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Sample records for hkii mrna expression

  1. Exercise increases hexokinase II mRNA, but not activity in obesity and type 2 diabetes.

    PubMed

    Cusi, K J; Pratipanawatr, T; Koval, J; Printz, R; Ardehali, H; Granner, D K; Defronzo, R A; Mandarino, L J

    2001-05-01

    Glucose phosphorylation, catalyzed by hexokinase, is the first committed step in glucose uptake in skeletal muscle. Hexokinase II (HKII) is the isoform that is present in muscle and is regulated by insulin and muscle contraction. Glucose phosphorylation and HKII expression are both reduced in obese and type 2 diabetic subjects. A single bout of exercise increases HKII mRNA and activity in muscle from healthy subjects. The present study was performed to determine if a moderate exercise increases HKII mRNA expression and activity in patients with type 2 diabetes. Muscle biopsies were performed before and 3 hours after a single bout of cycle ergometer exercise in obese and type 2 diabetic patients. HKII mRNA and activity and glycogen synthase activity were determined in the muscle biopsies. Exercise increased HKII mRNA in obese and diabetic subjects by 1.67 +/- 0.34 and 1.87 +/- 0.26-fold, respectively (P <.05 for both). Exercise did not significantly increase HKI mRNA. When HKII mRNA increases were compared with the 2.26 +/- 0.36-fold increase in HKII mRNA previously reported for healthy lean subjects, no statistically significant differences were found. In contrast to the increase in HKII activity observed after exercise by lean healthy controls, exercise did not increase HKII activity in obese nondiabetic or diabetic subjects. Exercise increased glycogen synthase activity (GS(0.1) and GS(FV)) significantly in both obese nondiabetic and type 2 diabetic patients. The present results indicate that there is a posttranscriptional defect in the response of HKII expression to exercise in obese and type 2 diabetic subjects. This defect may contribute to reduced HKII activity and glucose uptake in these patients.

  2. High lib mRNA expression in breast carcinomas.

    PubMed

    Satoh, Kazuki; Hata, Mitsumi; Yokota, Hiroshi

    2004-06-30

    Lib, first identified as a novel beta-amyloid responsive gene in rat astrocytes, has an extracellular domain of 15 leucine-rich repeats (LRRs) followed by a transmembrane domain and a short cytoplasmic region. It is a distinctly inducible gene and is thought to play a key role in inflammatory states via the LRR extracellular motif, an ideal structural framework for protein-protein and protein-matrix interactions. To evaluate potential roles of Lib, we screened various tumors for Lib expression. Lib mRNA expression was high and uniquely expressed in breast tumor tissues, compared to paired normal breast tissues. Lib mRNA was localized in the ductal carcinoma cells and Lib protein displayed a homophilic association on the surface of cultured cells. These data suggest that Lib may play a role in the progression of breast carcinomas and may be a diagnostic marker for breast tumors.

  3. The HK-II Survey: Kinematics of Metal-Poor Stars in the Galaxy

    NASA Astrophysics Data System (ADS)

    Rhee, J.; Beers, T. C.

    2003-12-01

    The digitized HK-II survey (Rhee 2000, Ph.D. thesis, MSU) was originated as a follow-on to the HK-I survey of Beers and colleagues (e.g., Beers et al. 1992, AJ, 103, 1987). HK-I was based on visually-selected candidate metal-poor stars from objective-prism plates. Unfortunately, in the absence of color information, this selection technique introduced a rather severe temperature-related bias. As a result, the HK-I candidates do not include large numbers of metal-deficient giants. In HK-II, candidate metal-poor stars are quantitatively selected from digitized objective-prism spectra with JHK color information from the recently completeted 2MASS catalog. This approach eliminates much of the temperature bias. We have begun to survey candidate very metal-poor ([Fe/H] ≤ -2.0) giants from HK-II, over the magnitude range 11.0 ≤ B ≤ 16.0, covering some ˜7000 deg2 of intermediate to high Galactic-latitudes. Ongoing medium-resolution ( ˜ 1-2Å ) spectroscopic follow-up using NOAO observing facilities has allowed us to obtain, to date, some 1000 spectra (400, 450, and 150 spectra for red giants, subgiants near the main-sequence turnoff, and FHB/A stars, respectively) for the HK-II metal-poor star candidates. In particular, the detection rate of bona fide very metal-poor giants is about 45 %, which is quite encouraging. Most of the "mistakes" are slightly more metal-rich giants, with -2.0 < [Fe/H] < -1.0. Metallicities and radial velocities are determined from our spectroscopy, and proper motions for most of the program stars are obtained from the recently released UCAC2 astrometric survey catalog. Here we present an analysis of the full space motions for numerous metal-poor stars from the HK-II survey. A comparision of the chemical and kinematic properties between high- and low-halo populations (that is, giants vs. sub-giants) will aid us in understanding the formation history of the Milky Way. J.R. acknowledges partial support for this work by NASA through the AAS

  4. 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.

  5. Prolyl carboxypeptidase mRNA expression in the mouse brain.

    PubMed

    Jeong, Jin Kwon; Diano, Sabrina

    2014-01-13

    Prolyl carboxypeptidase (PRCP), a serine protease, is widely expressed in the body including liver, lung, kidney and brain, with a variety of known substrates such as plasma prekallikrein, bradykinin, angiotensins II and III, and α-MSH, suggesting its role in the processing of tissue-specific substrates. In the brain, PRCP has been shown to inactivate hypothalamic α-MSH, thus modulating melanocortin signaling in the control of energy metabolism. While its expression pattern has been reported in the hypothalamus, little is known on the distribution of PRCP throughout the mouse brain. This study was undertaken to determine PRCP expression in the mouse brain. Radioactive in situ hybridization was performed to determine endogenous PRCP mRNA expression. In addition, using a gene-trap mouse model for PRCP deletion, X-gal staining was performed to further determine PRCP distribution. Results from both approaches showed that PRCP gene is broadly expressed in the brain.

  6. Cytokine mRNA expression in postischemic/reperfused myocardium.

    PubMed Central

    Herskowitz, A.; Choi, S.; Ansari, A. A.; Wesselingh, S.

    1995-01-01

    While the role of cytokines in mediating injury during hind limb skeletal muscle ischemia followed by reperfusion has recently been described, the role of cytokines in myocardial infarction and ischemia/reperfusion have remained relatively unexplored. We hypothesize that cytokines play an important role in the regulation of postischemic myocardial inflammation. This study reports the temporal sequence of proinflammatory cytokine gene expression in postischemic/reperfused myocardium and localizes interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha)-protein by immunostaining. Rats were subjected to either permanent left anterior descending (LAD) occlusion or to 35 minutes of LAD occlusion followed by reperfusion and sacrificed up to 7 days later. Rat-specific oligonucleotide probes were used to semiquantitatively assess the relative expression of mRNA for TNF-alpha, IL-1 beta, IL-2, IL-6, interferon-gamma (IFN-gamma), and transforming growth factor-beta 1 (TGF-beta 1) utilizing the reverse transcriptase-polymerase chain reaction amplification technique. Increased cardiac mRNA levels for all cytokines except IL-6 and IFN-gamma were measurable within 15 to 30 minutes of LAD occlusion and increased levels were generally sustained for 3 hours. During early reperfusion, mRNA levels for IL-6 and TGF-beta 1 were significantly reduced compared with permanent LAD occlusion. In both groups, cytokine mRNA levels all returned to baseline levels at 24 hours, while IL-1 beta, TNF-alpha, and TGF-beta 1 mRNA levels again rose significantly at 7 days only in animals with permanent LAD occlusion. Immunostaining for IL-1 beta and TNF-alpha protein revealed two patterns of reactivity: 1) microvascular staining for both IL-1 beta and TNF-alpha protein only in postischemic reperfused myocardium in early post-reperfusion time points; and 2) staining of infiltrating macrophages in healing infarct zones which was most prominent at 7 days after permanent LAD occlusion

  7. 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.

  8. Highest trkB mRNA expression in the entorhinal cortex among hippocampal subregions in the adult rat: contrasting pattern with BDNF mRNA expression.

    PubMed

    Tokuyama, W; Hashimoto, T; Li, Y X; Okuno, H; Miyashita, Y

    1998-11-20

    Brain-derived neurotrophic factor (BDNF) and its receptor, TrkB, regulate synaptic functions in the hippocampus of the adult rodent. In previous studies, in situ hybridization methods have been used to evaluate regional differences in BDNF and trkB mRNA expression levels in hippocampal subregions. However, these studies have failed to reach consensus regarding the regional differences in the mRNA expression levels. In the present study, we quantitated mRNA expression levels using two different methods, ribonuclease protection assays and a quantitative reverse-transcription polymerase chain reaction technique, in four hippocampal subregions: the entorhinal cortex, dentate gyrus (DG), CA3 and CA1. These two methods yielded the same results. We found that BDNF and trkB mRNA expression levels did not covary in the four subregions. BDNF and full length trkB (trkB FL) mRNA in the entorhinal cortex and the DG show contrasting expression patterns. The expression level of BDNF mRNA was highest in the DG among the hippocampal subregions and low in the entorhinal cortex and the CA1, whereas the trkB FL mRNA expression level was highest in the entorhinal cortex, low in the DG and lowest in the CA3. These results suggest regional differences in BDNF/TrkB signaling for maintenance and modifiability of neuronal connections in the hippocampal formation.

  9. Exercise and adrenaline increase PGC-1α mRNA expression in rat adipose tissue

    PubMed Central

    Sutherland, Lindsey N; Bomhof, Marc R; Capozzi, Lauren C; Basaraba, Susan A U; Wright, David C

    2009-01-01

    The purpose of the present investigation was to explore the effects of exercise and adrenaline on the mRNA expression of PGC-1α, a master regulator of mitochondrial biogenesis, in rat abdominal adipose tissue. We hypothesized that (1) exercise training would increase PGC-1α mRNA expression in association with increases in mitochondrial marker enzymes, (2) adrenaline would increase PGC-1α mRNA expression and (3) the effect of exercise on PGC-1α mRNA expression in white adipose tissue would be attenuated by a β-blocker. Two hours of daily swim training for 4 weeks led to increases in mitochondrial marker proteins and PGC-1α mRNA expression in epididymal and retroperitoneal fat depots. Additionally, a single 2 h bout of exercise led to increases in PGC-1α mRNA expression immediately following exercise cessation. Adrenaline treatment of adipose tissue organ cultures led to dose-dependent increases in PGC-1α mRNA expression. A supra-physiological concentration of adrenaline increased PGC-1α mRNA expression in epididymal but not retroperitoneal adipose tissue. β-Blockade attenuated the effects of an acute bout of exercise on PGC-1α mRNA expression in epididymal but not retroperitoneal fat pads. In summary, this is the first investigation to demonstrate that exercise training, an acute bout of exercise and adrenaline all increase PGC-1α mRNA expression in rat white adipose tissue. Furthermore it would appear that increases in circulating catecholamine levels may be one potential mechanism mediating exercise induced increases in PGC-1α mRNA expression in rat abdominal adipose tissue. PMID:19221126

  10. Transfection efficiency and transgene expression kinetics of mRNA delivered in naked and nanoparticle format.

    PubMed

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

    2013-03-28

    Transfection efficiencies and transgene expression kinetics of messenger RNA (mRNA), an emerging class of nucleic acid-based therapeutics, have been poorly characterized. In this study, we evaluated transfection efficiencies of mRNA delivered in naked and nanoparticle format in vitro and in vivo using GFP and luciferase as reporters. While mRNA nanoparticles transfect primary human and mouse dendritic cells (DCs) efficiently in vitro, naked mRNA could not produce any detectable gene product. The protein expression of nanoparticle-mediated transfection in vitro peaks rapidly within 5-7h and decays in a biphasic manner. In vivo, naked mRNA is more efficient than mRNA nanoparticles when administered subcutaneously. In contrast, mRNA nanoparticle performs better when administered intranasally and intravenously. Gene expression is most transient when delivered intravenously in nanoparticle format with an apparent half-life of 1.4h and lasts less than 24h, and most sustained when delivered in the naked format subcutaneously at the base of tail with an apparent half-life of 18h and persists for at least 6days. Notably, exponential decreases in protein expression are consistently observed post-delivery of mRNA in vivo regardless of the mode of delivery (naked or nanoparticle) or the site of administration. This study elucidates the performance of mRNA transfection and suggests a niche for mRNA therapeutics when predictable in vivo transgene expression kinetics is imperative.

  11. Expression of APOBEC3B mRNA in Primary Breast Cancer of Japanese Women

    PubMed Central

    Tokunaga, Eriko; Yamashita, Nami; Tanaka, Kimihiro; Inoue, Yuka; Akiyoshi, Sayuri; Saeki, Hiroshi; Oki, Eiji; Kitao, Hiroyuki; Maehara, Yoshihiko

    2016-01-01

    Recent studies have identified the apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3B (APOBEC3B) as a source of mutations in various malignancies. APOBEC3B is overexpressed in several human cancer types, including breast cancer. In this study, we analyzed APOBEC3B mRNA expression in 305 primary breast cancers of Japanese women using quantitative reverse transcription-PCR, and investigated the relationships between the APOBEC3B mRNA expression and clinicopathological characteristics, prognosis, and TP53 mutations. The expression of APOBEC3B mRNA was detected in 277 tumors and not detected in 28 tumors. High APOBEC3B mRNA expression was significantly correlated with ER- and PR-negativity, high grade and high Ki67 index. The APOBEC3B mRNA expression was highest in the triple-negative and lowest in the hormone receptor-positive/HER2-negative subtypes. The TP53 gene was more frequently mutated in the tumors with high APOBEC3B mRNA expression. High APOBEC3B mRNA expression was significantly associated with poor recurrence-free survival in all cases and the ER-positive cases. These findings were almost consistent with the previous reports from the Western countries. In conclusion, high APOBEC3B mRNA expression was related to the aggressive phenotypes of breast cancer, high frequency of TP53 mutation and poor prognosis, especially in ER-positive tumors. PMID:27977754

  12. Propionate induces mRNA expression of gluconeogenic genes in bovine calf hepatocytes.

    PubMed

    Zhang, Qian; Koser, Stephanie L; Donkin, Shawn S

    2016-05-01

    Hepatocytes monolayers from neonatal calves were used to determine the responses of the cytosolic phosphoenolpyruvate carboxykinase (PCK1) mRNA expression to propionate and direct hormonal cues including cyclic AMP (cAMP), dexamethasone, and insulin. The responses of other key gluconeogenic genes, including mitochondrial phosphoenolpyruvate carboxykinase (PCK2), pyruvate carboxylase (PC), and glucose-6-phosphotase (G6PC), were also measured. Expression of PCK1 was linearly induced with increasing propionate concentrations in media and 2.5 mM propionate increased PCK1 mRNA at 3 and 6h of incubation; however, the induction disappeared at 12 and 24 h. The induction of PCK1 mRNA by propionate was mimicked by 1 mM cAMP, or in combination with 5 µM dexamethasone, but not by dexamethasone alone. The induction of PCK1 mRNA by propionate or cAMP was eliminated by addition of 100 nM insulin. Additionally, expression of PCK2 and PC mRNA was also induced by propionate in a concentration-dependent manner. Consistent with PCK1, propionate-stimulated PCK2 and PC mRNA expression was inhibited by insulin. Expression of G6PC mRNA was neither affected by propionate nor cAMP, dexamethasone, insulin, or their combinations. These findings demonstrate that propionate can directly regulate its own metabolism in bovine calf hepatocytes through upregulation of PCK1, PCK2, and PC mRNA expression.

  13. Bioinspired Nanocomplex for Spatiotemporal Imaging of Sequential mRNA Expression in Differentiating Neural Stem Cells

    PubMed Central

    2015-01-01

    Messenger RNA plays a pivotal role in regulating cellular activities. The expression dynamics of specific mRNA contains substantial information on the intracellular milieu. Unlike the imaging of stationary mRNAs, real-time intracellular imaging of the dynamics of mRNA expression is of great value for investigating mRNA biology and exploring specific cellular cascades. In addition to advanced imaging methods, timely extracellular stimulation is another key factor in regulating the mRNA expression repertoire. The integration of effective stimulation and imaging into a single robust system would significantly improve stimulation efficiency and imaging accuracy, producing fewer unwanted artifacts. In this study, we developed a multifunctional nanocomplex to enable self-activating and spatiotemporal imaging of the dynamics of mRNA sequential expression during the neural stem cell differentiation process. This nanocomplex showed improved enzymatic stability, fast recognition kinetics, and high specificity. With a mechanism regulated by endogenous cell machinery, this nanocomplex realized the successive stimulating motif release and the dynamic imaging of chronological mRNA expression during neural stem cell differentiation without the use of transgenetic manipulation. The dynamic imaging montage of mRNA expression ultimately facilitated genetic heterogeneity analysis. In vivo lateral ventricle injection of this nanocomplex enabled endogenous neural stem cell activation and labeling at their specific differentiation stages. This nanocomplex is highly amenable as an alternative tool to explore the dynamics of intricate mRNA activities in various physiological and pathological conditions. PMID:25494492

  14. 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.

  15. 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.

  16. 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

    2016-05-25

    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.

  17. Transcription Expression and Clinical Significance of Dishevelled-3 mRNA and δ-Catenin mRNA in Pleural Effusions from Patients with Lung Cancer

    PubMed Central

    Li, Xiao-Yan; Liu, Shu-Li; Cha, Na; Zhao, Yu-Jie; Wang, Shao-Cheng; Li, Wei-Nan; Wang, En-Hua; Wu, Guang-Ping

    2012-01-01

    Objective. To evaluate diagnostic utility of Dishevelled-3 (DVL-3) mRNA and δ-catenin mRNA expression in pleural effusions of patients with lung cancer. Methods. DVL-3 mRNA and δ-catenin mRNA levels were assessed by performing RT-PCR on pleural effusion specimens from patients with lung cancer (n = 75) and with lung benign disease (n = 51). Results. The expressions of DVL-3 mRNA and δ-catenin mRNA were significantly higher in malignant than in benign lung disease (P < 0.01) and were obviously higher than cytology in adenocarcinoma (P < 0.01). In single use, DVL-3 mRNA had the highest specificity (94.1%) and PPV (95.7%), whereas δ-catenin mRNA had the highest sensitivity (92.0%) and NPV (88.5%). When combinations of markers were evaluated together, DVL-3 mRNA and δ-catenin mRNA gave a high-diagnostic performance: sensitivity of 100.0%, NPV of 100.0%, and accuracy of 96.0%, respectively. Conclusion. As molecular markers of detecting pleural micrometastasis, DVL-3 mRNA and δ-catenin mRNA are helpful to diagnose the cancer cells in pleural effusions of patients with lung cancer. PMID:22461838

  18. Reduced beta 2-microglobulin mRNA levels in transgenic mice expressing a designed hammerhead ribozyme.

    PubMed Central

    Larsson, S; Hotchkiss, G; Andäng, M; Nyholm, T; Inzunza, J; Jansson, I; Ahrlund-Richter, L

    1994-01-01

    We have generated three artificial hammerhead ribozymes, denoted 'Rz-b', 'Rz-c' and 'Rz-d', with different specificities for exon II of the mouse beta-2-microglobulin (beta 2M) mRNA. In this study we tested for ribozyme mediated reduction of beta 2M mRNA in a cell line and in transgenic mice. Transfections of either of the Rz-b, Rz-c or Rz-d plasmids into a mouse cell-line (NIH/3T3) revealed reductions of beta 2M mRNA substrate in each case. Ribozyme expression in individual transfected clones was accompanied with an up to 80% reduction of beta 2M mRNA levels. Rz-c was selected for a transgenic study. Seven Rz-c transgenic founder animals were identified from which three ribozyme expressing families were established and analysed. Expression of the ribozyme transgene was tested for and detected in lung, kidney and spleen. Expression was accompanied with reduction of the beta 2M mRNA levels of heterozygous (Rz+/-) animals compared to non-transgenic litter mates. The effect was most pronounced in lung with more than 90% beta 2M mRNA reduction in individual mice. In summary, expression of our ribozymes in a cell free system, in a cell-line and in transgenic mice were all accompanied with reductions of beta 2M mRNA levels. Images PMID:8036151

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

    SciTech Connect

    Milsted, A.; Barna, B.P.; Ransohoff, R.M.; Brosnihan, K.B.; Ferrario, C.M. )

    1990-08-01

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

  20. The effect of 3-bromopyruvate on human colorectal cancer cells is dependent on glucose concentration but not hexokinase II expression.

    PubMed

    Ho, Nelson; Morrison, Jodi; Silva, Andreza; Coomber, Brenda L

    2016-01-06

    Cancer cells heavily rely on the glycolytic pathway regardless of oxygen tension. Hexokinase II (HKII) catalyses the first irreversible step of glycolysis and is often overexpressed in cancer cells. 3-Bromopyruvate (3BP) has been shown to primarily target HKII, and is a promising anti-cancer compound capable of altering critical metabolic pathways in cancer cells. Abnormal vasculature within tumours leads to heterogeneous microenvironments, including glucose availability, which may affect drug sensitivity. The aim of the present study was to elucidate the mechanisms by which 3BP acts on colorectal cancer (CRC) cells with focus on the HKII/Akt signalling axis. High HKII-expressing cell lines were more sensitive to 3BP than low HKII-expressing cells. 3BP-induced rapid Akt phosphorylation at site Thr-308 and cell death via both apoptotic and necrotic mechanisms. Cells grown under lower glucose concentrations showed greater resistance towards 3BP. Cells with HKII knockdown showed no changes in 3BP sensitivity, suggesting the effects of 3BP are independent of HKII expression. These results emphasize the importance of the tumour microenvironment and glucose availability when considering therapeutic approaches involving metabolic modulation.

  1. The effect of 3-bromopyruvate on human colorectal cancer cells is dependent on glucose concentration but not hexokinase II expression

    PubMed Central

    Ho, Nelson; Morrison, Jodi; Silva, Andreza; Coomber, Brenda L.

    2016-01-01

    Cancer cells heavily rely on the glycolytic pathway regardless of oxygen tension. Hexokinase II (HKII) catalyses the first irreversible step of glycolysis and is often overexpressed in cancer cells. 3-Bromopyruvate (3BP) has been shown to primarily target HKII, and is a promising anti-cancer compound capable of altering critical metabolic pathways in cancer cells. Abnormal vasculature within tumours leads to heterogeneous microenvironments, including glucose availability, which may affect drug sensitivity. The aim of the present study was to elucidate the mechanisms by which 3BP acts on colorectal cancer (CRC) cells with focus on the HKII/Akt signalling axis. High HKII-expressing cell lines were more sensitive to 3BP than low HKII-expressing cells. 3BP-induced rapid Akt phosphorylation at site Thr-308 and cell death via both apoptotic and necrotic mechanisms. Cells grown under lower glucose concentrations showed greater resistance towards 3BP. Cells with HKII knockdown showed no changes in 3BP sensitivity, suggesting the effects of 3BP are independent of HKII expression. These results emphasize the importance of the tumour microenvironment and glucose availability when considering therapeutic approaches involving metabolic modulation. PMID:26740252

  2. Differential expression of IGF-1 mRNA isoforms in colorectal carcinoma and normal colon tissue.

    PubMed

    Kasprzak, Aldona; Szaflarski, Witold; Szmeja, Jacek; Andrzejewska, Małgorzata; Przybyszewska, Wiesława; Kaczmarek, Elżbieta; Koczorowska, Maria; Kościński, Tomasz; Zabel, Maciej; Drews, Michał

    2013-01-01

    The insulin-like growth factor (IGF)-1 gene consists of 6 exons resulting in the expression of 6 variant forms of mRNA (IA, IB, IC, IIA, IIB and IIC) due to an alternative splicing. The mechanisms of IGF-1 gene splicing and the role of local expression manifested by IGF-1 mRNA variants in colorectal carcinoma (CRC) have not been extensively investigated. Therefore, the aim of our study was to analyse the expression of IGF-1 mRNA isoforms [A, B, C, P1 (class I) and P2 (class II)], as well as the protein expression in CRC and control samples isolated from 28 patients. The expression of Ki-67 was also analysed and clinical data were obtained. For this purpose, we used quantitative real-time PCR (qPCR) and immunocytochemistry. The expression of mRNAs coding for all splicing isoforms of IGF-1 was observed in every tissue sample studied, with a significantly lower expression noted in the CRC as compared to the control samples. The cytoplasmic expression of IGF-1 protein was found in 50% of the CRC and in ~40% of the non-tumor tissues; however, no significant quantitative inter-group differences were observed. The expression of the IGF-1 gene in the 2 groups of tissues was controlled by the P1 and P2 promoters in a similar manner. No significant differences were detected in the expression of the IGF-1 A and B isoforms; however, their expression was significantly higher compared to that of isoform C. No significant differences were observed between the expression of Ki-67 mRNA in the CRC and control tissue even though the expression of the Ki-67 protein was higher in the CRC compared to the control samples. Ki-67 protein expression was associated with the macroscopic and microscopic aspects of CRC. A significant positive correlation was found between the local production of total mRNA and isoform A and the expression of Ki-67 mRNA, although only in the non-tumor tissues. In CRC samples, the local expression of the total IGF-1 mRNA and all splicing isoforms of IGF-1 mRNA

  3. Localization and differential regulation of angiotensinogen mRNA expression in the vessel wall.

    PubMed Central

    Naftilan, A J; Zuo, W M; Inglefinger, J; Ryan, T J; Pratt, R E; Dzau, V J

    1991-01-01

    Recent data demonstrate the existence of a vascular renin angiotensin system. In this study we examine the localization of angiotensinogen mRNA in the blood vessel wall of two rat strains, the Wistar and Wistar Kyoto (WKY), as well as the regulation of vascular angiotensinogen mRNA expression by dietary sodium. Northern blot analysis and in situ hybridization histochemistry demonstrate that in both strains angiotensinogen mRNA is detected in the aortic medial smooth muscle layer as well as the periaortic fat. In WKY rats fed a 1.6% sodium diet, angiotensinogen mRNA concentration is 2.6-fold higher in the periaortic fat than in the smooth muscle, as analyzed by quantitative slot blot hybridization. Angiotensinogen mRNA expression in the medial smooth muscle layer is sodium regulated. After 5 d of a low (0.02%) sodium diet, smooth muscle angiotensinogen mRNA levels increase 3.2-fold (P less than 0.005) as compared with the 1.6% sodium diet. In contrast, angiotensinogen mRNA level in the periaortic fat is not influenced by sodium diet. In summary, our data demonstrate regional (smooth muscle vs. periaortic fat) differential regulation of angiotensinogen mRNA levels in the blood vessel wall by sodium. This regional differential regulation by sodium may have important physiological implications. Images PMID:2010543

  4. Hypothalamic expression of NPY mRNA, vasopressin mRNA and CRF mRNA in response to food restriction and central administration of the orexigenic peptide GHRP-6.

    PubMed

    Johnstone, Louise E; Srisawat, Rungrudee; Kumarnsit, Ekkasit; Leng, Gareth

    2005-03-01

    In this study, we examined the effects of restricted feeding and of central administration of an orexigenic ghrelin agonist GHRP-6 on peptide mRNA expression in the hypothalamus. We compared rats fed ad libitum with rats that were allowed food for only 2?h every day, and treated with a continuous chronic i.c.v. infusion of GHRP-6 or vehicle. Ad libitum fed rats exposed to GHRP-6 increased their food intake and body weight over 6 days, but, at the end of this period, neuropeptide Y mRNA expression in the arcuate nucleus was not different to that in control rats. By contrast, expression of neuropeptide Y mRNA in the arcuate nucleus was elevated in food-restricted rats, consistent with the interpretation that increased expression reflects increased hunger. However, neuropeptide Y mRNA expression was no greater in food-restricted rats infused with GHRP-6 than in food-restricted rats infused with vehicle; thus if the drive to eat was stronger in rats infused with GHRP-6, this was not reflected by higher levels of neuropeptide Y mRNA expression. Expression of vasopressin mRNA and corticotrophin releasing factor (CRF) mRNA in the paraventricular nucleus (PVN) was not changed by food restriction. GHRP-6 infusion increased CRF mRNA expression in ad libitum rats only.

  5. Effect of taurine on mRNA expression of thioredoxin interacting protein in Caco-2 cells.

    PubMed

    Gondo, Yusuke; Satsu, Hideo; Ishimoto, Yoko; Iwamoto, Taku; Shimizu, Makoto

    2012-09-28

    Taurine (2-aminoethanesulfonic acid), a sulfur-containing β-amino acid, plays an important role in several essential biological processes; although, the underlying mechanisms for these regulatory functions remain to be elucidated, especially at the genetic level. We investigated the effects of taurine on the gene expression profile in Caco-2 cells using DNA microarray. Taurine increased the mRNA expression of thioredoxin interacting protein (TXNIP), which is involved in various metabolisms and diseases. β-Alanine or γ-aminobutyric acid (GABA), which are structurally or functionally related to taurine, did not increase TXNIP mRNA expression. These suggest the expression of TXNIP mRNA is induced specifically by taurine. β-Alanine is also known to be a substrate of taurine transporter (TAUT) and competitively inhibits taurine uptake. Inhibition of taurine uptake by β-alanine eliminated the up-regulation of TXNIP, which suggests TAUT is involved in inducing TXNIP mRNA expression. The up-regulation of TXNIP mRNA expression by taurine was also observed at the protein level. Furthermore, taurine significantly increased TXNIP promoter activity. Our present study demonstrated the taurine-specific phenomenon of TXNIP up-regulation, which sheds light on the physiological function of taurine.

  6. Sodium regulation of angiotensinogen mRNA expression in rat kidney cortex and medulla.

    PubMed Central

    Ingelfinger, J R; Pratt, R E; Ellison, K; Dzau, V J

    1986-01-01

    Rat liver angiotensinogen cDNA (pRang 3) and mouse renin cDNA (pDD-1D2) were used to identify angiotensinogen and renin mRNA sequences in rat kidney cortex and medulla in rats on high and low salt diet. Angiotensinogen mRNA sequences were present in renal cortex and medulla in apparently equal proportions, whereas renin mRNA sequences were found primarily in renal cortex. Average relative signal of rat liver to whole kidney angiotensinogen mRNA was 100:3. Densitometric analysis of Northern blots demonstrated that renal cortical angiotensinogen mRNA concentrations increased 3.5-fold (P less than 0.001) and medulla, 1.5-fold (P less than 0.005) on low sodium compared with high sodium diet, whereas renal cortex renin mRNA levels increased 6.8-fold (P less than 0.0005). Dietary sodium did not significantly influence liver angiotensinogen mRNA levels. These findings provide evidence for sodium regulation of renal renin and angiotensinogen mRNA expressions, which supports potential existence of an intrarenally regulated RAS and suggest that different factors regulate renal and hepatic angiotensinogen. Images PMID:3533999

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

    PubMed

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

    2003-02-01

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

  8. Expression of a streptomycete leaderless mRNA encoding chloramphenicol acetyltransferase in Escherichia coli.

    PubMed Central

    Wu, C J; Janssen, G R

    1997-01-01

    The chloramphenicol acetyltransferase (cat) gene from Streptomyces acrimycini encodes a leaderless mRNA. Expression of the cat coding sequence as a leaderless mRNA from a modified lac promoter resulted in chloramphenicol resistance in Escherichia coli. Transcript mapping with nuclease S1 confirmed that the 5' end of the cat message initiated at the A of the AUG translational start codon. Site-directed mutagenesis of the lac promoter or the cat start codon abolished chloramphenicol resistance, indicating that E. coli initiated translation at the 5' terminal AUG of the cat leaderless mRNA. Addition of 5'-AUGC-3' to the 5' end of the cat mRNA resulted in translation occurring also from the reading frame defined by the added AUG triplet, suggesting that a 5'-terminal start codon is an important recognition feature for initiation and establishing reading frame during translation of leaderless mRNA. Addition of an untranslated leader and Shine-Dalgarno sequence to the cat coding sequence increased cat expression in a cat:lacZ fusion; however, the level of expression was significantly lower than when a fragment of the bacteriophage lambda cI gene, also encoding a leaderless mRNA, was fused to lacZ. These results indicate that in the absence of an untranslated leader and Shine-Dalgarno sequence, the streptomycete cat mRNA is translated by E. coli; however, the cat translation signals, or other features of the cat mRNA, provide for only a low level of expression in E. coli. PMID:9352935

  9. Neuropeptide Y mRNA expression levels following chronic olanzapine, clozapine and haloperidol administration in rats.

    PubMed

    Huang, X-F; Deng, Chao; Zavitsanou, Katerina

    2006-06-01

    Using quantitative in situ hybridization, this study examined regional changes in rat brain mRNA levels encoding neuropeptide Y (NPY) following olanzapine, clozapine and haloperidol administration (1.2, 1.5 and 2.0 mg/kg, oral) for 36 days. The NPY mRNA expression levels and patterns were examined after the last drug administration at both time points enabling the measurement of immediate effect at 2h and the effects after 48 h of drug administration. It was found that all these drugs had an immediate effect on NPY mRNA expression, while virtually all these changes normalized 48 h after the drug treatments. A similarity in altered NPY mRNA expression patterns was seen between the olanzapine and clozapine groups; however, haloperidol was very different. Olanzapine and clozapine administration decreased NPY mRNA levels in the nucleus accumbens, striatum and anterior cingulate cortex (from -60% to -77%, p<0.05). Haloperidol decreased NPY mRNA expression in the amygdala and hippocampus (-69%, -64%, p<0.05). In the lateral septal nucleus, NPY mRNA levels significantly decreased in the olanzapine group (-66%, p<0.05), a trend toward a decrease was observed in the clozapine group, and no change was found in the haloperidol treated group. These results suggest that the different effects of atypical and typical antipsychotics on NPY systems may reflect the neural chemical mechanisms responsible for the differences between these drugs in their effects in treating positive and negative symptoms of schizophrenia. The immediate decrease of NPY mRNA levels suggests an immediate reduction of NPY biosynthesis in response to these drugs.

  10. 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.

  11. 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.

  12. Chemokines mRNA expression in relation to the Macrophage Migration Inhibitory Factor (MIF) mRNA and Vascular Endothelial Growth Factor (VEGF) mRNA expression in the microenvironment of endometrial cancer tissue and normal endometrium: a pilot study.

    PubMed

    Giannice, Raffaella; Erreni, Marco; Allavena, Paola; Buscaglia, Mauro; Tozzi, Roberto

    2013-11-01

    Tumor microenvironment inflammatory cells play a major role in cancer progression. Among these, the Tumor Associated Macrophages (TAMs) infiltration depends on the kind of chemokine, cytokines and growth factors secreted by the tumor cells and by the stroma in response to the cancer invasion. TAMs have been found to promote anti-tumor response in early stages and to stimulate neovascularization and metastases in advanced disease. In the microenvironment chemo-attractants of many human cancers, MIF and VEGF correlate with an increased TAMs recruitment. In addition, MIF enhances tumor cells metastases by modulating the immune responses and by promoting the angiogenesis related to VEGF. On the contrary the inhibition of MIF can lead to cell cycle arrest and apoptosis. Some chemokines (e.g. CXCL12, CXCL11, CXCL8) and their receptors, thanks to their ability to modulate migration and proliferation, are involved in the angiogenetic process. In this study we compared the expression of MIF mRNA with VEGF mRNA expression and with mRNA expression of other chemokines related to neo-angiogenesis, such as CXCL12, CXCL11, CXCL8 and CXCR4, in human endometrial cancer tissue (EC) and normal endometrium (NE). Fresh samples of EC tissue and NE were extracted from 15 patients with FIGO stage I-III undergoing primary surgery. Some of the tissue was sent for histology and part of it was treated with RNA later and stored at -80°C. Four patients dropped out. A significant up-regulation of MIF mRNA in EC tissue versus NE samples (P=0.01) was observed in all 11 patients. The MIF mRNA over-expression was coincident with a VEGF mRNA overexpression in 54% of patients (P=NS). MIF mRNA was inversely related to CXCL12 mRNA expression (P=0.01). MIF over-expression was significantly related to low grading G1-2 (P=0.01), endometrial type I (P=0.05), no lymphovascular spaces invasion (P=0.01) and 3years DFS (P=0.01). As reported in previous studies on patients with breast cancer, our data suggest

  13. Developmental expression of parvalbumin mRNA in the cerebral cortex and hippocampus of the rat.

    PubMed

    de Lecea, L; del Río, J A; Soriano, E

    1995-08-01

    Parvalbumin (PARV) belongs to the family of calcium-binding proteins bearing the EF hand domain. Immunocytochemical studies in the cerebral cortex have demonstrated that neurons containing PARV include two types of GABAergic interneurons, namely, basket and axo-axonic chandelier cells. The present study examines the onset and pattern of PARV mRNA expression during the development of rat neocortex and hippocampus by means of 'in situ' hybridization with an oligonucleotide probe corresponding to rat PARV cDNA. In animals aged P0-P6 no signal was detected above background in neocortex or hippocampus. At P8, a few cortical cells displayed a number of silver grains just above background levels. By P10 PARV mRNA-expressing cells in the neocortex were detected almost exclusively in layer V of somatosensory, frontal and cingulate cortices. At P12 PARV mRNA was mainly detected in layers IV, V and VIa. By P14 there was a marked overall increase in the entire neocortex, including layer II-III, both in the number of cells and in their intensity of labelling. Further maturation in the pattern of PARV mRNA concentration was observed between P16 and P21. In the hippocampus low hybridization was observed at P10-P12. In subsequent stages both the number of positive cells and the intensity of labelling increased steadily. No clear-cut radial gradients for the expression of PARV mRNA were observed in the hippocampal region. Our results show that the developmental radial gradient followed by PARV mRNA expression in the neocortex does not follow an 'inside-out' gradient, consistent with previous immunocytochemical findings. Taken together, these data indicate that the developmental sequence followed by the PARV protein directly reflects mRNA abundance and suggest that PARV mRNA expression correlates with the functional maturation of cortical interneurons.

  14. Myxovirus Resistance Protein A mRNA Expression Kinetics in Multiple Sclerosis Patients Treated with IFNβ

    PubMed Central

    Libertinova, Jana; Meluzinova, Eva; Tomek, Ales; Horakova, Dana; Kovarova, Ivana; Matoska, Vaclav; Kumstyrova, Simona; Zajac, Miroslav; Hyncicova, Eva; Liskova, Petra; Houzvickova, Eva; Martinkovic, Lukas; Bojar, Martin; Havrdova, Eva; Marusic, Petr

    2017-01-01

    Introduction Interferon-β (IFNß) is the first-line treatment for relapsing-remitting multiple sclerosis. Myxovirus resistance protein A (MxA) is a marker of IFNß bioactivity, which may be reduced by neutralizing antibodies (NAbs) against IFNß. The aim of the study was to analyze the kinetics of MxA mRNA expression during long-term IFNβ treatment and assess its predictive value. Methods A prospective, observational, open-label, non-randomized study was designed in multiple sclerosis patients starting IFNß treatment. MxA mRNA was assessed prior to initiation of IFNß therapy and every three months subsequently. NAbs were assessed every six months. Assessment of relapses was scheduled every three months during 24 months of follow up. The disease activity was correlated to the pretreatment baseline MxA mRNA value. In NAb negative patients, clinical status was correlated to MxA mRNA values. Results 119 patients were consecutively enrolled and 107 were included in the final analysis. There was no correlation of MxA mRNA expression levels between baseline and month three. Using survival analysis, none of the selected baseline MxA mRNA cut off points allowed prediction of time to first relapse on the treatment. In NAb negative patients, mean MxA mRNA levels did not significantly differ in patients irrespective of relapse status. Conclusion Baseline MxA mRNA does not predict the response to IFNß treatment or the clinical status of the disease and the level of MxA mRNA does not correlate with disease activity in NAb negative patients. PMID:28081207

  15. 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

  16. OPIATE EXPOSURE AND WITHDRAWAL DYNAMICALLY REGULATE mRNA EXPRESSION IN THE SEROTONERGIC DORSAL RAPHE NUCLEUS

    PubMed Central

    Lunden, Jason; Kirby, Lynn G.

    2013-01-01

    Previous results from our lab suggest that hypofunctioning of the serotonergic (5-HT) dorsal raphe nucleus (DRN) is involved in stress-induced opiate reinstatement. To further investigate the effects of morphine dependence and withdrawal on the 5-HT DRN system, we measured gene expression at the level of mRNA in the DRN during a model of morphine dependence, withdrawal and post withdrawal stress exposure in rats. Morphine pellets were implanted for 72h and then either removed or animals were injected with naloxone to produce spontaneous or precipitated withdrawal, respectively. Animals exposed to these conditions exhibited withdrawal symptoms including weight loss, wet dog shakes and jumping behavior. Gene expression for brain-derived neurotrophic factor (BDNF), TrkB, corticotrophin releasing-factor (CRF)-R1, CRF-R2, GABAA-α1, μ-opioid receptor (MOR), 5-HT1A, tryptophan hydroxylase2 and the 5-HT transporter was then measured using quantitative real-time PCR at multiple time-points across the model of morphine exposure, withdrawal and post withdrawal stress. Expression levels of BDNF, TrkB and CRF-R1 mRNA were decreased during both morphine exposure and following seven days of withdrawal. CRF-R2 mRNA expression was elevated after seven days of withdrawal. 5-HT1A receptor mRNA expression was decreased following 3 hours of morphine exposure, while TPH2 mRNA expression was decreased after seven days of withdrawal with swim stress. There were no changes in the expression of GABAA-α1, MOR or 5-HT transporter mRNA. Collectively these results suggest that alterations in neurotrophin support, CRF-dependent stress signaling, 5-HT synthesis and release may underlie 5-HT DRN hypofunction that can potentially lead to stress-induced opiate relapse. PMID:24055683

  17. mRNA expression profiling of neonatal rats after 16-day spaceflight

    NASA Astrophysics Data System (ADS)

    Miyake, M.; Ijiri, K.

    Some studies pointed out that postnatal development is the key to realize generation change of mammalians in space. For example, functional changes and hypoplasia in some organs after spaceflight during postnatal development were reported. Though profiling mRNA expression is useful to evaluate what happened in animals, these studies after spaceflight are limited to specific organs for understanding the relationship between phenotype and gene. The organ-wide analysis of mRNA expression is important to evaluate the condition of each animal, and it can find new phenomenon and help precise understanding for effect induced by spaceflight. In this experiment, we analyzed mRNA expression of liver, spleen and intestine in neonatal rats after 16-day spaceflight by Space Shuttle Columbia (STS-90).

  18. 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.

  19. 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

  20. mRNA Distribution and Heterologous Expression of Orphan Cytochrome P450 20A1

    PubMed Central

    Stark, Katarina; Wu, Zhong-Liu; Bartleson, Cheryl J.; Guengerich, F. Peter

    2015-01-01

    Cytochrome P450 (P450) 20A1 is one of the so-called “orphan” P450s without assigned biological function. mRNA expression was detected in human liver and extrahepatic expression was noted in several human brain regions, including substantia nigra, hippocampus, and amygdala, using conventional polymerase chain reaction and RNA dot blot analysis. Adult human liver contained 3-fold higher overall mRNA levels than whole brain, although specific regions (i.e., hippocampus and substantia nigra) exhibited higher mRNA expression levels than liver. Orthologous full-length and truncated transcripts of P450 20A1 were transcribed and sequenced from rat liver, heart, and brain. In rat, the concentrations of full-length transcripts were 3–4 fold higher in brain and heart than liver. In situ hybridization of rat whole brain sections showed a similar mRNA expression pattern as observed for human P450 20A1, indicating expression in substantia nigra, hippocampus, and amygdala. A number of N-terminal modifications of the codon-optimized human P450 20A1 sequence were prepared and expressed in Escherichia coli, and two of the truncated derivatives showed characteristic P450 spectra (200–280 nmol P450/l). Although the recombinant enzyme system oxidized NADPH, no catalytic activity was observed with the heterologously expressed protein when a number of potential steroids and biogenic amines were surveyed as potential substrates. The function of P450 20A1 remains unknown; however, the sites of mRNA expression in human brain and the conservation among species may suggest possible neurophysiological function. PMID:18541694

  1. Tissue-specific mRNA expression profiling in grape berry tissues

    PubMed Central

    Grimplet, Jerome; Deluc, Laurent G; Tillett, Richard L; Wheatley, Matthew D; Schlauch, Karen A; Cramer, Grant R; Cushman, John C

    2007-01-01

    Background Berries of grape (Vitis vinifera) contain three major tissue types (skin, pulp and seed) all of which contribute to the aroma, color, and flavor characters of wine. The pericarp, which is composed of the exocarp (skin) and mesocarp (pulp), not only functions to protect and feed the developing seed, but also to assist in the dispersal of the mature seed by avian and mammalian vectors. The skin provides volatile and nonvolatile aroma and color compounds, the pulp contributes organic acids and sugars, and the seeds provide condensed tannins, all of which are important to the formation of organoleptic characteristics of wine. In order to understand the transcriptional network responsible for controlling tissue-specific mRNA expression patterns, mRNA expression profiling was conducted on each tissue of mature berries of V. vinifera Cabernet Sauvignon using the Affymetrix GeneChip® Vitis oligonucleotide microarray ver. 1.0. In order to monitor the influence of water-deficit stress on tissue-specific expression patterns, mRNA expression profiles were also compared from mature berries harvested from vines subjected to well-watered or water-deficit conditions. Results Overall, berry tissues were found to express approximately 76% of genes represented on the Vitis microarray. Approximately 60% of these genes exhibited significant differential expression in one or more of the three major tissue types with more than 28% of genes showing pronounced (2-fold or greater) differences in mRNA expression. The largest difference in tissue-specific expression was observed between the seed and pulp/skin. Exocarp tissue, which is involved in pathogen defense and pigment production, showed higher mRNA abundance relative to other berry tissues for genes involved with flavonoid biosynthesis, pathogen resistance, and cell wall modification. Mesocarp tissue, which is considered a nutritive tissue, exhibited a higher mRNA abundance of genes involved in cell wall function and

  2. Deregulated expression of VHL mRNA variants in papillary thyroid cancer.

    PubMed

    Baldini, Enke; Tuccilli, Chiara; Arlot-Bonnemains, Yannick; Chesnel, Frank; Sorrenti, Salvatore; De Vito, Corrado; Catania, Antonio; D'Armiento, Eleonora; Antonelli, Alessandro; Fallahi, Poupak; Watutantrige-Fernando, Sara; Tartaglia, Francesco; Barollo, Susi; Mian, Caterina; Bononi, Marco; Arceri, Stefano; Mascagni, Domenico; Vergine, Massimo; Pironi, Daniele; Monti, Massimo; Filippini, Angelo; Ulisse, Salvatore

    2017-03-05

    Recent findings demonstrated that a subset of papillary thyroid cancers (PTCs) is characterized by reduced expression of the von Hippel-Lindau (VHL) tumor suppressor gene, and that lowest levels associated with more aggressive PTCs. In the present study, the levels of the two VHL mRNA splicing variants, VHL-213 (V1) and VHL-172 (V2), were measured in a series of 96 PTC and corresponding normal matched tissues by means of quantitative RT-PCR. Variations in the mRNA levels were correlated with patients' clinicopathological parameters and disease-free interval (DFI). The analysis of VHL mRNA in tumor tissues, compared to normal matched tissues, revealed that its expression was either up- or down-regulated in the majority of PTC. In particular, V1 and V2 mRNA levels were altered, respectively, in 78 (81.3%) and 65 (67.7%) out of the 96 PTCs analyzed. A significant positive correlation between the two mRNA variants was observed (p < 0.001). Univariate analysis documented the lack of association between each variant and clinicopathological parameters such as age, tumor size, histology, TNM stage, lymph node metastases, and BRAF mutational status. However, a strong correlation was found between altered V1 or V2 mRNA levels and DFI. Multivariate regression analysis indicated higher V1 mRNA values, along with lymph node metastases at diagnosis, as independent prognostic factors predicting DFI. In conclusion, the data reported demonstrate that VHL gene expression is deregulated in the majority of PTC tissues. Of particular interest is the apparent protective role exerted by VHL transcripts against PTC recurrences.

  3. CYP1A mRNA expression in redeye mullets (Liza haematocheila) from Bohai Bay, China.

    PubMed

    An, Lihui; Hu, Jianying; Yang, Min; Zheng, Binghui; Wei, An; Shang, Jingjing; Zhao, Xingru

    2011-04-01

    Induction of cytochrome P4501A (CYP1A) has been used as a biomarker in fish for monitoring aromatic and organic contaminants. In this study, a partial of CYP1A gene in redeye mullet (Liza haematocheila) was isolated and sequenced, and then a real-time quantitative reverse-transcription polymerase chain reaction assay was developed for quantification of CYP1A mRNA normalized to β-actin. The developed method was applied to detect CYP1A mRNA expression in redeye mullets collected from Nandaihe (reference site) and Dashentang (impacted site) in Bohai Bay, China. CYP1A mRNA expression values were significantly elevated in redeye mullets from Dashentang compared to a reference site--Nandaihe, which was correlated with the contents of different environmentally relevant pollutants in tissues, particularly with PCBs and PBDEs.

  4. An integrated analysis of differential miRNA and mRNA expressions in human gallstones.

    PubMed

    Yang, Bin; Liu, Bin; Bi, Pinduan; Wu, Tao; Wang, Qiang; Zhang, Jie

    2015-04-01

    Gallstone disease, including cholesterol precipitation in bile, increased bile salt hydrophobicity and gallbladder inflammation. Here, we investigated miRNA and mRNA involved in the formation of gallstones, and explored the molecular mechanisms in the development of gallstones. Differentially expressed 17 miRNAs and 525 mRNA were identified based on Illumina sequencing from gallbladder mucosa of patients with or without gallstones, and were validated by randomly selected 6 miRNAs and 8 genes using quantitative RT-PCR. 114 miRNA target genes were identified, whose functions and regulating pathways were related to gallstones. The differentially expressed genes were enriched upon lipoprotein binding and some metabolic pathways, and differentially expressed miRNAs enriched upon ABC transportation and cancer related pathways. A molecular regulatory network consisting of 17 differentially expressed miRNAs, inclusive of their target genes, was constructed. miR-210 and its potential target gene ATP11A were found to be differentially expressed in both miRNA and mRNA profiles. ATP11A was a direct target of miR-210, which was predicted to regulate the ABC-transporters pathway. The expression levels of ATP11A in the gallstone showed inverse correlation with miR-210 expression, and up-regulation of miR-210 could reduce ATP11A expression in HGBEC. This is the first report that indicates the existence of differences in miRNA and mRNA expression in patients with or without gallstones. Our data shed light on further investigating the mechanisms of gallstone formation.

  5. The influence of eccentric exercise on mRNA expression of skeletal muscle regulators.

    PubMed

    Jensky, Nicole E; Sims, Jennifer K; Rice, Judd C; Dreyer, Hans C; Schroeder, E Todd

    2007-11-01

    To evaluate change in myostatin, follistatin, MyoD and SGT mRNA gene expression using eccentric exercise to study mechanisms of skeletal muscle hypertrophy. Young (28+/-5 years) and older (68+/-6 years) men participated in a bout of maximal single-leg eccentric knee extension on an isokinetic dynamometer at 60 degrees /s: six sets, 12-16 maximal eccentric repetitions. Muscle biopsies of the vastus lateralis were obtained from the dominant leg before exercise and 24 h after exercise. Paired t tests were used to compare change (pre versus post-exercise) for normalized gene expression in all variables. Independent t tests were performed to test group differences (young vs. older). A probability level of PmRNA expression in young subjects 24 h after eccentric exercise. Similarly, we did not observe significant change in myostatin (-3.83+/-8.8; P=0.23), follistatin (-2.66+/-5.2; P=0.17), MyoD (-0.13+/-3.1; P=0.90), or SGT (-1.6+/-3.5; P=0.19) mRNA expression in older subjects. Furthermore, the non-significant changes in mRNA expression were not different between young and older subjects, P>0.23 for all variables. Our data suggests that a single bout of maximal eccentric exercise does not alter myostatin, follistatin, MyoD or SGT mRNA gene expression in young or older subjects.

  6. Differences in expression of retinal pigment epithelium mRNA between normal canines

    PubMed Central

    2004-01-01

    Abstract A reference database of differences in mRNA expression in normal healthy canine retinal pigment epithelium (RPE) has been established. This database identifies non-informative differences in mRNA expression that can be used in screening canine RPE for mutations associated with clinical effects on vision. Complementary DNA (cDNA) pools were prepared from mRNA harvested from RPE, amplified by PCR, and used in a subtractive hybridization protocol (representational differential analysis) to identify differences in RPE mRNA expression between canines. The effect of relatedness of the test canines on the frequency of occurrence of differences was evaluated by using 2 unrelated canines for comparison with 2 female sibling canines of blue heeler/bull terrier lineage. Differentially expressed cDNA species were cloned, sequenced, and identified by comparison to public database entries. The most frequently observed differentially expressed sequence from the unrelated canine comparison was cDNA with 21 base pairs (bp) identical to the human epithelial membrane protein 1 gene (present in 8 of 20 clones). Different clones from the same-sex sibling RPE contained repetitions of several short sequence motifs including the human epithelial membrane protein 1 (4 of 25 clones). Other prevalent differences between sibling RPE included sequences similar to a chicken genetic marker sequence motif (5 of 25), and 6 clones with homology to porcine major histocompatibility loci. In addition to identifying several repetitively occurring, noninformative, differentially expressed RPE mRNA species, the findings confirm that fewer differences occurred between siblings, highlighting the importance of using closely related subjects in representational difference analysis studies. PMID:15352545

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

    PubMed Central

    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. PMID:27199546

  8. 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...

  9. 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.

  10. 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

  11. 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.

  12. Expression of statherin mRNA and protein in nasal and vaginal secretions.

    PubMed

    Sakurada, Koichi; Akutsu, Tomoko; Watanabe, Ken; Fujinami, Yoshihito; Yoshino, Mineo

    2011-11-01

    Nasal secretion has been regarded as one of the most difficult body fluids to identify and is especially difficult to discriminate from vaginal secretions and saliva. At present, few specific markers are known for nasal secretions. The aim of this study is to find a new approach for the identification of nasal secretions. We examined expression levels of statherin and histatin, peptides which are commonly found in saliva, in nasal and vaginal secretions by real-time RT-PCR and ELISA assays. Statherin mRNA was highly expressed in all nasal samples (dCt value=-1.49±1.10, n=8) and was detected even in 1-day-old 0.1-μL stains. However, the stability of mRNA in nasal stains was significantly (P<0.01) lower than in saliva. Low levels of statherin mRNA were detected in 4 of the 17 vaginal samples (dCt value=11.65-14.72). Histatin mRNA was not detected in any nasal or vaginal samples, although it was highly expressed in all saliva samples. ELISA assays with anti-statherin goat polyclonal antibody showed that statherin peptide was detected in all nasal and saliva samples even after dilution of more than 1000-fold. The statherin peptide was not detected in any vaginal samples, including samples that expressed low levels of statherin mRNA. The amount of statherin peptide in vaginal samples might be less than the limit of detection of this assay. In the present study, statherin was highly expressed in nasal secretions, but histatin was not. These markers may be useful for discriminating nasal secretions from vaginal secretions and saliva. However, the usefulness of these markers in practical forensic case samples has not yet been examined. Therefore, further research is required to establish the utility of these assays for identification of nasal secretions.

  13. Moisturizers change the mRNA expression of enzymes synthesizing skin barrier lipids.

    PubMed

    Buraczewska, Izabela; Berne, Berit; Lindberg, Magnus; Lodén, Marie; Törmä, Hans

    2009-09-01

    In a previous study, 7-week treatment of normal human skin with two test moisturizers, Complex cream and Hydrocarbon cream, was shown to affect mRNA expression of certain genes involved in keratinocyte differentiation. Moreover, the treatment altered transepidermal water loss (TEWL) in opposite directions. In the present study, the mRNA expression of genes important for formation of barrier lipids, i.e., cholesterol, free fatty acids and ceramides, was examined. Treatment with Hydrocarbon cream, which increased TEWL, also elevated the gene expression of GBA, SPTLC2, SMPD1, ALOX12B, ALOXE3, and HMGCS1. In addition, the expression of PPARG was decreased. On the other hand, Complex cream, which decreased TEWL, induced only the expression of PPARG, although not confirmed at the protein level. Furthermore, in the untreated skin, a correlation between the mRNA expression of PPARG and ACACB, and TEWL was found, suggesting that these genes are important for the skin barrier homeostasis. The observed changes further demonstrate that long-term treatment with certain moisturizers may induce dysfunctional skin barrier, and as a consequence several signaling pathways are altered.

  14. Gastrointestinal hormone mRNA expression in human colonic adenocarcinomas, hepatic metastases and cell lines

    PubMed Central

    Monges, G; Biagini, P; Cantaloube, J F; De Micco, P; Parriaux, D; Seitz, J F; Delpero, J R; Hassoun, J

    1996-01-01

    Aims—(1) To investigate the expression of the four main hormones of the digestive tract by performing reverse transcription polymerase chain reaction (RT-PCR) on a series of samples, comprising tumoral and healthy colonic tissues, hepatic metastases and colonic cell line samples; and (2) to study the patterns of labelling obtained with serological and morphological markers. Methods—After extraction and reverse transcription, gastrin, somatostatin, cholecystokinin (CCK) and transforming growth factor α (TGFα) mRNAs were detected by PCR and nested PCR using specific primers. The corresponding proteins were detected by immunohistochemistry. Results—The cell lines expressed all four mRNAs. Gastrin mRNA was present in most tumoral and metastatic samples, while the somatostatin transcript was detected in all samples and was frequently overexpressed in the normal colon. TGFα mRNA was expressed systematically in tumours of the right and transverse colon, but not in those located in the left colon; the expression of CCK mRNA was systematically absent in the left colon. Conclusions—The data presented here shed some light on the transcriptional events involved in the production of the various hormones present in the gastrointestinal tract, in both healthy and tumoral tissues. The various mRNAs expressed in cell lines are therefore not systematically expressed in the human pathology. Images PMID:16696065

  15. 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

  16. Change of dopamine receptor mRNA expression in lymphocyte of schizophrenic patients

    PubMed Central

    Kwak, Yong T; Koo, Min-Seong; Choi, Chul-Hee; Sunwoo, IN

    2001-01-01

    Background Though the dysfunction of central dopaminergic system has been proposed, the etiology or pathogenesis of schizophrenia is still uncertain partly due to limited accessibility to dopamine receptor. The purpose of this study was to define whether or not the easily accessible dopamine receptors of peripheral lymphocytes can be the peripheral markers of schizophrenia. Results 44 drug-medicated schizophrenics for more than 3 years, 28 drug-free schizophrenics for more than 3 months, 15 drug-naïve schizophrenic patients, and 31 healthy persons were enrolled. Sequential reverse transcription and quantitative polymerase chain reaction of the mRNA were used to investigate the expression of D3 and D5 dopamine receptors in peripheral lymphocytes. The gene expression of dopamine receptors was compared in each group. After taking antipsychotics in drug-free and drug-naïve patients, the dopamine receptors of peripheral lymphocytes were sequentially studied 2nd week and 8th week after medication. In drug-free schizophrenics, D3 dopamine receptor mRNA expression of peripheral lymphocytes significantly increased compared to that of controls and drug-medicated schizophrenics, and D5 dopamine receptor mRNA expression increased compared to that of drug-medicated schizophrenics. After taking antipsychotics, mRNA of dopamine receptors peaked at 2nd week, after which it decreases but the level was above baseline one at 8th week. Drug-free and drug-naïve patients were divided into two groups according to dopamine receptor expression before medications, and the group of patients with increased dopamine receptor expression had more severe psychiatric symptoms. Conclusions These results reveal that the molecular biologically-determined dopamine receptors of peripheral lymphocytes are reactive, and that increased expression of dopamine receptor in peripheral lymphocyte has possible clinical significance for subgrouping of schizophrenis. PMID:11252158

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

    PubMed Central

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

    2016-01-01

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

  18. Expression of SART-1 mRNA in canine squamous cell carcinomas.

    PubMed

    Takaishi, Yumi; Yoshida, Yukari; Nakagaki, Kazuhide; Fujita, Michio; Taniguchi, Akiko; Orima, Hiromitsu

    2008-12-01

    SART-1, a squamous cell carcinoma (SCC) antigen recognized by cytotoxic T lymphocytes, has been useful in human cancer therapy. The SART-1(259) peptide is a potential candidate for vaccine. The present study examined an orthologue of the mRNA coding this peptide in canine SCCs. Specimens were obtained from seven canine patients with SCC, and the mRNA was isolated from the samples. The SART-1 and beta-actin genes were amplified by reverse-transcription polymerase chain reaction, using the isolated mRNA as a template. Canine SART-1 was amplified in six of the seven specimens, while beta-actin was detected in all the samples. In dogs, carcinomas expressing SART-1 could be a target for cytotoxic T lymphocyte mediated immunotherapy.

  19. Sprouty4 mRNA variants and protein expressions in breast and lung-derived cells

    PubMed Central

    Doriguzzi, Angelina; Salhi, Jihen; Sutterlüty-Fall, Hedwig

    2016-01-01

    Sprouty proteins are modulators of mitogen-induced signalling processes and are therefore hypothesized to affect malignant diseases. As a member of the Sprouty family, Sprouty4 has been previously shown to function as a tumour suppressor in lung and breast cancer. The present study analysed the expression of two known Sprouty4 splice variants in cells established from malignant and normal lung and breast tissues using semi-quantitative reverse transcription-polymerase chain reaction and immunoblotting. The results indicated that the expression of the two messenger RNA (mRNA) variants was reduced in the cells derived from malignant tissue in comparison to the normal counterparts. Although the expression of the two splice variants were associated in both tissue types, on average, the relative expression of the longer variant was slightly increased in malignant cells compared with normal tissues. Notably, the protein levels reflected the expression observed at the mRNA level only in breast-derived cells. Contrarily, with regards to the measured mRNA levels, Sprouty4 protein was disproportional augmented in lung cells known to harbour the mutated K-Ras gene. PMID:27895786

  20. Gallium nitrate regulates rat osteoblast expression of osteocalcin protein and mRNA levels.

    PubMed

    Guidon, P T; Salvatori, R; Bockman, R S

    1993-01-01

    Gallium nitrate, a group IIIa metal salt, has been found to be clinically effective for the treatment of accelerated bone resorption in cancer-related hypercalcemia and Paget's disease. Here we report the effects of gallium nitrate on osteocalcin mRNA and protein levels on the rat osteoblast-like cell line ROS 17/2.8. Gallium nitrate reduced both constitutive and vitamin D3-stimulated osteocalcin protein levels in culture medium by one-half and osteocalcin mRNA levels to one-third to one-tenth of control. Gallium nitrate also inhibited vitamin D3 stimulation of osteocalcin and osteopontin mRNA levels but did not affect constitutive osteopontin mRNA levels. Among several different metals examined, gallium was unique in its ability to reduce osteocalcin mRNA levels without decreasing levels of other mRNAs synthesized by ROS 17/2.8 cells. The effects of gallium nitrate on osteocalcin mRNA and protein synthesis mimic those seen when ROS 17/2.8 cells are exposed to transforming growth factor beta 1 (TGF beta 1); however, TGF-beta 1 was not detected in gallium nitrate-treated ROS 17/2.8 cell media. Use of the RNA polymerase II inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole demonstrated that gallium nitrate did not alter the stability of osteocalcin mRNA. Transient transfection assays using the rat osteocalcin promoter linked to the bacterial reporter gene chloramphenicol acetyltransferase indicated that gallium nitrate blocked reporter gene expression stimulated by the osteocalcin promoter. This is the first reported effect of gallium nitrate on isolated osteoblast cells.

  1. Embryonic and Postnatal Expression of Aryl Hydrocarbon Receptor mRNA in Mouse Brain

    PubMed Central

    Kimura, Eiki; Tohyama, Chiharu

    2017-01-01

    Aryl hydrocarbon receptor (AhR), a member of the basic helix-loop-helix-Per-Arnt-Sim transcription factor family, plays a critical role in the developing nervous system of invertebrates and vertebrates. Dioxin, a ubiquitous environmental pollutant, avidly binds to this receptor, and maternal exposure to dioxin has been shown to impair higher brain functions and dendritic morphogenesis, possibly via an AhR-dependent mechanism. However, there is little information on AhR expression in the developing mammalian brain. To address this issue, the present study analyzed AhR mRNA expression in the brains of embryonic, juvenile, and adult mice by reverse transcription (RT)-PCR and in situ hybridization. In early brain development (embryonic day 12.5), AhR transcript was detected in the innermost cortical layer. The mRNA was also expressed in the hippocampus, cerebral cortex, cerebellum, olfactory bulb, and rostral migratory stream on embryonic day 18.5, postnatal days 3, 7, and 14, and in 12-week-old (adult) mice. Hippocampal expression was abundant in the CA1 and CA3 pyramidal and dentate gyrus granule cell layers, where expression level of AhR mRNA in 12-week old is higher than that in 7-day old. These results reveal temporal and spatial patterns of AhR mRNA expression in the mouse brain, providing the information that may contribute to the elucidation of the physiologic and toxicologic significance of AhR in the developing brain. PMID:28223923

  2. Alpha-synuclein mRNA expression in oligodendrocytes in MSA.

    PubMed

    Asi, Yasmine T; Simpson, Julie E; Heath, Paul R; Wharton, Stephen B; Lees, Andrew J; Revesz, Tamas; Houlden, Henry; Holton, Janice L

    2014-06-01

    Multiple system atrophy (MSA) is a progressive neurodegenerative disease presenting clinically with parkinsonian, cerebellar, and autonomic features. α-Synuclein (αsyn), encoded by the gene SNCA, is the main constituent of glial cytoplasmic inclusion (GCI) found in oligodendrocytes in MSA, but the methods of its accumulation have not been established. The aim of this study is to investigate alterations in regional and cellular SNCA mRNA expression in MSA as a possible substrate for GCI formation. Quantitative reverse transcription polymerase chain reaction (qPCR) was performed on postmortem brain samples from 15 MSA, 5 IPD, and 5 control cases to investigate regional expression in the frontal and occipital regions, dorsal putamen, pontine base, and cerebellum. For cellular expression analysis, neurons and oligodendrocytes were isolated by laser-capture microdissection from five MSA and five control cases. SNCA mRNA expression was not significantly different between the MSA, IPD and control cases in all regions (multilevel model, P = 0.14). After adjusting for group effect, the highest expression was found in the occipital cortex while the lowest was in the putamen (multilevel model, P < 0.0001). At the cellular level, MSA oligodendrocytes expressed more SNCA than control oligodendrocytes and expression in MSA neurons was slightly lower than that in controls, however, these results did not reach statistical significance. We have demonstrated regional variations in SNCA expression, which is higher in cortical than subcortical regions. This study is the first to demonstrate SNCA mRNA expression by oligodendrocytes in human postmortem tissue using qPCR and, although not statistically significant, could suggest that this may be increased in MSA compared to controls.

  3. Integrated analysis of microRNA and mRNA expression profiles in HBx-expressing hepatic cells

    PubMed Central

    Chen, Ruo-Chan; Wang, Juan; Kuang, Xu-Yuan; Peng, Fang; Fu, Yong-Ming; Huang, Yan; Li, Ning; Fan, Xue-Gong

    2017-01-01

    AIM To identify the miRNA-mRNA regulatory network in hepatitis B virus X (HBx)-expressing hepatic cells. METHODS A stable HBx-expressing human liver cell line L02 was established. The mRNA and miRNA expression profiles of L02/HBx and L02/pcDNA liver cells were identified by RNA-sequencing analysis. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis was performed to investigate the function of candidate biomarkers, and the relationship between miRNA and mRNA was studied by network analysis. RESULTS Compared with L02/pcDNA cells, 742 unregulated genes and 501 downregulated genes were determined as differentially expressed in L02/HBx cells. Gene ontology analysis suggested that the differentially expressed genes were relevant to different biological processes. Concurrently, 22 differential miRNAs were also determined in L02/HBx cells. Furthermore, integrated analysis of miRNA and mRNA expression profiles identified a core miRNA-mRNA regulatory network that is correlated with the carcinogenic role of HBx. CONCLUSION Collectively, the miRNA-mRNA network-based analysis could be useful to elucidate the potential role of HBx in liver cell malignant transformation and shed light on the underlying molecular mechanism and novel therapy targets for hepatocellular carcinoma. PMID:28348484

  4. TLR2 and TLR4 polymorphisms influence mRNA and protein expression in colorectal cancer

    PubMed Central

    Proença, Marcela Alcântara; de Oliveira, Juliana Garcia; Cadamuro, Aline Cristina Targa; Succi, Maysa; Netinho, João Gomes; Goloni-Bertolo, Eny Maria; Pavarino, Érika Cristina; Silva, Ana Elizabete

    2015-01-01

    AIM: To evaluate the effect of promoter region polymorphisms of toll-like receptor (TLR)2-196 to -174del and TLR4-1607T/C (rs10759932) on mRNA and protein expression in tumor tissue and of TLR4+896A/G (rs4986790) on colorectal cancer (CRC) risk. METHODS: The TLR2-196 to -174del polymorphism was investigated using allele-specific polymerase chain reaction (PCR) and the TLR4-1607T/C and TLR4+896A/G by PCR-restriction fragment length polymorphism (RFLP). We genotyped 434 DNA samples from 194 CRC patients and 240 healthy individuals. The mRNA relative quantification (RQ) was performed in 40 tumor tissue samples by quantitative PCR TaqMan assay, using specific probes for TLR2 and TLR4 genes, and ACTB and GAPDH reference genes were used as endogenous controls. Protein expression was analyzed by immunohistochemistry with specific primary antibodies. RESULTS: No association was found for TLR4-1607T/C and TLR4+896A/G by three statistical models (log-additive, dominant and recessive). However, based on dominant and log-additive models, the polymorphic variant TLR2-196 to -174del was associated with increased CRC risk [dominant: odds ratio (OR) = 1.72, 95%CI: 1.03-2.89; P = 0.038 and log-additive: OR =1.59, 95%CI: 1.02-2.48; P = 0.039]. TLR2 mRNA expression was increased in tumor tissue (RQ = 2.36) when compared to adjacent normal tissue (RQ = 1; P < 0.0001), whereas the TLR4 mRNA showed a basal expression (RQ = 0.74 vs RQ = 1, P = 0.452). Immunohistochemistry analysis of TLR2 and TLR4 protein expression was concordant with the findings of mRNA expression. In addition, the TLR2-196 to -174del variant carriers showed mRNA relative expression 2.19 times higher than wild-genotype carriers. The TLR2 protein expression was also higher for the TLR2-196 to -174del variant carriers [117 ± 10 arbitrary unit (a.u.) vs 95 ± 4 a.u., P = 0.03]. However, for the TLR4 -1607T/C polymorphism no significant difference was found for both mRNA (P = 0.56) and protein expression (P = 0

  5. The mRNA Decay Pathway Regulates the Expression of the Flo11 Adhesin and Biofilm Formation in Saccharomyces cerevisiae

    PubMed Central

    Lo, Tricia L.; Qu, Yue; Uwamahoro, Nathalie; Quenault, Tara; Beilharz, Traude H.; Traven, Ana

    2012-01-01

    Regulation of the FLO11 adhesin is a model for gene expression control by extracellular signals and developmental switches. We establish that the major mRNA decay pathway regulates FLO11 expression. mRNA deadenylation of transcriptional repressors of FLO11 by the exonuclease Ccr4 keeps their levels low, thereby allowing FLO11 transcription. PMID:22595243

  6. 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

  7. 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

  8. Control of Cytokine mRNA Expression by RNA-binding Proteins and microRNAs

    PubMed Central

    Palanisamy, V.; Jakymiw, A.; Van Tubergen, E.A.; D’Silva, N.J.; Kirkwood, K.L.

    2012-01-01

    Cytokines are critical mediators of inflammation and host defenses. Regulation of cytokines can occur at various stages of gene expression, including transcription, mRNA export, and post- transcriptional and translational levels. Among these modes of regulation, post-transcriptional regulation has been shown to play a vital role in controlling the expression of cytokines by modulating mRNA stability. The stability of cytokine mRNAs, including TNFα, IL-6, and IL-8, has been reported to be altered by the presence of AU-rich elements (AREs) located in the 3′-untranslated regions (3′UTRs) of the mRNAs. Numerous RNA-binding proteins and microRNAs bind to these 3′UTRs to regulate the stability and/or translation of the mRNAs. Thus, this paper describes the cooperative function between RNA-binding proteins and miRNAs and how they regulate AU-rich elements containing cytokine mRNA stability/degradation and translation. These mRNA control mechanisms can potentially influence inflammation as it relates to oral biology, including periodontal diseases and oral pharyngeal cancer progression. PMID:22302144

  9. 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

  10. Cytokine mRNA Expression in Lesions in Cats with Chronic Gingivostomatitis

    PubMed Central

    Harley, R.; Helps, C. R.; Harbour, D. A.; Gruffydd-Jones, T. J.; Day, M. J.

    1999-01-01

    Semiquantitative reverse transcription-PCR assays were developed to measure feline interleukin-2 (IL-2), IL-4, IL-5, IL-6, IL-10, and IL-12 (p35 & p40); gamma interferon (IFN-γ); and glyceraldehyde-3-phosphate dehydrogenase mRNA concentrations in biopsies of feline oral mucosa. Biopsies were collected from 30 cats with chronic gingivostomatitis (diseased) prior to each cat receiving one of four treatments. In 23 cases replicate biopsies were collected 3 months after treatment commenced. Biopsies were also analyzed from 11 cats without clinical disease (nondiseased). Expression of IL-2, IL-10, IL-12 (p35 and p40), and IFN-γ was detected in most nondiseased biopsies, while IL-6 was detected in a minority, and IL-4 and IL-5 were both undetectable. Compared to nondiseased cats, the diseased population showed a significant increase in the relative mRNA expression of IL-2, IL-4, IL-6, IL-10, IL-12 (p35 and p40), and IFN-γ. In contrast, IL-5 mRNA expression was unchanged and was only detected in one case. No significant relationship was demonstrable between the change in relative expression of specific cytokine mRNA and the change in clinical severity of the local mucosal lesions over the treatment period. The results demonstrate that the normal feline oral mucosa is biased towards a predominantly (Th) type 1 profile of cytokine expression and that during the development of lesions seen in feline chronic gingivostomatitis there is a shift in the cytokine profile from a type 1 to a mixed type 1 and type 2 response. PMID:10391845

  11. Cytokine mRNA expression in lesions in cats with chronic gingivostomatitis.

    PubMed

    Harley, R; Helps, C R; Harbour, D A; Gruffydd-Jones, T J; Day, M J

    1999-07-01

    Semiquantitative reverse transcription-PCR assays were developed to measure feline interleukin-2 (IL-2), IL-4, IL-5, IL-6, IL-10, and IL-12 (p35 & p40); gamma interferon (IFN-gamma); and glyceraldehyde-3-phosphate dehydrogenase mRNA concentrations in biopsies of feline oral mucosa. Biopsies were collected from 30 cats with chronic gingivostomatitis (diseased) prior to each cat receiving one of four treatments. In 23 cases replicate biopsies were collected 3 months after treatment commenced. Biopsies were also analyzed from 11 cats without clinical disease (nondiseased). Expression of IL-2, IL-10, IL-12 (p35 and p40), and IFN-gamma was detected in most nondiseased biopsies, while IL-6 was detected in a minority, and IL-4 and IL-5 were both undetectable. Compared to nondiseased cats, the diseased population showed a significant increase in the relative mRNA expression of IL-2, IL-4, IL-6, IL-10, IL-12 (p35 and p40), and IFN-gamma. In contrast, IL-5 mRNA expression was unchanged and was only detected in one case. No significant relationship was demonstrable between the change in relative expression of specific cytokine mRNA and the change in clinical severity of the local mucosal lesions over the treatment period. The results demonstrate that the normal feline oral mucosa is biased towards a predominantly (Th) type 1 profile of cytokine expression and that during the development of lesions seen in feline chronic gingivostomatitis there is a shift in the cytokine profile from a type 1 to a mixed type 1 and type 2 response.

  12. Rift Valley fever virus NSS gene expression correlates with a defect in nuclear mRNA export.

    PubMed

    Copeland, Anna Maria; Van Deusen, Nicole M; Schmaljohn, Connie S

    2015-12-01

    We investigated the localization of host mRNA during Rift Valley fever virus (RVFV) infection. Fluorescence in situ hybridization revealed that infection with RVFV altered the localization of host mRNA. mRNA accumulated in the nuclei of RVFV-infected but not mock-infected cells. Further, overexpression of the NSS gene, but not the N, GN or NSM genes correlated with mRNA nuclear accumulation. Nuclear accumulation of host mRNA was not observed in cells infected with a strain of RVFV lacking the gene encoding NSS, confirming that expression of NSS is likely responsible for this phenomenon.

  13. Genome-wide analysis of microRNA and mRNA expression signatures in cancer

    PubMed Central

    Li, Ming-hui; Fu, Sheng-bo; Xiao, Hua-sheng

    2015-01-01

    Cancer is an extremely diverse and complex disease that results from various genetic and epigenetic changes such as DNA copy-number variations, mutations, and aberrant mRNA and/or protein expression caused by abnormal transcriptional regulation. The expression profiles of certain microRNAs (miRNAs) and messenger RNAs (mRNAs) are closely related to cancer progression stages. In the past few decades, DNA microarray and next-generation sequencing techniques have been widely applied to identify miRNA and mRNA signatures for cancers on a genome-wide scale and have provided meaningful insights into cancer diagnosis, prognosis and personalized medicine. In this review, we summarize the progress in genome-wide analysis of miRNAs and mRNAs as cancer biomarkers, highlighting their diagnostic and prognostic roles. PMID:26299954

  14. Anesthesia for Euthanasia Influences mRNA Expression in Healthy Mice and after Traumatic Brain Injury

    PubMed Central

    Staib-Lasarzik, Irina; Kriege, Oliver; Timaru-Kast, Ralph; Pieter, Dana; Werner, Christian; Engelhard, Kristin

    2014-01-01

    Abstract Tissue sampling for gene expression analysis is usually performed under general anesthesia. Anesthetics are known to modulate hemodynamics, receptor-mediated signaling cascades, and outcome parameters. The present study determined the influence of anesthetic paradigms typically used for euthanization and tissue sampling on cerebral mRNA expression in mice. Naïve mice and animals with acute traumatic brain injury induced by controlled cortical impact (CCI) were randomized to the following euthanasia protocols (n=10–11/group): no anesthesia (NA), 1 min of 4 vol% isoflurane in room air (ISO), 3 min of a combination of 5 mg/kg midazolam, 0.05 mg/kg fentanyl, and 0.5 mg/kg medetomidine intraperitoneally (COMB), or 3 min of 360 mg/kg chloral hydrate intraperitoneally (CH). mRNA expression of actin-1-related gene (Act1), FBJ murine osteosarcoma viral oncogene homolog B (FosB), tumor necrosis factor alpha (TNFα), heat shock protein beta-1 (HspB1), interleukin (IL)-6, tight junction protein 1 (ZO-1), IL-1ß, cyclophilin A, micro RNA 497 (miR497), and small cajal body-specific RNA 17 were determined by real-time polymerase chain reaction (PCR) in hippocampus samples. In naïve animals, Act1 expression was downregulated in the CH group compared with NA. FosB expression was downregulated in COMB and CH groups compared with NA. CCI reduced Act1 and FosB expression, whereas HspB1 and TNFα expression increased. After CCI, HspB1 expression was significantly higher in ISO, COMB, and CH groups, and TNFα expression was elevated in ISO and COMB groups. MiR497, IL-6, and IL-1ß were upregulated after CCI but not affected by anesthetics. Effects were independent of absolute mRNA copy numbers. The data demonstrate that a few minutes of anesthesia before tissue sampling are sufficient to induce immediate mRNA changes, which seem to predominate in the early-regulated gene cluster. Anesthesia-related effects on gene expression might explain limited

  15. Anesthesia for euthanasia influences mRNA expression in healthy mice and after traumatic brain injury.

    PubMed

    Staib-Lasarzik, Irina; Kriege, Oliver; Timaru-Kast, Ralph; Pieter, Dana; Werner, Christian; Engelhard, Kristin; Thal, Serge C

    2014-10-01

    Tissue sampling for gene expression analysis is usually performed under general anesthesia. Anesthetics are known to modulate hemodynamics, receptor-mediated signaling cascades, and outcome parameters. The present study determined the influence of anesthetic paradigms typically used for euthanization and tissue sampling on cerebral mRNA expression in mice. Naïve mice and animals with acute traumatic brain injury induced by controlled cortical impact (CCI) were randomized to the following euthanasia protocols (n=10-11/group): no anesthesia (NA), 1 min of 4 vol% isoflurane in room air (ISO), 3 min of a combination of 5 mg/kg midazolam, 0.05 mg/kg fentanyl, and 0.5 mg/kg medetomidine intraperitoneally (COMB), or 3 min of 360 mg/kg chloral hydrate intraperitoneally (CH). mRNA expression of actin-1-related gene (Act1), FBJ murine osteosarcoma viral oncogene homolog B (FosB), tumor necrosis factor alpha (TNFα), heat shock protein beta-1 (HspB1), interleukin (IL)-6, tight junction protein 1 (ZO-1), IL-1ß, cyclophilin A, micro RNA 497 (miR497), and small cajal body-specific RNA 17 were determined by real-time polymerase chain reaction (PCR) in hippocampus samples. In naïve animals, Act1 expression was downregulated in the CH group compared with NA. FosB expression was downregulated in COMB and CH groups compared with NA. CCI reduced Act1 and FosB expression, whereas HspB1 and TNFα expression increased. After CCI, HspB1 expression was significantly higher in ISO, COMB, and CH groups, and TNFα expression was elevated in ISO and COMB groups. MiR497, IL-6, and IL-1ß were upregulated after CCI but not affected by anesthetics. Effects were independent of absolute mRNA copy numbers. The data demonstrate that a few minutes of anesthesia before tissue sampling are sufficient to induce immediate mRNA changes, which seem to predominate in the early-regulated gene cluster. Anesthesia-related effects on gene expression might explain limited reproduciblity of real

  16. 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.

  17. Progressive APOBEC3B mRNA expression in distant breast cancer metastases

    PubMed Central

    Dalm, Simone U.; de Weerd, Vanja; Moelans, Cathy B.; ter Hoeve, Natalie; van Diest, Paul J.; Martens, John W. M.; van Deurzen, Carolien H. M.

    2017-01-01

    Background APOBEC3B was recently identified as a gain-of-function enzymatic source of mutagenesis, which may offer novel therapeutic options with molecules that specifically target this enzyme. In primary breast cancer, APOBEC3B mRNA is deregulated in a substantial proportion of cases and its expression is associated with poor prognosis. However, its expression in breast cancer metastases, which are the main causes of breast cancer-related death, remained to be elucidated. Patients and methods RNA was isolated from 55 primary breast cancers and paired metastases, including regional lymph node (N = 20) and distant metastases (N = 35). APOBEC3B mRNA levels were measured by RT-qPCR. Expression levels of the primary tumors and corresponding metastases were compared, including subgroup analysis by estrogen receptor (ER/ESR1) status. Results Overall, APOBEC3B mRNA levels of distant metastases were significantly higher as compared to the corresponding primary breast tumor (P = 0.0015), an effect that was not seen for loco-regional lymph node metastases (P = 0.23). Subgroup analysis by ER-status showed that increased APOBEC3B levels in distant metastases were restricted to metastases arising from ER-positive primary breast cancers (P = 0.002). However, regarding ER-negative primary tumors, only loco-regional lymph node metastases showed increased APOBEC3B expression when compared to the corresponding primary tumor (P = 0.028). Conclusion APOBEC3B mRNA levels are significantly higher in breast cancer metastases as compared to the corresponding ER-positive primary tumors. This suggests a potential role for APOBEC3B in luminal breast cancer progression, and consequently, a promising role for anti-APOBEC3B therapies in advanced stages of this frequent form of breast cancer. PMID:28141868

  18. Developmental and tissue-specific expression of prosaposin mRNA in murine tissues.

    PubMed Central

    Sun, Y.; Witte, D. P.; Grabowski, G. A.

    1994-01-01

    Prosaposin is a multifunctional locus in humans and mice that encodes in tandem and in the same reading frame four glycoprotein activators, or saposins, of lysosomal hydrolases. These ubiquitously expressed glycoproteins and the precursor, prosaposin, have been proposed to function in glycosphingolipid catabolic pathways and glycolipid transport. To characterize the temporal and spatial expression of the prosaposin locus, prenatal and postnatal mouse tissues were screened by in situ hybridization with a mouse antisense riboprobe for prosaposin. Prenatally, prosaposin mRNA was expressed differentially in the placenta and prominently in the decidua basalis and capsularis where expression was gestational age dependent. No other region of high-level expression was detectable in the prenatal mouse. In comparison, high-level differential expression of prosaposin was clearly evident postnatally in a variety of organs, including secretory epithelial cells of the choroid plexus, ependymal lining, upper trachea, esophagus, cortical tubules of the kidney, sertoli cells of the testes and epididymis. Discrete localization of prosaposin mRNA expression was also found in neurons of the cerebral cortex, cerebellar cortex, and lateral columns of the spinal cord as well as in hepatocytes of the mature liver. Very high levels of expression were found in specialized tissues including the Harderian glands and macrophages of lymph nodes, lungs, splenic tissue, and thymus. These studies indicate that the expression of the prosaposin locus, a presumed "housekeeping" gene, is under tissue- and cell-specific differential control. The spatial organization of expression suggests a role for this locus in the expression of glycosphingolipid-storage diseases. Images Figure 2 PMID:7992842

  19. Nutritional control of mRNA isoform expression during developmental arrest and recovery in C. elegans.

    PubMed

    Maxwell, Colin S; Antoshechkin, Igor; Kurhanewicz, Nicole; Belsky, Jason A; Baugh, L Ryan

    2012-10-01

    Nutrient availability profoundly influences gene expression. Many animal genes encode multiple transcript isoforms, yet the effect of nutrient availability on transcript isoform expression has not been studied in genome-wide fashion. When Caenorhabditis elegans larvae hatch without food, they arrest development in the first larval stage (L1 arrest). Starved larvae can survive L1 arrest for weeks, but growth and post-embryonic development are rapidly initiated in response to feeding. We used RNA-seq to characterize the transcriptome during L1 arrest and over time after feeding. Twenty-seven percent of detectable protein-coding genes were differentially expressed during recovery from L1 arrest, with the majority of changes initiating within the first hour, demonstrating widespread, acute effects of nutrient availability on gene expression. We used two independent approaches to track expression of individual exons and mRNA isoforms, and we connected changes in expression to functional consequences by mining a variety of databases. These two approaches identified an overlapping set of genes with alternative isoform expression, and they converged on common functional patterns. Genes affecting mRNA splicing and translation are regulated by alternative isoform expression, revealing post-transcriptional consequences of nutrient availability on gene regulation. We also found that phosphorylation sites are often alternatively expressed, revealing a common mode by which alternative isoform expression modifies protein function and signal transduction. Our results detail rich changes in C. elegans gene expression as larvae initiate growth and post-embryonic development, and they provide an excellent resource for ongoing investigation of transcriptional regulation and developmental physiology.

  20. 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.

  1. 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.

  2. 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.

  3. 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

  4. 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

  5. Antisense oligodeoxynucleotides targeted to MAG mRNA profoundly alter BP and PLP mRNA expression in differentiating oligodendrocytes: a caution.

    PubMed

    Laszkiewicz, I; Wiggins, R C; Konat, G W

    1999-09-01

    The applicability of antisense technology to suppress the expression of myelin associated glycoprotein (MAG) in cultured oligodendrocytes was evaluated. Differentiating oligodendrocyte precursor cells obtained by the shake-off method were exposed to nine unmodified antisense oligodeoxynucleotides (ODNs) targeted to the first seven exons of MAG mRNA. After four days, steady-state levels of MAG, proteolipid protein (PLP) and basic protein (BP) mRNAs were determined by Northern blot analysis. Only ODN annealing to 599-618 nt of the MAG mRNA (the junction of exon 5 and 6) resulted in a significant, 75% decrease in the MAG mRNA level. Unexpectedly, six other anti-MAG ODNs which had no significant effect on the MAG message, greatly increased the level of BP mRNA. The highest upregulation of approximately 12 fold was observed with ODN annealing to 139-168 nt (junction of exon 3 and 4). On the other hand, the 997-1016 ODN decreased the levels of BP and PLP messages by 70-80%. The 599-618 ODN also decreased the PLP mRNA by 85%. The results demonstrate that antisense ODNs targeted to one gene may profoundly alter the expression of other genes, and hence, complicate functional analysis of the targeted protein.

  6. DDR2 polymorphisms and mRNA expression in lung cancers of Japanese patients.

    PubMed

    Sasaki, Hidefumi; Shitara, Masayuki; Yokota, Keisuke; Okuda, Katsuhiro; Hikosaka, Yu; Moriyama, Satoru; Yano, Motoki; Fujii, Yoshitaka

    2012-07-01

    Discoidin domain receptor 2, DDR2, is a tyrosine kinase receptor for fibrillar collagen that is involved in postnatal development, tissue repair and primary and metastatic cancer progression. Recently, mutations in the DDR2 kinase gene were identified in squamous cell lung cancer from large-scale Sanger sequencing. The present study investigated the DDR2 gene mutations and mRNA expression in surgically treated non-small cell lung cancer (NSCLC) of squamous histology cases. The presence or absence of DDR2 mutations at the kinase and discoidin domain was analyzed by direct sequencing. In this cohort, DDR2 mutations were not observed in the 166 patients with lung cancer, although DDR2 polymorphisms were observed (H136H, n=14) at the discoidin domain. mRNA levels of DDR2 in lung tumor samples and the adjacent normal lung samples were simultaneously analyzed. DDR2 mRNA levels were significantly decreased in tumor samples compared with normal lung samples. However, the DDR2 mRNA levels were elevated in the DDR2 polymorphism cases.

  7. 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

  8. Heterogeneous expression of protein and mRNA in pyruvate dehydrogenase deficiency.

    PubMed Central

    Wexler, I D; Kerr, D S; Ho, L; Lusk, M M; Pepin, R A; Javed, A A; Mole, J E; Jesse, B W; Thekkumkara, T J; Pons, G

    1988-01-01

    Deficiency of pyruvate dehydrogenase [pyruvate:lipoamide 2-oxidoreductase (decarboxylating and acceptor-acetylating), EC 1.2.4.1], the first component of the pyruvate dehydrogenase complex, is associated with lactic acidosis and central nervous system dysfunction. Using both specific antibodies to pyruvate dehydrogenase and cDNAs coding for its two alpha and beta subunits, we characterized pyruvate dehydrogenase deficiency in 11 patients. Three different patterns were found on immunologic and RNA blot analyses. (i) Seven patients had immunologically detectable crossreactive material for the alpha and beta proteins of pyruvate dehydrogenase. (ii) Two patients had no detectable crossreactive protein for either the alpha or beta subunit but had normal amounts of mRNA for both alpha and beta subunits. (iii) The remaining two patients also had no detectable crossreactive protein but had diminished amounts of mRNA for the alpha subunit of pyruvate dehydrogenase only. These results indicate that loss of pyruvate dehydrogenase activity may be associated with either absent or catalytically inactive proteins, and in those cases in which this enzyme is absent, mRNA for one of the subunits may also be missing. When mRNA for one of the subunits is lacking, both protein subunits are absent, suggesting that a mutation affecting the expression of one of the subunit proteins causes the remaining uncomplexed subunit to be unstable. The results show that several different mutations account for the molecular heterogeneity of pyruvate dehydrogenase deficiency. Images PMID:3140238

  9. 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.

  10. The Role of Neuropeptide Y mRNA Expression Level in Distinguishing Different Types of Depression

    PubMed Central

    Yue, Yingying; Jiang, Haitang; Yin, Yingying; Zhang, Yuqun; Liang, Jinfeng; Li, Shenghua; Wang, Jun; Lu, Jianxin; Geng, Deqin; Wu, Aiqin; Yuan, Yonggui

    2016-01-01

    Previous studies demonstrate that the protein of neuropeptide Y (NPY) is abnormal in depression patients, but the changes of NPY in different types of depression are unclear. This study was aimed to examine protein and mRNA expression levels of NPY in 159 cases with four groups including post-stroke depression (PSD) group, stroke without depression (Non-PSD) group, major depressive disorder (MDD) group and normal control (NC) group. The protein and gene expression analysis were performed by enzyme-linked immunosorbent assay (ELISA) and quantitative polymerase chain reaction-based methods. One way analysis of variance (ANOVA), chi-square tests and nonparametric test were used to evaluate general characteristics, clinical and biological materials. In order to explore the role of NPY in different types of depression, the partial correlations, binary logistic regression analysis and receiver operating characteristic (ROC) curve were calculated for PSD and MDD groups. There are significant differences of NPY protein (Fdf(3) = 5.167, P = 0.002) and mRNA expression levels (χKruskal2-Wallis, df(3) = 20.541, P < 0.001) among four groups. Bonferroni multiple comparisons found that the NPY protein was significantly decreased in PSD (FBonferroni = −7.133, P = 0.002) and Non-PSD group (FBonferroni = −5.612, P = 0.018) compared with NC group. However, contrasted with MDD group, the mRNA expression was increased in PSD and Non-PSD group by nonparametric test (all P < 0.05). In binary logistic analyses, NPY mRNA expression was independent predictors of PSD (odds ratio: 1.452, 95% CI, 1.081–1.951, P = 0.013). The ROC curve showed NPY mRNA had a general prognostic accuracy (area under the curve: 0.766, 95% CI, 0.656–0.876, P < 0.001). This is the first study to explore the distinguishing function of NPY in different types of depression. It will provide help in the identification of different subtypes of depression. PMID:28082897

  11. The Role of Neuropeptide Y mRNA Expression Level in Distinguishing Different Types of Depression.

    PubMed

    Yue, Yingying; Jiang, Haitang; Yin, Yingying; Zhang, Yuqun; Liang, Jinfeng; Li, Shenghua; Wang, Jun; Lu, Jianxin; Geng, Deqin; Wu, Aiqin; Yuan, Yonggui

    2016-01-01

    Previous studies demonstrate that the protein of neuropeptide Y (NPY) is abnormal in depression patients, but the changes of NPY in different types of depression are unclear. This study was aimed to examine protein and mRNA expression levels of NPY in 159 cases with four groups including post-stroke depression (PSD) group, stroke without depression (Non-PSD) group, major depressive disorder (MDD) group and normal control (NC) group. The protein and gene expression analysis were performed by enzyme-linked immunosorbent assay (ELISA) and quantitative polymerase chain reaction-based methods. One way analysis of variance (ANOVA), chi-square tests and nonparametric test were used to evaluate general characteristics, clinical and biological materials. In order to explore the role of NPY in different types of depression, the partial correlations, binary logistic regression analysis and receiver operating characteristic (ROC) curve were calculated for PSD and MDD groups. There are significant differences of NPY protein (Fdf(3) = 5.167, P = 0.002) and mRNA expression levels ([Formula: see text] = 20.541, P < 0.001) among four groups. Bonferroni multiple comparisons found that the NPY protein was significantly decreased in PSD (FBonferroni = -7.133, P = 0.002) and Non-PSD group (FBonferroni = -5.612, P = 0.018) compared with NC group. However, contrasted with MDD group, the mRNA expression was increased in PSD and Non-PSD group by nonparametric test (all P < 0.05). In binary logistic analyses, NPY mRNA expression was independent predictors of PSD (odds ratio: 1.452, 95% CI, 1.081-1.951, P = 0.013). The ROC curve showed NPY mRNA had a general prognostic accuracy (area under the curve: 0.766, 95% CI, 0.656-0.876, P < 0.001). This is the first study to explore the distinguishing function of NPY in different types of depression. It will provide help in the identification of different subtypes of depression.

  12. MRNA and miRNA expression patterns associated to pathways linked to metal mixture health effects.

    PubMed

    Martínez-Pacheco, M; Hidalgo-Miranda, A; Romero-Córdoba, S; Valverde, M; Rojas, E

    2014-01-10

    Metals are a threat to human health by increasing disease risk. Experimental data have linked altered miRNA expression with exposure to some metals. MiRNAs comprise a large family of non-coding single-stranded molecules that primarily function to negatively regulate gene expression post-transcriptionally. Although several human populations are exposed to low concentrations of As, Cd and Pb as a mixture, most toxicology research focuses on the individual effects that these metals exert. Thus, this study aims to evaluate global miRNA and mRNA expression changes induced by a metal mixture containing NaAsO2, CdCl2, Pb(C2H3O2)2·3H2O and to predict possible metal-associated disease development under these conditions. Our results show that this metal mixture results in a miRNA expression profile that may be responsible for the mRNA expression changes observed under experimental conditions in which coding proteins are involved in cellular processes, including cell death, growth and proliferation related to the metal-associated inflammatory response and cancer.

  13. Prognostic value of amphiregulin and epiregulin mRNA expression in metastatic colorectal cancer patients

    PubMed Central

    You, Zhai; Qiong, Qian; Jun, Zhou

    2016-01-01

    Epidermal growth factor receptor (EGFR) and its ligands amphiregulin (AREG) and epiregulin (EREG) play a central role in the development of colorectal cancer, but the prognostic values of AREG and EREG are controversial. We conducted a meta-analysis of studies that investigated AREG and/or EREG mRNA levels in primary tumors to determine their prognostic value in metastatic colorectal cancer (mCRC). In addition, RAS status was assessed. Relevant articles were identified by searching the EMBASE, PubMed, and Cochrane Library databases. Hazard ratios (HR) with 95% confidence intervals (CIs) were calculated using a random-effects model. Nine studies involving 2167 patients were included in this meta-analysis. High AREG expression was associated with longer overall survival (OS) and progression-free survival (PFS). High EREG expression was also associated with prolonged OS and PFS. In RAS wild-type (WT) patients who received anti-EGFR therapy, high AREG and EREG expression was associated with longer OS. Our results indicate that high AREG and EREG mRNA expression are independent favorable prognostic biomarkers in mCRC. The expression of these ligands should be considered when evaluating prognoses in RAS-WT patients receiving anti-EGFR therapy. PMID:27344184

  14. Apigenin prevents UVB-induced cyclooxygenase 2 expression: coupled mRNA stabilization and translational inhibition.

    PubMed

    Tong, Xin; Van Dross, Rukiyah T; Abu-Yousif, Adnan; Morrison, Aubrey R; Pelling, Jill C

    2007-01-01

    Cyclooxygenase 2 (COX-2) is a key enzyme in the conversion of arachidonic acid to prostaglandins, and COX-2 overexpression plays an important role in carcinogenesis. Exposure to UVB strongly increased COX-2 protein expression in mouse 308 keratinocytes, and this induction was inhibited by apigenin, a nonmutagenic bioflavonoid that has been shown to prevent mouse skin carcinogenesis induced by both chemical carcinogens and UV exposure. Our previous study suggested that one pathway by which apigenin inhibits UV-induced and basal COX-2 expression is through modulation of USF transcriptional activity in the 5' upstream region of the COX-2 gene. Here, we found that apigenin treatment also increased COX-2 mRNA stability, and the inhibitory effect of apigenin on UVB-induced luciferase reporter gene activity was dependent on the AU-rich element of the COX-2 3'-untranslated region. Furthermore, we identified two RNA-binding proteins, HuR and the T-cell-restricted intracellular antigen 1-related protein (TIAR), which were associated with endogenous COX-2 mRNA in 308 keratinocytes, and apigenin treatment increased their localization to cell cytoplasm. More importantly, reduction of HuR levels by small interfering RNA inhibited apigenin-mediated stabilization of COX-2 mRNA. Cells expressing reduced TIAR showed marked resistance to apigenin's ability to inhibit UVB-induced COX-2 expression. Taken together, these results indicate that in addition to transcriptional regulation, another mechanism by which apigenin prevents COX-2 expression is through mediating TIAR suppression of translation.

  15. 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

  16. Distinct prognostic values of S100 mRNA expression in breast cancer

    PubMed Central

    Zhang, Shizhen; Wang, Zhen; Liu, Weiwei; Lei, Rui; Shan, Jinlan; Li, Ling; Wang, Xiaochen

    2017-01-01

    S100 family genes encode low molecular weight, acidic-Ca2+ binding proteins implicating in a wide spectrum of biological processes. S100 family contains at least 20 members, most of which are frequently dysregulated in human malignancies including breast cancer. However, the prognostic roles of each individual S100, especially the mRNA level, in breast cancer patients remain elusive. In the current study, we used “The Kaplan-Meier plotter” (KM plotter) database to investigate the prognostic values of S100 mRNA expression in breast cancer. Our results indicated that high mRNA expression of S100A8, S100A9, S100A11 and S100P were found to be significantly correlated to worse outcome, while S100A1 and S100A6 were associated with better prognosis in all breast cancer patients. We further assessed the prognostic value of S100 in different intrinsic subtypes and clinicopathological features of breast cancer. The associated results will elucidate the role of S100 in breast cancer and may further lead the research to explore the S100-targeting reagents for treating breast cancer patients. PMID:28051137

  17. Combinatorial analysis of mRNA expression patterns in mouse embryos using hybridization chain reaction.

    PubMed

    Huss, David; Choi, Harry M T; Readhead, Carol; Fraser, Scott E; Pierce, Niles A; Lansford, Rusty

    2015-03-02

    Multiplexed fluorescent hybridization chain reaction (HCR) and advanced imaging techniques can be used to evaluate combinatorial gene expression patterns in whole mouse embryos with unprecedented spatial resolution. Using HCR, DNA probes complementary to mRNA targets trigger chain reactions in which metastable fluorophore-labeled DNA HCR hairpins self-assemble into tethered fluorescent amplification polymers. Each target mRNA is detected by a probe set containing one or more DNA probes, with each probe carrying two HCR initiators. For multiplexed experiments, probe sets for different target mRNAs carry orthogonal initiators that trigger orthogonal DNA HCR amplification cascades labeled by spectrally distinct fluorophores. As a result, in situ amplification is performed for all targets simultaneously, and the duration of the experiment is independent of the number of target mRNAs. We have used multiplexed fluorescent in situ HCR and advanced imaging technologies to address questions of cell heterogeneity and tissue complexity in craniofacial patterning and anterior neural development. In the sample protocol presented here, we detect three different mRNA targets: Tg(egfp), encoding the enhanced green fluorescent protein (GFP) transgene (typically used as a control); Twist1, encoding a transcription factor involved in cell lineage determination and differentiation; and Pax2, encoding a transcription factor expressed in the mid-hindbrain region of the mouse embryo.

  18. Induction of Ski protein expression upon luteinization in rat granulosa cells without a change in its mRNA expression.

    PubMed

    Kim, Hyun; Yamanouchi, Keitaro; Matsuwaki, Takashi; Nishihara, Masugi

    2012-01-01

    The Ski protein is implicated in the proliferation/differentiation of a variety of cells. We previously reported that the Ski protein is present in granulosa cells of atretic follicles, but not in preovulatory follicles, suggesting that Ski has a role in apoptosis of granulosa cells. However, granulosa cells cannot only undergo apoptosis but can alternatively differentiate into luteal cells. It is unknown whether Ski is expressed and has a role in granulosa cells undergoing luteinization. Thus, the aim of the present study was to determine the localization of the Ski protein in the rat ovary during luteinization to examine if Ski might play a role in this process. In order to examine the Ski protein expression during the progression of luteinization, follicular growth was induced in immature female rats by administration of equine chorionic gonadotropin, and luteinization was induced by human chorionic gonadotropin treatment to mimic the luteinizing hormone (LH) surge. While no Ski-positive granulosa cells were present in the preovulatory follicle, Ski protein expression was induced in response to the LH surge and was maintained after formation of the corpus luteum (CL). Although the Ski protein is absent from the granulosa cells of the preovulatory follicle, its mRNA (c-ski) was expressed, and the level of c-ski mRNA was unchanged even after the LH surge. The combined results demonstrated that Ski protein expression is induced in granulosa cells upon luteinization, and suggested that its expression is regulated posttranscriptionally.

  19. Cloning and expression analysis of prohibitin mRNA in canine mammary tumors

    PubMed Central

    MATSUYAMA, Satoshi; NAKANO, Yuko; NAKAMURA, Mieko; YAMAMOTO, Ryohei; SHIMADA, Terumasa; OHASHI, Fumihito; KUBO, Kihei

    2014-01-01

    Prohibitin is an antiproliferative protein that is a product of a putative tumor suppressor gene. However, there is little information on prohibitins in companion animals. In this study, we cloned canine prohibitin mRNA using RT-PCR and 3′-RACE (Rapid Amplification of cDNA Ends). The sequence was well conserved compared with those of other mammals, including human. The deduced amino acid sequence translated from the open reading frame completely corresponded to the human sequence. Canine prohibitin mRNA was expressed in all normal mammary and tumor samples examined. These results suggest that this protein plays a vital role in cell growth mechanisms and may be related to the occurrence of canine mammary tumors. PMID:25312047

  20. Effect of running training on uncoupling protein mRNA expression in rat brown adipose tissue

    NASA Astrophysics Data System (ADS)

    Yamashita, Hitoshi; Yamamoto, Mikio; Sato, Yuzo; Izawa, Tetsuya; Komabayashi, Takao; Saito, Daizo; Ohno, Hideki

    1993-03-01

    The effect was investigated of endurance training on the expression of uncoupling protein (UCP) mRNA in brown adipose tissue (BAT) of rats. The exercised rats were trained on a rodent treadmill for 5 days per week and a total of 9 weeks. After the training programme, a marked decrease in BAT mass was found in terms of weight or weight per unit body weight; there was a corresponding decrease in DNA content and a downward trend in RNA and glycogen levels. The UCP mRNA was present at a markedly decreased level in BAT of trained animals. In consideration of the reduced levels of mRNAs for hormone-sensitive lipase and acylCoA synthetase, the brown adipose tissue investigated appeared to be in a relatively atrophied and thermogenically quiescent state.

  1. Zinc transporter mRNA expression in the RWPE-1 human prostate epithelial cell line.

    PubMed

    Albrecht, Amy L; Somji, Seema; Sens, Mary Ann; Sens, Donald A; Garrett, Scott H

    2008-08-01

    The human prostate gland undergoes a prominent alteration in Zn+2 homeostasis during the development of prostate cancer. The goal of the present study was to determine if the immortalized human prostate cell line (RWPE-1) could serve as a model system to study the role of zinc in prostate cancer. The study examined the expression of mRNA for 19 members of the zinc transporter gene family in normal prostate tissue, the prostate RWPE-1 cell line, and the LNCaP, DU-145 and PC-3 prostate cancer cell lines. The study demonstrated that the expression of the 19 zinc transporters was similar between the RWPE-1 cell line and the in situ prostate gland. Of the 19 zinc transporters, only 5 had levels that were different between the RWPE-1 cells and the tissue samples; all five being increased (ZnT-6, Zip-1, Zip-3A, Zip-10, and Zip-14). The response of the 19 transporters was also determined when the cell lines were exposed to 75 microM Zn+2 for 24 h. It was shown for the RWPE-1 cells that only 5 transporters responded to Zn+2 with mRNA for ZnT-1 and ZnT-2 being increased while mRNA for ZnT-7, Zip-7 and Zip-10 transporters were decreased. It was shown for the LNCaP, DU-145 and PC-3 cells that Zn+2 had no effect on the mRNA levels of all 19 transporters except for an induction of ZnT-1 in PC-3 cells. Overall, the study suggests that the RWPE-1 cells could be a valuable model for the study of the zinc transporter gene family in the prostate.

  2. Chondrogenic mRNA expression in prechondrogenic cells after blue laser irradiation.

    PubMed

    Kushibiki, Toshihiro; Tajiri, Takako; Ninomiya, Yoshihisa; Awazu, Kunio

    2010-03-08

    Low-level laser therapy (LLLT) has been used as a method for biostimulation. Cartilage develops through the differentiation of mesenchymal cells into chondrocytes, and differentiated chondrocytes in articular cartilage maintain cartilage homeostasis by synthesizing cartilage-specific extracellular matrix. The aim of this study is to evaluate the enhancement of chondrocyte differentiation and the expression levels of chondrogenic mRNA in prechondrogenic ATDC5 cells after laser irradiation. For chondrogenic induction, ATDC5 cells were irradiated with a blue laser (405 nm, continuous wave) at 100 mW/cm(2) for 180 s following incubation in chondrogenic differentiation medium. Differentiation after laser irradiation was quantitatively evaluated by the measurement of total collagen contents and chondrogenesis-related mRNAs. The total amount of collagen and mRNA levels of aggrecan, collagen type II, SOX-9, and DEC-1 were increased relative to those of a non-laser irradiated group after 14 days of laser irradiation. On the other hand, Ap-2alpha mRNA, a negative transcription factor of chondrogenesis, was dramatically decreased after laser irradiation. In addition, intracellular reactive oxygen species (ROS) were generated after laser irradiation. These results, for the first time, provide functional evidence that mRNA expression relating to chondrogenesis is increased, and Ap-2alpha is decreased immediately after laser irradiation. As this technique could readily be applied in situ to control the differentiation of cells at an implanted site within the body, this approach may have therapeutic potential for the restoration of damaged or diseased tissue.

  3. 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.

  4. Identification of prostate cancer mRNA markers by averaged differential expression and their detection in biopsies, blood, and urine

    PubMed Central

    Bai, V. Uma; Kaseb, Ahmed; Tejwani, Sheela; Divine, George W.; Barrack, Evelyn R.; Menon, Mani; Pardee, Arthur B.; Reddy, G. Prem-Veer

    2007-01-01

    The advent of serum prostate-specific antigen (PSA) as a biomarker has enabled early detection of prostate cancer and, hence, improved clinical outcome. However, a low PSA is not a guarantee of disease-free status, and an elevated PSA is frequently associated with a negative biopsy. Therefore, our goal is to identify molecular markers that can detect prostate cancer with greater specificity in body fluids such as urine or blood. We used the RT-PCR differential display method to first identify mRNA transcripts differentially expressed in tumor vs. patient-matched nontumor prostate tissue. This analysis led to the identification of 44 mRNA transcripts that were expressed differentially in some but not all tumor specimens examined. To identify mRNA transcripts that are differentially expressed in most tumor specimens, we turned to differential display of pooled tissue samples, a technique we name averaged differential expression (ADE). We performed differential display of mRNA from patient-matched nontumor vs. tumor tissue, each pooled from 10 patients with various Gleason scores. Differentially expressed mRNA transcripts identified by ADE were fewer in number, but were expressed in a greater percentage of tumors (>75%) than those identified by differential display of mRNA from individual patient samples. Differential expression of these mRNA transcripts was also detected by RT-PCR in mRNA isolated from urine and blood samples of prostate cancer patients. Our findings demonstrate the principle that specific cDNA probes of frequently differentially expressed mRNA transcripts identified by ADE can be used for the detection of prostate cancer in urine and blood samples. PMID:17283334

  5. Isoeugenol destabilizes IL-8 mRNA expression in THP-1 cells through induction of the negative regulator of mRNA stability tristetraprolin.

    PubMed

    Galbiati, Valentina; Carne, Alice; Mitjans, Montserrat; Galli, Corrado Lodovico; Marinovich, Marina; Corsini, Emanuela

    2012-02-01

    We previously demonstrated in the human promyelocytic cell line THP-1 that all allergens tested, with the exception of the prohapten isoeugenol, induced a dose-related release of interleukin-8 (IL-8). In the present study, we investigated whether this abnormal behavior was regulated by the AU-rich element-binding proteins HuR and tristetraprolin (TTP) or by the downstream molecule suppressor of cytokine signaling (SOCS)-3. The contact allergens isoeugenol, diethylmaleate (DEM), and 2,4-dinitrochlorobenzene (DNCB), and the irritant salicylic acid were used as reference compounds. Chemicals were used at concentrations that induced a 20% decrease in cell viability as assessed by propidium iodide staining, namely 100 μg/ml (0.61 mM) for isoeugenol, 100 μg/ml (0.58 mM) for DEM, 3 μg/ml (14.8 μM) for DNCB, and 250 μg/ml (1.81 mM) for salicylic acid. Time course experiments of IL-8 mRNA expression and assessment of IL-8 mRNA half-life, indicated a decreased IL-8 mRNA stability in isoeugenol-treated cells. We could demonstrate that a combination and regulation of HuR and TTP following exposure to contact allergens resulted in a different modulation of IL-8 mRNA half-life and release. The increased expression of TTP in THP-1 cells treated with isoeugenol results in destabilization of the IL-8 mRNA, which can account for the lack of IL-8 release. In contrast, the strong allergen DNCB failing to up-regulate TTP, while inducing HuR, resulted in longer IL-8 mRNA half-life and protein release. SOCS-3 was induced only in isoeugenol-treated cells; however, its modulation did not rescue the lack of IL-8 release, indicating that it is unlikely to be involved in the lack of IL-8 production. Finally, the destabilization effect of isoeugenol on IL-8 mRNA expression together with SOCS-3 expression resulted in an anti-inflammatory effect, as demonstrated by the ability of isoeugenol to modulate LPS or ionomycin-induced cytokine release.

  6. Robust Transgene Expression from Bicistronic mRNA in the Green Alga Chlamydomonas reinhardtii

    PubMed Central

    Onishi, Masayuki; Pringle, John R.

    2016-01-01

    The unicellular green alga Chlamydomonas reinhardtii is a model organism that provides an opportunity to understand the evolution and functional biology of the lineage that includes the land plants, as well as aspects of the fundamental core biology conserved throughout the eukaryotic phylogeny. Although many tools are available to facilitate genetic, molecular biological, biochemical, and cell biological studies in Chlamydomonas, expression of unselected transgenes of interest (GOIs) has been challenging. In most methods used previously, the GOI and a selectable marker are expressed from two separate mRNAs, so that their concomitant expression is not guaranteed. In this study, we developed constructs that allow expression of an upstream GOI and downstream selectable marker from a single bicistronic mRNA. Although this approach in other systems has typically required a translation-enhancing element such as an internal ribosome entry site for the downstream marker, we found that a short stretch of unstructured junction sequence was sufficient to obtain adequate expression of the downstream gene, presumably through post-termination reinitiation. With this system, we obtained robust expression of both endogenous and heterologous GOIs, including fluorescent proteins and tagged fusion proteins, in the vast majority of transformants, thus eliminating the need for tedious secondary screening for GOI-expressing transformants. This improved efficiency should greatly facilitate a variety of genetic and cell-biological studies in Chlamydomonas and also enable new applications such as expression-based screens and large-scale production of foreign proteins. PMID:27770025

  7. miRNA and mRNA expression analysis reveals potential sex-biased miRNA expression

    PubMed Central

    Guo, Li; Zhang, Qiang; Ma, Xiao; Wang, Jun; Liang, Tingming

    2017-01-01

    Recent studies suggest that mRNAs may be differentially expressed between males and females. This study aimed to perform expression analysis of mRNA and its main regulatory molecule, microRNA (miRNA), to discuss the potential sex-specific expression patterns using abnormal expression profiles from The Cancer Genome Atlas database. Generally, deregulated miRNAs and mRNAs had consistent expression between males and females, but some miRNAs may be oppositely expressed in specific diseases: up-regulated in one group and down-regulated in another. Studies of miRNA gene families and clusters further confirmed that these sequence or location related miRNAs might have opposing expression between sexes. The specific miRNA might have greater expression divergence across different groups, suggesting flexible expression across different individuals, especially in tumor samples. The typical analysis regardless of the sex will ignore or balance these sex-specific deregulated miRNAs. Compared with flexible miRNAs, their targets of mRNAs showed relative stable expression between males and females. These relevant results provide new insights into miRNA-mRNA interaction and sex difference. PMID:28045090

  8. FKBP5, SERT and COMT mRNA expressions in the peripheral leukocytes during menstruation cycle in healthy reproductive females.

    PubMed

    Kinouchi, Sawako; Iga, Jun-Ichi; Ueno, Shu-Ichi; Yamauchi, Ken; Numata, Shusuke; Song, Hongwei; Sumitani, Satsuki; Shibuya-Tayoshi, Sumiko; Haku, Mari; Yasui, Toshiyuki; Irahara, Minoru; Morita, Kyoko; Rokutan, Kazuhito; Ohmori, Tetsuro

    2008-03-21

    There have been several evidences that the mRNA expressions in the peripheral leukocytes may indicate not only physical but also psychological states. The purpose of this study is whether the mRNA expressional changes in the leukocytes are related to the mental states across the menstrual cycle in reproductive healthy female subjects. Thirty-eight female subjects (22.4+/-1.4 year-old) were participated in this study at three menstruation cycle periods (menstrual, follicular and luteal phase). The FKBP5 (FK506-binding protein gene), SERT (serotonin transporter gene) and COMT (catechol-o-methyltransferase gene) mRNA expressions in the leukocytes were determined with hormonal data. The psychological changes were assessed with self-rating hospital anxiety and depression scale (HADS). Only one thirds of subjects (n=12) had regular menstrual cycles during the experiment. So we analyzed the data from these 12 subjects. The anxiety score of each subject was changed across the menstrual cycle (Friedman test: P<0.05). The FKBP5 mRNA expression was significantly lower in the follicular phase than in the other phases but no changes were seen in either SERT or COMT mRNA expressions among the phases. In conclusion, there are differences of HADS anxiety score and FKBP5 mRNA expression in the leukocytes across the menstrual cycle but there is no correlation between anxiety scores and FKBP5 mRNA.

  9. Changes in superoxide dismutase mRNA expression by streptozotocin-induced diabetes.

    PubMed Central

    Kamata, K.; Kobayashi, T.

    1996-01-01

    1. Experiments were designed to investigate the involvement of superoxide anions in the attenuated endothelium-dependent relaxation of the rat aorta from streptozotocin (STZ)-induced diabetic rats. 2. The endothelium-dependent relaxation responses to acetylcholine (ACh, 10(-7) M) in helical strips of the aorta precontracted with noradrenaline (NA, 5 x 10(-3) approximately 3 x 10(-7) M) were significantly decreased in STZ-induced diabetic rats. The recovery phase of the relaxation after single administration of ACh in the STZ-induced diabetic rats was more rapid than those in control vessels. 3. Preincubation of aortic strips with superoxide dismutase (SOD, 60 u ml-1) normalized the recovery phase of the relaxation of diabetic aorta after single administration of ACh, whereas catalase (150 u ml-1) or indomethacin (10(-5) M) had no effects on the relaxation. 4. SOD (180 u ml-1) caused relaxation in NA precontracted aortic strips and the degree of the SOD-induced relaxation was significantly greater in diabetic aorta as compared with age-matched control vessels. 5. When the changes in mRNA expressions of Mn-SOD or Cu-Zn-SOD were observed, Mn-SOD mRNA expression was markedly decreased, and Cu-Zn-SOD was slightly decreased in diabetic aorta. 6. These results suggest that the rapid destruction of NO by superoxide anions may occur in the STZ-induced diabetic rats, and this may be due to a decrease in mRNA expression of Mn-SOD or Cu-Zn-SOD. Images Figure 4 PMID:8894182

  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. BCL6 mRNA Expression Level in Invasive Duct Carcinoma not otherwise Specified

    PubMed Central

    Badr, Eman; Masoud, Eman; Eldien, Marwa Serag

    2016-01-01

    Introduction B-Cell Lymphoma 6 (BCL6) has an oncogenic role in tumourigenesis of various malignancies. It represses genes involved in terminal differentiation and plays complementary role with Signal Transducer and Activator of Transcription 3 (STAT3) in triple-negative breast cancer cellular function. Aim To evaluate the expression of BCL6 in cancer breast and determine its correlation with the clinico-pathological features including the molecular subtype of breast carcinoma. Materials and Methods This prospective case control study was carried out on 150 patients, divided into 100 cases of invasive duct carcinoma not otherwise specified and 50 benign breast lesions including fibroadenoma and fibrocystic disease. Fresh tissues were excised, which were then subjected to RNA extraction. The BCL6 mRNA level was assessed using real-time reverse transcription Polymerase Chain Reaction (PCR). Results There was a significant higher levels of BCL6 mRNA in malignant cases compared to benign ones (p<0.001). The level of BCL6 mRNA was higher in cases showing advanced tumor stage (p<0.04), triple negative subtype and associated in situ component (p<0.001) compared to cases with an early stage, luminal or Her 2-neu positive subtypes and those lacking in situ component. Conclusion BCL6 is up-regulated in breast cancer and is associated with poor prognostic features such as advanced stage and triple negative molecular subtype. BCL6 inhibitors might be considered as targeted therapy for breast cancer. PMID:28208987

  12. Keratin14 mRNA expression in human pneumocytes during quiescence, repair and disease

    PubMed Central

    Confalonieri, Marco; Buratti, Emanuele; Grassi, Gabriele; Bussani, Rossana; Chilosi, Marco; Farra, Rossella; Abrami, Michela; Stuani, Cristiana; Salton, Francesco; Ficial, Miriam; Confalonieri, Paola; Zandonà, Lorenzo; Romano, Maurizio

    2017-01-01

    The lung alveoli slowly self-renew pneumocytes, but their facultative regeneration capacity is rapidly efficient after an injury, so fibrosis infrequently occurs. We recently observed Keratin 14 (KRT14) expression during diffuse alveolar damage (DAD), but not in controls. We wonder if KRT14 may be a marker of pneumocyte transition from quiescence to regeneration. Quantitative PCR and Western blot analyses highlighted the presence of KRT14 (mRNA and protein) only in human lung samples with DAD or interstitial lung disease (ILD). In the exponentially growing cell lines A549 and H441, the mRNA and protein levels of KRT14 peaked at day one after cell seeding and decreased at day two, opposite to what observed for the proliferation marker E2F1. The inverse relation of KRT14 versus E2F1 expression holds true also for other proliferative markers, such as cyclin E1 and cyclin D1. Of interest, we also found that E2F1 silencing caused cell cycle arrest and increased KRT14 expression, whilst E2F1 stimulation induced cell cycle progression and decreased KRT14. KRT14 also increased in proliferative pneumocytes (HPAEpiC) just before transdifferentiation. Overall, our results suggest that KRT14 is a viable biomarker of pneumocyte activation, and repair/regeneration. The involvement of KRT14 in regenerative process may suggest a novel pharmaceutical target to accelerate lung repair. PMID:28199407

  13. On a stochastic gene expression with pre-mRNA, mRNA and protein contribution.

    PubMed

    Rudnicki, Ryszard; Tomski, Andrzej

    2015-12-21

    In this paper we develop a model of stochastic gene expression, which is an extension of the model investigated in the paper [T. Lipniacki, P. Paszek, A. Marciniak-Czochra, A.R. Brasier, M. Kimmel, Transcriptional stochasticity in gene expression, J. Theor. Biol. 238 (2006) 348-367]. In our model, stochastic effects still originate from random fluctuations in gene activity status, but we precede mRNA production by the formation of pre-mRNA, which enriches classical transcription phase. We obtain a stochastically regulated system of ordinary differential equations (ODEs) describing evolution of pre-mRNA, mRNA and protein levels. We perform mathematical analysis of a long-time behavior of this stochastic process, identified as a piece-wise deterministic Markov process (PDMP). We check exact results using numerical simulations for the distributions of all three types of particles. Moreover, we investigate the deterministic (adiabatic) limit state of the process, when depending on parameters it can exhibit two specific types of behavior: bistability and the existence of the limit cycle. The latter one is not present when only two kinds of gene expression products are considered.

  14. Analysis of the sequence and embryonic expression of chicken neurofibromin mRNA.

    PubMed

    Schafer, G L; Ciment, G; Stocker, K M; Baizer, L

    1993-04-01

    Neurofibromatosis type 1 (NF1) is a common inherited disorder that primarily affects tissues derived from the neural crest. Recent identification and characterization of the human NF1 gene has revealed that it encodes a protein (now called neurofibromin) that is similar in sequence to the ras-GTPase activator protein (or ras-GAP), suggesting that neurofibromin may be a component of cellular signal transduction pathways regulating cellular proliferation and/or differentiation. To initiate investigations on the role of the NF1 gene product in embryonic development, we have isolated a partial cDNA for chicken neurofibromin. Sequence analysis reveals that the predicted amino acid sequence is highly conserved between chick and human. The chicken cDNA hybridizes to a 12.5-kb transcript on RNA blots, a mol wt similar to that reported for the human and murine mRNAs. Ribonuclease protection assays indicate that NF1 mRNA is expressed in a variety of tissues in the chick embryo; this is confirmed by in situ hybridization analysis. NF1 mRNA expression is detectable as early as embryonic stage 18 in the neural plate. This pattern of expression may suggest a role for neurofibromin during normal development, including that of the nervous system.

  15. 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

  16. Time-course of 5-HT(6) receptor mRNA expression during memory consolidation and amnesia.

    PubMed

    Huerta-Rivas, A; Pérez-García, G; González-Espinosa, C; Meneses, A

    2010-01-01

    Growing evidence indicates that antagonists of the 5-hydroxytryptamine (serotonin) receptor(6) (5-HT(6)) improve memory and reverse amnesia although the mechanisms involved are poorly understood. Hence, in this paper RT-PCR was used to evaluate changes in mRNA expression of 5-HT(6) receptor in trained and untrained rats treated with the 5-HT(6) receptor antagonist SB-399885 and amnesic drugs scopolamine or dizocilpine. Changes in mRNA expression of 5-HT(6) receptor were investigated at different times in prefrontal cortex, hippocampus and striatum. Data indicated that memory in the Pavlovian/instrumental autoshaping task was a progressive process associated to reduced mRNA expression of 5-HT(6) receptor in the three structures examined. SB-399885 improved long-term memory at 48h, while the muscarinic receptor antagonist scopolamine or the non-competitive NMDA receptor antagonist dizocilpine impaired it at 24h. Autoshaping training and treatment with SB-399885 increased 5-HT(6) receptor mRNA expression in (maximum increase) prefrontal cortex and striatum, 24 or 48h. The scopolamine-induced amnesia suppressed 5-HT(6) receptor mRNA expression while the dizocilpine-induced amnesia did not modify 5-HT(6) receptor mRNA expression. SB-399885 and scopolamine or dizocilpine were able to reestablish memory and 5-HT(6) receptor mRNA expression. These data confirmed previous memory evidence and of more interest is the observation that training, SB-399885 and amnesic drugs modulated 5-HT(6) receptor mRNA expression in prefrontal cortex, hippocampus and striatum. Further investigation in different memory tasks, times and amnesia models together with more complex control groups might provide further clues.

  17. Mycobacterium tuberculosis ESAT6 and CPF10 Induce Adenosine Deaminase 2 mRNA Expression in Monocyte-Derived Macrophages

    PubMed Central

    Bae, Mi Jung; Ryu, Suyeon; Kim, Ha-Jeong; Cha, Seung Ick

    2017-01-01

    Background Delayed hypersensitivity plays a large role in the pathogenesis of tuberculous pleural effusion (TPE). Macrophages infected with live Mycobacterium tuberculosis (MTB) increase the levels of adenosine deaminase2 (ADA2) in the pleural fluid of TPE patients. However, it is as yet unclear whether ADA2 can be produced by macrophages when challenged with MTB antigens alone. This study therefore evaluated the levels of ADA2 mRNA expression, using monocyte-derived macrophages (MDMs) stimulated with MTB antigens. Methods Purified monocytes from the peripheral blood mononuclear cells of healthy volunteers were differentiated into macrophages using granulocyte-macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF). The MDMs were stimulated with early secretory antigenic target protein 6 (ESAT6) and culture filtrate protein 10 (CFP10). The mRNA expression levels for the cat eye syndrome chromosome region, candidate 1 (CECR1) gene encoding ADA2 were then measured. Results CECR1 mRNA expression levels were significantly higher in MDMs stimulated with ESAT6 and CFP10, than in the unstimulated MDMs. When stimulated with ESAT6, M-CSF-treated MDMs showed more pronounced CECR1 mRNA expression than GM-CSF-treated MDMs. Interferon-γ decreased the ESAT6- and CFP10-induced CECR1 mRNA expression in MDMs. CECR1 mRNA expression levels were positively correlated with mRNA expression of tumor necrosis factor α and interleukin 10, respectively. Conclusion ADA2 mRNA expression increased when MDMs were stimulated with MTB antigens alone. This partly indicates that pleural fluid ADA levels could increase in patients with culture-negative TPE. Our results may be helpful in improving the understanding of TPE pathogenesis. PMID:28119750

  18. Expression and autoregulation of transforming growth factor beta receptor mRNA in small-cell lung cancer cell lines.

    PubMed Central

    Nørgaard, P.; Spang-Thomsen, M.; Poulsen, H. S.

    1996-01-01

    In small-cell lung cancer cell lines resistance to growth inhibition by transforming growth factor (TGF)-beta 1, was previously shown to correlate with lack of TGF-beta receptor I (RI) and II (RII) proteins. To further investigate the role of these receptors, the expression of mRNA for RI, RII and beta-glycan (RIII) was examined. The results showed that loss of RII mRNA correlated with TGF-beta 1 resistance. In contrast, RI-and beta-glycan mRNA was expressed by all cell lines, including those lacking expression of these proteins. According to Southern blot analysis, the loss of type II mRNA was not due to gross structural changes in the gene. The effect of TGF-beta 1 on expression of TGF-beta receptor mRNA (receptor autoregulation) was examined by quantitative Northern blotting in four cell lines with different expression of TGF-beta receptor proteins. In two cell lines expressing all three TGF-beta receptor proteins beta-glycan mRNA was rapidly down-regulated and this effect was sustained throughout the 24 h observation period. RI and RII mRNAs were slightly increased 24 h after treatment. In one cell line sensitive to growth inhibition by TGF-beta, 1 but lacking beta-glycan expression, and one cell line expressing only beta-glycan and thus TGF-beta 1 -resistant, no autoregulation of mRNA of either TGF-beta receptor was demonstrated. The results suggest that TGF-beta 1 regulates the expression of its receptors, in particular beta-glycan, and that this effect is dependent on co-expression of beta-glycan, RI and RII. Images Figure 1 Figure 2 Figure 4 PMID:8624260

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

    PubMed

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

    2017-02-16

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

  20. 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

  1. Increased 8-hydroxy-2'-deoxyguanosine in plasma and decreased mRNA expression of human 8-oxoguanine DNA glycosylase 1, anti-oxidant enzymes, mitochondrial biogenesis-related proteins and glycolytic enzymes in leucocytes in patients with systemic lupus erythematosus.

    PubMed

    Lee, H-T; Lin, C-S; Lee, C-S; Tsai, C-Y; Wei, Y-H

    2014-04-01

    We measured plasma levels of the oxidative DNA damage marker 8-hydroxy-2'-deoxyguanosine (8-OHdG) and leucocyte mRNA expression levels of the genes encoding the 8-OHdG repair enzyme human 8-oxoguanine DNA glycosylase 1 (hOGG1), the anti-oxidant enzymes copper/zinc superoxide dismutase (Cu/ZnSOD), manganese superoxide dismutase (MnSOD), catalase, glutathione peroxidase-1 (GPx-1), GPx-4, glutathione reductase (GR) and glutathione synthetase (GS), the mitochondrial biogenesis-related proteins mtDNA-encoded ND 1 polypeptide (ND1), ND6, ATPase 6, mitochondrial transcription factor A (Tfam), nuclear respiratory factor 1(NRF-1), pyruvate dehydrogenase E1 component alpha subunit (PDHA1), pyruvate dehydrogenase kinase isoenzyme 1 (PDK-1) and hypoxia inducible factor-1α (HIF-1α) and the glycolytic enzymes hexokinase-II (HK-II), glucose 6-phosphate isomerase (GPI), phosphofructokinase (PFK), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and lactate dehydrogenase A (LDHa). We analysed their relevance to oxidative damage in 85 systemic lupus erythematosus (SLE) patients, four complicated SLE patients undergoing rituximab treatment and 45 healthy individuals. SLE patients had higher plasma 8-OHdG levels (P < 0·01) but lower leucocyte expression of the genes encoding hOGG1(P < 0·01), anti-oxidant enzymes (P < 0·05), mitochondrial biogenesis-related proteins (P < 0·05) and glycolytic enzymes (P < 0·05) than healthy individuals. The increase in plasma 8-OHdG was correlated positively with the elevation of leucocyte expression of the genes encoding hOGG1 (P < 0·05), anti-oxidant enzymes (P < 0·05), several mitochondrial biogenesis-related proteins (P < 0·05) and glycolytic enzymes (P < 0·05) in lupus patients. The patients, whose leucocyte mtDNA harboured D310 heteroplasmy, exhibited a positive correlation between the mtDNA copy number and expression of ND1, ND6 and ATPase 6 (P < 0·05) and a negative correlation between mt

  2. Decline in c-myc mRNA expression but not the induction of c-fos mRNA expression is associated with differentiation of SH-SY5Y human neuroblastoma cells

    SciTech Connect

    Jalava, A.M.; Heikkilae, J.E.; Akerman, K.E.O. )

    1988-11-01

    The induction of differentiation in SH-SY5Y human neuroblastoma cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) is accompanied by a rapid and a transient expression of c-fos mRNA and a down-regulation of c-myc RNA. The TPA-induced expression of c-fos mRNA was inhibited by H-7, a specific inhibitor of protein kinase C (PK-C). Dioctanoylglycerol (DiC{sub 8}) failed to induce differentiation of SH-SY5Y cells or to down-regulate c-myc mRNA but it did induce the expression of c-fos mRNA. Treatment of IMR-32 human neuroblastoma cells with TPA did not cause differentiation although c-fos mRNA was induced. Since PK-C in SH-SY5Y cells was activated by both TPA and DiC{sub 8} it is suggested that the activation of PK-C alone is not sufficient to induce differentiation in SH-SY5Y cells. The down-regulation of c-myc mRNA rather than the induction of c-fos mRNA seems to be associated with differentiation process in SH-SY5Y cells.

  3. Expression of cytokine mRNA in lentivirus-induced arthritis.

    PubMed Central

    Lechner, F.; Vogt, H. R.; Seow, H. F.; Bertoni, G.; Cheevers, W. P.; von Bodungen, U.; Zurbriggen, A.; Peterhans, E.

    1997-01-01

    Infection of goats with the lentivirus caprine arthritis encephalitis virus (CAEV) leads to persistent infection and development of chronic arthritis. We analyzed the expression of cytokines and viral RNA in the joints of goats at early time points after experimental infection with CAEV and in those of animals suffering from chronic arthritis as a result of natural infection. In situ hybridization experiments showed that the pattern of cytokine expression in caprine arthritis was similar to that found in rheumatoid arthritis (RA), with a few cells expressing the lymphocyte-derived cytokines interferon (IFN)-gamma and interleukin (IL)-2 and rather more cells expressing monocyte chemoattractant protein (MCP)-1, IL-6, and tumor necrosis factor (TNF)-alpha. IFN-gamma mRNA expression in experimentally infected joints peaked at day 12 and was mostly detected in areas containing viral RNA. At later time points, no IFN-gamma- or virus-expressing cells were found in inflamed joints but both were again detected in goats with severe arthritis. Interestingly, at the clinical stage of arthritis reflecting the chronic stage of infection, the inflammatory lesion was found to be immunologically compartmentalized. Humoral immune responses and cell-mediated immune responses appeared to concurrently occur in distinct areas of the synovial membrane. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:9327739

  4. The Expression of Antibiotic Resistance Methyltransferase Correlates with mRNA Stability Independently of Ribosome Stalling

    PubMed Central

    Dzyubak, Ekaterina

    2016-01-01

    Members of the Erm methyltransferase family modify 23S rRNA of the bacterial ribosome and render cross-resistance to macrolides and multiple distantly related antibiotics. Previous studies have shown that the expression of erm is activated when a macrolide-bound ribosome stalls the translation of the leader peptide preceding the cotranscribed erm. Ribosome stalling is thought to destabilize the inhibitory stem-loop mRNA structure and exposes the erm Shine-Dalgarno (SD) sequence for translational initiation. Paradoxically, mutations that abolish ribosome stalling are routinely found in hyper-resistant clinical isolates; however, the significance of the stalling-dead leader sequence is largely unknown. Here, we show that nonsense mutations in the Staphylococcus aureus ErmB leader peptide (ErmBL) lead to high basal and induced expression of downstream ErmB in the absence or presence of macrolide concomitantly with elevated ribosome methylation and resistance. The overexpression of ErmB is associated with the reduced turnover of the ermBL-ermB transcript, and the macrolide appears to mitigate mRNA cleavage at a site immediately downstream of the ermBL SD sequence. The stabilizing effect of antibiotics on mRNA is not limited to ermBL-ermB; cationic antibiotics representing a ribosome-stalling inducer and a noninducer increase the half-life of specific transcripts. These data unveil a new layer of ermB regulation and imply that ErmBL translation or ribosome stalling serves as a “tuner” to suppress aberrant production of ErmB because methylated ribosome may impose a fitness cost on the bacterium as a result of misregulated translation. PMID:27645242

  5. 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

  6. 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.

  7. Regional expression of inducible heat shock protein-70 mRNA in the rat brain following administration of convulsant drugs.

    PubMed

    Planas, A M; Soriano, M A; Ferrer, I; Rodríguez Farré, E

    1994-11-01

    Expression of inducible heat shock protein-70 mRNA (hsp-70 mRNA) was studied in the rat brain following systemic administration of different convulsant agents: an L-type voltage-dependent calcium channel agonist, (+/-)-BAY K 8644 (BAY-K); the excitotoxic glutamate agonists kainic acid and N-methyl-D-aspartic acid (NMDA); and the GABAA receptor complex antagonists pentylenetetrazole (PTZ) and lindane (gamma-hexaclorocyclohexane). BAY-K induced minimal hsp-70 mRNA expression in the hippocampus of convulsant rats, localized in the dentate gyrus and the pyramidal cell layer of Ammon's horn. Kainic acid treatment in rats, showing severe limbic convulsions, caused intense expression of hsp-70 mRNA and protein (HSP-70). Expression was localized in select cerebral regions, notably the pyramidal cell layer of the hippocampal CA3 field of Ammon's horn and the piriform cortex, and also the subicular complex and the amygdala, and, to a lesser extent, the entorhinal cortex, the pyramidal cell layer of CA1, several thalamic nuclei, and the parietal cortex. In contrast, systemic administration of NMDA, PTZ or lindane led to no detectable induction of hsp-70 mRNA in the rat brain, despite producing convulsions. Histological examination revealed cell injury only following kainic acid treatment. Damage was most apparent in the piriform and entorhinal cortices, pyramidal cell layer of the CA1 field, and cortical amygdaloid nuclei. BAY-K, NMDA, PTZ and lindane did not lead to any observable histopathological changes. These results show that convulsions of different aetiology do not inevitably induce hsp-70 mRNA expression or cell damage. Intense expression of hsp-70 mRNA was generally associated with regions that later showed variable degrees of nerve cell damage, although hsp-70 mRNA expression was not always predictive of subsequent cell death or survival.

  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. Evolution of Steroid-Inducible RP2 mRNA Expression in the Mouse Kidney

    PubMed Central

    Tseng-Crank, Julie; Berger, Franklin G.

    1987-01-01

    We have examined the structure and expression of mRNAs encoded by the androgen-inducible RP2 gene in the kidneys of nine mouse species within the genus Mus. There is considerable interspecies variation in the lengths of the major RP2 transcripts; some of this variation is due to the presence or absence of a B1 repetitive element in the 3'-untranslated region of the gene. In addition, the extent of RP2 mRNA induction by testosterone differs among the species. Two species show 10-20-fold induction, while others display a reduced response or none at all. Analysis of an interspecific hybrid indicates that the inducibility phenotype is inherited in an additive fashion. A correlation between RP2 inducibility and the time of formation of lineages within the Mus genus suggests that induction evolved in a stepwise fashion, with the acquisition of a modest hormonal response being followed by the appearance of a greater response. The interspecies variations in RP2 mRNA structure and regulation provide a useful model for the identification and study of genetic elements that elicit evolutionary alterations in steroid-modulated gene expression. PMID:3623081

  10. Streamlining gene expression analysis: integration of co-culture and mRNA purification.

    PubMed

    Berry, Scott M; Singh, Chandresh; Lang, Jessica D; Strotman, Lindsay N; Alarid, Elaine T; Beebe, David J

    2014-02-01

    Co-culture of multiple cell types within a single device enables the study of paracrine signaling events. However, extracting gene expression endpoints from co-culture experiments is laborious, due in part to pre-PCR processing of the sample (i.e., post-culture cell sorting and nucleic acid purification). Also, a significant loss of nucleic acid may occur during these steps, especially with microfluidic cell culture where lysate volumes are small and difficult to access. Here, we describe an integrated platform for performing microfluidic cell culture and extraction of mRNA for gene expression analysis. This platform was able to recover 30-fold more mRNA than a similar, non-integrated system. Additionally, using a breast cancer/bone marrow stroma co-culture, we recapitulated stromal-dependent, estrogen-independent growth of the breast cancer cells, coincident with transcriptional changes. We anticipate that this platform will be used for streamlined analysis of paracrine signaling events as well as for screening potential drugs and/or patient samples.

  11. Circulating irisin levels and muscle FNDC5 mRNA expression are independent of IL-15 levels in mice.

    PubMed

    Quinn, LeBris S; Anderson, Barbara G; Conner, Jennifer D; Wolden-Hanson, Tami

    2015-11-01

    Interleukin-15 (IL-15) and irisin are exercise-induced myokines that exert favorable effects on energy expenditure and metabolism. IL-15 can induce PGC-1α expression, which in turn induces expression of irisin and its precursor, FNDC5. Therefore, the present study tested the hypothesis that increases in circulating irisin levels and muscle FNDC5 mRNA expression are dependent on IL-15. Circulating irisin levels and gastrocnemius muscle FNDC5 mRNA expression were examined following acute exercise in control and IL-15-deleted (IL-15 KO) mice, following injection of IL-15 into IL-15 KO mice, and in transgenic mice with elevated circulating IL-15 levels (IL-15 Tg mice). Circulating IL-15 levels and muscle PGC-1α and PPARδ mRNA expressions were determined as positive controls. No effect of IL-15 deletion on post-exercise serum irisin levels or muscle FNDC5 mRNA expression was detected. While serum IL-15 levels and muscle PGC-1α expression were elevated post-exercise in control mice, both serum irisin levels and muscle FNDC5 expression decreased shortly after exercise in both control and IL-15 KO mice. A single injection of recombinant IL-15 into IL-15 KO mice that significantly increased muscle PPARδ and PGC-1α mRNA expressions had no effect on circulating irisin release, but modestly induced muscle FNDC5 expression. Additionally, serum irisin and gastrocnemius muscle FNDC5 expression in IL-15 Tg mice were similar to those of control mice. Muscle FNDC5 mRNA expression and irisin release are not IL-15-dependent in mice.

  12. 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

  13. Imipramine induces brain-derived neurotrophic factor mRNA expression in cultured astrocytes.

    PubMed

    Takano, Katsura; Yamasaki, Hiroshi; Kawabe, Kenji; Moriyama, Mitsuaki; Nakamura, Yoichi

    2012-01-01

    Depression is one of the most prevalent and livelihood-threatening forms of mental illnesses and the neural circuitry underlying depression remains incompletely understood. Recent studies suggest that the neuronal plasticity involved with brain-derived neurotrophic factor (BDNF) plays an important role in the recovery from depression. Some antidepressants are reported to induce BDNF expression in vivo; however, the mechanisms have been considered solely in neurons and not fully elucidated. In the present study, we evaluated the effects of imipramine, a classic tricyclic antidepressant drug, on BDNF expression in cultured rat brain astrocytes. Imipramine dose-dependently increased BDNF mRNA expression in astrocytes. The imipramine-induced BDNF increase was suppressed with inhibitors for protein kinase A (PKA) or MEK/ERK. Moreover, imipramine exposure activated transcription factor cAMP response element binding protein (CREB) in a dose-dependent manner. These results suggested that imipramine induced BDNF expression through CREB activation via PKA and/or ERK pathways. Imipramine treatment in depression might exert antidepressant action through BDNF production from astrocytes, and glial BDNF expression might be a target of developing novel antidepressants.

  14. Differential expression of tyrosine hydroxylase mRNA in the developing rat mesencephalon.

    PubMed

    Solberg, Y; Pollack, Y; Silverman, W F

    1992-12-01

    1. With respect to the mesostriatal projection, the mesencephalon is composed of two dopaminergic (DA) cell populations, called dorsal tier and ventral tier. Strong evidence suggests differences in both the spatial and the temporal sequence of the innervation of the striatum between the two groups, with the ventral tier neurons innervating striatal patches prenatally and dorsal tier cells innervating striatal matrix postnatally. 2. Using in situ hybridization, we have examined the expression of the gene coding for tyrosine hydroxylase (TH) in mesencephalic DA neurons with respect to their postnatal development. Two ontogenic patterns of expression were observed: (a) dorsal tier neurons of the medial mesencephalon exhibited a sharp increase in expression beginning after birth, peaking on day 14, then decreasing and, finally, stabilizing; and (b) ventral tier neurons and dorsal tier cells from the lateral and the medial-dorsal mesencephalon showed only a slight increase in TH mRNA, reaching a plateau at P10. 3. The time course of the observed increase in TH gene expression in the first group, generally parallels the innervation of their target cells in the striatal matrix, suggesting that TH gene expression in these cells may be influenced by their postsynaptic cells or by the innervation process.

  15. In situ hybridization analysis of leucomyosuppressin mRNA expression in the cockroach, Diploptera punctata.

    PubMed

    Fusé, M; Bendena, W G; Donly, B C; Tobe, S S; Orchard, I

    1998-06-08

    In the cockroach Diploptera punctata, sequencing of the cDNA for the insect myoinhibitory neuropeptide, leucomyosuppressin (LMS), has demonstrated that LMS is the only Phe-Met-Arg-Phe-amide (NH2) (FMRFamide)-related peptide to be encoded by this gene (Donly et al. [1996] Insect Biochem. Mol. Biol. 26:627-637). However, in the present study, high performance liquid chromatography analysis of brain extracts showed six discrete FMRFamide-like immunoreactive fractions, one of which co-eluted with LMS. This study compared the distribution of FMRFamide-related peptides visualized by immunohistochemistry with LMS mRNA expression demonstrated by in situ hybridization in D. punctata. Immunohistochemistry with a polyclonal antiserum generated against FMRFamide, but which recognizes extended RFamide peptides, demonstrated numerous RFamide-like immunoreactive cells and processes in both nervous and nonnervous tissues. RFamide-like immunoreactivity was found in cells and processes of the brain and optic lobes, the stomatogastric nervous system, including the frontal and ingluvial ganglia, and the suboesophageal ganglion. Immunoreactivity was also present in all ganglia of the ventral nerve cord and in the alimentary canal. Within the alimentary canal, positively stained processes were found in the crop, midgut, and hindgut, and immunoreactive endocrinelike cells were located in the midgut. In situ hybridization with a digoxigenin-labeled RNA probe spanning the entire LMS coding region showed cell bodies containing LMS mRNA in all ganglia studied, other than the ingluvial ganglion. Expression was most abundant in the brain and optic lobes and in the frontal and suboesophageal ganglia. LMS mRNA was also apparent, although less intensely, in all other ganglia of the ventral nerve cord. Within the alimentary canal, LMS mRNA-positive cells were only visible in the anterior portion of the midgut, in the endocrinelike cells. The appearance of LMS mRNA in the central nervous system

  16. p73 expression is regulated by ribosomal protein RPL26 through mRNA translation and protein stability

    PubMed Central

    Yan, Wensheng; Chen, Xinbin

    2016-01-01

    p73, a p53 family tumor suppressor, is regulated by multiple mechanisms, including transcription and mRNA and protein stability. However, whether p73 expression is regulated via mRNA translation has not been explored. To test this, we examined whether ribosomal protein 26 (RPL26) plays a role in p73 expression. Here, we showed that p73 expression is controlled by RPL26 via protein stability and mRNA translation. To examine whether MDM2 mediates RPL26 to regulate p73 protein stability, we generated multiple MDM2-knockout cell lines by CRISPR-cas9. We found that in the absence of MDM2, the half-life of p73 protein is markedly increased. Interestingly, we also found that RPL26 is still capable of regulating p73 expression, albeit to a lesser extent, in MDM2-KO cells compared to that in isogenic control cells, suggesting that RPL26 regulates p73 expression via multiple mechanisms. Indeed, we found that RPL26 is necessary for efficient assembly of polysomes on p73 mRNA and de novo synthesis of p73 protein. Consistently, we found that RPL26 directly binds to p73 3′ untranslated region (3′UTR) and that RPL26 is necessary for efficient expression of an eGFP reporter that carries p73 3′UTR. We also found that RPL26 interacts with cap-binding protein eIF4E and enhances the association of eIF4E with p73 mRNA, leading to increased p73 mRNA translation. Finally, we showed that knockdown of RPL26 promotes, whereas ectopic expression of RPL26 inhibits, cell growth in a TAp73-dependent manner. Together, our data indicate that RPL26 regulates p73 expression via two distinct mechanisms: protein stability and mRNA translation. PMID:27825141

  17. BENZO(A)PYRENE DECREASES BRAIN AND OVARIAN AROMATASE mRNA EXPRESSION IN FUNDULUS HETEROCLITUS

    PubMed Central

    Dong, Wu; Wang, Lu; Thornton, Cammi; Scheffler, Brian E.; Willett, Kristine L.

    2008-01-01

    The higher molecular weight polycyclic aromatic hydrocarbons (PAHs) such as benzo(a)pyrene (BaP) are typically associated with genotoxicity, however, newer evidence suggests that these compounds may also act as endocrine system disruptors. We hypothesized that altered expression of the P450 enzyme aromatase genes could be a target for reproductive or developmental dysfunction caused by BaP exposure. Aromatase is at least partially responsible for estrogen homeostasis by converting androgens into estrogens. In fish, there are two isoforms of aromatase, a predominantly ovarian form, CYP19A1, and a brain form, CYP19A2. CYP19 mRNA expression was measured following BaP exposure (0, 10, 100 µg/L waterborne for 10 or 15 days) in Fundulus adults, juveniles and embryos by in situ hybridization. The CYP19A1 expression was significantly decreased after BaP exposure in the 3 month old Fundulus immature oocytes, but BaP did not affect CYP19A1 expression at any stage in adult oocytes. In embryo brains, BaP significantly decreased CYP19A2 compared to controls by 3.6-fold at 14 days post-fertilization. In adults, CYP19A2 expression was decreased significantly in the pituitary and hypothalamus (81% and 85% of controls, respectively). Promoter regions of Fundulus CYP19s were cloned, and putative response elements in the CYP19A1 and CYP19A2 promoters such as CRE, AhR and ERE may be involved in BaP-mediated changes in CYP19 expression. In order to compare the mechanism of BaP-mediated inhibition with that of a known aromatase inhibitor, fish were also exposed to fadrozole (20 and 100 µg/L). Fadrozole did not significantly decrease the mRNA expression in embryos or adult Fundulus. However, aromatase enzyme activity was significantly decreased in adult ovary and brain tissues. These studies provide a greater molecular understanding of the mechanisms of action of BaP and its potential to impact reproduction or development. PMID:18571745

  18. Pattern of expression of transforming growth factor-beta 4 mRNA and protein in the developing chicken embryo.

    PubMed

    Jakowlew, S B; Ciment, G; Tuan, R S; Sporn, M B; Roberts, A B

    1992-12-01

    Expression of TGF-beta 4 mRNA and protein was studied in the developing chicken embryo using specific cDNA probes and antibodies for chicken TGF-beta 4. Expression of TGF-beta 4 mRNA was detected by day 4 of incubation (Hamburger and Hamilton stage 22, E4) by RNA Northern blot analysis and increased with developmental age until day 12 of incubation (stage 38, E12) where it was detected in every embryonic tissue examined, with expression being highest in smooth muscle and lowest in the kidney. The steady-state level of expression of TGF-beta 4 mRNA remained relatively constant in most embryonic tissues through day 19 (stage 45, E19). In situ hybridization analysis detected TGF-beta 4 mRNA as early as the "definitive primitive streak" stage (stage 4); during neurulation (stage 10), TGF-beta 4 mRNA was detected in all three germ layers, including neuroectoderm. Following neurulation, TGF-beta 4 mRNA was detected in the neural tube, notochord, ectoderm, endoderm, sclerotome, and myotome, but not dermotome at stage 16. By day 6 of incubation (stage 29, E6), TGF-beta 4 mRNA was localized in several tissues including heart, lung, and gizzard. Immunohistochemical staining analysis also showed expression of TGF-beta 4 protein in all three germ layers as early as stage 4 in various cell types in qualitatively similar locations as TGF-beta 4 mRNA. These results suggest that TGF-beta 4 may play an important role in the development of many tissues in the chicken.

  19. Expression of c-Kit receptor mRNA and protein in the developing, adult and irradiated rodent testis.

    PubMed

    Prabhu, Sridurga Mithra; Meistrich, Marvin L; McLaughlin, Eileen A; Roman, Shaun D; Warne, Sam; Mendis, Sirisha; Itman, Catherine; Loveland, Kate Lakoski

    2006-03-01

    Germ cell proliferation, migration and survival during all stages of spermatogenesis are affected by stem cell factor signalling through the c-Kit receptor, the expression and function of which are vital for normal male reproductive function. The present study comprehensively describes the c-Kit mRNA and protein cellular expression profiles in germ cells of the postnatal and adult rodent testis, revealing their significant elevation in synthesis at the onset of spermatogenesis. Real-time PCR analysis for both mice and rats matched the cellular mRNA expression profile where examined. Localization studies in normal mouse testes indicated that both c-Kit mRNA and protein are first detectable in differentiating spermatogonia. In addition, all spermatogonia isolated from 8-day-old mice displayed detectable c-Kit mRNA, but 30-50% of these lacked protein expression. The c-Kit mRNA and protein profile in normal rat testes indicated expression in gonocytes, in addition to differentiating spermatogonia. However, in the irradiated adult rat testes, in which undifferentiated spermatogonia are the only germ cell type, mRNA was also detected in the absence of protein. This persisted at 3 days and 1 and 2 weeks following treatment with gonadotrophin-releasing hormone (GnRH) antagonist to stimulate spermatogenesis recovery. By 4 weeks of GnRH antagonist treatment, accompanying the emergence of differentiating spermatogonia, both mRNA and protein were detected. Based on these observations, we propose that c-Kit mRNA and protein synthesis are regulated separately, possibly by influences linked to testis maturation and circulating hormone levels.

  20. Effect of Radix Isatidis on the expression of moesin mRNA induced by LPS in the tissues of mice.

    PubMed

    Li, Jing; Liu, Yunhai; Fang, Jianguo; Chen, Xin; Xie, Wei

    2007-04-01

    To investigate the effect of the anti-endotoxic part of Radix Isatidis on the expression of moesin mRNA in murine tissues induced by lipopolysaccharide (LPS), the sample solution of F(022) part from Radix Isatidis was intraperitoneally administered to experimental mice, and the lipopoly-saccharide (LPS) were injected into the tail vein, and then the tissues of liver, kidney and spleen were colleted and cut into slices. The mRNA was detected by moesin mRNA hybridization in situ. The staining results were observed under microscope. It was found that moesin mRNA expression was increased in the tissues of liver, kidndy and spleen in mice treated with LPS, while in the mice pre-treated with F(022) part from Radix Isatidis, the LPS-induced moesin mRNA expressions in these tissues were inhibited in a dose-dependant manner. Our study showed that F(022) part from Radix Isatidis can inhibit the LPS-induced expression of moesin mRNA in the tissues of liver, kidney and spleen in mice.

  1. Identify signature regulatory network for glioblastoma prognosis by integrative mRNA and miRNA co-expression analysis.

    PubMed

    Bing, Zhi-Tong; Yang, Guang-Hui; Xiong, Jie; Guo, Ling; Yang, Lei

    2016-12-01

    Glioblastoma multiforme (GBM) is the most common and aggressive type of primary brain tumor in adults. Patients with this disease have a poor prognosis. The objective of this study is to identify survival-related individual genes (or miRNAs) and miRNA -mRNA pairs in GBM using a multi-step approach. First, the weighted gene co-expression network analysis and survival analysis are applied to identify survival-related modules from mRNA and miRNA expression profiles, respectively. Subsequently, the role of individual genes (or miRNAs) within these modules in GBM prognosis are highlighted using survival analysis. Finally, the integration analysis of miRNA and mRNA expression as well as miRNA target prediction is used to identify survival-related miRNA -mRNA regulatory network. In this study, five genes and two miRNA modules that significantly correlated to patient's survival. In addition, many individual genes (or miRNAs) assigned to these modules were found to be closely linked with survival. For instance, increased expression of neuropilin-1 gene (a member of module turquoise) indicated poor prognosis for patients and a group of miRNA -mRNA regulatory networks that comprised 38 survival-related miRNA -mRNA pairs. These findings provide a new insight into the underlying molecular regulatory mechanisms of GBM.

  2. 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

  3. Increased ceramide synthase 2 and 6 mRNA levels in breast cancer tissues and correlation with sphingosine kinase expression.

    PubMed

    Erez-Roman, Racheli; Pienik, Reut; Futerman, Anthony H

    2010-01-01

    Intervention in the ceramide metabolic pathway is emerging as a novel means to regulate cancer and to modify the activity of chemotherapeutic drugs. We now study mRNA expression levels of the six ceramide synthase (CerS) genes in breast cancer tissue. CerS2 and CerS6 mRNA was significantly elevated in breast cancer tissue compared to paired normal tissue, with approximately half of the individuals showing elevated CerS2 and CerS6 mRNA. A significant correlation was found between CerS2 and CerS6 expression, and between CerS4 and CerS2/CerS6 expression. Moreover, patients that expressed higher CerS2 or 4 mRNA levels tended to show no changes in sphingosine kinase 1 levels, and likewise patients that expressed no change in CerS2 or CerS4 mRNA levels tended to express higher levels of sphingosine kinase 1. Together these results suggest an important role for the CerS genes in breast cancer etiology or diagnosis.

  4. A study on mRNA expressions of fibronectin in dermal and cerebral wound healing for wound age estimation.

    PubMed

    Takamiya, Masataka; Kumagai, Reiko; Nakayashiki, Nori; Aoki, Yasuhiro

    2006-07-01

    We investigated mRNA expressions of fibronectin for wound age estimation during dermal and cerebral wound healing. Fibronectin mRNA expressions in the injured skin peaked at 8h post-injury. The expressions were detected in endothelial cells before and after injury, whereas they were detectable in the epidermal cells at 1-240 h, in fibroblasts at 1-72 h, in neutrophils and macrophages at 8-72 h, respectively. However, the expressions in epidermal cells became relatively weak in the subacute phase. Fibronectin mRNA expressions of the injured cerebrum increased after the intervention and peaked at 48 h, whereas there was a slight decrease during 24h post-injury. Although fibronectin mRNA was seen exclusively in the endothelial cells of the intact cerebrum, it was also detected in astrocytes during wound healing. From these findings, it was considered that fibronectin played an important role in dermal and cerebral wound healing. Expression of fibronectin mRNA was considered to indicate the acute phase of dermal wound healing, and the subacute phase of cerebral wound healing.

  5. Heat stress stimulates hepcidin mRNA expression and C/EBPα protein expression in aged rodent liver.

    PubMed

    Bloomer, Steven A; Kregel, Kevin C; Brown, Kyle E

    2014-01-01

    Elevations in hepatic iron content occur with aging and physiological stressors, which may promote oxidative injury to the liver. Since dysregulation of the iron regulatory hormone, hepcidin, can cause iron accumulation, our goal was to characterize the regulation of hepcidin in young (6 mo) and old (24 mo) Fischer 344 rats exposed to environmental heat stress. Liver and blood samples were taken in the control condition and after heating. Hepcidin expression did not differ between young and old rats in the control condition, despite higher levels of hepatic iron and IL-6 mRNA in the latter. Following heat stress, pSTAT3 increased in both groups, but C/EBPα and hepcidin mRNA increased only in old rats. Despite this, serum iron decreased in both age groups 2 h after heat stress, suggesting hepcidin-independent hypoferremia in the young rats. The differential regulation of hepcidin between young and old rats after hyperthermia may be due to the enhanced expression of C/EBPα protein in old rats. These data support the concept of "inflammaging" and suggest that repeated exposures to stressors may contribute to the development of anemia in older individuals.

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

    PubMed Central

    Patani, Neill; Jiang, Wen; Mansel, Robert; Newbold, Robert; Mokbel, Kefah

    2009-01-01

    Background SATB1 is a nuclear protein that has been recently reported to be a 'genome organizer' which delineates specific epigenetic modifications at target gene loci, directly up-regulating metastasis-associated genes while down-regulating tumor-suppressor genes. In this study, the level of mRNA expression of SATB1 and SATB2 were assessed in normal and malignant breast tissue in a cohort of women with breast cancer and correlated to conventional clinico-pathological parameters. Materials and methods Breast cancer tissues (n = 115) and normal background tissues (n = 31) were collected immediately after excision during surgery. Following RNA extraction, reverse transcription was carried out and transcript levels were determined using real-time quantitative PCR and normalized against β-actin expression. Transcript levels within the breast cancer specimens were compared to the normal background tissues and analyzed against TNM stage, nodal involvement, tumour grade and clinical outcome over a 10 year follow-up period. Results The levels of SATB1 were higher in malignant compared with normal breast tissue (p = 0.0167). SATB1 expression increased with increasing TNM stage (TNM1 vs. TNM2 p = 0.0264), increasing tumour grade (grade1 vs. grade 3 p = 0.017; grade 2 vs. grade 3 p = 0.0437; grade 1 vs. grade 2&3 p = 0.021) and Nottingham Prognostic Index (NPI) (NPI-1 vs. NPI-3 p = 0.0614; NPI-2 vs. NPI-3 p = 0.0495). Transcript levels were associated with oestrogen receptor (ER) positivity (ER(-) vs. ER(+) p = 0.046). SABT1 expression was also significantly correlated with downstream regulated genes IL-4 and MAF-1 (Pearson's correlation coefficient r = 0.21 and r = 0.162) and SATB2 (r = 0.506). After a median follow up of 10 years, there was a trend for higher SATB1 expression to be associated with shorter overall survival (OS). Higher levels of SATB2 were also found in malignant compared to background tissue (p = 0.049). SATB2 expression increased with increasing tumour

  7. Tobamovirus infection is independent of HSP101 mRNA induction and protein expression.

    PubMed

    Carr, Tyrell; Wang, Yongzeng; Huang, Zhonglian; Yeakley, Joanne M; Fan, Jian-Bing; Whitham, Steven A

    2006-10-01

    Heat shock protein 101 (HSP101) has been implicated in tobamovirus infections by virtue of its ability to enhance translation of mRNAs possessing the 5'Omega-leader of Tobacco mosaic virus (TMV). Enhanced translation is mediated by HSP101 binding to a CAA-repeat motif in TMV Omega leader. CAA repeat sequences are present in the 5' leaders of other tobamoviruses including Oilseed rape mosaic virus (ORMV), which infects Arabidopsis thaliana. HSP101 is one of eight HSP100 gene family members encoded by the A. thaliana genome, and of these, HSP101 and HSP98.7 are predicted to encode proteins localized to the cytoplasm where they could potentially interact with TMV RNA. Analysis of the expression of the HSP100s showed that only HSP101 mRNA transcripts were induced significantly by ORMV in A. thaliana. The induction of HSP101 mRNA was also correlated with an increase in its protein levels and was independent of defense-related signaling pathways involving salicylic acid, jasmonic acid, or ethylene. A. thaliana mutants lacking HSP101, HSP98.7, or both supported wild-type levels of ORMV replication and movement. Similar results were obtained for TMV infection in Nicotiana benthamiana plants silenced for HSP101, demonstrating that HSP101 is not necessary for efficient tobamovirus infection.

  8. Rift Valley fever virus NS{sub S} gene expression correlates with a defect in nuclear mRNA export

    SciTech Connect

    Copeland, Anna Maria; Van Deusen, Nicole M.; Schmaljohn, Connie S.

    2015-12-15

    We investigated the localization of host mRNA during Rift Valley fever virus (RVFV) infection. Fluorescence in situ hybridization revealed that infection with RVFV altered the localization of host mRNA. mRNA accumulated in the nuclei of RVFV-infected but not mock-infected cells. Further, overexpression of the NS{sub S} gene, but not the N, G{sub N} or NS{sub M} genes correlated with mRNA nuclear accumulation. Nuclear accumulation of host mRNA was not observed in cells infected with a strain of RVFV lacking the gene encoding NS{sub S}, confirming that expression of NS{sub S} is likely responsible for this phenomenon. - Highlights: • Rift Valley fever virus (RVFV) infection alters the localization of host mRNA. • mRNA accumulates in the nuclei of RVFV-infected but not mock-infected cells. • NS{sub S} is likely responsible for mRNA relocalization to the nucleus.

  9. 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

  10. Changes of expression of stretch-activated potassium channel TREK-1 mRNA and protein in hypertrophic myocardium.

    PubMed

    Cheng, Longxian; Su, Fengcheng; Ripen, Nsenga; Fan, Hong; Huang, Kai; Wang, Min; Peng, Hongyu; Mei, Chunli; Zhao, Fang; Liao, Yuhua

    2006-01-01

    The expression of stretch-activated potassium channel TREK-1 mRNA and protein of hypertrophic myocardium was measured. Using a model of hypertrophy induced by coarctation of abdominal aorta in male Wistar rats, the expression of TREK-1 mRNA and protein was detected by using semi-quantitative RT PCR and Western blot respectively. At 4th and 8th week after constriction of the abdominal aorta, rats developed significant left ventricular hypertrophy. As compared to sham-operated group, stretch-activated potassium channel TREK-1 mRNA was strongly expressed and protein was up-regulated in operation groups (P < 0.05). It was concluded that the expression of TREK-1 was up-regulated in hypertrophic myocardium induced by chronic pressure overload in Wistar rats.

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

    PubMed Central

    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. © 1999 Cancer Research Campaign PMID:9888482

  12. Comprehensive expression analysis of FSHD candidate genes at the mRNA and protein level.

    PubMed

    Klooster, Rinse; Straasheijm, Kirsten; Shah, Bharati; Sowden, Janet; Frants, Rune; Thornton, Charles; Tawil, Rabi; van der Maarel, Silvère

    2009-12-01

    In facioscapulohumeral muscular dystrophy (FSHD) the majority of patients carry a D4Z4 macrosatellite repeat contraction in the subtelomere of chromosome 4q. Several disease mechanisms have been proposed to explain how repeat contraction causes muscular dystrophy. All proposed mechanisms foresee a change from a closed to a more open chromatin structure followed by loss of control over expression of genes in or proximal to D4Z4. Initially, a distance and residual repeat size-dependent upregulation of the candidate genes FRG2, FRG1 and ANT1 was observed, but most successive expression studies failed to support transcriptional upregulation of 4qter genes. Moreover, chromatin studies do not provide evidence for a cis-spreading mechanism operating at 4qter in FSHD. In part, this inconsistency may be explained by differences in the techniques used, and the use of RNA samples obtained from different muscle groups. The aim of this study is to comprehensively and uniformly study the expression of the FSHD candidate genes FRG1, FRG2, CRYM, ANT1, ALP, PITX1 and LRP2BP at the RNA and protein level in identically processed primary myoblasts, myotubes and quadriceps muscle. Expression was compared between samples obtained from FSHD patients and normal controls with samples from myotonic dystrophy type 1 patients as disease controls. No consistent changes in RNA or protein expression levels were observed between the samples. The one exception was a selective increase in FRG2 mRNA expression in FSHD myotubes. This study provides further evidence that there is no demonstrable consistent, large magnitude, overexpression of any of the FSHD candidate genes.

  13. 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...

  14. Metallothionein mRNA expression and cadmium tolerance in metal-stressed and reference populations of the springtail Orchesella cincta.

    PubMed

    Timmermans, Martijn J T N; Ellers, Jacintha; Roelofs, Dick; van Straalen, Nico M

    2005-10-01

    Metal contamination in soil ecosystems is a permanent and often strong selection pressure. The present study investigates metal tolerance in 17 Orchesella cincta (Collembola) populations from metal-contaminated and reference sites, and combines analyses at the phenotypic and molecular level. Metal tolerance was phenotypically assayed by measuring survival times of laboratory cultures during exposure to cadmium. Comparisons of survival curves showed that five out of eight metal-stressed populations tested evolved increased cadmium tolerance (Stolberg, Plombieres, Hoboken, Hygum and Gusum). In addition, the role of the metallothionein (MT) gene in cadmium tolerance of O. cincta was studied by means of quantitative RT-PCR. The constitutive and Cd-induced MT mRNA expression of the laboratory cultures was measured. Results show that the mean constitutive MT mRNA expression of populations from polluted sites was significantly higher than of populations from reference sites. However, no correlation between MT mRNA expression levels after laboratory exposure to cadmium and field cadmium concentrations was observed. Furthermore, no relation between survival rate during exposure to cadmium and MT mRNA expression was detected. Our results suggest that constitutive MT mRNA expression plays a role in early protection against cadmium toxicity, and indicate that mechanisms other then MT up-regulation are involved in tolerance to prolonged exposure to cadmium.

  15. Olive Leaf Extract Elevates Hepatic PPAR α mRNA Expression and Improves Serum Lipid Profiles in Ovariectomized Rats.

    PubMed

    Yoon, Leena; Liu, Ya-Nan; Park, Hyunjin; Kim, Hyun-Sook

    2015-07-01

    We hypothesized that olive leaf extract might alleviate dyslipidemia resulting from estrogen deficiency. Serum lipid profile and mRNA expression of the related genes in the liver and adipose tissue were analyzed after providing olive leaf extract (200 or 400 mg/kg body weight; n=7 for each group) to ovariectomized rats for 10 weeks. After 10 weeks' administration, the rats in the olive leaf extract-administered groups showed significantly lower levels of serum triglyceride and very-low-density lipoprotein (VLDL)-cholesterol compared with the rats in the control group, whereas the administration of olive leaf extract did not significantly change the elevated low-density lipoprotein cholesterol levels. In addition, administration of high dose of olive leaf extract significantly decreased the liver triglyceride and increased serum estradiol levels. mRNA expressions of peroxisome proliferator-activated receptor alpha (PPAR α) and acyl-CoA oxidase (ACO) were not affected by ovariectomy, however, administration of olive leaf extract significantly increased both PPAR α and ACO mRNA expression. Expression of adiponectin mRNA in adipose tissue was significantly decreased in the ovariectomized control group. Rats administered low-dose olive leaf extract showed significantly elevated adiponectin mRNA expression compared with rats in the ovariectomized control group. Even though dose-dependent effects were not observed in most of the measurements, these results suggest that genes involved in lipid metabolism may be regulated by olive leaf extract administration in ovariectomized rats.

  16. Brain and heart sodium channel subtype mRNA expression in rat cerebral cortex.

    PubMed Central

    Yarowsky, P J; Krueger, B K; Olson, C E; Clevinger, E C; Koos, R D

    1991-01-01

    The expression of mRNAs coding for the alpha subunit of rat brain and rat heart sodium channels has been studied in adult and neonatal rat cerebral cortex using the reverse transcription-polymerase chain reaction (RT-PCR). Rat brain sodium channel subtype I, II, IIA, and III sequences were simultaneously amplified in the same PCR using a single oligonucleotide primer pair matched to all four subtype sequences. Identification of each subtype-specific product was inferred from the appearance of unique fragments when the product was digested with specific restriction enzymes. By using this RT-PCR method, products arising from mRNAs for all four brain sodium channel subtypes were identified in RNA extracted from adult rat cerebral cortex. The predominant component was type IIA with lesser levels of types I, II, and III. In contrast, the type II and IIA sequences were the predominant RT-PCR products in neonatal rat cortex, with slightly lower levels of type III and undetectable levels of type I. Thus, from neonate to adult, type II mRNA levels decrease relative to type IIA levels. Using a similar approach, we detected mRNA coding for the rat heart sodium channel in neonatal and adult rat cerebral cortex and in adult rat heart. These results reveal that mRNAs coding for the heart sodium channel and all four previously sequenced rat brain sodium channel subtypes are expressed in cerebral cortex and that type II and IIA channels may be differentially regulated during development. Images PMID:1658783

  17. 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.

  18. Development × environment interactions control tph2 mRNA expression.

    PubMed

    Lukkes, J L; Kopelman, J M; Donner, N C; Hale, M W; Lowry, C A

    2013-05-01

    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 rodent 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 4h 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.

  19. 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

  20. CTCF and CTCFL mRNA expression in 17β-estradiol-treated MCF7 cells

    PubMed Central

    DEL CAMPO, EDUARDO PORTILLO; MÁRQUEZ, JOSÉ JORGE TALAMÁS; REYES-VARGAS, FRANCIANELLA; INTRIAGO-ORTEGA, MARÍA DEL PILAR; QUINTANAR-ESCORZA, MARTHA ANGÉLICA; BURCIAGA-NAVA, JORGE ALBERTO; SIFUENTES-ALVAREZ, ANTONIO; REYES-ROMERO, MIGUEL

    2014-01-01

    Estrogens play a key role in breast cancer, with 60–70% of the cases expressing estrogen receptors (ERs), which are encoded by the ESR1 gene. CTCFL, a paralogue of the chromatin organizer CTCF, is a potential biomarker of breast cancer, but its expression in this disease is currently controversial. A positive correlation has been reported between CTCFL and ERs in breast tumors and there also exists a coordinated interaction between CTCF and ERs in breast cancer cells. Therefore, there appears to be an association between CTCF, CTCFL and estrogens in breast cancer; however, there has been no report on the effects of estrogens on CTCF and CTCFL expression. The aim of this study was to determine the effect of 17β-estradiol (E2) on the CTCF and CTCFL mRNA expression in the MCF7 breast cancer cell line. The promoter methylation status of CTCFL and data mining for estrogen response elements in promoters of the CTCF and CTCFL genes were also determined. The transcription of CTCF and CTCFL was performed by quantitative polymerase chain reaction (qPCR) and the promoter methylation status of CTCFL was determined by methylation-specific PCR. The MCF7 cells exhibited basal transcription of CTCF, which was significantly downregulated to 0.68 by 1 μM E2; basal or E2-regulated transcription of CTCFL was not detected. Under basal conditions, the CTCFL promoter was methylated. Through data mining, an estrogen response element was identified in the CTCF promoter, but no such element was found in CTCFL. These results suggested that estrogens may modulate CTCF expression, although there was no apparent association between ERs and CTCFL. PMID:24649078

  1. Specific responses in rat small intestinal epithelial mRNA expression and protein levels during chemotherapeutic damage and regeneration.

    PubMed

    Verburg, Melissa; Renes, Ingrid B; Van Nispen, Danielle J P M; Ferdinandusse, Sacha; Jorritsma, Marieke; Büller, Hans A; Einerhand, Alexandra W C; Dekker, Jan

    2002-11-01

    The rapidly dividing small intestinal epithelium is very sensitive to the cytostatic drug methotrexate. We investigated the regulation of epithelial gene expression in rat jejunum during methotrexate-induced damage and regeneration. Ten differentiation markers were localized on tissue sections and quantified at mRNA and protein levels relative to control levels. We analyzed correlations in temporal expression patterns between markers. mRNA expression of enterocyte and goblet cell markers decreased significantly during damage for a specific period. Of these, sucrase-isomaltase (-62%) and CPS (-82%) were correlated. Correlations were also found between lactase (-76%) and SGLT1 (-77%) and between I-FABP (-52%) and L-FABP (-45%). Decreases in GLUT5 (-53%), MUC2 (-43%), and TFF3 (-54%) mRNAs occurred independently of any of the other markers. In contrast, lysozyme mRNA present in Paneth cells increased (+76%). At the protein level, qualitative and quantitative changes were in agreement with mRNA expression, except for Muc2 (+115%) and TFF3 (+81%), which increased significantly during damage, following independent patterns. During regeneration, expression of each marker returned to control levels. The enhanced expression of cytoprotective molecules (Muc2, TFF3, lysozyme) during damage represents maintenance of goblet cell and Paneth cell functions, most likely to protect the epithelium. Decreased expression of enterocyte-specific markers represents decreased enterocyte function, of which fatty acid transporters were least affected.

  2. Expression of klotho mRNA and protein in rat brain parenchyma from early postnatal development into adulthood.

    PubMed

    Clinton, Sarah M; Glover, Matthew E; Maltare, Astha; Laszczyk, Ann M; Mehi, Stephen J; Simmons, Rebecca K; King, Gwendalyn D

    2013-08-21

    Without the age-regulating protein klotho, mouse lifespan is shortened and the rapid onset of age-related disorders occurs. Conversely, overexpression of klotho extends mouse lifespan. Klotho is most abundant in kidney and expressed in a limited number of other organs, including the brain, where klotho levels are highest in choroid plexus. Reports vary on where klotho is expressed within the brain parenchyma, and no data is available as to whether klotho levels change across postnatal development. We used in situ hybridization to map klotho mRNA expression in the developing and adult rat brain and report moderate, widespread expression across grey matter regions. mRNA expression levels in cortex, hippocampus, caudate putamen, and amygdala decreased during the second week of life and then gradually rose to adult levels by postnatal day 21. Immunohistochemistry revealed a protein expression pattern similar to the mRNA results, with klotho protein expressed widely throughout the brain. Klotho protein co-localized with both the neuronal marker NeuN, as well as, oligodendrocyte marker olig2. These results provide the first anatomical localization of klotho mRNA and protein in rat brain parenchyma and demonstrate that klotho levels vary during early postnatal development.

  3. Quantitative tissue-specific dynamics of in vivo GILZ mRNA expression and regulation by endogenous and exogenous glucocorticoids

    PubMed Central

    Ayyar, Vivaswath S; Almon, Richard R; Jusko, William J; DuBois, Debra C

    2015-01-01

    Glucocorticoids (GC) are steroid hormones, which regulate metabolism and immune function. Synthetic GCs, or corticosteroids (CS), have appreciable clinical utility via their ability to suppress inflammation in immune-mediated diseases like asthma and rheumatoid arthritis. Recent work has provided insight to novel GC-induced genes that mediate their anti-inflammatory effects, including glucocorticoid-induced leucine zipper (GILZ). Since GILZ comprises an important part of GC action, its regulation by both drug and hormone will influence CS therapy. In addition, GILZ expression is often employed as a biomarker of GC action, which requires judicious selection of sampling time. Understanding the in vivo regulation of GILZ mRNA expression over time will provide insight into both the physiological regulation of GILZ by endogenous GC and the dynamics of its enhancement by CS. A highly quantitative qRT-PCR assay was developed for measuring GILZ mRNA expression in tissues obtained from normal and CS-treated rats. This assay was applied to measure GILZ mRNA expression in eight tissues; to determine its endogenous regulation over time; and to characterize its dynamics in adipose tissue, muscle, and liver following treatment with CS. We demonstrate that GILZ mRNA is expressed in several tissues. GILZ mRNA expression in adipose tissue displayed a robust circadian rhythm that was entrained with the circadian oscillation of endogenous corticosterone; and is strongly enhanced by acute and chronic dosing. Single dosing also enhanced GILZ mRNA in muscle and liver, but the dynamics varied. In conclusion, GILZ is widely expressed in the rat and highly regulated by endogenous and exogenous GCs. PMID:26056061

  4. CXCR4 mRNA expression in colon, esophageal and gastric cancers and hepatitis C infected liver.

    PubMed

    Mitra, P; Shibuta, K; Mathai, J; Shimoda, K; Banner, B F; Mori, M; Barnard, G F

    1999-05-01

    We have recently demonstrated by Northern blot and RT-PCR that the mRNA expression of the alpha-chemokine hIRH/SDF-1alpha is reduced in hepatocellular carcinoma (HCC), several digestive tract cancers and premalignant colon adenomas, and that its receptor CXCR4 mRNA expression is reduced in HCC. Here we investigate the expression of CXCR4 mRNA expression in several digestive tract cancers and hepatitis C viral (HCV) infected liver, a premalignant condition. There was no difference in the CXCR4 mRNA expression in colon, esophageal or gastric cancers compared to non-cancerous tissues. This is significantly different from the reduced expression we have seen with hepatocellular carcinoma (p<0.05). To better refine regional tumor or hepatic cytokine mRNA analysis within a biopsy sample we describe a micro-isolation technique for RNA extraction from portal and triad areas of liver biopsies or other small malignant or non-malignant biopsy samples suitable for use in RT-PCR and differential display reactions. In HCV liver biopsies, the expression of hIRH and its receptor CXCR4 mRNA, corrected for G3PDH, was not significantly different from that of control non-HCV (steatosis) biopsies. CXCR4 is expressed on leukocytes and its expression was predicted to correlate with hepatic inflammation. CXCR4 receptor mRNA expression did correlate significantly with that of its ligand hIRH/SDF-1alpha (p=0.001), and with the severity of fibrosis (p<0.05), but not with portal inflammation (p<0.10), piecemeal necrosis (p<0.10), lobular inflammation (p>0.10), the presence of lymphoid aggregates (p>0.10), or the total histological activity index (p=0.07). There was no difference in expression of hIRH or CXCR4 between responders and non-responders to interferon (IFN) treatment, while as a control, the responder group of patients did show a higher expression of IFNalpha receptor than the non-responder group (p=0.05).

  5. Predominant expression of Fas ligand mRNA in CD8+ T lymphocytes in patients with HTLV-1 associated myelopathy.

    PubMed

    Kawahigashi, N; Furukawa, Y; Saito, M; Usuku, K; Osame, M

    1998-10-01

    To determine if Fas ligand (FasL) mediated apoptosis is involved in the pathogenesis of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), we examined the expression of FasL mRNA in fresh uncultured peripheral blood mononuclear cells (PBMC) from 17 Japanese patients with HAM/TSP, four adult T-cell leukemia/lymphoma (ATL) patients, three asymptomatic HTLV-1 carriers and three normal individuals. Using competitive PCR with primers specific for FasL mRNA, we demonstrated that nine of 17 HAM/TSP and one of four ATL patients expressed significant levels of FasL mRNA, whereas asymptomatic carriers, normal controls and both HTLV-1 infected and uninfected T-cell lines did not. Cell separation analysis following PCR revealed that FasL mRNA was expressed in CD8 + T lymphocytes. FasL mRNA was preferentially expressed in patients with increased proviral load and longer duration of clinical illness. These results suggest that FasL mediated mechanisms contribute to the pathogenesis of HAM/TSP.

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

    PubMed

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

    2011-07-01

    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 1h (single isolation) or 1h 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 vasopressin, might

  7. FOXA2 mRNA expression is associated with relapse in patients with Triple-Negative/Basal-like breast carcinoma.

    PubMed

    Perez-Balaguer, Ariadna; Ortiz-Martínez, Fernando; García-Martínez, Araceli; Pomares-Navarro, Critina; Lerma, Enrique; Peiró, Gloria

    2015-09-01

    The FOXA family of transcription factors regulates chromatin structure and gene expression especially during embryonic development. In normal breast tissue FOXA1 acts throughout mammary development; whereas in breast carcinoma its expression promotes luminal phenotype and correlates with good prognosis. However, the role of FOXA2 has not been previously studied in breast cancer. Our purpose was to analyze the expression of FOXA2 in breast cancer cells, to explore its role in breast cancer stem cells, and to correlate its mRNA expression with clinicopathological features and outcome in a series of patients diagnosed with breast carcinoma. We analyzed FOXA2 mRNA expression in a retrospective cohort of 230 breast cancer patients and in cell lines. We also knocked down FOXA2 mRNA expression by siRNA to determine the impact on cell proliferation and mammospheres formation using a cancer stem cells culture assay. In vitro studies demonstrated higher FOXA2 mRNA expression in Triple-Negative/Basal-like cells. Further, when it was knocked down, cells decreased proliferation and its capability of forming mammospheres. Similarly, FOXA2 mRNA expression was detected in 10% (23/230) of the tumors, especially in Triple-Negative/Basal-like phenotype (p < 0.001, Fisher's test). Patients whose tumors expressed FOXA2 had increased relapses (59 vs. 79%, p = 0.024, log-rank test) that revealed an independent prognostic value (HR = 3.29, C.I.95% = 1.45-7.45, p = 0.004, Cox regression). Our results suggest that FOXA2 promotes cell proliferation, maintains cancer stem cells, favors the development of Triple-Negative/Basal-like tumors, and is associated with increase relapses.

  8. Cavernous nerve injury elicits GAP-43 mRNA expression but not regeneration of injured pelvic ganglion neurons.

    PubMed

    Kato, Ryuichi; Kiryu-Seo, Sumiko; Sato, Yoshikazu; Hisasue, Shinichi; Tsukamoto, Taiji; Kiyama, Hiroshi

    2003-10-03

    Recovery of erectile dysfunction after cavernous nerve injury takes a long period. To elucidate this mechanism, unilateral cavernous nerve of male rat was cut, and the expression level of a nerve regeneration marker, the growth associated protein-43 (GAP-43) mRNA was evaluated by in situ hybridization and RT-PCR. While GAP-43 mRNA expression was transiently increased in the injured neurons of the major pelvic ganglion (MPG) at 7 days after nerve injury, continuous increase of GAP-43 mRNA was observed in the contralateral MPG from 7 days to 6 months after the nerve injury. Histochemical double-labeling studies for either neuronal NOS (nNOS) or tyrosine hydroxylase (TH) and the GAP-43 mRNA expression demonstrated that in injured MPG the transient up-regulation of GAP-43 mRNA was mainly seen in nNOS negative and/or TH positive neurons, suggesting non-parasympathetic post-ganglionic neurons, and also demonstrated that in contralateral MPG GAP-43 mRNA positive neurons were gradually increased in nNOS positive but TH negative neurons, suggesting parasympathetic post-ganglionic neurons. When a retrograde tracer Fluorogold (FG) was injected into the penile crus 7 days before histological experiments, FG-positive neurons were, if any, hardly seen in nNOS-positive neurons of the injured MPG for at least 6 months, whereas numerous FG-positive cells were seen in nNOS-positive neurons of the contralateral MPG. These results suggest that post-ganglionic projecting neurons of the intact side, which express increased GAP-43 mRNA, would be most likely to contribute to the recovery of the erectile function after unilateral cavernous nerve injury possibly by a plastic change such as nerve sprouting.

  9. 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

  10. Expression of brain-derived neurotrophic factor mRNA in rat hippocampus after treatment with antipsychotic drugs.

    PubMed

    Bai, Ou; Chlan-Fourney, Jennifer; Bowen, Rudy; Keegan, David; Li, Xin-Min

    2003-01-01

    Typical and atypical antipsychotic drugs, though both effective, act on different neurotransmitter receptors and are dissimilar in some clinical effects and side effects. The typical antipsychotic drug haloperidol has been shown to cause a decrease in the expression of brain-derived neurotrophic factor (BDNF), which plays an important role in neuronal cell survival, differentiation, and neuronal connectivity. However, it is still unknown whether atypical antipsychotic drugs similarly regulate BDNF expression. We examined the effects of chronic (28 days) administration of typical and atypical antipsychotic drugs on BDNF mRNA expression in the rat hippocampus using in situ hybridization. Quantitative analysis revealed that the typical antipsychotic drug haloperidol (1 mg/kg) down-regulated BDNF mRNA expression in both CA1 (P < 0.05) and dentate gyrus (P < 0.01) regions compared with vehicle control. In contrast, the atypical antipsychotic agents clozapine (10 mg/kg) and olanzapine (2.7 mg/kg) up-regulated BDNF mRNA expression in CA1, CA3, and dentate gyrus regions of the rat hippocampus compared with their respective controls (P < 0.01). These findings demonstrate that the typical and atypical antipsychotic drugs differentially regulate BDNF mRNA expression in rat hippocampus.

  11. 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

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

    PubMed Central

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

    2017-01-01

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

  13. Relationship between IL-4 and IL-5 mRNA expression and disease severity in atopic asthma.

    PubMed

    Humbert, M; Corrigan, C J; Kimmitt, P; Till, S J; Kay, A B; Durham, S R

    1997-09-01

    Atopic asthma is characterized by chronic inflammation of the bronchial mucosa in which eosinophil- and immunoglobulin E (IgE)-dependent mechanisms are believed to be prominent. Therefore, specific proeosinophilic mediators such as interleukin (IL)-5 and essential cofactors for IgE switching in B-lymphocytes such as IL-4 could play a pivotal role in asthma. However, the exact role that individual inflammatory mediators play in the development of the disease in humans is still unknown. Using semiquantitative reverse transcriptase-polymerase chain reaction amplification in bronchial biopsies from 10 atopic asthmatics, we have tested the hypothesis that IL-4 and IL-5 mRNA expression relative to beta-actin mRNA correlates with validated indicators of disease severity. IL-4 and IL-5 mRNA copies relative to beta-actin mRNA were detected in bronchial biopsies from atopic asthmatics. The numbers of IL-5 mRNA copies relative to beta-actin mRNA correlated with disease severity assessed by the Aas asthma score (r = 0.70, p = 0.01), baseline FEV1 (r = -0.94, p = 0.001), baseline peak expiratory flow rate (r = -0.77, p = 0.01), peak expiratory flow rate variability over 2 wk (r = 0.69, p = 0.028), and the histamine PC20 (r = -0.72, p = 0.018). Conversely, the numbers of IL-4 mRNA copies relative to beta-actin mRNA did not correlate with asthma severity, but they positively correlated with total serum IgE concentrations (r = -0.90, p = 0.001). Our present results support the concept that IL-5 may determine asthma clinical expression and severity, and by inference they support the development of IL-5 targeted therapies.

  14. Fucoxanthin regulates adipocytokine mRNA expression in white adipose tissue of diabetic/obese KK-Ay mice.

    PubMed

    Hosokawa, Masashi; Miyashita, Tatsuya; Nishikawa, Sho; Emi, Shingo; Tsukui, Takayuki; Beppu, Fumiaki; Okada, Tomoko; Miyashita, Kazuo

    2010-12-01

    Fucoxanthin, a marine carotenoid found in edible brown seaweeds, attenuates white adipose tissue (WAT) weight gain and hyperglycemia in diabetic/obese KK-A(y) mice, although it does not affect these parameters in lean C57BL/6J mice. In perigonadal and mesenteric WATs of KK-A(y) mice fed fucoxanthin, mRNA expression levels of monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α), which are considered to induce insulin resistance, were markedly reduced compared to control mice. In contrast to KK-A(y) mice, fucoxanthin did not alter MCP-1 and TNF-α mRNA expression levels in the WAT of lean C57BL/6J mice. Interleukin-6 (IL-6) and plasminogen activator inhibitor-1 mRNA expression levels in WAT were also decreased by fucoxanthin in KK-A(y) mice. In differentiating 3T3-F442A adipocytes, fucoxanthinol, which is a fucoxanthin metabolite found in WAT, attenuated TNF-α-induced MCP-1 and IL-6 mRNA overexpression and protein secretion into the culture medium. In addition, fucoxanthinol decreased TNF-α, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) mRNA expression in RAW264.7 macrophage-like cells stimulated by palmitic acid. These findings indicate that fucoxanthin regulates mRNA expression of inflammatory adipocytokines involved in insulin resistance, iNOS, and COX-2 in WAT and has specific effects on diabetic/obese KK-A(y) mice, but not on lean C57BL/6J mice.

  15. 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.

  16. 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.

  17. 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.

  18. 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

  19. Expression of RANTES mRNA in skin lesions of feline eosinophilic plaque.

    PubMed

    Kimura, Tomoe; Kano, Rui; Maeda, Sadatoshi; Tsujimoto, Hajime; Nagata, Masahiko; Hasegawa, Atsuhiko

    2003-10-01

    One of the mechanisms of eosinophil infiltration is its induction by chemoattractants such as regulated upon activation, normal T-expressed and secreted (RANTES) which is a cysteine-cysteine chemokine that mediates chemotaxis and activation of eosinophils in humans and mice. Skin lesions of feline eosinophilic plaque are characterized by a predominant infiltration of eosinophils. The mechanism(s) of eosinophilic infiltration in the skin and/or mucosa of cats is unknown. It is possible that RANTES is involved. To investigate the presence of RANTES in the skin of cats with eosinophilic plaques and nonaffected skin, we cloned and sequenced the full-length feline RANTES cDNA gene, in order to determine whether it is present in the skin of cats with eosinophilic plaques and/or if it is present in normal adjacent skin. We were able to document the the expression of RANTES mRNAs in skin with feline eosinophilic plaque as well as in normal cat skin. The full-length cDNA sequence of the RANTES gene (742 bp) contained a single open reading frame of 276 bp encoding a protein of 92 amino acids. The amino acid sequence of feline RANTES shared 67 and 74% sequence identity with that of bovine and mouse RANTES genes, respectively. RT-PCR analysis on RANTES mRNA in the skin of cats with eosinophilic plaque revealed that its expression was higher in the eosinophilic plaque skin lesions than in the normal skin. The result suggested that RANTES might play a role to induce eosinophil infiltration in feline eosinophilic plaque lesions.

  20. Differential expression of hypothalamic CART mRNA in response to body weight change following different dietary interventions.

    PubMed

    Yu, Yinghua; South, Tim; Wang, Qing; Huang, Xu-Feng

    2008-06-01

    Cocaine- and amphetamine-regulated transcript (CART) peptide is widely expressed in the hypothalamus and is involved in the central regulation of energy balance. Using in situ hybridization, this study examined the roles of CART peptide in the hypothalamus of diet-induced obese (DIO) or diet-resistant (DR) mice under different dietary interventions including high-fat (HF), low-fat (LF) and pair-feeding (PF) diet for 6 weeks. Pair feeding the energy intake of the DIO and DR mice was used to determine whether there is an inherent difference in baseline CART expression that may cause the DIO and DR phenotypes. The results demonstrated that CART mRNA expression in the hypothalamus of the DIO mice responded differently on the high-fat diet compared to DR mice. The arcuate nucleus and paraventricular nucleus showed a significant reduction in CART mRNA expression in DIO mice compared to DR mice on the HF diet (-19.6%, p=0.019; -26.1%, p=0.003); whilst a profound increase in CART mRNA expression was observed in the dorsomedial nucleus and lateral hypothalamic area (+44.5%, p=0.007; +37.4%, p=0.033). Our study suggests that the decrease of CART mRNA expression in Arc and PVN regions of DIO mice may contribute to the development of high-fat diet-induced obesity. In addition, CART in the dorsomedial nucleus (DM) of hypothalamus and lateral hypothalamus (LH) may be involved in the activation of an orexigenic effect. Since pair feeding of the high-fat diet eliminated both the body weight and CART mRNA differences between the DIO and DR mice, it is likely that their alterations in gene expression were a consequence of their dissimilar body weight levels.

  1. 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

    Hollerer, Ina; Curk, Tomaz; Haase, Bettina; Benes, Vladimir; Hauer, Christian; Neu-Yilik, Gabriele; Bhuvanagiri, Madhuri; Hentze, Matthias W; Kulozik, Andreas E

    2016-09-01

    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.

  2. 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.

  3. Diurnal expression of Dnmt3b mRNA in mouse liver is regulated by feeding and hepatic clockwork.

    PubMed

    Maekawa, Fumihiko; Shimba, Shigeki; Takumi, Shota; Sano, Tomoharu; Suzuki, Takehiro; Bao, Jinhua; Ohwada, Mika; Ehara, Tatsuya; Ogawa, Yoshihiro; Nohara, Keiko

    2012-09-01

    DNA methyltransferase 3B (DNMT3B) is critically involved in de novo DNA methylation and genomic stability, while the regulatory mechanism in liver is largely unknown. We previously reported that diurnal variation occurs in the mRNA expression of Dnmt3b in adult mouse liver. The aim of this study was to determine the mechanism underlying the diurnal expression pattern. The highest level and the lowest level of Dnmt3b mRNA expression were confirmed to occur at dawn and in the afternoon, respectively, and the expression pattern of Dnmt3b closely coincided with that of Bmal1. Since the diurnal pattern of Dnmt3b mRNA expression developed at weaning and scheduled feeding to separate the feeding cycle from the light/dark cycle led to a phase-shift in the expression, it could be assumed that feeding plays a critical role as an entrainment signal. In liver-specific Bmal1 knockout (L-Bmal1 KO) mice, L-Bmal1 deficiency resulted in significantly higher levels of Dnmt3b at all measured time points, and the time when the expression was the lowest in wild-type mice was shifted to earlier. Investigation of global DNA methylation revealed a temporal decrease of 5-methyl-cytosine percentage in the genome of wild-type mice in late afternoon. By contrast, no such decrease in 5-methyl-cytosine percentage was detected in L-Bmal1 KO mice, suggesting that altered Dnmt3b expression affects the DNA methylation state. Taken together, the results suggest that the feeding and hepatic clockwork generated by the clock genes, including Bmal1, regulate the diurnal variation in Dnmt3b mRNA expression and the consequent dynamic changes in global DNA methylation.

  4. Ya-fish (Schizothorax prenanti) spexin: identification, tissue distribution and mRNA expression responses to periprandial and fasting.

    PubMed

    Wu, Hongwei; Lin, Fangjun; Chen, Hu; Liu, Ju; Gao, Yundi; Zhang, Xin; Hao, Jin; Chen, Defang; Yuan, Dengyue; Wang, Tao; Li, Zhiqiong

    2016-02-01

    Spexin (SPX) is a novel peptide which was known for its role in physiological homeostasis. A recent study has confirmed that SPX plays an important role in the feeding regulation. However, the reports about SPX are very limited. In the present study, we characterized the structure, distribution and mRNA expression responses to feeding status of SPX in Ya-fish (Schizothorax prenanti). The full-length cDNA of Ya-fish SPX was 1330 base pairs (bp), which encoded 106 amino acid residues. These residues contained a 31-amino acid signal peptide region and a 14-amino acid mature peptide. The sequence alignment demonstrated that the Ya-fish SPX showed high conservation with other species. Our data revealed that SPX was widely expressed in all test tissues. The highest expression of SPX mRNA was observed in Ya-fish forebrain. Compared with the Ya-fish SPX mRNA expression in the forebrain between the preprandial and postprandial groups, the fed group was prominently increased than unfed groups after a meal, while the unfed group at 1 and 3 h substantially decreased than preprandial groups (P < 0.01). In addition, SPX mRNA expression in forebrain was significantly decreased (P < 0.01) during fasting for a week and sharply increased (P < 0.01) after refeeding on the 7th day, and then return to normal level on the 9th day. These results point toward that SPX mRNA expression is regulated by metabolic status or feeding conditions in Ya-fish.

  5. Long form leptin receptor mRNA expression in the brain, pituitary, and other tissues in the pig.

    PubMed

    Lin, J; Barb, C R; Matteri, R L; Kraeling, R R; Chen, X; Meinersmann, R J; Rampacek, G B

    2000-07-01

    Much effort has focused recently on understanding the role of leptin, the obese gene product secreted by adipocytes, in regulating growth and reproduction in rodents, humans and domestic animals. We previously demonstrated that leptin inhibited feed intake and stimulated growth hormone (GH) and luteinizing hormone (LH) secretion in the pig. This study was conducted to determine the location of long form leptin receptor (Ob-Rl) mRNA in various tissues of the pig. The leptin receptor has several splice variants in the human and mouse, but Ob-Rl is the major form capable of signal transduction. The Ob-Rl is expressed primarily in the hypothalamus of the human and rodents, but has been located in other tissues as well. In the present study, a partial porcine Ob-Rl cDNA, cloned in our laboratory and specific to the intracellular domain, was used to evaluate the Ob-Rl mRNA expression by RT-PCR in the brain and other tissues in three 105 d-old prepuberal gilts and in a 50 d-old fetus. In 105 d-old gilts, Ob-Rl mRNA was expressed in the hypothalamus, cerebral cortex, amygdala, thalamus, cerebellum, area postrema and anterior pituitary. In addition, Ob-Rl mRNA was expressed in ovary, uterine body, liver, kidney, pancreas, adrenal gland, heart, spleen, lung, intestine, bone marrow, muscle and adipose tissue. However, expression was absent in the thyroid, thymus, superior vena cava, aorta, spinal cord, uterine horn and oviduct. In the 50 d-old fetus, Ob-Rl mRNA was expressed in brain, intestine, muscle, fat, heart, liver and umbilical cord. These results support the idea that leptin might play a role in regulating numerous physiological functions.

  6. TP53 Promoter Methylation in Primary Glioblastoma: Relationship with TP53 mRNA and Protein Expression and Mutation Status

    PubMed Central

    Szybka, Malgorzata; Malachowska, Beata; Fendler, Wojciech; Potemski, Piotr; Piaskowski, Sylwester; Jaskolski, Dariusz; Papierz, Wielislaw; Skowronski, Wieslaw; Och, Waldemar; Kordek, Radzislaw

    2014-01-01

    Reduced expression of TP53 by promoter methylation has been reported in several neoplasms. It remains unclear whether TP53 promoter methylation is associated with reduced transcriptional and protein expression in glioblastoma (GB). The aim of our work was to study the impact of TP53 methylation and mutations on TP53 mRNA level and protein expression in 42 molecularly characterized primary GB tumors. We also evaluate the impact of all molecular alterations on the overall patient survival. The frequency of TP53 promoter methylation was found in 21.4%. To the best of our knowledge, this is the first report showing such high frequency of TP53 promoter methylation in primary GB. There was no relation between TP53 promoter methylation and TP53 mRNA level (p=0.5722) and between TP53 promoter methylation and TP53 protein expression (p=0.2045). No significant associations were found between TP53 mRNA expression and mutation of TP53 gene (p=0.9076). However, significant association between TP53 mutation and TP53 protein expression was found (p=0.0016). Our data suggest that in primary GB TP53 promoter methylation does not play a role in silencing of TP53 transcriptional and protein expression and is probably regulated by other genetic and epigenetic mechanisms associated with genes involved in the TP53 pathway. PMID:24506545

  7. Changes in mRNA expression of arcuate nucleus appetite-regulating peptides during lactation in rats.

    PubMed

    Suzuki, Yoshihiro; Nakahara, Keiko; Maruyama, Keisuke; Okame, Rieko; Ensho, Takuya; Inoue, Yoshiyuki; Murakami, Noboru

    2014-04-01

    The contribution of hypothalamic appetite-regulating peptides to further hyperphagia accompanying the course of lactation in rats was investigated by using PCR array and real-time PCR. Furthermore, changes in the mRNA expression for appetite-regulating peptides in the hypothalamic arcuate nucleus (ARC) were analyzed at all stages of pregnancy and lactation, and also after weaning. Food intake was significantly higher during pregnancy, lactation, and after weaning than during non-lactation periods. During lactation, ARC expression of mRNAs for agouti-related protein (AgRP) and peptide YY was increased, whereas that of mRNAs for proopiomelanocortin (POMC) and cholecystokinin (CCK) was decreased, in comparison with non-lactation periods. The increase in AgRP mRNA expression during lactation was especially marked. The plasma level of leptin was significantly decreased during the course of lactation, whereas that of acyl-ghrelin was unchanged. In addition, food intake was negatively correlated with the plasma leptin level during lactation. This study has clarified synchronous changes in the expression of many appetite-regulating peptides in ARC of rats during lactation. Our results suggest that hyperphagia during lactation in rats is caused by decreases in POMC and CCK expression and increases in AgRP expression in ARC, the latter being most notable. Together with the decrease in the blood leptin level, such changes in mRNA expression may explain the further hyperphagia accompanying the course of lactation.

  8. Expression of synaptophysin and its mRNA in bovine corpus lutea during different stages of pregnancy.

    PubMed

    Zhang, Wenhua; Chen, Shulin; Wang, Zhonghui; Tang, Caiyan; Meng, Xia; Li, Feihu; Zhao, Shanting

    2013-06-01

    In order to investigate the expression of mRNA and protein for synaptophysin (SYP) in bovine corpus luteum (CL) during different stages of pregnancy, we chose Holstein cows during various pregnancy stages. The CL was divided into two parts, then immunohistochemical streptavidin-perosidase and RT-PCR were used to determine the levels of protein and mRNA for SYP respectively. SYP immunoreactive products mainly located in large luteal cells; much less or no immunoreactivity was found in small luteal cells. The expression levels of SYP were different in various stages of pregnancy. In the CL of mid pregnancy, the levels of protein and mRNA for SYP were both significantly higher than those in early and late stage of pregnancy (P<0.05). After parturition, compared with late stage of pregnancy, the protein level of SYP decreased (P<0.05), but its mRNA increased (P<0.05). In conclusion, SYP has the strongest expression in mid stage of pregnancy, and its regular expression in bovine CL indicates that SYP may play important roles in maintaining the function of bovine CL and in the regulation of production.

  9. Type VII and XVII Collagen mRNA Expressions in Regenerated Epidermal Laminae in Chronic Equine Laminitis.

    PubMed

    Kuwano, Atsutoshi; Hasegawa, Telhisa; Arai, Katsuhiko

    2008-01-01

    To confirm ability forming the basement membrane of the regenerated laminar epidermis (rLE) in chronic laminitis, expression of type VII and type XVII collagen mRNAs in the rLE was studied applying sequences of two type of murine collagens. On northern blot analysis, complement DNA (cDNA) probes adjusted from the murine type VII and type XVII collagen could hybridize with the equine mRNAs, and each signal was detected as single-bands at approximately 9.5 kb and 5.6 kb, respectively. Contrasting with the expression level of equine glyceraldehyde-3-phosphate dehydrogenease mRNA, the band of type VII collagen mRNA in laminitis was stronger than normal, but the type XVII collagen mRNA in laminitis was less than normal. By in situ hybridization, positive signals in response to the murine type VII and type XVII collagen mRNA probes could be detected in the equine laminitic rLE region. From these results, it is concluded that the keratinocytes constructing the rLE in chronic stage of laminitis can express type VII and type XVII collagen mRNAs and these expression patterns were different from the normal.

  10. 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.

  11. Serum leptin concentrations, leptin mRNA expression, and food intake during the estrous cycle in rats.

    PubMed

    Fungfuang, Wirasak; Nakada, Tomoaki; Nakao, Nobuhiro; Terada, Misao; Yokosuka, Makoto; Gizurarson, Sveinbjorn; Hau, Jann; Moon, Changjong; Saito, Toru R

    2013-03-01

    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.

  12. TAK1 mRNA expression in the tumor tissue of locally advanced head and neck cancer patients.

    PubMed

    Honorato, Beatriz; Alcalde, Juan; Martinez-Monge, Rafael; Zabalegui, Natalia; Garcia-Foncillas, Jesús

    2008-02-14

    Resistance to radio and chemotherapy is one of the major drawbacks in the progression of head and neck squamous cell cancer (HNSCC) patients, evidencing the importance of finding optimum molecular prognosis markers to develop personalized treatment schedules. TGF-beta effector TAK1 activity has been related to a greater aggressiveness in several types of cancer (Kondo et al. 1998; Edlund et al. 2003; Kaur et al. 2005) and, although there has been described no significant implication of TAK1 in HNSCC development, we have further examined the role of its mRNA expression as a marker of prognosis in HNSCC. Fifty-nine advanced HNSCC patients were recruited for the study. The tumor expression of TAK1 mRNA was analyzed with RT-PCR using Taqman technology and its relationship with the clinical outcome of the patients studied. TAK1 mRNA expression was lower in patients that relapsed than in those that did not, but the difference was only significant between the patients that showed response to treatment (p < 0.001). ROC curve analyses pointed a 0.5 expression ratio TAK1/B2M value as an optimum cut-off point for relapse and response. Our data suggest the TAK1 mRNA analysis by Taqman RT-PCR can predict the risk of relapse in HNSCC patients.

  13. TAK1 mRNA Expression in the Tumor Tissue of Locally Advanced Head and Neck Cancer Patients

    PubMed Central

    Honorato, Beatriz; Alcalde, Juan; Martinez-Monge, Rafael; Zabalegui, Natalia; Garcia-Foncillas, Jesús

    2008-01-01

    Resistance to radio and chemotherapy is one of the major drawbacks in the progression of head and neck squamous cell cancer (HNSCC) patients, evidencing the importance of finding optimum molecular prognosis markers to develop personalized treatment schedules. TGF-β effector TAK1 activity has been related to a greater aggressiveness in several types of cancer (Kondo et al. 1998; Edlund et al. 2003; Kaur et al. 2005) and, although there has been described no significant implication of TAK1 in HNSCC development, we have further examined the role of its mRNA expression as a marker of prognosis in HNSCC. Fifty-nine advanced HNSCC patients were recruited for the study. The tumor expression of TAK1 mRNA was analyzed with RT-PCR using Taqman technology and its relationship with the clinical outcome of the patients studied. TAK1 mRNA expression was lower in patients that relapsed than in those that did not, but the difference was only significant between the patients that showed response to treatment (p < 0.001). ROC curve analyses pointed a 0.5 expression ratio TAK1/B2M value as an optimum cut-off point for relapse and response. Our data suggest the TAK1 mRNA analysis by Taqman RT-PCR can predict the risk of relapse in HNSCC patients. PMID:19787075

  14. cAMP analogs and their metabolites enhance TREK-1 mRNA and K+ current expression in adrenocortical cells.

    PubMed

    Enyeart, Judith A; Liu, Haiyan; Enyeart, John J

    2010-03-01

    bTREK-1 K(+) channels set the resting membrane potential of bovine adrenal zona fasciculata (AZF) cells and function pivotally in the physiology of cortisol secretion. Adrenocorticotropic hormone controls the function and expression of bTREK-1 channels through signaling mechanisms that may involve cAMP and downstream effectors including protein kinase A (PKA) and exchange protein 2 directly activated by cAMP (Epac2). Using patch-clamp and Northern blot analysis, we explored the regulation of bTREK-1 mRNA and K(+) current expression by cAMP analogs and several of their putative metabolites in bovine AZF cells. At concentrations sufficient to activate both PKA and Epac2, 8-bromoadenosine-cAMP enhanced the expression of both bTREK-1 mRNA and K(+) current. N(6)-Benzoyladenosine-cAMP, which activates PKA but not Epac, also enhanced the expression of bTREK-1 mRNA and K(+) current measured at times from 24 to 96 h. An Epac-selective cAMP analog, 8-(4-chlorophenylthio)-2'-O-methyl-cAMP (8CPT-2'-OMe-cAMP), potently stimulated bTREK-1 mRNA and K(+) current expression, whereas the nonhydrolyzable Epac activator 8-(4-chlorophenylthio)-2'-O-methyl-cAMP, Sp-isomer was ineffective. Metabolites of 8CPT-2'-OMe-cAMP, including 8-(4-chlorophenylthio)-2'-O-methyladenosine-5'-O-monophosphate and 8CPT-2'-OMe-adenosine, promoted the expression of bTREK-1 transcripts and ion current with a temporal pattern, potency, and effectiveness resembling that of the parent compound. Likewise, at low concentrations, 8-(4-chlorophenylthio)-cAMP (8CPT-cAMP; 30 microM) but not its nonhydrolyzable analog 8-(4-chlorophenylthio)-cAMP, Sp-isomer, enhanced the expression of bTREK-1 mRNA and current. 8CPT-cAMP metabolites, including 8CPT-adenosine and 8CPT-adenine, also increased bTREK-1 expression. These results indicate that cAMP increases the expression of bTREK-1 mRNA and K(+) current through a cAMP-dependent but Epac2-independent mechanism. They further demonstrate that one or more metabolites of 8

  15. Genome-Wide Screening of mRNA Expression in Leprosy Patients

    PubMed Central

    Belone, Andrea de Faria F.; Rosa, Patrícia S.; Trombone, Ana P. F.; Fachin, Luciana R. V.; Guidella, Cássio C.; Ura, Somei; Barreto, Jaison A.; Pinilla, Mabel G.; de Carvalho, Alex F.; Carraro, Dirce M.; Soares, Fernando A.; Soares, Cleverson T.

    2015-01-01

    Leprosy, an infectious disease caused by Mycobacterium leprae, affects millions of people worldwide. However, little is known regarding its molecular pathophysiological mechanisms. In this study, a comprehensive assessment of human mRNA was performed on leprosy skin lesions by using DNA chip microarrays, which included the entire spectrum of the disease along with its reactional states. Sixty-six samples from leprotic lesions (10TT, 10BT, 10BB, 10BL, 4LL, 14R1, and 10R2) and nine skin biopsies from healthy individuals were used as controls (CC) (ages ranged from 06 to 83 years, 48 were male and 29 female). The evaluation identified 1580 differentially expressed mRNAs [Fold Change (FC) ≥ 2.0, p ≤ 0.05] in diseased lesions vs. healthy controls. Some of these genes were observed in all forms of the disease (CD2, CD27, chit1, FA2H, FAM26F, GZMB, MMP9, SLAMF7, UBD) and others were exclusive to reactional forms (Type “1” reaction: GPNMB, IL1B, MICAL2, FOXQ1; Type “2” reaction: AKR1B10, FAM180B, FOXQ1, NNMT, NR1D1, PTX3, TNFRSF25). In literature, these mRNAs have been associated with numerous pathophysiological processes and signaling pathways and are present in a large number of diseases. The role of these mRNAs maybe studied in the context of developing new diagnostic markers and therapeutic targets for leprosy. PMID:26635870

  16. 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.

  17. Predicting gene targets of perturbations via network-based filtering of mRNA expression compendia

    PubMed Central

    Cosgrove, Elissa J.; Zhou, Yingchun; Gardner, Timothy S.; Kolaczyk, Eric D.

    2008-01-01

    Motivation: DNA microarrays are routinely applied to study diseased or drug-treated cell populations. A critical challenge is distinguishing the genes directly affected by these perturbations from the hundreds of genes that are indirectly affected. Here, we developed a sparse simultaneous equation model (SSEM) of mRNA expression data and applied Lasso regression to estimate the model parameters, thus constructing a network model of gene interaction effects. This inferred network model was then used to filter data from a given experimental condition of interest and predict the genes directly targeted by that perturbation. Results: Our proposed SSEM–Lasso method demonstrated substantial improvement in sensitivity compared with other tested methods for predicting the targets of perturbations in both simulated datasets and microarray compendia. In simulated data, for two different network types, and over a wide range of signal-to-noise ratios, our algorithm demonstrated a 167% increase in sensitivity on average for the top 100 ranked genes, compared with the next best method. Our method also performed well in identifying targets of genetic perturbations in microarray compendia, with up to a 24% improvement in sensitivity on average for the top 100 ranked genes. The overall performance of our network-filtering method shows promise for identifying the direct targets of genetic dysregulation in cancer and disease from expression profiles. Availability: Microarray data are available at the Many Microbe Microarrays Database (M3D, http://m3d.bu.edu). Algorithm scripts are available at the Gardner Lab website (http://gardnerlab.bu.edu/SSEMLasso). Contact: kolaczyk@math.bu.edu Supplementary information: Supplementary Data are available at Bioinformatics on line. PMID:18779235

  18. Increased litter size and suckling intensity inhibit KiSS-1 mRNA expression in rat arcuate nucleus

    PubMed Central

    Noroozi, Atefeh; Shirazi, Mohammad Reza Jafarzadeh; Zamiri, Mohammad Javad; Tamadon, Amin; Akhlaghi, Amir; Tanideh, Nader; Niazi, Ali; Moghadam, Ali

    2014-01-01

    Objective(s): The effect of litter size and suckling intensity on the expression of KiSS-1 mRNA in the arcuate nucleus (ARC) of rats were evaluated. Materials and Methods: Thirty two pregnant and four non-lactating ovariectomized (as control group) rats were used in this experiment. Lactating rats were allotted to eight equal groups. In three groups, litter size was adjusted to 5, 10, or 15 pups upon parturition and allowed to suckle their pups continuously by 8 days postpartum. In the other three groups, litter size was adjusted to five upon birth; the pups were separated from the dams for 6 hr on day 8 postpartum, after which the pups were allowed to suckle their dams for 2.5, 5, or 7.5 min prior to killing the dams. Two groups of lactating rats with either 10 or 15 pups were separated from their pups for 6 hr on day 8 postpartum, after which the pups were allowed to suckle their dams for 5 min before the dams were killed on day 8 postpartum. The ARC was removed and the expression of KiSS-1 mRNA was evaluated, using real-time PCR. Results: The expression of KiSS-1 mRNA in the ARC was decreased as the litter size and intensity of suckling stimulus were increased. The effect of suckling intensity on the expression of KiSS-1 mRNA was more pronounced than that of litter size. Conclusion: Increased litter size and suckling intensity decreased KiSS-1 mRNA expression in the ARC which may contribute to lactation anestrus in rat. PMID:25422754

  19. Altered EphA5 mRNA expression in rat brain with a single methamphetamine treatment.

    PubMed

    Numachi, Yohtaro; Yoshida, Sumiko; Yamashita, Motoyasu; Fujiyama, Ko; Toda, Shigenobu; Matsuoka, Hiroo; Kajii, Yasushi; Nishikawa, Toru

    2007-09-07

    Methamphetamine is a potent and indirect dopaminergic agonist which can cause chronic brain dysfunctions including drug abuse, drug dependence and drug-induced psychosis. Methamphetamine is known to trigger molecular mechanisms involved in associative learning and memory, and thereby alter patterns of synaptic connectivity. The persistent risk of relapse in methamphetamine abuse, dependence and psychosis may be caused by such alterations in synaptic connectivity. EphA5 receptors constitute large families of tyrosine kinase receptor and are expressed almost exclusively in the nervous system, especially in the limbic structures. Recent studies suggest EphA5 to be important in the topographic projection, development, and plasticity of limbic structures, and to be involved in dopaminergic neurotransmission. We used in situ hybridization to examine whether methamphetamine alters EphA5 mRNA expression in the brains of adult male Wister rats. EphA5 mRNA was widely distributed in the medial frontal cortex, cingulate cortex, piriform cortex, hippocampus, habenular nucleus and amygdala. Compared to baseline expression at 0h, EphA5 mRNA was significantly decreased (by 20%) in the medial frontal cortex at 24h, significantly increased (by 30%) in the amygdala at 9 and 24h, significantly but transiently decreased (by 30%) in the habenular nucleus at 1h after a single injection of methamphetamine. Methamphetamine did not change EphA5 mRNA expression in the cingulate cortex, piriform cortex or hippocampus. Our results that methamphetamine altered EphA5 mRNA expression in rat brain suggest methamphetamine could affect patterns of synaptic connectivity, which might be responsible for methamphetamine-induced chronic brain dysfunctions.

  20. Breast Cancer Resistance Protein Abundance, but Not mRNA Expression, Correlates With Estrone-3-Sulfate Transport in Caco-2.

    PubMed

    Harwood, Matthew D; Neuhoff, Sibylle; Rostami-Hodjegan, Amin; Warhurst, Geoffrey

    2016-04-01

    Transporter mRNA and protein expression data are used to extrapolate in vitro transporter kinetics to in vivo drug disposition predictions. Breast cancer resistance protein (BCRP) possesses broad substrate specificity; therefore, understanding BCRP expression-activity relationships are necessary for the translation to in vivo. Bidirectional transport of estrone-3-sulfate (E-3-S), a BCRP probe, was evaluated with respect to relative BCRP mRNA expression and absolute protein abundance in 10- and 29-day cultured Caco-2 cells. BCRP mRNA expression was quantified by real-time PCR against a housekeeper gene, Cyclophilin A. The BCRP protein abundance in total membrane fractions was quantified by targeted proteomics, and [(3)H]-E-3-S bidirectional transport was determined in the presence or absence of Ko143, a potent BCRP inhibitor. BCRP mRNA expression was 1.5-fold higher in 29- versus 10-day cultured cells (n = 3), whereas a 2.4-fold lower (p < 0.001) BCRP protein abundance was observed in 29- versus 10-day cultured cells (1.28 ± 0.33 and 3.06 ± 0.22 fmol/μg protein, n = 6, respectively). This correlated to a 2.45-fold lower (p < 0.01) efflux ratio for E-3-S in 29- versus 10-day cultured cells (8.97 ± 2.51 and 3.32 ± 0.66, n = 6, respectively). Caco-2 cell BCRP protein abundance, but not mRNA levels, correlates with BCRP activity, suggesting that extrapolation strategies incorporating BCRP protein abundance-activity relationships may be more successful.

  1. 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.

  2. Detection of full-length and truncated neurokinin-1 receptor mRNA expression in human brain regions.

    PubMed

    Lai, Jian-Ping; Cnaan, Avital; Zhao, Huaqing; Douglas, Steven D

    2008-02-15

    We have applied a newly developed SYBR green-based real-time RT-PCR assay for quantification of full-length and truncated neurokinin-1 receptor (NK1R) mRNA expression in nine regions of human brain tissues obtained from 23 subjects who died with no evidence of neurological or neurodegenerative disease. The following brain regions were examined: cingulate cortex, cerebellum, nucleus accumbens, caudate nucleus, putamen, pons, hippocampus, locus coeruleus, and basal ganglia. The SYBR green-based real-time PCR was more sensitive than TaqMan probe-based real-time PCR in amplifying both full-length and truncated NK1R mRNA. The real-time RT-PCR assay had excellent specificity and sensitivity, with a dynamic range of detection between 100 and 1,000,000 copies of the NK1R cDNA per reaction. The truncated NK1R mRNA levels were more abundant than those of the full-length NK1R in most of the regions examined and there was no significant difference in the truncated NK1R mRNA levels among the nine regions studied. There was, however, a significant difference in the expression of full-length NK1R mRNA levels among the nine regions (P=0.0024), and the putamen region expressed the highest full-length NK1R mRNA. Further studies are needed in order to examine the differences between full-length and truncated NK1R in signal transduction and functional consequences in order to delineate the significance of the co-presence of the two forms of NK1R in the human brain.

  3. Detection of Full-Length and Truncated Neurokinin-1 Receptor mRNA Expression in Human Brain Regions

    PubMed Central

    Lai, Jian-Ping; Cnaan, Avital; Zhao, Huaqing; Douglas, Steven D.

    2008-01-01

    We have applied a newly developed SYBR green based real-time RT-PCR assay for quantification of full-length and truncated neurokinin-1 receptor (NK1R) mRNA expression in 9 regions of human brain tissues obtained from 23 subjects who died with no evidence of neurological or neurodegenerative disease. The following brain regions were examined: cingulate cortex, cerebellum, nucleus accumbens, caudate nucleus, putamen, pons, hippocampus, locus coeruleus, and basal ganglia. The SYBR green based-real-time PCR was more sensitive than TaqMan probe based real-time PCR in amplifying both full-length and truncated NK1R mRNA. The real-time RT-PCR assay had excellent specificity and sensitivity, with a dynamic range of detection between 100 and 1000,000 copies of the NK1R cDNA per reaction. The truncated NK1R mRNA levels were more abundant than those of the full-length NK1R in most of the regions examined and there was no significant difference in the truncated NK1R mRNA levels among the nine regions studied. There was, however, a significant difference in the expression of full-length NK1R mRNA levels among the nine regions (P=0.0024), and the putamen region expressed the highest full-length NK1R mRNA. Further studies are needed in order to examine the differences between full-length and truncated NK1R in signal transduction and functional consequences in order to delineate the significance of the copresence of the two forms of NK1R in the human brain. PMID:18035424

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

    PubMed

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

    2006-09-01

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

  5. 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

  6. Relationship Between the DPD and TS mRNA Expression and the Response to S-1-Based Chemotherapy and Prognosis in Patients with Advanced Gastric Cancer.

    PubMed

    Shen, Xiao-Ming; Zhou, Chong; Lian, Lian; Li, Li-Qun; Li, Wei; Tao, Min

    2015-04-01

    The aim was to determine changes in dihydropyrimidine dehydrogenase (DPD) and thymidylate synthase (TS) mRNAs in the blood of advanced gastric cancer (AGC) patients to see whether these enzymes affected the patients' response to S-1-based chemotherapy and prognosis. For this purpose, pretreatment DPD/TS mRNA expressions were determined in 40 AGC patients using RT-PCR. The patients were then administered with S-1-based regimen (S-1 + cisplatin) and toxicities were recorded. The relationship between the DPD/TS mRNA expressions and the chemotherapy response, drug resistance, and prognosis was analyzed. The data show that DPD mRNA expression correlated significantly with Lauren type while TS mRNA expression correlated with distant metastasis. Patients with higher DPD and/or TS mRNA expression(s) showed poor response, while those with low DPD mRNA expression showed better response to the chemotherapy. Pooled analysis showed that the patients with low DPD/TS mRNA expressions had better therapeutic response. The incidence of bone marrow suppression, diarrhea, and oral mucositis was high in patients with low DPD mRNA expression. Median overall survival (OS) in 40 patients was 13.5 months. It was 17 months for low and 10 months for high DPD (P = 0.044) and TS mRNA expression (P = 0.047). Pooled analysis showed that the patients with both low DPD/TS mRNA expressions had longer OS (P = 0.001). In conclusion, the detection of DPD and/or TS mRNA expression can be used to predict the response to S-1-based chemotherapy, drug resistance, and prognosis in AGC patients as well as to help guide the individualized treatment of gastric cancer.

  7. 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

  8. Expression and stability of c-sis mRNA in human glioblastoma cells

    SciTech Connect

    Press, R.D.; Samols, D.; Goldthwait, D.A.

    1988-07-26

    The production of platelet-derived growth factor like (PDGF-like) material by glioblastomas may be involved in the conversion of normal cells to tumor cells. In an investigation of this problem, the authors have examined some of the properties of the platelet-derived growth factor B-chain mRNA (c-sis mRNA) by a sensitive and quantitative RNA-RNA solution hybridization method. In 5 out of 8 human glioblastoma cell lines, c-sis mRNA was present, and in the line with the highest level, there were approximately 4-10 molecules per cell. The half-lives of the c-sis mRNA in two glioblastoma cell lines were 2.6 and 3.4 h, while in human umbilical vein endothelial (HUVE) and bladder carcinoma (T24) cells they were 1.6 and 2.5 h, respectively. Inhibiting protein synthesis produced no significant alteration of the c-sis mRNA half-lives in the glioblastoma or HUVE cells. The A-U-rich sequence at the 3' end of the c-sis mRNA therefore does not appear to affect the mRNA stability in the presence of cycloheximide as it does in other transcripts. The similarity of the c-sis mRNA half-lives in normal and tumor cells suggests that regulation of stability of c-sis mRNA is not a major factor in tumorigenesis in the glioblastoma cell lines examined.

  9. Expression of fragile X mental retardation protein and Fmr1 mRNA during folliculogenesis in the rat.

    PubMed

    Ferder, Ianina; Parborell, Fernanda; Sundblad, Victoria; Chiauzzi, Violeta; Gómez, Karina; Charreau, Eduardo H; Tesone, Marta; Dain, Liliana

    2013-04-01

    Fragile X mental retardation protein (FMRP) belongs to a small family of RNA-binding proteins. Its absence or inactivity is responsible for fragile X syndrome, the most common cause of inherited mental retardation. Despite its ubiquitous expression, FMRP function and expression remain almost understudied in non-neuronal tissues, though previous studies on germline development during oogenesis may suggest a special function of this protein also in ovarian tissue. In addition, the well-documented association of FMR1 premutation state with fragile X-related premature ovarian insufficiency adds interest to the role of FMRP in ovarian physiology. The aim of the present work was to investigate the expression of Fmr1 mRNA and its protein, FMRP, at different stages of rat follicular development. By immunohistochemical studies we demonstrated FMRP expression in granulosa, theca and germ cells in all stages of follicular development. In addition, changes in Fmr1 expression, both at the protein and mRNA levels, were observed. FMRP levels increased upon follicular development while preantral and early antral follicles presented similar levels of Fmr1 transcripts with decreased expression in preovulatory follicles. These observations suggest that Fmr1 expression in the ovary is regulated at different and perhaps independent levels. In addition, our results show expression of at least four different isoforms of FMRP during all stages of follicular growth with expression patterns that differ from those observed in brain and testis. Our study shows a regulated expression of Fmr1, both at mRNA and protein levels, during rat follicular development.

  10. Amphotericin B severely affects expression and activity of the endothelial constitutive nitric oxide synthase involving altered mRNA stability

    PubMed Central

    Suschek, Christoph Viktor; Bonmann, Eckhard; Kleinert, Hartmut; Wenzel, Michael; Mahotka, Csaba; Kolb, Hubert; Förstermann, Ulrich; Gerharz, Claus-Dieter; Kolb-Bachofen, Victoria

    2000-01-01

    The therapeutic use of the antifungal drug amphotericin B (AmB) is limited due to severe side effects like glomerular vasoconstriction and risk of renal failure during AmB administration. As nitric oxide (NO) has substantial functions in renal autoregulation, we have determined the effects of AmB on endothelial constitutive NO synthase (ecNOS) expression and activity in human and rat endothelial cell cultures.AmB used at concentrations of 0.6 to 1.25 μg ml−1 led to increases in ecNOS mRNA and protein expression as well as NO production. This was the result of an increased ecNOS mRNA half-life. In contrast, incubation of cells with higher albeit subtoxic concentrations of AmB (2.5–5.0 μg ml−1) resulted in a decrease or respectively in completely abolished ecNOS mRNA and protein expression with a strongly reduced or inhibited ecNOS activity, due to a decrease of ecNOS mRNA half-life. None of the AmB concentrations affected promoter activity as found with a reporter gene construct stably transfected into ECV304 cells.Thus, our experiments show a concentration-dependent biphasic effect of AmB on expression and activity of ecNOS, an effect best explained by AmB influencing ecNOS mRNA stability. In view of the known renal accumulation of this drug the results reported here could help to elucidate its renal toxicity. PMID:11015297

  11. Effect of a transpositional muscle flap on VEGF mRNA expression in a canine fracture model.

    PubMed

    Khodaparast, Omeed; Coberly, Dana M; Mathey, Jonathon; Rohrich, Rod J; Levin, L Scott; Brown, Spencer A

    2003-07-01

    The effect of sepsis on neovascularization in fractures that follows open fractures is important to the understanding of bone and soft-tissue healing. An animal model was designed that mimics the open fracture and the clinical repair of the human, high-energy open fracture. Vascular endothelial growth factor (VEGF) mRNA levels in canine bone samples were determined in samples from days 0 and 7. Canine right tibiae were fractured with a penetrating, captive-bolt device and then repaired in a standard clinical fashion using an interlocking intramedullary nail. Animals were subject to one of the following experimental protocols: tibial fracture (group I, n = 3); tibial fracture and Staphylococcus aureus inoculation at the fracture site (group II, n = 3); and tibial fracture and S. aureus inoculation with a rotational gastrocnemius muscle flap (group III, n = 3). Bone samples were harvested on days 0 and 7 and prepared for reverse transcriptase polymerase chain reaction assay. Primers for VEGF were commercially prepared and assay products were sequenced. The assay products were associated with Genebank VEGF mRNA sequences. VEGF mRNA levels increased significantly in the fracture-alone group from day 0 to day 7 (n = 3, p < 0.05). In the fracture and S. aureus group (group I), VEGF mRNA expression decreased 79 percent (p < 0.05). In animals with fractures inoculated with S. aureus and a transpositional muscle flap (group III), VEGF mRNA expression was increased 38 percent from day 0 to day 7 (p < 0.05) and was similar to the increase observed in the fracture-alone group. These results demonstrate that S. aureus decreased the normal increase of VEGF mRNA expression during bone wound healing. Use of the transpositional muscle flap in the presence of S. aureus increased VEGF mRNA expression over time to the expression pattern observed in the fracture-alone group. This experimental model demonstrates that specific biological signals and cellular pathways are influenced by

  12. Expression and localization of the cystic fibrosis transmembrane conductance regulator mRNA and its protein in rat brain.

    PubMed

    Mulberg, A E; Resta, L P; Wiedner, E B; Altschuler, S M; Jefferson, D M; Broussard, D L

    1995-07-01

    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.

  13. 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

  14. Local anesthetics inhibit tissue factor expression in activated monocytes via inhibition of tissue factor mRNA synthesis.

    PubMed

    Kim, Ji-Eun; Kim, Ki Jun; Ahn, Wonsik; Han, Kyou-Sup; Kim, Hyun Kyung

    2011-01-01

    Local anesthetics have been reported to have anticoagulant properties, but the mechanisms responsible for this action are poorly understood. Here, we evaluated the in vitro effects of 3 local anesthetics--lidocaine, ropivacaine, and bupivacaine--on the tissue factor expression by monocytes. Monocytes from peripheral blood were stimulated with lipopolysaccharide (LPS) in the presence or absence of local anesthetics. All 3 local anesthetics inhibited the expression of tissue factor antigen and tissue factor activity in LPS-stimulated monocytes in a dose- and time-dependent manner and reduced tissue factor messenger RNA (mRNA) expression in endothelial cells and a monocytic cell line. None of the 3 drugs induced apoptosis or affected the viability of monocytes. Our findings that local anesthetics inhibited the tissue factor induction in activated monocytes by inhibiting tissue factor mRNA level may demonstrate the feasibility of using local anesthetics in hypercoagulable and inflammatory conditions.

  15. An mRNA expression analysis of stimulation and blockade of 5-HT7 receptors during memory consolidation.

    PubMed

    Pérez-García, Georgina; Gonzalez-Espinosa, Claudia; Meneses, Alfredo

    2006-04-25

    Despite the compelling support for 5-hydroxytryptamine (5-HT) receptors participation in learning and memory in mammal species, the molecular basis had been largely absent from any discussion of its mechanistic underpinnings. Here, we report that reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed that there was a higher level of expression of the investigated 5-HT receptor mRNAs in autoshaping-trained relative to untrained groups. Actually, pharmacological naïve untrained and autoshaping-trained rats showed significant differences, the latter groups expressing, in decreasing order, 5-HT1A < 5-HT6 < 5-HT4 < or = 5-HT7 receptors mRNA in prefrontal cortex and hippocampus. In order to determine more precisely mRNA expression and memory consolidation, we combined selective 5-HT7 receptors stimulation or blockade in the same animals, and brain areas individually analyzed. 5-HT7 receptors were strongly expressed in all the three brain areas of vehicle-trained rats relative to untrained group. The potential selective 5-HT7 receptor agonist AS 19 enhanced memory consolidation, attenuated mRNA receptors expression, and the facilitatory memory effect was reversed by SB-269970. Finally, pharmacological stimulation of 5-HT7 receptors reversed scopolamine- or dizocilpine-induced amnesia and receptor down-regulation.

  16. Dopamine receptor D3 mRNA expression in human lymphocytes is negatively correlated with the personality trait of persistence.

    PubMed

    Czermak, Christoph; Lehofer, Michael; Renger, Helmut; Wagner, Elke M; Lemonis, Leonidas; Rohrhofer, Alfred; Schauenstein, Konrad; Liebmann, Peter M

    2004-05-01

    It has been proposed that neurotransmitter receptor expression in peripheral immune cells reflects expression of these receptors in the brain. To test this "peripheral marker hypothesis", we compared mRNA expression of the dopamine receptors D3 (DRD3) and D4 (DRD4) in peripheral blood lymphocytes (PBL) to personality traits assessed with the Temperament and Character Inventory (TCI) in 50 healthy and unmedicated Caucasian individuals. A shared variance of at least 17% (p=0.016) between DRD3 mRNA expression in PBL and the personality trait of persistence was found. As personality traits have been generally assumed polygenic with a single gene accounting for rarely more than 1-2% of observed variance in a trait, this result lends further support to the peripheral marker hypothesis for DRD3 mRNA expression in PBL. It may also suggest a significant role for the DRD3 in the neurobiology of persistence and point to an interesting link between personality and functioning of the immune system.

  17. BDNF and trkB mRNA expression in the rat hippocampus following entorhinal cortex lesions.

    PubMed

    Lapchak, P A; Araujo, D M; Hefti, F

    1993-02-01

    Quantitative in situ hybridization was used to determine whether the prevalence or topographical distribution of brain-derived neurotrophic factor (BDNF) or tyrosine receptor kinase (trk) B mRNA is altered in the hippocampal formation following lesions of excitatory afferents from the entorhinal cortex which provides an external source of innervation for the hippocampal formation. BDNF mRNA levels were not altered in the hippocampal formation up to 10 days following entorhinal cortex lesions (ECLs). The levels of mRNA coding for all known forms of trkB receptors also remained unchanged. The prevalence of the synaptic plasticity marker SNAP-25 mRNA was increased in the CA2 and CA3 pyramidal cell layers and the dentate gyrus by 6 days following ECLs and remained elevated at 10 days following ECLs. Our findings indicate that hippocampal neuron sprouting which occurs in response to ECLs is not the result of changes in the expression of the BDNF or trkB mRNA.

  18. microRNA expression in autonomous thyroid adenomas: Correlation with mRNA regulation.

    PubMed

    Floor, Sébastien L; Trésallet, Christophe; Hébrant, Aline; Desbuleux, Alice; Libert, Frédérick; Hoang, Catherine; Capello, Matteo; Andry, Guy; van Staveren, Wilma C G; Maenhaut, Carine

    2015-08-15

    The objective of the study was to identify the deregulated miRNA in autonomous adenoma and to correlate the data with mRNA regulation. Seven autonomous adenoma with adjacent healthy thyroid tissues were investigated. Twelve miRNAs were downregulated and one was upregulated in the tumors. Combining bioinformatic mRNA target prediction and microarray data on mRNA regulations allowed to identify mRNA targets of our deregulated miRNAs. A large enrichment in mRNA encoding proteins involved in extracellular matrix organization and different phosphodiesterases were identified among these putative targets. The direct interaction between miR-101-3p and miR-144-3p and PDE4D mRNA was experimentally validated. The global miRNA profiles were not greatly modified, confirming the definition of these tumors as minimal deviation tumors. These results support a role for miRNA in the regulation of extracellular matrix proteins and tissue remodeling occurring during tumor development, and in the important negative feedback of the cAMP pathway, which limits the consequences of its constitutive activation in these tumors.

  19. 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.

  20. Pyruvate dehydrogenase complex: mRNA and protein expression patterns of E1α subunit genes in human spermatogenesis.

    PubMed

    Pinheiro, Ana; Silva, Maria João; Graça, Inês; Silva, Joaquina; Sá, Rosália; Sousa, Mário; Barros, Alberto; Tavares de Almeida, Isabel; Rivera, Isabel

    2012-09-10

    During spermatogenesis, germ cells undergo a complex process of cell differentiation and morphological restructuring, which depends on the coordinated expression of different genes. Some vital examples are those involved in cell energy metabolism, namely the genes encoding the E1α subunit of pyruvate dehydrogenase complex: the somatic PDHA1 (X-linked) and the testis-specific PDHA2 (autosomal). There are no data related to the study at the RNA and protein levels of PDHA genes during human spermatogenesis. The present study aimed to describe the mRNA and protein expression patterns of the human PDHA genes during spermatogenesis. Expression profiles of the PDHA1 and PDHA2 genes were characterized using different human tissues and cells. Diploid and haploid germ cells fractions were obtained from testis tissues. The mRNA profiles were analyzed by quantitative RT-PCR, whereas the protein profiles were evaluated by immunohistochemistry, western blotting and two-dimensional electrophoresis. Expression of the PDHA1 gene was found in all somatic cells, whereas expression of PDHA2 gene was restricted to germ cells. The switch from X-linked to autosomic gene expression occurred in spermatocytes. Data suggest the activation of PDHA2 gene expression is most probably a mechanism to ensure the continued expression of the protein, thus allowing germ cell viability and functionality.

  1. Electrical activation and c-fos mRNA expression in rat neurosecretory neurones after systemic administration of cholecystokinin.

    PubMed Central

    Hamamura, M; Leng, G; Emson, P C; Kiyama, H

    1991-01-01

    1. The expression of c-fos mRNA in the rat hypothalamus was examined by in situ hybridization following systemic administration of cholecystokinin (CCK), a procedure known to activate magnocellular oxytocin neurons but not magnocellular vasopressin neurones. 2. Conscious male rats were given a single I.P. injection of 50 micrograms/kg CCK, c-fos mRNA signal was apparent in the supraoptic and paraventricular nuclei in rats killed 10 min after injection but not in uninjected or saline-(vehicle) injected rats. The density of c-fos mRNA at both sites was further elevated in rats killed 30 min or 60 min following injection, and was absent in rats killed 4 h after injection. 3. In the paraventricular nucleus the most dense expression of c-fos mRNA following CCK administration was in the medial, mainly parvocellular portion of the nucleus, in an area corresponding to the distribution of corticotrophin-releasing factor mRNA determined by in situ hybridization in adjacent sections. 4. The I.P. injection of CCK increased plasma oxytocin concentrations, measured by specific radioimmunoassay from 13 +/- 5 pg/ml in control rats to 107 +/- 9 pg/ml in the rats killed 10 min after injection, a similar response to that observed previously in urethane-anaesthetized rats. 5. In each of six urethane-anaesthetized rats, recordings were made from single neurones in the supraoptic nucleus, identified antidronomically as projecting to the posterior pituitary and identified electrophysiologically as putative oxytocin neurones. Following I.P. injection of 50 micrograms/kg CCK, the neurones increased their firing rate by a mean of 1.3 +/- 0.2 spikes/s averaged over the 10 min following injection. 6. From the appearance of c-fos mRNA in supraoptic neurones following CCK administration we conclude that this message is expressed in magnocellular oxytocin neurones, since vasopressin neuronal activity and vasopressin release is known to be unaffected by this stimulus, and since the supraoptic

  2. Luteotropic and luteolytic factors regulate mRNA and protein expression of progesterone receptor isoforms A and B in the bovine endometrium.

    PubMed

    Rekawiecki, Robert; Kowalik, Magdalena Karolina; Kotwica, Jan

    2014-12-17

    The aim of the present study was to examine the effects of luteotropic and luteolytic factors on the mRNA and protein levels of progesterone receptor isoforms A (PGRA) and B (PGRB) in the bovine endometrium. Endometrial slices from Days 6-10 and 17-20 of the oestrous cycle were treated with LH (100ngmL-1), oestradiol (E2; 1×10-8M), prostaglandin (PG) E2 (1×10-6M) and PGF2? (1×10-6M) and the nitric oxide donor NONOate (1×10-4M); these treatments lasted for 6h for mRNA expression analysis and 24h for protein expression analysis. On Days 6-10 of the oestrous cycle PGRAB (PGRAB; the entire PGRA mRNA sequence is common to the PGRB mRNA sequence) mRNA expression in endometrial slices was enhanced by E2 treatment (PPGRB mRNA expression was increased by LH (PPPPGRAB mRNA expression increased after E2 (P2 (PPGRB mRNA expression was increased by PGE2 (P2? (PPPPPP2? (P2 (P2? (P<0.001). These data suggest that luteotropic and luteolytic factors affect PGRA and PGRB mRNA and protein levels, and this may regulate the effects of progesterone on endometrial cells.

  3. Unique expression features of cancer-type organic anion transporting polypeptide 1B3 mRNA expression in human colon and lung cancers

    PubMed Central

    2014-01-01

    Background We have previously identified the cancer-type organic anion transporting polypeptide 1B3 (Ct-OATP1B3) mRNA in several human colon and lung cancer tissues. Ct-OATP1B3 is a variant of the liver-type OATP1B3 (Lt-OATP1B3) mRNA, which is a hepatocyte plasma membrane transporter with broad substrate specificity. However, in cancer tissues, both the detailed characteristics of Ct-OATP1B3 mRNA expression and its biological functions remain unclear. With this point in mind, we sought to characterize Ct-OATP1B3 mRNA expression in colon and lung cancer tissues. In addition, we attempted to obtain functional implication of Ct-OATP1B3 in cancer cells. Methods Matched pairs of cancer and normal tissues were collected from 39 colon cancer and 28 lung cancer patients. The OATP1B3 mRNA expression levels in each of these tissues were separately determined by quantitative real-time polymerase chain reaction. Mann–Whitney U test and Fisher’s exact test were used in statistical analysis. The Ct-OATP1B3 functional expression in colon cancer cells was then examined by Western blotting and transport analyses. Results Ct-OATP1B3 mRNA, but not Lt-OATP1B3 mRNA, was abundantly expressed in colon cancer tissues at a higher detection frequency (87.2%) than that of the adjacent normal tissues (2.6%). Furthermore, it was found that Ct-OATP1B3 mRNA expression was often detected in early colon cancer stages (88.9%, n = 18), and that its expression was associated with well-differentiated colon cancer statuses. On the other hand, Ct-OATP1B3 mRNA also showed a predominant and cancer-associated expression profile in lung tissues, although at frequencies and expression levels that were lower than those obtained from colon cancer. As for attempts to clarify the Ct-OATP1B3 functions, neither protein expression nor transport activity could be observed in any of the cell lines examined. Conclusions Based on the unique characteristics of the Ct-OATP1B3 mRNA expression profile identified in

  4. Appetite regulating peptides in red-bellied piranha, Pygocentrus nattereri: cloning, tissue distribution and effect of fasting on mRNA expression levels.

    PubMed

    Volkoff, Hélène

    2014-06-01

    cDNAs encoding the appetite regulating peptides apelin, cocaine and amphetamine regulated transcript (CART), cholecystokinin (CCK), peptide YY (PYY) and orexin were isolated in red-bellied piranha and their mRNA tissue and brain distributions examined. When compared to other fish, the sequences obtained for all peptides were most similar to that of other Characiforme fish, as well as to Cypriniformes. All peptides were widely expressed within the brain and in several peripheral tissues, including gastrointestinal tract. In order to assess the role of these peptides in the regulation of feeding of red-bellied piranha, we compared the brain mRNA expression levels of these peptides, as well as the gut mRNA expression of CCK and PYY, between fed and 7-day fasted fish. Within the brain, fasting induced a significant increase in both apelin and orexin mRNA expressions and a decrease in CART mRNA expression, but there where were no significant differences for either PYY or CCK brain mRNA expressions between fed and fasted fish. Within the intestine, PYY mRNA expression was lower in fasted fish compared to fed fish but there was no significant difference for CCK intestine mRNA expression between fed and fasted fish. Our results suggest that these peptides, perhaps with the exception of CCK, play a major role in the regulation of feeding of red-bellied piranha.

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

    PubMed

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

    2016-12-01

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

  6. Effects of AFP gene silencing on Survivin mRNA expression inhibition in HepG2 cells.

    PubMed

    Fang, Z L; Fang, N; Han, X N; Huang, G; Fu, X J; Xie, G S; Wang, N R; Xiong, J P

    2015-04-10

    We investigated the effects of alpha-fetoprotein (AFP) gene silencing on Survivin expression in HepG2 cells. Small interfering RNA technology was used to downregulate AFP expression in HepG2 cells. An enzyme-linked immunosorbent assay was used to measure AFP concentration in the supernatant before and after transfection. An MTT assay was used to detect cell proliferation activity before and after transfection. We performed flow cytometric analysis to detect the cell apoptosis rate, and reverse transcription-polymerase chain reaction to detect Survivin mRNA levels before and after transfection. Forty-eight hours after transfection, AFP concentration in the supernatant of the experimental group significantly decreased, hepatocellular carcinoma cell growth was inhibited by 43.1%, and the apoptosis rate increased by 24.3%. Survivin mRNA expression was reduced by 78.0% in HepG2 cells. These indicators in the control group and in the blank group did not change significantly. Silencing of AFP expression in HepG2 cells can effectively inhibit the growth of hepatoma cells and promote apoptosis, which may be useful for reducing intracellular Survivin mRNA levels.

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

    PubMed

    Sullivan, Bridget E; Carroll, Chad C; Jemiolo, Bozena; Trappe, Scott W; Magnusson, S Peter; Døssing, Simon; Kjaer, Michael; Trappe, Todd A

    2009-02-01

    Tendon is mainly composed of collagen and an aqueous matrix of proteoglycans that are regulated by enzymes called matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). Although it is known that resistance exercise (RE) and sex influence tendon metabolism and mechanical properties, it is uncertain what structural and regulatory components contribute to these responses. We measured the mRNA expression of tendon's main fibrillar collagens (type I and type III) and the main proteoglycans (decorin, biglycan, fibromodulin, and versican) and the regulatory enzymes MMP-2, MMP-9, MMP-3, and TIMP-1 at rest and after RE. Patellar tendon biopsy samples were taken from six individuals (3 men and 3 women) before and 4 h after a bout of RE and from a another six individuals (3 men and 3 women) before and 24 h after RE. Resting mRNA expression was used for sex comparisons (6 men and 6 women). Collagen type I, collagen type III, and MMP-2 were downregulated (P < 0.05) 4 h after RE but were unchanged (P > 0.05) 24 h after RE. All other genes remained unchanged (P > 0.05) after RE. Women had higher resting mRNA expression (P < 0.05) of collagen type III and a trend (P = 0.08) toward lower resting expression of MMP-3 than men. All other genes were not influenced (P > 0.05) by sex. Acute RE appears to stimulate a change in collagen type I, collagen type III, and MMP-2 gene regulation in the human patellar tendon. Sex influences the structural and regulatory mRNA expression of tendon.

  8. LDOC1 mRNA is differentially expressed in chronic lymphocytic leukemia and predicts overall survival in untreated patients

    PubMed Central

    Duzkale, Hatice; Schweighofer, Carmen D.; Coombes, Kevin R.; Barron, Lynn L.; Ferrajoli, Alessandra; O'Brien, Susan; Wierda, William G.; Pfeifer, John; Majewski, Tadeusz; Czerniak, Bogdan A.; Jorgensen, Jeffrey L.; Medeiros, L. Jeffrey; Freireich, Emil J; Keating, Michael J.

    2011-01-01

    We previously identified LDOC1 as one of the most significantly differentially expressed genes in untreated chronic lymphocytic leukemia (CLL) patients with respect to the somatic mutation status of the immunoglobulin heavy-chain variable region genes. However, little is known about the normal function of LDOC1, its contribution to the pathophysiology of CLL, or its prognostic significance. In this study, we have investigated LDOC1 mRNA expression in a large cohort of untreated CLL patients, as well as in normal peripheral blood B-cell (NBC) subsets and primary B-cell lymphoma samples. We have confirmed that LDOC1 is dramatically down-regulated in mutated CLL cases compared with unmutated cases, and have identified a new splice variant, LDOC1S. We show that LDOC1 is expressed in NBC subsets (naive > memory), suggesting that it may play a role in normal B-cell development. It is also expressed in primary B-cell lymphoma samples, in which its expression is associated with somatic mutation status. In CLL, we show that high levels of LDOC1 correlate with biomarkers of poor prognosis, including cytogenetic markers, unmutated somatic mutation status, and ZAP70 expression. Finally, we demonstrate that LDOC1 mRNA expression is an excellent predictor of overall survival in untreated CLL patients. PMID:21310924

  9. Differential effects of binge methamphetamine injections on the mRNA expression of histone deacetylases (HDACs) in the rat striatum

    PubMed Central

    Omonijo, Oluwaseyi; Wongprayoon, Pawaris; Ladenheim, Bruce; McCoy, Michael T.; Govitrapong, Piyarat; Jayanthi, Subramaniam; Cadet, Jean Lud

    2014-01-01

    Methamphetamine use disorder is characterized by recurrent binge episodes. Humans addicted to methamphetamine experience various degrees of cognitive deficits and show evidence of neurodegenerative processes in the brain. Binge injections of METH to rodents also cause significant toxic changes in the brain. In addition, this pattern of METH injections can alter gene expression in the dorsal striatum. Gene expression is regulated, in part, by histone deacetylation. We thus tested the possibility that METH toxic doses might cause changes in the mRNA levels of histone deacetylases (HDACs). We found that METH did produce significant decreases in the mRNA expression of HDAC8, which is a class I HDAC. METH also decreased expression of HDAC6, HDAC9, and HDAC10 that are class II HDACs. The expression of the class IV HDAC, HDAC11, was also suppressed by METH. The expression of Sirt2, Sirt5, and Sirt6 that are members of class III HDACs was also downregulated by METH injections. Our findings implicate changes in HDAC expression may be an early indicator of impending METH-induced neurotoxicity in the striatum. This idea is consistent with the accumulated evidence that some HDACs are involved in neurodegenerative processes in the brain. PMID:25452209

  10. Electroacupuncture-regulated neurotrophic factor mRNA expression in the substantia nigra of Parkinson's disease rats.

    PubMed

    Wang, Shuju; Fang, Jianqiao; Ma, Jun; Wang, Yanchun; Liang, Shaorong; Zhou, Dan; Sun, Guojie

    2013-02-25

    Acupuncture for the treatment of Parkinson's disease has a precise clinical outcome. This study investigated the effect of electroacupuncture at Fengfu (GV16) and Taichong (LR3) acupoints in rat models of Parkinson's disease induced by subcutaneous injection of rotenone into rat neck and back. Reverse transcription-PCR demonstrated that brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor mRNA expression was significantly increased in the substantia nigra of rat models of Parkinson's disease, and that abnormal behavior of rats was significantly improved following electroacupuncture treatment. These results indicated that electroacupuncture treatment upregulated brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor mRNA expression in the substantia nigra of rat models of Parkinson's disease. Thus, electroacupuncture may be useful in the treatment of Parkinson's disease.

  11. Calcium CaV1 channel subtype mRNA expression in Parkinson's disease examined by in situ hybridization.

    PubMed

    Hurley, Michael J; Gentleman, Steve M; Dexter, David T

    2015-03-01

    The factors which make some neurons vulnerable to neurodegeneration in Parkinson's disease while others remain resistant are not fully understood. Studies in animal models of Parkinson's disease suggest that preferential use of CaV1.3 subtypes by neurons may contribute to the neurodegenerative process by increasing mitochondrial oxidant stress. This study quantified the level of mRNA for the CaV1 subtypes found in the brain by in situ hybridization using CaV1 subtype-specific [(35)S]-radiolabelled oligonucleotide probes. In normal brain, the greatest amount of messenger RNA (mRNA) for each CaV1 subtype was found in the midbrain (substantia nigra), with a moderate level in the pons (locus coeruleus) and lower quantities in cerebral cortex (cingulate and primary motor). In Parkinson's disease, the level of CaV1 subtype mRNA was maintained in the midbrain and pons, despite cell loss in these areas. In cingulate cortex, CaV1.2 and CaV1.3 mRNA increased in cases with late-stage Parkinson's disease. In primary motor cortex, the level of CaV1.2 mRNA increased in late-stage Parkinson's disease. The level of CaV1.3 mRNA increased in primary motor cortex of cases with early-stage Parkinson's disease and normalized to near the control level in cases from late-stage Parkinson's disease. The finding of elevated CaV1 subtype expression in cortical brain regions supports the view that disturbed calcium homeostasis is a feature of Parkinson's disease throughout brain and not only a compensatory consequence to the neurodegenerative process in areas of cell loss.

  12. 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.

  13. 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

  14. FLT3-ITD and MLL-PTD influence the expression of MDR-1, MRP-1, and BCRP mRNA but not LRP mRNA assessed with RQ-PCR method in adult acute myeloid leukemia.

    PubMed

    Nasilowska-Adamska, Barbara; Solarska, Iwona; Paluszewska, Monika; Malinowska, Iwona; Jedrzejczak, Wieslaw W; Warzocha, Krzysztof

    2014-04-01

    Fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) and mixed-lineage leukemia gene-partial tandem duplication (MLL-PTD) are aberrations associated with leukemia which indicate unsatisfactory prognosis. Downstream regulatory targets of FLT3-ITD and MLL-PTD are not well defined. We have analyzed the expression of MDR-1, multidrug resistant protein-1 (MRP-1), breast cancer resistance protein (BCRP), and lung resistance protein (LRP) messenger RNA (mRNA) in relation to the mutational status of FLT3-ITD and MLL-PTD in 185 acute myeloid leukemia (AML) adult patients. The real-time quantitative polymerase chain reaction method was performed to assess the expression of the MDR-1, MRP-1, BCRP, and LRP mRNA, and the results were presented as coefficients calculated using an intermediate method according to Pfaffl's rule. Significantly higher expressions of MDR-1 mRNA were found in patients who did not harbor FLT3-ITD (0.20 vs. 0.05; p = 0.0001) and MRP-1 mRNA in patients with this mutation (0.96 vs. 0.70; p = 0.002) and of BCRP mRNA in patients with MLL-PTD (0.61 vs. 0.38; p = 0.03). In univariate analysis, the high expression of MDR-1 mRNA (≥0.1317) negatively influenced the outcome of induction therapy (p = 0.05), whereas the high expression of BCRP mRNA (≥1.1487) was associated with a high relapse rate (RR) (p = 0.013). We found that the high expression of MDR-1 (≥0.1317), MRP-1 (≥0.8409), and BCRP mRNA (≥1.1487) significantly influenced disease-free survival (DFS; p = 0.059, 0.032, and 0.009, respectively) and overall survival (0.048, 0.014, and 0.059, respectively). Moreover, a high expression of BCRP mRNA (≥1.1487) proved to be an independent prognostic factor for RR (p = 0.01) and DFS (p = 0.002) in multivariate analysis. The significant correlation between the expression of MDR-1, MRP-1, and BCRP mRNA and FLT3-ITD or MLL-PTD in AML patients requires further investigation.

  15. Expression of trkB mRNA is altered in rat hippocampus after experimental brain trauma.

    PubMed

    Hicks, R R; Zhang, L; Dhillon, H S; Prasad, M R; Seroogy, K B

    1998-08-31

    Recent investigations have shown that expression of mRNAs for the neurotrophins brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) is differentially altered in the hippocampus following traumatic brain injury. In the present study, modulation of neurotrophin receptor expression was examined in the hippocampus in a rat model of traumatic brain injury using in situ hybridization. Messenger RNA for trkB, the high-affinity receptor for BDNF and neurotrophin-4 (NT-4), was increased between 3 and 6 h bilaterally in the dentate gyrus following a lateral fluid-percussion brain injury of moderate severity (2.0-2.1 atm). No time-dependent alterations were observed for trkB mRNA in hippocampal subfields CA1 and CA3. Levels of mRNA for trkC, the high-affinity receptor for NT-3, did not change in any region of the hippocampus. These data demonstrate that lateral fluid-percussion injury modulates expression of trkB mRNA in the hippocampus and support a role for BDNF/trkB signalling mechanisms in secondary events associated with traumatic brain injury.

  16. Human skeletal muscle creatine transporter mRNA and protein expression in healthy, young males and females.

    PubMed

    Murphy, Robyn M; Tunstall, Rebecca J; Mehan, Kate A; Cameron-Smith, David; McKenna, Michael J; Spriet, Lawrence L; Hargreaves, Mark; Snow, Rodney J

    2003-02-01

    The present study investigated whether there were any differences between males and females in respect to creatine transporter (CreaT) gene expression and/or total creatine (TCr) content in human vastus lateralis muscle. Skeletal muscle obtained from young healthy male (n = 13, age: 23.2 +/- 5.0 years) and female subjects (n = 12, age: 21.7 +/- 4.3 years) was analyzed for CreaT mRNA, CreaT protein and TCr content. Total CreaT protein content in the muscle was similar (p > 0.05) between the sexes. Two bands (approximately 55 and 73 kDa) of the CreaT protein were detected in all muscle samples. Both the 55 and the 73 kDa bands were present in similar (p > 0.05) amounts in males compared with females. The 73 kDa band was in greater abundance (p < 0.05) than the 55 kDa band, irrespective of gender. In addition, CreaT mRNA expression relative to beta-actin mRNA and the TCr content (males: 117.8 +/- 2.2, females: 125.3 +/- 4.3 mmol.kg(-1) dry mass) were also unaffected (p > 0.05) by gender. These data demonstrate that gender does not influence skeletal muscle TCr content and CreaT gene expression in young human subjects.

  17. Differential expression of equine myosin heavy-chain mRNA and protein isoforms in a limb muscle.

    PubMed

    Eizema, Karin; van den Burg, Maarten; Kiri, Arpna; Dingboom, Elizabeth G; van Oudheusden, Hans; Goldspink, Geoffrey; Weijs, Wim A

    2003-09-01

    The horse is one of the few animals kept and bred for its athletic performance and is therefore an interesting model for human sports performance. The regulation of the development of equine locomotion in the first year of life, and the influence of early training on later performance, are largely unknown. The major structural protein in skeletal muscle, myosin heavy-chain (MyHC), is believed to be primarily transcriptionally controlled. To investigate the expression of the MyHC genes at the transcriptional level, we isolated cDNAs encoding the equine MyHC isoforms type 1 (slow), type 2a (fast oxidative), and type 2d/x (fast glycolytic). cDNAs encoding the 2b gene were not identified. The mRNA expression was compared to the protein expression on a fiber-to-fiber basis using in situ hybridization (non-radioactive) and immunohistochemistry. Marked differences were detected between the expression of MyHC transcripts and MyHC protein isoforms in adult equine gluteus medius muscle. Mismatches were primarily due to the presence of hybrid fibers expressing two fast (2ad) MyHC protein isoforms, but only one fast (mainly 2a) MyHC RNA isoform. This discrepancy was most likely not due to differential mRNA expression of myonuclei.

  18. PD-1 mRNA expression in peripheral blood cells and its modulation characteristics in cancer patients.

    PubMed

    Wang, Wei; Shen, Ge; Wu, Shikai; Song, Shiping; Ni, Yanli; Suo, Zhuoyao; Meng, Xiangying; Li, Dan; Zhou, Lin; Hao, Rimin; Zhao, Yaowei; Bai, Li; Hou, Lili; Liu, Bing; Liu, Guangxian

    2017-02-02

    Immune checkpoint inhibitors that block the PD-1/PD-L1 signaling pathway have been used to treat a wide variety of cancers. Although results have been promising, significant inter-individual and inter-tumor variability has been observed. It is believed that better clinical outcome could be achieved if the treatment was individually designed based on the functional status of the PD-1/PD-L1 signaling and the cellular immunity. In this study, we analyzed the mRNA expression of PD-1 and other immunomodulatory genes in peripheral blood from cancer patients, and immunomodulatory gene expression during radiotherapy and immunomodulation therapy with cytokines. Our results show that the PD-1 mRNA expression is significantly increased in peripheral blood in cancer patients. Anti-cancer treatments can significantly modulate the PD-1 expression, but this is largely dependent on the initial immune status. Moreover, the PD-1 expression on peripheral lymphocytes can be immunoactivation-derived. These results suggest that the regulation and expression pattern of PD-1/PD-L1 signal is complicated which will influence the effect of blockade of the PD-1/PD-L1 signaling pathway for cancer treatment. Through combined analysis of PD-1, CTLA-4, and other immune markers in peripheral blood, we may accurately evaluate the functional status of PD-1/PD-L1 signaling and cellular immunity, thereby providing clues for guiding anti-PD-1 or anti-PD-L1 treatment.

  19. 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.

  20. 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.

  1. Strain differences in cytochrome P450 mRNA and protein expression, and enzymatic activity among Sprague Dawley, Wistar, Brown Norway and Dark Agouti rats.

    PubMed

    Nishiyama, Yoshihiro; Nakayama, Shouta M M; Watanabe, Kensuke P; Kawai, Yusuke K; Ohno, Marumi; Ikenaka, Yoshinori; Ishizuka, Mayumi

    2016-05-03

    Rat cytochrome P450 (CYP) exhibits inter-strain differences, but their analysis has been scattered across studies under different conditions. To identify these strain differences in CYP more comprehensively, mRNA expression, protein expression and metabolic activity among Wistar (WI), Sprague Dawley (SD), Dark Agouti (DA) and Brown Norway (BN) rats were compared. The mRNA level and enzymatic activity of CYP1A1 were highest in SD rats. The rank order of Cyp3a2 mRNA expression mirrored its protein expression, i.e., DA>BN>SD>WI, and was similar to the CYP3A2-dependent warfarin metabolic activity, i.e., DA>SD>BN>WI. These results suggest that the strain differences in CYP3A2 enzymatic activity are caused by differences in mRNA expression. Cyp2b1 mRNA levels, which were higher in DA rats, did not correlate with its protein expression or enzymatic activity. This suggests that the strain differences in enzymatic activity are not related to Cyp2b1 mRNA expression. In conclusion, WI rats tended to have the lowest CYP1A1, 2B1 and 3A2 mRNA expression, protein expression and enzymatic activity among the strains. In addition, SD rats had the highest CYP1A1 mRNA expression and activity, while DA rats had higher CYP2B1 and CYP3A2 mRNA and protein expression. These inter-strain differences in CYP could influence pharmacokinetic considerations in preclinical toxicological studies.

  2. 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.

  3. Effect of water accommodated fraction of 0# diesel oil and crude oil on EROD activity of liver of Sparus macrocephlus and its mRNA expression.

    PubMed

    Lei, Li; Shen, Xinqiang; Jiang, Mei

    2016-12-01

    We studied the effect of water accommodated fractions (WAF) of 0# diesel and crude oil on ethoxy resorufin o-deethylase (EROD) activity and CYP1A1 mRNA expression quantity in the liver of Sparus macrocephlus. We found that there were some differences in the EROD activity and CYP1A1 mRNA induction between these two petroleum hydrocarbons. Both the EROD activity and CYP1A1 mRNA expression of fish exposed to 0# diesel WAF were higher than those of crude oil WAF fish. The EROD activities and CYP1A1 mRNA expressions in the livers 0# diesel WAF exposed group declined faster than those of crude oil WAF and the recovery of EROD activity and CYP1A1 mRNA expression in the crude oil group was higher than that of 0# diesel group.

  4. 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...

  5. 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.

  6. Towards a multi protein and mRNA expression of biological predictive and distinguish model for post stroke depression

    PubMed Central

    Yue, Yingying; Jiang, Haitang; Liu, Rui; Yin, Yingying; Zhang, Yuqun; Liang, Jinfeng; Li, Shenghua; Wang, Jun; Lu, Jianxin; Geng, Deqin; Wu, Aiqin; Yuan, Yonggui

    2016-01-01

    Previous studies suggest that neurotrophic factors participate in the development of stroke and depression. So we investigated the utility of these biomarkers as predictive and distinguish model for post stroke depression (PSD). 159 individuals including PSD, stroke without depression (Non-PSD), major depressive disorder (MDD) and normal control groups were recruited and examined the protein and mRNA expression levels of vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptors (VEGFR2), placental growth factor (PIGF), insulin-like growth factor (IGF-1) and insulin-like growth factor receptors (IGF-1R). The chi-square test was used to evaluate categorical variable, while nonparametric test and one-way analysis of variance were applied to continuous variables of general characteristics, clinical and biological changes. In order to explore the predictive and distinguish role of these factors in PSD, discriminant analysis and receiver operating characteristic curve were calculated. The four groups had statistical differences in these neurotrophic factors (all P < 0.05) except VEGF concentration and IGF-1R mRNA (P = 0.776, P = 0.102 respectively). We identified these mRNA expression and protein analytes with general predictive performance for PSD and Non-PSD groups [area under the curve (AUC): 0.805, 95% CI, 0.704-0.907, P < 0.001]. Importantly, there is an excellent predictive performance (AUC: 0.984, 95% CI, 0.964-1.000, P < 0.001) to differentiate PSD patients from MDD patients. This was the first study to explore the changes of neurotrophic factors family in PSD patients, the results intriguingly demonstrated that the combination of protein and mRNA expression of biological factors could use as a predictive and discriminant model for PSD. PMID:27527872

  7. 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

  8. The effect of hypothermia on the expression of neurotrophin mRNA in the hippocampus following transient cerebral ischemia in the rat.

    PubMed

    Boris-Möller, F; Kamme, F; Wieloch, T

    1998-12-10

    The expression of the mRNAs of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT3) and the neurotrophin receptor, TrkB, was studied in the rat hippocampus by in situ hybridization following normothermic (37 degreesC) and protective hypothermic (33 degreesC) transient cerebral ischemia of 15 min duration. In the resistant dentate gyrus, normothermic ischemia transiently induced NGF mRNA at around 8 h of recovery, while the NT3 mRNA levels were depressed over at least a 24-h recovery period. The levels of BDNF and TrkB were transiently and markedly elevated with a maximal expression at 24 h of recovery. Intraischemic hypothermia reduced the induction of NGF mRNA, while the increase of BDNF mRNA expression occurred earlier during recovery, and the post-ischemic NT3 mRNA depression was not affected. Also, the expression of TrkB mRNA was enhanced, and occurred concomitantly with the elevation of BDNF mRNA. In contrast, there were no changes in neurotrophin and TrkB mRNA in the CA3 and CA1 regions. The expression of BDNF mRNA at 24 h after normothermic ischemia, was attenuated by intraischemic hypothermia. We conclude that, the expressions of NGF, BDNF, NT3 or TrkB mRNA in ischemia-sensitive hippocampal subregions are not increased by protective hypothermia. In contrast, hypothermia induces neurotrophin mRNA alterations in the ischemia-resistant dentate gyrus that may convey protection to sensitive regions.

  9. Sex Differences in mRNA Expression of Reduced Folate Carrier-1, Folypolyformyl Glutamate Synthase, and γ-Glutamyl Hydrolase in a Healthy Japanese Population.

    PubMed

    Hashiguchi, Masayuki; Tanaka, Takanori; Shimizu, Mikiko; Tsuru, Tomomi; Chiyoda, Takeshi; Miyawaki, Kumika; Irie, Shin; Takeuchi, Osamu; Hakamata, Jun; Mochizuki, Mayumi

    2016-12-01

    Sex differences in the prevalence of autoimmune diseases such as rheumatoid arthritis (RA) are well known, but little is known about those differences in relation to therapeutic response. Reduced folate carrier-1 (RFC-1), folypolyformyl glutamate synthase (FPGS), and γ-glutamyl hydrolase (GGH) are important transporters and enzymes that convert methotrexate (MTX) in the body. This study investigated the sex differences in mRNA expression of RFC-1, FPGS, and GGH in 190 unrelated healthy Japanese people. The genotypes and mRNA expression were determined using the real-time PCR method. Significant differences between men and women were observed in RFC-1, FPGS, and GGH mRNA expression. The mRNA expression of FPGS and GGH was greater in women than that in men, but the expression of RFC-1 was less in the former than the latter. In stratified analysis by genotype, significant differences in sex-specific mRNA expression were observed in G/G of FPGS, C/C of GGH 452, and C/C of GGH -401. All showed greater mRNA expression in women than in men. In the 5 single-nucleotide polymorphisms RFC-1 80G>A, RFC-1 -43T>C, FPGS 1994G>A, GGH 452C>T, and GGH -401C>T examined, the FPGS 1994 G/G (1.46-fold), GGH 452 C/C (2.16-fold), and GGH -401 C/C (2.68-fold) genotypes showed significantly higher mRNA expression in women than in men. Healthy Japanese adults in this study showed sex-specific differences in mRNA expression that differed among RFC-1, FPGS, and GGH. Therefore, the relationship between genetic polymorphisms and mRNA expression including sex differences might contribute to the variation in the efficacy/toxicity of MTX in patients with RA.

  10. Effect of natural antioxidants on superoxide dismutase and glutathione peroxidase mRNA expression in leukocytes from periparturient dairy cows.

    PubMed

    Colitti, M; Stefanon, B

    2006-01-01

    During the peripartum period, high-yielding dairy cows experience metabolic stress, which alters their homeostasis and exposes the cows to illness. The aim of this study was to quantify the expression levels of genes involved in antioxidant defences during the transition period in the blood of dairy cows and to evaluate the regulative activity on these genes of natural antioxidants in the diet. Three groups of 7 heifers each, at the 7th month of pregnancy, were used. Starting from 3 weeks before the expected calving date (-22 days), the three groups were allotted to the following experimental treatments: control (CTR, basal diet); lycopene (LYC, basal diet + lycopene 540 mg/day) and grape polyphenols (POL, basal diet + grape polyphenols 10 g/day). Blood was sampled at 22 and 8 days before and 8, 15 and 22 after calving and analysed for the expression level of glutathione peroxidase (GPx) and superoxide dismutase (Cu/ZnSOD) using the real-time PCR technique with LUX (Light Upon eXtension) fluorogenic primers. During the peripartum period (-22 days until + 22 days from calving), Cu/ ZnSOD mRNA expression decreased (p<0.05) in the CTR and LYC groups, but increased at 15 days after calving in the POL group. No significant differences were found in GPx mRNA expression. The results suggest that grape polyphenols may have a controlling effect on peripartum metabolic stress through modulation of superoxide dismutase expression.

  11. Striatal mRNA expression patterns underlying peak dose L-DOPA-induced dyskinesia in the 6-OHDA hemiparkinsonian rat.

    PubMed

    Smith, L M; Parr-Brownlie, L C; Duncan, E J; Black, M A; Gemmell, N J; Dearden, P K; Reynolds, J N J

    2016-06-02

    L-DOPA is the primary pharmacological treatment for relief of the motor symptoms of Parkinson's disease (PD). With prolonged treatment (⩾5 years) the majority of patients will develop abnormal involuntary movements as a result of L-DOPA treatment, known as L-DOPA-induced dyskinesia. Understanding the underlying mechanisms of dyskinesia is a crucial step toward developing treatments for this debilitating side effect. We used the 6-hydroxydopamine (6-OHDA) rat model of PD treated with a three-week dosing regimen of L-DOPA plus the dopa decarboxylase inhibitor benserazide (4 mg/kg and 7.5 mg/kgs.c., respectively) to induce dyskinesia in 50% of individuals. We then used RNA-seq to investigate the differences in mRNA expression in the striatum of dyskinetic animals, non-dyskinetic animals, and untreated parkinsonian controls at the peak of dyskinesia expression, 60 min after L-DOPA administration. Overall, 255 genes were differentially expressed; with significant differences in mRNA expression observed between all three groups. In dyskinetic animals 129 genes were more highly expressed and 14 less highly expressed when compared with non-dyskinetic and untreated parkinsonian controls. In L-DOPA treated animals 42 genes were more highly expressed and 95 less highly expressed when compared with untreated parkinsonian controls. Gene set cluster analysis revealed an increase in expression of genes associated with the cytoskeleton and phosphoproteins in dyskinetic animals compared with non-dyskinetic animals, which is consistent with recent studies documenting an increase in synapses in dyskinetic animals. These genes may be potential targets for drugs to ameliorate L-DOPA-induced dyskinesia or as an adjunct treatment to prevent their occurrence.

  12. ALDH1A1 mRNA expression in association with prognosis of triple-negative breast cancer.

    PubMed

    Liu, Yan; Baglia, Michelle; Zheng, Ying; Blot, William; Bao, Ping-Ping; Cai, Hui; Nechuta, Sarah; Zheng, Wei; Cai, Qiuyin; Shu, Xiao Ou

    2015-12-01

    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 expression and 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.

  13. Gonadal mRNA expression levels of TGFbeta superfamily signaling factors correspond with post-hatching morphological development in American alligators.

    PubMed

    Moore, B C; Hamlin, H J; Botteri, N L; Guillette, L J

    2010-01-01

    Paracrine factor signaling regulates many aspects of vertebrate gonadal development. We investigated key ovarian and testicular morphological markers of the American alligator (Alligator mississippiensis) during the first 5 months post-hatching and correlated gonadal development with mRNA expression levels of a suite of regulatory factors. In both sexes, we observed significant morphology changes, including ovarian follicle assembly and meiotic progression of testicular germ cells. Concomitant with these changes were sexually dimorphic and ontogenetically variable mRNA expressions. In ovaries, FOXL2, aromatase, and follistatin mRNA expression was greater than in testes at all ages. At one week after hatching, we observed ovarian medullary remodeling in association with elevated activin/inhibin beta A subunit, follistatin, and aromatase mRNA expressions. Three and 5 months following hatching and concomitant with follicle assembly, ovaries showed increased mRNA expression levels of GDF9 and the mitotic factor PCNA. In testes, the activin/inhibin alpha and beta B subunit transcript levels were greater than in ovaries at all ages. Elevated testicular expression of GDF9 mRNA levels at 5 months after hatching aligned with increased spermatogenic activity. We propose that the mRNA expression levels and concomitant morphological changes observed here affect the establishment of alligator reproductive health and later fertility.

  14. Molecular Cloning, mRNA Expression, and Localization of the G-protein Subunit Galphaq in Sheep Testis and Epididymis

    PubMed Central

    Li, Zhen; Lu, Jieli; Sun, Xiaowei; Pang, Quanhai; Zhao, Yiwen

    2016-01-01

    The reproductive function of G-protein subunit Galphaq (GNAQ), a member of the G protein alpha subunit family, has been extensively studied in humans and rats. However, no data is available on its status in ruminants. The objectives of this study were to evaluate the expression pattern of the GNAQ in the testis and epididymis of sheep by polymerase chain reaction (PCR). The mRNA expression levels were detected by real-time fluorescent quantitative PCR, and cellular localization of GNAQ in the testis and epididymis was examined by immunohistochemistry. Additionally, GNAQ protein was qualitatively evaluated via western blot, with the results indicating that similarities between GNAQ mRNA levels from sheep was highly conserved with those observed in Bos taurus and Sus scrofa. Our results also indicated that GNAQ exists in the caput and cauda epididymis of sheep, while GNAQ in the testis and epididymis was localized to Leydig cells, spermatogonial stem cells, spermatocytes, Sertoli cells, spermatid, principal cells, and epididymis interstitial cells. The concentrations of GNAQ mRNA and protein in the caput and cauda epididymis were significantly greater than those observed in the corpus epididymis (p<0.01) and testis (p<0.05). Our results indicated that GNAQ exists at high concentrations in the caput and cauda epididymis of sheep, suggesting that GNAQ may play an important role in gonad development and sperm maturation. PMID:27004818

  15. 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.

  16. A genome-wide survey demonstrates widespread non-linear mRNA in expressed sequences from multiple species

    PubMed Central

    Dixon, Richard J.; Eperon, Ian C.; Hall, Laurence; Samani, Nilesh J.

    2005-01-01

    We describe here the results of the first genome-wide survey of candidate exon repetition events in expressed sequences from human, mouse, rat, chicken, zebrafish and fly. Exon repetition is a rare event, reported in <10 genes, in which one or more exons is tandemly duplicated in mRNA but not in the gene. To identify candidates, we analysed database sequences for mRNA transcripts in which the order of the spliced exons does not follow the linear genomic order of the individual gene [events we term rearrangements or repetition in exon order (RREO)]. Using a computational approach, we have identified 245 genes in mammals that produce RREO events. RREO in mRNA occurs predominantly in the coding regions of genes. However, exon 1 is never involved. Analysis of the open reading frames suggests that this process may increase protein diversity and regulate protein expression via nonsense-mediated RNA decay. The sizes of the exons and introns involved around these events suggest a gene model structure that may facilitate non-linear splicing. These findings imply that RREO affects a significant subset of genes within a genome and suggests that non-linear information encoded within the genomes of complex organisms could contribute to phenotypic variation. PMID:16237125

  17. 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.

  18. 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.

  19. Amitriptyline induces brain-derived neurotrophic factor (BDNF) mRNA expression through ERK-dependent modulation of multiple BDNF mRNA variants in primary cultured rat cortical astrocytes and microglia.

    PubMed

    Hisaoka-Nakashima, Kazue; Kajitani, Naoto; Kaneko, Masahiro; Shigetou, Takahiro; Kasai, Miho; Matsumoto, Chie; Yokoe, Toshiki; Azuma, Honami; Takebayashi, Minoru; Morioka, Norimitsu; Nakata, Yoshihiro

    2016-03-01

    A significant role of brain-derived neurotrophic factor (BDNF) has been previously implicated in the therapeutic effect of antidepressants. To ascertain the contribution of specific cell types in the brain that produce BDNF following antidepressant treatment, the effects of the tricyclic antidepressant amitriptyline on rat primary neuronal, astrocytic and microglial cortical cultures were examined. Amitriptyline increased the expression of BDNF mRNA in astrocytic and microglial cultures but not neuronal cultures. Antidepressants with distinct mechanisms of action, such as clomipramine, duloxetine and fluvoxamine, also increased BDNF mRNA expression in astrocytic and microglial cultures. There are multiple BDNF mRNA variants (exon I, IIA, IV and VI) expressed in astrocytes and microglia and the variant induced by antidepressants has yet to be elaborated. Treatment with antidepressants increased the expression of exon I, IV and VI in astrocyte and microglia. Clomipramine alone significantly upregulated expression of exon IIA. The amitriptyline-induced expression of both total and individual BDNF mRNA variants (exon I, IV and VI) were blocked by MEK inhibitor U0126, indicating MEK/ERK signaling is required in the expression of BDNF. These findings indicate that non-neural cells are a significant target of antidepressants and further support the contention that glial production of BDNF is crucial role in the therapeutic effect of antidepressants. The current data suggest that targeting of glial function could lead to the development of antidepressants with a truly novel mechanism of action.

  20. CDKN2A (p16) mRNA decreased expression is a marker of poor prognosis in malignant high-grade glioma.

    PubMed

    Sibin, M K; Bhat, Dhananjaya I; Narasingarao, K V L; Lavanya, Ch; Chetan, G K

    2015-09-01

    Human high-grade glioma is heterogeneous in nature based on pathological and genetic profiling. Various tumour suppressor gene alterations are considered as prognostic markers in high-grade glioma. Gene expression of CDKN2A (p16) is used in various cancers as a prognostic biomarker along with methylation and deletion status of this gene. Expression levels of p16 mRNA were not studied as a biomarker in gliomas before. In this study, we have performed mRNA quantification analysis on 48 high-grade glioma tissues and checked for a possible prognostic role. The decreased expression of p16 mRNA in majority of the tumour tissues (57.1 %) was observed when compared to control tissues (P = 0.02). mRNA expression level was correlated with clinical variables also. p16 deletion status and BMI1 mRNA expression were also considered for comparison. p16 mRNA was negatively correlated with the BMI1 mRNA (P = <0.0001) but not with p16 deletion. p16 mRNA expression, midline shift in MRI and tumour type were able to predict patient survival in overall survival (OS) and progression-free survival (PFS). p16 mRNA could independently predict prognosis of OS (P = 0.0146) and PFS (P = 0.0305) in multivariate analysis. We have shown that p16 mRNA expression can act as an independent prognostic biomarker in high-grade glioma.

  1. The up-regulation of ferritin expression using a small-molecule ligand to the native mRNA

    PubMed Central

    Tibodeau, Jennifer D.; Fox, Paige M.; Ropp, Patricia A.; Theil, Elizabeth C.; Thorp, H. Holden

    2006-01-01

    The binding of small molecules to distinctive three-dimensional structures in mRNA provides a new dimension in RNA control, previously limited to the targeting of secondary structures with antisense and RNA interference; such targeting can modulate mRNA function and rates of protein biosynthesis. Small molecules that selectively bind the iron-responsive element (IRE), a specific three-dimensional structure in the noncoding region of the ferritin mRNA model that is recognized by the iron-regulatory protein repressor, were identified by using chemical footprinting. The assay used involved an oxoruthenium(IV) complex that oxidizes guanine bases in RNA sequences. Small molecules that blocked oxidation of guanines in the internal loop region were expected to selectively increase the rate of ferritin synthesis, because the internal loop region of the ferritin IRE is distinctive from those of other IREs. The natural product yohimbine was found (based on gel mobility shifts) to block cleavage of the internal loop RNA site by >50% and seemed to inhibit protein binding. In the presence of yohimbine, the rate of biosynthesis of ferritin in a cell-free expression system (rabbit reticulocyte lysate) increased by 40%. Assignment of the IRE–yohimbine interaction as the origin of this effect was supported by a similar increase in synthesis of luciferase protein in a chimera of the IRE and luciferase gene. The identification of a small, drug-like molecule that recognizes a naturally occurring three-dimensional mRNA structure and regulates protein biosynthesis rates raises the possibility that small molecules can regulate protein biosynthesis by selectively binding to mRNA. PMID:16381820

  2. Improved Method for Recovery of mRNA from Aquatic Samples and Its Application to Detection of mer Expression

    PubMed Central

    Jeffrey, Wade H.; Nazaret, Sylvie; Von Haven, Robin

    1994-01-01

    Previously described methods for extraction of mRNA from environmental samples may preclude detecting transcripts from genes that were present in low abundance in aquatic bacterial communities. By combining a boiling sodium dodecyl sulfate-diethylpyrocarbonate lysis step with acid-guanidinium extraction, we improved recovery of target mRNA from both pure cultures and environmental samples. The most significant advantage of the new protocol is that it is easily adapted to yield high recovery of mRNA from 142-mm-diameter flat filters and high-capacity cartridge filters. The lysis and extraction procedures are more rapid than previously described methods, and many samples can be handled at once. RNA extracts have been shown to be free of contaminating DNA. The lysis procedure does not damage target mRNA sequences, and mRNA can be detected from fewer than 106 bacterial cells. We used the new method to examine transcripts of genes responsible for detoxification of mercurial compounds. Induction of merA (specifying mercuric reductase) transcripts in stationary-phase Pseudomonas aeruginosa containing Tn501 occurred within 60 s of HgCl2 addition and was proportional to the amount of Hg(II) added. The new technique also allowed the detection of merA transcripts from the microbial community of a mercury-contaminated pond (Reality Lake, Oak Ridge, Tenn.). Significant differences in merA transcript abundance were observed between different locations associated with the lake. The results indicate that the new method is simple and rapid and can be applied to the study of mer gene expression of aquatic communities in their natural habitats. PMID:16349274

  3. 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.

  4. Integrated Analysis of Long Noncoding RNA and mRNA Expression Profile in Advanced Laryngeal Squamous Cell Carcinoma

    PubMed Central

    Lian, Meng; Ma, Hongzhi; He, Ning; Liu, Honggang; Wang, Haizhou; Fang, Jugao

    2016-01-01

    Long non-coding RNA (lncRNA) plays an important role in tumorigenesis. However, the expression pattern and function of lncRNAs in laryngeal squamous cell carcinoma (LSCC) are still unclear. To investigate the aberrantly expressed lncRNAs and mRNAs in advanced LSCC, we screened lncRNA and mRNA expression profiles in 9 pairs of primary Stage IVA LSCC tissues and adjacent non-neoplastic tissues by lncRNA and mRNA integrated microarrays. Gene Ontology and pathway analysis were performed to find out the significant function and pathway of the differentially expressed mRNAs, gene-gene functional interaction network and ceRNA network were constructed to select core mRNAs, and lncRNA-mRNA expression correlation network was built to identify the interactions between lncRNA and mRNA. qRT-PCR was performed to further validate the expressions of selected lncRNAs and mRNAs in advanced LSCC. We found 1459 differentially expressed lncRNAs and 2381 differentially expressed mRNAs, including 846 up-regulated lncRNAs and 613 down-regulated lncRNAs, 1542 up-regulated mRNAs and 839 down-regulated mRNAs. The mRNAs ITGB1, HIF1A, and DDIT4 were selected as core mRNAs, which are mainly involved in biological processes, such as matrix organization, cell cycle, adhesion, and metabolic pathway. LncRNA-mRNA expression correlation network showed LncRNA NR_027340, MIR31HG were positively correlated with ITGB1, HIF1A respectively. LncRNA SOX2-OT was negatively correlated with DDIT4. qRT-PCR further validated the expression of these lncRNAs and mRNAs. The work provides convincing evidence that the identified lncRNAs and mRNAs are potential biomarkers in advanced LSCC for further future studies. PMID:28033431

  5. The Expression and Significance of Feces Cyclooxygensae-2 mRNA in Colorectal Cancer and Colorectal Adenomas

    PubMed Central

    Li, Xiaofeng; Kong, Lixia; Liao, Suhuan; Lu, Jing; Ma, Lin; Long, Xiaohua

    2017-01-01

    Background/Aim: This study aims to explore the expression and significance of feces cyclooxygensae-2 (COX-2) mRNA in colorectal cancer and colorectal adenomas. Materials and Methods: The expression of feces COX-2 mRNA in colorectal cancer (n = 28), colorectal adenomas (n = 54), and normal control group (n = 11) were examined by reverse transcriptase polymerase chain reaction (RT-PCR). The positive rate of fecal occult blood test (FOBT) were detected in colorectal cancer (n = 30), colorectal adenomas (n = 56), and normal control group (n = 11); the sensitivity of the two methods was also compared. Results: The positive rate of feces COX-2 mRNA in colorectal cancer was 82.1% (25/28), which was significantly higher than colorectal adenomas 59.3% (32/54), and normal tissues 18.2% (2/11), the difference being significant between the three groups (χ2= 13.842, P = 0.001). The positive rate of FOBT in colorectal cancer was 73.3% (10/30), which was significantly higher than colorectal adenomas 10.7% (6/56) and normal tissues 9.1% (1/11), the difference being significant between these three groups (χ2= 7.525, P = 0.023). There was no significant association between feces COX-2 expression and various clinical pathological features of colorectal cancer and colorectal adenomas (P > 0.05). The sensitivity of the RT-PCR method is higher than FOBT, however, the specificity of FOBT is slightly higher than RT-PCR. Conclusions: High expression of feces COX-2 mRNA in colorectal adenomas and colorectal cancer is a common event; it is an early event in the development of colorectal adenomas to colorectal cancer. Feces COX-2 mRNA has a high sensitivity for detect colorectal cancer; combination with FOBT will be the best alternative. Feces COX-2 can be potentially used in the early diagnosis and screening of colorectal cancer. PMID:28139497

  6. 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.

  7. 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.

  8. Evidence for tissue-specific activation of renal angiotensinogen mRNA expression in chronic stable experimental heart failure.

    PubMed Central

    Schunkert, H; Ingelfinger, J R; Hirsch, A T; Tang, S S; Litwin, S E; Talsness, C E; Dzau, V J

    1992-01-01

    The intrarenal renin-angiotensin system (RAS) may contribute to the pathophysiology of heart failure by the generation of angiotensin II at local sites within the kidneys. Angiotensin II may directly influence renal hemodynamics, glomerular contractility, and tubular sodium reabsorption, thereby promoting sodium and fluid retention in this syndrome. In the present study, we examined components of the circulating RAS as well as the intrarenal expressions of renin and angiotensinogen mRNA in rats with stable compensated heart failure (HF) 12 wk after experimental myocardial infarction. Renal angiotensinogen mRNA level in vehicle-treated HF rats increased 47%, as compared with sham control rats (P = 0.001). The increase in angiotensinogen mRNA levels was more pronounced in animals with medium (46%, P < 0.05) and large (66%, P < 0.05) infarcts than in those with small infarcts (31%, P = NS). There were no differences in liver angiotensinogen mRNA, circulating angiotensinogen, angiotensin II, plasma renin concentration (PRC), kidney renin content (KRC), and renal renin mRNA level between sham and HFv. In addition, in a separate group of rats with heart failure, we demonstrated that renal angiotensin II concentration increased twofold (P < 0.05) as compared with that of age-matched sham operated controls. A parallel group of heart failure rats (HFe, n = 11) was treated with enalapril (25 mg/kg per d) in drinking water for 6 wk before these measurements. Blood pressure decreased significantly during treatment (91 vs. 103 mm Hg, P < 0.05). Enalapril treatment in HF rats increased renin mRNA level (2.5-fold, P < 0.005), KRC (5.6-fold, P = 0.005), and PRC (15.5-fold, P < 0.005). The increase in renal angiotensinogen mRNA level observed in HFv rats was markedly attenuated in enalapril treated HF rats (P < 0.001), suggesting a positive feedback of angiotensin II on renal angiotensinogen synthesis. These findings demonstrate an activation of intrarenal RAS, but no changes in

  9. 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

  10. 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.

  11. Increased GAD67 mRNA expression in cerebellar interneurons in autism: implications for Purkinje cell dysfunction.

    PubMed

    Yip, Jane; Soghomonian, Jean-Jacques; Blatt, Gene J

    2008-02-15

    It has been widely reported that in autism, the number of Purkinje cells (PCs) is decreased, and recently, decreased expression of glutamic acid decarboxylase 67 (GAD67) mRNA in Purkinje cells also has been observed. However, the autism literature has not addressed key GABAergic inputs into Purkinje cells. Inhibitory basket and stellate cell interneurons in the molecular layer of the cerebellar cortex provide direct key GABAergic input into Purkinje cells and could potently influence the output of Purkinje cells to deep cerebellar nuclei. We investigated the capacity for interneuronal synthesis of gamma-amino butyric acid (GABA) in both types of interneurons that innervate the remaining PCs in the posterolateral cerebellar hemisphere in autism. The level of GAD67 mRNA, one of the isoforms of the key synthesizing enzymes for GABA, was quantified at the single-cell level using in situ hybridization in brains of autistic and aged-matched controls. The National Institutes of Health imaging system showed that expression of GAD67 mRNA in basket cells was significantly up-regulated, by 28%, in eight autistic brains compared with that in eight control brains (mean +/- SEM pixels per cell, 1.03 +/- 0.05 versus 0.69 +/- 0.05, respectively; P < 0.0001 by independent t test). Stellate cells showed a trend toward a small increase in GAD67 mRNA levels, but this did not reach significance. The results suggest that basket cells likely provide increased GABAergic feed-forward inhibition to PCs in autism, directly affecting PC output to target neurons in the dentate nucleus and potentially disrupting its modulatory role in key motor and/or cognitive behaviors in autistic individuals.

  12. Dexamethasone suppresses iNOS yet induces GTPCH and CAT-2 mRNA expression in rat lungs.

    PubMed

    Skimming, Jeffrey W; Nasiroglu, Omer; Huang, Chun-Jen; Wood, Charles E; Stevens, Bruce R; Haque, Ikram U L; Scumpia, Philip O; Sarcia, Paul J

    2003-08-01

    The in vivo mechanisms by which glucocorticoids inhibit nitric oxide expression await detailed investigation. In cell culture experiments, glucocorticoids have been shown to inhibit inducible nitric oxide synthase (iNOS) formation and activity. Glucocorticoids can inhibit iNOS activity in cultured cells by blocking arginine transport and inhibiting tetrahydrobiopterin biosynthesis. We recently reported that changes in intrapulmonary formation of nitric oxide in endotoxemic rats correspond with changes in transcription of the predominant arginine transporter cationic amino acid transporter (CAT)-2. Realizing that hemorrhagic shock induces nitric oxide overproduction in intact animals, we sought to explore whether glucocorticoids attenuate hemorrhagic shock-induced increases in intrapulmonary nitric oxide formation and whether they might do so by inhibiting the formation of tetrahydrobiopterin, iNOS protein, and CAT-2. We randomly assigned 10 male Sprague-Dawley rats to receive dexamethasone or normal saline. Bleeding the animals to a mean systemic blood pressure of between 40 and 45 mmHg created the hemorrhagic shock. Dexamethasone abrogated the increase in exhaled nitric oxide concentrations caused by hemorrhagic shock. At the end of the experiment, plasma nitrate/nitrite values were lower in the dexamethasone group than in the control group. The iNOS protein concentrations were also lower in the dexamethasone group than in the control group. Dexamethasone decreased the intrapulmonary iNOS mRNA concentrations yet increased both guanosine triphosphate cyclohydrolase I mRNA and CAT-2 mRNA. Our results support the idea that dexamethasone inhibits nitric oxide formation in a manner that is independent of tetrahydrobiopterin and arginine transport yet dependent on downregulation of iNOS mRNA expression.

  13. Enhancing effect of taurine on CYP7A1 mRNA expression in Hep G2 cells.

    PubMed

    Lam, N V; Chen, W; Suruga, K; Nishimura, N; Goda, T; Yokogoshi, H

    2006-02-01

    Taurine has been reported to enhance cholesterol 7alpha-hydroxylase (CYP7A1) mRNA expression in animal models. However, no in vitro studies of this effect have been reported. The Hep G2 human hepatoma cell line has been recognized as a good model for studying the regulation of human CYP7A1. This work characterizes the effects of taurine on CYP7A1 mRNA levels of Hep G2 cells in a dose- and time-dependent manner. In the dose-dependent experiment, Hep G2 cells were treated with 0, 2, 10 or 20 mM taurine in the presence or absence of cholesterol 0.2 mM for 48 h. In the time-dependent experiment, Hep G2 cells were treated with 0 or 20 mM taurine for 4, 24 and 48 h with and without cholesterol 0.2 mM. Our data revealed that taurine showed time- and dose-response effects on CYP7A1 mRNA levels in Hep G2 cells. However, glycine - a structural analogue of taurine - did not have an effect on CYP7A1 gene expression. These results show that, in agreement to previous studies on animal models, taurine induces the mRNA levels of CYP7A1 in Hep G2 cells, which could enhance cholesterol conversion into bile acids. Also, Hep G2 cell line may be an appropriate model to study the effects of taurine on human cholesterol metabolism.

  14. 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.

  15. Biological analysis of chronic lymphocytic leukemia: integration of mRNA and microRNA expression profiles.

    PubMed

    Dong, L; Bi, K H; Huang, N; Chen, C Y

    2016-01-08

    Chronic lymphocytic leukemia (CLL) is a disease that involves progressive accumulation of nonfunctioning lymphocytes and has a low cure rate. There is an urgent requirement to determine the molecular mechanism underlying this disease in order to improve the early diagnosis and treatment of CLL. In this study, genes differentially expressed between CLL samples and age-matched controls were identified using microRNA (miRNA) and mRNA expression profiles. Differentially expressed (DE) miRNA targets were predicted by combining five algorithms. Common genes were obtained on overlapping the DE mRNA and DE miRNA targets. Then, network and module analyses were performed. A total of 239 miRNA targets were predicted and 357 DE mRNAs were obtained. On intersecting miRNA targets and DE mRNAs, 33 common genes were obtained. The protein-protein interaction network and module analysis identified several crucial genes and modules that might be associated with the development of CLL. These DE mRNAs were significantly enriched in the hematopoietic cell lineage (P = 2.58E-4), mitogen-activated protein kinase signaling pathway (P = 0.0025), and leukocyte transendothelial migration pathway (P = 0.0026). Thus, we conducted biological analysis on integration of DE mRNAs and DE miRNAs in CLL, determined gene expression patterns, and screened out several important genes that might be related to CLL.

  16. Incorporation of beta-globin untranslated regions into a Sindbis virus vector for augmentation of heterologous mRNA expression.

    PubMed

    Strong, T V; Hampton, T A; Louro, I; Bilbao, G; Conry, R M; Curiel, D T

    1997-06-01

    Polynucleotide immunization has been employed as a means of inducing immune responses through the introduction of antigen-encoding DNA. While immunization against specific tumor antigens may be achieved through this strategy, various candidate tumor antigens may not be approached via DNA-based vaccines as they represent transforming oncogenes. As an alternative approach, we have explored the utility of mRNA vectors for polynucleotide immunization. The transient expression achieved by mRNA may provide an efficient and safe system for stimulating immune responses to tumor-specific antigens. Our previous work demonstrated that a self-replicating RNA enhances the magnitude and duration of transgene expression for this application. Here we further modify the vector for optimal use in gene therapy through the incorporation of untranslated regions flanking the encoded transgene. The beta-globin 5' and 3' untranslated regions (UTRs) were inserted directly flanking the luciferase gene in both nonreplicative and replicative RNA constructs. In both cases, elevated and prolonged levels of luciferase expression were detected from the beta-globin UTR-flanked luciferase as compared to luciferase without these sequences. These modifications improve the ability of replicative RNA vectors to produce high, yet transient transgene expression for cancer immunotherapy strategies.

  17. Acute physiological stress down-regulates mRNA expressions of growth-related genes in coho salmon.

    PubMed

    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.

  18. 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

  19. Assessment of cathepsin mRNA expression and enzymatic activity during early embryonic development in the yellowtail kingfish Seriola lalandi.

    PubMed

    Palomino, Jaime; Herrera, Giannina; Torres-Fuentes, Jorge; Dettleff, Phillip; Patel, Alok; Martínez, Víctor

    2017-02-21

    In pelagic species such as Seriola lalandi, survival of both the eggs and embryos depends on yolk processing during oocyte maturation and embryo development. The main enzymes involved in these processes are the cathepsins, which are essential for the hydration process, acquiring buoyancy and nutrition of the embryo before hatching. This study aimed to investigate the mRNA expression profiles of cathepsins B, D and L (catb, catd and catl) and the activity of these enzymes during early development in S. lalandi. We included previtellogenic oocytes (PO). All three enzymes were highly expressed in PO, but the expression was reduced throughout development. Between PO and recently spawned eggs (E1) the transcript to catb and catd decreased, unlike catl. Cathepsin B activity, showed stable levels between PO until blastula stage (E4). High activities levels of cathepsins D and L were observed in E1 in comparison with later developmental stages. Cathepsin L activity remained constant until E1, consistent with observations in other pelagic spawners, where its participation in a second protolithic cleavage of the yolk proteins, has been proposed for this enzyme. Their profiles of both mRNA expression and enzymatic activity indicate the importance of these enzymes during early development and suggest different roles in egg yolk processing for the hydration process and nutrition in early embryos in this species.

  20. Clinical significance of high-Km 5'-nucleotidase (cN-II) mRNA expression in high-risk myelodysplastic syndrome.

    PubMed

    Suzuki, Keijiro; Sugawara, Takeshi; Oyake, Tatsuo; Uchiyama, Toshiyuki; Aoki, Yusei; Tsukushi, Yasuhiko; Onodera, Shima; Ito, Shigeki; Murai, Kazunori; Ishida, Yoji

    2007-10-01

    We analyzed cytosolic high-Km 5'-nucleotidase (cN-II) and deoxycytidine kinase (dCK) mRNA expression in bone marrow mononuclear cells (BMMNC) of patients with high-risk myelodysplastic syndrome (MDS) using quantitative real-time polymerase chain reaction (rt-PCR). At diagnosis, the cN-II mRNA expression of patients was higher than that of healthy volunteers, but the dCK mRNA expression showed no significant difference. Patients with ara-C-containing chemotherapies whose BMMNC showed a high level of cN-II expression (greater than the median value) had shorter median overall survival (15 months versus 22 months, p<0.01) and shorter median post-chemotherapy survival (10 months versus 16 months, p=0.012). These data suggest that the expression level of cN-II mRNA might be a prognostic factor of high-risk MDS.

  1. 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.

  2. 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.

  3. 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.

  4. 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

  5. Prognostic impact of HER3 based on protein and mRNA expression in high-grade serous ovarian carcinoma.

    PubMed

    Unger, Ulrike; Denkert, Carsten; Braicu, Ioana; Sehouli, Jalid; Dietel, Manfred; Loibl, Sibylle; Darb-Esfahani, Silvia

    2017-02-01

    HER3 is a member of the epidermal growth factor family and was predominantly described as a negative prognostic factor in various solid tumors as well as in ovarian cancer. In this study, we investigated HER3 on protein and mRNA expression in histologically defined subtypes of ovarian cancer looking for an influence on patient's survival. Altogether, we examined HER3 in ovarian high-grade serous (HGSC, n = 320), low-grade serous (LGSC, n = 55), endometrioid (EC, n = 33), and clear cell (CCC, n = 48) carcinomas using immunohistochemistry (IHC) and quantitative real-time reverse transcription PCR (qRT-PCR). Univariate and multivariate analyses were performed to explore the association between HER3 and overall survival (OS) as well as progression-free survival (PFS). In HGSC, high HER3 mRNA expression was a favorable prognostic factor for PFS (P = 0.008) and OS (P = 0.052), while for high HER3 protein expression, a trend towards better survival was seen (OS P = 0.064; PFS P = 0.099). A subgroup of HGSC with negative HER3 staining and negative HER3 mRNA levels showed most unfavorable OS and PFS (P = 0.002 and P = 0.004, respectively). Using the multivariate Cox regression model, HER3 was predictive for prolonged PFS (HR, 0.48; 95% CI, 0.26-0.88; P = 0.018). All in all, we cannot confirm the reported negative prognostic impact of HER3 expression in high-grade serous ovarian carcinoma and moreover find a rather positive prognostic implication of HER3 in this major ovarian cancer histological subtype.

  6. Analysis by in situ hybridization of cells expressing mRNA for interleukin 4 in the developing thymus and in peripheral lymphocytes from mice.

    PubMed Central

    Sideras, P; Funa, K; Zalcberg-Quintana, I; Xanthopoulos, K G; Kisielow, P; Palacios, R

    1988-01-01

    We have made use of RNA.RNA in situ hybridization to study the presence of cells producing mRNA for interleukin 4 (IL-4) in the developing thymus, spleen, and T-cell line 2.19. Approximately 1 of 300-400 spleen cells expressed detectable IL-4 mRNA 24 hr after their stimulation by the lectin concanavalin A. Spleen cells were also induced to express mRNA for IL-4 by stimulation with alloantigens. Splenocytes producing mRNA for IL-4 were detected 4 hr after stimulation by concanavalin A; the response peaked at approximately equal to 24 hr and was undetectable by 72 hr. Cyclosporin A inhibited the synthesis of IL-4 mRNA in the T-cell line 2.19, which had been induced by concanavalin A. Approximately 1 of 10 fetal thymocytes at day 14 of gestation expressed mRNA for IL-4 after their stimulation by phorbol 12-myristate 13-acetate and ionomycin. Both the frequency of fetal thymocytes expressing IL-4 mRNA and the amount of mRNA for IL-4 synthesized per cell sharply decreased at day 16 of gestation, and less than 1 of 1800 fetal thymocytes at day 18 of gestation expressed detectable IL-4 mRNA. Our results define the relative frequency of cells capable of expressing IL-4 mRNA after stimulation in vitro in the spleen and in the developing thymus. The data strongly argue for an important role of IL-4 in growth and differentiation of lymphoid cells, notably during T-cell development within the thymus. Images PMID:3257564

  7. Posttranscriptional regulation of GAP-43 gene expression in PC12 cells through protein kinase C-dependent stabilization of the mRNA

    PubMed Central

    1993-01-01

    We have previously shown that nerve growth factor (NGF) selectively stabilizes the GAP-43 mRNA in PC12 cells. To study the cellular mechanisms for this post-transcriptional control and to determine the contribution of mRNA stability to GAP-43 gene expression, we examined the effects of several agents that affect PC12 cell differentiation on the level of induction and rate of degradation of the GAP-43 mRNA. The NGF-mediated increase in GAP-43 mRNA levels and neurite outgrowth was mimicked by the phorbol ester TPA, but not by dibutyryl cAMP or the calcium ionophore A12783. Downregulation of protein kinase C (PKC) by high doses of phorbol esters or selective PKC inhibitors prevented the induction of this mRNA by NGF, suggesting that NGF and TPA act through a common PKC-dependent pathway. In mRNA decay studies, phorbol esters caused a selective 6-fold increase in the half-life of the GAP-43 mRNA, which accounts for most of the induction of this mRNA by TPA. The phorbol ester-induced stabilization of GAP-43 mRNA was blocked by the protein kinase inhibitor polymyxin B and was partially inhibited by dexamethasone, an agent that blocks GAP-43 expression and neuronal differentiation in PC12 cells. In contrast, the rates of degradation and the levels of the GAP-43 mRNA in control and TPA-treated cells were not affected by cycloheximide treatment. Thus, changes in GAP-43 mRNA turnover do not appear to require continuous protein synthesis. In conclusion, these data suggest that PKC activity regulates the levels of the GAP-43 mRNA in PC12 cells through a novel, translation- independent mRNA stabilization mechanism. PMID:8436593

  8. Developmental Expression of CYP2B6: A Comprehensive Analysis of mRNA Expression, Protein Content and Bupropion Hydroxylase Activity and the Impact of Genetic Variation.

    PubMed

    Pearce, Robin E; Gaedigk, Roger; Twist, Greyson P; Dai, Hongying; Riffel, Amanda K; Leeder, J Steven; Gaedigk, Andrea

    2016-07-01

    Although CYP2B6 catalyzes the biotransformation of many drugs used clinically for children and adults, information regarding the effects of development on CYP2B6 expression and activity are scarce. Utilizing a large panel of human liver samples (201 donors: 24 fetal, 141 pediatric, and 36 adult), we quantified CYP2B6 mRNA and protein expression levels, characterized CYP2B6 (bupropion hydroxylase) activity in human liver microsomes (HLMs), and performed an extensive genotype analysis to differentiate CYP2B6 haplotypes such that the impact of genetic variation on these parameters could be assessed. Fetal livers contained extremely low levels of CYP2B6 mRNA relative to postnatal samples and fetal HLMs did not appear to catalyze bupropion hydroxylation; however, fetal CYP2B6 protein levels were not significantly different from postnatal levels. Considerable interindividual variation in CYP2B6 mRNA expression, protein levels, and activity was observed in postnatal HLMs (mRNA, ∼40,000-fold; protein, ∼300-fold; activity, ∼600-fold). The extremely wide range of interindividual variability in CYP2B6 expression and activity was significantly associated with age (P < 0.01) following log transformation of the data. Our data suggest that CYP2B6 activity appears as early as the first day of life, increases through infancy, and by 1 year of age, CYP2B6 levels and activity may approach those of adults. Surprisingly, CYP2B6 interindividual variability was not significantly associated with genetic variation in CYP2B6, nor was it associated with differences in gender or ethnicity, suggesting that factors other than these are largely responsible for the wide range of variability in CYP2B6 expression and activity observed among a large group of individuals/samples.

  9. 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

  10. Selenium Deficiency Affects the mRNA Expression of Inflammatory Factors and Selenoprotein Genes in the Kidneys of Broiler Chicks.

    PubMed

    Zhang, Jiu-Li; Xu, Bo; Huang, Xiao-Dan; Gao, Yu-Hong; Chen, Yu; Shan, An-Shan

    2016-05-01

    The aim of this study was to investigate the influence of Se deficiency on the transcription of inflammatory factors and selenoprotein genes in the kidneys of broiler chicks. One hundred fifty 1-day-old broiler chicks were randomly assigned to two groups fed with either a low-Se diet (L group, 0.033 mg/kg Se) or an adequate Se diet (C group, 0.2 mg/kg Se). The levels of uric acid (UA) and creatinine (Cr) in the serum and the mRNA levels of 6 inflammatory factors and 25 selenoprotein genes in the kidneys were measured as the clinical signs of Se deficiency occurred at 20 days old. The results indicated that the contents of UA and Cr in the serum increased in L group (p < 0.05), and the mRNA levels of the inflammatory factors (NF-κB, iNOS, COX-2, and TNF-α) increased in L group (p < 0.05). Meanwhile, the mRNA levels of PTGEs and HO-1 were not changed. In addition, 25 selenoprotein transcripts displayed ubiquitous expression in the kidneys of the chicks. The mRNA levels of 14 selenoprotein genes (Dio1, Dio2, GPx3, Sepp1, SelH, SelI, SelK, Sepn1, SelO, SelW, Sep15, SelT, SelU, and SelS) decreased, and 9 selenoprotein genes (GPx1, GPx2, GPx4, SelPb, Txnrd1, Txnrd2, Txnrd3, SPS2, and SelM) increased in L group (p < 0.05), but the Dio3 and Sepx1 mRNA levels did not change. The results indicated that Se deficiency resulted in kidney dysfunction, activation of the NF-κB pathway, and a change in selenoprotein gene expression. The changes of inflammatory factor and selenoprotein gene expression levels were directly related to the abnormal renal functions induced by Se deficiency.

  11. Effect of insulin on the mRNA expression of procollagen N-proteinases in chondrosarcoma OUMS-27 cells

    PubMed Central

    AKYOL, SUMEYYA; CÖMERTOĞLU, İSMAIL; FIRAT, RIDVAN; ÇAKMAK, ÖZLEM; YUKSELTEN, YUNUS; ERDEN, GÖNÜL; UGURCU, VELI; DEMIRCAN, KADIR

    2015-01-01

    Chondrosarcoma is one of the most common bone tumors, and at present, there is no non-invasive treatment option for this cancer. The chondrosarcoma OUMS-27 cell line produces proteoglycan and type II, IX, and XI collagens, which constitutes cartilage tissue. A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) proteases are a group of secreted proteases, which include the procollagen N-proteinases ADAMTS-2, -3 and -14. These procollagen N-proteinases perform a role in the processing of procollagens to collagen and the maturation of type I collagen. The present study aimed to improve the understanding of the causes of metastasis, local invasion and resistance to chemo- and radiotherapy in chondrosarcoma, as well as the effect of insulin on cancer cells. The present study was designed to reveal the effects of insulin on procollagen N-proteinases in chondrosarcoma OUMS-27 cells. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) alone or in DMEM containing 10 µg/ml insulin. The medium was changed every other day for 11 days. The cells were harvested on days 1, 3, 7 and 11, and total RNA isolation was performed immediately following harvesting. The expression levels of ADAMTS2, ADAMTS3 and ADAMTS14 mRNA were estimated by reverse transcription-quantitative polymerase chain reaction using appropriate primers. ADAMTS2 mRNA expression was found to be decreased on day 7 (P=0.028) and increased at day 11 compared with the control group (P=0.016). The increase in mRNA concentration at day 11 was significantly different compared to the concentrations on days 3 (P=0.047) and 7 (P=0.008). The expression of ADAMTS3 mRNA decreased immediately subsequent to insulin induction on day 1 compared with the control group (P=0.008). The most evident decrease in mRNA concentration was seen at day 7 subsequent to insulin induction (P=0.008). The present results demonstrated that ADAMTS2 and ADAMTS3 may perform a role in the invasion and metastasis of

  12. 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

  13. Chtop (Chromatin target of Prmt1) auto-regulates its expression level via intron retention and nonsense-mediated decay of its own mRNA

    PubMed Central

    Izumikawa, Keiichi; Yoshikawa, Harunori; Ishikawa, Hideaki; Nobe, Yuko; Yamauchi, Yoshio; Philipsen, Sjaak; Simpson, Richard J; Isobe, Toshiaki; Takahashi, Nobuhiro

    2016-01-01

    Chtop (chromatin target of Prmt1) regulates various aspects of gene expression including transcription and mRNA export. Despite these important functions, the regulatory mechanism underlying Chtop expression remains undetermined. Using Chtop-expressing human cell lines, we demonstrate that Chtop expression is controlled via an autoregulatory negative feedback loop whereby Chtop binds its own mRNA to retain intron 2 during splicing; a premature termination codon present at the 5′ end of intron 2 leads to nonsense-mediated decay of the mRNA. We also show that Chtop interacts with exon 2 of Chtop mRNA via its arginine-glycine-rich (RG) domain, and with intron 2 via its N-terminal (N1) domain; both are required for retention of intron 2. In addition, we show that hnRNP H accelerates intron 2 splicing of Chtop mRNA in a manner dependent on Chtop expression level, suggesting that Chtop and hnRNP H regulate intron 2 retention of Chtop mRNA antagonistically. Thus, the present study provides a novel molecular mechanism by which mRNA and protein levels are constitutively regulated by intron retention. PMID:27683223

  14. 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

  15. 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. PMID:27818993

  16. Effect of acute restraint on hypothalamic pro-vasotocin mRNA expression in flounder, Platichthys flesus.

    PubMed

    Bond, H; Warne, J M; Balment, R J

    2007-01-01

    Arginine vasotocin (AVT) stimulates release of adenocorticotrophin hormone (ACTH) in trout. However, AVT's role in fish hypothalamic-pituitary-interrenal-axis (HPIA) is not fully understood. Here, we examined distribution of AVT and glucocorticoid receptor (GR) in the magnocellular preoptic nucleus (PM) and the AVT/cortisol response to acute restraint in flounder. The GR/AVT distribution in the PM was determined using double immunohistochemistry (IHC). Flounder were confined in nets, immersed in water for 30m, with plasma and tissue samples taken prior to, 3, 24 and 48h post-confinement. Plasma osmolality, Na(+), Cl(-) and cortisol were taken as indicators of HPIA activation. Plasma AVT was measured proVT mRNA expression in the PM was detected using in situ hybridisation (ISH) with a S35 labelled oligoprobe for homologous flounder proVT. Double IHC showed the presence of GR in AVT synthesising neurones of the PM. Plasma Na(+), Cl(-), osmolality and cortisol (1.0+/-0.9 to 183.6+/-3.1mM; p<0.001) increased significantly 3h post-restraint: recovering to control levels after 48h. Plasma AVT levels did not change. However, a concomitant increase in proVT mRNA expression in the magnocellular (PMm) and gigantocellular (PMg) neurones of the PM was observed (11.1+/-1.8 to 55.2+/-9.1% 24h post-restraint; p<0.001) and levels still remained significantly elevated at 48h (p<0.01). This suggests that PMm and PMg AVT neurones are associated with HPIA activation following acute restraint, including potential cortisol negative feedback. The extended elevation of hypothalamus proAVT mRNA expression following a single acute stressor affords a possible mechanism to moderate sensitivity of the HPIA to subsequent challenges.

  17. Transcriptomic Biomarkers for Tuberculosis: Evaluation of DOCK9. EPHA4, and NPC2 mRNA Expression in Peripheral Blood

    PubMed Central

    de Araujo, Leonardo S.; Vaas, Lea A. I.; Ribeiro-Alves, Marcelo; Geffers, Robert; Mello, Fernanda C. Q.; de Almeida, Alexandre S.; Moreira, Adriana da S. R.; Kritski, Afrânio L.; Lapa e Silva, José R.; Moraes, Milton O.; Pessler, Frank; Saad, Maria H. F.

    2016-01-01

    Lately, much effort has been made to find mRNA biomarkers for tuberculosis (TB) disease/infection with microarray-based approaches. In a pilot investigation, through RNA sequencing technology, we observed a prominent modulation of DOCK9, EPHA4, and NPC2 mRNA abundance in the blood of TB patients. To corroborate these findings, independent validations were performed in cohorts from different areas. Gene expression levels in blood were evaluated by quantitative real-time PCR (Brazil, n = 129) or reanalysis of public microarray data (UK: n = 96; South Africa: n = 51; Germany: n = 26; and UK/France: n = 63). In the Brazilian cohort, significant modulation of all target-genes was observed comparing TB vs. healthy recent close TB contacts (rCt). With a 92% specificity, NPC2 mRNA high expression (NPC2high) showed the highest sensitivity (85%, 95% CI 65%–96%; area under the ROC curve [AUROC] = 0.88), followed by EPHA4 (53%, 95% CI 33%–73%, AUROC = 0.73) and DOCK9 (19%, 95% CI 7%–40%; AUROC = 0.66). All the other reanalyzed cohorts corroborated the potential of NPC2high as a biomarker for TB (sensitivity: 82–100%; specificity: 94–97%). An NPC2high profile was also observed in 60% (29/48) of the tuberculin skin test positive rCt, and additional follow-up evaluation revealed changes in the expression levels of NPC2 during the different stages of Mycobacterium tuberculosis infection, suggesting that further studies are needed to evaluate modulation of this gene during latent TB and/or progression to active disease. Considering its high specificity, our data indicate, for the first time, that NPC2high might serve as an accurate single-gene biomarker for TB. PMID:27826286

  18. 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.

  19. p19INK4d mRNA and protein expression as new prognostic factors in ovarian cancer patients

    PubMed Central

    Felisiak-Golabek, Anna; Dansonka-Mieszkowska, Agnieszka; Rzepecka, Iwona K; Szafron, Lukasz; Kwiatkowska, Ewa; Konopka, Bozena; Podgorska, Agnieszka; Rembiszewska, Alina; Kupryjanczyk, Jolanta

    2013-01-01

    p19INK4d (CDKN2D) is a negative regulator of the cell cycle. Little is known of its role in cancer development and prognosis. We aimed to evaluate the clinical significance of p19INK4d expression in ovarian carcinomas with respect to the TP53 accumulation status, as well as the frequency of CDKN2D mutations. p19INK4d and TP53 expression was evaluated immunohistochemically in 445 ovarian carcinomas: 246 patients were treated with platinum–cyclophosphamide (PC/PAC), while 199 were treated with taxane–platinum agents (TP). CDKN2D gene expression (mRNA) was examined in 106 carcinomas, while CDKN2D mutations in 68 tumors. Uni- and multivariate statistical analyses (logistic regression and the Cox proportional hazards model) were performed for patient groups divided according to the chemotherapeutic regimen administered, and in subgroups with and without TP53 accumulation. High p19INK4d expression increased the risk of death, but only in patients with the TP53-negative carcinomas (HR 1.61, P = 0.049 for PC/PAC-treated patients, HR 2.00, P = 0.015 for TP-treated patients). This result was confirmed by the mRNA analysis (HR 4.24, P = 0.001 for TP-treated group). High p19INK4d protein expression associated with adverse clinicopathological factors. We found no alterations in the CDKN2D gene; the c.90C>G (p.R30R; rs1968445) polymorphism was detected in 10% of tumors. Our results suggest that p19INK4d expression is a poor prognostic factor in ovarian cancer patients. Analyses of tumor groups according to the TP53 accumulation status facilitate the identification of cancer biomarkers. PMID:24022213

  20. Correlation analysis of the mRNA and miRNA expression profiles in the nascent synthetic allotetraploid Raphanobrassica

    PubMed Central

    Ye, Bingyuan; Wang, Ruihua; Wang, Jianbo

    2016-01-01

    Raphanobrassica is an allopolyploid species derived from inter-generic hybridization that combines the R genome from R. sativus and the C genome from B. oleracea var. alboglabra. In the present study, we used a high-throughput sequencing method to identify the mRNA and miRNA profiles in Raphanobrassica and its parents. A total of 33,561 mRNAs and 283 miRNAs were detected, 9,209 mRNAs and 134 miRNAs were differentially expressed respectively, 7,633 mRNAs and 39 miRNAs showed ELD expression, 5,219 mRNAs and 57 miRNAs were non-additively expressed in Raphanobrassica. Remarkably, differentially expressed genes (DEGs) were up-regulated and maternal bias was detected in Raphanobrassica. In addition, a miRNA-mRNA interaction network was constructed based on reverse regulated miRNA-mRNAs, which included 75 miRNAs and 178 mRNAs, 31 miRNAs were non-additively expressed target by 13 miRNAs. The related target genes were significantly enriched in the GO term ‘metabolic processes’. Non-additive related target genes regulation is involved in a range of biological pathways, like providing a driving force for variation and adaption in this allopolyploid. The integrative analysis of mRNA and miRNA profiling provides more information to elucidate gene expression mechanism and may supply a comprehensive and corresponding method to study genetic and transcription variation of allopolyploid. PMID:27874043

  1. Profiles of oxidative stress-related microRNA and mRNA expression in auditory cells.

    PubMed

    Wang, Zhi; Liu, Yimin; Han, Ning; Chen, Xuemei; Yu, Wei; Zhang, Weisen; Zou, Fei

    2010-07-30

    Oxidative stress and high levels of reactive oxygen species (ROS) are risk factors of auditory cell injury and hearing impairment. MicroRNAs (miRNAs) are critical for the post-transcriptional regulation of gene expression and cell proliferation and survival. However, little is known about the impact of oxidative stress on the expression of miRNAs and their targeted mRNAs in auditory cells. We employed a cell model of oxidative stress by treatment of House Ear Institute-Organ of Corti 1 (HEI-OC1) cells with different concentrations of tert-butyl hydroperoxide (t-BHP) to examine the t-BHP-induced production of ROS and to determine the impact of t-BHP treatment on the relative levels of miRNA and mRNA transcripts in HEI-OC1 cells. We found that treatment with different concentrations of t-BHP promoted the production of ROS, but inhibited the proliferation of HEI-OC1 cells in a dose- and time-dependent manner. Furthermore, treatment with t-BHP induced HEI-OC1 cell apoptosis. Further microarray analyses revealed that treatment with t-BHP increased the transcription of 35 miRNAs, but decreased the expression of 40 miRNAs. In addition, treatment with t-BHP up-regulated the transcription of 2076 mRNAs, but down-regulated the levels of 580 mRNA transcripts. Notably, the up-regulated (or down-regulated) miRNAs were associated with the decreased (or increased) expression of predicted targeted mRNAs. Importantly, these differentially expressed mRNAs belonged to different functional categories, forming a network participating in the oxidative stress-related process in HEI-OC1 cells. Therefore, our findings may provide new insights into understanding the regulation of miRNAs on the oxidative stress-related gene expression and function in auditory cells.

  2. Detection of Mutant NPM1 mRNA in Acute Myeloid Leukemia Using Custom Gene Expression Arrays

    PubMed Central

    van Vliet, Martin H.; Dumee, Belinda; Simons, Erik; Bullinger, Lars; Döhner, Konstanze; Döhner, Hartmut; Viëtor, Henk; Löwenberg, Bob; Valk, Peter J.M.

    2013-01-01

    Mutations in the gene encoding nucleophosmin (NPM1) carry a prognostic value for patients with acute myeloid leukemia (AML). Various techniques are currently being used to detect these mutations in routine molecular diagnostics. Incorporation of accurate NPM1 mutation detection on a gene expression platform would enable simultaneous detection with various other expression biomarkers. Here we present an array-based mutation detection using custom probes for NPM1 WT mRNA and NPM1 type A, B, and D mutant mRNA. This method was 100% accurate on a training cohort of 505 newly diagnosed unselected AML cases. Validation on an independent cohort of 143 normal-karyotype AML cases revealed no false-negative results, and one false positive (sensitivity 100.0% and specificity 98.7%). Based on this, we conclude that this method provides a reliable method for NPM1 mutation detection. The method can be applied to other genes/mutations as long as the mutant alleles are sufficiently highly expressed. PMID:23530539

  3. An mRNA encoding a putative GABA-gated chloride channel is expressed in the human cardiac conduction system.

    PubMed

    Garret, M; Bascles, L; Boue-Grabot, E; Sartor, P; Charron, G; Bloch, B; Margolskee, R F

    1997-04-01

    GABA-gated chloride channels are the main inhibitory neurotransmitter receptors in the CNS. Conserved domains among members of previously described GABAA receptor subunits were used to design degenerate sense and antisense oligonucleotides. A PCR product from this amplification was used to isolate a full-length cDNA. The predicted protein has many of the features shared by other members of the ligand-gated ion channel family. This channel subunit has significant amino acid identity (25-40%) with members of GABAA and GABAC receptor subunits and thus may represent a new subfamily of the GABA receptor channel. Although we cannot rule out that this clone encodes a receptor for an unidentified ligand, it was termed GABA chi. This gene is mainly expressed in placenta and in heart; however, placenta appears to express only an unspliced mRNA. In situ hybridization reveals that the GABA chi subunit mRNA is present in the electrical conduction system of the human heart. Our results suggest that novel GABA receptors expressed outside of the CNS may regulate cardiac function.

  4. Local Context Finder (LCF) reveals multidimensional relationships among mRNA expression profiles of Arabidopsis responding to pathogen infection.

    PubMed

    Katagiri, Fumiaki; Glazebrook, Jane

    2003-09-16

    A major task in computational analysis of mRNA expression profiles is definition of relationships among profiles on the basis of similarities among them. This is generally achieved by pattern recognition in the distribution of data points representing each profile in a high-dimensional space. Some drawbacks of commonly used pattern recognition algorithms stem from their use of a globally linear space and/or limited degrees of freedom. A pattern recognition method called Local Context Finder (LCF) is described here. LCF uses nonlinear dimensionality reduction for pattern recognition. Then it builds a network of profiles based on the nonlinear dimensionality reduction results. LCF was used to analyze mRNA expression profiles of the plant host Arabidopsis interacting with the bacterial pathogen Pseudomonas syringae. In one case, LCF revealed two dimensions essential to explain the effects of the NahG transgene and the ndr1 mutation on resistant and susceptible responses. In another case, plant mutants deficient in responses to pathogen infection were classified on the basis of LCF analysis of their profiles. The classification by LCF was consistent with the results of biological characterization of the mutants. Thus, LCF is a powerful method for extracting information from expression profile data.

  5. [Measurement of gene expression of CYP mRNA in liver of rats exposed to toluene and 1-butanol vapors].

    PubMed

    Ishidao, Toru; Ishimatsu, Sumiyo; Hirohashi, Masami; Morimoto, Yasuo; Hori, Hajime

    2006-12-01

    A reduction of the biological half life of toluene in blood in the simultaneous exposure to toluene and alcohol vapors has been reported. To clarify the cause of this reduction, gene expression of CYP mRNA in liver of rats exposed to bi-component organic vapors was investigated. Wistar male rats were repeatedly exposed to 500 ppm of toluene and 300 ppm of 1-butanol vapors individually and simultaneously by inhalation 6 hours a day, five days a week for 4 weeks. After the exposure, the rats were sacrificed and the livers were collected and homogenized. RNA was extracted from the livers, and gene expression of CYP mRNA was observed by reverse transcription-polymerase chain reaction (RT-PCR). The gene expression of CYP3A2 in the simultaneous exposure group was significantly higher than that in the toluene exposure group. However, there was no significant difference in that of CYP1A2, CYP2B1, CYP2C11, CYP2E1 and CYP4A1.

  6. 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

  7. Arginine vasopressin regulates CFTR and ClC-2 mRNA expression in rat kidney cortex and medulla.

    PubMed

    Morales, M M; Nascimento, D S; Capella, M A; Lopes, A G; Guggino, W B

    2001-11-01

    The presence of both CFTR and ClC-2 proteins in the kidney suggest that they are involved in chloride transport along the nephron but their physiological roles in this organ are not known. To further understand the role of these chloride channels we studied Wistar rats subjected to dehydration for 2 days and also the homozygous Brattleboro rats, a strain of Long-Evans rats carrying an autosomal recessive mutation that leads to a deficiency of arginine-vasopressin (AVP) secretion in the plasma. The expression of CFTR was increased in the medulla of dehydrated Wistar rats and no variation was observed in the cortex. The expression of both ClC-2 and CFTR mRNAs was low in the renal cortex and medulla of the homozygous Brattleboro rats but returned to normal levels after AVP reposition. By the use of Madine-Darby canine kidney (MDCK) type I epithelial cells, it was observed that AVP (10(-8), 10(-7) and 10(-6) M) increased CFTR mRNA expression "in vitro" but no effect was observed when changes in the medium tonicity were caused by the addition of sucrose, NaCl, manitol or urea. The modulation of both CFTR and ClC-2 mRNA by AVP, the main hormone involved in the regulation of body fluid osmolality, suggests the participation of these two chloride channels in the renal tubule transcellular chloride transport modulated by AVP.

  8. The Andes hantavirus NSs protein is expressed from the viral small mRNA by a leaky scanning mechanism.

    PubMed

    Vera-Otarola, Jorge; Solis, Loretto; Soto-Rifo, Ricardo; Ricci, Emiliano P; Pino, Karla; Tischler, Nicole D; Ohlmann, Théophile; Darlix, Jean-Luc; López-Lastra, Marcelo

    2012-02-01

    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.

  9. 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

  10. Disorders in barrier protein mRNA expression and placenta secretory activity under the influence of polychlorinated biphenyls in vitro.

    PubMed

    Wojciechowska, A; Mlynarczuk, J; Kotwica, J

    2017-02-01

    Pregnancy disorders are often correlated with the presence of organic pollutants in the tissues of living bodies. The aim of this study was to investigate the effects (over 24 and 48 hours) of polychlorinated biphenyls (PCBs) 153, 126, and 77 at doses of 1, 10, and 100 ng/mL on barrier function and secretory activity in cow placentome sections collected during the second trimester of pregnancy. None of the PCBs affected the viability of the sections (P > 0.05). Polychlorinated biphenyl 153 decreased (P < 0.05) connexin 26 (Cx 26) mRNA expression, and all three PCBs reduced (P < 0.05) Cx 43 mRNA expression. Cx 32 mRNA expression showed a downward trend (P > 0.05) under the influence of PCBs 126 and 77. Moreover, PCBs 153 and 126 increased keratin 8 (KRT8) mRNA expression, whereas all PCBs decreased (P < 0.05) placenta specific protein 1 (PLAC-1) mRNA expression without changing (P > 0.05) hypoxia inducible factor 1α (HIF1α) mRNA expression. Concomitantly, PCBs 153 and 126 stimulated (P < 0.05) cyclooxygenase 2 (COX-2) mRNA expression, all PCBs increased (P < 0.05) prostaglandin E2 synthase (PGES) mRNA expression, and PCBs 126 and 77 increased prostaglandin E2 (PGE2) secretion. All three PCBs decreased (P < 0.05) prostaglandin F2α synthase (PGFS) mRNA expression and prostaglandin F2α (PGF2α) secretion. In addition, all three PCBs increased (P < 0.05) neurophysin I/oxytocin (NP-I/OT) mRNA expression and OT secretion but did not affect peptidyl-glycine-α-amidating monooxygenase (PGA) mRNA expression (P > 0.05). Moreover, the PCBs increased (P < 0.05) estradiol (E2) secretion, whereas progesterone (P4) secretion remained unchanged (P > 0.05). These changes could affect trophoblast invasion and uterine contractility and thus impact the course of gestation and/or fetal development in the cow.

  11. Glutamine and glutamic acid enhance thyroid-stimulating hormone β subunit mRNA expression in the rat pars tuberalis.

    PubMed

    Aizawa, Sayaka; Sakai, Takafumi; Sakata, Ichiro

    2012-03-01

    Thyroid-stimulating hormone (TSH)-producing cells of the pars tuberalis (PT) display distinct characteristics that differ from those of the pars distalis (PD). The mRNA expression of TSHβ and αGSU in PT has a circadian rhythm and is inhibited by melatonin via melatonin receptor type 1; however, the detailed regulatory mechanism for TSHβ expression in the PT remains unclear. To identify the factors that affect PT, a microarray analysis was performed on laser-captured PT tissue to screen for genes coding for receptors that are abundantly expressed in the PT. In the PT, we found high expression of the KA2, which is an ionotropic glutamic acid receptor (iGluR). In addition, the amino acid transporter A2 (ATA2), also known as the glutamine transporter, and glutaminase (GLS), as well as GLS2, were highly expressed in the PT compared to the PD. We examined the effects of glutamine and glutamic acid on TSHβ expression and αGSU expression in PT slice cultures. l-Glutamine and l-glutamic acid significantly stimulated TSHβ expression in PT slices after 2- and 4-h treatments, and the effect of l-glutamic acid was stronger than that of l-glutamine. In contrast, treatment with glutamine and glutamic acid did not affect αGSU expression in the PT or the expression of TSHβ or αGSU in the PD. These results strongly suggest that glutamine is taken up by PT cells through ATA2 and that glutamic acid locally converted from glutamine by Gls induces TSHβ expression via the KA2 in an autocrine and/or paracrine manner in the PT.

  12. Changes in the mRNA expression of structural proteins, hormone synthesis and secretion from bovine placentome sections after DDT and DDE treatment.

    PubMed

    Wojciechowska, A; Mlynarczuk, J; Kotwica, J

    2017-01-15

    Disorders in the barrier function and secretory activity of the placenta can be caused by xenobiotics (XB) present in the environment and their accumulation in tissues of living organisms. Thus, the aim of this study was to investigate the effect of 1,1,1-trichloro-2,2,-bis-4-chlorophenyl-ethane (DDT) and its metabolite 1,1-dichloro-2,2-bis-4-chlorophenyl-ethene (DDE) (for 24 or 48h) at doses of 1, 10 or 100ng/ml on the function of cow placentome sections in the second trimester of pregnancy. DDT and DDE affected neither (P>0.05) the viability nor hypoxia inducible factor 1 (HIF1α) mRNA expression of the sections. XB decreased (P<0.05) connexin (Cx) 26, 32, 43 and placenta-specific 1 (PLAC-1) mRNA expression but did not affect (P>0.05) keratin 8 (KRT8) mRNA expression. DDT and DDE also reduced (P<0.05) prostaglandin F2α (PGF2α) synthase (PGFS) mRNA expression, while DDT increased (P<0.05) prostaglandin E2 (PGE2) synthase (PGES) mRNA expression. Neither cyclooxygenase 2 (COX-2) mRNA expression nor PGF2α and PGE2 secretion were affected. Both DDT and DDE increased (P<0.05) neurophysin I/oxytocin (NP1/OT) mRNA expression and oxytocin (OT), oestradiol (E2) and progesterone (P4) secretion while DDT stimulated only 3β-hydroxysteroid dehydrogenase (3βHSD) and cholesterol side-chain cleavage enzyme (CYP11A1) mRNA expression (P<0.05). In summary, DDT and DDE impaired the barrier function and secretory activity of the placenta. Thus, these compounds can disrupt trophoblast invasion, myometrium contractility and gas/nutrient exchange throughout pregnancy in cows.

  13. Profiles of mRNA expression of related genes in the duck hypothalamus-pituitary growth axis during embryonic and early post-hatch development.

    PubMed

    Hu, Yan; Liu, Hongxiang; Song, Chi; Xu, Wenjuan; Ji, Gaige; Zhu, Chunhong; Shu, Jingting; Li, Huifang

    2015-03-15

    In this study, the ontogeny of body and liver weight and the pattern of related gene mRNA expression in the hypothalamus-pituitary growth axis (HPGA) of two different duck breeds (Anas platyrhynchos domestica) were compared during embryonic and post-hatch development. Duck hypothalamic growth hormone release hormone (GHRH), somatostatin (SS), pituitary growth hormone (GH), liver growth hormone receptor (GHR) and insulin-like growth factor-I (IGF-1) mRNA were first detected on the 13th embryonic day. During early duck development, SS maintained a lower expression status, whereas the other four genes exhibited highly significant variations in an age-specific manner. Highly significant breed specificity was observed with respect to hepatic IGF-1 mRNA expression, which showed a significant breed-age interaction effect. Compared with previous studies on chickens, significant species differences were observed regarding the mRNA expression of bird embryonic HPGA-related genes. During early development, highly significant breed and age specificity were observed with respect to developmental changes in body and liver weight, and varying degrees of significant linear correlation were found between these performances and the mRNA expression of HPGA-related genes in the duck HPGA. These results suggest that different genetic backgrounds may lead to differences in duck growth and HPGA-related gene mRNA expression, and the differential mRNA expression of related genes in the duck HPGA may be particularly important in the early growth of ducks. Furthermore, hepatic IGF-1 mRNA expression presented highly significant breed specificity, and evidence suggests the involvement of hepatic IGF-1 in mediating genetic effects on embryo and offspring growth in ducks.

  14. Increased hexokinase II expression in the renal glomerulus of mice in response to arsenic

    SciTech Connect

    Pysher, Michele D.; Sollome, James J.; Regan, Suzanne; Cardinal, Trevor R.; Hoying, James B.; Brooks, Heddwen L.; Vaillancourt, Richard R.

    2007-10-01

    Epidemiological studies link arsenic exposure to increased risks of cancers of the skin, kidney, lung, bladder and liver. Additionally, a variety of non-cancerous conditions such as diabetes mellitus, hypertension, and cardiovascular disease have been associated with chronic ingestion of low levels of arsenic. However, the biological and molecular mechanisms by which arsenic exerts its effects remain elusive. Here we report increased renal hexokinase II (HKII) expression in response to arsenic exposure both in vivo and in vitro. In our model, HKII was up-regulated in the renal glomeruli of mice exposed to low levels of arsenic (10 ppb or 50 ppb) via their drinking water for up to 21 days. Additionally, a similar effect was observed in cultured renal mesangial cells exposed to arsenic. This correlation between our in vivo and in vitro data provides further evidence for a direct link between altered renal HKII expression and arsenic exposure. Thus, our data suggest that alterations in renal HKII expression may be involved in arsenic-induced pathological conditions involving the kidney. More importantly, these results were obtained using environmentally relevant arsenic concentrations.

  15. Phospholipase C-{delta}{sub 1} regulates interleukin-1{beta} and tumor necrosis factor-{alpha} mRNA expression

    SciTech Connect

    Chung, Eric; Jakinovich, Paul; Bae, Aekyung; Rebecchi, Mario

    2012-10-01

    Phospholipase C-{delta}{sub 1} (PLC{delta}{sub 1}) is a widely expressed highly active PLC isoform, modulated by Ca{sup 2+} that appears to operate downstream from receptor signaling and has been linked to regulation of cytokine production. Here we investigated whether PLC{delta}{sub 1} modulated expression of the pro-inflammatory cytokines interleukin-1{beta} (IL-1{beta}), tumor necrosis factor-{alpha} (TNF-{alpha}) and interleukin-6 (IL-6) in rat C6 glioma cells. Expression of PLC{delta}{sub 1} was specifically suppressed by small interfering RNA (siRNA) and the effects on cytokine mRNA expression, stimulated by the Toll-like receptor (TLR) agonist, lipopolysaccharide (LPS), were examined. Real-time polymerase chain reaction (RT-PCR) results showed that PLC{delta}{sub 1} knockdown enhanced expression IL-1{beta} and tumor necrosis factor-{alpha} (TNF-{alpha}) mRNA by at least 100 fold after 4 h of LPS stimulation compared to control siRNA treatment. PLC{delta}{sub 1} knock down caused persistently high Nf{kappa}b levels at 4 h of LPS stimulation compared to control siRNA-treated cells. PLC{delta}{sub 1} knockdown was also associated with elevated nuclear levels of c-Jun after 30 min of LPS stimulation, but did not affect LPS-stimulated p38 or p42/44 MAPK phosphorylation, normally associated with TLR activation of cytokine gene expression; rather, enhanced protein kinase C (PKC) phosphorylation of cellular proteins was observed in the absence of LPS stimulation. An inhibitor of PKC, bisindolylmaleimide II (BIM), reversed phosphorylation, prevented elevation of nuclear c-Jun levels, and inhibited LPS-induced increases of IL-1{beta} and TNF-{alpha} mRNA's induced by PLC{delta}{sub 1} knockdown. Our results show that loss of PLC{delta}{sub 1} enhances PKC/c-Jun signaling and up-modulates pro-inflammatory cytokine gene transcription in concert with the TLR-stimulated p38MAPK/Nf{kappa}b pathway. Our findings are consistent with the idea that PLC{delta}{sub 1} is a

  16. Effect of Gestational Age on mRNA and Protein Expression of Polyspecific Organic Cation Transporters during Pregnancy

    PubMed Central

    Lee, Nora; Hebert, Mary F.; Prasad, Bhagwat; Easterling, Thomas R.; Kelly, Edward J.; Unadkat, Jashvant D.

    2013-01-01

    Polyspecific organic cation (OC) transporters play important roles in the disposition of clinically used drugs, including drugs used during pregnancy. Pregnancy is known to alter the expression of drug-metabolizing enzymes and transporters, but its specific effect on OC transporters has not been well defined. Using quantitative polymerase chain reaction and liquid chromatography coupled with tandem mass spectrometry targeted proteomics, we determined the effect of pregnancy and gestational age on mRNA and protein expression of major OC transporters in the kidney, liver, and placenta in mice with timed pregnancies. Human organic cation transporter 3 (hOCT3) expression was further investigated in human placentas from the first and second trimesters and at term. Our results showed that pregnancy had a marginal effect on renal mouse organic cation transporter 1/2 (mOct1/2) expression but significantly reduced mouse multidrug and toxin extrusion transporter 1 (mMate1) expression by 20%–40%. Hepatic expression of mOct1 and mMate1 was minimally affected by pregnancy. Human and mouse placentas predominantly expressed OCT3 with little expression of OCT1/2, MATE1/2, and plasma membrane monoamine transporter (PMAT). The hOCT3 protein in first and second trimester and term placentas was quantified to be 0.23 ± 0.033, 0.38 ± 0.072, and 0.36 ± 0.099 fmol/μg membrane protein, respectively. In contrast with the moderate increase in hOCT3 protein during human pregnancy, mOct3 expression in the mouse placenta was highly dependent on gestational age. Compared with gestational day (gd) 10, placental mOct3 mRNA increased by 37-fold and 46-fold at gd 15 and 19, leading to a 56-fold and 128-fold increase in mOct3 protein, respectively. Our study provides new insights into the effect of pregnancy on the expression of polyspecific OC transporters and supports an important role of OCT3 in OC transport at the placental barrier. PMID:24101703

  17. Cytochrome p450 mRNA expression in the rodent brain: species-, sex-, and region-dependent differences.

    PubMed

    Stamou, Marianna; Wu, Xianai; Kania-Korwel, Izabela; Lehmler, Hans-Joachim; Lein, Pamela J

    2014-02-01

    Cytochrome P450 (P450) enzymes play a critical role in the activation and detoxication of many neurotoxic chemicals. Although research has largely focused on P450-mediated metabolism in the liver, emerging evidence suggests that brain P450s influence neurotoxicity by modulating local metabolite levels. As a first step toward better understanding the relative role of brain P450s in determining neurotoxic outcome, we characterized mRNA expression of specific P450 isoforms in the rodent brain. Adult mice (male and female) and rats (male) were treated with vehicle, phenobarbital, or dexamethasone. Transcripts for CYP2B, CYP3A, CYP1A2, and the orphan CYP4X1 and CYP2S1 were quantified in the liver, hippocampus, cortex, and cerebellum by quantitative (real-time) polymerase chain reaction. These P450s were all detected in the liver with the exception of CYP4X1, which was detected in rat but not mouse liver. P450 expression profiles in the brain varied regionally. With the exception of the hippocampus, there were no sex differences in regional brain P450 expression profiles in mice; however, there were marked species differences. In the liver, phenobarbital induced CYP2B expression in both species. Dexamethasone induced hepatic CYP2B and CYP3A in mice but not rats. In contrast, brain P450s did not respond to these classic hepatic P450 inducers. Our findings demonstrate that P450 mRNA expression in the brain varies by region, regional brain P450 profiles vary between species, and their induction varies from that of hepatic P450s. These novel data will be useful for designing mechanistic studies to examine the relative role of P450-mediated brain metabolism in neurotoxicity.

  18. Gene microarray analysis of lncRNA and mRNA expression profiles in patients with hypopharyngeal squamous cell carcinoma

    PubMed Central

    Zhou, Jieyu; Li, Wenming; Jin, Tong; Xiang, Xuan; Li, Maocai; Wang, Juan; Li, Guojun; Pan, Xinliang; Lei, Dapeng

    2015-01-01

    Background: Studies have shown that long noncoding RNAs (lncRNAs) are involved in the development and progression of many types of cancer. However, the mechanisms by which lncRNAs influence development and progression of hypopharyngeal squamous cell carcinoma (HSCC) are unclear. Method: We investigated differences in lncRNA and mRNA expression profiles between 3 pairs of HSCC tissues and adjacent nontumor tissues by microarray analysis. Results: In HSCC tissues, 1299 lncRNAs were significantly upregulated (n=669) or downregulated (n=630) compared to levels in adjacent nontumor tissues. Moreover, 1432 mRNAs were significantly upregulated (n=684) or downregulated (n=748) in HSCC tissues. We randomly selected 2 differentially expressed lncRNAs (AB209630, AB019562) and 2 differentially expressed mRNAs (SPP1, TJP2) for confirmation of microarray results using qRT-PCR. The qRT-PCR results matched well with the microarray data. The differentially expressed lncRNAs and mRNAs were distributed on each of the chromosomes, including the X and Y chromosomes. Pathway analysis indicated that the biological functions of differentially expressed mRNAs were related to 48 cellular pathways that may be associated with HSCC development. GO analysis revealed that 593 mRNAs involved in biological processes, 50 mRNAs involved in cellular components, and 46 mRNAs involved in molecular functions were upregulated in the carcinomas; 280 mRNAs involved in biological processes, 58 mRNAs involved in cellular components, and 71 mRNAs involved in molecular functions were downregulated in the carcinomas. In addition, 8 enhancer-like lncRNAs and 21 intergenic lncRNAs with their adjacent mRNA pairs were identified as coregulated transcripts. Conclusion: These findings provide insight into the mechanisms underlying HSCC tumorigenesis and will facilitate identification of new therapeutic targets and diagnostic biomarkers for this disease. PMID:26131061

  19. Identification of reference housekeeping-genes for mRNA expression studies in patients with type 1 diabetes.

    PubMed

    Kar, Parmita; Chawla, Himika; Saha, Soma; Tandon, Nikhil; Goswami, Ravinder

    2016-06-01

    Selection of appropriate housekeeping-genes as reference is important in mRNA expression-related experiments. It is more important in diabetes since hyperglycemia per se can influence expression of housekeeping-genes. RNA expression of Glyceraldehyde-3-phosphate-dehydrogenase, β-actin and 18S-ribosomal-RNA, Hypoxanthine-phosphoribosyl-transferase (HPRT), Tyrosine-3-monooxygenase/tryptophan (YHWAZ), β2-microglobin (β2M), TATA-binding-protein (TBP), and Ubiquitin C and cytochrome1 (CYC1) assessed in circulating-lymphocytes-(PBMC) of patients with type-1-diabetes and healthy controls. The stability ('M' value <1.02) and number of housekeeping-genes required for normalization in qRT-PCR were determined by 'ge-norm software.' Vitamin-D-receptor (VDR) was used as a target gene. All the nine genes tested had sufficient 'M' value in diabetes and healthy controls. However, housekeeping-genes indicated a relatively higher stability of expression in healthy controls in comparison to diabetes. Use of single housekeeping-genes brought gross variation in the calculation of VDR-mRNA copies. The ge-norm software suggested geometric mean of five housekeeping-genes for ideal normalization in diabetes (CYC1, β-actin, YHWAZ, HPRT, and β2M) and only three in controls (CYC1, β-actin, and TBP). HbA1c did not correlate with expression of any of the nine housekeeping-genes. Thus, geometric mean of CYC1, β-actin, YHWAZ, HPRT, and β2M needs to be used for ideal normalization of mRNA in type-1-diabetes. Similar studies are required in other population.

  20. Effect of hyperosmotic conditions on flavin-containing monooxygenase activity, protein and mRNA expression in rat kidney

    PubMed Central

    Rodríguez-Fuentes, Gabriela; Coburn, Cary; Currás-Collazo, Margarita; Guillén, Gabriel; Schlenk, Daniel

    2010-01-01

    Flavin-containing monooxigenases (FMOs) are a polymorphic family of drug and pesticide metabolizing enzymes, found in the smooth endoplasmatic reticulum that catalyze the oxidation of soft nucleophilic heteroatom substances to their respective oxides. Previous studies in euryhaline fishes have indicated induction of FMO expression and activity in vivo under hyperosmotic conditions. In this study we evaluated the effect of hypersaline conditions in rat kidney. Male Sprague–Dawley rats were injected intraperitoneal with 3.5 M NaCl at a doses ranging from 0.3 cm3/100 g to 0.6 cm3/100 g in two separate treatments. Three hours after injection, FMO activities and FMO1 protein was examined in the first experiment, and the expression of FMO1 mRNA was measured in the second experiment from kidneys after treatment with NaCl. A positive significant correlation was found between FMO1 protein expression and plasma osmolarity (p < 0.05, r = 0.6193). Methyl-p-tolyl sulfide oxidase showed a statistically significant increase in FMO activity, and a positive correlation was observed between plasma osmolarity and production of FMO1-derived (R)-methyl-p-tolyl sulfoxide (p < 0.05, r = 0.6736). Expression of FMO1 mRNA was also positively correlated with plasma osmolality (p < 0.05, r = 0.8428). Similar to studies in fish, these results suggest that expression and activities of FMOs may be influenced by hyperosmotic conditions in the kidney of rats. PMID:19429252

  1. Expression of cytokines by human astrocytomas following stimulation by C3a and C5a anaphylatoxins: specific increase in interleukin-6 mRNA expression.

    PubMed

    Sayah, S; Ischenko, A M; Zhakhov, A; Bonnard, A S; Fontaine, M

    1999-06-01

    C3a and C5a anaphylatoxins are two proinflammatory peptides generated during complement activation that act through distinct Gi protein-coupled receptors named C3aR and C5aR, respectively. We have demonstrated previously that human astrocytes expressed C3aR and C5aR constitutively and were able to produce a functional complement. In this study, we examined the effect of an anaphylatoxin stimulation on cytokine expression by human astrocyte cell lines. Interleukin (IL)-1beta, IL-6, tumor necrosis factor-alpha, and transforming growth factor-beta mRNA expression was studied by quantitative RT-PCR. Whereas IL-1beta, tumor necrosis factor-alpha, and transforming growth factor-beta mRNA levels remained unchanged, stimulation of astrocytoma cells (T98G, CB193, U118MG) by C3a, C5a, and peptidic C3aR and C5aR agonists induced an increase in the IL-6 mRNA level. The amount of IL-6 was markedly increased at 3 and 6 h and returned to the basal level at 9 h of stimulation. This response was specific, because pretreatment of cells with pertussis toxin or with polyclonal anti-C3aR or anti-C5aR antibodies completely blocked the IL-6 mRNA increase. The IL-6 response was also investigated at the protein level, but IL-6 protein was detected neither in cell lysates nor in supernatants of stimulated cells. The anaphylatoxin-mediated transcriptional activation of IL-6 gene suggests that C3a and C5a could play a role in priming glial cells during the inflammatory process in the brain.

  2. Differential detection of cytoplasmic Wilms tumor 1 expression by immunohistochemistry, western blotting and mRNA quantification.

    PubMed

    Maki, Takehiro; Ikeda, Hiroaki; Kuroda, Aki; Kyogoku, Noriaki; Yamamura, Yoshiyuki; Tabata, Yukiko; Abiko, Takehiro; Tsuchikawa, Takahiro; Hida, Yasuhiro; Shichinohe, Toshiaki; Tanaka, Eiichi; Kaga, Kichizo; Hatanaka, Kanako; Matsuno, Yoshihiro; Imai, Naoko; Hirano, Satoshi

    2017-01-01

    Wilms tumor 1 (WT1) is considered to be a promising target of cancer treatment because it has been reported to be frequently expressed at high levels in various malignancies. Although WT1-targeted cancer treatment has been initiated, conclusive detection methods for WT1 are not established. The present study aimed to consolidate immunohistochemistry for WT1 with statistical basis. Transfected cells with forced WT1 expression yielded specific western blot bands and nuclear immunostaining; cytoplasmic immunostaining was not specifically recognized. Immunohistochemistry, western blotting, and quantitative reverse transcriptase-polymerase chain reaction were performed in 35 human cell lines using multiple WT1 antibodies and their results were quantified. Relationships among the quantified results were statistically analyzed; the nuclear immunostaining positively correlated with western blot bands and mRNA expression levels, whereas cytoplasmic immunostaining did not. These results indicate that nuclear immunostaining reflects WT1 expression but cytoplasmic immunostaining does not. The nuclear immunostaining was barely (3/541) observed in primary cancer of esophagus, bile duct, pancreas and lung. Although the present study has some limitations, the results indicate that the cytoplasmic immunostaining does not correlate with actual WT1 expression and prompts researchers to carefully evaluate target molecule expression in treatment of cancer.

  3. A Systematic Analysis on mRNA and MicroRNA Expression in Runting and Stunting Chickens.

    PubMed

    Zhang, Li; Li, Ying; Xie, Xiujuan; Xu, Haiping; Xu, Zhenqiang; Ma, Jinge; Li, Bixiao; Lin, Shudai; Nie, Qinghua; Luo, Qingbin; Zhang, Xiquan

    2015-01-01

    Runting and stunting syndrome (RSS), which is characterized by lower body weight, widely occurs in broilers. Some RSS chickens simply exhibit slow growth without pathological changes. An increasing number of studies indicate that broiler strains differ in susceptibility to infectious diseases, most likely due to their genetic differences. The objective of this study was to detect the differentially expressed miRNAs and mRNAs in RSS and normal chickens. By integrating miRNA with mRNA expression profiling, potential molecular mechanisms involved in RSS could be further explored. Twenty-two known miRNAs and 1,159 genes were differentially expressed in RSS chickens compared with normal chickens (P < 0.05). qPCR validation results displayed similar patterns. The differentially expressed genes were primarily involved in energy metabolism pathways. The antisense transcripts were extensively expressed in chicken liver albeit with reduced abundance. Dual-luciferase reporter assay indicated that gga-miR-30b/c directly target CARS through binding to its 3'UTR. The miR-30b/c: CARS regulation mainly occurred in liver. In thigh muscle and the hypothalamus, miR-30b/c are expressed at higher levels in RSS chickens compared with normal chickens from 2 to 6 w of age, and notably significant differences are observed at 4 w of age.

  4. Vitellogenin mRNA expression in Cherax quadricarinatus during secondary vitellogenic at first maturation females.

    PubMed

    Serrano-Pinto, Vania; Landais, Igor; Ogliastro, Marie-Helene; Gutiérrez-Ayala, Meliza; Mejía-Ruíz, Humberto; Villarreal-Colmenares, Humberto; García-Gasca, Alejandra; Vázquez-Boucard, Celia

    2004-09-01

    PCR products of 1.1 and 0.9 kb were generated using Cherax quadricarinatus genomic DNA in the first case, and hepatopancreas and ovary cDNAs in the second case. These PCR products were cloned and analyzed for nucleotide sequences. The 1.1 kb fragment was used as a probe for Northern hybridization, revealing a transcript of approximately 8 kb in both tissues. Results from both Northern blot and RT-PCR analyses showed that the mRNA enconding the 3' end of the vitellogenin cDNA was present simultaneously in both hepatopancreas and ovary tissues in secondary vitellogenic at first maturation females, but was not detected in male hepatopancreas. The deduced amino acid sequences of Vitellogenin (Vg) cDNAs from ovary and hepatopancreas confirmed the existence at least two different Vg genes, and two different sites of synthesis.

  5. LARP6 Meets Collagen mRNA: Specific Regulation of Type I Collagen Expression

    PubMed Central

    Zhang, Yujie; Stefanovic, Branko

    2016-01-01

    Type I collagen is the most abundant structural protein in all vertebrates, but its constitutive rate of synthesis is low due to long half-life of the protein (60–70 days). However, several hundred fold increased production of type I collagen is often seen in reparative or reactive fibrosis. The mechanism which is responsible for this dramatic upregulation is complex, including multiple levels of regulation. However, posttranscriptional regulation evidently plays a predominant role. Posttranscriptional regulation comprises processing, transport, stabilization and translation of mRNAs and is executed by RNA binding proteins. There are about 800 RNA binding proteins, but only one, La ribonucleoprotein domain family member 6 (LARP6), is specifically involved in type I collagen regulation. In the 5′untranslated region (5’UTR) of mRNAs encoding for type I and type III collagens there is an evolutionally conserved stem-loop (SL) structure; this structure is not found in any other mRNA, including any other collagen mRNA. LARP6 binds to the 5′SL in sequence specific manner to regulate stability of collagen mRNAs and their translatability. Here, we will review current understanding of how is LARP6 involved in posttranscriptional regulation of collagen mRNAs. We will also discuss how other proteins recruited by LARP6, including nonmuscle myosin, vimentin, serine threonine kinase receptor associated protein (STRAP), 25 kD FK506 binding protein (FKBP25) and RNA helicase A (RHA), contribute to this process. PMID:27011170

  6. Hyaluronan tetrasaccharides stimulate ceramide production through upregulated mRNA expression of ceramide synthesis-associated enzymes.

    PubMed

    Kage, Madoka; Tokudome, Yoshihiro

    2016-03-01

    It has been reported that hyaluronan has different physiological functions as suggested by variation in molecular weight. In addition, it has also been reported that CD44, the major hyaluronan receptor, was demonstrated to induce keratinocyte differentiation and lipid synthesis of cholesterol. We focus attention on the hyaluronan tetrasaccharides (HA4) which is the smallest unit of hyaluronan. We previously reported that HA4 induced keratinocyte differentiation and that CD44 may be involved. For the purpose of clarifying the influence of HA4 on ceramide synthesis, we evaluated both of these factors in keratinocytes in vitro and in vivo. The mRNA expression of ceramide synthesis-associated enzymes and intracellular ceramide content were evaluated after HA4 treatment in normal human epidermal keratinocytes. In addition, the ceramide increasing effect of HA4 on skin in UVA-irradiated hairless mice was assessed by water content of stratum corneum (SC) and transepidermal water loss (TEWL) methods. The mRNA expression of ceramide synthesis-associated enzymes and intracellular ceramide content after HA4 treatment were increased compared with the control. Furthermore, HA4 treatment increased water content of SC and decreased TEWL. These findings suggest that HA4 affected ceramide synthesis and is involved in the improvement of UV-induced skin damage.

  7. Lack of expression of the liver-type glutaminase (LGA) mRNA in human malignant gliomas.

    PubMed

    Szeliga, Monika; Sidoryk, Marta; Matyja, Ewa; Kowalczyk, Paweł; Albrecht, Jan

    2005-02-21

    In the central nervous system (CNS), liver-type glutaminase (LGA) shows a unique nuclear localization suggesting its role in the regulation of transcription rather than in the cellular glutamine metabolism. RT-PCR analysis of RNA derived from postoperative tissue samples revealed the absence or only traces of LGA mRNA in all (9) cases of malignant gliomas (astrocytoma anaplasticum, AA, WHO grade III; glioblastoma multiforme, WHO grade IV) examined. The RNA was strongly expressed in the non-neoplastic tissue derived from the same patients (6 cases), and in most of the brain metastases from different organs (5 out of 7 cases). By contrast, the mRNAs coding for the kidney-type glutaminase (KGA) and its less ubiquitous isoform GAC, which catalyze degradation of the cytoplasmic pool of Gln, were expressed in all the tissues examined. The lack of LGA may be thus considered as a useful negative diagnostic marker of highly malignant gliomas in situ.

  8. Gene expression profiling by mRNA array reveals different pattern in Chinese glioblastoma patients between Uygur and Han populations

    PubMed Central

    Liu, Liang; Xia, Haichen; Luan, Xinping; Dun, Zhiping; Zhu, Zhengquan; Dushan, Bieke; Li, Wenting

    2015-01-01

    Objective: To identify differentially expressed genes in Chinese glioblastoma patients of Uygur and Han populations, and investigate their potential clinical value for pathogenesis determination and progress prediction. Methods: Gene expression profiling was obtained from three patients of each Uygur and Han nationalities, respectively, by mRNA expression array. Data were processed by the GenomeStudio software and language R of the Lumi package, followed by GO (Gene Ontology) term and KEGG pathway annotation analysis by the Web Gestalt software. Results: The comparative analysis of genome-scale gene expression in glioblastomas revealed 1,475 differentially expressed genes, with 669 and 807 genes up-regulated and down-regulated, respectively. These included the STRC gene, which has two transcripts, one up-regulated and one down-regulated. GO term analysis suggested that 1,175 out of 1,475 key genes were involved in small GTPase mediated signal transduction, Ras protein signal transduction, bioprocess of neuronal response regulation, and central nervous system myelination. The KEGG pathway enrichment analysis showed that the differentially expressed genes were covered by 28 signaling pathways associated with tumorigenesis, including metabolic pathways, tumor suppressor pathways, MAP kinase signaling pathways, TGF-β signaling pathway, neurotrophin signaling pathways, and mTOR signaling pathway. Conclusion: The comparative study of gene expression profiling in glioblastomas between Uygur and Han nationalities revealed differentially expressed genes, whose functions and expression localization were analyzed by GO term analysis and KEGG pathway enrichment analysis. Different pathogenesis mechanisms were proposed for glioblastomas in Chinese patients of Uygur and Han nationalities from a molecular biology perspective. PMID:26309555

  9. Leptin in Whales: Validation and Measurement of mRNA Expression by Absolute Quantitative Real-Time PCR

    PubMed Central

    Ball, Hope C.; Holmes, Robert K.; Londraville, Richard L.; Thewissen, Johannes G. M.; Duff, Robert Joel

    2013-01-01

    Leptin is the primary hormone in mammals that regulates adipose stores. Arctic adapted cetaceans maintain enormous adipose depots, suggesting possible modifications of leptin or receptor function. Determining expression of these genes is the first step to understanding the extreme physiology of these animals, and the uniqueness of these animals presents special challenges in estimating and comparing expression levels of mRNA transcripts. Here, we compare expression of two model genes, leptin and leptin-receptor gene-related product (OB-RGRP), using two quantitative real-time PCR (qPCR) methods: “relative” and “absolute”. To assess the expression of leptin and OB-RGRP in cetacean tissues, we first examined how relative expression of those genes might differ when normalized to four common endogenous control genes. We performed relative expression qPCR assays measuring the amplification of these two model target genes relative to amplification of 18S ribosomal RNA (18S), ubiquitously expressed transcript (Uxt), ribosomal protein 9 (Rs9) and ribosomal protein 15 (Rs15) endogenous controls. Results demonstrated significant differences in the expression of both genes when different control genes were employed; emphasizing a limitation of relative qPCR assays, especially in studies where differences in physiology and/or a lack of knowledge regarding levels and patterns of expression of common control genes may possibly affect data interpretation. To validate the absolute quantitative qPCR methods, we evaluated the effects of plasmid structure, the purity of the plasmid standard preparation and the influence of type of qPCR “background” material on qPCR amplification efficiencies and copy number determination of both model genes, in multiple tissues from one male bowhead whale. Results indicate that linear plasmids are more reliable than circular plasmid standards, no significant differences in copy number estimation based upon background material used, and

  10. Effects of mRNA amplification on gene expression ratios in cDNA experiments estimated by analysis of variance

    PubMed Central

    Nygaard, Vigdis; Løland, Anders; Holden, Marit; Langaas, Mette; Rue, Håvard; Liu, Fang; Myklebost, Ola; Fodstad, Øystein; Hovig, Eivind; Smith-Sørensen, Birgitte

    2003-01-01

    Background A limiting factor of cDNA microarray technology is the need for a substantial amount of RNA per labeling reaction. Thus, 20–200 micro-grams total RNA or 0.5–2 micro-grams poly (A) RNA is typically required for monitoring gene expression. In addition, gene expression profiles from large, heterogeneous cell populations provide complex patterns from which biological data for the target cells may be difficult to extract. In this study, we chose to investigate a widely used mRNA amplification protocol that allows gene expression studies to be performed on samples with limited starting material. We present a quantitative study of the variation and noise present in our data set obtained from experiments with either amplified or non-amplified material. Results Using analysis of variance (ANOVA) and multiple hypothesis testing, we estimated the impact of amplification on the preservation of gene expression ratios. Both methods showed that the gene expression ratios were not completely preserved between amplified and non-amplified material. We also compared the expression ratios between the two cell lines for the amplified material with expression ratios between the two cell lines for the non-amplified material for each gene. With the aid of multiple t-testing with a false discovery rate of 5%, we found that 10% of the genes investigated showed significantly different expression ratios. Conclusion Although the ratios were not fully preserved, amplification may prove to be extremely useful with respect to characterizing low expressing genes. PMID:12659661

  11. Final report: FASEB Summer Research Conference on ''Post-transcriptional control of gene expression: Effectors of mRNA decay'' [agenda and attendees list

    SciTech Connect

    Maquat, Lynne

    2002-12-01

    The goal of this meeting was to provide an interactive forum for scientists working on prokaryotic and eukaryotic mRNA decay. A special seminar presented by a leader in the field of mRNA decay in S. cerevisiae focused on what is known and what needs to be determined, not only for yeast but for other organisms. The large attendance (110 participants) reflects the awareness that mRNA decay is a key player in gene regulation in a way that is affected by the many steps that precede mRNA formation. Sessions were held on the following topics: mRNA transport and mRNP; multicomponent eukaryotic nucleases; nonsense-mediated mRNA decay and nonsense-associated altered splicing; Cis-acting sequences/Trans-acting factors of mRNA decay; translational accuracy; multicomponent bacterial nucleases; interplay between mRNA polyadenylation, translation and decay in prokaryotes and prokaryotic organelles; and RNA interference and other RNA mediators of gene expression. In addition to the talks and two poster sessions, there were three round tables: (1) Does translation occur in the nucleus? (2) Differences and similarities in the mechanisms of mRNA decay in different eukaryotes, and (3) RNA surveillance in bacteria?

  12. Platelet-derived growth factor activity and mRNA expression in healing vascular grafts in baboons. Association in vivo of platelet-derived growth factor mRNA and protein with cellular proliferation.

    PubMed Central

    Golden, M A; Au, Y P; Kirkman, T R; Wilcox, J N; Raines, E W; Ross, R; Clowes, A W

    1991-01-01

    In a baboon graft model of arterial intimal thickening, smooth muscle cells (SMC) have been observed to proliferate underneath an intact monolayer of endothelium and in the absence of platelet adherence. Because platelets are not present and therefore cannot be a major source of growth stimulus, we have proposed that the vascular wall cells in the graft intima express mitogens and regulate SMC proliferation. To test this hypothesis, we assayed the grafts for mitogenic activity and expression of growth factor genes. Segments of healing graft and of normal artery, when perfused ex vivo, released mitogenic activity into the perfusate. The graft released more mitogen than the normal arterial segment, and some of the activity was inhibitable with an antibody to human platelet-derived growth factor (PDGF). In addition, Northern analysis of total RNA demonstrated higher expression of PDGF-A chain mRNA in the graft intima compared to normal artery. PDGF-B chain mRNA was barely detectable in both tissues. PDGF mRNA levels within the graft interstices were not measured. In situ hybridization of 7.5- or 12-wk grafts indicated that some luminal endothelial cells and adjacent intimal SMC contained PDGF-A chain mRNA. By thymidine autoradiography, intimal SMC were observed to be proliferating in the inner third of the intima. These data demonstrate a difference in the pattern of PDGF transcript expression and luminal perfusate activity in graft as compared with control arteries. The association of intimal smooth muscle cell proliferation with intimal PDGF mRNA expression and release of PDGF-like protein supports the hypothesis that factors from cells that have grown into the graft or populated its surface rather than platelets may regulate intimal smooth muscle cell proliferation in this model. Images PMID:1825089

  13. Expression of cadherin-8 mRNA in the developing mouse central nervous system.

    PubMed

    Korematsu, K; Redies, C

    1997-10-20

    The expression of cadherin-8 was mapped by in situ hybridization in the embryonic and postnatal mouse central nervous system (CNS). From embryonic day 18 (E18) to postnatal day 6 (P6), cadherin-8 expression is restricted to a subset of developing brain nuclei and cortical areas in all major subdivisions of the CNS. The anlagen of some of the cadherin-8-positive structures also express this molecule at earlier developmental stages (E12.5-E16). The cadherin-8-positive neuroanatomical structures are parts of several functional systems in the brain. In the limbic system, cadherin-8-positive regions are found in the septal region, habenular nuclei, amygdala, interpeduncular nucleus, raphe nuclei, and hippocampus. Cerebral cortex shows expression in several limbic areas at P6. In the basal ganglia and related nuclei, cadherin-8 is expressed by parts of the striatum, globus pallidus, substantia nigra, entopeduncular nucleus, subthalamic nucleus, zona incerta, and pedunculopontine nuclei. A third group of cadherin-8-positive gray matter structures has functional connections with the cerebellum (superior colliculus, anterior pretectal nucleus, red nucleus, nucleus of posterior commissure, inferior olive, pontine, pontine reticular, and vestibular nuclei). The cerebellum itself shows parasagittal stripes of cadherin-8 expression in the Purkinje cell layer. In the hindbrain, cadherin-8 is expressed by several cranial nerve nuclei. Results from this study show that cadherin-8 expression in the embryonic and postnatal mouse brain is restricted to specific developing gray matter structures. These data support the idea that cadherins are a family of molecules whose expression provides a molecular code for the regionalization of the developing vertebrate brain.

  14. Antiatherogenic effect of Pistacia lentiscus via GSH restoration and downregulation of CD36 mRNA expression.

    PubMed

    Dedoussis, George V Z; Kaliora, Andriana C; Psarras, Stellios; Chiou, Antonia; Mylona, Anastasia; Papadopoulos, Nikolaos G; Andrikopoulos, Nikolaos K

    2004-06-01

    Pistacia lentiscus var. Chia (Anacardiaceae) grows almost exclusively on Chios Island, Greece, and gives a resinous exudate resin used for culinary purposes by Mediterranean people. We investigated the molecular mechanisms through which total polar extract of the resin inhibits oxidized low-density lipoprotein (oxLDL) cytotoxic effect on peripheral blood mononuclear cell (PBMC). Cells exposed to oxLDL underwent apoptosis and necrosis, dependent on the duration of exposure. When culturing cells with oxLDL and the polar extract concurrently, we observed inhibition of both the phenomena. Because under oxidative stress the pro-oxidant systems outbalance the antioxidant, potentially producing oxidative damage and ultimately leading to cell death, we measured the levels of intracellular antioxidant glutathione (GSH). Additionally, we measured CD36 expression, a class B scavenger receptor, on CD14-positive cells, as CD36 has been identified as the oxLDL receptor in macrophages and may play a pivotal role in atherosclerotic foam cell formation. oxLDL decreased GSH levels and upregulated CD36 expression. P. lentiscus extract restored GSH levels and downregulated CD36 expression, even at the mRNA level. In order to find out the biologically drastic constituents of the resin's polar extract, fractions derived from RP-HPLC analysis were examined for their antioxidant effect on oxidatively stressed PBMC. The triterpenoid fraction revealed remarkable increase in intracellular GSH. We suggest GSH restoration and downregulation of CD36 mRNA expression as the pathways via which P. lentiscus triterpenes exert antioxidant/antiatherogenic effect. Additionally, our results provide strong evidence of the resin's antiatherogenic effect; therefore it is credited with beneficial health aspects.

  15. Knockdown of PKD1 in normal human epidermal keratinocytes increases mRNA expression of keratin 10 and involucrin: early markers of keratinocyte differentiation.

    PubMed

    Ivanova, Petya; Atanasova, Ganka; Poumay, Yves; Mitev, Vanyo

    2008-03-01

    Subconfluent normal human keratinocytes exhibit autonomous (autocrine growth factor driven) proliferation and express the specific markers for keratinocyte proliferation K5 (keratin 5) and K14 (keratin 14). Utilizing this model the effects of PKD1 (Protein kinase D1) knockdown on activation of differentiation was studied. siRNA approach was applied to achieve specific knockdown of PKD1 and the mRNA levels of different keratinocyte markers -- K14 and PCNA (markers of basal proliferating keratinocytes), involucrin and K10 (early differentiation markers) were analyzed. Treatment of cultured keratinocytes with siRNA for PKD1 resulted in reduction of mRNA levels of PKD1, altered cell phenotype and promotion of keratinocyte differentiation, demonstrated by increased expression of involucrin and K10 mRNAs. No significant changes in K14 mRNA expression levels were detected, but the expression of PCNA mRNA was markedly diminished. This study was the first to show that mRNA expression of PKD1 in subconfluent normal human keratinocytes is very low, the PKD1 mRNA levels were more than 8-fold lower than the same ones in hTert keratinocytes. These findings suggest antidifferentiative role of PKD1 in normal human keratinocytes, contrary to the prodiferentiative role of PKD1 in human hTert keratinocytes. We came to the conclusion that there are differences between transduction pathways involving PKD1 in primary human keratinocyte cultures and these in immortalized hTert keratinocytes.

  16. Amphiphysin I but not dynamin I nor synaptojanin mRNA expression increased after repeated methamphetamine administration in the rat cerebrum and cerebellum.

    PubMed

    Hamamura, Mitsuko; Okouchi, Jiro; Ozawa, Hidetoshi; Kimuro, Yoshihiko; Iwaki, Akiko; Fukumaki, Yasuyuki

    2013-07-01

    Dopamine increases/decreases synaptic vesicle recycling and in schizophrenia the proteins/mRNA is decreased. We isolated cDNA clone, similar to amphiphysin 1 (vesicle protein) mRNA from the neocortex of rats injected repeatedly with methamphetamine using polymerase chain reaction (PCR) differential display. This clone is highly homologous to the 3' region of the human amphiphysin gene. PCR extension study using a primer specific for the rat amphiphysin 1 gene and a primer located within the clone revealed that it is the 3' UTR region of the rat amphiphysin 1 gene. Furthermore, in situ hybridization revealed that amphiphysin 1 mRNA is expressed in the cerebrum, medial thalamus, hippocampus and cerebellum. In the cerebellum, amphiphysin mRNA expression was confined to upper granule cell layer. Repeated methamphetamine administration increased amphiphysin I mRNA expression in both anterior part of the cerebrum, and the cerebellum. However, the repeated administration did not alter mRNA expression of the other vesicle proteins, synaptotagmin I, synapsin I, synaptojanin and dynamin I, we conclude that the repeated administration selectively increased amphiphysin 1 mRNA expression. Thus, amphiphysin 1 does not work as synaptic recycling, but it is suggested, as a part of pathogenesis of brain tissue injury (under Ca²⁺ and Mg²⁺ devoid environment) in repeated methamphetamine-injected states, the gene regulate actin-asssembly, learning, cell stress signaling and cell polarity.

  17. Complex Control of GABA(A) Receptor Subunit mRNA Expression: Variation, Covariation, and Genetic Regulation

    PubMed Central

    Mulligan, Megan K.; Wang, Xusheng; Adler, Adrienne L.; Mozhui, Khyobeni; Lu, Lu; Williams, Robert W.

    2012-01-01

    GABA type-A receptors are essential for fast inhibitory neurotransmission and are critical in brain function. Surprisingly, expression of receptor subunits is highly variable among individuals, but the cause and impact of this fluctuation remains unknown. We have studied sources of variation for all 19 receptor subunits using massive expression data sets collected across multiple brain regions and platforms in mice and humans. Expression of Gabra1, Gabra2, Gabrb2, Gabrb3, and Gabrg2 is highly variable and heritable among the large cohort of BXD strains derived from crosses of fully sequenced parents—C57BL/6J and DBA/2J. Genetic control of these subunits is complex and highly dependent on tissue and mRNA region. Remarkably, this high variation is generally not linked to phenotypic differences. The single exception is Gabrb3, a locus that is linked to anxiety. We identified upstream genetic loci that influence subunit expression, including three unlinked regions of chromosome 5 that modulate the expression of nine subunits in hippocampus, and that are also associated with multiple phenotypes. Candidate genes within these loci include, Naaa, Nos1, and Zkscan1. We confirmed a high level of coexpression for subunits comprising the major channel—Gabra1, Gabrb2, and Gabrg2—and identified conserved members of this expression network in mice and humans. Gucy1a3, Gucy1b3, and Lis1 are novel and conserved associates of multiple subunits that are involved in inhibitory signaling. Finally, proximal and distal regions of the 3′ UTRs of single subunits have remarkably independent expression patterns in both species. However, corresponding regions of different subunits often show congruent genetic control and coexpression (proximal-to-proximal or distal-to-distal), even in the absence of sequence homology. Our findings identify novel sources of variation that modulate subunit expression and highlight the extraordinary capacity of biological networks to buffer 4–100 fold

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

    PubMed Central

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

    2014-01-01

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

  19. Thyroid Hormone Regulates the mRNA Expression of Small Heterodimer Partner through Liver Receptor Homolog-1

    PubMed Central

    Ahn, Hwa Young; Kim, Hwan Hee; Kim, Ye An; Kim, Min; Ohn, Jung Hun; Chung, Sung Soo; Lee, Yoon-Kwang; Park, Do Joon; Park, Kyong Soo

    2015-01-01

    Background Expression of hepatic cholesterol 7α-hydroxylase (CYP7A1) is negatively regulated by orphan nuclear receptor small heterodimer partner (SHP). In this study, we aimed to find whether thyroid hormone regulates SHP expression by modulating the transcriptional activities of liver receptor homolog-1 (LRH-1). Methods We injected thyroid hormone (triiodothyronine, T3) to C57BL/6J wild type. RNA was isolated from mouse liver and used for microarray analysis and quantitative real-time polymerase chain reaction (PCR). Human hepatoma cell and primary hepatocytes from mouse liver were used to confirm the effect of T3 in vitro. Promoter assay and electrophoretic mobility-shift assay (EMSA) were also performed using human hepatoma cell line Results Initial microarray results indicated that SHP expression is markedly decreased in livers of T3 treated mice. We confirmed that T3 repressed SHP expression in the liver of mice as well as in mouse primary hepatocytes and human hepatoma cells by real-time PCR analysis. LRH-1 increased the promoter activity of SHP; however, this increased activity was markedly decreased after thyroid hormone receptor β/retinoid X receptor α/T3 administration. EMSA revealed that T3 inhibits specific LRH-1 DNA binding. Conclusion We found that thyroid hormone regulates the expression of SHP mRNA through interference with the transcription factor, LRH-1. PMID:26485468

  20. A survey of carbonic anhydrase mRNA expression in enamel cells

    PubMed Central

    LACRUZ, Rodrigo S.; HILVO, Mika; KURTZ, Ira; PAINE, Michael L.

    2010-01-01

    Enamel formation requires rigid control of pH homeostasis during all stages of development to prevent disruptions to crystal growth. The acceleration of the generation of bicarbonate by carbonic anhydrases (CA) has been suggested as one of the pathways used by ameloblasts cells to regulate extracellular pH yet only two isozymes (CA II and CA VI) have been reported to date during enamel formation. The mammalian CA family contains 16 different isoforms of which 13 are enzymatically active. We have conducted a systematic screening by RT-PCR on the expression of all known CA isoforms in mouse enamel organ epithelium (EOE) cells dissected from new born, in secretory ameloblasts derived from 7-day old animals, and in the LS8 ameloblast cell line. Results show that all CA isoforms are expressed by EOE/ameloblast cells in vivo. The most highly expressed are the catalytic isozymes CA II, VI, IX, and XIII, and the acatalytic CA XI isoform. Only minor differences were found in CA expression levels between 1-day EOE cells and 7-day old secretory stage ameloblasts, whereas LS8 cells expressed fewer CA isoforms than both of these. The broad expression of CAs by ameloblasts reported here contributes to our understanding of pH homeostasis during enamel development and demonstrates its complexity. Our results also highlight the critical role that regulation of pH plays during the development of enamel. PMID:20175995

  1. Transcriptional Profiling of mRNA Expression in the Mouse Distal Colon

    PubMed Central

    HOOGERWERF, WILLEMIJNTJE A.; SINHA, MALA; CONESA, ANA; LUXON, BRUCE A.; SHAHINIAN, VAHAKN B.; CORNÉLISSEN, GERMAINE; HALBERG, FRANZ; BOSTWICK, JONATHON; TIMM, JOHN; CASSONE, VINCENT M.

    2009-01-01

    Background & Aims Intestinal epithelial cells and the myenteric plexus of the mouse gastrointestinal tract contain a circadian clock–based intrinsic timekeeping system. Because disruption of the biological clock has been associated with increased susceptibility to colon cancer and gastrointestinal symptoms, we aimed to identify rhythmically expressed genes in the mouse distal colon. Methods Microarray analysis was used to identify genes that were rhythmically expressed over a 24-hour light/dark cycle. The transcripts were then classified according to expression pattern, function, and association with physiologic and pathophysiologic processes of the colon. Results A circadian gene expression pattern was detected in approximately 3.7% of distal colonic genes. A large percentage of these genes were involved in cell signaling, differentiation, and proliferation and cell death. Of all the rhythmically expressed genes in the mouse colon, approximately 7% (64/906) have been associated with colorectal cancer formation (eg, B-cell leukemia/lymphoma-2 [Bcl2]) and 1.8% (18/906) with various colonic functions such as motility and secretion (eg, vasoactive intestinal polypeptide, cystic fibrosis transmembrane conductance regulator). Conclusions A subset of genes in the murine colon follows a rhythmic expression pattern. These findings may have significant implications for colonic physiology and pathophysiology. PMID:18848557

  2. Sequence analysis of choriogenin H gene of medaka (Oryzias latipes) and mRNA expression.

    PubMed

    Lee, Chulwoo; Jeon, Seong Hwan; Na, Jin Gyun; Park, Kwangsik

    2002-08-01

    Zona radiata proteins of medaka (Oryzias latipes) consist of two major subunit groups, ZI-1,2 and ZI-3. The precursor of ZI-1,2, which is a glycoprotein with a molecular weight of 74,000 to 76,000 Da, is called choriogenin H, and the precursor of ZI-3, with a molecular weight of 49,000 Da, is called choriogenin L. The precursor proteins are synthesized in the liver in response to estrogen in sexually mature female medaka. However, they are also induced in the male medaka when fish are exposed to estrogenic chemicals. Therefore, choriogenin is known as a possible sensitive biomarker for endocrine disruption in fish. In this study, the choriogenin H cDNA sequence was reanalyzed and the genomic DNA sequence was newly analyzed. This was done for the selection of proper reverse transcription-polymerase chain reaction (RT-PCR) primers to measure the choriogenin mRNA induction by estrogenic chemicals. In the results, the full cDNA sequence was found to be 2,109 bp long and the size of the open reading frame (ORF) was found to be a total of 1,998 bp encoding 666 predicted amino acids, which was found to be different from previously reported cDNA sequence of medaka choriogenin. In the choriogenin H cDNA, a repetitive domain and a nonrepetitive domain were shown. Regarding the repetitive domain, seven complete repeats of the 45 bp of 5'-ccc cag tac cca tca aag cct cag ccc cct cag aat cct cag gtc-3' encoding PQYPSKPQPPQNPQV were found. Medaka choriogenin H gene was found to possess seven exons and six introns, and the total length was 2,643 bp long. The seven repetitive nucleotide residues described above existed in exon 1, which was found to be 1,033 bp long. Based on the full sequence information, proper primers were synthesized for RT-PCR to detect choriogenin H mRNA induction in male and juvenile medaka by 17alpha-ethinylestradiol, and this type of measurement system was found to be effective as a simple tool for the screening of endocrine-disrupting chemicals.

  3. P-selectin mRNA is expressed at a later phase of megakaryocyte maturation than mRNAs for von Willebrand factor and glycoprotein Ib-alpha.

    PubMed

    Schick, P K; Konkle, B A; He, X; Thornton, R D

    1993-05-01

    The assembly of alpha-granules occurs exclusively in megakaryocytes because platelets have limited capacity for the synthesis of macromolecules. Thus far, alpha-granule development in megakaryocytes has been primarily evaluated by ultrastructural studies. The aim of the study was to obtain molecular and biochemical evidence for the expression of selected alpha-granule proteins in megakaryocytes. Guinea pig megakaryocytes were purified and separated into subgroups at different phases of maturation by the Celsep procedure (Schick et al. Blood 1989;73:1801-8). Guinea pig-specific probes for P-selectin, von Willebrand factor (vWF), glycoprotein Ib-alpha (GpIb-alpha), and phosphoglycerate kinase were prepared by using the polymerase chain reaction. By Northern blot analysis, P-selectin messenger ribonucleic acid (mRNA) was primarily expressed in the mature megakaryocyte Celsep subgroup, whereas vWF and GpIb-alpha mRNA were expressed at all phases of megakaryocyte maturation. In situ hybridization confirmed that P-selectin mRNA was primarily expressed at later stages of cytoplasmic maturation: 14% +/- 6.2% of stage I, 35.5% +/- 6.1% of stage II, 72% +/- 5.2% of stage III, and 47.0% +/- 3.3% of stage IV megakaryocytes expressed P-selectin mRNA. Thus, the expression of mRNA for P-selectin appeared to peak in stage III cells. In contrast vWF mRNA was expressed in immature megakaryocytes and persisted throughout megakaryocyte maturation. In situ hybridization did not demonstrate a relationship between the expression of mRNA for P-selectin or vWF with megakaryocyte ploidy.(ABSTRACT TRUNCATED AT 250 WORDS)

  4. Effect of cortisol on neurophysin I/oxytocin and peptidyl glycine-alpha-amidating mono-oxygenase mRNA expression in bovine luteal and granulosa cells.

    PubMed

    Ziolkowska, A; Mlynarczuk, J; Kotwica, J

    2013-01-01

    Cortisol stimulates the synthesis and secretion of oxytocin (OT) from bovine granulosa and luteal cells, but the molecular mechanisms of cortisol action remain unknown. In this study, granulosa cells or luteal cells from days 1-5 and 11-15 of the oestrous cycle were incubated for 4 or 8 h with cortisol (1 x 10(-5), 1 x 10(-7) M). After testing cell viability and hormone secretion (OT, progesterone, estradiol), we studied the effect of cortisol on mRNA expression for precursor of OT (NP-I/OT) and peptidyl glycine-alpha-amidating mono-oxygenase (PGA). The influence of RU 486 (1 x 10(-5) M), a progesterone receptor blocker and inhibitor of the glucocorticosteroid receptor (GR), on the expression for both genes was tested. Cortisol increased the mRNA expression for NP-I/OT and PGA in granulosa cells and stimulated the expression for NP-I/OT mRNA in luteal cells obtained from days 1-5 and days 11-15 of the oestrous cycle. Expression for PGA mRNA was increased only in luteal cells from days 11-15 of the oestrous cycle. In addition, RU 486 blocked the cortisol-stimulated mRNA expression for NP-I/OT and PGA in both types of cells. These data suggest that cortisol affects OT synthesis and secretion in bovine ovarian cells, by acting on the expression of key genes, that may impair ovary

  5. Isoform-specific regulation of transforming growth factor-β mRNA expression in macrophages in response to adrenoceptor stimulation.

    PubMed

    Yanagawa, Yoshiki; Hiraide, Sachiko; Iizuka, Kenji

    2016-01-01

    Transforming growth factor-beta (TGF-β) is a multifunctional cytokine responsible for both immune regulation and tissue repair. Although TGF-β consists of TGF-β1, -β2, and -β3 in mammals, isoform-selective transcriptional regulation is less well documented in myeloid linage cells such as macrophages. In the present study, the effect of the stress-related catecholamine adrenaline on the expression of TGF-β isoforms in RAW264.7 macrophages and murine bone marrow-derived macrophages was examined. Treatment with adrenaline markedly increased the mRNA expression of TGF-β3 but not of TGF-β1 and -β2. Agonist and antagonist studies indicated that adrenaline-induced TGF-β3 mRNA expression is mediated via β2 -adrenoceptor. Protein kinase A (PKA) inhibitor H89 was found to block an increase in adrenoceptor-mediated TGF-β3 mRNA expression. The membrane-permeable cAMP analog 8-Br-cAMP increased the mRNA expression of TGF-β3 but not of TGF-β1 and -β2. Thus, the β2 -adrenoceptor-mediated cAMP-PKA pathway appears to enhance TGF-β3 mRNA expression in macrophages. Adrenoceptor-mediated TGF-β3 expression by macrophages may influence immune regulation and tissue repair in conditions of stress, during which the sympathetic-nervous system releases catecholamines.

  6. Expression of Ets-1 and FOXP3 mRNA in CD4(+)CD25 (+) T regulatory cells from patients with systemic lupus erythematosus.

    PubMed

    Xiang, Nan; Li, Xiang-Pei; Li, Xiao-Mei; Wang, Guo-Sheng; Tao, Jin-Hui; Pan, Hai-Feng; Fang, Xuan; Ma, Qian; Yu, Ning

    2014-11-01

    Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease with complex genetic predisposing factors involved. Ets-1 transcription factor plays an important role in the suppressive activity of CD4(+)CD25(+) Treg cells and stable expression of FOXP3. To find its potential role in the pathogenesis of SLE, we investigate the mRNA expression of Ets-1 and FOXP3 mRNA in CD4(+)CD25(+) Treg cells from patients with SLE. Real-time transcription-polymerase chain reaction analysis was used to determine the expression of Ets-1 and FOXP3 mRNA in CD4(+)CD25(+) Treg cells from 36 patients with SLE and 18 sex-and-age-matched healthy controls. The Ets-1 mRNA expression level was decreased in patients with SLE [0.225 (0.135, 0.337)] than healthy controls [0.528 (0.303, 0.681)] (P < 0.001). The expression levels of FOXP3 mRNA were lower in SLE patients [0.608 (0.272, 1.164)] than healthy controls [0.919 (0.690, 1.223)], but the difference was not significant (P = 0.106). Significant reduction in Ets-1 and FOXP3 expression was also found in new-onset SLE subgroup when compared with healthy controls (P < 0.001). The level of Ets-1 and FOXP3 mRNA was not significantly different in hyperactive and lower active SLE group when compared with inactive SLE group, respectively (P > 0.05). There were no significant differences between SLE with lupus nephritis (LN) and SLE without LN either (P > 0.05). Associations of Ets-1 and FOXP3 mRNA expression levels with major clinical and laboratory parameters of SLE patients were also analyzed. However, no significant association was found. Significant positive correlation was found between Ets-1 and FOXP3 mRNA expression in CD4(+)CD25(+) Treg cells from SLE patients (r = 0.698, P < 0.001). Our results found that the expression levels of Ets-1 mRNA were decreased in SLE patients and Ets-1 expression was positively correlated with the expression of FOXP3. It indicated that Ets-1 may play an important role in the stable expression of FOXP3 in

  7. Regulation of mRNA expression in drug-sensitive and drug-resistant gastric carcinoma cells is independent of YB-1 expression.

    PubMed

    Kurucz, Reka; Belian, Elisa; Treue, Denise; Lage, Hermann

    2010-02-01

    Y-Box protein 1 (YB-1) is a multifunctional cellular protein expressed in a range of mammalian cells, including human cancer cells. It is involved in the regulation of various genes including cancer-associated genes, but the full range of target genes and regulatory mechanisms have not been fully elucidated. To identify global mRNA expression patterns that are potentially regulated by YB-1, a previously established and well-characterized cell model derived from drug-sensitive (EPG85-257P/tetR/YB-1) and multidrug-resistant (EPG85-257RDB/tetR/YB-1) gastric carcinoma cells in which the expression of YB-1 can be inhibited by tetracycline-dependent activation of the RNA interference (RNAi) pathway, was analyzed by microarray technology. By this approach, various potentially regulated genes encoding members of important cellular pathways such as the Jak/STAT, VEGF and the MAP-kinase signaling pathways were identified. Independent validation of these findings by quantitative real-time reverse transcriptase polymerase chain reaction and Western blot did not confirm these regulatory effects. In conclusion, the findings suggest that YB-1 is not directly involved in the regulation of mRNA expression in drug-sensitive or drug-resistant gastric carcinoma cells.

  8. Cyclic mRNA expression of thyrotropin subunits and deiodinases in red drum, Sciaenops ocellatus.

    PubMed

    Jones, R A; Cohn, W B; Miller, T C; Jaques, J T; Mackenzie, D S

    2013-12-01

    The role of thyrotropin (thyroid-stimulating hormone, TSH) in driving peripheral thyroid function in non-mammalian species is still poorly understood. Thyroxine (T₄), the principal hormone released from the thyroid gland in response to TSH stimulation, circulates with a robust daily rhythm in the teleost fish the red drum. Previous research suggests that the red drum T₄ cycle is circadian in nature, driven by TSH secretion in the early photophase and inhibited by T₄ feedback in the early scotophase. To determine whether TSH is produced in a pattern consistent with feedback inhibition by this T₄ cycle, we used quantitative real time PCR (qPCR) to quantify the daily cycle of expression of the pituitary TSH subunits GSUα, and TSHβ. We found that TSH expression cycled inversely to, and 6-12 h out of phase with, the T₄ cycle, consistent with the hypothesis that TSH secretion drives the T₄ cycle. To examine the potential role of deiodinases in negative feedback regulation of this TSH cycle, we also utilized qPCR to assess the pituitary expression patterns of the TH activating enzyme outer-ring deiodinase (Dio2) and the TH deactivating enzyme inner-ring deiodinase (Dio3). Dio2 was not expressed with an obvious daily cycle, whereas Dio3 expression mirrored the expression of TSH. These results are consistent with circulating T₄ providing the negative feedback signal controlling both TSH production and Dio3 expression in the pituitary, and suggest that TH inactivation by inner ring deiodination is an important component of TSH negative feedback control.

  9. [mRNA expression of notch ligand-delta-like-1 and jagged-1 in mesenchymal stem cells of MDS patients].

    PubMed

    Fei, Cheng-Ming; Gu, Shu-Cheng; Zhao, You-Shan; Guo, Juan; Li, Xiao; Chang, Chun-Kang

    2014-12-01

    This study was aimed to investigated the mRNA expression levels of Notch ligands- Delta-like-1 and Jagged-1 in bone marrow mesenchymal stem cells of patients with myelodysplastic syndrome (MDS), and to explore their relation with onset of MDS. Bone marrow mesenchymal stem cells of 38 patients with MDS and 16 normal subjects as control were collected to detect mRNA expression of Delta-like-1 and Jagged-1 by using real-time quantitative polymerase chain reaction. The results showed that the expression levels of Delta-like-1 and Jagged-1 in mesenchymal stem cells of MDS patients were significantly higher than that in normal controls (P < 0.05). According to WHO criteria, the mRNA expression of Delta-like-1 in RA/RAS, RCMD and RAEB groups were significantly higher than that in normal controls (P < 0.05), the mRNA expression of Jagged-1 in RAEB group was also significantly higher than that in normal controls (P < 0.05). The mRNA expression of Delta-like-1 was significantly correlated with the proportion of blasts in the bone marrow of MDS patients (r = 0.502, P < 0.05). The expression levels of Delta-like-1 and Jagged-1 in MDS patients with abnormal karyotypes were significantly higher than those in MDS patients with normal karyotypes (P < 0.05). The mRNA expression of Delta-like-1 in higher risk group according to International Prognostic Scoring System was significantly higher than that in lower risk group (P < 0.05), there was no significant difference in Jagged-1 expression levels between higher risk group and lower risk group (P > 0.05). It is concluded that the changes of Delta-like-1 and Jagged-1 expression level in MSC may play a role in the pathogenesis of myelodysplastic syndrome.

  10. The Integrative Analysis of microRNA and mRNA Expression in Mouse Uterus under Delayed Implantation and Activation

    PubMed Central

    Liu, Ji-Long; Zhang, Zhi-Rong; Jia, Bo; Feng, Xu-Hui; Ren, Gang; Hu, Shi-Jun; Yang, Zeng-Ming

    2010-01-01

    Background Delayed implantation is a developmental arrest at the blastocyst stage and a good model for embryo implantation. MicroRNAs (miRNAs) have been shown to be involved in mouse embryo implantation through regulating uterine gene expression. This study was to have an integrative analysis on global miRNA and mRNA expression in mouse uterus under delayed implantation and activation through Illumina sequencing. Methodology/Principal Findings By deep sequencing and analysis, we found that there are 20 miRNAs up-regulated and 42 miRNAs down-regulated at least 1.2 folds, and 268 genes up-regulated and 295 genes down-regulated at least 2 folds under activation compared to delayed implantation, respectively. Many different forms of editing in mature miRNAs are detected. The percentage of editing at positions 4 and 5 of mature miRNAs is significantly higher under delayed implantation than under activation. Although the number of miR-21 reference sequence under activation is slightly lower than that under delayed implantation, the total level of miR-21 under activation is higher than that under delayed implantation. Six novel miRNAs are predicted and confirmed. The target genes of significantly up-regulated miRNAs under activation are significantly enriched. Conclusions miRNA and mRNA expression patterns are closely related. The target genes of up-regulated miRNAs are significantly enriched. A high level of editing at positions 4 and 5 of mature miRNAs is detected under delayed implantation than under activation. Our data should be valuable for future study on delayed implantation. PMID:21124741

  11. Use of mRNA expression to detect the induction of drug metabolising enzymes in rat and human hepatocytes

    SciTech Connect

    Richert, L. Tuschl, G.; Pekthong, D.; Mantion, G.; Weber, J.-C.; Mueller, S.O.

    2009-02-15

    It is important to investigate the induction of cytochrome P450 (CYP) enzymes by drugs. The most relevant end point is enzyme activity; however, this requires many cells and is low throughput. We have compared the CYP1A, CYP2B and CYP3A induction response to eight inducers in rat and human hepatocytes using enzyme activities (CYP1A2 (ethoxyresorufin), 2B (benzoxyresorufin for rat and bupropion for human) and CYP3A (testosterone)) and Taqman{sup TM} Low Density Array (TLDA) analysis. There was a good correlation between the induction of CYP1A2, CYP2B6 and CYP3A4 enzyme activities and mRNA expression in human hepatocytes. In contrast, BROD activities and mRNA expression in rat hepatocytes correlated poorly. However, bupropion hydroxylation correlated well with Cyp2b1 expression in rat hepatocytes. TLDA analysis of a panel of mRNAs encoding for CYPs, phase 2 enzymes, nuclear receptors and transporters revealed that the main genes induced by the 8 compounds tested were the CYPs. AhR ligands also induced UDP-glucuronosyltransferases and glutathione S-transferases in rat and human hepatocytes. The transporters, MDR1, MDR3 and OATPA were the only transporter genes significantly up-regulated in human hepatocytes. In rat hepatocytes Bsep, Mdr2, Mrp2, Mrp3 and Oatp2 were up-regulated. We could then show a good in vivo:in vitro correlation in the induction response of isolated rat hepatocytes and ex-vivo hepatic microsomes for the drug development candidate, EMD392949. In conclusion, application of TLDA methodology to investigate the potential of compounds to induce enzymes in rat and human hepatocytes increases the throughput and information gained from one assay, without reducing the predictive capacity.

  12. Differential regulation of cytokine and cytokine receptor mRNA expression upon infection of bone marrow-derived macrophages with Listeria monocytogenes.

    PubMed Central

    Demuth, A; Goebel, W; Beuscher, H U; Kuhn, M

    1996-01-01

    Cytokine and cytokine receptor mRNA expression was analyzed by PCR-assisted amplification of RNA extracted from bone marrow-derived macrophages (BMM phi) at different time points after infection with Listeria monocytogenes. The mRNAs for the cytokines interleukin-1 alpha (IL-1 alpha), IL-1 beta, and tumor necrosis factor alpha (TNF-alpha) were induced early after infection, whereas IL-6 mRNA appeared later and even nonhemolytic Listeria strains, which are unable to grow inside eukaryotic cells, induced the same cytokine mRNAs at levels similar to those of the wild-type strain. In most cases, the amounts of cytokines determined by various bioassays correlated with the level of mRNA induction. Inhibition of phagocytic uptake of L. monocytogenes by cytochalasin D treatment resulted in adherent bacteria which still induced the proinflammatory cytokines. In BMM phi, the level of IL-1 receptor II mRNA was unaffected, whereas mRNA expression of the two subtypes of tumor necrosis factor receptors (TNF-RI and TNF-RII) was differentially regulated upon infection: transcription of TNF-RI was reduced, and that of TNF-RII mRNA was induced. Similar to the decreased TNF-RI mRNA expression, gamma interferon receptor mRNA was downregulated in L. monocytogenes-infected BMM phi. This dose- and time-dependent induction or downregulation of cytokine receptor mRNA following L. monocytogenes infection of BMM phi was not observed upon infection of established macrophage-like cell lines J774 and P388D1. Induction of IL-6 mRNA as well as IL-1 alpha/beta and TNF-alpha mRNAs upon L. monocytogenes infection of BMM phi occurs independently of autocrine TNF-alpha signaling via TNF-RI or TNF-RII, as shown by infection of TNF-RI- and TNF-RII-deficient macrophages derived from mutant B6 x 129 mice. In contrast to gamma interferon receptor mRNA, both TNF receptor subtype mRNAs were not influenced by L. monocytogenes infection of hybrid (B6 x 129) mouse macrophages. Whereas the proinflammatory

  13. Effect of infliximab on mRNA expression profiles in synovial tissue of rheumatoid arthritis patients

    PubMed Central

    Lindberg, Johan; af Klint, Erik; Catrina, Anca Irinel; Nilsson, Peter; Klareskog, Lars; Ulfgren, Ann-Kristin; Lundeberg, Joakim

    2006-01-01

    We examined the gene expression profiles in arthroscopic biopsies retrieved from 10 rheumatoid arthritis patients before and after anti-TNF treatment with infliximab to investigate whether such profiles can be used to predict responses to the therapy, and to study effects of the therapy on the profiles. Responses to treatment were assessed using European League Against Rheumatism response criteria. Three patients were found to be good responders, five patients to be moderate responders and two patients to be nonresponders. The TNF-α status of the biopsies from each of the patients before treatment was also investigated immunohistochemically, and it was detected in biopsies from four of the patients, including all three of the good responders. The gene expression data demonstrate that all patients had unique gene expression signatures, with low intrapatient variability between biopsies. The data also revealed significant differences between the good responding and nonresponding patients (279 differentially expressed genes were detected, with a false discovery rate < 0.025). Among the identified genes we found that MMP-3 was significantly upregulated in good responders (log2 fold change, 2.95) compared with nonresponders, providing further support for the potential of MMP-3 as a marker for good responses to therapy. An even more extensive list of 685 significantly differentially expressed genes was found between patients in whom TNF-α was found and nonresponders, indicating that TNF-α could be an important biomarker for successful infliximab treatment. Significant differences were also observed between biopsies taken before and after anti-TNF treatment, including 115 differentially expressed genes in the good responding group. Interestingly, the effect was even stronger in the group in which TNF-α was immunohistochemically detected before therapy. Here, 1,058 genes were differentially expressed, including many that were novel in this context (for example, CXCL3

  14. Medium dose ultraviolet A1 phototherapy and mRNA expression of interleukin 8, interferon γ, and chemokine receptor 4 in acute skin lesions in atopic dermatitis

    PubMed Central

    Malinowska, Karolina; Sysa-Jedrzejowska, Anna; Wozniacka, Anna

    2016-01-01

    Introduction Mechanisms responsible for UVA1 efficacy in atopic dermatitis (AD) are not fully elucidated. Aim To investigate IL-8, CCR-4, and IFN-γ mRNA expression in AD before and after UVA1, to identify correlations among them, and to determine whether and to what degree mRNA expression is influenced by UVA1. Material and methods Twenty-five patients with AD underwent medium dose UVA1-phototherapy at daily dosages of 10, 20, 30, 45, and then continuing 45 J/cm2 up to 20 days, from Monday to Friday for 4 weeks. Before and after UVA1, biopsies from acute skin lesions were studied using reverse-transcription and RT-PCR. Results The levels of CCR-4 mRNA correlated with those of IFN-γ, both before and after UVA1 phototherapy (p < 0.05). A significant correlation was found after UVA1 between mRNA levels of IL-8 and IFN-γ (p < 0.05). After UVA1 an increase in IL-8 mRNA expression in comparison to the baseline assessment (p = 0.02) was found, while no significant difference was revealed in the expression of CCR-4 and IFN-γ mRNA. UVA1 improved both SCORAD and severity of AD (p < 0.001). SCORAD and the severity of AD did not correlate with the degree of expression of measured cytokine mRNA, neither before nor after UVA1. Conclusions CCR-4 is expressed in parallel with IFN-γ in acute skin lesions of patients with AD both before and after UVA1 phototherapy. UVA1 significantly improves SCORAD index, lessens the severity of AD and increases the expression of IL-8, with no direct effects on other studied molecules. PMID:27512350

  15. Effect of sodium hydrosulfide on mRNA expression of prostaglandin E2 receptors in response to mucosal acidification and distention-induced gastric acid secretion in rats

    PubMed Central

    Mard, Seyyed Ali; Mahini, Simin; Dianat, Mahin; Farbood, Yaghoob

    2017-01-01

    Objective(s): Prostaglandins have been shown to mediate the gastro-protective effect of sodium hydrosulfide (NaHS) but effect of NaHS on mRNA expression of prostaglandin E2 receptors (EP1, 3-4; EPs) has not been investigated. Therefore, this study designed to evaluate the effect of NaHS on mRNA expression of EPs receptors in response to mucosal acidification and distention-induced gastric acid secretion in rats. Materials and Methods: Fasted rats were randomly assigned into 4 groups (n=6/group). They were control, and NaHS-treated groups. To evaluate the effect of NaHS on mucosal mRNA expression of EPs receptors, the gastric mucosa exposed to stimulated gastric acid output and mucosal acidification. The pylorus sphincter catheterized for instillation of isotonic neutral saline or acidic solution. Ninety min after beginning the experiments, animals sacrificed and the gastric mucosa collected to determine the pH, mucus secretion and to quantify the mRNA expression of EPs receptors by quantitative real-time PCR. Results: present results showed that a) NaHS increased the mucus secretion, mRNA expression of EP3 and EP4 receptors in response to distention-induced expression; b) The mRNA expression of EP1 receptors increased while EP4 mRNA receptors decreased in response to mucosal acidification in NaHS-pretreated rats; and c) NaHS increased pH of gastric contents both in response to distention-induced gastric acid secretion and mucosal acidification. Conclusion: NaHS behaves in a different manner. It effectively only increased the pH of gastric contents to reinforce the gastric mucosa against a highly acidic solution but modulated both acid and mucus secretion when the rate of acid increase in the stomach was slower. PMID:28293390

  16. Impact of post-synaptic block of neuromuscular transmission, muscle unloading and mechanical ventilation on skeletal muscle protein and mRNA expression.

    PubMed

    Norman, H; Nordquist, J; Andersson, P; Ansved, T; Tang, X; Dworkin, B; Larsson, L

    2006-10-01

    To analyse mechanisms of muscle wasting in intensive care unit patients, we developed an experimental model where rats were pharmacologically paralysed by post-synaptic block of neuromuscular transmission (NMB) and mechanically ventilated for 9+/-2 days. Specific interest was focused on the effects on protein and mRNA expression of sarcomeric proteins, i.e., myosin heavy chain (MyHC), actin, myosin-binding protein C (MyBP-C) and myosin-binding protein H (MyBP-H) in fast- and slow-twitch limb, respiratory and masticatory muscles. Muscle-specific differences were observed in response to NMB at both the protein and mRNA levels. At the protein level, a decreased MyHC-to-actin ratio was observed in all muscles excluding the diaphragm, whereas at the mRNA level a decreased expression of the dominating MyHC isoform(s) was observed in the hind limb and intercostal muscles, but not in the diaphragm and masseter muscles. MyBP-C mRNA expression was decreased in the limb muscles, but it otherwise remained unaffected. MyBP-H conversely increased in all muscles. Furthermore, we found myofibrillar protein and mRNA expression to be affected differently when comparing NMB animals with peripherally denervated (DEN) ambulatory rats. We report that NMB has both a larger and different impact on muscle, at the protein and mRNA levels, than DEN has.

  17. Expression of homothorax and extradenticle mRNA in the legs of the crustacean Parhyale hawaiensis: evidence for a reversal of gene expression regulation in the pancrustacean lineage.

    PubMed

    Prpic, Nikola-Michael; Telford, Maximilian J

    2008-06-01

    In Drosophila leg development, the extradenticle (exd) gene is expressed ubiquitously and its co-factor homothorax (hth) is restricted to the proximal leg portion. This condition is conserved in other insect species but is reversed in chelicerates and myriapods. As the region of co-expression does not differ in the two groups and transcripts from both are necessary for function, this difference in expression is likely to be functionally neutral. Here, we report the expression patterns of exd and hth in a crustacean, the amphipod shrimp Parhyale hawaiensis. The patterns in P. hawaiensis are similar to the insect patterns, supporting the close relationship between crustaceans and insects in the taxon Tetraconata. However, mRNA expression of exd in P. hawaiensis is weak in the distal leg parts, thus being intermediate between the complete lack of distal exd expression in chelicerates and myriapods and the strong distal exd expression in insects. Our data suggest that the reversal of the gene expression regulation of hth and exd occurred in the pancrustacean lineage.

  18. Expression of mRNA in the frontal cortex and hypothalamus in a rat model of acute carbon dioxide poisoning.

    PubMed

    Sato, Kazuo; Tsuji, Akiko; Usumoto, Yosuke; Kudo, Keiko; Yokoyama, Takeshi; Ikeda, Noriaki

    2016-03-01

    Acute carbon dioxide (CO2) poisoning causes no specific features that are revealed upon autopsy, and the pathophysiological mechanism of this syndrome is unclear. To address this issue, in the present study, we exposed rats to CO2 concentrations ranging from 10% to 60% and determined the effects on mRNA expression. According to the results of Gene Ontology (GO) and cluster analyses of microarrays data, we selected the following genes for further analysis: alkylglycerone phosphate synthase (Agps), hypocretin (Hcrt), tyrosine hydroxylase (Th), heat shock protein beta 2 (Hspb2), and opioid receptor delta 1 (Oprd1) expressed in the frontal cortex and renin (Ren), pancreatic polypeptide (Ppy), corticotropin releasing hormone receptor 2 (Crhr2), carbonic anhydrase 1 (Car1), and hypocretin receptor 1 (Hcrtr1) expressed in the hypothalamus. We found significant differences between the expression levels of Agps and Hspb2 mRNAs in the frontal cortex and that of Ppy, Crhr2 mRNAs in the hypothalamus in the presence of high concentrations of CO2. Further investigation of these genes may clarify the pathophysiology of acute CO2 poisoning and facilitate the development of novel forensic tests that can diagnose the cause of death.

  19. Ammonia upregulates kynurenine aminotransferase II mRNA expression in rat brain: a role for astrocytic NMDA receptors?

    PubMed

    Obara-Michlewska, Marta; Tuszyńska, Paulina; Albrecht, Jan

    2013-06-01

    Kynurenine aminotransferase II (KAT-II) is the astrocytic enzyme catalyzing the synthesis of kynurenic acid (KYNA), an endogenous inhibitor of the α7-nicotinic receptor and the NMDA receptor (NMDAr). A previous study demonstrated an increase of KYNA synthesis in the brain of rats with thioacetamide (TAA)-induced acute liver failure. Here we show that TAA administration increases KAT-II expression in the rat cerebral cortex and the effect is mimicked in cerebral cortical astrocytes in culture treated with high (5 mM) concentration of ammonia. KAT-II expression in control and TAA-treated rats was increased by NMDAr antagonist memantine, and the effects of TAA and memantine appeared additive. In astrocytes, the NMDAr antagonist MK-801 raised KAT-II expression as well, while NMDA added alone had no effect. Glutamate decreased KAT-II mRNA level, which was attenuated by MK-801. The results suggest that stimulation of KAT-II expression during hepatic encephalopathy may be associated with a partial inactivation of astrocytic NMDAr by ammonia.

  20. Kisspeptin mRNA expression is increased in the posterior hypothalamus in the rat model of polycystic ovary syndrome.

    PubMed

    Matsuzaki, Toshiya; Tungalagsuvd, Altankhuu; Iwasa, Takeshi; Munkhzaya, Munkhsaikhan; Yanagihara, Rie; Tokui, Takako; Yano, Kiyohito; Mayila, Yiliyasi; Kato, Takeshi; Kuwahara, Akira; Matsui, Sumika; Irahara, Minoru

    2017-01-30

    Hypersecretion of luteinizing hormone (LH) is a common endocrinological finding of polycystic ovary syndrome (PCOS). This derangement might have a close relationship with hypothalamic kisspeptin expression that is thought to be a key regulator of gonadotropin-releasing hormone (GnRH). We evaluated the relationship between the hypothalamic-pituitary-gonadal axis (HPG axis) and kisspeptin using a rat model of PCOS induced by letrozole. Letrozole pellets (0.4 mg/day) and control pellets were placed subcutaneously onto the backs of 3-week-old female Wistar rats. Body weight, vaginal opening and vaginal smear were checked daily. Blood and tissues of ovary, uterus and brain were collected at 12-weeks of age. An hypothalamic block was cut into anterior and posterior blocks, which included the anteroventral periventricular nucleus (AVPV) and the arcuate nucleus (ARC), respectively, in order to estimate hypothalamic kisspeptin expression in each area. The letrozole group showed a similar phenotype to human PCOS such as heavier body weight, heavier ovary, persistent anovulatory state, multiple enlarged follicles with no corpus luteum and higher LH and testosterone (T) levels compared to the control group. Kisspeptin mRNA expression in the posterior hypothalamic block including ARC was higher in the letrozole group than in the control group although its expression in the anterior hypothalamic block was similar between groups. These results suggest that enhanced KNDy neuron activity in ARC contributes to hypersecretion of LH in PCOS and might be a therapeutic target to rescue ovulatory disorder of PCOS in the future.

  1. Expression of 6-phosphofructo-2-kinase/ fructose-2,6-bisphosphatase-3 and VEGF mRNA in rat liver, lung and heart: effect of methyl tertbutyl ether.

    PubMed

    Minchenko, D O; Kundieva, A V; Tsuchihara, K; Yavorovsky, O P; Paustovsky, Y O; Esumi, H; Minchenko, O H

    2009-01-01

    The main goal of this work was investigation of the effect of methyl tertbutyl ether, ecologically dangerous chemical compound, on the expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB-3) and vascular endothelial growth factor (VEGF) mRNA in different rat organs. Expression of PFKFB-3 and VEGF is a hypoxia inducible factor (HIF)-dependent process which significantly increases under hypoxia, in malignant tumors and other pathology. In this study we have shown that PFKFB-3 and VEGF mRNA expression in the liver, lung, and heart changes in rats, treated with methyl tertbutyl ether for two months, in organ-specific manner. Expression of alternative splice variants of PFKFB-3 mRNA as well as VEGF mRNA also changes in organ-specific manner in rats, treated with methyl tertbutyl ether. The effect of methyl tertbutyl ether on the expression of PFKFB-3 and VEGF mRNA and its alternative splice variants is dose-dependent. Results of this investigation clearly demonstrated that methyl tertbutyl ether affects the expression of PFKFB-3, a key regulatory enzyme of glycolysis, as well as VEGF, very important factor of angiogenesis, in an organ-specific and dose-dependent manner.

  2. mRNA expression profiling of laser microbeam microdissected cells from slender embryonic structures.

    PubMed

    Scheidl, Stefan J; Nilsson, Sven; Kalén, Mattias; Hellström, Mats; Takemoto, Minoru; Håkansson, Joakim; Lindahl, Per

    2002-03-01

    Microarray hybridization has rapidly evolved as an important tool for genomic studies and studies of gene regulation at the transcriptome level. Expression profiles from homogenous samples such as yeast and mammalian cell cultures are currently extending our understanding of biology, whereas analyses of multicellular organisms are more difficult because of tissue complexity. The combination of laser microdissection, RNA amplification, and microarray hybridization has the potential to provide expression profiles from selected populations of cells in vivo. In this article, we present and evaluate an experimental procedure for global gene expression analysis of slender embryonic structures using laser microbeam microdissection and laser pressure catapulting. As a proof of principle, expression profiles from 1000 cells in the mouse embryonic (E9.5) dorsal aorta were generated and compared with profiles for captured mesenchymal cells located one cell diameter further away from the aortic lumen. A number of genes were overexpressed in the aorta, including 11 previously known markers for blood vessels. Among the blood vessel markers were endoglin, tie-2, PDGFB, and integrin-beta1, that are important regulators of blood vessel formation. This demonstrates that microarray analysis of laser microbeam micro-dissected cells is sufficiently sensitive for identifying genes with regulative functions.

  3. Single-Cell mRNA Profiling Reveals Cell-Type Specific Expression of Neurexin Isoforms

    PubMed Central

    Fuccillo, Marc V.; Földy, Csaba; Gökce, Özgün; Rothwell, Patrick E.; Sun, Gordon L.; Malenka, Robert C.; Südhof, Thomas C.

    2016-01-01

    Summary Neurexins are considered central organizers of synapse architecture that are implicated in neuropsychiatric disorders. Expression of neurexins in hundreds of alternatively spliced isoforms suggested that individual neurons might exhibit a cell type-specific neurexin expression pattern (a neurexin code). To test this hypothesis, we quantified the single-cell levels of neurexin isoforms and other trans-synaptic cell-adhesion molecules by microfluidics-based RT-PCR. We show that the neurexin repertoire displays pronounced cell-type specificity that is remarkably consistent within each type of neuron. Furthermore, we uncovered region-specific regulation of neurexin transcription and splice-site usage. Finally, we demonstrate that the transcriptional profiles of neurexins can be altered in an experience-dependent fashion by exposure to a drug of abuse. Our data provide evidence of cell type-specific expression patterns of multiple neurexins at the single-cell level, and suggest that expression of synaptic cell-adhesion molecules overlaps with other key features of cellular identity and diversity. PMID:26182417

  4. Customized Treatment in Non-Small-Cell Lung Cancer Based on EGFR Mutations and BRCA1 mRNA Expression

    PubMed Central

    Rosell, Rafael; Perez-Roca, Laia; Sanchez, Jose Javier; Cobo, Manuel; Moran, Teresa; Chaib, Imane; Provencio, Mariano; Domine, Manuel; Sala, Maria Angeles; Jimenez, Ulpiano; Diz, Pilar; Barneto, Isidoro; Macias, Jose Antonio; de las Peñas, Ramon; Catot, Silvia; Isla, Dolores; Sanchez, Jose Miguel; Ibeas, Rafael; Lopez-Vivanco, Guillermo; Oramas, Juana; Mendez, Pedro; Reguart, Noemi; Blanco, Remei; Taron, Miquel

    2009-01-01

    Background Median survival is 10 months and 2-year survival is 20% in metastatic non-small-cell lung cancer (NSCLC) treated with platinum-based chemotherapy. A small fraction of non-squamous cell lung cancers harbor EGFR mutations, with improved outcome to gefitinib and erlotinib. Experimental evidence suggests that BRCA1 overexpression enhances sensitivity to docetaxel and resistance to cisplatin. RAP80 and Abraxas are interacting proteins that form complexes with BRCA1 and could modulate the effect of BRCA1. In order to further examine the effect of EGFR mutations and BRCA1 mRNA levels on outcome in advanced NSCLC, we performed a prospective non-randomized phase II clinical trial, testing the hypothesis that customized therapy would confer improved outcome over non-customized therapy. In an exploratory analysis, we also examined the effect of RAP80 and Abraxas mRNA levels. Methodology/Principal Findings We treated 123 metastatic non-squamous cell lung carcinoma patients using a customized approach. RNA and DNA were isolated from microdissected specimens from paraffin-embedded tumor tissue. Patients with EGFR mutations received erlotinib, and those without EGFR mutations received chemotherapy with or without cisplatin based on their BRCA1 mRNA levels: low, cisplatin plus gemcitabine; intermediate, cisplatin plus docetaxel; high, docetaxel alone. An exploratory analysis examined RAP80 and Abraxas expression. Median survival exceeded 28 months for 12 patients with EGFR mutations, and was 11 months for 38 patients with low BRCA1, 9 months for 40 patients with intermediate BRCA1, and 11 months for 33 patients with high BRCA1. Two-year survival was 73.3%, 41.2%, 15.6% and 0%, respectively. Median survival was influenced by RAP80 expression in the three BRCA1 groups. For example, for patients with both low BRCA1 and low RAP80, median survival exceeded 26 months. RAP80 was a significant factor for survival in patients treated according to BRCA1 levels (hazard ratio, 1

  5. Himasthla elongata: effect of infection on expression of the LUSTR-like receptor mRNA in common periwinkle haemocytes.

    PubMed

    Gorbushin, A M; Klimovich, A V; Iakovleva, N V

    2009-09-01

    The first mollusc mRNA coding G-protein-coupled transmembrane receptor (GPcapital ES, CyrillicR), homologous to human receptors LUSTR 1 (GPR107) and LUSTR 2 (GPR108), was isolated from haemocytes of common periwinkle Littorina littorea. The analyses showed that the full-length cDNA is 1935 bp long and is predicted to encode a 614 amino acid protein (named Lit-LUSTR) with a calculated molecular mass of 69.6 kDa and theoretical isoelectric point 7.59. Pair-wise comparisons between Lit-LUSTR and LUSTR proteins from human or mouse have approximately 38% identity and 56% similarity. Lit-LUSTR clusters with LUSTR-A sub-family proteins and is a first characterization of proteins containing Lung7TM-R domain in Mollusca. Significant differences were found between the Lit-LUSTR mRNA levels in haemocytes of healthy periwinkles and those naturally infected with the echinostome trematode Himasthla elongata. Down regulated expression of the LUSTR-like receptor caused by infection illustrates modification of the haemocyte receptor system and may be attributed to the previously demonstrated greater numbers of "immature" haemocytes in the circulation of infected snails.

  6. Deduced primary structure of rat hepatocyte growth factor and expression of the mRNA in rat tissues.

    PubMed Central

    Tashiro, K; Hagiya, M; Nishizawa, T; Seki, T; Shimonishi, M; Shimizu, S; Nakamura, T

    1990-01-01

    The primary structure of rat hepatocyte growth factor (HGF) was elucidated by determining the base sequence of the complementary DNA (cDNA) of HGF. The cDNA for rat HGF was isolated by screening a liver cDNA library with oligonucleotides based on the partial N-terminal amino acid sequence of the beta subunit of purified rat HGF. HGF is encoded in an mRNA of about 6 kilobases. Both alpha and beta subunits of HGF are specified in a single open reading frame for a 728-amino acid protein with a calculated molecular weight of 82,904. The N-terminal part of HGF has a signal sequence and a prosequence with 30 and 25 amino acid residues, respectively. The mature heterodimer structure is derived proteolytically from this single pre-pro precursor polypeptide. The calculated molecular weights of the alpha and beta subunits are 50,664 and 25,883, respectively, and each subunit has two potential N-linked glycosylation sites. The amino acid sequence of HGF is 38% identical with that of plasminogen. The alpha subunit of HGF contains four "kringle" structures, and the beta subunit has 37% amino acid identity with the serine protease domain of plasmin. Northern blot analysis revealed that HGF mRNA was expressed in rat various tissues, including the liver, kidney, lung, and brain. Images PMID:2139229

  7. mRNA stabilization controls the expression of a class of developmentally regulated genes in Dictyostelium discoideum

    PubMed Central

    Mangiarotti, Giorgio; Giorda, Roberto; Ceccarelli, Adriano; Perlo, Carla

    1985-01-01

    During the development of Dictyostelium discoideum, several thousand new mRNA species appear in the cytoplasm after the cells have formed stable aggregates. Here we show that six of these late mRNAs, corresponding to six clones randomly chosen from a genomic library, are synthesized from the very beginning of development at a rate comparable to that observed late in development but that transcripts do not accumulate until after aggregation. The early- and late-synthesized mRNAs are identical in size and compete with each other for hybridization to the genomic clones. The early-synthesized mRNAs do not accumulate in the cytoplasm in the preaggregation stage because they are very unstable. Their stability, estimated from the kinetics of incorporation during continuous labeling with 32P, increases by perhaps an order of magnitude in the postaggregation stage. We conclude that mRNA stabilization is the major controlling factor of the expression of these genes. Images PMID:16593597

  8. Brain cytokine and chemokine mRNA expression in mice induced by intranasal instillation with ultrafine carbon black.

    PubMed

    Tin-Tin-Win-Shwe; Yamamoto, Shoji; Ahmed, Sohel; Kakeyama, Masaki; Kobayashi, Takahiro; Fujimaki, Hidekazu

    2006-05-25

    Ambient air ultrafine particles (UFPs) have gained enormous attention to many researchers with recent evidence showing them to have more hazardous effects on human health than larger ambient particles. Studies focusing the possibility of effects on brain are quite limited. To examine the effect of ultrafine carbon black (ufCB) on mice brain, we instilled 125 microg of 14 nm or 95 nm CB into the nostrils of 8-week-old male BALB/c mice, once a week for 4 weeks. Four hours after the last instillation, we collected olfactory bulb and hippocampus and detected the expression of cytokine and chemokine mRNA by quantitative real-time PCR method. In this study, we found the induction of proinflammatory cytokines (interleukin-1 beta and tumor necrosis factor-alpha and chemokines (monocyte chemoattractant protein-1/CCL2, macrophage inflammatory protein-1 alpha/CCL3), and monokine induced interferon-gamma/CXC chemokine ligand (CXCL9) mRNA in brain olfactory bulb, not in the hippocampus of mice instilled with 14 nm ufCB intranasally. We suggest that the intranasal instillation of ufCB may influence the brain immune function depending on their size. To our knowledge, this is the first study to demonstrate region-specific brain cytokine and chemokine mRNA-induction in mice triggered by intranasal instillation of specific-sized ufCB, in a physiologically relevant condition.

  9. Differential expression of viral PAMP receptors mRNA in peripheral blood of patients with chronic hepatitis C infection

    PubMed Central

    Atencia, Rafael; Bustamante, Francisco J; Valdivieso, Andrés; Arrieta, Arantza; Riñón, Marta; Prada, Alvaro; Maruri, Natalia

    2007-01-01

    Background Pathogen-associated molecular patterns (PAMP) receptors play a key role in the early host response to viruses. In this work, we determined mRNA levels of two members of the Toll-like Receptors family, (TLR3 and TLR7) and the helicase RIG-I, all of three recognizing viral RNA products, in peripheral blood of healthy donors and hepatitis C virus (HCV) patients, to observe if their transcripts are altered in this disease. Methods IFN-α, TLR3, TLR7 and RIG-I levels in peripheral blood from healthy controls (n = 18) and chronic HCV patients (n = 18) were quantified by real-time polymerase chain reaction. Results Our results show that IFN-α, TLR3, TLR7 and RIG-I mRNA levels are significantly down-regulated in patients with chronic HCV infection when compared with healthy controls. We also found that the measured levels of TLR3 and TLR7, but not RIG-I, correlated significantly with those of IFN-α Conclusion Monitoring the expression of RNA-sensing receptors like TLR3, TLR7 and RIG-I during the different clinical stages of infection could bring a new source of data about the prognosis of disease. PMID:18021446

  10. PCB-associated changes in mRNA expression in killer whales (Orcinus orca) from the NE Pacific Ocean.

    PubMed

    Buckman, Andrea H; Veldhoen, Nik; Ellis, Graeme; Ford, John K B; Helbing, Caren C; Ross, Peter S

    2011-12-01

    Killer whales in the NE Pacific Ocean are among the world's most PCB-contaminated marine mammals, raising concerns about implications for their health. Sixteen health-related killer whale mRNA transcripts were analyzed in blubber biopsies collected from 35 free-ranging killer whales in British Columbia using real-time quantitative polymerase chain reaction. We observed PCB-related increases in the expression of five gene targets, including the aryl hydrocarbon receptor (AhR; r(2) = 0.83; p < 0.001), thyroid hormone α receptor (TRα; r(2) = 0.64; p < 0.001), estrogen α receptor (ERα; r(2) = 0.70; p < 0.001), interleukin 10 (IL-10; r(2) = 0.74 and 0.68, males and females, respectively; p < 0.001), and metallothionein 1 (MT1; r(2) = 0.58; p < 0.001). Best-fit models indicated that population (dietary preference), age, and sex were not confounding factors, except for IL-10, where males differed from females. While the population-level consequences are unclear, the PCB-associated alterations in mRNA abundance of such pivotal end points provide compelling evidence of adverse physiological effects of persistent environmental contaminants in these endangered killer whales.

  11. Ventral tegmental area dopamine and GABA neurons: Physiological properties and expression of mRNA for endocannabinoid biosynthetic elements.

    PubMed

    Merrill, Collin B; Friend, Lindsey N; Newton, Scott T; Hopkins, Zachary H; Edwards, Jeffrey G

    2015-11-10

    The ventral tegmental area (VTA) is involved in adaptive reward and motivation processing and is composed of dopamine (DA) and GABA neurons. Defining the elements regulating activity and synaptic plasticity of these cells is critical to understanding mechanisms of reward and addiction. While endocannabinoids (eCBs) that potentially contribute to addiction are known to be involved in synaptic plasticity mechanisms in the VTA, where they are produced is poorly understood. In this study, DA and GABAergic cells were identified using electrophysiology, cellular markers, and a transgenic mouse model that specifically labels GABA cells. Using single-cell RT-qPCR and immunohistochemistry, we investigated mRNA and proteins involved in eCB signaling such as diacylglycerol lipase α, N-acyl-phosphatidylethanolamine-specific phospholipase D, and 12-lipoxygenase, as well as type I metabotropic glutamate receptors (mGluRs). Our results demonstrate the first molecular evidence of colocalization of eCB biosynthetic enzyme and type I mGluR mRNA in VTA neurons. Further, these data reveal higher expression of mGluR1 in DA neurons, suggesting potential differences in eCB synthesis between DA and GABA neurons. These data collectively suggest that VTA GABAergic and DAergic cells have the potential to produce various eCBs implicated in altering neuronal activity or plasticity in adaptive motivational reward or addiction.

  12. Intervention of rosiglitazone on myocardium Glut-4 mRNA expression during ischemia-reperfusion injury in cardio-pulmonary bypass in dogs.

    PubMed

    Liu, Bin; Liang, Guiyou; Xu, Gang; Liu, Daxin; Cai, Qingyong; Gao, Zhenyu

    2013-01-01

    During cardiac pulmonary bypass (CPB), myocardial ischemia-reperfusion (I/R) induces heart glucose metabolism impairment. Our previous research showed that the decreased glucose utilization is due to decreased glucose transporter-4 (Glut-4) expression and translocation to myocyte surface membranes. This study further examined whether rosiglitazone, a synthetic agonist of peroxisome proliferator-activated receptor γ, could intervene glucose metabolism by regulating Glut-4 mRNA during I/R in dogs. Cardiac ischemia was induced by cardiopulmonary bypass for 30 or 120 min. Plasma insulin and glucose concentrations were measured at pre-bypass (control), aortic cross-clamp off (I/R) at 15, 45, and 75 min. The left ventricle biopsies were taken for the expression of Glut-4 mRNA by real-time RT-PCR. In dogs receiving 120 min ischemia, coronary arterial, venous glucose concentrations, plasma insulin levels, and insulin resistant index (IRI) were increased, but the expression of Glut-4 mRNA was decreased obviously at 15 min of reperfusion, and recovered gradually. On the other hand, these changes were relatively mild in dogs treated with rosiglitazone in cardioplegic solution and expression of Glut-4 mRNA was increased remarkably. It is concluded that the decrease in total amount of Glut-4 mRNA expression could be one of the important molecular mechanisms, which causes the myocardium insulin resistance. The longer the ischemia period, the decrease in amount of Glut-4 mRNA was more dramatic. Adding rosiglitazone into the cardioplegic solution during I/R can increase the amount of Glut-4 mRNA expression, mitigate the myocardium insulin resistance and improve the myocardium I/R injury during CPB.

  13. Heterogeneity of Kv2.1 mRNA expression and delayed rectifier current in single isolated myocytes from rat left ventricle.

    PubMed

    Schultz, J H; Volk, T; Ehmke, H

    2001-03-16

    Expression of the voltage-gated K(+) channel Kv2.1, a possible molecular correlate for the cardiac delayed rectifier current (I(K)), has recently been shown to vary between individual ventricular myocytes. The functional consequences of this cell-to-cell heterogeneity in Kv2.1 expression are not known. Using multiplex single-cell reverse transcriptase-polymerase chain reaction (RT-PCR), we detected Kv2.1 mRNA in 47% of isolated midmyocardial myocytes from the rat left ventricular free wall that were positive for alpha-myosin heavy chain mRNA (n=74). Whole-cell patch-clamp recordings demonstrated marked differences in the magnitude of I(K) (200 to 1450 pA at V(Pip)=40 mV) between individual myocytes of the same origin. Furthermore, the tetraethylammonium (TEA)-sensitive outward current (I(TEA)), known to be partly encoded by Kv2.1 in mice, revealed a wide range of current magnitudes between single cells (150 to 1130 pA at V(Pip)=40 mV). Combined patch-clamp recordings and multiplex single-cell RT-PCR analysis of the same myocytes, however, showed no differences in I(K) or I(TEA) magnitude or inactivation kinetics between myocytes expressing Kv2.1 mRNA and those that did not express Kv2.1 mRNA. In contrast, in all midmyocardial myocytes expressing the transient outward potassium current (I(to1)), Kv4 mRNA, which has been shown to underlie I(to1), was detected (n=10). These results indicate that I(K) heterogeneity among individual left ventricular myocytes cannot be explained by the distribution pattern of Kv2.1 mRNA. Other mechanisms besides Kv2.1 mRNA expression appear to determine magnitude and kinetics of I(K) in rat ventricular myocytes.

  14. Effect of dietary lead on intestinal nutrient transporters mRNA expression in broiler chickens.

    PubMed

    Ebrahimi, Roohollah; Faseleh Jahromi, Mohammad; Liang, Juan Boo; Soleimani Farjam, Abdoreza; Shokryazdan, Parisa; Idrus, Zulkifli

    2015-01-01

    Lead- (Pb-) induced oxidative stress is known to suppress growth performance and feed efficiency in broiler chickens. In an attempt to describe the specific underlying mechanisms of such phenomenon we carried out the current study. Ninety-six one-day-old broiler chicks were randomly assigned to 2 dietary treatment groups of 6 pen replicates, namely, (i) basal diet containing no lead supplement (control) and (ii) basal diet containing 200 mg lead acetate/kg of diet. Following 3 weeks of experimental period, jejunum samples were collected to examine the changes in gene expression of several nutrient transporters, antioxidant enzymes, and heat shock protein 70 (Hsp70) using quantitative real-time PCR. The results showed that addition of lead significantly decreased feed intake, body weight gain, and feed efficiency. Moreover, with the exception of GLUT5, the expression of all sugar, peptide, and amino acid transporters was significantly downregulated in the birds under Pb induced oxidative stress. Exposure to Pb also upregulated the antioxidant enzymes gene expression together with the downregulation of glutathione S-transferase and Hsp70. In conclusion, it appears that Pb-induced oxidative stress adversely suppresses feed efficiency and growth performance in chicken and the possible underlying mechanism for such phenomenon is downregulation of major nutrient transporter genes in small intestine.

  15. DNA methylation and mRNA expression of HLA-DQA1 alleles in type 1 diabetes mellitus.

    PubMed

    Cepek, Pavel; Zajacova, Marta; Kotrbova-Kozak, Anna; Silhova, Elena; Cerna, Marie

    2016-06-01

    Type 1 diabetes (T1D) belongs among polygenic multifactorial autoimmune diseases. The highest risk is associated with human leucocyte antigen (HLA) class II genes, including HLA-DQA1 gene. Our aim was to investigate DNA methylation of HLA-DQA1 promoter alleles (QAP) and correlate methylation status with individual HLA-DQA1 allele expression of patients with T1D and healthy controls. DNA methylation is one of the epigenetic modifications that regulate gene expression and is known to be shaped by the environment.Sixty one patients with T1D and 39 healthy controls were involved in this study. Isolated DNA was treated with sodium bisulphite and HLA-DQA1 promoter sequence was amplified using nested PCR. After sequencing, DNA methylation of HLA-DQA1 promoter alleles was analysed. Individual mRNA HLA-DQA1 relative allele expression was assessed using two different endogenous controls (PPIA, DRA). We have found statistically significant differences in HLA-DQA1 allele 02:01 expression (PPIA normalization, Pcorr = 0·041; DRA normalization, Pcorr = 0·052) between healthy controls and patients with T1D. The complete methylation profile of the HLA-DQA1 promoter was gained with the most methylated allele DQA1*02:01 and the least methylated DQA1*05:01 in both studied groups. Methylation profile observed in patients with T1D and healthy controls was similar, and no correlation between HLA-DQA1 allele expression and DNA methylation was found. Although we have not proved significant methylation differences between the two groups, detailed DNA methylation status and its correlation with expression of each HLA-DQA1 allele in patients with T1D have been described for the first time.

  16. Abnormal expression of mRNA, microRNA alteration and aberrant DNA methylation patterns in rectal adenocarcinoma

    PubMed Central

    Liu, Xianglong; Yuan, Xiangfei; Qin, Hai; Zhang, Xipeng

    2017-01-01

    Aim Rectal adenocarcinoma (READ) is a malignancy cancer with the high morbidity and motility worldwide. Our study aimed to explore the potential pathogenesis of READ through integrated analysis of gene expression profiling and DNA methylation data. Methods The miRNA, mRNA expression profiling and corresponding DNA methylation data were downloaded from The Cancer Genome Atlas (TCGA) database. Differentially expressed mRNAs/ miRNAs/methylated regions (DEmRNA/DEmiRNAs) were identified in READ. The negatively correlation of DEmiRNA-DEmRNAs and DNA methylation-DEmRNAs were obtained. DEmRNAs expression was validated through quantitative real-time polymerase chain reaction (qRT-PCR) and microarray expression profiling analyses. Results 1192 dysregulated DEmRNAs, 27 dysregulated DEmiRNAs and 6403 aberrant methylation CpG sites were screened in READ compared to normal controls. 1987 negative interaction pairs among 27 DEmiRNAs and 668 DEmRNAs were predicted. 446 genes with aberrant methylation were annotated. Eventually, 50 DEmRNAs (39 down- and 11 up-regulated DEmRNAs) with hypermethylation, synchronously negatively targeted by DEmiRNAs, were identified through the correlation analysis among 446 genes with aberrant methylation and 668 DEmRNAs. 50 DEmRNAs were significantly enriched in cAMP signaling pathway, circadian entrainment and glutamatergic synapse. The validation results of expression levels of DEmRNAs through qRT-PCR and microarray analyses were compatible with our study. Conclusion 7 genes of SORCS1, PDZRN4, LONRF2, CNGA3, HAND2, RSPO2 and GNAO1 with hypermethylation and negatively regulation by DEmiRNAs might contribute to the tumorigenesis of READ. Our work might provide valuable foundation for the READ in mechanism elucidation, early diagnosis and therapeutic target identification. PMID:28350845

  17. miR-143 decreases COX-2 mRNA stability and expression in pancreatic cancer cells

    SciTech Connect

    Pham, Hung; Ekaterina Rodriguez, C.; Donald, Graham W.; Hertzer, Kathleen M.; Jung, Xiaoman S.; Chang, Hui-Hua; Moro, Aune; Reber, Howard A.; Hines, O. Joe; Eibl, Guido

    2013-09-13

    Highlights: •Pancreatic cancer cells express low miR-143 levels and elevated p-MEK, p-MAPK and RREB1. •MEK inhibitors U0126 and PD98059 increase miR-143 expression. •miR-143 decreases COX-2 mRNA stability and expression and PGE{sub 2}. •miR-143 decreases p-p38MAPK, p-MEK, p-MAPK and RREB1 expression. -- Abstract: Small non-coding RNAs, microRNAs (miRNA), inhibit the translation or accelerate the degradation of message RNA (mRNA) by targeting the 3′-untranslated region (3′-UTR) in regulating growth and survival through gene suppression. Deregulated miRNA expression contributes to disease progression in several cancers types, including pancreatic cancers (PaCa). PaCa tissues and cells exhibit decreased miRNA, elevated cyclooxygenase (COX)-2 and increased prostaglandin E{sub 2} (PGE{sub 2}) resulting in increased cancer growth and metastases. Human PaCa cell lines were used to demonstrate that restoration of miRNA-143 (miR-143) regulates COX-2 and inhibits cell proliferation. miR-143 were detected at fold levels of 0.41 ± 0.06 in AsPC-1, 0.20 ± 0.05 in Capan-2 and 0.10 ± 0.02 in MIA PaCa-2. miR-143 was not detected in BxPC-3, HPAF-II and Panc-1 which correlated with elevated mitogen-activated kinase (MAPK) and MAPK kinase (MEK) activation. Treatment with 10 μM of MEK inhibitor U0126 or PD98059 increased miR-143, respectively, by 187 ± 18 and 152 ± 26-fold in BxPC-3 and 182 ± 7 and 136 ± 9-fold in HPAF-II. miR-143 transfection diminished COX-2 mRNA stability at 60 min by 2.6 ± 0.3-fold in BxPC-3 and 2.5 ± 0.2-fold in HPAF-II. COX-2 expression and cellular proliferation in BxPC-3 and HPAF-II inversely correlated with increasing miR-143. PGE{sub 2} levels decreased by 39.3 ± 5.0% in BxPC-3 and 48.0 ± 3.0% in HPAF-II transfected with miR-143. Restoration of miR-143 in PaCa cells suppressed of COX-2, PGE{sub 2}, cellular proliferation and MEK/MAPK activation, implicating this pathway in regulating miR-143 expression.

  18. Effects of codon usage versus putative 5'-mRNA structure on the expression of Fusarium solani cutinase in the Escherichia coli cytoplasm.

    PubMed

    Griswold, Karl E; Mahmood, Nadir A; Iverson, Brent L; Georgiou, George

    2003-01-01

    Matching the codon usage of recombinant genes to that of the expression host is a common strategy for increasing the expression of heterologous proteins in bacteria. However, while developing a cytoplasmic expression system for Fusarium solani cutinase in Escherichia coli, we found that altering codons to those preferred by E. coli led to significantly lower expression compared to the wild-type fungal gene, despite the presence of several rare E. coli codons in the fungal sequence. On the other hand, expression in the E. coli periplasm using a bacterial PhoA leader sequence resulted in high levels of expression for both the E. coli optimized and wild-type constructs. Sequence swapping experiments as well as calculations of predicted mRNA secondary structure provided support for the hypothesis that differential cytoplasmic expression of the E. coli optimized versus wild-type cutinase genes is due to differences in 5(') mRNA secondary structures. In particular, our results indicate that increased stability of 5(') mRNA secondary structures in the E. coli optimized transcript prevents efficient translation initiation in the absence of the phoA leader sequence. These results underscore the idea that potential 5(') mRNA secondary structures should be considered along with codon usage when designing a synthetic gene for high level expression in E. coli.

  19. A retrospective study on TS mRNA expression and prediction of the effects of adjuvant oral 5-fluorouracil in breast cancer

    PubMed Central

    AKI, FUMINORI; BANDO, YOSHIMI; TAKAHASHI, TETSUYUKI; UEHARA, HISANORI; NUMOTO, SATOSHI; ITO, SUEYOSHI; SASA, MITSUNORI; IZUMI, KEISUKE

    2010-01-01

    Nucleic acid-metabolizing enzymes, such as thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), thymidine phosphorylase (TP) and orotate phosphoribosyl transferase (OPRT), have attracted attention as candidates for response determinants of 5-fluorouracil (5-FU). Whether the expression levels of these enzymes can be adopted as valuable parameters for 5-FU sensitivity in breast cancer has yet to be elucidated. In the present study, intratumoral mRNA expression of TS, DPD, TP and OPRT were determined in formalin-fixed paraffin-embedded surgical specimens collected from 217 breast cancer patients, using the Danenberg Tumor Profile method, which combines microdissection and real-time-polymerase chain reaction. The significance of these enzymes as prognostic and 5-FU efficacy-predicting factors was evaluated. Our data showed that a low DPD expression is related to a high nuclear grade and other factors including hormone receptor-negativity. Low expression levels of TP were found in hormone receptor-negative tumors. TS and OPRT expression were not related to various clinicopathological factors, but patients with a high TS mRNA expression showed a significantly poorer prognosis in cases where 5-FU was not administered. The efficacy of 5-FU was more significant when administered for more than 6 months in the group with a high TS mRNA expression. These data suggest that TS mRNA expression in breast cancer tissue is an ideal predictor of outcomes for patients with no administration of 5-FU, and of the efficacy of 5-FU. PMID:22870098

  20. Actinidia DRM1--an intrinsically disordered protein whose mRNA expression is inversely correlated with spring budbreak in kiwifruit.

    PubMed

    Wood, Marion; Rae, Georgina M; Wu, Rong-Mei; Walton, Eric F; Xue, Bin; Hellens, Roger P; Uversky, Vladimir N

    2013-01-01

    Intrinsically disordered proteins (IDPs) are a relatively recently defined class of proteins which, under native conditions, lack a unique tertiary structure whilst maintaining essential biological functions. Functional classification of IDPs have implicated such proteins as being involved in various physiological processes including transcription and translation regulation, signal transduction and protein modification. Actinidia DRM1 (Ade DORMANCY ASSOCIATED GENE 1), represents a robust dormancy marker whose mRNA transcript expression exhibits a strong inverse correlation with the onset of growth following periods of physiological dormancy. Bioinformatic analyses suggest that DRM1 is plant specific and highly conserved at both the nucleotide and protein levels. It is predicted to be an intrinsically disordered protein with two distinct highly conserved domains. Several Actinidia DRM1 homologues, which align into two distinct Actinidia-specific families, Type I and Type II, have been identified. No candidates for the Arabidopsis DRM1-Homologue (AtDRM2) an additional family member, has been identified in Actinidia.

  1. cAMP stimulation of StAR expression and cholesterol metabolism is modulated by co-expression of labile suppressors of transcription and mRNA turnover.

    PubMed

    Jefcoate, Colin R; Lee, Jinwoo; Cherradi, Nadia; Takemori, Hiroshi; Duan, Haichuan

    2011-04-10

    The steroidogenic acute regulatory (StAR) protein is generated in rodents from 1.6 kb and 3.5 kb mRNA formed by alternative polyadenylation. The zinc finger protein, TIS11B (also Znf36L1), is elevated by cAMP in adrenal cells in parallel with StAR mRNA. TIS11b selectively destabilizes the 3.5 kb mRNA through AU-rich sequences at the end of the 3'UTR. siRNA suppression shows that TIS11b surprisingly increases StAR protein and cholesterol metabolism. StAR transcription is directly activated by PKA phosphorylation. cAMP responsive element binding (CREB) protein 1 phosphorylation is a key step leading to recruitment of the co-activator, CREB binding protein (CBP). A second protein, CREB regulated transcription coactivator (TORC/CRTC), enhances this recruitment, but is inhibited by salt inducible kinase (SIK). Basal StAR transcription is constrained through this phosphorylation of TORC. PKA provides an alternative stimulation by phosphorylating SIK, which prevents TORC inactivation. PKA stimulation of StAR nuclear transcripts substantially precedes TORC recruitment to the StAR promoter, which may, therefore, mediate a later step in mRNA production. Inhibition of SIK by staurosporine elevates StAR transcription and TORC recruitment to maximum levels, but without CREB phosphorylation. TORC suppression by SIK evidently limits basal StAR transcription. Staurosporine and cAMP stimulate synergistically. SIK targets the phosphatase, PP2a (activation), and Type 2 histone de-acetylases (inhibition), which may each contribute to suppression. Staurosporine stimulation through SIK inhibition is repeated in cAMP stimulation of many steroidogenic genes regulated by steroidogenic factor 1 (SF-1) and CREB. TIS11b and SIK may combine to attenuate StAR expression when hormonal stimuli decline.

  2. Abnormal expression of ENaC and SGK1 mRNA induced by dietary sodium in Dahl salt-sensitively hypertensive rats.

    PubMed

    Aoi, Wataru; Niisato, Naomi; Sawabe, Yukinori; Miyazaki, Hiroaki; Tokuda, Shinsaku; Nishio, Kyosuke; Yoshikawa, Toshikazu; Marunaka, Yoshinori

    2007-10-01

    Epithelial sodium channel (ENaC) plays a crucial role in controlling sodium reabsorption in the kidney keeping the normal blood pressure. We previously reported that the expression of ENaC mRNA in the kidney of Dahl salt-sensitive (DS) rats was abnormally regulated by aldosterone, however it is unknown if dietary sodium affects the expression of ENaC and serum and glucocorticoid-regulated kinase 1 (SGK1), which plays an important role in ENaC activation, in DS rats. In the present study, we investigated whether dietary sodium abnormally affects the expression of ENaC and SGK1 mRNA in DS rats. DS and Dahl salt-resistant (DR) rats (8 weeks old) were divided into three different groups, respectively: (1) low sodium diet (0.005% NaCl), (2) normal sodium diet (0.3% NaCl), and (3) high sodium diet (8% NaCl). The high sodium diet for 4 weeks in DS rats elevated the systolic blood pressure, but did not in any other groups. The expression of alpha-ENaC mRNA in DS rats was abnormally increased by high sodium diet in contrast to DR rats, while it was normally increased by low sodium diet in DS rats similar to DR rats. The expression of beta- and gamma-ENaC mRNA in DS rats was also abnormally increased by high sodium diet unlike DR rats. The expression of SGK1 mRNA was elevated by high sodium diet in DS rats, but it was decreased in DR rats. These observations indicate that the expression of ENaC and SGK1 mRNA is abnormally regulated by dietary sodium in salt-sensitively hypertensive rats, and that this abnormal expression would be one of the factors causing salt-sensitive hypertension.

  3. Regulation of DM-20 mRNA expression and intracellular translocation of glutathione-S-transferase pi isoform during oligodendrocyte differentiation in the adult rat spinal cord.

    PubMed

    Kitada, Masaaki; Takeda, Kazuya; Dezawa, Mari

    2016-07-01

    We previously demonstrated that NG2-positive oligodendrocyte precursor cells (OPCs) do not express DM-20 mRNA and identified a distinct DM-20 mRNA-positive cell population expressing glutathione-S-transferase pi isoform (GST-pi) in the nucleus (GST-pi(Nuc)) of the adult rat spinal cord. As GST-pi intranuclear localization correlates with progenitor cell properties, we examined the differentiation status of this cell population under the intensive 5-bromo-2'-deoxyuridine (BrdU) administration method, consisting of intraperitoneal BrdU injections every 2 h for 48 h. We observed that a certain population of proliferating/proliferated cells expressed DM-20 mRNA, and sometimes two proliferating/proliferated cells were observed still attached to each other. We performed triple staining for BrdU, DM-20 mRNA, and NG2 and found pairs of neighboring BrdU-positive cells, which were considered to originate from the same progenitor cells and where both cells expressed DM-20 mRNA. Triple staining for BrdU, DM-20 mRNA, and GST-pi detected proliferating/proliferated cells exhibiting the GST-pi(Nuc)/DM-20 mRNA-positive expression pattern. These findings suggested the presence of a GST-pi(Nuc)/DM-20 mRNA-positive oligodendrocyte-lineage progenitor cell population in the adult rat spinal cord. However, we did not find any pair of neighboring BrdU-positive cells with this expression pattern. These observations collectively support the idea that GST-pi(Nuc)/DM-20 mRNA-expressing cells are the progeny of NG2-positive OPCs rather than a novel type of oligodendrocyte-lineage progenitor cells and that DM-20 mRNA expression is dynamically regulated during differentiation of OPCs into oligodendrocytes.

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

    PubMed Central

    2016-01-01

    Purpose. This study aimed to evaluate the clinical efficacy of using expression levels of CCL24 (eotaxin-2) mRNA on the ocular surface as a biomarker in patients with vernal keratoconjunctivitis (VKC) and atopic keratoconjunctivitis (AKC). Methods. Eighteen patients with VKC or AKC (VKC/AKC group) and 12 control subjects (control group) were enrolled in this study. The VKC/AKC clinical score was determined by objective findings in patients by using the 5-5-5 exacerbation grading scale. All subjects underwent modified impression cytology and specimens were obtained from the upper tarsal conjunctiva. Expression levels of CCL24 (eotaxin-2) mRNA on the ocular surface were determined using real-time reverse transcription polymerase chain reaction. Results. The VKC group was divided into two subgroups, depending on the clinical score: the active stage subgroup with 100 points or more of clinical scores and the stable stage subgroup with 100 points or less. CCL24 (eotaxin-2) mRNA expression levels in the active VKC/AKC stage subgroup were significantly higher than those in the stable VKC/AKC subgroup and the control group. Clinical scores correlated significantly with CCL24 (eotaxin-2) mRNA expression levels in the VKC group. Conclusions. CCL24 (eotaxin-2) mRNA expression levels on the ocular surface are a useful biomarker for clinical severity of VKC/AKC. PMID:27721987

  5. [In vitro effect of total flavones of Fructus Chorspondiatis on expression of collagen type I and type III mRNA and protein of cultured rat cardiac fibroblasts].

    PubMed

    Bao, Jun-Ping; Jin, Ming; Yang, Yu-Min; Gao, Xiao-Hui; Shu, Liang; Xing, Hui-Hui; Jia, Lei

    2014-01-01

    This study aims to investigate the effect of total flavones of Fructus Chorspondiatis (TFFC) on the mRNA and protein expression of collagen type I and III of rat cardiac fibroblasts (CFs) induced by angiotensin II (Ang II), and explore its anti-myocardial fibrosis molecular mechanism. Neonatal rat CFs were prepared from Sprague-Dawley rats (1-3 d after birth). The expression of collagen type I and III mRNA and protein were measured by RT-PCR and Western blotting, respectively. The study showed that stimulation of neonatal rat CFs with 100 nmol.L-1 of Ang II for 72 h resulted in a significant increase of the expression of collagen type I and III mRNA and protein. The changes on the expression level were blocked by TFFC. The results demonstrated that TFFC can inhibit myocardial fibrosis induced by Ang II in rats, which is probably associated with the collagen type I and III mRNA and protein levels up-regulated by Ang II, and TFFC was shown to decrease the expression levels of collagen type I and III mRNA and protein.

  6. Semi-quantitative analysis of cytokine mRNA expression induced by the herbal medicine Sho-saiko-to (TJ-9) using a Gel Doc system.

    PubMed

    Huang, X X; Yamashiki, M; Nakatani, K; Nobori, T; Mase, A

    2001-01-01

    The RT-PCR method was employed to determine the cytokine mRNA expression of human peripheral lymphocytes induced by the Japanese herbal medicine Sho-saiko-to (TJ-9). The results showed that the mRNA expression of IL-12, IL-1beta, IL-10, TNF-alpha, G-CSF, and IFN-gamma increased after 6 hr in culture. This is the first reported finding that TJ-9 is an IFN-gamma inducer. Next, cytokine mRNA expression was semi-quantitatively measured using the Gel Doc system with a CCD camera and then statistically analyzed in order to determine which component of TJ-9 was the true cytokine inducer. The results showed that the scutellaria root is the main component inducing the cytokines, while the glycyrrhiza root is the secondary component. When the cytokine concentrations in the supernatants of cell cultures were measured by ELISA, the levels of IL-12, IL-1beta, IL-10, TNF-alpha, and G-CSF reflected mRNA expression levels in the cell fraction. However, the level of IFN-gamma was below the detectable limit. The effects of various reagents on many different kinds of cytokine mRNA expression could be analyzed objectively in a short time using the Gel Doc system. Many important findings could be demonstrated by this simple, easy, sensitive, and cheap method. After the clinical significance of cytokine analysis is confirmed, this method may become a useful clinical examination tool.

  7. Molecular cloning of the SMAD4 gene and its mRNA expression analysis in ovarian follicles of the Yangzhou goose (Anser cygnoides).

    PubMed

    Huang, Z; Yuan, X; Wang, M; Wu, N; Song, Y; Chen, Y; Zhang, Y; Xu, Q; Chen, G; Zhao, W

    2016-08-01

    Mothers against decapentaplegic homolog 4 (SMAD4) is an important protein in animal reproduction. It plays pivotal roles in cellular pathways, including apoptosis. The expression profile of the SMAD4 gene in goose ovarian follicles has not been reported. In this study, the SMAD4 coding sequence was cloned from the Yangzhou goose. A phylogenetic analysis was performed and mRNA expression was examined in various tissues using quantitative real-time PCR. An alternative splice form of SMAD4, SMAD4-b having 1656 bp, was identified. SMAD4-a mRNA was widely expressed in various healthy tissues, whereas SMAD4-b was very weakly expressed. SMAD4 mRNA in the ovary and oviduct was significantly higher than that in the pituitary and hypothalamus. SMAD4 mRNA expression analysis in hierarchical follicles showed that the level of SMAD4 mRNA was higher in large white follicles and post-ovulatory follicles than in the other follicles. The results indicate that SMAD4 might be involved in the recruitment of hierarchical follicles.

  8. Effects of acute and chronic cigarette smoking on the expression of endothelin-1 mRNA of the cardiovascular tissues in rats.

    PubMed

    Adachi, C; Naruse, M; Ishihara, Y; Tanabe, A; Takagi, S; Yoshimoto, T; Naruse, K; Kagawa, J; Takano, K

    2000-11-01

    Although smoking has been suggested to be involved in the development of cardiovascular diseases, details of the mechanism still need to be revealed. We investigated the effects of cigarette smoking on the tissue mRNA expression of endothelin-1 (ET-1). Male Wistar rats of 4 weeks of age were exposed to smoke from six cigarettes for 30 min (acute exposure) and six cigarettes for 30 min/day, 5 days a week for 6 months (chronic exposure). Half of the rats exposed to 6 months smoking were kept in clean-air conditions for a further 3 months to clear the effects. Tissue expression of ET-1 mRNA in the kidney, aorta, heart and lung was determined by reverse transcriptase polymerase chain reaction (RT-PCR) followed by Southern blot analysis. There was no significant difference in body and organ weight of the heart and kidney between the control and smoking group in either the acute or chronic experiment. In the acute-exposure experiment, expression of ET-1 mRNA was increased in the heart and lung, while that in the kidney and aorta was unchanged. In the chronic-exposure experiment, however, there was no significant difference in the expression of ET-1 mRNA in all the tissues between the smoking and control groups. These results suggest that cigarette smoking could cause cardiovascular and pulmonary diseases by modulating ET-1 mRNA expression in the tissues.

  9. Cafeteria diet inhibits insulin clearance by reduced insulin-degrading enzyme expression and mRNA splicing.

    PubMed

    Brandimarti, P; Costa-Júnior, J M; Ferreira, S M; Protzek, A O; Santos, G J; Carneiro, E M; Boschero, A C; Rezende, L F

    2013-11-01

    Insulin clearance plays a major role in glucose homeostasis and insulin sensitivity in physiological and/or pathological conditions, such as obesity-induced type 2 diabetes as well as diet-induced obesity. The aim of the present work was to evaluate cafeteria diet-induced obesity-induced changes in insulin clearance and to explain the mechanisms underlying these possible changes. Female Swiss mice were fed either a standard chow diet (CTL) or a cafeteria diet (CAF) for 8 weeks, after which we performed glucose tolerance tests, insulin tolerance tests, insulin dynamics, and insulin clearance tests. We then isolated pancreatic islets for ex vivo glucose-stimulated insulin secretion as well as liver, gastrocnemius, visceral adipose tissue, and hypothalamus for subsequent protein analysis by western blot and determination of mRNA levels by real-time RT-PCR. The cafeteria diet induced insulin resistance, glucose intolerance, and increased insulin secretion and total insulin content. More importantly, mice that were fed a cafeteria diet demonstrated reduced insulin clearance and decay rate as well as reduced insulin-degrading enzyme (IDE) protein and mRNA levels in liver and skeletal muscle compared with the control animals. Furthermore, the cafeteria diet reduced IDE expression and alternative splicing in the liver and skeletal muscle of mice. In conclusion, a cafeteria diet impairs glucose homeostasis by reducing insulin sensitivity, but it also reduces insulin clearance by reducing IDE expression and alternative splicing in mouse liver; however, whether this mechanism contributes to the glucose intolerance or helps to ameliorate it remains unclear.

  10. Membrane-attached Cytokines Expressed by mRNA Electroporation Act as Potent T-Cell Adjuvants.

    PubMed

    Weinstein-Marom, Hadas; Pato, Aviad; Levin, Noam; Susid, Keren; Itzhaki, Orit; Besser, Michal J; Peretz, Tamar; Margalit, Alon; Lotem, Michal; Gross, Gideon

    2016-01-01

    Proinflammatory cytokines are widely explored in different adoptive cell therapy protocols for enhancing survival and function of the transferred T cells, but their systemic administration is often associated with severe toxicity which limits their clinical use. To confine cytokine availability to the therapeutic T cells, we expressed 3 key cytokines, IL-2, IL-12, and IL-15, as integral T-cell membrane proteins. To prevent permanent activation of growth signaling pathways, we delivered these genes to T cells through mRNA electroporation. The engineered cytokines could be detected on the surface of mRNA-transfected cells and binding to their cell-surface receptors mainly occurred in cis. The 3 human cytokines supported the ex vivo growth of activated human CD8 and CD4 T cells for at least 6 days posttransfection, comparably to high-dose soluble IL-2. Similarly, membrane IL-2, membrane IL-12, and, to a lesser extent, membrane IL-15, were comparable with their soluble counterparts in supporting proliferation of splenic mouse CD8 T cells. Following electroporation of human CD8 T cells and antimelanoma tumor-infiltrating lymphocytes, membrane cytokines synergized with constitutively active toll-like receptor 4 in inducing interferon-γ secretion. Efficient cooperation with TLR4 was also evident in the upregulation of the activation molecules CD25, CD69, CD137 (4-1BB), and CD134 (OX40). Taken together, membrane cytokines expressed through mRNA transfection emerge as effective tools for enhancing T-cell proliferation and function and may have potential use in adoptive T-cell therapy.

  11. Arginase-1 mRNA expression correlates with myeloid-derived suppressor cell levels in peripheral blood of NSCLC patients.

    PubMed

    Heuvers, Marlies E; Muskens, Femke; Bezemer, Koen; Lambers, Margaretha; Dingemans, Anne-Marie C; Groen, Harry J M; Smit, Egbert F; Hoogsteden, Henk C; Hegmans, Joost P J J; Aerts, Joachim G J V

    2013-09-01

    Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature and progenitor myeloid cells with immunosuppressive activity that are increased in cancer patients. Until now, the characterization of MDSC in humans was very challenging. The aim of this study was to determine the characterization and optimal assessment of MDSC and to investigate their presence and function in blood of advanced-stage NSCLC patients. We determined MDSC and lymphocyte populations in peripheral blood mononuclear cells (PBMC) samples of 185 treatment-naïve NSCLC patients and 20 healthy controls (HC). NSCLC patients had an increased population of PMN-MDSC compared to HC (p < 0.0001). Frequencies of CD4(+) and CD8(+) T-cells were significantly decreased in NSCLC patients (p < 0.0001 and p = 0.05). We found that PMN-MDSC were able to suppress T-cell proliferation in vitro. qRT-PCR showed that arginase-1 (Arg-1) mRNA is mainly expressed by MDSC and that the level of Arg-1 in PBMC correlates with the frequency of MDSC in PBMC (Spearman's rho: 0.797). There were significant differences in MDSC and lymphocyte populations between NSCLC patients and HC. We found that MDSC frequencies are stable up to six hours at room temperature after blood was drawn and that cryopreservation leads to a strong decrease of MDSC in PBMC. We show that Arg-1 mRNA expression is a valuable method to determine the levels of MDSC in peripheral blood of cancer patients. This method is therefore a useful alternative for the complex flowcytometric analysis in large multicenter patient studies.

  12. Impact of nonsense-mediated mRNA decay on the global expression profile of budding yeast.

    PubMed

    Guan, Qiaoning; Zheng, Wei; Tang, Shijie; Liu, Xiaosong; Zinkel, Robert A; Tsui, Kam-Wah; Yandell, Brian S; Culbertson, Michael R

    2006-11-24

    Nonsense-mediated mRNA decay (NMD) is a eukaryotic mechanism of RNA surveillance that selectively eliminates aberrant transcripts coding for potentially deleterious proteins. NMD also functions in the normal repertoire of gene expression. In Saccharomyces cerevisiae, hundreds of endogenous RNA Polymerase II transcripts achieve steady-state levels that depend on NMD. For some, the decay rate is directly influenced by NMD (direct targets). For others, abundance is NMD-sensitive but without any effect on the decay rate (indirect targets). To distinguish between direct and indirect targets, total RNA from wild-type (Nmd(+)) and mutant (Nmd(-)) strains was probed with high-density arrays across a 1-h time window following transcription inhibition. Statistical models were developed to describe the kinetics of RNA decay. 45% +/- 5% of RNAs targeted by NMD were predicted to be direct targets with altered decay rates in Nmd(-) strains. Parallel experiments using conventional methods were conducted to empirically test predictions from the global experiment. The results show that the global assay reliably distinguished direct versus indirect targets. Different types of targets were investigated, including transcripts containing adjacent, disabled open reading frames, upstream open reading frames, and those prone to out-of-frame initiation of translation. Known targeting mechanisms fail to account for all of the direct targets of NMD, suggesting that additional targeting mechanisms remain to be elucidated. 30% of the protein-coding targets of NMD fell into two broadly defined functional themes: those affecting chromosome structure and behavior and those affecting cell surface dynamics. Overall, the results provide a preview for how expression profiles in multi-cellular eukaryotes might be impacted by NMD. Furthermore, the methods for analyzing decay rates on a global scale offer a blueprint for new ways to study mRNA decay pathways in any organism where cultured cell lines are

  13. Pipping success, isomer-specific accumulation, and hepatic mRNA expression in chicken embryos exposed to HBCD.

    PubMed

    Crump, Doug; Egloff, Caroline; Chiu, Suzanne; Letcher, Robert J; Chu, Shaogang; Kennedy, Sean W

    2010-06-01

    Hexabromocyclododecane (HBCD) is an additive flame retardant used primarily in polystyrene foams. HBCD is a persistent contaminant that has been detected in abiotic and biotic matrices, including wild avian species. The toxicological implications of exposure are not well characterized. We recently identified molecular end points responsive to HBCD exposure in chicken embryonic hepatocytes (CEHs) including genes involved in xenobiotic metabolism, thyroid hormone transport, and lipid metabolism. In the current study, a technical mixture of HBCD (HBCD-TM), comprising 12% alpha-, 11% beta-, and 77% gamma-stereoisomers, was injected into the air cell of chicken eggs prior to incubation. Embryonic viability to pipping, isomer-specific HBCD accumulation, and hepatic mRNA expression of the genes identified in the in vitro study were determined. Concentrations of 100 and 10,000 ng/g decreased pipping success while 50, 300, and 1000 ng/g had no effect. In contrast to HBCD-TM, the isomeric composition in liver tissue was significantly different for alpha- (31%) and gamma-HBCD (61%) demonstrating that isomer-specific processes were occurring in the egg and/or developing embryo. Exposure to 1000 ng/g HBCD-TM significantly upregulated cytochrome P450 (CYP) 2H1, CYP3A37, uridine 5'-diphospho-glucuronosyltransferase, and deiodinase 2, while liver fatty acid-binding protein and insulin-growth factor 1 expression were significantly decreased at 100 and 10,000 ng/g, respectively. The alterations in hepatic mRNA levels were in concordance with those observed in CEH highlighting the utility of both approaches for identifying molecular mechanisms of action. Research on the effects of HBCD in wild avian species is warranted.

  14. Effects of 12 Ca2+ antagonists on multidrug resistance, MDR1-mediated transport and MDR1 mRNA expression.

    PubMed

    Takara, Kohji; Sakaeda, Toshiyuki; Tanigawara, Yusuke; Nishiguchi, Kohshi; Ohmoto, Nobuko; Horinouchi, Masanori; Komada, Fusao; Ohnishi, Noriaki; Yokoyama, Teruyoshi; Okumura, Katsuhiko

    2002-08-01

    The effects of 12 Ca(2+) antagonists on MDR1 were examined by two independent models: the inhibitory effect on MDR1-mediated transport of [(3)H]digoxin using MDR1-overexpressing LLC-GA5-COL150 cell monolayers and the reversal effect on cytotoxicity of vinblastine or paclitaxel using MDR1-overexpressing Hvr100-6 cells. The inhibitory effects on [(3)H]digoxin transport were assessed as the 50% inhibitory concentration during 4 h exposure, and the values were the lowest for nicardipine (4.54 microM), manidipine (4.65 microM) and benidipine (4.96 microM), followed by bepridil (10.6 microM), barnidipine (12.6 microM), efonidipine (13.0 microM), verapamil (13.2 microM) and nilvadipine (18.0 microM). The reversal effect on cytotoxicity was assessed by the 50% growth inhibitory concentration after 3 days exposure, and the resistance to vinblastine or paclitaxel in Hvr100-6 cells was reversed by manidipine, verapamil, benidipine, barnidipine, and nicardipine, in that order. Bepridil, barnidipine, efonidipine, verapamil and nilvadipine showed similar inhibitory effects on [(3)H]digoxin transport, but barnidipine and verapamil showed a stronger effect in reversal of cytotoxicity. Real-time quantitative RT-PCR assay indicated a decrease in MDR1 mRNA expression by barnidipine and verapamil. It is concluded that Ca(2+) antagonists cannot only be direct inhibitors of MDR1 but that some may at the same time act as inhibitors of expression of MDR1 via down-regulation of MDR1 mRNA.

  15. Posttranscriptional regulation of hsp70 expression in human cells: effects of heat shock, inhibition of protein synthesis, and adenovirus infection on translation and mRNA stability.

    PubMed Central

    Theodorakis, N G; Morimoto, R I

    1987-01-01

    We have examined the posttranscriptional regulation of hsp70 gene expression in two human cell lines, HeLa and 293 cells, which constitutively express high levels of HSP70. HSP70 mRNA translates with high efficiency in both control and heat-shocked cells. Therefore, heat shock is not required for the efficient translation of HSP70 mRNA. Rather, the main effect of heat shock on translation is to suppress the translatability of non-heat shock mRNAs. Heat shock, however, has a marked effect on the stability of HSP70 mRNA; in non-heat-shocked cells the half-life of HSP70 mRNA is approximately 50 min, and its stability increases at least 10-fold upon heat shock. Moreover, HSP70 mRNA is more stable in cells treated with protein synthesis inhibitors, suggesting that a heat shock-sensitive labile protein regulates its turnover. An additional effect on posttranscriptional regulation of hsp70 expression can be found in adenovirus-infected cells, in which HSP70 mRNA levels decline precipititously late during infection although hsp70 transcription continues unabated. Images PMID:3437893

  16. Untranslated regions of mRNA and their role in regulation of gene expression in protozoan parasites.

    PubMed

    Rao, Shilpa J; Chatterjee, Sangeeta; Pal, Jayantapal K

    2017-03-01

    Protozoan parasites are one of the oldest living entities in this world that throughout their existence have shown excellent resilience to the odds of survival and have adapted beautifully to ever changing rigors of the environment. In view of the dynamic environment encountered by them throughout their life cycle, and in establishing pathogenesis, it is unsurprising that modulation of gene expression plays a fundamental role in their survival. In higher eukaryotes, untranslated regions (UTRs) of transcripts are one of the crucial regulators of gene expression (influencing mRNA stability and translation efficiency). Parasitic protozoan genome studies have led to the characterization (in silico, in vitro and in vivo) of a large number of their genes. Comparison of higher eukaryotic UTRs with parasitic protozoan UTRs reveals the existence of several similar and dissimilar facets of the UTRs. This review focuses on the elements of UTRs of medically important protozoan parasites and their regulatory role in gene expression. Such information may be useful to researchers in designing gene targeting strategies linked with perturbation of host-parasite relationships leading to control of specific parasites.

  17. Integrated Analysis of DNA Methylation and mRNA Expression Profiles Data to Identify Key Genes in Lung Adenocarcinoma

    PubMed Central

    Jin, Xiang; Li, Xiaodan; Guan, Yinghui

    2016-01-01

    Introduction. Lung adenocarcinoma (LAC) is the most frequent type of lung cancer and has a high metastatic rate at an early stage. This study is aimed at identifying LAC-associated genes. Materials and Methods. GSE62950 downloaded from Gene Expression Omnibus included a DNA methylation dataset and an mRNA expression profiles dataset, both of which included 28 LAC tissue samples and 28 adjacent normal tissue samples. The differentially expressed genes (DEGs) were screened by Limma package in R, and their functions were predicted by enrichment analysis using TargetMine online tool. Then, protein-protein interaction (PPI) network was constructed using STRING and Cytoscape. Finally, LAC-associated methylation sites were identified by CpGassoc package in R and mapped to the DEGs to obtain LAC-associated DEGs. Results. Total 913 DEGs were identified in LAC tissues. In the PPI networks, MAD2L1, AURKB, CCNB2, CDC20, and WNT3A had higher degrees, and the first four genes might be involved in LAC through interaction. Total 8856 LAC-associated methylation sites were identified and mapped to the DEGs. And there were 29 LAC-associated methylation sites located in 27 DEGs (e.g., SH3GL2, BAI3, CDH13, JAM2, MT1A, LHX6, and IGFBP3). Conclusions. These key genes might play a role in pathogenesis of LAC. PMID:27610375

  18. Quantification of diatom gene expression in the sea by selecting uniformly transcribed mRNA as the basis for normalization.

    PubMed

    Kang, Lee-Kuo; Tsui, Feng-Hsiu; Chang, Jeng

    2012-09-01

    To quantify gene expressions by quantitative reverse transcription-PCR (Q-RT-PCR) in natural diatom assemblages, it is necessary to seek a biomass reference specific to the target species. Two housekeeping genes, TBP (encoding the TATA box-binding protein) and EFL (encoding the translation elongation factor-like protein), were evaluated as candidates for reference genes in Q-RT-PCR assays. Transcript levels of TBP and EFL were relatively stable under various test conditions including growth stages, light-dark cycle phases, and nutrient stresses in Skeletonema costatum and Chaetoceros affinis, and TBP expression was more stable than that of EFL. Next, the sequence diversity of diatom assemblages was evaluated by obtaining 32 EFL and 29 TBP homologous gene fragments from the East China Sea (ECS). Based on sequence alignments, EFL and TBP primer sets were designed for Chaetoceros and Skeletonema groups in the ECS. An evaluation of primer specificity and PCR efficiency indicated that the EFL primer sets performed better. To demonstrate the applicability of EFL primer sets in the ECS, they were employed to measure mRNA levels of the FcpB (fucoxanthin-chlorophyll protein) gene in diatoms. The results correctly revealed prominent diel variations in FcpB expression and confirmed EFL as a good reference gene.

  19. Loss of CD4 membrane expression and CD4 mRNA during acute human immunodeficiency virus replication

    PubMed Central

    1988-01-01

    Using mAbs and genomic probe to the CD4 molecule, the HIV receptor, we demonstrated that HIV replication induces the disappearance of its functional receptor from the cell surface by two distinct mechanisms. First, after being expressed onto the cell surface, HIV envelope gp110 will complex CD4, efficiently masking the CD4 epitope used by the virus to bind its receptor. This phenomenon occurs on the surface of each infected cell and is not due to the release of soluble gp110; infection with recombinant HIV/vaccinia viruses expressing a mutated HIV env gene designed to prevent gp110 release from the cell surface induces a similar gp/CD4 complexes formation. Second, virus replication induces a dramatic and rapid loss of CD4 mRNA transcripts, preventing new CD4 molecules from being synthesized. These two mechanisms of receptor modulation could have been developed to avoid reinfection of cells replicating the virus as well as to produce more infectious particles. These results suggest that the classical virus interference documented for other retroviruses might not only be due to receptor/envelope interaction, but might also depend on receptor gene expression. PMID:3264318

  20. Vanillin Suppresses Cell Motility by Inhibiting STAT3-Mediated HIF-1α mRNA Expression in Malignant Melanoma Cells.

    PubMed

    Park, Eun-Ji; Lee, Yoon-Mi; Oh, Taek-In; Kim, Byeong Mo; Lim, Beong-Ou; Lim, Ji-Hong

    2017-03-01

    Recent studies have shown that vanillin has anti-cancer, anti-mutagenic, and anti-metastatic activity; however, the precise molecular mechanism whereby vanillin inhibits metastasis and cancer progression is not fully elucidated. In this study, we examined whether vanillin has anti-cancer and anti-metastatic activities via inhibition of hypoxia-inducible factor-1α (HIF-1α) in A2058 and A375 human malignant melanoma cells. Immunoblotting and quantitative real time (RT)-PCR analysis revealed that vanillin down-regulates HIF-1α protein accumulation and the transcripts of HIF-1α target genes related to cancer metastasis including fibronectin 1 (FN1), lysyl oxidase-like 2 (LOXL2), and urokinase plasminogen activator receptor (uPAR). It was also found that vanillin significantly suppresses HIF-1α mRNA expression and de novo HIF-1α protein synthesis. To understand the suppressive mechanism of vanillin on HIF-1α expression, chromatin immunoprecipitation was performed. Consequently, it was found that vanillin causes inhibition of promoter occupancy by signal transducer and activator of transcription 3 (STAT3), but not nuclear factor-κB (NF-κB), on HIF1A. Furthermore, an in vitro migration assay revealed that the motility of melanoma cells stimulated by hypoxia was attenuated by vanillin treatment. In conclusion, we demonstrate that vanillin might be a potential anti-metastatic agent that suppresses metastatic gene expression and migration activity under hypoxia via the STAT3-HIF-1α signaling pathway.

  1. Vanillin Suppresses Cell Motility by Inhibiting STAT3-Mediated HIF-1α mRNA Expression in Malignant Melanoma Cells

    PubMed Central

    Park, Eun-Ji; Lee, Yoon-Mi; Oh, Taek-In; Kim, Byeong Mo; Lim, Beong-Ou; Lim, Ji-Hong

    2017-01-01

    Recent studies have shown that vanillin has anti-cancer, anti-mutagenic, and anti-metastatic activity; however, the precise molecular mechanism whereby vanillin inhibits metastasis and cancer progression is not fully elucidated. In this study, we examined whether vanillin has anti-cancer and anti-metastatic activities via inhibition of hypoxia-inducible factor-1α (HIF-1α) in A2058 and A375 human malignant melanoma cells. Immunoblotting and quantitative real time (RT)-PCR analysis revealed that vanillin down-regulates HIF-1α protein accumulation and the transcripts of HIF-1α target genes related to cancer metastasis including fibronectin 1 (FN1), lysyl oxidase-like 2 (LOXL2), and urokinase plasminogen activator receptor (uPAR). It was also found that vanillin significantly suppresses HIF-1α mRNA expression and de novo HIF-1α protein synthesis. To understand the suppressive mechanism of vanillin on HIF-1α expression, chromatin immunoprecipitation was performed. Consequently, it was found that vanillin causes inhibition of promoter occupancy by signal transducer and activator of transcription 3 (STAT3), but not nuclear factor-κB (NF-κB), on HIF1A. Furthermore, an in vitro migration assay revealed that the motility of melanoma cells stimulated by hypoxia was attenuated by vanillin treatment. In conclusion, we demonstrate that vanillin might be a potential anti-metastatic agent that suppresses metastatic gene expression and migration activity under hypoxia via the STAT3-HIF-1α signaling pathway. PMID:28257048

  2. Microarray analysis of long noncoding RNA and mRNA expression profiles in human macrophages infected with Mycobacterium tuberculosis

    PubMed Central

    Yang, Xiaofan; Yang, Jiahui; Wang, Jinli; Wen, Qian; Wang, Hui; He, Jianchun; Hu, Shengfeng; He, Wenting; Du, Xialin; Liu, Sudong; Ma, Li

    2016-01-01

    Macrophages play a crucial role in the control and elimination of invading Mycobacterium tuberculosis (Mtb), and also serve as the major residence for Mtb. However, the interaction between macrophages and Mtb remains to be clearly determined. Although long noncoding RNAs (lncRNAs) have emerged as key regulators in many biological processes, their roles in anti-mycobacterial responses of macrophages remain to be elucidated. Here, we applied microarray analysis to examine lncRNA and mRNA expression profiles in human primary macrophages after 72 h of infection with H37Ra or H37Rv. Our results revealed that many lncRNAs were differentially expressed in macrophages after H37Ra or H37Rv infection, indicating a possible role for lncRNAs in immune responses induced by Mtb infection and providing important cues for further functional studies. Furthermore, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) biological pathway analysis of the differentially expressed mRNAs showed the potential functions and pathways related to the pathogenesis of Mtb infection. Finally, two lncRNAs, MIR3945HG V1 and MIR3945HG V2, were identified as novel candidate diagnostic markers for tuberculosis. Our results provide novel insight into the mechanisms of the pivotal Mtb-macrophage interactions, and reveal potential targets for diagnostics and the treatment of tuberculosis. PMID:27966580

  3. Effect of doxycycline and Lactobacillus probiotics on mRNA expression of ABCC2 in small intestines of chickens

    PubMed Central

    Milanova, A.; Pavlova, I.; Yordanova, V.; Danova, S.

    2016-01-01

    Probiotics and antibiotics are widely used in poultry and may alter drug bioavailability by affecting the expression of intestinal ATP-binding cassette (ABC) efflux transporters. Therefore the aim of the present investigation was to evaluate the effect of Lactobacilli probiotics, administered alone or in combination with doxycycline, on the expression of ABCB1 (gene, encoding P-glycoprotein), ABCC2 (gene, encoding multidrug resistance protein 2, MRP2) and ABCG2 (gene, encoding breast cancer resistance protein) mRNAs in chicken using RT-PCR. Duc one-day-old chicks (n=24) were divided equally in four groups: untreated control, probiotics supplemented group, probiotics plus doxycycline treated chickens and antibiotic administered group. Expression of ABCC2 mRNA was affected by doxycycline or by combination of Lactobacillus plantarum, L. brevis and L. bulgaricus and the antibiotic in the intestines. These results can be used as a basis for further functional studies to prove the beneficial effect on limitation of the absorption of toxins and improvement of efflux of endogenous substances and xenobiotics when the combination of doxycycline and Lactobacillus spp. probiotics are administered to poultry. PMID:28224011

  4. HER-2 and EGFR mRNA Expression and Its Relationship with Versican in Malignant Matrix-Producing Tumors of the Canine Mammary Gland

    PubMed Central

    Damasceno, Karine Araújo; Ferreira, Enio; Estrela-Lima, Alessandra; Gamba, Conrado de Oliveira; Miranda, Fernanda Freitas; Alves, Mariana Rezende; Rocha, Rafael Malagoli; de Barros, André Luís Branco; Cassali, Geovanni Dantas

    2016-01-01

    Versican expression promotes tumor growth by destabilizing focal cell contacts, thus impeding cell adhesion and facilitating cell migration. It not only presents or recruits molecules to the cell surface, but also modulates gene expression levels and coordinates complex signal pathways. Previously, we suggested that the interaction between versican and human epidermal growth factor receptors may be directly associated with tumor aggressiveness. Thus, the expression of EGFR and HER-2 in these neoplasms may contribute to a better understanding of the progression mechanisms in malignant mammary tumors. The purpose of this study was to correlate the gene and protein expressions of EGFR and HER2 by RNA In Situ Hybridization (ISH) and immunohistochemistry (IHC), respectively, and their relationship with the versican expression in carcinomas in mixed tumors and carcinosarcomas of the canine mammary gland. The results revealed that EGFR mRNA expression showed a significant difference between in situ and invasive carcinomatous areas in low and high versican expression groups. Identical results were observed in HER-2 mRNA expression. In immunohistochemistry analysis, neoplasms with low versican expression showed greater EGFR immunostaining in the in situ areas than in invasive areas, even as the group presenting high versican expression displayed greater EGFR and HER-2 staining in in situ areas. Significant EGFR and HER-2 mRNA and protein expressions in in situ carcinomatous sites relative to invasive areas suggest that these molecules play a role during the early stages of tumor progression. PMID:27490467

  5. Sodium arsenite-induced inhibition of cell proliferation is related to inhibition of IL-2 mRNA expression in mouse activated T cells.

    PubMed

    Conde, Patricia; Acosta-Saavedra, Leonor C; Goytia-Acevedo, Raquel C; Calderon-Aranda, Emma S

    2007-04-01

    A proposed mechanism for the As-induced inhibition of cell proliferation is the inhibition of IL-2 secretion. However, the effects of arsenite on IL-2 mRNA expression or on the ERK pathway in activated-T cells have not yet been described. We examined the effect of arsenite on IL-2 mRNA expression, cell activation and proliferation in PHA-stimulated murine lymphocytes. Arsenite (1 and 10 microM) decreased IL-2 mRNA expression, IL-2 secretion and cell proliferation. Arsenite (10 microM) strongly inhibited ERK-phosphorylation. However, the partial inhibition (50%) of IL-2 mRNA produced by 1 microM, consistent with the effects on IL-2 secretion and cell proliferation, could not be explained by the inhibition of ERK-phosphorylation, which was not affected at this concentration. The inhibition of IL-2 mRNA expression caused by 1 microM could be associated to effects on pathways located downstream or parallel to ERK. Arsenite also decreased early activation (surface CD69+ expression) in both CD4+ and CD8+, and decreased total CD8+ count without significantly affecting CD4+, supporting that the cellular immune response mediated by cytotoxic T cells is an arsenic target. Thus, our results suggest that arsenite decreases IL-2 mRNA levels and T-cell activation and proliferation. However, further studies on the effects of arsenite on IL-2 gene transcription and IL-2 mRNA stability are needed.

  6. Temporal changes in the expression of brain-derived neurotrophic factor mRNA in the ventromedial nucleus of the hypothalamus of the developing rat brain.

    PubMed

    Sugiyama, Nobuhiro; Kanba, Shigenobu; Arita, Jun

    2003-07-04

    Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family, which is important for the growth, differentiation, and survival of neurons during development. We have performed a detailed mapping of BDNF mRNA in the neonatal rat brain using a quantitative in situ hybridization technique. At postnatal day (PND) 4, hypothalamic structures showed only modest expression of BDNF mRNA, with the exception of the ventromedial nucleus (VMN), where expression was higher than that detected in the hippocampus. Abundant BDNF mRNA was also found in the bed nucleus of the anterior commissure, retrosplenial granular cortex, and the posteroventral part of the medial amygdaloid nucleus. Messenger RNAs encoding other neurotrophins, including nerve growth factor (NGF) and neurotrophin-3 (NT-3) and the BDNF receptor trkB, were not selectively localized in neonatal VMN. During subsequent developmental stages, BDNF mRNA expression in the VMN changed dynamically, peaking at PND 4 and falling to minimal levels in the adult brain. In contrast, the low levels of BDNF mRNA observed in the CA3 region of the hippocampus increased to adult levels following PND 10. As the VMN undergoes sexual differentiation, we compared BDNF, NGF, NT-3, and trkB mRNA expression in the VMN in males and females at embryonic day 20 and PND 4, but found no differences between them. These results suggest that localized and high level expression of BDNF mRNA in the neonatal VMN plays an important role in its neural organization and functional development.

  7. Seasonal changes in peptide, receptor and ion channel mRNA expression in the caudal neurosecretory system of the European flounder (Platichthys flesus).

    PubMed

    Lu, Weiqun; Worthington, Jonathan; Riccardi, Daniela; Balment, Richard J; McCrohan, Catherine R

    2007-01-01

    The caudal neurosecretory system (CNSS) of the euryhaline flounder Platichthys flesus has suggested roles in osmoregulatory, reproductive and nutritional adaptation, as fish migrate between seawater (winter) and brackish/freshwater (summer) environments. This study examined seasonal changes in mRNA expression profile of functionally important genes in the CNSS. cDNAs encoding neuropeptides, receptors and ion channels were cloned by reverse transcriptase polymerase chain reaction (RT-PCR) and screening of a flounder CNSS cDNA library. The expression profile of cloned genes was determined by real-time RT-PCR at 2-month intervals throughout the year in CNSS from seawater-adapted fish. Plasma cortisol (measured by radioimmunoassay) showed a peak in April, the time of spawning. Expression levels of mRNA for peptides urotensins I and II (UI, UII) and corticotropin releasing factor (CRF) all showed a seasonal cycle, with lowest expression in April and highest in August-October. The expression of CRF2(UI), UT(UII) and CRF1 receptors was not correlated with corresponding peptide expression. Receptors for potential neuromodulators of CNSS activity also displayed a seasonal mRNA expression profile. Glucocorticoid, 5-hydroxytryptamine, kappa-opioid and glutamate receptor expression peaked around April, suggesting that modulation of electrical activity of the neurosecretory Dahlgren cells is of particular importance at this time. Expression of mRNA for L-type Ca(2+) and Ca-activated K(+) channels was lower during the summer months. These channels underlie electrical bursting activity in Dahlgren cells. Ion channel mRNA expression was also lower in CNSS from flounder fully adapted to freshwater as opposed to seawater, consistent with previously reported observations of reduced bursting activity in Dahlgren cells from freshwater-adapted CNSS. These findings support the hypothesis that the CNSS is functionally reprogrammed to cope with changes in physiological challenge as fish

  8. Tissue-specific mRNA expression profiles of porcine Toll-like receptors at different ages in germ-free and conventional pigs

    PubMed Central

    Shao, Lulu; Fischer, David D.; Kandasamy, Sukumar; Saif, Linda J.; Vlasova, Anastasia N.

    2016-01-01

    Toll-like receptors (TLRs), key initiators of innate immune responses, recognize antigens and are essential in linking innate and adaptive immune responses. Misrecognition and over-stimulation/expression of TLRs may contribute to the development of chronic inflammatory diseases and autoimmune diseases. However, appropriate and mature TLR responses are associated with the establishment of resistance against some infectious diseases. In this study, we assessed the mRNA expression profile of TLRs 1-10 in splenic and ileal mononuclear cells (MNCs) and dendritic cells (DCs) of germ-free (GF) and conventional pigs at different ages. We found that the TLR mRNA expression profiles were distinct between GF and conventional pigs. The expression profiles were also significantly different between splenic and ileal MNCs/DCs. Comparison of the TLR expression profiles in GF and conventional newborn and young pigs demonstrated that exposure to commensal microbiota may play a more important role than age in TLR mRNA expression profiles. To our knowledge, this is the first report that systematically assesses porcine TLRs 1-10 mRNA expression profiles in MNCs and DCs from GF and conventional pigs at different ages. These results further highlighted that the commensal microbiota of neonates play a critical role through TLR signaling in the development of systemic and mucosal immune systems. PMID:26964712

  9. Toll-like receptor 4 and CD14 mRNA expression are lower in resistive exercise-trained elderly women.

    PubMed

    Flynn, Michael G; McFarlin, Brian K; Phillips, Melody D; Stewart, Laura K; Timmerman, Kyle L

    2003-11-01

    The purpose of this study was to examine the influence of resistive exercise training and hormone status on mRNA expression of toll-like receptor 4 (TLR4), CD14, IL-1beta, IL-6, and TNF-alpha. Resistive exercise-trained women on "traditional" hormone replacements [hormone replacement therapy (HRT), n = 9], not taking hormones (NHR, n = 6), or taking medications known to influence bone (MIB, n = 7) were compared with untrained subjects not taking supplemental hormones (Con, n = 6). Blood was taken from trained subjects before, immediately after, and 2 h after resistive exercise (same time points for resting Con). TLR4 mRNA expression (RT-PCR) was not different among groups or across time but was significantly (P = 0.044) lower (1.9-fold) when trained groups were collapsed and compared with Con. There was also a significant group effect (P < 0.0001) for TLR4 mRNA when expressed per monocyte. CD14 expression was significantly (P = 0.006) lower (2.3-fold) for training groups collapsed and compared with Con. CD14 mRNA, expressed per monocyte, was significantly lower immediately after resistive exercise for NHR, HRT, and MIB compared with Con. There were few significant effects detected for IL-6, IL-1beta, and TNF-alpha mRNA, but there was a significant group effect (P < 0.0001) for TNF-alpha mRNA expressed per monocyte (Con > HRT, NHR, MIB). These findings suggest that there may be a resistive exercise training-induced reduction in TLR4/CD14 expression in older women. Further research is needed to determine whether lower TLR4/CD14 could explain the lower LPS-stimulated inflammatory cytokines observed in these women.

  10. Intrahepatic mRNA Expression of FAS, FASL, and FOXP3 Genes Is Associated with the Pathophysiology of Chronic HCV Infection

    PubMed Central

    Amoras, Ednelza da Silva Graça; Gomes, Samara Tatielle Monteiro; Freitas, Felipe Bonfim; Santana, Bárbara Brasil; Ishak, Geraldo; Ferreira de Araújo, Marialva Tereza; Demachki, Sâmia; Conde, Simone Regina Souza da Silva; Ishak, Marluísa de Oliveira Guimarães; Ishak, Ricardo; Vallinoto, Antonio Carlos Rosário

    2016-01-01

    This study aimed to evaluate the relative mRNA expression of Fas receptor (FAS), Fas ligand (FASL), and forkhead box protein 3 (FOXP3) in liver biopsy specimens obtained from patients with viral and non-viral chronic hepatitis and correlate their expression with the fibrosis stage. A total of 51 liver biopsy specimens obtained from HBV (n = 6), HCV (n = 28), and non-viral hepatic disease (NVHD) (n = 9) patients and from individuals with normal liver histology (n = 8) (control—CT) were analyzed. Quantifications of the target genes were assessed using qPCR, and liver biopsies according to the METAVIR classification. The mRNA expression levels of FAS and FASL were lower in the CT group compared to the groups of patients. The increase in the mRNA expression of FAS and FASL was correlated with higher levels of inflammation and disease progression, followed by a decline in tissues with cirrhosis, and it was also associated with increased levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Higher mRNA expression of FOXP3 was observed in the HCV and NVHD groups, with the peak observed among patients with cirrhosis. The increased FOXP3 mRNA expression was positively correlated with increased FAS and FASL mRNA expression and the AST and ALT levels in all patients. Conclusions: These results suggest that regardless of the cause, the course of chronic liver disease may be modulated by the analyzed genes and correlated with an increase in regulatory T cells during the liver damage followed by hepatocyte destruction by Fas/FasL system and subsequent non specific lymphocytic infiltrate accumulation. PMID:27243827

  11. mRNA expression of corticotropin-releasing factor and urocortin 1 after restraint and foot shock together with alprazolam administration.

    PubMed

    Cespedes, Isabel C; de Oliveira, Amanda R; da Silva, Joelcimar M; da Silva, André V; Sita, Luciane V; Bittencourt, Jackson C

    2010-12-01

    Corticotropin-releasing factor (CRF) is expressed in the paraventricular nucleus of the hypothalamus (PVN), and act centrally to provoke stress-like autonomic and behavioral responses. Urocortins 1-3 are additional ligands to the CRF receptors 1 and 2. Ucn 1 neurons are primarily concentrated in the Edinger-Westphal (EW) nucleus and also have been associated with stress responses. It is also known that UCN 1 respond in different ways depending on the stressor presented. Benzodiazepines can act via the CRF peptidergic system and chronic administration of alprazolam does not interfere with CRF mRNA expression in the PVN, but significantly increase Ucn 1 mRNA expression in the EW. The aim of our study was to investigate the relationship between different stressor stimuli, foot shock (FS) and restraint (R), and the mRNA expression of CRF and Ucn 1 in the PVN and EW using alprazolam (A). We employed fos activation and in situ hybridization. Restraint group presented increased fos-ir and CRF mRNA expression in the PVN compared to FS group. The stress responses of R group were prevented by A. In the EW, fos-ir was higher in the FS group than in the R group, whereas Ucn 1 mRNA expression was higher in the R group than in the FS group. Alprazolam significantly increased fos-ir and Ucn 1 mRNA expression in both groups. Our results show that PVN and EW respond in different ways to the same stressors. Furthermore, EW of stressed animals replies in a complementary way comparing to PVN with the use of Alprazolam.

  12. The developmental changes and effect on IMF content of H-FABP and PPARgamma mRNA expression in sheep muscle.

    PubMed

    Huang, Zhi-Guo; Xiong, Li; Liu, Zhen-Shan; Qiao, Yong; Liu, Shou-Ren; Ren, Hang-Xing; Xie, Zhuang; Liu, Guo-Qing; Li, Xue-Bin

    2006-06-01

    Male Kazak sheep and Xinjiang fine wool sheep of different ages were selected to investigate the developmental changes and effect on intramuscular fat (IMF) content of heart fatty acid-binding protein (H-FABP) and peroxisome proliferator-activated receptor gamma (PPARgamma) mRNA expression in muscle. Longissimus dorsal muscle was sampled to measure IMF content; and total RNA was extracted to determine H-FABP and PPARgamma mRNA expression levels by real-time PCR. The results showed that: (1) The IMF content increased continuously with growing and showed significant differences (P<0.05) between ages in male Kazak sheep, but no such differences (P>0.05) existed in Xinjiang fine wool sheep. Furthermore, the IMF content in Kazak sheep was very much higher (P<0.01) than that of the other breed from day 30 to 90; (2) H-FABP mRNA expression level was the highest on day 2 and showed significant differences (P<0.05) between ages in male Kazak sheep as well as in Xinjiang fine wool sheep. In the former breed, the expression reached the lowest point at day 30, and then rose continuously. But in the latter breed, it declined continuously from day 2 to 90, and then increased; (3) Significant differences (P<0.05) of PPARgamma mRNA expression between ages occurred in both breeds. In male Kazak sheep, PPARgamma mRNA expression declined from day 2 to 90, while in the other breed it increased continuously from day 2 to 60, but reached the lowest level at day 90, then increased; (4) In male Kazak sheep, the mRNA expression level of H-FABP was highly positively correlated (r=0.737, P<0.01) with IMF content from day 30 to 90, but that of PPARgamma was highly negatively correlated (r=-0.835, P<0.01) with IMF content from day 2 to 90.

  13. Effects of growth differentiation factor-9 and FSH on in vitro development, viability and mRNA expression in bovine preantral follicles.

    PubMed

    Vasconcelos, G L; Saraiva, M V A; Costa, J J N; Passos, M J; Silva, A W B; Rossi, R O D S; Portela, A M L R; Duarte, A B G; Magalhães-Padilha, D M; Campelo, C C; Figueiredo, J R; van den Hurk, R; Silva, J R V

    2013-01-01

    The present study investigated the role of growth differentiation factor (GDF)-9 and FSH, alone or in combination, on the growth, viability and mRNA expression of FSH receptor, proliferating cell nuclear antigen (PCNA) and proteoglycan-related factors (i.e., hyaluronan synthase (HAS) 1, HAS2, versican, perlecan) in bovine secondary follicles before and after in vitro culture. After 12 days culture, sequential FSH (100 ng mL⁻¹) from Days 0 to 6 and 500 ng mL⁻¹ from Days 7 to 12) increased follicular diameter and resulted in increased antrum formation (P<0.05). Alone, 200 ng mL⁻¹ GDF-9 significantly reduced HAS1 mRNA levels, but increased versican and perlecan mRNA levels in whole follicles, which included the oocyte, theca and granulosa cells. Together, FSH and GDF-9 increased HAS2 and versican (VCAN) mRNA levels, but decreased PCNA mRNA expression, compared with levels in follicles cultured in α-minimum essential medium supplemented with 3.0 mg mL⁻¹ bovine serum albumin, 10 µg mL⁻¹ insulin, 5.5 µg mL⁻¹ transferrin, 5 ng mL⁻¹ selenium, 2 mM glutamine, 2mM hypoxanthine and 50 μg mL⁻¹ ascorbic acid (α-MEM⁺). Comparisons of uncultured (0.2 mm) and α-MEM⁺ cultured follicles revealed that HAS1 mRNA expression was higher, whereas VCAN expression was lower, in cultured follicles (P<0.05). Expression of HAS1, VCAN and perlecan (HSPG2) was higher in cultured than in vivo-grown (0.3 mm) follicles. In conclusion, FSH and/or GDF-9 promote follicular growth and antrum formation. Moreover, GDF-9 stimulates expression of versican and perlecan and interacts positively with FSH to increase HAS2 expression.

  14. Volcano plots in analyzing differential expressions with mRNA microarrays.

    PubMed

    Li, Wentian

    2012-12-01

    A volcano plot displays unstandardized signal (e.g. log-fold-change) against noise-adjusted/standardized signal (e.g. t-statistic or -log(10)(p-value) from the t-test). We review the basic and interactive use of the volcano plot and its crucial role in understanding the regularized t-statistic. The joint filtering gene selection criterion based on regularized statistics has a curved discriminant line in the volcano plot, as compared to the two perpendicular lines for the "double filtering" criterion. This review attempts to provide a unifying framework for discussions on alternative measures of differential expression, improved methods for estimating variance, and visual display of a microarray analysis result. We also discuss the possibility of applying volcano plots to other fields beyond microarray.

  15. A Novel Actin mRNA Splice Variant Regulates ACTG1 Expression

    PubMed Central

    Drummond, Meghan C.; Friderici, Karen H.

    2013-01-01

    Cytoplasmic actins are abundant, ubiquitous proteins in nucleated cells. However, actin expression is regulated in a tissue- and development-specific manner. We identified a novel cytoplasmic-γ-actin (Actg1) transcript that includes a previously unidentified exon (3a). Inclusion of this exon introduces an in-frame termination codon. We hypothesized this alternatively-spliced transcript down-regulates γ-actin production by targeting these transcripts for nonsense-mediated decay (NMD). To address this, we investigated conservation between mammals, tissue-specificity in mice, and developmental regulation using C2C12 cell culture. Exon 3a is 80% similar among mammals and varies in length from 41 nucleotides in humans to 45 in mice. Though the predicted amino acid sequences are not similar between all species, inclusion of exon 3a consistently results in the in the introduction of a premature termination codon within the alternative Actg1 transcript. Of twelve tissues examined, exon 3a is predominantly expressed in skeletal muscle, cardiac muscle, and diaphragm. Splicing to include exon 3a is concomitant with previously described down-regulation of Actg1 in differentiating C2C12 cells. Treatment of differentiated C2C12 cells with an inhibitor of NMD results in a 7-fold increase in exon 3a-containing transcripts. Therefore, splicing to generate exon 3a-containing transcripts may be one component of Actg1 regulation. We propose that this post-transcriptional regulation occurs via NMD, in a process previously described as “regulated unproductive splicing and translation” (RUST). PMID:24098136

  16. Phase I Metabolic Genes and Risk of Lung Cancer: Multiple Polymorphisms and mRNA Expression

    PubMed Central

    Rotunno, Melissa; Yu, Kai; Lubin, Jay H.; Consonni, Dario; Pesatori, Angela C.; Goldstein, Alisa M.; Goldin, Lynn R.; Wacholder, Sholom; Burdette, Laurie; Chanock, Stephen J.; Bertazzi, Pier Alberto; Tucker, Margaret A.; Caporaso, Neil E.; Chatterjee, Nilanjan; Bergen, Andrew W.; Landi, Maria Teresa

    2009-01-01

    Polymorphisms in genes coding for enzymes that activate tobacco lung carcinogens may generate inter-individual differences in lung cancer risk. Previous studies had limited sample sizes, poor exposure characterization, and a few single nucleotide polymorphisms (SNPs) tested in candidate genes. We analyzed 25 SNPs (some previously untested) in 2101 primary lung cancer cases and 2120 population controls from the Environment And Genetics in Lung cancer Etiology (EAGLE) study from six phase I metabolic genes, including cytochrome P450s, microsomal epoxide hydrolase, and myeloperoxidase. We evaluated the main genotype effects and genotype-smoking interactions in lung cancer risk overall and in the major histology subtypes. We tested the combined effect of multiple SNPs on lung cancer risk and on gene expression. Findings were prioritized based on significance thresholds and consistency across different analyses, and accounted for multiple testing and prior knowledge. Two haplotypes in EPHX1 were significantly associated with lung cancer risk in the overall population. In addition, CYP1B1 and CYP2A6 polymorphisms were inversely associated with adenocarcinoma and squamous cell carcinoma risk, respectively. Moreover, the association between CYP1A1 rs2606345 genotype and lung cancer was significantly modified by intensity of cigarette smoking, suggesting an underling dose-response mechanism. Finally, increasing number of variants at CYP1A1/A2 genes revealed significant protection in never smokers and risk in ever smokers. Results were supported by differential gene expression in non-tumor lung tissue samples with down-regulation of CYP1A1 in never smokers and up-regulation in smokers from CYP1A1/A2 SNPs. The significant haplotype associations emphasize that the effect of multiple SNPs may be important despite null single SNP-associations, and warrants consideration in genome-wide association studies (GWAS). Our findings emphasize the necessity of post-GWAS fine mapping and

  17. The Hobby-Eberly Telescope "Chemical Abundances Of Stars In The Halo" (CASH) Project. I. The Lithium-, r-, and s-enhanced Metal-poor Giant HK-II 17435-00532

    NASA Astrophysics Data System (ADS)

    Roederer, Ian U.; Frebel, A.; Shetrone, M.; Allende Prieto, C.; Rhee, J.; Gallino, R.; Bisterzo, S.; Sneden, C.; Beers, T. C.; Cowan, J. J.

    2007-12-01

    We present the first detailed abundance analysis of the metal-poor giant HK-II 17435-00532. This star was observed as part of the University of Texas Long-Term "Chemical Abundances of Stars in the Halo" (CASH) Project. A spectrum was obtained with the High Resolution Spectrograph (HRS) on the Hobby-Eberly Telescope with a resolving power of R 15,000. Our analysis reveals that this star may be located on the red giant branch, red horizontal branch, or early asymptotic giant branch. We find that this metal-poor ([Fe/H]=-2.2) star has an unusually high lithium abundance (log ɛ (Li)=+2.1), mild carbon ([C/Fe]=+0.7) and sodium ([Na/Fe]=+0.6) enhancement, as well as enhancement of both s-process ([Ba/Fe]=+0.8) and r-process ([Eu/Fe]=+0.5) material. The high Li abundance can be explained by self-enrichment through extra mixing mechanisms that connect the convective envelope with the outer regions of the H-burning shell. If so, HK-II 17435-00532 is the most metal-poor star in which this short-lived phase of Li enrichment has been observed. The r- and s-process material was not produced in this star but was either present in the gas from which HK-II 17435-00532 formed or was transferred to it from a more massive binary companion. Despite the current non-detection of radial velocity variations (over a time span of 180 days), it is possible that HK-II 17435-00532 is in a long-period binary system, similar to other stars with both r and s enrichment. We acknowledge support from the W.J. McDonald Fellowship of McDonald Observatory (to A.F), NASA's AAS Small Research Grant Program and the GALEX GI grant 05-GALEX05-27 (to J.R.), the Italian MIUR-PRIN06 Project "Late phases of Stellar Evolution: Nucleosynthesis in Supernovae, AGB Stars, Planetary Nebulae" (to R.G.), and the U.S. National Science Foundation (grants AST06-07708 to C.S., AST04-06784, AST07-07776 and PHY02-15783 to T.C.B., and AST 07-07447 to J.J.C.).

  18. In vitro neuropeptide Y mRNA expressing model for screening essences that may affect appetite using Rolf B1.T cells.

    PubMed

    Chen, Shiau-Wei; Wu, Po-Ju; Chiang, Been-Huang

    2012-08-15

    Neuropeptide Y (NPY) is the most important appetite regulator. This study aimed to establish an in vitro NPY mRNA expression model for screening essences to determine if they are an appetite stimulator or inhibitor. We cultured the olfactory nerve cells Rolf B1.T for 2 days and then treated the cells with the known appetite inhibitor limonene and stimulator linalool. It was found that linalool could significantly stimulate NPY mRNA expression in 10 min, and limonene had the opposite effect. Similar results were also found in primary olfactory ensheathing cells isolated from rats. Further clinical trials using human subjects found that, when 10 min of treatment was applied, linalool indeed increased the serum NPY level in human peripheral blood. Limonene, on the other hand, decreased the serum NPY level. Thus, NPY mRNA expression in Rolf B1.T cells could be used as an in vitro model for screening essences that may affect appetite.

  19. Erythromycin and clarithromycin modulation of growth factor-induced expression of heparanase mRNA on human lung cancer cells in vitro.

    PubMed Central

    Sasaki, M; Ito, T; Kashima, M; Fukui, S; Izumiyama, N; Watanabe, A; Sano, M; Fujiwara, Y; Miura, M

    2001-01-01

    Heparanase activity is correlated with the metastatic potential of several cancer cells and is a key enzyme in the breakdown of tissue barriers. It is also involved in the regulation of growth factor and cytokine activity. However, little is known about the factors that induce heparanase in cancer cells. We investigated the effect of three growth factors, platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF) and basic fibroblast growth factor (bFGF), on heparanase mRNA induction in lung cancer cells in vitro. In addition, we examined the effect of erythromycin (EM) and clarithromycin (CAM), which are 14-membered ring macrolide antibiotics that act as biological response modifiers, on the expression of heparanase mRNA induced by growth factors. PDGF, HGF and bFGF stimulated cell migration activity and enhanced the expression of heparanase mRNA in the human lung adenocarcinoma cell line A549. Via different mechanisms, EM and CAM modulate the induction by these factors of heparanase mRNA expression on A549 cells. EM also significantly suppressed A549 cell migration induced by PDGF and HGF, and CAM significantly suppressed A549cell migration induced by bFGF. The results suggest that the growth factors PDGF, HGF and bFGF are important inducers of heparanase in potentially invasive and metastatic cancer cells. The suppressive effect of heparanase mRNA expression by EM and CAM may have interestingtherapeutic applications in the prevention of metastasis. PMID:11759110

  20. Expression of mRNA of apolipoprotein E, apolipoprotein A-IV, and matricellular proteins in the myocardium and intensity of fibroplastic processes during experimental hypercholesterolemia.

    PubMed

    Lushnikova, E L; Nepomnyashchikh, L M; Pichigin, V I; Klinnikova, M G; Nepomnyashchikh, R D; Sergeevichev, D S

    2013-12-01

    The expression of mRNA of matricellular proteins (osteopontin, and lumican), apolipoproteins E and A-IV, and microsomal triglyceride transfer protein, and the intensity of fibroplastic processes were studied in the myocardium of rats during experimental chronic hypercholesterolemia. We have found that the development of chronic hypercholesterolemia was followed by an increase in volume density of interstitial connective tissue in the myocardium reflecting the activation of fibroplastic processes. A slight positive correlation was observed between the connective tissue density in the myocardium and expression of osteopontin mRNA (r=0.408) and lumican mRNA (r=0.470). Myocardium remodeling during hypercholesterolemia is realized against the background of increased expression of apolipoproteins E and A-IV mRNA and microsomal triglyceride transfer protein mRNA involved in transport and metabolism of lipoproteins in several tissues and probably play a pivotal role in the regulation of lipoprotein transport and metabolism in the myocardium. We concluded that the increase in the expression of apolipoproteins (E and A-IV) and microsomal triglyceride transfer protein play adaptive and compensatory role and is related to the increase in lipoprotein utilization by macrophages.

  1. Si Shen Wan Inhibits mRNA Expression of Apoptosis-Related Molecules in p38 MAPK Signal Pathway in Mice with Colitis

    PubMed Central

    Zhao, Hai-Mei; Huang, Xiao-Ying; Zhou, Feng; Tong, Wen-Ting; Wan, Pan-Ting; Huang, Min-Fang; Ye, Qing; Liu, Duan-Yong

    2013-01-01

    Si Shen Wan (SSW) is used to effectively treat ulcerative colitis (UC) as a formula of traditional Chinese medicine. To explore the mechanism of SSW-inhibited apoptosis of colonic epithelial cell, the study observed mRNA expression of apoptosis-related molecules in p38 MAPK signal pathway in colonic mucosa in colitis mice treated with SSW. Experimental colitis was induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS) in mice; meanwhile, the mice were administrated daily either SSW (5 g/kg) or p38 MAPK inhibitor (2 mg/kg) or vehicle (physiological saline) for 10 days. While microscopical evaluation was observed, apoptosis rate of colonic epithelial cell and mRNA expression of apoptosis-related molecules were tested. Compared with colitis mice without treatment, SSW alleviated colonic mucosal injuries and decreased apoptosis rate of colonic epithelial cell, while the mRNA expressions of p38 MAPK, p53, caspase-3, c-jun, c-fos, Bax, and TNF-α were decreased in the colonic mucosa in colitis mice treated with SSW, and Bcl-2 mRNA and the ratio of Bcl-2/Bax were increased. The present study demonstrated that SSW inhibited mRNA expression of apoptosis-related molecules in p38 MAPK signal pathway to downregulate colonic epithelial cells apoptosis in colonic mucosa in mice with colitis. PMID:24223057

  2. Involvement of second messengers in the signaling pathway of vitellogenesis-inhibiting hormone and their effects on vitellogenin mRNA expression in the whiteleg shrimp, Litopenaeus vannamei.

    PubMed

    Bae, Sun-Hye; Okutsu, Tomoyuki; Tsutsui, Naoaki; Kang, Bong Jung; Chen, Hsiang-Yin; Wilder, Marcy N

    2017-01-03

    We incubated fragments of Litopenaeus vannamei ovary to investigate second messengers involved in the regulation of vitellogenin (vg) mRNA levels. The use of 100nM recombinant vitellogenesis-inhibiting hormone (VIH) (corresponding to recombinant L. vannamei sinus gland peptide-G: rLiv-SGP-G) significantly reduced vg mRNA expression in sub-adults after 8h incubation to less than 20% of the control. The concentration of intracellular cyclic guanosine monophosphate (cGMP) increased 3.2-fold relative to the control after 2h incubation with rLiv-SGP-G. However, it reached levels 18-fold relative to the control after 0.5h incubation with rLiv-SGP-G where 3-isobutyl-1-methylxanthine (a phosphodiesterase inhibitor) was also added. Moreover, vg mRNA expression was significantly reduced to less than 50% of the control after 24h incubation with 1μM A23187 (a calcium ionophore). Thus, rLiv-SGP-G and calcium ionophore reduced vg mRNA expression in in vitro-cultured ovary, and cGMP may be involved in the signaling pathway of VIH. Overall, the above results suggest that vg mRNA expression might be inhibited in vitro by increasing intracellular cGMP and Ca(2+) in L. vannamei ovary.

  3. Use of expression-enhancing terminators in Saccharomyces cerevisiae to increase mRNA half-life and improve gene expression control for metabolic engineering applications.

    PubMed

    Curran, Kathleen A; Karim, Ashty S; Gupta, Akash; Alper, Hal S

    2013-09-01

    Control of gene and protein expression of both endogenous and heterologous genes is a key component of metabolic engineering. While a large amount of work has been published characterizing promoters for this purpose, less effort has been exerted to elucidate the role of terminators in yeast. In this study, we characterize over 30 terminators for use in metabolic engineering applications in Saccharomyces cerevisiae and determine mRNA half-life changes to be the major cause of the varied protein and transcript expression level. We demonstrate that the difference in transcript level can be over 6.5-fold even for high strength promoters. The influence of terminator selection is magnified when coupled with a low-expression promoter, with a maximum difference in protein expression of 11-fold between an expression-enhancing terminator and the parent plasmid terminator and over 35-fold difference when compared with a no-terminator baseline. This is the first time that terminators have been investigated in the context of multiple promoters spanning orders of magnitude in activity. Finally, we demonstrate the utility of terminator selection for metabolic engineering by using a mutant xylose isomerase gene as a proof-of-concept. Through pairing an expression-enhancing terminator with a low-expression promoter, we were able to achieve the same phenotypic result as with a promoter considerably higher in strength. Moreover, we can further boost the phenotype of the high-strength promoter by pairing it with an expression-enhancing terminator. This work highlights how terminator elements can be used to control metabolic pathways in the same way that promoters are traditionally used in yeast. Together, this work demonstrates that terminators will be an important part of heterologous gene expression and metabolic engineering for yeast in the future.

  4. Stress and withdrawal from d-amphetamine alter 5-HT2A receptor mRNA expression in the prefrontal cortex.

    PubMed

    Murray, Ryan C; Hebbard, John C; Logan, Anna S; Vanchipurakel, Golda A; Gilbert, Yamiece E; Horner, Kristen A

    2014-01-24

    Psychostimulant withdrawal results in emotional, behavioral, and cognitive impairments, which may be exacerbated by stress. However, little is known about the neurochemical changes that occur when these two conditions are experienced concomitantly. 5-HT2A receptor (5-HT2AR) mRNA expression in the prefrontal cortex (PFC) is diminished following withdrawal from d-amphetamine (AMPH) and may underlie the emotional and cognitive impairments observed in psychostimulant withdrawal, but whether stress affects 5-HT2AR mRNA expression during psychostimulant withdrawal is unknown. The goal of this study was to examine the impact of forced swim test (FST) exposure during AMPH withdrawal on 5-HT2AR mRNA expression in PFC. Animals were treated 3 times a day for 4 days with escalating doses of AMPH (1-10mg/kg) and 24h or 4 days after the final injection, animals were subjected to FST. At 24h of withdrawal, AMPH-treated animals showed greater immobility in FST and at 4 days of withdrawal, AMPH-treated animals did not show immobility. At 24h of withdrawal, animals showed lower 5-HT2AR mRNA expression in the PFC relative to saline-treated animals, and exposure to FST did not further decrease expression in these animals. At 4 days of withdrawal, AMPH-treated animals showed greater 5-HT2AR mRNA expression relative to saline-treated animals in the PFC, an effect that was diminished by exposure to FST. These data indicate that stress and short-term AMPH withdrawal affect prefrontal 5-HT2AR mRNA expression to a similar degree, and stress experienced during long-term AMPH withdrawal can diminish the recovery of 5-HT2AR mRNA expression. Together, these data suggest that exposure to stress during extended AMPH withdrawal could prolong withdrawal-induced, 5-HT2AR mRNA