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Sample records for persistent mrna expression

  1. Transient and persistent expression of NT-3/HDNF mRNA in the rat brain during postnatal development.

    PubMed

    Friedman, W J; Ernfors, P; Persson, H

    1991-06-01

    Neurotrophin-3 (NT-3) is closely related to two known neurotrophic agents, NGF and brain-derived neurotrophic factor (BDNF), and acts upon overlapping, yet distinct, populations of peripheral ganglia. NT-3 mRNA expression in the adult rat brain is largely confined to the hippocampus. In this study, we have used in situ hybridization to examine expression of this novel neurotrophic factor during postnatal development. The striking observation was made that NT-3 mRNA was transiently expressed at high levels in the cingulate cortex during the first 2 weeks of age. In the hippocampus, the adult pattern of expression, in the CA2, medial CA1, and granule layer of the dentate gyrus, was detected at all ages examined. However, there were two major differences in NT-3 mRNA expression in the developing hippocampus: Labeled cells were detected in the hilar region of the dentate gyrus at postnatal day 1 (P1) and 1 week that were absent by 2 weeks of age. Further, the caudal hippocampus, which has a lower intensity of labeling than the rostral region in the adult, was devoid of NT-3-expressing cells in the P1 and 1-week-old rat brain. These data indicate a substantial plasticity in NT-3 mRNA expression and suggest that the spectrum of neurons supported by NT-3 during development is partially different from that in the mature rat brain. PMID:2045877

  2. Cytokine mRNA expression in foot-and-mouth disease virus (FMDV) persistently infected bovine pharynx cultures: effect of IFNgamma on replication of persistent virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Foot-and-mouth disease virus (FMDV), a member of the family Picornaviridae, genus Aphtovirus, causes a highly contagious disease in livestock. Following acute infection in ruminants, up to 50% of both vaccinated and non-vaccinated animals become persistently infected asymptomatic carriers with low-l...

  3. A clinically relevant BTX-A injection protocol leads to persistent weakness, contractile material loss, and an altered mRNA expression phenotype in rabbit quadriceps muscles.

    PubMed

    Fortuna, Rafael; Vaz, Marco A; Sawatsky, Andrew; Hart, David A; Herzog, Walter

    2015-07-16

    Botulinum toxin type-A (BTX-A) injections have become a common treatment modality for patients suffering from muscle spasticity. Despite its benefits, BTX-A treatments have been associated with adverse effects on target muscles. Currently, application of BTX-A is largely based on clinical experience, and research quantifying muscle structure following BTX-A treatment has not been performed systematically. The purpose of this study was to evaluate strength, muscle mass, and contractile material six months following a single or repeated (2 and 3) BTX-A injections into the quadriceps femoris of New Zealand white rabbits. Twenty three skeletally mature rabbits were divided into four groups: experimental group rabbits received 1, 2, or 3 injections at intervals of 3 months (1-BTX-A, 2-BTX-A, 3-BTX-A, respectively) while control group rabbits received volume-matched saline injections. Knee extensor strength, quadriceps muscle mass, and quadriceps contractile material of the experimental group rabbits were expressed as a percentage change relative to the control group rabbits. One-way ANOVA was used to determine group differences in outcome measures (α=0.05). Muscle strength and contractile material were significantly reduced in experimental compared to control group rabbits but did not differ between experimental groups. Muscle mass was the same in experimental BTX-A and control group rabbits. We concluded from these results that muscle strength and contractile material do not fully recover within six months of BTX-A treatment. PMID:26087882

  4. mRNA modifications: Dynamic regulators of gene expression?

    PubMed Central

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

    2016-01-01

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

  5. Anger Expression and Persistence in Young Children

    ERIC Educational Resources Information Center

    He, Jie; Xu, Qinmei; Degnan, Kathryn Amey

    2012-01-01

    This study investigated anger expression during toy removal (TR) in 92 young Chinese children, two to five years of age, and its relations to their persistence in responding to obstacles during two challenging tasks with highly desirable goals [TR and locked box (LB)] and one challenging task with a less desirable goal [impossible perfect circles…

  6. mRNA Composition and Control of Bacterial Gene Expression

    PubMed Central

    Liang, S.-T.; Xu, Y.-C.; Dennis, P.; Bremer, H.

    2000-01-01

    The expression of any given bacterial protein is predicted to depend on (i) the transcriptional regulation of the promoter and the translational regulation of its mRNA and (ii) the synthesis and translation of total (bulk) mRNA. This is because total mRNA acts as a competitor to the specific mRNA for the binding of initiation-ready free ribosomes. To characterize the effects of mRNA competition on gene expression, the specific activity of β-galactosidase expressed from three different promoter-lacZ fusions (Pspc-lacZ, PRNAI-lacZ, and PRNAII-lacZ) was measured (i) in a relA+ background during exponential growth at different rates and (ii) in relA+ and ΔrelA derivatives of Escherichia coli B/r after induction of a mild stringent or a relaxed response to raise or lower, respectively, the level of ppGpp. Expression from all three promoters was stimulated during slow exponential growth or at elevated levels of ppGpp and was reduced during fast exponential growth or at lower levels of ppGpp. From these observations and from other considerations, we propose (i) that the concentration of free, initiation-ready ribosomes is approximately constant and independent of the growth rate and (ii) that bulk mRNA made during slow growth and at elevated levels of ppGpp is less efficiently translated than bulk mRNA made during fast growth and at reduced levels of ppGpp. These features lead to an indirect enhancement in the expression of LacZ (or of any other protein) during growth in media of poor nutritional quality and at increased levels of ppGpp. PMID:10809680

  7. 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.

  8. Leptin mRNA expresses in the bull reproductive organ.

    PubMed

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

    2009-12-01

    Leptin, a 167-amino acid hormone, is secreted mainly by fat tissue. It has some powerful effects on the regulation of metabolism and reproductive function through endocrine and probably paracrine mechanisms. The contribution rate of leptin function on the male reproductive system is not still clear. Characterization of leptin expression in reproductive organs will suggest that in addition to its endocrine action, leptin has also paracrine/autocrine effects on reproduction. The expression of functional leptin receptor mRNA has been already recognized in testis of rodents, human and cattle. Thus, the aim of the present study was to investigate the presence of leptin mRNA in the bovine testis, because it will be the first step for understanding of its paracrine/autocrine effects on the male reproductive organs in cattle. The present study was the first to showed leptin mRNA expression in the testis of Holstein cattle using reverse transcription and polymerase chain reaction (RT-PCR) analysis. RT-PCR products were amplified with nested PCR using inner leptin primer pairs to emphasis the first results. Besides, bovine beta actin gene was acted as an internal positive control as well as RNA purification marker. Our findings suggest that in addition to its endocrine actions at the hypothalamic-pituitary axis, leptin can has an autocrine and/or paracrine role in bull testicular function. PMID:19466574

  9. 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. PMID:24161824

  10. Bacterial persistence and expression of disease.

    PubMed Central

    Domingue, G J; Woody, H B

    1997-01-01

    A considerable body of experimental and clinical evidence supports the concept that difficult-to-culture and dormant bacteria are involved in latency of infection and that these persistent bacteria may be pathogenic. This review includes details on the diverse forms and functions of individual bacteria and attempts to make this information relevant to the care of patients. A series of experimental studies involving host-bacterium interactions illustrates the probability that most bacteria exposed to a deleterious host environment can assume a form quite different from that of a free-living bacterium. A hypothesis is offered for a kind of reproductive cycle of morphologically aberrant bacteria as a means to relate their diverse tissue forms to each other. Data on the basic biology of persistent bacteria are correlated with expression of disease and particularly the mechanisms of both latency and chronicity that typify certain infections. For example, in certain streptococcal and nocardial infections, it has been clearly established that wall-defective forms can be induced in a suitable host. These organisms can survive and persist in a latent state within the host, and they can cause pathologic responses compatible with disease. A series of cases illustrating idiopathic conditions in which cryptic bacteria have been implicated in the expression of disease is presented. These conditions include nephritis, rheumatic fever, aphthous stomatitis, idiopathic hematuria, Crohn's disease, and mycobacterial infections. By utilizing PCR, previously nonculturable bacilli have been identified in patients with Whipple's disease and bacillary angiomatosis. Koch's postulates may have to be redefined in terms of molecular data when dormant and nonculturable bacteria are implicated as causative agents of mysterious diseases. PMID:9105757

  11. 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.

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

    PubMed

    Uchida, Satoshi; Kataoka, Kazunori; Itaka, Keiji

    2015-01-01

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

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

    PubMed Central

    Uchida, Satoshi; Kataoka, Kazunori; Itaka, Keiji

    2015-01-01

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

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

    PubMed Central

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

    1996-01-01

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

  15. Persistent induction of c-fos and c-jun expression by asbestos

    SciTech Connect

    Heintz, N.H.; Mossman, B.T. ); Janssen, Y.M. Univ. of Limburg, Maastricht )

    1993-04-15

    To investigate the mechanisms of asbestos-induced carcinogenesis, expression of c-fos and c-jun protooncogenes was examined in rat pleural mesothelial cells and hamster tracheal epithelial cells after exposure to crocidolite or chrysotile asbestos. In contrast to phorbol 12-myristate 13-acetate, which induces rapid and transient increases in c-fos and c-jun mRNA, asbestos causes 2- to 5-fold increases in c-fos and c-jun mRNA that persist for at least 24 hr in mesothelial cells. The induction of c-fos and c-jun mRNA by asbestos in mesothelial cells is dose-dependent and is most pronounced with crocidolite, the type of asbestos most pathogenic in the causation of pleural mesothelioma. Induction of c-jun gene expression by asbestos occurs in tracheal epithelial cells but is not accompanied by a corresponding induction of c-fos gene expression. In both cell types, asbestos induces increases in protein factors that bind specifically to the DNA sites that mediate gene expression by the AP-1 family of transcription factors. The persistent induction of AP-1 transcription factors by asbestos suggests a model of asbestos-induced carcinogenesis involving chronic stimulation of cell proliferation through activation of the early response gene pathway that includes c-jun and/or c-fos. 30 refs., 5 figs.

  16. Prophylactic mRNA Vaccination against Allergy Confers Long-Term Memory Responses and Persistent Protection in Mice

    PubMed Central

    Hattinger, E.; Scheiblhofer, S.; Roesler, E.; Thalhamer, T.; Thalhamer, J.; Weiss, R.

    2015-01-01

    Recently, mRNA vaccines have been introduced as a safety-optimized alternative to plasmid DNA-based vaccines for protection against allergy. However, it remained unclear whether the short persistence of this vaccine type would limit memory responses and whether the protective immune response type would be maintained during recurrent exposure to allergen. We tested the duration of protective memory responses in mice vaccinated with mRNA encoding the grass pollen allergen Phl p 5 by challenging them with recombinant allergen, 3.5, 6, and 9 months after vaccination. In a second experiment, vaccinated mice were repeatedly challenged monthly with aerosolized allergen over a period of 7 months. Antibody and cytokine responses as well as lung inflammation and airway hyperresponsiveness were assessed. mRNA vaccination induced robust TH1 memory responses for at least 9 months. Vaccination efficiently suppressed TH2 cytokines, IgE responses, and lung eosinophilia. Protection was maintained after repeated exposure to aerosolized allergen and no TH1 associated pathology was observed. Lung function remained improved compared to nonvaccinated controls. Our data clearly indicate that mRNA vaccination against Phl p 5 induces robust, long-lived memory responses, which can be recalled by allergen exposure without side effects. mRNA vaccines fulfill the requirements for safe prophylactic vaccination without the need for booster immunizations. PMID:26557723

  17. Developmentally Regulated Expression of HDNF/NT-3 mRNA in Rat Spinal Cord Motoneurons and Expression of BDNF mRNA in Embryonic Dorsal Root Ganglion.

    PubMed

    Ernfors, Patrik; Persson, Håkan

    1991-01-01

    Northern blot analysis was used to demonstrate high levels of hippocampus-derived neurotrophic factor/neurotrophin-3 (HDNF/NT-3) mRNA in the embryonic day (E) 13 - 14 and 15 - 16 spinal cord. The level decreased at E18 - 19 and remained the same until postnatal day (P) 1, after which it decreased further to a level below the detection limit in the adult. In situ hybridization revealed that the NT-3 mRNA detected in the developing spinal cord was derived from motoneurons and the decrease seen at E18 - 19 was caused by a reduction in the number of motoneurons expressing NT-3 mRNA. The distribution of NT-3 mRNA-expressing cells in the E15 spinal cord was very similar to the distribution of cells expressing choline acetyltransferase or nerve growth factor receptor (NGFR) mRNA. Moreover, a striking similarity between the developmentally regulated expression of NT-3 and NGFR mRNA was noted in spinal cord motoneurons. A subpopulation of all neurons in the dorsal root ganglia expressed brain-derived neurotrophic factor (BDNF) mRNA from E13, the earliest time examined, to adulthood. These results are consistent with a trophic role of NT-3 for proprioceptive sensory neurons innervating the ventral horn, and imply a local action of BDNF for developing sensory neurons within the dorsal root ganglia. PMID:12106253

  18. Associations of MMP-2, BAX, and Bcl-2 mRNA and Protein Expressions with Development of Atrial Fibrillation.

    PubMed

    Diao, Shu-Ling; Xu, Hui-Pu; Zhang, Bei; Ma, Bao-Xin; Liu, Xian-Liang

    2016-01-01

    BACKGROUND To examine changes of mRNA and protein expressions of MMP-2, Bcl-2, and BAX in atrial fibrillation (AF) patients, and investigate the correlations among these 3 biomarkers. MATERIAL AND METHODS Rheumatic heart disease patients (n=158) undergoing cardiac surgical procedures for mitral valve repair or replacement were included as the AF group (n=123), containing paroxysmal AF (n=42), persistent AF (n=36), and permanent AF (n=45). Rheumatic heart disease patients with sinus rhythm (SR) (n=35) were enrolled as the SR group (control group). Immunohistochemistry, Western blot, and real-time polymerase chain reaction (PCR) were applied to detect the protein and mRNA expression levels of MMP-2, Bcl-2, and BAX. Apoptosis was observed with light and electron microscopes and detected by TdT-mediated dUTP nick-end labeling (TUNEL). RESULTS Compared with the SR group, the left atrial diameters (LADs), protein and mRNA expression levels of MMP-2 and BAX, apoptotic index (AI), and Bcl-2/BAX ratio were evidently increased in the 3 AF groups, but protein and mRNA expression levels of Bcl-2 decreased in the AF groups (all P<0.05). Correlation analysis found that MMP-2 protein expression levels was positively correlated with BAX expression, but negatively correlated with Bcl-2 expression levels. CONCLUSIONS Our study results suggest that elevated MMP-2 expression and disturbance balance of Bcl-2/BAX expressions may be associated with the development and maintenance of AF. MMP-2 may be involved in the development of AF through promoting BAX expressions and inhibiting Bcl-2. PMID:27141955

  19. Associations of MMP-2, BAX, and Bcl-2 mRNA and Protein Expressions with Development of Atrial Fibrillation

    PubMed Central

    Diao, Shu-Ling; Xu, Hui-Pu; Zhang, Bei; Ma, Bao-Xin; Liu, Xian-Liang

    2016-01-01

    Background To examine changes of mRNA and protein expressions of MMP-2, Bcl-2, and BAX in atrial fibrillation (AF) patients, and investigate the correlations among these 3 biomarkers. Material/Methods Rheumatic heart disease patients (n=158) undergoing cardiac surgical procedures for mitral valve repair or replacement were included as the AF group (n=123), containing paroxysmal AF (n=42), persistent AF (n=36), and permanent AF (n=45). Rheumatic heart disease patients with sinus rhythm (SR) (n=35) were enrolled as the SR group (control group). Immunohistochemistry, Western blot, and real-time polymerase chain reaction (PCR) were applied to detect the protein and mRNA expression levels of MMP-2, Bcl-2, and BAX. Apoptosis was observed with light and electron microscopes and detected by TdT-mediated dUTP nick-end labeling (TUNEL). Results Compared with the SR group, the left atrial diameters (LADs), protein and mRNA expression levels of MMP-2 and BAX, apoptotic index (AI), and Bcl-2/BAX ratio were evidently increased in the 3 AF groups, but protein and mRNA expression levels of Bcl-2 decreased in the AF groups (all P<0.05). Correlation analysis found that MMP-2 protein expression levels was positively correlated with BAX expression, but negatively correlated with Bcl-2 expression levels. Conclusions Our study results suggest that elevated MMP-2 expression and disturbance balance of Bcl-2/BAX expressions may be associated with the development and maintenance of AF. MMP-2 may be involved in the development of AF through promoting BAX expressions and inhibiting Bcl-2. PMID:27141955

  20. Elevated TREM2 mRNA expression in leukocytes in schizophrenia but not major depressive disorder.

    PubMed

    Yoshino, Yuta; Kawabe, Kentaro; Yamazaki, Kiyohiro; Watanabe, Shinya; Numata, Shusuke; Mori, Yoko; Yoshida, Taku; Iga, Junichi; Ohmori, Tetsuro; Ueno, Shu-Ichi

    2016-06-01

    The pathological mechanisms of schizophrenia (SCZ) have not been clarified, but the microglia hypothesis has recently been discussed. We previously reported that the mRNA for a protein related to activation of microglia, triggering receptor expressed on myeloid cell 2 (TREM2), is expressed higher in peripheral leukocytes in SCZ than controls. In this study, we analyzed TREM2 mRNA expression in leukocytes from both SCZ and major depressive disorder (MDD) patients. We compared 50 SCZ patients and 42 MDD patients with age-matched controls. Levels of TREM2 mRNA in leukocytes were analyzed with quantitative real-time PCR method using TaqMan probe. TREM2 mRNA expression was significantly higher in leukocytes of SCZ subjects than controls, but the expression level was non-significantly different in MDD subjects. We observed a decrease in TREM2 mRNA expression in leukocytes from one SCZ patient after clozapine treatment. The expression did not change following ECT, but the expression level in this patient was still significantly higher than that in controls. We conclude that the high amount of TREM2 mRNA expression in leukocytes is specific to SCZ but not MDD and that changes in TREM2 mRNA expression may be a trait biomarker for SCZ. PMID:27130565

  1. Negative regulation of neuromedin U mRNA expression in the rat pars tuberalis by melatonin.

    PubMed

    Aizawa, Sayaka; Sakata, Ichiro; Nagasaka, Mai; Higaki, Yuriko; Sakai, Takafumi

    2013-01-01

    The pars tuberalis (PT) is part of the anterior pituitary gland surrounding the median eminence as a thin cell layer. The characteristics of PT differ from those of the pars distalis (PD), such as cell composition and gene expression, suggesting that the PT has a unique physiological function compared to the PD. Because the PT highly expresses melatonin receptor type 1, it is considered a mediator of seasonal and/or circadian signals of melatonin. Expression of neuromedin U (NMU) that is known to regulate energy balance has been previously reported in the rat PT; however, the regulatory mechanism of NMU mRNA expression and secretion in the PT are still obscure. In this study, we examined both the diurnal change of NMU mRNA expression in the rat PT and the effects of melatonin on NMU in vivo. In situ hybridization and quantitative PCR analysis of laser microdissected PT samples revealed that NMU mRNA expression in the PT has diurnal variation that is high during the light phase and low during the dark phase. Furthermore, melatonin administration significantly suppressed NMU mRNA expression in the PT in vivo. On the other hand, 48 h fasting did not have an effect on PT-NMU mRNA expression, and the diurnal change of NMU mRNA expression was maintained. We also found the highest expression of neuromedin U receptor type 2 (NMUR2) mRNA in the third ventricle ependymal cell layer, followed by the arcuate nucleus and the spinal cord. These results suggest that NMU mRNA expression in the PT is downregulated by melatonin during the dark phase and shows diurnal change. Considering that NMU mRNA in the PT showed the highest expression level in the brain, PT-NMU may act on NMUR2 in the brain, especially in the third ventricle ependymal cell layer, with a circadian rhythm. PMID:23843987

  2. Negative Regulation of Neuromedin U mRNA Expression in the Rat Pars Tuberalis by Melatonin

    PubMed Central

    Aizawa, Sayaka; Sakata, Ichiro; Nagasaka, Mai; Higaki, Yuriko; Sakai, Takafumi

    2013-01-01

    The pars tuberalis (PT) is part of the anterior pituitary gland surrounding the median eminence as a thin cell layer. The characteristics of PT differ from those of the pars distalis (PD), such as cell composition and gene expression, suggesting that the PT has a unique physiological function compared to the PD. Because the PT highly expresses melatonin receptor type 1, it is considered a mediator of seasonal and/or circadian signals of melatonin. Expression of neuromedin U (NMU) that is known to regulate energy balance has been previously reported in the rat PT; however, the regulatory mechanism of NMU mRNA expression and secretion in the PT are still obscure. In this study, we examined both the diurnal change of NMU mRNA expression in the rat PT and the effects of melatonin on NMU in vivo. In situ hybridization and quantitative PCR analysis of laser microdissected PT samples revealed that NMU mRNA expression in the PT has diurnal variation that is high during the light phase and low during the dark phase. Furthermore, melatonin administration significantly suppressed NMU mRNA expression in the PT in vivo. On the other hand, 48 h fasting did not have an effect on PT-NMU mRNA expression, and the diurnal change of NMU mRNA expression was maintained. We also found the highest expression of neuromedin U receptor type 2 (NMUR2) mRNA in the third ventricle ependymal cell layer, followed by the arcuate nucleus and the spinal cord. These results suggest that NMU mRNA expression in the PT is downregulated by melatonin during the dark phase and shows diurnal change. Considering that NMU mRNA in the PT showed the highest expression level in the brain, PT-NMU may act on NMUR2 in the brain, especially in the third ventricle ependymal cell layer, with a circadian rhythm. PMID:23843987

  3. 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

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

    PubMed

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

    1995-02-10

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

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

    PubMed

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

    2016-01-01

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

  6. Prefrontal cortical-striatal dopamine receptor mRNA expression predicts distinct forms of impulsivity.

    PubMed

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

    2013-06-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2011-01-01

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

  9. Impact of STAT/SOCS mRNA Expression Levels after Major Injury

    PubMed Central

    Brumann, M.; Matz, M.; Kusmenkov, T.; Stegmaier, J.; Biberthaler, P.; Kanz, K.-G.; Mutschler, W.; Bogner, V.

    2014-01-01

    Background. Fulminant changes in cytokine receptor signalling might provoke severe pathological alterations after multiple trauma. The aim of this study was to evaluate the posttraumatic imbalance of the innate immune system with a special focus on the STAT/SOCS family. Methods. 20 polytraumatized patients were included. Blood samples were drawn 0 h–72 h after trauma; mRNA expression profiles of IL-10, STAT 3, SOCS 1, and SOCS 3 were quantified by qPCR. Results. IL-10 mRNA expression increased significantly in the early posttraumatic period. STAT 3 mRNA expressions showed a significant maximum at 6 h after trauma. SOCS 1 levels significantly decreased 6 h–72 h after trauma. SOCS 3 levels were significantly higher in nonsurvivors 6 h after trauma. Conclusion. We present a serial, sequential investigation in human neutrophil granulocytes of major trauma patients evaluating mRNA expression profiles of IL-10, STAT 3, SOCS 1, and SOCS 3. Posttraumatically, immune disorder was accompanied by a significant increase of IL-10 and STAT 3 mRNA expression, whereas SOCS 1 mRNA levels decreased after injury. We could demonstrate that death after trauma was associated with higher SOCS 3 mRNA levels already at 6 h after trauma. To support our results, further investigations have to evaluate protein levels of STAT/SOCS family in terms of posttraumatic immune imbalance. PMID:24648661

  10. 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.

  11. Expression of lipoprotein lipase mRNA and secretion in macrophages isolated from human atherosclerotic aorta.

    PubMed

    Mattsson, L; Johansson, H; Ottosson, M; Bondjers, G; Wiklund, O

    1993-10-01

    The expression of lipoprotein lipase (LPL) mRNA and the LPL activity were studied in macrophages (CD14 positive) from human atherosclerotic tissue. Macrophages were isolated after collagenase digestion by immunomagnetic isolation. About 90% of the cells were foam cells with oil red O positive lipid droplets. To analyze the mRNA expression, PCR with specific primers for LPL was used. Arterial macrophages were analyzed directly after isolation and the data showed low expression of LPL mRNA when compared with monocyte-derived macrophages. To induce the expression of LPL mRNA in macrophages, PMA was used. When incubating arterial macrophages with PMA for 24 h we could not detect any increase in LPL mRNA levels. Similarly, the cells secreted very small amounts of LPL even after PMA stimulation. In conclusion, these studies show a very low expression of LPL mRNA in the CD14-positive macrophage-derived foam cells isolated from human atherosclerotic tissue. These data suggest that the CD14-positive cells are a subpopulation of foam cells that express low levels of lipoprotein lipase, and the lipid content could be a major factor for downregulation of LPL. However, the cells were isolated from advanced atherosclerotic lesions, and these findings may not reflect the situation in early fatty streaks. PMID:8408628

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

    PubMed

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

    1992-11-01

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

  13. Versican gene expression in human articular cartilage and comparison of mRNA splicing variation with aggrecan.

    PubMed Central

    Grover, J; Roughley, P J

    1993-01-01

    The chondrocytes in human articular cartilage from subjects of all ages express mRNAs for both of the aggregating proteoglycans aggrecan and versican, although the level of expression of versican mRNA is much lower than that of aggrecan mRNA. Aggrecan shows alternative splicing of the epidermal growth factor (EGF)-like domain within its C-terminal globular region, but there is no evidence for a major difference in situ in the relative expression of this domain with age. At all ages studied from birth to the mature adult, a greater proportion of transcripts lacked the EGF domain. The relative proportions of the two transcripts did not change upon culture and passage of isolated chondrocytes. In contrast, the neighbouring complement regulatory protein (CRP)-like domain was predominantly expressed irrespective of age, but cell culture did result in variation of the splicing of this domain. Versican possesses two EGF-like domains and one CRP-like domain, but at all ages the three domains were predominantly present in all transcripts. This situation persisted upon culture and passage of the chondrocytes. Thus, unlike aggrecan, the versican expressed by human articular cartilage does not appear to undergo alternative splicing of its C-terminal globular region, either in cartilage in situ or in chondrocytes in culture. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:8484718

  14. Ribozyme cleaves rex/tax mRNA and inhibits bovine leukemia virus expression.

    PubMed Central

    Cantor, G H; McElwain, T F; Birkebak, T A; Palmer, G H

    1993-01-01

    Bovine leukemia virus (BLV) encodes at least two regulatory proteins, Rex and Tax. Tax, the transactivating protein, stimulates the long terminal repeat to promote viral transcription and may be involved in tumorigenesis. Rex is involved in the transition from early expression of regulatory proteins to later expression of viral structural proteins. We have targeted ribozymes against the mRNA encoding Rex and Tax. The ribozymes consist of the hammer-head catalytic motif flanked by antisense sequences that hybridize with the complementary rex/tax mRNA. To evaluate cleavage in a cell-free system, we transcribed portions of rex/tax mRNA and incubated them with synthetic RNA ribozymes. A ribozyme was identified that cleaves > 80% of the target RNA. Synthetic DNA encoding this ribozyme was cloned into the expression vector pRc/RSV and transfected into BLV-infected bat lung cells. Intracellular cleavage of rex/tax mRNA was confirmed by reverse transcriptase PCR. In cells expressing the ribozyme, viral expression was markedly inhibited. Expression of the BLV core protein p24 was inhibited by 61%, and reverse transcriptase activity in supernatant was inhibited by 92%. Ribozyme inhibition of BLV expression suggests that cattle expressing these sequences may be able to control BLV replication. Images Fig. 2 Fig. 4 Fig. 5 Fig. 6 PMID:7504287

  15. Unification of gene expression data applying standard mRNA quantification references for comparable analyses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    High throughput quantitative measurements of gene expression data have problems of reproducibility and comparability due to a lack of standard mRNA quantification references. Efforts have been made to safeguard data fidelity, yet generating quality expression data of inherent value remains a challe...

  16. Stability regulation of mRNA and the control of gene expression.

    PubMed

    Cheadle, Chris; Fan, Jinshui; Cho-Chung, Yoon S; Werner, Thomas; Ray, Jill; Do, Lana; Gorospe, Myriam; Becker, Kevin G

    2005-11-01

    Microarray technology has become highly valuable for identifying complex global changes in gene expression patterns. Standard techniques measure changes in total cellular poly(A) mRNA levels. The assumption that changes in gene expression as measured by these techniques are directly and well correlated with changes in rates of new gene synthesis form the basis of attempts to connect coordinated changes in gene expression with shared transcription regulatory elements. Yet systematic attempts at this approach remain difficult to demonstrate convincingly. One reason for this difficulty may result from the intricate convergence of both transcriptional and mRNA turnover events which, together, directly influence steady-state mRNA levels. Recent technical advances have led to the successful scale-up and application of nuclear run-on procedures directly to microarrays. This development has allowed a gene-by-gene comparison between new gene synthesis in the nucleus and measured changes in total cellular polyA mRNA. Results from these studies have begun to challenge the strict interpretation of changes in gene expression measured by conventional microarrays as being closely correlated with changes in mRNA transcription rate, but rather they tend to support the significant expansion of the role played by changes in mRNA stability regulation to standard analyses of gene expression. Gene expression profiles obtained from both polyA mRNA (whole-cell) and nuclear run-on (newly transcribed) RNA across a time course of one hour following the activation of human Jurkat T cells with PMA plus ionomycin revealed that regulation of mRNA stability may account for as much as 50% of all measurements of changes in total cellular polyA mRNA in this system. Stability regulation was inferred by the absence of corresponding regulation of nuclear gene transcription activity for groups of genes strongly regulated at the whole cell level and which were also resistant to inhibition by Actinomycin

  17. Gemin5 Binds to the Survival Motor Neuron mRNA to Regulate SMN Expression.

    PubMed

    Workman, Eileen; Kalda, Caitlin; Patel, Aalapi; Battle, Daniel J

    2015-06-19

    Reduced expression of SMN causes spinal muscular atrophy, a severe neurodegenerative disease. Despite the importance of maintaining SMN levels, relatively little is known about the mechanisms by which SMN levels are regulated. We show here that Gemin5, the snRNA-binding protein of the SMN complex, binds directly to the SMN mRNA and regulates SMN expression. Gemin5 binds with high specificity, both in vitro and in vivo, to sequence and structural elements in the SMN mRNA 3'-untranslated region that are reminiscent of the snRNP code to which Gemin5 binds on snRNAs. Reduction of Gemin5 redistributes the SMN mRNA from heavy polysomes to lighter polysomes and monosomes, suggesting that Gemin5 functions as an activator of SMN translation. SMN protein is not stoichiometrically present on the SMN mRNA with Gemin5, but the mRNA-binding activity of Gemin5 is dependent on SMN levels, providing a feedback mechanism for SMN to regulate its own expression via Gemin5. This work both reveals a new autoregulatory pathway governing SMN expression, and identifies a new mechanism through which SMN can modulate specific mRNA expression via Gemin5. PMID:25911097

  18. 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.

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

    PubMed

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

    2016-04-20

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

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

    PubMed Central

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

    2016-01-01

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

  1. Variations in cytokine mRNA expression during normal human pregnancy

    PubMed Central

    Kruse, N; Greif, M; Moriabadi, N F; Marx, L; Toyka, K V; Rieckmann, P

    2000-01-01

    Epidemiological data provide evidence that disease activity of T cell-mediated, organ-specific autoimmune diseases is reduced during pregnancy. Although there are several experimental animal studies on the effect of pregnancy on the immune system, the situation in humans is less clear. We therefore performed a prospective analysis of cytokine mRNA expression in whole blood by a new on-line reverse transcriptase-polymerase chain reaction technique and of serum hormone levels during pregnancy in healthy women. The control group included age-matched non-pregnant healthy women. Quantitativecytokine mRNA expression revealed significantly reduced IL-18, interferon-gamma (IFN-γ), and IL-2 mRNA levels in the first and second trimester in pregnancy compared with non-pregnant women. No difference between groups was detected for tumour necrosis factor-alpha (TNF-α) mRNA. IL-4 and IL-10 mRNA were detected at low levels in only 20% of pregnant women and were reduced to a statistically significant extent in the second and third trimester compared with the control group. Changes in IL-18 mRNA expression correlated inversely with serum values for human choriogonadotropin (HCG) and IL-10 serum levels correlated with increases in serum 17β-oestradiol levels. These data indicate immunomodulatory effects of pregnancy at the cytokine level which may be related to the variations in the clinical course of organ-specific, T cell-mediated autoimmune diseases during pregnancy. PMID:10632669

  2. 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

  3. 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

  4. Glucose induces FGF21 mRNA expression through ChREBP activation in rat hepatocytes.

    PubMed

    Iizuka, Katsumi; Takeda, Jun; Horikawa, Yukio

    2009-09-01

    Fibroblast growth factor 21 (FGF21) has beneficial effects of improving the plasma glucose and lipid profiles in diabetic rodents. Here, we investigated carbohydrate response element binding protein (ChREBP) involvement in the regulation of FGF21 mRNA expression in liver. Glucose stimulation and adenoviral overexpression of dominant active ChREBP increased FGF21 mRNA. Consistently, adenoviral expression of dominant negative Mlx inhibited glucose induction of FGF21 mRNA. Furthermore, deletion studies of mouse FGF21 gene promoter (-2000 to +65 bp) revealed a glucose responsive region between -74 and -52 bp. These findings suggest that FGF21 expression is regulated by ChREBP. PMID:19660458

  5. 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.

  6. 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

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

    PubMed

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

    2016-08-01

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

  8. mRNA expression pattern of gonadotropin receptors in bovine follicular cysts.

    PubMed

    Marelli, Belkis E; Diaz, Pablo U; Salvetti, Natalia R; Rey, Florencia; Ortega, Hugo H

    2014-12-01

    Follicular growth and steroidogenesis are dependent on gonadotropin binding to their receptors in granulosa and theca cells of ovarian follicles. The aim of the present study was to evaluate the expression patterns of follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHCGR) in ovarian follicular structures from cows with cystic ovarian disease (COD) as compared with those of regularly cycling cows. Relative real-time RT-PCR analysis showed that the expression of FSHR mRNA in granulosa cells was highest in small antral follicles, then decreased significantly as follicles increased in size, and was lowest in cysts. FSHR mRNA was not detected in the theca cells of any follicular category, including cysts. LHCGR mRNA expression in granulosa cells was significantly higher in large antral follicles than in cysts, and not detected in granulosa cells of small and medium antral follicles. In theca cells, the expression level of LHCGR mRNA in medium antral follicles was higher than in small and large antral follicles, whereas that in follicular cysts it was similar to those in small and medium antral follicles, but higher than that in large antral follicles. Our findings provide evidence that there is an altered gonadotropin receptor expression in bovine cystic follicles, and suggest that in conditions characterized by altered ovulation, such as COD, changes in the signaling system of gonadotropins may play a fundamental role in their pathogenesis. PMID:25454493

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

    PubMed

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

    2016-05-01

    Angiopoietin-like protein 4 (ANGPTL4) is involved in a variety of functions, including lipoprotein metabolism and angiogenesis. To reveal the role of ANGPTL4 in fat metabolism of sheep, ovine ANGPTL4 mRNA expression was analyzed in seven adipose tissues from two breeds with distinct tail types. Forty-eight animals with the gender ratio of 1:1 for both Guangling Large Tailed (GLT) and Small Tailed Han (STH) sheep were slaughtered at 2, 4, 6, 8, 10, and 12 months of age, respectively. Adipose tissues were collected from greater and lesser omental, subcutaneous, retroperitoneal, perirenal, mesenteric, and tail fats. Ontogenetic mRNA expression of ANGPTL4 in these adipose tissues from GTL and STH was studied by quantitative real time polymerase chain reaction. The results showed that ANGPTL4 mRNA expressed in all adipose tissues studied with the highest in subcutaneous and the lowest in mesenteric fat depots. Months of age, tissue and breed are the main factors that significantly influence the mRNA expression. These results provide new insights into ovine ANGPTL4 gene expression and clues for its function mechanism. PMID:26954186

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

    PubMed Central

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

    2016-01-01

    Angiopoietin-like protein 4 (ANGPTL4) is involved in a variety of functions, including lipoprotein metabolism and angiogenesis. To reveal the role of ANGPTL4 in fat metabolism of sheep, ovine ANGPTL4 mRNA expression was analyzed in seven adipose tissues from two breeds with distinct tail types. Forty-eight animals with the gender ratio of 1:1 for both Guangling Large Tailed (GLT) and Small Tailed Han (STH) sheep were slaughtered at 2, 4, 6, 8, 10, and 12 months of age, respectively. Adipose tissues were collected from greater and lesser omental, subcutaneous, retroperitoneal, perirenal, mesenteric, and tail fats. Ontogenetic mRNA expression of ANGPTL4 in these adipose tissues from GTL and STH was studied by quantitative real time polymerase chain reaction. The results showed that ANGPTL4 mRNA expressed in all adipose tissues studied with the highest in subcutaneous and the lowest in mesenteric fat depots. Months of age, tissue and breed are the main factors that significantly influence the mRNA expression. These results provide new insights into ovine ANGPTL4 gene expression and clues for its function mechanism. PMID:26954186

  11. Steroid receptor mRNA expression in the ovarian follicles of cows with cystic ovarian disease.

    PubMed

    Alfaro, Natalia S; Salvetti, Natalia R; Velazquez, Melisa M; Stangaferro, Matías L; Rey, Florencia; Ortega, Hugo H

    2012-06-01

    Steroid receptors have been demonstrated to be important intra-ovarian regulators of follicular development and ovulatory processes. The aim of the present study was to determine the expression of steroid receptor mRNA in ovarian follicular structures from cows with cystic ovarian disease (COD) compared with ovarian structures from regularly cycling cows using reverse transcription polymerase chain reaction (RT-PCR). The cystic follicles showed a higher estrogen receptor α (ESR1) mRNA expression in the theca and granulosa and a lower estrogen receptor β (ESR2) expression. The cystic follicles also showed a strong expression of androgen receptor mRNA in the granulosa. No changes were observed in total progesterone receptor mRNA, but a very significant increase in the B isoform was found in the granulosa of the cystic follicles. The findings of the current study provide evidence that an altered steroid signaling system may be present in bovine follicular cysts, and we suggest that in conditions characterized by altered ovulation, such as COD, changes in the expression of ovarian steroid receptors could play a fundamental role in the pathogeny of this disease. PMID:21536311

  12. EWS represses cofilin 1 expression by inducing nuclear retention of cofilin 1 mRNA.

    PubMed

    Huang, L; Kuwahara, I; Matsumoto, K

    2014-06-01

    In Ewing's sarcoma family tumors (ESFTs), the proto-oncogene EWS that encodes an RNA-binding protein is fused by chromosomal translocation to the gene encoding one of the E-twenty six (ETS) family of transcription factors, most commonly friend leukemia virus integration 1 (FLI-1). Although EWS/FLI-1 chimeric proteins are necessary for carcinogenesis, additional events seem to be required for transformation to occur. We have previously reported that a protein product of an EWS mRNA target, whose expression is negatively regulated by EWS but not by EWS/FLI-1, contributes to ESFT development. However, the mechanism by which EWS represses protein expression remains to be elucidated. Here, we report that overexpression of full-length EWS repressed protein expression and induced nuclear retention of reporter mRNAs in a tethering assay. In contrast, when a mutant lacking the EWS C-terminal nuclear localization signal (classified as a PY-NLS) was expressed, reporter protein expression was upregulated, and the number of cells exporting reporter mRNA to the cytoplasm increased. EWS binds to the 3'-untranslated region in another mRNA target, cofilin 1 (CFL1), and negatively regulates the expression of CFL1. Overexpression of EWS induced nuclear retention of CFL1 mRNA. Furthermore, ESFT cell proliferation and metastatic potential were suppressed by small interfering RNA-mediated CFL1 knockdown. Together, our findings suggest that EWS induces nuclear retention of CFL1 mRNA, thereby suppressing expression of CFL1, and that CFL1 promotes development of ESFT. Targeting CFL1 might therefore provide another novel approach for treatment of this aggressive disease. PMID:23831569

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

    NASA Astrophysics Data System (ADS)

    Elgart, Vlad; Jia, Tao; Fenley, Andrew T.; Kulkarni, Rahul

    2011-08-01

    The intrinsic stochasticity of gene expression can lead to large variability in protein levels for genetically identical cells. Such variability in protein levels can arise from infrequent synthesis of mRNAs which in turn give rise to bursts of protein expression. Protein expression occurring in bursts has indeed been observed experimentally and recent studies have also found evidence for transcriptional bursting, i.e. production of mRNAs in bursts. Given that there are distinct experimental techniques for quantifying the noise at different stages of gene expression, it is of interest to derive analytical results connecting experimental observations at different levels. In this work, we consider stochastic models of gene expression for which mRNA and protein production occurs in independent bursts. For such models, we derive analytical expressions connecting protein and mRNA burst distributions which show how the functional form of the mRNA burst distribution can be inferred from the protein burst distribution. Additionally, if gene expression is repressed such that observed protein bursts arise only from single mRNAs, we show how observations of protein burst distributions (repressed and unrepressed) can be used to completely determine the mRNA burst distribution. Assuming independent contributions from individual bursts, we derive analytical expressions connecting means and variances for burst and steady-state protein distributions. Finally, we validate our general analytical results by considering a specific reaction scheme involving regulation of protein bursts by small RNAs. For a range of parameters, we derive analytical expressions for regulated protein distributions that are validated using stochastic simulations. The analytical results obtained in this work can thus serve as useful inputs for a broad range of studies focusing on stochasticity in gene expression.

  14. Replenishment of RANTES mRNA expression in activated eosinophils fromatopic asthmatics

    PubMed Central

    Velazquez, J R; Lacy, P; Moqbel, R

    2000-01-01

    Eosinophils have been shown to express the gene encoding regulated upon activation, normal T‐cell expressed and secreted (RANTES), a potent eosinophilotactic chemokine. RANTES protein expression in eosinophils has previously been shown to be up‐regulated by a number of agonists, including complement‐dependent factors (C3b/iC3b) and interferon‐γ (IFN‐γ). We hypothesized that gene expression of RANTES is regulated in these cells by eosinophil‐specific agonists. We analysed RANTES mRNA expression by reverse‐transcription polymerase chain reaction (RT‐PCR) in human peripheral blood eosinophils obtained from mild atopic asthmatics following stimulation over time. In resting eosinophils, a low level of RANTES mRNA was found to be constitutively expressed in all the atopic donors tested in this study (n = 6). Following stimulation with C3b/iC3b (serum‐coated surfaces), eosinophils released measurable levels of RANTES, while sustained transcript expression was detected for up to 24 hr of stimulation. In contrast, IFN‐γ (5 ng/ml) transiently and significantly (P < 0·05, n = 3) depleted relative amounts of RANTES PCR product (compared with β2‐microglobulin) after 1–4 hr of stimulation. RANTES transcript was again detectable after 24 hr of IFN‐γ incubation, suggesting that the pool of RANTES mRNA had been replenished. Other eosinophil‐active cytokines, interleukin‐3 (IL‐3), IL‐4, IL‐5 and granulocyte–macrophage colony‐stimulating factor, did not appear to modulate RANTES mRNA expression after 1 hr of incubation. The effect of IFN‐γ on RANTES mRNA was reversed by cycloheximide, suggesting that IFN‐γ may act by increasing the rate of translation of RANTES mRNA. These findings indicate that IFN‐γ may induce a rapid and transient effect on the translation and replenishment of RANTES mRNA in eosinophils. This novel observation supports the notion that eosinophils have the potential to replenish their stored and released

  15. RANKL, OPG and CTR mRNA expression in the temporomandibular joint in rheumatoid arthritis

    PubMed Central

    LIU, WEI-WEI; XU, ZHI-MIN; LI, ZHENG-QIANG; ZHANG, YAN; HAN, BING

    2015-01-01

    The calcitonin receptor (CTR) and receptor activator of nuclear factor κB ligand (RANKL) have been found to be involved in the differentiation of osteoclasts. The association between the RANKL:osteoprotegerin (OPG) expression ratio and the pathogenesis of bone-destructive rheumatoid arthritis (RA) has been described in several joints, but the available data for the temporomandibular joint (TMJ) are limited. The aim of the present study was to investigate the involvement of osteoclasts at sites of bone erosion by determining the CTR expression and the RANKL:OPG expression ratio in the TMJ in a collagen-induced arthritis (CIA) model. Forty-eight male Wistar rats were randomly divided into two groups: Control group, injected with saline solution for 6 weeks; and CIA group, injected with emulsion. The RANKL and OPG mRNA expression was significantly increased in immunized rats compared with that in non-immunized rats. The RANKL:OPG expression ratio on the trabecular bone surface was 9.0 and 6.4 in the CIA group at weeks 4 and 6, respectively, while the RANKL:OPG expression ratio in the controls was 1.0:2. CTR mRNA expression was significantly upregulated in immunized rats compared with that in non-immunized rats; the level of CTR mRNA in the CTR-positive osteoclasts on the trabecular bone surface was 10.9- and 7.8-fold higher in the CIA rats than that in the control rats at weeks 4 and 6, respectively. In conclusion, focal bone destruction in an experimental model of arthritis in the TMJ can be attributed to cells expressing CTR, a defining feature of osteoclasts. The expression of RANKL and OPG mRNA within the inflamed synovium provides an insight into the mechanism of osteoclast differentiation and function at the border of bone erosion in arthritis. PMID:26622411

  16. Serum KIBRA mRNA and Protein Expression and Cognitive Functions in Depression

    PubMed Central

    Talarowska, Monika; Szemraj, Janusz; Kowalczyk, Małgorzata; Gałecki, Piotr

    2016-01-01

    Background Genes participating in synaptic signalling or plasticity in brain regions such as the prefrontal cortex (PFC) and the hippocampus have been implicated in cognition. Recently, a new gene (KIBRA, WWC1) has been added to this group due to its impact on memory performance. Recurrent depressive disorder (rDD) is a multifactorial disease, that one of the typical features is cognitive impairment. The main objective of this study was to perform an analysis of the KIBRA gene on both mRNA and protein levels in patients suffering from rDD and to investigate the relationship between KIBRA expression and cognitive performance. Material/Methods The study comprised 236 subjects: patients with rDD (n=131) and healthy subjects (n=105, HS). Cognitive function assessment was based on: Trail Making Test, The Stroop Test, Verbal Fluency Test and Auditory Verbal Learning Test. Results Both mRNA and protein expression levels of KIBRA gene were significantly higher in healthy subjects when compared to rDD. The presented relationship is clear even after taking age, education and sex of the examined subjects into consideration. No statistically significant relationship was found in the experiments between any of the conducted tests and KIBRA gene expression on mRNA level for both the rDD and HS groups. The presented study has limitations related to the fact that patients were being treated with antidepressant. This is relevant due to the fact that some antidepressants may affect mRNA expression. Number of patients and healthy subjects may result in the lack of statistical significance in some cases. Conclusions 1. The results of our study show decreased expression of the KIBRA gene on both mRNA and protein levels in depression. 2. We did not find any significant relationship between KIBRA gene expression and cognitive functions in case of both the healthy subjects and the patients affected by rDD. PMID:26768155

  17. Expression of CCT6A mRNA in chicken granulosa cells is regulated by progesterone.

    PubMed

    Wei, Qingqing; Zhu, Guiyu; Cui, Xinxing; Kang, Li; Cao, Dingguo; Jiang, Yunliang

    2013-08-01

    CCT6A, the zeta subunit of the chaperonin containing TCP1 complex, is the only cytosolic chaperonin in eukaryotes and is estimated to assist in the folding of multiple proteins including actin, tubulin, cyclin E, myosin, transducin and the Von Hippel Lindau tumor suppressor. In this study, we examined the expression of CCT6A and progesterone receptor (PGR) mRNA in various tissues of chickens and the regulation of CCT6A and PGR mRNA in ovarian granulosa cells. Northern blot analysis revealed that CCT6A had one transcript and was highly expressed in the ovary tissues from chickens at both the sexually immature and mature stages. CCT6A mRNA expression was increased maximally from pre-hierarchy follicles to F5 follicles and subsequently declined in pre-ovulatory and post-ovulatory follicles. The expression of PGR mRNA exhibited the similar pattern to CCT6A. In granulosa cells isolated from pre-ovulatory follicles, follicle-stimulating hormone (FSH) inhibited the expression of CCT6A mRNA, whereas progesterone activated CCT6A and suppressed PGR expression in a time-dependent manner. We further investigated the regulation of CCT6A transcription by progesterone by constructing various progressive deletions and mutants and identified the core promoter element of CCT6A and the binding region of progesterone, which is located from -2056 to -2051. Taken together, our results indicate that CCT6A likely plays an important role in follicle growth, and in granulosa cells, progesterone activates CCT6A transcription via a progesterone response element (PRE) located in the distal promoter of CCT6A. PMID:23644154

  18. 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

  19. CYP4 mRNA expression in marine polychaete Perinereis aibuhitensis in response to petroleum hydrocarbon and deltamethrin.

    PubMed

    Chen, Xue; Zhou, Yibing; Yang, Dazuo; Zhao, Huan; Wang, Lili; Yuan, Xiutang

    2012-09-01

    A CYP4 cDNA was cloned and characterized to identify the relationship between persistent organic pollutants and stress response in marine polychaete Perinereis aibuhitensis. The full length of PaCYP4 cDNA is 1857bp and encodes 481 amino acids. The deduced amino acid sequence showed 73% identity with CYP4BB1 from polychaete Nereis virens and shared high homology to other known CYP4 sequences. The expression level of PaCYP4 under petroleum hydrocarbon (PH) and deltamethrin (DM) exposure was detected using Real-time PCR. PH and combined toxicity treatments elevated the mRNA level of PaCYP4 in a dose- and time-dependent manner. The mRNA transcripts of PaCYP4 increased at the beginning of DM exposure and then eventually decreased, and the expression level of PaCYP4 down-regulated with increasing concentration of DM. CYP4 in P. aibuhitensis plays an important role in the metabolism of petroleum hydrocarbon and organochlorine pesticide. PMID:22768804

  20. 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...

  1. Real time imaging of mRNA expression dynamics in live cells using protein complementation methods

    NASA Astrophysics Data System (ADS)

    Meller, Amit

    2009-03-01

    Traditional methods for mRNA quantification in cells, such as northern blots, quantitative PCR or microarrays assays, require cell lysis and therefore do not preserve its dynamics. These methods cannot be used to probe the spatio-temporal localization of mRNA in cells, which provide useful information for a wide range biomolecular process, including RNA metabolizim, expression kinetics and RNA interference. To probe mRNA dynamics in live prokaryotic and eukaryotic cells, we develop a method, which exploit the strong affinity of the eukaryotic initiation factor 4A (eIF4A) to specific RNA aptamers. Two parts of the eIF4A are fused to a split Green Fluorescence Protein (GFP), and are expressed in the cells at high abundance. However, only when the RNA apatmer is also present, the two protein parts complement and become fluorescent. Thus, the fluorescent background remains low, allowing us to directly image the expression of mRNA molecules in live e-coli cells from its early onset, over hours. We find that the expression kinetics can be classified in one out of at least three forms, which also display distinct spatial distributions. I will discuss the possible biological origin for these distributions and their time evolution.

  2. Sodium lauryl sulphate alters the mRNA expression of lipid-metabolizing enzymes and PPAR signalling in normal human skin in vivo.

    PubMed

    Törmä, Hans; Berne, Berit

    2009-12-01

    Detergents irritate skin and affect skin barrier homeostasis. In this study, healthy skin was exposed to 1% sodium lauryl sulphate (SLS) in water for 24 h. Biopsies were taken 6 h to 8 days post exposure. Lipid patterns were stained in situ and real-time polymerase chain reaction (PCR) was used to examine mRNA expression of enzymes synthesizing barrier lipids, peroxisome proliferator-activated receptors (PPAR) and lipoxygenases. The lipid pattern was disorganized from 6 h to 3 days after SLS exposure. Concomitant changes in mRNA expression included: (i) reduction, followed by induction, of ceramide-generating beta-glucocerebrosidase, (ii) increase on day 1 of two other enzymes for ceramide biosynthesis and (iii) persistent reduction of acetyl-CoA carboxylase-B, a key enzyme in fatty acid synthesis. Surprisingly, the rate-limiting enzyme in cholesterol synthesis, HMG-CoA reductase, was unaltered. Among putative regulators of barrier lipids synthesis, PPARalpha and PPARgamma exhibited reduced mRNA expression, while PPARbeta/delta and LXRbeta were unaltered. Epidermal lipoxygenase-3, which may generate PPARalpha agonists, exhibited reduced expression. In conclusion, SLS induces reorganization of lipids in the stratum corneum, which play a role in detergents' destruction of the barrier. The changes in mRNA expression of enzymes involved in synthesizing barrier lipids are probably important for the restoration of the barrier. PMID:19366370

  3. hnRNP-U enhances the expression of specific genes by stabilizing mRNA.

    PubMed

    Yugami, Masato; Kabe, Yasuaki; Yamaguchi, Yuki; Wada, Tadashi; Handa, Hiroshi

    2007-01-01

    Heterogeneous nuclear ribonucleoproteins (hnRNPs) are thought to be involved in pre-mRNA processing. hnRNP-U, also termed scaffold attachment factor A (SAF-A), binds to pre-mRNA and nuclear matrix/scaffold attachment region DNA elements. However, its role in the regulation of gene expression is as yet poorly understood. In the present study, we show that hnRNP-U specifically enhances the expression of tumor necrosis factor alpha mRNA by increasing its stability, possibly through binding to the 3' untranslated region. We also show that hnRNP-U enhances the expression of several other genes as well, including GADD45A, HEXIM1, HOXA2, IER3, NHLH2, and ZFY, by binding to and stabilizing these mRNAs. These results suggest that hnRNP-U enhances the expression of specific genes by regulating mRNA stability. PMID:17174306

  4. 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.

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

    PubMed

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

    2004-01-01

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

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

    PubMed

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

    2003-04-10

    Inducible-cAMP early repressor (ICER) is a potent inhibitor of CRE (cAMP-related element)-driven gene transcription. In the rat pineal gland, it has been proposed to be part of the mechanisms involved in the shutting down of the transcription of the gene coding for arylalkylamine N-acetyltransferase (AA-NAT, the melatonin rhythm-generating enzyme). In this study, we report that ICER is expressed in the pineal gland of the photoperiodic rodent Syrian hamster although with some difference compared to the rat. In the Syrian hamster pineal, Icer mRNA levels, low at daytime, displayed a 20-fold increase during the night. Nighttime administration of a beta-adrenergic antagonist, propranolol, significantly reduced Icer mRNA levels although daytime administration of a beta-adrenergic agonist, isoproterenol, was unable to raise the low amount of Icer mRNA. These observations indicate that Icer mRNA expression is induced by the clock-driven norepinephrine release and further suggest that this stimulation is restricted to nighttime, as already observed for Aa-nat gene transcription. Furthermore, we found that the daily profile of Icer mRNA displayed photoperiodic variation with a lengthening of the nocturnal peak in short versus long photoperiod. These data indicate that ICER may be involved in both daily and seasonal regulation of melatonin synthesis in the Syrian hamster. PMID:12670714

  7. 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

  8. Decreased drebrin mRNA expression in Alzheimer disease: correlation with tau pathology.

    PubMed

    Julien, Carl; Tremblay, Cyntia; Bendjelloul, Farid; Phivilay, Alix; Coulombe, Marie-Andrée; Emond, Vincent; Calon, Frédéric

    2008-08-01

    To investigate the mRNA expression of the dendritic spine protein drebrin in Alzheimer's disease (AD), we performed post-mortem in situ hybridization studies in brain sections from 20 AD patients and 21 controls. AD diagnosis was confirmed by decreased drebrin protein and increased Abeta(40) (+464%; P < 0.05), Abeta(42) (+369%; P < 0.0001), Abeta(42/40) ratio (+226%; P < 0.01), total tau (+2,725%; P < 0.0001), and paired helical filament tau (PHFtau; +867%; P < 0.001) compared with controls. We found significant decreases in drebrin mRNA in the parietal cortex (-27%; P < 0.01), the temporal cortex (-22%; P < 0.05), and the hippocampus (-25%; P < 0.05) of AD patients compared with controls. Cortical levels of drebrin mRNA correlated positively with soluble total tau (r(2) = +0.244) but negatively with duration of symptoms (r(2) = -0.357) and PHFtau (r(2) = -0.248). Drebrin mRNA levels were correlated to a lesser degree with the drebrin protein content (r(2) = +0.136) and with sim2 (r(2) = +0.176), a potential modulator of drebrin transcription. Our results suggest that the down-regulation of drebrin mRNA expression plays an important role in AD and is closely related to the progression of the disease. PMID:18338803

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

    PubMed Central

    2013-01-01

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

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

    PubMed

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

    2001-06-01

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

  11. Expression of interleukin 6 receptors and interleukin 6 mRNA by bovine leukaemia virus-induced tumour cells.

    PubMed

    Droogmans, L; Cludts, I; Cleuter, Y; Kerkhofs, P; Adam, E; Willems, L; Kettmann, R; Burny, A

    1994-11-01

    Bovine leukaemia virus (BLV) is the aetiologic agent of bovine leucosis. The virus induces malignancies of the B-cell lineage (leukaemia/lymphoma). The role played by interleukin 6 (IL-6) in the BLV-induced leukemogenesis process was evaluated. Six cell lines derived from BLV-induced tumours were tested for the expression of IL-6 receptors. Two cell lines (LB155 and YR2) display 250-300 receptor per cell (kd = 1.7 10(-10) M and 1.4 10(-10) M, respectively) whereas the other four (LB159, LB167, YR1 and M51) do not display detectable amounts of receptors. Very low (if any) expression of IL-6 receptors has been found in the case of the B lymphocytes of animals in persistent lymphocytosis (PL). Despite the presence of IL-6 receptors on the surface of LB155 and YR2 cells, no influence of exogenous IL-6 on their growth has been observed. Northern analyses indicated the presence of IL-6 transcripts only in the case of mRNA isolated from LB155 cells. Since this cell line also expresses receptors for the cytokine, an autocrine loop may exist in these cells. Experiments in which bovine and bovine epithelial cell lines were transfected with a plasmid containing the bovine IL-6 promoter controlling the expression of the reporter cat gene failed to indicate any influence of the viral transactivator p34tax on the activity of this promoter. We conclude that IL-6 receptors and IL-6 mRNA can be found in some BLV-induced tumours, but this does not correlate with viral expression in BLV-induced leukaemia/lymphoma. PMID:7893972

  12. Expression of tilapia prepro-melanin-concentrating hormone mRNA in hypothalamic and neurohypophysial cells.

    PubMed

    Gröneveld, D; Eckhardt, E R; Coenen, A J; Martens, G J; Balm, P H; Wendelaar Bonga, S E

    1995-04-01

    Melanin-concentrating hormone (MCH) is a neuropeptide involved in background adaptation in teleost fish, and in multiple regulatory functions in mammals and fish. To study the expression of the MCH preprohormone (ppMCH) in teleosts, we first cloned a hypothalamic cDNA encoding the complete ppMCH of tilapia (Oreochromis mossambicus), and a cRNA probe derived from a 270 bp ppMCH cDNA fragment was used for the expression studies. The level of ppMCH mRNA expression in tilapia hypothalamus, measured by dot blot analysis, was significantly higher in fish adapted to a white background than in black-adapted animals, which is in accordance with the reported MCH plasma and tissue concentrations in fish. Northern blot analysis not only revealed a strong ppMCH mRNA signal in the hypothalamus, but also the presence of ppMCH mRNA in the neurointermediate lobe (NIL) of the pituitary. In situ hybridization and immunocytochemistry showed that ppMCH mRNA as well as MCH immunoreactivity are located in perikarya of two hypothalamic regions, namely in the nucleus lateralis tuberis (NLT) and the nucleus recessus lateralis (NRL). Quantitative analysis by dot blot hybridization revealed about eight times more ppMCH mRNA in the NLT than in the NRL and NIL of mature tilapias. ppMCH mRNA in the NIL could be localized to cell bodies of the neurohypophysis, which were also MCH immunoreactive. PMID:7619209

  13. 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

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

    PubMed

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

    2005-10-01

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

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

    PubMed

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

    2015-07-01

    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

  16. Dithranol downregulates expression of Id1 mRNA in human keratinocytes in vitro.

    PubMed

    Ronpirin, C; Tencomnao, T

    2012-01-01

    The precise causes of psoriasis, a chronic skin disorder characterized by hyperproliferation of keratinocytes and incomplete keratinization, are unclear. It is known that expression of helix-loop-helix transcription factor Id1, which functions as an inhibitor of differentiation, is upregulated in psoriatic skin. We investigated the effect of the antipsoriatic drug dithranol on mRNA and protein expression levels of Id1 in the HaCaT keratinocyte cell line. Cultured HaCaT cells were treated with 0-0.5 μg/mL dithranol for 30 min. After 2 and 4 h, total cellular RNA and total proteins were isolated from HaCaT cells, and quantitative real-time reverse transcriptase (RT-PCR) and Western blot were used to determine the mRNA and protein levels of Id1, respectively. Changes in normalized Id1 mRNA levels were observed only after 4 h of dithranol treatment. There was reduced expression of Id1 mRNA transcripts in the HaCaT cells treated with 0.1 μg/mL dithranol, but the reduction was not significant. The expression of Id1 mRNA was significantly downregulated (almost 50%) when 0.25 or 0.5 μg/mL dithranol was applied to the HaCaT cells. However, the normalized Id1 protein levels were not significantly affected. The molecular mechanisms underlying this finding should be investigated further to help determine the therapeutic action of this drug. PMID:23079823

  17. c-kit mRNA expression in human and murine hematopoietic cell lines.

    PubMed

    André, C; d'Auriol, L; Lacombe, C; Gisselbrecht, S; Galibert, F

    1989-08-01

    The c-kit proto-oncogene belongs to the tyrosine kinase receptor family. Although its ligand is still unknown, there is increasing evidence to suggest its involvement in hematopoiesis. In order to detect lineage or differentiation related specificity, we have studied c-kit mRNA expression in both human and murine hematopoietic organs and cell lines. We show that c-kit mRNA expression is found at early stages of erythroid and myeloid differentiation. There is however, no evidence of c-kit expression in the lymphoid lineage. Our results suggest a possible role for c-kit as a receptor in the early stages of the erythroid/myeloid differentiation. PMID:2474787

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

    PubMed

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

    2007-06-01

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

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

    SciTech Connect

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

    1991-10-01

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

  20. mRNA as gene therapeutic: how to control protein expression.

    PubMed

    Tavernier, Geertrui; Andries, Oliwia; Demeester, Jo; Sanders, Niek N; De Smedt, Stefaan C; Rejman, Joanna

    2011-03-30

    For many years, it was generally accepted that mRNA is too unstable to be efficiently used for gene therapy purposes. In the last decade, however, several research groups faced this challenge and not only proved the feasibility of mRNA-mediated transfection with surprising results regarding transfection efficiency and duration of protein expression, but also were able to demonstrate major advantages over the use of pDNA. These advantages will be the first issue discussed in this review, which first of all addresses the notions that mRNA does not need to cross the nuclear barrier to exert its biological activity and in addition lacks CpG motifs, which reduces its immunogenicity. Secondly, it provides insight in the (in)stability of the mRNA molecule, in how mRNA can be modified to increase its half-life and in the necessities of exogenously produced mRNA to be successfully used in transfection protocols. Furthermore, this review gives an in-depth overview of the different techniques and vehicles for intracellular mRNA delivery exploited by us and other groups, comprising electroporation, gene gun injection, lipo- and polyplexes. Finally, it covers recent literature describing specific applications for mRNA based gene delivery, showing that until now most attention has been paid to vaccination strategies. This review offers a comprehensive overview of current knowledge of the major theoretical as well as practical aspects of mRNA-mediated transfection, showing both its possibilities and its pitfalls and should therefore be useful for a diverse scientific audience. PMID:20970469

  1. Aromatase and estrogen receptor alpha mRNA expression as prognostic biomarkers in patients with astrocytomas.

    PubMed

    Dueñas Jiménez, J M; Candanedo Arellano, A; Santerre, A; Orozco Suárez, S; Sandoval Sánchez, H; Feria Romero, I; López-Elizalde, R; Alonso Venegas, M; Netel, B; de la Torre Valdovinos, B; Dueñas Jiménez, S H

    2014-09-01

    Estrogens are oncogenic hormones at a high level in breast, prostate, endometrial and lung cancer. Estrogens are synthesized by aromatase which has been used as a biomarker both in breast and lung cancer. Estrogen biological activities are executed by their classic receptors (ERα and ERβ). ERα has been described as a cancer promoter and ERβ, as a possible tumor suppressor. Both receptors are present at low levels in primary multiforme glioblastoma (GBM). The GBM frequency is 50 % higher in men than in women. The GBM patient survival period ranges from 7 to 18 months. The purpose of this pilot study was to evaluate aromatase and estrogen receptor expression, as well as 17ß-estradiol concentration in astrocytoma patients biopsies to obtain a prognosis biomarker for these patients. We analyzed 36 biopsies of astrocytoma patients with a different grade (I-IV) of malignity. Aromatase and estrogen receptor mRNA expression were analyzed by semiquantitative RT-PCR, and the E2 levels, by ELISA. E2 concentration was higher in GBM, compared to grade II or III astrocytomas. The number of cells immunoreactive to aromatase and estrogen receptors decreased as the grade of tumor malignity increased. Aromatase mRNA expression was present in all biopsies, regardless of malignity grade or patient age or gender. The highest expression of aromatase mRNA in GBM patients was associated to the worst survival prognostic (6.28 months). In contrast lowest expression of ERα mRNA in astrocytoma patients had a worst prognosis. In conclusion, aromatase and ERα expression could be used as prognosis biomarkers for astrocytoma patients. PMID:25005528

  2. 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

  3. Genetic organization and mRNA expression of enolase genes of Candida albicans.

    PubMed

    Postlethwait, P; Sundstrom, P

    1995-04-01

    In previous work, we cloned a Candida albicans cDNA for the glycolytic enzyme enolase and found a single, abundant enolase transcript on Northern (RNA) blots and a single protein on immunoblots, using antiserum raised against a recombinant enolase fusion protein. Because C. albicans enolase is abundantly produced during infection and elicits strong host immune responses, the mechanisms regulating enolase production are important for understanding the growth of C. albicans in vivo. To obtain more information on enolase gene expression by C. albicans, we used the enolase cDNA clone to investigate the genetic organization of enolase genes and the steady-state levels of enolase mRNA under several growth conditions. Gene disruption techniques in combination with Southern blot analyses of genomic DNA showed the presence of two enolase gene loci that could be distinguished by the locations of ClaI and Mn/I sites in their 3' flanking regions. Enolase steady-state mRNA levels were greatest during the middle phase of the logarithmic growth curve and were low during stationary phase. Minimal differences in enolase mRNA levels between yeast cells and hyphae were found. Propagation of C. albicans in glucose did not cause increased enolase mRNA levels compared with growth in a nonfermentable carbon source (pyruvate). It was concluded that two gene loci exist for C. albicans enolase and that enolase mRNA is constitutively produced at high levels during active metabolism. PMID:7896700

  4. Alpha-synuclein mRNA expression in oligodendrocytes in MSA

    PubMed Central

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

    2014-01-01

    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. PMID:24590631

  5. Regulation of luteinizing hormone receptor mRNA expression by a specific RNA binding protein in the ovary*

    PubMed Central

    Menon, K.M.J.; Nair, Anil K.; Wang, Lei; Peegel, Helle

    2009-01-01

    Summary The expression of LH receptor mRNA shows significant changes during different physiological states of the ovary. Previous studies from our laboratory have identified a post-transcriptional mechanism by which LH receptor mRNA is regulated following preovulatory LH surge or in response to hCG administration. A specific binding protein, identified as mevalonate kinase, binds to the open reading frame of LH receptor mRNA. The protein binding site is localized to nucleotides 203–220 of the LH receptor mRNA and exhibits a high degree of specificity. The expression levels of the protein show an inverse relationship to the LH receptor mRNA levels. The hCG-induced down-regulation of LH receptor mRNA can be mimicked by increasing the intracellular levels of cyclic AMP by a phosphodiesterase inhibitor. An in vitro mRNA decay assay showed that addition of the binding protein to the decay system caused accelerated LH receptor mRNA decay. Our results therefore show that LH receptor mRNA expression in the ovary is regulated post-transcriptionally by altering the rate of mRNA degradation by a specific mRNA binding protein. PMID:17055149

  6. 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

  7. 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. PMID:21985468

  8. Expression2Kinases: mRNA profiling linked to multiple upstream regulatory layers

    PubMed Central

    Gordonov, Simon; Lim, Maribel P.; Perkins, Matthew H.; Ma'ayan, Avi

    2012-01-01

    Motivation: Genome-wide mRNA profiling provides a snapshot of the global state of cells under different conditions. However, mRNA levels do not provide direct understanding of upstream regulatory mechanisms. Here, we present a new approach called Expression2Kinases (X2K) to identify upstream regulators likely responsible for observed patterns in genome-wide gene expression. By integrating chromatin immuno-precipitation (ChIP)-seq/chip and position weight matrices (PWMs) data, protein–protein interactions and kinase–substrate phosphorylation reactions, we can better identify regulatory mechanisms upstream of genome-wide differences in gene expression. We validated X2K by applying it to recover drug targets of food and drug administration (FDA)-approved drugs from drug perturbations followed by mRNA expression profiling; to map the regulatory landscape of 44 stem cells and their differentiating progeny; to profile upstream regulatory mechanisms of 327 breast cancer tumors; and to detect pathways from profiled hepatic stellate cells and hippocampal neurons. The X2K approach can advance our understanding of cell signaling and unravel drugs mechanisms of action. Availability: The software and source code are freely available at: http://www.maayanlab.net/X2K. Contact: avi.maayan@mssm.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:22080467

  9. Elongation factor 1 gamma mRNA expression in oesophageal carcinoma.

    PubMed Central

    Mimori, K; Mori, M; Inoue, H; Ueo, H; Mafune, K; Akiyoshi, T; Sugimachi, K

    1996-01-01

    Elongation factor 1 gamma (EF1 gamma) is known to be a subunit of EF1, one of the G proteins that mediate the transport of aminoacyl tRNA to 80S ribosomes during translation. As little is known regarding the expression of EF1 gamma in human oesophageal carcinoma, this study looked at its expression using a northern blot analysis. Thirty six cases of oesophageal carcinoma and 15 oesophageal carcinoma cell lines were studied. The EF1 gamma mRNA overexpression at a level of twofold or more was seen in five (14%) of 36 carcinomatous tissues compared with the normal counterparts. All five overexpressed cases showed severe lymph node metastases compared with the non-overexpressed cases, and the difference was significant (p = 0.028). The stage of the disease of these five cases was far advanced compared with the nonoverexpressed cases (p = 0.012). All 15 oesophageal carcinoma cells expressed EF1 gamma mRNA relatively lower than the gastric or pancreatic carcinoma cell lines, in which EF1 gamma was originally isolated. As the expression of EF1 gamma mRNA could be detected even in the biopsy specimens, its overexpression in tumour tissue may provide preoperative useful information for predicting the aggressiveness of tumours. Images Figure 1 Figure 2 Figure 3 PMID:8566862

  10. 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

  11. Regional distribution of solute carrier mRNA expression along the human intestinal tract.

    PubMed

    Meier, Yvonne; Eloranta, Jyrki J; Darimont, Jutta; Ismair, Manfred G; Hiller, Christian; Fried, Michael; Kullak-Ublick, Gerd A; Vavricka, Stephan R

    2007-04-01

    Intestinal absorption of drugs, nutrients, and other compounds is mediated by uptake transporters expressed at the apical enterocyte membrane. These compounds are returned to the intestinal lumen or released into portal circulation by intestinal efflux transporters expressed at apical or basolateral membranes, respectively. One important transporter superfamily, multiple members of which are intestinally expressed, are the solute carriers (SLCs). SLC expression levels may determine the pharmacokinetics of drugs that are substrates of these transporters. In this study we characterize the distribution of 15 human SLC transporter mRNAs in histologically normal biopsies from five regions of the intestine of 10 patients. The mRNA expression levels of CNT1, CNT2, apical sodium-dependent bile acid transporter (ABST), serotonin transporter (SERT), PEPT1, and OCTN2 exhibit marked differences between different regions of the intestine: the first five are predominantly expressed in the small intestine, whereas OCTN2 exhibits strongest expression in the colon. Two transporter mRNAs studied (OCTN1, OATP2B1) are expressed at similar levels in all gut sections. In addition, ENT2 mRNA is present at low levels across the colon, but not in the small intestine. The other six SLC mRNAs studied are not expressed in the intestine. Quantitative knowledge of transporter expression levels in different regions of the human gastrointestinal tract could be useful for designing intestinal delivery strategies for orally administered drugs. Furthermore, changes in transporter expression that occur in pathological states, such as inflammatory bowel disease, can now be defined more precisely by comparison with the expression levels measured in healthy individuals. PMID:17220238

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

    PubMed

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

    1994-06-01

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

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

    PubMed

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

    2014-05-01

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

  14. Requirement for nuclear autoantigenic sperm protein mRNA expression in bovine preimplantation development.

    PubMed

    Nagatomo, Hiroaki; Kohri, Nanami; Akizawa, Hiroki; Hoshino, Yumi; Yamauchi, Nobuhiko; Kono, Tomohiro; Takahashi, Masashi; Kawahara, Manabu

    2016-03-01

    Nuclear autoantigenic sperm protein (NASP) is associated with DNA replication, cell proliferation, and cell cycle progression through its specific binding to histones. The aim of this study was to examine the roles of NASP in bovine preimplantation embryonic development. Using NASP gene knockdown (KD), we confirmed the reduction of NASP messenger RNA (mRNA) expression during preimplantation development. NASP KD did not affect cleavage but significantly decreased development of embryos into the blastocyst stage. Furthermore, blastocyst hatching was significantly decreased in NASP KD embryos. Cell numbers in the inner cell mass of NASP KD blastocysts were also decreased compared to those of controls. These results suggest that NASP mRNA expression is required for preimplantation development into the blastocyst stage in cattle. PMID:26690724

  15. 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

  16. Expression profiling of Drosophila mitochondrial genes via deep mRNA sequencing

    PubMed Central

    Torres, Tatiana Teixeira; Dolezal, Marlies; Schlötterer, Christian; Ottenwälder, Birgit

    2009-01-01

    Mitochondria play an essential role in several cellular processes. Nevertheless, very little is known about patterns of gene expression of genes encoded by the mitochondrial DNA (mtDNA). In this study, we used next-generation sequencing (NGS) for transcription profiling of genes encoded in the mitochondrial genome of Drosophila melanogaster and D. pseudoobscura. The analysis of males and females in both species indicated that the expression pattern was conserved between the two species, but differed significantly between both sexes. Interestingly, mRNA levels were not only different among genes encoded by separate transcription units, but also showed significant differences among genes located in the same transcription unit. Hence, mRNA abundance of genes encoded by mtDNA seems to be heavily modulated by post-transcriptional regulation. Finally, we also identified several transcripts with a noncanonical structure, suggesting that processing of mitochondrial transcripts may be more complex than previously assumed. PMID:19843606

  17. Stochastic induction of persister cells by HipA through (p)ppGpp-mediated activation of mRNA endonucleases

    PubMed Central

    Germain, Elsa; Roghanian, Mohammad; Gerdes, Kenn; Maisonneuve, Etienne

    2015-01-01

    The model organism Escherichia coli codes for at least 11 type II toxin–antitoxin (TA) modules, all implicated in bacterial persistence (multidrug tolerance). Ten of these encode messenger RNA endonucleases (mRNases) inhibiting translation by catalytic degradation of mRNA, and the 11th module, hipBA, encodes HipA (high persister protein A) kinase, which inhibits glutamyl tRNA synthetase (GltX). In turn, inhibition of GltX inhibits translation and induces the stringent response and persistence. Previously, we presented strong support for a model proposing (p)ppGpp (guanosine tetra and penta-phosphate) as the master regulator of persistence. Stochastic variation of [(p)ppGpp] in single cells induced TA-encoded mRNases via a pathway involving polyphosphate and Lon protease. Polyphosphate activated Lon to degrade all known type II antitoxins of E. coli. In turn, the activated mRNases induced persistence and multidrug tolerance. However, even though it was known that activation of HipA stimulated (p)ppGpp synthesis, our model did not explain how hipBA induced persistence. Here we show that, in support of and consistent with our initial model, HipA-induced persistence depends not only on (p)ppGpp but also on the 10 mRNase-encoding TA modules, Lon protease, and polyphosphate. Importantly, observations with single cells convincingly show that the high level of (p)ppGpp caused by activation of HipA does not induce persistence in the absence of TA-encoded mRNases. Thus, slow growth per se does not induce persistence in the absence of TA-encoded toxins, placing these genes as central effectors of bacterial persistence. PMID:25848049

  18. 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.

  19. Comparison of IgE expression at the mRNA and protein levels in vitro.

    PubMed Central

    Turner, K J; Creany, J; Coelen, R J; Cameron, K J; Holt, B J; Beilharz, M W

    1991-01-01

    The regulating effects of IL-4 and pokeweed mitogen on IgE synthesis in vitro by human peripheral blood leucocytes has been compared with the corresponding effect of these regulators on the expression of IgE mRNA. The latter was measured by dot blot hybridization with an oligonucleotide coding for a unique six amino acid region of the CH epsilon 2 domain. Specificity of the oligonucleotide probe was established by its inability to hybridize with RNA extracted from HMY-2 (IgG) and XQ-15 (IgM) secreting cell lines whilst producing intense signals with RNA extracted from the IgE secreting cell line U266. Whilst IgE mRNA was detected in RNA extracted from PBL of both atopic and control subjects, spontaneous IgE synthesis was restricted to atopic PBL. IL-4 increased both IgE mRNA and IgE synthesis in all PBL samples but PWM, while significantly increasing IgE mRNA expression either failed to modify IgE synthesis or actively suppressed it. The assay system employed to quantitate IgE synthesis in vitro was shown to be inhibited by both IgE binding factors and IgG anti-IgE autoantibodies which are produced in PBL cultures. IgE mRNA levels might therefore more accurately monitor the regulatory effects of IL-4 and PWM on IgE synthesis than quantitation of the IgE by radioimmunoassay. Images Figure 1 PMID:1783428

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

    Boël, Grégory; Letso, Reka; Neely, Helen; Price, W Nicholson; Wong, Kam-Ho; Su, Min; Luff, Jon D; Valecha, Mayank; Everett, John K; Acton, Thomas B; Xiao, Rong; Montelione, Gaetano T; Aalberts, Daniel P; Hunt, John F

    2016-01-21

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

  2. 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

  3. 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.

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

    PubMed

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

    2016-07-01

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

  5. 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

  6. Trichinella spiralis thymidylate synthase: cDNA cloning and sequencing, and developmental pattern of mRNA expression.

    PubMed

    Dabrowska, M; Jagielska, E; Cieśla, J; Płucienniczak, A; Kwiatowski, J; Wranicz, M; Boireau, P; Rode, W

    2004-02-01

    The persistent expression of thymidylate synthase activity has previously been demonstrated not only in adult forms, but also in non-developing muscle larvae of Trichinella spiralis and T. pseudospiralis, pointing to an unusual pattern of cell cycle regulation, and prompting further studies on the developmental pattern of T. spiralis thymidylate synthase gene expression. The enzyme cDNA was cloned and sequenced, allowing the characterization of a single open reading frame of 307 amino acids coding for a putative protein of 35,582 Da molecular weight. The amino acid sequence of the parasite enzyme was analysed, the consensus phylogenetic tree built and its stability assessed. The aa sequence identity with thymidylate synthase was confirmed by the enzymatic activity of the recombinant protein expressed in E. coli. As compared with the enzyme purified from muscle larvae, it showed apparently similar Vmax value, but higher Km(app) values desscribing interactions with dUMP (28.8 microM vs. 3.9 microM) and (6RS,alphaS)-N(5,10)-methylenetetrahydrofolate (383 microM vs. 54.7 microM). With the coding region used as a probe, thymidylate synthase mRNA levels, relative to 18S rRNA, were found to be similar in muscle larvae, adult forms and newborn larvae, in agreement with muscle larvae cells being arrested in the cell cycle. PMID:15030008

  7. Humoral immunity to human breast cancer: antigen definition and quantitative analysis of mRNA expression.

    PubMed

    Scanlan, M J; Gout, I; Gordon, C M; Williamson, B; Stockert, E; Gure, A O; Jäger, D; Chen, Y T; Mackay, A; O'Hare, M J; Old, L J

    2001-03-30

    The ability of the immune system to recognize structurally altered, amplified or aberrantly expressed proteins can be used to identify molecules of etiologic relevance to cancer and to define targets for cancer immunotherapy. In the current study, ninety-four distinct antigens reactive with serum IgG from breast cancer patients were identified by immunoscreening breast cancer-derived cDNA expression libraries (SEREX). A serological profile was generated for each antigen on the basis of reactivity with allogeneic sera from normal individuals and cancer patients, and mRNA expression profiles for coding sequences were assembled based upon the tissue distribution of expressed sequence tags, Northern blots and real-time RT-PCR. Forty antigens reacted exclusively with sera from cancer patients. These included well-characterized tumor antigens, e.g. MAGE-3, MAGE-6, NY-ESO-1, Her2neu and p53, as well as newly-defined breast cancer antigens, e.g. kinesin 2, TATA element modulatory factor 1, tumor protein D52 and MAGE D, and novel gene products, e.g. NY-BR-62, NY-BR-75, NY-BR-85, and NY-BR-96. With regard to expression profiles, two of the novel gene products, NY-BR-62 and NY-BR-85, were characterized by a high level of testicular mRNA expression, and were overexpressed in 60% and 90% of breast cancers, respectively. In addition, mRNA encoding tumor protein D52 was overexpressed in 60% of breast cancer specimens, while transcripts encoding SNT-1 signal adaptor protein were downregulated in 70% of these cases. This study adds to the growing list of breast cancer antigens defined by SEREX and to the ultimate objective of identifying the complete repertoire of immunogenic gene products in human cancer (the cancer immunome). PMID:12747765

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

    PubMed

    Fagergren, Pernilla; Hurd, Yasmin

    2007-09-10

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

  9. 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

  10. Differential dynamics of the mammalian mRNA and protein expression response to misfolding stress.

    PubMed

    Cheng, Zhe; Teo, Guoshou; Krueger, Sabrina; Rock, Tara M; Koh, Hiromi W L; Choi, Hyungwon; Vogel, Christine

    2016-01-01

    The relative importance of regulation at the mRNA versus protein level is subject to ongoing debate. To address this question in a dynamic system, we mapped proteomic and transcriptomic changes in mammalian cells responding to stress induced by dithiothreitol over 30 h. Specifically, we estimated the kinetic parameters for the synthesis and degradation of RNA and proteins, and deconvoluted the response patterns into common and unique to each regulatory level using a new statistical tool. Overall, the two regulatory levels were equally important, but differed in their impact on molecule concentrations. Both mRNA and protein changes peaked between two and eight hours, but mRNA expression fold changes were much smaller than those of the proteins. mRNA concentrations shifted in a transient, pulse-like pattern and returned to values close to pre-treatment levels by the end of the experiment. In contrast, protein concentrations switched only once and established a new steady state, consistent with the dominant role of protein regulation during misfolding stress. Finally, we generated hypotheses on specific regulatory modes for some genes. PMID:26792871

  11. 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

  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. PMID:24080485

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

    PubMed

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

    2012-01-01

    Calmodulin (CALM), a calcium-binding protein, is expressed in the hypothalamic-pituitary-gonadal axis; it plays a pivotal role in the reproductive system by regulating gonadotropin-releasing hormone signaling. Downstream of hypothalamic-pituitary-gonadal signaling pathways, liver receptor homolog-1 (LRH-1) is involved in female gonadal hormone synthesis. In the chicken, although the two genes are known to be associated with reproductive traits, the interaction between gonadotropins and gonadal steroids remains unclear. We used quantitative real-time PCR to quantify the tissular (hypothalamus, pituitary, ovary, liver, kidney, oviduct, heart) and ontogenetic (12, 18, 32, and 45 weeks) mRNA expression profiles of CALM and LRH-1 in Erlang Mountainous chickens to determine their roles in the endocrine control of fertility, and compared these profiles with expression in Roman chickens. We found that the relative expressions of CALM and LRH-1 genes had the highest levels in the pituitary and ovary at 32 weeks. The expression level of CALM mRNA in the pituitary of Roman chickens was significantly higher than that in Erlang Mountainous chickens at 32 and 45 weeks, while the LRH-1 transcript level in the ovaries of Roman chickens was significantly lower than that of Erlang Mountainous chickens at 32 and 45 weeks. In summary, the transcript levels of CALM and LRH-1 genes are associated with chicken reproductive traits; in addition, we found that the CALM gene is the key regulator in the hypothalamic-pituitary-gonadal signaling network. PMID:23079841

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

    PubMed

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

    2014-04-01

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

  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. Role of T-helper type 2 cytokines in down-modulation of fas mRNA and receptor on the surface of activated CD4(+) T cells: molecular basis for the persistence of the allergic immune response.

    PubMed

    Spinozzi, F; Agea, E; Fizzotti, M; Bassotti, G; Russano, A; Droetto, S; Bistoni, O; Grignani, F; Bertotto, A

    1998-12-01

    The mechanisms responsible for persistence of T lymphocytes at the sites of allergic inflammation are not completely understood. Activated T cells, usually expressing Fas on their surface, undergo activation-induced apoptotic death, thus limiting the dangerous consequences of a persistent immune reaction. We have previously shown that pulmonary T lymphocytes from untreated asthmatic subjects do not express surface Fas receptors nor do they contain Fas mRNA, yet they display normal levels of Fas ligand. This is not an inherited defect and is confined to mucosal T cells. To gain insights into the mechanism responsible for these findings, we performed a set of experiments with both purified Dermatophagoides pteronyssinus allergen and recombinant human cytokines: interleukin 2 (IL-2), IL-4, IL-5, transforming growth factor beta1, interferon gamma, and granulocyte-macrophage colony-stimulating factor (GM-CSF). In vitro exposure of purified CD4(+) lymphocytes to allergen yielded only transient up-regulation of surface Fas but did not influence susceptibility to Fas-mediated cell death. T-helper type 2 cytokines (IL-4, IL-5, and GM-CSF) had a dose-dependent and specific inhibitory effect on Fas mRNA, suggesting a new fundamental biological role in the survival of inflammatory cells during allergen exposure. PMID:9837865

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

    PubMed

    Matsuyama, Satoshi; Nakano, Yuko; Nakamura, Mieko; Yamamoto, Ryohei; Shimada, Terumasa; Ohashi, Fumihito; Kubo, Kihei

    2015-01-01

    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

  18. 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.

  19. 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. PMID:25967984

  20. Increased expression of C5a receptor (CD88) mRNA in canine mammary tumors.

    PubMed

    Hezmee, Mohd Noor Mohd; Kyaw-Tanner, Myat; Lee, Jia Yu Peppermint; Shiels, Ian A; Rolfe, Barbara; Woodruff, Trent; Mills, Paul C

    2011-01-01

    Mammary tumors are among the most common neoplastic conditions in dogs, and there is evidence that inflammation plays a role in the development of some tumor types in dogs. The complement system is a major participant in the inflammatory process and the complement activation component, C5a, is a potent inflammatory peptide. This study investigated the mRNA expression of the major receptor for C5a (C5aR; CD88) in histopathological samples of canine mammary tumors by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) using canine-specific primers for CD88. A total of seven canine mammary tumors (four malignant carcinomas, two benign mixed mammary tumors, and one myoepithelioma) and eight normal mammary glands were analysed. All the tumor samples expressed low levels of CD88 mRNA, while none of the normal mammary tissues showed any detectable expression. These preliminary results suggest that C5a-CD88 interaction may play a contributory role in the inflammatory response associated with mammary tumor development in dogs. Further studies investigating the mechanisms behind complement activation and C5a receptor expression in canine mammary tumors are warranted. PMID:20846729

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

    PubMed Central

    Günthner, Roman; Kumar, Vankayala Ramaiah Santhosh; Lorenz, Georg; Anders, Hans-Joachim; Lech, Maciej

    2013-01-01

    The cell type-, organ-, and species-specific expression of the pattern-recognition receptors (PRRs) are well described but little is known about the respective expression profiles of their negative regulators. We therefore determined the mRNA expression levels of A20, CYLD, DUBA, ST2, CD180, SIGIRR, TANK, SOCS1, SOCS3, SHIP, IRAK-M, DOK1, DOK2, SHP1, SHP2, TOLLIP, IRF4, SIKE, NLRX1, ERBIN, CENTB1, and Clec4a2 in human and mouse solid organs. Humans and mice displayed significant differences between their respective mRNA expression patterns of these factors. Additionally, we characterized their expression profiles in mononuclear blood cells upon bacterial endotoxin, which showed a consistent induction of A20, SOCS3, IRAK-M, and Clec4a2 in human and murine cells. Furthermore, we studied the expression pattern in transient kidney ischemia-reperfusion injury versus post-ischemic atrophy and fibrosis in mice. A20, CD180, ST2, SOCS1, SOCS3, SHIP, IRAK-M, DOK1, DOK2, IRF4, CENTB1, and Clec4a2 were all induced, albeit at different times of injury and repair. Progressive fibrosis was associated with a persistent induction of these factors. Thus, the organ- and species-specific expression patterns need to be considered in the design and interpretation of studies related to PRR-mediated innate immunity, which seems to be involved in tissue injury, tissue regeneration and in progressive tissue scarring. PMID:24009023

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

    PubMed

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

    1998-08-01

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

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

    PubMed Central

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

    2013-01-01

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

  4. 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. PMID:21969073

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

    PubMed Central

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

    2005-01-01

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

  6. Exercise training does not increase muscle FNDC5 protein or mRNA expression in pigs

    PubMed Central

    Fain, John N.; Company, Joseph M.; Booth, Frank W.; Laughlin, M. Harold; Padilla, Jaume; Jenkins, Nathan T.; Bahouth, Suleiman W.; Sacks, Harold S.

    2013-01-01

    Background Exercise training elevates circulating irisin and induces the expression of the FNDC5 gene in skeletal muscles of mice. Our objective was to determine whether exercise training also increases FNDC5 protein or mRNA expression in the skeletal muscles of pigs as well as plasma irisin. Methods Castrated male pigs of the Rapacz familial hypercholesterolemic (FHM) strain and normal (Yucatan miniature) pigs were sacrificed after 16–20 weeks of exercise training. Samples of cardiac muscle, deltoid and triceps brachii muscle, subcutaneous and epicardial fat were obtained and FNDC5 mRNA, along with that of 6 other genes, was measured in all tissues of FHM pigs by reverse transcription polymerase chain reaction. FNDC protein in deltoid and triceps brachii was determined by Western blotting in both FHM and normal pigs. Citrate synthase activity was measured in the muscle samples of all pigs as an index of exercise training. Irisin was measured by an ELISA assay. Results There was no statistically significant effect of exercise training on FNDC5 gene expression in epicardial or subcutaneous fat, deltoid muscle, triceps brachii muscle or heart muscle. Exercise-training elevated circulating levels of irisin in the FHM pigs and citrate synthase activity in deltoid and triceps brachii muscle. A similar increase in citrate synthase activity was seen in muscle extracts of exercise-trained normal pigs but there was no alteration in circulating irisin. Conclusion Exercise training in pigs does not increase FNDC5 mRNA or protein in the deltoid or triceps brachii of FHM or normal pigs while increasing circulating irisin only in the FHM pigs. These data indicate that the response to exercise training in normal pigs is not comparable to that seen in mice. PMID:23831442

  7. Posttraumatic temporal TGF-β mRNA expression in lens epithelial cells of paediatric patients.

    PubMed

    Berezowski, P; Strzalka-Mrozik, B; Forminska-Kapuscik, M; Mazurek, U; Filipek, E; Nawrocka, L; Pieczara, E; Banasiak, P; Kimsa, M

    2012-01-01

    The aim of the study was to determine temporal TGFB1, TGFB2 and TGFB3 gene expression profiles in the anterior lens capsule of paediatric patients with posttraumatic cataract. The patient group comprised 22 children selected with a fragment of anterior lens capsule obtained during elective cataract surgery and sampled for molecular analysis. The levels of TGF-β isoforms in the anterior lens capsule were determined based on the number of mRNA copies per 1 μg total RNA by real-time qRTPCR. Three time-related result clusters were identified based on hierarchical cluster analysis: 2.2, 4.4 and 15.0 months (time span from injury to anterior capsule sampling during surgery) and compared with regard to temporal gene expression profile and quantitative relations of TGF-β1, 2 and 3 mRNAs. TGF-β1, TGF-β2, and TGF-β3 mRNAs were detected in all anterior lens capsule samples. A comparative analysis revealed: TGF-β1>TGF-β2>TGF-β3 during the entire observation period. The TGF-β mRNA levels continued to increase up to four months after injury, then returning close to the base levels after around 15 months. The expression patterns of TGF-β isoforms showed a similar tendency. Differences in the expression levels of TGF-β1 and TGF-β2 between the particular clusters were statistically significant. Posttraumatic transcriptional activities of TGF-β1 and TGF-β2 in the anterior lens capsule of paediatric patients depend on the time elapsing from injury. Our findings indicate that the transcriptional activities of TGFB family genes show a transient period of over-expression during the months after injury. TGF-β1 is a dominant isoform expressed in lens epithelial cells following injury. PMID:22464821

  8. Cloning, characterization and mRNA expression of interleukin-6 in blunt snout bream (Megalobrama amblycephala).

    PubMed

    Zhang, Chun-Nuan; Zhang, Ji-Liang; Liu, Wen-Bin; Wu, Qiu-Jue; Gao, Xiao-Chan; Ren, Hong-Tao

    2016-07-01

    In the present study, the interleukin-6 gene (IL-6) cDNA in blunt snout bream (Megalobrama amblycephala) was identified and its expression profiles under ammonia stress and bacterial challenge were investigated. The IL-6 sequence consisted of 1045 bp, including a 696 bp ORF which translated into a 232 amino acid (AA) protein. The protein contained a putative signal peptide of 24 AA in length. IL-6 expression analysis showed that the it is differentially expressed in various tissues under normal conditions and the highest IL-6 level was observed in the intestine tissue, followed by the liver, and then in the gills. Under ammonia stress, the IL-6 mRNA level both in spleens and intestine increased significantly (P < 0.05), with the maximum levels attained at 6 h, 12 h (72, 10-fold, respectively). Thereafter, they all significantly decreased (P < 0.01) and returned to the basal value within 48 h. Whereas, in livers it slightly decreased at 3 h firstly (0.5-fold), and then significantly (P < 0.05) increased with the maximum level attained 12 h (3-fold). Further expression analysis showed that the mRNA level of IL-6 in spleens, intestine and livers of blunt snout bream all increased significantly (P < 0.05), with maximum values attained at 6 h, 3 h, 6 h (10, 6, 18-fold, respectively) after Aeromonas hydrophila (A. hydrophila) injection, and then decreased to the basal value within 24 h which suggested that IL-6 was involved in the immune response to A. hydrophila. The cloning and expression analysis of the IL-6 provide theoretical basis to further study the mechanism of anti-adverseness and expression characteristics under stress conditions in blunt snout bream. PMID:26965748

  9. Embedding mRNA Stability in Correlation Analysis of Time-Series Gene Expression Data

    PubMed Central

    Farina, Lorenzo; De Santis, Alberto; Salvucci, Samanta; Morelli, Giorgio; Ruberti, Ida

    2008-01-01

    Current methods for the identification of putatively co-regulated genes directly from gene expression time profiles are based on the similarity of the time profile. Such association metrics, despite their central role in gene network inference and machine learning, have largely ignored the impact of dynamics or variation in mRNA stability. Here we introduce a simple, but powerful, new similarity metric called lead-lag R2 that successfully accounts for the properties of gene dynamics, including varying mRNA degradation and delays. Using yeast cell-cycle time-series gene expression data, we demonstrate that the predictive power of lead-lag R2 for the identification of co-regulated genes is significantly higher than that of standard similarity measures, thus allowing the selection of a large number of entirely new putatively co-regulated genes. Furthermore, the lead-lag metric can also be used to uncover the relationship between gene expression time-series and the dynamics of formation of multiple protein complexes. Remarkably, we found a high lead-lag R2 value among genes coding for a transient complex. PMID:18670596

  10. Lesion of the substantia nigra pars compacta downregulates striatal glutamate receptor subunit mRNA expression.

    PubMed

    Fan, X D; Li, X M; Ashe, P C; Juorio, A V

    1999-12-11

    This is a study of the effect of the unilateral administration of dopamine (DA) in the pars compacta of the substantia nigra (SN) of the rat on striatal glutamate receptor subunit (GluR1, GluR2 and NMDAR1) gene expression determined by in situ hybridization. The location of the nigral lesion was determined by tyrosine hydroxylase (TH) immunohistochemistry and its extent by the striatal DA and 3,4-dihydroxyphenylacetic acid (DOPAC) concentrations. The DA-induced lesions produce significant bilateral reductions in the expression of GluR1 and NMDAR1 subunit mRNA in the medio-lateral striatum, whereas the expression of striatal GluR2 receptors was not changed. The reduction in GluR1 and NMDAR1 subunit mRNA may be the consequence of glutamatergic hyperactivity developed in the presence of a damaged nigro-striatal system and these may be associated with the genesis of some neurodegenerative diseases. PMID:10629751

  11. Down-regulated expression of transforming growth factor beta 1 mRNA in endometrial carcinoma.

    PubMed Central

    Perlino, E.; Loverro, G.; Maiorano, E.; Giannini, T.; Cazzolla, A.; Napoli, A.; Fiore, M. G.; Ricco, R.; Marra, E.; Selvaggi, L.

    1998-01-01

    Transforming growth factor beta1 (TGF-beta1) is a potent modulator of cell proliferation in vitro, and recent studies have demonstrated its overexpression in several different tumours; nevertheless, the molecular mechanisms of TGF-beta1 action on cell growth and differentiation have not been fully elucidated. To clarify the role of TGF-beta and its receptor in human endometrial proliferation and differentiation, TGF-beta1 expression at both the mRNA and protein levels has been evaluated by using Northern blotting and immunohistochemistry, in both normal (atrophic, proliferative and secretory) and neoplastic (adenocarcinoma) endometrial samples. This study demonstrates that TGF-beta1 mRNA expression is dramatically reduced in endometrial carcinomas with respect to non-neoplastic tissues, whereas the immunohistochemical expression of TGF-beta1 is enhanced in the epithelial component of endometrial carcinomas compared with non-neoplastic tissues. These data suggest that TGF-beta1 acts as a paracrine regulator of endometrial cell proliferation and that it may contribute to the carcinogenic mechanisms of endometrial carcinoma. Images Figure 1 Figure 5 Figure 6 Figure 8 PMID:9579831

  12. Down-regulated expression of transforming growth factor beta 1 mRNA in endometrial carcinoma.

    PubMed

    Perlino, E; Loverro, G; Maiorano, E; Giannini, T; Cazzolla, A; Napoli, A; Fiore, M G; Ricco, R; Marra, E; Selvaggi, L

    1998-04-01

    Transforming growth factor beta1 (TGF-beta1) is a potent modulator of cell proliferation in vitro, and recent studies have demonstrated its overexpression in several different tumours; nevertheless, the molecular mechanisms of TGF-beta1 action on cell growth and differentiation have not been fully elucidated. To clarify the role of TGF-beta and its receptor in human endometrial proliferation and differentiation, TGF-beta1 expression at both the mRNA and protein levels has been evaluated by using Northern blotting and immunohistochemistry, in both normal (atrophic, proliferative and secretory) and neoplastic (adenocarcinoma) endometrial samples. This study demonstrates that TGF-beta1 mRNA expression is dramatically reduced in endometrial carcinomas with respect to non-neoplastic tissues, whereas the immunohistochemical expression of TGF-beta1 is enhanced in the epithelial component of endometrial carcinomas compared with non-neoplastic tissues. These data suggest that TGF-beta1 acts as a paracrine regulator of endometrial cell proliferation and that it may contribute to the carcinogenic mechanisms of endometrial carcinoma. PMID:9579831

  13. 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

  14. Attenuated mRNA expression of inflammatory mediators in neonatal rat lung following lipopolysaccharide treatment

    PubMed Central

    Le Rouzic, Valerie; Wiedinger, Kari; Zhou, Heping

    2012-01-01

    Neonates are known to exhibit increased susceptibility to bacterial and viral infections and increasing evidence demonstrates that the increased susceptibility is related to their attenuated immune response to infections. The lung is equipped with an innate defense system involving both cellular and humoral mediators. The present study was performed to characterize the expression of inflammatory mediators in the lung of neonatal rats in comparison with older animals. Rats at postnatal day 1 (P1), P21, and P70 were treated with saline or 0.25 mg/kg lipopolysaccharide (LPS) via intraperitoneal injection. Two hours later, animals were sacrificed and the transcriptional response of key inflammatory mediators and enzyme activity of myeloperoxidase (MPO) in the lung of these animals were examined. LPS-induced messenger RNA (mRNA) expression of pro-inflammatory cytokines, namely interleukin (IL)-1β, IL-6, and tumor necrosis factor-α, antiinflammatory cytokines, namely IL-10 and IL-1 receptor antagonist (IL-1ra), and chemokines, namely macrophage inflammatory protein (MIP)-1β, MIP-2, and monocyte chemotactic protein-1, in P1 lung was much reduced compared to that in P21 and P70 animals at 2 hours postinjection. These data suggest that LPS-induced transcriptional response of cytokines and chemokines was much reduced in P1 lung even though the protein levels of these genes were not ascertained and mRNA levels of these genes may not reflect their final protein levels. MPO activity in LPS-treated P1 lung was also significantly attenuated compared to that in LPS-treated P70 lung, suggesting impaired neutrophil infiltration in P1 lung at 2 hours following LPS treatment. In parallel, the baseline mRNA expression of LPS-binding protein (LBP) in P1 lung was much lower than that in P21 and P70 lungs. While the protein level of LBP was not examined and the mRNA level of LBP may not reflect its final protein level, the reduced transcriptional response of cytokines and chemokines in

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

    PubMed Central

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

    2016-01-01

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

  16. Dynamic expression of tyrosine hydroxylase mRNA and protein in neurons of the striatum and amygdala of mice, and experimental evidence of their multiple embryonic origin.

    PubMed

    Bupesh, Munisamy; Vicario, Alba; Abellán, Antonio; Desfilis, Ester; Medina, Loreta

    2014-05-01

    Emotional and motivational dysfunctions observed in Parkinson's disease, schizophrenia, and drug addiction are associated to an alteration of the mesocortical and mesolimbic dopaminergic pathways, which include axons projecting to the prefrontal cortex, the ventral striatum, and the amygdala. Subpopulations of catecholaminergic neurons have been described in the cortex and striatum of several mammals, but the presence of such cells in the adult amygdala is unclear in murine rodents, and in other rodents appears to show variations depending on the species. Moreover, the embryonic origin of telencephalic tyrosine hydroxylase (TH) cells is unknown, which is essential for trying to understand aspects of their evolution, distribution and function. Herein we investigated the expression of TH mRNA and protein in cells of the striatum and amygdala of developing and adult mice, and analyzed the embryonic origin of such cells using in vitro migration assays. Our results showed the presence of TH mRNA and protein expressing cells in the striatum (including nucleus accumbens), central and medial extended amygdala during development, which are persistent in adulthood although they are less numerous, generally show weak mRNA expression, and some appear to lack the protein. Fate mapping analysis showed that these cells include at least two subpopulations with different embryonic origin in either the commissural preoptic area of the subpallium or the supraopto-paraventricular domain of the alar hypothalamus. These data are important for future studies trying to understand the role of catecholamines in modulation of emotion, motivation, and reward. PMID:23479178

  17. Electroacupuncture Enhances Preproenkephalin mRNA Expression in Rostral Ventrolateral Medulla of Rats

    PubMed Central

    Li, Min; Tjen-A-Looi, Stephanie C.; Longhurst, John C.

    2010-01-01

    Electroacupuncture (EA) causes prolonged suppression of reflex elevations in blood pressure for at least 60 minutes in anesthetized preparations. Thus, EA can modify sympathetic outflow and elevated blood pressure through actions in a number of hind brain regions, including the rostral ventrolateral medulla (rVLM). Since our previous data show that the opioid system plays a role in EA-related prolonged inhibition of presympathetic neuronal activity in the rVLM, we postulated that EA increases preproenkephalin (PPE) mRNA in this region, possibly for prolonged periods of time. Under α–chloralose anesthesia, rats received EA (1-2 mA, 2 Hz, 0.5 ms) at P5-P6 acupoints (overlying median nerves) or sham (needle placement without electrical stimulation) for 30 min. PPE mRNA in the rVLM also was evaluated in control rats that received surgery but no EA or sham treatment. 20 min, 1.5 h or 4 h following EA or sham treatment, PPE mRNA in the rVLM was analyzed by reverse transcription and quantitative real-time PCR. Relative ratios of PPE mRNA levels (normalized with 18s house keeping gene) were increased 1.5 h after EA stimulation (7.77 ± 1.39, n=6) relative to sham (2.84 ± 0.37, n=5) but were unchanged both 20 min and 4 h after EA, compared to the sham or surgery groups at the same time points. Thus, 30 min of EA transiently stimulates the production of enkephalin in a region of the brain that importantly regulates sympathetic outflow suggesting that even a single brief acupuncture treatment can increase the expression of this modulatory neuropeptide. PMID:20399834

  18. IPLA2 mRNA expression by human neutrophils in type 2 diabetes and chronic periodontitis.

    PubMed

    Ayilavarapu, Srinivas; Kantarci, Alpdogan; Hasturk, Hatice; Van Dyke, Thomas E

    2014-10-01

    Type 2 diabetes mellitus (T2D) is becoming increasingly prevalent worldwide and complications of T2D cause significant systemic and dental morbidity in the susceptible individual. Although T2D has been linked as a significant risk factor for chronic periodontitis (CP), molecular mechanisms explaining the pathogenesis and inflammatory impact of CP in T2D are lacking. iPLA2 is the calcium-independent form of phospholipase A2. In previous studies, we demonstrated that iPLA2 enzyme activity is altered in T2D. The purpose of this study was to elucidate the level of the iPLA2 abnormality in T2D by measuring messenger RNA levels in T2D-associated CP. A total of 53 healthy and T2D subjects with CP were recruited for this study. The clinical periodontal exam included probing pocket depth, clinical attachment levels and bleeding on probing. Peripheral venous blood was collected and neutrophils were isolated. Real time polymerase chain reaction was used to quantify iPLA2 mRNA in neutrophils from healthy controls and people with diabetes. Results revealed that the prevalence of moderate to severe CP was increased in people with T2D. The iPLA, mRNA levels in diabetics with different severity of CP were not significantly different compared to healthy controls; 1.07 vs 0.97 (mild CP), 1.07 vs 0.85 (moderate CP) and 1.07 vs 1.05 (severe CP). Collectively, the data suggest that levels of iPLA2 mRNA in T2D are not different than in health and are not directly influenced by periodontal disease status. The impact of inflammation on iPLA2 regulation is at the level of activation of the enzyme rather than expression at the mRNA level. PMID:25654966

  19. 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. PMID:19733250

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

    PubMed

    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. 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

  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. 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

  4. Short-lived recombinant adeno-associated virus transgene expression in dystrophic muscle is associated with oxidative damage to transgene mRNA

    PubMed Central

    Dupont, Jean-Baptiste; Tournaire, Benoit; Georger, Christophe; Marolleau, Béatrice; Jeanson-Leh, Laurence; Ledevin, Mireille; Lindenbaum, Pierre; Lecomte, Emilie; Cogné, Benjamin; Dubreil, Laurence; Larcher, Thibaut; Gjata, Bernard; Van Wittenberghe, Laetitia; Le Guiner, Caroline; Penaud-Budloo, Magalie; Snyder, Richard O; Moullier, Philippe; Léger, Adrien

    2015-01-01

    Preclinical gene therapy strategies using recombinant adeno-associated virus (AAV) vectors in animal models of Duchenne muscular dystrophy have shown dramatic phenotype improvements, but long-lasting efficacy remains questionable. It is believed that in dystrophic muscles, transgene persistence is hampered, notably by the progressive loss of therapeutic vector genomes resulting from muscle fibers degeneration. Intracellular metabolic perturbations resulting from dystrophin deficiency could also be additional factors impacting on rAAV genomes and transgene mRNA molecular fate. In this study, we showed that rAAV genome loss is not the only cause of reduced transgene mRNA level and we assessed the contribution of transcriptional and post-transcriptional factors. We ruled out the implication of transgene silencing by epigenetic mechanisms and demonstrated that rAAV inhibition occurred mostly at the post-transcriptional level. Since Duchenne muscular dystrophy (DMD) physiopathology involves an elevated oxidative stress, we hypothesized that in dystrophic muscles, transgene mRNA could be damaged by oxidative stress. In the mouse and dog dystrophic models, we found that rAAV-derived mRNA oxidation was increased. Interestingly, when a high expression level of a therapeutic transgene is achieved, oxidation is less pronounced. These findings provide new insights into rAAV transductions in dystrophic muscles, which ultimately may help in the design of more effective clinical trials. PMID:26029721

  5. Effect of radiofrequency radiation on MRNA expression in cultured rodent cells

    SciTech Connect

    Parker, J.E.; Kiel, J.L.; Winters, W.D.

    1988-01-01

    Radiofrequency radiation (RFR) has been reported to induce adverse effects in biological systems, such as teratogenic and embryo lethal effects in mammals particularly during exposures producing significant hyperthermia. Other studies have implicated microwave exposure with causing changes in chromosome number and structure, formation of cataracts in humans rabbits and dogs; and promoting malignant tumor formation in rats, as well as increasing tumor production and leukemias. In addition, microwave exposures have been reported to change the structure of purified double-stranded plasmid DNA, causing it to become nicked and increasing the proportion of relaxed to super coiled molecules. In view of these reports of changes at different levels of cellular function and structure of mammalian systems to microwaves, the authors asked themselves if changes at the level of mRNA expression could be detected after microwave exposure of cultured rodent cells. They chose to look at the mRNA expression of certain oncogenes known to show elevated levels during cell replication, at the heat shock proteins known to respond to stresses other than heat, and at the long terminal repeat (LTR) region of mouse mammary tumor virus in four rodent cell lines.

  6. Circadian control of mRNA polyadenylation dynamics regulates rhythmic protein expression

    PubMed Central

    Kojima, Shihoko; Sher-Chen, Elaine L.; Green, Carla B.

    2012-01-01

    Poly(A) tails are 3′ modifications of eukaryotic mRNAs that are important in the control of translation and mRNA stability. We identified hundreds of mouse liver mRNAs that exhibit robust circadian rhythms in the length of their poly(A) tails. Approximately 80% of these are primarily the result of nuclear adenylation coupled with rhythmic transcription. However, unique decay kinetics distinguish these mRNAs from other mRNAs that are transcribed rhythmically but do not exhibit poly(A) tail rhythms. The remaining 20% are uncoupled from transcription and exhibit poly(A) tail rhythms even though the steady-state mRNA levels are not rhythmic. These are under the control of rhythmic cytoplasmic polyadenylation, regulated at least in some cases by cytoplasmic polyadenylation element-binding proteins (CPEBs). Importantly, we found that the rhythmicity in poly(A) tail length is closely correlated with rhythmic protein expression, with a several-hour delay between the time of longest tail and the time of highest protein level. Our study demonstrates that the circadian clock regulates the dynamic polyadenylation status of mRNAs, which can result in rhythmic protein expression independent of the steady-state levels of the message. PMID:23249735

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

    PubMed Central

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

    2012-01-01

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

  8. 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

  9. IONOTROPIC GLUTAMATE RECEPTORS mRNA EXPRESSION IN THE HUMAN THALAMUS: ABSENCE OF CHANGE IN SCHIZOPHRENIA

    PubMed Central

    Dracheva, Stella; Byne, William; Chin, Benjamin; Haroutunian, Vahram

    2009-01-01

    Abnormalities in glutamate neurotransmission are thought to be among the major contributing factors to the pathophysiology of schizophrenia. Although schizophrenia has been regarded mostly as a disorder of higher cortical function, the cortex and thalamus work as a functional unit. Existing data regarding alterations of glutamate receptor subunit expression in the thalamus in schizophrenia remain equivocal. This postmortem study examined mRNA expression of ionotropic glutamate receptor (iGluR) subunits and PSD95 in 5 precisely defined and dissected thalamic subdivisions (medial and lateral sectors of the mediodorsal nucleus; and the ventrolateral posterior, ventral posterior, and centromedian nuclei) of persons with schizophrenia and matched controls using quantitative PCR with normalization to multiple endogenous controls. Among 15 genes examined (NR1 and NR2A-D subunits of NMDA receptor; GluR1-4 subunits of AMPA receptor; GluR5-7 and KA1-2 subunits of kainate receptor; PSD95), all but two (GluR4 and KA1) were expressed at quantifiable levels. Differences in iGluR gene expression were seen between different nuclei but not between diagnostic groups. The relative abundance of transcripts was: NR1≫NR2A>NR2B>NR2D>NR2C for NMDA, GluR2>GluR1>GluR3 for AMPA, and KA2>GluR5>GluR7>GluR6 for kainate receptors. The expression of PSD95 correlated with the expression of NR1, NR2A, NR2B, NR2D and GluR6 in all nuclei. These results provide detailed and quantitative information on iGluR subunit expression in multiple nuclei of the human thalamus but suggest that alterations in their expression are not a prominent feature of schizophrenia. PMID:18462708

  10. A distinct ERCC1 haplotype is associated with mRNA expression levels in prostate cancer patients.

    PubMed

    Woelfelschneider, Andreas; Popanda, Odilia; Lilla, Carmen; Linseisen, Jakob; Mayer, Claudia; Celebi, Oktay; Debus, Jürgen; Bartsch, Helmut; Chang-Claude, Jenny; Schmezer, Peter

    2008-09-01

    Both genetic variants and messenger RNA (mRNA) expression of DNA repair and tumor suppressor genes have been investigated as molecular markers for therapy outcome. However, the phenotypic impact of genetic variants often remained unclear, thus the rationale of their use in risk prediction may be limited. We therefore analyzed genetic variants together with anthropometric and lifestyle factors to see how these affect mRNA levels of ERCC1, MDM2 and TP53 in primary blood lymphocytes. mRNA expression was measured in 376 prostate cancer patients by quantitative real-time polymerase chain reaction after reverse transcription, and ERCC1 rs11615 T>C, ERCC1 rs3212986 C>A, MDM2 rs2279744 T>G and TP53 rs17878362 (p53PIN3) polymorphisms were determined. Considerable interindividual differences in mRNA expression were found (coefficients of variation: ERCC1, 45%; MDM2, 43% and TP53, 35%). ERCC1 expression was positively correlated with plasma levels of beta-carotene (P = 0.03) and negatively correlated with canthaxanthin (P = 0.02) and lutein (P = 0.02). Overall, the polymorphisms affected mRNA expression only weakly. Carriers of a distinct ERCC1 haplotype (CC) showed, however, significantly lower expression values than non-carriers (P = 0.001). Applying logistic regression, we found that CC haplotype carriers had a 1.69-fold increased odds ratio (95% confidence interval: 1.06-2.71) for reduced ERCC1 mRNA levels. This low ERCC1 expression might be associated with reduced DNA repair and better therapy response. In summary, the association we have found between ERCC1 genotype and mRNA expression supports recent clinical observations that genetic variation in ERCC1 can affect treatment outcome and prognosis. Our study further revealed a modulating effect by nutritional factors. PMID:18332046

  11. 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

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

    PubMed

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

    1999-05-01

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

  13. Antagonistic regulation of mRNA expression and splicing by CELF and MBNL proteins

    PubMed Central

    Wang, Eric T.; Ward, Amanda J.; Cherone, Jennifer M.; Giudice, Jimena; Wang, Thomas T.; Treacy, Daniel J.; Lambert, Nicole J.; Freese, Peter; Saxena, Tanvi; Cooper, Thomas A.; Burge, Christopher B.

    2015-01-01

    RNA binding proteins of the conserved CUGBP1, Elav-like factor (CELF) family contribute to heart and skeletal muscle development and are implicated in myotonic dystrophy (DM). To understand their genome-wide functions, we analyzed the transcriptome dynamics following induction of CELF1 or CELF2 in adult mouse heart and of CELF1 in muscle by RNA-seq, complemented by crosslinking/immunoprecipitation-sequencing (CLIP-seq) analysis of mouse cells and tissues to distinguish direct from indirect regulatory targets. We identified hundreds of mRNAs bound in their 3′ UTRs by both CELF1 and the developmentally induced MBNL1 protein, a threefold greater overlap in target messages than expected, including messages involved in development and cell differentiation. The extent of 3′ UTR binding by CELF1 and MBNL1 predicted the degree of mRNA repression or stabilization, respectively, following CELF1 induction. However, CELF1's RNA binding specificity in vitro was not detectably altered by coincubation with recombinant MBNL1. These findings support a model in which CELF and MBNL proteins bind independently to mRNAs but functionally compete to specify down-regulation or localization/stabilization, respectively, of hundreds of mRNA targets. Expression of many alternative 3′ UTR isoforms was altered following CELF1 induction, with 3′ UTR binding associated with down-regulation of isoforms and genes. The splicing of hundreds of alternative exons was oppositely regulated by these proteins, confirming an additional layer of regulatory antagonism previously observed in a handful of cases. The regulatory relationships between CELFs and MBNLs in control of both mRNA abundance and splicing appear to have evolved to enhance developmental transitions in major classes of heart and muscle genes. PMID:25883322

  14. KIF14 mRNA expression is a predictor of grade and outcome in breast cancer.

    PubMed

    Corson, Timothy W; Gallie, Brenda L

    2006-09-01

    Gain of chromosome 1q is a hallmark of breast cancer, and likely reflects oncogene amplification. We previously identified mitotic kinesin KIF14 (kinesin family member 14) as an overexpressed candidate oncogene in the 1q31.3-1q32.1 minimal region of genomic gain in breast cancer cell lines. KIF14 also showed high expression in other cancers, notably an association with survival in lung tumors. We now report KIF14 expression in 99 primary breast tumors and 10 normal breast controls. Measured by real-time RT-PCR, KIF14 was overexpressed 10-fold on average in tumors relative to normals (t test p = 0.000054); expression increased with grade (ANOVA p = 0.000006). Infiltrating ductal carcinomas had higher KIF14 levels than lobular (p = 0.017), and estrogen receptor (ER) negative tumors had higher KIF14 levels than ER positive tumors (t test p = 0.030). KIF14 expression correlated positively with Ki-67 mRNA level (Spearman r = 0.692, p = 0.000001), fraction of positive nodes (r = 0.227, p = 0.024) and percent invasive cells (r = 0.360, p = 0.0002), and negatively with percent fatty stroma (r = -0.258, p = 0.010) and percent normal epithelium (r = -0.291, p = 0.003). KIF14 expression is thus tumor-specific and increased in more aggressive tumors. Indeed, KIF14 expression predicted overall survival (univariate Cox p = 0.010), with an odds ratio of 3.60 (1.37-9.48), in 50 tumors with available outcome data. KIF14 overexpression also predicted decreased disease-free survival (log-rank p = 0.049). These findings are the first evidence of association between expression of a mitotic kinesin and prognostic variables in breast cancer. PMID:16570270

  15. mRNA expression levels of hypoxia-induced and stem cell-associated genes in human glioblastoma.

    PubMed

    Bache, Matthias; Rot, Swetlana; Keßler, Jacqueline; Güttler, Antje; Wichmann, Henri; Greither, Thomas; Wach, Sven; Taubert, Helge; Söling, Ariane; Bilkenroth, Udo; Kappler, Matthias; Vordermark, Dirk

    2015-06-01

    The roles of hypoxia-induced and stem cell-associated genes in the development of malignancy and tumour progression are well known. However, there are a limited number of studies analysing the impact of mRNA expression levels of hypoxia-induced and stem cell-associated genes in the tissues of brain tumours and glioblastoma patients. In this study, tumour tissues from patients with glioblastoma multiforme and tumour adjacent tissues were analysed. We investigated mRNA expression levels of hypoxia-inducible factor-1α (HIF-1α), hypoxia-inducible factor-2α (HIF-2α), carbonic anhydrase 9 (CA9), vascular endothelial growth factor (VEGF), glucose transporter-1 (GLUT-1) and osteopontin (OPN), and stem cell-associated genes survivin, epidermal growth factor receptor (EGFR), human telomerase reverse transcriptase (hTERT), Nanog and octamer binding transcription factor 4 (OCT4) using quantitative real-time polymerase chain reaction (qRT-PCR). Our data revealed higher mRNA expression levels of hypoxia-induced and stem cell-associated genes in tumour tissue than levels in the tumour adjacent tissues in patients with glioblastoma multiforme. A strong positive correlation between the mRNA expression levels of HIF-2α, CA9, VEGF, GLUT-1 and OPN suggests a specific hypoxia-associated profile of mRNA expression in glioblastoma multiforme. Additionally, the results indicate the role of stem-cell-related genes in tumour hypoxia. Kaplan-Maier analysis revealed that high mRNA expression levels of hypoxia-induced markers showed a trend towards shorter overall survival in glioblastoma patients (P=0.061). Our data suggest that mRNA expression levels of hypoxia-induced genes are important tumour markers in patients with glioblastoma multiforme. PMID:25963717

  16. Adverse early life experience and social stress during adulthood interact to increase serotonin transporter mRNA expression

    PubMed Central

    Gardner, Katherine L.; Hale, Matthew W.; Lightman, Stafford L.; Plotsky, Paul M.; Lowry, Christopher A.

    2009-01-01

    Anxiety disorders, depression and animal models of vulnerability to a depression-like syndrome have been associated with dysregulation of serotonergic systems in the brain. To evaluate the effects of early life experience, adverse experiences during adulthood, and potential interactions between these factors on serotonin transporter (slc6a4) mRNA expression, we investigated in rats the effects of maternal separation (180 min/day from days 2–14 of life; MS180), neonatal handing (15 min/day from days 2–14 of life; MS15), or normal animal facility rearing control conditions (AFR) with or without subsequent exposure to adult social defeat on slc6a4 mRNA expression in the dorsal raphe nucleus (DR) and caudal linear nucleus. At the level of specific subdivisions of the DR, there were no differences in slc6a4 mRNA expression between MS15 and AFR rats. Among rats exposed to a novel cage control condition, increased slc6a4 mRNA expression was observed in the dorsal part of the DR in MS180 rats, relative to AFR control rats. In contrast, MS180 rats exposed to social defeat as adults had increased slc6a4 mRNA expression throughout the DR compared to both MS15 and AFR controls. Social defeat increased slc6a4 mRNA expression, but only in MS180 rats and only in the “lateral wings” of the DR. Overall these data demonstrate that early life experience and stressful experience during adulthood interact to determine slc6a4 mRNA expression. These data support the hypothesis that early life experience and major stressful life events contribute to dysregulation of serotonergic systems in stress-related neuropsychiatric disorders. PMID:19781533

  17. Helicobacter pylori cag Pathogenicity Island Is Associated with Reduced Expression of Interleukin-4 (IL-4) mRNA and Modulation of the IL-4δ2 mRNA Isoform in Human Gastric Mucosa

    PubMed Central

    Orsini, Barbara; Ottanelli, Barbara; Amedei, Amedeo; Surrenti, Elisabetta; Capanni, Marco; Del Prete, Gianfranco; Amorosi, Andrea; Milani, Stefano; D'Elios, Mario Milco; Surrenti, Calogero

    2003-01-01

    Interleukin-4 (IL-4) and IL-4δ2 mRNA gastric expression was evaluated in healthy subjects and patients who did not have ulcers but were infected with Helicobacter pylori with or without the cag pathogenicity island (cag PAI). IL-4 mRNA was physiologically expressed by gastric epithelium and negatively influenced by H. pylori. Also, nonepithelial cells in the lamina propria of H. pylori-infected patients expressed IL-4 mRNA, whereas IL-4δ2 mRNA was found only in cag PAI-negative patients. Thus, gastric IL-4 takes part in the local immune response to H. pylori. PMID:14573693

  18. Altered expression of mRNA profiles in blood of early-onset schizophrenia

    PubMed Central

    Xu, Yong; Yao Shugart, Yin; Wang, Guoqiang; Cheng, Zaohuo; Jin, Chunhui; Zhang, Kai; Wang, Jun; Yu, Hao; Yue, Weihua; Zhang, Fuquan; Zhang, Dai

    2016-01-01

    To identify gene expression abnormalities in schizophrenia (SZ), we generated whole-genome gene expression profiles using microarrays on peripheral blood mononuclear cells (PBMCs) from 18 early-onset SZ cases and 12 controls. We detected 84 transcripts differentially expressed by diagnostic status, with 82 genes being upregulated and 2 downregulated. We identified two SZ associated gene coexpression modules (green and red), including 446 genes . The green module is positively correlated with SZ, encompassing predominantly up-regulated genes in SZ; while the red module was negatively correlated with disease status, involving mostly nominally down-regulated genes in SZ. The olfactory transduction pathway was the most enriched pathways for the genes within the two modules. The expression levels of several hub genes, including AKT1, BRCA1, CCDC134, UBD, and ZIC2 were validated using real-time quantitative PCR. Our findings indicate that mRNA coexpression abnormalities may serve as a promising mechanism underlying the development of SZ. PMID:26733343

  19. Amphetamine-induced c-fos mRNA expression is altered in rats with neonatal ventral hippocampal damage.

    PubMed

    Lillrank, S M; Lipska, B K; Bachus, S E; Wood, G K; Weinberger, D R

    1996-08-01

    To further characterize the mechanisms underlying enhanced dopamine-related behaviors expressed during adulthood in rats with neonatal excitotoxic ventral hippocampal (VH) damage, we studied the expression of c-fos mRNA in these rats after a single saline or amphetamine (AMPH) (10 mg/kg, i.p.) injection using in situ hybridization. The VH of rat pups was lesioned with ibotenic acid on postnatal day 7 (PD7). At the age of 90 days, rats were challenged with AMPH or saline, and the expression of c-fos mRNA using an oligonucleotide probe was assessed 30, 90, and 180 min later. AMPH significantly increased c-fos mRNA expression in medial prefrontal cortex, piriform cortex, cingulate cortex, septal region, and dorsolateral and ventromedial striatum in control and lesioned rats. However, this response to AMPH was attenuated 30 min after AMPH injection in all of these regions in the lesioned as compared to the sham-operated rats. No significant changes were seen at other time points. These results indicate that the neonatal VH lesion alters time-dependent intracellular signal transduction mechanisms measured by AMPH-induced c-fos mRNA expression in cortical and subcortical brain regions. Changes in c-fos mRNA expression in this putative animal model of schizophrenia may have implications for long-term alterations in cellular phenotype because of altered regulation of certain target genes. PMID:8855514

  20. Enhancement of the in vivo persistence and antitumor efficacy of CD19 chimeric antigen receptor T cells through the delivery of modified TERT mRNA

    PubMed Central

    Bai, Yun; Kan, Shifeng; Zhou, Shixin; Wang, Yuting; Xu, Jun; Cooke, John P; Wen, Jinhua; Deng, Hongkui

    2015-01-01

    Chimeric antigen receptor T cell immunotherapy is a promising therapeutic strategy for treating tumors, demonstrating its efficiency in eliminating several hematological malignancies in recent years. However, a major obstacle associated with current chimeric antigen receptor T cell immunotherapy is that the limited replicative lifespan of chimeric antigen receptor T cells prohibits the long-term persistence and expansion of these cells in vivo, potentially hindering the long-term therapeutic effects of chimeric antigen receptor T cell immunotherapy. Here we showed that the transient delivery of modified mRNA encoding telomerase reverse transcriptase to human chimeric antigen receptor T cells targeting the CD19 antigen (CD19 chimeric antigen receptor T cells) would transiently elevate the telomerase activity in these cells, leading to increased proliferation and delayed replicative senescence without risk of insertion mutagenesis or immortalization. Importantly, compared to conventional CD19 chimeric antigen receptor T cells, after the transient delivery of telomerase reverse transcriptase mRNA, these CD19 chimeric antigen receptor T cells showed improved persistence and proliferation in mouse xenograft tumor models of human B-cell malignancies. Furthermore, the transfer of CD19 chimeric antigen receptor T cells after the transient delivery of telomerase reverse transcriptase mRNA enhanced long-term antitumor effects in mouse xenograft tumor models compared with conventional CD19 chimeric antigen receptor T cell transfer. The results of the present study provide an effective and safe method to improve the therapeutic potential of chimeric antigen receptor T cells, which might be beneficial for treating other types of cancer, particularly solid tumors. PMID:27462436

  1. Expression of preproenkephalin-like mRNA and its peptide products in mammalian testis and ovary.

    PubMed Central

    Kilpatrick, D L; Howells, R D; Noe, M; Bailey, L C; Udenfriend, S

    1985-01-01

    The distribution of preproenkephalin mRNA and proenkephalin-derived peptides have been examined in gonadal tissues from rats, hamsters, and cattle. A preproenkephalin mRNA band was detected in the ovaries of all three species and in hamster testis that is identical in size to the 1450-nucleotide mRNA typically found in tissues that express proenkephalin. Rat testis, on the other hand, expresses at least one preproenkephalin-like mRNA that is substantially greater in size (1900 nucleotides). [Met]enkephalin-containing peptides were also detected in each of the gonadal tissues examined. Although the abundance of preproenkephalin-like mRNA in rat testis was comparable to that in rat brain, the testicular content of proenkephalin-derived [Met]enkephalin sequences was less than 4% of the rat brain content. Together these data suggest that preproenkephalin-like mRNA in rat testis is not efficiently translated, proenkephalin-derived peptides undergo rapid turnover in this tissue, or the mRNA in rat testis has a frameshift resulting in an altered coding sequence. Images PMID:3864164

  2. 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

  3. Exposure to ionizing radiation induced persistent gene expression changes in mouse mammary gland

    PubMed Central

    2012-01-01

    Background Breast tissue is among the most sensitive tissues to the carcinogenic actions of ionizing radiation and epidemiological studies have linked radiation exposure to breast cancer. Currently, molecular understanding of radiation carcinogenesis in mammary gland is hindered due to the scarcity of in vivo long-term follow up data. We undertook this study to delineate radiation-induced persistent alterations in gene expression in mouse mammary glands 2-month after radiation exposure. Methods Six to eight week old female C57BL/6J mice were exposed to 2 Gy of whole body γ radiation and mammary glands were surgically removed 2-month after radiation. RNA was isolated and microarray hybridization performed for gene expression analysis. Ingenuity Pathway Analysis (IPA) was used for biological interpretation of microarray data. Real time quantitative PCR was performed on selected genes to confirm the microarray data. Results Compared to untreated controls, the mRNA levels of a total of 737 genes were significantly (p<0.05) perturbed above 2-fold of control. More genes (493 genes; 67%) were upregulated than the number of downregulated genes (244 genes; 33%). Functional analysis of the upregulated genes mapped to cell proliferation and cancer related canonical pathways such as ‘ERK/MAPK signaling’, ‘CDK5 signaling’, and ‘14-3-3-mediated signaling’. We also observed upregulation of breast cancer related canonical pathways such as ‘breast cancer regulation by Stathmin1’, and ‘HER-2 signaling in breast cancer’ in IPA. Interestingly, the downregulated genes mapped to fewer canonical pathways involved in cell proliferation. We also observed that a number of genes with tumor suppressor function (GPRC5A, ELF1, NAB2, Sema4D, ACPP, MAP2, RUNX1) persistently remained downregulated in response to radiation exposure. Results from qRT-PCR on five selected differentially expressed genes confirmed microarray data. The PCR data on PPP4c, ELF1, MAPK12, PLCG1, and E2F

  4. 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...

  5. Vesnarinone suppresses TNFα mRNA expression by inhibiting valosin-containing protein.

    PubMed

    Hotta, Kentaro; Nashimoto, Akihiro; Yasumura, Eiji; Suzuki, Masafumi; Azuma, Motoki; Iizumi, Yosuke; Shima, Daisuke; Nabeshima, Ryusuke; Hiramoto, Masaki; Okada, Akira; Sakata-Sogawa, Kumiko; Tokunaga, Makio; Ito, Takumi; Ando, Hideki; Sakamoto, Satoshi; Kabe, Yasuaki; Aizawa, Shinichi; Imai, Takeshi; Yamaguchi, Yuki; Watanabe, Hajime; Handa, Hiroshi

    2013-05-01

    Vesnarinone is a synthetic quinolinone derivative used in the treatment of cardiac failure and cancer. It is also known to cause agranulocytosis as a side effect, which restricts its use, although the mechanism underlying agranulocytosis is not well understood. Here, we show that vesnarinone binds to valosin-containing protein (VCP), which interacts with polyubiquitinated proteins and is essential for the degradation of IκBα to activate nuclear factor (NF)κB. We show that vesnarinone impairs the degradation of IκBα, and that the impairment of the degradation of IκBα is the result of the inhibition of the interaction between VCP and the 26S proteasome by vesnarinone. These results suggest that vesnarinone suppresses NFκB activation by inhibiting the VCP-dependent degradation of polyubiquitinated IκBα, resulting in the suppression of tumor necrosis factor-α mRNA expression. PMID:23393163

  6. 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. PMID:25714618

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

    Yoon, Leena; Liu, Ya-Nan; Park, Hyunjin

    2015-01-01

    Abstract 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. PMID:25714618

  9. 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.

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

    PubMed Central

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

    1989-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  12. Localized expression of mRNA for phagocyte-specific chemotactic cytokines in human periodontal infections.

    PubMed Central

    Tonetti, M S; Imboden, M A; Gerber, L; Lang, N P; Laissue, J; Mueller, C

    1994-01-01

    In bacterial infections, mononuclear and polymorphonuclear phagocytes are key components of host defenses. Recent investigations have indicated that chemokines are able to recruit and activate phagocytes. In particular, interleukin-8 (IL-8) attracts polymorphonuclear leukocytes (PMNs), while monocyte chemoattractant protein-1 (MCP-1) is selective for cells of the monocyte/macrophage lineage. In this investigation, we analyzed the in situ expression of IL-8 and MCP-1 mRNAs in human periodontal infections. Specific mRNA was detected by in situ hybridization using 35S-labeled riboprobes in frozen tissue sections. Phagocytes (PMNs and macrophages) were specifically detected as elastase-positive or CD68+ cells by a three-stage immunoperoxidase technique. Results indicated that expression of phagocyte-specific cytokines was confined to selected tissue locations and, in general, paralleled phagocyte infiltration. In particular, IL-8 expression was maximal in the junctional epithelium adjacent to the infecting microorganisms; PMN infiltration was more prominent in the same area. MCP-1 was expressed in the chronic inflammatory infiltrate and along the basal layer of the oral epithelium. Cells of the monocyte/macrophage lineage were demonstrated to be present in the same areas. The observed expression pattern may be the most economic way to establish a cell-type-selective chemotactic gradient within the tissue that is able to effectively direct polymorphonuclear phagocyte migration toward the infecting microorganisms and modulate mononuclear phagocyte infiltration in the surrounding tissues. This process may optimize host defenses and contribute to containing leukocyte infiltration to the infected and inflamed area, thus limiting tissue damage. Images PMID:8063420

  13. Isolation of an mRNA binding protein homologue that is expressed in nociceptors.

    PubMed

    Eilers, Helge; Trilk, Sharon L; Lee, Sook Young; Xue, Qing; Jong, Beverly E; Moff, Irene; Levine, Jon D; Schumacher, Mark A

    2004-11-01

    The peripheral detection of painful stimuli requires the activation of small-diameter primary afferent neurons known as nociceptors. We have exploited two features of nociceptor biology, expression of the high affinity receptor for nerve growth factor (TrkA) and sensitivity to capsaicin, to isolate novel proteins using a differential display cloning scheme. A resulting approximately 4.3-kb cDNA was isolated and sequence analysis inferred a approximately 157-kDa protein containing a signal/mitochondrial targeting peptide sequence. Due to its molecular weight and significant amino acid identity with 'human leucine-rich protein 130'[leucine-rich pentatricopeptide motif containing (LRPPRC)], we termed the cDNA candidate leucine-rich protein 157 (rLRP157). Western blot analysis of HEK293 cells over-expressing the candidate cDNA showed a single protein product of similar size to that found in rat dorsal root ganglion as well as in other neuronal tissues and cell lines. Although expressed in a wide variety of tissues, in situ hybridization and immunohistochemistry in dorsal root ganglion revealed that rLRP157 expression was restricted to the small-diameter neurons. Sequence identity with previously characterized mRNA binding proteins and its subcellular localization in sensory neurons suggest that rLRP157 is associated with mitochondrial function. Moreover, the genetic basis of French-Canadian Leigh syndrome, which confers a loss of mitochondrial cytochrome c oxidase and is characterized by neurodegeneration, was recently mapped to a mutation in the LRPPRC gene. Taken together with its expression in small-diameter sensory neurons, we hypothesize that rLRP157, the rat orthologue of the human LRPPRC, may play a role in the modulation of peripheral pain transduction and serve as a novel marker for nociceptor subtypes. PMID:15525270

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

    PubMed Central

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

    2013-01-01

    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. PMID:23838326

  15. 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. PMID:12417619

  16. The angiotensin II receptor antagonist, losartan, enhances regulator of G protein signaling 2 mRNA expression in vascular smooth muscle cells of Wistar rats.

    PubMed

    Wu, Yaqiong; Nakagawa, Suguru; Takahashi, Hidenori; Kawabata, Yukari; Suzuki, Etsu; Uehara, Yoshio

    2016-05-01

    Angiotensin II (Ang II) reportedly enhances regulator of G-protein signaling 2 (RGS2), thus making a negative feedback loop for Ang II signal transduction. However, few studies have reported whether Ang II receptor (ATR) antagonists influence RGS2 mRNA expression. We investigated RGS2 mRNA expression when Ang II binding to ATR was blocked with Ang II subtype-1 receptor (AT1R) blockers using vascular smooth muscle cells from the thoracic aorta of male Wistar rats. RGS2 mRNA expression significantly increased with Ang II stimulation, and this increase was almost completely abolished by olmesartan, a potent AT1R-specific blocker. Ang II subtype-2 receptor (AT2R) was not involved in Ang II-mediated RGS expression. In contrast, the AT1R blocker, losartan, partially decreased Ang II-mediated RGS2 mRNA expression because this antagonist directly stimulated RGS2 mRNA expression in Ang II-free medium. EXP3174, which is an active metabolite of losartan, almost completely blunted Ang II-mediated RGS2 mRNA expression without direct stimulation of RGS2 mRNA expression. Moreover, pretreatment with olmesartan abolished Ang II-mediated RGS2 mRNA expression. Treatment with a protein kinase C inhibitor partially decreased losartan-mediated RGS2 mRNA expression. These results suggest that AT1R blockers inhibit RGS2 mRNA expression in response to Ang II via an AT1R-mediated mechanism. However, the AT1R blocker, losartan, behaves as a direct agonist for RGS2 mRNA expression via AT1R through protein kinase C-dependent and -independent pathways. In conclusion, losartan exhibits dual effects on RGS2 mRNA expression, and the direct upregulation of RGS2 mRNA expression may provide a new strategy for the treatment of hypertension. PMID:26763849

  17. Expression of the mRNA encoding truncated PPAR alpha does not correlate with hepatic insensitivity to peroxisome proliferators.

    PubMed

    Hanselman, J C; Vartanian, M A; Koester, B P; Gray, S A; Essenburg, A D; Rea, T J; Bisgaier, C L; Pape, M E

    2001-01-01

    Two alternatively spliced forms of human PPAR alpha mRNA, PPAR alpha1 and PPAR alpha2, have been identified. PPAR alpha1 mRNA gives rise to an active PPAR alpha protein while PPAR alpha2 mRNA gives rise to a form of PPAR which lacks the ligand-binding domain. PPAR alpha2 is unable to activate a peroxisome proliferator response element (PPRE) reporter gene construct in transient transfection assays. Both PPAR alpha1 and PPAR alpha2 mRNA are present in human liver, kidney, testes, heart, small intestine, and smooth muscle. In human liver, PPAR alpha2 mRNA abundance is approximately half that of PPAR alpha1 mRNA; a correlation analysis of PPAR alpha1 and PPAR alpha2 mRNA mass revealed an r-value of 0.75 (n = 18). Additional studies with intact liver from various species, showed that the PPAR alpha2/PPAR alpha1 mRNA ratios in rat, rabbit, and mouse liver were less than 0.10; significantly lower than the 0.3 and 0.5 ratios observed in monkey and human livers, respectively. To determine if a high PPAR alpha2/PPAR alpha1 mRNA ratio was associated with insensitivity to peroxisome proliferators, we treated human, rat, and rabbit hepatocytes with WY14643, a potent PPAR alpha activator, and measured acyl CoA oxidase (ACO) mRNA levels. Rat ACO mRNA levels increased markedly in response to WY14643 while human and rabbit hepatocytes were unresponsive. Thus, although the PPAR alpha2/PPAR alpha1 mRNA ratio is low in rabbits, this species is not responsive to peroxisome proliferators. Further studies with male and female rats, which vary significantly in their response to peroxisome proliferators, showed little difference in the ratio of PPAR alpha2/PPAR alpha1 mRNA. These data suggest that selective PPAR alpha2 mRNA expression is not the basis for differential species or gender responses to peroxisome proliferators. PMID:11269670

  18. 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. PMID:26298189

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

    PubMed Central

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

    2014-01-01

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

  20. Altered prefrontal cortical MARCKS and PPP1R9A mRNA expression in schizophrenia and bipolar disorder

    PubMed Central

    Konopaske, Glenn T.; Subburaju, Sivan; Coyle, Joseph T.; Benes, Francine M.

    2015-01-01

    Background We previously observed dendritic spine loss in the dorsolateral prefrontal cortex (DLPFC) from schizophrenia and bipolar disorder subjects. In the current study, we sought to determine if the mRNA expression of genes known to regulate the actin cytoskeleton and spines correlated with spine loss. Methods Five candidate genes were identified using previously obtained microarray data from the DLPFC from schizophrenia and control subjects. The relative mRNA expression of the genes linked to dendritic spine growth and function, i.e. IGF1R, MARCKS, PPP1R9A, PTPRF, and ARHGEF2, were assessed using quantitative real-time PCR (qRT-PCR) in the DLPFC from a second cohort including schizophrenia, bipolar disorder, and control subjects. Functional pathway analysis was conducted to determine which actin cytoskeleton-regulatory pathways the genes of interest interact with. Results MARCKS mRNA expression was increased in both schizophrenia and bipolar disorder subjects. PPP1R9A mRNA expression was increased in bipolar disorder subjects. For IGF1R, mRNA expression did not differ significantly among groups; however, it did show a significant, negative correlation with dendrite length. MARCKS and PPP1R9A mRNA expression did not correlate with spine loss, but interact with NMDA receptor signaling pathways that regulate the actin cytoskeleton and spines. Conclusions MARCKS and PPP1R9A might contribute to spine loss in schizophrenia and bipolar disorder through their interactions, possibly indirect ones, with NMDA signaling pathways that regulate spine structure and function. PMID:25757715

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

    PubMed

    Spencer, C M; Houpt, T A

    2001-09-30

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

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

    PubMed Central

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

    2009-01-01

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

  3. Impact of persistent cytomegalovirus infection on human neuroblastoma cell gene expression.

    PubMed

    Hoever, Gerold; Vogel, Jens-Uwe; Lukashenko, Polina; Hofmann, Wolf-Karsten; Komor, Martina; Doerr, Hans Wilhelm; Cinatl, Jindrich

    2005-01-14

    In a model of human neuroblastoma (NB) cell lines persistently infected with human cytomegalovirus (HCMV) we previously showed that persistent HCMV infection is associated with an increased malignant phenotype, enhanced drug resistance, and invasive properties. To gain insights into the mechanisms of increased malignancy we analyzed the global changes in cellular gene expression induced by persistent HCMV infection of human neuroblastoma cells by use of high-density oligonucleotide microarrays (HG-U133A, Affymetrix) and RT-PCR. Comparing the gene expression of different NB cell lines with persistently infected cell sub-lines revealed 11 host cell genes regulated in a similar manner throughout all infected samples. Nine of these 11 genes may contribute to the previously observed changes in malignant phenotype of persistently HCMV infected NB cells by influencing invasive growth, apoptosis, angiogenesis, and proliferation. Thus, this work provides the basis for further functional studies. PMID:15582591

  4. Integrated Expression Profiles of mRNA and miRNA in Polarized Primary Murine Microglia

    PubMed Central

    Freilich, Robert W.; Woodbury, Maya E.; Ikezu, Tsuneya

    2013-01-01

    Neuroinflammation contributes to many neurologic disorders including Alzheimer’s disease, multiple sclerosis, and stroke. Microglia is brain resident myeloid cells and have emerged as a key driver of the neuroinflammatory responses. MicroRNAs (miRNAs) provide a novel layer of gene regulation and play a critical role in regulating the inflammatory response of peripheral macrophages. However, little is known about the miRNA in inflammatory activation of microglia. To elucidate the role that miRNAs have on microglial phenotypes under classical (M1) or alternative (M2) activation under lipopolysaccharide (‘M1’-skewing) and interleukin-4 (‘M2a’-skewing) stimulation conditions, we performed microarray expression profiling and bioinformatics analysis of both mRNA and miRNA using primary cultured murine microglia. miR-689, miR-124, and miR-155 were the most strongly associated miRNAs predicted to mediate pro-inflammatory pathways and M1-like activation phenotype. miR-155, the most strongly up-regulated miRNA, regulates the signal transducer and activator of transcription 3 signaling pathway enabling the late phase response to M1-skewing stimulation. Reduced expression in miR-689 and miR-124 are associated with dis-inhibition of many canonical inflammatory pathways. miR-124, miR-711, miR-145 are the strongly associated miRNAs predicted to mediate anti-inflammatory pathways and M2-like activation phenotype. Reductions in miR-711 and miR-124 may regulate inflammatory signaling pathways and peroxisome proliferator-activated receptor-gamma pathway. miR-145 potentially regulate peripheral monocyte/macrophage differentiation and faciliate the M2-skewing phenotype. Overall, through combined miRNA and mRNA expression profiling and bioinformatics analysis we have identified six miRNAs and their putative roles in M1 and M2-skewing of microglial activation through different signaling pathways. PMID:24244499

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

    PubMed Central

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

    2014-01-01

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

  6. mRNA expression of DOK1-6 in human breast cancer

    PubMed Central

    Ghanem, Tamara; Bracken, James; Kasem, Abdul; Jiang, Wen G; Mokbel, Kefah

    2014-01-01

    AIM: To examine the expression of downstream of tyrosine kinase (DOK)1-6 genes in normal and breast cancer tissue and correlated this with several clinico-pathological and prognostic factors. METHODS: DOK1-6 mRNA extraction and reverse transcription were performed on fresh frozen breast cancer tissue samples (n = 112) and normal background breast tissue (n = 31). Tissues were collected between 1991 and 1996 at two centres and all patients underwent mastectomy and ipsilateral axillary node dissection. All tissues were randomly numbered and the details were only made known after all analyses were completed. Transcript levels of expression were determined using real-time polymerase chain reaction and analyzed against TNM stage, tumour grade and clinical outcome over a 10-year follow-up period. RESULTS: DOK-2 and DOK-6 expression decreased with increasing TNM stage. DOK-6 expression decreased with increasing Nottingham Prognostic Index (NPI) [NPI-1 vs NPI-3 (mean copy number 15.4 vs 0.22, 95%CI: 2.7-27.6, P = 0.018) and NPI-2 vs NPI-3 (mean copy number 7.6 vs 0.22, 95%CI: 0.1-14.6, P = 0.048)]. After a median follow up period of 10 years, higher levels of DOK-2 expression were found among patients who remained disease-free compared to those who developed local or distant recurrence (mean copy number 3.94 vs 0.0000096, 95%CI: 1.0-6.85, P = 0.0091), and distant recurrence (mean copy number 3.94 vs 0.0025, 95%CI: 1.0-6.84, P = 0.0092). Patients who remained disease-free had higher levels of DOK-6 expression compared to those who died from breast cancer. CONCLUSION: Decreasing expression levels of DOK-2 and DOK-6 with increased breast tumour progression supports the notion that DOK-2 and DOK-6 behave as tumour suppressors in human breast cancer. PMID:24829863

  7. 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

  8. Prognostic values of four Notch receptor mRNA expression in gastric cancer

    PubMed Central

    Wu, Xiaoyu; Liu, Wentao; Tang, Ding; Xiao, Haijuan; Wu, Zhenfeng; Chen, Che; Yao, Xuequan; Liu, Fukun; Li, Gang

    2016-01-01

    Notch ligands and receptors are frequently deregulated in several human malignancies including gastric cancer. The activation of Notch signaling has been reported to contribute to gastric carcinogenesis and progression. However, the prognostic roles of individual Notch receptors in gastric cancer patients remain elusive. In the current study, we accessed the prognostic roles of four Notch receptors, Notch 1–4, in gastric cancer patients through “The Kaplan-Meier plotter” (KM plotter) database, in which updated gene expression data and survival information include a total of 876 gastric cancer patients. All four Notch receptors’ high mRNA expression was found to be correlated to worsen overall survival (OS) for all gastric cancer patients followed for 20 years. We further accessed the prognostic roles of individual Notch receptors in different clinicopathological features using Lauren classification, pathological grades, clinical grades, HER2 status and different choices of treatments of gastric cancer patients. These results indicate that there are critical prognostic values of the four Notch receptors in gastric cancer. This information will be useful for better understanding of the heterogeneity and complexity in the molecular biology of gastric cancer and to develop tools to more accurately predict their prognosis. PMID:27363496

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

    PubMed

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

    2006-07-01

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

  10. Persistent Foot-and-Mouth Disease Virus Infection in the Nasopharynx of Cattle; Tissue-Specific Distribution and Local Cytokine Expression

    PubMed Central

    Pacheco, Juan M.; Smoliga, George R.; O’Donnell, Vivian; Brito, Barbara P.; Stenfeldt, Carolina; Rodriguez, Luis L.; Arzt, Jonathan

    2015-01-01

    Tissues obtained post-mortem from cattle persistently infected with foot-and-mouth disease virus (FMDV) were analyzed to characterize the tissue-specific localization of FMDV and partial transcriptome profiles for selected immunoregulatory cytokines. Analysis of 28 distinct anatomic sites from 21 steers infected with FMDV serotype A, O or SAT2, had the highest prevalence of overall viral detection in the dorsal nasopharynx (80.95%) and dorsal soft palate (71.43%). FMDV was less frequently detected in laryngeal mucosal tissues, oropharyngeal mucosal sites, and lymph nodes draining the pharynx. Immunomicroscopy indicated that within persistently infected mucosal tissues, FMDV antigens were rarely detectable within few epithelial cells in regions of mucosa-associated lymphoid tissue (MALT). Transcriptome analysis of persistently infected pharyngeal tissues by qRT-PCR for 14 cytokine genes indicated a general trend of decreased mRNA levels compared to uninfected control animals. Although, statistically significant differences were not observed, greatest suppression of relative expression (RE) was identified for IP-10 (RE = 0.198), IFN-β (RE = 0.269), IL-12 (RE = 0.275), and IL-2 (RE = 0.312). Increased relative expression was detected for IL-6 (RE = 2.065). Overall, this data demonstrates that during the FMDV carrier state in cattle, viral persistence is associated with epithelial cells of the nasopharynx in the upper respiratory tract and decreased levels of mRNA for several immunoregulatory cytokines in the infected tissues. PMID:25996935

  11. Effects of oral exposure to naturally-occurring and synthetic deoxynivalenol congeners on proinflammatory cytokine and chemokine mRNA expression in the mouse

    SciTech Connect

    Wu, Wenda; He, Kaiyu; Zhou, Hui-Ren; Berthiller, Franz; Adam, Gerhard; Sugita-Konishi, Yoshiko; Watanabe, Maiko; Krantis, Anthony; Durst, Tony; Zhang, Haibin; Pestka, James J.

    2014-07-15

    The foodborne mycotoxin deoxynivalenol (DON) induces a ribotoxic stress response in mononuclear phagocytes that mediate aberrant multi-organ upregulation of TNF-α, interleukins and chemokines in experimental animals. While other DON congeners also exist as food contaminants or pharmacologically-active derivatives, it is not known how these compounds affect expression of these cytokine genes in vivo. To address this gap, we compared in mice the acute effects of oral DON exposure to that of seven relevant congeners on splenic expression of representative cytokine mRNAs after 2 and 6 h. Congeners included the 8-ketotrichothecenes 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), fusarenon X (FX), nivalenol (NIV), the plant metabolite DON-3-glucoside (D3G) and two synthetic DON derivatives with novel satiety-inducing properties (EN139528 and EN139544). DON markedly induced transient upregulation of TNF-α IL-1β, IL-6, CXCL-2, CCL-2 and CCL-7 mRNA expressions. The two ADONs also evoked mRNA expression of these genes but to a relatively lesser extent. FX induced more persistent responses than the other DON congeners and, compared to DON, was: 1) more potent in inducing IL-1β mRNA, 2) approximately equipotent in the induction of TNF-α and CCL-2 mRNAs, and 3) less potent at upregulating IL-6, CXCL-2, and CCL-2 mRNAs. EN139528's effects were similar to NIV, the least potent 8-ketotrichothecene, while D3G and EN139544 were largely incapable of eliciting cytokine or chemokine mRNA responses. Taken together, the results presented herein provide important new insights into the potential of naturally-occurring and synthetic DON congeners to elicit aberrant mRNA upregulation of cytokines associated with acute and chronic trichothecene toxicity. - Highlights: • We compared effects of DON congeners on biomarker proinflammatory genes in mice. • Oral DON induced splenic IL-1β, IL-6, TNF-α,CXCL-2, CCL-2 and CCL-7 mRNAs. • 8-Ketotrichothecene ranking for

  12. 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.

  13. 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.

  14. BayMiR: inferring evidence for endogenous miRNA-induced gene repression from mRNA expression profiles

    PubMed Central

    2013-01-01

    Background Popular miRNA target prediction techniques use sequence features to determine the functional miRNA target sites. These techniques commonly ignore the cellular conditions in which miRNAs interact with their targets in vivo. Gene expression data are rich resources that can complement sequence features to take into account the context dependency of miRNAs. Results We introduce BayMiR, a new computational method, that predicts the functionality of potential miRNA target sites using the activity level of the miRNAs inferred from genome-wide mRNA expression profiles. We also found that mRNA expression variation can be used as another predictor of functional miRNA targets. We benchmarked BayMiR, the expression variation, Cometa, and the TargetScan “context scores” on two tasks: predicting independently validated miRNA targets and predicting the decrease in mRNA abundance in miRNA overexpression assays. BayMiR performed better than all other methods in both benchmarks and, surprisingly, the variation index performed better than Cometa and some individual determinants of the TargetScan context scores. Furthermore, BayMiR predicted miRNA target sets are more consistently annotated with GO and KEGG terms than similar sized random subsets of genes with conserved miRNA seed regions. BayMiR gives higher scores to target sites residing near the poly(A) tail which strongly favors mRNA degradation using poly(A) shortening. Our work also suggests that modeling multiplicative interactions among miRNAs is important to predict endogenous mRNA targets. Conclusions We develop a new computational method for predicting the target mRNAs of miRNAs. BayMiR applies a large number of mRNA expression profiles and successfully identifies the mRNA targets and miRNA activities without using miRNA expression data. The BayMiR package is publicly available and can be readily applied to any mRNA expression data sets. PMID:24001276

  15. Uncoupling protein-2 mRNA expression in mice subjected to intermittent hypoxia*

    PubMed Central

    Vieira, Luciana Rodrigues; Martinez, Denis; Forgiarini, Luiz Felipe; da Rosa, Darlan Pase; de Muñoz, Gustavo Alfredo Ochs; Fagundes, Micheli; Martins, Emerson Ferreira; Montanari, Carolina Caruccio; Fiori, Cintia Zappe

    2015-01-01

    Objective: To investigate the effect of intermittent hypoxia-a model of obstructive sleep apnea (OSA)-on pancreatic expression of uncoupling protein-2 (UCP2), as well as on glycemic and lipid profiles, in C57BL mice. Methods: For 8 h/day over a 35-day period, male C57BL mice were exposed to intermittent hypoxia (hypoxia group) or to a sham procedure (normoxia group). The intermittent hypoxia condition involved exposing mice to an atmosphere of 92% N and 8% CO2 for 30 s, progressively reducing the fraction of inspired oxygen to 8 ± 1%, after which they were exposed to room air for 30 s and the cycle was repeated (480 cycles over the 8-h experimental period). Pancreases were dissected to isolate the islets. Real-time PCR was performed with TaqMan assays. Results: Expression of UCP2 mRNA in pancreatic islets was 20% higher in the normoxia group than in the hypoxia group (p = 0.11). Fasting serum insulin was higher in the hypoxia group than in the normoxia group (p = 0.01). The homeostasis model assessment of insulin resistance indicated that, in comparison with the control mice, the mice exposed to intermittent hypoxia showed 15% lower insulin resistance (p = 0.09) and 21% higher pancreatic β-cell function (p = 0.01). Immunohistochemical staining of the islets showed no significant differences between the two groups in terms of the area or intensity of α- and β-cell staining for insulin and glucagon. Conclusions: To our knowledge, this is the first report of the effect of intermittent hypoxia on UCP2 expression. Our findings suggest that UCP2 regulates insulin production in OSA. Further study of the role that UCP2 plays in the glycemic control of OSA patients is warranted. PMID:25909153

  16. 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

  17. Mechanical stretch of human uterine smooth muscle cells increases IL-8 mRNA expression and peptide synthesis.

    PubMed

    Loudon, J A Z; Sooranna, S R; Bennett, P R; Johnson, M R

    2004-12-01

    Labour is associated with increased synthesis of interleukin-8 (IL-8) by the fetal membranes and myometrium, which leads to an inflammatory infiltrate. Stretch has been shown to increase the expression of contraction-associated proteins in animal models of labour and in human myocytes in vitro. In this study, we tested the hypothesis that mechanical stretch of human myometrial cells increases IL-8 messenger ribonucleic acid (mRNA) expression. We isolated myocytes from non-pregnant women undergoing hysterectomy and pregnant women undergoing Caesarean section before and after the onset of labour. Myocytes in culture were subjected to stretch of varying intensity (6-16%) and duration (1 or 6 h) using the Flexercell system. IL-8 mRNA expression was lowest in myocytes from pregnant women not in labour, intermediate in those from non-pregnant women and greatest in those from pregnant women in labour. Stretch increased IL-8 mRNA expression independent of reproductive state. The stretch-induced increase in IL-8 mRNA expression was associated with higher IL-8 levels in the culture supernatant and enhanced promoter activity. These data suggest that stretch contributes to the increase in myometrial IL-8 synthesis associated with the onset of labour in humans. PMID:15489245

  18. Expressions and clinical significances of CD133 protein and CD133 mRNA in primary lesion of gastric adenocacinoma

    PubMed Central

    2010-01-01

    Background To study on expressions and clinical significances of CD133 protein and CD133 mRNA in primary lesion of gastric adenocarcinoma (GC). Methods Expressions of CD133 protein by immunostaining (99 cases) and CD133 mRNA by semi-quantitative RT-PCR (31 cases) were detected in primary lesion and in noncancerous gastric mucosa tissue (NCGT). Correlations of CD133 protein expression with clinicopathological parameters and post-operative survival were analyzed. Relations of CD133 mRNA level with Ki-67 labeling index (LI), and lymphatic metastasis were assessed too. Results Brown particles indicating CD133 protein positivity occurred in some parts of tumor cells and epithelium. Expressive percentage of CD133 protein positivity was significantly higher in subgroups with >5 cm diameter (P = 0.041), later TNM stage (P = 0.044), severer lymph node metastasis (P = 0.017), occurrences of lymphatic invasion (P = 0.000) and vascular invasion (P = 0.000) respectively. Severer invasion depth (P = 0.011), lymph node metastasis occurrence (P = 0.043) and later TNM stage (P = 0.049) were the independent risk factors for CD133 protein expression. Average brightness scale value (BSV) of CD133 mRNA was significantly higher in subgroups with >5 cm diameter (P = 0.041), lymph node metastasis occurrence (P = 0.004) and in lower Ki-67 LI (P = 0.02). Relative analysis revealed that BSV of CD133 mRNA related positively to metastatic lymphatic nodes ratio (P = 0.008) and metastatic lymph node number (P = 0.009), but negatively to Ki-67 LI (P = 0.009). Survival of positive subgroup of CD 133 protein was significantly poorer (P = 0.047). Lymph node metastasis occurrence (P = 0.042), later TNM stage (P = 0.046) and CD 133 protein positive expression (P = 0.046) were respectively the independent risk factors to survival. Conclusion Higher expressive level of CD133 mRNA is associated to lower Ki-67 LI and severer lymphatic metastasis. Therefore, the expressive level of CD133 mRNA can play an

  19. The differential expression of alternatively polyadenylated transcripts is a common stress-induced response mechanism that modulates mammalian mRNA expression in a quantitative and qualitative fashion

    PubMed Central

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

    2016-01-01

    Stress adaptation plays a pivotal role in biological processes and requires tight regulation of gene expression. In this study, we explored the effect of cellular stress on mRNA polyadenylation and investigated the implications of regulated polyadenylation site usage on mammalian gene expression. High-confidence polyadenylation site mapping combined with global pre-mRNA and mRNA expression profiling revealed that stress induces an accumulation of genes with differentially expressed polyadenylated mRNA isoforms in human cells. Specifically, stress provokes a global trend in polyadenylation site usage toward decreased utilization of promoter-proximal poly(A) sites in introns or ORFs and increased utilization of promoter-distal polyadenylation sites in intergenic regions. This extensively affects gene expression beyond regulating mRNA abundance by changing mRNA length and by altering the configuration of open reading frames. Our study highlights the impact of post-transcriptional mechanisms on stress-dependent gene regulation and reveals the differential expression of alternatively polyadenylated transcripts as a common stress-induced mechanism in mammalian cells. PMID:27407180

  20. 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. PMID:27407180

  1. Clinical usefulness of WT1 mRNA expression in bone marrow detected by a new WT1 mRNA assay kit for monitoring acute myeloid leukemia: a comparison with expression of WT1 mRNA in peripheral blood.

    PubMed

    Kitamura, Kunio; Nishiyama, Takahiro; Ishiyama, Ken; Miyawaki, Shuichi; Miyazaki, Kanji; Suzuki, Kenshi; Masaie, Hiroaki; Okada, Masaya; Ogawa, Hiroyasu; Imai, Kiyotoshi; Kiyoi, Hitoshi; Naoe, Tomoki; Yokoyama, Yasuhisa; Chiba, Shigeru; Hata, Tomoko; Miyazaki, Yasushi; Hatta, Yoshihiro; Takeuchi, Jin; Nannya, Yasuhito; Kurokawa, Mineo; Ueda, Yasunori; Koga, Daisuke; Sugiyama, Haruo; Takaku, Fumimaro

    2016-01-01

    We have previously shown the clinical usefulness of Wilms' tumor 1 gene (WT1) mRNA expression in peripheral blood (PB) as a minimal residual disease (MRD) monitoring marker in 191 acute myeloid leukemia (AML) patients using the WT1 mRNA assay kit "Otsuka" (Otsuka Pharmaceutical Co., Ltd.; "former kit"). In contrast, the usefulness of WT1 mRNA expression in bone marrow (BM) has been investigated in only a limited number of subjects using former kit. Following that previous study, a next-generation kit, WT1 mRNA assay kit II "Otsuka" (Otsuka Pharmaceutical Co., Ltd.; "new kit") has been newly developed. In the present study, we aimed to evaluate the performance of the new kit and to investigate the clinical usefulness of WT1 mRNA expression in BM. The PB and BM were collected on the same day from 164 blood disease patients, including 118 AML patients. WT1 mRNA expression was determined using the new and former kits and the values obtained were compared. The performance of new kit was shown to be equivalent to that of former kit. As reported in PB, WT1 mRNA expression in BM was found to be a useful marker for monitoring disease status as well as for a diagnosis of early stage relapse in AML patients. PMID:26520650

  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. Chronic neonatal nicotine exposure increases mRNA expression of neurotrophic factors in the postnatal rat hippocampus.

    PubMed

    Son, Jong-Hyun; Winzer-Serhan, Ursula H

    2009-06-30

    Nicotine, the psychoactive ingredient in tobacco, can be neuroprotective but the mechanism is unknown. In the adult hippocampus, chronic nicotine can increase expression of growth factors which could contribute to nicotine's neuroprotective effects. During development, nicotine could also increase expression of neurotrophic factors. Therefore, we determined whether chronic neonatal nicotine (CNN) exposure increased mRNA expression levels of brain-derived neurotrophic factor (BDNF), nerve-growth factor (NGF), neurotrophin-3 (NT-3), fibroblast growth factor-2 (FGF-2), and insulin-like growth factor-1 (IGF-1). Nicotine (6 mg/kg/day in milk formula) or milk formula (controls) were delivered in three daily doses via oral gastric intubation to rat pups from postnatal day (P)1 to P8, and then sacrificed. Brains were processed for in situ hybridization using specific (35)S-labeled cRNA probes. At P8, CNN had a significant stimulant treatment effect on the expression of BDNF, FGF-2, NT-3 and IGF-1 [p<0.01], but not NGF. Specifically, BDNF mRNA expression, detected in CA1, CA3 stratum (s.) pyramidal and granule cell layer of the dentate gyrus (DG), was increased by 27.4%, 23.26% and 27.3%, respectively. FGF-2 mRNA expression, detected in neurons and astrocytes in CA1 s. radiatum, CA2 and CA3 s. pyramidale, and molecular layer of the DG, was increased by 34.0%, 8.9%, 31.0% and 23.1%, respectively. NT-3 mRNA expression in CA2 s. pyramidale was increased by 80.0%, and CNN increased the number of IGF-1-expressing cells in CA1 (18.0%), CA3 (20.9%) and DG (17.7%). Thus, nicotine exposure during early postnatal development differentially up-regulated expression of neurotrophic factor mRNAs in the hippocampus, which could increase neurotrophic tone and alter developmental processes. PMID:19410565

  4. Redirecting T-Cell Specificity to EGFR Using mRNA to Self-limit Expression of Chimeric Antigen Receptor.

    PubMed

    Caruso, Hillary G; Torikai, Hiroki; Zhang, Ling; Maiti, Sourindra; Dai, Jianliang; Do, Kim-Anh; Singh, Harjeet; Huls, Helen; Lee, Dean A; Champlin, Richard E; Heimberger, Amy B; Cooper, Laurence J N

    2016-06-01

    Potential for on-target, but off-tissue toxicity limits therapeutic application of genetically modified T cells constitutively expressing chimeric antigen receptors (CARs) from tumor-associated antigens expressed in normal tissue, such as epidermal growth factor receptor (EGFR). Curtailing expression of CAR through modification of T cells by in vitro-transcribed mRNA species is one strategy to mitigate such toxicity. We evaluated expression of an EGFR-specific CAR coded from introduced mRNA in human T cells numerically expanded ex vivo to clinically significant numbers through coculture with activating and propagating cells (AaPC) derived from K562 preloaded with anti-CD3 antibody. The density of AaPC could be adjusted to affect phenotype of T cells such that reduced ratio of AaPC resulted in higher proportion of CD8 and central memory T cells that were more conducive to electrotransfer of mRNA than T cells expanded with high ratios of AaPC. RNA-modified CAR T cells produced less cytokine, but demonstrated similar cytolytic capacity as DNA-modified CAR T cells in response to EGFR-expressing glioblastoma cells. Expression of CAR by mRNA transfer was transient and accelerated by stimulation with cytokine and antigen. Loss of CAR abrogated T-cell function in response to tumor and normal cells expressing EGFR. We describe a clinically applicable method to propagate and modify T cells to transiently express EGFR-specific CAR to target EGFR-expressing tumor cells that may be used to limit on-target, off-tissue toxicity to normal tissue. PMID:27163741

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

    PubMed Central

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

    2014-01-01

    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. PMID:24299740

  6. Sorting Live Stem Cells Based on Sox2 mRNA Expression

    PubMed Central

    Larsson, Hans M.; Lee, Seung Tae; Roccio, Marta; Velluto, Diana; Lutolf, Matthias P.; Frey, Peter; Hubbell, Jeffrey A.

    2012-01-01

    While cell sorting usually relies on cell-surface protein markers, molecular beacons (MBs) offer the potential to sort cells based on the presence of any expressed mRNA and in principle could be extremely useful to sort rare cell populations from primary isolates. We show here how stem cells can be purified from mixed cell populations by sorting based on MBs. Specifically, we designed molecular beacons targeting Sox2, a well-known stem cell marker for murine embryonic (mES) and neural stem cells (NSC). One of our designed molecular beacons displayed an increase in fluorescence compared to a nonspecific molecular beacon both in vitro and in vivo when tested in mES and NSCs. We sorted Sox2-MB+SSEA1+ cells from a mixed population of 4-day retinoic acid-treated mES cells and effectively isolated live undifferentiated stem cells. Additionally, Sox2-MB+ cells isolated from primary mouse brains were sorted and generated neurospheres with higher efficiency than Sox2-MB− cells. These results demonstrate the utility of MBs for stem cell sorting in an mRNA-specific manner. PMID:23209609

  7. Expression of fluorescent proteins in Branchiostoma lanceolatum by mRNA injection into unfertilized oocytes.

    PubMed

    Hirsinger, Estelle; Carvalho, João Emanuel; Chevalier, Christine; Lutfalla, Georges; Nicolas, Jean-François; Peyriéras, Nadine; Schubert, Michael

    2015-01-01

    We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the cephalochordate amphioxus, Branchiostoma lanceolatum. We use constructs for membrane and nuclear targeted mCherry and eGFP that have been modified to accommodate amphioxus codon usage and Kozak consensus sequences. We describe the type of injection needles to be used, the immobilization protocol for the unfertilized oocytes, and the overall injection set-up. This technique generates fluorescently labeled embryos, in which the dynamics of cell behaviors during early development can be analyzed using the latest in vivo imaging strategies. The development of a microinjection technique in this amphioxus species will allow live imaging analyses of cell behaviors in the embryo as well as gene-specific manipulations, including gene overexpression and knockdown. Altogether, this protocol will further consolidate the basal chordate amphioxus as an animal model for addressing questions related to the mechanisms of embryonic development and, more importantly, to their evolution. PMID:25650764

  8. Molecular cloning and mRNA expression analysis of sheep MYL3 and MYL4 genes.

    PubMed

    Zhang, Chunlan; Wang, Jianmin; Wang, Guizhi; Ji, Zhibin; Hou, Lei; Liu, Zhaohua; Chao, Tianle

    2016-02-15

    Using longissimus dorsi muscles of Dorper sheep as the experimental materials, the complete cDNAs of ovine MYL3 (Myosin light chain 3) and MYL4 (Myosin light chain 4) genes were cloned using RT-PCR, 5' RACE and 3' RACE. We obtained 925-bp and 869-bp full-length cDNAs and submitted their sequences to GenBank as accession numbers of KJ710703 and KJ768855, respectively. The cDNAs contained 600-bp and 582-bp open reading frames (ORFs) and encoded proteins comprising 199 and 193 amino acid residues, respectively. Neither protein was predicted to have a signal peptide, but both were predicted to have several N-glycosylation, O-glycosylation, and phosphorylation sites. The secondary structures of MYL3 and MYL4 were predicted to be 40.70% and 48.70% α- helical, respectively. Sequence alignment showed that the MYL3 and MYL4 proteins of Ovis aries both shared more than 91% amino acid sequence similarity with those of Mus musculus, Homo sapiens, Rattus norvegicus, Bos taurus, and Sus scrofa. The levels of MYL3 and MYL4 mRNA in various sheep tissues were determined using qRT-PCR. The results showed that both mRNAs were highly expressed in the heart. This study has established a foundation for further investigation of the ovine MYL3 and MYL4 genes. PMID:26656596

  9. 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

  10. REDUCED NITRIC OXIDE PRODUCTION AND INOS MRNA EXPRESSION IN IFN-G STIMULATED CHICKEN MACROPHAGES TRANSFECTED WITH INOS SIRNAS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Utilizing RNA interference technology with siRNA in the HD-11 macrophage cell line, we determined how the inhibition or knock-down of the iNOS (inducible nitric oxide synthase) gene affected IFN-y' induced macrophage production of nitric oxide (NO) and mRNA expression of genes involved in this biolo...

  11. RELATIONSHIP BETWEEN BRAIN AND OVARY AROMATASE ACTIVITY AND ISOFORM-SPECIFIC AROMATASE MRNA EXPRESSION IN THE FATHEAD MINNOW (PIMEPHALES PROMELAS)

    EPA Science Inventory

    There is growing evidence that some chemicals present in the environment have the capacity to inhibit, or potentially induce, aromatase activity. This study compared aromatase activities and isoform-specific mRNA expression in brain and ovary tissue from non-exposed fathead min...

  12. 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.

  13. Effect of atmospheric fine particles on epidermal growth factor receptor mRNA expression in mouse skin tissue.

    PubMed

    Han, X; Liang, W L; Zhang, Y; Sun, L D; Liang, W Y

    2016-01-01

    We investigated the effect of atmospheric fine particles on epidermal growth factor receptor (Egfr) mRNA expression in mouse skin tissue and explored the effect of atmospheric fine particles on skin aging. Forty female BALB/c mice were randomly divided into four groups (each comprising 10 mice) as follows: a saline control group and low-, medium-, and high-dose atmospheric fine particle groups (1.6, 8.0, and 40.0 mg/kg, respectively) (fine particles were defined as those with a diameter of £2.5 mm, i.e., PM2.5). Each dose group was exposed to intratracheal instillation for 3 days. Twenty-four hours after the last exposure, real-time quantitative polymerase chain reaction was used to detect the expression of Egfr mRNA in the skin tissue of each mouse. The expression levels of Egfr mRNA in the medium- and high-dose PM2.5 groups were significantly higher (P < 0.05) than that in the control group, and were positively correlated with the dose. Medium and high concentrations of PM2.5 can induce the expression of Egfr mRNA and promote skin aging. PMID:27050971

  14. 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. PMID:23573101

  15. Expression levels of estrogen receptor α mRNA in peripheral blood cells are an independent biomarker for postmenopausal osteoporosis☆

    PubMed Central

    Chou, Chi-Wen; Chiang, Tsay-I; Chang, I-Chang; Huang, Chung-Hung; Cheng, Ya-Wen

    2016-01-01

    Background The up- and down-regulation of the osteoclastogenesis response depends on the estrogen/estrogen receptor (ER) signaling pathway. Previous reports have shown that the promoter hypermethylation and gene polymorphism of ERα are risks for menopausal osteoporosis. No previous study has evaluated the expression levels of ERα mRNA in menopausal osteoporosis using human subjects. We hypothesized that ERα mRNA expression may show less resistance to postmenopausal osteoporosis. Methods In this study, we enrolled 107 women older than 45 years without menstruation and classified them into control, osteopenia, and osteoporosis groups depending on their T-scores. The ERα mRNA levels in peripheral blood cells (PBCs) were analyzed via quantitative real-time reverse-transcription polymerase chain reaction (QRT-PCR), and estrogen in the serum was detected via ELISA. Results ERα mRNA levels in PBCs had a negative correlation with age and a positive correlation with estrogen and BAP in the osteopenia and osteoporosis groups, but not in the control group. Additionally, multivariate analysis showed that older age (> 55 years), and low ERα mRNA levels in PBLs (≦ 250.39 copies/μg DNA) were associated with an approximately 9.188-, and 31.25-fold risk of osteoporosis. Conclusion We conclude that ERα mRNA levels in PBLs could be used as an independent risk factor for postmenopausal osteoporosis. General significance Our findings suggested that ERα mRNA levels in PBLs may be more important than age and serum estrogen levels. PMID:27051599

  16. Effects of jump training on procollagen alpha(1)(i) mRNA expression and its relationship with muscle collagen concentration.

    PubMed

    Ducomps, Christophe; Larrouy, Dominique; Mairal, Aline; Doutreloux, Jean-Paul; Lebas, Francois; Mauriege, Pascale

    2004-04-01

    The aim of this study was to examine the effects of a prolonged high-intensity exercise, jumping, on procollagen alpha(1)(I) mRNA level and collagen concentration in different muscles of trained (T) and control (C) rabbits. Procollagen alpha(1)(I) mRNA expression was much higher (2.8 to 23.5 times) in semimembranosus proprius (SMP), a slow-twitch oxidative muscle, than in extensor digitorum longus (EDL), rectus femoris (RF), and psoas major (Psoas) muscles, both fast-twitch mixed and glycolytic, whatever group was considered (p < 0.001). Procollagen alpha(1)(I) mRNA level also decreased significantly between 50 and 140 days in all muscles (0.001< p < 0.01). However, mRNA levels were 16 to 97% greater at 140 days in all muscles of T animals compared to C ones (0.01< p <0.05). Collagen concentrations of EDL and RF muscles were also higher (14 to 19%) in T than in C rabbits at 90 and 140 days (0.001 < p < 0.05). In the whole sample, collagen concentration was negatively associated with the procollagen alpha(1)(I) mRNA level in EDL and RF muscles (- 0.49 < r < (- 0.44, p < 0.05), while being positively related to mRNA expression in SMP and Psoas muscles (0.65 < r < 0.85, p < 0.01). It is concluded that jump training clearly restricts the decrease of procollagen (I) mRNA level and probably affects collagen synthesis level. In trained rabbit muscles, the maintenance of a better synthesis level could partly explain the higher collagen concentrations found in EDL and RF at 140 days. Nevertheless, the collagen degradation process seems to play the main role in the increase of total collagen concentration with age in EDL and RF muscles. PMID:15064425

  17. Brain-derived peptides increase blood-brain barrier GLUT1 glucose transporter gene expression via mRNA stabilization.

    PubMed

    Boado, R J

    1998-10-23

    The present investigation studied the effect of the brain-derived peptide preparation Cerebrolysin (CI, EBEWE, Austria) on the turnover rate and gene expression of the blood-brain barrier (BBB) GLUT1 glucose transporter mRNA. Studies were performed in brain endothelial cultured cells transfected with the human (h) GLUT1 transcript. In control cells, the full length 2.8 Kb hGLUT1 mRNA was rapidly degraded following transfection, and the abundance of this transcript at 4 and 6 h was comparable to background mRNA levels seen in cells transfected without hGLUT1 mRNA. On the contrary, the decay of the hGLUT1 mRNA was stabilized in CI-treated cells resulting in a marked reduction in the fractional turnover rate (72.4 and 4.0%/h, control and CI, respectively). In parallel experiments, CI induced a significant increase in the levels of immunoreactive GLUT1 protein measured by enzyme-linked immunosorbent assay (ELISA). In conclusion, data presented here demonstrate that factors in CI increase BBB-GLUT1 transcript stability, and that this is associated with an induction of BBB-GLUT1 gene expression in brain endothelial cultured cells. PMID:9832194

  18. Phase Variation Mediates Reductions in Expression of Surface Proteins during Persistent Meningococcal Carriage

    PubMed Central

    Alamro, Mohamed; Bidmos, Fadil A.; Chan, Hannah; Oldfield, Neil J.; Newton, Emma; Bai, Xilian; Aidley, Jack; Care, Rory; Mattick, Claire; Turner, David P. J.; Neal, Keith R.; Ala'Aldeen, Dlawer A. A.; Feavers, Ian; Borrow, Ray

    2014-01-01

    Asymptomatic and persistent colonization of the upper respiratory tract by Neisseria meningitidis occurs despite elicitation of adaptive immune responses against surface antigens. A putative mechanism for facilitating host persistence of this bacterial commensal and pathogen is alterations in expression of surface antigens by simple sequence repeat (SSR)-mediated phase variation. We investigated how often phase variation occurs during persistent carriage by analyzing the SSRs of eight loci in multiple isolates from 21 carriers representative of 1 to 6 months carriage. Alterations in repeat number were detected by a GeneScan analysis and occurred at 0.06 mutations/gene/month of carriage. The expression states were determined by Western blotting and two genes, fetA and nadA, exhibited trends toward low expression states. A critical finding from our unique examination of combinatorial expression states, “phasotypes,” was for significant reductions in expression of multiple phase-variable surface proteins during persistent carriage of some strains. The immune responses in these carriers were examined by measuring variant-specific PorA IgG antibodies, capsular group Y IgG antibodies and serum bactericidal activity in concomitant serum samples. Persistent carriage was associated with high levels of specific IgG antibodies and serum bactericidal activity while recent strain acquisition correlated with a significant induction of antibodies. We conclude that phase-variable genes are driven into lower expression states during long-term persistent meningococcal carriage, in part due to continuous exposure to antibody-mediated selection, suggesting localized hypermutation has evolved to facilitate host persistence. PMID:24686058

  19. Chronic stress induces persistent changes in global DNA methylation and gene expression in the medial prefrontal cortex, orbitofrontal cortex, and hippocampus.

    PubMed

    Mychasiuk, R; Muhammad, A; Kolb, B

    2016-05-13

    Chronic stress is associated with a plethora of cognitive symptoms such as emotional dysregulation and impaired executive function that have been attributed to modifications in neuroanatomy in the orbitofrontal cortex (OFC), medial prefrontal cortex (mPFC), and hippocampus (HPC). While many studies have examined stress-induced changes in neuronal morphology, synaptic plasticity, and cellular function, there has been little investigation into persistent changes in gene expression that may be responsible for the maintenance of these changes. This study exposed adult rats to a chronic stressor and then examined changes in mRNA gene expression in the OFC, mPFC and HPC following a two-week withdrawal period. mRNA bio-sequencing results revealed sex- and region-dependent changes. Surprisingly the greatest changes in gene expression were found in the OFC, and similar to anatomical studies, analysis of gene changes with Ingenuity Pathway Analysis software demonstrated that the mPFC and OFC exhibited contrasting activation of canonical pathways and functional networks. The HPC demonstrated the largest degree of sex-dependent change in gene expression. In general, chronic stress induced persistent changes in gene expression in the three brain regions we examined and these changes could be associated with the commonly reported cognitive symptoms. The current study highlights the region- and sex-dependent nature of the brain's response to chronic stress and the difficulty we face when attempting to develop treatment options. PMID:26946265

  20. The effects of high fat diet and estradiol on hypothalamic prepro-QRFP mRNA expression in female rats.

    PubMed

    Schreiber, Allyson L; Arceneaux, Kenneth P; Malbrue, Raphael A; Mouton, Alan J; Chen, Christina S; Bench, Elias M; Braymer, H Douglas; Primeaux, Stefany D

    2016-08-01

    Estradiol (E2) is a potent regulator of feeding behavior, body weight and adiposity in females. The hypothalamic neuropeptide, QRFP, is an orexigenic peptide that increases the consumption of high fat diet (HFD) in intact female rats. Therefore, the goal of the current series of studies was to elucidate the effects of E2 on the expression of hypothalamic QRFP and its receptors, QRFP-r1 and QRFP-r2, in female rats fed a HFD. Alterations in prepro-QRFP, QRFP-r1, and QRFP-r2 expression across the estrous cycle, following ovariectomy (OVX) and following estradiol benzoate (EB) treatment were assessed in the ventral medial nucleus of the hypothalamus/arcuate nucleus (VMH/ARC) and the lateral hypothalamus. In intact females, consumption of HFD increased prepro-QRFP and QRFP-r1 mRNA levels in the VMH/ARC during diestrus, a phase associated with increased food intake and low levels of E2. To assess the effects of diminished endogenous E2, rats were ovariectomized. HFD consumption and OVX increased prepro-QRFP mRNA in the VMH/ARC. Ovariectomized rats consuming HFD expressed the highest levels of QRFP. In the third experiment, all rats received EB replacement every 4days following OVX to examine the effects of E2 on QRFP expression. Prepro-QRFP, QRFP-r1 and QRFP-r2 mRNA were assessed prior to and following EB administration. EB replacement significantly reduced prepro-QRFP mRNA expression in the VMH/ARC. Overall these studies support a role for E2 in the regulation of prepro-QRFP mRNA in the VMH/ARC and suggest that E2's effects on food intake may be via a direct effect on the orexigenic peptide, QRFP. PMID:26823127

  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. Hormonal change and cytokine mRNA expression in peripheral blood mononuclear cells during the development of canine autoimmune thyroiditis.

    PubMed

    Choi, E-W; Shin, I-S; Bhang, D-H; Lee, D-H; Bae, B-K; Kang, M-S; Kim, D-Y; Hwang, C-Y; Lee, C-W; Youn, H-Y

    2006-10-01

    To elucidate the hormonal change and alteration in cytokine expression in peripheral blood mononuclear cells (PBMC) during the early stage of autoimmune thyroiditis, we have developed a canine model of this disease, in which normal dogs were immunized with bovine thyroglobulin (Tg) and/or canine thyroid extract. Serum samples were collected weekly, anti-canine Tg antibody was measured by enzyme-linked immunosorbent assay (ELISA) and thyroid stimulating hormone (TSH) and total T4 levels by radioimmunoassay. We also assayed T lymphocyte proliferation in response to Tg, as well as measuring cytokine mRNA by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). All six dogs immunized with bovine Tg had both canine Tg autoantibody and anti-T4 antibody. When the sample from the highest TgAA titre time-point was compared with baseline the expression of mRNA encoding the Th1-type cytokine such as interferon (IFN)-gamma, interleukin (IL)-18 and IL-15 was increased during the development of autoimmune thyroiditis. Expression of the Th2-type cytokine, IL-6 showed minimal change and IL-4 expression was not detected in any of the samples. Expression of the T suppressive cytokine, IL-10 and transforming growth factor (TGF)-beta was increased in the presence of antigen stimulation. These findings suggest that, although autoimmune thyroiditis is an organ-specific autoimmune disease, systemic cytokine mRNA expression is also changed. PMID:16968404

  3. Enhancement of mRNA expression of survivin and human beta-defensin-3 in lesions of psoriasis vulgaris.

    PubMed

    Wang, Fang; Zhang, Xia; Xia, Ping; Zhang, Li; Zhang, Zhenghua

    2016-02-01

    Suppression of apoptosis is one of the pathogenetic mechanisms for psoriasis vulgaris (PV). Survivin has the function of regulating cell division and inhibiting apoptosis. Patients with PV have an increased resistance to cutaneous infections. Human β-defensin-3 (hBD-3) is a kind of antimicrobial peptide with antimicrobial activities. To assess and compare the transcript levels of survivin and hBD-3 in pairwise skin from PV. A total of 20 patients, 10 with mild PV and 10 with severe PV, and 10 healthy control donors were recruited in the study. Real-time PCR was conducted to determine survivin and hBD-3 mRNA expression in skin lesions and normal-appearing skin of PV patients, and normal skin of healthy controls. Compared with normal control skin, the survivin mRNA expression of normal-appearing skin in the mild PV group, lesions of the mild PV group and the severe PV group were significantly elevated (P<0.05). hBD-3 mRNA expression was statistically increased in both normal-appearing skin and in lesions in mild and severe PV groups, in contrast to normal skin (P<0.001). Significant differences of hBD-3 mRNA were also found between lesions and non-lesional skin in the mild PV group and severe PV group (P<0.05). Survivin mRNA levels were mildly correlated with hBD-3 mRNA levels (rs = 0.398; P<0.05) in skin lesions from 20 PV patients. Survivin and hBD-3 may be involved in the pathogenesis of psoriasis. PMID:26771657

  4. [Regulatory effect of thymosin α1 on expression of tlr9/ido mRNA in bone marrow mesenchymal stem cells from children with aplastic anemia].

    PubMed

    Hou, Fang; Huang, Jian-Ming; Li, Ge

    2010-12-01

    The purpose of this study was to explore the regulatory effect of thymosin α1 (Tα1) on expression of TOLL-like receptor 9 (TLR9)/indoleamine2, 3-dioxygenase (ido) mRNA in bone marrow mesenchymal stem cells (MSC) from children with aplastic anemia (AA). Culture system of bone marrow MSC from AA children and normal children in vitro was established, and the effects of Tα1 on expressions of tlr9 mRNA and ido mRNA of MSC from AA children and normal children were determined by RT-PCR. The results showed that the bone marrow MSC from normal children did not express tlr9 and ido mRNA. Bone marrow MSC from children with AA obviously expressed tlr9 mRNA , but did not express ido mRNA; AA children's MSC treated with Tα1 for 18 hours markedly down-regulated tlr9 mRNA expression, but up-regulated ido mRNA expression in the concentration- and time-dependent ways. It is concluded that Tα1 can up-regulate the expression of ido mRNA in bone marrow MSC from children with AA. PMID:21176371

  5. Effect of propionate on mRNA expression of key genes for gluconeogenesis in liver of dairy cattle.

    PubMed

    Zhang, Qian; Koser, Stephanie L; Bequette, Brian J; Donkin, Shawn S

    2015-12-01

    Elevated needs for glucose in lactating dairy cows are met through a combination of increased capacity for gluconeogenesis and increased supply of gluconeogenic precursors, primarily propionate. This study evaluated the effects of propionate on mRNA expression of cytosolic phosphoenolpyruvate carboxykinase (PCK1), mitochondrial phosphoenolpyruvate carboxykinase (PCK2), pyruvate carboxylase (PC), and glucose-6-phosphatase (G6PC), key gluconeogenic enzymes, and capacity for glucose synthesis in liver of dairy cattle. In experiment 1, six multiparous mid-lactation Holstein cows were used in a replicated 3×3 Latin square design consisting of a 6-d acclimation or washout phase followed by 8h of postruminal infusion of either propionate (1.68mol), glucose (0.84mol), or an equal volume (10mL/min) of water. In experiment 2, twelve male Holstein calves [39±4 kg initial body weight (BW)] were blocked by birth date and assigned to receive, at 7d of age, either propionate [2mmol·h(-1)·(BW(0.75))(-1)], acetate [3.5mmol·h(-1)·(BW(.75))(-1)], or an equal volume (4mL/min) of saline. In both experiments, blood samples were collected at 0, 2, 4, 6, and 8h relative to the start of infusion and liver biopsy samples were collected at the end of the infusion for mRNA analysis. Liver explants from experiment 1 were used to measure tricarboxylic acid cycle flux and gluconeogenesis using (13)C mass isotopomer distribution analysis from (13)C3 propionate. Dry matter intake and milk yield were not altered by infusions in cows. Serum insulin concentration in cows receiving propionate was elevated than cows receiving water, but was not different from cows receiving glucose. Hepatic expression of PCK1 and G6PC mRNA and glucose production in cows receiving propionate were not different from cows receiving water, but tended to be higher compared with cows receiving glucose. Hepatic expression of PCK2 and PC mRNA was not altered by propionate infusion in cows. Blood glucose, insulin, and

  6. Expression of CD134 and CXCR4 mRNA in term placentas from FIV-infected and control cats.

    PubMed

    Scott, Veronica L; Burgess, Shane C; Shack, Leslie A; Lockett, Nikki N; Coats, Karen S

    2008-05-15

    Feline immunodeficiency virus (FIV) causes a natural infection of domestic cats that resembles HIV-1 in pathogenesis and disease progression. Feline AIDS is characterized by depression of the CD4+ T cell population and fatal opportunistic infections. Maternal-fetal transmission of FIV readily occurs under experimental conditions, resulting in infected viable kittens and resorbed or arrested fetal tissues. Although both FIV and HIV use the chemokine receptor CXCR4 as a co-receptor, FIV does not utilize CD4 as the primary receptor. Rather, CD134 (OX40), a T cell activation antigen and co-stimulatory molecule, is the primary receptor for FIV. We hypothesized that placental expression of CD134 and CXCR4 may render the placenta vulnerable to FIV infection, possibly facilitating efficient vertical transmission of FIV, and impact pregnancy outcome. The purpose of this project was to quantify the relative expression of CD134 and CXCR4 mRNA from the term placentas of three groups of cats: uninfected queens producing viable offspring, experimentally-infected queens producing only viable offspring, and experimentally-infected queens producing viable offspring among mostly non-viable fetuses. Total RNA was extracted from term placental tissues from all groups of cats. Real-time one-step reverse transcriptase-PCR was used to measure gene expression. The FIV receptors CD134 and CXCR4 were expressed in all late term feline placental tissues. Placentas from FIV-infected queens producing litters of only viable offspring expressed more CD134 and CXCR4 mRNA than those from uninfected queens, suggesting that infection may cause upregulation of the receptors. On the other hand, placentas from FIV-infected cats with non-successful pregnancies expressed similar levels of CD134 mRNA and slightly less CXCR4 mRNA than those from uninfected queens. Thus, it appears that cells expressing these receptors may play a role in pregnancy maintenance. PMID:18295905

  7. Distribution of Androgen Receptor mRNA Expression in Vocal, Auditory, and Neuroendocrine Circuits in a Teleost Fish

    PubMed Central

    Forlano, Paul M.; Marchaterre, Margaret; Deitcher, David L.; Bass, Andrew H.

    2010-01-01

    Across all major vertebrate groups, androgen receptors (ARs) have been identified in neural circuits that shape reproductive-related behaviors, including vocalization. The vocal control network of teleost fishes presents an archetypal example of how a vertebrate nervous system produces social, context-dependent sounds. We cloned a partial cDNA of AR that was used to generate specific probes to localize AR expression throughout the central nervous system of the vocal plainfin midshipman fish (Porichthys notatus). In the forebrain, AR mRNA is abundant in proposed homologs of the mammalian striatum and amygdala, and in anterior and posterior parvocellular and magnocellular nuclei of the preoptic area, nucleus preglomerulosus, and posterior, ventral and anterior tuberal nuclei of the hypothalamus. Many of these nuclei are part of the known vocal and auditory circuitry in midshipman. The midbrain periaqueductal gray, an essential link between forebrain and hindbrain vocal circuitry, and the lateral line recipient nucleus medialis in the rostral hindbrain also express abundant AR mRNA. In the caudal hindbrain-spinal vocal circuit, high AR mRNA is found in the vocal prepacemaker nucleus and along the dorsal periphery of the vocal motor nucleus congruent with the known pattern of expression of aromatase-containing glial cells. Additionally, abundant AR mRNA expression is shown for the first time in the inner ear of a vertebrate. The distribution of AR mRNA strongly supports the role of androgens as modulators of behaviorally defined vocal, auditory, and neuroendocrine circuits in teleost fish and vertebrates in general. PMID:20020540

  8. 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

  9. The metastatic potential of canine mammary tumours can be assessed by mRNA expression analysis of connective tissue modulators.

    PubMed

    Lamp, O; Honscha, K U; Schweizer, S; Heckmann, A; Blaschzik, S; Einspanier, A

    2013-03-01

    Metastases are the crucial factor for the prognosis of canine mammary tumours (CMTs). In women, the peptide hormone relaxin is linked with metastatic breast cancer. Therefore, the impact of relaxin and its receptors on matrix metalloproteinase (MMP) expression, metastatic disease and survival was analysed using qRT-PCR and immunohistochemistry of CMT samples from 59 bitches. The expression of relaxin and its receptor RXFP1 (relaxin family peptide receptor 1) was discovered on gene and protein levels. Intratumoural relaxin mRNA expression and relaxin plasma levels had no prognostic value. High mRNA levels RXFP1 were an independent marker of metastatic potential, with a more than 15-fold risk increase, and a predictor for shorter survival. Also, MMP-2 expression was associated with early death because of CMT. The mRNA expressions of relaxin, RXFP1 and MMP-2 were positively correlated indicating a common pathogenetic linkage. Thus, RXFP1 is proposed as a new early marker of metastatic potential in CMT and a possible therapeutic target. PMID:22235833

  10. Regulation of NMDA receptor subunit mRNA expression in the guinea pig vestibular nuclei following unilateral labyrinthectomy.

    PubMed

    Sans, N; Sans, A; Raymond, J

    1997-10-01

    The localization of neurons expressing mRNAs for the NR1 and NR2A-D subunits of the glutamatergic NMDA receptor was examined by non-radioactive in situ hybridization throughout the guinea pig vestibular nuclei. After deafferentation of the vestibular nuclei by unilateral labyrinthectomy, modifications of the mRNA distributions were followed for 30 days. A quantitative analysis was performed in the medial vestibular nucleus by comparison of the labelled neurons in the ipsi- and contra-lateral nuclei. In vestibular nuclei, the NR1 subunit mRNA was found in various populations of neurons. The NR2A and NR2C subunit mRNAs were less widely distributed, whereas little NR2D mRNA was detected and only rare cells contained NR2B mRNA. NR1 and NR2A-D mRNAs were colocalized in some but not other neuronal types. Twenty hours after the lesion, there was a transient ipsilateral increase of NR1 mRNA level in the medial vestibular nucleus, followed by a decrease 48 h after the lesion and, at 3 days, by recovery to the control level. An ipsilateral increase in the mRNA level of NR2C subunit was detected 20 h after lesion and maintained at 48 h. No significant changes were apparent in NR2A, NR2B and NR2D mRNA levels. The distributions and the differential signal intensities of NR2A-D mRNAs suggest various subunit organizations of the NMDA receptors in different neurons of the vestibular nuclei. Neuronal plasticity reorganizations in the vestibular nuclei following unilateral labyrinthectomy appear to include only changes in NR1 and NR2C mRNA levels modifying the functional diversity of the NMDA receptor in the ipsilateral medial vestibular nucleus neurons. The transient changes in NR1 and the NR2C subunit mRNA expressions in response to sensory deprivation are consistent with an active role for NMDA receptors in the appearance and development of the vestibular compensatory process. PMID:9421163

  11. Effects of arginine supplementation on splenocyte cytokine mRNA expression in rats with gut-derived sepsis

    PubMed Central

    Shang, Huey-Fang; Hsu, Chun-Sen; Yeh, Chiu-Li; Pai, Man-Hui; Yeh, Sung-Ling

    2005-01-01

    AIM: To investigate the effects of arginine (Arg)-enriched diets before sepsis and/or Arg-containing total parenteral nutrition (TPN) after sepsis or both on cytokine mRNA expression levels in splenocytes of rats with gut-derived sepsis. METHODS: Rats were assigned to four experimental groups. Groups 1 and 2 were fed with a semipurified diet, while groups 3 and 4 had part of the casein replaced by Arg which provided 2% of the total calories. After the rats were fed with these diets for 10 d, sepsis was induced by cecal ligation and puncture (CLP), at the same time an internal jugular vein was cannulated. All rats were maintained on TPN for 3 d. Groups 1 and 3 were infused with conventional TPN, while groups 2 and 4 were supplemented with Arg which provided 2% of the total calories in the TPN solution. All rats were killed 3 d after CLP to examine their splenocyte subpopulation distribution and cytokine expression levels. RESULTS: Plasma interleukin (IL)-2, IL-4, tumor necrosis factor-α (TNF-α) and interferon (IFN-γ) were not detectable 3 d after CLP. There were no differences in the distributions of CD45Ra+, CD3+, CD4+, and CD8+ cells in whole blood and splenocytes among the four groups. The splenocyte IL-2 mRNA expression in the Arg-supplemented groups was significantly higher than that in group 1. IL-4 mRNA expression in groups 3 and 4 was significantly higher than that in groups 1 and 2. The mRNA expression of IL-10 and IFN-γ was significantly higher in group 4 than in the other three groups. There was no difference in TNF-α mRNA expression among the four groups. CONCLUSION: The influence of Arg on the whole blood and splenic lymphocyte subpopulation distribution is not obvious. However, Arg administration, especially before and after CLP, significantly enhances the mRNA expression levels of Th1 and Th2 cytokines in the spleen of rats with gut-derived sepsis. PMID:16437653

  12. FATHEAD MINNOW VITELLOGENIN: CDNA SEQUENCE AND MRNA AND PROTEIN EXPRESSION AFTER 17 BETA-ESTRADIOL TREATMENT

    EPA Science Inventory

    In the present study, a sensitive ribonuclease protection assay (RPA) for VTG mRNA was developed for the fathead minnow (Pimephales promelas), a species proposed for routine endocrine-disrupting chemical (EDC) screening.

  13. The Prognostic Value of BRCA1 mRNA Expression Levels Following Neoadjuvant Chemotherapy in Breast Cancer

    PubMed Central

    Margeli, Mireia; Cirauqui, Beatriz; Castella, Eva; Tapia, Gustavo; Costa, Carlota; Gimenez-Capitan, Ana; Barnadas, Agusti; Ronco, Maria Sanchez; Benlloch, Susana; Taron, Miquel; Rosell, Rafael

    2010-01-01

    Background A fraction of sporadic breast cancers has low BRCA1 expression. BRCA1 mutation carriers are more likely to achieve a pathological complete response with DNA-damage-based chemotherapy compared to non-mutation carriers. Furthermore, sporadic ovarian cancer patients with low levels of BRCA1 mRNA have longer survival following platinum-based chemotherapy than patients with high levels of BRCA1 mRNA. Methodology/Principal Findings Tumor biopsies were obtained from 86 breast cancer patients who were candidates for neoadjuvant chemotherapy, treated with four cycles of neoadjuvant fluorouracil, epirubicin and cyclophosphamide. Estrogen receptor (ER), progesterone receptor (PR), HER2, cytokeratin 5/6 and vimentin were examined by tissue microarray. HER2 were also assessed by chromogenic in situ hybridization, and BRCA1 mRNA was analyzed in a subset of 41 patients for whom sufficient tumor tissue was available by real-time quantitative PCR. Median time to progression was 42 months and overall survival was 55 months. In the multivariate analysis for time to progression and overall survival for 41 patients in whom BRCA1 could be assessed, low levels of BRCA1 mRNA, positive PR and negative lymph node involvement predicted a significantly lower risk of relapse, low levels of BRCA1 mRNA and positive PR were the only variables associated with significantly longer survival. Conclusions/Significance We provide evidence for a major role for BRCA1 mRNA expression as a marker of time to progression and overall survival in sporadic breast cancers treated with anthracycline-based chemotherapy. These findings can be useful for customizing chemotherapy. PMID:20209131

  14. Chronic morphine exposure during puberty induces long-lasting changes in opioid-related mRNA expression in the mediobasal hypothalamus.

    PubMed

    Byrnes, Elizabeth M

    2008-01-23

    Substance abuse in developing females may have significant long-term effects on reproductive competency. Chronic morphine exposure during puberty has been shown to reduce prolactin secretion in lactating rats. Opioid activity within the mediobasal hypothalamus (MBH) regulates suckling-induced prolactin secretion. Thus, the current study was conducted to determine whether chronic pubertal morphine exposure alters the expression of mu- and/or kappa-opioid receptor mRNA or pro-opioimelanocortin (POMC) mRNA within the MBH. Using an increasing dose regimen, female Sprague-Dawley rats were injected twice daily for a total of 20 days with morphine sulfate or saline beginning at 30 days of age. Several weeks later, quantitative RT-PCR was used to determine mRNA expression within the MBH in diestrus, never pregnant (nulliparous) controls, postpartum day 5 (PPD5), PPD10, PPD18, and diestrus, reproductively experienced (primiparous) females. Pubertal morphine exposed females had increased mu- and kappa-receptor mRNA expression as well as decreased POMC mRNA expression on diestrus. During lactation, mu- and kappa-receptor mRNA expression in the MBH decreased while POMC mRNA expression increased in similarly treated females. No changes in mRNA expression were observed during lactation in pubertal saline-treated females; however, increased mu- and kappa-receptor mRNA expression as well as decreased POMC mRNA expression was observed in primiparous, pubertal saline-treated females when compared to nulliparous controls. Thus, chronic morphine exposure during puberty results in long-term alterations in mu- and kappa-receptor as well as POMC mRNA expression in the MBH which are similar to the changes observed following reproductive experience. These changes do not correlate with the decreased prolactin secretion observed during early lactation; however, they do demonstrate the enduring nature of the effects of chronic opiate exposure during puberty on hypothalamic opioid systems in

  15. Gravitational loading of a simulated launch alters mRNA expression in osteoblasts

    NASA Technical Reports Server (NTRS)

    Fitzgerald, J.; Hughes-Fulford, M.

    1996-01-01

    Serum-deprived mouse osteoblastic cells (MC3T3-E1a) were centrifuged under a regime designed to simulate a space shuttle launch (maximum of 3g). Messenger RNA levels for eight genes involved in bone growth and maintenance were determined using RT-PCR. Following 30 min of centrifugation, mRNA level for early response gene c-fos was significantly increased 89% (P < 0.05). The c-fos induction was transient and returned to control levels after 3 h. The mRNA level for the mineralization marker gene osteocalcin was significantly decreased to 44% of control level (P < 0.005) 3 h after centrifugation. No changes in mRNA levels were detected for c-myc, TGFbeta1, TGFbeta2, cyclophilin A, or actin. No basal mRNA level for TGFbeta3 was detected. In addition, no change in the steady-state synthesis of prostaglandin E2 was detected, possibly due to lack of lipid substrates in serum-deprived cells, suggesting that the increase in c-fos mRNA in response to gravitational loading is a result of mechanical stimulation. These results indicate that a small magnitude mechanical loading, such as that experienced during a shuttle launch, can alter mRNA levels in quiescent osteoblastic cells.

  16. Infusion of ACTH stimulates expression of adrenal ACTH receptor and steroidogenic acute regulatory protein mRNA in fetal sheep.

    PubMed

    Carey, Luke C; Su, Yixin; Valego, Nancy K; Rose, James C

    2006-08-01

    The late-gestation plasma cortisol surge in the sheep fetus is critical for stimulating organ development and parturition. Increased adrenal responsiveness is one of the key reasons for the surge; however, the underlying mechanisms are not fully understood. Our recent studies suggest that ACTH-mediated increased expression of ACTH receptor (ACTH-R) and steroid acute regulatory protein (StAR) may play a role in enhancing responsiveness. Hence, we examined effects of ACTH infusion in fetal sheep on mRNA expression of these two mediators of adrenal responsiveness and assessed the functional consequences of this treatment in vitro. Fetuses of approximately 118 and 138 days of gestational age (dGA) were infused with ACTH-(1-24) for 24 h. Controls received saline infusion. Arterial blood was sampled throughout the infusion. Adrenals were isolated and analyzed for ACTH-R and StAR mRNA, or cells were cultured for 48 h. Cells were stimulated with ACTH, and medium was collected for cortisol measurement. Fetal plasma ACTH and cortisol concentrations increased over the infusion period in both groups. ACTH-R mRNA levels were significantly higher in ACTH-infused fetuses in both the 118 and 138 dGA groups. StAR mRNA increased significantly in both the 118 and 138 dGA groups. Adrenal cells from ACTH-infused fetuses were significantly more responsive to ACTH stimulation in terms of cortisol secretion than those from saline-infused controls. These findings demonstrate that increases in circulating ACTH levels promote increased expression of ACTH-R and StAR mRNA and are coupled to heightened adrenal responsiveness. PMID:16478774

  17. mRNA expression is a relevant tool to identify developmental neurotoxicants using an in vitro approach.

    PubMed

    Hogberg, Helena T; Kinsner-Ovaskainen, Agnieszka; Coecke, Sandra; Hartung, Thomas; Bal-Price, Anna K

    2010-01-01

    So far, only a few industrial chemicals have been identified as developmental neurotoxicants. Because the current developmental neurotoxicity (DNT) guideline (Organisation for Economic Co-operation and Development TG 426) is based entirely on in vivo studies that are both time consuming and costly, there is a need to develop alternative in vitro methods for initial screening to prioritize chemicals for further DNT testing. In this study, gene expression at the mRNA level was evaluated to determine whether this could be a suitable endpoint to detect potential developmental neurotoxicants. Primary cultures of rat cerebellar granule cells (CGCs) were exposed to well known (developmental) neurotoxicants (methyl mercury chloride, lead chloride, valproic acid, and tri-methyl tin chloride) for different time periods. A significant downregulation of the mRNA level for the neuronal markers (NF-68, NF-200, N-methyl D-aspartate glutamate receptor, and gamma-amino butyric acid receptor) was observed after exposure to methyl mercury chloride, valproic acid, and tri-methyl tin chloride. Moreover, a significant increase of the neural precursor marker nestin mRNA was also observed. The mRNA expression of the astrocytic markers (glial fibrillary acidic protein [GFAP] and S100beta) was unchanged. In contrast, exposure to lead chloride significantly decreased the mRNA level of the astrocytic marker GFAP, whereas the neuronal markers were less affected. These results suggest that gene expression could be used as a sensitive tool for the initial identification of DNT effects induced by different mechanisms of toxicity in both cell types (neuronal and glial) and at various stages of cell development and maturation. PMID:19651682

  18. Mutation-dependent effects on mRNA and protein expressions in cultured keratinocytes of Hailey-Hailey disease.

    PubMed

    Matsuda, Mitsuhiro; Hamada, Takahiro; Numata, Sanae; Teye, Kwesi; Okazawa, Hiromi; Imafuku, Shinichi; Ohata, Chika; Furumura, Minao; Hashimoto, Takashi

    2014-07-01

    Hailey-Hailey disease (HHD) is a dominantly inherited skin disease caused by mutations in ATP2C1 gene, which encodes secretory pathway Ca(2+) /Mn(2+) -ATPase protein 1. The precise mechanism remains unclear. In this study, to understand molecular basis of HHD, we examined expression of mRNA and protein in cultured keratinocytes derived from three HHD patients with different mutations. We showed that reduced expression of mRNA and protein in patient with p.Gln504X, but not in patients with p.Pro307His and c.1308+1G>A. RT-PCR analysis for patient with c.1308+1G>A revealed in-frame exon skipping. Reduction of mRNA and protein in p.Gln504X was considered to be caused by nonsense-mediated mRNA decay. p.Pro307His located adjacent to Ca(2+) -binding residue may induced conformational change, which leads to defective Ca(2+) transport. In-frame shorter transcript caused by c.1308+1G>A may have slightly reduced activity, which accounted for mild phenotype of the patient. These results clarified the pathogenic effects of different causative mutations in development of skin lesions. PMID:24698124

  19. Persistent increases in rat hypothalamic POMC gene expression following chronic withdrawal from chronic “binge” pattern escalating-dose, but not steady-dose, cocaine

    PubMed Central

    Zhou, Yan; Kreek, Mary Jeanne

    2016-01-01

    Recent research suggests an involvement of pro-opiomelanocortin (POMC) gene products (e.g., beta-endorphin) in modulating cocaine-induced reward and addiction-like behaviors in rodents. In this study, we investigated whether chronic “binge” cocaine and its withdrawal altered POMC gene expression in the brain of rats. Male Fischer rats were treated with two different chronic (14-day) “binge” pattern cocaine administration regimens (3 injections at 1-h intervals, i.p.): steady-dose (45 mg/kg/day) and escalating-dose (90 mg/kg on the last day). Although there was no POMC mRNA alteration after chronic steady-dose cocaine, a significant decrease in POMC mRNA levels in the hypothalamus was found after chronic escalating-dose cocaine. In contrast, after acute (1-day) withdrawal from chronic “binge” escalating-dose regimen, but not steady-dose regimen, there were increased hypothalamic POMC mRNA levels that persisted into 14 days of protracted withdrawal. To study the role of the endogenous opioid systems in the cocaine withdrawal effects, we administered a single naloxone injection (1 mg/kg) that caused an elevated POMC mRNA levels observed 24 h later in cocaine naïve rats, but it did not lead to further increases in cocaine-withdrawn rats. Our results suggest that during withdrawal from chronic “binge” escalating-dose cocaine: (1) there was a persistent increase in hypothalamic POMC gene expression; and (2) hyposensitivity of the POMC gene expression to naloxone indicates altered opioidergic tone at or above the hypothalamic level. PMID:25595971

  20. Expression of BDNF and TH mRNA in the brain following inhaled administration of α-pinene.

    PubMed

    Kasuya, Hikaru; Okada, Narumi; Kubohara, Mika; Satou, Tadaaki; Masuo, Yoshinori; Koike, Kazuo

    2015-01-01

    Essential oils are mainly administered by inhalation. Administration by inhalation is considered to occur through two pathways, neurological transfer and pharmacological transfer. However, the relationship between the two routes is not clear. To clarify this relationship, we administered α-pinene, which has an anxiolytic-like effect, to mice. Emotional behavior and accumulation and expression of relevant mRNAs in the brain (brain-derived neurotrophic factor (BDNF); tyrosine hydroxylase (TH)) were examined following inhaled administration of α-pinene (10 μL/L air for 60 or 90min). To evaluate the anxiolytic-like effect, the elevated plus maze (EPM) test was used. Inhalation of α-pinene for 60 min produced a significant increase in the total distance traveled in the EPM test compared with control (water). The concentration of α-pinene in the brain after 60 min of inhalation was significantly increased compared with that after 90 min of inhalation. The expression of BDNF mRNA in the olfactory bulb and in the hippocampus was almost the same after 60 min of inhalation compared to that after 90 min of inhalation. The expression of TH mRNA in the midbrain after 60 min of inhalation was significantly increased compared with that of the control. Thus, an increase in α-pinene in the brain induces an increase in TH mRNA expression and increases locomotor activity. The anxiolytic-like effect may be related to both neurological transfer and pharmacological transfer. PMID:25230317

  1. Oligodendrocyte Morphometry and Expression of Myelin – Related mRNA in Ventral Prefrontal White Matter in Major Depressive Disorder

    PubMed Central

    Rajkowska, Grazyna; Mahajan, Gouri; Maciag, Dorota; Sathyanesan, Monica; Iyo, Abiye H.; Moulana, Mohadetheh; Kyle, Patrick B.; Woolverton, William L.; Miguel-Hidalgo, Jose Javier; Stockmeier, Craig A.; Newton, Samuel S.

    2015-01-01

    White matter disturbance in the ventral prefrontal cortex (vPFC) in major depressive disorder (MDD) has been noted with diffusion tensor imaging (DTI). However, the cellular and molecular pathology of prefrontal white matter in MDD and potential influence of antidepressant medications is not fully understood. Oligodendrocyte morphometry and myelin-related mRNA and protein expression was examined in the white matter of the vPFC in MDD. Sections of deep and gyral white matter from the vPFC were collected from 20 subjects with MDD and 16 control subjects. Density and size of CNPase-immunoreactive (−IR) oligodendrocytes were estimated using 3-dimensional cell counting. While neither density nor soma size of oligodendrocytes was significantly affected in deep white matter, soma size was significantly decreased in the gyral white matter in MDD. In rhesus monkeys treated chronically with fluoxetine there was no significant effect on oligodendrocyte morphometry. Using quantitative RTPCR to measure oligodendrocyte-related mRNA for CNPase, PLP1, MBP, MOG, MOBP, Olig1 and Olig2, in MDD there was a significantly reduced expression of PLP1 mRNA (which positively correlated with smaller sizes) and increased expression of mRNA for CNPase, OLIG1 and MOG. The expression of CNPase protein was significantly decreased in MDD. Altered expression of four myelin genes and CNPase protein suggests a mechanism for the degeneration of cortical axons and dysfunctional maturation of oligodendrocytes in MDD. The change in oligodendrocyte morphology in gyral white matter may parallel altered axonal integrity as revealed by DTI. PMID:25930075

  2. Calcyon mRNA expression in the frontal-striatal circuitry and its relationship to vesicular processes and ADHD

    PubMed Central

    Heijtz, Rochellys Diaz; Alexeyenko, Andrey; Castellanos, F Xavier

    2007-01-01

    Background Calcyon is a single transmembrane protein predominantly expressed in the brain. Very recently, calcyon has been implicated in clathrin mediated endocytosis, a critical component of synaptic plasticity. At the genetic level, preliminary evidence supports an association between attention-deficit/hyperactivity disorder (ADHD) and polymorphisms in the calcyon gene. As little is known about the potential role of calcyon in ADHD, animal models may provide important insights into this issue. Methods We examined calcyon mRNA expression in the frontal-striatal circuitry of three-, five-, and ten-week-old Spontaneously Hypertensive Rats (SHR), the most commonly used animal model of ADHD, and Wistar-Kyoto (WKY; the strain from which SHR were derived). As a complement, we performed a co-expression network analysis using a database of mRNA gene expression profiles of multiple brain regions in order to explore potential functional links of calcyon to other genes. Results In all age groups, SHR expressed significantly more calcyon mRNA in the medial prefrontal and orbital frontal cortices than WKY rats. In contrast, in the motor cortex, dorsal striatum and nucleus accumbens, calcyon mRNA expression was only significantly elevated in SHR in younger animals. In both strains, calcyon mRNA levels decreased significantly with age in all regions studied. In the co-expression network analysis, we found a cluster of genes (many of them poorly studied so far) strongly connected to calcyon, which may help elucidate its role in the brain. The pair-wise relations of calcyon with other genes support its involvement in clathrin mediated endocytosis and, potentially, some other membrane/vesicular processes. Interestingly, no link was found between calcyon and the dopamine D1 receptor, which was previously shown to interact with the C-terminal of calcyon. Conclusion The results indicate an alteration in calcyon expression within the frontal-striatal circuitry of SHR, especially in

  3. Changes in apoptotic microRNA and mRNA expression profiling in Caenorhabditis elegans during the Shenzhou-8 mission.

    PubMed

    Gao, Ying; Li, Shuai; Xu, Dan; Wang, Junjun; Sun, Yeqing

    2015-11-01

    Radiation and microgravity exposure have been proven to induce abnormal apoptosis in microRNA (miRNA) and mRNA expression, but whether space conditions, including radiation and microgravity, activate miRNAs to regulate the apoptosis is undetermined. For that purpose, we investigated miRNome and mRNA expression in the ced-1 Caenorhabditis elegans mutant vs the wild-type, both of which underwent spaceflight, spaceflight 1g-centrifuge control and ground control conditions during the Shenzhou-8 mission. Results showed that no morphological changes in the worms were detected, but differential miRNA expression increased from 43 (ground control condition) to 57 and 91 in spaceflight and spaceflight control conditions, respectively. Microgravity altered miRNA expression profiling by decreasing the number and significance of differentially expressed miRNA compared with 1 g incubation during spaceflight. Alterations in the miRNAs were involved in alterations in apoptosis, neurogenesis larval development, ATP metabolism and GTPase-mediated signal transduction. Among these, 17 altered miRNAs potentially involved in apoptosis were screened and showed obviously different expression signatures between space conditions. By integrated analysis of miRNA and mRNA, miR-797 and miR-81 may be involved in apoptosis by targeting the genes ced-10 and both drp-1 and hsp-1, respectively. Compared with ground condition, space conditions regulated apoptosis though a different manner on transcription, by altering expression of seven core apoptotic genes in spaceflight condition, and eight in spaceflight control condition. Results indicate that, miRNA of Caenorhabditis elegans probably regulates apoptotic gene expression in response to space environmental stress, and shows different behavior under microgravity condition compared with 1 g condition in the presence of space radiation. PMID:26286471

  4. Changes in apoptotic microRNA and mRNA expression profiling in Caenorhabditis elegans during the Shenzhou-8 mission

    PubMed Central

    Gao, Ying; Li, Shuai; Xu, Dan; Wang, Junjun; Sun, Yeqing

    2015-01-01

    Radiation and microgravity exposure have been proven to induce abnormal apoptosis in microRNA (miRNA) and mRNA expression, but whether space conditions, including radiation and microgravity, activate miRNAs to regulate the apoptosis is undetermined. For that purpose, we investigated miRNome and mRNA expression in the ced-1 Caenorhabditis elegans mutant vs the wild-type, both of which underwent spaceflight, spaceflight 1g-centrifuge control and ground control conditions during the Shenzhou-8 mission. Results showed that no morphological changes in the worms were detected, but differential miRNA expression increased from 43 (ground control condition) to 57 and 91 in spaceflight and spaceflight control conditions, respectively. Microgravity altered miRNA expression profiling by decreasing the number and significance of differentially expressed miRNA compared with 1 g incubation during spaceflight. Alterations in the miRNAs were involved in alterations in apoptosis, neurogenesis larval development, ATP metabolism and GTPase-mediated signal transduction. Among these, 17 altered miRNAs potentially involved in apoptosis were screened and showed obviously different expression signatures between space conditions. By integrated analysis of miRNA and mRNA, miR-797 and miR-81 may be involved in apoptosis by targeting the genes ced-10 and both drp-1 and hsp-1, respectively. Compared with ground condition, space conditions regulated apoptosis though a different manner on transcription, by altering expression of seven core apoptotic genes in spaceflight condition, and eight in spaceflight control condition. Results indicate that, miRNA of Caenorhabditis elegans probably regulates apoptotic gene expression in response to space environmental stress, and shows different behavior under microgravity condition compared with 1 g condition in the presence of space radiation. PMID:26286471

  5. Modelling the role of Tax expression in HTLV-I persistence in vivo.

    PubMed

    Li, Michael Y; Lim, Aaron G

    2011-12-01

    Human T-lymphotropic virus type I (HTLV-I) is a persistent human retrovirus characterized by life-long infection and risk of developing HAM/TSP, a progressive neurological and inflammatory disease, and adult T-cell leukemia (ATL). Chronically infected individuals often harbor high proviral loads despite maintaining a persistently activated immune response. Based on a new hypothesis for the persistence of HTLV-I infection, a three-dimensional compartmental model is constructed that describes the dynamic interactions among latently infected target cells, target-cell activation, and immune responses to HTLV-I, with an emphasis on understanding the role of Tax expression in the persistence of HTLV-I. PMID:21509627

  6. 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

  7. The expression of histamine H4 receptor mRNA in the skin and other tissues of normal dogs.

    PubMed

    Eisenschenk, Melissa N C; Torres, Sheila M F; Oliveira, Simone; Been, Clint S

    2011-10-01

    The histamine 4 (H(4)) receptor was first cloned and characterized in 2000 using the human H(3) receptor DNA sequence. The H(4) receptor has been shown to participate in various aspects of inflammation, such as chemotaxis, upregulation of adhesion molecule expression and modulation of cytokine secretion. The primary goal of this study was to determine whether H(4) receptor mRNA is expressed in normal canine skin by performing an RT-PCR. An additional goal was to determine the expression of this receptor in the colon, liver, spleen and kidney. Tissues were collected from five healthy, young-adult pit bull dogs. Samples were immediately placed in RNAlater(®) solution and stored at -20°C until processed. The amplified products in all skin samples in addition to the colon, liver, spleen and kidney (variable expression) had the expected size of 400-500 bp. The sequenced amplicons matched the National Center for Biotechnology Information published sequence for the canine H(4) receptor. The study results showed that canine normal skin expresses the H(4) receptor mRNA. Further studies using immunohistochemistry should be conducted to demonstrate the expression of the H(4) receptor at the protein level and to localize the expression of this receptor in the skin. PMID:21392139

  8. Lifelong Parental Voluntary Wheel Running Increases Offspring Hippocampal Pgc-1α mRNA Expression But Not Mitochondrial Content or Bdnf Expression

    PubMed Central

    Venezia, Andrew C.; Guth, Lisa M.; Spangenburg, Espen E.; Roth, Stephen M.

    2015-01-01

    When exercise is initiated during pregnancy, offspring of physically active mothers have higher hippocampal expression of brain derived neurotrophic factor (Bdnf) and other plasticity and mitochondrial-associated genes, resulting in hippocampal structural and functional adaptations. In the present study, we examined the effects of lifelong parental voluntary wheel running (before, during, and after pregnancy) on offspring hippocampal mRNA expression of genes implicated in the exercise-induced improvement of cognitive function. C57BL/6 mice were individually housed at 8 weeks of age with (EX; n=20) or without (SED; n=20) access to a computer-monitored voluntary running wheel (VRW) for 12 weeks prior to breeding. EX breeders maintained access to the VRW throughout breeding, pregnancy, and lactation. Male offspring were housed in sedentary cages, regardless of parental group, and were sacrificed at 8 (n=18) or 28 weeks (n=19). PCR was used to assess mRNA expression of several genes and mitochondrial content (ratio of mitochondrial to nuclear DNA) in hippocampal homogenates. We found significantly higher peroxisome proliferator-activated receptor γ coactivator 1 alpha (Pgc-1α) mRNA expression in EX offspring compared to SED offspring at 8 wks (p=0.04), though the effect was no longer present at 28 wks. There was no difference in mitochondrial content or expression of Bdnf or any other mRNA targets between offspring at 8 or 28 wks. In contrast to exercise initiated during pregnancy, parental voluntary physical activity initiated early in life and maintained throughout pregnancy has little effect on offspring mRNA expression of genes implicated in exercise-induced hippocampal plasticity. PMID:25919993

  9. 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. PMID:25206697

  10. merA gene expression in aquatic environments measured by mRNA production and Hg(II) volatilization.

    PubMed Central

    Nazaret, S; Jeffrey, W H; Saouter, E; Von Haven, R; Barkay, T

    1994-01-01

    The relationship of merA gene expression (specifying the enzyme mercuric reductase) to mercury volatilization in aquatic microbial communities was investigated with samples collected at a mercury-contaminated freshwater pond, Reality Lake, in Oak Ridge, Tenn. Levels of merA mRNA transcripts and the rate of inorganic mercury [Hg(II)] volatilization were related to the concentration of mercury in the water and to heterotrophic activity in field samples and laboratory incubations of pond water in which microbial heterotrophic activity and Hg(II) concentration were manipulated. Levels of merA-specific mRNA and Hg(II) volatilization were influenced more by microbial metabolic activity than by the concentration of mercury. merA-specific transcripts were detected in some samples which did not reduce Hg(II), suggesting that rates of mercury volatilization in environmental samples may not always be proportional to merA expression. PMID:7527625

  11. Structure and expression of the human L-myc gene reveal a complex pattern of alternative mRNA processing

    SciTech Connect

    Kaye, F.; Battey, J.; Nau, M.; Brooks, B.; Seifter, E.; De Greve, J.; Birrer, M.; Sausville, E.; Minna, J.

    1988-01-01

    The authors' analyzed in detail the structure of the L-myc gene isolated from human placental DNA and characterized its expression in several small-cell lung cancer cell lines. The gene is composed of three exons and two introns spanning 6.6 kilobases in human DNA. Several distinct mRNA species are produced in all small-cell lung cancer cell lines that express L-myc. These transcripts are generated from a single gene by alternative splicing of introns 1 and 2 and by use of alternative polyadenylation signals. In some mRNAs that is a long open reading frame with a predicted translated protein of 364 residues. Amino acid sequence comparison with c-myc and N-myc demonstrated multiple discrete regions with extensive homology. In contrast, other mRNA transcripts, generated by alternative processing, could encode a truncated protein with a novel carboxy-terminal end.

  12. Higher decidual EBI3 and HLA-G mRNA expression in preeclampsia: Cause or consequence of preeclampsia.

    PubMed

    Prins, J R; van der Hoorn, M L P; Keijser, R; Ris-Stalpers, C; van Beelen, E; Afink, G B; Claas, F H J; van der Post, J A M; Scherjon, S A

    2016-01-01

    The maternal immune system must adapt to tolerate the invasion of the allogeneic feto-placental unit. It is generally accepted that improper adaptation causes pregnancy complications like preeclampsia. The Epstein-Barr virus-induced gene 3 (EBI3) protein is a subunit of immune-modulatory cytokines interleukin 27 (IL-27) and IL-35. EBI3 has been reported to associate with HLA-G. In this small pilot study we find higher decidual EBI3 (p<0.05) and HLA-G (p<0.01) mRNA expression in preeclampsia (n=7) compared to normotensive (n=8) pregnancies. Whether the higher EBI3 and HLA-G mRNA expression is a consequence or cause of preeclampsia remains to be answered. Further research to determine the effects on IL-27 and IL-35 is needed. PMID:26472010

  13. Differential Expression of mRNA Encoding Cytokines and Chemokines in the Reproductive Tract after Infection of Mice with Chlamydia trachomatis

    PubMed Central

    Cerny, Katheryn L.; Van Fleet, Maranda; Slepenkin, Anatoly; Peterson, Ellena M.; Bridges, Phillip J.

    2015-01-01

    Infection with Chlamydia trachomatis targets epithelial cells within the genital tract which respond by secreting chemokines and cytokines. Persistent inflammation can lead to fibrosis, tubal infertility and/or ectopic pregnancy; many infections are asymptomatic. Most studies have investigated the inflammatory response in the initial stages of infection, less is known about the later stages of infection, especially with a low, potentially asymptomatic, bacterial load. Our objective was to determine the inflammatory mediators involved in clearance of low-grade infection and the potential involvement in chronic inflammation. Six to eight week old C3H/HeJ mice were pretreated with 2.5 mg medroxyprogesterone acetate on day -10 and -3 before infection. Mice (n=3 for 28 d, n=3 for 35 d) were infected with 5 × 102 inclusion-forming units of C. trachomatis, serovar D; vaginal cultures were obtained weekly to monitor infection. Control mice (n=3 for 28 d, n=3 for 35 d) were sham infected. Mice were killed on day 28 (experiment 1) and day 35 (experiment 2) post-infection and vaginal tissue, uterine horns and oviducts collected for analysis of mRNAs encoding inflammatory cytokines and chemokines. Total RNA was isolated and a superarray analysis performed using mouse Cytokines and Chemokines PCR arrays (Qiagen, Valencia, CA). Statistical differences in gene expression were determined using a paired Students t-test. At 28 days after infection, the expression of mRNA encoding 6, 35 and 3 inflammatory genes differed from controls in vaginal, uterine and oviductal tissues, respectively (P<0.05). At 35 days after infection, the expression of mRNA encoding 16, 38 and 14 inflammatory genes differed from controls in vaginal, uterine and oviductal tissues, respectively (P<0.05). Understanding the mechanisms involved in the inflammatory response at later stages of infection should aid in the development of treatment options that minimize the development of asymptomatic, chronic

  14. Expression of insulin-like growth factors at mRNA levels during the metamorphic development of turbot (Scophthalmus maximus).

    PubMed

    Meng, Zhen; Hu, Peng; Lei, Jilin; Jia, Yudong

    2016-09-01

    Insulin-like growth factors I and II (IGF-I and IGF-II) are important regulators of vertebrate growth and development. This study characterized the mRNA expressions of igf-i and igf-ii during turbot (Scophthalmus maximus) metamorphosis to elucidate the possible regulatory role of the IGF system in flatfish metamorphosis. Results showed that the mRNA levels of igf-i significantly increased at the early-metamorphosis stage and then gradually decreased until metamorphosis was completed. By contrast, mRNA levels of igf-ii significantly increased at the pre-metamorphosis stage and then substantially decreased during metamorphosis. Meanwhile, the whole-body thyroxine (T4) levels varied during larval metamorphosis, and the highest value was observed in the climax-metamorphosis. The mRNA levels of igf-i significantly increased and decreased by T4 and thiourea (TU, inhibitor of endogenous thyroid hormone) during metamorphosis, respectively. Conversely, the mRNA levels of igf-ii remained unchanged. Furthermore, TU significantly inhibited the T4-induced mRNA up-regulation of igf-i during metamorphosis. The whole-body thyroxine (T4) levels were significantly increased and decreased by T4 and TU during metamorphosis, respectively. These results suggested that igf-i and igf-ii may play different functional roles in larval development stages, and igf-i may have a crucial function in regulating the early metamorphic development of turbot. These findings may enhance our understanding of the potential roles of the IGF system to control flatfish metamorphosis and contribute to the improvement of broodstock management for larvae. PMID:27255364

  15. Posttranscriptional regulation of sodium-iodide symporter mRNA expression in the rat thyroid gland by acute iodide administration.

    PubMed

    Serrano-Nascimento, Caroline; Calil-Silveira, Jamile; Nunes, Maria Tereza

    2010-04-01

    Iodide is an important regulator of thyroid activity. Its excess elicits the Wolff-Chaikoff effect, characterized by an acute suppression of thyroid hormone synthesis, which has been ascribed to serum TSH reduction or TGF-beta increase and production of iodolipids in the thyroid. These alterations take hours/days to occur, contrasting with the promptness of Wolff-Chaikoff effect. We investigated whether acute iodide administration could trigger events that precede those changes, such as reduction of sodium-iodide symporter (NIS) mRNA abundance and adenylation, and if perchlorate treatment could counteract them. Rats subjected or not to methylmercaptoimidazole treatment (0.03%) received NaI (2,000 microg/0.5 ml saline) or saline intraperitoneally and were killed 30 min up to 24 h later. Another set of animals was treated with iodide and perchlorate, in equimolar doses. NIS mRNA content was evaluated by Northern blotting and real-time PCR, and NIS mRNA poly(A) tail length by rapid amplification of cDNA ends-poly(A) test (RACE-PAT). We observed that NIS mRNA abundance and poly(A) tail length were significantly reduced in all periods of iodide treatment. Perchlorate reversed these effects, indicating that iodide was the agent that triggered the modifications observed. Since the poly(A) tail length of mRNAs is directly associated with their stability and translation efficiency, we can assume that the rapid decay of NIS mRNA abundance observed was due to a reduction of its stability, a condition in which its translation could be impaired. Our data show for the first time that iodide regulates NIS mRNA expression at posttranscriptional level, providing a new mechanism by which iodide exerts its autoregulatory effect on thyroid. PMID:20107044

  16. Regulation of VEGF and bFGF mRNA expression and other proliferative compounds in skeletal muscle cells.

    PubMed

    Jensen, L; Schjerling, P; Hellsten, Y

    2004-01-01

    The role of muscle contraction, prostanoids, nitric oxide and adenosine in the regulation of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and endothelial cell proliferative compounds in skeletal muscle cell cultures was examined. VEGF and bFGF mRNA, protein release as well as the proliferative effect of extracellular medium was determined in non-stimulated and electro-stimulated rat and human skeletal muscle cells. In rat skeletal muscle cells these aspects were also determined after treatment with inhibitors and/or donors of nitric oxide (NO), prostanoids and adenosine. Electro-stimulation caused an elevation in the VEGF and bFGF mRNA levels of rat muscle cells by 33% and 43% (P < 0.05), respectively, and in human muscle cells VEGF mRNA was elevated by 24%. Medium from electro-stimulated human, but not rat muscle cells induced a 126% higher (P < 0.05) endothelial cell proliferation than medium from non-stimulated cells. Cyclooxygenase inhibition of rat muscle cells induced a 172% increase (P < 0.05) in VEGF mRNA and a 104% increase in the basal VEGF release. Treatment with the NO donor SNAP (0.5 microM) decreased (P < 0.05) VEGF and bFGF mRNA by 42 and 38%, respectively. Medium from SNAP treated muscle cells induced a 45% lower (P < 0.05) proliferation of endothelial cells than control medium. Adenosine enhanced the basal VEGF release from muscle cells by 75% compared to control. The present data demonstrate that contractile activity, NO, adenosine and products of cyclooxygenase regulate the expression of VEGF and bFGF mRNA in skeletal muscle cells and that contractile activity and NO regulate endothelial cell proliferative compounds in muscle extracellular fluid. PMID:15609080

  17. Amylase mRNA transcripts in normal tissues and neoplasms: the implication of different expressions of amylase isogenes.

    PubMed

    Seyama, K; Nukiwa, T; Takahashi, K; Takahashi, H; Kira, S

    1994-01-01

    To understand the cellular origin and mechanism of gene expression in amylase-producing cancers, the phenotyping of amylase isogenes by the polymerase chain reaction and restriction-fragment-length polymorphism using restriction endonucleases TaqI, DdeI, HinfI, and AfaI were performed for 3 amylase-producing lung adenocarcinomas, 16 lung cancers without hyperamylasemia, other human malignant neoplasms, cultured cell lines, and normal tissues. In addition, amylase mRNA transcripts were semi-quantified by the limited polymerase chain reaction. Amylase mRNA transcripts were detected in all of the tissues examined. The AMY1 gene (salivary type) was exclusively and highly expressed in the salivary glands and the amylase-producing lung adenocarcinomas. Coexpression of the AMY1 gene and AMY2 gene (pancreatic type) was observed in most of the lung cancers without hyperamylasemia, lung tissue, and cells scraped from the tracheal epithelium, thyroid, and female genital tract (ovary, fallopian tube, and uterus cervix), while minimal levels of mRNA transcripts of the AMY2 gene were detected in other malignant neoplasms, various normal tissues, and the cultured cell lines. All mRNA transcripts identified as being those of the AMY2 gene were further identified as being from the AMY2B gene except for the transcripts from the pancreas, in which the AMY2A gene and AMY2B gene were coexpressed. On the basis of these results, the clinical occurrence of amylase-producing cancer likely relates to the tissues expressing the AMY1 gene, while the AMY2B gene, which evolutionarily is the oldest gene among human amylase isogenes, is constitutively expressed in various tissues. PMID:7507116

  18. Lung squamous cell carcinoma mRNA expression subtypes are reproducible, clinically important and correspond to different normal cell types

    PubMed Central

    Wilkerson, Matthew D.; Yin, Xiaoying; Hoadley, Katherine A.; Liu, Yufeng; Hayward, Michele C.; Cabanski, Christopher R.; Muldrew, Kenneth; Miller, C. Ryan; Randell, Scott H.; Socinski, Mark A.; Parsons, Alden M.; Funkhouser, William K.; Lee, Carrie B.; Roberts, Patrick J.; Thorne, Leigh; Bernard, Philip S.; Perou, Charles M.; Hayes, D. Neil

    2010-01-01

    Purpose Lung squamous cell carcinoma (SCC) is clinically and genetically heterogeneous and current diagnostic practices do not adequately substratify this heterogeneity. A robust, biologically-based SCC subclassification may describe this variability and lead to more precise patient prognosis and management. We sought to determine if SCC mRNA expression subtypes exist, are reproducible across multiple patient cohorts, and are clinically relevant. Experimental Design Subtypes were detected by unsupervised consensus clustering in five published discovery cohorts of mRNA microarrays, totaling 382 SCC patients. An independent validation cohort of 56 SCC patients was collected and assayed by microarrays. A nearest-centroid subtype predictor was built using discovery cohorts. Validation cohort subtypes were predicted and evaluated for confirmation. Subtype survival outcome, clinical covariates, and biological processes were compared by statistical and bioinformatic methods. Results Four lung SCC mRNA expression subtypes, named primitive, classical, secretory, and basal, were detected and independently validated (P < 0.001). The primitive subtype had the worst survival outcome (P < 0.05) and is an independent predictor of survival (P < 0.05). Tumor differentiation and patient sex were associated with subtype. The subtypes’ expression profiles contained distinct biological processes (primitive – proliferation, classical – xeniobiotics metabolism, secretory – immune response, basal – cell adhesion) and suggested distinct pharmacologic interventions. Comparison to lung model systems revealed distinct subtype to cell type correspondence. Conclusions Lung SCC consists of four mRNA expression subtypes that have different survival outcomes, patient populations, and biological processes. The subtypes stratify patients for more precise prognosis and targeted research. PMID:20643781

  19. The Negative Sign and Exponential Expressions: Unveiling Students' Persistent Errors and Misconceptions

    ERIC Educational Resources Information Center

    Cangelosi, Richard; Madrid, Silvia; Cooper, Sandra; Olson, Jo; Hartter, Beverly

    2013-01-01

    The purpose of this study was to determine whether or not certain errors made when simplifying exponential expressions persist as students progress through their mathematical studies. College students enrolled in college algebra, pre-calculus, and first- and second-semester calculus mathematics courses were asked to simplify exponential…

  20. 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.

  1. Effects of ketamine exposure on dopamine concentrations and dopamine type 2 receptor mRNA expression in rat brain tissue

    PubMed Central

    Li, Bing; Liu, Mei-Li; Wu, Xiu-Ping; Jia, Juan; Cao, Jie; Wei, Zhi-Wen; Wang, Yu-Jin

    2015-01-01

    Objective: To explore the effects of ketamine abuse on the concentration of dopamine (DA), a monoamine neurotransmitter, and the mRNA expression of dopamine type 2 (D2) receptors in brain tissue, we used male Wistar rats to model ketamine abuse through chronic intraperitoneal infusion of ketamine across different doses. Methods: The rats were sacrificed 45 minutes and 1, 2, and 3 weeks after initiating the administration of ketamine or normal saline, as well as 3 days following discontinuation. Brain tissue was harvested to examine the concentration of 2,5-dihydroxyphenylacetic acid and homovanillic acid, the primary metabolites of DA, as well as the expression of D2 receptor mRNA. In addition, behavioral changes were observed within 30 minutes of administration, and withdrawal symptoms were also documented. A factorial experimental design was used to investigate variations and correlations in the primary outcome measures across the four doses and five time points. Brain DA concentrations were significantly higher in the ketamine-treated groups compared with the saline-treated group, with 30 mg/kg > 10 mg/kg > 60 mg/kg > saline (P < 0.05). The D2 receptor mRNA expression exhibited an inverse downregulation pattern, with 30 mg/kg < 10 mg/kg < 60 mg/kg < saline (P < 0.05). In the 10 mg/kg and 30 mg/kg ketamine-treated groups, the DA concentration and D2 receptor mRNA level in the brain tissue correlated with the dose of ketamine (r = 0.752, r = -0.806), but no significant correlation was found in the 60 mg/kg group. Result: These findings indicated that chronic dosing with ketamine increased the concentration of DA in rat brain tissue by increasing DA release or interrupting DA degradation. D2 receptor mRNA expression likely decreased because of stimulation with excessive DA. Conclusion: High-dose (60 mg/kg) ketamine had potent paralyzing effects on the central nervous system of rats and weakened the excitatory effects of the limbic system. Brain DA and D2 receptor mRNA

  2. mRNA and microRNA expression profiles of the NCI-60 integrated with drug activities

    PubMed Central

    Liu, Hongfang; D’Andrade, Petula; Fulmer-Smentek, Stephanie; Lorenzi, Philip; Kohn, Kurt W.; Weinstein, John N.; Pommier, Yves; Reinhold, William C.

    2010-01-01

    As part of the Spotlight on Molecular Profiling series, we present here new profiling studies of mRNA and microRNA expression for the 60 cell lines of the NCI DTP drug screen (NCI-60) using the 41,000-probe Agilent Whole Human Genome Oligo Microarray and the 15,000-feature Agilent Human microRNA Microarray V2. The expression levels of ~21,000 genes and 723 human microRNAs were measured. These profiling studies include quadruplicate technical replicates for six and eight cell lines for mRNA and microRNA, respectively, and duplicates for the remaining cell lines. The resulting data sets are freely available and searchable online in our CellMiner database. The result indicates high reproducibility for both platforms and an essential biological similarity across the various cell types. The mRNA and microRNA expression levels were integrated with our previously published 1,429-compound database of anticancer activity obtained from the NCI DTP drug screen. Large blocks of both mRNAs and microRNAs were identified with predominately unidirectional correlations to ~1,300 drugs including 121 drugs with known mechanisms of action. The data sets presented here will facilitate the identification of groups of mRNAs, microRNAs and drugs that potentially affect and interact with one another. PMID:20442302

  3. Limb segment vibration modulates spinal reflex excitability and muscle mRNA expression after spinal cord injury

    PubMed Central

    Chang, Shuo-Hsiu; Tseng, Shih-Chiao; McHenry, Colleen L.; Littmann, Andrew E.; Suneja, Manish; Shields, Richard K.

    2012-01-01

    Objective We investigated the effect of various doses of vertical oscillation (vibration) on soleus H-reflex amplitude and post-activation depression in individuals with and without SCI. We also explored the acute effect of short-term limb vibration on skeletal muscle mRNA expression of genes associated with spinal plasticity. Methods Six healthy adults and five chronic complete SCI subjects received vibratory stimulation of their tibia over three different gravitational accelerations (0.3g, 0.6g, and 1.2g) at a fixed frequency (30 Hz). Soleus H-reflexes were measured before, during, and after vibration. Two additional chronic complete SCI subjects had soleus muscle biopsies 3 h following a single bout of vibration. Results H-reflex amplitude was depressed over 83% in both groups during vibration. This vibratory-induced inhibition lasted over 2 min in the control group, but not in the SCI group. Post-activation depression was modulated during the long-lasting vibratory inhibition. A single bout of mechanical oscillation altered mRNA expression from selected genes associated with synaptic plasticity. Conclusions Vibration of the lower leg inhibits the H-reflex amplitude, influences post-activation depression, and alters skeletal muscle mRNA expression of genes associated with synaptic plasticity. Significance Limb segment vibration may offer a long term method to reduce spinal reflex excitability after SCI. PMID:21963319

  4. Chronic stress alters glucocorticoid receptor and mineralocorticoid receptor mRNA expression in the European starling (Sturnus vulgaris) brain.

    PubMed

    Dickens, M; Romero, L M; Cyr, N E; Dunn, I C; Meddle, S L

    2009-10-01

    Although the glucocorticoid response to acute short-term stress is an adaptive physiological mechanism that aids in the response to and survival of noxious stimuli, chronic stress is associated with a negative impact on health. In wild-caught European starlings (Sturnus vulgaris), chronic stress alters the responsiveness of hypothalamic-pituitary-adrenal (HPA) axis as measured by the acute corticosterone response. In the present study, we investigated potential underlying neuroendocrine mechanisms by comparing glucocorticoid receptor and mineralocorticoid receptor mRNA expression in the brains of chronically and nonchronically-stressed starlings. Hypothalamic paraventricular nucleus, but not hippocampal, glucocorticoid receptor mRNA expression in chronically-stressed birds was significantly lower compared to controls, suggesting changes in the efficacy of corticosterone negative feedback. In addition, chronically-stressed birds showed a significant decrease in hippocampal MR mRNA expression. Together, these results suggest that chronic stress changes the brain physiology of wild birds and provides important information for the understanding of the underlying mechanisms that result in dysregulation of the HPA axis in wild animals by chronic stress. PMID:19686439

  5. Altered mRNA expression of hepatic lipogenic enzyme and PPARalpha in rats fed dietary levan from Zymomonas mobilis.

    PubMed

    Kang, Soon Ah; Hong, Kyunghee; Jang, Ki-Hyo; Kim, Yun-Young; Choue, Ryowon; Lim, Yoongho

    2006-06-01

    Levan or high molecular beta-2,6-linked fructose polymer is produced extracellularly from sucrose-based substrates by bacterial levansucrase. In the present study, to investigate the effect of levan feeding on serum leptin, hepatic lipogenic enzyme and peroxisome proliferation-activated receptor (PPAR) alpha expression in high-fat diet-induced obese rats, 4-week-old Sprague-Dawley male rats were fed high-fat diet (beef tallow, 40% of calories as fat), and, 6 weeks later, the rats were fed 0%, 1%, 5% or 10% levan-supplemented diets for 4 weeks. Serum leptin and insulin level were dose dependently reduced in levan-supplemented diet-fed rats. The mRNA expressions of hepatic fatty acid synthase and acetyl CoA carboxylase, which are the key enzymes in fatty acid synthesis, were down-regulated by dietary levan. However, dietary levan did not affect the gene expression of hepatic malic enzyme, phosphatidate phosphohydrolase and HMG CoA reductase. Also, the lipogenic enzyme gene expression in the white adipose tissue (WAT) was not affected by the diet treatments. However, hepatic PPARalpha mRNA expression was dose dependently up-regulated by dietary levan, whereas PPARgamma in the WAT was not changed. The results suggest that the in vivo hypolipidemic effect of dietary levan, including anti-obesity and lipid-lowering, may result from the inhibition of lipogenesis and stimulation of lipolysis, accompanied with regulation of hepatic lipogenic enzyme and PPARalpha gene expression. PMID:16214330

  6. An mRNA expression signature for prognostication in de novo acute myeloid leukemia patients with normal karyotype

    PubMed Central

    Chou, Wen-Chien; Hou, Hsin-An; Tseng, Mei-Hsuan; Kuo, Yi-Yi; Chen, Yidong; Chuang, Eric Y.; Tien, Hwei-Fang

    2015-01-01

    Although clinical features, cytogenetics, and mutations are widely used to predict prognosis in patients with acute myeloid leukemia (AML), further refinement of risk stratification is necessary for optimal treatment, especially in cytogenetically normal (CN) patients. We sought to generate a simple gene expression signature as a predictor of clinical outcome through analyzing the mRNA arrays of 158 de novo CN AML patients. We compared the gene expression profiles of patients with poor response to induction chemotherapy with those who responded well. Forty-six genes expressed differentially between the two groups. Among them, expression of 11 genes was significantly associated with overall survival (OS) in univariate Cox regression analysis in 104 patients who received standard intensive chemotherapy. We integrated the z-transformed expression levels of these 11 genes to generate a risk scoring system. Higher risk scores were significantly associated with shorter OS (median 17.0 months vs. not reached, P < 0.001) in ours and another 3 validation cohorts. In addition, it was an independent unfavorable prognostic factor by multivariate analysis (HR 1.116, 95% CI 1.035~1.204, P = 0.004). In conclusion, we developed a simple mRNA expression signature for prognostication in CN-AML patients. This prognostic biomarker will help refine the treatment strategies for this group of patients. PMID:26517675

  7. Lack of correlation between mRNA expression and enzymatic activity of the aspartate aminotransferase isoenzymes in various tissues of the rat.

    PubMed

    Abruzzese, F; Greco, M; Perlino, E; Doonan, S; Marra, E

    1995-06-12

    Little is known about control of expression of basal levels of the aspartate aminotransferases which are ubiquitous 'house keeping' enzymes in vertebrates. We have measured both mRNA and activity levels for both isoenzymes in various rat tissues as a function of age. Patterns of mRNA expression for the two isoenzymes were similar in a particular tissue about differed widely between tissues. Surprisingly, there was no simple correlation between mRNA levels and specific activities of the enzyme products. We conclude that translation for mRNA for these two isoenzymes is subject to tissue-specific, and in some cases age-related, regulation. PMID:7789537

  8. Distribution of PTPN22 polymorphisms in SLE from western Mexico: correlation with mRNA expression and disease activity.

    PubMed

    Machado-Contreras, Jesús René; Muñoz-Valle, José Francisco; Cruz, Alvaro; Salazar-Camarena, Diana Celeste; Marín-Rosales, Miguel; Palafox-Sánchez, Claudia Azucena

    2016-08-01

    Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune inflammatory disease characterized by loss of self-tolerance with hyperactivation of autoreactive T and B cells. Protein tyrosine phosphatase non-receptor type 22 (PTPN22) encodes for lymphoid-specific phosphatase (Lyp), which is a key negative regulator of T lymphocyte activation. The aim of this study was to evaluate the genetic contribution of PTPN22 -1123G>C and +1858C>T polymorphisms and their haplotypes in SLE patients, as well as mRNA expression according to -1123G>C promoter polymorphism and disease activity. One hundred and fifty SLE patients and 150 unrelated healthy controls (HC), both Mexican mestizos, were genotyped by PCR-RFLP technique for the PTPN22 -1123G>C and +1858C>T polymorphisms. PTPN22 mRNA expression levels were determined by real-time PCR from PBMCs of thirty patients with SLE and fifteen HC carrying different genotypes. Distributions of genotype and allelic frequencies were similar between SLE and HC. The most frequent alleles were -1123 G and +1858 C in both groups (69 vs. 66 % and 97 vs. 98 %, in SLE and HC, respectively). However, the recessive model of inheritance analysis showed a lower frequency of -1123 CC genotype in SLE patients (7 vs. 15 %), suggesting a protection effect to develop SLE (OR 0.41, CI 1.10-5.28, p = 0.02). Haplotype analysis showed strong linkage disequilibrium D' = 0.98 for PTPN22 -1123G>C and +1858C>T polymorphisms, but haplotypes were not associated with SLE. The PTPN22 mRNA expression did not show differences among -1123G>C genotypes; nevertheless, a significant negative correlation with disease activity was found (r = -0.64, p < 0.01). SLE inactive patients showed similar PTPN22 mRNA expression levels to healthy controls, whereas in patients with severe flare, the expression was nearly depleted. In conclusion, we found a lack of association of PTPN22 -1123G>C and +1858C>T polymorphisms with the risk of developing SLE in a Mexican

  9. Characteristics of mRNA dynamic expression related to spinal cord ischemia/reperfusion injury: a transcriptomics study.

    PubMed

    Qi, Zhi-Ping; Xia, Peng; Hou, Ting-Ting; Li, Ding-Yang; Zheng, Chang-Jun; Yang, Xiao-Yu

    2016-03-01

    Following spinal cord ischemia/reperfusion injury, an endogenous damage system is immediately activated and participates in a cascade reaction. It is difficult to interpret dynamic changes in these pathways, but the examination of the transcriptome may provide some information. The transcriptome reflects highly dynamic genomic and genetic information and can be seen as a precursor for the proteome. We used DNA microarrays to measure the expression levels of dynamic evolution-related mRNA after spinal cord ischemia/reperfusion injury in rats. The abdominal aorta was blocked with a vascular clamp for 90 minutes and underwent reperfusion for 24 and 48 hours. The simple ischemia group and sham group served as controls. After rats had regained consciousness, hindlimbs showed varying degrees of functional impairment, and gradually improved with prolonged reperfusion in spinal cord ischemia/reperfusion injury groups. Hematoxylin-eosin staining demonstrated that neuronal injury and tissue edema were most severe in the 24-hour reperfusion group, and mitigated in the 48-hour reperfusion group. There were 8,242 differentially expressed mRNAs obtained by Multi-Class Dif in the simple ischemia group, 24-hour and 48-hour reperfusion groups. Sixteen mRNA dynamic expression patterns were obtained by Serial Test Cluster. Of them, five patterns were significant. In the No. 28 pattern, all differential genes were detected in the 24-hour reperfusion group, and their expressions showed a trend in up-regulation. No. 11 pattern showed a decreasing trend in mRNA whereas No. 40 pattern showed an increasing trend in mRNA from ischemia to 48 hours of reperfusion, and peaked at 48 hours. In the No. 25 and No. 27 patterns, differential expression appeared only in the 24-hour and 48-hour reperfusion groups. Among the five mRNA dynamic expression patterns, No. 11 and No. 40 patterns could distinguish normal spinal cord from pathological tissue. No. 25 and No. 27 patterns could distinguish simple

  10. Characteristics of mRNA dynamic expression related to spinal cord ischemia/reperfusion injury: a transcriptomics study

    PubMed Central

    Qi, Zhi-ping; Xia, Peng; Hou, Ting-ting; Li, Ding-yang; Zheng, Chang-jun; Yang, Xiao-yu

    2016-01-01

    Following spinal cord ischemia/reperfusion injury, an endogenous damage system is immediately activated and participates in a cascade reaction. It is difficult to interpret dynamic changes in these pathways, but the examination of the transcriptome may provide some information. The transcriptome reflects highly dynamic genomic and genetic information and can be seen as a precursor for the proteome. We used DNA microarrays to measure the expression levels of dynamic evolution-related mRNA after spinal cord ischemia/reperfusion injury in rats. The abdominal aorta was blocked with a vascular clamp for 90 minutes and underwent reperfusion for 24 and 48 hours. The simple ischemia group and sham group served as controls. After rats had regained consciousness, hindlimbs showed varying degrees of functional impairment, and gradually improved with prolonged reperfusion in spinal cord ischemia/reperfusion injury groups. Hematoxylin-eosin staining demonstrated that neuronal injury and tissue edema were most severe in the 24-hour reperfusion group, and mitigated in the 48-hour reperfusion group. There were 8,242 differentially expressed mRNAs obtained by Multi-Class Dif in the simple ischemia group, 24-hour and 48-hour reperfusion groups. Sixteen mRNA dynamic expression patterns were obtained by Serial Test Cluster. Of them, five patterns were significant. In the No. 28 pattern, all differential genes were detected in the 24-hour reperfusion group, and their expressions showed a trend in up-regulation. No. 11 pattern showed a decreasing trend in mRNA whereas No. 40 pattern showed an increasing trend in mRNA from ischemia to 48 hours of reperfusion, and peaked at 48 hours. In the No. 25 and No. 27 patterns, differential expression appeared only in the 24-hour and 48-hour reperfusion groups. Among the five mRNA dynamic expression patterns, No. 11 and No. 40 patterns could distinguish normal spinal cord from pathological tissue. No. 25 and No. 27 patterns could distinguish simple

  11. The Promoter Methylation Status and mRNA Expression Levels of CTCF and SIRT6 in Sporadic Breast Cancer

    PubMed Central

    Wang, Da; Zhang, Xuemei

    2014-01-01

    Promoter hypermethylation causes gene silencing and is thought to be an early event in carcinogenesis. This study was to detect promoter methylation status and mRNA expression levels of CCCTC-binding factor (CTCF) and sirtuin 6 (SIRT6), and to explore the relationship between methylation and mRNA expression in breast cancer patient samples. Promoter methylation analysis and expression profile analysis of two genes were performed by methylation-specific PCR, bisulfite sequencing PCR, and quantitative real-time PCR in cancer lesions and matched normal tissues. The promoter region of CTCF has not been hypermethylated in all patient samples. In contrast, methylation of SIRT6 gene was present in invasive cancers (93.5%) and matched normal tissues (96.8%) from 62 patients. Promoter hypermethylation of SIRT6 was also observed in ductal carcinoma in situ (three of three) and matched normal tissues (two of three). mRNA expression of CTCF and SIRT6 in invasive tumors showed a lower level than that in paired normal tissues (p=0.008 and p=0.030, respectively). The fold change values of CTCF expression were significantly lower in invasive ductal cancer lesions with Ki-67-positive status (p=0.042). In conclusion, our data showed that the methylation status of CTCF and SIRT6 promoter regions was not statistically different in cancer lesions compared with matched normal tissues. No significant association between promoter methylation status and expression profiles of CTCF and SIRT6 was found in invasive breast cancers. PMID:24842653

  12. Molecular cloning, mRNA expression, and characterization of HSP90 gene from Chinese mitten crab Eriocheir japonica sinensis.

    PubMed

    Li, Peng; Zha, Jie; Zhang, Zhenhua; Huang, Hua; Sun, Hongying; Song, Daxiang; Zhou, Kaiya

    2009-07-01

    HSP90 is a highly conserved molecular chaperone important in the maturation of a broad spectrum of proteins. Using expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) techniques, an HSP90 gene designated as EjsHSP90 was cloned and characterized from the Chinese mitten crab Eriocheir japonica sinensis. The full-length cDNA of EjsHSP90 is 2,517 bp and contains an open reading frame of 2157 bp which encodes a 718 amino acid polypeptide (82.8 kDa) bearing characteristics of the HSP90 family and an ATP binding domain. Sequence alignment shows that EjsHSP90 shared 79%-96% identity with HSP90 sequences reported in other animals, and it shares identical structural features. Fluorescent real-time quantitative RT-PCR approach was performed to examine the expression profiles of EjsHSP90 mRNA by testing its relative level in three types of tissues at three different developmental stages, respectively. We found that EjsHSP90 is expressed throughout the three developmental stages but expression levels varied among different body parts of crabs. EjsHSP90 mRNA expression in the abdomen of the first crab stage is consistently higher than that of the other two stages, suggesting that EjsHSP90 gene is involved in the crabs' early developmental process, especially in the crab brachyurization process. Results from quantitative RT-PCR excluded the possibility that the expression of EjsHSP90 mRNA is induced primarily by osmotic stress. Phylogenetic analyses reveal that HSP90 gene is informative and complementary for reconstruction of arthropod phylogenetic relationships. PMID:19166961

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

    PubMed Central

    NISHIYAMA, Yoshihiro; NAKAYAMA, Shouta M.M.; WATANABE, Kensuke P.; KAWAI, Yusuke K.; OHNO, Marumi; IKENAKA, Yoshinori; ISHIZUKA, Mayumi

    2016-01-01

    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. PMID:26806536

  14. 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...

  15. Effects of starter feeding and early weaning on GHR mRNA expression in liver and rumen of lambs from birth to 84 days of age.

    PubMed

    Wang, Fangbin; Li, Chong; Li, Fadi; Wang, Weimin; Wang, Xiaojuan; Liu, Ting; Ma, Zhiyuan; Li, Baosheng

    2016-06-01

    The growth hormone receptor (GHR) is associated with animal growth and development. To investigate such effects on GHR gene expression, a total of 102 Hu lambs were randomly allocated to one of three groups (Group 1: starter diet from 7 d of age, weaning at 56 d of age; Group 2: starter diet from 42 d of age, weaning at 56 d of age; Group 3: starter diet from 7 d of age; weaning at 28 d of age). Six lambs from each group were sacrificed every 14 d to investigate the effects of starter feeding and weaning age on GHR mRNA expression in the liver and rumen. The results revealed that GHR mRNA expression was significantly higher in the liver and rumen (p < 0.05) than in other tissues. Early starter feeding up-regulated hepatic GHR mRNA expression on days 14, 28, 42 and 56 and ruminal GHR mRNA expression on days 28, 42, 70, and 84 (p < 0.05). Early weaning up-regulated hepatic GHR mRNA expression on days 56, 70 and 84 and ruminal GHR mRNA expression on days 42, 56, 70 and 84 (p < 0.05). Dietary and weaning regimes and age affected the hepatic and ruminal GHR mRNA expression. PMID:27032032

  16. Expression of beta 3-adrenoceptor mRNA in rat brain.

    PubMed Central

    Summers, R. J.; Papaioannou, M.; Harris, S.; Evans, B. A.

    1995-01-01

    The reverse transcription/polymerase chain reaction was used to demonstrate beta 3-adrenoceptor mRNA in rat brain regions. Levels were highest in hippocampus, cerebral cortex and striatum and lower in hypothalamus, brainstem and cerebellum. Images Figure 1 PMID:8590968

  17. Genome-wide identification and analysis of mRNA expression in fibroblasts, ES cells, and iPS cells.

    PubMed

    Hirai, Hiroyuki; Kikyo, Nobuaki

    2016-03-01

    Genome-wide expression patterns of mRNA were compared between mouse embryonic fibroblasts (MEFs), embryonic stem cells (ESCs), and various types of induced pluripotent stem cells (iPSCs). iPSCs were established and maintained using modified Oct4 with or without exogenous leukemia inhibitory factor (LIF) and used to identify mRNAs that were potentially involved in the LIF-independence. The data have been deposited in the NCBI's Gene Expression Omnibus (GEO) database with the accession number GSE65563. PMID:26981399

  18. Genome-wide identification and analysis of mRNA expression in fibroblasts, ES cells, and iPS cells

    PubMed Central

    Hirai, Hiroyuki; Kikyo, Nobuaki

    2015-01-01

    Genome-wide expression patterns of mRNA were compared between mouse embryonic fibroblasts (MEFs), embryonic stem cells (ESCs), and various types of induced pluripotent stem cells (iPSCs). iPSCs were established and maintained using modified Oct4 with or without exogenous leukemia inhibitory factor (LIF) and used to identify mRNAs that were potentially involved in the LIF-independence. The data have been deposited in the NCBI's Gene Expression Omnibus (GEO) database with the accession number GSE65563. PMID:26981399

  19. Basal expression of nucleoside transporter mRNA differs among small intestinal epithelia of beef steers and is differentially altered by ruminal or abomasal infusion of starch hydrolysate.

    PubMed

    Liao, S F; Alman, M J; Vanzant, E S; Miles, E D; Harmon, D L; McLeod, K R; Boling, J A; Matthews, J C

    2008-04-01

    In ruminants, microbial-derived nucleic acids are a major source of N and are absorbed as nucleosides by small intestinal epithelia. Although the biochemical activities of 2 nucleoside transport systems have been described for cattle, little is known regarding the regulation of their gene expression. This study was conducted to test 2 hypotheses: (1) the small intestinal epithelia of beef cattle differentially express mRNA for 3 concentrative (CNT1, 2, 3) and 2 equilibrative (ENT1, 2) nucleoside transporters (NT), and (2) expression of these NT is responsive to small intestine luminal supply of rumen-derived microbes (hence, nucleosides), energy (cornstarch hydrolysate, SH), or both. Eighteen ruminally and abomasally catheterized Angus steers (260 +/- 17 kg of BW) were fed an alfalfa cube-based diet at 1.33x NE(m) requirement. Six steers in each of 3 periods were blocked by BW (heavy vs. light). Within each block, 3 steers were randomly assigned to 3 treatments (n = 6): ruminal and abomasal water infusion (control), ruminal SH infusion/abomasal water infusion, or ruminal water infusion/abomasal SH infusion. The dosage of SH infusion amounted to 20% of ME intake. After a 14-or 16-d infusion period, steers were slaughtered, and duodenal, jejunal, and ileal epithelia were harvested for total RNA extraction and the relative amounts of mRNA expressed were determined using real-time RT-PCR quantification methodologies. All 5 NT mRNA were found expressed by each epithelium, but their abundance differed among epithelia. Specifically, jejunal expression of all 5 NT mRNA was higher than that by the ileum, whereas jejunal expression of CNT1, CNT3, and ENT1 mRNA was higher, or tended to be higher, than duodenal expression. Duodenal expression of CNT2, CNT3, and ENT2 mRNA was higher than ileal expression. With regard to SH infusion treatments, ruminal infusion increased duodenal expression of CNT3 (67%), ENT1 (51%), and ENT2 (39%) mRNA and ileal expression of CNT3 (210%) and

  20. 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

  1. [Effects of PTK787 on cell proliferation and expression of fak mRNA in K562].

    PubMed

    Di, Xiao-Hua; Chen, Ri-Ling; Liu, Xiao-Li; Tian, Chuan; Guo, Ya-Nan

    2010-06-01

    The aim of this study was to investigate the effects of tyrosine kinase inhibitor PTK787 on cell proliferation, cell cycle and the expression of fak mRNA of human chronic myeloid leukemia (CML) cell line K562, and to explore the mechanism of PTK787 against acute myeloid leukemia. The MTT method was used to detect the effects of PTK787 in various concentrations and at different time points on proliferation of K562 cells; the flow cytometry was used to determine the effects of PTK787 in different concentrations on cell cycle of K562 cells; the RT-PCR was used to assay the expression of fak mRNA in K562 cells treated with PTK787 for 48 hours. The results showed that along with increasing of the concentration and prolonging of time, the inhibitory rate of PTK787 on K562 proliferation was gradually enhanced. The comparison between various concentration groups at same time or comparison between various time groups in same concentration showed significant differences (p < 0.05), in which the effect of 320 micromol/L PTK787 on cells was strongest, while the continuous increase of PTK787 concentration or prolong of action time did not enhance the inhibitory rate on K562 proliferation. With increasing of drug concentration, the cell proportion in G(1) phase gradually increased, the cell proportion in S phase gradually decreased, the comparison between various groups revealed significant differences (p < 0.05), however the continuous increase of drug concentration from 160 micromol/L did not obviously change the cell proportion in phases of cell cycle. With increasing of drug concentration, the expression of fak mRNA in K562 cells gradually reduced with significant differences between various groups (p < 0.05), but with continuous increase of drug concentration from 160 micromol/L, the effect of PTK787 on the expression of fak mRNA in K562 cells also did not obviously change. It is concluded that the PTK787 shows effect of anti-leukemia cells through inhibiting transformation

  2. Changes in neurotransmitter levels and proinflammatory cytokine mRNA expressions in the mice olfactory bulb following nanoparticle exposure.

    PubMed

    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 microg) intranasally. Six hours after the nanoparticle instillation, the mice were intraperitoneally injected with normal saline or 50 mug 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 in the

  3. Runx3 Expression Inhibits Proliferation and Distinctly Alters mRNA Expression of Bax in AGS and A549 Cancer Cells

    PubMed Central

    Torshabi, Maryam; Faramarzi, Mohammad Ali; Tabatabaei Yazdi, Mojtaba; Ostad, Seyyed Naser; Gharemani, Mohammad Hosein

    2011-01-01

    Runx3, a member of Runt-related transcription factor (Runx) proteins with tumor suppressor effect, is a tissue–restricted and cancer related transcription factor that regulate cell proliferation and growth, as well as differentiation. In the present study, exogenous Run3 was transiently expressed in AGS (human gastric adenocarcinoma), with undetectable Runx3 protein and in A549 (human lung carcinoma) with low levels of endogenous Runx3 protein. The GFP tagged Runx3 was transfected into AGS and A549 cells using fugene6 and PolyFect and Runx3 expression was confirmed by fluorescent microscopy and RT-PCR. The effect of Runx3 transfection on cell proliferation was determined by MTT assay and the results were confirmed by the trypan blue dye exclusion method. The effect of Runx3 expression on mRNA expression of BCL2-associated X protein (Bax) was evaluated using RT-PCR. In AGS and A549 cells, Runx3 expression inhibited cell proliferation (p < 0.01). The growth inhibition was less in A549 cells. We show that Runx3 expression increases Bax mRNA expression in AGS cells when compared with control (p < 0.05), but no significant differences in mRNA expression was observed in both examined cells. Runx3 expression has antiproliferative effect in AGS cell perhaps via increase in expression of Bax. The effect of Runx3 on A549 cells’ viability which has endogenous level of Runx3 is not related to Bax. These findings implicate a complex regulation by Runx3 in inhibition of cell proliferation utilizing Bax. PMID:24250365

  4. 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

    2016-06-01

    l-DOPA is the primary pharmacological treatment for relief of the motor symptoms of Parkinson's disease (PD). With prolonged treatment (⩾5years) 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 (4mg/kg and 7.5mg/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, 60min 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. PMID:26968766

  5. 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.

  6. 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

    2016-01-01

    Bacterial adaptation requires large-scale regulation of gene expression. We have performed a genome-wide analysis of the Csr system, which regulates many important cellular functions. The Csr system is involved in post-transcriptional regulation, but a role in transcriptional regulation has also been suggested. Two proteins, an RNA-binding protein CsrA and an atypical signaling protein CsrD, participate in the Csr system. Genome-wide transcript stabilities and levels were compared in wildtype E. coli (MG1655) and isogenic mutant strains deficient in CsrA or CsrD activity demonstrating for the first time that CsrA and CsrD are global negative and positive regulators of transcription, respectively. The role of CsrA in transcription regulation may be indirect due to the 4.6-fold increase in csrD mRNA concentration in the CsrA deficient strain. Transcriptional action of CsrA and CsrD on a few genes was validated by transcriptional fusions. In addition to an effect on transcription, CsrA stabilizes thousands of mRNAs. This is the first demonstration that CsrA is a global positive regulator of mRNA stability. For one hundred genes, we predict that direct control of mRNA stability by CsrA might contribute to metabolic adaptation by regulating expression of genes involved in carbon metabolism and transport independently of transcriptional regulation. PMID:27112822

  7. Upregulation of metabotropic glutamate receptor 8 mRNA expression in the rat forebrain after repeated amphetamine administration

    PubMed Central

    Parelkar, Nikhil K; Wang, John Q.

    2008-01-01

    Metabotropic glutamate receptors (mGluRs) are G-protein-coupled receptors and are densely expressed in the forebrain of adult rats. Accumulative evidence suggests a critical role of mGluRs in the regulation of normal physiological activity of neurons and pathogenesis of mental illnesses such as schizophrenia, depression, and substance addiction. In this study, we investigated alterations in mGluR8 subtype mRNA expression in the rat forebrain in response to repeated intraperitoneal administration of amphetamine (twice daily for 12 days, 5 mg/kg per injection) using quantitative in situ hybridization. We found that mGluR8 mRNA levels were profoundly increased in the dorsal (caudate putamen) and ventral (nucleus accumbens) striatum 1 day after the discontinuation of amphetamine treatments. Such increases were sustained up to 21 days of withdrawal. Increases in mGluR8 mRNAs were also found in the cerebral cortex, including the cingulate and sensory cortex but not the piriform cortex, at 1 and 21 days. These data demonstrate a positive response of mGluR8 in mRNA abundance in most forebrain regions to repeated stimulant exposure. PMID:18255232

  8. The Csr system regulates genome-wide mRNA stability and transcription and thus gene expression in Escherichia coli

    PubMed Central

    Esquerré, Thomas; Bouvier, Marie; Turlan, Catherine; Carpousis, Agamemnon J.; Girbal, Laurence; Cocaign-Bousquet, Muriel

    2016-01-01

    Bacterial adaptation requires large-scale regulation of gene expression. We have performed a genome-wide analysis of the Csr system, which regulates many important cellular functions. The Csr system is involved in post-transcriptional regulation, but a role in transcriptional regulation has also been suggested. Two proteins, an RNA-binding protein CsrA and an atypical signaling protein CsrD, participate in the Csr system. Genome-wide transcript stabilities and levels were compared in wildtype E. coli (MG1655) and isogenic mutant strains deficient in CsrA or CsrD activity demonstrating for the first time that CsrA and CsrD are global negative and positive regulators of transcription, respectively. The role of CsrA in transcription regulation may be indirect due to the 4.6-fold increase in csrD mRNA concentration in the CsrA deficient strain. Transcriptional action of CsrA and CsrD on a few genes was validated by transcriptional fusions. In addition to an effect on transcription, CsrA stabilizes thousands of mRNAs. This is the first demonstration that CsrA is a global positive regulator of mRNA stability. For one hundred genes, we predict that direct control of mRNA stability by CsrA might contribute to metabolic adaptation by regulating expression of genes involved in carbon metabolism and transport independently of transcriptional regulation. PMID:27112822

  9. Integration of mRNA expression profile, copy number alterations, and microRNA expression levels in breast cancer to improve grade definition.

    PubMed

    Cava, Claudia; Bertoli, Gloria; Ripamonti, Marilena; Mauri, Giancarlo; Zoppis, Italo; Della Rosa, Pasquale Anthony; Gilardi, Maria Carla; Castiglioni, Isabella

    2014-01-01

    Defining the aggressiveness and growth rate of a malignant cell population is a key step in the clinical approach to treating tumor disease. The correct grading of breast cancer (BC) is a fundamental part in determining the appropriate treatment. Biological variables can make it difficult to elucidate the mechanisms underlying BC development. To identify potential markers that can be used for BC classification, we analyzed mRNAs expression profiles, gene copy numbers, microRNAs expression and their association with tumor grade in BC microarray-derived datasets. From mRNA expression results, we found that grade 2 BC is most likely a mixture of grade 1 and grade 3 that have been misclassified, being described by the gene signature of either grade 1 or grade 3. We assessed the potential of the new approach of integrating mRNA expression profile, copy number alterations, and microRNA expression levels to select a limited number of genomic BC biomarkers. The combination of mRNA profile analysis and copy number data with microRNA expression levels led to the identification of two gene signatures of 42 and 4 altered genes (FOXM1, KPNA4, H2AFV and DDX19A) respectively, the latter obtained through a meta-analytical procedure. The 42-based gene signature identifies 4 classes of up- or down-regulated microRNAs (17 microRNAs) and of their 17 target mRNA, and the 4-based genes signature identified 4 microRNAs (Hsa-miR-320d, Hsa-miR-139-5p, Hsa-miR-567 and Hsa-let-7c). These results are discussed from a biological point of view with respect to pathological features of BC. Our identified mRNAs and microRNAs were validated as prognostic factors of BC disease progression, and could potentially facilitate the implementation of assays for laboratory validation, due to their reduced number. PMID:24866763

  10. Global RT-PCR and RT-qPCR Analysis of the mRNA Expression of the Human PTPome.

    PubMed

    Nunes-Xavier, Caroline E; Pulido, Rafael

    2016-01-01

    Comprehensive comparative gene expression analysis of the tyrosine phosphatase superfamily members (PTPome) under cell- or tissue-specific growth conditions may help to define their individual and specific role in physiology and disease. Semi-quantitative and quantitative PCR are commonly used methods to analyze and measure gene expression. Here, we describe technical aspects of PTPome mRNA expression analysis by semi-quantitative RT-PCR and quantitative RT-PCR (RT-qPCR). We provide a protocol for each method consisting in reverse transcription followed by PCR using a global platform of specific PTP primers. The chapter includes aspects from primer validation to the setup of the PTPome RT-qPCR platform. Examples are given of PTP-profiling gene expression analysis using a human breast cancer cell line upon long-term or short-term treatment with cell signaling-activation agents. PMID:27514798

  11. 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. PMID:26764533

  12. In situ expression of intercellular adhesion molecule-1 (ICAM-1) mRNA in calves with acute Pasteurella haemolytica pneumonia.

    PubMed

    Radi, Z A; Register, K B; Lee, E K; Kehrli, M E; Brogden, K A; Gallup, J M; Ackermann, M R

    1999-09-01

    The in situ expression of intercellular adhesion molecule-1 (ICAM-1) mRNA in normal and pneumonic lung tissues of Holstein calves with bovine leukocyte adhesion deficiency (BLAD) was compared with that of age-matched non-BLAD Holstein calves by in situ hybridization. Twenty-four Holstein calves (both BLAD and non-BLAD) were randomly assigned to one of two experimental groups and inoculated intrabronchially with Pasteurella haemolytica or pyrogen-free saline. Lung tissues were collected and fixed in 10% neutral formalin at 2 or 4 hours postinoculation (PI). The expression and distribution of ICAM-1 mRNA in the different cell types of the lung tissue was detected by in situ hybridization with a 307-base-pair bovine ICAM-1 riboprobe. In lungs of both non-BLAD and BLAD saline-inoculated calves, ICAM-1 expression was present in epithelial cells but occurred in <30% of cells in bronchi, bronchioles, and alveoli. ICAM-1 expression in vascular endothelial cells was present in <30% of cells in pulmonary arteries and veins. The expression of ICAM-1 was significantly greater (>60% of cells) in bronchiolar and alveolar epithelial cells and pulmonary endothelial cells of arteries and veins in both BLAD and non-BLAD calves inoculated with P. haemolytica. Bronchiolar epithelium had the highest intensity of mRNA expression and highest percentage of cells that were stained, whereas bronchial epithelium had the lowest intensity and percentage of cells stained. Most alveolar macrophages and neutrophils in infected lungs also expressed ICAM-1. ICAM-1 expression was generally increased in infected BLAD calves at 2 hours PI as compared with non-BLAD calves but not at 4 hours PI. The increased expression of ICAM-1 during acute P. haemolytica pneumonia in calves suggests that ICAM-1 is upregulated and may play a role in leukocyte infiltration. The extent of ICAM-1 expression in P. haemolytica-inoculated calves with BLAD was initially enhanced but otherwise similar to that in non

  13. Elevated Intracellular Calcium Increases Ferritin H Expression Through an NFAT-Independent Posttranscriptional Mechanism Involving mRNA Stabilization

    PubMed Central

    MacKenzie, Elizabeth L.; Tsuji, Yoshiaki

    2009-01-01

    An increase in intracellular Ca2+ is one of the initiating events in T cell activation. A calcium-mediated signaling cascade in T cells involves activation of calcineurin and the dephosphorylation and translocation of Nuclear Factor of Activated T-cells (NFAT), resulting in the transcriptional activation of target genes such as IL-2. In the present study, we found that increased intracellular calcium leads to induction of the antioxidant protein ferritin H. We previously reported that the ferritin H gene is transcriptionally activated under oxidative stress conditions through an antioxidant responsive element (ARE). The facts that the ferritin H ARE contains a composite AP1 site, and that NFAT collaborates with AP1 transcription factors, led us to test whether calcium-activated NFAT is involved in the ferritin H induction through the ARE. Treatment of Jurkat T cells with the calcium ionophore, ionomycin, increased ferritin H mRNA and protein expression. Though NFAT translocated to the nucleus and bound a consensus NFAT sequence located in the IL-2 promoter following ionomycin treatment, it did not activate ferritin H transcription despite the presence of a putative NFAT binding sequence in the ferritin H ARE. In addition, the calcineurin inhibitor cyclosporin A treatment blocked ionomycin-mediated NFAT nuclear translocation but failed to abrogate the increase in ferritin H mRNA. Analysis of mRNA stability following actinomycin D treatment revealed that ionomycin prolongs ferritin H mRNA half-life. Taken together, these results suggest that ionomycin-mediated induction of ferritin H may occur in an NFAT-independent manner but through posttranscriptional stabilization of the ferritin H mRNA. PMID:18076382

  14. Occupational Toluene Exposure Induces Cytochrome P450 2E1 mRNA Expression in Peripheral Lymphocytes

    PubMed Central

    Mendoza-Cantú, Ania; Castorena-Torres, Fabiola; de León, Mario Bermúdez; Cisneros, Bulmaro; López-Carrillo, Lizbeth; Rojas-García, Aurora E.; Aguilar-Salinas, Alberto; Manno, Maurizio; Albores, Arnulfo

    2006-01-01

    Print workers are exposed to organic solvents, of which the systemic toxicant toluene is a main component. Toluene induces expression of cytochrome P450 2E1 (CYP2E1), an enzyme involved in its own metabolism and that of other protoxicants, including some procarcinogens. Therefore, we investigated the association between toluene exposure and the CYP2E1 response, as assessed by mRNA content in peripheral lymphocytes or the 6-hydroxychlorzoxazone (6OH-CHZ)/chlorzoxazone (CHZ) quotient (known as CHZ metabolic ratio) in plasma, and the role of genotype (5′-flanking region RsaI/PstI polymorphic sites) in 97 male print workers. The geometric mean (GM) of toluene concentration in the air was 52.80 ppm (10–760 ppm); 54% of the study participants were exposed to toluene concentrations that exceeded the maximum permissible exposure level (MPEL). The GM of urinary hippuric acid at the end of a work shift (0.041 g/g creatinine) was elevated relative to that before the shift (0.027 g/g creatinine; p < 0.05). The GM of the CHZ metabolic ratio was 0.33 (0–9.3), with 40% of the subjects having ratios below the GM. However, the average CYP2E1 mRNA level in peripheral lymphocytes was 1.07 (0.30–3.08), and CYP2E1 mRNA levels within subjects correlated with the toluene exposure ratio (environmental toluene concentration:urinary hippuric acid concentration) (p = 0.014). Genotype did not alter the association between the toluene exposure ratio and mRNA content. In summary, with further validation, CYP2E1 mRNA content in peripheral lymphocytes could be a sensitive and noninvasive biomarker for the continuous monitoring of toluene effects in exposed persons. PMID:16581535

  15. Late-Postnatal Cannabinoid Exposure Persistently Increases FoxP2 Expression within Zebra Finch Striatum

    PubMed Central

    Soderstrom, Ken; Luo, Bin

    2010-01-01

    Prior work has shown that cannabinoid exposure of zebra finches during sensorimotor stages of vocal development alters song patterns produced in adulthood. We are currently working to identify physiological substrates for this altered song learning. FoxP2 is a transcription factor associated with altered vocal development in both zebra finches and humans. This protein shows a distinct pattern of expression within Area X of striatum that coincides with peak expression of CB1 cannabinoid receptors during sensorimotor learning. Coincident expression in a brain region essential for song learning led us to test for a potential signaling interaction. We have found that cannabinoid agonists acutely increase expression of FoxP2 throughout striatum. When administered during sensorimotor song learning, cannabinoids increase basal levels of striatal FoxP2 expression in adulthood. Thus, song-altering cannabinoid treatments are associated with persistent increases in basal expression of FoxP2 in zebra finch striatum. PMID:20017118

  16. Complex control of GABA(A) receptor subunit mRNA expression: variation, covariation, and genetic regulation.

    PubMed

    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

  17. 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.

  18. The expression and localization of mRNA for colony-stimulating factor (CSF)-1 in human term placenta.

    PubMed

    Kanzaki, H; Yui, J; Iwai, M; Imai, K; Kariya, M; Hatayama, H; Mori, T; Guilbert, L J; Wegmann, T G

    1992-04-01

    A 4-kb mRNA for colony-stimulating factor 1 (CSF-1) was detected in normal human placenta at term by Northern blot analysis. In-situ hybridization revealed that the mRNA for CSF-1 was localized in the mesenchymal cells of the chorionic villous stroma, but not in the trophoblasts or capillary epithelial cells. Because there are significant numbers of tissue macrophages (Hofbauer cells) in the placental stroma and because the receptor for CSF-1 (the c-fms proto-oncogene product) is known to be expressed by trophoblasts, our results suggest that CSF-1 produced by placental stromal cells may act as a growth and survival factor for human placental macrophages and trophoblasts. PMID:1522204

  19. Predominant mucosal IL-8 mRNA expression in non-cagA Thais is risk for gastric cancer

    PubMed Central

    Yamada, Sirikan; Kato, Shunji; Matsuhisa, Takeshi; Makonkawkeyoon, Luksana; Yoshida, Masaru; Chakrabandhu, Thiraphat; Lertprasertsuk, Nirush; Suttharat, Pawit; Chakrabandhu, Bandhuphat; Nishiumi, Shin; Chongraksut, Wilaiwan; Azuma, Takeshi

    2013-01-01

    AIM: To study gastric mucosal interleukine-8 (IL-8) mRNA expression, the cytotoxin-associated gene A (cagA) mutation, and serum pepsinogen (PG) I/II ratio related risk in Thai gastric cancer. METHODS: There were consent 134 Thai non-cancer volunteers who underwent endoscopic narrow band imaging examination, and 86 Thais advance gastric cancer patients who underwent endoscopic mucosal biopsies and gastric surgery. Tissue samples were taken by endoscopy with 3 points biopsies. The serum PG I, II, and Helicobacter pylori (H. pylori) immunoglobulin G (IgG) antibody for H. pylori were tested by enzyme-linked immunosorbent assay technique. The histopathology description of gastric cancer and non-cancer with H. pylori detection was defined with modified Sydney Score System. Gastric mucosal tissue H. pylori DNA was extracted and genotyped for cagA mutation. Tissue IL-8 and cyclooxygenase-2 (COX-2) mRNA expression were conducted by real time relative quantitation polymerase chain reaction. From 17 Japanese advance gastric cancer and 12 benign gastric tissue samples, all were tested for genetic expression with same methods as well as Thai gastric mucosal tissue samples. The multivariate analysis was used for the risk study. Correlation and standardized t-test were done for quantitative data, P value < 0.05 was considered as a statistically significant. RESULTS: There is a high non cagA gene of 86.8 per cent in Thai gastric cancer although there are high yields of the East Asian type in the positive cagA. The H. pylori infection prevalence in this study is reported by combined histopathology and H. pylori IgG antibody test with 77.1% and 97.4% of sensitivity and specificity, respectively. The serum PG I/II ratio in gastric cancer is significantly lower than in the non-cancer group, P = 0.045. The serum PG I/II ratio of less than 3.0 and IL-8 mRNA expression ≥ 100 or log10 ≥ 2 are significant cut off risk differences between Thai cancer and non-cancer, P = 0.03 and

  20. 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

  1. Regulation of CYP27B1 mRNA Expression in Primary Human Osteoblasts.

    PubMed

    van der Meijden, K; van Essen, H W; Bloemers, F W; Schulten, E A J M; Lips, P; Bravenboer, N

    2016-08-01

    The enzyme 1α-hydroxylase (gene CYP27B1) catalyzes the synthesis of 1,25(OH)2D in both renal and bone cells. While renal 1α-hydroxylase is tightly regulated by hormones and 1,25(OH)2D itself, the regulation of 1α-hydroxylase in bone cells is poorly understood. The aim of this study was to investigate in a primary human osteoblast culture whether parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23), calcitonin, calcium, phosphate, or MEPE affect mRNA levels of CYP27B1. Our results show that primary human osteoblasts in the presence of high calcium concentrations increase their CYP27B1 mRNA levels by 1.3-fold. CYP27B1 mRNA levels were not affected by PTH1-34, rhFGF23, calcitonin, phosphate, and rhMEPE. Our results suggest that the regulation of bone 1α-hydroxylase is different from renal 1α-hydroxylase. High calcium concentrations in bone may result in an increased local synthesis of 1,25(OH)2D leading to an enhanced matrix mineralization. In this way, the local synthesis of 1,25(OH)2D may contribute to the stimulatory effect of calcium on matrix mineralization. PMID:27016371

  2. Lentiviral vectors with CMV or MHCII promoters administered in vivo: immune reactivity versus persistence of expression.

    PubMed

    Kimura, Takahiro; Koya, Richard C; Anselmi, Laura; Sternini, Catia; Wang, He-Jing; Comin-Anduix, Begonya; Prins, Robert M; Faure-Kumar, Emmanuelle; Rozengurt, Nora; Cui, Yan; Kasahara, Noriyuki; Stripecke, Renata

    2007-07-01

    Lentiviral vectors (LVs) are potential tools for genetic vaccination. To improve the safety of LV vaccines, we evaluated the selectivity, bio-distribution, persistence of expression, and immune potency of vesicular stomatitis virus G (VSV-G)-pseudotyped vectors transcriptionally targeted to antigen presenting cells (APCs) through a major histocompatibility complex class II (MHCII) promoter. Control vectors contained the ubiquitous cytomegalovirus (CMV) promoter. Bio-distribution studies after intravenous injections of LVs expressing green fluorescent protein (GFP) or luciferase were conducted by a combination of flow cytometry, immunofluorescence, real-time quantitative polymerase chain reaction (RT-Q-PCR) and whole-body bioluminescence analyses. GFP-expressing vectors showed selective expression in MHCII(+) cells of spleen and LV-CMV-GFP administration produced noticeable spleen inflammation, whereas LV-MHCII-GFP did not. Long-term optical imaging analyses of C57BL/6 mice injected with LV-CMV-LUC showed diminishing luciferase expression in the liver and spleen over time. Vaccination/boost with LV-CMV expressing the melanoma antigen tyrosinase-related protein 2 (TRP2) yielded dose-dependent antigen-specific CD8(+) T-cell reactivity and high protection against B16 melanoma challenge. Unexpectedly, administration of LVs containing the MHCII promoter resulted in persistence of luciferase expression and viral integration in MHCII(+) splenocytes and virtually no CD8(+) T-cell responses against TRP2. These studies reveal that APC transduction by LVs could lead to immune reactivity (LV-CMV) or persistence of transgene expression (LV-MHCII), providing a relevant paradigm for vaccination and gene replacement approaches. PMID:17505480

  3. Naproxen sodium decreases prostaglandins secretion from cultured human endometrial stromal cells modulating metabolizing enzymes mRNA expression.

    PubMed

    Carrarelli, Patrizia; Funghi, Lucia; Bruni, Simone; Luisi, Stefano; Arcuri, Felice; Petraglia, Felice

    2016-04-01

    Dysmenorrhea, defined as painful cramps occurring immediately before or during the menstrual period, is a common symptom of different gynecological diseases. An acute uterine inflammatory response driven by prostaglandins (PGs) is responsible for painful symptoms. Progesterone withdrawal is responsible for activation of cyclooxygenase (COX-2) enzyme and decrease of hydroxyprostaglandin dehydrogenase (HPDG) with consequent increased secretion of PGs secretion, inducing uterine contractility and pain. The most widely used drugs for the treatment of pelvic pain associated with menstrual cycle are non steroidal anti-inflammatory drugs (NSAIDs). The uterine site of action of these drugs is still not defined and the present study evaluated the effect of naproxen sodium in cultured human endometrial stromal cells (HESC) collected from healthy women. PGE2 release was measured by ELISA; COX-2 and HPDG mRNA expression were assessed by qRT-PCR. Naproxen sodium did not affect HESC vitality. Naproxen sodium significantly decreased PGE2 secretion (p < 0.01) and COX-2 mRNA expression (p < 0.01). TNF-α induced PGE2 release was reduced in presence of naproxen sodium (p < 0.05), in association with decreased COX-2 and increased HPDG mRNAs expression. Naproxen sodium decreases endometrial PGE2 release induced by inflammatory stimulus acting on endometrial COX-2 and HPDG expression, suggesting endometrial synthesis of prostaglandins as a possible target for reduction of uterine inflammatory mechanism in dysmenorrhea. PMID:26634864

  4. Influence of beta-blockers on the myocardial mRNA expressions of circadian clock- and metabolism-related genes.

    PubMed

    Ushijima, Kentarou; Maekawa, Tomohiro; Ishikawa-Kobayashi, Eiko; Ando, Hitoshi; Shiga, Tsuyoshi; Fujimura, Akio

    2013-01-01

    Daily rhythms are regulated by a master clock-system in the suprachiasmatic nucleus and by a peripheral clock-system in each organ. Because norepinephrine is one of the timekeepers for the myocardial circadian clock that influences cardiac metabolism, it is speculated that a beta-blocker may affect the circadian clock and metabolism in heart tissue. In this study, thirty mg/kg/day of propranolol (a lipophilic beta-blocker) or atenolol (a hydrophilic beta-blocker) was given orally to Wistar rats for 4 weeks. The mRNA expressions of Bmal1 and E4BP4 in heart tissue were suppressed by the beta-blockers. However, the mRNA expressions of these clock genes in the suprachiasmatic nucleus were unchanged. Myocardial mRNA expressions of lactate dehydrogenase a and pyruvate dehydrogenase kinase 4 were also suppressed by the beta-blockers. In addition, ATP content in heart tissue was significantly elevated by the beta-blockers throughout 24 hours. The effects of propranolol and atenolol did not differ significantly. This study showed for the first time that a beta-blocker affects myocardial clock gene expression. Propranolol and atenolol increased ATP content in heart tissue throughout 24 hours. The influences of beta-blockers may be negligible on the SCN, and may be independent of lipid solubility on heart tissue. It is well known that these drugs exert a protective effect against myocardial ischemia, which may be mediated by an increase in the preservation of myocardial ATP. PMID:23394803

  5. 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. PMID:27129231

  6. CYP2C9 Promoter Variable Number Tandem Repeat Polymorphism Regulates mRNA Expression in Human Livers

    PubMed Central

    Sun, Xiaochun; Gong, Yan; Gawronski, Brian E.; Langaee, Taimour Y.; Shahin, Mohamed Hossam A.; Khalifa, Sherief I.; Johnson, Julie A.

    2012-01-01

    CYP2C9 is involved in metabolism of nearly 25% of clinically used drugs. Coding region polymorphisms CYP2C9*2 and *3 contribute to interperson variability in drug dosage and clinical outcomes, whereas the role of a regulatory polymorphism remains uncertain. Measuring allelic RNA expression in 87 human liver samples, combined with genotyping, sequencing, and reporter gene assays, we identified a promoter variable number tandem repeat polymorphism (pVNTR) that fully accounted for allelic CYP2C9 mRNA expression differences. Present in three different variant forms [short (pVNTR-S), medium (pVNTR-M), and long (pVNTR-L)], only the pVNTR-S allele reduced the CYP2C9 mRNA level compared with the pVNTR-M (reference) allele. pVNTR-S is in linkage disequilibrium with *3, with linkage disequilibrium r2 of 0.53 to 0.75 in different populations. In patients who were taking a maintenance dose of warfarin, the mean warfarin dose was associated with the copies of pVNTR-S (p = 0.0001). However, in multivariate regression models that included the CYP2C9*3, pVNTR-S was no longer a significant predictor of the warfarin dose (p = 0.60). These results indicate that although pVNTR-S reduced CYP2C9 mRNA expression, the in vivo effects of pVNTR-S on warfarin metabolism cannot be separated from the effects of *3. Therefore, it is not necessary to consider pVNTR-S as an additional biomarker for warfarin dosing. Larger clinical studies are needed to define whether the pVNTR-S has a minimal effect in vivo, or whether the effect attributed to *3 is really a combination of effects on expression by the pVNTR-S along with effects on catalytic activity from the nonsynonymous *3 variant. PMID:22289258

  7. The human lactase persistence-associated SNP -13910*T enables in vivo functional persistence of lactase promoter-reporter transgene expression.

    PubMed

    Fang, Lin; Ahn, Jong Kun; Wodziak, Dariusz; Sibley, Eric

    2012-07-01

    Lactase is the intestinal enzyme responsible for digestion of the milk sugar lactose. Lactase gene expression declines dramatically upon weaning in mammals and during early childhood in humans (lactase nonpersistence). In various ethnic groups, however, lactase persists in high levels throughout adulthood (lactase persistence). Genetic association studies have identified that lactase persistence in northern Europeans is strongly associated with a single nucleotide polymorphism (SNP) located 14 kb upstream of the lactase gene: -13910*C/T. To determine whether the -13910*T SNP can function in vivo to mediate lactase persistence, we generated transgenic mice harboring human DNA fragments with the -13910*T SNP or the ancestral -13910*C SNP cloned upstream of a 2-kb rat lactase gene promoter in a luciferase reporter construct. We previously reported that the 2-kb rat lactase promoter directs a post-weaning decline of luciferase transgene expression similar to that of the endogenous lactase gene. In the present study, the post-weaning decline directed by the rat lactase promoter is impeded by addition of the -13910*T SNP human DNA fragment, but not by addition of the -13910*C ancestral SNP fragment. Persistence of transgene expression associated with the -13910*T SNP represents the first in vivo data in support of a functional role for the -13910*T SNP in mediating the human lactase persistence phenotype. PMID:22258180

  8. Upregulation of Arc mRNA Expression in the Prefrontal Cortex Following Cue-Induced Reinstatement of Extinguished Cocaine-Seeking Behavior

    PubMed Central

    ZAVALA, ARTURO R.; OSREDKAR, TRACY; JOYCE, JEFFREY N.; NEISEWANDER, JANET L.

    2010-01-01

    Cocaine-associated cues acquire incentive motivational effects that manifest as cue-elicited craving in humans and cocaine-seeking behavior in rats. Here we examine the hypothesis that neuronal processes associated with incentive motivational effects of cocaine cues involve increased expression of the plasticity-associated gene, Arc. Rats trained to self-administer cocaine subsequently underwent extinction training, during which cocaine-seeking behavior (i.e., responses without cocaine reinforcement) progressively decreased. Rats were then tested for cocaine-seeking behavior either with or without response-contingent presentations of light/tone cues that had been previously paired with cocaine infusions during self-administration training. Cues elicited reinstatement of cocaine-seeking behavior and were accompanied by increased Arc mRNA levels in the orbitofrontal, prelimbic, and anterior cingulate cortices, suggesting Arc involvement in conditioned plasticity associated with incentive motivational effects of cocaine cues. Additionally, rats with a history of cocaine self-administration and extinction exhibited upregulation of Arc expression in several limbic and cortical regions relative to saline-yoked controls regardless of cue exposure condition, suggesting persistent neuroadaptations involving Arc within these regions. PMID:18361437

  9. 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. PMID:26608082

  10. Exposure to morphine-associated cues increases mu opioid receptor mRNA expression in the nucleus accumbens of Wistar Kyoto rats.

    PubMed

    Dennis, Torry S; Beck, Kevin D; Cominski, Tara P; Bobzean, Samara A M; Kuzhikandathil, Eldo V; Servatius, Richard J; Perrotti, Linda I

    2016-10-15

    The Wistar-Kyoto (WKY) rat has been proposed as a model of anxiety vulnerability as it exhibits pronounced behavioral inhibition, passive avoidance, exaggerated startle response, enhanced HPA-axis activation, and active avoidance that is resistant to extinction. Accumulating evidence suggests that WKY rats respond differently to rewarding stimuli when compared to outbred strains of rat. Conditioned responding to drug-associated cues is linked with alterations in the activation of mu opioid receptors (MOR) and kappa opioid receptors (KOR) in the nucleus accumbens (NAc). Furthermore, alterations in KOR expression/activation in the NAc of WKY rats are implicated in the regulation of some of the components that make up the unique behavioral phenotype of this strain. The purpose of this study was to extend upon previous work from our laboratory by investigating conditioned morphine reward in adult male WKY and SD rats, and to examine levels of KOR mRNA and MOR mRNA in the NAc at baseline and after acquisition of morphine CPP. Our results demonstrate that SD rats displayed morphine-induced CPP to each of the six doses of morphine tested (0.5, 1.25, 2.5, 5, 7.5, or 10mg/kg). Interestingly, WKY rats demonstrated CPP for only the 1.25, 2.5, and 5mg/kg doses, yet no preference at the lowest (0.5mg/kg) or highest (7.5 and 10mg/kg) doses. qPCR analysis of MOR and KOR in the NAc revealed no strain differences in basal levels of MOR, but higher levels of KOR in WKY rats compared to those of SD rats. Interestingly, after completion of the CPP task, WKY rats had overall higher levels of NAc MOR mRNA compared to SD rats; the initial basal differences in NAc KOR levels persisted without change due to CPP in either strain. These results demonstrate that the WKY rat exhibits a unique pattern of behavioral responding to morphine and implicates differences in NAc KOR signaling as a potential source of aversion to higher doses of morphine. Additionally, the CPP-induced upregulation of

  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. Exploiting aberrant mRNA expression in autism for gene discovery and diagnosis.

    PubMed

    Guan, Jinting; Yang, Ence; Yang, Jizhou; Zeng, Yong; Ji, Guoli; Cai, James J

    2016-07-01

    Autism spectrum disorder (ASD) is characterized by substantial phenotypic and genetic heterogeneity, which greatly complicates the identification of genetic factors that contribute to the disease. Study designs have mainly focused on group differences between cases and controls. The problem is that, by their nature, group difference-based methods (e.g., differential expression analysis) blur or collapse the heterogeneity within groups. By ignoring genes with variable within-group expression, an important axis of genetic heterogeneity contributing to expression variability among affected individuals has been overlooked. To this end, we develop a new gene expression analysis method-aberrant gene expression analysis, based on the multivariate distance commonly used for outlier detection. Our method detects the discrepancies in gene expression dispersion between groups and identifies genes with significantly different expression variability. Using this new method, we re-visited RNA sequencing data generated from post-mortem brain tissues of 47 ASD and 57 control samples. We identified 54 functional gene sets whose expression dispersion in ASD samples is more pronounced than that in controls, as well as 76 co-expression modules present in controls but absent in ASD samples due to ASD-specific aberrant gene expression. We also exploited aberrantly expressed genes as biomarkers for ASD diagnosis. With a whole blood expression data set, we identified three aberrantly expressed gene sets whose expression levels serve as discriminating variables achieving >70 % classification accuracy. In summary, our method represents a novel discovery and diagnostic strategy for ASD. Our findings may help open an expression variability-centered research avenue for other genetically heterogeneous disorders. PMID:27131873

  13. 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

  14. Puerperal influence of bovine uterine health status on the mRNA expression of pro-inflammatory factors.

    PubMed

    Peter, S; Michel, G; Hahn, A; Ibrahim, M; Lubke-Becker, A; Jung, M; Einspanier, R; Gabler, C

    2015-06-01

    After parturition, uterine bacterial infections lead to inflammatory processes such as subclinical/clinical endometritis with high prevalence in dairy cows. Endometrial epithelial cells participate in this immune response with the production of pro-inflammatory factors. The objective of the present study was to evaluate the endometrial mRNA expression pattern of pro-inflammatory factors during a selected postpartum (pp) period. Dairy cows with three different uterine health conditions on days 24-30 pp (healthy: n = 11, subclinical endometritis: n = 10, clinical endometritis: n = 10) were sampled using the cytobrush technique. Subsequently, each cow was sampled 3 more times in weekly intervals (days 31-37 pp; days 38-44 pp; days 45-51 pp). Samples were subjected to mRNA analysis performed by RT-qPCR. Additionally, an analysis of cultivable bacteria was performed at the early/late stage of the selected puerperal period. mRNA expression of 16 candidate genes was analyzed by using two different approaches. The first approach referred to the initial grouping on days 24-30 pp to reveal long-term effects of the uterine health on the subsequent puerperal period. The second approach considered the current uterine health status at each sampling to elucidate the impact of different points in time. Long-term effects seem to appear for chemokines, prostacyclin synthase and prostaglandin D2 synthase. If related to the current uterine health, the majority of candidate genes were significantly higher expressed in endometritic cows on days 45-51 pp in contrast to earlier stages of the puerperium. Microbiological analysis revealed the significantly higher prevalence of Trueperella pyogenes findings in cows with clinical endometritis on days 24-30 pp, but no correlations were found on days 45-51 pp. In conclusion, a strong immune response to subclinical/clinical endometritis in the late puerperium may be related to the negative impact of these conditions on reproductive performance

  15. Vasoactive peptides upregulate mRNA expression and secretion of vascular endothelial growth factor in human airway smooth muscle cells.

    PubMed

    Alagappan, Vijay K T; Willems-Widyastuti, Anna; Seynhaeve, Ann L B; Garrelds, Ingrid M; ten Hagen, Timo L M; Saxena, Pramod R; Sharma, Hari S

    2007-01-01

    Airway remodeling and associated angiogenesis are documented features of asthma, of which the molecular mechanisms are not fully understood. Angiotensin (ANG)II and endothelin (ET)-1 are potent vasoconstricting circulatory hormones implicated in asthma. We investigated the effects of ANG II and ET-1 on human airway smooth muscle (ASM) cells proliferation and growth and examined the mRNA expression and release of the angiogenic peptide, vascular endothelial growth factor (VEGF). Serum deprived (48 h) human ASM cells were incubated with ANG II (100 nM) or ET-1 (10 nM) for 30 min, 1, 2, 4, 8, 16, and 24 h and the endogenous synthesis of VEGF was examined in relation to control cells receiving serum free culture medium. ET-1 induced time dependent DNA biosynthesis as determined by [3H]-thymidine incorporation assay. Using northern blot hybridization, we detected two mRNA species of 3.9 and 1.7 kb encoding VEGF in the cultured smooth muscle cells. Both ANG II and ET-1 induced the mRNA expression (two- to threefold) and secretion (1.8- to 2.8-fold) of VEGF reaching maximal levels between 4-8 h of incubation. Induced expression and release of VEGF declined after 8 h of ANG II incubation while levels remained elevated in the case of ET-1. The conditioned medium derived from ET-1-treated ASM cells induced [3H]-thymidine incorporation and cell number in porcine pulmonary artery endothelial as well as human umbilical vein endothelial cells. Moreover, the VEGF tyrosine kinase receptor inhibitor blocked the conditioned medium induced mitogenesis in endothelial cells. Our results suggest a potential role for ANG II and ET-1 in ASM cell growth and upregulation of VEGF that may participate in endothelial cell proliferation via paracrine mechanisms and thus causing pathological angiogenesis and vascular remodelling seen during asthma. PMID:17406064

  16. mRNA expression profiles of primary high-grade central osteosarcoma are preserved in cell lines and xenografts

    PubMed Central

    2011-01-01

    Background Conventional high-grade osteosarcoma is a primary malignant bone tumor, which is most prevalent in adolescence. Survival rates of osteosarcoma patients have not improved significantly in the last 25 years. Aiming to increase this survival rate, a variety of model systems are used to study osteosarcomagenesis and to test new therapeutic agents. Such model systems are typically generated from an osteosarcoma primary tumor, but undergo many changes due to culturing or interactions with a different host species, which may result in differences in gene expression between primary tumor cells, and tumor cells from the model system. We aimed to investigate whether gene expression profiles of osteosarcoma cell lines and xenografts are still comparable to those of the primary tumor. Methods We performed genome-wide mRNA expression profiling on osteosarcoma biopsies (n = 76), cell lines (n = 13), and xenografts (n = 18). Osteosarcoma can be subdivided into several histological subtypes, of which osteoblastic, chondroblastic, and fibroblastic osteosarcoma are the most frequent ones. Using nearest shrunken centroids classification, we generated an expression signature that can predict the histological subtype of osteosarcoma biopsies. Results The expression signature, which consisted of 24 probes encoding for 22 genes, predicted the histological subtype of osteosarcoma biopsies with a misclassification error of 15%. Histological subtypes of the two osteosarcoma model systems, i.e. osteosarcoma cell lines and xenografts, were predicted with similar misclassification error rates (15% and 11%, respectively). Conclusions Based on the preservation of mRNA expression profiles that are characteristic for the histological subtype we propose that these model systems are representative for the primary tumor from which they are derived. PMID:21933437

  17. A circadian neuropeptide PDF in the honeybee, Apis mellifera: cDNA cloning and expression of mRNA.

    PubMed

    Sumiyoshi, Miho; Sato, Seiji; Takeda, Yukimasa; Sumida, Kazunori; Koga, Keita; Itoh, Tsunao; Nakagawa, Hiroyuki; Shimohigashi, Yasuyuki; Shimohigashi, Miki

    2011-12-01

    Pigment-dispersing factor (PDF) is a pacemaker hormone regulating the locomotor rhythm in insects. In the present study, we cloned the cDNAs encoding the Apis PDF precursor protein, and found that there are at least seven different pdf mRNAs yielded by an alternative splicing site and five alternative polyadenylation sites in the 5'UTR and 3'UTR regions. The amino acid sequence of Apis PDF peptide has a characteristic novel amino acid residue, aspargine (Asn), at position 17. Quantitative real-time PCR of total and 5'UTR insertion-type pdf mRNAs revealed, for the first time, that the expression levels change in a circadian manner with a distinct trough at the beginning of night in LD conditions, and at the subjective night under DD conditions. In contrast, the expression level of 5'UTR deletion-type pdf mRNAs was about half of that of the insertion type, and the expression profile failed to show a circadian rhythm. As the expression profile of the total pdf mRNA exhibited a circadian rhythm, transcription regulated at the promoter region was supposed to be controlled by some of the clock components. Whole mount in situ hybridization revealed that 14 lateral neurons at the frontal margin of the optic lobe express these mRNA isoforms. PDF expressing cells examined with a newly produced antibody raised against Apis PDF were also found to have a dense supply of axon terminals in the optic lobes and the central brain. PMID:22132787

  18. Comparative Analysis of Xenorhabdus koppenhoeferi Gene Expression during Symbiotic Persistence in the Host Nematode

    PubMed Central

    An, Ruisheng; Grewal, Parwinder S.

    2016-01-01

    Species of Xenorhabdus and Photorhabdus bacteria form mutualistic associations with Steinernema and Heterorhabditis nematodes, respectively and serve as model systems for studying microbe-animal symbioses. Here, we profiled gene expression of Xenorhabdus koppenhoeferi during their symbiotic persistence in the newly formed infective juveniles of the host nematode Steinernema scarabaei through the selective capture of transcribed sequences (SCOTS). The obtained gene expression profile was then compared with other nematode-bacteria partnerships represented by Steinernema carpocapsae—Xenorhabdus nematophila and Heterorhabditis bacteriophora—Photorhabdus temperata. A total of 29 distinct genes were identified to be up-regulated and 53 were down-regulated in X. koppenhoeferi while in S. scarabaei infective juveniles. Of the identified genes, 8 of the up-regulated and 14 of the down-regulated genes were similarly expressed in X. nematophila during persistence in its host nematode S. carpocapsae. However, only one from each of these up- and down-regulated genes was common to the mutualistic partnership between the bacterium P. temperata and the nematode H. bacteriophora. Interactive network analysis of the shared genes between X. koppenhoeferi and X. nematophila demonstrated that the up-regulated genes were mainly involved in bacterial survival and the down-regulated genes were more related to bacterial virulence and active growth. Disruption of two selected genes pta (coding phosphotransacetylase) and acnB (coding aconitate hydratase) in X. nematophila with shared expression signature with X. koppenhoeferi confirmed that these genes are important for bacterial persistence in the nematode host. The results of our comparative analyses show that the two Xenorhabdus species share a little more than a quarter of the transcriptional mechanisms during persistence in their nematode hosts but these features are quite different from those used by P. temperata bacteria in their

  19. 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

  20. Expression and cellular localization of rab28 mRNA and Rab28 protein during maize embryogenesis.

    PubMed

    Niogret, M F; Culiáñez-Macià, F A; Goday, A; Mar Albà, M; Pagès, M

    1996-04-01

    The maize abscisic acid (ABA) responsive gene rab28, has been shown to be ABA-inducible in embryos and vegetative tissues. A polyclonal antiserum was raised against Rab28 protein. Using immunoblotting and immunoprecipitation, the antiserum specifically recognized a protein of about 30 kDa and pl 6 which is in close agreement with the molecular weight and pl predicted by the deduced amino acid sequence. The rab28 gene product accumulated during late embryogenesis. In vegetative tissues, dehydration stress induced rab28 gene expression both in the light and in the dark. The spatial and temporal pattern of rab28 mRNA expression during embryogenesis was investigated by in situ hybridization using digoxigenin-labelled rab28 probes, and the immunochemical localization of Rab28 protein using anti-Rab28 antibodies. Expression of rab28 mRNA is restricted to provascular tissues in young embryos, and at later stages of development the most prevalent accumulation occurred in meristem and in the vascular elements of the plumule, root and scutellum. Using immunoelectron microscopy the Rab28 protein has been located in the nucleolus of different cell types. In light of these results the stress regulation of rab28 and a likely role for this protein during late embryogenesis are discussed. PMID:8624517

  1. Anatomical characterization of bombesin receptor subtype-3 mRNA expression in the rodent central nervous system.

    PubMed

    Zhang, Li; Parks, Gregory S; Wang, Zhiwei; Wang, Lien; Lew, Michelle; Civelli, Olivier

    2013-04-01

    Bombesin receptor subtype-3 (BRS-3) is an orphan G-protein-coupled receptor (GPCR) involved in the regulation of energy homeostasis. Mice deficient in BRS-3 develop late-onset mild obesity with metabolic defects, while synthetic agonists activating BRS-3 show antiobesity profiles by inhibiting food intake and increasing metabolic rate in rodent models. The molecular mechanisms and the neural circuits responsible for these effects, however, remain elusive and demand better characterization. We report here a comprehensive mapping of BRS-3 mRNA in the rat and mouse brain through in situ hybridization. Furthermore, to investigate the neurochemical characteristics of the BRS-3-expressing neurons, double in situ hybridization was performed to determine whether BRS-3 colocalizes with other neurotransmitters or neuropeptides. Many, but not all, of the BRS-3-expressing neurons were found to be glutamatergic, while few were found to be cholinergic or GABAergic. BRS-3-containing neurons do not express some of the well-characterized neuropeptides, such as neuropeptide Y (NPY), proopiomelanocortin (POMC), orexin/hypocretin, melanin-concentrating hormone (MCH), thyrotropin-releasing hormone (TRH), gonadotropin-releasing hormone (GnRH), and kisspeptin. Interestingly, BRS-3 mRNA was found to partially colocalize with corticotropin-releasing factor (CRF) and growth hormone-releasing hormone (GHRH), suggesting novel interactions of BRS-3 with stress- and growth-related endocrine systems. Our study provides important information for evaluating BRS-3 as a potential therapeutic target for the treatment of obesity. PMID:22911445

  2. Resveratrol Decreases TXNIP mRNA and Protein Nuclear Expressions With an Arterial Function Improvement in Old Mice.

    PubMed

    Bedarida, Tatiana; Baron, Stephanie; Vibert, Françoise; Ayer, Audrey; Henrion, Daniel; Thioulouse, Elizabeth; Marchiol, Carmen; Beaudeux, Jean-Louis; Cottart, Charles-Henry; Nivet-Antoine, Valerie

    2016-06-01

    Aging leads to a high prevalence of glucose intolerance and cardiovascular diseases, with oxidative stress playing a potential role. Resveratrol has shown promising effects on glucose tolerance and tends to improve endothelial function in elderly patients. Thioredoxin-interacting protein (TXNIP) was recently proposed as a potential link connecting glucose metabolism to oxidative stress. Here, we investigated the resveratrol-induced improvement of arterial aging phenotype in old mice and the expression of aortic TXNIP. Using an in vivo model of old mice with or without 3-month resveratrol treatment, we investigated the effects of resveratrol on age-related impairments from a cardiovascular Doppler analysis, to a molecular level, by studying inflammation and oxidative stress factors. We found a dual effect of resveratrol, with a decrease of age-related glucose intolerance and oxidative stress imbalance leading to reduced matrix remodeling that forestalls arterial aging phenotype in terms of intima-media thickness and arterial distensibility. These results provide the first evidence that aortic TXNIP mRNA and protein nuclear expressions are increased in the arterial aging and decreased by resveratrol treatment. In conclusion, we demonstrated that resveratrol helped to restore several aging impaired processes in old mice, with a decrease of aortic TXNIP mRNA and protein nuclear expressions. PMID:26041427

  3. Altered mRNA editing and expression of ionotropic glutamate receptors after kainic acid exposure in cyclooxygenase-2 deficient mice.

    PubMed

    Caracciolo, Luca; Barbon, Alessandro; Palumbo, Sara; Mora, Cristina; Toscano, Christopher D; Bosetti, Francesca; Barlati, Sergio

    2011-01-01

    Kainic acid (KA) binds to the AMPA/KA receptors and induces seizures that result in inflammation, oxidative damage and neuronal death. We previously showed that cyclooxygenase-2 deficient (COX-2(-/-)) mice are more vulnerable to KA-induced excitotoxicity. Here, we investigated whether the increased susceptibility of COX-2(-/-) mice to KA is associated with altered mRNA expression and editing of glutamate receptors. The expression of AMPA GluR2, GluR3 and KA GluR6 was increased in vehicle-injected COX-2(-/-) mice compared to wild type (WT) mice in hippocampus and cortex, whereas gene expression of NMDA receptors was decreased. KA treatment decreased the expression of AMPA, KA and NMDA receptors in the hippocampus, with a significant effect in COX-2(-/-) mice. Furthermore, we analyzed RNA editing levels and found that the level of GluR3 R/G editing site was selectively increased in the hippocampus and decreased in the cortex in COX-2(-/-) compared with WT mice. After KA, GluR4 R/G editing site, flip form, was increased in the hippocampus of COX-2(-/-) mice. Treatment of WT mice with the COX-2 inhibitor celecoxib for two weeks decreased the expression of AMPA/KA and NMDAR subunits after KA, as observed in COX-2(-/-) mice. After KA exposure, COX-2(-/-) mice showed increased mRNA expression of markers of inflammation and oxidative stress, such as cytokines (TNF-α, IL-1β and IL-6), inducible nitric oxide synthase (iNOS), microglia (CD11b) and astrocyte (GFAP). Thus, COX-2 gene deletion can exacerbate the inflammatory response to KA. We suggest that COX-2 plays a role in attenuating glutamate excitotoxicity by modulating RNA editing of AMPA/KA and mRNA expression of all ionotropic glutamate receptor subunits and, in turn, neuronal excitability. These changes may contribute to the increased vulnerability of COX-2(-/-) mice to KA. The overstimulation of glutamate receptors as a consequence of COX-2 gene deletion suggests a functional coupling between COX-2 and the

  4. The effect of copper on the mRNA expression profile of xenobiotic-metabolizing enzymes in cultured rat H4-II-E cells.

    PubMed

    Darwish, Wageh Sobhy; Ikenaka, Yoshinori; Nakayama, Shouta; Ishizuka, Mayumi

    2014-05-01

    Copper (Cu(2+)) is an essential element that plays important roles in physiological functions of the body. However, high Cu(2+) levels can have toxic implications. This study aims to investigate the constitutive response to Cu(2+) exposure of xenobiotic-metabolizing enzymes in cultured rat liver (H4-II-E) cell lines. Rat cells were exposed to copper sulfate (0-500 μM) for 24 h. The effects of Cu(2+) on the messenger RNA (mRNA) expressions of phase I and II enzymes and regulatory elements were examined using real-time PCR. Metallothionein mRNA expression was induced in a dose-dependent manner after treatment with Cu(2+). mRNA expressions of phase I enzymes such as cytochrome P450 1A1 and 1A2 (CYP1A1 and CYP1A2) were slightly induced after exposure to low concentrations of Cu(2+); however, CYP1A1 and CYP1A2 mRNA expressions were significantly downregulated at higher Cu(2+) concentrations. These effects corresponded with expression of aryl hydrocarbon receptor mRNA. The mRNA expressions of phase II enzymes were reduced upon exposure to Cu(2+). In conclusion, phase I and II enzyme expressions were significantly modulated upon Cu(2+) exposure. These results indicated that Cu(2+) exposure had toxicological implications for cultured H4-II-E cells. PMID:24599699

  5. Differential regulation of mRNA stability controls the transient expression of genes encoding Drosophila antimicrobial peptide with distinct immune response characteristics

    PubMed Central

    Wei, Youheng; Xiao, Qianghai; Zhang, Ting; Mou, Zongchun; You, Jia; Ma, Wei-Jun

    2009-01-01

    The tight regulation of transiently expressed antimicrobial peptides (AMPs) with a distinct antimicrobial spectrum and different expression kinetics contributes greatly to the properly regulated immune response for resistance to pathogens and for the maintenance of mutualistic microbiota in Drosophila. The important role of differential regulation of AMP expression at the posttranscriptional level needs to be elucidated. It was observed that the highly expressed Cecropin A1 (CecA1) mRNA encoding a broad antimicrobial spectrum AMP against both bacteria and fungi decayed more quickly than did the moderately expressed Diptericin mRNA encoding AMP against Gram negative bacteria. The mRNA stability of AMPs is differentially regulated and is attributed to the specific interaction between cis-acting ARE in 3′-UTR of AMP mRNA and the RNA destabilizing protein transactor Tis11 as shown in co-immunoprecipitation of the Tis11 RNP complex with CecA1 mRNA but not other AMP mRNA. The p38MAPK was further demonstrated to play a crucial role in stabilizing ARE-bearing mRNAs by inhibiting Tis11-mediated degradation in LPS induced AMP expression. This evidence suggests an evolutionarily conserved and functionally important molecular basis for and effective approach to exact control of AMP gene expression. These mechanisms thereby orchestrate a well balanced and dynamic antimicrobial spectrum of innate immunity to resist infection and maintain resident microbiota properly. PMID:19726583

  6. Differential regulation of mRNA stability controls the transient expression of genes encoding Drosophila antimicrobial peptide with distinct immune response characteristics.

    PubMed

    Wei, Youheng; Xiao, Qianghai; Zhang, Ting; Mou, Zongchun; You, Jia; Ma, Wei-Jun

    2009-10-01

    The tight regulation of transiently expressed antimicrobial peptides (AMPs) with a distinct antimicrobial spectrum and different expression kinetics contributes greatly to the properly regulated immune response for resistance to pathogens and for the maintenance of mutualistic microbiota in Drosophila. The important role of differential regulation of AMP expression at the posttranscriptional level needs to be elucidated. It was observed that the highly expressed Cecropin A1 (CecA1) mRNA encoding a broad antimicrobial spectrum AMP against both bacteria and fungi decayed more quickly than did the moderately expressed Diptericin mRNA encoding AMP against Gram negative bacteria. The mRNA stability of AMPs is differentially regulated and is attributed to the specific interaction between cis-acting ARE in 3'-UTR of AMP mRNA and the RNA destabilizing protein transactor Tis11 as shown in co-immunoprecipitation of the Tis11 RNP complex with CecA1 mRNA but not other AMP mRNA. The p38MAPK was further demonstrated to play a crucial role in stabilizing ARE-bearing mRNAs by inhibiting Tis11-mediated degradation in LPS induced AMP expression. This evidence suggests an evolutionarily conserved and functionally important molecular basis for and effective approach to exact control of AMP gene expression. These mechanisms thereby orchestrate a well balanced and dynamic antimicrobial spectrum of innate immunity to resist infection and maintain resident microbiota properly. PMID:19726583

  7. Cyclosporine A inhibits the mRNA expressions of IL-2, IL-4 and IFN-gamma, but not TNF-alpha, in canine mononuclear cells.

    PubMed

    Kobayashi, Tetsuro; Momoi, Yasuyuki; Iwasaki, Toshiroh

    2007-09-01

    The effects of the calcineurin inhibitors cyclosporine A (CsA) and FK506 on the mRNA expressions of various cytokines were evaluated in dogs to determine whether the effects of CsA and FK506 in dogs were similar to those in humans. The mRNA expression levels of the cytokines IL-2, IL-4, IFN-gamma and TNF-alpha were measured in PHA-stimulated canine PBMC using real-time RT-PCR after incubation with CsA or FK506 for 5 hr. Both reagents inhibited IL-2, IL-4 and IFN-gamma mRNA expressions in a dose-dependent manner. However, CsA hardly inhibited the mRNA expression of TNF-alpha. These findings are important for assessing the indications of CsA treatment in dogs. PMID:17917372

  8. The expression of a plant genome in hnRNA and mRNA.

    PubMed

    Kiper, M; Bartels, D; Herzfeld, F; Richter, G

    1979-01-01

    Representation of genomic kinetic sequence classes and sequence complexities were investigated in nuclear and polysomal RNA of the higher plant Petroselinum sativum (parsley). Two different methods indicated that most if not all polysomal poly(A) -RNA is transcribed from unique sequences. As measured by saturation hybridization in root callus and young leaves 8.7% and 6.2%, respectively, of unique DNA were transcribed in mRNA corresponding to 13.700 and 10.000 average sized genes. Unique nuclear DNA hybridized with an excess of polysomal poly(A)mRNA to the same extent as with total polysomal RNA. 3H-cDNA - poly(A)mRNA hybridization kinetics revealed the presence of two abundance classes with 9.200 and about 30 different mRNAs in leaves and two abundance classes with 10.500 and 960 different mRNAs in callus cells. The existence of plant poly(A)hnRNA was proven both by its fast kinetics of appearance, its length distribution larger than mRNA, and its sequence complexity a few times that of polysomal RNA. PMID:450719

  9. Sulindac increases the expression of APC mRNA in malignant colonic epithelial cells: an in vitro study.

    PubMed Central

    Schnitzler, M; Dwight, T; Robinson, B G

    1996-01-01

    BACKGROUND--Sulindac is a non-steroidal anti-inflammatory drug which induces regression of colonic polyps in patients with familial adenomatous polyposis. Animal and in vitro studies have shown that both the sulphide metabolite of sulindac, which is able to inhibit cyclo-oxygenase, and the sulphone metabolite, which lacks this ability, are able to inhibit the growth of colonic carcinoma cells. The exact mechanism by which these effects occurs is not known. AIMS--To examine the effect of sulindac sulphide and sulindac sulphone on the expression of APC messenger RNA (mRNA), and on the proliferation of colonic carcinoma cells in vitro. METHODS--The colonic carcinoma cell line LIM 1215 was treated with sulindac sulphide and sulindac sulphone (10 microM or 100 microM) for 24 hours. Total RNA was extracted and APC mRNA was quantitated using competitive reverse transcription polymerase chain reaction. Measurements of cell number, cell proliferation, and prostaglandin E2 concentrations were also made. RESULTS--A significant increase in APC mRNA was observed after treatment with 10 microM of both sulindac sulphide and sulindac sulphone (control: 37.2 (19.7); 10 microM sulindac sulphide: 129 (112.8); 10 microM sulindac sulphone: 207.7 (102.9) pg/(g total RNA) (p < 0.05). Prostaglandin E2 concentrations were significantly reduced after treatment with sulindac sulphide, but not after sulindac sulphone. Both agents produced a dose dependent reduction in cell numbers and cell proliferation, which was more noticeable after treatment with sulindac sulphide. CONCLUSIONS--Both sulindac sulphide and sulindac sulphone inhibit the growth of carcinoma cells in vitro and cause an increase in APC mRNA. The effect of these agents on colonic carcinogenesis is not mediated entirely by means of an inhibition of prostaglandin biosynthesis. Images Figure 1 PMID:8707116

  10. Profiles of mRNA expression for genes involved in implantation, early and middle phases of secretion stage in human endometrium.

    PubMed

    Maslova, M A; Smol'nikova, V Yu; Savilova, A M; Burmenskaya, O V; Bystritskii, A A; Tabolova, V K; Korneeva, I E; Demura, T A; Donnikov, A E

    2015-04-01

    The expression of mRNA of 36 genes involved in implantation was studied by reverse transcription and real-time PCR. Significant differences in mRNA expression during the early and middle stages of the secretion phase were detected for genes mmp7, vegf, il2m, il1β, il8, il18, tnfα, il10, tgfβ, igfbp2, etc. PMID:25894777

  11. Gene expression profiling reveals differences in microenvironment interaction between patients with chronic lymphocytic leukemia expressing high versus low ZAP70 mRNA

    PubMed Central

    Stamatopoulos, Basile; Haibe-Kains, Benjamin; Equeter, Carole; Meuleman, Nathalie; Sorée, Anne; De Bruyn, Cécile; Hanosset, Delphine; Bron, Dominique; Martiat, Philippe; Lagneaux, Laurence

    2009-01-01

    Background Zeta-associated protein 70 (ZAP70) is a widely recognized prognostic factor in chronic lymphocytic leukemia, but mechanisms by which its higher expression leads to a poor outcome must still be fully explained. Design and Methods In an attempt to unveil unfavorable cellular properties linked to high ZAP70 expression, we used gene expression profiling to identify genes associated with disparities in B cells from chronic lymphocytic leukemia patients expressing high versus low ZAP70 mRNA, measured by quantitative real-time PCR. Two groups of 7 patients were compared, selected on the basis of either high or low ZAP70 mRNA expression. Results Twenty-seven genes were differentially expressed with an FDR<10%, and several genes were significant predictors of treatment-free survival (TFS) and/or overall survival; PDE8A and FCRL family genes (down-regulated in ZAP70+ patients) could predict TFS and overall survival; ITGA4 mRNA (up-regulated in ZAP70+ patients) could significantly predict overall survival. Importantly, gene set enrichment analysis revealed overrepresentation of adhesion/migration genes. We therefore investigated in vitro adhesion/migration capacity of chronic lymphocytic leukemia cells into a stromal microenvironment or in response to conditioned medium. We showed that ZAP70+ cells had better adhesion/migration capacities and only ZAP70+ patient cells responded to microenvironment contact by CXCR4 downregulation. Conclusions We concluded that several prognostic factors are the reflection of microenvironment interactions and that the increased adhesion/migratory capacity of ZAP70+ cells in their microenvironment can explain their better survival and thus the aggressiveness of the disease. PMID:19377082

  12. 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. PMID:27160934

  13. 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

  14. Cytochrome P450 mRNA Expression in the Rodent Brain: Species-, Sex-, and Region-Dependent Differences

    PubMed Central

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

    2014-01-01

    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. PMID:24255117

  15. 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. PMID:24255117

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

    PubMed Central

    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. PMID:26010155

  17. Persistent Gene Expression in Mouse Nasal Epithelia following Feline Immunodeficiency Virus-Based Vector Gene Transfer

    PubMed Central

    Sinn, Patrick L.; Burnight, Erin R.; Hickey, Melissa A.; Blissard, Gary W.; McCray, Paul B.

    2005-01-01

    Gene transfer development for treatment or prevention of cystic fibrosis lung disease has been limited by the inability of vectors to efficiently and persistently transduce airway epithelia. Influenza A is an enveloped virus with natural lung tropism; however, pseudotyping feline immunodeficiency virus (FIV)-based lentiviral vector with the hemagglutinin envelope protein proved unsuccessful. Conversely, pseudotyping FIV with the envelope protein from influenza D (Thogoto virus GP75) resulted in titers of 106 transducing units (TU)/ml and conferred apical entry into well-differentiated human airway epithelial cells. Baculovirus GP64 envelope glycoproteins share sequence identity with influenza D GP75 envelope glycoproteins. Pseudotyping FIV with GP64 from three species of baculovirus resulted in titers of 107 to 109 TU/ml. Of note, GP64 from Autographa californica multicapsid nucleopolyhedrovirus resulted in high-titer FIV preparations (∼109 TU/ml) and conferred apical entry into polarized primary cultures of human airway epithelia. Using a luciferase reporter gene and bioluminescence imaging, we observed persistent gene expression from in vivo gene transfer in the mouse nose with A. californica GP64-pseudotyped FIV (AcGP64-FIV). Longitudinal bioluminescence analysis documented persistent expression in nasal epithelia for ∼1 year without significant decline. According to histological analysis using a LacZ reporter gene, olfactory and respiratory epithelial cells were transduced. In addition, methylcellulose-formulated AcGP64-FIV transduced mouse nasal epithelia with much greater efficiency than similarly formulated vesicular stomatitis virus glycoprotein-pseudotyped FIV. These data suggest that AcGP64-FIV efficiently transduces and persistently expresses a transgene in nasal epithelia in the absence of agents that disrupt the cellular tight junction integrity. PMID:16188984

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

    PubMed

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

    2015-09-01

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

  19. Molecular cloning of amyloid cDNA derived from mRNA of the Alzheimer disease brain: coding and noncoding regions of the fetal precursor mRNA are expressed in the cortex

    SciTech Connect

    Zain, S.B.; Salim, M.; Chou, W.G.; Sajdel-Sulkowska, E.M.; Majocha, R.E.; Marotta, C.A.

    1988-02-01

    To gain insight into factors associated with the excessive accumulation of ..beta..-amyloid in the Alzheimer disease (AD) brain, the present studies were initiated to distinguish between a unique primary structure of the AD-specific amyloid precursor mRNA vis a vis other determinants that may affect amyloid levels. Previous molecular cloning experiments focused on amyloid derived from sources other than AD cases. In the present work, the authors cloned and characterized amyloid cDNA derived directly from AD brain mRNA. Poly(A)/sup +/ RNA from AD cortices was used for the preparation of lambdagt11 recombinant cDNA libraries. An insert of 1564 nucleotides was isolated that included the ..beta..-amyloid domain and corresponded to 75% of the coding region and approx. = 70% of the 3'-noncoding region of the fetal precursor amyloid cDNA reported by others. On RNA blots, the AD amyloid mRNA consisted of a doublet of 3.2 and 3.4 kilobases. In control and AD cases, the amyloid mRNA levels were nonuniform and were independent of glial-specific mRNA levels. Based on the sequence analysis data, they conclude that a segment of the amyloid gene is expressed in the AD cortex as a high molecular weight precursor mRNA with major coding and 3'-noncoding regions that are identical to the fetal brain gene product.

  20. 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. PMID:15278899

  1. 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

  2. Expression of GABA A receptor alpha1 subunit mRNA and protein in rat neocortex following photothrombotic infarction.

    PubMed

    Kharlamov, Elena A; Downey, Kathy L; Jukkola, Peter I; Grayson, Dennis R; Kelly, Kevin M

    2008-05-19

    Photothrombotic infarcts of the neocortex result in structural and functional alterations of cortical networks, including decreased GABAergic inhibition, and can generate epileptic seizures within 1 month of lesioning. In our study, we assessed the involvement and potential changes of cortical GABA A receptor (GABA AR) alpha1 subunits at 1, 3, 7, and 30 days after photothrombosis. Quantitative competitive reverse transcription-polymerase chain reaction (cRT-PCR) and semi-quantitative Western blot analysis were used to investigate GABA AR alpha1 subunit mRNA and protein levels in proximal and distal regions of perilesional cortex and in homotopic areas of young adult Sprague-Dawley rats. GABA AR alpha1 subunit mRNA levels were decreased ipsilateral and contralateral to the infarct at 7 days, but were increased bilaterally at 30 days. GABA AR alpha1 subunit protein levels revealed no significant change in neocortical areas of both hemispheres of lesioned animals compared with protein levels of sham-operated controls at 1, 3, 7, and 30 days. At 30 days, GABA AR alpha1 subunit protein expression was significantly increased in lesioned animals within proximal and distal regions of perilesional cortex compared with distal neocortical areas contralaterally (Student's t-test, p<0.05). Short- and long-term alterations of mRNA and protein levels of the GABA AR alpha1 subunit ipsilateral and contralateral to the lesion may influence alterations in cell surface receptor subtype expression and GABA AR function following ischemic infarction and may be associated with formative mechanisms of poststroke epileptogenesis. PMID:18407248

  3. 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

  4. Leptin in whales: validation and measurement of mRNA expression by absolute quantitative real-time PCR.

    PubMed

    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 that the use of

  5. Differential Expression and Clinical Significance of Glioblastoma mRNA Expression Profiles in Uyghur and Han Patients in Xinjiang Province, China

    PubMed Central

    Liu, Liang; Li, Wenting; Xia, Haicheng; Zhu, Zhengquan; Luan, Xinping

    2014-01-01

    Background The aim of this study was to investigate differences in glioblastoma RNA gene expression profiles between Uyghur and Han patients in Xinjiang province and to screen and compare differentially expressed genes with respect to their clinical significance in the pathogenesis of high-grade glioma and their relationship to disease prognosis. Material/Methods Illumina HT-12mRNA expression profiles microarray was employed to measure the gene expression profiles of 6 patients with advanced glioma and to screen for differentially expressed genes. Results GO and KEGG analyses were performed on the differentially expressed genes using Web Gestalt software (P<0.05). Comparison of glioblastoma RNA expression profiles in the Uyghur and Han patients indicated that 1475 genes were significantly differentially expressed, of which 669 showed increased expression, while 807 showed decreased expression. One gene (STRC) corresponded to 2 transcripts, 1 of which showed increased expression and the other showed decreased expression. The differentially expressed genes participate in metabolic processes, biological regulation, stress response, and multi-cellular organic processes, including small GTPase regulatory signaling pathways, Ras signaling pathway, neuronal reactive protein regulation, and myelination of the central nervous system. The genes are also involved in tumor-related signaling pathways, including metabolic pathways, cancer pathways, MAPK signaling pathway, TGF-β signaling pathway, neurotrophic factor signal transduction pathway, and mTOR signaling pathway. Conclusions Differentially expressed genes were screened by studying the gene expression profiles in glioblastoma from Uyghur and Han patients. The cellular function and location of these genes were further investigated. Based on related molecular markers of glioblastoma, the differences in the mechanism of initiation and development of glioblastoma between Uyghur and Han patients were investigated for

  6. The use of logic relationships to model colon cancer gene expression networks with mRNA microarray data.

    PubMed

    Ruan, Xiaogang; Wang, Jinlian; Li, Hui; Perozzi, Rhoda E; Perozzi, Edmund F

    2008-08-01

    The ultimate goal of genomics research is to describe the network of molecules and interactions that govern all biological functions and disease processes in cells. Nonlinear interactions among genes in terms of their logic relationships play a key role for deciphering the networks of molecules that underlie cellular function. We present a method based on a graph coloring scheme and information theory to identify the gene expression network with lower and higher order logic interactions of genes. The analysis of oncogenes and suppressor genes from a colon cancer mRNA microarray dataset identifies a gene expression network with directionality and weights that reflects intracellular communication pathways. The success of the proposed method in mining hidden, complicated gene interactions and reliably interpreting experimental results suggests that the proposed method is a useful tool for understanding cancer systems. Extension of this method holds the potential to be fruitful for understanding other complex, nonsymmetric systems. PMID:18249040

  7. Bistable Expression of CsgD in Salmonella enterica Serovar Typhimurium Connects Virulence to Persistence

    PubMed Central

    MacKenzie, Keith D.; Wang, Yejun; Shivak, Dylan J.; Wong, Cynthia S.; Hoffman, Leia J. L.; Lam, Shirley; Kröger, Carsten; Cameron, Andrew D. S.; Townsend, Hugh G. G.; Köster, Wolfgang

    2015-01-01

    Pathogenic bacteria often need to survive in the host and the environment, and it is not well understood how cells transition between these equally challenging situations. For the human and animal pathogen Salmonella enterica serovar Typhimurium, biofilm formation is correlated with persistence outside a host, but the connection to virulence is unknown. In this study, we analyzed multicellular-aggregate and planktonic-cell subpopulations that coexist when S. Typhimurium is grown under biofilm-inducing conditions. These cell types arise due to bistable expression of CsgD, the central biofilm regulator. Despite being exposed to the same stresses, the two cell subpopulations had 1,856 genes that were differentially expressed, as determined by transcriptome sequencing (RNA-seq). Aggregated cells displayed the characteristic gene expression of biofilms, whereas planktonic cells had enhanced expression of numerous virulence genes. Increased type three secretion synthesis in planktonic cells correlated with enhanced invasion of a human intestinal cell line and significantly increased virulence in mice compared to the aggregates. However, when the same groups of cells were exposed to desiccation, the aggregates survived better, and the competitive advantage of planktonic cells was lost. We hypothesize that CsgD-based differentiation is a form of bet hedging, with single cells primed for host cell invasion and aggregated cells adapted for persistence in the environment. This allows S. Typhimurium to spread the risks of transmission and ensures a smooth transition between the host and the environment. PMID:25824832

  8. Effects of chronic noise on mRNA and protein expression of CRF family molecules and its relationship with p-tau in the rat prefrontal cortex.

    PubMed

    Gai, Zhihui; Li, Kang; Sun, Huanrui; She, Xiaojun; Cui, Bo; Wang, Rui

    2016-09-15

    Chronic noise exposure has been associated with Alzheimer's disease (AD)-like pathological changes, such as tau hyperphosphorylation and β-amyloid peptide accumulation in the prefrontal cortex (PFC). Corticotropin-releasing factor (CRF) is the central driving force in the stress response and a regulator of tau phosphorylation via binding to CRF receptors (CRFR). Little is known about the CRF system in relation to noise-induced AD-like changes in the PFC. The aim of this study was to explore the effects of chronic noise exposure on the CRF system in the PFC of rats and its relationship to tau phosphorylation. Male Wistar rats were randomly divided into control and noise exposure groups. The CRF system was evaluated following chronic noise exposure (95dB sound pressure level white noise, 4h/day×30days). Chronic noise significantly accelerated the progressive overproduction of corticosterone and upregulated CRF and CRFR1 mRNA and protein, both of which persisted 7-14days after noise exposure. In contrast, CRFR2 was elevated 3-7days following the last stimulus. Double-labeling immunofluorescence co-localized p-tau with CRF in PFC neurons. The results suggest that chronic noise exposure elevates the expression of the CRF system, which may contribute to AD-like changes. PMID:27538655

  9. 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

  10. 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?

  11. The vasopressin mRNA poly(A) tract is unusually long and increases during stimulation of vasopressin gene expression in vivo.

    PubMed Central

    Carrazana, E J; Pasieka, K B; Majzoub, J A

    1988-01-01

    We developed a method, termed an H-blot, by which the poly(A) tract of any specific mRNA may be detected by RNA filter hybridization after its removal from the body of the mRNA by a RNase H-catalyzed endonucleolytic cleavage in the 3' untranslated region. Using this method, we studied the modulation of the length of the poly(A) tract of rat vasopressin mRNA in vivo during changes in the levels of this mRNA resulting from a physiologic stimulus, osmotic stress. The poly(A) tract of hypothalamic vasopressin mRNA in hydrated rats was, quite remarkably, approximately 250 nucleotides in length, in contrast to that of somatostatin mRNA, which was approximately 30 nucleotides long. Vasopressin mRNA poly(A) tail length increased progressively from approximately 250 to approximately 400 nucleotides with the application of the hyperosmotic stimulus and declined to base line after its removal; somatostatin mRNA poly(A) tail length did not change during osmotic stress. The poly(A) tract length of total hypothalamic mRNA was between 35 and 140 nucleotides and also did not change with osmotic stress. Modulation of poly(A) tract length of specific mRNAs during stimulation of gene expression may provide an additional level of genetic regulation. Images PMID:2841576

  12. The vasopressin mRNA poly(A) tract is unusually long and increases during stimulation of vasopressin gene expression in vivo

    SciTech Connect

    Carrazana, E.J.; Pasieka, K.B.; Majzoub, J.A.

    1988-06-01

    The authors developed a method, termed an H-blot, by which the poly(A) tract of any specific mRNA may be detected by RNA filter hybridization after its removal from the body of the mRNA by a RNase H-catalyzed endonucleolytic cleavage in the 3' untranslated region. Using this method, they studied the modulation of the length of the poly(A) tract of rat vasopressin mRNA in vivo during changes in the levels of this mRNA resulting from a physiologic stimulus, osmotic stress. The poly(A) tract of hypothalamic vasopressin mRNA in hydrated rats was, quite remarkably, --250 nucleotides in length, in contrast to that of somatostatin mRNA, which was --30 nucleotides long. Vasopressin mRNA poly(A) tail length increased progressively from --250 to --400 nucleotides with the application of the hyperosmotic stimulus and declined to base line after its removal; somatostatin mRNA poly(A) tail length did not change during osmotic stress. The poly(A) tract length of total hypothalamic mRNA was between 35 and 140 nucleotides and also did not change with osmotic stress. Modulation of poly(A) tract length of specific mRNAs during stimulation of gene expression may provide an additional level of genetic regulation.

  13. Efficient delivery and functional expression of transfected modified mRNA in human embryonic stem cell-derived retinal pigmented epithelial cells.

    PubMed

    Hansson, Magnus L; Albert, Silvia; González Somermeyer, Louisa; Peco, Rubén; Mejía-Ramírez, Eva; Montserrat, Núria; Izpisua Belmonte, Juan Carlos

    2015-02-27

    Gene- and cell-based therapies are promising strategies for the treatment of degenerative retinal diseases such as age-related macular degeneration, Stargardt disease, and retinitis pigmentosa. Cellular engineering before transplantation may allow the delivery of cellular factors that can promote functional improvements, such as increased engraftment or survival of transplanted cells. A current challenge in traditional DNA-based vector transfection is to find a delivery system that is both safe and efficient, but using mRNA as an alternative to DNA can circumvent these major roadblocks. In this study, we show that both unmodified and modified mRNA can be delivered to retinal pigmented epithelial (RPE) cells with a high efficiency compared with conventional plasmid delivery systems. On the other hand, administration of unmodified mRNA induced a strong innate immune response that was almost absent when using modified mRNA. Importantly, transfection of mRNA encoding a key regulator of RPE gene expression, microphthalmia-associated transcription factor (MITF), confirmed the functionality of the delivered mRNA. Immunostaining showed that transfection with either type of mRNA led to the expression of roughly equal levels of MITF, primarily localized in the nucleus. Despite these findings, quantitative RT-PCR analyses showed that the activation of the expression of MITF target genes was higher following transfection with modified mRNA compared with unmodified mRNA. Our findings, therefore, show that modified mRNA transfection can be applied to human embryonic stem cell-derived RPE cells and that the method is safe, efficient, and functional. PMID:25555917

  14. 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. PMID:27245757

  15. 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

  16. Vascular activation of adhesion molecule mRNA and cell surface expression by ionizing radiation.

    PubMed

    Heckmann, M; Douwes, K; Peter, R; Degitz, K

    1998-01-10

    During cutaneous inflammatory reactions the recruitment of circulating leukocytes into the tissue critically depends on the regulated expression of endothelial cell adhesion molecules (CAMs). Various proinflammatory stimuli upregulate endothelial CAMs, including cytokines and UV irradiation. We have investigated the effects of ionizing radiation (IR) on endothelial CAM expression. Organ cultures of normal human skin as well as cultured human dermal microvascular endothelial cells (HDMEC) were exposed to IR. Expression of three major endothelial CAMs was studied in skin organ cultures by immunohistochemistry and in cell culture by Northern blot analysis and flow cytometry. In skin organ cultures vascular immunoreactivity for ICAM-1, E-selectin, and VCAM-1 was strongly induced 24 h after exposure to 5 or 10 Gy of IR, while immunoreactivity for CD31/PECAM-1, a constitutively expressed endothelial cell adhesion molecule, remained unchanged. In cultured HDMEC IR upregulated ICAM-1, VCAM-1, and E-selectin mRNAs and cell surface expression in a time- and dose-dependent fashion. Cellular morphology and viability remained unaltered by IR up to 24 h postirradiation. This study characterizes microvascular activation of adhesion molecule expression in response to ionizing radiation in a clinically relevant IR dose range. The findings also underscore the ability of endothelial cells to integrate environmental electromagnetic stimuli. PMID:9457067

  17. 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

  18. Early and persistent up-regulated expression of renal cortical osteopontin in experimental hydronephrosis.

    PubMed Central

    Diamond, J. R.; Kees-Folts, D.; Ricardo, S. D.; Pruznak, A.; Eufemio, M.

    1995-01-01

    The mechanical disturbance after unilateral ureteral obstruction (UUO) is a nonimmune stimulus that is capable of eliciting a florid macrophage infiltration of the kidney and subsequent post-inflammatory renal scarring. Osteopontin has potential chemoattractant activity and, for this reason, we delineated the kinetics of its expression in the renal cortex of rats with UUO. Whole body X-irradiation and reversal of UUO were utilized as interventional maneuvers to give additional pathobiological insight into this protein's role in the response of the kidneys to ureteral obstruction. Increased osteopontin mRNA levels in obstructed kidneys versus contralateral unobstructed specimens were evident as early as 4 hours after UUO and steadily increased at 12, 24, 48, and 96 hours after UUO. Both X-irradiation and reversal of UUO failed to significantly modulate renal cortical osteopontin mRNA expression at all of the above time points. Paralleling the increments in renal cortical osteopontin mRNA levels were significant elevations in the cortical renal interstitial macrophage number, which was significantly diminished by previous X-irradiation but not reversal of UUO. Focal labeling of osteopontin was noted in both tubular and Bowman's capsular epithelium in obstructed kidneys as early as 4 hours after UUO, whereas, in the contralateral unobstructed specimens, there was only faint staining in Bowman's capsule. By 96 hours after UUO, obstructed kidneys exhibited intense, diffuse staining for osteopontin in both tubules and Bowman's capsule. Osteopontin's immunolocalization was not modulated by X-irradiation or reversal of UUO. These data support the contention that osteopontin is involved in the accumulation of macrophages within the peritubular and periglomerular interstitium in the obstructed renal cortex. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:7778684

  19. Dogs with patent Dirofilaria immitis infection have higher expression of circulating IL-4, IL-10 and iNOS mRNA than those with occult infection.

    PubMed

    Morchón, R; López-Belmonte, J; Bazzocchi, C; Grandi, G; Kramer, L; Simón, F

    2007-01-15

    Dirofilaria immitis is the agent of canine heartworm disease, in which adult worms reside in the pulmonary arteries, producing first stage larvae (microfilariae) that are released into the bloodstream. The present work describes the cytokine and iNOS mRNA expression in the peripheral blood of naturally infected dogs classified as either microfilariemic or amicrofilariemic. Results show that microfilariemic dogs had higher expression of IL-4 and iNOS mRNA than amicrofilariemic dogs. Furthermore, IL-10 mRNA expression was strongly expressed in dogs with circulating microfilariae, compared to only negligible expression in amicrofilariemic dogs. Finally, mf+ status was associated with a predominance in IgG1 production against worm antigens. These results would suggest that circulating mf may stimulate, like in other filarial infections, an immune bias towards unresponsiveness in D. immitis-infected dogs, consenting long-term adult worm survival. PMID:17112598

  20. Synaptophysin: molecular organization and mRNA expression as determined from cloned cDNA.

    PubMed Central

    Leube, R E; Kaiser, P; Seiter, A; Zimbelmann, R; Franke, W W; Rehm, H; Knaus, P; Prior, P; Betz, H; Reinke, H

    1987-01-01

    Synaptophysin is a major glycoprotein of Mr approximately 38,000 (in deglycosylated form: Mr approximately 34,000) characteristic of a certain class of small (30-80 nm diameter) neurosecretory vesicles, including presynaptic vesicles, but also vesicles of various neuroendocrine cells of both neuronal and epithelial phenotype. Using synaptophysin-specific antibodies we have isolated cDNA clones from rat nervous tissue libraries, which identify an approximately 2.5-kb mRNA in rat and human cells, including neuroendocrine tumours, that contains a reading frame for a polypeptide of 307 amino acids with a total mol. wt of 33 312. The deduced amino acid sequence, which was partly confirmed by comparison with sequences of two tryptic peptides obtained from purified synaptophysin, revealed four hydrophobic regions of 24 amino acids each, which are characterized, according to conformation prediction analyses, by marked alpha-helicity. The sequence shows a single potential N-glycosylation site, which is assigned to the vesicle interior, and a carboxy-terminal tail of 89 amino acids which contains glycine-rich tetrapeptide repeats, the epitope of monoclonal antibody SY38, and a number of collagenase-sensitive sites accessible on the surface of the intact vesicles. These features suggest that the polypeptide spans the vesicle membrane four times, with both N and C termini located on the outer, i.e. cytoplasmic, surface of the vesicles. Images Fig. 2. Fig. 6. PMID:3123215

  1. Presence of claudins mRNA in the brain. Selective modulation of expression by kindling epilepsy.

    PubMed

    Lamas, Mónica; González-Mariscal, Lorenza; Gutiérrez, Rafael

    2002-08-15

    In the central nervous system, the junctional types that establish and maintain tissue architecture include gap junctions, for cytoplasmic connectivity, and tight junctions, for paracellular and/or cell polarity barriers. Connexins are the integral membrane proteins of gap junctions, whereas occludin and members of the multigene family of claudins form tight junctions. In the brain, there are no transendothelial pathways, as continuous tight junctions are present between the endothelial cells. Thus, they provide a continuous cellular barrier between the blood and the insterstitial fluid. However, several brain pathologies, including epilepsy, are known to alter the permeability of the blood-brain barrier and to cause edema. Therefore, since claudins, as constitutive proteins of tight junctions are likely candidates for modulation under pathological states, we explored their normal pattern of expression in the brain and its modulation by seizures. We found that several members of this family are normally expressed in the hippocampus and cortex. Interestingly, claudin-7 is expressed in the hippocampus but not in the cortex. On the other hand, the expression of claudin-8 is selectively down-regulated in the hippocampus as kindling evolves. These results link for the first time the modulation of expression of a tight junction protein to abnormal neuronal synchronization that could probably be reflected in permeability changes of the blood-brain barrier or edema. PMID:12225881

  2. Gene (mRNA) expression in canine atopic dermatitis: microarray analysis.

    PubMed

    Merryman-Simpson, Annemarie E; Wood, Shona H; Fretwell, Neale; Jones, Paul G; McLaren, William M; McEwan, Neil A; Clements, Dylan N; Carter, Stuart D; Ollier, William E; Nuttall, Tim

    2008-04-01

    Genes potentially involved in the pathology of canine atopic dermatitis (AD) were identified using gene expression microarrays. Total RNA extracted from skin biopsies was hybridized to an Agilent Technologies custom-designed 22K canine array. The arrays were analysed using Genedata Analyst software. Data were corrected for multiple hypothesis testing and tested for significance using the National Institute on Aging array analysis tool. For comparison, data were divided into separate groups: lesional atopic (n = 16), nonlesional atopic (n = 17) and healthy controls (n = 9). Fifty-four genes were differentially expressed at a significance level of 0.05 in canine AD compared to healthy controls. Sixteen genes were differentially expressed in both nonlesional and lesional atopic skin, 26 genes only in nonlesional skin and 12 only in lesional skin. These genes were associated with innate immune and inflammatory responses, cell cycle, apoptosis, barrier formation and transcriptional regulation. The most dysregulated gene in lesional skin was S100A8, which showed an almost 23-fold increase in expression. This is a pro-inflammatory cytokine located in the epidermal differentiation complex. Microarray analysis is a novel technique in canine AD. Significant changes in gene expression were identified in atopic skin. These were relevant to skin barrier formation and the immune response, suggesting that they both participate in AD. Gene expression restricted to lesional skin may be involved in inflammatory changes, whereas those shared or restricted to nonlesional skin may reflect the atopic phenotype. Investigating gene polymorphisms in the targets identified in this study will help improve our understanding of the genetic basis of this disease. PMID:18336422

  3. Modeling Genome-Scale mRNA Expression Datasets: From Matrix Algebra to Genetic Networks

    NASA Astrophysics Data System (ADS)

    Alter, Orly

    2003-03-01

    DNA microarray genome-wide expression data promise to enhance fundamental understanding of life on the molecular level, and may prove useful in medical diagnosis, treatment and drug design. Analysis of these new data requires mathematical tools that use large quantities of data and reduce the complexity of the data to make them comprehensible. These tools should provide predictive models, i.e., mathematical frameworks for the description of the data, in which the mathematical variables and operations may be assigned biological meaning. Such models will facilitate the unraveling of the cellular machineries that generate, sense and react to the expression signal. I will start with a description of the use of singular value decomposition (SVD) to construct the first model for genome-wide expression data. SVD is a unique data-driven linear transformation of the expression data from the genes × arrays space to the reduced ``eigengenes'' × ``eigenarrays'' space, where the eigengenes (eigenarrays) are unique orthonormal superpositions of the genes (arrays). Normalizing the data, by detecting and filtering out additive and multiplicative experimental artifacts and irrelevant biological processes, enables meaningful comparison of the expression of different genes across different arrays in different experiments. Sorting the data, according to a chosen subset of eigengenes (and eigenarrays), rather than by overall expression, gives a global picture of gene expression in which individual genes and arrays appear to be classified into groups of similar regulation and function, or similar cellular state and biological phenotype, respectively. In some experiments, the significant eigengenes and eigenarrays can be associated with genome-wide effects of regulators, or with measured samples in which these regulators are overactive or underactive, respectively. I will then describe the use of generalized singular value decomposition (GSVD) to construct the first comparative model

  4. 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.

  5. FGFR1 mRNA and Protein Expression, not Gene Copy Number, Predict FGFR TKI Sensitivity Across All Lung Cancer Histologies

    PubMed Central

    Wynes, Murry W.; Hinz, Trista K.; Gao, Dexiang; Martini, Michael; Marek, Lindsay A.; Ware, Kathryn E.; Edwards, Michael G.; Böhm, Diana; Perner, Sven; Helfrich, Barbara A.; Dziadziuszko, Rafal; Jassem, Jacek; Wojtylak, Szymon; Sejda, Aleksandra; Gozgit, Joseph M.; Bunn, Paul A.; Camidge, D. Ross; Tan, Aik-Choon; Hirsch, Fred R.; Heasley, Lynn E.

    2014-01-01

    Purpose FGFR1 gene copy number (GCN) is being evaluated as a biomarker for FGFR tyrosine kinase inhibitor (TKI) response in squamous-cell lung cancers (SCC). The exclusive use of FGFR1 GCN for predicting FGFR TKI sensitivity assumes increased GCN is the only mechanism for biologically-relevant increases in FGFR1 signaling. Herein, we tested whether FGFR1 mRNA and protein expression may serve as better biomarkers of FGFR TKI sensitivity in lung cancer. Experimental Design Histologically diverse lung cancer cell lines were submitted to assays for ponatinib sensitivity, a potent FGFR TKI. A tissue microarray comprised of resected lung tumors was submitted to FGFR1 GCN and mRNA analyses and the results were validated with TCGA lung cancer data. Results 14/58 cell lines exhibited ponatinib sensitivity (IC50 values ≤ 50 nM) that correlated with FGFR1 mRNA and protein expression, but not with FGFR1 GCN or histology. Moreover, ponatinib sensitivity associated with mRNA expression of the ligands, FGF2 and FGF9. In resected tumors, 22% of adenocarcinomas and 28% of SCCs expressed high FGFR1 mRNA. Importantly, only 46% of SCCs with increased FGFR1 GCN expressed high mRNA. Lung cancer TCGA data validated these findings and unveiled overlap of FGFR1 mRNA positivity with KRAS and PIK3CA mutations. Conclusions FGFR1 dependency is frequent across various lung cancer histologies and FGFR1 mRNA may serve as a better biomarker of FGFR TKI response in lung cancer than FGFR1 GCN. The study provides important and timely insight into clinical testing of FGFR TKIs in lung cancer and other solid tumor types. PMID:24771645

  6. 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

  7. The effect of exercise on expression of myokine and angiogenesis mRNA in skeletal muscle of high fat diet induced obese rat

    PubMed Central

    Shin, Ki Ok; Bae, Ju Yong; Woo, Jinhee; Jang, Ki Soeng; Kim, Keun Su; Park, Jung Sub; Kim, In Ki; Kang, Sunghwun

    2015-01-01

    [Purpose] The purpose of this study was to investigate the effect of regular treadmill exercise on the mRNA expressions of myokines and angiogenesis factors in the skeletal muscle of obese rats. [Methods] Thirty two male Sprague-Dawley rats (4weeks old) were divided into the CO (control) and HF (high fat diet) groups. Obesity was induced in the HF group by consumption of 45% high-fat diet for 15 weeks. These groups were further subdivided into training groups (COT and HFT); the training groups conducted moderate intensity treadmill training for 8 weeks. Soleus muscles were excised and analyzed by real-time quantitative PCR. [Results] mRNA expression of myokines, such as PGC-1α, IL-6, and IL-15, in the COT and HFT groups (which conducted regular exercise), were higher as compared with the CO and HF groups (p < 0.05). Also, the levels in the HF group were significantly lower when compared with CO group (p < 0.05). Expression of angiogenesis mRNA, namely mTOR, VEGF, and FLT1, were significantly lower in the HF group, as compared to the CO group (p < 0.05). In addition, COT group had a higher expression of mTORC1, mTORC2, VEGF and FLT mRNA, than the CO group (p < 0.05); the HFT group also had higher expressions of mTOR, VEGF and FLT1 mRNA than the HF group (p < 0.05). [Conclusion] These results indicate that mRNA expression of myokines was increased through the activity of muscle contraction, and it also promoted the mRNA expression of angiogenesis due to activation of mTOR. Thus, we conclude that not only under normal health conditions, but in obesity and excess nutritional circumstances also, regular exercise seems to act positively on the glycemic control and insulin sensitivity through the angiogenesis signaling pathway. PMID:26244127

  8. Spatial behavior and seizure-induced changes in c-fos mRNA expression in young and old rats.

    PubMed

    Chawla, Monica K; Penner, Marsha R; Olson, Kathy M; Sutherland, Vicki L; Mittelman-Smith, Melinda A; Barnes, Carol A

    2013-04-01

    The subcellular processes of gene induction and expression in the hippocampus are likely to underlie some of the known age-related impairments in spatial learning and memory. It is well established that immediate-early genes are rapidly and transiently induced in response to neuronal activity and this expression is required for stabilization of durable memories. To examine whether age-related memory impairment might be caused, in part, by differences in the level of cellular activation or subcellular processing, c-fos expression in CA1 pyramidal and dentate gyrus granule cells in the dorsal hippocampus of young and old rats was determined using fluorescence in situ hybridization and reverse transcription polymerase chain reaction. No significant age differences were found in the numbers of pyramidal or granule cells that show c-fos expression; however, c-fos mRNA transcripts were altered in these 2 cell types in aged animals. These findings suggest that though the networks of cells that participate in behavior or seizure-induced activity are largely maintained in aged rats, their RNA transcript levels are altered. This might, in part, contribute to cognitive deficits frequently observed with advancing age. PMID:23158763

  9. Gonadotropin-releasing hormone receptor mRNA expression by human pituitary tumors in vitro.

    PubMed Central

    Alexander, J M; Klibanski, A

    1994-01-01

    An important question in the pathogenesis and regulation of human gonadotroph adenomas is whether heterogeneous gonadotropin responses to gonadotropin-releasing hormone (GnRH) are due to dysregulation of GnRH receptor biosynthesis and/or cell-signaling pathways. We investigated gonadotropin responsiveness to pulsatile GnRH in 13 gonadotroph adenomas. All tumors had evidence of follicle-stimulating hormone (FSH) beta and alpha subunit biosynthesis using reverse transcriptase/polymerase chain reaction (RTPCR) techniques. Four tumors significantly increased gonadotropin and/or free subunit secretion during pulsatile 10(-8) M GnRH administration. The GnRH antagonist Antide (10(-6) to 10(-8) M) blocked secretory increases in all GnRH-responsive tumors. Gonadotropin and/or free subunit secretion increased after 60 mM KCl, confirming that GnRH nonresponsiveness was not due to intracellular gonadotropin depletion. We hypothesized that GnRH nonresponsiveness in these tumors may be due to GnRH receptor (GnRH-Rc) biosynthetic defects. RTPCR analyses detected GnRH-Rc transcripts only in responsive tumors and normal human pituitary. This is the first demonstration of a cell-surface receptor biosynthetic defect in human pituitary tumors. We conclude (a) one third of gonadotroph tumors respond to pulsatile GnRH in vitro, (b) GnRH-Rc mRNA is detected in human gonadotroph adenomas and predicts GnRH responsiveness, and (c) GnRH-Rc biosynthetic defects may underlie GnRH nonresponsiveness in gonadotroph tumors. Images PMID:8200967

  10. Delayed Correlation of mRNA and Protein Expression in Rapamycin-treated Cells and a Role for Ggc1 in Cellular Sensitivity to Rapamycin*

    PubMed Central

    Fournier, Marjorie L.; Paulson, Ariel; Pavelka, Norman; Mosley, Amber L.; Gaudenz, Karin; Bradford, William D.; Glynn, Earl; Li, Hua; Sardiu, Mihaela E.; Fleharty, Brian; Seidel, Christopher; Florens, Laurence; Washburn, Michael P.

    2010-01-01

    To identify new molecular targets of rapamycin, an anticancer and immunosuppressive drug, we analyzed temporal changes in yeast over 6 h in response to rapamycin at the transcriptome and proteome levels and integrated the expression patterns with functional profiling. We show that the integration of transcriptomics, proteomics, and functional data sets provides novel insights into the molecular mechanisms of rapamycin action. We first observed a temporal delay in the correlation of mRNA and protein expression where mRNA expression at 1 and 2 h correlated best with protein expression changes after 6 h of rapamycin treatment. This was especially the case for the inhibition of ribosome biogenesis and induction of heat shock and autophagy essential to promote the cellular sensitivity to rapamycin. However, increased levels of vacuolar protease could enhance resistance to rapamycin. Of the 85 proteins identified as statistically significantly changing in abundance, most of the proteins that decreased in abundance were correlated with a decrease in mRNA expression. However, of the 56 proteins increasing in abundance, 26 were not correlated with an increase in mRNA expression. These protein changes were correlated with unchanged or down-regulated mRNA expression. These proteins, involved in mitochondrial genome maintenance, endocytosis, or drug export, represent new candidates effecting rapamycin action whose expression might be post-transcriptionally or post-translationally regulated. We identified GGC1, a mitochondrial GTP/GDP carrier, as a new component of the rapamycin/target of rapamycin (TOR) signaling pathway. We determined that the protein product of GGC1 was stabilized in the presence of rapamycin, and the deletion of the GGC1 enhanced growth fitness in the presence of rapamycin. A dynamic mRNA expression analysis of Δggc1 and wild-type cells treated with rapamycin revealed a key role for Ggc1p in the regulation of ribosome biogenesis and cell cycle progression

  11. Characterization and mRNA expression profile of the TbNre1 gene of the ectomycorrhizal fungus Tuber borchii.

    PubMed

    Guescini, Michele; Stocchi, L; Sisti, D; Zeppa, S; Polidori, E; Ceccaroli, P; Saltarelli, R; Stocchi, V

    2009-02-01

    This study focuses on the cloning and characterization of the major nitrogen regulator element from the ectomycorrhizal fungus Tuber borchii, TbNre1. Sequence analysis of the predicted protein and complementation experiments in Neurospora crassa demonstrated that the cloned gene is orthologous to areA/nit-2 gene. Transcriptional expression investigations by real-time RT-PCR showed TbNre1 up-regulation in the presence of nitrate or in the absence of nitrogen during free-living mycelium growth. On the contrary, TbNre1 mRNA levels remained at basal values in the presence of preferred nitrogen sources like ammonium and glutamine. Furthermore, TbNre1 mRNA was found to be up-regulated during T. borchii and T. platyphyllos interaction. All these data suggest that the regulatory protein TBNRE1 could play a major role in regulating N metabolism genes of T. borchii in the free living mycelium and in T. borchii-T. platyphyllos interaction. Finally, the possible role of the transcription factor TBNRE1 in the induction of proteases and virulence-like genes, necessary in ectomycorrhizal establishment, was also discussed. PMID:19115055

  12. 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. PMID:19460375

  13. 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. PMID:16293374

  14. E5HPV16 mRNA EXPRESSION PATTERN ANALYSIS IN PATIENTS WITH CERVICAL LESIONS IN VIRAL STATUS CONTEXT.

    PubMed

    Iancu, Iulia V; Pleşa, Adriana; Botezatu, Anca; Huică, Irina; Stănescu, Anca D; Socolov, Demetra; Anton, Gabriela

    2015-01-01

    Human papilloma virus (HPV) may cause mostly transient infections of cutaneous and mucous epithelia. Persistent HPV genital infections may induce pre-malignant or malignant lesions. While E6 and E7 HPV genes' malignant character is known, E5 is still under debate. We evaluated the possible role of E5 gene in cervix oncogenesis, in patients with abnormal cytology and HPV1 6 positive, in the context of viral status correlated with potential targets (p21, EGFR). HPV DNA was detected and genotyped using Linear Array HPV Genotyping Test (Roche Molecular Biochemicals, Mannheim, Germany) and E2, E6, E5 HPV16, p21 and EGFR transcripts levels were investigated by qRT-PCR. Our results indicate a significantly high E5 expression in low grade cytology, expression correlated with a moderated E6 and low p21 levels. All HSIL specimens presented integrated/mixed viral forms; mixed forms presented moderate E5 expression, high levels of p21 correlates with E6 oncogene high expression. These findings indicate a potential role for E5 pattern of expression in discriminating be-tween lesions that may progress to cancer. PMID:26727852

  15. The regulation of gene expression in transformed maize aleurone and endosperm protoplasts. Analysis of promoter activity, intron enhancement, and mRNA untranslated regions on expression.

    PubMed Central

    Gallie, D R; Young, T E

    1994-01-01

    Gene expression in the aleurone and endosperm is highly regulated during both seed development and germination. Studies of alpha-amylase expression in the aleurone of barley (Hordeum vulgare) have generated the current paradigm for hormonal control of gene expression in germinating cereal grain. Gene expression studies in both the aleurone and endosperm tissues of maize (Zea mays) seed have been hampered because of a lack of an efficient transformation system. We report here the rapid isolation of protoplasts from maize aleurone and endosperm tissue, their transformation using polyethylene glycol or electroporation, and the regulation of gene expression in these cells. Adh1 promoter activity was reduced relative to the 35S promoter in aleurone and endosperm protoplasts compared to Black Mexican Sweet suspension cells in which it was nearly as strong as the 35S promoter. Intron-mediated stimulation of expression was substantially higher in transformed aleurone or endosperm protoplasts than in cell-suspension culture protoplasts, and the data suggest that the effect of an intron may be affected by cell type. To examine cytoplasmic regulation, the 5' and 3' untranslated regions from a barley alpha-amylase were fused to the firefly luciferase-coding region, and their effect on translation and mRNA stability was examined following the delivery of in vitro synthesized mRNA to aleurone and endosperm protoplasts. The alpha-amylase untranslated regions regulated translational efficiency in a tissue-specific manner, increasing translation in aleurone or endosperm protoplasts but not in maize or carrot cell-suspension protoplasts, in animal cells, or in in vitro translation lysates. PMID:7824660

  16. Association between Toll-like Receptors expression and Human Papillomavirus Type 16 Persistence

    PubMed Central

    Daud, Ibrahim I.; Scott, Mark E.; Ma, Yifei; Shiboski, Stephen; Farhat, Sepideh; Moscicki, Anna-Barbara

    2010-01-01

    The mechanisms involved in mucosal immune control of cervical human papillomavirus (HPV) infection remain ill-defined. Because Toll-like receptors (TLRs) are key players in innate immune responses, we investigated the association between TLR expression and viral persistence or clearance in young women with incident infections with oncogenic HPV types 16 or 51. Messenger RNA expression of TLR1, TLR2, TLR3, TLR4, TLR6, TLR7, TLR8, and TLR9 was measured by quantitative reverse transcription-PCR using human endocervical specimens, collected before and following viral acquisition, in a cohort well characterized for HPV infections. Wilcoxon rank sum test was used to compare the change seen from pre-infection to incident infection between women who subsequently cleared infection with those who did not. HPV 16 infections that cleared were significantly (P < 0.05) associated with an increase in expression of the four viral nucleic acid-sensing TLRs (TLR3, TLR7, TLR8 and TLR9) as well as TLR2 upon viral acquisition. Similar associations were not observed for HPV 51. In women who subsequently cleared their HPV 16 infection, changes in TLR1, TLR3, TLR7, and TLR8 expression levels between pre-incident and incident visits were significantly correlated with parallel changes in the levels of interferon-α2, measured by immunoassay in cervical lavage specimens. This study suggests that dampened TLR expression in the cervical mucosa is a type-specific mechanism by which HPV 16 interferes with innate immune responses, contributing to viral persistence, and that TLR upregulation and resultant cytokine induction is important in subsequent viral clearance. PMID:20473890

  17. Human oocytes and preimplantation embryos express mRNA for growth hormone receptor.

    PubMed

    Ménézo, Y J; el Mouatassim, S; Chavrier, M; Servy, E J; Nicolet, B

    2003-11-01

    Human genetic expression of growth hormone receptor (GHR) gene was qualitatively analysed using reverse transcription polymerase chain reaction (RT-PCR) in cumulus cells, immature germinal vesicle (GV) and mature metaphase II (MII) stage oocytes and preimplantation human embryos. The transcripts encoding GHR were detected in cumulus cells and also in naked oocytes, either mature or not. In this case, a nested PCR is needed, as for early embryo preimplantation stages, before genomic activation. The GHR gene is highly expressed from the 4-day morula onwards. This suggests that GHR transcription follows a classical scheme associated with genomic activation. It is probable that, in human, growth hormone plays a role in the final stages of oocyte maturation and early embryogenesis as it does for several other mammalian species. PMID:15085728

  18. mRNA expression profile of mouse oligodendrocytes in inflammatory conditions.

    PubMed

    Kudriaeva, A A; Khaustova, N A; Maltseva, D V; Kuzina, E S; Glagoleva, I S; Surina, E A; Knorre, V D; Belogurov, A A; Tonevitsky, A G; Gabibov, A G

    2016-07-01

    In this study, we performed transcriptome profiling of oligodendrocyte culture of mice treated with the remyelinating therapeutic agent benztropine in the presence and absence of interferon gamma (IFNγ). The results of this work are important for understanding the expression profile of oligodendrocytes under conditions of systemic inflammation in the central nervous system in multiple sclerosis as well as the mechanisms of cellular response to benztropine in light of its possible use for the treatment of multiple sclerosis. PMID:27599508

  19. Modulation of immunoglobulin production and cytokine mRNA expression in peripheral blood mononuclear cells by intravenous immunoglobulin.

    PubMed

    Toyoda, M; Zhang, X; Petrosian, A; Galera, O A; Wang, S J; Jordan, S C

    1994-05-01

    Intravenous immunoglobulin (IVIG) has the potential to regulate Ig production, but the mechanism(s) responsible for this effect is unknown. In experiments reported here, we examined the ability of IVIG to regulate Ig production in human peripheral blood mononuclear cells (PBMCs) stimulated with pokeweed mitogen (PWM). IVIG (2-10 mg/ml) showed a potent (80-85%) inhibition of PWM-stimulated IgG, IgM, and IgA production. To determine more precisely how IVIG mediated the inhibition of Ig production, we studied Ig promoting cytokine gene expression after PWM stimulation with or without IVIG (2 and 10 mg/ml) using dot-blot techniques. RNA was isolated from PBMCs at predetermined time points and probed with cDNAs specific for human cytokines (IL-1 beta, IL-2, IL-2R, IL-4, IL-5, IL-6, gamma-IFN, and TNF-alpha). IL-6 mRNA accumulation was maximal at 4.5 hr post-PWM stimulation and was inhibited 64-75% when IVIG (10 mg/ml) was present. gamma-IFN mRNA levels peaked at 72 hr poststimulation and were also 68-75% inhibited by IVIG. IL-2 mRNA levels peaked at 4.5 hr and were 23-46% inhibited by IVIG. The inhibitory effect of IVIG on production of these cytokines (IL-6 and gamma-IFN) was also observed at the protein level in sonicated PBMCs after incubation with PWM and IVIG. The mRNA levels for other cytokines were not or only minimally inhibited by IVIG. Addition of IL-6, gamma-IFN, or IL-2 partially restored Ig production in IVIG-treated PWM-stimulated cultures, suggesting that inhibition of other cytokines or another mechanism(s) independent of cytokine inhibition might also be involved, although inhibition of IL-6, gamma-IFN, and IL-2 may be one of the critical factors in the suppression of Ig production by IVIG. PMID:7523434

  20. Effect of Dietary Lead on Intestinal Nutrient Transporters mRNA Expression in Broiler Chickens

    PubMed Central

    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. PMID:25695048

  1. A protein and mRNA expression-based classification of gastric cancer.

    PubMed

    Setia, Namrata; Agoston, Agoston T; Han, Hye S; Mullen, John T; Duda, Dan G; Clark, Jeffrey W; Deshpande, Vikram; Mino-Kenudson, Mari; Srivastava, Amitabh; Lennerz, Jochen K; Hong, Theodore S; Kwak, Eunice L; Lauwers, Gregory Y

    2016-07-01

    The overall survival of gastric carcinoma patients remains poor despite improved control over known risk factors and surveillance. This highlights the need for new classifications, driven towards identification of potential therapeutic targets. Using sophisticated molecular technologies and analysis, three groups recently provided genetic and epigenetic molecular classifications of gastric cancer (The Cancer Genome Atlas, 'Singapore-Duke' study, and Asian Cancer Research Group). Suggested by these classifications, here, we examined the expression of 14 biomarkers in a cohort of 146 gastric adenocarcinomas and performed unsupervised hierarchical clustering analysis using less expensive and widely available immunohistochemistry and in situ hybridization. Ultimately, we identified five groups of gastric cancers based on Epstein-Barr virus (EBV) positivity, microsatellite instability, aberrant E-cadherin, and p53 expression; the remaining cases constituted a group characterized by normal p53 expression. In addition, the five categories correspond to the reported molecular subgroups by virtue of clinicopathologic features. Furthermore, evaluation between these clusters and survival using the Cox proportional hazards model showed a trend for superior survival in the EBV and microsatellite-instable related adenocarcinomas. In conclusion, we offer as a proposal a simplified algorithm that is able to reproduce the recently proposed molecular subgroups of gastric adenocarcinoma, using immunohistochemical and in situ hybridization techniques. PMID:27032689

  2. Identification of novel isoforms of dairy goat EEF1D and their mRNA expression characterization.

    PubMed

    Zhang, Sihuan; Wu, Xianfeng; Pan, Chuanying; Lei, Chuzhao; Dang, Ruihua; Chen, Hong; Lan, Xianyong

    2016-04-25

    Eukaryotic translation elongation factor 1 delta (EEF1D) gene encodes guanine nucleotide exchange protein eEF1Bδ, which participates in the eukaryotic protein synthesis, and plays important roles in regulating cell cycling and milk production. This study firstly focused on detecting the isoforms of dairy goat EEF1D gene and their mRNA expression characterization. Herein, two novel isoforms, EEF1Da and EEF1Dc, were identified in dairy goat. The entire coding sequences of EEF1Da and EEF1Dc isoforms were 843bp and 267bp in length, respectively. Goat EEF1Da had complete conserved domains of elongation factor 1 (EF1) family, and the evolution of goat EEF1Da isoform was agreed with the evolution of species. Expression pattern analysis of different isoforms revealed relatively ubiquitous expression of EEF1D and EEF1Da. While EEF1Dc only expressed in heart, lung, kidney, adipose and muscle. Combining with the analysis results of cloning, qRT-PCR and bioinformatics, EEF1Da is the major alternative splicing form of EEF1D gene. Interestingly, qRT-PCR result showed that the highest expression of EEF1D was in adipose, which is the major component of mammary. This result was consistent with the early research that EEF1D expressed highly in the mammary, which indicated that EEF1D played a potential key role in regulating adipose development and milk production. All these findings would provide a foundation for the further research of EEF1D gene and development of dairy goat industry. PMID:26794801

  3. 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. PMID:26854762

  4. Increased serum hepcidin-25 level and increased tumor expression of hepcidin mRNA are associated with metastasis of renal cell carcinoma

    PubMed Central

    2009-01-01

    Background Hepcidin has an important role in iron metabolism. We investigated whether hepcidin was involved in renal cell carcinoma (RCC). Methods We measured serum hepcidin-25 levels in 32 patients by liquid chromatograpy (LC)-mass spectrometry (MS)/MS, and assessed hepcidin mRNA expression in paired tumor and non-tumor tissue samples from the surgical specimens of 53 consecutive patients with RCC by real-time reverse transcription polymerase chain reaction. Results The serum hepcidin-25 level was higher in patients with metastatic RCC than nonmetastatic RCC (P < 0.0001), and was positively correlated with the serum interleukin-6 and C-reactive protein levels (P < 0.001). Expression of hepcidin mRNA was lower in tumor tissues than in non-tumor tissues (P < 0.0001). The serum hepcidin-25 level was not correlated with the expression of hepcidin mRNA in the corresponding tumor tissue specimens from 32 patients. Hepcidin mRNA expression in tumor tissue was correlated with metastatic potential, but not with histological differentiation or tumor stage. Kaplan-Meier analysis showed that over expression of hepcidin mRNA was related to shorter overall survival in RCC patients. Univariate analysis (Cox proportional hazards model) showed that the hepcidin mRNA level was an independent prognostic factor for overall survival. Conclusion Our findings suggest that a high serum hepcidin-25 level may indicate the progression of RCC, and that upregulation of hepcidin mRNA expression in tumor tissue may be related to increased metastatic potential. PMID:19656379

  5. 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.

  6. Association Analysis of Myosin Heavy-chain Genes mRNA Transcription with the Corresponding Proteins Expression of Longissimus Muscle in Growing Pigs.

    PubMed

    Men, X M; Deng, B; Tao, X; Qi, K K; Xu, Z W

    2016-04-01

    The goal of this work was to investigate the correlations between MyHC mRNA transcription and their corresponding protein expressions in porcine longissimus muscle (LM) during postnatal growth of pigs. Five DLY (Duroc×Landrace×Yorkshire) crossbred pigs were selected, slaughtered and sampled at postnatal 7, 30, 60, 120, and 180 days, respectively. Each muscle was subjected to quantity MyHCs protein contents through an indirect enzyme-linked immunosorbent assay (ELISA), to quantity myosin heavy-chains (MyHCs) mRNA abundances using real-time polymerase chain reaction. We calculated the proportion (%) of each MyHC to total of four MyHC for two levels, respectively. Moreover, the activities of several key energy metabolism enzymes were determined in LM. The result showed that mRNA transcription and protein expression of MyHC I, IIa, IIx and IIb in LM all presented some obvious changes with postnatal aging of pigs, especially at the early stage after birth, and their mRNA transcriptions were easy to be influenced than their protein expressions. The relative proportion of each MyHC mRNA was significantly positively related to that of its corresponding protein (p<0.01), and MyHC I mRNA proportion was positively correlated with creatine kinase (CK), succinate dehydrogenase (SDH), malate dehydrogenase (MDH) activities (p<0.05). These data suggested that MyHC mRNA transcription can be used to reflect MyHC expression, metabolism property and adaptive plasticity of porcine skeletal muscles, and MyHC mRNA composition could be a molecular index reflecting muscle fiber type characteristics. PMID:26949945

  7. Association Analysis of Myosin Heavy-chain Genes mRNA Transcription with the Corresponding Proteins Expression of Longissimus Muscle in Growing Pigs

    PubMed Central

    Men, X. M.; Deng, B.; Tao, X.; Qi, K. K.; Xu, Z. W.

    2016-01-01

    The goal of this work was to investigate the correlations between MyHC mRNA transcription and their corresponding protein expressions in porcine longissimus muscle (LM) during postnatal growth of pigs. Five DLY (Duroc×Landrace×Yorkshire) crossbred pigs were selected, slaughtered and sampled at postnatal 7, 30, 60, 120, and 180 days, respectively. Each muscle was subjected to quantity MyHCs protein contents through an indirect enzyme-linked immunosorbent assay (ELISA), to quantity myosin heavy-chains (MyHCs) mRNA abundances using real-time polymerase chain reaction. We calculated the proportion (%) of each MyHC to total of four MyHC for two levels, respectively. Moreover, the activities of several key energy metabolism enzymes were determined in LM. The result showed that mRNA transcription and protein expression of MyHC I, IIa, IIx and IIb in LM all presented some obvious changes with postnatal aging of pigs, especially at the early stage after birth, and their mRNA transcriptions were easy to be influenced than their protein expressions. The relative proportion of each MyHC mRNA was significantly positively related to that of its corresponding protein (p<0.01), and MyHC I mRNA proportion was positively correlated with creatine kinase (CK), succinate dehydrogenase (SDH), malate dehydrogenase (MDH) activities (p<0.05). These data suggested that MyHC mRNA transcription can be used to reflect MyHC expression, metabolism property and adaptive plasticity of porcine skeletal muscles, and MyHC mRNA composition could be a molecular index reflecting muscle fiber type characteristics. PMID:26949945

  8. Transforming growth factor-β1 induces cholesterol synthesis by increasing HMG-CoA reductase mRNA expression in keratinocytes.

    PubMed

    Yamane, Takumi; Muramatsu, Aimi; Shimura, Mari; Kobayashi-Hattori, Kazuo; Oishi, Yuichi

    2016-07-01

    In this study, we investigated the effect of TGF-β1 on cholesterol synthesis in human keratinocytes. TGF-β1 increased the level of cholesterol and the mRNA level of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase in human keratinocytes. These results show that TGF-β1 induces cholesterol synthesis by increasing HMG-CoA reductase mRNA expression in human keratinocytes. PMID:26932266

  9. Explore the dynamic alternation of gene PLAC4 mRNA expression levels in maternal plasma in second trimester for nonivasive detection of trisomy 21

    PubMed Central

    Sun, Hai-Yan; Chen, Dao-Zhen; Lu, Mu-Dan; Tang, Ye; Xiao, Jian-Pin

    2015-01-01

    Objective Noninvasive prenatal detection of trisomy 21 (T21) has been achieved by measuring the ratio of two alleles of a single nucleotide polymorphism in circulating placenta specific 4 (PLAC4) mRNA in maternal plasma with a few assays in recent years. Our research is to explore the variations of PLAC4 mRNA expression level in maternal plasma with normal pregnancies in second trimester, which can provide pregnant women deeper insights with suitable detection period for the non-invasive prenatal detection of T21. Methods We measured a serial plasma PLAC4 mRNA concentrations weekly from the same 25 singleton normal pregnant women. We recruited maternal plasma samples from 45 singleton pregnant women, comprising of 25 euploid pregnancies (control group; range, 17 to 21 weeks) and 20 T21 pregnancies (T21 group; range, 19 to 24 weeks). With the application of reverse transcription polymerase chain reaction, we achieved an insight of PLAC4 mRNA expression levels in maternal plasma during second trimester with euploid pregnancies. Results Among the control group, the levels of PLAC4 mRNA expression in the gestation of 17 to 18 weeks were significantly less than those in the gestation of 18 to 21 weeks (P<0.05). The average PLAC4 mRNA concentration of the normal pregnant women was not higher than that of the T21 group (P>0.05). Conclusion The PLAC4 mRNA showed a higher level of expression in the gestation of 18 to 21 weeks with an euploid pregnancy of pregnant women. We also found that there was no significant difference in plasma PLAC4 mRNA concentration between the normal and the T21 pregnancies in second trimester. PMID:26217595

  10. The cell cycle-coupled expression of topoisomerase IIalpha during S phase is regulated by mRNA stability and is disrupted by heat shock or ionizing radiation.

    PubMed Central

    Goswami, P C; Roti Roti, J L; Hunt, C R

    1996-01-01

    Topoisomerase II is a multifunctional protein required during DNA replication, chromosome disjunction at mitosis, and other DNA-related activities by virtue of its ability to alter DNA supercoiling. The enzyme is encoded by two similar but nonidentical genes: the topoisomerase IIalpha and IIbeta genes. In HeLa cells synchronized by mitotic shake-off, topoisomeraseII alpha mRNA levels were found to vary as a function of cell cycle position, being 15-fold higher in late S phase (14 to 18 h postmitosis) than during G1 phase. Also detected was a corresponding increase in topoisomerase IIalpha protein synthesis at 14 to 18 h postmitosis which resulted in significantly higher accumulation of the protein during S and G2 phases. Topoisomerase IIalpha expression was not dependent on DNA synthesis during S phase, which could be inhibited without effect on the timing or level of mRNA expression. Mechanistically, topoisomerase IIalpha expression appears to be coupled to cell cycle position mainly through associated changes in mRNA stability. When cells are in S phase and mRNA levels are maximal, the half-life of topoisomerase IIalpha mRNA was determined to be approximately 30 min. A similar decrease in mRNA stability was also induced by two external factors known to delay cell cycle progression. Treatment of S-phase cells, at the time of maximum topoisomerase IIalpha mRNA stability, with either ionizing radiation (5 Gy) or heat shock (45 degrees C for 15 min) caused the accumulated topoisomerase IIalpha mRNA to decay. This finding suggests a potential relationship between stress-induced decreases in topoisomerase IIalpha expression and cell cycle progression delays in late S/G2. PMID:8657123

  11. Mouse Endometrium Temporal and Spatial Expression mRNA and MicroRNA Associated With Embryo Implantation.

    PubMed

    Chen, Ke; Chen, Xuemei; He, Junlin; Ding, Yubin; Geng, Yanqing; Liu, Shangjing; Liu, Xueqing; Wang, Yingxiong

    2015-11-01

    Embryo implantation is a dynamic physiological process involving morphological and molecular changes in the endometrium during the pre-receptivity, receptivity, and implantation phases. A comprehensive analysis of messenger RNA (mRNA) and microRNA (miRNA) profiles during implantation will likely provide new clues to elucidate the underlying mechanisms governing embryo implantation. We characterized the mRNA and miRNA transcriptomes using next generation sequencing (NGS) of the endometrium 1 day postcoitum (dpc) and 4dpc and the implantation site (IMS) and inter-implantation (IIM) site of the endometrium on 5dpc. Real-time quantitative polymerase chain reaction was performed on selected miRNAs and their predicted target mRNAs to validate their negatively correlated expression. Statistical analysis of the data based on Gene Ontology (GO) group annotation and Kyoto Encyclopedia of Genes and Genomes pathway analysis demonstrated that the genes with significant expression at the IIM site were primarily involved in glucose, protein, and lipoprotein metabolism to provide energy for embryo implantation, while the genes identified at the IMS were involved in RNA functions to produce proteins in support of embryo development and trophoblast invasion. Extracellular matrix (ECM)-receptor interactions between cells and the ECM was the most remarkable event during implantation. The miRNA-mRNA interaction network unraveled the regulatory relationship between miRNAs and mRNAs. Hub miRNAs (mmu-miR-96 and mmu-miR-200b) were identified to target B-cell lymphoma 2 (Bcl-2), Kruppel-like factor 13 (Klf13), and Progesterone receptor (PGR), which are associated with the preparation of the receptive condition or the maintenance of early pregnancy. PMID:25862677

  12. Decreased mRNA and protein expression of TWIST1 in myocardial tissue of fetuses with ventricular septal defects.

    PubMed

    Wang, Yuting; Wang, Qidi; Guo, Changlong; Wang, Shuo; Qiu, Yue; Li, Hui; Ma, Xu

    2015-08-01

    Ventricular septal defect (VSD) is the most common type of congenital heart disease (CHD). The single gene mutations or absences that contribute to VSD development are well established; however, the aim of the present study was to measure gene expression variation between VSDs and normal fetal myocardial tissue. TWIST1, an important tumor biomarker, is a basic helix-loop-helix transcription factor that regulates cell proliferation, migration and differentiation in embryonic development and transformed tumor cells. Although growing evidence demonstrates that TWIST1 participates in a variety of human neoplastic diseases, the role of TWIST1 in VSD has remained elusive. Twenty-six VSD fetal myocardial tissue samples and 12 normal samples at matched gestational weeks (22-28 weeks) were included in the present study. Using reverse transcription quantitative polymerase chain reaction (PCR) and real-time PCR, it was demonstrated that TWIST1 mRNA was reduced by almost two-fold in the VSD samples compared with the normal samples. Western blot analysis also revealed that TWIST1 expression was decreased by ~three-fold (P=0.001) in the VSD samples compared with that in the normal samples. Of note, five complete ventricular (also called functionally univentricular or single ventricular) septal ageneses were identified among the specimens. For the five complete ventricular septal agenesis samples, similar results to those for other VSD fetal myocardial tissues were obtained. In conclusion, the results of the present study showed that TWIST1 mRNA and protein levels were reduced in VSDs. The present study was the first, to the best of our knowledge, to report that TWIST1 is not only a tumor biomarker, but may also be involved in the pathogenesis of VSD. PMID:25955272

  13. 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. PMID:26849075

  14. Disruption of subcellular Arc/Arg 3.1 mRNA expression in striatal efferent neurons following partial monoamine loss induced by methamphetamine

    PubMed Central

    Barker-Haliski, Melissa L.; Oldenburger, Katharina; Keefe, Kristen A.

    2012-01-01

    The immediate-early gene Arc (activity-regulated cytoskeleton-associated protein) is provocative in the context of neuroplasticity because of its experience-dependent regulation and mRNA transport to and translation at activated synapses. Normal rats have more preproenkephalin-negative (ppe-neg; presumed striatonigral) neurons with cytoplasmic Arc mRNA than ppe-positive (ppe-pos; striatopallidal) neurons, despite equivalent numbers of these neurons showing novelty-induced transcriptional activation of Arc. Furthermore, rats with partial monoamine loss induced by methamphetamine (METH) show impaired Arc mRNA expression in both ppe-neg and ppe-pos neurons relative to normal animals following response-reversal learning. In this study, Arc expression induced by exposure to a novel environment was used to assess transcriptional activation and cytoplasmic localization of Arc mRNA in striatal efferent neuron subpopulations subsequent to METH-induced neurotoxicity. Partial monoamine depletion significantly altered Arc expression. Specifically, basal Arc expression was elevated, but novelty-induced transcriptional activation was abolished. Without novelty-induced Arc transcription, METH-pretreated rats also had fewer neurons with cytoplasmic Arc mRNA expression, with the effect being greater for ppe-neg neurons. Thus, METH-induced neurotoxicity substantially alters striatal efferent neuron function at the level of Arc transcription, suggesting a long-term shift in basal ganglia neuroplasticity processes subsequent to METH-induced neurotoxicity. Such changes potentially underlie striatally-based learning deficits associated with METH-induced neurotoxicity. PMID:22978492

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

    PubMed Central

    Kripp, Melanie; Merx, Kirsten; Wirtz, Ralph Markus; Gaiser, Timo; Eidt, Sebastian; Schwaab, Juliana; Post, Stefan; Wenz, Frederik; Hochhaus, Andreas

    2016-01-01

    Background. No predictive or prognostic biomarker is available for patients with locally advanced rectal cancer (LARC) undergoing perioperative chemoradiotherapy (CRT). Members of the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases EGFR (HER1, ERBB1), HER2 (ERBB2), HER3 (ERBB3), and HER4 (ERBB4) are therapeutic targets in several cancers. The analysis was performed to assess expression levels and study the potential prognostic impact for disease-free and overall survival in patients with LARC. Patients and Methods. ERBB1–4 mRNA expression and tumor proliferation using Ki-67 (MKI67) mRNA were evaluated using RT-quantitative PCR in paraffin-embedded tumor samples from 86 patients (median age: 63) treated with capecitabine or 5-fluorouracil-based CRT within a phase 3 clinical trial. Results. A positive correlation of HER4 and HER2, HER3 and HER2, and HER4 and HER3 with each other was observed. Patients with high mRNA expression of ERBB1 (EGFR, HER1) had significantly increased risk for recurrence and death. Patients with high mRNA expression of MKI67 had reduced risk for relapse. Conclusion. This analysis suggests a prognostic impact of both ERBB1 and MKi67 mRNA expression in LARC patients treated with capecitabine or fluorouracil-based chemoradiotherapy. PMID:27610130

  16. The mRNA Expression Status of Dopamine Receptor D2, Dopamine Receptor D3 and DARPP-32 in T Lymphocytes of Patients with Early Psychosis

    PubMed Central

    Cui, Yin; Prabhu, Vishwanath; Nguyen, Thong Ba; Yadav, Binod Kumar; Chung, Young-Chul

    2015-01-01

    Peripheral blood lymphocytes are an attractive tool because there is accumulating evidence indicating that lymphocytes may be utilized as a biomarker in the field of psychiatric study as they could reveal the condition of cells distributed in the brain. Here, we measured the mRNA expression status of dopamine receptor D2 (DRD2), DRD3, and dopamine and cyclic adenosine 3′,5′-monophosphate regulated phosphoprotein-32 (DARPP-32) in T lymphocytes of patients with early psychosis by quantitative real-time polymerase chain reaction (q-PCR) and explored the relationships between their mRNA levels and the psychopathological status of patients. The present study demonstrated that the mRNA expression levels of DRD3 in T lymphocytes were significantly different among controls, and in patients with psychotic disorder not otherwise specified (NOS) and schizophrenia/schizophreniform disorder. However, no significant differences in mRNA expression levels of DRD2 and DARPP-32 were found among the three groups. We found a significant positive correlation between the DRD2 mRNA level and the score of the excited factor of the Positive and Negative Syndrome Scale (PANSS) in patients with schizophrenia/schizophreniform disorder. These findings suggest that DRD3 mRNA levels may serve as a potential diagnostic biomarker differentiating patients with early psychosis from controls. PMID:26561806

  17. cDNA cloning and mRNA expression of canine pancreatic and duodenum homeobox 1 (Pdx-1).

    PubMed

    Takemitsu, Hiroshi; Yamamoto, Ichiro; Lee, Peter; Ohta, Taizo; Mori, Nobuko; Arai, Toshiro

    2012-10-01

    Pancreatic and duodenal homeobox 1 (Pdx-1) is a critical insulin transcription factor expressed by pancreatic β-cells, and is crucial in the early stage of pancreas development. Unfortunately, nothing concerning Pdx-1 in canine has been elucidated yet. In this study, full length canine Pdx-1 cDNA was cloned and it was 1498 bp in length, consisting of a 99 bp 5'-untranslated region (UTR), a 849 bp coding region, and a 550 bp 3'-UTR region. A deduced 282 amino acid sequence of canine PDX-1 displayed high overall sequence identity with human, bovine, and mouse PDX-1. qRT-PCR analysis revealed that a high level of Pdx1 mRNA expression is exists in the duodenum and pancreas of canines. In addition, functional canine insulin promoter-luciferase reporter constructs with various canine insulin promoter region fragments revealed that our Pdx-1 isolated cDNA sequence encodes for a functionally active PDX-1 protein. Significant promoter activity was observed within the -583 bp 5'-upstream region of canine insulin gene with Chinese hamster ovary cells. In addition, Pdx-1 appears to have a synergistic effect with beta cell transactivator 2 (BETA2) and V-maf avian musculoaponeurotic fibrosarcoma oncogene homolog A (MafA), which also have important roles in the activation of the insulin gene promoter. Our results confirm that similar to humans, Pdx1 plays an important role in expression of insulin gene in canines. PMID:22172402

  18. Integrated Analysis of DNA Methylation and mRNA Expression Profiles Data to Identify Key Genes in Lung Adenocarcinoma.

    PubMed

    Jin, Xiang; Liu, Xingang; 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

  19. 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

  20. 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. PMID:22706063

  1. Quantification of Diatom Gene Expression in the Sea by Selecting Uniformly Transcribed mRNA as the Basis for Normalization

    PubMed Central

    Kang, Lee-Kuo; Tsui, Feng-Hsiu

    2012-01-01

    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. PMID:22706063

  2. Metagenome and mRNA expression analyses of anaerobic methanotrophic archaea of the ANME-1 group.

    PubMed

    Meyerdierks, Anke; Kube, Michael; Kostadinov, Ivaylo; Teeling, Hanno; Glöckner, Frank Oliver; Reinhardt, Richard; Amann, Rudolf

    2010-02-01

    Microbial consortia mediating the anaerobic oxidation of methane with sulfate are composed of methanotrophic Archaea (ANME) and Bacteria related to sulfate-reducing Deltaproteobacteria. Cultured representatives are not available for any of the three ANME clades. Therefore, a metagenomic approach was applied to assess the genetic potential of ANME-1 archaea. In total, 3.4 Mbp sequence information was generated based on metagenomic fosmid libraries constructed directly from a methanotrophic microbial mat in the Black Sea. These sequence data represent, in 30 contigs, about 82-90% of a composite ANME-1 genome. The dataset supports the hypothesis of a reversal of the methanogenesis pathway. Indications for an assimilatory, but not for a dissimilatory sulfate reduction pathway in ANME-1, were found. Draft genome and expression analyses are consistent with acetate and formate as putative electron shuttles. Moreover, the dataset points towards downstream electron-accepting redox components different from the ones known from methanogenic archaea. Whereas catalytic subunits of [NiFe]-hydrogenases are lacking in the dataset, genes for an [FeFe]-hydrogenase homologue were identified, not yet described to be present in methanogenic archaea. Clustered genes annotated as secreted multiheme c-type cytochromes were identified, which have not yet been correlated with methanogenesis-related steps. The genes were shown to be expressed, suggesting direct electron transfer as an additional possible mode to shuttle electrons from ANME-1 to the bacterial sulfate-reducing partner. PMID:19878267

  3. 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

  4. HER-2 and EGFR mRNA Expression and Its Relationship with Versican in Malignant Matrix-Producing Tumors of the Canine Mammary Gland.

    PubMed

    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. The p38 MAPK Regulates IL-24 Expression by Stabilization of the 3′ UTR of IL-24 mRNA

    PubMed Central

    Otkjaer, Kristian; Holtmann, Helmut; Kragstrup, Tue Wenzel; Paludan, Søren Riis; Johansen, Claus; Gaestel, Matthias; Kragballe, Knud; Iversen, Lars

    2010-01-01

    Background IL-24 (melanoma differentiation-associated gene-7 (mda-7)), a member of the IL-10 cytokine family, possesses the properties of a classical cytokine as well as tumor suppressor effects. The exact role of IL-24 in the immune system has not been defined but studies have indicated a role for IL-24 in inflammatory conditions such as psoriasis. The tumor suppressor effects of IL-24 include inhibition of angiogenesis, sensitization to chemotherapy, and p38 mitogen-activated protein kinase (MAPK)-mediated apoptosis. Current knowledge on the regulation of IL-24 expression is sparse. Previous studies have suggested that mRNA stabilization is of major importance to IL-24 expression. Yet, the mechanisms responsible for the regulation of IL-24 mRNA stability remain unidentified. As p38 MAPK is known to regulate gene expression by interfering with mRNA degradation we examined the role of p38 MAPK in the regulation of IL-24 gene expression in cultured normal human keratinocytes. Methodology/Principal Findings In the present study we show that anisomycin- and IL-1β- induced IL-24 expression is strongly dependent on p38 MAPK activation. Studies of IL-24 mRNA stability in anisomycin-treated keratinocytes reveal that the p38 MAPK inhibitor SB 202190 accelerates IL-24 mRNA decay suggesting p38 MAPK to regulate IL-24 expression by mRNA-stabilizing mechanisms. The insertion of the 3′ untranslated region (UTR) of IL-24 mRNA in a tet-off reporter construct induces degradation of the reporter mRNA. The observed mRNA degradation is markedly reduced when a constitutively active mutant of MAPK kinase 6 (MKK6), which selectively activates p38 MAPK, is co-expressed. Conclusions/Significance Taken together, we here report p38 MAPK as a regulator of IL-24 expression and determine interference with destabilization mediated by the 3′ UTR of IL-24 mRNA as mode of action. As discussed in the present work these findings have important implications for our understanding of IL-24 as a

  6. The N Terminus of Andes Virus L Protein Suppresses mRNA and Protein Expression in Mammalian Cells

    PubMed Central

    Heinemann, Patrick; Schmidt-Chanasit, Jonas

    2013-01-01

    Little is known about the structure and function of the 250-kDa L protein of hantaviruses, although it plays a central role in virus genome transcription and replication. When attempting to study Andes virus (ANDV) L protein in mammalian cells, we encountered difficulties. Even in a strong overexpression system, ANDV L protein could not be detected by immunoblotting. Deletion analysis revealed that the 534 N-terminal amino acid residues determine the low-expression phenotype. Inhibition of translation due to RNA secondary structures around the start codon, rapid proteasomal degradation, and reduced half-life time were excluded. However, ANDV L protein expression could be rescued upon mutation of the catalytic PD-E-K motif and further conserved residues of the putative endonuclease at the N terminus of the protein. In addition, wild-type ANDV L rather than expressible L mutants suppressed the level of L mRNA, as well as reporter mRNAs. Wild-type L protein also reduced the synthesis of cellular proteins in the high-molecular-weight range. Using expressible ANDV L mutants as a tool for localization studies, we show that L protein colocalizes with ANDV N and NSs but not Gc protein. A fraction of L protein also colocalized with the cellular processing (P) body component DCP1a. Overall, these data suggest that ANDV L protein possesses a highly active endonuclease at the N terminus suppressing the level of its own as well as heterologous mRNAs upon recombinant expression in mammalian cells. PMID:23576516

  7. Feather follicles express two classes of pro-opiomelanocortin (POMC) mRNA using alternative promoters in chickens.

    PubMed

    Yoshihara, Chihiro; Tashiro, Yuichi; Taniuchi, Shusuke; Katayama, Harumi; Takahashi, Sumio; Takeuchi, Sakae

    2011-03-01

    Feather coloration in chickens mainly depends on melanin produced by melanocytes located in the feather follicles. The melanocortin 1 receptor (MC1R) on follicular melanocytes regulates melanin synthesis; however, the source of the melanocortins that interact with the receptors remains unclear. In this study, we examine the potential expression of melanocortins and characterize the mRNAs for the precursor pro-opiomelanocortin (POMC) in chicken feather follicles. Reverse transcription-polymerase chain reaction (RT-PCR) revealed the expression of mRNAs for POMC, prohormone convertase 1 (PC1) and PC2, and western blotting detected adrenocorticotropic hormone (ACTH)-related products of POMC processing in feather follicles, suggesting that melanocortins are produced locally in the tissues of chickens. A combination of 5'RACE (rapid amplification of cDNA 5' end), 3'RACE and RT-PCR analyzes identified two classes of POMC mRNA, class a and class b, which encode the same full-length POMC protein but have different non-coding leader exons. Class a mRNAs were expressed specifically in feather follicles, whereas class b mRNAs were expressed in the pituitary, hypothalamus, and various peripheral tissues that we examined. Within the feather follicles, the class a mRNAs were distributed in epidermal layers from middle to distal locations, whereas the class b mRNAs were mainly expressed in pulp at proximal locations. Our findings suggest that feather pigmentation is regulated by locally produced melanocortins, and indicate that the melanocortins encoded by the different classes of POMC mRNAs may play different intra-follicular roles in chickens. This is the first report that demonstrates alternative promoter usage generating different full-length POMC mRNAs in vertebrates. PMID:21187100

  8. Thymidine phosphorylase mRNA expression may be a predictor of response to post-operative adjuvant chemotherapy with S-1 in patients with stage III colorectal cancer.

    PubMed

    Ogawa, Masaichi; Watanabe, Michiaki; Mitsuyama, Yoshinobu; Anan, Tadashi; Ohkuma, Masahisa; Kobayashi, Tetsuya; Eto, Ken; Yanaga, Katsuhiko

    2014-12-01

    The aim of the present study was to investigate markers in surgically resected specimens of colorectal cancer that can be used to predict the response to chemotherapy. The mRNA expression levels of enzymes involved in 5-fluorouracil (5-FU) metabolism and folate metabolism were measured in formalin-fixed, paraffin-embedded tumor sections obtained from the primary tumors of 54 patients with resected stage II or III colorectal cancer who received S-1 for one year. The 5-FU metabolizing enzymes studied were thymidylate synthase, dihydropyrimidine dehydrogenase and thymidine phosphorylase (TP). The folate metabolizing enzymes studied were folypolyglutamate synthetase, γ-glutamyl hydrolase and dihydrofolate reductase. The associations between the mRNA expression levels of these enzymes and clinical variables were investigated. Tumors were classified as exhibiting high or low expression as compared with the median mRNA expression level of each metabolizing enzyme defined as the cutoff value. The associations between the high and low expression levels of each enzyme and disease-free survival (DFS) were analyzed with the use of Kaplan-Meier curves and the log-rank test. DFS was not significantly associated with the relative mRNA expression level of any metabolizing enzyme in the study group as a whole, but there was a trend toward longer DFS in patients with high TP expression (P=0.066). In patients with stage III colorectal cancer, high TP expression was associated with significantly improved outcomes compared with low TP expression (P=0.039). These results indicate that the mRNA expression of TP, a metabolizing enzyme of 5-FU, is a significant predictor of response to post-operative chemotherapy with S-1 in patients with stage III colorectal cancer. PMID:25364408

  9. Analysis of Differential Expression Patterns of mRNA and Protein During Cold-acclimation and De-acclimation in Arabidopsis*

    PubMed Central

    Nakaminami, Kentaro; Matsui, Akihiro; Nakagami, Hirofumi; Minami, Anzu; Nomura, Yuko; Tanaka, Maho; Morosawa, Taeko; Ishida, Junko; Takahashi, Satoshi; Uemura, Matsuo; Shirasu, Ken; Seki, Motoaki

    2014-01-01

    Overwintering plants are capable of exhibiting high levels of cold tolerance, which is acquired through the process of cold acclimation (CA). In contrast to CA, the acquired freezing tolerance is rapidly reduced during cold de-acclimation (DA) and plants resume growth after sensing warm temperatures. In order to better understand plant growth and development, and to aid in the breeding of cold-tolerant plants, it is important to decipher the functional mechanisms of the DA process. In this study, we performed comparative transcriptomic and proteomic analyses during CA and DA. As revealed by shotgun proteomics, we identified 3987 peptides originating from 1569 unique proteins and the corresponding mRNAs were analyzed. Among the 1569 genes, 658 genes were specifically induced at the transcriptional level during the process of cold acclimation. In order to investigate the relationship between mRNA and the corresponding protein expression pattern, a Pearson correlation was analyzed. Interestingly, 199 genes showed a positive correlation of mRNA and protein expression pattern, indicating that both their transcription and translation occurred during CA. However, 226 genes showed a negative correlation of mRNA and protein expression pattern, indicating that their mRNAs were transcribed during CA and were stored for the subsequent DA step. Under this scenario, those proteins were specifically increased during DA without additional transcription of mRNA. In order to confirm the negative correlation of mRNA and protein expression patterns, qRT-PCR and western blot analyses were performed. Mitochondrial malate dehydrogenase 1 (mMDH1) exhibited a negative correlation of mRNA and protein levels, which was characterized by CA-specific mRNA induction and protein accumulation specifically during DA. These data indicate that the expression of specific mRNAs and subsequent accumulation of corresponding proteins are not always in accordance under low temperature stress conditions in

  10. Oocyte-Specific Expression of Mouse MEX3C652AA in the Ovary and Its Potential Role in Regulating Maternal Fos mRNA.

    PubMed

    Li, Xue; Li, Yan; Liu, Chunlian; Jin, Mulan; Lu, Baisong

    2016-05-01

    Currently, the human MEX3C gene is known to encode an RNA-binding protein of 659 amino acid residues. Here we show that the MEX3C gene has alternative splicing forms giving rise to multiple MEX3C variants, and some cells express MEX3C transcripts coding for short MEX3C isoforms but not transcripts for MEX3C(659AA) MEX3C(659AA) functions as an adaptor protein for Exportin 1 (XPO1)-mediated nuclear export since it increases the cytoplasmic distribution of poly(A)(+) RNA and since addition of the nuclear export signal (NES) sequence to a short MEX3C isoform MEX3C(464AA) confers similar cytoplasmic poly(A)(+) RNA accumulation activity as MEX3C(659AA) FOS mRNA is a potential MEX3C target mRNA. One mechanism by which MEX3C(659AA) could regulate FOS mRNA is by promoting its nuclear export. Overexpressing MEX3C(659AA) significantly increased FOS mRNA expression, whereas mutating the NES of MEX3C(659AA) and treating cells with leptomycin B to inhibit XPO1-mediated nuclear export attenuated FOS upregulation. FOS mRNA is unstable in somatic cells but less so in oocytes; how it is stabilized in the oocytes is unknown. Transcripts for the mouse counterpart of human MEX3C(659AA) (MEX3C(652AA)) are specifically expressed in developing oocytes in the ovary, although total Mex3c transcripts are expressed in both granulosa cells and oocytes. The specific expression of this long MEX3C isoform in oocytes and its ability to enhance FOS mRNA nuclear export and stability all suggest that MEX3C(659AA) is an RNA-binding protein that preserves maternal FOS mRNA in oocytes. PMID:27053362

  11. Augmentation of Metastin/Kisspeptin mRNA Expression by the Proestrous Luteinizing Hormone Surge in Granulosa Cells of Rats: Implications for Luteinization.

    PubMed

    Laoharatchatathanin, Titaree; Terashima, Ryota; Yonezawa, Tomohiro; Kurusu, Shiro; Kawaminami, Mitsumori

    2015-07-01

    Variations in mRNA levels and sources of metastin/kisspeptin, neurokinin B (NKB), dynorphin, and kisspeptin receptor GPR54 were examined in the ovaries of cycling rats. Kisspeptin and dynorphin mRNAs dramatically increased at 2000 h of the proestrous day. NKB mRNA also increased, but the peak was delayed by 6 h. GPR54 mRNA declined inversely with kisspeptin. Whole-ovary expressions of kisspeptin and dynorphin mRNAs, but not of NKB mRNA, were augmented by the administration of human chorionic gonadotropin (hCG). By means of laser-capture microdissection, kisspeptin mRNA was shown mostly in follicles at 2000 h of proestrus, whereas NKB and dynorphin were expressed mainly in interstitial tissues. GPR54 mRNA was detected equally in follicles, corpora lutea, and interstitial tissues. The hCG stimulated the follicular expression of kisspeptin and interstitial tissue expression of dynorphin mRNA. In primary cultures of granulosa cells prepared from equine chorionic gonadotropin-pretreated immature rats, hCG stimulated the expression of kisspeptin, dynorphin, and NKB mRNAs. Distortion of the corpus luteum and surrounding tissue borders was sometimes seen after intra-ovarian bursa administration of kisspeptin antagonist p234 for 3 days from proestrus. Progesterone production stimulated by hCG in granulosa cell culture was suppressed by p234. These data demonstrate that significant amounts of kisspeptin are synthesized in granulosa cells and dynorphin in interstitial tissues, in response to the proestrous luteinizing hormone surge, whereas granulosa cells also contain dynorphin and NKB, suggesting at least a role for kisspeptin in the luteinization of granulosa cells. PMID:25995272

  12. Prenylation differentially inhibits insulin-dependent immediate early gene mRNA expression.

    PubMed

    Franklin, J Lee; Amsler, Maggie O; Messina, Joseph L

    2016-06-01

    Increased activity of prenyl transferases is observed in pathological states of insulin resistance, diabetes, and obesity. Thus, functional inhibitors of farnesyl transferase (FTase) and geranylgeranyl transferase (GGTase) may be promising therapeutic treatments. We previously identified insulin responsive genes from a rat H4IIE hepatoma cell cDNA library, including β-actin, EGR1, Pip92, c-fos, and Hsp60. In the present study, we investigated whether acute treatment with FTase and GGTase inhibitors would alter insulin responsive gene initiation and/or elongation rates. We observed differential regulation of insulin responsive gene expression, suggesting a differential sensitivity of these genes to one or both of the specific protein prenylation inhibitors. PMID:27086854

  13. Evaluation of the mRNA and Protein Expressions of Nutritional Biomarkers in the Gastrointestinal Mucosa of Patients with Small Intestinal Disorders.

    PubMed

    Nakamura, Masanao; Hirooka, Yoshiki; Watanabe, Osamu; Yamamura, Takeshi; Funasaka, Kohei; Ohno, Eizaburo; Miyahara, Ryoji; Kawashima, Hiroki; Shimoyama, Yoshie; Goto, Hidemi

    2016-01-01

    Objective The objectives of this study were to investigate the mRNA and protein expression of biomarkers related to absorption in the small intestinal mucosa of humans and determine the relationships between small intestinal diseases and nutrition. Methods The study subjects consisted of patients scheduled to undergo double-balloon endoscopy (DBE) or total colonoscopy for suspected gastrointestinal disorder in a clinical practice. Biopsies were taken from apparently normal mucosa in the visible areas of 6 parts of the intestines from the duodenum to the colon. The mRNA expression of specific biomarkers (SGLT1, SGLT5, GIP, GLP, LAT1, LAT2, and NPC1L1) in the mucosa was compared among three patient groups: Inflammation, Tumor, and Control. Results Sixty-six patients participated in this study. Both routes of DBE were performed in 20 patients, in whom biopsy samples were obtained from the mucosa for all sections. There were no remarkable differences in the mRNA expression levels among the 3 groups. However, SGLT1, GIP, GLP, and NPC1L1 exhibited specific distribution patterns. The expression levels of GIP and NPC1L1 were highest in the upper jejunum, but were extremely low in the terminal ileum and colon. A comparison of the mRNA expression profile in each intestinal section revealed that the SGLT1 mRNA expression in the Tumor group and the GIP mRNA expression in the Inflammation group were significantly higher than the corresponding levels in the Control group in the upper jejunum. Conclusion The gastrointestinal mucosa of patients with small bowel diseases can maintain proper nutrient absorption, except in the upper jejunum. PMID:27522989

  14. Relationship between mRNA expression of splice forms of the zeta1 subunit of the N-methyl-D-aspartate receptor and spatial memory in aged mice.

    PubMed

    Das, Siba R; Magnusson, Kathy R

    2008-05-01

    Age-related changes in the protein and mRNA expression of some of the splice forms of the zeta1 (NR1) subunit of the NMDA receptor have been seen in mice and rats. The present study was designed to determine whether individual splice forms of the zeta1 subunit of the NMDA receptor within prefrontal/frontal cortical regions contribute to memory deficits during aging and whether experience in learning tasks can influence the expression of the splice forms. mRNA expression of 4 splice forms (zeta1-1, zeta1-3, zeta1-a and zeta1-b) and mRNA for all known splice forms (zeta1-pan) were examined by in situ hybridization. mRNA for C-terminal splice forms, zeta1-1 (+ C1 and + C2 cassettes) and zeta1-3 (+ C1 and + C2'), showed significant declines during aging in several brain regions even though overall zeta1-pan mRNA expression was not significantly affected by aging. This suggests that these splice forms are more influenced by aging than the subunit as a whole. There was an increase in the expression of zeta1-a (-N1 cassette) splice form in the behaviorally-experienced old mice relative to the younger groups. Old mice with high levels of mRNA expression for the zeta1-a splice form in orbital cortex showed the best performances in the working memory task, but the poorest performances in the cued, associative learning task. These results suggest that there is a complex interaction between zeta1 splice form expression and performance of memory tasks during aging. PMID:18374315

  15. 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

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

    PubMed Central

    Jee, Hyeon-Gun; Kim, Young A; Kim, Ju Han; Xing, Mingzhao; Lee, Kyu Eun

    2016-01-01

    Background BRAFV600E is the most common mutation in papillary thyroid carcinoma (PTC), and it is associated with high-risk prognostic factors. However, the significance of the BRAF mRNA level in PTC remains unknown. We evaluated the significance of BRAF mRNA expression level by analyzing PTC data from The Cancer Genome Atlas (TCGA) database. Methods Data from 499 patients were downloaded from the TCGA database. After excluding other PTC variants, we selected 353 cases of classic PTC, including 193 cases with BRAFV600E and 160 cases with the wild-type BRAF. mRNA abundances were measured using RNA-Seq with the Expectation Maximization algorithm. Results The mean BRAF mRNA level was significantly higher in BRAFV600E patients than in patients with wild-type BRAF (197.6 vs. 179.3, p = 0.031). In wild-type BRAF patients, the mean BRAF mRNA level was higher in cases with a tumor > 2 cm than those with a tumor ≤ 2.0 cm (189.4 vs. 163.8, p = 0.046), and was also higher in cases with lymph node metastasis than in those without lymph node metastasis (188.5 vs. 157.9, p = 0.040). Within BRAFV600E patients, higher BRAF mRNA expression was associated with extrathyroidal extension (186.4 vs. 216.4, p = 0.001) and higher T stage (188.1 vs. 210.2, p = 0.016). Conclusions A higher BRAF mRNA expression level was associated with tumor aggressiveness in classic PTC regardless of BRAF mutational status. Evaluation of BRAF mRNA level may be helpful in prognostic risk stratification of PTC. PMID:27410688

  17. Distinct pattern of matrix metalloproteinase 9 and tissue inhibitor of metalloproteinase 1 mRNA expression in human colorectal cancer and liver metastases.

    PubMed Central

    Zeng, Z. S.; Guillem, J. G.

    1995-01-01

    The matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are perceived as essential for tumour invasion and metastasis. In the present study, we compare the topographical pattern of MMP-9 and TIMP-1 expression in colorectal cancer and liver metastasis by in situ hybridisation. TIMP-1 mRNA was detected in all 26 colorectal cancers examined, while only 18 out of 26 (69.2%) were positive for MMP-9. Both MMP-9 and TIMP-1 mRNA were observed in all ten liver metastases but were absent in three adenomas and in all normal colonic mucosa and liver. There was no association between MMP-9 or TIMP-1 mRNA expression and degree of differentiation or size of Tumours. MMP-9 and TIMP-1 mRNA were similarly observed in the peritumour stroma cells rather than in tumour cells themselves. MMP-9 mRNA positive cells were round and identified as macrophages by immunostaining with an anti-macrophage antibody (KP1), while TIMP-1, mRNA was detected in spindle-shaped stromal cells. In liver metastases, MMP-9 localised within peritumour stroma or at the interface between the tumour stroma and normal liver, whereas TIMP-1 mRNA was located throughout the malignant tumour stroma. Our data demonstrate a distinct pattern of MMP-9 and TIMP-1 mRNA expression in colorectal cancer and liver metastases suggesting distinct cellular origins as well as separate patterns of regulation. Images Figure 1 Figure 2 Figure 4 Figure 5 Figure 6 PMID:7669564

  18. Expression of insulin-like growth factor-II (IGF-II) messenger ribonucleic acid (mRNA), but not IGF-I mRNA, in human preovulatory granulosa cells.

    PubMed

    Geisthovel, F; Moretti-Rojas, I; Asch, R H; Rojas, F J

    1989-11-01

    Increasing evidence suggests that insulin-like growth factors (IGFs) play an important role as intra-ovarian regulators in several mammalian species. Recently, we and others have reported the presence of both IGF-I and IGF-II in human follicular fluid. The source of these follicular IGFs, however, has not been determined. In this study, we have evaluated the possibility that human ovarian granulosa cells are a production site of IGFs in vivo. We used cDNA probes to analyse directly IGF-I and IGF-II gene expression at the level of mRNA content in granulosa cells from preovulatory follicles of women undergoing either gamete intra-Fallopian transfer or in-vitro fertilization. Samples of granulosa cell RNA enriched for polyadenylated RNA [poly(A)+RNA] were hybridized with probes for human IGF-I, human IGF-II and human actin (as a control). Transfer blot analysis revealed that the enriched poly(A)+RNA of human granulosa cells from preovulatory follicles contained no detectable IGF-I mRNA. In contrast, three species of IGF-II mRNA of approximately 6.1, 4.9 and 2.1 kb were detected. These data suggest that IGF-II mRNA, but not IGF-I mRNA, is expressed in human granulosa cells collected immediately before ovulation. Our results support the concept that human ovarian IGF-II is produced locally and may function in an autocrine or paracrine fashion in the human ovary in vivo. PMID:2613863

  19. Significance of Matrix Metalloproteinase 9 Expression as Supporting Marker to Cytokeratin 19 mRNA in Sentinel Lymph Nodes in Breast Cancer Patients

    PubMed Central

    Murawski, Marek; Woźniak, Marta; Duś-Szachniewicz, Kamila; Kołodziej, Paweł; Rzeszutko, Marta; Ziółkowski, Piotr

    2016-01-01

    One-step nucleic acid amplification (OSNA) detects and quantifies, with the use of a polymerase chain reaction, the presence of cytokeratin 19 mRNA in sentinel lymph nodes. The main advantage of the OSNA assay is the avoidance of second surgery in case of positive sentinel lymph node diagnosis. The objective of this study was to evaluate the significance of matrix metalloproteinase 9 expression by immunohistochemistry as supporting marker to cytokeratin 19 mRNA in sentinel lymph nodes in breast cancer patients and to relate this expression with clinicopathological data. This study was conducted on fresh sentinel lymph nodes obtained from 40 patients with tumors classified as carcinoma of no special type. The presence of metastatic cells in the slices of lymph nodes was evaluated by immunohistochemistry using antibodies for CK19 and MMP-9. Expression of CK19 and MMP-9 in lymph nodes was also confirmed by means of Western blot analysis. Results indicated that the strongest correlation with CK19 mRNA was displayed by MMP-9, CK19 (by immunohistochemistry, IHC), and nodal metastases (p < 0.001). Higher histological grading also positively correlated with CK19 mRNA, however that correlation was less significant. Since MMP-9 shows very strong correlation with CK19 mRNA in breast carcinoma of no special type metastases, expression of MMP-9 in sentinel lymph nodes should be considered as useful method whenever OSNA analysis is not available. PMID:27110764

  20. 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

  1. mRNA expression in different developmental stages of the chicken bursa of Fabricius.

    PubMed

    Liu, Xiao-Dong; Zhang, Fubo; Shan, Hu; Wang, Shu-Bai; Chen, Pu-Yan

    2016-08-01

    The bursa of Fabricius, the central humoral immune organ unique to birds, plays an important role in B-lymphocyte differentiation. In order to gain a better understanding of the molecular mechanism of critical biological processes like B-cell immigration, differentiation, and final emigration, the transcriptional changes during embryonic and posthatch development of this organ were investigated. We generated a cDNA library from total RNA isolated from 3 representative developmental stages (embryonic day [ED] 10, posthatch d 2 and d 21). We generated over 70 million high-quality reads from the cDNA library by using deep sequencing. The uniquely mapped sequences of ED 10, d 2 and d 21 were 71087280, 59167491 and 70263675 respectively. All of the differential expressed genes were involved in Vitamin A metabolism, regulation of actin cytoskeleton, Wnt signaling pathway, MAPK signaling pathway, Jak-STAT signaling pathway, Notch signaling pathway, Toll-like receptor signaling pathway. The RNA-seq analysis provides a powerful method for analyzing the transcriptome and investigating the transcriptional changes of different development stages of bursa of Fabricius. The assembled bursa transcriptome provides an essential resource for future investigations about chicken Bursa development. PMID:26994188

  2. Persistent neuronal Ube3a expression in the suprachiasmatic nucleus of Angelman syndrome model mice.

    PubMed

    Jones, Kelly A; Han, Ji Eun; DeBruyne, Jason P; Philpot, Benjamin D

    2016-01-01

    Mutations or deletions of the maternal allele of the UBE3A gene cause Angelman syndrome (AS), a severe neurodevelopmental disorder. The paternal UBE3A/Ube3a allele becomes epigenetically silenced in most neurons during postnatal development in humans and mice; hence, loss of the maternal allele largely eliminates neuronal expression of UBE3A protein. However, recent studies suggest that paternal Ube3a may escape silencing in certain neuron populations, allowing for persistent expression of paternal UBE3A protein. Here we extend evidence in AS model mice (Ube3a(m-/p+)) of paternal UBE3A expression within the suprachiasmatic nucleus (SCN), the master circadian pacemaker. Paternal UBE3A-positive cells in the SCN show partial colocalization with the neuropeptide arginine vasopressin (AVP) and clock proteins (PER2 and BMAL1), supporting that paternal UBE3A expression in the SCN is often of neuronal origin. Paternal UBE3A also partially colocalizes with a marker of neural progenitors, SOX2, implying that relaxed or incomplete imprinting of paternal Ube3a reflects an overall immature molecular phenotype. Our findings highlight the complexity of Ube3a imprinting in the brain and illuminate a subpopulation of SCN neurons as a focal point for future studies aimed at understanding the mechanisms of Ube3a imprinting. PMID:27306933

  3. Persistent neuronal Ube3a expression in the suprachiasmatic nucleus of Angelman syndrome model mice

    PubMed Central

    Jones, Kelly A.; Han, Ji Eun; DeBruyne, Jason P.; Philpot, Benjamin D.

    2016-01-01

    Mutations or deletions of the maternal allele of the UBE3A gene cause Angelman syndrome (AS), a severe neurodevelopmental disorder. The paternal UBE3A/Ube3a allele becomes epigenetically silenced in most neurons during postnatal development in humans and mice; hence, loss of the maternal allele largely eliminates neuronal expression of UBE3A protein. However, recent studies suggest that paternal Ube3a may escape silencing in certain neuron populations, allowing for persistent expression of paternal UBE3A protein. Here we extend evidence in AS model mice (Ube3am–/p+) of paternal UBE3A expression within the suprachiasmatic nucleus (SCN), the master circadian pacemaker. Paternal UBE3A-positive cells in the SCN show partial colocalization with the neuropeptide arginine vasopressin (AVP) and clock proteins (PER2 and BMAL1), supporting that paternal UBE3A expression in the SCN is often of neuronal origin. Paternal UBE3A also partially colocalizes with a marker of neural progenitors, SOX2, implying that relaxed or incomplete imprinting of paternal Ube3a reflects an overall immature molecular phenotype. Our findings highlight the complexity of Ube3a imprinting in the brain and illuminate a subpopulation of SCN neurons as a focal point for future studies aimed at understanding the mechanisms of Ube3a imprinting. PMID:27306933

  4. Global miRNA expression and correlation with mRNA levels in primary human bone cells

    PubMed Central

    Laxman, Navya; Rubin, Carl-Johan; Mallmin, Hans; Nilsson, Olle; Pastinen, Tomi; Grundberg, Elin; Kindmark, Andreas

    2015-01-01

    MicroRNAs (miRNAs) are important post-transcriptional regulators that have recently introduced an additional level of intricacy to our understanding of gene regulation. The aim of this study was to investigate miRNA–mRNA interactions that may be relevant for bone metabolism by assessing correlations and interindividual variability in miRNA levels as well as global correlations between miRNA and mRNA levels in a large cohort of primary human osteoblasts (HOBs) obtained during orthopedic surgery in otherwise healthy individuals. We identified differential expression (DE) of 24 miRNAs, and found 9 miRNAs exhibiting DE between males and females. We identified hsa-miR-29b, hsa-miR-30c2, and hsa-miR-125b and their target genes as important modulators of bone metabolism. Further, we used an integrated analysis of global miRNA–mRNA correlations, mRNA-expression profiling, DE, bioinformatics analysis, and functional studies to identify novel target genes for miRNAs with the potential to regulate osteoblast differentiation and extracellular matrix production. Functional studies by overexpression and knockdown of miRNAs showed that, the differentially expressed miRNAs hsa-miR-29b, hsa-miR-30c2, and hsa-miR-125b target genes highly relevant to bone metabolism, e.g., collagen, type I, α1 (COL1A1), osteonectin (SPARC), Runt-related transcription factor 2 (RUNX2), osteocalcin (BGLAP), and frizzled-related protein (FRZB). These miRNAs orchestrate the activities of key regulators of osteoblast differentiation and extracellular matrix proteins by their convergent action on target genes and pathways to control the skeletal gene expression. PMID:26078267

  5. Molecular cloning, characterization, tissue distribution and mRNA expression changes during the hibernation and reproductive periods of estrogen receptor alpha (ESR1) in Chinese alligator, Alligator sinensis.

    PubMed

    Zhang, Ruidong; Hu, Yuehong; Wang, Huan; Yan, Peng; Zhou, Yongkang; Wu, Rong; Wu, Xiaobing

    2016-10-01

    Chinese alligator, Alligator sinensis, is a critically endangered reptile species unique to China. Little is known about the mechanism of growth- and reproduction-related hormones gene expression in Chinese alligator. Estrogens play important roles in regulating multiple reproduction- and non-reproduction-related functions by binding to their corresponding receptors. Here, the full-length cDNA of estrogen receptor alpha (ERα/ESR1) was cloned and sequenced from Chinese alligator for the first time, which comprises 1764bp nucleotides and encodes a predicted protein of 587 amino acids. Phylogenetic analysis of ESR1 showed that crocodilians and turtles were the sister-group of birds. The results of real-time quantitative PCR indicated that the ESR1 mRNA was widely expressed in the brain and peripheral tissues. In the brain and pituitary gland, ESR1 was most highly transcribed in the cerebellum. But in other peripheral tissues, ESR1 mRNA expression level was the highest in the ovary. Compared with hibernation period, ESR1 mRNA expression levels were increased significantly in the reproductive period (P<0.05) in cerebellum, pituitary gland, liver, spleen, lung, kidney and ovary, while no significant change in other examined tissues (P>0.05). The ESR1 mRNA expression levels changes during the two periods of different tissues suggested that ESR1 might play an important role in mediation of estrogenic multiple reproductive effects in Chinese alligator. Furthermore, it was the first time to quantify ESR1 mRNA level in the brain of crocodilians, and the distribution and expression of ESR1 mRNA in the midbrain, cerebellum and medulla oblongata was also reported for the first time in reptiles. PMID:27212643

  6. Effects of tris(1,3-dichloro-2-propyl) phosphate and triphenyl phosphate on receptor-associated mRNA expression in zebrafish embryos/larvae.

    PubMed

    Liu, Chunsheng; Wang, Qiangwei; Liang, Kang; Liu, Jingfu; Zhou, Bingsheng; Zhang, Xiaowei; Liu, Hongling; Giesy, John P; Yu, Hongxia

    2013-03-15

    Tris(1,3-dichloro-2-propyl) phosphate (TDCPP) and triphenyl phosphate (TPP) are frequently detected in biota, including fish. However, knowledge of the toxicological and molecular effects of these currently used flame retardants is limited. In the present study, an in vivo screening approach was developed to evaluate effects of TDCPP and TPP on developmental endpoints and receptor-associated expression of mRNA in zebrafish embryos/larvae. Exposure to TDCPP or TPP resulted in significantly smaller rates of hatching and survival, in dose- and time-dependent manners. The median lethal concentration (LC(50)) was 7.0 mg/L for TDCPP and 29.6 mg/L for TPP at 120 hour post-fertilization (hpf). Real-time PCR revealed alterations in expression of mRNAs involved in aryl hydrocarbon receptors (AhRs)-, peroxisome proliferator-activated receptor alpha (PPARα)-, estrogenic receptors (ERs)-, thyroid hormone receptor alpha (TRα)-, glucocorticoid receptor (GR)-, and mineralocorticoid receptor (MR)-centered gene networks. Exposure to positive control chemicals significantly altered abundances of mRNA in corresponding receptor-centered gene networks, a result that suggests that it is feasible to use zebrafish embryos/larvae to evaluate effects of chemicals on mRNA expression in these gene networks. Exposure to TDCPP altered transcriptional profiles in all six receptor-centered gene networks, thus exerting multiple toxic effects. TPP was easily metabolized and its potency to change expression of mRNA involved in receptor-centered gene networks was weaker than that of TDCPP. The PPARα- and TRα-centered gene networks migh