutations in microRNA (miRNA) processing proteins suggest a pivotal role of miRNAs in WT genesis. We performed miRNA expression profiling of 36 WTs of different subtypes and four normal kidney tissues using microarrays. Additionally, we determined the gene expression profile of 28 of these tumors to identify potentially correlated target genes and affected pathways. We identified 85 miRNAs and 2107 messenger RNAs (mRNA) differentially expressed in blastemal WT, and 266 miRNAs and 1267 mRNAs differentially expressed in regressive subtype. The hierarchical clustering of the samples, using either the miRNA or mRNA profile, showed the clear separation of WT from normal kidney samples, but the miRNA pattern yielded better separation of WT subtypes. A correlation analysis of the deregulated miRNA and mRNAs identified 13,026 miRNA/mRNA pairs with inversely correlated expression, of which 2844 are potential interactions of miRNA and their predicted mRNA targets. We found significant upregulation of miRNAs-183, -301a/b and -335 for the blastemal subtype, and miRNAs-181b, -223 and -630 for the regressive subtype. We found marked deregulation of miRNAs regulating epithelial to mesenchymal transition, especially in the blastemal subtype, and miRNAs influencing chemosensitivity, especially in regressive subtypes. Further research is needed to assess the influence of preoperative chemotherapy and tumor infiltrating lymphocytes on the miRNA and mRNA patterns in WT. PMID:27043538

  20. Elevated tph2 mRNA Expression in a Rat Model of Chronic Anxiety

    PubMed Central

    Donner, Nina C.; Johnson, Philip L.; Fitz, Stephanie D.; Kellen, Karen E.; Shekhar, Anantha; Lowry, Christopher A.

    2015-01-01

    Background Allelic variations in TPH2, the gene encoding tryptophan hydroxylase 2, the rate-limiting enzyme for brain serotonin (5-HT) biosynthesis, may be genetic predictors of panic disorder and panic responses to panicogenic challenges in healthy volunteers. To test the hypothesis that tph2 mRNA is altered in chronic anxiety states, we measured tph2 expression in an established rat model of panic disorder. Methods We implanted 16 adult, male rats with bilateral guide cannulae and then primed them with daily injections of the corticotropin-releasing factor (CRF) receptor agonist, urocortin 1 (UCN1, 6 fmoles/100 nl per side, n = 8) or vehicle (n = 8) into the basolateral amygdaloid complex (BL) for 5 consecutive days. Anxiety-like behavior was assessed, 24 hr prior to and 48 hr following priming, in the social interaction (SI) test. A third group (n = 7) served as undisturbed home cage controls. All rats were killed 3 days after the last intra-BL injection to analyze tph2 and slc6a4 (gene encoding the serotonin transporter, SERT) mRNA expression in the dorsal raphe nucleus (DR), the main source of serotonergic projections to anxiety-related brain regions, using in situ hybridization histochemistry. Results UCN1 priming increased anxiety-related behavior in the SI test compared to vehicle-injected controls and elevated tph2, but not slc6a4, mRNA expression in DR subregions, including the ventrolateral DR/ventrolateral periaqueductal gray (DRVL/VLPAG), a subregion previously implicated in control of panic-related physiologic responses. Tph2 mRNA expression in the DRVL/VLPAG was correlated with increased anxiety-related behavior. Conclusion Our data support the hypothesis that chronic anxiety states are associated with dysregulated tph2 expression. PMID:22511363

  1. Classification of human lung carcinomas by mRNA expression profiling reveals distinct adenocarcinoma subclasses.

    PubMed

    Bhattacharjee, A; Richards, W G; Staunton, J; Li, C; Monti, S; Vasa, P; Ladd, C; Beheshti, J; Bueno, R; Gillette, M; Loda, M; Weber, G; Mark, E J; Lander, E S; Wong, W; Johnson, B E; Golub, T R; Sugarbaker, D J; Meyerson, M

    2001-11-20

    We have generated a molecular taxonomy of lung carcinoma, the leading cause of cancer death in the United States and worldwide. Using oligonucleotide microarrays, we analyzed mRNA expression levels corresponding to 12,600 transcript sequences in 186 lung tumor samples, including 139 adenocarcinomas resected from the lung. Hierarchical and probabilistic clustering of expression data defined distinct subclasses of lung adenocarcinoma. Among these were tumors with high relative expression of neuroendocrine genes and of type II pneumocyte genes, respectively. Retrospective analysis revealed a less favorable outcome for the adenocarcinomas with neuroendocrine gene expression. The diagnostic potential of expression profiling is emphasized by its ability to discriminate primary lung adenocarcinomas from metastases of extra-pulmonary origin. These results suggest that integration of expression profile data with clinical parameters could aid in diagnosis of lung cancer patients. PMID:11707567

  2. Cryo-EM Analysis of the Conformational Landscape of Human P-glycoprotein (ABCB1) During its Catalytic Cycle

    PubMed Central

    Frank, Gabriel A.; Shukla, Suneet; Rao, Prashant; Borgnia, Mario J.; Bartesaghi, Alberto; Merk, Alan; Mobin, Aerfa; Esser, Lothar; Earl, Lesley A.; Gottesman, Michael M.; Xia, Di

    2016-01-01

    The multidrug transporter P-glycoprotein (P-gp, ABCB1) is an ATP-dependent pump that mediates the efflux of structurally diverse drugs and xenobiotics across cell membranes, affecting drug pharmacokinetics and contributing to the development of multidrug resistance. Structural information about the conformational changes in human P-gp during the ATP hydrolysis cycle has not been directly demonstrated, although mechanistic information has been inferred from biochemical and biophysical studies conducted with P-gp and its orthologs, or from structures of other ATP-binding cassette transporters. Using single-particle cryo-electron microscopy, we report the surprising discovery that, in the absence of the transport substrate and nucleotides, human P-gp can exist in both open [nucleotide binding domains (NBDs) apart; inward-facing] and closed (NBDs close; outward-facing) conformations. We also probe conformational states of human P-gp during the catalytic cycle, and demonstrate that, following ATP hydrolysis, P-gp transitions through a complete closed conformation to a complete open conformation in the presence of ADP. PMID:27190212

  3. Cryo-EM Analysis of the Conformational Landscape of Human P-glycoprotein (ABCB1) During its Catalytic Cycle.

    PubMed

    Frank, Gabriel A; Shukla, Suneet; Rao, Prashant; Borgnia, Mario J; Bartesaghi, Alberto; Merk, Alan; Mobin, Aerfa; Esser, Lothar; Earl, Lesley A; Gottesman, Michael M; Xia, Di; Ambudkar, Suresh V; Subramaniam, Sriram

    2016-07-01

    The multidrug transporter P-glycoprotein (P-gp, ABCB1) is an ATP-dependent pump that mediates the efflux of structurally diverse drugs and xenobiotics across cell membranes, affecting drug pharmacokinetics and contributing to the development of multidrug resistance. Structural information about the conformational changes in human P-gp during the ATP hydrolysis cycle has not been directly demonstrated, although mechanistic information has been inferred from biochemical and biophysical studies conducted with P-gp and its orthologs, or from structures of other ATP-binding cassette transporters. Using single-particle cryo-electron microscopy, we report the surprising discovery that, in the absence of the transport substrate and nucleotides, human P-gp can exist in both open [nucleotide binding domains (NBDs) apart; inward-facing] and closed (NBDs close; outward-facing) conformations. We also probe conformational states of human P-gp during the catalytic cycle, and demonstrate that, following ATP hydrolysis, P-gp transitions through a complete closed conformation to a complete open conformation in the presence of ADP.

  4. Interferon-alpha inhibits murine macrophage transforming growth factor-beta mRNA expression.

    PubMed

    Dhanani, S; Huang, M; Wang, J; Dubinett, S M

    1994-06-01

    Transforming growth factor-beta (TGF-beta), a multifunctional polypeptide is produced by a wide variety of cells and regulates a broad array of physiological and pathological functions. TGF-beta appears to play a central role in pulmonary fibrosis and may contribute to tumor-associated immunosuppression. Alveolar macrophages are a rich source of TGF-beta and are intimately involved in lung inflammation. We therefore chose to study TGF-beta regulation in murine alveolar macrophages as well as an immortalized peritoneal macrophage cell line (IC-21). Murine macrophages were incubated with cytokines to evaluate their role in regulating TGF-beta mRNA expression. We conclude that IFN-alpha downregulates TGF-beta mRNA expression in murine macrophages. PMID:8088926

  5. Evaluation of Parkia pendula lectin mRNA differentially expressed in seedlings.

    PubMed

    Rêgo, M J B M; Santos, P B; Carvalho-Junior, L B; Stirling, J; Beltrão, E I C

    2014-05-01

    Parkia pendula (Willd.) Walp. (Fabaceae) is a neotropical species of the genus Parkia more abundantly distributed in Central to South America. From the seeds of P. pendula a glucose/mannose specific lectin (PpeL) was isolated that has been characterised and used as a biotechnological tool but until now this is the first manuscript to analyse P. pendula mRNA expression in seedlings. For this porpoise a Differential display reverse transcription polimerase chain reaction (DDRT-PCR) was used to evaluate the expression of P. pendula lectin mRNAs in non-rooted seedlings. No bands were observed in the agarose gel, indicating the absence of mRNA of PpeL seedlings. our findings confirm that lectins mRNAs are differently regulated among species even if they are grouped in the same class. PMID:25166336

  6. Intragenic MBD5 familial deletion variant does not negatively impact MBD5 mRNA expression.

    PubMed

    Mullegama, Sureni V; Elsea, Sarah H

    2014-01-01

    2q23.1 deletion syndrome is characterized by intellectual disability, speech impairment, seizures, disturbed sleep pattern, behavioral problems, and hypotonia. Core features of this syndrome are due to haploinsufficiency of MBD5. Deletions that include coding and noncoding exons show reduced MBD5 mRNA expression. We report a patient with a neurological and behavioral phenotype similar to 2q23.1 deletion syndrome with an inherited intronic deletion in the 5-prime untranslated region of MBD5. Our data show that this patient has normal MBD5 mRNA expression; therefore, this deletion is likely not causative for 2q23.1 deletion syndrome. Overall, it is important to validate intronic deletions for pathogenicity.

  7. Gene expression during mammalian spermatogenesis. III. Changes in populations of mRNA during spermiogenesis.

    PubMed

    Stern, L; Kleene, K C; Gold, B; Hecht, N B

    1983-01-01

    Round spermatids and elongating spermatids were purified from a suspension of mouse testicular cells by sedimentation at unit gravity coupled with density gradient centrifugation through Percoll. Following separation, the two cell types were fractionated into polysomal and non-polysomal compartments. By comparison with round spermatids, elongating spermatids contain about one-half as much cytoplasmic RNA per cell, one sixth as much poly(A)+ RNA per cell and one-half the concentration of poly (A)+ mRNA in their cytoplasm. About two-thirds of the poly(A)+ messenger RNA (mRNA) was in the non-polysomal fraction in both cell types. Polypeptides whose synthesis was directed by cell-free translation of purified mRNA from each cell fraction were analyzed by two-dimensional gel electrophoresis. At the level of detection provided by the electrophoretic methods used, the majority of peptides from the polysomal and non-polysomal compartments for each cell type were similar. However, between the two cell types, approx. 5-10% of the polypeptides in the polysomal and non-polysomal fractions differed markedly in abundance. When the polypeptides encoded by the polysomal and non-polysomal mRNA from round spermatids were compared to the polypeptides encoded in the equivalent fractions from elongating spermatids, a significant reduction in number of polypeptides from elongating spermatids was seen. The presence of specific mRNAs in the non-polysomal fraction of round spermatids and in the polysomal fraction of elongating spermatids suggests that storage of mRNA in the cytoplasm and subsequent utilization provides a source of mRNA for proteins expressed at a time during spermiogenesis when transcription has terminated.

  8. Analysis of myosin heavy chain mRNA expression by RT-PCR

    NASA Technical Reports Server (NTRS)

    Wright, C.; Haddad, F.; Qin, A. X.; Baldwin, K. M.

    1997-01-01

    An assay was developed for rapid and sensitive analysis of myosin heavy chain (MHC) mRNA expression in rodent skeletal muscle. Only 2 microg of total RNA were necessary for the simultaneous analysis of relative mRNA expression of six different MHC genes. We designed synthetic DNA fragments as internal standards, which contained the relevant primer sequences for the adult MHC mRNAs type I, IIa, IIx, IIb as well as the embryonic and neonatal MHC mRNAs. A known amount of the synthetic fragment was added to each polymerase chain reaction (PCR) and yielded a product of different size than the amplified MHC mRNA fragment. The ratio of amplified MHC fragment to synthetic fragment allowed us to calculate percentages of the gene expression of the different MHC genes in a given muscle sample. Comparison with the traditional Northern blot analysis demonstrated that our reverse transcriptase-PCR-based assay was reliable, fast, and quantitative over a wide range of relative MHC mRNA expression in a spectrum of adult and neonatal rat skeletal muscles. Furthermore, the high sensitivity of the assay made it very useful when only small quantities of tissue were available. Statistical analysis of the signals for each MHC isoform across the analyzed samples showed a highly significant correlation between the PCR and the Northern signals as Pearson correlation coefficients ranged between 0.77 and 0.96 (P < 0.005). This assay has potential use in analyzing small muscle samples such as biopsies and samples from pre- and/or neonatal stages of development.

  9. Interleukin-1 receptor mRNA expression in activated bovine leukocytes in vitro.

    PubMed Central

    Yu, P W; Schuler, L A; Czuprynski, C J

    1997-01-01

    Interleukin-1 (IL-1) is a key player in inflammation and the immune response. To better understand the complex interactions of IL-1 and its receptors in inflammation, we need to investigate how type I and type II IL-1 receptors (IL-1RI and IL-1RII) are regulated by cytokines and other mediators. Using semiquantitative reverse transcriptase PCR and Northern analysis, we examined the regulation of IL-1RI and IL-1RII mRNA levels in bovine polymorphonuclear leukocytes (PMNs) (i.e., neutrophils) and peripheral blood mononuclear cells (PBMCs) in vitro. IL-1RI mRNA levels were up-regulated in PBMCs by recombinant bovine IL-1beta (rBoIL-1beta), recombinant bovine granulocyte-macrophage colony-stimulating factor (rBoGM-CSF), rBoIL-4, recombinant bovine gamma interferon (rBoIFN-gamma), and dexamethasone. IL-1RI mRNA was increased in bovine PMNs exposed to rBoGM-CSF, rBoIL-4, and dexamethasone but was down-regulated by rBoIL-1beta and rBoIFN-gamma. IL-1RII mRNA was increased in bovine PBMCs and PMNs after exposure to rBoIL-1beta, rBoGM-CSF, rBoIL-4, and dexamethasone. In contrast, rBoIFN-gamma down-regulated the expression of bovine IL-1RII mRNA in PBMCs. These findings suggest that the expression of bovine IL-1RI and IL-1RII mRNAs is regulated differently by certain soluble stimuli (e.g., IFN-gamma) in PMNs and PBMCs. PMID:9384305

  10. Codon influence on protein expression in E. coli correlates with mRNA levels

    PubMed Central

    Boël, Grégory; Wong, Kam-Ho; Su, Min; Luff, Jon; Valecha, Mayank; Everett, John K.; Acton, Thomas B.; Xiao, Rong; Montelione, Gaetano T.; Aalberts, Daniel P.; Hunt, John F.

    2016-01-01

    Degeneracy in the genetic code, which enables a single protein to be encoded by a multitude of synonymous gene sequences, has an important role in regulating protein expression, but substantial uncertainty exists concerning the details of this phenomenon. Here we analyze the sequence features influencing protein expression levels in 6,348 experiments using bacteriophage T7 polymerase to synthesize messenger RNA in Escherichia coli. Logistic regression yields a new codon-influence metric that correlates only weakly with genomic codon-usage frequency, but strongly with global physiological protein concentrations and also mRNA concentrations and lifetimes in vivo. Overall, the codon content influences protein expression more strongly than mRNA-folding parameters, although the latter dominate in the initial ~16 codons. Genes redesigned based on our analyses are transcribed with unaltered efficiency but translated with higher efficiency in vitro. The less efficiently translated native sequences show greatly reduced mRNA levels in vivo. Our results suggest that codon content modulates a kinetic competition between protein elongation and mRNA degradation that is a central feature of the physiology and also possibly the regulation of translation in E. coli. PMID:26760206

  11. An intronic RNA structure modulates expression of the mRNA biogenesis factor Sus1.

    PubMed

    AbuQattam, Ali; Gallego, José; Rodríguez-Navarro, Susana

    2016-01-01

    Sus1 is a conserved protein involved in chromatin remodeling and mRNA biogenesis. Unlike most yeast genes, the SUS1 pre-mRNA of Saccharomyces cerevisiae contains two introns and is alternatively spliced, retaining one or both introns in response to changes in environmental conditions. SUS1 splicing may allow the cell to control Sus1 expression, but the mechanisms that regulate this process remain unknown. Using in silico analyses together with NMR spectroscopy, gel electrophoresis, and UV thermal denaturation experiments, we show that the downstream intron (I2) of SUS1 forms a weakly stable, 37-nucleotide stem-loop structure containing the branch site near its apical loop and the 3' splice site after the stem terminus. A cellular assay revealed that two of four mutants containing altered I2 structures had significantly impaired SUS1 expression. Semiquantitative RT-PCR experiments indicated that all mutants accumulated unspliced SUS1 pre-mRNA and/or induced distorted levels of fully spliced mRNA relative to wild type. Concomitantly, Sus1 cellular functions in histone H2B deubiquitination and mRNA export were affected in I2 hairpin mutants that inhibited splicing. This work demonstrates that I2 structure is relevant for SUS1 expression, and that this effect is likely exerted through modulation of splicing.

  12. Expression of connexin 43 mRNA and protein in developing follicles of prepubertal porcine ovaries

    USGS Publications Warehouse

    Melton, C.M.; Zaunbrecher, G.M.; Yoshizaki, G.; Patio, R.; Whisnant, S.; Rendon, A.; Lee, V.H.

    2001-01-01

    A major form of cell-cell communication is mediated by gap junctions, aggregations of intercellular channels composed of connexins (Cxs), which are responsible for exchange of low molecular weight (< 1200 Da) cytosolic materials. These channels are a growing family of related proteins. This study was designed to determine the ontogeny of connexin 43 (Cx43) during early stages of follicular development in prepubertal porcine ovaries. A partial-length (412 base) cDNA clone was obtained from mature porcine ovaries and determined to have 98% identity with published porcine Cx43. Northern blot analysis demonstrated a 4.3-kb mRNA in total RNA isolated from prepubertal and adult porcine ovaries. In-situ hybridization revealed that Cx43 mRNA was detectable in granulosa cells of primary follicles but undetectable in dormant primordial follicles. The intensity of the signal increased with follicular growth and was greatest in the large antral follicles. Immunohistochemical evaluation indicated that Cx43 protein expression correlated with the presence of Cx43 mRNA. These results indicate that substantial amounts of Cx43 are first expressed in granulosa cells following activation of follicular development and that this expression increases throughout follicular growth and maturation. These findings suggest an association between the enhancement of intercellular gap-junctional communication and onset of follicular growth. ?? 2001 Elsevier Science Inc. All rights reserved.

  13. Promoter Methylation and mRNA Expression of Response Gene to Complement 32 in Breast Carcinoma

    PubMed Central

    Eskandari-Nasab, Ebrahim; Hashemi, Mohammad; Rafighdoost, Firoozeh

    2016-01-01

    Background. Response gene to complement 32 (RGC32), induced by activation of complements, has been characterized as a cell cycle regulator; however, its role in carcinogenesis is still controversial. In the present study we compared RGC32 promoter methylation patterns and mRNA expression in breast cancerous tissues and adjacent normal tissues. Materials and Methods. Sixty-three breast cancer tissues and 63 adjacent nonneoplastic tissues were included in our study. Design. Nested methylation-specific polymerase chain reaction (Nested-MSP) and quantitative PCR (qPCR) were used to determine RGC32 promoter methylation status and its mRNA expression levels, respectively. Results. RGC32 methylation pattern was not different between breast cancerous tissue and adjacent nonneoplastic tissue (OR = 2.30, 95% CI = 0.95–5.54). However, qPCR analysis displayed higher levels of RGC32 mRNA in breast cancerous tissues than in noncancerous tissues (1.073 versus 0.959; P = 0.001), irrespective of the promoter methylation status. The expression levels and promoter methylation of RGC32 were not correlated with any of patients' clinical characteristics (P > 0.05). Conclusion. Our findings confirmed upregulation of RGC32 in breast cancerous tumors, but it was not associated with promoter methylation patterns. PMID:27118972

  14. Hormone and metabolic factors associated with leptin mRNA expression in pre- and postmenopausal women.

    PubMed

    Fajardo, Martha E; Malacara, Juan M; Martínez-Rodríguez, Herminia G; Barrera-Saldaña, Hugo A

    2004-06-01

    Recent information has extended leptin's action, beyond the control of appetite, to various sites of metabolic regulation. To better understand leptin's role we studied its production in subcutaneous and visceral fat compartments before and after menopause. During elective abdominal surgery, biopsies of subcutaneous and omental tissues were taken from 20 women at pre- (BMI 28.4 +/- 4.5 kg/m2) and 10 at postmenopause (BMI 30.6 +/- 7.7 kg/m2). In both groups serum leptin levels were similar, and highly correlated with BMI. In subcutaneous adipose tissue, leptin mRNA expression was significantly higher in pre- than in postmenopausal women (50.4 +/- 20.5 amol/microg total RNA versus 34.5 +/- 24.9 amol/microg total RNA, respectively). Leptin mRNA expression in subcutaneous tissue was independently correlated with fasting glucose (R = 0.89, P < 0.006) at premenopause, and with serum estradiol (R = 0.77, P < 0.04) at postmenopause. Leptin mRNA expression in visceral fat was correlated with DHEAS (R = 0.86, P < 0.001), at premenopause. These results indicate that in both compartments, leptin production is sensitive to different but overlapping stimuli, conveying information about energy availability to central and peripheral sites under different conditions of estrogen exposure.

  15. An intronic RNA structure modulates expression of the mRNA biogenesis factor Sus1

    PubMed Central

    AbuQattam, Ali; Gallego, José; Rodríguez-Navarro, Susana

    2016-01-01

    Sus1 is a conserved protein involved in chromatin remodeling and mRNA biogenesis. Unlike most yeast genes, the SUS1 pre-mRNA of Saccharomyces cerevisiae contains two introns and is alternatively spliced, retaining one or both introns in response to changes in environmental conditions. SUS1 splicing may allow the cell to control Sus1 expression, but the mechanisms that regulate this process remain unknown. Using in silico analyses together with NMR spectroscopy, gel electrophoresis, and UV thermal denaturation experiments, we show that the downstream intron (I2) of SUS1 forms a weakly stable, 37-nucleotide stem–loop structure containing the branch site near its apical loop and the 3′ splice site after the stem terminus. A cellular assay revealed that two of four mutants containing altered I2 structures had significantly impaired SUS1 expression. Semiquantitative RT-PCR experiments indicated that all mutants accumulated unspliced SUS1 pre-mRNA and/or induced distorted levels of fully spliced mRNA relative to wild type. Concomitantly, Sus1 cellular functions in histone H2B deubiquitination and mRNA export were affected in I2 hairpin mutants that inhibited splicing. This work demonstrates that I2 structure is relevant for SUS1 expression, and that this effect is likely exerted through modulation of splicing. PMID:26546116

  16. Cerebral cortical amyloid protein precursor mRNA expression is similar in Alzheimer's disease and other neurodegenerative diseases.

    PubMed

    Ohyagi, Y; Takahashi, K; Satoh, Y; Makifuchi, T; Tabira, T

    1992-08-01

    The expression of 3 beta-amyloid protein precursor (APP) mRNAs (695, 751, and 770) in the cerebral cortex in Alzheimer's disease and other neurodegenerative diseases was analyzed by the S1 nuclease protection assay. We found no significant Alzheimer's disease-specific alteration of APP mRNA expression when compared to the other neurological diseases as controls. Since the expression of this mRNA was not correlated with amyloid deposition, it is possible that gliosis/neuronal loss may secondarily alter APP mRNA expression. However, the current study revealed no significant correlation between them.

  17. Cytokine mRNA expression in Peromyscus yucatanicus (Rodentia: Cricetidae) infected by Leishmania (Leishmania) mexicana.

    PubMed

    Loria-Cervera, Elsy Nalleli; Sosa-Bibiano, Erika Ivett; Van Wynsberghe, Nicole Raymonde; Saldarriaga, Omar Abdul; Melby, Peter C; Andrade-Narvaez, Fernando Jose

    2016-07-01

    Peromyscus yucatanicus, the main reservoir of Leishmania (Leishmania) mexicana in the Yucatan peninsula of Mexico, reproduces clinical and histological pictures of LCL in human as well as subclinical infection. Thus, we used this rodent as a novel experimental model. In this work, we analyzed cytokine mRNA expression in P. yucatanicus infected with L. (L.) mexicana. Animals were inoculated with either 2.5×10(6) or 1×10(2) promastigotes and cytokine expressions were analyzed by real-time RT-PCR in skin at 4 and 12weeks post-infection (wpi). Independently of the parasite inoculum none of the infected rodents had clinical signs of LCL at 4wpi and all expressed high IFN-γ mRNA. All P. yucatanicus inoculated with 2.5×10(6) promastigotes developed signs of LCL at 12wpi while the mice inoculated with 1×10(2) remained subclinical. At that time, both IFN-γ and IL-10 were expressed in P. yucatanicus with clinical and subclinical infections. Expressions of TNF-α and IL-4 were significantly higher in clinical animals (2.5×10(6)) compared with subclinical ones (1×10(2)). High TGF-β expression was observed in P. yucatanicus with clinical signs when compared with healthy animals. Results suggested that the clinical course of L. (L.) mexicana infection in P. yucatanicus was associated with a specific local pattern of cytokine production at 12wpi. PMID:27155064

  18. Distinct prognostic values of four-Notch-receptor mRNA expression in ovarian cancer.

    PubMed

    Zhou, Xinling; Teng, Lingling; Wang, Min

    2016-05-01

    Notch signaling pathway includes ligands and Notch receptors, which are frequently deregulated in several human malignancies including ovarian cancer. Aberrant activation of Notch signaling has been linked to ovarian carcinogenesis and progression. In the current study, we used the "Kaplan-Meier plotter" (KM plotter) database, in which updated gene expression data and survival information from a total of 1306 ovarian cancer patients were used to access the prognostic value of four Notch receptors in ovarian cancer patients. Hazard ratio (HR), 95 % confidence intervals, and log-rank P were calculated. Notch1 messenger RNA (mRNA) high expression was not found to be correlated to overall survival (OS) for all ovarian cancer, as well as in serous and endometrioid cancer patients followed for 20 years. However, Notch1 mRNA high expression is significantly associated with worsen OS in TP53 wild-type ovarian cancer patients, while it is significantly associated with better OS in TP53 mutation-type ovarian cancer patients. Notch2 mRNA high expression was found to be significantly correlated to worsen OS for all ovarian cancer patients, as well as in grade II ovarian cancer patients. Notch3 mRNA high expression was found to be significantly correlated to better OS for all ovarian cancer patients, but not in serous cancer patients and endometrioid cancer patients. Notch4 mRNA high expression was not found to be significantly correlated to OS for all ovarian cancer patients, serous cancer patients, and endometrioid cancer patients. These results indicate that there are distinct prognostic values of four Notch receptors in ovarian cancer. This information will be useful for better understanding of the heterogeneity and complexity in the molecular biology of ovarian cancer and for developing tools to more accurately predict their prognosis. Based on our results, Notch1 could be a potential drug target of TP53 wild-type ovarian cancer and Notch2 could be a potential drug

  19. Early and late stimulation of ob mRNA expression in meal-fed and overfed rats.

    PubMed Central

    Harris, R B; Ramsay, T G; Smith, S R; Bruch, R C

    1996-01-01

    ob protein is hypothesized to be a circulating feedback signal in the regulation of energy balance. Obese, overfed rats have high levels of ob mRNA expression and suppressed voluntary food intake, indicating the presence of a potent satiety factor. The objectives of this experiment were to determine whether feeding rats their normal daily intake in three meals, compared with ad libitum feeding, increased ob mRNA expression and to determine the degree of obesity required to stimulate expression of ob mRNA. Rats were fed ad libitum, were tube-fed their normal intake in three meals a day, or were tube-fed twice normal intake, ob mRNA was measured by Northern blot analysis after 0, 2, 7, 14, 21, and 32 d of tube-feeding. After only 2 d ob mRNA was threefold higher in tube-fed animals than in ad libitum controls. By day 21 there was a further increase in ob mRNA expression in overfed rats which were at 130% control weight. These results suggest that a metabolic consequence of meal-feeding increases ob mRNA expression in the absence of increased food intake or weight gain. There is a further increase in ob mRNA expression once significant obesity is established. PMID:8621790

  20. Nutritional regulation of insulin-like growth factor-I mRNA expression in barramundi, Lates calcarifer.

    PubMed

    Matthews, S J; Kinhult, A K; Hoeben, P; Sara, V R; Anderson, T A

    1997-06-01

    The effect of nutritional status on IGF-I mRNA expression in the liver and brain of juvenile barramundi (Lates calcarifer) was investigated. Fish were either fed a satiety ration (SAT) or starved (STV) for 6 weeks. Starved fish demonstrated significantly lower condition factor and hepatic IGF-I mRNA expression at 3 and 6 weeks, when compared with the SAT group. IGF-I mRNA expression in the brain was 10 fold lower than the liver and was not affected by ration size. These results suggest the liver is the major site of IGF-I mRNA synthesis and hepatic but not brain IGF-I mRNA expression is regulated by food availability in juvenile barramundi.

  1. CART mRNA expression in rat monkey and human brain: relevance to cocaine abuse.

    PubMed

    Fagergren, Pernilla; Hurd, Yasmin

    2007-09-10

    The neuropeptide CART (cocaine and amphetamine regulated transcript) is suggested to be regulated by psychostimulant administration. We review here the localization of CART mRNA expression in the human brain and its possible relevance to human cocaine abuse. Except for strong hypothalamic expression, the CART transcript is predominately expressed in target regions of the mesocorticolimbic dopamine system, such as the nucleus accumbens shell, amygdala complex, extended amygdala and orbitofrontal, enthorhinal and piriform cortices. The discrete limbic localization strongly implies involvement in reward and reinforcement behaviors. We therefore examined CART mRNA expression in both Sprague Dawley rats and Rhesus monkeys that had self-administered cocaine. Cocaine self-administration in the rat (1.5 mg/kg/inj, on a fixed ratio 1 schedule of reinforcement for 1 week) and monkey (0.03 or 0.3 mg/kg/inj on a fixed 3 min interval schedule of reinforcement for 5 or 100 days) did not alter transcript levels in CART expressing nucleus accumbens (monkey not studied), amygdala nuclei or cortical areas. However, in the monkey sublenticular extended amygdala, low dose cocaine self-administration resulted in increased CART transcript levels after both 5 and 100 days of self-administration, whereas no difference was found after high dose self-administration. In conclusion, we found no substantial alterations CART mRNA expression during cocaine self-administration, but this neuropeptide has the anatomical and functional potential to modulate brain areas relevant for cocaine abuse. Further studies are needed to evaluate the involvement of CART in other components of the cocaine abuse cycle. PMID:17631364

  2. CYP2C9, CYP2C19, ABCB1 genetic polymorphisms and phenytoin plasma concentrations in Mexican-Mestizo patients with epilepsy.

    PubMed

    Ortega-Vázquez, A; Dorado, P; Fricke-Galindo, I; Jung-Cook, H; Monroy-Jaramillo, N; Martínez-Juárez, I E; Familiar-López, I; Peñas-Lledó, E; LLerena, A; López-López, M

    2016-06-01

    We aimed to explore the possible influence of CYP2C9 (*2, *3 and IVS8-109 A>T), CYP2C19 (*2, *3 and *17) and ABCB1 (1236C>T, 2677G>A/T and 3435C>T) on phenytoin (PHT) plasma concentrations in 64 Mexican Mestizo (MM) patients with epilepsy currently treated with PHT in mono- (n=25) and polytherapy (n=39). Genotype and allele frequencies of these variants were also estimated in 300 MM healthy volunteers. Linear regression models were used to assess associations between the dependent variables (PHT plasma concentration and dose-corrected PHT concentration) with independent variables (CYP2C9, CYP2C19 and ABCB1 genotypes, ABCB1 haplotypes, age, sex, weight, and polytherapy). In multivariate models, CYP2C9 IVS8-109 T was significantly associated with higher PHT plasma concentrations (t(64)=2.27; P=0.03). Moreover, this allele was more frequent in the supratherapeutic group as compared with the subtherapeutic group (0.13 versus 0.03, respectively; P=0.05, Fisher's exact test). Results suggest that CYP2C9 IVS8-109 T allele may decrease CYP2C9 enzymatic activity on PHT. More research is needed to confirm findings.

  3. Hypoxia-induced gene expression results from selective mRNA partitioning to the endoplasmic reticulum

    PubMed Central

    Staudacher, Jonas J.; Naarmann-de Vries, Isabel S.; Ujvari, Stefanie J.; Klinger, Bertram; Kasim, Mumtaz; Benko, Edgar; Ostareck-Lederer, Antje; Ostareck, Dirk H.; Bondke Persson, Anja; Lorenzen, Stephan; Meier, Jochen C.; Blüthgen, Nils; Persson, Pontus B.; Henrion-Caude, Alexandra; Mrowka, Ralf; Fähling, Michael

    2015-01-01

    Protein synthesis is a primary energy-consuming process in the cell. Therefore, under hypoxic conditions, rapid inhibition of global mRNA translation represents a major protective strategy to maintain energy metabolism. How some mRNAs, especially those that encode crucial survival factors, continue to be efficiently translated in hypoxia is not completely understood. By comparing specific transcript levels in ribonucleoprotein complexes, cytoplasmic polysomes and endoplasmic reticulum (ER)-bound ribosomes, we show that the synthesis of proteins encoded by hypoxia marker genes is favoured at the ER in hypoxia. Gene expression profiling revealed that transcripts particularly increased by the HIF-1 transcription factor network show hypoxia-induced enrichment at the ER. We found that mRNAs favourably translated at the ER have higher conservation scores for both the 5′- and 3′-untranslated regions (UTRs) and contain less upstream initiation codons (uAUGs), indicating the significance of these sequence elements for sustained mRNA translation under hypoxic conditions. Furthermore, we found enrichment of specific cis-elements in mRNA 5′- as well as 3′-UTRs that mediate transcript localization to the ER in hypoxia. We conclude that transcriptome partitioning between the cytoplasm and the ER permits selective mRNA translation under conditions of energy shortage. PMID:25753659

  4. VGLUT1 and VGLUT2 mRNA expression in the primate auditory pathway

    PubMed Central

    Hackett, Troy A.; Takahata, Toru; Balaram, Pooja

    2011-01-01

    The vesicular glutamate transporters (VGLUTs) regulate storage and release of glutamate in the brain. In adult animals, the VGLUT1 and VGLUT2 isoforms are widely expressed and differentially distributed, suggesting that neural circuits exhibit distinct modes of glutamate regulation. Studies in rodents suggest that VGLUT1 and VGLUT2 mRNA expression patterns are partly complementary, with VGLUT1 expressed at higher levels in cortex and VGLUT2 prominent subcortically, but with overlapping distributions in some nuclei. In primates, VGLUT gene expression has not been previously studied in any part of the brain. The purposes of the present study were to document the regional expression of VGLUT1 and VGLUT2 mRNA in the auditory pathway through A1 in cortex, and to determine whether their distributions are comparable to rodents. In situ hybridization with antisense riboprobes revealed that VGLUT2 was strongly expressed by neurons in the cerebellum and most major auditory nuclei, including the dorsal and ventral cochlear nuclei, medial and lateral superior olivary nuclei, central nucleus of the inferior colliculus, sagulum, and all divisions of the medial geniculate. VGLUT1 was densely expressed in the hippocampus and ventral cochlear nuclei, and at reduced levels in other auditory nuclei. In auditory cortex, neurons expressing VGLUT1 were widely distributed in layers II – VI of the core, belt and parabelt regions. VGLUT2 was most strongly expressed by neurons in layers IIIb and IV, weakly by neurons in layers II – IIIa, and at very low levels in layers V – VI. The findings indicate that VGLUT2 is strongly expressed by neurons at all levels of the subcortical auditory pathway, and by neurons in the middle layers of cortex, whereas VGLUT1 is strongly expressed by most if not all glutamatergic neurons in auditory cortex and at variable levels among auditory subcortical nuclei. These patterns imply that VGLUT2 is the main vesicular glutamate transporter in subcortical

  5. Optimization of mRNA design for protein expression in the crustacean Daphnia magna.

    PubMed

    Törner, Kerstin; Nakanishi, Takashi; Matsuura, Tomoaki; Kato, Yasuhiko; Watanabe, Hajime

    2014-08-01

    The water flea Daphnia is a new model organism for ecological, evolutionary, and toxicological genomics. Detailed functional analysis of genes newly discovered through genomic approaches often requires overexpression of the identified protein. In the present study, we report the microinjection of in vitro-synthesized RNAs into the eggs as a method for overexpressing ubiquitous proteins in Daphnia magna. We injected a 1.3-kb mRNA that coded for the red fluorescent protein (DsRed2) flanked by UTRs from the ubiquitously expressed elongation factor 1α-1 (EF1α-1) into D. magna embryos. DsRed2 fluorescence in the embryos was measured 24 h after microinjection. Unexpectedly, the reporter RNA containing the 522-bp full-length EF1α-1 3' UTR failed to induce fluorescence. To assess reporter expression, the length of the 3' UTR that potentially contained negative regulatory elements of protein expression, including AU-rich regions and Musashi binding elements, was serially reduced from the 3' end. Assessing all injected RNA alternatives, mRNA containing the first 60 bp of the 3' UTR gave rise to the highest fluorescence, 14 times the Daphnia auto-fluorescence. In contrast, mRNA lacking the entire 3' UTR hardly induced any change in fluorescence intensity. This is the first evaluation of UTRs of mRNAs delivered into Daphnia embryos by microinjection for overexpressing proteins. The mRNA with truncated 3' UTRs of Daphnia EF1α-1 will be useful not only for gain-of-function analyses but also for labeling proteins and organelles with fluorescent proteins in Daphnia.

  6. CSD mRNA expression in rat testis and the effect of taurine on testosterone secretion.

    PubMed

    Yang, Jiancheng; Wu, Gaofeng; Feng, Ying; Sun, Changmian; Lin, Shumei; Hu, Jianmin

    2010-06-01

    In the present study, the cysteine sulfinate decarboxylase (CSD) mRNA expression was detected in rat testis by RT-PCR. The results showed that CSD mRNA was expressed in rat testis, and the putative encoded-amino acid sequence was exactly the same as that in rat liver which was already known. At the same time, the effects of taurine on testosterone secretion were investigated both in vivo and in vitro. In vivo, taurine were administered to male rats by tap water. The results showed that taurine obviously stimulated the secretion of FSH, LH and testosterone in serum, but showed no significant effect on the secretion of estradiol. Taurine administered in water could significantly increase the concentration of taurine in the blood and testis of rats. In vitro, cultured Leydig cells were treated with taurine independently or incubated with human chorionic gonadotropin (HCG) and progesterone. The results showed that taurine had biphasic effects on basal testosterone secretion in cultured Leydig cells. Low concentrations of taurine (0.1-100 microg/ml) could stimulate testosterone secretion, whereas high concentration of taurine (400 microg/ml) could inhibit testosterone secretion. Testosterone secretion stimulated by HCG was significantly increased by 10 and 100 microg/ml of taurine administration, and obviously decreased by treating with 400 microg/ml of taurine. Testosterone secretion induced by progesterone was significantly stimulated by treating with 1.0 and 10 microg/ml of taurine, however, it was significantly inhibited when treated with 400 microg/ml of taurine. Meanwhile, the effect of silencing CSD mRNA by siRNA on testosterone secretion was analyzed. The results showed that testosterone secretion was obviously decreased after the inhibition of CSD mRNA expression in cultured Leydig cells. These results indicated that taurine can be synthesized in rat testis by CSD pathway, and it plays important roles in testosterone secretion both in vivo and in vitro which

  7. mRNA expression profiles of calmodulin and liver receptor homolog-1 genes in chickens.

    PubMed

    Zhang, Z-C; Xiao, L-H; Wang, Y; Chen, S-Y; Yang, Z-Q; Zhao, X-L; Zhu, Q; Liu, Y-P

    2012-01-01

    Calmodulin (CALM), a calcium-binding protein, is expressed in the hypothalamic-pituitary-gonadal axis; it plays a pivotal role in the reproductive system by regulating gonadotropin-releasing hormone signaling. Downstream of hypothalamic-pituitary-gonadal signaling pathways, liver receptor homolog-1 (LRH-1) is involved in female gonadal hormone synthesis. In the chicken, although the two genes are known to be associated with reproductive traits, the interaction between gonadotropins and gonadal steroids remains unclear. We used quantitative real-time PCR to quantify the tissular (hypothalamus, pituitary, ovary, liver, kidney, oviduct, heart) and ontogenetic (12, 18, 32, and 45 weeks) mRNA expression profiles of CALM and LRH-1 in Erlang Mountainous chickens to determine their roles in the endocrine control of fertility, and compared these profiles with expression in Roman chickens. We found that the relative expressions of CALM and LRH-1 genes had the highest levels in the pituitary and ovary at 32 weeks. The expression level of CALM mRNA in the pituitary of Roman chickens was significantly higher than that in Erlang Mountainous chickens at 32 and 45 weeks, while the LRH-1 transcript level in the ovaries of Roman chickens was significantly lower than that of Erlang Mountainous chickens at 32 and 45 weeks. In summary, the transcript levels of CALM and LRH-1 genes are associated with chicken reproductive traits; in addition, we found that the CALM gene is the key regulator in the hypothalamic-pituitary-gonadal signaling network.

  8. Molecular cloning and mRNA expression of peroxiredoxin gene in black tiger shrimp (Penaeus monodon).

    PubMed

    Qiu, Lihua; Ma, Zhuojun; Jiang, Shigui; Wang, Weifang; Zhou, Falin; Huang, Jianhua; Li, Jianzhu; Yang, Qibin

    2010-07-01

    The techniques of homology cloning and anchored PCR were used to clone the peroxiredoxin (Prx) gene from black tiger shrimp (Penaeus monodon). The full length cDNA of black tiger shrimp Prx (PmPrx) contained a 5' untranslated region (UTR) of 51 bp, an ORF (open reading frame) of 582 bp encoding a polypeptide of 193 amino acids with an estimated molecular mass of 22.15 kDa and a 3' UTR of 948 bp. Sequence comparison showed that PmPrx shared higher identities with Prx IVs than that with other isoforms of Prx, indicating PmPrx was a member of the Prx IV family. A quantitative reverse transcriptase Real-Time PCR (qRT-PCR) assay was developed to assess the mRNA expression of PmPrx in different tissues and the temporal expression of PmPrx in the hepatopancreas challenged by lipopolyssacharide (LPS). Higher-level mRNA expression of PmPrx was detected in the tissues of hepatopancreas, gonad and heart. The expression of PmPrx in the hepatopancreas was up regulated after stimulated by LPS. The results indicated that PmPrx was a constitutive and inducible expressed protein and could be induced by LPS.

  9. Hepcidin expression in liver cells: evaluation of mRNA levels and transcriptional regulation.

    PubMed

    Kanamori, Yohei; Murakami, Masaru; Matsui, Tohru; Funaba, Masayuki

    2014-08-01

    Hepcidin produced in the liver negatively regulates intestinal iron absorption, and the bone morphogenetic protein (BMP) pathway is well-known to stimulate hepcidin expression. However, the regulation of hepcidin expression has not been fully elucidated. In this study, we evaluate different systems that can be used to determine how hepcidin expression is regulated. The basal expression of hepcidin in liver cell lines, such as HepG2 cells and Hepa1-6 cells, was lower than that in the liver and primary hepatocytes; the expression levels of hepcidin in the cell lines were near the limit of detection for RT-PCR and RT-qPCR analyses. Treatment with trichostatin A, RNAlater, or MG-132 enhanced the expression of hepcidin in HepG2 cells, suggesting that histone deacetylation, instability of mRNA, or proteosomal degradation of the protein(s) that positively regulate hepcidin expression may be responsible for the decreased expression of hepcidin in HepG2 cells. In luciferase-based reporter assays, BMP induced the transcription of a reporter, hepcidin(-2018)-luc, that contains nt -2018 through nt -35 of the hepcidin promoter in HepG2 cells and Hepa1-6 cells. However, BRE-luc, a representative reporter used to evaluate BMP signaling, was unresponsive to BMP in HepG2 cells. These results suggest that hepcidin transcription can be best evaluated in liver cell lines and that the hepcidin promoter senses BMP signaling with high sensitivity. The present study demonstrates that studies regarding the regulation of hepcidin expression at the mRNA level should be evaluated in primary hepatocytes, and liver cell lines are well-suited for studies examining the transcriptional regulation of hepcidin.

  10. Genomic Analysis and mRNA Expression of Equine Type I Interferon Genes

    PubMed Central

    Detournay, Olivier; Morrison, David A.; Wagner, Bettina; Zarnegar, Behdad

    2013-01-01

    This study aimed at identifying all of the type I interferon (IFN) genes of the horse and at monitoring their expression in equine cells on in vitro induction. We identified 32 putative type I IFN loci on horse chromosome 23 and an unplaced genomic scaffold. A phylogentic analysis characterized these into 8 different type I IFN classes, that is, putative functional genes for 6 IFN-α, 4 IFN-β, 8 IFN-ω (plus 4 pseudogenes), 3 IFN-δ (plus 1 pseudogene), 1 IFN-κ and 1 IFN-ɛ, plus 1 IFN-ν pseudogene, and 3 loci belonging to what has previously been called IFN-αω. Our analyses indicate that the IFN-αω genes are quite distinct from both IFN-α and IFN-ω, and we refer to this type I IFN as IFN-μ. Results from cell cultures showed that leukocytes readily expressed IFN-α, IFN-β, IFN-δ, IFN-μ, and IFN-ω mRNA on induction with, for example, live virus; while fibroblasts only expressed IFN-β mRNA on stimulation. IFN-κ or IFN-ɛ expression was not consistently induced in these cell cultures. Thus, the equine type I IFN family comprised 8 classes, 7 of which had putative functional genes, and mRNA expression of 5 was induced in vitro. Moreover, a relatively low number of IFN-α subtypes was found in the horse compared with other eutherian mammals. PMID:23772953

  11. Reduced mRNA expression levels of MBD2 and MBD3 in gastric carcinogenesis.

    PubMed

    Pontes, Thaís Brilhante; Chen, Elizabeth Suchi; Gigek, Carolina Oliveira; Calcagno, Danielle Queiroz; Wisnieski, Fernanda; Leal, Mariana Ferreira; Demachki, Samia; Assumpção, Paulo Pimentel; Artigiani, Ricardo; Lourenço, Laércio Gomes; Burbano, Rommel Rodriguez; Arruda Cardoso Smith, Marília

    2014-04-01

    Aberrant methylation has been reported in several neoplasias, including gastric cancer. The methyl-CpG-binding domain (MBD) family proteins have been implicated in the chromatin remodeling process, leading to the modulation of gene expression. To evaluate the role of MBD2 and MBD3 in gastric carcinogenesis and the possible association with clinicopathological characteristics, we assessed the mRNA levels and promoter methylation patterns in gastric tissues. In this study, MBD2 and MBD3 mRNA levels were determined by RT-qPCR in 28 neoplastic and adjacent nonneoplastic and 27 gastritis and non-gastritis samples. The promoter methylation status was determined by bisulfite sequencing, and we found reduced MBD2 and MBD3 levels in the neoplastic samples compared with the other groups. Moreover, a strong correlation between the MBD2 and MBD3 expression levels was observed in each set of paired samples. Our data also showed that the neoplastic tissues exhibited higher MBD2 promoter methylation than the other groups. Interestingly, the non-gastritis group was the only one with positive methylation in the MBD3 promoter region. Furthermore, a weak correlation between gene expression and methylation was observed. Therefore, our data suggest that DNA methylation plays a minor role in the regulation of MBD2 and MBD3 expression, and the presence of methylation at CpGs that interact with transcription factor complexes might also be involved in the modulation of these genes. Moreover, reduced mRNA expression of MBD2 and MBD3 is implicated in gastric carcinogenesis, and thus, further investigations about these genes should be conducted for a better understanding of the role of abnormal methylation involved in this neoplasia. PMID:24338710

  12. Molecular characterization and mRNA expression of catalase from pearl oyster Pinctada fucata.

    PubMed

    Guo, Huayang; Zhang, Dianchang; Cui, Shuge; Chen, Mingqiang; Wu, Kaichang; Li, Youning; Su, Tianfeng; Jiang, Shigui

    2011-12-01

    Catalase (EC 1.11.1.6) is an important antioxidant enzyme that protects aerobic organisms against oxidative damage by degrading hydrogen peroxide to water and oxygen. In the present study, a catalase cDNA of peal oyster Pincatada fucata (designated as PoCAT) is cloned and characterized by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) methods. PoCAT is 2428 bp long and consists of a 5'-UTR of 140 bp, an unusually long 3'-UTR of 749 bp, and an open reading frame (ORF) of 1539 bp. The ORF of PoCAT encodes a polypeptide of 512 amino acids with molecular weight of 58.1 kDa and the theoretical isoelectric point of 8.4. PoCAT shares 62.3-82.2% identity and 73.0-92.0% similarity to other catalase amino acid sequences. Sequence alignment indicates that PoCAT contains the proximal heme-ligand signature sequence (R³⁵¹LFSYSDT³⁵⁸), the proximal active site signature (F⁶¹NRERIPERVVHAKGGGA⁷⁸), and the three catalytic amino acid residues (His⁷², Asn¹⁴⁵, and Tyr³⁵⁵). PoCAT has two potential glycosylation sites (N⁴³⁶YS⁴³⁸ and N⁴⁷⁸FS⁴⁸⁰) and a peroxisome targeting signal (ASL). PoCAT mRNA was ubiquitously expressed in all detected tissues, and the expression level of PoCAT mRNA was higher in intestine and mantle. The expression profile analysis showed that the expression level of PoCAT mRNA in intestine was significantly up-regulated at 2, 4 and 12 h after Vibrio alginolyticus stimulation. These results demonstrated that PoCAT is a typical member of catalase family and might be involved in innate immune responses of pearl oyster.

  13. Expression of mRNA for interleukin-5 in mucosal bronchial biopsies from asthma.

    PubMed Central

    Hamid, Q; Azzawi, M; Ying, S; Moqbel, R; Wardlaw, A J; Corrigan, C J; Bradley, B; Durham, S R; Collins, J V; Jeffery, P K

    1991-01-01

    We have attempted to identify mRNA for IL-5 in endobronchial mucosal biopsies from asthmatics and controls, using the technique of in situ hybridization. Bronchial biopsies were obtained from 10 asthmatics and 9 nonatopic normal controls. A radio-labeled cRNA probe was prepared from an IL-5 cDNA and hybridized to permeabilized sections. These were washed extensively before processing for autoradiography. An IL-5-producing T cell clone derived from a patient with the hyper-IgE syndrome was used as a positive control. As a negative control, sections were also treated with a "sense" IL-5 probe. Specific hybridization signals for IL-5 mRNA were demonstrated within the bronchial mucosa in 6 out of the 10 asthmatic subjects. Cells exhibiting hybridization signals were located beneath the epithelial basement membrane. In contrast, there was no hybridization in the control group. No hybridization was observed with the sense probe. The six IL-5 mRNA-positive asthmatics tended to have more severe disease than the negative asthmatics, as assessed by symptoms and lung function, and showed a significant increase in the degree of infiltration of the bronchial mucosa by secreting (EG2+) eosinophils and activated (CD25+) T lymphocytes. Within the subjects who showed positive IL-5 mRNA, there was a correlation between IL-5 mRNA expression and the number of CD25+ and EG2+ cells and total eosinophil count. This study provides evidence for the cellular localization of IL-5 mRNA in the bronchial mucosa of asthmatics and supports the concept that this cytokine regulates eosinophil function in bronchial asthma. Images PMID:2022726

  14. Revealing the fate of cell surface human P-glycoprotein (ABCB1): The lysosomal degradation pathway.

    PubMed

    Katayama, Kazuhiro; Kapoor, Khyati; Ohnuma, Shinobu; Patel, Atish; Swaim, William; Ambudkar, Indu S; Ambudkar, Suresh V

    2015-10-01

    P-glycoprotein (P-gp) transports a variety of chemically dissimilar amphipathic compounds including anticancer drugs. Although mechanisms of P-gp drug transport are widely studied, the pathways involving its internalization are poorly understood. The present study is aimed at elucidating the pathways involved in degradation of cell surface P-gp. The fate of P-gp at the cell surface was determined by biotinylating cell surface proteins followed by flow cytometry and Western blotting. Our data shows that the half-life of endogenously expressed P-gp is 26.7±1.1 h in human colorectal cancer HCT-15 cells. Treatment of cells with Bafilomycin A1 (BafA1) a vacuolar H+ ATPase inhibitor increased the half-life of P-gp at the cell surface to 36.1±0.5 h. Interestingly, treatment with the proteasomal inhibitors MG132, MG115 or lactacystin alone did not alter the half-life of the protein. When cells were treated with both lysosomal and proteasomal inhibitors (BafA1 and MG132), the half-life was further prolonged to 39-50 h. Functional assays done with rhodamine 123 or calcein-AM, fluorescent substrates of P-gp, indicated that the transport function of P-gp was not affected by either biotinylation or treatment with BafA1 or proteasomal inhibitors. Immunofluorescence studies done with the antibody against lysosomal marker LAMP1 and the P-gp-specific antibody UIC2 in permeabilized cells indicated that intracellular P-gp is primarily localized in the lysosomal compartment. Our results suggest that the lysosomal degradation system could be targeted to increase the sensitivity of P-gp- expressing cancer cells towards chemotherapeutic drugs.

  15. Association of genotypes and haplotypes of multi-drug transporter genes ABCB1 and ABCG2 with clinical response to imatinib mesylate in chronic myeloid leukemia patients.

    PubMed

    Au, Anthony; Aziz Baba, Abdul; Goh, Ai Sim; Wahid Fadilah, S Abdul; Teh, Alan; Rosline, Hassan; Ankathil, Ravindran

    2014-04-01

    The introduction and success of imatinib mesylate (IM) has become a paradigm shift in chronic myeloid leukemia (CML) treatment. However, the high efficacy of IM has been hampered by the issue of clinical resistance that might due to pharmacogenetic variability. In the current study, the contribution of three common single nucleotide polymorphisms (SNPs) of ABCB1 (T1236C, G2677T/A and C3435T) and two SNPs of ABCG2 (G34A and C421A) genes in mediating resistance and/or good response among 215 CML patients on IM therapy were investigated. Among these patients, the frequency distribution of ABCG2 421 CC, CA and AA genotypes were significantly different between IM good response and resistant groups (P=0.01). Resistance was significantly associated with patients who had homozygous ABCB1 1236 CC genotype with OR 2.79 (95%CI: 1.217-6.374, P=0.01). For ABCB1 G2677T/A polymorphism, a better complete cytogenetic remission was observed for patients with variant TT/AT/AA genotype, compared to other genotype groups (OR=0.48, 95%CI: 0.239-0.957, P=0.03). Haplotype analysis revealed that ABCB1 haplotypes (C1236G2677C3435) was statistically linked to higher risk to IM resistance (25.8% vs. 17.4%, P=0.04), while ABCG2 diplotype A34A421 was significantly correlated with IM good response (9.1% vs. 3.9%, P=0.03). In addition, genotypic variant in ABCG2 421C>A was associated with a major molecular response (MMR) (OR=2.20, 95%CI: 1.273-3.811, P=0.004), whereas ABCB1 2677G>T/A variant was associated with a significantly lower molecular response (OR=0.49, 95%CI: 0.248-0.974, P=0.04). However, there was no significant correlation of these SNPs with IM intolerance and IM induced hepatotoxicity. Our results suggest the usefulness of genotyping of these single nucleotide polymorphisms in predicting IM response among CML patients. PMID:24581936

  16. Size selected mRNA induces expression of P-aminohippurate transport in Xenopus oocytes.

    PubMed

    Kwon, O; Kwon, H M; Hong, S K; Goldinger, J M

    1989-11-01

    Xenopus oocytes were injected with size-fractionated mRNA isolated from the renal cortex of rabbit kidney and after 4 days incubation, PAH uptake in oocytes injected with mRNA (0.7-1.3 kb) was 8 to 45 fold that of the water injected controls. The oocyte to medium ratio of accumulated PAH was 1.95. The Km and Vmax for transport were 333 microM and 66.6 nmoles.oocyte-1.min-1, respectively. This Km is similar to that reported for PAH transport in intact kidneys and slices. The uptake of PAH was unaffected by the absence of Na+ or the presence of probenecid. Expression of the transport represents the first step in an effort to clone and identify the gene for PAH transport.

  17. Oral and inhaled corticosteroids: Differences in P-glycoprotein (ABCB1) mediated efflux

    SciTech Connect

    Crowe, Andrew Tan, Ai May

    2012-05-01

    There is concern that P-glycoprotein mediated efflux contributes to steroid resistance. Therefore, this study examined bidirectional corticosteroid transport and induction capabilities for P-glycoprotein (P-gp) to understand which of the systemic and inhaled corticosteroids interacted with P-gp to the greatest extent. Hydrocortisone, prednisolone, prednisone, methylprednisolone, and dexamethasone represented systemically active drugs, while fluticasone propionate, beclomethasone dipropionate, ciclesonide and budesonide represented inhaled corticosteroids. Aldosterone and fludrocortisone represented mineralocorticoids. All drugs were detected using individually optimised HPLC protocols. Transport studies were conducted through Caco-2 monolayers. Hydrocortisone and aldosterone had efflux ratios below 1.5, while prednisone showed a P-gp mediated efflux ratio of only 1.8 compared to its active drug, prednisolone, with an efflux ratio of 4.5. Dexamethasone and beclomethasone had efflux ratios of 2.1 and 3.3 respectively, while this increased to 5.1 for methylprednisolone. Fluticasone showed an efflux ratio of 2.3. Protein expression studies suggested that all of the inhaled corticosteroids were able to induce P-gp expression, from 1.6 to 2 times control levels. Most of the systemic corticosteroids had higher passive permeability (> 20 × 10{sup −6} cm/s) compared to the inhaled corticosteroids (> 5 × 10{sup −6} cm/s), except for budesonide, with permeability similar to the systemic corticosteroids. Inhaled corticosteroids are not transported by P-gp to the same extent as systemic corticosteroids. However, they are able to induce P-gp production. Thus, inhaled corticosteroids may have greater interactions with other P-gp substrates, but P-gp itself is less likely to influence resistance to the drugs. -- Highlights: ► Inhaled corticosteroids are only weak substrates for P-gp, including budesonide. ► Inhaled corticosteroid potent P-gp inducers especially

  18. Contact call-driven zenk mRNA expression in the brain of the budgerigar (Melopsittacus undulatus).

    PubMed

    Brauth, Steven E; Tang, Ye-Zhong; Liang, Wenru; Roberts, Todd F

    2003-09-10

    Contact call-driven zenk (zif268, egr1, NGF1A, Krox 24) mRNA expression was mapped with in situ hybridization histochemistry in a vocal learning parrot, the budgerigar (M. undulatus). Relative to controls, call stimulation induced high zenk mRNA expression in all auditory areas including those closely associated with the vocal system within the anterior forebrain (Brauth et al. (2001) J. Comp. Neurol. 432, 481; (2002) Learn. Memory 9, 76). Thus there is a high correspondence between the distributions of neurons exhibiting contact call-driven zenk protein and mRNA expression in budgerigars. Field L2a, an area reported previously to express only perinucleolar zenk protein localization (Brauth et al. (2002) Learn. Memory 9, 76) also showed zenk mRNA expression.

  19. HUR mRNA expression in ovarian high-grade serous carcinoma effusions is associated with poor survival.

    PubMed

    Davidson, Ben; Holth, Arild; Hellesylt, Ellen; Hadar, Rivka; Katz, Betina; Tropé, Claes G; Reich, Reuven

    2016-02-01

    The objective of this study was to analyze the expression and clinical role of the RNA-binding molecule HuR in metastatic high-grade ovarian serous carcinoma (HGSC). HUR mRNA expression by reverse-transcription polymerase chain reaction was analyzed in 66 effusions from patients diagnosed with HGSC. Protein expression was analyzed in 262 HGSC effusions using immunohistochemistry. HUR mRNA was detected in all 66 effusions. HUR mRNA levels were unrelated to clinicopathological parameters. However, higher HUR mRNA levels were significantly related to poor overall survival in the entire cohort (P=.023), as well as in analysis limited to patients with prechemotherapy primary diagnosis specimens (P=.001) in univariate analysis. Cox multivariate analysis showed an independent prognostic role for HUR mRNA in the entire cohort (P=.033) and in patients with prechemotherapy primary diagnosis specimens (P=.002). HuR protein was detected in the nucleus and cytoplasm of tumor cells in 258 (98%) of 262 and 153 (58%) of 262 effusions, respectively. Higher HuR protein expression was associated with higher serum Cancer Antigen (CA) 125 levels at diagnosis (P=.01), but its presence at both cellular compartments was otherwise unrelated to clinicopathological parameters or survival. In conclusion, HuR is widely expressed in metastatic HGSC at both the mRNA and protein level. Higher HUR mRNA levels are associated with poor survival in metastatic HGSC, whereas protein expression has no prognostic value.

  20. Alterations in Somatostatin mRNA Expression in the Dorsolateral Prefrontal Cortex of Subjects with Schizophrenia or Schizoaffective Disorder

    PubMed Central

    Morris, Harvey M.; Hashimoto, Takanori; Lewis, David A.

    2010-01-01

    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. However, neither the cellular substrate nor the causal mechanisms for decreased SST mRNA levels in schizophrenia are known. We used in situ hybridization to quantify the compartmental, laminar, and cellular levels of SST mRNA expression in the DLPFC of 23 pairs of schizophrenia or schizoaffective disorder and control subjects. We also explored potential causal mechanisms by utilizing similar methods to analyze SST mRNA expression in 2 animal models. The expression of SST mRNA was significantly decreased in layers 2–superficial 6 of subjects with schizophrenia, but not in layer 1, deep 6 or the white matter. At the cellular level, both the density of cortical SST mRNA-positive neurons and the expression of SST mRNA per neuron were reduced in the subjects with schizophrenia. These alterations were not due to potential confounds and appeared to be a downstream consequence of impaired neurotrophin signaling through the trkB receptor. These findings support the hypothesis that a marked reduction in SST mRNA expression in a subset of GABA neurons contributes to DLPFC dysfunction in schizophrenia. PMID:18203698

  1. mRNA expression of dopamine receptors in peripheral blood lymphocytes of computer game addicts.

    PubMed

    Vousooghi, Nasim; Zarei, Seyed Zeinolabedin; Sadat-Shirazi, Mitra-Sadat; Eghbali, Fatemeh; Zarrindast, Mohammad Reza

    2015-10-01

    Excessive playing of computer games like some other behaviors could lead to addiction. Addictive behaviors may induce their reinforcing effects through stimulation of the brain dopaminergic mesolimbic pathway. The status of dopamine receptors in the brain may be parallel to their homologous receptors in peripheral blood lymphocytes (PBLs). Here, we have investigated the mRNA expression of dopamine D3, D4 and D5 receptors in PBLs of computer game addicts (n = 20) in comparison to normal subjects (n = 20), using a real-time PCR method. The results showed that the expression level of D3 and D4 dopamine receptors in computer game addicts were not statistically different from the control group. However, the expression of the mRNA of D5 dopamine receptor was significantly down-regulated in PBLs of computer game addicts and reached 0.42 the amount of the control group. It is concluded that unlike with drug addiction, the expression levels of the D3 and D4 dopamine receptors in computer game addicts are not altered compared to the control group. However, reduced level of the D5 dopamine receptor in computer game addicts may serve as a peripheral marker in studies where the confounding effects of abused drugs are unwanted.

  2. Murine metal-induced systemic autoimmunity: baseline and stimulated cytokine mRNA expression in genetically susceptible and resistant strains.

    PubMed

    Häggqvist, B; Hultman, P

    2001-10-01

    Cytokines play an important and complex role in the pathogenesis of systemic autoimmune diseases. In susceptible H-2s mice, inorganic mercury (Hg) induces lymphoproliferation, antinucleolar antibodies against the 34-kDa-protein fibrillarin, and systemic immune-complex (IC) deposits. Here, we report extensive analysis of cytokine mRNA levels in susceptible A.SW (H-2s) and resistant A.TL (H-2tl) mice under unstimulated conditions and during oral treatment with Hg and/or silver nitrate (Ag). Cytokine mRNA expression in lymphoid tissues was assessed using the ribonuclease protection assay and phosphorimaging. Baseline expression of IL-2 and IFN-gamma mRNA was higher in A.SW than in A.TL mice. In A.SW mice, Hg treatment caused early up-regulation of IL-2 and IFN-gamma levels, followed by substantial expression of IL-4 mRNA, which was significant compared to control A.SW and Hg-treated A.TL mice. Hg-exposed A.TL mice exhibited unchanged IFN-gamma, reduced IL-2 and greatly increased IL-10 mRNA expression. Ag-treated A.SW mice, which develop antifibrillarin antibodies (AFA) but exhibit minimal immune activation and no IC deposits, showed an early increase in IL-2 and IFN-gamma mRNA, but only a small and delayed rise in IL-4 mRNA. In conclusion, H-2-linked resistance to Hg-induced AFA is characterized by low constitutive expression of IL-2 and IFN-gamma mRNA, which is not increased by Hg, and a marked increase in IL-10 expression. Conversely, the key features of H-2-linked susceptibility to Hg- and Ag-induced AFA are up-regulation of IL-2, IFN-gamma and IL-4 mRNA expression, and down-regulation of IL-10 expression.

  3. Different structure and mRNA expression of Entamoeba invadens chitinases in the encystation and excystation.

    PubMed

    Makioka, Asao; Kumagai, Masahiro; Hiranuka, Kazushi; Kobayashi, Seiki; Takeuchi, Tsutomu

    2011-08-01

    Entamoeba histolytica forms chitin-walled cysts during encystation process, where formation of the cyst wall needs not only chitin synthase but also chitinase. During excystation, quadruplet amoebae emerge from the chitin-walled cysts by dissolving the wall, so that chitinase may be necessary for excystation process as well. There is, however, no report on chitinase expression during excystation. In this study, we used Entamoeba invadens, a reptilian amoeba, as a model for encystation and excystation of E. histolytica, and studied chitinase mRNA expression in those processes. Although expression of three E. invadens chitinases designated EiChit1, EiChit2, and EiChit3 during encystation has been reported, we identified another enzyme named as EiChit4 in the E. invadens genome database. Therefore, we investigated the primary structure and mRNA expression of these four chitinases of Ei in the excystation as well as the encystation by real-time reverse transcription polymerase chain reaction (RT-PCR). Like EiChit1, EiChit4 had an 8 × Cys chitin-binding domain (CBD) and a hydrophilic spacer between the CBD and catalytic domain, and was also closer to EiChit1 than EiChit2 and EiChit3 in the phylogenetic tree. During encystation, the expression of all four chitinases increased in the early phase; the increase in EiChit1 and EiChit4 was much higher than in EiChit2 and EiChit3. Then, the expression of all four chitinases sharply decreased in the later phase. In cysts, EiChit1 was most abundantly expressed and EiChit4 was at a lower level, while the expressions of EiChit2 and EiChit3 were virtually absent. Following the induction of excystation, mRNA levels of EiChit1 and EiChit4 in cysts 5 h after induction were significantly lower than those in cysts before induction, while those of EiChit2 and EiChit3 were remarkably higher than before induction. The mRNAs of only EiChit2 and EiChit3 remarkably increased when the excystation was induced in the presence of cytochalasin D

  4. Biochemical and pharmacological properties of an allosteric modulator site of the human P-glycoprotein (ABCB1).

    PubMed

    Maki, Nazli; Dey, Saibal

    2006-07-14

    The drug-transport function of the human P-glycoprotein (Pgp or ABCB1) is inhibited by a number of structurally unrelated compounds, known as modulators or reversing agents. Among them, the thioxanthene derivative flupentixol inhibits Pgp-mediated drug transport by an allosteric mechanism. Unlike most other Pgp modulators, the cis isomer of flupentixol [cis-(Z)-flupentixol] facilitates interaction of Pgp with its transport-substrate [125I]iodoarylazidoprazosin (or [125I]IAAP), yet inhibits transport. In this study, we show that the flupentixol site acts as a common site of interaction for the tricyclic ring-containing modulators thioxanthenes and phenothiazines. The allosteric stimulation of [125I]IAAP binding to Pgp occurs independent of the phosphorylation status of the transporter. Stimulation is retained in purified Pgp reconstituted into proteoliposomes, suggesting no involvement of any other cellular protein in the phenomenon. However, perturbation of the lipid environment of the reconstituted Pgp by nonionic detergent octylglucoside abolishes stimulation by cis-(Z)-flupentixol of [125I]IAAP binding. Extensive trypsin digestion of the [125I]IAAP-labeled Pgp generates a 5.5 kDa fragment with 80% of the stimulated level of labeling associated with it. Sensitivity to inhibition by transport-substrate vinblastine and competitive modulator cyclosporin A suggests that the elevated level of [125I]IAAP binding to the fragment represents a functionally relevant interaction with the substrate site of Pgp. In summary, we demonstrate that allosteric modulation by cis-(Z)-flupentixol is mediated through its interaction with Pgp at a site specific for tricyclic ring-containing Pgp modulators of thioxanthene and phenothiazine backbone, independent of other cellular components and the phosphorylation status of the protein.

  5. Expression of cytokine mRNA transcripts in renal cell carcinoma.

    PubMed

    Olive, C; Cheung, C; Nicol, D; Falk, M C

    1998-08-01

    Renal cell carcinoma (RCC) is a solid tumour of the kidney and is the most common renal neoplasm. Despite the presence of tumour infiltrating lymphocytes (TIL) in RCC, these tumours continue to progress in vivo suggesting a poor host immune response to the tumour, and the suppression of TIL effector function. Cytokines are key molecules that modulate the function of T cells. The possibility is investigated that the local production of cytokines in RCC contributes to immunosuppression of TIL. The expression of pro-inflammatory (IFN-gamma/IL-2) and immunosuppressive (IL-10/TGF-beta) cytokine mRNA transcripts was determined in RCC, normal kidney and peripheral blood of RCC patients using a semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) with cytokine-specific primers. Following Southern blot hybridization of the PCR products with internal radiolabelled oligonucleotide probes, cytokine transcript levels were measured by densitometry and expressed relative to the glyceraldehyde-3-phosphate dehydrogenase densitometry score. With the exception of IL-10, there were no differences in expression of cytokine mRNA transcripts between the peripheral blood of patients and normal healthy individuals. It was found that TGF-beta transcripts were well represented in normal kidney and RCC. In contrast, the expression of IFN-gamma transcripts, while low in the majority of samples, was significantly increased in RCC when compared to normal kidney (P=0.05). The IL-2 and IL-10 transcripts showed a more variable expression in normal kidney and RCC, with no significant differences in expression between the sample groups. The data demonstrating pro-inflammatory and immunosuppressive cytokine expression in RCC do not support a prominent immunosuppressive cytokine profile in these tumours. PMID:9723777

  6. Decreased parvalbumin mRNA expression in dorsolateral prefrontal cortex in Parkinson’s disease

    PubMed Central

    Lanoue, Amélie C.; Blatt, Gene J.; Soghomonian, Jean-Jacques

    2013-01-01

    It has recently been shown that expression of the rate-limiting GABA-synthesizing enzyme glutamic acid decarboxylase (GAD) is decreased in Brodmann area 9 (BA9) of the dorsolateral prefrontal cortex (DLPFC) in Parkinson’s disease (PD) compared to control brains (Lanoue, A.C., Dumitriu, A., Myers, R.H., Soghomonian, JJ., 2010. Exp Neurol. 206(1), 207–217). A subpopulation of cortical GABAergic interneurons expresses the calcium-binding protein parvalbumin and plays a critical role in the control of pyramidal neuron excitability and the generation of cortical gamma frequency oscillations. In view of its key role in the physiology of the cerebral cortex, we sought to determine whether the expression of parvalbumin and the number of parvalbumin-expressing neurons are altered in BA9 of PD brains. First, isotopic in situ hybridization histochemistry was used to examine mRNA expression of parvalbumin on post-mortem brain sections. Second, immunohistochemistry and design-based stereology were used to determine the density of parvalbumin-positive interneurons in BA9. Quantification of mRNA labeling at the single cell level showed a significant decrease in parvalbumin expression in PD cases. In contrast, neuronal density of parvalbumin-positive neurons was not significantly different between PD and controls. Results confirm that the GABAergic system is altered in the DLPFC in PD and identify the contribution of parvalbumin-expressing neurons in these alterations. We speculate that these effects could contribute to altered cortical excitability and oscillatory activity previously documented in PD. PMID:23891794

  7. Improvement of the cellular quality of cryopreserved bovine blastocysts accompanied by enhancement of the ATP-binding cassette sub-family B member 1 expression.

    PubMed

    Mori, Miyuki; Kasa, Shojiro; Isozaki, Yoshihiro; Kamori, Tsugumitsu; Yamaguchi, Shoichiro; Ueda, Shuji; Kuwano, Toshio; Eguchi, Minako; Isayama, Keishiro; Nishimura, Shotaro; Tabata, Shoji; Yamauchi, Nobuhiko; Hattori, Masa-aki

    2013-01-01

    The ATP-binding cassette sub-family B member 1 (ABCB1) plays a critical role in maintaining the metabolic capability of cells as an efflux transporter that pumps xenobiotics out of cells. We investigated the effects of highly expressed ABCB1 on the development and viability of cryopreserved bovine embryos. The ABCB1 level in cultured bovine embryos was decreased during development to blastocyst-stage compared to germinal vesicle- and second metaphase-stage oocytes. When bovine embryos were cultured with forskolin and/or rifampicin, the ABCB1 level was significantly increased in blastocysts but embryo development was not significantly improved. After embryo cryopreservation, highly ABCB1-expressed blastocysts exhibited significant increases in viability and hatching rates. The high viability of the cryopreserved blastocysts was accompanied by a significant increase in cell proliferation during culture for 48 h. Thus, ABCB1 is expressed in bovine oocytes and embryos, and the cellular quality of bovine blastocysts is improved by the enhancement of ABCB1 expression. PMID:23164983

  8. Effects of glutamine supplementation on splenocyte cytokine mRNA expression in rats with septic peritonitis

    PubMed Central

    Yeh, Sung-Ling; Lai, Yu-Ni; Shang, Huey-Fang; Lin, Ming-Tsan; Chiu, Wan-Chun; Chen, Wei-Jao

    2005-01-01

    AIM: To investigate the effects of glutamine (GLN)-enriched diets before and GLN-containing total parenteral nutrition (TPN) after sepsis or both on the secretion of cytokines and their mRNA expression levels in splenocytes of rats with septic peritonitis. METHODS: Rats were assigned to a control group and 4 experimental groups. The control group and experimental groups 1 and 2 were fed a semipurified diet, while experimental groups 3 and 4 had part of the casein replaced by GLN which provided 25% of the total nitrogen. After rats were fed with these diets for 10 d, sepsis was induced by cecal ligation and puncture (CLP), whereas the control group underwent a sham operation, at the same time, an internal jugular vein was cannulated. All rats were maintained on TPN for 3 d. The control group and experimental groups 1 and 3 were infused with conventional TPN, while the TPN in experimental groups 2 and 4 was supplemented with GLN, providing 25% of the total nitrogen in the TPN solution. All rats were kiued 3 d after sham operation or CLP to examine their splenocyte subpopulation distribution and cytokine expression levels. RESULTS: Most cytokines could not be detected in plasma except for IL-10. No difference in plasma IL-10 was observed among the 5 groups. The IL-2, IL-4, IL-10, and TNF-α mRNA expression levels in splenocytes were significantly higher in experimental groups 2 and 4 than in the control group and group 1. The mRNA expression of IFN-γ was significantly higher in the GLN-supplemented groups than in the control group and experimental group 1. The proportion of CD45Ra+ was increased, while those of CD3+ and CD4+ were decreased in experimental group 1 after CLP was performed. There were no differences in spleen CD3+ lymphocyte distributions between the control and GLN-supplemented groups. CONCLUSION: GLN supplementation can maintain T-lymphocyte populations in the spleen and significantly enhance the mRNA expression levels of Th1 and Th2 cytokines and TNF

  9. Hypergravity modulates vitamin D receptor target gene mRNA expression in mice.

    PubMed

    Ishizawa, Michiyasu; Iwasaki, Ken-Ichi; Kato, Shigeaki; Makishima, Makoto

    2009-09-01

    The possibility of pathological calcium metabolism is a critical health concern introduced by long-term space travel. Because vitamin D plays an important role in calcium homeostasis, we evaluated the effects of hypergravity on the expression of genes involved in vitamin D and calcium metabolism in ICR mice. When exposed to 2G hypergravity for 2 days, the mRNA expression of renal 25-hydroxyvitamin D 24-hydroxylase (Cyp24a1) was increased and that of 25-hydroxyvitamin D 1alpha-hydroxylase (Cyp27b1) was decreased. Although hypergravity decreased food intake and increased the expression of starvation-induced genes, the changes in Cyp24a1 and Cyp27b1 expression were not due to starvation, suggesting that hypergravity affects these genes directly. Hypergravity decreased plasma 1alpha,25-dihydroxyvitamin D(3) levels in ICR mice, suggesting a consequence of decreased Cyp27b1 and increased Cyp24a1 expression. Although 1alpha-hydroxyvitamin D(3) [1alpha(OH)D(3)] treatment induced the expression of vitamin D receptor (VDR) target genes in the kidney of 2G-exposed ICR mice to similar levels as controls, 1alpha(OH)D(3) increased the intestinal expression of Cyp24a1 in ICR mice. Hypergravity-dependent changes of Cyp24a1 and Cyp27b1 expression were diminished in mice exposed to hypergravity for 14 days, which may represent an adaptation to hypergravity stress. Hypergravity exposure also increased Cyp24a1 expression in the kidney of C57BL/6J mice. We examined the effects of hypergravity on VDR-null mice and found that renal Cyp27b1 expression in VDR-null mice was decreased by hypergravity while renal Cyp24a1 expression was not detected in VDR-null mice. Thus hypergravity modifies the expression of genes involved in vitamin D metabolism.

  10. GABAergic mRNA expression is upregulated in the prefrontal cortex of rats sensitized to methamphetamine.

    PubMed

    Wearne, Travis A; Parker, Lindsay M; Franklin, Jane L; Goodchild, Ann K; Cornish, Jennifer L

    2016-01-15

    Inhibitory gamma-aminobutyric acid (GABA)-mediated neurotransmission plays an important role in the regulation of the prefrontal cortex (PFC), with increasing evidence suggesting that dysfunctional GABAergic processing of the PFC may underlie certain deficits reported across psychotic disorders. Methamphetamine (METH) is a psychostimulant that induces chronic psychosis in a subset of users, with repeat administration producing a progressively increased vulnerability to psychotic relapse following subsequent drug administration (sensitization). The aim here was to investigate changes to GABAergic mRNA expression in the PFC of rats sensitized to METH using quantitative polymerase chain reaction (qPCR). Male Sprague-Dawley rats (n=12) underwent repeated methamphetamine (intraperitoneal (i.p.) or saline injections for 7 days. Following 14 days of withdrawal, rats were challenged with acute methamphetamine (1mg/kg i.p.) and RNA was isolated from the PFC to compare the relative mRNA expression of a range of GABA enzymes, transporters and receptors subunits. METH challenge resulted in a significant sensitized behavioral (locomotor) response in METH pre-treated animals compared with saline pre-treated controls. The mRNAs of transporters (GAT1 and GAT3), ionotropic GABAA receptor subunits (α3 and β1), together with the metabotropic GABAB1 receptor, were upregulated in the PFC of sensitized rats compared with saline controls. These findings indicate that GABAergic mRNA expression is significantly altered at the pre and postsynaptic level following sensitization to METH, with sensitization resulting in the transcriptional upregulation of several inhibitory genes. These changes likely have significant consequences on GABA-mediated neurotransmission in the PFC and may underlie certain symptoms conserved across psychotic disorders, such as executive dysfunction.

  11. p53-mediated control of gene expression via mRNA translation during Endoplasmic Reticulum stress.

    PubMed

    López, Ignacio; Tournillon, Anne-Sophie; Nylander, Karin; Fåhraeus, Robin

    2015-01-01

    p53 is activated by different stress and damage pathways and regulates cell biological responses including cell cycle arrest, repair pathways, apoptosis and senescence. Following DNA damage, the levels of p53 increase and via binding to target gene promoters, p53 induces expression of multiple genes including p21(CDKN1A) and mdm2. The effects of p53 on gene expression during the DNA damage response are well mimicked by overexpressing p53 under normal conditions. However, stress to the Endoplasmic Reticulum (ER) and the consequent Unfolded Protein Response (UPR) leads to the induction of the p53/47 isoform that lacks the first 40 aa of p53 and to an active suppression of p21(CDKN1A) transcription and mRNA translation. We now show that during ER stress p53 also suppresses MDM2 protein levels via a similar mechanism. These observations not only raise questions about the physiological role of MDM2 during ER stress but it also reveals a new facet of p53 as a repressor toward 2 of its major target genes during the UPR. As suppression of p21(CDKN1A) and MDM2 protein synthesis is mediated via their coding sequences, it raises the possibility that p53 controls mRNA translation via a common mechanism that might play an important role in how p53 regulates gene expression during the UPR, as compared to the transcription-dependent gene regulation taking place during the DNA damage response.

  12. Genome expression and mRNA maturation at late stages of productive adenovirus type 2 infection.

    PubMed

    Wold, W S; Green, M; Brackmann, K H; Cartas, M A; Devine, C

    1976-11-01

    RNA from adenovirus 2-infected KB cells was annealed in liquid with RNA in vast excess to viral heavy (l) and light (r) 32P-labeled DNA strands. Hybridization kinetics were analyzed by computer to estimate the number of viral RNA abundance classes, their relative concentrations, and the fraction of each DNA strand from which they originated. Early whole cell RNA extracted 5 h postinfection annealed rapidly to 10 to 15% of l and r strands and then slowly to final values of 60 and 40% of l and r strands. By 9 h postinfection the expression of late genes was apparent and whole cell RNA annealed to 20 and 75% of l and r strands. Whole cell RNA extracted between 12 and 36 h postinfection annealed to 7 to 15% and 75 to 90% of l and r strands. Late nuclear RNA hybridized to 10 and 90% of l and r strands, and late polyribosomal RNA hybridized to 20 and 75% of l and r strands. Based upon kinetic analyses, we estimate that mRNA synthesized exclusively during late stages arises from about 6 to 8% and 45 to 49% of l and r strands. This assumes that the early class I mRNA (in low concentration late) originates from 8 to 10% and 6 to 10% of l and r strands and that early class II mRNA (in high concentration late) is derived from 2% and 8 to 13% of l and r strands. Mixing experiments indicated that early mRNA is a subset of RNA extracted from polyribosomes late after infection and that late nuclear RNA contains sequences complementary to early l strand class I nRNA. RNA-RNA hybrids were isolated from late mRNA containing sequences from 60% of l and r strands, but it is not known when these were synthesized, and therefore whether complementary RNA transcripts are synthesized late after infection, as they are known to be synthesized early. These results demonstrate that portions of the genome are transcribed into RNA sequences that remain confined to the nucleus and are not exported to polyribosomes as mRNA.

  13. UU/UA dinucleotide frequency reduction in coding regions results in increased mRNA stability and protein expression.

    PubMed

    Al-Saif, Maher; Khabar, Khalid S A

    2012-05-01

    UU and UA dinucleotides are rare in mammalian genes and may offer natural selection against endoribonuclease-mediated mRNA decay. This study hypothesized that reducing UU and UA (UW) dinucleotides in the mRNA-coding sequence, including the codons and the dicodon boundaries, may promote resistance to mRNA decay, thereby increasing protein production. Indeed, protein expression from UW-reduced coding regions of enhanced green fluorescent protein (EGFP), luciferase, interferon-α, and hepatitis B surface antigen (HBsAg) was higher when compared to the wild-type protein expression. The steady-state level of UW-reduced EGFP mRNA was higher and the mRNA half-life was also longer. Ectopic expression of the endoribonuclease, RNase L, did not reduce the wild type or UW-reduced mRNA. A mutant form of the mRNA decay-promoting protein, tristetraprolin (TTP/ZFP36), which has a point mutation in the zinc-finger domain (C124R), was used. The wild-type EGFP mRNA but not the UW-reduced mRNA responded to the dominant negative action of the C124R ZFP36/TTP mutant. The results indicate the efficacy of the described rational approach to formulate a general scheme for boosting recombinant protein production in mammalian cells.

  14. Ustilago maydis natural antisense transcript expression alters mRNA stability and pathogenesis

    PubMed Central

    Donaldson, Michael E; Saville, Barry J

    2013-01-01

    Ustilago maydis infection of Zea mays leads to the production of thick-walled diploid teliospores that are the dispersal agent for this pathogen. Transcriptome analyses of this model biotrophic basidiomycete fungus identified natural antisense transcripts (NATs) complementary to 247 open reading frames. The U. maydis NAT cDNAs were fully sequenced and annotated. Strand-specific RT-PCR screens confirmed expression and identified NATs preferentially expressed in the teliospore. Targeted screens revealed four U. maydis NATs that are conserved in a related fungus. Expression of NATs in haploid cells, where they are not naturally occurring, resulted in increased steady-state levels of some complementary mRNAs. The expression of one NAT, as-um02151, in haploid cells resulted in a twofold increase in complementary mRNA levels, the formation of sense–antisense double-stranded RNAs, and unchanged Um02151 protein levels. This led to a model for NAT function in the maintenance and expression of stored teliospore mRNAs. In testing this model by deletion of the regulatory region, it was determined that alteration in NAT expression resulted in decreased pathogenesis in both cob and seedling infections. This annotation and functional analysis supports multiple roles for U. maydis NATs in controlling gene expression and influencing pathogenesis. PMID:23650872

  15. Ustilago maydis natural antisense transcript expression alters mRNA stability and pathogenesis.

    PubMed

    Donaldson, Michael E; Saville, Barry J

    2013-07-01

    Ustilago maydis infection of Zea mays leads to the production of thick-walled diploid teliospores that are the dispersal agent for this pathogen. Transcriptome analyses of this model biotrophic basidiomycete fungus identified natural antisense transcripts (NATs) complementary to 247 open reading frames. The U. maydis NAT cDNAs were fully sequenced and annotated. Strand-specific RT-PCR screens confirmed expression and identified NATs preferentially expressed in the teliospore. Targeted screens revealed four U. maydis NATs that are conserved in a related fungus. Expression of NATs in haploid cells, where they are not naturally occurring, resulted in increased steady-state levels of some complementary mRNAs. The expression of one NAT, as-um02151, in haploid cells resulted in a twofold increase in complementary mRNA levels, the formation of sense-antisense double-stranded RNAs, and unchanged Um02151 protein levels. This led to a model for NAT function in the maintenance and expression of stored teliospore mRNAs. In testing this model by deletion of the regulatory region, it was determined that alteration in NAT expression resulted in decreased pathogenesis in both cob and seedling infections. This annotation and functional analysis supports multiple roles for U. maydis NATs in controlling gene expression and influencing pathogenesis.

  16. Ustilago maydis natural antisense transcript expression alters mRNA stability and pathogenesis.

    PubMed

    Donaldson, Michael E; Saville, Barry J

    2013-07-01

    Ustilago maydis infection of Zea mays leads to the production of thick-walled diploid teliospores that are the dispersal agent for this pathogen. Transcriptome analyses of this model biotrophic basidiomycete fungus identified natural antisense transcripts (NATs) complementary to 247 open reading frames. The U. maydis NAT cDNAs were fully sequenced and annotated. Strand-specific RT-PCR screens confirmed expression and identified NATs preferentially expressed in the teliospore. Targeted screens revealed four U. maydis NATs that are conserved in a related fungus. Expression of NATs in haploid cells, where they are not naturally occurring, resulted in increased steady-state levels of some complementary mRNAs. The expression of one NAT, as-um02151, in haploid cells resulted in a twofold increase in complementary mRNA levels, the formation of sense-antisense double-stranded RNAs, and unchanged Um02151 protein levels. This led to a model for NAT function in the maintenance and expression of stored teliospore mRNAs. In testing this model by deletion of the regulatory region, it was determined that alteration in NAT expression resulted in decreased pathogenesis in both cob and seedling infections. This annotation and functional analysis supports multiple roles for U. maydis NATs in controlling gene expression and influencing pathogenesis. PMID:23650872

  17. Different Profile of mRNA Expression in Sinoatrial Node from Streptozotocin-Induced Diabetic Rat

    PubMed Central

    Ferdous, Zannatul; Qureshi, Muhammad Anwar; Jayaprakash, Petrilla; Parekh, Khatija; John, Annie; Oz, Murat; Raza, Haider; Dobrzynski, Halina; Adrian, Thomas Edward; Howarth, Frank Christopher

    2016-01-01

    Background Experiments in isolated perfused heart have shown that heart rate is lower and sinoatrial node (SAN) action potential duration is longer in streptozotocin (STZ)–induced diabetic rat compared to controls. In sino-atrial preparations the pacemaker cycle length and sino-atrial conduction time are prolonged in STZ heart. To further clarify the molecular basis of electrical disturbances in the diabetic heart the profile of mRNA encoding a wide variety of proteins associated with the generation and transmission of electrical activity has been evaluated in the SAN of STZ-induced diabetic rat heart. Methodology/Principal Findings Heart rate was measured in isolated perfused heart with an extracellular suction electrode. Expression of mRNA encoding a variety of intercellular proteins, intracellular Ca2+-transport and regulatory proteins, cell membrane transport proteins and calcium, sodium and potassium channel proteins were measured in SAN and right atrial (RA) biopsies using real-time reverse transcription polymerase chain reaction techniques. Heart rate was lower in STZ (203±7 bpm) compared to control (239±11 bpm) rat. Among many differences in the profile of mRNA there are some worthy of particular emphasis. Expression of genes encoding some proteins were significantly downregulated in STZ-SAN: calcium channel, Cacng4 (7-fold); potassium channel, Kcnd2 whilst genes encoding some other proteins were significantly upregulated in STZ-SAN: gap junction, Gjc1; cell membrane transport, Slc8a1, Trpc1, Trpc6 (4-fold); intracellular Ca2+-transport, Ryr3; calcium channel Cacna1g, Cacna1h, Cacnb3; potassium channels, Kcnj5, Kcnk3 and natriuretic peptides, Nppa (5-fold) and Nppb (7-fold). Conclusions/Significance Collectively, this study has demonstrated differences in the profile of mRNA encoding a variety of proteins that are associated with the generation, conduction and regulation of electrical signals in the SAN of STZ-induced diabetic rat heart. Data from this

  18. Correlation of Apobec Mrna Expression with overall Survival and pd-l1 Expression in Urothelial Carcinoma

    PubMed Central

    Mullane, Stephanie A.; Werner, Lillian; Rosenberg, Jonathan; Signoretti, Sabina; Callea, Marcella; Choueiri, Toni K.; Freeman, Gordon J.; Bellmunt, Joaquim

    2016-01-01

    Metastatic urothelial carcinoma (mUC) has a very high mutational rate and is associated with an APOBEC mutation signature. We examined the correlation of APOBEC expression with overall survival (OS) and PD-L1 expression in a cohort of 73 mUC patients. mRNA expression of APOBEC3 family of genes (A3A, A3B, A3C, A3F_a, A3F_b, A3G, A3H) was measured using Nanostring. PD-L1 expression, evaluated by immunohistochemistry, on tumor infiltrating mononuclear cells (TIMCs) and tumor cells was scored from 0 to 4, with 2–4 being positive. Wilcoxon’s non-parametric tests assessed the association of APOBEC and PD-L1. The Cox regression model assessed the association of APOBEC with OS. All APOBEC genes were expressed in mUC. Increased A3A, A3D, and A3H expression associates with PD-L1 positive TIMCs (p = 0.0009, 0.009, 0.06). Decreased A3B expression was marginally associated with PD-L1 positive TIMCs expression (p = 0.05). Increased A3F_a and A3F_b expression was associated with increased expression of PD-L1 on tumor cells (p = 0.05). Increased expression of A3D and A3H was associated with longer OS (p = 0.0009). Specific APOBEC genes have different effects on mUC in terms of survival and PD-L1 expression. A3D and A3H may have the most important role in mUC as they are associated with OS and PD-L1 TIMC expression. PMID:27283319

  19. Expression of myosin heavy-chain mRNA in cultured myoblasts induced by centrifugal force.

    PubMed

    Kurokawa, Katsuhide; Sakiyama, Koji; Abe, Shinichi; Hiroki, Emi; Naito, Kaoru; Nakajima, Kazunori; Takeda, Tomotaka; Inoue, Takashi; Ide, Yoshinobu; Ishigami, Keiichi

    2008-11-01

    Ballistic muscle training leads to hypertrophy of fast type fibers and training for endurance induces that of slow type fibers. Numerous studies have been conducted on electrical, extending and magnetic stimulation of cells, but the effect of centrifugal force on cells remains to be investigated. In this study, we investigated the effect of stimulating cultured myoblasts with centrifugal force at different speeds on cell proliferation and myosin heavy-chain (MyHC) mRNA expression in muscle fiber. Stimulation of myoblasts was carried out at 2 different speeds for 20 min using the Himac CT6D, a desk centrifuge, and cells were observed at 1, 3 and 5 days later. Number of cells 1 and 5 days after centrifugal stimulation was significantly larger in the 62.5 x g and 4,170 x g stimulation groups than in the control group. Expression of MyHC-2b mRNA 1 day after centrifugal stimulation was significantly higher in the 2 stimulation groups than in the control group. Almost no expression of MyHC-2a was observed in any group at 1 and 3 days after centrifugal stimulation. However, 5 days after stimulation, MyHC-2a was strongly expressed in the 2 stimulation groups in comparison to the control group. Three days after centrifugal stimulation, expression of MyHC-1 was significantly higher in the 2 stimulation groups than in the control group. The results of this study clarified the effect of different centrifugal stimulation speeds on muscle fiber characteristics, and suggest that centrifugal stimulation of myoblasts enhances cell proliferation.

  20. Myoepithelial mRNA expression profiling reveals a common tumor-suppressor phenotype.

    PubMed

    Barsky, Sanford H

    2003-04-01

    A series of myoepithelial cell lines and xenografts derived from benign human myoepithelial tumors of diverse sources (salivary gland, breast, and lung) exhibit common mRNA expression profiles indicative of a tumor-suppressor phenotype. Previously established myoepithelial cell lines and xenografts (HMS-#; HMS-#X) were compared to nonmyoepithelial breast carcinoma cells (MDA-MB-231 and MDA-MB-468, and inflammatory breast carcinoma samples, IBCr, and IBCw), a normal mammary epithelial cell line (HMEC) and individual cases of human breast cancer (zcBT#T), and matched normal human breast tissues (zcBT#N) (overall samples = 22). The global gene expression profile (22,000 genes) of these individual samples was examined using Affymetrix Microarray Gene Chips and subsequently analyzed with both Affymetrix and DChip algorithms. The myoepithelial cell lines/xenografts were distinct and very different from the nonmyoepithelial breast carcinoma cells and the normal breast and breast tumor biopsies. Two hundred and seven specifically selected genes represented a subset of genes that distinguished (P < 0.05) all the myoepithelial cell lines/xenografts from all the other samples and which themselves exhibited hierarchical clustering. Further analysis of these genes revealed increased expression in genes belonging to the classes of extracellular matrix proteins, angiogenic inhibitors, and proteinase inhibitors and decreased expression belonging to the classes of angiogenic factors and proteinases. Developmental genes were also differentially expressed (either over or underexpressed). These studies confirm our previous impression that human myoepithelial cells express a distinct tumor-suppressor phenotype.

  1. Bacteria and Toll-like receptor and cytokine mRNA expression profiles associated with canine arthritis.

    PubMed

    Riggio, Marcello P; Lappin, David F; Bennett, David

    2014-08-15

    The major forms of inflammatory canine arthritis are immune-mediated arthritis (IMA) and septic arthritis (SA), although some cases of cruciate disease (CD) are associated with significant levels of synovitis. In this study, the bacteria associated with canine arthritis were identified and mRNA expression levels of Toll-like receptors (TLRs) and pro-inflammatory cytokines determined. Of the 40 synovial fluid samples analysed, bacteria were isolated from 12 samples by culture (2 CD, 10 SA) and detected in 4 samples (3 CD, 1 SA) using culture-independent methods. Statistically significant increases in TLR2, tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-12 mRNA expression were seen in all disease groups compared to normal controls. All disease groups had decreased mRNA expression of other TLRs compared to normal controls, but this did not reach statistical significance. Synovial fluid cell counts revealed that the highest number and proportion of mononuclear cells and neutrophils were found in the IMA and SA samples, respectively. Age had an effect on the TLR and cytokine mRNA expression profiles: TNF-α (p=0.043) and IL-12 (p=0.025) mRNA expression was increased and TLR4 mRNA expression was reduced (p=0.033) in dogs up to 4 years of age compared to older animals. In the 10 SA samples from which bacteria were isolated, statistically significant increases in TLR2, TLR7, TNF-α and IL-6 mRNA expression were observed. It is concluded that canine arthritis is associated with increased mRNA levels of pro-inflammatory cytokines, which could in some cases be mediated by bacteria through activation of TLR2.

  2. mRNA Expression Signature of Gleason Grade Predicts Lethal Prostate Cancer

    PubMed Central

    Penney, Kathryn L.; Sinnott, Jennifer A.; Fall, Katja; Pawitan, Yudi; Hoshida, Yujin; Kraft, Peter; Stark, Jennifer R.; Fiorentino, Michelangelo; Perner, Sven; Finn, Stephen; Calza, Stefano; Flavin, Richard; Freedman, Matthew L.; Setlur, Sunita; Sesso, Howard D.; Andersson, Swen-Olof; Martin, Neil; Kantoff, Philip W.; Johansson, Jan-Erik; Adami, Hans-Olov; Rubin, Mark A.; Loda, Massimo; Golub, Todd R.; Andrén, Ove; Stampfer, Meir J.; Mucci, Lorelei A.

    2011-01-01

    Purpose Prostate-specific antigen screening has led to enormous overtreatment of prostate cancer because of the inability to distinguish potentially lethal disease at diagnosis. We reasoned that by identifying an mRNA signature of Gleason grade, the best predictor of prognosis, we could improve prediction of lethal disease among men with moderate Gleason 7 tumors, the most common grade, and the most indeterminate in terms of prognosis. Patients and Methods Using the complementary DNA–mediated annealing, selection, extension, and ligation assay, we measured the mRNA expression of 6,100 genes in prostate tumor tissue in the Swedish Watchful Waiting cohort (n = 358) and Physicians' Health Study (PHS; n = 109). We developed an mRNA signature of Gleason grade comparing individuals with Gleason ≤ 6 to those with Gleason ≥ 8 tumors and applied the model among patients with Gleason 7 to discriminate lethal cases. Results We built a 157-gene signature using the Swedish data that predicted Gleason with low misclassification (area under the curve [AUC] = 0.91); when this signature was tested in the PHS, the discriminatory ability remained high (AUC = 0.94). In men with Gleason 7 tumors, who were excluded from the model building, the signature significantly improved the prediction of lethal disease beyond knowing whether the Gleason score was 4 + 3 or 3 + 4 (P = .006). Conclusion Our expression signature and the genes identified may improve our understanding of the de-differentiation process of prostate tumors. Additionally, the signature may have clinical applications among men with Gleason 7, by further estimating their risk of lethal prostate cancer and thereby guiding therapy decisions to improve outcomes and reduce overtreatment. PMID:21537050

  3. Cytokine mRNA expression in normal skin of various age populations before and after engraftment onto nude mice.

    PubMed

    Gilhar, A; Ullmann, Y; Shalagino, R; Weisinger, G

    1998-01-01

    Whether the impact of skin biological age on cytokine expression is a result of this tissue's proliferation potential or not is an important issue in dermatology. We investigated these questions by monitoring cytokine marker mRNA expression from human skin samples from healthy groups of individuals. The skin samples studied represented three age groups: fetal (17-21 weeks), young (18-35 years) and aged (76-88 years). Furthermore, upon skin transplantation of tissue from different age groups onto nude mice, we investigated whether cytokine marker RNA levels would change or normalize. Interestingly, both TNF-alpha and P53 mRNA showed a similar pattern of expression. Both were significantly higher in fetal skin (p < 0.0001 and p < 0.05, respectively), and no difference was noted between aged versus young skin. In contrast to this, IL1-alpha mRNA was expressed at its lowest and highest levels in fetal and young skin, respectively. Following skin transplantation, cytokines and P53 mRNA expression were normalized to similar levels in all age groups. This study implies that when cytokine expression was determined directly at the mRNA level, post-natal expression was not significantly different at either age group. Furthermore, it seems that the environmental conditions surrounding the grafted human skin found on nude mice encouraged normalization of donor cytokine expression.

  4. mRNA expression profile of serotonin receptor subtypes and distribution of serotonergic terminations in marmoset brain

    PubMed Central

    Shukla, Rammohan; Watakabe, Akiya; Yamamori, Tetsuo

    2014-01-01

    To better understand serotonin function in the primate brain, we examined the mRNA expression patterns of all the 13 members of the serotonin receptor (5HTR) family, by in situ hybridization (ISH) and the distribution of serotonergic terminations by serotonin transporter (SERT) protein immunohistochemical analysis. Ten of the 13 5HTRs showed significant mRNA expressions in the marmoset brain. Our study shows several new features of the organization of serotonergic systems in the marmoset brain. (1) The thalamus expressed only a limited number of receptor subtypes compared with the cortex, hippocampus, and other subcortical regions. (2) In the cortex, there are layer-selective and area-selective mRNA expressions of 5HTRs. (3) Highly localized mRNA expressions of 5HT1F and 5HT3A were observed. (4) There was a conspicuous overlap of the mRNA expressions of receptor subtypes known to have somatodendritic localization of receptor proteins with dense serotonergic terminations in the visual cortex, the central lateral (CL) nucleus of the thalamus, the presubiculum, and the medial mammillary nucleus of the hypothalamus. This suggests a high correlation between serotonin availability and receptor expression at these locations. (5) The 5HTRs show differences in mRNA expression pattern between the marmoset and mouse cortices whereas the patterns of both the species were much similar in the hippocampus. We discuss the possible roles of 5HTRs in the marmoset brain revealed by the analysis of their overall mRNA expression patterns. PMID:24904298

  5. mRNA Expression in Papillary and Anaplastic Thyroid Carcinoma: Molecular Anatomy of a Killing Switch

    PubMed Central

    Hébrant, Aline; Dom, Geneviève; Dewaele, Michael; Andry, Guy; Trésallet, Christophe; Leteurtre, Emmanuelle; Dumont, Jacques E.; Maenhaut, Carine

    2012-01-01

    Anaplastic thyroid carcinoma (ATC) is the most lethal form of thyroid neoplasia and represents the end stage of thyroid tumor progression. No effective treatment exists so far. ATC frequently derive from papillary thyroid carcinomas (PTC), which have a good prognosis. In this study, we analyzed the mRNA expression profiles of 59 thyroid tumors (11 ATC and 48 PTC) by microarrays. ATC and PTC showed largely overlapping mRNA expression profiles with most genes regulated in all ATC being also regulated in several PTC. 43% of the probes regulated in all the PTC are similarly regulated in all ATC. Many genes modulations observed in PTC are amplified in ATC. This illustrates the fact that ATC mostly derived from PTC. A molecular signature of aggressiveness composed of 9 genes clearly separates the two tumors. Moreover, this study demonstrates gene regulations corresponding to the ATC or PTC phenotypes like inflammatory reaction, epithelial to mesenchymal transition (EMT) and invasion, high proliferation rate, dedifferentiation, calcification and fibrosis processes, high glucose metabolism and glycolysis, lactate generation and chemoresistance. The main qualitative differences between the two tumor types bear on the much stronger EMT, dedifferentiation and glycolytic phenotypes showed by the ATC. PMID:23115614

  6. Upregulation of PBR mRNA expression in human neuroblastoma cells by flavonoids.

    PubMed

    Ha, Jeoung-Hee; Lee, Jae-Tae; Cho, Ihn-ho; Chun, Kyung-Ah; Park, Gi-Eun; Choi, Hyung-Chul; Lee, Kwang-Youn; Kim, Sang-Hyun; Suk, Kyoungho; Kim, In-Kyeom; Lee, Maan-Gee

    2007-02-01

    To investigate the putative mediation of peripheral benzodiazepine receptor (PBR) in the cytotoxicity of flavonoids, in this study, modulatory effects of several flavonoids on the lipid peroxide (LPO) production and PBR mRNA expression of human neuroblastoma cells were observed. Elevated levels of peroxidated products in cancer cells may activate pro-apoptotic and anti-proliferative signaling pathways. Treatment of 10(-6) M 4'-chlorodiazepam and PK 11195 ligands of the PBR for 6 days enhanced the generation of LPO of the human neuroblastoma cells. Several flavonoids, well-known cytotoxic substances, potentiated the enhancement of LPO production by PBR ligands. Treatment of 10(-6) M flavonoids for 6 days elevated the expression of PBR mRNA in cells. These findings indicate that the potential of flavonoids to induce apoptosis in cancer cells is strongly associated with their PBR-inducing properties, thereby providing a new mechanism by which polyphenolic compounds may exert their cancer-preventive and anti-neoplastic effects.

  7. Joint analysis of miRNA and mRNA expression data.

    PubMed

    Muniategui, Ander; Pey, Jon; Planes, Francisco J; Rubio, Angel

    2013-05-01

    miRNAs are small RNA molecules ('22 nt) that interact with their target mRNAs inhibiting translation or/and cleavaging the target mRNA. This interaction is guided by sequence complentarity and results in the reduction of mRNA and/or protein levels. miRNAs are involved in key biological processes and different diseases. Therefore, deciphering miRNA targets is crucial for diagnostics and therapeutics. However, miRNA regulatory mechanisms are complex and there is still no high-throughput and low-cost miRNA target screening technique. In recent years, several computational methods based on sequence complementarity of the miRNA and the mRNAs have been developed. However, the predicted interactions using these computational methods are inconsistent and the expected false positive rates are still large. Recently, it has been proposed to use the expression values of miRNAs and mRNAs (and/or proteins) to refine the results of sequence-based putative targets for a particular experiment. These methods have shown to be effective identifying the most prominent interactions from the databases of putative targets. Here, we review these methods that combine both expression and sequence-based putative targets to predict miRNA targets. PMID:22692086

  8. The vitamin D receptor localization and mRNA expression in ram testis and epididymis.

    PubMed

    Jin, Hui; Huang, Yang; Jin, Guang; Xue, Yanrong; Qin, Xiaowei; Yao, Xiaolei; Yue, Wenbing

    2015-02-01

    The objectives of present study were to investigate the presence of vitamin D receptor (VDR) in testis and epididymis of ram by polymerase chain reaction (PCR), to locate VDR in testis and epididymis by immunohistochemistry and to compare difference of VDR expression between testis and epididymis before and after sexual maturation by Real time-PCR and Western blot. The results showed that VDR exists in the testis and epididymis of ram while VDR protein in testis and epididymis was localized in Leydig cells, spermatogonial stem cells, spermatocytes, Sertoli cells and principal cells. For the adult ram, the amounts of VDR mRNA and VDR protein were less (p < 0.01) in testis than compared with caput, corpus and cauda epididymis. For prepubertal ram, the result showed the same trend (p < 0.01). However, the expression levels of VDR mRNA and VDR protein in caput, corpus, cauda epididymis and testis showed no significant difference (p > 0.05) between adult and prepubertal. In conclusion, VDR exists in testis and epididymis of ram, suggesting 1α,25-(OH)(2)VD(3) may play a role in ram reproduction.

  9. Postnatal rat lung retinoic acid receptor (RAR) mRNA expression and effects of dexamethasone on RAR beta mRNA.

    PubMed

    Grummer, M A; Zachman, R D

    1995-10-01

    Retinoids exert multiple effects upon lung differentiation and growth. Although the mechanisms involved are presently poorly understood, increasing evidence points to a central role of nuclear retinoic acid receptors (RAR). The purpose of this study was to determine RAR mRNA expression profile during postnatal alveolarization, compared with the expression in prenatal and adult rat lung, and to describe the effects of dexamethasone (DEX) and oxygen on postnatal lung RAR gene expression. Total RNA was isolated from lungs of Sprague-Dawley rats on prenatal day 19, on postnatal days 1, 3, 7, 10, and 14 of life, and from adults. One subgroup of littermate pups was treated with DEX daily for 3 or 7 days. In a second experiment, rats were exposed to room air or to 95% oxygen for 72 hours, and received either DEX or saline. Northern hybridization showed that the levels of all RAR subtypes in fetal lung were 45% or less of levels at postnatal day 1. The 3.7 kb RAR alpha transcript levels were lower than day 1 on days 10 and 14 (relative to day 1, day 10 = 0.54 +/- 0.05; day 14 = 0.54 +/- 0.08), but there was no change in a 2.7 kb RAR alpha transcript over this time period. By contrast, RAR beta mRNA levels were significantly higher at days 3, 10, and 14 compared with day 1 (day 3 = 1.79 +/- 0.19; day 10 = 1.41 +/- 0.14; day 14 = 1.53 +/- 0.05). Similarly, RAR gamma mRNA expression levels were higher on day 10 (1.45 +/- 0.09), but by day 14 there was no difference from day 1. Adult lung 3.7 kb RAR alpha, 2.7 kb RAR alpha, and RAR gamma were lower than day 1, but RAR beta was significantly greater (3.7 alpha = 0.52 +/- 0.05; 2.7 alpha = 0.49 +/- 0.26; gamma = 0.74 +/- 0.06; beta = 1.63 +/- 0.22). Treatment with DEX prevented the rise in RAR beta mRNA occurring on day 3 and significantly lowered (0.65 +/- 0.06) the amount of RAR beta mRNA in day 7 lung. Exposure of rat pups to oxygen caused an increase in RAR beta mRNA (1.21 +/- 0.03). DEX treatment again decreased RAR beta mRNA

  10. Nilotinib (AMN107, Tasigna) reverses multidrug resistance by inhibiting the activity of the ABCB1/Pgp and ABCG2/BCRP/MXR transporters.

    PubMed

    Tiwari, Amit K; Sodani, Kamlesh; Wang, Si-Rong; Kuang, Ye-Hong; Ashby, Charles R; Chen, Xiang; Chen, Zhe-Sheng

    2009-07-15

    Nilotinib, a BCR-Abl tyrosine kinase inhibitor (TKI), was developed to surmount resistance or intolerance to imatinib in patients with Philadelphia positive chronic myelogenous leukemia. Recently, it was shown that several human multidrug resistance (MDR) ATP-binding cassette (ABC) proteins could be modulated by specific TKIs. MDR can produce cancer chemotherapy failure, typically due to overexpression of ABC transporters, which are involved in the extrusion of therapeutic drugs. Here, we report for the first time that nilotinib potentiates the cytotoxicity of widely used therapeutic substrates of ABCG2, such as mitoxantrone, doxorubicin, and ABCB1 substrates including colchicine, vincristine, and paclitaxel. Nilotinib also significantly enhances the accumulation of paclitaxel in cell lines overexpressing ABCB1. Similarly, nilotinib significantly increases the intracellular accumulation of mitoxantrone in cells transfected with ABCG2. Furthermore, nilotinib produces a concentration-dependent inhibition of the ABCG2-mediated transport of methotrexate (MTX), as well as E(2)17betaG a physiological substrate of ABCG2. Uptake studies in membrane vesicles overexpressing ABCG2 have indicated that nilotinib inhibits ABCG2 similar to other established TKIs as well as fumitremorgin C. Nilotinib is a potent competitive inhibitor of MTX transport by ABCG2 with a K(i) value of 0.69+/-0.083 microM as demonstrated by kinetic analysis of nilotinib. Overall, our results indicate that nilotinib could reverse ABCB1- and ABCG2-mediated MDR by blocking the efflux function of these transporters. These findings may be used to guide the design of present and future clinical trials with nilotinib, elucidating potential pharmacokinetic interactions. Also, these findings may be useful in clinical practice for cancer combination therapy with nilotinib.

  11. Ets-1 mRNA expression in effusions of serous ovarian carcinoma patients is a marker of poor outcome.

    PubMed

    Davidson, B; Risberg, B; Goldberg, I; Nesland, J M; Berner, A; Tropé, C G; Kristensen, G B; Bryne, M; Reich, R

    2001-12-01

    Ets-1 proto-oncogene is a transcription factor with a role in the activation of metastasis-associated molecules. We recently found that Ets-1 mRNA expression in solid tumors is a marker of poor prognosis in ovarian carcinoma. The objective of this study was to compare the expression of Ets-1 mRNA in effusions and primary and metastatic tumors of serous ovarian carcinoma patients and to evaluate its prognostic role in effusions. Sections from 67 malignant effusions and 90 primary and metastatic lesions were evaluated for expression of Ets-1 using mRNA in situ hybridization. Expression of Ets-1 mRNA was detected in carcinoma cells in 24 of 67 (36%) effusions. Expression in cancer cells was similar in peritoneal and pleural effusions. In solid lesions Ets-1 expression was detected in both tumor cells and stromal cells in 34 of 90 (38%) lesions. Ets-1 expression in tumor cells showed a strong association with that of stromal cells (p <0.001). Ets-1 expression in effusions showed an association with mRNA expression of basic fibroblast growth factor, previously studied in this patient cohort (p = 0.019). Ets-1 expression in solid lesions showed an association with mRNA expression of vascular endothelial growth factor (p <0.001 for both carcinoma and stromal cells), basic fibroblast growth factor (p = 0.007 for carcinoma cells, p = 0.006 for stromal cells), and interleukin-8 (IL-8) (p = 0.001 for tumor cells). Ets-1 mRNA showed upregulation in metastases when compared with effusion specimens (p = 0.028). In univariate survival analysis Ets-1 expression in carcinoma cells in effusions correlated with poor survival (p = 0.003). Our findings confirm the role of Ets-1 as a novel prognostic marker in advanced-stage ovarian carcinoma and extend it to effusion specimens. The elevated expression in solid metastases supports a central role in tumor progression as well. The association between Ets-1 mRNA expression and the expression of angiogenic genes, documented also in our

  12. Expression of cytochromes P450 3A and P-glycoprotein in human large intestine in paired tumour and normal samples.

    PubMed

    Canaparo, Roberto; Nordmark, Anna; Finnström, Niklas; Lundgren, Stefan; Seidegård, Janeric; Jeppsson, Bengt; Edwards, Robert J; Boobis, Alan R; Rane, Anders

    2007-04-01

    Our objective was to investigate the expression of different cytochromes P450 3A (CYP3A4, CYP3A5, and CYP3A7) and P-glycoprotein (ABCB1) genes along the human large intestine in paired tumour and normal samples. Real-time reverse transcriptase-polymerase chain reaction was used to measure CYP3A4-, CYP3A5-, CYP3A7- and ABCB1-specific mRNA expression, and Western blot analysis was used to measure membrane protein levels of CYP3A4/7, CYP3A5 and P-glycoprotein. Levels of mRNA and membrane protein fractions in the large intestine were compared with those of normal human liver. The mRNA expressions of CYP3A4, CYP3A5, CYP3A7 and ABCB1 in the large intestine were found to be highly variable, but overall the levels were significantly lower than those measured in liver (P < 0.0001, P < 0.001, P < 0.0001 and P < 0.01, respectively). At the membrane protein level, CYP3A4/7 was detected in all large intestine samples examined and the levels were substantially higher than those of the liver (P < 0.01). Although expression of CYP3A5 was detected in all large intestine samples, in most the levels were too low to allow quantification. P-glycoprotein was readily detected at levels slightly higher than those of liver (P < 0.05). Comparison between paired samples of normal and tumour in large intestine showed no significant differences in either the mRNA or membrane protein levels of these genes. In conclusion, this work suggests a potential role of the large intestine in the absorption and metabolism of xenobiotics and nutrients and no difference in the CYP3A and P-glycoprotein membrane protein fractions and mRNA expression between normal and tumour tissues.

  13. Chronic social subordination stress modulates glutamic acid decarboxylase (GAD) 67 mRNA expression in central stress circuits

    PubMed Central

    Makinson, Ryan; Lundgren, Kerstin H.; Seroogy, Kim B.; Herman, James P.

    2015-01-01

    Chronic social subordination is a well-known precipitant of numerous psychiatric and physiological health concerns. In this study, we examine the effects of chronic social stress in the visible burrow system (VBS) on the expression of glutamic acid decarboxylase (GAD) 67 and brain-derived neurotropic factor (BDNF) mRNA in forebrain stress circuitry. Male rats in the VBS system form a dominance hierarchy, whereby subordinate males exhibit neuroendocrine and physiological profiles characteristic of chronic exposure to stress. We found that social subordination decreases GAD67 mRNA in the peri-paraventricular nucleus region of the hypothalamus and the interfascicular nucleus of the bed nucleus of the stria terminalis (BNST), and increases in GAD67 mRNA in the hippocampus, medial prefrontal cortex, and dorsal medial hypothalamus. Expression of BDNF mRNA increased in the dorsal region of the BNST, but remained unchanged in all other regions examined. Results from this study indicate that social subordination is associated with several region-specific alterations in GAD67 mRNA expression in central stress circuits, whereas changes in the expression of BDNF mRNA are limited to the BNST. PMID:26066725

  14. Increased neutrophil adherence and adhesion molecule mRNA expression in endothelial cells during selenium deficiency.

    PubMed

    Maddox, J F; Aherne, K M; Reddy, C C; Sordillo, L M

    1999-05-01

    Leukocyte aggregation and activation on endothelial cells (EC) are important preliminary events in leukocyte migration into tissue and subsequent inflammation. Thus, an increase in leukocyte adherence has the potential to affect inflammatory disease outcome. Selenium (Se) is an integral part of the antioxidant enzyme glutathione peroxidase (GSH-Px) and plays an important role in the maintenance of the redox state of a cell. Se supplementation in the bovine has been shown to improve the outcome of acute mastitis caused by coliform bacteria, in part by enhancing the speed of neutrophil migration into the affected mammary gland. However, the mechanisms by which Se modulates neutrophil migration have not been elucidated. Therefore, an in vitro model of Se deficiency in primary bovine mammary artery EC was used to examine the impact of Se status on the adhesive properties of EC. The effect of Se on functional activities was examined by measuring neutrophil adherence to Se-deficient and Se-supplemented EC. Se-deficient EC showed significantly enhanced neutrophil adherence when stimulated with tumor necrosis factor alpha (TNF-alpha) for 4 or 24 h, interleukin-1 for 12 h, or H2O2 for 20 min (P < 0.05). To determine the mechanisms underlying these changes in neutrophil adherence, the expression of EC adhesion molecules, ICAM-1, E-selectin, and P-selectin were examined at the molecular level by a competitive reverse transcription-polymerase chain reaction. Results revealed higher mRNA expression for E-selectin and ICAM-1 in Se-deficient EC stimulated with TNF-alpha for 3 and 6 h, and greater expression of P-selectin mRNA in Se-supplemented EC with 3-h TNF-alpha stimulation. These studies provide new information to establish the role of Se nutrition in the initiation of leukocyte adherence to endothelium. PMID:10331495

  15. Expression analysis of mRNA in formalin-fixed, paraffin-embedded archival tissues by mRNA in situ hybridization.

    PubMed

    Henke, Ralf T; Eun Kim, Sung; Maitra, Anirban; Paik, Soonmyung; Wellstein, Anton

    2006-04-01

    Gene expression in diseased tissues can indicate the contribution to a disease process and potentially guide therapeutic decision-making. Archival tissues with associated clinical outcome may be useful to discover or validate the role of a candidate gene in a disease process or the response to therapy. Such archival tissues are commonly formalin-fixed and paraffin-embedded, restricting the methods available for gene expression analysis. Obviously, the detection of proteins in tissues requires adaptation for each protein and the detection of secreted proteins can prove difficult or of reduced value since the protein detected may not reflect the total amount produced. Thus, we describe here a reliable method for the detection of mRNA in archival tissues. The method for mRNA in situ hybridization (ISH) was adapted by us for >15 different genes and applied to several hundred tissue microarrays (TMAs) and full sections generating >10,000 expression data points. We also discuss the utility of TMAs to simultaneously analyze several hundred tissue samples on one slide to minimize variability and preserve valuable tissue samples. Experimental protocols are provided that can be implemented without major hurdles in a typical molecular pathology laboratory and we discuss quantitative analysis as well as advantages and limitations of ISH with a special focus on secreted proteins. We conclude that ISH is a reliable and cost effective approach to gene expression analysis in archival tissues that is amenable to screening of series of tissues or of genes of interest.

  16. Oncogenic kinase NPM/ALK induces expression of HIF1α mRNA.

    PubMed

    Marzec, M; Liu, X; Wong, W; Yang, Y; Pasha, T; Kantekure, K; Zhang, P; Woetmann, A; Cheng, M; Odum, N; Wasik, M A

    2011-03-17

    The mechanisms of malignant cell transformation mediated by the oncogenic anaplastic lymphoma kinase (ALK) tyrosine kinase remain only partially understood. In this study, we report that T-cell lymphoma (TCL) cells carrying the nucleophosmin (NPM)/ALK fusion protein (ALK+ TCL) strongly express hypoxia-induced factor 1α (HIF1α) mRNA, even under normoxic conditions, and markedly upregulate HIF1α protein expression under hypoxia. HIF1α expression is strictly dependent on the expression and enzymatic activity of NPM/ALK, as shown in BaF3 cells transfected with wild-type NPM/ALK and kinase-inactive NPM/ALK K210R mutant and by the inhibition of the NPM/ALK function in ALK+ TCL cells by a small-molecule ALK inhibitor. NPM/ALK induces HIF1α expression by upregulating its gene transcription through its key signal transmitter signal transducer and activator of transcription 3 (STAT3), which binds to the HIF1α gene promoter as shown by the chromatin immunoprecipitation assay and is required for HIF1α gene expression as demonstrated by its small interfering RNA-mediated depletion. In turn, depletion of HIF1α increases mammalian target of rapamycin complex 1 activation, cell growth and proliferation and decreases vascular endothelial growth factor synthesis. These results identify a novel cell-transforming property of NPM/ALK, namely its ability to induce the expression of HIF1α, a protein with an important role in carcinogenesis. These results also provide another rationale to therapeutically target NPM/ALK and STAT3 in ALK+ TCL.

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

    PubMed Central

    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 examined. Two of these had little if any XDH mRNA, but the third mutant had 1.3 times more XDH mRNA than wild type flies. Another mutant, ry2 , which contains no XDH protein and has a 9KB transposable element inserted into the XDH gene, has normal levels of XDH mRNA transcripts which are also the same size as those found in the wild type strain. Changes in XDH mRNA levels were measured during Drosophila development and found to parallel changes in the amount of XDH protein. In addition, there were no large changes in the size of XDH mRNA during development. Images PMID:6588363

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

    EPA Science Inventory

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

  19. Expression of vascular endothelial growth factor mRNA in non-small-cell lung carcinomas.

    PubMed

    Fontanini, G; Boldrini, L; Chinè, S; Pisaturo, F; Basolo, F; Calcinai, A; Lucchi, M; Mussi, A; Angeletti, C A; Bevilacqua, G

    1999-01-01

    The vascular endothelial growth factor (VEGF) has been shown to be strictly related to vascular permeability and endothelial cell growth under physiological and pathological conditions. In tumour development and progression, VEGF plays a pivotal role in the development of the tumoral vascular network, and useful information in the progression of human cancer can be obtained by analysing the vascular endothelial growth factor expression of the tumours. In this study, we investigated the vascular endothelial growth factor transcript expression in non-small-cell lung carcinomas to evaluate the significance of this factor in a group of cancers in which the vascular pattern has been shown to significantly affect progression. Surgical samples of 42 patients with NSCLC were studied using reverse transcription polymerase chain reaction (PCR) analysis and in situ hybridization. Thirty-three out of 42 cases (78.6%) showed VEGF transcript expression predominantly as transcripts for the secretory forms of VEGF (isoforms 121 and 165). In situ hybridization, performed on 24 out of 42 samples, showed that the VEGF transcript expression was in several cases present in the cytoplasm both of neoplastic and normal cells, even if the VEGF mRNA was less expressed in the corresponding non-tumoral part. The VEGF 121 expression was associated with hilar and/or mediastinal nodal involvement (P = 0.02), and, taken together, the VEGF isoforms were shown to significantly influence overall (P = 0.02) and disease-free survival (P = 0.03). As a regulator of tumour angiogenesis, VEGF may represent a useful indicator of progression and poor prognosis in non-small-cell lung carcinomas.

  20. [Effects of propofol, etomidate and ethanol on GPCR mRNA expression in Daphnia pulex ].

    PubMed

    Hu, Anmin; Dong, Changhong; Zuo, Yunxia; Li, Guohua

    2014-08-01

    The mechanisms of general anesthesia, which was introduced about 170 years ago, remain poorly under- stood. Even less well understood are the effects of general anesthesia on the human body. Recently we identified 18 G-protein coupled receptor (GPCR) genes of Daphnia pulex, an invertebrate model organism. Phylogenetic analysis identified these genes to be the homologs of the human γ-aminobutyric acid, type B (GABAB) receptor, metabotropic glutamate receptors (mGluR), adrenergic receptor, serotonin (5-HT) receptor, dopamine receptor and muscarinic acetylcholine receptor (mAChR). Using reverse transcription and quantitative PCR techniques, we systematically measured the effects of propofol, etomidate and ethanol on these 18 GPCR mRNA expressions in Daphnia pulex.

  1. Successful personalized chemotherapy for metastatic gastric cancer based on quantitative BRCA1 mRNA expression level: A case report

    PubMed Central

    HUANG, YING; WU, PUYUAN; LIU, BAORUI; DU, JUAN

    2016-01-01

    Personalized chemotherapy is based on the specific genetic profile of individual patients and is replacing the traditional ‘one size fits all’ medicine. Breast cancer 1 (BRCA1) plays a central role in the chemotherapy-induced DNA damage response. It has been repeatedly demonstrated that BRCA1 mRNA levels were negatively associated with cisplatin sensitivity, but positively associated with docetaxel sensitivity in patients with gastric cancer in experimental and clinical studies. This feature leads to customized chemotherapy based on the BRCA1 mRNA expression level and results in a high efficacy of treatment. The present study describes the case of a 77-year-old patient with metastatic gastric cancer who was treated with personalized chemotherapy based on quantitative BRCA1 mRNA expression level. This study and the available literature data suggest that the expression level of BRCA1 mRNA is dynamic to BRCA1-based chemotherapy. More importantly, de novo assessment of BRCA1 status is a preferable option for ciscisplatin- or docetaxel-resistant patients, since the expression levels of BRCA1 mRNA in certain patients may alter significantly following treatment. Therefore, BRCA1 expression should be assessed for predicting differential chemosensitivity and tailoring chemotherapy in gastric cancer. PMID:27313763

  2. Design and synthesis of human ABCB1 (P-glycoprotein) inhibitors by peptide coupling of diverse chemical scaffolds on carboxyl and amino termini of (S)-valine-derived thiazole amino acid.

    PubMed

    Singh, Satyakam; Prasad, Nagarajan Rajendra; Chufan, Eduardo E; Patel, Bhargav A; Wang, Yi-Jun; Chen, Zhe-Sheng; Ambudkar, Suresh V; Talele, Tanaji T

    2014-05-22

    P-glycoprotein (P-gp) serves as a therapeutic target for the development of multidrug resistance reversal agents. In this study, we synthesized 21 novel compounds by peptide coupling at corresponding carboxyl and amino termini of (S)-valine-based bis-thiazole and monothiazole derivatives with diverse chemical scaffolds. Using calcein-AM efflux assay, we identified compound 28 (IC50 = 1.0 μM) carrying 3,4,5-trimethoxybenzoyl and 2-aminobenzophenone groups, respectively, at the amino and carboxyl termini of the monothiazole zwitter-ion. Compound 28 inhibited the photolabeling of P-gp with [(125)I]-iodoarylazidoprazosin with IC50 = 0.75 μM and stimulated the basal ATP hydrolysis of P-gp in a concentration-dependent manner (EC50 ATPase = 0.027 μM). Compound 28 at 3 μM reduced resistance in cytotoxicity assay to paclitaxel in P-gp-expressing SW620/Ad300 and HEK/ABCB1 cell lines. Biochemical and docking studies showed site-1 to be the preferable binding site for 28 within the drug-binding pocket of human P-gp.

  3. Design and synthesis of human ABCB1 (P-glycoprotein) inhibitors by peptide coupling of diverse chemical scaffolds on carboxyl and amino termini of (S)-valine-derived thiazole amino acid.

    PubMed

    Singh, Satyakam; Prasad, Nagarajan Rajendra; Chufan, Eduardo E; Patel, Bhargav A; Wang, Yi-Jun; Chen, Zhe-Sheng; Ambudkar, Suresh V; Talele, Tanaji T

    2014-05-22

    P-glycoprotein (P-gp) serves as a therapeutic target for the development of multidrug resistance reversal agents. In this study, we synthesized 21 novel compounds by peptide coupling at corresponding carboxyl and amino termini of (S)-valine-based bis-thiazole and monothiazole derivatives with diverse chemical scaffolds. Using calcein-AM efflux assay, we identified compound 28 (IC50 = 1.0 μM) carrying 3,4,5-trimethoxybenzoyl and 2-aminobenzophenone groups, respectively, at the amino and carboxyl termini of the monothiazole zwitter-ion. Compound 28 inhibited the photolabeling of P-gp with [(125)I]-iodoarylazidoprazosin with IC50 = 0.75 μM and stimulated the basal ATP hydrolysis of P-gp in a concentration-dependent manner (EC50 ATPase = 0.027 μM). Compound 28 at 3 μM reduced resistance in cytotoxicity assay to paclitaxel in P-gp-expressing SW620/Ad300 and HEK/ABCB1 cell lines. Biochemical and docking studies showed site-1 to be the preferable binding site for 28 within the drug-binding pocket of human P-gp. PMID:24773054

  4. Effects of vitexin on the pharmacokinetics and mRNA expression of CYP isozymes in rats.

    PubMed

    Wang, Xin-shuai; Hu, Xiao-chen; Chen, Gui-ling; Yuan, Xiang; Yang, Rui-na; Liang, Shuo; Ren, Jing; Sun, Jia-chun; Kong, Guo-qiang; Gao, She-gan; Feng, Xiao-shan

    2015-03-01

    In traditional therapy with Chinese medicine, vitexin has several pharmacological properties, including antinociceptive, antispasmodic, antioxidant, antimyeloperoxidase, and α-glucosidase inhibitory activities. Recently, vitexin was shown to protect the heart against ischemia/reperfusion injury in an in vitro model by inhibiting apoptosis. The purpose of this study was to find out whether vitexin influences the effect on rat cytochrome P450 (CYP) enzymes (CYP1A2, CYP2C11, and CYP3A1) by using cocktail probe drugs in vivo; the influence on the levels of CYP mRNA was also studied. A cocktail solution at a dose of 5 mL/kg, which contained phenacetin (10 mg/kg), tolbutamide (1 mg/kg), and midazolam (5 mg/kg), was given as oral administration to rats treated with short or long period of intravenous vitexin via the caudal vein. Blood samples were collected at a series of time points, and the concentrations of probe drugs in plasma were determined by HPLC-mass spectrometry (MS)/MS. The corresponding pharmacokinetic parameters were calculated by the software of DAS 2.0. In addition, real-time reverse transcription-polymerase chain reaction was performed to determine the effects of vitexin on the mRNA expression of CYP1A2, CYP2C11, and CYP3A1 in rat liver. Treatment with short or long period of vitexin had no effects on rat CYP1A2. However, CYP3A1 enzyme activity was inhibited by vitexin in a concentration-dependent and time-dependent manner. Furthermore, CYP2C11 enzyme activity was induced after short period treatment but inhibited after long period of vitexin treatment. The mRNA expression results were in accordance with the pharmacokinetic results. In conclusion, vitexin can either inhibit or induce activities of CYP2C11 and CYP3A1. Therefore, caution is needed when vitexin is co-administered with some CYP2C11 or CYP3A1 substrates in clinic, which may result in treatment failure and herb-drug interactions.

  5. Translational control of germ cell-expressed mRNA imposed by alternative splicing: opioid peptide gene expression in rat testis.

    PubMed Central

    Garrett, J E; Collard, M W; Douglass, J O

    1989-01-01

    The three genes encoding the opioid peptide precursors (prodynorphin, proenkephalin, and proopiomelanocortin) are expressed in the rat testis. The sizes of the three opioid mRNAs in the testis differ from the sizes of the corresponding mRNAs in other rat tissues in which these genes are expressed. The smaller testicular proopiomelanocortin mRNA has previously been demonstrated to arise from alternative transcriptional initiation. In the present study, we found that the smaller testicular prodynorphin mRNA, expressed in Sertoli cells, results from alternative mRNA processing. Exon 2, which makes up 5' untranslated sequence, is removed from the mature transcript. Polysome analysis of brain and testis RNA indicates that the alteration of the prodynorphin leader sequence in the testis-specific transcript does not affect the efficiency of translation of this mRNA. The larger testicular proenkephalin transcript, expressed in developing germ cells, also results from alternative mRNA processing. Alternative acceptor site usage in the splicing of intron A results in a germ cell-specific proenkephalin transcript with a 491-nucleotide 5' untranslated leader sequence preceding the preproenkephalin-coding sequence. Polysome analysis indicates that this germ cell-specific proenkephalin mRNA is not efficiently translated. Mechanisms by which alternative mRNA splicing may serve to confer translational regulation upon the testicular proenkephalin transcript are discussed. Images PMID:2573832

  6. Rev-erb beta regulates the Srebp-1c promoter and mRNA expression in skeletal muscle cells

    SciTech Connect

    Ramakrishnan, Sathiya N.; Lau, Patrick; Crowther, Lisa M.; Cleasby, Mark E.; Millard, Susan; Leong, Gary M.; Cooney, Gregory J.; Muscat, George E.O.

    2009-10-30

    The nuclear hormone receptor, Rev-erb beta operates as a transcriptional silencer. We previously demonstrated that exogenous expression of Rev-erb{beta}{Delta}E in skeletal muscle cells increased Srebp-1c mRNA expression. We validated these in vitro observations by injection of an expression vector driving Rev-erb{beta}{Delta}E expression into mouse tibialis muscle that resulted in increased Srebp-1c mRNA expression. Paradoxically, Rev-erb{beta} siRNA expression in skeletal muscle cells repressed Srebp-1c expression, and indicated that Rev-erb{beta} expression was necessary for Srebp-1c expression. ChIP analysis demonstrated that Rev-erb{beta} was recruited to the Srebp-1c promoter. Moreover, Rev-erb{beta} trans-activated the Srebp-1c promoter, in contrast, Rev-erb{beta} efficiently repressed the Rev-erb{alpha} promoter, a previously characterized target gene. Finally, treatment with the Rev-erb agonist (hemin) (i) increased the trans-activation of the Srebp-1c promoter by Rev-erb{beta}; and (ii) increased Rev-erb{beta} and Srebp-1c mRNA expression. These data suggest that Rev-erb{beta} has the potential to activate gene expression, and is a positive regulator of Srebp-1c, a regulator of lipogenesis.

  7. Developmental, regional, and cellular expression of SFT/UbcH5A and DMT1 mRNA in brain.

    PubMed

    Knutson, Mitchell; Menzies, Sharon; Connor, James; Wessling-Resnick, Marianne

    2004-06-01

    Brain iron has marked developmental, regional, and cellular distribution patterns. To characterize better the potential mechanisms for iron transport into and within the brain, we have analyzed expression patterns of two factors: divalent metal transporter 1 (DMT1) and stimulator of Fe transport (SFT). DMT1 is known to participate in brain iron uptake although functional information is lacking. Even less clear is the possible role of SFT, which is related to a member of the ubiquitin-conjugating E2 family UbcH5A, but previous studies have found SFT/Ubc5Ha mRNA expressed abundantly in mouse brain. Like DMT1, SFT function has been implicated in transferrin and nontransferrin-bound iron uptake. Comparative Northern analysis indicates that SFT/UbcH5A mRNA levels are threefold higher in 3-day-old mice than at later ages, whereas levels of DMT1 mRNA do not change. In situ analysis of neonatal mouse brain reveals prominent SFT/UbcH5A mRNA expression in epithelial and ependymal cells in the choroid plexus and neurons of the olfactory bulb, hippocampus, and cortex. Adult mouse brain expresses SFT/UbcH5A mRNA mainly in white matter of the cerebellum and pons. Using a multiple tissue expression (MTE) array containing 20 different human brain regions, the highest levels of both SFT/UbcH5A and DMT1 mRNA are detected in the corpus callosum and cerebellum. The significantly elevated levels of SFT/UbcH5A mRNA in the neonatal mouse and its localization to choroid plexus, a major site of brain iron acquisition, suggest that this factor may contribute to the rapid rate of brain iron uptake that occurs in the early postnatal period.

  8. Angiopoietin-2 mRNA expression is increased in chronic lymphocytic leukemia patients with poor prognostic features.

    PubMed

    Vrbacky, F; Smolej, L; Vroblova, V; Pekova, S; Hrudkova, M; Cervinka, M; Pecka, M; Krejsek, J; Maly, J

    2010-08-01

    Several studies have demonstrated the potential prognostic importance of angiogenesis in chronic lymphocytic leukemia (CLL). Elevated expression of angiopoietin-2 (Ang-2), an angiogenic cytokine, was recently reported in CLL. However, data regarding prognostic significance of Ang-2 in CLL are limited. Therefore, we quantitated Ang-2 mRNA in purified mononuclear cells of 33 untreated CLL patients and compared the transcript levels to traditional as well as modern prognostic factors in patients with CLL (clinical stage, disease course, IgVH mutation status, CD38, and ZAP-70 expression). Elevated Ang-2 mRNA concentrations were detected in 12 cases; 21 patients had very low or undetectable levels of Ang-2 transcript. There was significant association between high Ang-2 mRNA levels and unmutated IgVH genes (n=27, P=0.010) and with CD38 expression (n=32, P=0.011), but not with ZAP-70 expression (n=32, P=0.784), Rai stage (n=33, P=0.305) or stable versus progressive clinical course (n=33, P=0.443). There was a trend towards shorter progression-free survival in patients with high Ang-2 expression; however, it did not reach statistical significance (P=0.090). Our pilot data show that Ang-2 mRNA is differentially expressed in patients with CLL and its increased expression appears to be associated with poor prognostic features. Further studies are needed to confirm the results in a larger patient cohort.

  9. Influence of cardiorespiratory fitness on PPARG mRNA expression using monozygotic twin case control.

    PubMed

    Queiroga, Marcos Roberto; Barbieri, Ricardo Augusto; Ferreira, Sandra Aires; Luchessi, André Ducati; Hirata, Rosario Dominguez Crespo; Hirata, Mario Hiroyuki; Kokubun, Eduardo

    2015-01-01

    The influence of cardiorespiratory fitness (VO2max) on anthropometric variables and PPARG mRNA expression was investigated. Monozygotic twin pairs aged 11-18 years were grouped into discordant (D) and concordant (C) high and low VO2max groups. VO2max was determined by progressive maximal exercise test on treadmill with gas exchange analysis. Body mass (BM), BMI, waist circumference (WC), triceps (TR), and subscapular (SB) skinfold thicknesses were measured. Twins from the discordant group had differences in VO2max values (D-high = 45.9 ± 10.0 versus D-low = 32.4 ± 10.6 mL·kg(-1)·min(-1), P = 0.025), while no differences were found in the concordant group (C-high = 42.4 ± 9.2 versus C-low = 38.8 ± 9.8 mL·kg(-1)·min(-1), P = 0.952). In discordant group, VO2max was negatively correlated with TR + SB (r = -0.540, P = 0.021) and positively correlated with PPARG expression in leukocytes (r = 0.952, P = 0.001). Moreover, PPARG expression was directly correlated with BM (r = 0.714, P = 0.047) and height (r = 0.762, P = 0.028). In concordant twins, VO2max was inversely correlated with BM (r = -0.290, P = 0.027), BMI (r = -0.472, P = 0.001), WC (r = -0.426, P = 0.001), and TR + SB (r = -0.739, P = 0.001). Twins D-high had 1.78-fold greater PPARG expression when compared with twins D-low (P = 0.048). In conclusion, the cardiorespiratory fitness may modulate PPARG expression in childhood and adolescence, independently of the genetic background. PMID:25879043

  10. Expression of neuropeptide receptor mRNA during osteoblastic differentiation of mouse iPS cells.

    PubMed

    Nagao, Satomi; Goto, Tetsuya; Kataoka, Shinji; Toyono, Takashi; Joujima, Takaaki; Egusa, Hiroshi; Yatani, Hirofumi; Kobayashi, Shigeru; Maki, Kenshi

    2014-12-01

    Various studies have shown a relationship between nerves and bones. Recent evidence suggests that both sensory and sympathetic nerves affect bone metabolism; however, little is known about how neuropeptides are involved in the differentiation of pluripotent stem cells into osteoblastic (OB) cells. To evaluate the putative effects of neuropeptides during the differentiation of mouse induced pluripotent stem (iPS) cells into calcified tissue-forming OB cells, we investigated the expression patterns of neuropeptide receptors at each differentiation stage. Mouse iPS cells were seeded onto feeder cells and then transferred to low-attachment culture dishes to form embryoid bodies (EBs). EBs were cultured for 4 weeks in osteoblastic differentiation medium. The expression of α1-adrenergic receptor (AR), α2-AR, β2-AR, neuropeptide Y1 receptor (NPY1-R), neuropeptide Y2 receptor (NPY2-R), calcitonin gene-related protein receptor (CGRP-R), and neurokinin 1-R (NK1-R) was assessed by reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR. Among these neuropeptide receptors, CGRP-R and β2-AR were expressed at all stages of cell differentiation, including the iPS cell stage, with peak expression occurring at the early osteoblastic differentiation stage. Another sensory nervous system receptor, NK1-R, was expressed mainly in the late osteoblastic differentiation stage. Furthermore, CGRP-R mRNA showed an additional small peak corresponding to EBs cultured for 3 days, suggesting that EBs may be affected by serum CGRP. These data suggest that the sensory nervous system receptor CGRP-R and the sympathetic nervous system receptor β2-AR may be involved in the differentiation of iPS cells into the osteoblastic lineage. It follows from these findings that CGRP and β2-AR may regulate cell differentiation in the iPS and EB stages, and that each neuropeptide has an optimal period of influence during the differentiation process. PMID:25464890

  11. Influence of Cardiorespiratory Fitness on PPARG mRNA Expression Using Monozygotic Twin Case Control

    PubMed Central

    Queiroga, Marcos Roberto; Barbieri, Ricardo Augusto; Ferreira, Sandra Aires; Luchessi, André Ducati; Hirata, Rosario Dominguez Crespo; Hirata, Mario Hiroyuki; Kokubun, Eduardo

    2015-01-01

    The influence of cardiorespiratory fitness (VO2max) on anthropometric variables and PPARG mRNA expression was investigated. Monozygotic twin pairs aged 11–18 years were grouped into discordant (D) and concordant (C) high and low VO2max groups. VO2max was determined by progressive maximal exercise test on treadmill with gas exchange analysis. Body mass (BM), BMI, waist circumference (WC), triceps (TR), and subscapular (SB) skinfold thicknesses were measured. Twins from the discordant group had differences in VO2max values (D-high = 45.9 ± 10.0 versus D-low = 32.4 ± 10.6 mL·kg−1·min−1, P = 0.025), while no differences were found in the concordant group (C-high = 42.4 ± 9.2 versus C-low = 38.8 ± 9.8 mL·kg−1·min−1, P = 0.952). In discordant group, VO2max was negatively correlated with TR + SB (r = −0.540, P = 0.021) and positively correlated with PPARG expression in leukocytes (r = 0.952, P = 0.001). Moreover, PPARG expression was directly correlated with BM (r = 0.714, P = 0.047) and height (r = 0.762, P = 0.028). In concordant twins, VO2max was inversely correlated with BM (r = −0.290, P = 0.027), BMI (r = −0.472, P = 0.001), WC (r = −0.426, P = 0.001), and TR + SB (r = −0.739, P = 0.001). Twins D-high had 1.78-fold greater PPARG expression when compared with twins D-low (P = 0.048). In conclusion, the cardiorespiratory fitness may modulate PPARG expression in childhood and adolescence, independently of the genetic background. PMID:25879043

  12. Development x environment interactions control tph2 mRNA expression

    PubMed Central

    Lukkes, Jodi L.; Kopelman, Jared M.; Donner, Nina C.; Hale, Matthew W.; Lowry, Christopher A.

    2013-01-01

    Adverse early life experience is thought to increase an individual's susceptibility to mental health disorders, including anxiety and affective disorders, later in life. Our previous studies have shown that post-weaning social isolation of female rats during a critical period of development sensitizes an anxiety-related serotonergic dorsal raphe nucleus (DR) system in adulthood. Therefore, we investigated how post-weaning social isolation, in combination with a challenge with the anxiogenic drug, N-methyl-beta-carboline-3-carboxamide (FG-7142; a partial inverse agonist at the benzodiazepine allosteric site on the GABAA receptor), affects home cage behavior and serotonergic gene expression in the DR of female rats using in situ hybridization histochemistry. Juvenile female rats were reared in isolation or groups of three for a 3-week period from weaning (postnatal day (PD) 21 to mid-adolescence (PD42)), after which all rats were group-reared for an additional 16 days until adulthood. Among vehicle-treated rats, isolation-reared rats had decreased tryptophan hydroxylase 2 (tph2) mRNA expression in ventral and ventrolateral subdivisions of the DR, a pattern observed previously in a rat model of panic disorder. Isolation-reared rats, but not group-reared rats, responded to FG-7142 with increased duration of vigilance and arousal behaviors. In addition, FG-7142 decreased tph2 expression, measured 4 h following treatment, in multiple subregions of the DR of group-reared rats but had no effect in isolation-reared rats. No treatment effects were observed on 5-HT1A receptor or serotonin transporter gene expression. These data suggest that adolescent social isolation alters tph2 expression in specific subregions of the DR and alters the effects of stress-related stimuli on behavior and serotonergic systems. PMID:23403177

  13. Corticosterone effects on BDNF mRNA expression in the rat hippocampus during morris water maze training.

    PubMed

    Schaaf, M J; Sibug, R M; Duurland, R; Fluttert, M F; Oitzl, M S; De Kloet, E R; Vreugdenhil, E

    1999-12-01

    Corticosterone and Brain-Derived Neurotrophic Factor (BDNF) have both been shown to be involved in spatial memory formation in rats. In the present study we have investigated the effect of corticosterone on hippocampal BDNF mRNA expression after training in the Morris water maze in young adult Wistar rats. Therefore, we first studied BDNF mRNA levels in the hippocampus in relation to corticosterone levels at several time points after 4 training trials in the Morris water maze. Corticosterone levels were significantly increased after this procedure, and hippocampal BDNF mRNA levels only displayed a minor change: an increase in CA1 at 1 hr after training. However, in a previous study we observed dramatically decreased hippocampal BDNF mRNA levels in dentate gyrus and CA1 at 3 hr after injection of corticosterone. In order to analyze this discrepancy, we subsequently investigated if hippocampal BDNF mRNA expression is affected by corticosterone at 3 hr after water maze training. Therefore, we incorporated ADX animals and ADX animals which were injected with corticosterone in our study. ADX animals which were subjected to water maze training displayed similar hippocampal BDNF mRNA levels 3 hr after training compared to control ADX animals. Furthermore, ADX animals which were injected with corticosterone showed decreased BDNF mRNA levels in all hippocampal regions compared to control ADX animals. Water maze training did not alter this effect. Thus, the increased corticosterone levels during water maze training do not affect hippocampal BDNF mRNA expression, although exogenous corticosterone is effective under these conditions. Hence, our results suggest that in this situation BDNF is resistant to regulation by endogenous corticosterone, which may be important for learning and memory processes.

  14. Effects of social isolation on mRNA expression for corticotrophin-releasing hormone receptors in prairie voles

    PubMed Central

    Pournajafi-Nazarloo, Hossein; Partoo, Leila; Yee, Jason; Stevenson, Jennifer; Sanzenbacher, Lisa; Kenkel, William; Mohsenpour, Seyed Ramezan; Hashimoto, Kozo; Carter, C. Sue

    2011-01-01

    Summary Previous studies have demonstrated that various type of stressors modulate messenger ribonucleic acid (mRNA) for type 1 corticotropin-releasing hormone (CRH) receptor (CRH-R1 mRNA) and type 2 CRH receptor (CRH-R2 mRNA). The purpose of this study was to explore the effect of social isolation stress of varying durations on the CRH, CRH-R1 and CRH-R2 mRNAs expression in the hypothalamus, hippocampus and pituitary of socially monogamous female and male prairie voles (Microtus ochrogaster). Isolation for 1 hr (single isolation) or 1 hr of isolation every day for 4 weeks (repeated isolation) was followed by a significant increase in plasma corticosterone levels. Single or repeated isolation increased hypothalamic CRH mRNA expression, but no changes in CRH-R1 mRNA in the hypothalamus were observed. Continuous isolation for 4 weeks (chronic isolation) showed no effect on hypothalamic CRH or CRH-R1 mRNAs in female or male animals. However, hypothalamic CRH-R2 mRNA was significantly reduced in voles exposed to chronic isolation. Single or repeated isolation, but not chronic isolation, significantly increased CRH-R1 mRNA and decreased CRH-R2 mRNA in the pituitary. Despite elevated CRH mRNA expression, CRH-R1 and CRH-R2 mRNAs were not modulated in the hippocampus following single or repeated isolation. Although, chronic isolation did not affect hippocampal CRH or CRH-R1 mRNAs, it did increase CRH-R2 mRNA expression in females and males. The results of the present study in prairie voles suggest that social isolation has receptor subtype and species-specific consequences for the modulation of gene expression for CRH and its receptors in brain and pituitary. Previous studies have revealed a female-biased increase in oxytocin in response to chronic isolation; however, we did not find a sex difference in CRH or its receptors following single, repeated or chronic social isolation, suggesting that sexually-dimorphic processes beyond the CRH system, possibly involving

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

    PubMed

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

    1992-05-25

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

  16. KIF14 and E2F3 mRNA expression in human retinoblastoma and its phenotype association

    PubMed Central

    Mitra, Moutushy; Mallikarjuna, Kandalam; Pranav, Oberoi; Srinivasan, Ramalingam; Nagpal, Amit; Venkatesan, Perumal; Kumaramanickavel, Govindasamy

    2009-01-01

    Purpose We quantified mRNA expression of candidate genes for proliferation (KIF14 and E2F3) in a large retinoblastoma tumor cohort and associated with disease phenotype. Methods KIF14 and E2F3 mRNA expression was quantified by real time PCR in 57 retinoblastoma (RB) tumors, 3 RB cell lines, and control samples that included 4 each fetal, age-matched, adult retinas. Immunohistochemistry was done to confirm KIF14 and E2F3 protein expression in tumor cells. The mRNA expression levels were correlated with disease phenotypes including the significance of chemotherapy on tumors. Results There was statistically significant overexpression of KIF14 and E2F3 mRNA in tumors compared with control retinas (p<0.0001). Further, E2F3 also showed a significant overexpression compared to RB cell lines (p=0.01). Immunohistochemistry confirmed KIF14 and E2F3 protein overexpression in tumor cells. KIF14 had significant mRNA overexpression with older age (p=0.01) in presenting patients and in unilateral RB patients (p=0.04). Chemotherapy-treated tumors showed a significant decrease in KIF14 and E2F3 expression compared to untreated tumors (p<0.01 and 0.001, respectively). Conclusions This report confirms significant mRNA overexpression of KIF14 and E2F3 together in a large cohort of RB tumors. The decreased expression in chemotherapy treated cases needs further validation in a large chemotherapy-treated cohort. PMID:19190782

  17. Dynamics of c-fos and ICER mRNA expression in rat forebrain following lithium chloride injection.

    PubMed

    Spencer, C M; Houpt, T A

    2001-09-30

    Lithium is commonly used as a treatment for affective disorders in humans and as a toxin to produce conditioned taste aversions in rats. LiCl administration in rats has been correlated with activation of c-fos and cAMP-mediated gene transcription in many brain regions; however, little is known about the timing or duration of gene activation. We hypothesized that c-fos gene transcription is rapidly stimulated by LiCl, followed later by the expression of the inducible cAMP early repressor (ICER) transcription factor, a negative modulator of cAMP-mediated gene transcription. By in situ hybridization, we analyzed the timecourse of c-fos and ICER mRNA expression in the central nucleus of the amygdala (CeA), the paraventricular nucleus of the hypothalamus (PVN) and the supraoptic nucleus (SON) at seven time points (0, 0.3, 1, 3, 6, 9 and 12 h) after intraperitoneal LiCl injection (0.15 M, 12 ml/kg, 76 mg/kg). Expression of c-fos mRNA peaked between 20 min and 1 h and returned to baseline by 3 h in the CeA, PVN and SON. ICER mRNA was detected in these regions at 20 min, peaked at 1-3 h and returned to nearly baseline 9 h following LiCl injection. The time lag between c-fos mRNA expression and ICER mRNA expression within the same regions is consistent with ICER terminating c-fos gene transcription. However, no refractory period was detected for restimulation of c-fos transcription by a second injection of LiCl during the period of peak ICER mRNA expression, suggesting the involvement of other transcriptional modulators. PMID:11589989

  18. Contributions of transcription and mRNA decay to gene expression dynamics of fission yeast in response to oxidative stress

    PubMed Central

    Marguerat, Samuel; Lawler, Katherine; Brazma, Alvis; Bähler, Jürg

    2014-01-01

    The cooperation of transcriptional and post-transcriptional levels of control to shape gene regulation is only partially understood. Here we show that a combination of two simple and non-invasive genomic techniques, coupled with kinetic mathematical modeling, affords insight into the intricate dynamics of RNA regulation in response to oxidative stress in the fission yeast Schizosaccharomyces pombe. This study reveals a dominant role of transcriptional regulation in response to stress, but also points to the first minutes after stress induction as a critical time when the coordinated control of mRNA turnover can support the control of transcription for rapid gene regulation. In addition, we uncover specialized gene expression strategies associated with distinct functional gene groups, such as simultaneous transcriptional repression and mRNA destabilization for genes encoding ribosomal proteins, delayed mRNA destabilization with varying contribution of transcription for ribosome biogenesis genes, dominant roles of mRNA stabilization for genes functioning in protein degradation, and adjustment of both transcription and mRNA turnover during the adaptation to stress. We also show that genes regulated independently of the bZIP transcription factor Atf1p are predominantly controlled by mRNA turnover, and identify putative cis-regulatory sequences that are associated with different gene expression strategies during the stress response. This study highlights the intricate and multi-faceted interplay between transcription and RNA turnover during the dynamic regulatory response to stress. PMID:25007214

  19. The Drosophila Tis11 Protein and Its Effects on mRNA Expression in Flies*

    PubMed Central

    Choi, Youn-Jeong; Lai, Wi S.; Fedic, Robert; Stumpo, Deborah J.; Huang, Weichun; Li, Leping; Perera, Lalith; Brewer, Brandy Y.; Wilson, Gerald M.; Mason, James M.; Blackshear, Perry J.

    2014-01-01

    Members of the mammalian tristetraprolin family of CCCH tandem zinc finger proteins can bind to certain AU-rich elements (AREs) in mRNAs, leading to their deadenylation and destabilization. Mammals express three or four members of this family, but Drosophila melanogaster and other insects appear to contain a single gene, Tis11. We found that recombinant Drosophila Tis11 protein could bind to ARE-containing RNA oligonucleotides with low nanomolar affinity. Remarkably, co-expression in mammalian cells with “target” RNAs demonstrated that Tis11 could promote destabilization of ARE-containing mRNAs and that this was partially dependent on a conserved C-terminal sequence resembling the mammalian NOT1 binding domain. Drosophila Tis11 promoted both deadenylation and decay of a target transcript in this heterologous cell system. We used chromosome deletion/duplication and P element insertion to produce two types of Tis11 deficiency in adult flies, both of which were viable and fertile. To address the hypothesis that Tis11 deficiency would lead to the abnormal accumulation of potential target transcripts, we analyzed gene expression in adult flies by deep mRNA sequencing. We identified 69 transcripts from 56 genes that were significantly up-regulated more than 1.5-fold in both types of Tis11-deficient flies. Ten of the up-regulated transcripts encoded probable proteases, but many other functional classes of proteins were represented. Many of the up-regulated transcripts contained potential binding sites for tristetraprolin family member proteins that were conserved in other Drosophila species. Tis11 is thus an ARE-binding, mRNA-destabilizing protein that may play a role in post-transcriptional gene expression in Drosophila and other insects. PMID:25342740

  20. Level of expression of E-cadherin mRNA in colorectal cancer correlates with clinical outcome.

    PubMed Central

    Dorudi, S.; Hanby, A. M.; Poulsom, R.; Northover, J.; Hart, I. R.

    1995-01-01

    A series of colorectal carcinomas (n = 49) resected from patients with known clinical outcomes were analysed for E-cadherin expression using in situ hybridisation to measure mRNA. Patients surviving 5 years or longer (n = 31) exhibited significantly higher levels of E-cadherin mRNA than those surviving less than 5 years (n = 18, P = 0.003). These preliminary results from this small sample suggest that E-cadherin expression may be a useful prognostic marker in colorectal cancer patients. Images Figure 1 PMID:7880746

  1. Neuropeptide Y in black seabream Acanthopagrus schlegelii: identification, distribution and mRNA expression responses to ghrelin.

    PubMed

    Ma, X L; Zheng, L W; Mao, L T; Zhou, L B; Wang, A L

    2013-04-01

    The coding region of neuropeptide Y (NPY) complementary (c)DNA was cloned from the hypothalamus RNA of black seabream Acanthopagrus schlegelii, including 297 bp coding for prepro-NPY of 98 amino acids. Real-time reverse transcriptase-polymerase chain reaction was performed to determine A. schlegelii npy gene expression; NPY messenger RNA (mRNA) was expressed highly in the brain and stomach. Treatment with acylated ghrelin significantly up-regulated NPY mRNA level in the hypothalamus, suggesting that NPY may be involved in regulating food intake of A. schlegelii.

  2. In vivo and in vitro CYP1B mRNA expression in channel catfish.

    PubMed

    Willett, Kristine L; Ganesan, Shobana; Patel, Monali; Metzger, Christine; Quiniou, Sylvie; Waldbieser, Geoff; Scheffler, Brian

    2006-07-01

    Our goal was to study the induction of CYP1B mRNA expression in channel catfish (Ictalurus punctatus). CYP1B belongs to the cytochrome P450 superfamily of genes, is involved in the oxidation of endogenous and exogenous compounds, and could potentially be a useful biomarker in fish for exposure to AhR ligands. The full-length catfish CYP1B cDNA is 2417 nt to the polyA tail and encodes a putative protein of 536 amino acids. It has 67% amino acid similarity to carp and zebrafish CYP1B and 68% similarity to carp CYP1B2. Male channel catfish were collected from three Mississippi Delta sites: Lake Roebuck, Itta Bena; Bee Lake, Thornton; and Sunflower River, Indianola. Total RNA was isolated from wild-caught catfish gill, blood, gonad and liver tissues. Quantitative real-time reverse transcriptase PCR was used to determine relative induction of CYP1B in wild catfish compared to laboratory control and BaP-exposed catfish (20mg/kg i.p. after 4 days). BaP exposure significantly induced CYP1B message in blood, gonad, and liver of laboratory catfish. In these same tissues of wild catfish from sites with relatively low sediment contaminants, CYP1B message was not statistically increased relative to laboratory control catfish. CYP1B transcript abundance was higher in gills compared to other tissues in both laboratory and wild catfish. When primary cultured gill cells were treated with increasing concentrations of BaP, TCDD, and PCBs 77, 126 and 169, CYP1B mRNA was induced more than 10-fold while PCB153 and 4,4'DDT did not cause significant CYP1B induction. Our results suggest that catfish CYP1B is induced by the classic AhR ligands. PMID:16697458

  3. Dysferlin expression in monocytes: a source of mRNA for mutation analysis.

    PubMed

    De Luna, N; Freixas, A; Gallano, P; Caselles, L; Rojas-García, R; Paradas, C; Nogales, G; Dominguez-Perles, R; Gonzalez-Quereda, L; Vílchez, J J; Márquez, C; Bautista, J; Guerrero, A; Salazar, J A; Pou, A; Illa, I; Gallardo, E

    2007-01-01

    Dysferlin protein is expressed in peripheral blood monocytes. The genomic analysis of the DYSF gene has proved to be time consuming because it has 55 exons. We designed a mutational screening strategy based on cDNA from monocytes to find out whether the mutational analysis could be performed in mRNA from a source less invasive than the muscle biopsy. We studied 34 patients from 23 families diagnosed with dysferlinopathy. The diagnosis was based on clinical findings and on the absence of protein expression using either immunohistochemistry or Western blot of skeletal muscle and/or monocytes. We identified 28 different mutations, 13 of which were novel. The DYSF mutations in both alleles were found in 30 patients and only in one allele in four. The results were confirmed using genomic DNA in 26/34 patients. This is the first report to furnish evidence of reliable mutational analysis using monocytes cDNA and constitutes a good alternative to genomic DNA analysis.

  4. Microarray-based mRNA expression profiling of leukemia cells treated with the flavonoid, casticin.

    PubMed

    Righeschi, Chiara; Eichhorn, Tolga; Karioti, Anastasia; Bilia, Anna Rita; Efferth, Thomas

    2012-01-01

    Natural polyphenols play an important role in tumor inhibition. We used a doxorubicin-sensitive acute T-lymphoblastic leukemia cell line (CCRF-CEM) and its multidrug-resistant subline (CEM/ADR5000) to evaluate the activity of 15 plant polyphenols isolated in our laboratory (hypericin and pseudohypericin, verbascoside, ellagic acid, casticin, kaempferol-3-O-(2'',6''-di-E-p-coumaroyl)-glucopyranoside, kaempferol-3-O-(3,4-diacetyl-2,6-di-E-p-coumaroyl) -glucopyranoside, tiliroside, salvianolic acid B, oleuropein, rosmarinic acid, bergenin) or of others from commercial sources (curcumin, epigallocatechin-3-gallate, silymarin). Casticin was the most potent compound (IC50 values of 0.28 ± 0.02 μM in CCRF-CEM and 0.44 ± 0.17 μM in CEM/ADR5000 cells. The IC50 values of the other compounds tested ranged from 1.52 μM to 164.1 μM. A microarray-based mRNA expression profiling of CCRF-CEM cells treated with casticin was performed in order to identify genes with altered expression following casticin treatment. Networks related to NF-κB, p38MAPK, histones H3 and H4, and follicle stimulating hormone were identified.

  5. Prognostic values of four Notch receptor mRNA expression in gastric cancer

    PubMed Central

    Wu, Xiaoyu; Liu, Wentao; Tang, Ding; Xiao, Haijuan; Wu, Zhenfeng; Chen, Che; Yao, Xuequan; Liu, Fukun; Li, Gang

    2016-01-01

    Notch ligands and receptors are frequently deregulated in several human malignancies including gastric cancer. The activation of Notch signaling has been reported to contribute to gastric carcinogenesis and progression. However, the prognostic roles of individual Notch receptors in gastric cancer patients remain elusive. In the current study, we accessed the prognostic roles of four Notch receptors, Notch 1–4, in gastric cancer patients through “The Kaplan-Meier plotter” (KM plotter) database, in which updated gene expression data and survival information include a total of 876 gastric cancer patients. All four Notch receptors’ high mRNA expression was found to be correlated to worsen overall survival (OS) for all gastric cancer patients followed for 20 years. We further accessed the prognostic roles of individual Notch receptors in different clinicopathological features using Lauren classification, pathological grades, clinical grades, HER2 status and different choices of treatments of gastric cancer patients. These results indicate that there are critical prognostic values of the four Notch receptors in gastric cancer. This information will be useful for better understanding of the heterogeneity and complexity in the molecular biology of gastric cancer and to develop tools to more accurately predict their prognosis. PMID:27363496

  6. Selenium Deficiency Influences the mRNA Expression of Selenoproteins and Cytokines in Chicken Erythrocytes.

    PubMed

    Luan, Yilin; Zhao, Jinxin; Yao, Haidong; Zhao, Xia; Fan, Ruifeng; Zhao, Wenchao; Zhang, Ziwei; Xu, Shiwen

    2016-06-01

    Selenium (Se) deficiency induces hemolysis in chickens, but the molecular mechanism for this effect remains unclear. Se primarily elicits its function through the activity of selenoproteins, which contain the unique amino acid selenocysteine (Sec). In this study, we aimed to investigate the effect of Se deficiency on the expression of 24 selenoproteins and 10 cytokines. One hundred eighty chickens were randomly divided into 2 groups (90 chickens per group). During the entire experimental period, chickens were allowed ad libitum consumption of feed and water. The chickens were fed either a Se-deficient diet (0.008 mg Se/kg; produced in the Se-deficient area of Heilongjiang, China) or a Se-supplemented diet (as sodium selenite) at 0.2 mg/kg for 35 days. At the 35th day, the messenger RNA (mRNA) levels of 24 selenoproteins and 10 cytokines were examined in erythrocytes of 5 chickens per group, and the correlation was analyzed. The results showed that the expression of 24 selenoproteins and 7 cytokines (IL-2, IL-4, IL-8, IL-10, IL-12β, TGF-β4, and IFN-γ) decreased (P < 0.05), and the expression of 3 cytokines (IL-1γ, IL-6 and IL-7) was higher in the Se-deficient group. In both groups, glutathione peroxidase (GPX), thioredoxin 1 (Txnrd1), selenoprotein P1 (SELP), and selenoprotein synthetase (SPS2) were highly expressed compared to the other selenoproteins in chicken erythrocytes (P < 0.05). These data suggest that GPXs, Txnrd1, SELP, and SPS2 possibly play a more important role than the other selenoproteins. The increase of pro-inflammatory cytokines (IL-1γ, IL-6, and IL-7) suggested that the immune system of chickens was damaged by the Se deficiency. Correlation analysis suggested that although the expression of 24 selenoproteins and 7 cytokines decreased and that of 3 cytokines increased, there was a close correlation between their expression levels and a Se diet. These results suggested that Se deficiency influenced the expressions of 24 selenoproteins

  7. Integrated miRNA and mRNA Expression Profiling in Inflamed Colon of Patients with Ulcerative Colitis

    PubMed Central

    Van der Goten, Jan; Vanhove, Wiebe; Lemaire, Katleen; Van Lommel, Leentje; Machiels, Kathleen; Wollants, Willem-Jan; De Preter, Vicky; De Hertogh, Gert; Ferrante, Marc; Van Assche, Gert; Rutgeerts, Paul; Schuit, Frans; Vermeire, Séverine; Arijs, Ingrid

    2014-01-01

    Background Ulcerative colitis (UC) is associated with differential colonic expression of genes involved in immune response (e.g. IL8) and barrier integrity (e.g. cadherins). MicroRNAs (miRNAs) are regulators of gene expression and are involved in various immune-related diseases. In this study, we investigated (1) if miRNA expression in UC mucosa is altered and (2) if any of these changes correlate with mucosal mRNA expression. Integration of mRNA and miRNA expression profiling may allow the identification of functional links between dysregulated miRNAs and their target mRNA. Methodology Colonic mucosal biopsies were obtained from 17 UC (10 active and 7 inactive) patients and 10 normal controls. Total RNA was used to analyze miRNA and mRNA expression via Affymetrix miRNA 2.0 and Affymetrix Human Gene 1.0ST arrays, respectively. Both miRNA and gene expression profiles were integrated by correlation analysis to identify dysregulated miRNAs with their corresponding predicted target mRNA. Microarray data were validated with qRT-PCR. Regulation of IL8 and CDH11 expression by hsa-miR-200c-3p was determined by luciferase reporter assays. Results When comparing active UC patients vs. controls, 51 miRNAs and 1543 gene probe sets gave significantly different signals. In contrast, in inactive UC vs. controls, no significant miRNA expression differences were found while 155 gene probe sets had significantly different signals. We then identified potential target genes of the significantly dysregulated miRNAs and genes in active UC vs. controls and found a highly significant inverse correlation between hsa-miR-200c-3p and IL8, an inflammatory marker, and between hsa-miR-200c-3p and CDH11, a gene related to intestinal epithelial barrier function. We could demonstrate that hsa-miR-200c-3p directly regulates IL8 and CDH11 expression. Conclusion Differential expression of immune- and barrier-related genes in inflamed UC mucosa may be influenced by altered expression of mi

  8. Triptolide inhibits COX-2 expression by regulating mRNA stability in TNF-{alpha}-treated A549 cells

    SciTech Connect

    Sun, Lixin; Zhang, Shuang; Jiang, Zhenzhou; Huang, Xin; Wang, Tao; Huang, Xiao; Li, Han; Zhang, Luyong

    2011-12-09

    Highlights: Black-Right-Pointing-Pointer Triptolide inhibited COX-2 expression and the half-life of COX-2 mRNA is decreased. Black-Right-Pointing-Pointer The HuR protein shuttling from nucleus to cytoplasm is inhibited by triptolide. Black-Right-Pointing-Pointer Triptolide inhibited 3 Prime -UTR fluorescence reporter gene activity. Black-Right-Pointing-Pointer COX-2 mRNA binding to HuR is decreased by triptolide in pull-down experiments. -- Abstract: Cyclooxygenase-2 (COX-2) over-expression is frequently associated with human non-small-cell lung cancer (NSCLC) and involved in tumor proliferation, invasion, angiogenesis and resistance to apoptosis. In the present study, the effects of triptolide on COX-2 expression in A549 cells were investigated and triptolide was found to inhibit TNF-{alpha}-induced COX-2 expression. In our further studies, it was found that triptolide decreased the half-life of COX-2 mRNA dramatically and that it inhibited 3 Prime -untranslated region (3 Prime -UTR) fluorescence reporter gene activity. Meanwhile, triptolide inhibited the HuR shuttling from nucleus to cytoplasm. After triptolide treatment, decreased COX-2 mRNA in pull-down experiments with anti-HuR antibodies was observed, indicating that the decreased cytoplasmic HuR is responsible for the decreased COX-2 mRNA. Taken together, our results provided evidence for the first time that triptolide inhibited COX-2 expression by COX-2 mRNA stability modulation and post-transcriptional regulation. These results provide a novel mechanism of action for triptolide which may be important in the treatment of lung cancer.

  9. Expression of angiotensin-converting enzyme mRNA gene in the kidneys of patients with glomerulonephrites.

    PubMed

    Alnahal, Alsayed Ahmed; Khalil, Usama Ahmed; Diab, Magada Alsayed; Zanaty, Ali Fahmy

    2012-09-01

    A little is known about the behavior of the renin-angiotensin system (RAS) in glomerulo-nephritis (GN), although it is activated in other models of injury. To study renal angiotensin-converting enzyme (ACE) messenger ribonucleic acid (mRNA) gene expression in patients with GN to determine its role in the disease process and other factors that may influence the course of the disease and the prognosis, e.g. treatment with ACE inhibitor (ACEI) drugs, we studied 20 patients with GN allocated to two groups: ten patients received an ACEI drug and ten patients did not receive ACEI in addition to a control group of ten healthy subjects. Routine and special laboratory investigation, histopathological studies and quantitative polymerase chain reaction analysis for renal ACE mRNA were done for both the study and the control groups. There was a statistically significant increase in ACE mRNA gene expression in the GN groups than in control group, but no statistically significant difference in ACE mRNA gene expression between the patients group that received and the group that did not receive ACEI. A significant correlation was found between the ACE mRNA gene expression and the mean blood pressure, serum creatinine, blood urea nitrogen and 24-h urinary protein. In conclusion, a higher level of ACE mRNA gene expression in patients suffering from GN may suggest a role of the RAS in the process of GN, perhaps contributing to glomerular hypertrophy and matrix overproduction. The use of ACEI drugs possibly slows the rate of progression of renal failure and plays a role in controlling the pathophysiology.

  10. Antioxidant enzyme activity and mRNA expression in reproductive tract of adult male European Bison (Bison bonasus, Linnaeus 1758).

    PubMed

    Koziorowska-Gilun, M; Gilun, P; Fraser, L; Koziorowski, M; Kordan, W; Stefanczyk-Krzymowska, S

    2013-02-01

    Antioxidants in the male reproductive tract are the main defence factors against oxidative stress caused by reactive oxygen species production, which compromises sperm function and male fertility. This study was designed to determine the activity of antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), in the testicular and epididymidal tissues of adult male European bison (Bison bonasus). The reproductive tract tissues were subjected to real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis to quantify mRNA expression levels of five antioxidant enzymes: copper/zinc SOD (Cu/Zn SOD), secretory extracellular SOD (Ec-SOD), CAT, phospholipid hydroperoxide glutathione peroxidase (PHGPx) and GPx5. The corpus and cauda epididymidal tissues displayed greater (p < 0.05) SOD activity compared with the testicular tissue. It was found that CAT activity was lowest (p < 0.05) in the cauda epididymidis, whereas negligible GPx activity was detected in the reproductive tract tissues. There were no detectable differences in the mRNA expression level of Cu/Zn SOD among the different reproductive tract tissues. Small amounts of Ec-SOD mRNA were found in the reproductive tract, particularly in the epididymides. The caput and cauda epididymides exhibited greater (p < 0.05) level of CAT mRNA expression, whereas PHGPx mRNA was more (p < 0.05) expressed in the testis. Furthermore, extremely large amounts of GPx5 mRNA were detected in the caput epididymidal tissue compared with other tissues of the reproductive tract. It can be suggested that the activity of the antioxidant enzymes and the relative gene expression of the enzymes confirm the presence of tissue-specific antioxidant defence systems in the bison reproductive tract, which are required for spermatogenesis, epididymal maturation and storage of spermatozoa.

  11. Assessment of potential biomarkers, metallothionein and vitellogenin mRNA expressions in various chemically exposed benthic Chironomus riparius larvae

    NASA Astrophysics Data System (ADS)

    Park, Kiyun; Kwak, Inn-Sil

    2012-12-01

    The objective of this study was conducted to identify the possibility of using Chironomus metallothionein (MT) and vitellogenin (VTG) as biomarkers of stress caused by endocrinedisrupting chemicals (EDCs), heavy metals, herbicides and veterinary antibiotics. We characterized the MT and VTG cDNA in Chironomus riparius and evaluated their mRNA expression profiles following exposure to different environmental pollutants. The gene expression analysis showed that the MT mRNA levels increased significantly after long-term exposure to cadmium (Cd), copper (Cu), Lead (Pb), di(2-ethylhexyl) phthalate (DEHP), and 2,4-dichlorophenoxyacetic acid (2,4-D). Moreover, the VTG mRNA expression increased significantly in C. riparius larvae exposed to BPA, NP, DEHP, Cd, 2,4-D and fenbendazole. Evaluation of the long-term effects of environmental pollutants revealed up regulation of Chironomus MT mRNA in response to DEHP exposure among EDCs, and the level of the VTG mRNA was increased significantly following treatment with Cd and herbicide 2,4-D at all concentrations in a dose-dependent manner. These results indicate that VTG could be used as a potential biomarker of herbicide and Cd as well as EDCs, while MT was a potential biomarker of heavy metals such as Cd, Cu, and Pb in aquatic environments.

  12. Astrocytes drive upregulation of the multidrug resistance transporter ABCB1 (P-Glycoprotein) in endothelial cells of the blood-brain barrier in mutant superoxide dismutase 1-linked amyotrophic lateral sclerosis.

    PubMed

    Qosa, Hisham; Lichter, Jessica; Sarlo, Mark; Markandaiah, Shashirekha S; McAvoy, Kevin; Richard, Jean-Philippe; Jablonski, Michael R; Maragakis, Nicholas J; Pasinelli, Piera; Trotti, Davide

    2016-08-01

    The efficacy of drugs targeting the CNS is influenced by their limited brain access, which can lead to complete pharmacoresistance. Recently a tissue-specific and selective upregulation of the multidrug efflux transporter ABCB1 or P-glycoprotein (P-gp) in the spinal cord of both patients and the mutant SOD1-G93A mouse model of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease that prevalently kills motor neurons has been reported. Here, we extended the analysis of P-gp expression in the SOD1-G93A ALS mouse model and found that P-gp upregulation was restricted to endothelial cells of the capillaries, while P-gp expression was not detected in other cells of the spinal cord parenchyma such as astrocytes, oligodendrocytes, and neurons. Using both in vitro human and mouse models of the blood-brain barrier (BBB), we found that mutant SOD1 astrocytes were driving P-gp upregulation in endothelial cells. In addition, a significant increase in reactive oxygen species production, Nrf2 and NFκB activation in endothelial cells exposed to mutant SOD1 astrocytes in both human and murine BBB models were observed. Most interestingly, astrocytes expressing FUS-H517Q, a different familial ALS-linked mutated gene, also drove NFκB-dependent upregulation of P-gp. However, the pathway was not dependent on oxidative stress but rather involved TNF-α release. Overall, these findings indicated that nuclear translocation of NFκB was a converging mechanism used by endothelial cells of the BBB to upregulate P-gp expression in mutant SOD1-linked ALS and possibly other forms of familial ALS. GLIA 2016 GLIA 2016;64:1298-1313.

  13. BayMiR: inferring evidence for endogenous miRNA-induced gene repression from mRNA expression profiles

    PubMed Central

    2013-01-01

    Background Popular miRNA target prediction techniques use sequence features to determine the functional miRNA target sites. These techniques commonly ignore the cellular conditions in which miRNAs interact with their targets in vivo. Gene expression data are rich resources that can complement sequence features to take into account the context dependency of miRNAs. Results We introduce BayMiR, a new computational method, that predicts the functionality of potential miRNA target sites using the activity level of the miRNAs inferred from genome-wide mRNA expression profiles. We also found that mRNA expression variation can be used as another predictor of functional miRNA targets. We benchmarked BayMiR, the expression variation, Cometa, and the TargetScan “context scores” on two tasks: predicting independently validated miRNA targets and predicting the decrease in mRNA abundance in miRNA overexpression assays. BayMiR performed better than all other methods in both benchmarks and, surprisingly, the variation index performed better than Cometa and some individual determinants of the TargetScan context scores. Furthermore, BayMiR predicted miRNA target sets are more consistently annotated with GO and KEGG terms than similar sized random subsets of genes with conserved miRNA seed regions. BayMiR gives higher scores to target sites residing near the poly(A) tail which strongly favors mRNA degradation using poly(A) shortening. Our work also suggests that modeling multiplicative interactions among miRNAs is important to predict endogenous mRNA targets. Conclusions We develop a new computational method for predicting the target mRNAs of miRNAs. BayMiR applies a large number of mRNA expression profiles and successfully identifies the mRNA targets and miRNA activities without using miRNA expression data. The BayMiR package is publicly available and can be readily applied to any mRNA expression data sets. PMID:24001276

  14. Graph based fusion of miRNA and mRNA expression data improves clinical outcome prediction in prostate cancer

    PubMed Central

    2011-01-01

    Background One of the main goals in cancer studies including high-throughput microRNA (miRNA) and mRNA data is to find and assess prognostic signatures capable of predicting clinical outcome. Both mRNA and miRNA expression changes in cancer diseases are described to reflect clinical characteristics like staging and prognosis. Furthermore, miRNA abundance can directly affect target transcripts and translation in tumor cells. Prediction models are trained to identify either mRNA or miRNA signatures for patient stratification. With the increasing number of microarray studies collecting mRNA and miRNA from the same patient cohort there is a need for statistical methods to integrate or fuse both kinds of data into one prediction model in order to find a combined signature that improves the prediction. Results Here, we propose a new method to fuse miRNA and mRNA data into one prediction model. Since miRNAs are known regulators of mRNAs we used the correlations between them as well as the target prediction information to build a bipartite graph representing the relations between miRNAs and mRNAs. This graph was used to guide the feature selection in order to improve the prediction. The method is illustrated on a prostate cancer data set comprising 98 patient samples with miRNA and mRNA expression data. The biochemical relapse was used as clinical endpoint. It could be shown that the bipartite graph in combination with both data sets could improve prediction performance as well as the stability of the feature selection. Conclusions Fusion of mRNA and miRNA expression data into one prediction model improves clinical outcome prediction in terms of prediction error and stable feature selection. The R source code of the proposed method is available in the supplement. PMID:22188670

  15. Maternal overnutrition enhances mRNA expression of adipogenic markers and collagen deposition in skeletal muscle of beef cattle fetuses.

    PubMed

    Duarte, M S; Gionbelli, M P; Paulino, P V R; Serão, N V L; Nascimento, C S; Botelho, M E; Martins, T S; Filho, S C V; Dodson, M V; Guimarães, S E F; Du, M

    2014-09-01

    Twenty-four pregnant Nellore cows were randomly assigned into 2 feeding level groups (control [CTL]; fed 1.0 times the maintenance requirement; n = 12; and overnourished [ON]; fed at 1.5 times the maintenance requirement; n = 12) to evaluate effects of maternal overnutrition on fetal skeletal muscle development. Cows were slaughtered at 135, 190, and 240 d of gestation and samples of fetal LM were collected for analysis of mRNA expression analysis and for histological evaluation of collagen content and number of muscle cells. There was no interaction between gestational period and maternal nutrition for the variables evaluated (P > 0.05). The mRNA expression of Cadherin-associated protein, β 1 (β-catenin) tended to be greater in fetuses from ON cows (P = 0.08), while myogenic differentiation 1 (MyoD; P = 0.56), myogenin (MyoG; P = 0.70), and the number of muscle cells (P = 0.90) were not affected by maternal overnutrition. Gestational period did not affect the mRNA expression of β-catenin (P = 0.60) and MyoG (P = 0.21). The mRNA expression of MyoD tended to increase with days of gestation (P = 0.06). The mRNA expression of zinc finger protein 423 (Zfp423; P < 0.0001), C/EBPα (P = 0.01), and PPARγ (P < 0.0001) were enhanced in ON fetuses. No effects of days of gestation were observed for mRNA expression of Zfp423 (P = 0.75) and C/EBPα (P = 0.48). The mRNA expression of PPARγ in fetuses at 190 d of gestation tended to be greater than those at 135 and 240 d of gestation (P = 0.06). The mRNA expression of transforming growth factor β (TGF-β; P < 0.0001), collagen type III, α I (COL3A1; P < 0.0001), and collagen content (P = 0.01) were increased in ON fetuses. Gestational period did not affect the mRNA expression of collagen type I, α I (COL1A1; P = 0.65). The mRNA expression of COL3A1 (P = 0.09) in fetuses at 190 d of gestation tended to be greater than fetuses at 135 and 240 d of gestation. The mRNA expression of TGF-β in fetuses at 190 d of gestation was

  16. DDAH2 mRNA Expression Is Inversely Associated with Some Cardiovascular Risk-Related Features in Healthy Young Adults

    PubMed Central

    Puchau, Blanca; Hermsdorff, Helen Hermana M.; Zulet, M. Ángeles; Martínez, J. Alfredo

    2009-01-01

    The purpose of this study was to evaluate whether the mRNA expression profiles of three genes (PRMT1, DDAH2 and NOS3) are related to ADMA metabolism and signalling, and the potential relationships with anthropometrical, biochemical, lifestyle and inflammatory indicators in healthy young adults. An emphasis on the putative effect of different mRNA expression on cardiovascular risk-related features was paid. Anthropometrical measurements as well as lifestyle features were analyzed in 120 healthy young adults. Fasting blood samples were collected for the measurement of glucose and lipid profiles as well as the concentrations of selected inflammatory markers. Profiles of mRNA expression were assessed for PRMT1, DDAH2 and NOS3 genes from peripheral blood mononuclear cells. Regarding inflammatory biomarkers, DDAH2 was inversely associated with IL-6 and TNF-α. Moreover, subjects in the highest quintile of DDAH2 mRNA expression showed a reduced risk to have higher values of waist circumference, and to be more prone to show higher values of HDL-c. Interestingly, DDAH2 gene expression seemed to be related with some anthropometrical, biochemical, lifestyle and inflammatory indicators linked to cardiovascular risk in apparently healthy young adults, emerging as a potential disease marker. PMID:19822957

  17. The differential expression of alternatively polyadenylated transcripts is a common stress-induced response mechanism that modulates mammalian mRNA expression in a quantitative and qualitative fashion

    PubMed Central

    Hollerer, Ina; Curk, Tomaz; Haase, Bettina; Benes, Vladimir; Hauer, Christian; Neu-Yilik, Gabriele; Bhuvanagiri, Madhuri; Hentze, Matthias W.; Kulozik, Andreas E.

    2016-01-01

    Stress adaptation plays a pivotal role in biological processes and requires tight regulation of gene expression. In this study, we explored the effect of cellular stress on mRNA polyadenylation and investigated the implications of regulated polyadenylation site usage on mammalian gene expression. High-confidence polyadenylation site mapping combined with global pre-mRNA and mRNA expression profiling revealed that stress induces an accumulation of genes with differentially expressed polyadenylated mRNA isoforms in human cells. Specifically, stress provokes a global trend in polyadenylation site usage toward decreased utilization of promoter-proximal poly(A) sites in introns or ORFs and increased utilization of promoter-distal polyadenylation sites in intergenic regions. This extensively affects gene expression beyond regulating mRNA abundance by changing mRNA length and by altering the configuration of open reading frames. Our study highlights the impact of post-transcriptional mechanisms on stress-dependent gene regulation and reveals the differential expression of alternatively polyadenylated transcripts as a common stress-induced mechanism in mammalian cells. PMID:27407180

  18. Expressions and clinical significances of CD133 protein and CD133 mRNA in primary lesion of gastric adenocacinoma

    PubMed Central

    2010-01-01

    Background To study on expressions and clinical significances of CD133 protein and CD133 mRNA in primary lesion of gastric adenocarcinoma (GC). Methods Expressions of CD133 protein by immunostaining (99 cases) and CD133 mRNA by semi-quantitative RT-PCR (31 cases) were detected in primary lesion and in noncancerous gastric mucosa tissue (NCGT). Correlations of CD133 protein expression with clinicopathological parameters and post-operative survival were analyzed. Relations of CD133 mRNA level with Ki-67 labeling index (LI), and lymphatic metastasis were assessed too. Results Brown particles indicating CD133 protein positivity occurred in some parts of tumor cells and epithelium. Expressive percentage of CD133 protein positivity was significantly higher in subgroups with >5 cm diameter (P = 0.041), later TNM stage (P = 0.044), severer lymph node metastasis (P = 0.017), occurrences of lymphatic invasion (P = 0.000) and vascular invasion (P = 0.000) respectively. Severer invasion depth (P = 0.011), lymph node metastasis occurrence (P = 0.043) and later TNM stage (P = 0.049) were the independent risk factors for CD133 protein expression. Average brightness scale value (BSV) of CD133 mRNA was significantly higher in subgroups with >5 cm diameter (P = 0.041), lymph node metastasis occurrence (P = 0.004) and in lower Ki-67 LI (P = 0.02). Relative analysis revealed that BSV of CD133 mRNA related positively to metastatic lymphatic nodes ratio (P = 0.008) and metastatic lymph node number (P = 0.009), but negatively to Ki-67 LI (P = 0.009). Survival of positive subgroup of CD 133 protein was significantly poorer (P = 0.047). Lymph node metastasis occurrence (P = 0.042), later TNM stage (P = 0.046) and CD 133 protein positive expression (P = 0.046) were respectively the independent risk factors to survival. Conclusion Higher expressive level of CD133 mRNA is associated to lower Ki-67 LI and severer lymphatic metastasis. Therefore, the expressive level of CD133 mRNA can play an

  19. Clinical usefulness of WT1 mRNA expression in bone marrow detected by a new WT1 mRNA assay kit for monitoring acute myeloid leukemia: a comparison with expression of WT1 mRNA in peripheral blood.

    PubMed

    Kitamura, Kunio; Nishiyama, Takahiro; Ishiyama, Ken; Miyawaki, Shuichi; Miyazaki, Kanji; Suzuki, Kenshi; Masaie, Hiroaki; Okada, Masaya; Ogawa, Hiroyasu; Imai, Kiyotoshi; Kiyoi, Hitoshi; Naoe, Tomoki; Yokoyama, Yasuhisa; Chiba, Shigeru; Hata, Tomoko; Miyazaki, Yasushi; Hatta, Yoshihiro; Takeuchi, Jin; Nannya, Yasuhito; Kurokawa, Mineo; Ueda, Yasunori; Koga, Daisuke; Sugiyama, Haruo; Takaku, Fumimaro

    2016-01-01

    We have previously shown the clinical usefulness of Wilms' tumor 1 gene (WT1) mRNA expression in peripheral blood (PB) as a minimal residual disease (MRD) monitoring marker in 191 acute myeloid leukemia (AML) patients using the WT1 mRNA assay kit "Otsuka" (Otsuka Pharmaceutical Co., Ltd.; "former kit"). In contrast, the usefulness of WT1 mRNA expression in bone marrow (BM) has been investigated in only a limited number of subjects using former kit. Following that previous study, a next-generation kit, WT1 mRNA assay kit II "Otsuka" (Otsuka Pharmaceutical Co., Ltd.; "new kit") has been newly developed. In the present study, we aimed to evaluate the performance of the new kit and to investigate the clinical usefulness of WT1 mRNA expression in BM. The PB and BM were collected on the same day from 164 blood disease patients, including 118 AML patients. WT1 mRNA expression was determined using the new and former kits and the values obtained were compared. The performance of new kit was shown to be equivalent to that of former kit. As reported in PB, WT1 mRNA expression in BM was found to be a useful marker for monitoring disease status as well as for a diagnosis of early stage relapse in AML patients. PMID:26520650

  20. Uncoupling protein-2 mRNA expression in mice subjected to intermittent hypoxia*

    PubMed Central

    Vieira, Luciana Rodrigues; Martinez, Denis; Forgiarini, Luiz Felipe; da Rosa, Darlan Pase; de Muñoz, Gustavo Alfredo Ochs; Fagundes, Micheli; Martins, Emerson Ferreira; Montanari, Carolina Caruccio; Fiori, Cintia Zappe

    2015-01-01

    Objective: To investigate the effect of intermittent hypoxia-a model of obstructive sleep apnea (OSA)-on pancreatic expression of uncoupling protein-2 (UCP2), as well as on glycemic and lipid profiles, in C57BL mice. Methods: For 8 h/day over a 35-day period, male C57BL mice were exposed to intermittent hypoxia (hypoxia group) or to a sham procedure (normoxia group). The intermittent hypoxia condition involved exposing mice to an atmosphere of 92% N and 8% CO2 for 30 s, progressively reducing the fraction of inspired oxygen to 8 ± 1%, after which they were exposed to room air for 30 s and the cycle was repeated (480 cycles over the 8-h experimental period). Pancreases were dissected to isolate the islets. Real-time PCR was performed with TaqMan assays. Results: Expression of UCP2 mRNA in pancreatic islets was 20% higher in the normoxia group than in the hypoxia group (p = 0.11). Fasting serum insulin was higher in the hypoxia group than in the normoxia group (p = 0.01). The homeostasis model assessment of insulin resistance indicated that, in comparison with the control mice, the mice exposed to intermittent hypoxia showed 15% lower insulin resistance (p = 0.09) and 21% higher pancreatic β-cell function (p = 0.01). Immunohistochemical staining of the islets showed no significant differences between the two groups in terms of the area or intensity of α- and β-cell staining for insulin and glucagon. Conclusions: To our knowledge, this is the first report of the effect of intermittent hypoxia on UCP2 expression. Our findings suggest that UCP2 regulates insulin production in OSA. Further study of the role that UCP2 plays in the glycemic control of OSA patients is warranted. PMID:25909153

  1. Associations of MMP-2, BAX, and Bcl-2 mRNA and Protein Expressions with Development of Atrial Fibrillation

    PubMed Central

    Diao, Shu-Ling; Xu, Hui-Pu; Zhang, Bei; Ma, Bao-Xin; Liu, Xian-Liang

    2016-01-01

    Background To examine changes of mRNA and protein expressions of MMP-2, Bcl-2, and BAX in atrial fibrillation (AF) patients, and investigate the correlations among these 3 biomarkers. Material/Methods Rheumatic heart disease patients (n=158) undergoing cardiac surgical procedures for mitral valve repair or replacement were included as the AF group (n=123), containing paroxysmal AF (n=42), persistent AF (n=36), and permanent AF (n=45). Rheumatic heart disease patients with sinus rhythm (SR) (n=35) were enrolled as the SR group (control group). Immunohistochemistry, Western blot, and real-time polymerase chain reaction (PCR) were applied to detect the protein and mRNA expression levels of MMP-2, Bcl-2, and BAX. Apoptosis was observed with light and electron microscopes and detected by TdT-mediated dUTP nick-end labeling (TUNEL). Results Compared with the SR group, the left atrial diameters (LADs), protein and mRNA expression levels of MMP-2 and BAX, apoptotic index (AI), and Bcl-2/BAX ratio were evidently increased in the 3 AF groups, but protein and mRNA expression levels of Bcl-2 decreased in the AF groups (all P<0.05). Correlation analysis found that MMP-2 protein expression levels was positively correlated with BAX expression, but negatively correlated with Bcl-2 expression levels. Conclusions Our study results suggest that elevated MMP-2 expression and disturbance balance of Bcl-2/BAX expressions may be associated with the development and maintenance of AF. MMP-2 may be involved in the development of AF through promoting BAX expressions and inhibiting Bcl-2. PMID:27141955

  2. Over-expression of corticotropin-releasing factor mRNA in inferior olivary neurons of rolling mouse Nagoya.

    PubMed

    Sawada, Kazuhiko; Kawano, Michihiro; Tsuji, Hiroshi; Sakata-Haga, Hiromi; Hisano, Setsuji; Fukui, Yoshihiro

    2003-10-01

    Expression of corticotropin-releasing factor (CRF) mRNA was examined in the inferior olivary nucleus (ION) of an ataxic mutant, rolling mouse Nagoya (RMN) by semi-quantitative in situ hybridization. The most marked difference in the level of CRF mRNA signals between RMN and non-ataxic littermates (control mice) was observed in the beta-subnucleus and ventrolateral protrusion of the ION. The level of signals in these subnuclei was about twofold higher in RMN than in the controls. Signal levels in the dorsal nucleus, principal nucleus and subnucleus A were slightly but significantly higher in RMN than in the controls. In the other subnuclei, there were no differences in signal level between RMN and controls. These results suggest a region-related over-expression of CRF mRNA in the ION of RMN. This may be responsible for the increased sensitivity of some Purkinje cells to glutamate, resulting in ataxic symptoms of RMN.

  3. Differential regulation of amyloid-. beta. -protein mRNA expression within hippocampal neuronal subpopulations in Alzheimer disease

    SciTech Connect

    Higgins, G.A.; Lewis, D.A.; Bahmanyar, S.; Goldgaber, D.; Gajdusek, D.C.; Young, W.G.; Morrison, J.H.; Wilson, M.C.

    1988-02-01

    The authors have mapped the neuroanatomical distribution of amyloid-..beta..-protein mRNA within neuronal subpopulations of the hippocampal formation in the cynomolgus monkey (Macaca fascicularis), normal aged human, and patients with Alzheimer disease. Amyloid-..beta..-protein mRNA appears to be expressed in all hippocampal neurons, but at different levels of abundance. In the central nervous system of monkey and normal aged human, image analysis shows that neurons of the dentate gyrus and cornu Ammonis fields contain a 2.5-times-greater hybridization signal than is present in neurons of the subiculum and entorhinal cortex. In contrast, in the Alzheimer disease hippocampal formation, the levels of amyloid-..beta..-protein mRNA in the cornu Ammonis field 3 and parasubiculum are equivalent. These findings suggest that within certain neuronal subpopulations cell type-specific regulation of amyloid-..beta..-protein gene expression may be altered in Alzheimer disease.

  4. Cellular localization of proenkephalin mRNA in rat brain: gene expression in the caudate-putamen and cerebellar cortex.

    PubMed Central

    Shivers, B D; Harlan, R E; Romano, G J; Howells, R D; Pfaff, D W

    1986-01-01

    The cellular locations of proenkephalin mRNA have been determined for the caudate-putamen and cerebellar cortex of the rat brain by in situ hybridization. In the caudate-putamen, more than half of the neurons express the proenkephalin gene. Morphologically, they are medium-sized cells that may represent projection neurons. In the cerebellar cortex, proenkephalin mRNA is present in a subpopulation of neurons in the granule layer that appear to be Golgi cells--i.e., inhibitory interneurons. The presence of [Met]enkephalin, a pentapeptide derived from proenkephalin, in these two brain areas is consistent with a synthetic role for this mRNA and implicates proenkephalin gene expression in the control of motor function. Images PMID:3461484

  5. Polymorphism of CYP3A4 and ABCB1 genes increase the risk of neuropathy in breast cancer patients treated with paclitaxel and docetaxel

    PubMed Central

    Kus, Tulay; Aktas, Gokmen; Kalender, Mehmet Emin; Demiryurek, Abdullah Tuncay; Ulasli, Mustafa; Oztuzcu, Serdar; Sevinc, Alper; Kul, Seval; Camci, Celaletdin

    2016-01-01

    Background Interindividual variability of pharmacogenetics may account for unpredictable neurotoxicities of taxanes. Methods From March 2011 to June 2015, female patients with operable breast cancer who had received docetaxel- or paclitaxel-containing adjuvant chemotherapy were included in this study. All patients were treated with single-agent paclitaxel intravenously (IV) 175 mg/m2 every 3 weeks for four cycles, or IV 80 mg/m2 weekly for 12 cycles, and IV 100 mg/m2 docetaxel for four cycles as adjuvant treatment. We evaluated the relationship between neurotoxicity of taxanes and single-nucleotide polymorphisms of ABCB1, CYP3A4, ERCC1, ERCC2, FGFR4, TP53, ERBB2, and CYP2C8 genes. Taxane-induced neurotoxicity during the treatment was evaluated according to the National Cancer Institute Common Toxicity Criteria version 4.03 prior to each cycle. Chi-squared tests were used to compare the two groups, and multivariate binary logistic regression models were used for determining possible risk factors of neuropathy. Results Pharmacogenetic analysis was performed in 219 females. ABCB1 3435 TT genotype had significantly higher risk for grade ≥2 neurotoxicity (odds ratio [OR]: 2.759, 95% confidence interval [CI]: 1.172–6.493, P: 0.017) compared to TC and CC genotype, and also CYP3A4 392 AA and AG genotype had significantly higher risk for grade ≥2 neurotoxicity (OR: 2.259, 95% CI: 1.033–4.941, P: 0.038) compared to GG genotype. For FDGF4 gene with AG and GG genotype, OR was 1.879 (95% CI: 1.001–3.525, P: 0.048) compared to AA genotype with regard to any grade of neuropathy risk. We could not find any other association of other genotypes with neurotoxicity grades. Conclusion ABCB1 3435 TT genotype and CYP3A4 392 AA/AG genotypes may be used as predictors of neurotoxicity during taxane chemotherapy. PMID:27574448

  6. Regulation of adeno-associated virus gene expression in 293 cells: control of mRNA abundance and translation

    SciTech Connect

    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.

  7. Serum leptin concentrations, leptin mRNA expression, and food intake during the estrous cycle in rats

    PubMed Central

    Fungfuang, Wirasak; Nakada, Tomoaki; Nakao, Nobuhiro; Terada, Misao; Yokosuka, Makoto; Gizurarson, Sveinbjorn; Hau, Jann; Moon, Changjong

    2013-01-01

    The aim of this study was to investigate food intake, serum leptin levels, and leptin mRNA expression during the sexual cycle in rats. Female Wistar-Imamichi rats aged 8-10 weeks were used in this experiment. Food intake was measured during the light and dark phases (light on at 07:00 and off at 19:00) of the 4-day estrous cycle in female rats. Serum leptin levels were measured by ELISA, and leptin mRNA expression levels were analyzed using real-time PCR on diestrous- and proestrous-stage rats. Our results revealed that during the sexual cycle, food intake was significantly higher in the dark phase compared with the light phase. Food intake in proestrous females was significantly lower in the light and dark phases compared with the other groups. Serum leptin concentrations were significantly higher in both phases in proestrous rats compared with diestrous rats. There was a significant increase in leptin mRNA expression in adipose tissue during the proestrous period compared with the diestrous period. These findings suggest that increased leptin mRNA expression and serum leptin levels, which are induced by estrogen during the proestrous stage, may play a role in regulating appetitive behavior. PMID:23573101

  8. Expression of fluorescent proteins in Branchiostoma lanceolatum by mRNA injection into unfertilized oocytes.

    PubMed

    Hirsinger, Estelle; Carvalho, João Emanuel; Chevalier, Christine; Lutfalla, Georges; Nicolas, Jean-François; Peyriéras, Nadine; Schubert, Michael

    2015-01-12

    We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the cephalochordate amphioxus, Branchiostoma lanceolatum. We use constructs for membrane and nuclear targeted mCherry and eGFP that have been modified to accommodate amphioxus codon usage and Kozak consensus sequences. We describe the type of injection needles to be used, the immobilization protocol for the unfertilized oocytes, and the overall injection set-up. This technique generates fluorescently labeled embryos, in which the dynamics of cell behaviors during early development can be analyzed using the latest in vivo imaging strategies. The development of a microinjection technique in this amphioxus species will allow live imaging analyses of cell behaviors in the embryo as well as gene-specific manipulations, including gene overexpression and knockdown. Altogether, this protocol will further consolidate the basal chordate amphioxus as an animal model for addressing questions related to the mechanisms of embryonic development and, more importantly, to their evolution.

  9. Expression of fluorescent proteins in Branchiostoma lanceolatum by mRNA injection into unfertilized oocytes.

    PubMed

    Hirsinger, Estelle; Carvalho, João Emanuel; Chevalier, Christine; Lutfalla, Georges; Nicolas, Jean-François; Peyriéras, Nadine; Schubert, Michael

    2015-01-01

    We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the cephalochordate amphioxus, Branchiostoma lanceolatum. We use constructs for membrane and nuclear targeted mCherry and eGFP that have been modified to accommodate amphioxus codon usage and Kozak consensus sequences. We describe the type of injection needles to be used, the immobilization protocol for the unfertilized oocytes, and the overall injection set-up. This technique generates fluorescently labeled embryos, in which the dynamics of cell behaviors during early development can be analyzed using the latest in vivo imaging strategies. The development of a microinjection technique in this amphioxus species will allow live imaging analyses of cell behaviors in the embryo as well as gene-specific manipulations, including gene overexpression and knockdown. Altogether, this protocol will further consolidate the basal chordate amphioxus as an animal model for addressing questions related to the mechanisms of embryonic development and, more importantly, to their evolution. PMID:25650764

  10. Sorting live stem cells based on Sox2 mRNA expression.

    PubMed

    Larsson, Hans M; Lee, Seung Tae; Roccio, Marta; Velluto, Diana; Lutolf, Matthias P; Frey, Peter; Hubbell, Jeffrey A

    2012-01-01

    While cell sorting usually relies on cell-surface protein markers, molecular beacons (MBs) offer the potential to sort cells based on the presence of any expressed mRNA and in principle could be extremely useful to sort rare cell populations from primary isolates. We show here how stem cells can be purified from mixed cell populations by sorting based on MBs. Specifically, we designed molecular beacons targeting Sox2, a well-known stem cell marker for murine embryonic (mES) and neural stem cells (NSC). One of our designed molecular beacons displayed an increase in fluorescence compared to a nonspecific molecular beacon both in vitro and in vivo when tested in mES and NSCs. We sorted Sox2-MB(+)SSEA1(+) cells from a mixed population of 4-day retinoic acid-treated mES cells and effectively isolated live undifferentiated stem cells. Additionally, Sox2-MB(+) cells isolated from primary mouse brains were sorted and generated neurospheres with higher efficiency than Sox2-MB(-) cells. These results demonstrate the utility of MBs for stem cell sorting in an mRNA-specific manner.

  11. Sorting Live Stem Cells Based on Sox2 mRNA Expression

    PubMed Central

    Larsson, Hans M.; Lee, Seung Tae; Roccio, Marta; Velluto, Diana; Lutolf, Matthias P.; Frey, Peter; Hubbell, Jeffrey A.

    2012-01-01

    While cell sorting usually relies on cell-surface protein markers, molecular beacons (MBs) offer the potential to sort cells based on the presence of any expressed mRNA and in principle could be extremely useful to sort rare cell populations from primary isolates. We show here how stem cells can be purified from mixed cell populations by sorting based on MBs. Specifically, we designed molecular beacons targeting Sox2, a well-known stem cell marker for murine embryonic (mES) and neural stem cells (NSC). One of our designed molecular beacons displayed an increase in fluorescence compared to a nonspecific molecular beacon both in vitro and in vivo when tested in mES and NSCs. We sorted Sox2-MB+SSEA1+ cells from a mixed population of 4-day retinoic acid-treated mES cells and effectively isolated live undifferentiated stem cells. Additionally, Sox2-MB+ cells isolated from primary mouse brains were sorted and generated neurospheres with higher efficiency than Sox2-MB− cells. These results demonstrate the utility of MBs for stem cell sorting in an mRNA-specific manner. PMID:23209609

  12. Lifelong ethanol consumption and brain regional GABAA receptor subunit mRNA expression in alcohol-preferring rats.

    PubMed

    Sarviharju, Maija; Hyytiä, Petri; Hervonen, Antti; Jaatinen, Pia; Kiianmaa, Kalervo; Korpi, Esa R

    2006-11-01

    Brain regional gamma-aminobutyric acid type A (GABAA) receptor subunit mRNA expression was studied in ethanol-preferring AA (Alko, Alcohol) rats after moderate ethanol drinking for up to 2 years of age. In situ hybridization with oligonucleotide probes specific for 13 different subunits was used with coronal cryostat sections of the brains. Selective alterations were observed by ethanol exposure and/or aging in signals for several subunits. Most interestingly, the putative highly ethanol-sensitive alpha4 and beta3 subunit mRNAs were significantly decreased in several brain regions. The age-related alterations in alpha4 subunit expression were parallel to those caused by lifelong ethanol drinking, whereas aging had no significant effect on beta3 subunit expression. The results suggest that prolonged ethanol consumption leading to blood concentrations of about 10 mM may downregulate the mRNA expression of selected GABAA receptor subunits and that aging might have partly similar effects.

  13. Effects of jump training on procollagen alpha(1)(i) mRNA expression and its relationship with muscle collagen concentration.

    PubMed

    Ducomps, Christophe; Larrouy, Dominique; Mairal, Aline; Doutreloux, Jean-Paul; Lebas, Francois; Mauriege, Pascale

    2004-04-01

    The aim of this study was to examine the effects of a prolonged high-intensity exercise, jumping, on procollagen alpha(1)(I) mRNA level and collagen concentration in different muscles of trained (T) and control (C) rabbits. Procollagen alpha(1)(I) mRNA expression was much higher (2.8 to 23.5 times) in semimembranosus proprius (SMP), a slow-twitch oxidative muscle, than in extensor digitorum longus (EDL), rectus femoris (RF), and psoas major (Psoas) muscles, both fast-twitch mixed and glycolytic, whatever group was considered (p < 0.001). Procollagen alpha(1)(I) mRNA level also decreased significantly between 50 and 140 days in all muscles (0.001< p < 0.01). However, mRNA levels were 16 to 97% greater at 140 days in all muscles of T animals compared to C ones (0.01< p <0.05). Collagen concentrations of EDL and RF muscles were also higher (14 to 19%) in T than in C rabbits at 90 and 140 days (0.001 < p < 0.05). In the whole sample, collagen concentration was negatively associated with the procollagen alpha(1)(I) mRNA level in EDL and RF muscles (- 0.49 < r < (- 0.44, p < 0.05), while being positively related to mRNA expression in SMP and Psoas muscles (0.65 < r < 0.85, p < 0.01). It is concluded that jump training clearly restricts the decrease of procollagen (I) mRNA level and probably affects collagen synthesis level. In trained rabbit muscles, the maintenance of a better synthesis level could partly explain the higher collagen concentrations found in EDL and RF at 140 days. Nevertheless, the collagen degradation process seems to play the main role in the increase of total collagen concentration with age in EDL and RF muscles. PMID:15064425

  14. Evaluation of CYP2C19, P2Y12, and ABCB1 polymorphisms and phenotypic response to clopidogrel in healthy Indian adults

    PubMed Central

    Sridharan, Kannan; Kataria, Rachna; Tolani, Drishti; Bendkhale, Shital; Gogtay, Nithya J.; Thatte, Urmila M.

    2016-01-01

    Introduction: CYP2C19 and P2Y12 polymorphisms have been claimed to alter the pharmacodynamic response to clopidogrel. ABCB1 polymorphism has been associated with the efflux of clopidogrel resulting in decreased bioavailability. Due to paucity of data from Indian population, the present study was undertaken to evaluate the association of genetic polymorphisms of CYP2C19, P2Y12, and ABCB1 with inhibition of platelet aggregation (IPA) by clopidogrel. Methods: Healthy adults (n = 90) of either gender were administered single dose of 300 mg clopidogrel. Baseline, 4 h postdose, and day 7 assessment of platelet aggregation and genotype of CYP2C19, P2Y12, and ABCB1 were carried out using standardized laboratory methods. The difference in the maximum platelet aggregation (MPA) between baseline and 4 h postdose was considered as delta-MPA (DMPA), and percentage change of MPA at 4 h from baseline was considered as IPA. Those with an IPA of <30% were considered as poor responders. Inferential statistics was applied to find out significant difference of these parameters between various groups of genetic polymorphisms. Results: Mean (standard deviation [SD]) of MPA (%) at baseline, 4 h postdose, and day 7 were 78 (5), 56 (16), and 71 (8), respectively. Similarly, mean (SD) of DMPA (%) and IPA (%) were 23 (17) and 29 (21), respectively. A total of 54/90 (60%) cases were found to be poor responders to clopidogrel. A wild genotype (*1/*1) of CYP2C19 was observed in 35 (40.2%), 42 (48.3%) had *1/*2, 2 (2.3%) individuals had *1/*3, and 8 (9.2%) had *2/*2 mutant genotypes. Although statistically not significant (P = 0.09), a trend was observed in having decreased inhibition values (both MPA and IPA) as we proceed from wild genotype (*1/*1) to mutant genotypes in the order of *1/*2, *1/*3, and *2/*2. Similarly, in P2Y12, a wild haplotype (H1/H1) was present in 77 (89.5%) and 9 (10.5%) individuals had H1/H2 type. A statistically significant difference in DMPA and IPA was observed with

  15. Identification of new M23A mRNA of mouse aquaporin-4 expressed in brain, liver, and kidney.

    PubMed

    Alikina, T Yu; Illarionova, N B; Zelenin, S M; Bondar, A A

    2012-05-01

    Aquaporins (AQPs) belong to a transmembrane protein family of water channels that are permeable to water by the osmotic gradient. There are two isoforms of mouse AQP4 - M1 and M23. Their balance in the cell determines water permeability of the plasma membrane. These two isoforms are encoded by three mRNAs: M1 isoform is encoded by M1 mRNA and M23 isoform is encoded by M23 and M23X mRNAs. Here we found a new fourth mRNA of mouse AQP4 - M23A mRNA. The start of transcription is different for M23A mRNA from all the known AQP4 mRNAs. The 5'-untranslated region (5'-UTR) of M23A mRNA is encoded by four new exons (A, B, C, and D), which are located in the 5' region from exon-0 of the AQP4 gene. Alternative splicing between the exons-A, -B, -C, and -D leads to formation of multiple variants of M23A mRNA. We cloned six of these variants, all of which code full length M23 isoform of AQP4. Using RT-PCR we detected tissue-specific expression of the new M23A and already known M23, M23X, and M1 mRNAs. The M23A mRNA is expressed mostly in kidney, liver, and brain. Analysis of mRNA 5'-UTR structure showed low translation efficacy for M1 mRNA in comparison with high translation efficacy for M23A, M23X, and M23 mRNAs. We propose that AQP4 expression is controlled tissue-specifically by independent promoters. Thus multiple AQP4 mRNAs may allow long-term regulation of the balance between M1 and M23 AQP4 isoforms in the cell and thus water permeability of the plasma membrane.

  16. Skeletal muscle myostatin mRNA expression is fiber-type specific and increases during hindlimb unloading

    NASA Technical Reports Server (NTRS)

    Carlson, C. J.; Booth, F. W.; Gordon, S. E.

    1999-01-01

    Transgenic mice lacking a functional myostatin (MSTN) gene demonstrate greater skeletal muscle mass resulting from muscle fiber hypertrophy and hyperplasia (McPherron, A. C., A. M. Lawler, and S. -J. Lee. Nature 387: 83-90, 1997). Therefore, we hypothesized that, in normal mice, MSTN may act as a negative regulator of muscle mass. Specifically, we hypothesized that the predominately slow (type I) soleus muscle, which demonstrates greater atrophy than the fast (type II) gastrocnemius-plantaris complex (Gast/PLT), would show more elevation in MSTN mRNA abundance during hindlimb unloading (HU). Surprisingly, MSTN mRNA was not detectable in weight-bearing or HU soleus muscle, which atrophied 42% by the 7th day of HU in female ICR mice. In contrast, MSTN mRNA was present in weight-bearing Gast/PLT muscle and was significantly elevated (67%) at 1 day but not at 3 or 7 days of HU. However, the Gast/PLT muscle had only atrophied 17% by the 7th day of HU. Because the soleus is composed only of type I and IIa fibers, whereas the Gast/PLT expresses type IId/x and IIb in addition to type I and IIa, it was necessary to perform a more careful analysis of the relationship between MSTN mRNA levels and myosin heavy-chain (MHC) isoform expression (as a marker of fiber type). A significant correlation (r = 0.725, P < 0. 0005) was noted between the percentage of MHC isoform IIb expression and MSTN mRNA abundance in several muscles of the mouse hindlimb. These results indicate that MSTN expression is not strongly associated with muscle atrophy induced by HU; however, it is strongly associated with MHC isoform IIb expression in normal muscle.

  17. The Influence of C3435T Polymorphism of the ABCB1 Gene on Genetic Susceptibility to Depression and Treatment Response in Polish Population - Preliminary Report

    PubMed Central

    Jeleń, Agnieszka Maria; Sałagacka, Aleksandra; Żebrowska, Marta Karolina; Mirowski, Marek; Talarowska, Monika; Gałecki, Piotr; Balcerczak, Ewa Izabela

    2015-01-01

    Background: Despite the high prevalence of depression, the mechanism of the origin of this disease as well as the causes of resistance to therapy in some patients are still not fully understood. Increasingly, the possible role of genetic factors is considered. One of them is polymorphisms in the ABCB1 (MDR1) gene which encodes P-glycoprotein, responsible for the transport of xenobiotics, including antidepressant drugs, through the blood-brain barrier. Methods: C3435T was evaluated in 90 patients with recurrent depressive disorders (rDD). Genotyping was performed using a polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). Results: The obtained results indicate that the TT genotype occurred more frequently among patients with rDD than in healthy volunteers (p=0.0441). Also, at least one C allele was present significantly less frequent in the study group than in healthy individuals (p=0.0300). The severity of depressive symptoms was higher among patient with the CC genotype in comparison with the other genotypes (p=0.0106) but treatment response to antidepressants was better in this group than among patients with CT or TT genotypes (p=0.0301). Likewise, patients with the T allele have a significantly lower severity of symptoms (p=0.0026) and decreased therapy effectiveness (p=0.0142) than C allele carriers. Conclusions: This study suggests that C3435T polymorphisms in the ABCB1 gene are strongly associated with a predisposition to depression development, the severity of depressive symptoms and the effectiveness of therapy with using different groups of antidepressant agents. PMID:26664259

  18. Pharmacokinetics of a Once-Daily Dose of Tacrolimus Early After Liver Transplantation: With Special Reference to CYP3A5 and ABCB1 Single Nucleotide Polymorphisms.

    PubMed

    Miyata, Yoichi; Akamatsu, Nobuhisa; Sugawara, Yasuhiko; Kaneko, Junichi; Yamamoto, Takehito; Suzuki, Hiroshi; Arita, Junichi; Sakamoto, Yoshihiro; Hasegawa, Kiyoshi; Tamura, Sumihito; Kokudo, Norihiro

    2016-01-01

    BACKGROUND The aim of the present study was to investigate the pharmacokinetics of the once-daily tacrolimus formulation (QD form) in relation to polymorphisms of the donor cytochrome P450 family 3 sub-family A polypeptide 5 (CYP3A5) gene and recipient adenosine triphosphate-binding cassette sub-family B member 1 (ABCB1) gene. MATERIAL AND METHODS A total of 80 consecutive living-donor liver transplant (LDLT) recipients were started on the QD form of tacrolimus (day 1), and 60 patients were completely followed for 7 days early after liver transplantation in order to evaluate the pharmacokinetics. RESULTS The concentration/dose (C/D) ratio in recipients with the donor CYP3A5 *1 allele was significantly lower throughout the observation period compared with those with the CYP3A5 genotype *3/*3 (p<0.001), while no effect of single-nucleotide polymorphisms (SNPs) of ABCB1 was observed. The administered doses required to achieve the target trough level were significantly higher on day 7 than on day 1 among all groups, regardless of the differences in the SNPs, especially among those with donor CYP3A5 *1 allele. The tacrolimus concentration was kept within the targeted level all through the study regardless of SNPs. CONCLUSIONS The donor CYP3A5 *1 allele correlated with the lower C/D ratio after administration of the QD form, and higher doses of QD-form tacrolimus and careful monitoring for the trough level should be considered, especially in recipients with the donor CYP3A5 *1 allele. PMID:27503662

  19. A case of cervical cancer expressed three mRNA variant of Hyaluronan-mediated motility receptor

    PubMed Central

    Villegas-Ruíz, Vanessa; Salcedo, Mauricio; Zentella-Dehesa, Alejandro; de Oca, Edén V Montes; Román-Basaure, Edgar; Mantilla-Morales, Alejandra; Dávila-Borja, Víctor M; Juárez-Méndez, Sergio

    2014-01-01

    Cervical cancer is the second malignancy in Mexico, little is known about the prognostic factors associated with this disease. Several cellular components are important in their transformation and progression. Alternative mRNA splice is an important mechanism for generating protein diversity, nevertheless, in cancer unknown mRNA diversity is expressed. Hyaluronan-mediated motility receptor (HMMR, RHAMM, CD168) is a family member of proteins, hyaluronan acid dependent, and has been associated with different malignant processes such as: angiogenesis, cell invasiveness, proliferation, metastasis and poor outcome in some tumors. In the present study we identified expression of HMMR in cervical cancer by means of RT-PCR and sequencing. Our results indicate co-expression of two HMMR variants in all samples, and one case expressed three alternative HMMR splice transcripts. These results showed the heterogeneity of mRNA transcripts of HMMR that could express in cancer and the expression of HMMR could be marker of malignancy in CC. PMID:24966934

  20. Effect of propionate on mRNA expression of key genes for gluconeogenesis in liver of dairy cattle.

    PubMed

    Zhang, Qian; Koser, Stephanie L; Bequette, Brian J; Donkin, Shawn S

    2015-12-01

    Elevated needs for glucose in lactating dairy cows are met through a combination of increased capacity for gluconeogenesis and increased supply of gluconeogenic precursors, primarily propionate. This study evaluated the effects of propionate on mRNA expression of cytosolic phosphoenolpyruvate carboxykinase (PCK1), mitochondrial phosphoenolpyruvate carboxykinase (PCK2), pyruvate carboxylase (PC), and glucose-6-phosphatase (G6PC), key gluconeogenic enzymes, and capacity for glucose synthesis in liver of dairy cattle. In experiment 1, six multiparous mid-lactation Holstein cows were used in a replicated 3×3 Latin square design consisting of a 6-d acclimation or washout phase followed by 8h of postruminal infusion of either propionate (1.68mol), glucose (0.84mol), or an equal volume (10mL/min) of water. In experiment 2, twelve male Holstein calves [39±4 kg initial body weight (BW)] were blocked by birth date and assigned to receive, at 7d of age, either propionate [2mmol·h(-1)·(BW(0.75))(-1)], acetate [3.5mmol·h(-1)·(BW(.75))(-1)], or an equal volume (4mL/min) of saline. In both experiments, blood samples were collected at 0, 2, 4, 6, and 8h relative to the start of infusion and liver biopsy samples were collected at the end of the infusion for mRNA analysis. Liver explants from experiment 1 were used to measure tricarboxylic acid cycle flux and gluconeogenesis using (13)C mass isotopomer distribution analysis from (13)C3 propionate. Dry matter intake and milk yield were not altered by infusions in cows. Serum insulin concentration in cows receiving propionate was elevated than cows receiving water, but was not different from cows receiving glucose. Hepatic expression of PCK1 and G6PC mRNA and glucose production in cows receiving propionate were not different from cows receiving water, but tended to be higher compared with cows receiving glucose. Hepatic expression of PCK2 and PC mRNA was not altered by propionate infusion in cows. Blood glucose, insulin, and

  1. Do drug transporter (ABCB1) SNPs and P-glycoprotein function influence cyclosporine and macrolides exposure in renal transplant patients? Results of the pharmacogenomic substudy within the symphony study.

    PubMed

    Llaudó, Inés; Colom, Helena; Giménez-Bonafé, Pepita; Torras, Joan; Caldés, Anna; Sarrias, Maria; Cruzado, Josep M; Oppenheimer, Federico; Sánchez-Plumed, Jaime; Gentil, Miguel Ángel; Ekberg, Henrik; Grinyó, Josep M; Lloberas, Núria

    2013-02-01

    The function of the efflux pump P-glycoprotein (Pgp) and ABCB1 single nucleotide polymorphisms (SNPs) should be considered as important tools to deepen knowledge of drug nephrotoxicity and disposition mechanisms. The aim of this study is to investigate the association of C3435T, G2677T, C1236T, and T129C ABCB1 SNPs with Pgp activity and exposure to different immunosuppressive drugs in renal transplant patients. Patients included in the Symphony Pharmacogenomic substudy were genotyped for ABCB1 SNPs. According to the design, patients were randomized into four immunosuppressive regimens: low and standard dose of cyclosporine (n = 30), tacrolimus (n = 13), and sirolimus (n = 23) concomitantly with mycophenolate and steroids. Pgp activity was evaluated in PBMC using the Rhodamine 123 efflux assay. TT carrier patients on C3435T, G2677T, and C1236T SNPs (Pgp-low pumpers) showed lower Pgp activity than noncarriers. Pgp-high pumpers treated with cyclosporine showed lower values of Pgp function than macrolides. There was a negative correlation between cyclosporine AUC and Pgp activity at 3 months. Results did not show any correlation between tacrolimus and sirolimus AUC and Pgp activity at 3 months. We found an important role of the ABCB1 SNPs Pgp function in CD3(+) peripheral blood lymphocytes from renal transplant recipients. Pgp activity was influenced by cyclosporine but not macrolides exposure.

  2. Food restriction during lactation suppresses Kiss1 mRNA expression and kisspeptin-stimulated LH release in rats.

    PubMed

    Ladyman, Sharon R; Woodside, Barbara

    2014-05-01

    Among the numerous physiological changes that accompany lactation is the suppression of the reproductive axis. The aim of this study was to investigate a possible role for the kisspeptin system in the restoration of the hypothalamic-pituitary-gonadal axis during late lactation in rats using a food restriction model that allows manipulation of the duration of lactational anovulation. Kiss1 mRNA expression and kisspeptin-immunoreactive cell counts were examined in both food-restricted dams and ad libitum (AL)-fed dams across late lactation when LH concentrations begin to increase. In the arcuate nucleus, Kiss1 mRNA expression and kisspeptin-positive cell counts were suppressed during late lactation. In the anteroventral periventricular (AVPV), day 15 food-restricted dams had significantly lower AVPV Kiss1 mRNA expression and a decreased LH response to exogenous kisspeptin compared with the AL-fed dams. Following 5 days of ad libitum food intake, these values were restored to levels similar to those in dams that had been fed ad libitum throughout lactation. In conclusion, this study shows that delayed restoration of the reproductive axis due to food restriction is associated with a decrease in kisspeptin sensitivity and low AVPV Kiss1 mRNA in late lactation.

  3. Myoglobin expression: early induction and subsequent modulation of myoglobin and myoglobin mRNA during myogenesis.

    PubMed Central

    Weller, P A; Price, M; Isenberg, H; Edwards, Y H; Jeffreys, A J

    1986-01-01

    We showed that myoglobin gene transcription and the appearance of myoglobin occur very early in myogenesis, in both humans and mice. In contrast to the contractile protein genes, there is a subsequent increase of 50- to 100-fold in myoglobin mRNA and protein levels during later muscle development. Myoglobin and myoglobin mRNA are present at elevated levels in fetal heart and are also detectable at low levels in adult smooth muscle. The absolute level of myoglobin mRNA in highly myoglobinized seal muscle is very high [2.8% of the total population of poly(A)+ RNAs]. Levels of myoglobin in seal skeletal muscle and in various human muscle types appear to be determined by the size of the myoglobin mRNA pool. In contrast, low levels of myoglobin in mouse skeletal muscle are not apparently correlated with low levels of myoglobin mRNA. As expected from the early appearance of myoglobin mRNA in embryonic skeletal muscle, both rat and mouse embryonic myoblasts accumulate myoglobin mRNA on fusion and differentiation in vitro. Images PMID:3796609

  4. Inducible expression of Pisum sativum xyloglucan fucosyltransferase in the pea root cap meristem, and effects of antisense mRNA expression on root cap cell wall structural integrity.

    PubMed

    Wen, Fushi; Celoy, Rhodesia M; Nguyen, Trang; Zeng, Weiqing; Keegstra, Kenneth; Immerzeel, Peter; Pauly, Markus; Hawes, Martha C

    2008-07-01

    Mitosis and cell wall synthesis in the legume root cap meristem can be induced and synchronized by the nondestructive removal of border cells from the cap periphery. Newly synthesized cells can be examined microscopically as they differentiate progressively during cap development, and ultimately detach as a new population of border cells. This system was used to demonstrate that Pisum sativum L. fucosyl transferase (PsFut1) mRNA expression is strongly expressed in root meristematic tissues, and is induced >2-fold during a 5-h period when mitosis in the root cap meristem is increased. Expression of PsFut1 antisense mRNA in pea hairy roots under the control of the CaMV35S promoter, which exhibits meristem localized expression in pea root caps, resulted in a 50-60% reduction in meristem localized endogenous PsFut1 mRNA expression measured using whole mount in situ hybridization. Changes in gross levels of cell wall fucosylated xyloglucan were not detected, but altered surface localization patterns were detected using whole mount immunolocalization with CCRC-M1, an antibody that recognizes fucosylated xyloglucan. Emerging hairy roots expressing antisense PsFut1 mRNA appeared normal macroscopically but scanning electron microscopy of tissues with altered CCRC-M1 localization patterns revealed wrinkled, collapsed cell surfaces. As individual border cells separated from the cap periphery, cell death occurred in correlation with extrusion of cellular contents through breaks in the wall.

  5. Eukaryotic mRNA decay: methodologies, pathways, and links to other stages of gene expression.

    PubMed

    Pérez-Ortín, José E; Alepuz, Paula; Chávez, Sebastián; Choder, Mordechai

    2013-10-23

    mRNA concentration depends on the balance between transcription and degradation rates. On both sides of the equilibrium, synthesis and degradation show, however, interesting differences that have conditioned the evolution of gene regulatory mechanisms. Here, we discuss recent genome-wide methods for determining mRNA half-lives in eukaryotes. We also review pre- and posttranscriptional regulons that coordinate the fate of functionally related mRNAs by using protein- or RNA-based trans factors. Some of these factors can regulate both transcription and decay rates, thereby maintaining proper mRNA homeostasis during eukaryotic cell life.

  6. Expression and localization of the cystic fibrosis transmembrane conductance regulator mRNA and its protein in rat brain.

    PubMed Central

    Mulberg, A E; Resta, L P; Wiedner, E B; Altschuler, S M; Jefferson, D M; Broussard, D L

    1995-01-01

    In previous studies we have characterized the expression of the cystic fibrosis transmembrane conductance regulator (CFTR) protein in clathrin-coated vesicles derived from bovine brain and in neurons of rat brain. In this study we have further characterized the expression of the CFTR protein mRNA and protein in rat brain with reverse transcriptase polymerase chain reaction amplification (RT-PCR), in situ hybridization, and immunocytochemistry. The expression of CFTR mRNA and protein in discrete areas of brain, including the hypothalamus, thalamus, and amygdaloid nuclei, which are involved in regulation of appetite and resting energy expenditure, is identical. The presence of CFTR in neurons localized to these regions of brain controlling homeostasis and energy expenditure may elucidate the pathogenesis of other nonpulmonary and gastrointestinal manifestations which commonly are observed in children with cystic fibrosis. Dysregulation of normal neuropeptide vesicle trafficking by mutant CFTR in brain may serve as a pathogenic mechanism for disruption of homeostasis. Images PMID:7542288

  7. Quantification of the mRNA expression of G protein-coupled receptors in human adipose tissue.

    PubMed

    Amisten, Stefan

    2016-01-01

    G protein-coupled receptors (GPCRs) are important regulators of human physiology and therefore the targets of a large number of modern therapeutics. Although GPCRs are important regulators of adipose tissue endocrine and energy storage functions, the expression and function of a majority of GPCRs in adipose tissue is poorly characterized. A first step in the functional characterization of adipose tissue GPCRs is to accurately quantify the expression of GPCRs in adipose tissue. In this methods chapter, a detailed, step-by-step protocol is presented for the isolation of adipose tissue total RNA, its conversion into cDNA and the real-time PCR quantification of human GPCR mRNA expression relative to the mRNA expression of the stable adipose tissue housekeeping gene peptidylprolyl isomerase A (PPIA). A comprehensive list of 377 manually validated, commercially available GPCR qPCR primers allows facilitated swift quantification of either the entire human GPCRome or individual GPCRs, thus providing a sensitive, flexible, and cost-effective means of determining the mRNA expression of GPCRs in adipose tissue. PMID:26928540

  8. The metastatic potential of canine mammary tumours can be assessed by mRNA expression analysis of connective tissue modulators.

    PubMed

    Lamp, O; Honscha, K U; Schweizer, S; Heckmann, A; Blaschzik, S; Einspanier, A

    2013-03-01

    Metastases are the crucial factor for the prognosis of canine mammary tumours (CMTs). In women, the peptide hormone relaxin is linked with metastatic breast cancer. Therefore, the impact of relaxin and its receptors on matrix metalloproteinase (MMP) expression, metastatic disease and survival was analysed using qRT-PCR and immunohistochemistry of CMT samples from 59 bitches. The expression of relaxin and its receptor RXFP1 (relaxin family peptide receptor 1) was discovered on gene and protein levels. Intratumoural relaxin mRNA expression and relaxin plasma levels had no prognostic value. High mRNA levels RXFP1 were an independent marker of metastatic potential, with a more than 15-fold risk increase, and a predictor for shorter survival. Also, MMP-2 expression was associated with early death because of CMT. The mRNA expressions of relaxin, RXFP1 and MMP-2 were positively correlated indicating a common pathogenetic linkage. Thus, RXFP1 is proposed as a new early marker of metastatic potential in CMT and a possible therapeutic target. PMID:22235833

  9. A comparison of eukaryotic viral 5'-leader sequences as enhancers of mRNA expression in vivo.

    PubMed Central

    Gallie, D R; Sleat, D E; Watts, J W; Turner, P C; Wilson, T M

    1987-01-01

    The 5'-untranslated leader sequences of several plant RNA viruses, and a portion of the 5'-leader of an animal retrovirus, were tested for their ability to enhance expression of contiguous open reading frames for chloramphenicol acetyltransferase (CAT) or beta-glucuronidase (GUS) in tobacco mesophyll protoplasts, Escherichia coli and oocytes of Xenopus laevis. Translation of capped or uncapped transcripts was substantially enhanced in almost all systems by the leader sequence of either the U1 or SPS strain of TMV. All leader sequences, except that of TYMV, stimulated expression of 5'-capped GUS mRNA with the native prokaryotic initiation codon context, in electroporated protoplasts. Only the TMV leaders enhanced translation of uncapped GUS mRNAs in protoplasts and increased expression of uncapped CAT mRNA in microinjected X. laevis oocytes. In oocytes, the TYMV leader sequence was inhibitory. In transformed E. coli, the TMV-U1 leader enhanced expression of both the native and eukaryotic context forms of GUS mRNA about 7.5-fold, despite the absence of a Shine-Dalgarno region in any of the transcripts. The absolute levels of GUS activity were all about 6-fold higher with mRNAs containing the native initiation codon context. In E. coli, the leaders of AlMV RNA4 and TYMV were moderately stimulatory whereas those of BMV RNA3, RSV and the SPS strain of TMV enhanced GUS expression by only 2- to 3-fold. Images PMID:2825117

  10. Effects of arginine supplementation on splenocyte cytokine mRNA expression in rats with gut-derived sepsis

    PubMed Central

    Shang, Huey-Fang; Hsu, Chun-Sen; Yeh, Chiu-Li; Pai, Man-Hui; Yeh, Sung-Ling

    2005-01-01

    AIM: To investigate the effects of arginine (Arg)-enriched diets before sepsis and/or Arg-containing total parenteral nutrition (TPN) after sepsis or both on cytokine mRNA expression levels in splenocytes of rats with gut-derived sepsis. METHODS: Rats were assigned to four experimental groups. Groups 1 and 2 were fed with a semipurified diet, while groups 3 and 4 had part of the casein replaced by Arg which provided 2% of the total calories. After the rats were fed with these diets for 10 d, sepsis was induced by cecal ligation and puncture (CLP), at the same time an internal jugular vein was cannulated. All rats were maintained on TPN for 3 d. Groups 1 and 3 were infused with conventional TPN, while groups 2 and 4 were supplemented with Arg which provided 2% of the total calories in the TPN solution. All rats were killed 3 d after CLP to examine their splenocyte subpopulation distribution and cytokine expression levels. RESULTS: Plasma interleukin (IL)-2, IL-4, tumor necrosis factor-α (TNF-α) and interferon (IFN-γ) were not detectable 3 d after CLP. There were no differences in the distributions of CD45Ra+, CD3+, CD4+, and CD8+ cells in whole blood and splenocytes among the four groups. The splenocyte IL-2 mRNA expression in the Arg-supplemented groups was significantly higher than that in group 1. IL-4 mRNA expression in groups 3 and 4 was significantly higher than that in groups 1 and 2. The mRNA expression of IL-10 and IFN-γ was significantly higher in group 4 than in the other three groups. There was no difference in TNF-α mRNA expression among the four groups. CONCLUSION: The influence of Arg on the whole blood and splenic lymphocyte subpopulation distribution is not obvious. However, Arg administration, especially before and after CLP, significantly enhances the mRNA expression levels of Th1 and Th2 cytokines in the spleen of rats with gut-derived sepsis. PMID:16437653

  11. Molecular properties and regulation of mRNA expression for murine T cell-replacing factor/IL-5.

    PubMed

    Tominaga, A; Matsumoto, M; Harada, N; Takahashi, T; Kikuchi, Y; Takatsu, K

    1988-02-15

    We previously cloned cDNA for a T cell-replacing factor (TRF) that has been defined as a T cell-derived lymphokine that acts on activated B cells as a B cell growth and differentiation factor. Based on the diverse activities of rTRF on different target cells, we proposed that TRF be called IL-5. In this study, the molecular characteristics of TRF/IL-5 prepared by rDNA technology and TRF/IL-5 mRNA expression in various T cell lines and normal T cells have been studied. Specific immunoassay showed that rTRF/IL-5, which is transiently translated in vitro by rabbit reticulocyte lysate, has an apparent m.w. of 14,000. By contrast, active forms of rTRF/IL-5 translated in Xenopus oocytes has an apparent m.w. of 45,000 to 50,000 in the nonreducing condition and migrates to the m.w. of 25,000 to 30,000 under the reducing condition, indicating that active form of rTRF/IL-5 consists of dimer forms. The rTRF/IL-5 does not show detectable levels of IL-2, IL-3, and B-cell stimulatory factor 1 (IL-4) activities. Northern blot hybridization of poly (A)+ RNA from constitutively TRF-producing B151K12 T cell hybridoma revealed a single 1.7-kb band hybridizing to the cloned murine TRF/IL-5 cDNA. The expression of TRF/IL-5 mRNA in B151K12 was augmented by the stimulation with PMA plus calcium ionophore. In contrast, neither thymoma BW5147 nor IL-2-producing T cell hybridoma A55, both of which produced an undetectable level of TRF, expressed detectable levels of TRF/IL-5 mRNA. Stimulation of EL 4 and D9 cells with PMA and Con A, respectively, induced an increase in the levels of TRF/IL-5 mRNA expression accompanied by TRF/IL-5 production, whereas both cell lines did not show significant gene expression in the absence of the stimulation. In spleen cells from Mycobacterium tuberculosis-primed mice, significant expression of TRF/IL-5 mRNA was detected only when the cells were stimulated with relevant Ag, PPD. Normal spleen cells stimulated with Con A showed a significant, but approximately

  12. Sirolimus induces apoptosis and reverses multidrug resistance in human osteosarcoma cells in vitro via increasing microRNA-34b expression

    PubMed Central

    Zhou, Yan; Zhao, Rui-hua; Tseng, Kuo-Fu; Li, Kun-peng; Lu, Zhi-gang; Liu, Yuan; Han, Kun; Gan, Zhi-hua; Lin, Shu-chen; Hu, Hai-yan; Min, Da-liu

    2016-01-01

    Aim: Multi-drug resistance poses a critical bottleneck in chemotherapy. Given the up-regulation of mTOR pathway in many chemoresistant cancers, we examined whether sirolimus (rapamycin), a first generation mTOR inhibitor, might induce human osteosarcoma (OS) cell apoptosis and increase the sensitivity of OS cells to anticancer drugs in vitro. Methods: Human OS cell line MG63/ADM was treated with sirolimus alone or in combination with doxorubicin (ADM), gemcitabine (GEM) or methotrexate (MTX). Cell proliferation and apoptosis were detected using CCK-8 assay and flow cytometry, respectively. MiRNAs in the cells were analyzed with miRNA microarray. The targets of miR-34b were determined based on TargetScan analysis and luciferase reporter assays. The expression of relevant mRNA and proteins was measured using qRT-PCR and Western blotting. MiR-34, PAK1 and ABCB1 levels in 40 tissue samples of OS patients were analyzed using qRT-PCR and in situ hybridization assays. Results: Sirolimus (1–100 nmol/L) dose-dependently suppressed the cell proliferation (IC50=23.97 nmol/L) and induced apoptosis. Sirolimus (10 nmol/L) significantly sensitized the cells to anticancer drugs, leading to decreased IC50 values of ADM, GEM and MTX (from 25.48, 621.41 and 21.72 μmol/L to 4.93, 73.92 and 6.77 μmol/L, respectively). Treatment of with sirolimus increased miR-34b levels by a factor of 7.5 in the cells. Upregulation of miR-34b also induced apoptosis and increased the sensitivity of the cells to the anticancer drugs, whereas transfection with miR-34b-AMO, an inhibitor of miR-34b, reversed the anti-proliferation effect of sirolimus. Two key regulators of cell cycle, apoptosis and multiple drug resistance, PAK1 and ABCB1, were demonstrated to be the direct targets of miR-34b. In 40 tissue samples of OS patients, significantly higher miR-34 ISH score and lower PAK5 and ABCB1 scores were detected in the chemo-sensitive group. Conclusion: Sirolimus increases the sensitivity of human OS

  13. Prenatal auditory stimulation alters the levels of CREB mRNA, p-CREB and BDNF expression in chick hippocampus.

    PubMed

    Chaudhury, Sraboni; Wadhwa, Shashi

    2009-10-01

    Prenatal auditory stimulation influences the development of the chick auditory pathway and the hippocampus showing an increase in various morphological parameters as well as expression of calcium-binding proteins. Calcium regulates the activity of cyclic adenosine monophosphate-response element binding (CREB) protein. CREB is known to play a role in development, undergo phosphorylation with neural activity as well as regulate transcription of BDNF. BDNF is important for the survival of neurons and regulates synaptic strength. Hence in the present study, we have evaluated the levels of CREB mRNA and protein along with p-CREB protein as well as BDNF mRNA and protein levels in the chick hippocampus at embryonic days (E) 12, E16, E20 and post-hatch day (PH) 1 following activation by prenatal auditory stimulation. Fertilized eggs were exposed to species-specific sound or sitar music (frequency range: 100-6300Hz) at 65dB levels for 15min/h over 24h from E10 till hatching. The control chick hippocampus showed higher CREB mRNA and p-CREB protein in the early embryonic stages, which later decline whereas BDNF mRNA and BDNF protein levels increase until PH1. The CREB mRNA and p-CREB protein were significantly increased at E12, E16 and PH1 in the auditory stimulated groups as compared to control group. A significant increase in the level of BDNF mRNA was observed from E12 and the protein expression from E16 onwards in both auditory stimulated groups. Therefore, enhanced phosphorylation of CREB during development following prenatal sound stimulation may be responsible for cell survival. Increased levels of p-CREB again at PH1 may trigger synthesis of proteins necessary for synaptic plasticity. Further, the increased levels of BDNF may also help in regulating synaptic plasticity. PMID:19559781

  14. The Prognostic Value of BRCA1 mRNA Expression Levels Following Neoadjuvant Chemotherapy in Breast Cancer

    PubMed Central

    Margeli, Mireia; Cirauqui, Beatriz; Castella, Eva; Tapia, Gustavo; Costa, Carlota; Gimenez-Capitan, Ana; Barnadas, Agusti; Ronco, Maria Sanchez; Benlloch, Susana; Taron, Miquel; Rosell, Rafael

    2010-01-01

    Background A fraction of sporadic breast cancers has low BRCA1 expression. BRCA1 mutation carriers are more likely to achieve a pathological complete response with DNA-damage-based chemotherapy compared to non-mutation carriers. Furthermore, sporadic ovarian cancer patients with low levels of BRCA1 mRNA have longer survival following platinum-based chemotherapy than patients with high levels of BRCA1 mRNA. Methodology/Principal Findings Tumor biopsies were obtained from 86 breast cancer patients who were candidates for neoadjuvant chemotherapy, treated with four cycles of neoadjuvant fluorouracil, epirubicin and cyclophosphamide. Estrogen receptor (ER), progesterone receptor (PR), HER2, cytokeratin 5/6 and vimentin were examined by tissue microarray. HER2 were also assessed by chromogenic in situ hybridization, and BRCA1 mRNA was analyzed in a subset of 41 patients for whom sufficient tumor tissue was available by real-time quantitative PCR. Median time to progression was 42 months and overall survival was 55 months. In the multivariate analysis for time to progression and overall survival for 41 patients in whom BRCA1 could be assessed, low levels of BRCA1 mRNA, positive PR and negative lymph node involvement predicted a significantly lower risk of relapse, low levels of BRCA1 mRNA and positive PR were the only variables associated with significantly longer survival. Conclusions/Significance We provide evidence for a major role for BRCA1 mRNA expression as a marker of time to progression and overall survival in sporadic breast cancers treated with anthracycline-based chemotherapy. These findings can be useful for customizing chemotherapy. PMID:20209131

  15. Gravitational loading of a simulated launch alters mRNA expression in osteoblasts

    NASA Technical Reports Server (NTRS)

    Fitzgerald, J.; Hughes-Fulford, M.

    1996-01-01

    Serum-deprived mouse osteoblastic cells (MC3T3-E1a) were centrifuged under a regime designed to simulate a space shuttle launch (maximum of 3g). Messenger RNA levels for eight genes involved in bone growth and maintenance were determined using RT-PCR. Following 30 min of centrifugation, mRNA level for early response gene c-fos was significantly increased 89% (P < 0.05). The c-fos induction was transient and returned to control levels after 3 h. The mRNA level for the mineralization marker gene osteocalcin was significantly decreased to 44% of control level (P < 0.005) 3 h after centrifugation. No changes in mRNA levels were detected for c-myc, TGFbeta1, TGFbeta2, cyclophilin A, or actin. No basal mRNA level for TGFbeta3 was detected. In addition, no change in the steady-state synthesis of prostaglandin E2 was detected, possibly due to lack of lipid substrates in serum-deprived cells, suggesting that the increase in c-fos mRNA in response to gravitational loading is a result of mechanical stimulation. These results indicate that a small magnitude mechanical loading, such as that experienced during a shuttle launch, can alter mRNA levels in quiescent osteoblastic cells.

  16. Differential mRNA display cloning and characterization of a Cryptosporidium parvum gene expressed during intracellular development.

    PubMed

    Schroeder, A A; Lawrence, C E; Abrahamsen, M S

    1999-04-01

    Differential mRNA display was used to detect differences in gene expression between mock-infected and Cryptosporidium parvum-infected human adenocarcinoma cells. A reproducible band present only in C. parvum-infected cells, ddHC-10 was isolated and cloned. Northern blot analysis was used to confirm the differential expression of the HC-10 mRNA. As differential mRNA display does not differentiate between parasite and host mRNAs, Southern blot analysis was used to demonstrate that ddHC-10 represented a C. parvum gene. Northern blot analysis demonstrated that HC-10 mRNA is expressed by sporozoites prior to invasion of host cells. Screening of a C. parvum genomic library identified 2 different genomic clones, HC-10-13C and HC-10-6C. The combined genomic sequence contained a predicted open reading frame of 2,952 base pairs (bp), coding for a protein of 984 amino acids with a predicted molecular weight of approximately 106 kDa. Reverse transcription polymerase chain reaction mapping of the HC-10 transcript demonstrated that the HC-10 gene lacks introns, and the approximately 4,789-bp mRNA contains relatively large 5' (approximately 1,390-bp) and 3' (approximately 440-bp) untranslated regions. The predicted polypeptide contained a high proportion of polar amino acids, with the most abundant amino acids being serine (10.5%), threonine (9.8%), and cysteine (7.6%). The C-terminal region of the predicted polypeptide is characterized by a threonine-rich region containing multiple repeats of the sequence TTTTRP. This repeat motif is similar to that found in the mucin-like genes of vertebrates and lower eukaryotes that have been shown to play important roles in cell-cell interactions in multicellular organisms and invasion of host cells by unicellular parasites. PMID:10219298

  17. mRNA expression and DNA methylation in three key genes involved in caste differentiation in female honeybees (Apis mellifera)

    PubMed Central

    SHAO, Xin-Liang; HE, Shao-Yu; ZHUANG, Xin-Ying; FAN, Ying; LI, Ya-Hui; YAO, Yong-Gang

    2014-01-01

    In honeybee (Apis mellifera) colonies, queens and workers are alternative forms of the adult female honeybee that develop from genetically identical zygotes but that depend on differential nourishment. Queens and workers display distinct morphologies, anatomies and behavior, better known as caste differentiation. Despite some basic insights, the exact mechanism responsible for this phenomenon, especially at the molecular level, remains unclear although some progress has been achieved. In this study, we examined mRNA levels of the TOR (target of rapamycin) and Dnmt3 (DNA methyltransferase 3) genes, closely related to caste differentiation in honeybees. We also investigated mRNA expression of the S6K (similar to RPS6-p70-protein kinase) gene linked closely to organismal growth and development in queen and worker larvae (1-day and 3-day old). Last, we investigated the methylation status of these three genes in corresponding castes. We found no difference in mRNA expression for the three genes between 1st instar queen and worker larvae; however, 3rd instar queen larvae had a higher level of TOR mRNA than worker larvae. Methylation levels of all three genes were lower in queen larvae than worker larvae but the differences were not statistically significant. These findings provide basic data for broadening our understanding of caste differentiation in female honeybees. PMID:24668651

  18. Using DNA sequencing electrophoresis compression artifacts as reporters of stable mRNA structures affecting gene expression.

    PubMed

    Kapoor, Divya; Chandrayan, Sanjeev Kumar; Ahmed, Shubbir; Guptasarma, Purnananda

    2007-11-01

    The formation of secondary structure in oligonucleotide DNA is known to lead to "compression" artifacts in electropherograms produced through DNA sequencing. Separately, the formation of secondary structure in mRNA is known to suppress translation; in particular, when such structures form in a region covered by the ribosome either during, or shortly after, initiation of translation. Here, we demonstrate how a DNA sequencing compression artifact provides important clues to the location(s) of translation-suppressing secondary structural elements in mRNA. Our study involves an engineered version of a gene sourced from Rhodothermus marinus encoding an enzyme called Cel12A. We introduced this gene into Escherichia coli with the intention of overexpressing it, but found that it expressed extremely poorly. Intriguingly, the gene displayed a remarkable compression artifact during DNA sequencing electrophoresis. Selected "designer" silent mutations destroyed the artifact. They also simultaneously greatly enhanced the expression of the cel12A gene, presumably by destroying stable mRNA structures that otherwise suppress translation. We propose that this method of finding problem mRNA sequences is superior to software-based analyses, especially if combined with low-temperature CE.

  19. Inhibition of Multidrug Resistance-Linked P-Glycoprotein (ABCB1) Function by 5′-Fluorosulfonylbenzoyl 5′-Adenosine: Evidence for an ATP Analog That Interacts With Both Drug-Substrate- and Nucleotide-Binding Sites†

    PubMed Central

    Ohnuma, Shinobu; Chufan, Eduardo; Nandigama, Krishnamachary; Miller Jenkins, Lisa M.; Durell, Stewart R.; Appella, Ettore; Sauna, Zuben E.; Ambudkar, Suresh V.

    2011-01-01

    5′-fluorosulfonylbenzonyl 5′-adenosine (FSBA) is an ATP analog that covalently modifies several residues in the nucleotide-binding domains (NBDs) of several ATPases, kinases and other proteins. P-glycoprotein (P-gp, ABCB1) is a member of the ATP-binding cassette (ABC) transporter superfamily that utilizes energy from ATP hydrolysis for the efflux of amphipathic anticancer agents from cancer cells. We investigated the interactions of FSBA with P-gp to study the catalytic cycle of ATP hydrolysis. Incubation of P-gp with FSBA inhibited ATP hydrolysis (IC50= 0.21 mM) and the binding of 8-azido[α–32P]ATP (IC50= 0.68 mM). In addition, 14C-FSBA crosslinks to P-gp, suggesting that FSBA-mediated inhibition of ATP hydrolysis is irreversible due to covalent modification of P-gp. However, when the NBDs were occupied with a saturating concentration of ATP prior to treatment, FSBA stimulated ATP hydrolysis by P-gp. Furthermore, FSBA inhibited the photocrosslinking of P-gp with [125I]-Iodoaryl-azidoprazosin (IAAP; IC50 = 0.17 mM). As IAAP is a transport substrate for P-gp, this suggests that FSBA affects not only the NBDs, but also the transport-substrate site in the transmembrane domains. Consistent with these results, FSBA blocked efflux of rhodamine 123 from P-gp-expressing cells. Additionally, mass spectrometric analysis identified FSBA crosslinks to residues within or nearby the NBDs but not in the transmembrane domains and docking of FSBA in a homology model of human P-gp NBDs supports the biochemical studies. Thus, FSBA is an ATP analog that interacts with both the drug-binding and ATP-binding sites of P-gp, but fluorosulfonyl-mediated crosslinking is observed only at the NBDs. PMID:21452853

  20. Ethanol enhancement of cocaine- and amphetamine-regulated transcript mRNA and peptide expression in the nucleus accumbens.

    PubMed

    Salinas, Armando; Wilde, Jennifer D; Maldve, Regina E

    2006-04-01

    Cocaine- and amphetamine-regulated transcript (CART) is a peptide neurotransmitter that has been implicated in drug reward and reinforcement. CART mRNA and peptide expression are highly concentrated in several compartments of the mesolimbic reward pathway. Several lines of evidence suggest that CART peptides may contribute to rewarding behaviors and the addiction liability of psychostimulants; however, there are no reports of basic work concerning CART in relation to alcohol and mechanisms of alcohol dependence development. Therefore, in this study we investigated the response of CART transcript and peptide to acute ethanol administration in vivo. Rats were administered ethanol (1 g/kg or 3.5 g/kg, 1 h, ip) and CART expression was measured by RT-PCR in the nucleus accumbens (NAcc). Ethanol (3.5 g/kg) increased CART transcription markedly. The interactions of dopamine on ethanol-induced CART expression were further evaluated pharmacologically using D1 and D2/D3 receptor antagonists. Both SCH 23390 (0.25 mg/kg) or raclopride (0.2 mg/kg) pre-treatment significantly suppressed ethanol-enhancement of CART mRNA transcription. Confocal immunofluorescence microscopy revealed that CART peptide immunoreactivity was also enhanced in both the core and the shell of the NAcc by ethanol administration. These findings demonstrate that CART mRNA and peptide expression are responsive to acute ethanol administrated in vivo and suggests that CART peptides may be important in regulating the rewarding and reinforcing properties of ethanol. PMID:16539670

  1. Oligodendrocyte morphometry and expression of myelin - Related mRNA in ventral prefrontal white matter in major depressive disorder.

    PubMed

    Rajkowska, Grazyna; Mahajan, Gouri; Maciag, Dorota; Sathyanesan, Monica; Iyo, Abiye H; Moulana, Mohadetheh; Kyle, Patrick B; Woolverton, William L; Miguel-Hidalgo, Jose Javier; Stockmeier, Craig A; Newton, Samuel S

    2015-06-01

    White matter disturbance in the ventral prefrontal cortex (vPFC) in major depressive disorder (MDD) has been noted with diffusion tensor imaging (DTI). However, the cellular and molecular pathology of prefrontal white matter in MDD and potential influence of antidepressant medications is not fully understood. Oligodendrocyte morphometry and myelin-related mRNA and protein expression was examined in the white matter of the vPFC in MDD. Sections of deep and gyral white matter from the vPFC were collected from 20 subjects with MDD and 16 control subjects. Density and size of CNPase-immunoreactive (-IR) oligodendrocytes were estimated using 3-dimensional cell counting. While neither density nor soma size of oligodendrocytes was significantly affected in deep white matter, soma size was significantly decreased in the gyral white matter in MDD. In rhesus monkeys treated chronically with fluoxetine there was no significant effect on oligodendrocyte morphometry. Using quantitative RT-PCR to measure oligodendrocyte-related mRNA for CNPase, PLP1, MBP, MOG, MOBP, Olig1 and Olig2, in MDD there was a significantly reduced expression of PLP1 mRNA (which positively correlated with smaller sizes) and increased expression of mRNA for CNPase, OLIG1 and MOG. The expression of CNPase protein was significantly decreased in MDD. Altered expression of four myelin genes and CNPase protein suggests a mechanism for the degeneration of cortical axons and dysfunctional maturation of oligodendrocytes in MDD. The change in oligodendrocyte morphology in gyral white matter may parallel altered axonal integrity as revealed by DTI.

  2. Oligodendrocyte Morphometry and Expression of Myelin – Related mRNA in Ventral Prefrontal White Matter in Major Depressive Disorder

    PubMed Central

    Rajkowska, Grazyna; Mahajan, Gouri; Maciag, Dorota; Sathyanesan, Monica; Iyo, Abiye H.; Moulana, Mohadetheh; Kyle, Patrick B.; Woolverton, William L.; Miguel-Hidalgo, Jose Javier; Stockmeier, Craig A.; Newton, Samuel S.

    2015-01-01

    White matter disturbance in the ventral prefrontal cortex (vPFC) in major depressive disorder (MDD) has been noted with diffusion tensor imaging (DTI). However, the cellular and molecular pathology of prefrontal white matter in MDD and potential influence of antidepressant medications is not fully understood. Oligodendrocyte morphometry and myelin-related mRNA and protein expression was examined in the white matter of the vPFC in MDD. Sections of deep and gyral white matter from the vPFC were collected from 20 subjects with MDD and 16 control subjects. Density and size of CNPase-immunoreactive (−IR) oligodendrocytes were estimated using 3-dimensional cell counting. While neither density nor soma size of oligodendrocytes was significantly affected in deep white matter, soma size was significantly decreased in the gyral white matter in MDD. In rhesus monkeys treated chronically with fluoxetine there was no significant effect on oligodendrocyte morphometry. Using quantitative RTPCR to measure oligodendrocyte-related mRNA for CNPase, PLP1, MBP, MOG, MOBP, Olig1 and Olig2, in MDD there was a significantly reduced expression of PLP1 mRNA (which positively correlated with smaller sizes) and increased expression of mRNA for CNPase, OLIG1 and MOG. The expression of CNPase protein was significantly decreased in MDD. Altered expression of four myelin genes and CNPase protein suggests a mechanism for the degeneration of cortical axons and dysfunctional maturation of oligodendrocytes in MDD. The change in oligodendrocyte morphology in gyral white matter may parallel altered axonal integrity as revealed by DTI. PMID:25930075

  3. Adenovirus-mediated interference of FABP4 regulates mRNA expression of ADIPOQ, LEP and LEPR in bovine adipocytes.

    PubMed

    Wei, S; Zan, L S; Wang, H B; Cheng, G; Du, M; Jiang, Z; Hausman, G J; McFarland, D C; Dodson, M V

    2013-01-01

    Fatty acid binding protein 4 (FABP4) is an important adipocyte gene, with roles in fatty acid transport and fat deposition in animals as well as human metabolic syndrome. However, little is known about the functional regulation of FABP4 at the cellular level in bovine. We designed and selected an effective shRNA (small hairpin RNA) against bovine FABP4, constructed a corresponding adenovirus (AD-FABP4), and then detected its influence on mRNA expression of four differentiation-related genes (PPAR(y), CEBPA, CEBPB, and SREBF1) and three lipid metabolism-related genes (ADIPOQ, LEP and LEPR) of adipocytes. The FABP4 mRNA content, derived from bovine adipocytes, decreased by 41% (P < 0.01) after 24 h and 66% (P < 0.01) after 72 h of AD-FABP4 infection. However, lower mRNA content of FABP4 did not significantly alter levels of differentiation-related gene expression at 24 h following AD-FABP4 treatment of bovine-derived preadipocytes (P = 0.54, 0.78, 0.89, and 0.94, respectively). Meanwhile, knocking down (partially silencing) FABP4 significantly decreased ADIPOQ (P < 0.05) and LEP (P < 0.01) gene expression after 24 h of AD-FABP4 treatment, decreased ADIPOQ (P < 0.01) and LEP (P < 0.01) gene expression, but increased LEPR mRNA expression (P < 0.01) after a 72-h treatment of bovine preadipocytes. We conclude that FABP4 plays a role in fat deposition and metabolic syndrome by regulating lipid metabolism-related genes (such as ADIPOQ, LEP and LEPR), without affecting the ability of preadipocytes to differentiate into adipocytes.

  4. Heterogeneous expression of melatonin receptor MT1 mRNA in the rat intestine under control and fasting conditions.

    PubMed

    Soták, Matús; Mrnka, Libor; Pácha, Jirí

    2006-09-01

    Melatonin is found in mammalian central nervous system and various peripheral tissues including gastrointestinal tract (GIT) where it participates in the regulation of intestinal motility, blood flow, immunomodulation, ion transport, cell proliferation and scavenging of free radicals. Some of these effects are achieved via melatonin binding to specific receptors, MT1 and MT2. As no thorough study on the expression of these receptors in the GIT has yet been done, the aim of this study was to determine the MT1 mRNA expression in the rat intestine under both control and fasting conditions. Our results suggest that MT1 mRNA is present in epithelial as well as subepithelial layer, with higher expression in the latter in all intestinal segments studied. The highest signal of the MT1 transcript along the rostro-caudal intestinal axis was found both in epithelial and subepithelial layers of the duodenum. Nevertheless, duodenal MT1 mRNA expression did not reach the level found in pituitary gland. In a 12:12-hr light:dark cycle a MT1 receptor expression in the subepithelial layer of rat distal colon did not manifest a significant diurnal rhythm. Short-term fasting increased the expression of MT1 transcript in the subepithelial layer of both the small and large intestine. During long-term fasting the increase persisted only in distal colon while a return to control levels was observed in small intestinal segments. In conclusion we demonstrated heterogeneous expression of MT1 receptor in the rat intestine and showed that its expression is up-regulated by nutritional deprivation.

  5. Changes in apoptotic microRNA and mRNA expression profiling in Caenorhabditis elegans during the Shenzhou-8 mission

    PubMed Central

    Gao, Ying; Li, Shuai; Xu, Dan; Wang, Junjun; Sun, Yeqing

    2015-01-01

    Radiation and microgravity exposure have been proven to induce abnormal apoptosis in microRNA (miRNA) and mRNA expression, but whether space conditions, including radiation and microgravity, activate miRNAs to regulate the apoptosis is undetermined. For that purpose, we investigated miRNome and mRNA expression in the ced-1 Caenorhabditis elegans mutant vs the wild-type, both of which underwent spaceflight, spaceflight 1g-centrifuge control and ground control conditions during the Shenzhou-8 mission. Results showed that no morphological changes in the worms were detected, but differential miRNA expression increased from 43 (ground control condition) to 57 and 91 in spaceflight and spaceflight control conditions, respectively. Microgravity altered miRNA expression profiling by decreasing the number and significance of differentially expressed miRNA compared with 1 g incubation during spaceflight. Alterations in the miRNAs were involved in alterations in apoptosis, neurogenesis larval development, ATP metabolism and GTPase-mediated signal transduction. Among these, 17 altered miRNAs potentially involved in apoptosis were screened and showed obviously different expression signatures between space conditions. By integrated analysis of miRNA and mRNA, miR-797 and miR-81 may be involved in apoptosis by targeting the genes ced-10 and both drp-1 and hsp-1, respectively. Compared with ground condition, space conditions regulated apoptosis though a different manner on transcription, by altering expression of seven core apoptotic genes in spaceflight condition, and eight in spaceflight control condition. Results indicate that, miRNA of Caenorhabditis elegans probably regulates apoptotic gene expression in response to space environmental stress, and shows different behavior under microgravity condition compared with 1 g condition in the presence of space radiation. PMID:26286471

  6. Lipoprotein lipase and hepatic lipase mRNA tissue specific expression, developmental regulation, and evolution.

    PubMed

    Semenkovich, C F; Chen, S H; Wims, M; Luo, C C; Li, W H; Chan, L

    1989-03-01

    Lipoprotein lipase (LPL) and hepatic lipase (HL) enzyme activities were previously reported to be regulated during development, but the underlying molecular events are unknown. In addition, little is known about LPL evolution. We cloned and sequenced a complete mouse LPL cDNA. Comparison of sequences from mouse, human, bovine, and guinea pig cDNAs indicated that the rates of evolution of mouse, human, and bovine LPL are quite low, but guinea pig LPL has evolved several times faster than the others. 32P-Labeled mouse LPL and rat HL cDNAs were used to study lipase mRNA tissue distribution and developmental regulation in the rat. Northern gel analysis revealed the presence of a single 1.87 kb HL mRNA species in liver, but not in other tissues including adrenal and ovary. A single 4.0 kb LPL mRNA species was detected in epididymal fat, heart, psoas muscle, lactating mammary gland, adrenal, lung, and ovary, but not in adult kidney, liver, intestine, or brain. Quantitative slot-blot hybridization analysis demonstrated the following relative amounts of LPL mRNA in rat tissues: adipose, 100%; heart, 94%; adrenal, 6.6%; muscle, 3.8%; lung, 3.0%; kidney, 0%; adult liver, 0%. The same quantitative analysis was used to study lipase mRNA levels during development. There was little postnatal variation in LPL mRNA in adipose tissue; maximal levels were detected at the earliest time points studied for both inguinal and epididymal fat. In heart, however, LPL mRNA was detected at low levels 6 days before birth and increased 278-fold as the animals grew to adulthood.(ABSTRACT TRUNCATED AT 250 WORDS)

  7. Increased mRNA expression of selected antimicrobial peptides around ovulation and during inflammatory processes in the bovine endometrium postpartum.

    PubMed

    Ibrahim, M; Peter, S; Gärtner, M A; Michel, G; Jung, M; Einspanier, R; Gabler, C

    2016-11-01

    In the uterus, the first pathogen confrontations take place at the luminal endometrial epithelium. Therefore, it is required that these cells have the potential to recognize and respond to a bacterial infection. Antimicrobial peptides (AMP), part of the innate immune system in addition to cytokines, are principal effector molecules of mucosal immunity against pathogens. One important family of AMP that can permeabilize bacterial membranes is the beta-defensin (DEFB) family, which includes the following members: DEFB1, DEFB4A, and DEFB5, lingual AMP, and tracheal AMP. The bactericidal/permeability-increasing protein is also a cationic AMP that results in the death of bacteria. Another AMP family is the S100 calcium-binding protein (S100A) family including the following members: S100A8, S100A9, S100A11, and S100A12. These AMP exert their antimicrobial action through chelation of several ions. The aim of the present study was to evaluate mRNA expression patterns of selected AMP in bovine endometrial cells collected (1) at different stages of the estrous cycle (postovulatory, early-to-mid luteal, late luteal, and pre-ovulatory phase); (2) during the puerperium depending on uterine health status (healthy, subclinical, or clinical endometritis) starting on Day 24 to 30 postpartum for 3 weeks on a weekly basis; and (3) in vitro after co-culturing with Bacillus pumilus at three different multiplicities of infection (MOI 1, 5, and 10) up to 6 hours. The results reported that the mRNA expression of all candidate AMP, except DEFB1, S100A8, and S100A9, was estrous cycle dependent. In particular, around the time of ovulation, the transcription level of most AMP was higher (P < 0.05) compared with the luteal phase. Almost all candidate AMP mRNA expression was dependent on uterine health status, with a higher transcription level (P < 0.05) in inflamed endometrial tissues, especially during the late stage of the puerperium (Day 45-51 postpartum). Members of the DEFB family

  8. The expression of histamine H4 receptor mRNA in the skin and other tissues of normal dogs.

    PubMed

    Eisenschenk, Melissa N C; Torres, Sheila M F; Oliveira, Simone; Been, Clint S

    2011-10-01

    The histamine 4 (H(4)) receptor was first cloned and characterized in 2000 using the human H(3) receptor DNA sequence. The H(4) receptor has been shown to participate in various aspects of inflammation, such as chemotaxis, upregulation of adhesion molecule expression and modulation of cytokine secretion. The primary goal of this study was to determine whether H(4) receptor mRNA is expressed in normal canine skin by performing an RT-PCR. An additional goal was to determine the expression of this receptor in the colon, liver, spleen and kidney. Tissues were collected from five healthy, young-adult pit bull dogs. Samples were immediately placed in RNAlater(®) solution and stored at -20°C until processed. The amplified products in all skin samples in addition to the colon, liver, spleen and kidney (variable expression) had the expected size of 400-500 bp. The sequenced amplicons matched the National Center for Biotechnology Information published sequence for the canine H(4) receptor. The study results showed that canine normal skin expresses the H(4) receptor mRNA. Further studies using immunohistochemistry should be conducted to demonstrate the expression of the H(4) receptor at the protein level and to localize the expression of this receptor in the skin. PMID:21392139

  9. ProteoMirExpress: Inferring MicroRNA and Protein-centered Regulatory Networks from High-throughput Proteomic and mRNA Expression Data*

    PubMed Central

    Qin, Jing; Li, Mulin Jun; Wang, Panwen; Wong, Nai Sum; Wong, Maria P.; Xia, Zhengyuan; Tsao, George S. W.; Zhang, Michael Q.; Wang, Junwen

    2013-01-01

    MicroRNAs (miRNAs) regulate gene expression through translational repression and RNA degradation. Recently developed high-throughput proteomic methods measure gene expression changes at protein level and therefore can reveal the direct effects of miRNAs' translational repression. Here, we present a web server, ProteoMirExpress, that integrates proteomic and mRNA expression data together to infer miRNA-centered regulatory networks. With both types of high-throughput data from the users, ProteoMirExpress is able to discover not only miRNA targets that have decreased mRNA, but also subgroups of targets with suppressed proteins whose mRNAs are not significantly changed or with decreased mRNA whose proteins are not significantly changed, which are usually ignored by most current methods. Furthermore, both direct and indirect targets of miRNAs can be detected. Therefore, ProteoMirExpress provides more comprehensive miRNA-centered regulatory networks. We used several published data to assess the quality of our inferred networks and prove the value of our server. ProteoMirExpress is available online, with free access to academic users. PMID:23924514

  10. High-throughput differential screening of mRNAs by serial analysis of gene expression: decreased expression of trefoil factor 3 mRNA in thyroid follicular carcinomas.

    PubMed

    Takano, T; Miyauchi, A; Yoshida, H; Kuma, K; Amino, N

    2004-04-19

    To find mRNAs whose expression differs between thyroid follicular adenomas and carcinomas, a high-throughput analysis of mRNAs in these two tumours was performed. This method, named high-throughput differential screening by serial analysis of gene expression (HDSS), combines a modified method of serial analysis of gene expression (SAGE) and real-time quantitative reverse transcription polymerase chain reaction (RT-PCR). A total of 40 candidate tag sequences that showed extremely different expression levels between a follicular carcinoma and a follicular adenoma in the SAGE analysis were analysed by real-time quantitative RT-PCR, using RNAs from an additional four typical follicular carcinomas and adenomas. One sequence tag that represents trefoil factor 3 (TFF3) mRNA showed a clear difference in its expression level between adenomas and carcinomas. The expression levels of TFF3 mRNA in 48 follicular adenomas and 29 follicular carcinomas were measured by real-time quantitative RT-PCR using a specific probe for TFF3. They were significantly decreased in follicular carcinomas, especially in widely invasive types and those with evident metastases. These results indicate that the decreased expression of TFF3 mRNA is a marker of follicular carcinomas, especially those with a high risk of invasion or metastasis.

  11. Structure and expression of the human L-myc gene reveal a complex pattern of alternative mRNA processing

    SciTech Connect

    Kaye, F.; Battey, J.; Nau, M.; Brooks, B.; Seifter, E.; De Greve, J.; Birrer, M.; Sausville, E.; Minna, J.

    1988-01-01

    The authors' analyzed in detail the structure of the L-myc gene isolated from human placental DNA and characterized its expression in several small-cell lung cancer cell lines. The gene is composed of three exons and two introns spanning 6.6 kilobases in human DNA. Several distinct mRNA species are produced in all small-cell lung cancer cell lines that express L-myc. These transcripts are generated from a single gene by alternative splicing of introns 1 and 2 and by use of alternative polyadenylation signals. In some mRNAs that is a long open reading frame with a predicted translated protein of 364 residues. Amino acid sequence comparison with c-myc and N-myc demonstrated multiple discrete regions with extensive homology. In contrast, other mRNA transcripts, generated by alternative processing, could encode a truncated protein with a novel carboxy-terminal end.

  12. Expression of insulin-like growth factors at mRNA levels during the metamorphic development of turbot (Scophthalmus maximus).

    PubMed

    Meng, Zhen; Hu, Peng; Lei, Jilin; Jia, Yudong

    2016-09-01

    Insulin-like growth factors I and II (IGF-I and IGF-II) are important regulators of vertebrate growth and development. This study characterized the mRNA expressions of igf-i and igf-ii during turbot (Scophthalmus maximus) metamorphosis to elucidate the possible regulatory role of the IGF system in flatfish metamorphosis. Results showed that the mRNA levels of igf-i significantly increased at the early-metamorphosis stage and then gradually decreased until metamorphosis was completed. By contrast, mRNA levels of igf-ii significantly increased at the pre-metamorphosis stage and then substantially decreased during metamorphosis. Meanwhile, the whole-body thyroxine (T4) levels varied during larval metamorphosis, and the highest value was observed in the climax-metamorphosis. The mRNA levels of igf-i significantly increased and decreased by T4 and thiourea (TU, inhibitor of endogenous thyroid hormone) during metamorphosis, respectively. Conversely, the mRNA levels of igf-ii remained unchanged. Furthermore, TU significantly inhibited the T4-induced mRNA up-regulation of igf-i during metamorphosis. The whole-body thyroxine (T4) levels were significantly increased and decreased by T4 and TU during metamorphosis, respectively. These results suggested that igf-i and igf-ii may play different functional roles in larval development stages, and igf-i may have a crucial function in regulating the early metamorphic development of turbot. These findings may enhance our understanding of the potential roles of the IGF system to control flatfish metamorphosis and contribute to the improvement of broodstock management for larvae.

  13. Expression of insulin-like growth factors at mRNA levels during the metamorphic development of turbot (Scophthalmus maximus).

    PubMed

    Meng, Zhen; Hu, Peng; Lei, Jilin; Jia, Yudong

    2016-09-01

    Insulin-like growth factors I and II (IGF-I and IGF-II) are important regulators of vertebrate growth and development. This study characterized the mRNA expressions of igf-i and igf-ii during turbot (Scophthalmus maximus) metamorphosis to elucidate the possible regulatory role of the IGF system in flatfish metamorphosis. Results showed that the mRNA levels of igf-i significantly increased at the early-metamorphosis stage and then gradually decreased until metamorphosis was completed. By contrast, mRNA levels of igf-ii significantly increased at the pre-metamorphosis stage and then substantially decreased during metamorphosis. Meanwhile, the whole-body thyroxine (T4) levels varied during larval metamorphosis, and the highest value was observed in the climax-metamorphosis. The mRNA levels of igf-i significantly increased and decreased by T4 and thiourea (TU, inhibitor of endogenous thyroid hormone) during metamorphosis, respectively. Conversely, the mRNA levels of igf-ii remained unchanged. Furthermore, TU significantly inhibited the T4-induced mRNA up-regulation of igf-i during metamorphosis. The whole-body thyroxine (T4) levels were significantly increased and decreased by T4 and TU during metamorphosis, respectively. These results suggested that igf-i and igf-ii may play different functional roles in larval development stages, and igf-i may have a crucial function in regulating the early metamorphic development of turbot. These findings may enhance our understanding of the potential roles of the IGF system to control flatfish metamorphosis and contribute to the improvement of broodstock management for larvae. PMID:27255364

  14. The effects of a single bout pilates exercise on mRNA expression of bone metabolic cytokines in osteopenia women

    PubMed Central

    Kim, Chang Sun; Kim, Ji Yeon; Kim, Hyo Jin

    2014-01-01

    [Purpose] The purpose of this study was to examine the effect of a single bout pilates exercise on mRNA expression of bone metabolic cytokines in elderly osteopenia women. [Methods] We selected 11 people of elderly osteopenia women and loaded a single bout pilates exercise about RPE 10-14 level. The blood samples were collected before, immediately after and 60 minute after pilates exercise, then examined calcium metabolic markers in serum and extracted peripheral blood mononuclear cell (PBMC) from whole blood and confirmed mRNA expression of bone metabolic cytokines from PBMC. To clarify the changes during exercise, we designed repeated measure ANOVA as the control group to perform blood sampling without exercise. [Results] As a result, serum P showed significant interaction effect between group and time (p<.001), the pilates exercise group decreased about 9% at immediately after exercise and 13% during recovery after exercise (p<.05), while the control group showed a tendency to increase. Serum CK also showed a significant interaction between group and time (p<.05), the pilates group significantly increased at immediately after exercise and during recovery after exercise (p<.05) but the control group didn’t have changes. TNF-α and IL-6 mRNA expression in PBMC was significantly increased in the pilates group (p<.01, p<.05), although INF-γ mRNA expression didn’t show statistically significant difference, it tended to increase in the pilates group (NS). [Conclusion] These results suggested that a single bout pilates exercise of elderly osteopenia women cause hypophosphatemia with temporary muscle damage, and it leading high turnover bone metabolic state with to activate both of bone formation and bone resorption. PMID:25566441

  15. Effects of simulated microgravity on microRNA and mRNA expression profile of rat soleus

    NASA Astrophysics Data System (ADS)

    Xu, Hongjie; Wu, Feng; Cao, Hongqing; Kan, Guanghan; Zhang, Hongyu; Yeung, Ella W.; Shang, Peng; Dai, Zhongquan; Li, Yinghui

    2015-02-01

    Spaceflight induces muscle atrophy but mechanism is not well understood. Here, we quantified microRNAs (miRNAs) and mRNA shifts of rat soleus in response to microgravity. MiRNAs and mRNA microarray of soleus after tail suspension (TS) for 7 and 14 days were performed followed by target gene and function annotation analysis and qRT-PCR. Relative muscle mass lost by 37.0% in TS-7 but less than 10% in the following three weeks. TS altered 23 miRNAs and 1313 mRNAs with at least 2-fold. QRT-PCR confirmed some of these changes. MiR-214, miR-486-5p and miR-221 continuously decreased. MiR-674 and Let-7e decreased only in TS-7, while miR-320b and miR-187 decreased only in TS-14. But there was no alteration of miR-320 and miR-206 in both time point. For mRNA detection, actn3 (5.1-fold and 13.8-fold) and myh4 (38-fold and 51.6-fold) increased abundantly and a3galt2 decreased. Predicted targeted genes (whyz, ywhaz and SFRP2) of altered miRNAs decreased. GO terms and cellular pathway of these alteration showed enrichment in regulation of muscle metabolism. Integration analysis of the miRNA and mRNA expression profiles confirmed that eleven genes were differently regulated by four miRNAs. This is the first study that showed expression pattern and synergistical regulation of miRNA and mRNA in rat soleus of TS for up to 14 days.

  16. Effects of ketamine exposure on dopamine concentrations and dopamine type 2 receptor mRNA expression in rat brain tissue

    PubMed Central

    Li, Bing; Liu, Mei-Li; Wu, Xiu-Ping; Jia, Juan; Cao, Jie; Wei, Zhi-Wen; Wang, Yu-Jin

    2015-01-01

    Objective: To explore the effects of ketamine abuse on the concentration of dopamine (DA), a monoamine neurotransmitter, and the mRNA expression of dopamine type 2 (D2) receptors in brain tissue, we used male Wistar rats to model ketamine abuse through chronic intraperitoneal infusion of ketamine across different doses. Methods: The rats were sacrificed 45 minutes and 1, 2, and 3 weeks after initiating the administration of ketamine or normal saline, as well as 3 days following discontinuation. Brain tissue was harvested to examine the concentration of 2,5-dihydroxyphenylacetic acid and homovanillic acid, the primary metabolites of DA, as well as the expression of D2 receptor mRNA. In addition, behavioral changes were observed within 30 minutes of administration, and withdrawal symptoms were also documented. A factorial experimental design was used to investigate variations and correlations in the primary outcome measures across the four doses and five time points. Brain DA concentrations were significantly higher in the ketamine-treated groups compared with the saline-treated group, with 30 mg/kg > 10 mg/kg > 60 mg/kg > saline (P < 0.05). The D2 receptor mRNA expression exhibited an inverse downregulation pattern, with 30 mg/kg < 10 mg/kg < 60 mg/kg < saline (P < 0.05). In the 10 mg/kg and 30 mg/kg ketamine-treated groups, the DA concentration and D2 receptor mRNA level in the brain tissue correlated with the dose of ketamine (r = 0.752, r = -0.806), but no significant correlation was found in the 60 mg/kg group. Result: These findings indicated that chronic dosing with ketamine increased the concentration of DA in rat brain tissue by increasing DA release or interrupting DA degradation. D2 receptor mRNA expression likely decreased because of stimulation with excessive DA. Conclusion: High-dose (60 mg/kg) ketamine had potent paralyzing effects on the central nervous system of rats and weakened the excitatory effects of the limbic system. Brain DA and D2 receptor mRNA

  17. Decreased HLA-DR antigen-associated invariant chain (CD74) mRNA expression predicts mortality after septic shock

    PubMed Central

    2013-01-01

    Introduction Septic syndromes remain the leading cause of mortality in intensive care units (ICU). Septic patients rapidly develop immune dysfunctions, the intensity and duration of which have been linked with deleterious outcomes. Decreased mRNA expressions of major histocompatibility complex (MHC) class II-related genes have been reported after sepsis. We investigated whether their mRNA levels in whole blood could predict mortality in septic shock patients. Methods A total of 93 septic shock patients were included. On the third day after shock, the mRNA expressions of five MHC class II-related genes (CD74, HLA-DRA, HLA-DMB, HLA-DMA, CIITA) were measured by qRT-PCR and monocyte human leukocyte antigen-DR (mHLA-DR) by flow cytometry. Results A significant correlation was found among MHC class II related gene expressions. Among mRNA markers, the best prognostic value was obtained for CD74 (HLA-DR antigen-associated invariant chain). For this parameter, the area under the receiver operating characteristic curve (AUC) was calculated (AUC = 0.67, 95% confidence interval (CI) = 0.55 to 0.79; P = 0.01) as well as the optimal cut-off value. After stratification based on this threshold, survival curves showed that a decreased CD74 mRNA level was associated with increased mortality after septic shock (Log rank test, P = 0.0043, Hazard Ratio = 3.0, 95% CI: 1.4 to 6.5). Importantly, this association remained significant after multivariate logistic regression analysis including usual clinical confounders (that is, severity scores, P = 0.026, Odds Ratio = 3.4, 95% CI: 1.2 to 9.8). Conclusion Decreased CD74 mRNA expression significantly predicts 28-day mortality after septic shock. After validation in a larger multicentric study, this biomarker could become a robust predictor of death in septic patients. PMID:24321376

  18. The effect of manganese-induced toxicity on the cytokine mRNA expression of chicken spleen lymphocytes in vitro.

    PubMed

    Lu, Xinxing; Zhu, Yihao; Bai, Rensheng; Li, Shu; Teng, Xiaohua

    2015-08-01

    Manganese (Mn) is essential for life, but excess Mn exposure is harmful. This study investigated the effect of excess Mn on the cytokines of spleen lymphocytes in chicken. Lymphocytes were incubated with or without MnCl2 (2, 4, 6, and 8×10(-4) mmol/L) for 12, 24, 36, and 48 h, respectively. The mRNA expression of interleukin (IL) -2, IL-4, IL-6, IL-12β, and IL-17 and interferon (INF) -γ was examined using RT-PCR. Excess Mn inhibited IL-2, IL-4, IL-6, IL-12β, and IL-17 mRNA expression in chicken spleen lymphocytes in a dose-dependent manner. IFN-γ was inhibited by 8×10(-4) mmol/L Mn for 48 h. This study demonstrates that excess Mn affects cytokine mRNA expression and causes immunosuppression in chicken spleen lymphocytes. The relationships between IL-6 and IL-17 and between IL-2 and IL-12β were strong under immunosuppression caused by excess Mn in lymphocytes.

  19. Effects of alfalfa saponin extract on mRNA expression of Ldlr, LXRα, and FXR in BRL cells*

    PubMed Central

    Liang, Xin-ping; Zhang, Dong-qiang; Chen, Yan-yan; Guo, Rui; Wang, Jie; Wang, Cheng-zhang; Shi, Ying-hua

    2015-01-01

    We studied the effects of alfalfa saponin extract (ASE) on low density lipoprotein receptor (Ldlr), liver X receptor α (LXRα), and farnesoid X receptor (FXR) in normal and hyperlipidemic Buffalo rat liver (BRL) cells. Normal and hyperlipidemic BRL cells were divided into eight groups: normal, or normal cells treated with 50, 100, and 150 mg/L ASE, hyperlipidemic, or hyperlipidemic cells treated with 50, 100, and 150 mg/L ASE. After treatment for 24 h, Ldlr, LXRα, and FXR mRNA expression levels were measured by quantitative real-time polymerase chain reaction (qRT-PCR). Data showed that mRNA expression of Ldlr in normal BRL cells was significantly up-regulated by ASE treatment and mRNA expressions of LXRα and FXR were significantly down-regulated both in normal and hyperlipidemic BRL cells after ASE treatment. Thus, ASE might ameliorate hepatic steatosis by regulating genes involved in cholesterol metabolism, including up-regulation of Ldlr as well as down-regulation of LXRα and FXR. PMID:26055909

  20. Chronic stress alters glucocorticoid receptor and mineralocorticoid receptor mRNA expression in the European starling (Sturnus vulgaris) brain.

    PubMed

    Dickens, M; Romero, L M; Cyr, N E; Dunn, I C; Meddle, S L

    2009-10-01

    Although the glucocorticoid response to acute short-term stress is an adaptive physiological mechanism that aids in the response to and survival of noxious stimuli, chronic stress is associated with a negative impact on health. In wild-caught European starlings (Sturnus vulgaris), chronic stress alters the responsiveness of hypothalamic-pituitary-adrenal (HPA) axis as measured by the acute corticosterone response. In the present study, we investigated potential underlying neuroendocrine mechanisms by comparing glucocorticoid receptor and mineralocorticoid receptor mRNA expression in the brains of chronically and nonchronically-stressed starlings. Hypothalamic paraventricular nucleus, but not hippocampal, glucocorticoid receptor mRNA expression in chronically-stressed birds was significantly lower compared to controls, suggesting changes in the efficacy of corticosterone negative feedback. In addition, chronically-stressed birds showed a significant decrease in hippocampal MR mRNA expression. Together, these results suggest that chronic stress changes the brain physiology of wild birds and provides important information for the understanding of the underlying mechanisms that result in dysregulation of the HPA axis in wild animals by chronic stress. PMID:19686439

  1. Characteristics of mRNA dynamic expression related to spinal cord ischemia/reperfusion injury: a transcriptomics study.

    PubMed

    Qi, Zhi-Ping; Xia, Peng; Hou, Ting-Ting; Li, Ding-Yang; Zheng, Chang-Jun; Yang, Xiao-Yu

    2016-03-01

    Following spinal cord ischemia/reperfusion injury, an endogenous damage system is immediately activated and participates in a cascade reaction. It is difficult to interpret dynamic changes in these pathways, but the examination of the transcriptome may provide some information. The transcriptome reflects highly dynamic genomic and genetic information and can be seen as a precursor for the proteome. We used DNA microarrays to measure the expression levels of dynamic evolution-related mRNA after spinal cord ischemia/reperfusion injury in rats. The abdominal aorta was blocked with a vascular clamp for 90 minutes and underwent reperfusion for 24 and 48 hours. The simple ischemia group and sham group served as controls. After rats had regained consciousness, hindlimbs showed varying degrees of functional impairment, and gradually improved with prolonged reperfusion in spinal cord ischemia/reperfusion injury groups. Hematoxylin-eosin staining demonstrated that neuronal injury and tissue edema were most severe in the 24-hour reperfusion group, and mitigated in the 48-hour reperfusion group. There were 8,242 differentially expressed mRNAs obtained by Multi-Class Dif in the simple ischemia group, 24-hour and 48-hour reperfusion groups. Sixteen mRNA dynamic expression patterns were obtained by Serial Test Cluster. Of them, five patterns were significant. In the No. 28 pattern, all differential genes were detected in the 24-hour reperfusion group, and their expressions showed a trend in up-regulation. No. 11 pattern showed a decreasing trend in mRNA whereas No. 40 pattern showed an increasing trend in mRNA from ischemia to 48 hours of reperfusion, and peaked at 48 hours. In the No. 25 and No. 27 patterns, differential expression appeared only in the 24-hour and 48-hour reperfusion groups. Among the five mRNA dynamic expression patterns, No. 11 and No. 40 patterns could distinguish normal spinal cord from pathological tissue. No. 25 and No. 27 patterns could distinguish simple

  2. Profiles of mRNA expression for prolactin, growth hormone, and somatolactin in Japanese eels, Anguilla japonica: The effect of salinity, silvering and seasonal change.

    PubMed

    Sudo, Ryusuke; Suetake, Hiroaki; Suzuki, Yuzuru; Aoyama, Jun; Tsukamoto, Katsumi

    2013-01-01

    For understanding the functions of the growth hormone (GH)/prolactin (PRL)/somatolactin (SL) family of hormones, we examined pituitary mRNA expression of these hormones in anguillid eels in relation to salinity difference, silvering, and seasonal change. Female Japanese eels (Anguilla japonica) were collected in the brackish Hamana Lake and its freshwater rivers from July to December. To clarify the effect of salinity, the habitat use history of the eels were determined using otolith microchemistry. Expression levels of mRNA of each hormone were determined using real time PCR. Although GH and PRL have been known to be osmoregulatory hormones, there were no consistent differences in expression levels of these hormones between different salinity habitats. In contrast, SL mRNA expression was higher in eels from freshwater rivers than from the brackish lake. GH mRNA expression clearly decreased during silvering, whereas PRL and SL mRNA expression did not change. We also showed that PRL mRNA and SL mRNA decreased in the brackish lake and PRL mRNA increased in freshwater rivers from autumn to early winter. These findings provide basic knowledge for a further understanding of the role of these hormones. PMID:23047050

  3. Leptin and cholecystokinin in Schizothorax prenanti: molecular cloning, tissue expression, and mRNA expression responses to periprandial changes and fasting.

    PubMed

    Yuan, Dengyue; Wang, Tao; Zhou, Chaowei; Lin, Fangjun; Chen, Hu; Wu, Hongwei; Wei, Rongbin; Xin, Zhiming; Li, Zhiqiong

    2014-08-01

    In the present study, full-length cDNA sequences of leptin and cholecystokinin (CCK) were cloned from Schizothorax prenanti (S. prenanti), and applied real-time quantitative PCR to characterize the tissue distribution, and appetite regulatory effects of leptin and CCK in S. prenanti. The S. prenanti leptin and CCK full-length cDNA sequences were 1121 bp and 776 bp in length, encoding the peptide of 171 and 123 amino acid residues, respectively. Tissue distribution analysis showed that leptin mRNA was mainly expressed in the liver of S. prenanti. CCK was widely expressed, with the highest levels of expression in the hypothalamus, myelencephalon, telencephalon and foregut of S. prenanti. The CCK mRNA expression was highly elevated after feeding, whereas the leptin mRNA expression was not affected by single meal. These results suggested that CCK is a postprandial satiety signal in S. prenanti, but leptin might not be. In present study, leptin and CCK gene expression were both decreased after fasting and increased after refeeding, which suggested leptin and CCK might be involved in regulation of appetite in S. prenanti. This study provides an essential groundwork to further elucidate the appetite regulatory systems of leptin and CCK in S. prenanti as well as in other teleosts.

  4. ABC-Transporter Expression Does Not Correlate with Response to Irinotecan in Patients with Metastatic Colorectal Cancer

    PubMed Central

    Trumpi, K.; Emmink, B.L.; Prins, A.M.; van Oijen, M.G.H.; van Diest, P.J.; Punt, C.J.A.; Koopman, M.; Kranenburg, O.; Rinkes, I.H.M. Borel

    2015-01-01

    Background: Active efflux of irinotecan by ATP-binding cassette (ABC)-transporters, in particular ABCB1 and ABCG2, is a well-established drug resistance mechanism in vitro and in pre-clinical mouse models, but its relevance in colorectal cancer (CRC) patients is unknown. Therefore, we assessed the association between ABC-transporter expression and tumour response to irinotecan in patients with metastatic CRC. Methods: Tissue microarrays of a large cohort of metastatic CRC patients treated with irinotecan in a prospective study (CAIRO study; n=566) were analysed for expression of ABCB1 and ABCG2 by immunohistochemistry. Kaplan-Meier and Cox proportional hazard regression analyses were performed to assess the association of ABC transporter expression with irinotecan response. Gene expression profiles of 17 paired tumours were used to assess the concordance of ABCB1/ABCG2 expression in primary CRC and corresponding metastases. Results: The response to irinotecan was not significantly different between primary tumours with positive versus negative expression of ABCB1 (5.8 vs 5.7 months, p=0.696) or ABCG2 (5.7 vs 6.1 months, p=0.811). Multivariate analysis showed neither ABCB1 nor ABCG2 were independent predictors for progression free survival. There was a mediocre to poor concordance between ABC-transporter expression in paired tumours. Conclusion: In metastatic CRC, ABC-transporter expression in the primary tumour does not predict irinotecan response. PMID:26516354

  5. Effects of starter feeding and early weaning on GHR mRNA expression in liver and rumen of lambs from birth to 84 days of age.

    PubMed

    Wang, Fangbin; Li, Chong; Li, Fadi; Wang, Weimin; Wang, Xiaojuan; Liu, Ting; Ma, Zhiyuan; Li, Baosheng

    2016-01-01

    The growth hormone receptor (GHR) is associated with animal growth and development. To investigate such effects on GHR gene expression, a total of 102 Hu lambs were randomly allocated to one of three groups (Group 1: starter diet from 7 d of age, weaning at 56 d of age; Group 2: starter diet from 42 d of age, weaning at 56 d of age; Group 3: starter diet from 7 d of age; weaning at 28 d of age). Six lambs from each group were sacrificed every 14 d to investigate the effects of starter feeding and weaning age on GHR mRNA expression in the liver and rumen. The results revealed that GHR mRNA expression was significantly higher in the liver and rumen (p < 0.05) than in other tissues. Early starter feeding up-regulated hepatic GHR mRNA expression on days 14, 28, 42 and 56 and ruminal GHR mRNA expression on days 28, 42, 70, and 84 (p < 0.05). Early weaning up-regulated hepatic GHR mRNA expression on days 56, 70 and 84 and ruminal GHR mRNA expression on days 42, 56, 70 and 84 (p < 0.05). Dietary and weaning regimes and age affected the hepatic and ruminal GHR mRNA expression. PMID:27032032

  6. Effects of starter feeding and early weaning on GHR mRNA expression in liver and rumen of lambs from birth to 84 days of age.

    PubMed

    Wang, Fangbin; Li, Chong; Li, Fadi; Wang, Weimin; Wang, Xiaojuan; Liu, Ting; Ma, Zhiyuan; Li, Baosheng

    2016-01-01

    The growth hormone receptor (GHR) is associated with animal growth and development. To investigate such effects on GHR gene expression, a total of 102 Hu lambs were randomly allocated to one of three groups (Group 1: starter diet from 7 d of age, weaning at 56 d of age; Group 2: starter diet from 42 d of age, weaning at 56 d of age; Group 3: starter diet from 7 d of age; weaning at 28 d of age). Six lambs from each group were sacrificed every 14 d to investigate the effects of starter feeding and weaning age on GHR mRNA expression in the liver and rumen. The results revealed that GHR mRNA expression was significantly higher in the liver and rumen (p < 0.05) than in other tissues. Early starter feeding up-regulated hepatic GHR mRNA expression on days 14, 28, 42 and 56 and ruminal GHR mRNA expression on days 28, 42, 70, and 84 (p < 0.05). Early weaning up-regulated hepatic GHR mRNA expression on days 56, 70 and 84 and ruminal GHR mRNA expression on days 42, 56, 70 and 84 (p < 0.05). Dietary and weaning regimes and age affected the hepatic and ruminal GHR mRNA expression.

  7. Relation between mRNA expression and sequence information in Desulfovibrio vulgaris: Combinatorial contributions of upstream regulatory motifs and coding sequence features to variations in mRNA abundance

    SciTech Connect

    Wu, Gang; Nie, Lei; Zhang, Weiwen

    2006-05-26

    ABSTRACT-The context-dependent expression of genes is the core for biological activities, and significant attention has been given to identification of various factors contributing to gene expression at genomic scale. However, so far this type of analysis has been focused whether on relation between mRNA expression and non-coding sequence features such as upstream regulatory motifs or on correlation between mRN abundance and non-random features in coding sequences (e.g. codon usage and amino acid usage). In this study multiple regression analyses of the mRNA abundance and all sequence information in Desulfovibrio vulgaris were performed, with the goal to investigate how much coding and non-coding sequence features contribute to the variations in mRNA expression, and in what manner they act together...

  8. mRNA expression of diacylglycerol kinase isoforms in insulin-sensitive tissues: effects of obesity and insulin resistance.

    PubMed

    Mannerås-Holm, Louise; Kirchner, Henriette; Björnholm, Marie; Chibalin, Alexander V; Zierath, Juleen R

    2015-04-01

    Diacylglycerol kinase (DGK) isoforms regulate signal transduction and lipid metabolism. DGKδ deficiency leads to hyperglycemia, peripheral insulin resistance, and metabolic inflexibility. Thus, dysregulation of other DGK isoforms may play a role in metabolic dysfunction. We investigated DGK isoform mRNA expression in extensor digitorum longus (EDL) and soleus muscle, liver as well as subcutaneous and epididymal adipose tissue in C57BL/6J mice and obese and insulin-resistant ob/ob mice. All DGK isoforms, except for DGKκ, were detectable, although with varying mRNA expression. Liver DGK expression was generally lowest, with several isoforms undetectable. In soleus muscle, subcutaneous and epididymal adipose tissue, DGKδ was the most abundant isoform. In EDL muscle, DGKα and DGKζ were the most abundant isoforms. In liver, DGKζ was the most abundant isoform. Comparing obese insulin-resistant ob/ob mice to lean C57BL/6J mice, DGKβ, DGKι, and DGKθ were increased and DGKε expression was decreased in EDL muscle, while DGKβ, DGKη and DGKθ were decreased and DGKδ and DGKι were increased in soleus muscle. In liver, DGKδ and DGKζ expression was increased in ob/ob mice. DGKη was increased in subcutaneous fat, while DGKζ was increased and DGKβ, DGKδ, DGKη and DGKε were decreased in epididymal fat from ob/ob mice. In both adipose tissue depots, DGKα and DGKγ were decreased and DGKι was increased in ob/ob mice. In conclusion, DGK mRNA expression is altered in an isoform- and tissue-dependent manner in obese insulin-resistant ob/ob mice. DGK isoforms likely have divergent functional roles in distinct tissues, which may contribute to metabolic dysfunction. PMID:25847921

  9. Molecular cloning, mRNA expression, and characterization of HSP90 gene from Chinese mitten crab Eriocheir japonica sinensis.

    PubMed

    Li, Peng; Zha, Jie; Zhang, Zhenhua; Huang, Hua; Sun, Hongying; Song, Daxiang; Zhou, Kaiya

    2009-07-01

    HSP90 is a highly conserved molecular chaperone important in the maturation of a broad spectrum of proteins. Using expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) techniques, an HSP90 gene designated as EjsHSP90 was cloned and characterized from the Chinese mitten crab Eriocheir japonica sinensis. The full-length cDNA of EjsHSP90 is 2,517 bp and contains an open reading frame of 2157 bp which encodes a 718 amino acid polypeptide (82.8 kDa) bearing characteristics of the HSP90 family and an ATP binding domain. Sequence alignment shows that EjsHSP90 shared 79%-96% identity with HSP90 sequences reported in other animals, and it shares identical structural features. Fluorescent real-time quantitative RT-PCR approach was performed to examine the expression profiles of EjsHSP90 mRNA by testing its relative level in three types of tissues at three different developmental stages, respectively. We found that EjsHSP90 is expressed throughout the three developmental stages but expression levels varied among different body parts of crabs. EjsHSP90 mRNA expression in the abdomen of the first crab stage is consistently higher than that of the other two stages, suggesting that EjsHSP90 gene is involved in the crabs' early developmental process, especially in the crab brachyurization process. Results from quantitative RT-PCR excluded the possibility that the expression of EjsHSP90 mRNA is induced primarily by osmotic stress. Phylogenetic analyses reveal that HSP90 gene is informative and complementary for reconstruction of arthropod phylogenetic relationships. PMID:19166961

  10. Expression of RFamide-Related Peptide-3 (RFRP-3) mRNA in Dorsomedial Hypothalamic Nucleus and KiSS-1 mRNA in Arcuate Nucleus of Rat during Pregnancy

    PubMed Central

    Sabet Sarvestani, Fatemeh; Tamadon, Amin; Koohi-Hosseinabadi, Omid; Mohammadi Nezhad, Saeed; Rahmanifar, Farhad; Jafarzadeh Shirazi, Mohammad Reza; Tanideh, Nader; Moghadam, Ali; Niazi, Ali

    2014-01-01

    Background RFamide-related peptide-3 (RFRP-3) and kisspeptin (KiSS-1) are known to respectively inhibit and stimulate gonadotropin releasing hormone (GnRH) and lute- inizing hormone (LH) secretion in rat. The aim of the present study was to evaluate the relative mRNA expression of RFRP-3 and KiSS-1 in the hypothalamus of pregnant rats. Materials and Methods In a randomized controlled experimental study, the exact preg- nancy day of 18 Sprague-Dawley rats were confirmed using the vaginal smear method and were equally assigned to three groups of days 7, 14 and 21 of pregnancy. Four non- pregnant female rats were ovariectomized and assigned as the control group. All rats were decapitated, and the dorsomedial hypothalamic nucleus (DMH) and the arcuate nucleus (ARC) for detection of KiSS-1 mRNA were separated from their hypothalamus to detect RFRP-3 and KiSS-1 mRNA respectively. Then, their relative expressions were compared between control and pregnant groups using real-time polymerase chain reac- tion (PCR). Results The relative expression of RFRP-3 mRNA in DMH did not change significantly during pregnancy (p>0.01). However, the relative expression of KiSS-1 mRNA in ARC was at its highest in day 7 of pregnancy and decreased until day 21 of pregnancy (p<0.01). Conclusion Decrease in GnRH and LH secretion during the pregnancy of rat may be controlled by constant expression of RFRP-3 mRNA and reduced expression of KiSS-1 mRNA in hypothalamus. PMID:25379163

  11. A Candida albicans gene expressed in Saccharomyces cerevisiae results in a distinct pattern of mRNA processing.

    PubMed

    Iborra, A; Sentandreu, R; Gozalbo, D

    1996-09-01

    Two plasmids (derived from YCplac22 and YEplac112) carrying a Candida albicans gene (including the 5' non-coding promoter sequences) coding for a 30 kDa membrane-bound protein, were used to transform Saccharomyces cerevisiae cells. A 30 kDa protein was immunodetected by Western blot in the membrane fraction of transformants. Northern analysis showed the presence of three mRNA species (of about 1.1, 0.7 and 0.5 kb) hybridizing with the C. albicans gene as a probe. The same result was obtained using the 5' and 3' regions of the gene as probes, whereas only a 1.1 kb mRNA was found in C. albicans and none was detected in S. cerevisiae control transformants. Thus, heterologous expression of this gene in S. cerevisiae results in a distinct pattern of mRNA processing, either due to the location on plasmid vectors and/or to differences in the mRNA processing systems in the two microorganisms.

  12. Changes in neurotransmitter levels and proinflammatory cytokine mRNA expressions in the mice olfactory bulb following nanoparticle exposure

    SciTech Connect

    Tin-Tin-Win-Shwe Mitsushima, Dai; Yamamoto, Shoji; Fukushima, Atsushi; Funabashi, Toshiya; Kobayashi, Takahiro; Fujimaki, Hidekazu

    2008-01-15

    Recently, there have been increasing reports that nano-sized component of particulate matter can reach the brain and may be associated with neurodegenerative diseases. Previously, our laboratory has studied the effect of intranasal instillation of nano-sized carbon black (CB) (14 nm and 95 nm) on brain cytokine and chemokine mRNA expressions and found that 14-nm CB increased IL-1{beta}, TNF-{alpha}, CCL2 and CCL3 mRNA expressions in the olfactory bulb, not in the hippocampus of mice. To investigate the effect of a single administration of nanoparticles on neurotransmitters and proinflammatory cytokines in a mouse olfactory bulb, we performed in vivo microdialysis and real-time PCR methods. Ten-week-old male BALB/c mice were implanted with guide cannula in the right olfactory bulb and, 1 week later, were instilled vehicle or CB (14 nm, 250 {mu}g) intranasally. Six hours after the nanoparticle instillation, the mice were intraperitoneally injected with normal saline or 50 {mu}g of bacteria cell wall component lipoteichoic acid (LTA), which may potentiate CB-induced neurologic effect. Extracellular glutamate and glycine levels were significantly increased in the olfactory bulb of CB-instilled mice when compared with vehicle-instilled control mice. Moreover, we found that LTA further increased glutamate and glycine levels. However, no alteration of taurine and GABA levels was observed in the olfactory bulb of the same mice. We also detected immunological changes in the olfactory bulb 11 h after vehicle or CB instillation and found that IL-1{beta} mRNA expression was significantly increased in CB- and LTA-treated mice when compared with control group. However, TNF-{alpha} mRNA expression was increased significantly in CB- and saline-treated mice when compared with control group. These findings suggest that nanoparticle CB may modulate the extracellular amino acid neurotransmitter levels and proinflammatory cytokine IL-1 {beta} mRNA expressions synergistically with LTA

  13. [Effects of PTK787 on cell proliferation and expression of fak mRNA in K562].

    PubMed

    Di, Xiao-Hua; Chen, Ri-Ling; Liu, Xiao-Li; Tian, Chuan; Guo, Ya-Nan

    2010-06-01

    The aim of this study was to investigate the effects of tyrosine kinase inhibitor PTK787 on cell proliferation, cell cycle and the expression of fak mRNA of human chronic myeloid leukemia (CML) cell line K562, and to explore the mechanism of PTK787 against acute myeloid leukemia. The MTT method was used to detect the effects of PTK787 in various concentrations and at different time points on proliferation of K562 cells; the flow cytometry was used to determine the effects of PTK787 in different concentrations on cell cycle of K562 cells; the RT-PCR was used to assay the expression of fak mRNA in K562 cells treated with PTK787 for 48 hours. The results showed that along with increasing of the concentration and prolonging of time, the inhibitory rate of PTK787 on K562 proliferation was gradually enhanced. The comparison between various concentration groups at same time or comparison between various time groups in same concentration showed significant differences (p < 0.05), in which the effect of 320 micromol/L PTK787 on cells was strongest, while the continuous increase of PTK787 concentration or prolong of action time did not enhance the inhibitory rate on K562 proliferation. With increasing of drug concentration, the cell proportion in G(1) phase gradually increased, the cell proportion in S phase gradually decreased, the comparison between various groups revealed significant differences (p < 0.05), however the continuous increase of drug concentration from 160 micromol/L did not obviously change the cell proportion in phases of cell cycle. With increasing of drug concentration, the expression of fak mRNA in K562 cells gradually reduced with significant differences between various groups (p < 0.05), but with continuous increase of drug concentration from 160 micromol/L, the effect of PTK787 on the expression of fak mRNA in K562 cells also did not obviously change. It is concluded that the PTK787 shows effect of anti-leukemia cells through inhibiting transformation

  14. Gene expression analysis in sections and tissue microarrays of archival tissues by mRNA in situ hybridization.

    PubMed

    Henke, R T; Maitra, A; Paik, S; Wellstein, A

    2005-01-01

    Altered expression of genes in diseased tissues can prognosticate a distinct natural progression of the disease as well as predict sensitivity or resistance to particular therapies. Archival tissues from patients with a known medical history and treatments are an invaluable resource to validate the utility of candidate genes for prognosis and prediction of therapy outcomes. However, stored tissues with associated long-term follow-up information typically are formalin-fixed, paraffin-embedded specimen and this can severely restrict the methods applicable for gene expression analysis. We report here on the utility of tissue microarrays (TMAs) that use valuable tissues sparingly and provide a platform for simultaneous analysis of gene expression in several hundred samples. In particular, we describe a stable method applicable to mRNA expression screening in such archival tissues. TMAs are constructed from sections of small drill cores, taken from tissue blocks of archival tissues and multiple samples can thus be arranged on a single microscope slide. We used mRNA in situ hybridization (ISH) on >500 full sections and >100 TMAs for >10 different cDNAs that yielded >10,000 data points. We provide detailed experimental protocols that can be implemented without major hurdles in a molecular pathology laboratory and discuss quantitative analysis and the advantages and limitations of ISH. We conclude that gene expression analysis in archival tissues by ISH is reliable and particularly useful when no protein detection methods are available for a candidate gene.

  15. Molecular beacons can assess changes in expression and 3'-polyadenylation of human eNOS mRNA.

    PubMed

    Jones, Rachel; Baker, Meredith B; Weber, Martina; Harrison, David G; Bao, Gang; Searles, Charles D

    2009-03-01

    The endothelium plays an essential role in maintaining vascular homeostasis, and it fulfills this role by modulating intracellular signaling and gene expression in response to chemical and mechanical stimuli. Assessing changes in endothelial gene expression is essential to understanding how physiological and pathophysiological processes modulate vascular homeostasis. Here we describe the use of molecular beacons to rapidly and quantitatively assess expression and 3'-polyadenylation of a gene that is important for vascular homeostasis, endothelial nitric oxide synthase (eNOS). Single- and dual-fluorescence resonance energy transfer (FRET) molecular beacon hybridization assays were developed to measure changes in mRNA levels and 3'-polyadenylation, respectively, in primary human endothelial cell cultures subjected to laminar shear stress or statin treatment. Optimized beacon hybridization assays took approximately 15 min to perform, and eNOS mRNA levels were validated by quantitative real-time RT-PCR. Competitive inhibition assays and posttranscriptional silencing of eNOS expression were used to verify the specificity of molecular beacon fluorescence. Finally, the dual-FRET method was used to assess eNOS polyadenylation in tissues isolated from mice subjected to exercise training. These data demonstrate that molecular beacons can be used to rapidly and efficiently measure endothelial gene expression and 3'-polyadenylation. This approach could easily be adapted for studies of other endothelial genes and has promise for applications in live endothelial cells.

  16. Catalog of mRNA expression patterns for DNA methylating and demethylating genes in developing mouse lower urinary tract.

    PubMed

    Keil, Kimberly P; Altmann, Helene M; Mehta, Vatsal; Abler, Lisa L; Elton, Erik A; Vezina, Chad M

    2013-12-01

    The mouse prostate develops from a component of the lower urinary tract (LUT) known as the urogenital sinus (UGS). This process requires androgens and signaling between mesenchyme and epithelium. Little is known about DNA methylation during prostate development, including which factors are expressed, whether their expression changes over time, and if DNA methylation contributes to androgen signaling or influences signaling between mesenchyme and epithelium. We used in situ hybridization to evaluate the spatial and temporal expression pattern of mRNAs which encode proteins responsible for establishing, maintaining or remodeling DNA methylation. These include DNA methyltransferases, DNA deaminases, DNA glycosylases, base excision repair and mismatch repair pathway members. The mRNA expression patterns were compared between male and female LUT prior to prostatic bud formation (14.5 days post coitus (dpc)), during prostatic bud formation (17.5 dpc) and during prostatic branching morphogenesis (postnatal day (P) 5). We found dramatic changes in the patterns of these mRNAs over the course of prostate development and identified examples of sexually dimorphic mRNA expression. Future investigation into how DNA methylation patterns are established, maintained and remodeled during the course of embryonic prostatic bud formation may provide insight into prostate morphogenesis and disease.

  17. Molecular cloning and mRNA expression of cyclophilin A gene in black tiger shrimp (Penaeus monodon).

    PubMed

    Qiu, Lihua; Jiang, Shigui; Huang, Jianhua; Wang, Weifang; Zhu, Caiyan; Su, Tianfeng

    2009-01-01

    The techniques of homology cloning and anchored PCR were used to clone the cyclophilin A (CypA) gene from black tiger shrimp (Penaeus monodon). The full-length cDNA of black tiger shrimp CypA (btsCypA) contained a 5' untranslated region (UTR) of 81 bp, an ORF (open reading frame) of 495 bp encoding a polypeptide of 164 amino acids with an estimated molecular mass of 17.68 kDa and a 3' UTR of 308 bp. The predicted amino acid sequence of btsCypA shared high identity with CypA in other organisms. A quantitative reverse transcriptase Real-Time PCR (qRT-PCR) assay was developed to assess the mRNA expression of btsCypA in different tissues and the temporal expression of btsCypA in the hepatopancreas challenged by lipopolyssacharide (LPS). Higher-level mRNA expression of btsCypA was detected in the tissues of hepatopancreas and blood. The expression of btsCypA in the hepatopancreas was up regulated after stimulated by LPS. The results indicated that btsCypA was a constitutive and inducible expressed protein and could be induced by LPS.

  18. Runx3 Expression Inhibits Proliferation and Distinctly Alters mRNA Expression of Bax in AGS and A549 Cancer Cells

    PubMed Central

    Torshabi, Maryam; Faramarzi, Mohammad Ali; Tabatabaei Yazdi, Mojtaba; Ostad, Seyyed Naser; Gharemani, Mohammad Hosein

    2011-01-01

    Runx3, a member of Runt-related transcription factor (Runx) proteins with tumor suppressor effect, is a tissue–restricted and cancer related transcription factor that regulate cell proliferation and growth, as well as differentiation. In the present study, exogenous Run3 was transiently expressed in AGS (human gastric adenocarcinoma), with undetectable Runx3 protein and in A549 (human lung carcinoma) with low levels of endogenous Runx3 protein. The GFP tagged Runx3 was transfected into AGS and A549 cells using fugene6 and PolyFect and Runx3 expression was confirmed by fluorescent microscopy and RT-PCR. The effect of Runx3 transfection on cell proliferation was determined by MTT assay and the results were confirmed by the trypan blue dye exclusion method. The effect of Runx3 expression on mRNA expression of BCL2-associated X protein (Bax) was evaluated using RT-PCR. In AGS and A549 cells, Runx3 expression inhibited cell proliferation (p < 0.01). The growth inhibition was less in A549 cells. We show that Runx3 expression increases Bax mRNA expression in AGS cells when compared with control (p < 0.05), but no significant differences in mRNA expression was observed in both examined cells. Runx3 expression has antiproliferative effect in AGS cell perhaps via increase in expression of Bax. The effect of Runx3 on A549 cells’ viability which has endogenous level of Runx3 is not related to Bax. These findings implicate a complex regulation by Runx3 in inhibition of cell proliferation utilizing Bax. PMID:24250365

  19. Effect of vitamin C and lipoic acid on streptozotocin-induced diabetes gene expression: mRNA and protein expressions of Cu-Zn SOD and catalase.

    PubMed

    Sadi, Gökhan; Yilmaz, Okkes; Güray, Tülin

    2008-02-01

    The involvement of oxidative stress in the pathogenesis of diabetes mellitus has been confirmed by numerous studies. In this study, the expression of two antioxidant enzymes, superoxide dismutase (SOD), and catalase which are involved in the detoxification of reactive oxygen species was studied in the streptozotocin-induced diabetic rat liver tissues. The enzyme assays showed a significant decrease in both enzymes activities compared to control animals. The RT-PCR and Western-blot analysis results demonstrated that this decrease in activity is regulated at the level of gene expression, as both catalase and Cu-Zn SOD mRNA and protein expressions were also suppressed. Supplementing the animals with vitamin C, a powerful antioxidant increased both SOD and catalase activities with no change in both mRNA and protein expressions suggesting a role of post-translational modification. However, even though mRNA expressions of both catalase and Cu-Zn SOD were not changed, the protein levels increased in parallel to activities in the case of another antioxidant, alpha-lipoic acid. An increase in the rate of translation, without changing the rate of transcription indicates a translational effect of lipoic acid in changing the activities of antioxidant enzymes to prevent the oxidative damage in diabetes.

  20. Differences in correlation of mRNA gene expression in mice sensitive and resistant to radiation-induced pulmonary fibrosis

    SciTech Connect

    Johnston, C.J.; Piedboeuf, B.; Finkelstein, J.N.; Baggs, R.; Rubin, P.

    1995-05-01

    Fibrosis, characterized by the accumulation of collagen, is a late result of thoracic irradiation. The purpose of this study was to determine if extracellular matrix protein and transforming growth factor {beta} mRNA expression are altered late in the course of pulmonary fibrosis after irradiation, and then to determine if these changes differ between two strains of mice which vary in their sensitivity to radiation. Radiation-sensitive (C57BL/6) and radiation-resistant (C3H/HeJ) mice were irradiated with a single dose of 5 or 12.5 Gy to the thorax. Total lung RNA was prepared and immobilized by Northern and slot blotting and hybridized with radiolabeled cDNA probes for collagens I, III and IV, fibronectin, and transforming growth factor {beta}{sub 1} and {beta}{sub 3}. Autoradiographic data were quantified by video densitometry and results normalized to a control probe encoding for glyceralde-hyde-3-phosphate dehydrogenase. Alterations in mRNA abundance were observed in the sensitive mice at all times, while levels in the resistant mice were unaffected until 26 weeks after irradiation. The relationship between extracellular matrix protein per se and increased mRNA abundance suggests that late matrix protein accumulation may be a function of gene expression. Differences in levels of transforming growth factor {beta}mRNA may lead to strain-dependent variation in fibrotic response and may also contribute to the radiation-induced component of pulmonary fibrosis. 32 refs., 5 figs.

  1. The Csr system regulates genome-wide mRNA stability and transcription and thus gene expression in Escherichia coli.

    PubMed

    Esquerré, Thomas; Bouvier, Marie; Turlan, Catherine; Carpousis, Agamemnon J; Girbal, Laurence; Cocaign-Bousquet, Muriel

    2016-04-26

    Bacterial adaptation requires large-scale regulation of gene expression. We have performed a genome-wide analysis of the Csr system, which regulates many important cellular functions. The Csr system is involved in post-transcriptional regulation, but a role in transcriptional regulation has also been suggested. Two proteins, an RNA-binding protein CsrA and an atypical signaling protein CsrD, participate in the Csr system. Genome-wide transcript stabilities and levels were compared in wildtype E. coli (MG1655) and isogenic mutant strains deficient in CsrA or CsrD activity demonstrating for the first time that CsrA and CsrD are global negative and positive regulators of transcription, respectively. The role of CsrA in transcription regulation may be indirect due to the 4.6-fold increase in csrD mRNA concentration in the CsrA deficient strain. Transcriptional action of CsrA and CsrD on a few genes was validated by transcriptional fusions. In addition to an effect on transcription, CsrA stabilizes thousands of mRNAs. This is the first demonstration that CsrA is a global positive regulator of mRNA stability. For one hundred genes, we predict that direct control of mRNA stability by CsrA might contribute to metabolic adaptation by regulating expression of genes involved in carbon metabolism and transport independently of transcriptional regulation.

  2. Effect of silicon dioxide on expression of poly (ADP-ribose) polymerase mRNA and protein.

    PubMed

    Gao, Ai; Song, Shanshan; Wang, Danlin; Peng, Wei; Tian, Lin

    2009-07-01

    Silicon dioxide induces acute injury and chronic pulmonary fibrosis. International Agency for Research on Cancer (IARC) listed it as a human carcinogen in 1996. However, the molecular mechanisms to induce cancer are not understood yet. The content of poly (ADP-ribose) polymerases (PARP) mRNA and protein in Hela cells treated with concentrations of silicon dioxide up to 400microg/ml was determined by real-time fluorogenetic quantitative PCR (RQ-PCR) and immunofluorescence assay, respectively. MTT assay was used to determine cell viability. The results showed that viability at 400microg/ml silica was significantly decreased but not at lower concentrations. The protein content of gamma-H2AX in silica-treated group was significantly higher than the controls. The PARP mRNA and protein levels were significantly reduced with a dose response manner from the lowest silicon dioxide level. Our findings suggested that silicon dioxide increased the expression of gamma-H2AX and inhibited the expression of PARP mRNA and protein in Hela cells.

  3. The Csr system regulates genome-wide mRNA stability and transcription and thus gene expression in Escherichia coli

    PubMed Central

    Esquerré, Thomas; Bouvier, Marie; Turlan, Catherine; Carpousis, Agamemnon J.; Girbal, Laurence; Cocaign-Bousquet, Muriel

    2016-01-01

    Bacterial adaptation requires large-scale regulation of gene expression. We have performed a genome-wide analysis of the Csr system, which regulates many important cellular functions. The Csr system is involved in post-transcriptional regulation, but a role in transcriptional regulation has also been suggested. Two proteins, an RNA-binding protein CsrA and an atypical signaling protein CsrD, participate in the Csr system. Genome-wide transcript stabilities and levels were compared in wildtype E. coli (MG1655) and isogenic mutant strains deficient in CsrA or CsrD activity demonstrating for the first time that CsrA and CsrD are global negative and positive regulators of transcription, respectively. The role of CsrA in transcription regulation may be indirect due to the 4.6-fold increase in csrD mRNA concentration in the CsrA deficient strain. Transcriptional action of CsrA and CsrD on a few genes was validated by transcriptional fusions. In addition to an effect on transcription, CsrA stabilizes thousands of mRNAs. This is the first demonstration that CsrA is a global positive regulator of mRNA stability. For one hundred genes, we predict that direct control of mRNA stability by CsrA might contribute to metabolic adaptation by regulating expression of genes involved in carbon metabolism and transport independently of transcriptional regulation. PMID:27112822

  4. Effect of long real space flight on the whole genome mRNA expression properties in medaka Oryzias latipes

    NASA Astrophysics Data System (ADS)

    Kozlova, Olga; Gusev, Oleg; Levinskikh, Margarita; Sychev, Vladimir; Poddubko, Svetlana

    The current study is addressed to the complex analysis of whole genome mRNA expression profile and properties of splicing variants formation in different organs of medaka fish exposed to prolonged space flight in the frame of joint Russia-Japan research program “Aquarium-AQH”. The fish were kept in the AQH joint-aquariums system in October-December 2013, followed by fixation in RNA-preserving buffers and freezing during the space flight. The samples we returned to the Earth frozen in March 2013 and mRNAs from four fish were sequenced in organ-specific manner using HiSeq Illumina sequencing platform. The ground group fish treated in the same way was used as a control. The comparison between the groups revealed space group-specific specific mRNA expression pattern. More than 50 genes (including several types of myosins) were down-regulated in the space group. Moreover, we found an evidence for formation of space group-specific splicing variants of mRNA. Taking together, the data suggest that in spite of aquatic environment, space flight-associated factors have a strong effect on the activity of fish genome. This work was supported in part by subsidy of the Russian Government to support the Program of competitive growth of Kazan Federal University among world class academic centres and universities.

  5. Integration of mRNA expression profile, copy number alterations, and microRNA expression levels in breast cancer to improve grade definition.

    PubMed

    Cava, Claudia; Bertoli, Gloria; Ripamonti, Marilena; Mauri, Giancarlo; Zoppis, Italo; Della Rosa, Pasquale Anthony; Gilardi, Maria Carla; Castiglioni, Isabella

    2014-01-01

    Defining the aggressiveness and growth rate of a malignant cell population is a key step in the clinical approach to treating tumor disease. The correct grading of breast cancer (BC) is a fundamental part in determining the appropriate treatment. Biological variables can make it difficult to elucidate the mechanisms underlying BC development. To identify potential markers that can be used for BC classification, we analyzed mRNAs expression profiles, gene copy numbers, microRNAs expression and their association with tumor grade in BC microarray-derived datasets. From mRNA expression results, we found that grade 2 BC is most likely a mixture of grade 1 and grade 3 that have been misclassified, being described by the gene signature of either grade 1 or grade 3. We assessed the potential of the new approach of integrating mRNA expression profile, copy number alterations, and microRNA expression levels to select a limited number of genomic BC biomarkers. The combination of mRNA profile analysis and copy number data with microRNA expression levels led to the identification of two gene signatures of 42 and 4 altered genes (FOXM1, KPNA4, H2AFV and DDX19A) respectively, the latter obtained through a meta-analytical procedure. The 42-based gene signature identifies 4 classes of up- or down-regulated microRNAs (17 microRNAs) and of their 17 target mRNA, and the 4-based genes signature identified 4 microRNAs (Hsa-miR-320d, Hsa-miR-139-5p, Hsa-miR-567 and Hsa-let-7c). These results are discussed from a biological point of view with respect to pathological features of BC. Our identified mRNAs and microRNAs were validated as prognostic factors of BC disease progression, and could potentially facilitate the implementation of assays for laboratory validation, due to their reduced number. PMID:24866763

  6. Occupational Toluene Exposure Induces Cytochrome P450 2E1 mRNA Expression in Peripheral Lymphocytes

    PubMed Central

    Mendoza-Cantú, Ania; Castorena-Torres, Fabiola; de León, Mario Bermúdez; Cisneros, Bulmaro; López-Carrillo, Lizbeth; Rojas-García, Aurora E.; Aguilar-Salinas, Alberto; Manno, Maurizio; Albores, Arnulfo

    2006-01-01

    Print workers are exposed to organic solvents, of which the systemic toxicant toluene is a main component. Toluene induces expression of cytochrome P450 2E1 (CYP2E1), an enzyme involved in its own metabolism and that of other protoxicants, including some procarcinogens. Therefore, we investigated the association between toluene exposure and the CYP2E1 response, as assessed by mRNA content in peripheral lymphocytes or the 6-hydroxychlorzoxazone (6OH-CHZ)/chlorzoxazone (CHZ) quotient (known as CHZ metabolic ratio) in plasma, and the role of genotype (5′-flanking region RsaI/PstI polymorphic sites) in 97 male print workers. The geometric mean (GM) of toluene concentration in the air was 52.80 ppm (10–760 ppm); 54% of the study participants were exposed to toluene concentrations that exceeded the maximum permissible exposure level (MPEL). The GM of urinary hippuric acid at the end of a work shift (0.041 g/g creatinine) was elevated relative to that before the shift (0.027 g/g creatinine; p < 0.05). The GM of the CHZ metabolic ratio was 0.33 (0–9.3), with 40% of the subjects having ratios below the GM. However, the average CYP2E1 mRNA level in peripheral lymphocytes was 1.07 (0.30–3.08), and CYP2E1 mRNA levels within subjects correlated with the toluene exposure ratio (environmental toluene concentration:urinary hippuric acid concentration) (p = 0.014). Genotype did not alter the association between the toluene exposure ratio and mRNA content. In summary, with further validation, CYP2E1 mRNA content in peripheral lymphocytes could be a sensitive and noninvasive biomarker for the continuous monitoring of toluene effects in exposed persons. PMID:16581535

  7. Global RT-PCR and RT-qPCR Analysis of the mRNA Expression of the Human PTPome.

    PubMed

    Nunes-Xavier, Caroline E; Pulido, Rafael

    2016-01-01

    Comprehensive comparative gene expression analysis of the tyrosine phosphatase superfamily members (PTPome) under cell- or tissue-specific growth conditions may help to define their individual and specific role in physiology and disease. Semi-quantitative and quantitative PCR are commonly used methods to analyze and measure gene expression. Here, we describe technical aspects of PTPome mRNA expression analysis by semi-quantitative RT-PCR and quantitative RT-PCR (RT-qPCR). We provide a protocol for each method consisting in reverse transcription followed by PCR using a global platform of specific PTP primers. The chapter includes aspects from primer validation to the setup of the PTPome RT-qPCR platform. Examples are given of PTP-profiling gene expression analysis using a human breast cancer cell line upon long-term or short-term treatment with cell signaling-activation agents. PMID:27514798

  8. In situ expression of intercellular adhesion molecule-1 (ICAM-1) mRNA in calves with acute Pasteurella haemolytica pneumonia.

    PubMed

    Radi, Z A; Register, K B; Lee, E K; Kehrli, M E; Brogden, K A; Gallup, J M; Ackermann, M R

    1999-09-01

    The in situ expression of intercellular adhesion molecule-1 (ICAM-1) mRNA in normal and pneumonic lung tissues of Holstein calves with bovine leukocyte adhesion deficiency (BLAD) was compared with that of age-matched non-BLAD Holstein calves by in situ hybridization. Twenty-four Holstein calves (both BLAD and non-BLAD) were randomly assigned to one of two experimental groups and inoculated intrabronchially with Pasteurella haemolytica or pyrogen-free saline. Lung tissues were collected and fixed in 10% neutral formalin at 2 or 4 hours postinoculation (PI). The expression and distribution of ICAM-1 mRNA in the different cell types of the lung tissue was detected by in situ hybridization with a 307-base-pair bovine ICAM-1 riboprobe. In lungs of both non-BLAD and BLAD saline-inoculated calves, ICAM-1 expression was present in epithelial cells but occurred in <30% of cells in bronchi, bronchioles, and alveoli. ICAM-1 expression in vascular endothelial cells was present in <30% of cells in pulmonary arteries and veins. The expression of ICAM-1 was significantly greater (>60% of cells) in bronchiolar and alveolar epithelial cells and pulmonary endothelial cells of arteries and veins in both BLAD and non-BLAD calves inoculated with P. haemolytica. Bronchiolar epithelium had the highest intensity of mRNA expression and highest percentage of cells that were stained, whereas bronchial epithelium had the lowest intensity and percentage of cells stained. Most alveolar macrophages and neutrophils in infected lungs also expressed ICAM-1. ICAM-1 expression was generally increased in infected BLAD calves at 2 hours PI as compared with non-BLAD calves but not at 4 hours PI. The increased expression of ICAM-1 during acute P. haemolytica pneumonia in calves suggests that ICAM-1 is upregulated and may play a role in leukocyte infiltration. The extent of ICAM-1 expression in P. haemolytica-inoculated calves with BLAD was initially enhanced but otherwise similar to that in non

  9. Photoperiod history-dependent responses to intermediate day lengths engage hypothalamic iodothyronine deiodinase type III mRNA expression.

    PubMed

    Kampf-Lassin, August; Prendergast, Brian J

    2013-04-15

    Perihypothalamic thyroid hormone signaling features prominently in the seasonal control of reproductive physiology. Triiodothyronine (T(3)) signaling stimulates gonadal development, and decrements in T(3) signaling are associated with gonadal regression. Type 3 iodothyronine deiodinase (DIO3) converts the prohormone thyroxine (T(4)) into biologically inactive 3,3',5'-triiodothyronine, and in long-day breeding Siberian hamsters exposure to long (LD) and short (SD) photoperiods, respectively, inhibit and stimulate hypothalamic dio3 mRNA expression. Reproductive responses to intermediate-duration photoperiods (IntD) occur in a history-dependent manner; IntDs are interpreted as inhibitory only when preceded by longer photoperiods. Because dio3 expression has only been evaluated under LD or SD photoperiods, it is not known whether hypothalamic dio3 encodes absolute photoperiod duration or the reproductive interpretation of photoperiod. Male Siberian hamsters with and without a prior history of LD were exposed to IntD photoperiods, and hypothalamic dio3 mRNA expression was measured 6 wk later. Hamsters with a LD photoperiod history exhibited gonadal regression in IntD and a marked upregulation of hypothalamic dio3 expression, whereas in hamsters without prior exposure to LD, gonadal responses to IntD were absent, and dio3 expression remained low. Patterns of deiodinase expression in hamsters maintained in chronic IntD photoperiods did not appear to reflect feedback effects of gonadal status. Hypothalamic expression of dio3 does not exclusively reflect ambient photoperiod, but rather the context-dependent reproductive interpretation of photoperiod. Neuroendocrine mechanisms that compare current and prior photoperiods, which permit detection of directional changes in day length, occur either upstream, or at the level, of hypothalamic dio3 expression.

  10. Quantification of low-expressed mRNA using 5' LNA-containing real-time PCR primers

    SciTech Connect

    Malgoyre, A.; Banzet, S.; Mouret, C.; Bigard, A.X.; Peinnequin, A. . E-mail: andrepeinnequin@crssa.net

    2007-03-02

    Real-time RT-PCR is the most sensitive and accurate method for mRNA quantification. Using specific recombinant DNA as a template, real-time PCR allows accurate quantification within a 7-log range and increased sensitivity below 10 copies. However, when using RT-PCR to quantify mRNA in biological samples, a stochastic off-targeted amplification can occur. Classical adjustments of assay parameters have minimal effects on such amplification. This undesirable amplification appears mostly to be dependent on specific to non-specific target ratio rather than on the absolute quantity of the specific target. This drawback, which decreases assay reliability, mostly appears when quantifying low-expressed transcript in a whole organ. An original primer design using properties of LNA allows to block off-target amplification. 5'-LNA substitution strengthens 5'-hybridization. Consequently on-target hybridization is stabilized and the probability for the off-target to lead to amplification is decreased.

  11. Promoter methylation and mRNA expression of HLA-G in relation to HLA-G protein expression in colorectal cancer.

    PubMed

    Swets, Marloes; Seneby, Lina; Boot, Arnoud; van Wezel, Tom; Gelderblom, Hans; van de Velde, Cornelis J H; van den Elsen, Peter J; Kuppen, Peter J K

    2016-09-01

    Expression of human leukocyte antigen-G (HLA-G) is a suggested mechanism used by tumor cells to escape from host immune recognition and destruction. Advances in the field have made it evident that HLA-G is expressed in different types of malignancies including colorectal cancer (CRC). We analyzed HLA-G expression in 21 low passage CRC cell lines. The level of DNA methylation of the HLA-G gene and the presence of mRNA encoding HLA-G was measured. Moreover, HLA-G protein expression was determined by flow cytometry and immunohistochemistry (IHC). IHC was performed with three different monoclonal antibodies (mAbs) (4H84, MEM-G/1 and MEM-G/2). In addition, HLA-G protein expression was measured in matching primary tumor tissues. RNA analysis using RT-PCR followed by sequencing in 6 samples indicated strong homology of the PCR product with HLA-G3 in 5 samples. In accordance, in none of the cell lines, HLA-G1 expression was detected by flow-cytometry. Furthermore, no association between HLA-G DNA methylation patterns and HLA-G mRNA expression was observed. In addition, different immunohistochemical staining profiles among various anti-HLA-G mAbs were observed. In conclusion, the results of this study show that the HLA-G3 isoform was expressed in some of the CRC cell lines irrespective of the level of DNA methylation of HLA-G.

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

    PubMed Central

    Shvedova, Anna A.; Yanamala, Naveena; Kisin, Elena R.; Khailullin, Timur O.; Birch, M. Eileen; Fatkhutdinova, Liliya M.

    2016-01-01

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

  13. Characterizing dose-responses of catalase to nitrofurazone exposure in model ciliated protozoan Euplotes vannus for ecotoxicity assessment: enzyme activity and mRNA expression.

    PubMed

    Li, Jiqiu; Zhou, Liang; Lin, Xiaofeng; Yi, Zhenzhen; Al-Rasheid, Khaled A S

    2014-02-01

    In environmental studies, some biological responses, known as biomarkers, have been used as a powerful bioassay tool for more than four decades. Disparity between enzyme activity and mRNA abundance leads to correlation equivocality, which makes the application of biomarkers for environmental risk assessment more complicated. This study investigates this disparity in the case of catalase when used as a biomarker for detecting ecotoxicity induced by antibiotics in aquatic ecosystems. In particular, dose-responses for catalase activity and mRNA expression abundance were investigated in Euplotes vannus which were exposed to graded doses of nitrofurazone for several discrete durations, and dose-response models were developed to characterize the dose-response dynamics. Significant differences were found in both catalase activity and mRNA expression abundance among the E. vannus treated with nitrofurazone. Catalase activity showed a hormetic-like effect in terms of dose-response, characterized by a biphasic relationship which was more clearly evident after a longer exposure period, while mRNA expression abundance increased linearly with the exposure duration. Additionally, the correlation between catalase activity and mRNA expression abundance reversed along with the duration of exposure to nitrofurazone. Taken together, our results demonstrate that catalase mRNA expression offers a more straightforward dose-response model than enzyme activity. Our findings suggest that both catalase enzyme activity and mRNA expression abundance can be used jointly as bioassay tools for detecting ecotoxicity induced by nitrofurazone in aquatic ecosystems.

  14. Distinct cytokine mRNA expression pattern in immunoglobulin G4-related kidney disease associated with renal cell carcinoma.

    PubMed

    Watanabe, Renya; Yasuno, Tetsuhiko; Hisano, Satoshi; Sasatomi, Yoshie; Nakashima, Hitoshi

    2014-06-01

    We treated a 61-year-old man with immunoglobulin (Ig)G4-related kidney disease (IgG4-RKD). He had a history of allergic diseases and an allergic reaction and had received a diagnosis of autoimmune pancreatitis (AIP). He had also received a diagnosis of renal cell carcinoma (RCC) and had undergone segmental resection of the left kidney at 59 years of age. His serum amylase level and number of peripheral eosinophils increased after RCC development. We hypothesized that the RCC may have induced AIP and IgG4-RKD and we therefore examined the excised RCC tissue; typical findings of IgG4-RKD associated with RCC were recognized. We next evaluated the mRNA expression of cytokines in the excised tissues of this case and ten other ordinary RCC cases. In all cases, notable levels of IL-10 mRNA and high levels of TGF-β mRNA were seen. Although prominent differences were not observed in the mRNA expression of Th1, Th17 and Treg cytokines in all cases, the present case alone showed increased production of the Th2 cytokines IL-4 and IL-5, which were not detected in ordinary RCC cases. Although the mechanism underlying IgG4-RKD development has not yet been determined, Th2 and Treg cells are thought to play a prominent role in the pathogenesis. It is therefore likely that in this case, the association of these two diseases was not coincidental, and a distinct immune response against RCC may trigger IgG4-RKD development.

  15. ABCB1, ABCC2, SCN1A, SCN2A, GABRA1 gene polymorphisms and drug resistant epilepsy in the Chinese Han population.

    PubMed

    Zhou, Luo; Cao, Yuze; Long, Hongyu; Long, Lili; Xu, Lin; Liu, Zhaoqian; Zhang, Ying; Xiao, Bo

    2015-06-01

    Drug resistance is common in epilepsy despite multiple available medications. Single nucleotide polymorphisms (SNP) may influence drug efficacy in epilepsy. We therefore aimed to clarify the association between polymorphisms of several controversial SNP loci and drug resistance in Chinese Han epilepsy patients from central China. Among all the 391 recruited subjects, 235 and 156 patients were classified into a drug responsive and resistant group, respectively, according to the definition of drug resistance proposed by the International League Against Epilepsy. The candidate SNP loci, including ATP-binding cassette (ABC) subfamily gene ABCB1 rs2032582 and rs1045642; ABC subfamily gene ABCC2 rs717620 and rs2273697; sodium channel subunit gene SCN1A rs3812718, SCN2A rs2304016; γ-amino butyric acid type A (GABAA) receptor subunit subtype gene GABRA1 rs2279020 were genotyped following the Illumina protocols. There were no significant differences in allelic or genotypic frequencies between the drug responsive and resistant patients. The polymorphisms of the above SNP loci may not be associated with drug resistance of epilepsy in the Chinese Han population.

  16. [Effects of electroacupuncture on the expression of estrogen receptor protein and mRNA in rat brain].

    PubMed

    Chen, B Y; Cheng, L H; Gao, H; Ji, S Z

    1998-10-01

    In order to show some light on the possible molecular mechanism of the effects of acupuncture on the hypothalamic-pituitary-ovarian axial function, radioimmunoassay (RIA), RNA dot blot and Northern blot, monoclonal antibody immuno-histochemistry and computer image analysis technique were used to study the effects of electroacupuncture (EA) on the blood level of estradiol (E2) and the expression of estrogen receptor protein and mRNA in the ovariectomized rat brain. The results show that ovariectomy induced a decrease of blood E2 level and increase of the expression of estrogen receptor protein and mRNA in the brain. All these effects could be remarkably reversed by previous EA treatment of ovariectomized rats. EA in intact rat, however, had no effects on blood E2 and expression of estrogen receptor. The above results suggest that EA at some effective acupoints may activate the production of body estrogen, resulting in a long term increase or suppression at the level of gene(s) expression and consequent normalization on the dysfunction of hypothalamic-pituitary-ovarian axis.

  17. Expression of neurotrophin receptors in rat testis. Upregulation of TrkA mRNA with hCG treatment.

    PubMed

    Schultz, R; Metsis, M; Hökfelt, T; Parvinen, M; Pelto-Huikko, M

    2001-08-20

    We report the expression of TrkA, TrkB and TrkC mRNAs in adult rat testis. With in situ hybridisation a low signal for TrkB and TrkC could be seen in postmeiotic cells of the seminiferous epithelium, whereas no signal for TrkA could be observed in untreated animals. Animals treated with hCG showed an induction of TrkA mRNA in premeiotic cells 12 h after the treatment, whereas an injection with EDS had no effect on the expression of Trk mRNAs. With the RNAse protection assay a low signal for TrkA was seen in whole testis of hCG treated animals. In staged tubules low expression was seen at stages VII-XI of untreated animals. Animals injected with hCG revealed that TrkA induction was highest during stages VIIcd and VIII of the cycle. The distinct expression pattern of these high-affinity neurotrophin receptors suggests different roles for neurotrophins during spermatogenesis. Induction of TrkA mRNA by hCG suggests that high-affinity binding of NGF during stages VIIcd-VIII in premeiotic cells is under control of the hypothalamic-pituitary-testicular axis.

  18. Synchronized expression of retinoid X receptor mRNA with reproductive tract recrudescence in an imposex-susceptible mollusc.

    PubMed

    Sternberg, Robin M; Hotchkiss, Andrew K; Leblanc, Gerald A

    2008-02-15

    The biocide tributyltin (TBT) causes the development of male sex characteristics in females of some molluscan species, a phenomenon known as imposex. Recent evidence suggests that the retinoid X receptor (RXR) participates in TBT-induced imposex. Accordingly, we hypothesized that RXR may contribute to the seasonal development of the male reproductive tract in molluscs and would be expressed in concert with this phenomenon. RXR was cloned and sequenced from an imposex-susceptble species, the eastern mud snail Ilyanassa obsoleta. The DNA-binding domain of the receptor protein was 100 and 97% identical to those of the rock shell Thais clavigera and the freshwater snail Biomphalaria glabrata. The ligand-binding domain was 93 and 92% identicalto the LBD of these two molluscan species, respectively. Phylogenetic analyses revealed that RXR is an ancient nuclear receptor whose origin predates the emergence of the Bilateria. Interestingly, though inexplicably, the molluscan RXRs were more similar to sequences of vertebrate RXRs than to the RXRs of other lophotrochozoan invertebrates. Next, the expression of RXR mRNA levels in the reproductive tract was determined through the reproductive cycle. RXR mRNA levels increased commensurate with reproductive tract recrudescence in both sexes. However, the timing of coordinate recrudescence-RXR expression differed between sexes. Results demonstrate that RXR expression is associated with reproductive tract recrudescence in both sexes; although, the timing of recrudescence may dictate sex-specific development. Retinoid signaling initiated by TBT during an inappropriate time in females may result in imposex.

  19. Acute Physiological Stress Down-Regulates mRNA Expressions of Growth-Related Genes in Coho Salmon

    PubMed Central

    Nakano, Toshiki; Afonso, Luis O. B.; Beckman, Brian R.; Iwama, George K.; Devlin, Robert H.

    2013-01-01

    Growth and development in fish are regulated to a major extent by growth-related factors, such as liver-derived insulin-like growth factor (IGF) -1 in response to pituitary-secreted growth hormone (GH) binding to the GH receptor (GHR). Here, we report on the changes in the expressions of gh, ghr, and igf1 genes and the circulating levels of GH and IGF-1 proteins in juvenile coho salmon (Oncorhynchus kisutch) in response to handling as an acute physiological stressor. Plasma GH levels were not significantly different between stressed fish and prestressed control. Plasma IGF-1 concentrations in stressed fish 1.5 h post-stress were the same as in control fish, but levels in stressed fish decreased significantly 16 h post-stress. Real-time quantitative PCR (qPCR) analysis showed that ghr mRNA levels in pituitary, liver, and muscle decreased gradually in response to the stressor. After exposure to stress, hepatic igf1 expression transiently increased, whereas levels decreased 16 h post-stress. On the other hand, the pituitary gh mRNA level did not change in response to the stressor. These observations indicate that expression of gh, ghr, and igf1 responded differently to stress. Our results show that acute physiological stress can mainly down-regulate the expressions of growth-related genes in coho salmon in vivo. This study also suggests that a relationship between the neuroendocrine stress response and growth-related factors exists in fish. PMID:23990952

  20. Transforming Growth Factor β1 (TGF-β1) Activates Hepcidin mRNA Expression in Hepatocytes.

    PubMed

    Chen, Simeng; Feng, Teng; Vujić Spasić, Maja; Altamura, Sandro; Breitkopf-Heinlein, Katja; Altenöder, Jutta; Weiss, Thomas S; Dooley, Steven; Muckenthaler, Martina U

    2016-06-17

    The hepatic hormone hepcidin is the master regulator of systemic iron homeostasis. Its expression level is adjusted to alterations in iron levels, inflammatory cues, and iron requirements for erythropoiesis. Bone morphogenetic protein 6 (BMP6) contributes to the iron-dependent control of hepcidin. In addition, TGF-β1 may stimulate hepcidin mRNA expression in murine hepatocytes and human leukocytes. However, receptors and downstream signaling proteins involved in TGF-β1-induced hepcidin expression are still unclear. Here we show that TGF-β1 treatment of mouse and human hepatocytes, as well as ectopic expression of TGF-β1 in mice, increases hepcidin mRNA levels. The hepcidin response to TGF-β1 depends on functional TGF-β1 type I receptor (ALK5) and TGF-β1 type II receptor (TβRII) and is mediated by a noncanonical mechanism that involves Smad1/5/8 phosphorylation. Interestingly, increasing availability of canonical Smad2/3 decreases TGF-β1-induced hepcidin regulation, whereas the BMP6-hepcidin signal was enhanced, indicating a signaling component stoichiometry-dependent cross-talk between the two pathways. Although ALK2/3-dependent hepcidin activation by BMP6 can be modulated by each of the three hemochromatosis-associated proteins: HJV (hemojuvelin), HFE (hemochromatosis protein), and TfR2 (transferrin receptor 2), these proteins do not control the ALK5-mediated hepcidin response to TGF-β1. TGF-β1 mRNA levels are increased in mouse models of iron overload, indicating that TGF-β1 may contribute to hepcidin synthesis under these conditions. In conclusion, these data demonstrate that a complex regulatory network involving TGF-β1 and BMP6 may control the sensing of systemic and/or hepatic iron levels.

  1. High intensity interval training favourably affects antioxidant and inflammation mRNA expression in early-stage chronic kidney disease.

    PubMed

    Tucker, Patrick S; Briskey, David R; Scanlan, Aaron T; Coombes, Jeff S; Dalbo, Vincent J

    2015-12-01

    Increased levels of oxidative stress and inflammation have been linked to the progression of chronic kidney disease. To reduce oxidative stress and inflammation related to chronic kidney disease, chronic aerobic exercise is often recommended. Data suggests high intensity interval training may be more beneficial than traditional aerobic exercise. However, appraisals of differing modes of exercise, along with explanations of mechanisms responsible for observed effects, are lacking. This study assessed effects of eight weeks of high intensity interval training (85% VO2max), versus low intensity exercise (45-50% VO2max) and sedentary behaviour, in an animal model of early-stage chronic kidney disease. We examined kidney-specific mRNA expression of genes related to endogenous antioxidant enzyme activity (glutathione peroxidase 1; Gpx1, superoxide dismutase 1; Sod1, and catalase; Cat) and inflammation (kidney injury molecule 1; Kim1 and tumour necrosis factor receptor super family 1b; Tnfrsf1b), as well as plasma F2-isoprostanes, a marker of lipid peroxidation. Compared to sedentary behaviour, high intensity interval training resulted in increased mRNA expression of Sod1 (p=0.01) and Cat (p<0.001). Compared to low intensity exercise, high intensity interval training resulted in increased mRNA expression of Cat (p<0.001) and Tnfrsf1b (p=0.047). In this study, high intensity interval training was superior to sedentary behaviour and low intensity exercise as high intensity interval training beneficially influenced expression of genes related to endogenous antioxidant enzyme activity and inflammation.

  2. ALDH1A1 mRNA expression in association with prognosis of triple-negative breast cancer

    PubMed Central

    Liu, Yan; Baglia, Michelle; Zheng, Ying; Blot, William; Bao, Ping-Ping; Cai, Hui; Nechuta, Sarah; Zheng, Wei; Cai, Qiuyin; Shu, Xiao Ou

    2015-01-01

    ALDH1 is a crucial element in the retinoic acid signaling pathway regulating the self-renewal and differentiation of normal stem cells, and may play an important role in cancer progression. However, research on ALDH1 gene expressionand breast cancer prognosis has yielded conflicting results. We evaluated the association between tumor tissue ALDH1A1/ALDH1A3 mRNA expression and triple-negative breast cancer (TNBC) prognosis in the Shanghai Breast Cancer Survival Study (SBCSS, N=463), Nashville Breast Health Study (NBHS, N=86), and Southern Community Cohort Study (SCCS, N=47). Gene expression was measured in RNA isolated from breast cancer tissues. In the SBCSS, higher ALDH1A1 mRNA level was associated with improved disease-free (HR=0.87, 95% CI: 0.80-0.95, per log unit change) and overall survival (HR=0.85, 95% CI: 0.78-0.93 per log unit change) independent of age at diagnosis, TNM stage and treatment. We replicated the findings for overall survival in the NBHS and SCCS (HR = 0.27, 95% CI: 0.10-0.73) and for disease-free survival by a meta-analysis of four publicly-available gene expression datasets (HR = 0.86, 95% CI: 0.76-0.97). No significant association was found for ALDH1A3. Our study suggests high expression of ALDH1A1 mRNA in tumor tissues may be an independent predictor of a favorable TNBC outcome. PMID:26462023

  3. Regional and Duration of Illness Differences in the Alteration of NCAM-180 mRNA Expression within the Cortex of Subjects with Schizophrenia

    PubMed Central

    Gibbons, A. S.; Thomas, E. A.; Dean, B

    2009-01-01

    Schizophrenia has been proposed to have a neurodevelopmental aetiology. Neural Cell Adhesion Molecule 1 (NCAM1) is involved in several neurodevelopmental processes and abnormal expression of this gene has been associated in the pathology of schizophrenia and, thus, altered NCAM1 expression may be characteristic of the early stages of the illness. Alternative splicing of the NCAM1 transcript produces 3 major isoforms. Using qPCR we analysed mRNA expression of one of these isoforms; the 180 kDa isoform of NCAM1 (NCAM-180), in Brodmann Area (BA) 46, BA10 and BA17, postmortem, from 15 subjects with a short duration of illness of schizophrenia (<7 years) and 15 control subjects. NCAM-180 mRNA expression was increased in BA46 from subjects with schizophrenia compared to controls (P=0.013). By contrast, there were no significant differences in the expression of NCAM-180 mRNA in BA10 (P=0.575) or BA17 (P=0.772). We then analysed NCAM-180 mRNA expression in BA46 from 15 subjects with a longer duration of illness of schizophrenia (>22 years) and 15 controls. There was no significant difference in NCAM-180 mRNA expression in this second cohort. This data suggests NCAM-180 mRNA expression is altered in a regionally-specific manner in schizophrenia and these changes are associated with the early period following diagnosis. PMID:19411161

  4. Enhanced stability of microRNA expression facilitates classification of FFPE tumour samples exhibiting near total mRNA degradation

    PubMed Central

    Hall, J S; Taylor, J; Valentine, H R; Irlam, J J; Eustace, A; Hoskin, P J; Miller, C J; West, C M L

    2012-01-01

    Background: As degradation of formalin-fixed paraffin-embedded (FFPE) samples limits the ability to profile mRNA expression, we explored factors predicting the success of mRNA expression profiling of FFPE material and investigated an approach to overcome the limitation. Methods: Bladder (n=140, stored 3–8 years) and cervix (n=160, stored 8–23 years) carcinoma FFPE samples were hybridised to Affymetrix Exon 1.0ST arrays. Percentage detection above background (%DABG) measured technical success. Biological signal was assessed by distinguishing cervix squamous cell carcinoma (SCC) and adenocarcinoma (AC) using a gene signature. As miR-205 had been identified as a marker of SCC, precursor mir-205 was measured by Exon array and mature miR-205 by qRT–PCR. Genome-wide microRNA (miRNA) expression (Affymetrix miRNA v2.0 arrays) was compared in eight newer FFPE samples with biological signal and eight older samples without. Results: RNA quality controls (QCs) (e.g., RNA integrity (RIN) number) failed to predict profiling success, but sample age correlated with %DABG in bladder (R=−0.30, P<0.01) and cervix (R=−0.69, P<0.01). Biological signal was lost in older samples and neither a signature nor precursor mir-205 separated samples by histology. miR-205 qRT–PCR discriminated SCC from AC, validated by miRNA profiling (26-fold higher in SCC; P=1.10 × 10−5). Genome-wide miRNA (R=0.95) and small nucleolar RNA (R=0.97) expression correlated well in the eight newer vs older FFPE samples and better than mRNA expression (R=0.72). Conclusion: Sample age is the best predictor of successful mRNA profiling of FFPE material, and miRNA profiling overcomes the limitation of age and copes well with older samples. PMID:22805332

  5. Biological mechanism analysis of acute renal allograft rejection: integrated of mRNA and microRNA expression profiles

    PubMed Central

    Huang, Shi-Ming; Zhao, Xia; Zhao, Xue-Mei; Wang, Xiao-Ying; Li, Shan-Shan; Zhu, Yu-Hui

    2014-01-01

    Objectives: Renal transplantation is the preferred method for most patients with end-stage renal disease, however, acute renal allograft rejection is still a major risk factor for recipients leading to renal injury. To improve the early diagnosis and treatment of acute rejection, study on the molecular mechanism of it is urgent. Methods: MicroRNA (miRNA) expression profile and mRNA expression profile of acute renal allograft rejection and well-functioning allograft downloaded from ArrayExpress database were applied to identify differentially expressed (DE) miRNAs and DE mRNAs. DE miRNAs targets were predicted by combining five algorithm. By overlapping the DE mRNAs and DE miRNAs targets, common genes were obtained. Differentially co-expressed genes (DCGs) were identified by differential co-expression profile (DCp) and differential co-expression enrichment (DCe) methods in Differentially Co-expressed Genes and Links (DCGL) package. Then, co-expression network of DCGs and the cluster analysis were performed. Functional enrichment analysis for DCGs was undergone. Results: A total of 1270 miRNA targets were predicted and 698 DE mRNAs were obtained. While overlapping miRNA targets and DE mRNAs, 59 common genes were gained. We obtained 103 DCGs and 5 transcription factors (TFs) based on regulatory impact factors (RIF), then built the regulation network of miRNA targets and DE mRNAs. By clustering the co-expression network, 5 modules were obtained. Thereinto, module 1 had the highest degree and module 2 showed the most number of DCGs and common genes. TF CEBPB and several common genes, such as RXRA, BASP1 and AKAP10, were mapped on the co-expression network. C1R showed the highest degree in the network. These genes might be associated with human acute renal allograft rejection. Conclusions: We conducted biological analysis on integration of DE mRNA and DE miRNA in acute renal allograft rejection, displayed gene expression patterns and screened out genes and TFs that may

  6. Molecular Cloning, Characterization, and mRNA Expression of Hemocyanin Subunit in Oriental River Prawn Macrobrachium nipponense

    PubMed Central

    Kong, Youqin; Ding, Zhili; Sun, Shengming; Wang, Ligai; Ye, Jinyun

    2016-01-01

    Hemocyanin is a copper-containing protein with immune function against disease. In this study, a hemocyanin subunit named MnHc-1 was cloned from Macrobrachium nipponense. The full-length cDNA of MnHc-1 was 2,163 bp with a 2,028-bp open reading frame (ORF) encoding a polypeptide of 675 amino acids. The MnHc-1 mRNA was expressed in the hepatopancreas, gill, hemocytes, intestine, ovary, and stomach, with the highest level in the hepatopancreas. In the infection trial, the MnHc-1 mRNA transcripts in the hemocytes were significantly downregulated at 3 h after injection of Aeromonas hydrophila and then upregulated at 6 h and 12 h, followed by a gradual recovery from 24 to 48 h. The MnHc-1 transcriptional expression in the hepatopancreas was measured after M. nipponense were fed seven diets with 2.8, 12.2, 20.9, 29.8, 43.1, 78.9, and 157.1 mg Cu kg−1 for 8 weeks, respectively. The level of MnHc-1 mRNA was significantly higher in the prawns fed 43.1–157.1 mg Cu kg−1 diet than in that fed 2.8–29.8 mg Cu kg−1 diet. This study indicated that the MnHc-1 expression can be affected by dietary copper and the hemocyanin may potentially participate in the antibacterial defense of M. nipponense.

  7. Effects of long-term smoking on the activity and mRNA expression of CYP isozymes in rats

    PubMed Central

    He, Xiao-Meng; Zhou, Ying; Xu, Ming-Zhen; Li, Yang; Li, Hu-Qun

    2015-01-01

    Background To investigate the effect of long-term smoking on the activity and mRNA expression of cytochrome P450 (CYP) enzymes. Methods Sprague-Dawley rats were exposed to passive smoking 6 cigarettes per day for 180 days. A cocktail solution which contained phenacetin (20 mg/kg), tolbutamide (5 mg/kg), chlorzoxazone (20 mg/kg) and midazolam (10 mg/kg) was given orally to rats. Blood samples were collected at pre-specified time points and the concentrations of probe drugs in plasma were determined by HPLC-MS/MS. The corresponding pharmacokinetic parameters were calculated by DAS 3.0. In addition, real-time RT-PCR was used to analyze the mRNA expression of CYP1A2, CYP2C11, CYP2E1 and CYP3A1 in rat liver. Results There were no significant influences of pharmacokinetic profiles of chlorzoxazone in long-term smoking pretreated rats. But many pharmacokinetic profiles of phenacetin, tolbutamide, and midazolam in long-term smoking pretreated rats were affected significantly (P<0.05). The results suggested that long-term smoking had significant inhibition effects on CYP2C11 and CYP3A1 while CYP1A2 enzyme activity was induced. Furthermore, Long-term smoking had no effects on rat CYP2E1. The mRNA expression results were consistent with the pharmacokinetic results. Conclusions Alterations of CYP450 enzyme activities may fasten or slow down excretion with corresponding influence on drug efficacy or toxicity in smokers compared to nonsmokers, which may lead to clinical failures of lung cancer therapy or toxicity in smokers. PMID:26623094

  8. Na(+):K(+):ATPase mRNA expression in the kidney during adaptation to sodium intake and furosemide treatment.

    PubMed

    Merino, A; Moreno, G; Mercado, A; Bobadilla, N A; Gamba, G

    2000-01-01

    Nephron tubular epithelium possesses the capacity of adaptation to any salt ingestion condition. The mechanism of adaptation is due in part to an increase in the activity of Na(+):K(+):ATPase at the basolateral membrane. The goal of the present study was to analyze the long-term regulation of the Na(+):K(+):ATPase alpha(1)-subunit mRNA expression during changes in NaCl metabolism. Male Wistar rats given a normal, high, or low NaCl diet, and intraperitoneal administration of the loop diuretic furosemide from 12 h to 7 days were studied. Rats were kept in metabolic cages 4 days before and throughout the study to determine daily urinary electrolyte excretion and osmolarity. At the end of each experimental period, creatinine clearance and serum electrolytes were also measured. Total RNA was extracted from each individual cortex or outer medulla and from pooled inner medullas using the guanidine/cesium chloride method. Na(+):K(+):ATPase alpha(1)-subunit mRNA expression was assessed by nonradioactive dot-blot analysis. Experimental maneuvers were well tolerated and all groups developed the appropriate renal response to each experimental condition. Urinary sodium excretion was significantly higher in rats administered a high sodium diet or furosemide and lower in rats treated with a low sodium diet after 7 days of treatment. Glomerular filtration rate was similar among all groups. However, the level of expression of the Na(+):K(+):ATPase alpha(1)-subunit did not change in any model. Nephron adaptation to the modification in NaCl intake or furosemide administration over 7 days did not include changes in Na(+):K(+):ATPase alpha(1)-subunit mRNA levels.

  9. Bovine respiratory syncytial virus nucleocapsid protein: mRNA sequence analysis and expression from recombinant vaccinia virus vectors.

    PubMed

    Amann, V L; Lerch, R A; Anderson, K; Wertz, G W

    1992-04-01

    The nucleotide sequence of the mRNA encoding the nucleocapsid (N) protein of bovine respiratory syncytial (BRS) virus, strain 391-2, was determined. Recombinant vectors containing a cDNA of the complete N gene were constructed, and expression of the N protein in eukaryotic cells was demonstrated using two different vector systems. The BRS virus N mRNA was 1197 nucleotides in length, exclusive of poly(A), and had a single major open reading frame that encoded a polypeptide of 391 amino acids with a calculated M(r) of 42.6K. The nucleotide and amino acid sequences of the BRS virus N gene were compared to those of human respiratory syncytial (HRS) virus strains A2 and 18537, and to BRS virus strain A51908. The level of nucleic acid identity between the N mRNA of BRS virus 391-2 and both HRS virus subtypes was 80 to 81%, whereas the identity between the two BRS virus strains was 97%. A 93 to 94% level of identity was observed between the deduced amino acid sequences of the N protein of BRS virus 391-2 and the corresponding sequences of the two HRS virus strains. The two BRS virus N proteins differed in amino acid sequence at only three positions. Recombinant BRS virus N protein was expressed using two different vector systems: in cells from a plasmid using the vaccinia virus/T7 polymerase expression system or from a recombinant vaccinia virus. N proteins synthesized by the two vector systems migrated with an electrophoretic mobility identical to that of authentic BRS virus N protein, and were precipitated by anti-BRS virus antibodies.

  10. A circadian neuropeptide PDF in the honeybee, Apis mellifera: cDNA cloning and expression of mRNA.

    PubMed

    Sumiyoshi, Miho; Sato, Seiji; Takeda, Yukimasa; Sumida, Kazunori; Koga, Keita; Itoh, Tsunao; Nakagawa, Hiroyuki; Shimohigashi, Yasuyuki; Shimohigashi, Miki

    2011-12-01

    Pigment-dispersing factor (PDF) is a pacemaker hormone regulating the locomotor rhythm in insects. In the present study, we cloned the cDNAs encoding the Apis PDF precursor protein, and found that there are at least seven different pdf mRNAs yielded by an alternative splicing site and five alternative polyadenylation sites in the 5'UTR and 3'UTR regions. The amino acid sequence of Apis PDF peptide has a characteristic novel amino acid residue, aspargine (Asn), at position 17. Quantitative real-time PCR of total and 5'UTR insertion-type pdf mRNAs revealed, for the first time, that the expression levels change in a circadian manner with a distinct trough at the beginning of night in LD conditions, and at the subjective night under DD conditions. In contrast, the expression level of 5'UTR deletion-type pdf mRNAs was about half of that of the insertion type, and the expression profile failed to show a circadian rhythm. As the expression profile of the total pdf mRNA exhibited a circadian rhythm, transcription regulated at the promoter region was supposed to be controlled by some of the clock components. Whole mount in situ hybridization revealed that 14 lateral neurons at the frontal margin of the optic lobe express these mRNA isoforms. PDF expressing cells examined with a newly produced antibody raised against Apis PDF were also found to have a dense supply of axon terminals in the optic lobes and the central brain. PMID:22132787

  11. Anatomical characterization of bombesin receptor subtype-3 mRNA expression in the rodent central nervous system.

    PubMed

    Zhang, Li; Parks, Gregory S; Wang, Zhiwei; Wang, Lien; Lew, Michelle; Civelli, Olivier

    2013-04-01

    Bombesin receptor subtype-3 (BRS-3) is an orphan G-protein-coupled receptor (GPCR) involved in the regulation of energy homeostasis. Mice deficient in BRS-3 develop late-onset mild obesity with metabolic defects, while synthetic agonists activating BRS-3 show antiobesity profiles by inhibiting food intake and increasing metabolic rate in rodent models. The molecular mechanisms and the neural circuits responsible for these effects, however, remain elusive and demand better characterization. We report here a comprehensive mapping of BRS-3 mRNA in the rat and mouse brain through in situ hybridization. Furthermore, to investigate the neurochemical characteristics of the BRS-3-expressing neurons, double in situ hybridization was performed to determine whether BRS-3 colocalizes with other neurotransmitters or neuropeptides. Many, but not all, of the BRS-3-expressing neurons were found to be glutamatergic, while few were found to be cholinergic or GABAergic. BRS-3-containing neurons do not express some of the well-characterized neuropeptides, such as neuropeptide Y (NPY), proopiomelanocortin (POMC), orexin/hypocretin, melanin-concentrating hormone (MCH), thyrotropin-releasing hormone (TRH), gonadotropin-releasing hormone (GnRH), and kisspeptin. Interestingly, BRS-3 mRNA was found to partially colocalize with corticotropin-releasing factor (CRF) and growth hormone-releasing hormone (GHRH), suggesting novel interactions of BRS-3 with stress- and growth-related endocrine systems. Our study provides important information for evaluating BRS-3 as a potential therapeutic target for the treatment of obesity. PMID:22911445

  12. Expression and Presence of OPG and RANKL mRNA and Protein in Human Periodontal Ligament with Orthodontic Force

    PubMed Central

    Otero, Liliana; García, Dabeiba Adriana; Wilches-Buitrago, Liseth

    2016-01-01

    OBJECTIVE The objective of this study is to investigate the expression and concentration of ligand receptor activator of NFkB (RANKL) and osteoprotegerin (OPG) in human periodontal ligament (hPDL) with orthodontic forces of different magnitudes. METHODS Right premolars in 32 patients were loaded with 4oz or 7oz of orthodontic force for 7 days. Left first premolars were not loaded. After 7 days, premolars were extracted for treatment as indicated. OPG and RANKL mRNA expressions were measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR), and ELISA was used to assess OPG and RANKL protein concentration in compression and tension sides of PDL. Data were subjected to analysis of variance and Tukey tests. RESULTS There was statistically significant difference in RANKL concentration on comparing control teeth with tension and compression sides of the experimental teeth (P < 0.0001). The expression of mRNA RANKL was increased in the tension and compression sides with 4oz (P < 0.0001). OPG did not show statistically significant association with any group. Changes in RANKL/OPG protein ratio in experimental and control groups showed statistically significant difference (P < 0.0001). CONCLUSIONS RANKL protein levels are elevated in hPDL loaded with orthodontic forces, suggesting that RANKL protein contributes to bone modeling in response to the initial placement of orthodontic force. PMID:26823650

  13. Pituitary hormone mRNA expression in European sea bass Dicentrarchus labrax in seawater and following acclimation to fresh water.

    PubMed

    Varsamos, Stamatis; Xuereb, Benoît; Commes, Thérèse; Flik, Gert; Spanings-Pierrot, Céline

    2006-11-01

    The mRNA expression of pituitary prolactin (prl), growth hormone (gh), somatolactin (sl), proopiomelanocortin (pomc), and gonadotropins (gthI and gthII) was quantified by real-time PCR, in sea bass, Dicentrarchus labrax, adapted for 1 month to seawater (SW) or freshwater (FW). In addition, IGF-I (igfI) mRNA expression in liver and branchial Na+/K+ -ATPase activity were determined. L17 ribosomal protein (rpL17) and elongation factor 1alpha (ef1alpha) were validated as reference genes in real-time PCR in the experimental context. The real-time PCR assays were validated for the different hormone genes considered. Expression of pituitary pomc, gthI, gthII, gh, and liver igfI was not significantly different between FW and SW fish. Pituitary prlwas 4.5-foldhigher in FWthan in SW, whereas pituitary sl was 1.8-fold higher in SW- compared with FW-adapted fish. Gill Na+/K+ -ATPase specific activity was 2.3-fold higher in FW sea bass compared with SW fish. Plasma cortisol levels were 6.5-fold lower in SW- than in FW-adapted specimens. The results are discussed in relation to the osmoregulatory strategy of this euryhaline SW species, which displays features that do not fit present models based on salmonids and FWeuryhaline teleosts.

  14. Efficient Downregulation of Multiple mRNA Targets with a Single shRNA-Expressing Lentiviral Vector

    PubMed Central

    Chumakov, Stepan P.; Kravchenko, Julia E.; Prassolov, Vladimir S.; Frolova, Elena I.; Chumakov, Peter M.

    2010-01-01

    Gene silencing based on RNA interference is widely used in fundamental research and in practical applications. However, a commonly incomplete functional suppression represents a serious drawback of this technology. We describe a series of lentiviral vectors each containing a single or multiple shRNA expression cassette(s) driven by a RNA polymerase III specific promoter and localized within the 3′-LTR of the lentiviral DNA backbone. The vectors also contain an antibiotic-resistance gene that allows positive selection of recipient cells. The combined expression of three different shRNAs specific to a single mRNA was shown to improve dramatically the level of mRNA inhibition, while the use of three different RNA polymerase III specific promoters avoids the loss of shRNA expression cassettes through the homologous recombination. The vector system was used for successful simultaneous suppression of three related SESN1, SESN2 and SESN3 genes, which suggests its particular value for testing phenotypes of functionally redundant genes. PMID:20064551

  15. Efficient downregulation of multiple mRNA targets with a single shRNA-expressing lentiviral vector.

    PubMed

    Chumakov, Stepan P; Kravchenko, Julia E; Prassolov, Vladimir S; Frolova, Elena I; Chumakov, Peter M

    2010-05-01

    Gene silencing based on RNA interference is widely used in fundamental research and in practical applications. However, a commonly incomplete functional suppression represents a serious drawback of this technology. We describe a series of lentiviral vectors each containing a single or multiple shRNA-expression cassette(s) driven by a RNA-polymerase III specific promoter and localized within the 3'-LTR of the lentiviral DNA backbone. The vectors also contain an antibiotic-resistance gene that allows positive selection of recipient cells. The combined expression of three different shRNAs specific to a single mRNA was shown to improve dramatically the level of mRNA inhibition, while the use of three different RNA-polymerase III specific promoters avoids the loss of shRNA-expression cassettes through the homologous recombination. The vector system was used for successful simultaneous suppression of three related SESN1, SESN2 and SESN3 genes, which suggests its particular value for testing phenotypes of functionally redundant genes.

  16. Altered mRNA editing and expression of ionotropic glutamate receptors after kainic acid exposure in cyclooxygenase-2 deficient mice.

    PubMed

    Caracciolo, Luca; Barbon, Alessandro; Palumbo, Sara; Mora, Cristina; Toscano, Christopher D; Bosetti, Francesca; Barlati, Sergio

    2011-01-01

    Kainic acid (KA) binds to the AMPA/KA receptors and induces seizures that result in inflammation, oxidative damage and neuronal death. We previously showed that cyclooxygenase-2 deficient (COX-2(-/-)) mice are more vulnerable to KA-induced excitotoxicity. Here, we investigated whether the increased susceptibility of COX-2(-/-) mice to KA is associated with altered mRNA expression and editing of glutamate receptors. The expression of AMPA GluR2, GluR3 and KA GluR6 was increased in vehicle-injected COX-2(-/-) mice compared to wild type (WT) mice in hippocampus and cortex, whereas gene expression of NMDA receptors was decreased. KA treatment decreased the expression of AMPA, KA and NMDA receptors in the hippocampus, with a significant effect in COX-2(-/-) mice. Furthermore, we analyzed RNA editing levels and found that the level of GluR3 R/G editing site was selectively increased in the hippocampus and decreased in the cortex in COX-2(-/-) compared with WT mice. After KA, GluR4 R/G editing site, flip form, was increased in the hippocampus of COX-2(-/-) mice. Treatment of WT mice with the COX-2 inhibitor celecoxib for two weeks decreased the expression of AMPA/KA and NMDAR subunits after KA, as observed in COX-2(-/-) mice. After KA exposure, COX-2(-/-) mice showed increased mRNA expression of markers of inflammation and oxidative stress, such as cytokines (TNF-α, IL-1β and IL-6), inducible nitric oxide synthase (iNOS), microglia (CD11b) and astrocyte (GFAP). Thus, COX-2 gene deletion can exacerbate the inflammatory response to KA. We suggest that COX-2 plays a role in attenuating glutamate excitotoxicity by modulating RNA editing of AMPA/KA and mRNA expression of all ionotropic glutamate receptor subunits and, in turn, neuronal excitability. These changes may contribute to the increased vulnerability of COX-2(-/-) mice to KA. The overstimulation of glutamate receptors as a consequence of COX-2 gene deletion suggests a functional coupling between COX-2 and the

  17. The Andes Hantavirus NSs Protein Is Expressed from the Viral Small mRNA by a Leaky Scanning Mechanism

    PubMed Central

    Vera-Otarola, Jorge; Solis, Loretto; Soto-Rifo, Ricardo; Ricci, Emiliano P.; Pino, Karla; Tischler, Nicole D.; Ohlmann, Théophile; Darlix, Jean-Luc

    2012-01-01

    The small mRNA (SmRNA) of all Bunyaviridae encodes the nucleocapsid (N) protein. In 4 out of 5 genera in the Bunyaviridae, the smRNA encodes an additional nonstructural protein denominated NSs. In this study, we show that Andes hantavirus (ANDV) SmRNA encodes an NSs protein. Data show that the NSs protein is expressed in the context of an ANDV infection. Additionally, our results suggest that translation initiation from the NSs initiation codon is mediated by ribosomal subunits that have bypassed the upstream N protein initiation codon through a leaky scanning mechanism. PMID:22156529

  18. Increased mRNA expression levels of ERCC1, OGG1 and RAI in colorectal adenomas and carcinomas

    PubMed Central

    Sæbø, Mona; Skjelbred, Camilla Furu; Nexø, Bjørn Andersen; Wallin, Håkan; Hansteen, Inger-Lise; Vogel, Ulla; Kure, Elin H

    2006-01-01

    Background The majority of colorectal cancer (CRC) cases develop through the adenoma-carcinoma pathway. If an increase in DNA repair expression is detected in both early adenomas and carcinomas it may indicate that low repair capacity in the normal mucosa is a risk factor for adenoma formation. Methods We have examined mRNA expression of two DNA repair genes, ERCC1 and OGG1 as well as the putative apoptosis controlling gene RAI, in normal tissues and lesions from 36 cases with adenomas (mild/moderat n = 21 and severe n = 15, dysplasia) and 9 with carcinomas. Results Comparing expression levels of ERCC1, OGG1 and RAI between normal tissue and all lesions combined yielded higher expression levels in lesions, 3.3-fold higher (P = 0.005), 5.6-fold higher(P < 3·10-5) and 7.7-fold higher (P = 0.0005), respectively. The levels of ERCC1, OGG1 and RAI expressions when comparing lesions, did not differ between adenomas and CRC cases, P = 0.836, P = 0.341 and P = 0.909, respectively. When comparing expression levels in normal tissue, the levels for OGG1 and RAI from CRC cases were significantly lower compared to the cases with adenomas, P = 0.012 and P = 0.011, respectively. Conclusion Our results suggest that increased expression of defense genes is an early event in the progression of colorectal adenomas to carcinomas. PMID:16914027

  19. SEIZURE ACTIVITY INVOLVED IN THE UP-REGULATION OF BDNF mRNA EXPRESSION BY ACTIVATION OF CENTRAL MU OPIOID RECEPTORS

    PubMed Central

    ZHANG, H. N.; KO, M. C.

    2009-01-01

    Chemical-induced seizures up-regulated brain-derived neurotrophic factor (BDNF) mRNA expression. Intracerebroventricular (i.c.v.) administration of endogenous opioids preferentially activating μ opioid receptor (MOR) could also increase BDNF mRNA expression. The aim of this study was to determine to what extent i.c.v. administration of synthetic MOR-selective agonists in rats can modulate both seizure activity and up-regulation of BDNF mRNA expression. Effects and potencies of i.c.v. administration of morphine and [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO), were directly investigated by scoring behavioral seizures and measuring BDNF mRNA expression. In addition, effects of the opioid receptor antagonist naloxone and antiepileptic drugs, diazepam, phenobarbital, and valproate, on i.c.v. MOR agonist-induced behavioral seizures and up-regulation of BDNF mRNA expression were determined. A single i.c.v. administration of morphine (10–100 μg) or DAMGO (0.15–1.5 μg) dose-dependently elicited behavioral seizures and increased BDNF mRNA expression in the widespread brain regions. However, subcutaneous administration of MOR agonists neither produced behavioral seizures nor increased BDNF mRNA expression. Pretreatment with naloxone 1 mg/kg significantly reduced behavioral seizure scores and the up-regulation of BDNF mRNA expression elicited by i.c.v. morphine or DAMGO. Similarly, diazepam 10 mg/kg and phenobarbital 40 mg/kg significantly blocked i.c.v. MOR agonist-induced actions. Pretreatment with valproate 300 mg/kg only attenuated behavioral seizures, but it did not affect morphine-induced increase of BDNF mRNA expression. This study provides supporting evidence that seizure activity plays an important role in the up-regulation of BDNF mRNA expression elicited by central MOR activation and that decreased inhibitory action of GABAergic system through the modulation on GABA receptor synaptic function by central MOR activation is involved in its regulation of BDNF mRNA

  20. Profiles of mRNA expression of related genes in the duck hypothalamus-pituitary growth axis during embryonic and early post-hatch development.